MINIATURIZED METHODS AND SYSTEMS FOR SAMPLE SCREENING

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/380,709, filed October 24, 2022, which application is incorporated herein by reference in its entirety.

BACKGROUND

[0002] Sample screening may often be a time consuming and expensive process requiring large quantities of precious samples. Miniaturizing may facilitate efficiently performing sample screening in a high-throughput manner, using small sample volumes, at a high speed. Such methods and systems may be highly valuable and have vast applications in research, diagnostics, and drug discovery.

SUMMARY

[0003] Presented herein are methods and systems for miniaturized high-throughput screening. In some embodiments, the methods comprise providing or obtaining encoded effectors. Encoded effectors may be bound to solid supports. The methods and systems presented herein further facilitate screening encoded effectors against one or more targets, in some cases, by performing assays, for the effects of the effectors on the targets to be assessed and/or profiled. The methods and systems have vast applications in drug discovery, diagnostics, clinical applications, and beyond.

[0004] In an aspect, provided herein is a screening method comprising providing or obtaining a plurality of compartments, wherein a first subset of the plurality of compartments each comprises an effector bound to a scaffold and a barcode for identifying the effector; performing an assay in the plurality of compartments, thereby generating a signal indicative of the activity of the assay or an effect of the effector on the assay; processing a second subset of the plurality of compartments based on the signal to substantially block liquid flow thereto, thereby yielding a third subset of the plurality of compartments, wherein the third subset of the plurality of compartments are substantially open to liquid entry; flowing a liquid into a compartment of the third subset of the plurality of compartments to collect or sort the effector or the barcode, or to elucidate the identity or structure of the effector. [0005] In some embodiments, the compartments are immobilized on a solid surface. In some embodiments, the processing comprises selecting the second subset based on the signal. In some embodiments, the compartments are wells. In some embodiments, the effector and the barcode are bound to the scaffold. In some embodiments, the scaffold is a particle. In some embodiments, the particle comprises a diameter of at least 1 micrometer (pm). In some embodiments, the barcode is a nucleic acid molecule, a DNA, an RNA, a peptide, a molecular weight barcode, or a peptide nucleic acid (PNA). In some embodiments, the compartment further comprises a first reagent for detecting the signal.

[0006] In some embodiments, the method further comprises providing a polymerizable monomer or hydrogel in the plurality of compartments, and processing comprises selectively polymerizing the polymerizable monomer in the second subset of the plurality of compartments by providing a stimulus thereto. In some embodiments, the stimulus comprises an energy, a chemical, or both. In some embodiments, energy comprises at least one of: electrical energy, electromagnetic energy, light, or heat. In some embodiments, the polymerizable monomer or hydrogel is photopolymerizable upon exposure to light.

[0007] In some embodiments, the method further comprises exposing a pattern of light to the plurality of compartments to selectively polymerize the second subset of the plurality of compartments. In some embodiments, the pattern of light is defined based on the signal. In some embodiments, the pattern of light is defined using a digital mask, a digital mirror device (DMD), a 3D printer (e.g., 3D laser printer), an optomechanical device, or any combination thereof.

[0008] In some embodiments, the processing comprises using a membrane or a blocking material based on the signal to substantially block liquid flow to the second subset of the plurality of compartments.

[0009] In some embodiments, the compartment further comprises a cell. In some embodiments, the cell is adhered to the compartment. In some embodiments, the effector is covalently bound to the scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker. In some embodiments, the effector comprises a plurality of subunits of the effector and the barcode comprises a plurality of subunits of the barcode corresponding to the effector or the subunits of the effector. In some embodiments, the liquid comprises an oil immiscible with the contents of the compartment, and the oil collects the effector from a compartment of the third subset of the compartments.

[0010] In one aspect, provided herein is a screening system comprising: (a) a miniaturized device comprising a plurality of compartments, a first subset of the plurality of compartments each comprising an effector bound to a scaffold and a barcode for identifying the effector; (b) a selector; (c) a blocking medium comprising a flowing state configured to allow for liquid flow to the plurality of compartments and a blocking state configured to block liquid flow to the plurality of compartments; and (d) a detector configured to generate a signal indicative of activity or structure of the effector. The selector may be configured to activate the blocking state of the blocking medium in proximity of each compartment of a second subset of the plurality of compartments, based on the signal. In some embodiments, the selector comprises a light source, a microoptoelectromechanical system, or both. In some embodiments, the microoptoelectromechanical system comprises a digital micromirror device. In some embodiments, the light source comprises a UV light source.

[0011] In an aspect, provided herein is a screening system comprising: (a) a device comprising a plurality of compartments, wherein a first subset of the plurality of compartments each comprise an effector bound to a scaffold and a barcode for identifying the effector; (b) a detector configured to generate a signal indicative of activity or structure of the effector; (c) a selector configured to block liquid flow to a second subset of the plurality of compartments based on the signal, thereby yielding a third subset of the plurality of compartments, wherein the third subset of the plurality of compartments are substantially open to liquid entry; and (d) a fluidics module configured to flow a liquid into a compartment of the third subset of the plurality of compartments to collect or sort the effector or the barcode, or to elucidate the identity or structure of the effector.

[0012] In some embodiments, the selector comprises a light source and a microelectromechanical system. In some embodiments, the fluidics module is configured to provide a polymerizable monomer or hydrogel to the plurality of compartments. In some embodiments, the selector is configured to provide a stimulus to the polymerizable monomer or hydrogel located in proximity of the plurality of compartments. In some embodiments, the stimulus is selected from the group consisting of an energy, electrical energy, electromagnetic energy, light, heat, and a chemical. In some embodiments, the polymerizable monomer or hydrogel is photopolymerizable upon exposure to light. In some embodiments, the selector is configured to expose a pattern of light to the plurality of compartments to selectively polymerize the second subset of the plurality of compartments. In some embodiments, the pattern of light is defined based on the signal. In some embodiments, the pattern of light is defined using a digital mask, a digital mirror device (DMD), or both.

[0013] In an aspect, provided herein is a kit comprising: (a) a miniaturized device comprising a plurality of compartments; (b) a library of different unique effectors, wherein each different unique effector is bound to a bead, and wherein the bead further comprises a barcode corresponding to and for identifying the effector; and (c) a blocking medium comprising a polymerizable monomer. The blocking medium may be configured to block liquid flow to the plurality of compartments upon exposure of the blocking medium to a stimulus.

[0014] In an aspect, provided herein is a screening method comprising: (a) providing a plurality of compartments, a subset of the plurality of compartments each comprising: (i) a sample; (ii) an effector; and (iii) a barcode corresponding to and identifying the effector. The method further comprises detecting a signal indicative of an effect of the effector on the sample, wherein the signal is a result of a change of phase in the sample.

[0015] In some embodiments, the method comprises screening a plurality of unique effectors. In some embodiments, the plurality of unique encoded effectors comprises at least 1,000 unique encoded effectors. In some embodiments, the plurality of unique encoded effectors comprises at least 5,000 unique encoded effectors. In some embodiments, the plurality of unique encoded effectors comprises at least 80,000 unique encoded effectors. [0016] In some embodiments, the change of phase is a liquid-liquid phase separation (LLPS). In some embodiments, the signal is indicative of formation of one or more condensates or stress granules (SG) in the sample or in the compartment. In some embodiments, the signal is further indicative of a protein-protein interaction (ppi) or protein- RNA interaction taking place in the sample. In some embodiments, the effect comprises an effect on a protein-protein interaction or protein-RNA interaction. In some embodiments, the signal is further indicative of a protein-RNA interaction taking place in the sample. In some embodiments, the method comprises drug screening and/or drug design for a protein-protein interaction network. In some embodiments, the method comprises drug screening and/or drug design for a protein-RNA interaction network.

[0017] In some embodiments, the sample comprises a biological target. In some embodiments, the sample comprises a cell, and wherein the change in phase takes place inside the cell. In some embodiments, the plurality of compartments comprise a plurality of droplets or a plurality of wells. In some embodiments, the plurality of compartments are a plurality of droplets generated with the aid of a microfluidic device, and the sample is incubated in an incubation line of the microfluidic device. In some embodiments, the change in phase is detected as a spike in the signal over time (e.g., droplet time trace digital or analog signal). [0018] In some embodiments, the barcode is an optical barcode. In some embodiments, the barcode is a nucleic acid molecule. In some embodiments, the barcode comprises an optical barcode and a nucleic acid molecule barcode. In some embodiments, the barcode comprises a deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a peptide, or a peptide nucleic acid (PNA). In some embodiments, the effector is bound to a scaffold. In some embodiments, the effector is covalently bound to a scaffold via a cleavable linker and is releasable upon cleavage of the cleavable linker.

INCORPORATION BY REFERENCE

[0019] U.S. Ser. No. 17/067,534, filed Oct. 9, 2020, is incorporated by reference herein in its entirety and for all purposes. In addition, all publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety and for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0021] FIG. 1 provides a depiction of a bead-bound encoded effector. The bead-bound encoded effector comprises a bead scaffold comprising an effector bound thereto via a cleavable linker and a barcode corresponding to and identifying the effector. Both the effector and the barcode are covalently bound to the bead scaffold.

[0022] FIG. 2A shows an exemplary workflow for screening a bead-bound encoded effector, such as the bead-bound encoded effector shown in FIG. 1.

[0023] FIG. 2B shows an exemplary workflow for screening an encoded effector using an integrated droplet microfluidic device and downstream barcode decoding by sequencing (e.g., NGS decoding analysis).

[0024] FIG. 3 illustrates an exemplary method for amplifying a primer to maximize cellular nucleic acid capture. [0025] FIG. 4 provides a depiction of an exemplary droplet microfluidic device for performing the methods of the present disclosure.

[0026] FIG. 5A provides a depiction of a library of encoded effector beads, wherein the effector is a fluorophore bound to the bead via a cleavable linker, also referred to as an effector- fluorophore.

[0027] FIG. 5B schematically illustrates encapsulating an encoded effector bead in a droplet compartment, wherein the effector is a fluorophore bound to the bead via a photocleavable inker (effector-fluorophore). The workflow illustrates releasing the effector- fluorophore from the bead into the droplet upon photocleavage of the photocleavable linker by exposing the droplet to UV light.

[0028] FIG. 5C provides a depiction of the released encoded effector-fluorophore from FIG. 5B.

[0029] FIG. 5D provides a depiction of the cleavage region or exposure region of a microfluidic device described herein. A UV waveguide is inserted into a channel to expose droplets passing through the exposure region to UV light.

[0030] FIGs. 6A and 6B provide exemplary chemical reactions for activating molecules for photocleavage.

[0031] FIG. 7A depicts an exemplary embodiment of a top/down light source which may be used as part of or in connection with the methods and systems of the present disclosure such as microfluidic devices, arrays, or other systems for screening or bead generation (e.g., photopolymerization).

[0032] FIG. 7B depicts the top/down light source from FIG. 7A over an empty stage on which any one of the devices or systems of the present disclosure may be placed.

[0033] FIG. 7C depicts a cutaway view of the top/down light source from FIG. 7A.

[0034] FIGs. 8A-8C provide, respectively, view from the side, view from the top, and three-dimensional view of an exemplary miniaturized array for sample screening.

[0035] FIG. 9 schematically illustrates an exemplary workflow of a method for surface treatment of a miniaturized array platform to facilitate cell seeding or cell adhesion.

[0036] FIG. 10 depicts a miniaturized array comprising a plurality of wells each comprising a plurality of cells seeded therein and adhered to the bottom surface of the well.

[0037] FIG. 11A depicts a flow cell comprising a plurality of wells immobilized on a solid substrate, each of the wells comprising one or more cells seeded therein, adhered to the bottom surface of the wells, and the direction of fluid flow into and out of the flow cell. [0038] FIG. 11B schematically illustrates a plurality of miniaturized flow cells (e.g., similar to the flow cell shown in FIG. 11 A). The flow cells are immobilized on a solid substrate, each of the flow cells comprises one or more inlet and outlet port(s) connected to fluid lines such as tubes.

[0039] FIG. 12 schematically illustrates an exemplary system and workflow for using an optoelectromechanical system comprising a digital mirror device (DMD) and a flow cell for selective light patterning.

[0040] FIG. 13A depicts an exemplary digital mask for selective light exposure.

[0041] FIG. 13B illustrates an exemplary digital mask overlayed on a miniaturized array using a computer system, a microscope, and a digital mirror device (DMD).

[0042] FIG. 14 provides an example image of a miniaturized platform comprising a plurality of wells, wherein a subset of the plurality of wells are selectively substantially blocked to liquid flow using selective polymerization of a polymerizable monomer in the wells according to the methods of the present disclosure.

[0043] FIG. 15 provides an example image demonstrating compound/fluorophore release from scaffolds (beads) inside the wells of a miniaturized array platform using selective light patterning according to the methods of the present disclosure.

[0044] FIG. 16 provides an example image demonstrating the selective polymerization method in a miniaturized array platform using an optoelectromechanical light exposure system. [0045] FIG. 17 schematically illustrates an exemplary method for enzymatic cleavage and collection of nucleic acid barcodes from beads.

[0046] FIG. 18 schematically illustrates an exemplary method for DNA barcode extension, stripping, and collection from a subset of compartments (e.g., positive compartments) following assay screening and selective polymerization through light patterning.

[00022] FIGs. 19A and 19B provide exemplary data demonstrating condensate/stress granule (SG) formation through liquid-liquid phase separation (LLPS) leading to fluorescence signal redistribution in space as detected from a static compartment via fluorescence microscopy using the methods and systems of the present disclosure. FIG. 19C schematically illustrates LLPS and condensate formation in an assay.

[0047] FIG. 20 provides exemplary data demonstrating condensate/stress granule (SG) formation through liquid-liquid phase separation (LLPS) leading to fluorescence signal redistribution in space as detected from a droplet compartment via laser-induced fluorescence spectroscopy in a system comprising a droplet microfluidic device. [0048] FIGs. 21A and 21B provide an example of condensate detection and quantification (counting) using the methods and systems of the present disclosure.

DETAILED DESCRIPTION

Screening Methods and Systems

[0049] Provided herein are methods and systems for sample screening. In some cases, a sample may comprise a biological/biochemical target, a cell, one or more cellular constituents inside cells or extracted from cells (e.g., cell lysates), deoxyribonucleic-acid (DNA), ribonucleic acid (RNA), messenger RNA, proteins, enzymes, cell-free samples, or other kinds of targets. In some examples, the methods may comprise screening the effects of one or more effectors against one or more samples in a high-throughput, low-material manner. The methods and systems provided herein comprise devices such as miniaturized screening platforms, screening instrumentation (e.g., hardware), computer systems, software programs, reagents, and workflows, which may be used individually or in concert (e.g., using an integrated platform and workflow), to facilitate screening the samples which may in some cases comprise cells. In some examples, screening platforms may comprise droplet microfluidic devices. In some examples, screening platforms may comprise a plurality of wells (e.g., a well array system). Sample screening may be performed for any application, in some cases, for diagnostics, drug discovery and/or development, or various combinations of both such as personalized medicine, precision medicine, and beyond.

[0050] In some cases, sample screening may be performed for drug discovery purposes. For example, to screen one or more drugs, or a library of effectors on one or more samples comprising one or more targets such as to screen effectors to discover drug candidates (e.g., effectors) which may have an intended effect on the target. The sample may be compartmentalized into the compartments of a system (e.g., wells of a plate and/or an array) or into compartments generated by a system (e.g., droplets generated by a microfluidic device or through bulk emulsification). The methods and systems provided herein may be particularly useful for screening encoded effector libraries against targets in miniaturized systems such as droplet microfluidics and/or well array platforms. In some cases, screening may comprise high- throughput screening wherein large numbers of effectors are screened in a shorter period compared to preceding technologies.

[0051] In some embodiments, the present disclosure provides methods and systems for screening encoded effector libraries in miniaturized and compartmentalized platforms. Encoded effector libraries may be according to any encoded effector library described anywhere herein. The miniaturized and compartmentalized screening system may be any screening system described anywhere herein which may comprise a droplet microfluidic device, a miniaturized well array platform, or another platform/device comprising a plurality of discrete or semi-discrete compartments/partitions described anywhere herein. The present disclosure provides a set of bioanalytical toolkits which can be used individually and/or in various combinations to achieve various goals.

[0052] Encoded libraries may comprise bead-bound encoded effector (e.g., chemical effector, molecular effectors, compound/small molecule, peptide, macrocyclic molecules, polymers, RNA, DNA, genes or other types of effectors) libraries in which scaffolds such as beads are used as synthetic substrates and carriers for immobilizing, delivering, locating, tracking, extracting, or otherwise manipulating effectors in order to test or measure their effect on a sample or a target. Effectors and barcodes can be attached to the bead or encapsulated therein to be linked together in space, such that the barcode can provide information regarding the identity or structure of the effector. The effector and barcode may be directly attached to one another. Alternatively, the effector and barcode may be immobilized on the bead but not necessarily bound or attached to one another. The scaffold (e.g., bead resin), effector, and barcode may have many different modalities, versions, and embodiments, as described anywhere herein, which may be mixed and matched together and with the various embodiments of the screening/detection system to perform the methods of the present disclosure.

[0053] In some examples, a scaffold may comprise on or more effectors bound thereto. The one or more effectors may be similar or different. For example, a scaffold may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different effectors attached thereto. In some cases, the synergistic effects of more than one effector can be screened on the sample in the same compartment. For example, a scaffold (e.g., a bead) may comprise effector A and effector B attached thereto via a cleavable linker. Effector A and effector may be attached to one another. Alternatively, effector A and effector B may not be attached to one another. The scaffold may further comprise one or more barcodes. The one or more barcodes may be encoding the structures and/or identities of effector A and effector B. The barcode may comprise one or more sequences each of which may encode each of the effectors. This embodiment may be referred to as “combinatorial effector screening”.

[0054] The methods provided herein may comprise screening encoded effector libraries which comprise a scaffold such as a bead. The bead/scaffold may comprise a bead resin (e.g., hydrogel bead, core-shell bead, TentaGel bead, or any other suitable bead). The bead may further comprise an effector covalently bound to the bead via a cleavable linker and a barcode (oligonucleotide such as DNA or RNA, peptide, or peptide nucleic acid (PNA)) corresponding to and for identifying the effector. In some cases, the barcode may also be linked to the bead via a cleavable linker. In some cases, the barcode may be inside the bead. In some cases, the barcode may comprise or be an optical barcode (e.g., one or more fluorophore(s)/dye, optical particles, and/or both). In some examples, the effector on the bead can be released (e.g., selectively released) to assay its activity against a sample or target. In some examples, the target of a screen may be a protein contained in either a biochemical assay or expressed by a cell.

[0055] Screening encoded effector libraries against targets present in live cells may be challenging. Many conventional cell-based high-throughput assays are limited in the number of compounds that can be economically screened due to the time required to process samples as well as the ability to store and manage a large library of effectors. Additionally, combining encoded effector libraries with conventional microtiter plate-based high-throughput screening (HTS) platforms may be challenging. The volume of reagent required to fill a microtiter plate can place an upper limit on the effective concentration of effector released from a bead. Also, sorting individual beads into a microtiter plate is technically challenging and time consuming. [0056] In some embodiments, provided herein are methods and systems for performing high-throughput cell screening and perturbation analysis at a scale greater than existing/conventional high-throughput screening (HTS) systems. This may be facilitated by miniaturizing the screening system. The methods presented herein provide a plurality of miniaturized compartments/partitions such as droplet-based systems or arrays of wells (e.g., miniaturized wells, microwells, nanowells, picowells, or wells of any size). The plurality of compartments may comprise a plurality of droplets, a plurality of wells, a plurality of fabricated pens (e.g., nano pens), a plurality of miniaturized constructs of miniaturized size, nanovials, container particles (e.g., lab on a particle nanovials). In some cases, by miniaturizing the compartments, the effective concentration of compounds released from a single bead of a given size inside the assay compartment can be increased (e.g., compared to releasing the contents of the same scaffold into a larger compartment). Additionally, the number of assay conditions that can be performed in a unit area can increase by miniaturization of the screening platform.

[0057] In some cases, a miniaturized droplet-based microfluidic platform may be used to perform the screens. Alternatively or in addition, platforms such as array -based platforms, wellbased platforms, micro-raft arrays, and other compartmentalized screening platforms may be used. Some cell types (e.g., some adherent cells) may be more suitably screened while adhered to a solid surface in presence of specific adhesion/ signaling molecules or receptors. Such cells may function more properly when adhered to a solid/semi-solid surface. Such condition may be facilitated by seeding the cells in a static compartment such as a well. Alternatively or in addition, a hydrogel matrix may be used to encapsulate the cell inside a well and/or in a droplet (e.g., in a microfluidic device) to provide a natural micro-environment for the cell.

[0058] In some cases, a hydrogel matrix may provide support for cell adhesion. In some cases, an artificial tumor spheroid may be generated. For example, one or more cells (e.g., suspension cells or adherent cells) may be suspended in a polymerizable monomer. The polymerizable monomer may be subjected to polymerization and gelati on/solidificati on. This may generate an artificial tumor microenvironment made of a hydrogel material surrounding the cells suspended therein and may be referred to as a tumor spheroid. The tumor spheroid may then be compartmentalized into a plurality of compartments such as droplets, wells, or any other compartment described anywhere herein. The encoded effectors may be screened against the tumor spheroids. In some cases, the cells may be seeded on the surface of a hydrogel. The cells seeded inside a hydrogel or on its surface may grow over time. In some cases, more than one cell type may be co-cultured using the described methods and systems and may be perturbed and/or screened.

[0059] The methods presented herein may further comprise or be useful for phenotypic image-based analysis and intracellular measurements and observations via live cell microscopy (e.g., high resolution fluorescence microscopy or confocal microscopy). In some examples, one or more beads can be encapsulated in a compartment such as a well with a cell or a population of cells. The bead (scaffold) may comprise an effector (e.g., compound). The effector can be released from the bead into the solution inside the compartment and interact with the cell(s). The cell response to the effector can be measured to determine the potential effect of the released effector on the cell and/or a target therein. Stated differently, the effector may perturb the cell, and the cell may be screened in presence and absence of the perturbation. Perturbation may be at a genomic level, transcriptome level, translational level, protein function level, morphological level, or secretion, functional level, or any combination thereof. For example, perturbation may comprise gene therapy. Perturbation may comprise perturbing transfection, translation, differentiation, homeostasis, spatial reorganization of the contents of the cell, cellular phenotype, or any combination thereof.

[0060] The methods of the present disclosure comprise performing an assay in a plurality of compartments and measuring a signal indicative of an effect (e.g., perturbation) of an effector on the sample or the contents of the wells which may comprise a cell or constituents of a cell (e.g., cell lysates or intracellular components, elements, organelles, molecules, or beyond). In some cases, based on the detected signal, a threshold may be defined to identify a condition or set of conditions as defined criteria for denoting an effector as a hit. The hits or the population having defined criteria may be processed and/or sorted according to the signal at some point during or after a screen. Alternatively or in addition, in some cases, a threshold may not necessarily be defined for the signal. Signals may be measured for a subset of, many, most, or all of the compartments of the screening platform and/or any contents therein (e.g., one or more scaffolds in the compartment, cells, particles, encoded effector), the data may be aggregated. For example, a plurality of signals such as images or digital/analog signals may be collected and/or aggregated from the compartments using computer systems, detectors, signal detection devices, hardware, and software. Decisions about hit definition or identification (e.g., the conditions to be called hits, such as to have an effect on a sample, such as a cell, constituent of a cell, or a target in the sample or in the cell) may be performed during the screen or at a later stage. Stated in other words, the effects of the effectors encapsulated or trapped in the plurality of the compartments measured may be mapped for at least a subset of the compartments (e.g., wells), most, or all of the compartments.

Miniaturized screening platforms

[0061] In some examples, the present disclosure provides a system comprising a plurality of partitions or compartments for screening (e.g., high-throughput screening in a miniaturized platform), which can be used to compartmentalize a sample into discrete volumes in open or closed partitions of various shapes, sizes, and materials. A partition may comprise, be, or termed as a compartment, chamber, confinement, encapsulation, raft, or other type of partitions, such as to, for example, keep multiple discrete or semi-discrete sub-samples substantially separate from one another, and in some applications, test different perturbation conditions in each compartment/partition, in a parallelized and/or high-throughput fashion. In some cases, each compartment can act as an independent reaction vessel. In some cases, the terms compartment, partition, encapsulation, raft, chamber, microfluidic compartment, may be used interchangeably. The methods and systems provided herein can facilitate sample screening in a relatively short period of time (i.e., in high throughput) compared to conventional methods, and/or using small sample sizes. In some cases, the systems provided herein may comprise or be bench-time instruments which may effectively replace large HTS facilities.

[0062] The systems provided herein may comprise a plurality of partitions/compartments (e.g., wells or arrays) built in or immobilized on a solid support, surface, or substrate. Alternatively, the compartments may not be immobilized on a solid support. For example, the compartments may be suspended in a liquid, such as a droplet in oil emulsion (i.e., a plurality of droplets suspended in an immiscible oil fluid) generated using a microfluidic device or using a vortexer or shaking platform (e.g., bulk emulsification and/or particle-templated emulsification (PTE)). In case droplets are used as compartments, droplet formation may be performed in a variety ways.

[0063] In some examples, in the methods of the present disclosure, the compartments may comprise or be droplets, wells, or combinations of both (e.g., droplets trapped in wells). Among the applications of such screening platforms are single cell analysis, cell culture, cell perturbation analysis, encoded effector library screening, cell perturbation analysis, phenotypic cell screening, drug screening and drug discovery, diagnostics, personalized medicine, synthetic biology, discovery of biologies or antibodies, stem cell research, cell co-culture (e.g., culturing multiple cell types and screening them for a variety of metrics), cell signaling pathway analysis, protein degradation analysis, cellular cross-talk analysis, cell morphology analysis, cell viability assays, tissue engineering, directed evolution (e.g., enzyme evolution), a plethora of genomics and proteomics, secretomics, metabolomics, and other applications.

[0064] The methods and systems of the present disclosure may comprise obtaining, providing, and using encoded effector libraries. Encoded effector libraries may be designed and synthesized using a variety of methods. Encoded effector libraries may further be prepared and integrated with a variety of screening platforms. Screening platforms may be according to the screening systems provided anywhere herein or other screening platforms beyond this disclosure which may be used for screening encoded effector libraries provided herein.

[0065] Screening in a miniaturized platform may comprise screening an assay in a system provided herein. Screening systems may be high-throughput and miniaturized screening platforms configured to measure the activity of an assay. An assay may be configured to measure an activity or condition of a biological target, such as any kind of target mentioned anywhere herein. In some cases, samples may comprise live cells. In other cases, samples may not comprise live cells. The condition of the sample may be screened using the screening methods, systems, and workflows provided herein. In some cases, the screening systems, the assays, and the effector libraries may be integrated into a workflow, such as to assess the effect of the members of the encoded effector library on the sample, through the assay, detected via the screening system. In other cases, assays may be performed in absence of effector libraries. The methods, systems, system components, workflows, and all of their parts, pieces, and components may be used individually or in concert to achieve various goals.

[0066] In some examples, encoded effectors may comprise or be molecules whose structures can be measured or identified by measuring a property of the corresponding encoding. For example, in an integrated workflow comprising a screening system, an assay, and an effector library, the members of the encoded effector library may be incubated with an assay in a plurality of compartments or encapsulations of a screening platform (e.g., wells of an array-based system) or generated by a screening platform (e.g., droplets generated by a microfluidic device or vortexing system).

[0067] The assay may generate a signal which can be detected by a screening system (e.g., a detector). In case the compartment comprises an effector (e.g., a member of an encoded effector library), the effector may interact with the sample or a target therein and may have an effect or suspected effect on the target, the sample, and/or the signal measured from the assay (e.g., in each compartment). For example, the signal may be indicative of an effect of the effector on the assay, the target, or the sample.

[0068] In some cases, based on the signal, the effector can be determined to have efficacy against the sample in inducing a particular response. The systems and methods described herein, in some embodiments, utilize small encapsulations, such as droplets, wells, channels, miniaturized confinements, or any other type of compartment mentioned anywhere herein. The terms compartment and encapsulation may be used interchangeably. In some instances, at least a subset of the plurality of the encapsulations of a system or generated by the system may each individual carry out an assay. The encapsulation may comprise a unique effector of the encoded effector library. The encoded effector library may comprise a plurality of unique encoded effectors described in further detail elsewhere herein. In some cases, a plurality of unique and/or different encoded effectors (e.g., a large encoded effector library) may be screened in presence of a sample (e.g., against one or more targets) in a parallelized and high-throughput fashion, consuming small quantities of assay reagents, using the miniaturized systems provided herein. In some cases, the miniaturized systems may comprise automated benchtop instruments.

[0069] In some cases, it may be intended (e.g., by a user or researcher) to achieve an effect from an effector. For example, it may be intended to find a drug for a disease target. It may be intended to find an effector which may decrease or increase an activity of a biological target in a sample. The effect may be screened for using the methods and systems of the present disclosure. In some cases, the effects of a multi-member library (e.g., a one-million-member library of encoded effectors) may be screened against a sample/target. The effect may manifest in the signal detected from the compartments, using the screening platforms and assays of the present disclosure. If the intended effect is achieved, detected, or observed through the system, the compartment containing the effector may be selected by the system for further processing. Selection may be performed using computer systems, hardware, and software. For example, a rule may be defined on a software of the system which is in communication with the miniaturized screening chip through a computer. In some cases, the rule may comprise defining a threshold for the signal. Based on the rule, the effector and/or the compartment it is in may be selectively processed. Selective processing may comprise pulsing, separation in space, sorting, and detecting a property of the barcode to elucidate the identity of the effector which led to the intended effect (e.g., the subject of the screen/search). Processing may comprise additional embodiments beyond sorting, as mentioned elsewhere herein.

Encoded Effectors

[0070] The systems and methods provided herein may comprise providing, synthesizing, making, obtaining, and/or screening encoded effectors. An encoded effector may comprise or be an effector that has been linked with, associated with, or barcoded with an encoding/barcode such that ascertaining a property of the encoding allows for readily determining the structure of the effector. The terms encoding and barcode may be used interchangeably.

[0071] An effector can be any type of molecule or substance whose effect on a sample may be investigated. In examples, the effector may comprise or be a compound, a protein, a peptide, an enzyme, a nucleic acid, a gene, or any other substance. In some instances, the encoding allows a user to determine the structure of the effector by measuring/detecting a property of the encoding. Thus, each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded. Many different encoding modalities can be used. Encoding modalities may comprise nucleic acids, DNA, RNA, peptides, peptide nucleic acids (PNA). In some examples, encoding modalities may comprise optical barcodes, nanoparticles, luminescent materials, and/or quantum dot particles. In some examples, various combinations of encoding modalities may be used. For example, encoding modalities may comprise both nucleic acid molecules and optical barcodes. [0072] In some examples, encodings may comprise nucleic acid molecules such as DNA, RNA, or PNA. In some cases, the encoding may comprise a sequence unique to the structure of the effector, a sequence unique to the scaffold that is bound to, comprised therein, and/or both. When the encoding modalities are nucleic acids, the sequence of the nucleic acid may provide information about the structure of its corresponding effector. In some instances, the encoded effectors are described by what kind of molecules is used in the encoding. For example, “nucleic acid encoded effectors” comprise an effector encoded by a nucleic acid.

[0073] In some instances, the effectors and their corresponding encodings are bound to a scaffold. The effector may be covalently bound to the scaffold via a cleavable linker. The encoding may be covalently bound to the scaffold. The effector may comprise a plurality of subunits which may be covalently bound to one another. The encoding may comprise a plurality of subunits which may also be covalently bound to one another. The effector and the encoding may form an effector/ encoding pair linked in space bound to the scaffold, and in some cases, bound to one another. Alternatively, the effector and the encoding may be separately bound to the scaffold but not bound to one another. In some instances, when encoded effectors are placed into solutions or other environments, the link between the pairing is not lost. Many materials can be used as scaffolds, as any material capable of binding both the effector and the encoding may accomplish the desired goal of keeping the pair linked in space. In some cases, the scaffold may be a bead.

[0074] Various methods for preparing encoded effectors linked to scaffolds can be used. In some embodiments, the methods use orthogonal, compatible methodologies to create an effector and its encoding in a parallel synthesis scheme. This is sometimes referred to as “split and pool synthesis.” For illustrative purposes only, an exemplary, non-limiting, workflow for the preparation of a scaffold containing an effector and encoding is described as follows: A first effector subunit is attached at an attachment point of a scaffold. The scaffold is then washed to remove unreacted and excess reagents from the scaffold. A first encoding subunit is then attached at another attachment point on the scaffold, and a wash step performed. Following this, a second effector subunit is then attached to the first effector subunit, followed by another wash step. Then, a second encoding subunit is attached to the first encoding subunit, followed by a wash step. This process is repeated as many times as desired to prepare the desired effectors and corresponding encodings. This process can be repeated on a massively parallel scale in small volumes to prepare vast libraries of compounds at low cost and with low amounts of reagents. In some instances, pre-synthesized compounds are loaded onto scaffolds which contain encodings. The encodings may be pre-synthesized and loaded onto the scaffolds or are synthesized directly onto the scaffolds using methods analogous to the split and pool synthesis described above. In some instances, each scaffold comprises numerous copies of a unique effector and its corresponding encoding. The encoding may comprise information related to the synthetic history (a plurality of synthetic steps) of the effector.

[0075] An example of a nucleic acid encoded effector linked with a bead is shown in FIG. 1. A bead linked encoded effector 100 comprises a bead 101. Attached at one position is a nucleic acid encoding 102, which is covalently attached to the scaffold in this example. The nucleic acid encoding comprises encoding subunits A, B, and C. The encoding subunits correspond with effector subunits A, B, and C, which make up effector 103. The effector 103 is linked to the bead 101 through a linker 104. The linker 104 may be a cleavable linker, such a linker cleavable by electromagnetic radiation (photocleavable) or selectively cleavable by a cleaving reagent (chemically cleavable). Cleavable linkers can be used to liberate effectors from a bead or other scaffold to allow the effector to interact with a sample.

[0076] In some embodiments, the scaffolds further comprise impurities in the effector and/or its encoding. For example, the subunits of the effector may comprise or be building blocks of a small molecule. The effector may be a small molecule. The effector may further comprise building block fragments. In some instances, impurities of the effector and its corresponding encoding occur due to damage during a screen, during manufacturing of the bead, effector, or encoding combination, or during storage. In some embodiments, impurities of the effector and its corresponding encoding are present due to defects in the methodologies used to synthesize the encoded effectors. In some embodiments, scaffolds as described herein can comprise a single encoder, an encoding and its impurities, or combinations thereof. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the effectors attached to a scaffold comprise an identical structure. In some embodiments, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the encodings attached to a scaffold comprise a substantially identical structure or sequence.

Sample Screening in Droplet Microfluidic Platforms

[0077] Provided herein are methods and systems for screening encoded effectors on samples using encapsulations (e.g., droplets or compartments). In some embodiments, methods and systems for screening encoded effectors on samples are capable of being performed in a high-throughput manner. In some embodiments, the methods and systems provided herein allow for screening large libraries of encoded effectors using small volumes, minimal amounts of reagents, and small amounts of the effectors being screened. In some embodiments, the methods and systems provided herein allow for uniform dosing of effectors in a library against samples. In some embodiments, the methods and systems described herein allow for measurement of cellular properties, behaviors, or responses, in a high throughput manner. In some embodiments, the methods and systems provided herein measure genomic, metabolomic, and/or proteomic data from cells screened against the encoded effectors. In some embodiments, the methods and systems provided herein allow for detecting synergistic effects of using multiple effectors against a particular sample. In some embodiments, the methods and systems provided herein allow for a library of mutant proteins to be screened for a desired activity or improvement in activity of a target.

[0078] An example workflow of a screen utilizing a single encoded effector bound to a scaffold is shown in FIG. 2A. The nucleic acid encoded effector bound to a scaffold is encapsulated with a target, in this case a cell or a target inside a cell. In step 1, the effector, in this case a drug, is then cleaved from the bead within the encapsulation. In step 2, the effector is allowed to interact with the cell. If the drug has an intended effect on the cell, a reporter signal indicates that the drug is a positive hit. If there is no reporter signal detected, then the result for that drug is negative. In step 3, positive and negative results are sorted based on the detection of the signal. At the end of a screen, in step 4, the positive hits, which have been pooled together, are then sequenced (in the case of nucleic acid encodings) to reveal which effectors had the intended effect. In step 5, this information can then be used to guide synthesis of further libraries or identify lead molecules for further development.

[0079] FIG. 2B shows an additional exemplary workflow of an effector screen performed in a microfluidic device. In the exemplary workflow shown, a nucleic acid encoded effector bound to a bead is placed in an inlet and merged with an additional aqueous stream, which, in some embodiments, contains a sample to be screened. The merged fluids are driven through an “extrusion region” or “droplet formation region,” wherein beads and sample are encapsulated in droplets. Droplets/encapsulations are discrete aqueous volumes surrounded by a continuous immiscible oil. An effector is then cleaved from bead at the effector cleavage region or dosing region, which in some embodiments utilizes a light source to cleave a photocleavable linker. The encapsulations containing cleaved effectors are then allowed to continue flowing along the flow path of the device through the incubation region, which in some embodiments contains widened or enlarged chambers to control flow rate or residence time of the encapsulations. As the encapsulations travel through the incubation region, a detectable signal is generated by the assay (e.g., a cleavage of a fluorophore from an assay probe). The signal may increase over time. The signal can be measured dynamically throughout the device. Different regions in the device may correspond to a given incubation time (e.g., the duration of time which takes the encapsulation to arrive at that location). As such, the signal increase over time can be detected and characterized. The signal may also be detected in a detection region of the device. In some embodiments, this detectable signal is a fluorescent signal, though any detectable signal can be employed. This signal is then measured or detected at a detection region, which is in some embodiments equipped with a light source (e.g., a laser or LED) and a detector (e.g., a photomultiplier tube (PMT), a charged coupled device (CCD), or a photodiode) coupled to a sorting device (e.g., a dielectrophoresis electrode or any other sorting mechanism). In some embodiments, the detection region comprises an interrogation region, which is coupled to a sensor or an array of sensors. Based on the signal, the encapsulations are sorted into a waste outlet or a hit outlet. For example, the negative encapsulations may be defaulted into the waste stream. The positive encapsulations may be deflected into a hit stream, and thereby separated from the negative encapsulations. Following completion of the screen, the encodings of the hits are amplified (e.g., by PCR or emulsion PCR) and the encodings sequenced (e.g., by next generation sequencing (NGS)). The sequenced encodings can then be decoded to reveal the effectors which had the desired activity. In some embodiments, each bead further comprises barcode unique to the bead itself (independent of the effector). Thus, in some embodiments, it is possible to ascertain if multiple beads bearing identical effectors were selected as hits within multiple encapsulations.

[0080] Provided herein are methods and systems for screening encoded effectors on samples using encapsulations, wherein the sample and an encoded effector are encapsulated or co-localized in a compartment (e.g., a droplet or a well). In some cases, a plurality of unique encoded effectors are screened. Each unique encoded effector may comprise a unique effector the effect of which on a sample may be screened using any screening system provided herein. [0081] In some examples, screening the sample in presence and/or absence of encoded effectors may be performed in a droplet microfluidic device. The droplet microfluidic device may comprise a droplet generation region/j unction for forming droplets/encapsulations. The droplet generation junction may comprise or be a microfluidic flow focusing junction or a microfluidic T-junction. In some cases, the microfluidic device may comprise one or more droplet generation junction, such as 1, 2, 3, 4, 5, 6, or more droplet generation junctions integrated in the droplet microfluidic platform.

[0082] Each droplet formation region may comprise a plurality aqueous inlet streams. Each aqueous inlet stream may be connected to a separate reservoir holding a liquid, through a tube and one or more tube connections. The separate reservoirs may each comprise a sample, a portion of a sample, assay reagents, assay probes, fluorophores, targets, cells, and/or encoded effectors. The materials held in separate reservoirs may be set up and adjusted to accomplish intended purposes for screening a sample in presence or absence of a plurality of unique encoded effectors. In a particular example, a droplet generation junction of a microfluidic device may comprise 3 inlet aqueous streams. The first aqueous inlet stream may comprise a first portion of an assay reagent. The first aqueous inlet stream may comprise a target. The second aqueous inlet stream may comprise a second portion of an assay reagent. The second aqueous inlet stream may comprise a probe or materials which may result in a signal as a result of interacting with the target. As long as the materials of the first stream and second stream are held in separate reservoirs, the assay does not start. The assay will start once the two streams meet a microfluidic channel of the microfluidic device that is connected to the first and second streams and to the droplet generation region. Once the first and second streams meet and start mixing, the assay starts to take place, generating a signal which may continue to increase over time inside the formed droplet containing the assay materials. The droplet may flow through the chip for the assay to be incubated. In some cases, the assay may be screened in presence of the encoded effectors. The encoded effectors may be introduced through the first inlet or the second inlet. Alternatively, the microfluidic device may comprise a third inlet stream for introduction of the encoded effectors. The encoded effectors may be bound to beads according to the information presented elsewhere herein. The third inlet stream may be holding a solution comprising a suspension of encoded effector beads. The third aqueous stream may enter the device, co-flow along with the first and second streams, and reach the droplet generation junction.

[0083] The droplet generation junction may be a flow focusing droplet generation junction. An oil phase immiscible with the aqueous solutions may enter the droplet generation junction and break up droplets which may contain assay reagents, encoded effectors, and/or both. The oil may comprise a surfactant. The percentage of the surfactant in oil may be at least about 0.5%, 1%, 2%, 3% (v/v), or more. The surfactant may form as a barrier surrounding the droplet, reducing or eliminating material transfer from the aqueous environment of the droplet into the surrounding continuous oil. A plurality of droplets may be formed at a predetermined frequency. The droplets may encapsulate a plurality of different/unique scaffolds (beads comprising a plurality of different encoded effectors such that each bead comprises a unique effector encoded using an optical or a nucleic acid barcode). In some cases, a subset of the plurality of droplets may each comprise at least one scaffold/bead. Another subset of the plurality of droplets may be empty. Another subset of the plurality of droplets may comprise more than one scaffold, such as 2, 3, 4, 5, 6, or more scaffolds (e.g., multiple scaffolds). The number of scaffolds (e.g., beads) entrapped in the droplets may be quantified using a parameter termed “droplet occupancy”. Droplet occupancy may characterize the percentage of droplets containing 0 beads, 1 bead, 2 beads, or more than 2 beads. Bead occupancy among the droplets may follow a Poisson distribution. For example, the percentage of droplets containing 1, 2, or more beads may be calculated according to the Poisson distribution using the size of the droplet. Droplet characteristics

[0084] In some examples, the diameter of the droplets formed in the droplet generation junction may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 micrometers/microns (um) or larger. In some cases, droplet diameter may be at most about 100, 90, 80, 70, 60, 50, 40, 30, 20 microns or smaller. The droplet may be of any suitable volume. In some cases, the droplet may be from about 1 picolitres to 500 picolitres.

[0085] examples, the droplets are placed in an oil emulsion. In some examples, the oil comprises a silicone oil, a fluorosilicone oil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenated oil, a fluorocarbon oil, or any combination thereof. In some examples, the oil comprises a silicone oil. In some examples, the oil comprises a fluorosilicone oil. In some examples, the oil comprises a hydrocarbon oil. In some examples, the oil comprises a mineral oil. In some examples, the oil comprises a paraffin oil. In some examples, the oil comprises a halogenated oil. In some examples, the oil comprises a fluorocarbon oil.

[0086] In some examples a population of substantially monodispersed droplets may be formed. In some embodiments, each encapsulation is within 5%, 10%, 15%, 20%, or 25% of the average size encapsulation within the plurality. In some embodiments, at least 80%, 85%, 90%, or 95% of the encapsulations are within about 5%, 10%, 15%, 20%, or 25% of the average size encapsulation within the plurality.

[0087] The droplets may be formed by any method. In some examples, a droplet is formed by flowing an aqueous stream into an immiscible carrier fluid. In some examples, the aqueous stream flows into an immiscible carrier fluid at a junction of microfluidic channels. In some embodiments, the junction is a T-junction. In some examples, the junction is a meeting of two perpendicular microfluidic channels. The junction may be a meeting of any number of microfluidic channels. The junction may be at any angle. The aqueous stream may be formed by an upstream junction of two or more aqueous streams. In some examples, sample solutions and effector solutions are joined upstream of the aqueous stream junction with the immiscible carrier fluid. The size of the droplets may be controlled by modulating a variety of parameters. These parameters include the geometry of the junction of two microfluidic channels, the flow rate of the two streams, the type of oil used, the presence of surfactants, the pressure applied to the flow streams, or any combination thereof.

[0088] In some examples, a single encoded effector is present in a compartment of encapsulation of the present disclosure (e.g., a droplet or a well). In some examples, a single scaffold comprising an encoded effector and its encoding are present in an encapsulation. In some examples, a plurality of scaffolds, each scaffold comprising a different encoded effector and its respective encoding, are present in a compartment.

[0089] In some examples, encapsulations comprise biological samples. In some embodiments, encapsulations comprise single cells. In some embodiments, encapsulations comprise one or more cells. In some embodiments, the encapsulations comprise nucleic acids. In some embodiments, the encapsulations comprise proteins.

[0090] In some examples, the percentage of droplets each containing exactly one bead may be at least 5% of the total droplets formed. In other examples, this percentage may be at least about 8%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80%, 90%, or 99.9%. In some cases, this percentage may be at most about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or less of the total number of droplets formed per unit time.

[0091] In some examples, the percentage of empty droplets among the total droplets formed may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more. In some examples, the percentage of empty droplets among the total droplets formed may be at most about 99%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, or less of the total number of droplets formed per unit time. In some examples, the percentage of droplets containing 2 or more beads may be at least about 0.05%, 0.1%, 0.5%, 1%, 1.5%, 2%, 3%, 4%, 5%, 6%, 10%, 20%, 30%, 40% or more of the total droplets formed. I some examples, this percentage may be at most about 50%, 40%, 30%, 20%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1.5%, 1%, or less of the total number of droplets formed per unit time.

Screening encoded effector libraries in compartmentalized systems

[0092] Provided herein are methods and systems for screening a library of encoded effectors, such as a plurality of different/unique encoded effectors using the screening platform provided anywhere herein on any sample or target provided anywhere herein. In some examples, a plurality of scaffolds (e.g., beads or any other kind of scaffold or solid support mentioned anywhere herein) may be encapsulated or otherwise localized, loaded, dispensed (manually or automatically by a person, machine, or robot, or placed in a plurality of compartments according to any compartment described anywhere herein, such as droplets, wells, rafts, encapsulations, channels, or microfluidic confinements. The compartment may further comprise a sample, a target, a reagent, assay probes, fluorophores, and any other components which may facilitate screening the sample/target in presence and/or absence of the effector(s)s. [0093] The library of unique encoded effectors may comprise a predetermined number of unique and/or different encoded effectors (e.g., effectors with different structures and/or chemical properties and features). The different structure of the effector may lead to a different effect on the assay or the target. The number of unique encoded effector libraries may be designed according to the applications and objectives to be accomplished. In some examples, the library may comprise at least 2, 3, 4, 5, 6, 7, 8 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 10000 or more unique encoded effectors. In some examples, the library may comprise at least 10 ⁴ , 2 x 10 ⁴ , 3 x 10 ⁴ , 4 x 10 ⁴ , 5 x 10 ⁴ , 6 x 10 ⁴ , 7 x 10 ⁴ , 8 x 10 ⁴ , 9 x 10 ⁴ , or more unique encoded effectors. In some examples, the library may comprise at least 10 ⁵ , 2 x 10 ⁵ , 3 x 10 ⁵ , 4 x 10 ⁵ , 5 x 10 ⁵ , 6 x 10 ⁵ , 7 x 10 ⁵ , 8 x 10 ⁵ , 9 x

10 ⁵ , or more unique encoded effectors. In some examples, the library may comprise at least 10 ⁶ , 2 x 10 ⁶ , 3 x 10 ⁶ , 4 x 10 ⁶ , 5 x 10 ⁶ , 6 x 10 ⁶ , 7 x 10 ⁶ , 8 x 10 ⁶ , 9 x 10 ⁶ , or more unique encoded effectors.

[0094] In some examples, the library may comprise at most 9 x 10 ⁷ , 8 x 10 ⁷ , 7 x 10 ⁷ , 6 x 10 ⁷ , 5 x 10 ⁷ , 4 x 10 ⁷ , 3 x 10 ⁷ , 2 x 10 ⁷ , 10 ⁷ or less unique encoded effectors. In some examples, the library may comprise at most 9 x 10 ⁶ , 8 x 10 ⁶ , 7 x 10 ⁶ , 6 x 10 ⁶ , 5 x 10 ⁶ , 4 x

10 ⁶ , 3 x 10 ⁶ , 2 x 10 ⁶ , 10 ⁶ or less unique encoded effectors. In some examples, the library may comprise 9 x 10 ⁵ , 8 x 10 ⁵ , 7 x 10 ⁵ , 6 x 10 ⁵ , 5 x 10 ⁵ , 4 x 10 ⁵ , 3 x 10 ⁵ , 2 x 10 ⁵ , 10 ⁵ or a smaller number of unique encoded effectors. In some examples, the library may comprise 9 x 10 ⁴ , 8 x 10 ⁴ , 7 x 10 ⁴ , 6 x 10 ⁴ , 5 x 10 ⁴ , 4 x 10 ⁴ , 3 x 10 ⁴ , 2 x 10 ⁴ , 10 ⁴ or less unique encoded effectors. In some examples, the library may comprise 10000, 9000, 8000, 7000, 6000, 5000, 4000, 3000, 2000, 1000, or a smaller number of unique encoded effectors.

[0095] The encoded effector libraries are described in detail throughout the present disclosure. The unique encoded effectors may be bound to solid supports or scaffolds (e.g., bead resin) via a cleavable linker. In some examples, the encoded effectors libraries may comprise or be a One Bead One Compound (OBOC) encoded effector library, such that each scaffold comprises a unique effector bound to the bead via a cleavable linker and releasable from the bead upon cleavage of the cleavable linker and encoded with an encoded on or in the scaffold which corresponds to and identifies the effector (e.g., as shown FIG. 1).

Post-screen processing

[0096] Upon detection of the signal from the compartments during or after the screen, rules may be defined to process the compartments or the contents thereof (e.g., based on the detected signals). In some cases, the signals may be aggregated and process after the screen. Alternatively or in addition, signals may be analyzed or processed in real-time. For example, the systems may comprise computer systems and software systems in communication with the compartmentalized screening platform. Data may be collected from the screening platform and transferred to the memory of the computer systems for storage. In some cases, rules may be defined (e.g., using software, in real-time screen run environment), based on the signals. Such software programs may comprise LAB VIEW, Python, or any other programming languages. In some cases, a hit population or positive population may be defined. Alternatively or in addition, other populations may be defined and selected for further processing, based on the observations during the screen or based on the signal.

[0097] The selected population (e.g., positive population) may then go through one or more processes, such as experiencing one or more stimuli (e.g., energy/energy pulse, current, wave, electric field, magnetic field, heat, light), being injected with a chemical or reagent, being separated in space, a change of state, a temporary or permanent opening and/or closing of the compartment, or other processes. An opening and closing of the compartment may depend on the type of compartment used in each screen/condition. For example, in case the compartment is a droplet, a stimulus may temporarily destabilize the boundaries of the droplet as facilitated by the surfactant, such as to temporarily open the droplet to reagent addition or extraction. Reagent addition my comprise pico-inj ection to inject a reagent into the droplet. Reagent extraction may comprise extracting the bead from the droplet. For example, in some cases, the bead may be magnetic, such as having magnetic properties. The bead can be made of magnetic materials. In some cases, magnetic particles may be mixed with a material which makes up the bead to magnetize the bead. A magnetic bead can be extracted from the compartment using a magnetic field. The magnetic field may be generated by a component of the system or by a device/module external to the system. The compartment may be temporarily opened to facilitate pulling the bead out of the compartment using the magnetic field. The opening and the separation may be based on the signal as described above.

[0098] In some embodiments, a selected plurality of encapsulations may be sorted based on a corresponding signal being detected from such encapsulation. A plurality of compartments (droplets or wells) may be selected using the computer systems based on the screening results, during or after the screen. The selected compartments may go through post-processing according to the methods described anywhere herein. In some cases, post-processing may comprise separation in space and/or sorting. [0099] In some examples, processing of the selected compartments may comprise sorting. Sorting may comprise sorting the compartment (droplet, well, or another kind of compartment), sorting the contents of the compartment either with the compartment or separate from the compartment, and/or sorting the scaffold in the compartment. Selection of the compartment or its contents for sorting may be performed based on the signal or other observations during the screen as described anywhere herein. For example, a subset of the plurality of compartments (droplets or wells) may be selected based on an observation or data acquired, encountered, or collected, during the screen (e.g., the assay signal measured from the compartment in presence and/or absence of bead-bound encoded effector). In some cases, the selected compartments may be positive compartments (a compartment containing at least one positive scaffold or a hit). In other examples, the selected compartments may have any criteria, a user/researcher may select/define. The selected compartments or contents thereof may then be sorted.

[0100] Compartment sorting may be performed in a variety of ways. For example, sorting may be performed using an integrated microfluidic circuit presented herein. Sorting may comprise generating a pulse of energy (e.g., electrical pulse generated by a waveform pulse generator) and exposing it to the compartment. The energy or other kind of stimulus may affect the selected droplet and may be configured to sort it. For example, the pulse of energy may deflect the droplet into a separate stream, thereby sorting the droplet into a separate channel/container from the non-selected droplets. As such, the selected droplet/compartment population may be separated it in space from the non-selected droplets.

[0101] Separation may comprise a variety of separation modalities. In some examples, compartments may comprise droplets. In some examples, compartments may be wells of an array. Assays may be performed in well compartments in presence and/or absence of a target and an encoded effector presented anywhere herein. In some cases, the wells of an array may be separable from the array. Based on an indication, observation, measurement, or data detected through the screen (e.g., a property of the assay signal), a population may be selected to be processed. Processing may comprise sorting. Sorting may comprise sorting the compartments. For example, individual wells in an array system may be magnetic, and may be individually separable. A magnetic field may be applied to separate the selected wells from the rest of the platform. In some cases, a magnetic pen may be used to separate an individual well from the array. Sorting may comprise sorting the contents of the compartment with or without sorting the compartment. If a compartment is sorted, its contents may be sorted with it. Alternatively, the contents of the compartment may be sorted according to any method provided anywhere herein. In one example, the contents of the compartment may be magnetic. The bead may be magnetic and separable via a magnetic field

[0102] In some examples, processing of the selected compartments may comprise barcoding the compartment, barcoding a scaffold in the selected compartment, or barcoding the encoding of the selected compartment. Barcoding may be performed inside the compartment or after extracting the scaffold from the compartment. Barcoding may be performed during the screen in real-time, or after a screen. The systems provided herein may facilitate performing and controlling scaffold barcoding and/or encoding barcoding (barcode barcoding/tagging or barcoding/tagging the barcode). In some cases, the encoding(s) associated with the encapsulations having a detected signal(s) are barcoded, as an alternative or in addition to sorting the encapsulation. In some embodiments, encapsulations (e.g., droplets) are formed with the aid of microfluidic devices. In some embodiments, encapsulations flow through a microfluidic device. In other examples, encapsulations/compartments may be wells of an arraybased platform or confinements of a miniaturize multi-compartment screening platform.

[0103] Provided herein are methods and systems for screening encoded effectors on samples using encapsulations, wherein a signal may be detected from the encapsulation. In some embodiments, the signal may be indicative of an interaction between an effector and the sample. For example, an assay may measure an activity of a biological target in a sample. The interaction of the effector with the sample may cause a change in the signal which may be indicative of the effect of the effector on the assay and/or the target. In some examples, the signal may be detected with a detector. In some examples, detecting the signal may comprise generating the encapsulation (droplet) with the aid of a microfluidic device which may comprise a droplet generation junction connected to one or more channels and/or a flow path. The flow path may comprise or be an incubation line in which the encapsulations can be incubated for a duration of time for the assay to take place and generate a signal. In some examples, detecting the signal may comprise providing the encapsulation using a system comprising a microfluidic device equipped with or connected to a detector. In some examples, the detector may be configured to detect the signal. The detector may comprise or be a camera, a signal processing device, an image processing device, a PMT, any combination thereof, on any other detector mentioned elsewhere herein.

[0104] Signals of the methods and systems provided herein can be any detectable signal which can be suitably detected from any kind of compartment or encapsulation described herein. In some examples, the signal may comprise or be electromagnetic radiation, thermal radiation, a visual change in the sample, or combinations thereof. Electromagnetic radiation may comprise or be fluorescence or luminescence. Electromagnetic radiation may be in the visible spectrum. In other examples, electromagnetic radiation may not be in the visible spectrum. Fluorescence may be induced or generated by applying a stimulus to the sample in the compartment. In some examples, a laser with a specified energy and wavelength may pass through a filter which allows an intended wavelength of light to pass through and expose the sample, such as to excite the sample. The system may comprise equipment for exposing the sample to the stimulus (light or other kind of stimulus) to excite the sample to generate the signal. The sample or a component therein (e.g., a probe, a fluorophore, a fluorogenic substrate, any combination thereof, or other kind of assay reagents) may be excited as a result of the stimulus such as to generate a response in form of a signal. For example, the sample may emit an emitted light or generate a signal of any type which may be detectable using the system (e.g., the detection, PMT, camera, signal processing device, or any suitable detector based on the type of the signal). The emitted light/signal/response may pass through an emission filter before being detected. The components of the system, such as the stimulus (e.g., a light or laser used for exciting the sample), the excitation filter, the emission filter, the PMTs, and other devices and systems may be adjusted to optimize the conditions for each assay.

Alternative signal detection

[0105] Provided herein are methods and systems for screening encoded effectors, wherein various alternative signal detection methods and systems may be used to identify activity by an effector which may in some cases be placed and/or screened within a compartment or encapsulation of any kind. The compartment or encapsulation may be any kind of compartment described anywhere herein. In some examples, the signal may be a thermal radiation. The thermal radiation may in some cases be detected using an infrared camera. In some cases, the signal may comprise or be a change in thermal radiation emitted by a sample. The change in thermal radiation may be due to metabolic activity in the sample in presence or absence of an effect from an encoded effector of the present disclosure. For example, an encoded effector may alter a metabolic activity in a sample, and thereby cause a change in the thermal radiation detected from the sample as the signal.

[0106] Provided herein or screening a sample for a change in metabolic activity in presence and/or absence of an encoded effector. The methods may further comprise studying the effect of the encoded effector on the sample. In some cases, the change in metabolic activity may be detected through measuring a change in thermal radiation emitted from the sample in a screen system of the present disclosure in presence and/or absence of treatment with an encoded effector. In some examples, the sample may comprise a cell and the signal may comprise or be thermal radiation and/or a change thereof. For example, the sample may display a change in emission of thermal radiation compared to a sample not encapsulated or treated/perturbed with the effector.

[0107] In some cases, the change in thermal radiation may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% change in the emission of thermal radiation, for example, when comparing a no-treatment control to a sample treated with an encoded effector. In some cases, such change may be at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% emission of thermal radiation relative to sample not treated with the effector. In some examples, the change in thermal radiation may be at least 2-fold, 3 -fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold emission of thermal radiation relative to a sample not treated with the effector. Alternatively or in addition, the signal may comprise luminescence.

[0108] In some examples, signals may be detected upon or after incubating the sample for a duration of time. For example, detecting the signal may comprises monitoring the signal (e.g., fluorescence, luminescence, thermal radiation, or any other form of signal) from the sample over a period of time. In some examples, the signal (e.g., luminescence) may be integrated over a period of time. In some examples, the luminescence is integrated over a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60 or more minutes (min). In some cases, the sample may be incubated for at least about 1, 2, 3, 4, 5, 6, hours (hr) or more.

[0109] The sample may be incubated in any kind of compartment described anywhere herein, such as a well of any size, a droplet, a nanopen or miniaturized feature, channel or compartment, or any other suitable compartment. The compartment may be static or dynamic. In case of incubating the sample in a dynamic compartment (e.g., droplets or encapsulations), droplets may be travelling through a microfluidic chip (e.g., FIG. 4). The microfluidic chip may comprise an incubation line comprising a number of loops. It may take the droplets a known duration of time to travel through the chip and reach a known location/loop in the chip. Therefore, the incubation time corresponding to each chip location can be measured and be known. In other examples, the sample may be incubated in a static compartment such as a well. The incubation time in a static compartment such as a well can be precisely controlled and known. The sample may be loaded, incubate, measured, and post-processed according to any suitable method.

Nucleic acid detection

[0110] In some embodiments, detecting the signal comprises detecting the presence of a target nucleic acid. In some embodiments, the encapsulation further comprises a molecular beacon. In some embodiments, the molecular beacon is complementary to a portion of the target nucleic acid sequence of the sample. In some embodiments, the methods further comprise adding a molecular beacon to the encapsulation. In some embodiments, the target nucleic acid is detected by a molecular beacon. In some embodiments, the encapsulation further comprises a probe and a polymerase. In some embodiments, the encapsulation further comprises a TaqMan probe and a Taq polymerase. In some embodiments, the methods further comprise adding a TaqMan probe and a Taq polymerase to the encapsulation. In some embodiments, the TaqMan probe is complementary to a portion of the target nucleic acid sequence. In some embodiments, the TaqMan probe and Taq polymerase are added to the encapsulation at the same time. In some embodiments, the TaqMan probe and Taq polymerase are added sequentially. In some embodiments, the signal is fluorescence emitted by a molecular beacon. In some embodiments, the signal is fluorescence emitted by TaqMan probe. In some embodiments, the signal is fluorescence emitted by a molecular beacon or TaqMan probe.

[OHl] Various molecular beacons can be used with the methods and systems described herein. In general, a molecular beacon comprises a nucleic acid binding region that binds to a complementary nucleic acid of interest. The molecular beacon can typically have a secondary structure wherein a fluorophore and a quencher are in proximity when the nucleic acid binding region is not bound to the complementary nucleic acid of interest. Upon binding of the nucleic acid binding region to the complementary nucleic acid of interest, the fluorophore and quencher may be separated in space such that a fluorescent signal can be detected. Thus, the amount of fluorescence detected can be used to quantify the amount of nucleic acid of interest present in a sample. In some embodiments, an inhibitor is used wherein activity between an effector and a sample inhibits or limits the intensity of a fluorescence signal.

[0112] In some embodiments, two or more signal detection methods are used in combination for detecting a signal. In some embodiments, detecting a signal comprises detecting morphological changes in the sample as well as detecting fluorescence emitted by a molecular beacon or probe. For example, in some embodiments, fluorescence emission from a molecular beacon in the encapsulation (e.g., droplet) can be measured by PMT or Avalanche Photodiode (APD). In some embodiments, simultaneous image capture by transillumination can identify other features in the encapsulation (e.g., droplet), such as encoded effectors and cells. In some embodiments, these streams of information together determine outcome at the sorting junction.

[0113] In some embodiments, detecting the presence of the target nucleic acid comprises amplifying the target nucleic acid. In some embodiments, the target nucleic acid is amplified by an isothermal amplification method. In some embodiments, the isothermal amplification method is loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HAD), recombinase polymerase amplification (RPA), rolling circle replication (RCA) or nicking enzyme amplification reaction (NEAR). In some embodiments, the encapsulation further comprises reagents for isothermal amplification of the target nucleic acid. In some embodiments, the methods comprise adding reagents for isothermal amplification to the encapsulation. In some embodiments, the reagents for isothermal amplification are specific to the target nucleic acid sequence.

[0114] In some embodiments, the target nucleic acid is DNA. In some embodiments, the target nucleic acids are cellular DNA. In some embodiments, the target nucleic acids are genomic DNA. In some embodiments, the target nucleic acid is RNA. In some embodiments, the RNA is mRNA, ribosomal RNA, tRNA, non-protein-coding RNA (npcRNA), nonmessenger RNA, functional RNA (fRNA), long non-coding RNA (IncRNA), pre-mRNAs, or primary miRNAs (pri-miRNAs). In some embodiments, the target nucleic acids are mRNA.

Scaffold and beads

[0115] An exemplary embodiment of screening encoded effectors on samples using encapsulations comprises the use of a scaffold. In some embodiments, the effector is bound to a scaffold. In some embodiments, the scaffold acts as a solid support and keeps the encoded effector molecules linked in space to their encodings. In some embodiments, the scaffold is a structure with a plurality of attachment points that allow linkage of one or more molecules. In some embodiments, the encoded effector is bound to a scaffold. In some embodiments, the scaffold is a solid support. In some embodiments, the scaffold is a bead, a fiber, nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valent molecular assembly.

[0116] In some embodiments, the scaffold is a bead. In some embodiments, the bead is a polymer bead, a glass bead, a metal bead, or a magnetic bead. In some embodiments, the bead is a polymer bead. In some embodiments, the bead is a glass bead. In some embodiments, the bead is a metal bead. In some embodiments, the bead is a magnetic bead.

[0117] The beads utilized in the methods provided herein may be made of any material. In some embodiments, the bead is a polymer bead. In some embodiments, the bead comprises a polystyrene core. In some embodiments, the beads are derivatized with polyethylene glycol. In some embodiments, the beads are grafted with polyethylene glycol. In some embodiments, the polyethylene glycol contains reactive groups for the attachment of other functionalities, such as effectors or encodings. In some embodiments, the reactive group is an amino or carboxylate group. In some embodiments, the reactive group is at the terminal end of the polyethylene glycol chain. In some embodiments, the bead is a TentaGel® bead.

[0118] The polyethylene glycol (PEG) attached to the beads may be any size. In some embodiments, the PEG is up to 20 kDa. In some embodiments, the PEG is up to 5 kDa. In some embodiments, the PEG is about 3 kDa. In some embodiments, the PEG is about 2 to 3 kDa.

[0119] In some embodiments, the PEG group is attached to the bead by an alkyl linkage. In some embodiments, the PEG group is attached to a polystyrene bead by an alkyl linkage. In some embodiments, the bead is a TentaGel® M resin.

[0120] In some embodiments, the bead comprises a PEG attached to a bead through an alkyl linkage and the bead comprises two bifunctional species. In some embodiments, the beads comprise surface modification on the outer surface of the beads that are orthogonally protected to reactive sites in the internal section of the beads. In some embodiments the beads comprise both cleavable and non-cleavable ligands. In some embodiments, the bead is a TentaGel® B resin.

[0121] The beads of the present disclosure may comprise any suitable size. The bead size may be optimize based on the application, the screening system, the effector, the barcode, the target, the assay, or other parameters involved in the integrated methods and systems.

[0122] In some examples, the bead diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less. The diameter of the bead may differ in different solvents. In some cases, the bead diameter may at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers (pm) in water.

[0123] In some examples, the effector may be covalently bound to the scaffold. In some examples, the effector may be non-covalently bound to the scaffold. In some examples, the effector may be bound to the scaffold through ionic interactions. In some examples, the effector is bound to the scaffold through hydrophobic interactions. In some cases, the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less.

[0124] In some examples, the bead mean diameter may be at least about 1 nm, 10 nm, 100 nm, 200 nm, 500 nm, 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 12 pm, 14 pm, 16 pm, 20 pm, 25 pm, 30 pm, 50 pm, 80 pm, 100 pm, 90 pm, 100 pm, 120 pm, 140 pm, 160 pm, 180 pm, 200 pm, 300 pm, or larger. The bead size may differ based on the application. In some examples, the bead size may be at most about 300 pm, 200 pm, 160 pm, 100 pm, 80 pm, 50 pm, 40 pm, 30 pm, 20 pm, 10 pm, 8 pm, 6 pm, 4 pm, 3 pm 2 pm, 1 pm, 500 nm, 200 nm, 100 nm, 10 nm, 1 nm, or less. The size of the bead may differ in different solvents. In some cases, the bead mean diameter may at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 micrometers (pm) in water.

[0125] Provided herein is a population of beads. The population of beads may comprise or be a plurality of beads according to the beads provided anywhere herein. The beads may comprise a resin suitable for generating an encoded effector library, such that the bead is compatible with synthesizing effectors and barcodes thereon and/or therein. In some cases, the beads may be used for synthesizing encoded effector libraries. In some examples, the beads may comprise effectors and/or barcodes bound thereto. The population of beads may comprise a bead diameter distribution. The bead diameter distribution may be narrow. The bead diameter of the bead population may be within a narrow range. This quality may be referred to as monodispersity. Bead monodispersity may lead to the homogeneity of effector loading (e.g., a low variability of loaded effectors) on the beads of the bead population. Bead diameter distribution may be characterized using coefficient of variance (CV %). In some cases, the CV of the bead population may be at most about 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 5%, 3%, 2%, 1%, or less. In some cases, the bead population may be substantially monodispersed.

Cleavable linker and effector release

[0126] Cleavable linkers can be used to attach effectors to scaffolds. In some embodiments, the effector is bound to a scaffold by a cleavable linker. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation, an enzyme, a chemical reagent, heat, pH adjustment, sound, or electrochemical reactivity. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation. In some embodiments, the cleavable linker is cleavable by electromagnetic radiation such as UV light. In some embodiments, the cleavable linker is a photocleavable linker. In some embodiments the photocleavable linker is cleavable by electromagnetic radiation. In some embodiments the photocleavable linker is cleavable through exposure to light. In some embodiments, the light comprises UV light. In some embodiments, the cleavable linker is cleavable by a cleaving reagent. In some embodiments, the cleavable linker must first be activated in order to be able to be cleaved. In some embodiments, the cleavable linker is activated through interaction with a reagent.

[0127] In some embodiments, the cleavable linker is a disulfide bond. In some embodiments, the cleavable linker is a disulfide bond, and the cleavable reagent is a reducing agent. In some embodiments, the reducing agent is a disulfide reducing agent. In some embodiments, the disulfide reducing agent is a phosphine. In some embodiments, the reducing agent is 2-mercapto ethanol, 2-mercaptoethylamine, tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, a combination thereof, or a derivative thereof.

[0128] In some embodiments, the cleavable linker and cleaving reagent are biorthogonal reagents. Bioorthogonal reagents are combinations of reagents that selectively react with each other, but do not have significant reactivity with other biological components. Such reagents allow for minimal cross-reactivity with other components of the reaction mixture, which allows for less off target events.

[0129] In some embodiments, the cleavable linker is a substituted trans-cyclooctene. In some embodiments, the cleavable linker is a substituted trans-cyclooctene and the cleaving reagent is a tetrazine. In some embodiments, the cleavable linker as the structure

Effector scaffold , wherein X is -C(=O)NR-, -C(=O)O-, -C(=O)- or a bond, and R is H or alkyl.

In some embodiments, the cleaving reagent is a tetrazine. In some embodiments, the cleaving reagent is dimethyl tetrazine (DMT). Further examples of tetrazine cleavable linkers and methods of use are described in Tetrazine-triggered release of carboxylic-acid-containing molecules for activation of an anti-inflammatory drug, ChemBioChem 2019, 20, 1541-1546, which is hereby incorporated by reference.

[0130] In some embodiments, the cleavable linker comprises an azido group attached to the same carbon as an ether linkage. In some embodiments, the cleavable linker has the

Scaffold Effector

Effector Scaffold structure In some embodiments, the cleaving reagent is a reagent that reduces an azido group. In some embodiments, the cleaving reagent is a phosphine. In some embodiments, the cleaving reagent is hydrogen and a palladium catalyst.

[0131] In some embodiments, the cleavable linker is cleaved by a transition metal catalyst. In some embodiments, the cleavage reagent is a transition metal catalyst. In some embodiments, the transition metal catalyst is a ruthenium metal complex. In some embodiments, the cleavable linker is an O-allylic alkene. In some embodiments, the cleavable

Scaffold linker has the structure . A non-limiting example of such a catalyst is described in Bioorthogonal catalysis: a general method to evaluate metal-catalyzed reaction in real time in living systems using a cellular luciferase reporter system, Bioconjugate Chem. 2016, 27, 376-382, which is hereby incorporated by reference. In some embodiments, the transition metal complex is a palladium complex. In some

Scaffold embodiments, the cleavable linker has the structure Effector

Such cleavable linkers are described in 3’-O-modified nucleotides as reversible terminators for pyrosequencing, PNAS October 16, 2007, 104 (42) 16462-16467, which is hereby incorporated by reference.

[0132] In some embodiments, the number of effectors cleaved from the scaffold is controlled. In some embodiments, the number of effectors cleaved from a scaffold is controlled by controlling the amount of stimulus used to cleave the cleavable linker. In this context, a “stimulus” is any method or chemical used to specifically cleave a cleavable linker. In some embodiments, the stimulus is a chemical reaction with a cleaving reagent. In some embodiments, the stimulus is electromagnetic radiation. In some embodiments, the stimulus is a change in pH. In some embodiments, the change in pH is acidification. In some embodiments, the change in pH is basification.

[0133] In some embodiments, methods described herein comprise cleaving the cleavable linker with a cleaving reagent. In some embodiments, the methods comprise adding the cleaving reagent to an encapsulation comprising an effector bound to a scaffold through a cleavable linker. In some embodiments, the methods comprise adding the cleaving reagent to an encapsulation comprising an encoding bound to a scaffold through a cleavable linker. [0134] In some embodiments, the number of effectors cleaved from the scaffold is controlled by controlling the concentration of the cleaving reagent. In some embodiments, the concentration of the cleavage reagent is controlled in an encapsulation containing an encoded effector bound to a scaffold. In some embodiments, the concentration of chemical reagent used to cleave the cleavable linker is at least 100 pM, at least 500 pM, at least 1 nM, at last 10 nM, at least 100 nM, at least 1 pM, at least 10 pM, at least 100 pM, at least 1 mM. at least 10 mM, at least 100 mM, or at least 500 mM. In some embodiments, the concentration of cleaving reagent used to cleave the cleavable linker is at most 100 pM, at most 500 pM, at most 1 nM, at most 10 nM, at most 100 nM, at most 1 pM, at most 10 pM, at most 100 pM, at most 1 mM, at most 10 mM, at most 100 mM, or at most 500 mM.

[0135] In some embodiments, the cleaving reagent is added to a plurality of encapsulations.

In some embodiments, the concentration of cleaving reagent added to the plurality of encapsulations is substantially uniform among individual encapsulations of the plurality. In some embodiments, the concentration of cleaving reagent used to cleave the cleavable linker in a plurality of encapsulations is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% identical in each individual encapsulation. In some embodiments, concentration of cleaving reagent used to cleave the cleavable linker in a plurality of encapsulations differs by no more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 50- fold, or 100-fold among each individual encapsulation of the plurality.

[0136] In some embodiments, the cleaving reagent is added to the encapsulation by picoinjection. In some embodiments, the encapsulation is passed through a microfluidic channel comprising a pico-inj ection site. In some embodiments, pico-inj ections are timed such that the rate of pico-inj ection matches the rate at which encapsulation cross the pico-inj ection site. In some embodiments, at least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a pico-inj ection site receive a pico-inj ection. In some embodiments, the pico-inj ections are at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 500-fold, or at least 1000-fold smaller in volume than the passing droplets. In some embodiments, the cleaving reagent is added to the encapsulation by droplet merging.

[0137] In some embodiments, the cleaving reagent is added from a stock solution to the encapsulation. In some embodiments, the stock solution is at least 2X, 5X, 10X, 20X, 30X, 50X, 100X, 500X, or 1000X more concentrated than the desired final concentration in the encapsulation. [0138] In some embodiments, methods and systems described herein comprise cleaving a photocleavable linker between an encoded effector and a scaffold. In some embodiments, the methods and systems described herein comprise exposing an encapsulation to electromagnetic radiation comprising an effector bound to a scaffold through a photocleavable linker. In some embodiments, the methods and systems described herein comprise exposing an encapsulation to light (for e.g., UV light) comprising an effector bound to a scaffold through a photocleavable linker. In some embodiments, the encapsulation is exposed to the light using a microfluidic device.

[0139] In some embodiments, the photocleavable linker is cleaved by exposure to light (e.g., UV light). In some embodiments, the concentration of the number of effector molecules released from a scaffold is controlled by controlling the intensity and/or duration of exposure to UV light. Any suitable UV light intensity may be used. In some cases, the intensity of the UV light used of exposing and cleaving the cleavable linker may be from about 0.1 J/cm ² to about 200 J/cm ² . Any suitable UV power may be used. In some examples, the UV power for cleaving the cleavable linker may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 2000, 4000, 5000 mW. The light may be calibrated and optimized as needed.

[0140] The cleavable linker may be cleaved by electromagnetic radiation. In some embodiments, the concentration of the number of effector molecules released from a scaffold is controlled by controlling the intensity or duration of electromagnetic radiation.

[0141] Any suitable photoreactive or photocleavable linker can be used as a cleavable linker cleaved by electromagnetic radiation (e.g., exposure to UV light). A list of example linkers cleavable by electromagnetic radiation may comprise: o-nitrobenzyloxy linkers, o- nitrobenzylamino linkers, a-substituted o-nitrobenzyl linkers, o-nitroveratryl linkers, (v) phenacyl linkers, p-alkoxyphenacyl linkers, benzoin linkers, pivaloyl linkers, and other photolabile linkers. Further examples of photocleavable linkers are described in Photolabile linkers for solid-phase synthesis, ACS Comb Sci. 2018 Jul 9;20(7):377-99, which is hereby incorporated by reference. In some examples, the cleavable linker is an o-nitrobenzyloxy linker, an o-nitrobenzylamino linker, an a-substituted o-nitrobenzyl linker, an o-nitroveratryl linker, a phenacyl linker, p-alkoxyphenacyl linker, a benzoin linker, or a pivaloyl linker.

[0142] In some examples, the photocleavable linker may be activatable by a stimulus before it is cleavable. For example, a first stimulus (light, heat, energy, chemical, or beyond) may be applied to activate the cleavable linker. A second stimulus (light, heat, energy, chemical, or beyond) may be applied to cleave the cleavable linker. UV exposure is an example of the second stimulus. Activation by a chemical is an example of activating the photocleavable linker. In some cases, the number of effectors released can be controlled by controlling and modulating the stimulus. Activatable photocleavable linkers that need to be activated before being cleaved through exposure to the second stimulus (e.g., UV light) may enable improved bead-handling, synthesis, storage, and preparation due to minimized or eliminated encoded effector release through the application of the second stimulus (e.g., incident UV exposure).

[0143] FIG. 6A provides an exemplary molecule configured to be transformed upon interaction with a reagent, such that it becomes activated for UV photocleavage (reference: J. AM. CHEM. SOC. 2003, 125, 8118-8119; 10.1021/j a035616d). As depicted, the azide group functionally reduces the sensitivity of the photocleavable-linker moiety, such that linker is more stable, thus advantageous for handling and storing under ambient lighting. As depicted in FIG. 6A, the azide can be converted upon reagent treatment (HOF-CH3CN) to generate the photo-sensitive Nitro-benzyl motif (molecule depicted in the middle), wherein the product photocleavable-linker can be calibrated to release a known quantity of effector upon UV- exposure. FIG. 6B provides another exemplary molecule configured to be transformed upon interaction with a reagent, such that it becomes activated for UV photocleavage (reference: J. Comb. Chem. 2000, 2, 3, 266-275). As depicted, the thio-phenol ester provides a stable covalent linker to compound (R). Specific oxidation of the thio-phenol (shown in middle molecule) can generate an “activated” linker-moiety. Kinetic control of the oxidation step may allow for quantitative “activation” to prescribe compound release. In some embodiments, base treatment causes linker scission through elimination, thereby generating a free acid compound, or with subsequent decarboxylation generates just a compound.

[0144] An active cleavable linker may be cleaved by a stimulus (e.g., the second stimulus in case a first stimulus is required for activating the linker). The stimulus for cleaving the cleavable linker may comprise a variety of modalities. Examples of stimuli which can be used for cleaving the linker may comprise an enzyme, a protease, a nuclease, a hydrolase, a chemical, an energy, light (e.g., UV light), heat, electromagnetic radiation, or another kind of stimulus. The cleavable linker may comprise or be a peptide, a nucleic acid molecule, a carbohydrate, a chemical moiety, a chemical bond and beyond. The stimulus may be optimized in terms of intensity or power (e.g., intensity of an energy) or concentration (e.g., in case of a chemical stimulus such as an enzyme capable of cleaving the linker).

[0145] The methods of screening may comprise cleaving the cleavable linker and thereby releasing the effector in a compartment to interact with a sample. The cleavable linker may be cleaved by any suitable stimulus described anywhere herein. In some cases, a chemical may be added to the compartment (e.g., droplet or well) by any suitable method (e.g., dispenser, robot, manually by a person, a microfluidic chip module, etc.) at a defined time point. For example, in case the screening system is a droplet microfluidic system, pico-inj ection may be used to inject a chemical into a droplet after its formation to cleave the cleavable linker. In another example, if the screening system is an array, the cleaving reagent may be added to the wells of the array manually by a person or using a machine or robot. The concentration of the released effector can be controlled by the intensity or concentration of the stimulus applied. In some examples, the activating reagent for activating the cleavable linker may comprise a disulfide reducing reagent. In an example, the activating reagent comprises tetrazine.

[0146] The activating reagent may be added to the encapsulation by pico-inj ection. In some examples, the encapsulation may pass through a microfluidic channel comprising a picoinjection site, in case the encapsulation is a droplet in a microfluidic device. Reagent addition (e.g., pico-inj ections) may be timed such that the rate of reagent addition (e.g., pico-inj ection) matches the rate at which encapsulation cross the pico-inj ection site. In some examples, least 80%, 85%, 90%, 95%, 98%, or 99% of encapsulations passing a pico-inj ection site receive a pico-inj ection. In some embodiments, the pico-inj ections are at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 50-fold, at least 100-fold, at least 500- fold, or at least 1000-fold smaller in volume than the passing droplets. In some embodiments, the activating reagent is added to the encapsulation by droplet merging.

[0147] Any suitable concentration of an activating reagent may be added to a compartment to activate a cleavable linker. In some examples, the concentration of the activating agent may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 millimolar (mM) or less. In some examples, the concentration of activating reagent may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 micromolar (pM). In some examples, the concentration of the activating reagent used to activate the cleavable linker may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100 picomolar (pM) or less.

[0148] The effector may be released from the scaffold to move freely in the compartment (e.g., in the solution). Free movement may allow the effector to interact with the sample or target being interrogated. The effector may be released in a controlled fashion. This controlled release may allow for a predetermined and/or known dose of effectors to be released form the scaffold. Such a procedure may allow for improved quantification and analysis of data (e.g., structure-activity relationship data or hits) from a screen, as dose response measurements can be detected or recorded. Additionally, releasing a known number of effectors across a library of effectors being screened may remove bias from the sample set. [0149] Bias can occur in library screens using encoded scaffolds when individual scaffolds possess attachments of effectors that vary in amount among the scaffolds of the library. For example, one scaffold may contain 10 copies of an effector molecule, and another scaffold may contain 1000 copies of an effector molecule. Consequently, different concentrations of effector being screened against a sample or target may be released. As a result, in some cases, making a determination of the efficacy of individual effectors may be difficult to ascertain. By releasing a uniform amount of effectors from each scaffold in a screen, a uniform dose across the screen may be employed, removing bias from lower potency, higher concentration effectors.

[0150] In some examples, the effectors may be released to a determined concentration in a compartment. The desired/intended concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 pM or higher. In some examples, the released effector concentration may be at least about 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 nM or higher. In some examples, the released effector concentration may be at least about 1,

2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 pM or higher. In some examples, the released effector concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mM or higher.

[0151] In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 mM or lower. In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4,

3, 2, 1 pM or lower. In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7,

6, 5, 4, 3, 2, 1 nM or lower. In some examples, the released effector concentration may be at most about 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 pM or lower. The concentration of the effector released in the compartments may be substantially uniform across the compartments. For example, the concentration of the released effector among the compartments may vary at most about 200%, 150%, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 8%, 9%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

[0152] Upon assay set-up in a compartment (e.g., a droplet or a well of any kind as mentioned anywhere herein), the compartment may be incubation for a determined period. The incubation time may be described anywhere herein. In some cases, incubation time may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 30, 40 minutes (min), or longer. In some cases, the incubation time may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, 30, 35, 40 hour(s) (hr) or longer. In some cases, the incubation time may be at most about 70, 60, 50, 40, 30, 24, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hour(s) (hr) or less. In some cases, the incubation time may be at most about 60, 50, 40, 30, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3 minutes (min) or less.

[0153] The effectors of the present disclosure may be of any kind or modality. The effector may be biochemical, chemical, or biological moiety. In some embodiments, an effector is a cell, a protein, a peptide, small molecule, a biological moiety, a small molecule fragment, a nucleic acid, or another kind of effector. In some cases, an effector may be molecule capable of interacting with a target. In an example, the effector is a small molecule. In an example, the effector is a compound. In an example, an effector is a small molecule comprising at least 1, 2, 3, 4, 5, 6, or more small molecule building blocks. The term “effector” is used broadly to encompass any moiety whose effect on a sample is being interrogated.

[0154] In some examples, the effectors may comprise a handle that allows for attachment to a scaffold. A handle may be a reactive functional group that can be used to tether the effector to an attachment site on a scaffold. This handle may be any functional group capable of forming a bond. Example of handles may comprise sulfhydryl groups, CLICK chemistry reagents, amino groups, carboxylate groups, or other groups.

[0155] The effectors may comprise subunits (e.g., individual subunits). Subunits may be joined using various chemical reactions to form the full effector. Iterative chemical processes may be used to generate the effectors, similar to methodologies used in peptide synthesis (e.g., solid-phase peptide synthesis (SPPS)). Similar methods can be used to create non-peptide effectors (e.g., small molecule effectors), wherein a first reaction may be performed to link two subunits, the two linked subunits may be subjected to a second reaction to activate the linked subunits, and a third subunit may then be attached, and so on. Any type of such an iterative chemical synthesis scheme may be employed to create the effectors used in the methods and systems provided herein.

[0156] In some examples, the effectors may elicit a response from the target being interrogated. The response elicited can take any form and may depend on the sample being interrogated. As an example, when the sample comprises a cell, the response may be a change in expression pattern, apoptosis, expression of a particular molecule, or a morphological change in the cell. As another example, when the sample comprises a protein, the effector may inhibit protein activity, enhance protein activity, alter protein folding, or measure protein activity.

[0157] In some examples, the effector may be a protein. The protein may be naturally occurring or mutant. The effector may be an antibody (AB) or antibody fragment. The effector may be an enzyme, a binding protein, an AB or AB-fragment, a structural protein, an enzyme, a binding protein, a storage protein, a transport protein, or any mutant or combinations thereof. In some examples, the effector may be a peptide, a non-natural peptide, a polymer, or an unnatural amino acid. In some examples, the peptide may comprise a non-peptide region. In some examples, the peptide may be a cyclic peptide. In some examples, the peptide may comprise a secondary structure that mimics a protein.

[0158] The peptide or polymer may be made of a number of units (e.g., a number of amino acids). In some examples, the peptides may comprise a number of amino acids. For example, a peptide effector may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 39, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 300 or more amino acids. In some examples, the peptide may comprise at most about 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5 or a smaller number of amino acids. In some cases, the peptide may comprise from about 3 to about 10, from about 6 to 20, from about 9 to 60, from about 12 to 180 amino acids.

[0159] The effector may be a compound, an organic molecule, a drug-like small molecule, an organic compound, an effector comprising organic and/or inorganic atoms or molecules, an effector comprising one or more metal atoms or molecules, a small molecule, or a macromolecule. The compound may be an organic molecule. The compound may be an inorganic molecule. The compounds used as effectors may contain organic and inorganic atoms. The compound may be a drug-like small molecule. In some embodiments, the compound may be an organic compound. The compound may comprise one or more inorganic atoms, such as one or more metal atoms. In some examples, an effector/compound may be a completed chemical that is synthesized by connecting a plurality of chemical monomers to each other. In some examples, the effector may be a pre-synthesized compound loaded onto a bead after synthesis. The compound may be a small molecule fragment. Small molecule fragments may be small organic molecules which are small in size and low in molecular weight. In some examples, the small molecule fragments may be less than about 500, 400, 300, 200, 100 Dalton (Da) or less in molecular weight (MW).

[0160] In some examples, the effector may be an effector nucleic acid. The effector nucleic acid may comprise a number of nucleotides. In some examples, the effector nucleic acid may comprise at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some examples, the number of nucleotides in the nucleic acid effector may be at least about 10 ³ , 2 x 10 ³ , 3 x 10 ³ , 4 x 10 ³ , 5 x 10 ³ , 6 x 10 ³ , 7 x 10 ³ , 8 x 10 ³ , 9 x 10 ³ or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least about 10 ⁴ , 2 x 10 ⁴ , 3 x 10 ⁴ , 4 x 10 ⁴ , 5 x 10 ⁴ , 6 x 10 ⁴ , 7 x 10 ⁴ , 8 x 10 ⁴ , 9 x 10 ⁴ , or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least about 10 ⁵ , 2 x 10 ⁵ , 3 x 10 ⁵ , 4 x 10 ⁵ , 5 x 10 ⁵ , 6 x 10 ⁵ , 7 x 10 ⁵ , 8 x 10 ⁵ , 9 x 10 ⁵ or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least 10 ⁶ , 2 x 10 ⁶ , 3 x 10 ⁶ , 4 x 10 ⁶ , 5 x 10 ⁶ , 6 x 10 ⁶ , 7 x 10 ⁶ , 8 x 10 ⁶ , 9 x 10 ⁶ or more. In some examples, the number of nucleotides in a nucleic acid effector may be at least about 10 ⁷ , 2 x 10 ⁷ , 3 x 10 ⁷ , 4 x 10 ⁷ , 5 x 10 ⁷ , 6 x 10 ⁷ , 7 x 10 ⁷ , 8 x 10 ⁷ , 9 x 10 ⁷ or more.

Barcode

[0161] The effectors provided herein can be linked with encodings. In some embodiments, the effectors are linked with an encoding. In some instances, the encoding allows a user to determine the structure of the effector by determining a property of the encoding. Thus, each encoding moiety has a measurable property that, when measured, can be used to determine the structure of the effector which is encoded.

[0162] In some examples, the encoding is a nucleic acid. The sequence of the nucleic acid may provide information about the structure and/or identity of the effector. The encoding may comprise or be a nucleic acid barcode. The terms encoding and barcode may be used interchangeably. The barcode may comprise a sequencing primer. Sequencing the nucleic acid encoding allows the user to ascertain the structure of the corresponding effector.

[0163] In some examples, the barcode/encoding may comprise or be DNA. An example barcode may comprise double-stranded DNA or single-stranded DNA. In some examples, the barcode/encoding may comprise or be RNA. An example barcode may comprise doublestranded RNA or single-stranded RNA. In some examples, the barcode may comprise or be a peptide or a peptide nucleic acid (PNA).

[0164] The barcode encoding the effector may comprise or be a nucleic acid molecule of any suitable length. For example, the barcode may comprise a number of nucleotides. The number of nucleotides in a barcode may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or more. In some examples, the number of nucleotides may be at least 10 ³ , 2 x 10 ³ , 3 x 10 ³ , 4 x 10 ³ , 5 x 10 ³ , 6 x 10 ³ , 7 x 10 ³ , 8 x 10 ³ , 9 x 10 ³ or more. In some examples, the number of nucleotides in a barcode may be at least IO ⁴ , 2 x IO ⁴ , 3 x IO ⁴ , 4 x IO ⁴ , 5 x IO ⁴ , 6 x IO ⁴ , 7 x IO ⁴ , 8 x IO ⁴ , 9 x IO ⁴ , or more. In some examples, the number of nucleotides in a barcode may be at least 10 ⁵ , 2 x 10 ⁵ , 3 x 10 ⁵ , 4 x 10 ⁵ , 5 x 10 ⁵ , 6 x 10 ⁵ , 7 x 10 ⁵ , 8 x 10 ⁵ , 9 x 10 ⁵ or more. In some examples, the number of nucleotides in a barcode may be at least 10 ⁶ , 2 x 10 ⁶ , 3 x 10 ⁶ , 4 x 10 ⁶ , 5 x 10 ⁶ , 6 x 10 ⁶ , 7 x 10 ⁶ , 8 x 10 ⁶ , 9 x 10 ⁶ or more. In some examples, the number of nucleotides in a barcode may be at least 10 ⁷ , 2 x 10 ⁷ , 3 x 10 ⁷ , 4 x 10 ⁷ , 5 x 10 ⁷ , 6 x 10 ⁷ , 7 x 10 ⁷ , 8 x 10 ⁷ , 9 x 10 ⁷ or more.

[0165] In some embodiments, the encoding is made up of individual subunits that encode a corresponding effector subunit. Consequently, an entire encoding can specify which individual subunits have been linked or combined to form the effector. In some embodiments, each subunit may comprise up to 5, 10, 15, 20, 25, 30, 40, 50, or more individual nucleotides. The full encoding sequence can comprise any number of these individual subunits. In some embodiments, the full encoding sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more encoding subunits. These encoding subunits can be ligated together using many known methods, including enzymatic ligation, template-free synthesis, templated polymerase extension, chemical ligation, recombination, or solid phase nucleic acid synthesis techniques.

[0166] In some examples, the encoding/barcode may be a molecular weight barcode. The molecular weight barcode may be a peptide. In some cases, molecular weight (MW) barcode may comprise a peptide with unnatural amino acids. The molecular weight (MW) of the barcode may be at least about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10000, 15000 Daltons or larger. In some examples, the molecular weight barcode peptide may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more amino acids. In some cases, the molecular weight barcode peptide may comprise at most about 60, 50, 40, 30, 20, 15, 10, 5, 4, 3, 2 or smaller number of amino acids.

[0167] Barcodes may be loaded onto and/or into the scaffold. In some examples, the barcode may comprise or be a DNA barcode. Alternatively or in addition, any suitable barcode mentioned anywhere herein may be used. A scaffold (e.g., an example bead with a porous material as described anywhere herein, in some cases a 10-micron bead) may be loaded with a predetermined number of barcodes or copies of the barcode. In some examples, the bead may be a TentaGel bead. Alternatively, any other kind of suitable bead may be used and loaded with the barcode and the effector. The number of copies of the barcode loaded onto/into the scaffold may be at least about 10, 100, 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ or more. In some examples, the number of copies of the barcode loaded onto/into the scaffold may be at most about IO ¹⁰ , 10 ⁹ , 10 ⁸ , 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ or less. The barcode may comprise one or more barcoding sequences. In some cases, the barcode may comprise a bead-specific barcode (BSB) that identifies the bead/ scaffold and may be used to count the scaffold.

Barcode non-sorting method

[0168] In some embodiments provided herein, the methods do not comprise a physical sorting step. For example, the deconvolution of which effectors had the desired effect on a sample is accomplished in a different manner. The method may further comprise the step of adding additional reagents to the encapsulation which may add a barcode to the encoding. In some examples, the method may further comprise the step of adding additional reagents to the encapsulation which add a barcode to a nucleic acid encoding. The additional reagents may add a barcode to the encoding by annealing the barcode to the encoding, ligating the barcode to the encoding, or amplifying the barcode onto the encoding. In some cases, the additional reagents may comprise a tagging nucleic acid comprising a sequence complementary to a sequence on the nucleic acid encoding which may act as a primer for the nucleic acid encoding and the barcode. In some embodiments, the additional reagents may comprise enzymes to add the barcode to the nucleic acid encoding.

[0169] Provided herein are methods for screening an encoded effector. In some examples, screening may be performed without a physical sorting step. Such method may comprise providing a sample, a nucleic acid encoded effector, and a nucleic acid encoding in an encapsulation. A signal may be detected which may be indicative of the activity of the assay and/or the target in presence and/or absence of perturbation by an encoded effector. In some examples, a first capping mixture may add a first nucleic acid cap to the nucleic acid encoding, a second capping mix is added to the encapsulation/compartment. In some examples, the second capping mixture may only be added if the first capping mixture is not added to the encapsulation/compartment. The first nucleic acid cap and the second nucleic acid cap may comprise different sequences. In some cases, only the first nucleic acid cap or only the second nucleic acid cap may be added to the nucleic acid encoding.

[0170] The first and second nucleic acid caps can have different significance and indicate different things when added to nucleic acid encodings. In some examples, the first nucleic acid cap may indicate that the effector comprises a given activity. The given activity may result in the signal being above a pre-determined level or threshold. The determined activity may have resulted in the signal being below a pre-determined level or threshold. In some examples, the second nucleic acid cap may indicate that the effector lacks a desired activity. The lack of desired activity may have resulted in the signal being below a pre-determined threshold. The lack of desired activity may have resulted in the signal being above a pre-determined threshold. [0171] The nucleic acid caps can be added to nucleic acid encodings by a variety of methods. Example methods for adding the nucleic acid cap to the nucleic acid encoding may comprise ligation, hybridization, extension of the nucleic acid encoding, chemical crosslinking of the nucleic acids, chemical crosslinking with psoralen, or other methods. In some examples, a sequence complementary with the nucleic acid cap is located on the terminal end of the nucleic acid encoding to allow for the addition of the nucleic acid cap. In some examples, the nucleic acid cap may comprise a barcode sequence. In some examples, the capping mixture may comprise additional reagents for adding the nucleic acid cap to the encoding. In some examples, the additional reagents may comprise an enzyme. The enzyme may comprise or be a polymerase, a ligase, a restriction enzyme, or a recombinase. In some examples, the enzyme may be a polymerase.

Bead capture of nucleic acids

[0172] In addition to measuring activity from detectable signals, additional information can be gathered from a screen by incorporating nucleic acids from the sample onto encodings. In some examples, the methods may comprise transferring one or more nucleic acids from the sample (e.g., from a cell or cell nucleolus) to the encoding. The transfer of nucleic acids from the sample to the encoding may allow for substantial information about the sample and the suspected effect of the effector on the sample or to be ascertained, for example, when the sample comprises a cell. The transfer of the nucleic acids from the sample can allow for quantification of expressed protein by quantifying the amount of target mRNA, as well as provide global proteomic and genomic data about the cell. This data can be collected and compared to cells that did not receive a dose of the indicated effector.

[0173] In some examples, provided herein is a method for detecting sample nucleic acids in a nucleic acid encoded effector screen. The method may comprises providing one or more cells, a nucleic acid encoded effector, and a nucleic acid encoding in a compartment. The compartment may be incubated for a period of time to allow for the effector and the sample (e.g., a sample comprising a cell) to interact. The interaction between the effector and the sample (e.g., a cell) may produce a signal. In some cases, the period of time may be sufficient to allow for changes in transcription and/or translation to occur in the sample (e.g., a sample comprising a cell) in presence and/or absence of to the effector (e.g., in response to the effector). The method may comprise transferring cellular nucleic acids from the sample (e.g., from a cell or constituent of a cell) to the nucleic acid encoding. The cellular nucleic acids may be quantified by sequencing the nucleic acid encodings after the cellular nucleic acids have been transferred. In some examples, the expression fingerprint of the cell can be generated in response to treatment with the effector. As described herein, the method may further comprise detecting a signal produced through interaction between the effector and one or more cells, and sorting the encapsulation based on the detection of the signal.

[0174] In order to release the cellular nucleic acids, the cell may be lysed. In some examples, the method may further comprise the step of lysing the cell. Lysing the cell may comprise adding lysis buffer to the compartment. In some cases, the lysis buffer may be added by pico-inj ection (e.g., in case the compartment is a droplet in a droplet microfluidic device). In case the compartment is a well, any suitable reagent addition method described anywhere herein (e.g., automatic dispensing by a robot, manual addition, or beyond) may be added. The lysis buffer may comprise a salt. In some examples, the lysis buffer may comprise a detergent. Examples of the detergent may comprise SDS, Triton, or Tween. The lysis buffer may comprise a chemical which causes cell lysis.

[0175] Any type of cellular nucleic acid can be transferred to the nucleic acid encoding. In some examples, the method may comprise transferring one or more cellular nucleic acids from the sample to the nucleic acid encoding. In some embodiments, the nucleic acids may comprise or be mRNA. In some cases, the nucleic acids may be mRNA that express a protein of interest. The nucleic acids may comprise or be genomic DNA. The nucleic acids may be added as antibody-DNA constructs. The nucleic acids added may be proximity ligation products. The nucleic acids may proximity extension products. In some examples, a plurality of different cellular nucleic acids may be attached to nucleic acid encodings.

[0176] The nucleic acids transferred to the encoding may comprise a complementary sequence to a sequence on the encoding. This may allow for the ligation of the sample nucleic acid with the encoding nucleic acid via various methods. These methods may comprise annealing, ligating, chemically cross-linking, or amplifying the cellular contents on to the nucleic acid encoding the effector. The nucleic acid encodings may comprise a sequence complementary to the nucleic acid of interest to be transferred to the encoding. This complementary sequence may allow for the nucleic acids to hybridize with the encoding, which in turn may allow for extension of the encoding with the cellular nucleic acid and/or vice versa. [0177] In some cases, additional reagents may be added to the compartment to facilitate the transfer of the nucleic acids to the encoding. The additional reagents may comprise an enzyme that may facilitates the transfer of the nucleic acids. The reagents for transferring the nucleic acids to the encoding may be added during the encapsulation step (e.g., loading the sample, the encoded effector, the reagents, and other components to the compartment). The reagents for transferring the nucleic acids to the encoding may be added during an incubation step. The reagents for transferring the nucleic acids to the encoding may be added after an incubation step.

[0178] In some embodiments, the additional reagents to facilitate the transfer of the nucleic acids comprise an enzyme. In some embodiments, the enzyme is a polymerase, a ligase, a restriction enzyme, or a recombinase. In some embodiments, the enzyme is a polymerase. In some embodiments, the additional reagents comprise a chemical cross-linking reagent. In some embodiments, the chemical cross-linking reagent is psoralen.

[0179] In some examples, the capture of nucleic acids from the sample, target, cell, and/or cellular constituents (e.g., nuclei) may be performed, facilitated, or improved by a second solid support or bead. The second solid support or bead may be a capture bead. In some cases, the second solid support or bead may be a nucleic acid encoded scaffold. The second bead (e.g., capture bead or nucleic acid encoded bead) may capture the released sample nucleic acids (e.g., cellular nucleic acid molecules). The second bead may be added into compartments at any point before, during, or after a screen, in some cases, prior to encoded effector release from the encoded effector bead (the first bead). In some examples, the second bead may be designed to capture the released sample nucleic acids, in addition to the encoded effector barcode, such that both the effector barcode and sample/cellular nucleic acids are captured on the second bead and co-localized or combined onto the second bead. Alternatively or in addition, in some cases, the nucleic acid barcode of the encoded effector bead (the first bead) may capture cellular nucleic acids thereon.

Adding reagents to a compartment

[0180] Methods and systems described herein may include adding one or more reagents to an compartment. In some examples, in case the compartment is a droplet in a microfluidic device, reagents can be added to the droplet by pico-inj ection. For example, reagents may be added to the droplet by pico-inj ection while a droplet passes through a pico-inj ection device.

[0181] In some embodiments, each encapsulation passing by a pico-inj ection site receives a pico-inj ection. In some examples, at least 80%, 90%, 95%, 97%, 98%, 99% or more of the encapsulation may pass through a pico-inj ection site may receive a pico-inj ection. In some examples, the encapsulations may be monitored by taking images in real time. In some cases, the encapsulations are monitored with a detector.

[0182] In some embodiments, the pico-inj ections are conditional. Conditional picoinjections may only occur after a certain condition is met. In some embodiments, a conditional pico-inj ection only occurs when a signal is detected. In some embodiments, a reagent is injected by pico-inj ection if a signal is detected. In some embodiments, a reagent is added to an encapsulation by pico-inj ections if a signal is detected. In some embodiments, the signal must be above a pre-determined threshold.

[0183] In some embodiments, a method for screening an encoded effector comprises providing an encapsulation comprising a sample and one or more scaffolds, wherein the scaffold comprises: an encoded effector bound to the scaffold by a cleavable linker and a nucleic acid encoding the effector; cleaving the cleavable linker to release a pre-determined amount of the effector; adding one or more reagents to the encapsulation through picoinjection or by droplet merging; detecting one or more signals from the encapsulation, wherein the signal results from an interaction between the encoded effector and the sample; and sorting the encapsulation based on the detection of the signal. In some embodiments, one or more reagents added to an encapsulation may comprise one or more fluorophores, one or more antibodies, one or more chemical compounds, or any combination thereof. The picoinjection or addition of reagents to the compartment or well can be performed at any point before, during, or after the assay and any other step mentioned in the workflow (e.g., the addition of the scaffold and the effector release, the detection of the signal, sorting, etc.) The steps of the workflow can be performed in any suitable order based on the application.

[0184] In some cases, the compartments are wells in an array. In some cases, wells of an array are labeled with barcodes (e.g., nucleic acid barcodes), specific for each compartment (e.g., each well in the array). This may be referred to as a compartment barcode. In some cases, the barcode labeling the compartment (e.g., nucleic acid labelling the well) may be attached to the surface of the compartment (e.g., well). In some cases, the compartment barcode may be attached to a scaffold introduced into the well. In some cases, the compartment barcode may be designed to anneal, ligate, or extend the nucleic acid barcode of the encoded-effectors (e.g., a nucleic acid molecule of a bead-bound encoded effector that is corresponding to and identifying the effector). As an example, wells of an array may be labeled with nucleic acid barcodes, samples and encoded effectors may be introduced into wells, wells may be covered, sealed, or encapsulated (this is to segregate the well contents from the surrounding environment outside the well), the effector may be released from the encoded-effector scaffold to interact with the sample in the well, a signal may be measured from the sample within the well, and the barcode corresponding to the encoded-effectors may be transferred to the compartment barcode, creating an extended compartment barcode that connects the location of each encoded effector in the miniaturized compartmentalized platform (e.g., well-array) to the effector barcode and thereby to the structure of the effector perturbing the sample in the corresponding compartment. In some examples, a well comprising a compartment barcode may be used for performing any method disclosed anywhere in the present disclosure, in some cases, such method may comprise performing light patterning, selective polymerization, barcode cleavage, barcode tagging, barcode extension, barcode/bead emulsification or retrieval from the compartment, barcode amplification, any combination thereof, or any barcode post screen processing method described elsewhere herein.

Post-sorting of encapsulations

[0185] After a sorting step or barcoding step based on the detection of the signal of interest, the results may be deconvoluted to determine which effectors displayed the activity of interest against the target sample. The methods presented herein may comprise the step of ascertaining which encodings are present in the samples sorted based on the detection of the signal. In some examples, the encoding may be a nucleic acid. The method may further comprise the step of sequencing the encodings. The encoding may be sequenced by next generation sequencing. The sequences may be compared to a reference to ascertain which effectors displayed the activity of interest in the screen.

[0186] Sequencing the nucleic acid encoding may comprise sequencing the encoding while the encoding is still attached to the scaffold. Sequencing the nucleic acid encoding may comprise cleaving the nucleic acid encoding from the scaffold. Sequencing the nucleic acid encoding may comprise cleaving the nucleic acid encoding from the scaffold prior to sequencing. Cleaving the nucleic acid encoding from the scaffold may comprise cleaving a cleavable linker with a stimulus (e.g., an energy or a chemical such as a cleaving reagent). Cleaving the nucleic acid encoding from the scaffold may comprise cleaving a cleavable linker with electromagnetic radiation. Any of the cleavable linkers and cleaving reagents described herein may work for this purpose. In some examples, a nicking enzyme or a restriction enzyme can be used to cleave. In some examples, enzymatic, chemical reagent, photocleavage, or other methods can be used to cleave the encodings.

[0187] In some examples, the nucleic acid encoding may comprise a sequencing primer. The sequencing primer may allow for facile amplification of the nucleic acid encoding. In some examples, the sequencing primer may be the same for each encoding. In some cases, the sequencing primer may differ among the encodings. The sequencing primer may be upstream of the encoding. The sequencing primer may be downstream of the encoding. [0188] The methods and systems provided herein may utilize libraries of encoded effectors. Libraries of encoded effectors may comprise a plurality of different effectors, each uniquely encoded by a known encoding modality, such as those described above. Libraries may contain any number of encoded effectors. In some embodiments, the libraries comprise at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ unique effectors. In some embodiments, the libraries comprise at least about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ unique effectors.

[0189] In some embodiments, libraries of encoded effectors are linked to scaffolds. These scaffolds may be referred to as “scaffold encoded libraries.” Scaffold encoded libraries comprise a plurality of encoded effector molecules linked to the scaffold. The scaffold acts as a solid support and keeps the encoded effector molecules linked in space to their encodings. In some embodiments, the libraries comprise at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ scaffolds. In some embodiments, the libraries comprise at least about 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , IO ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ scaffolds.

[0190] Any of the methods or systems described herein for a single encoded effector may be utilized by a library of encoded effectors. In some embodiments, provided herein, is a method of screening a library of encoded effectors, the method comprising using any of the methods previously described herein with a library of encoded effectors.

[0191] In some embodiments, libraries of encoded effectors comprise a plurality of different encoded effectors. In some embodiments, libraries comprise multiple copies of substantially identical effectors or scaffold encoded effectors.

Microfluidic devices

[0192] The methods and systems provided herein may be performed on a microfluidic device. Device architecture and methods may be accomplished in a variety of ways. An analyzer or sorter device according to the disclosure comprises at least one analysis unit having an inlet region in communication with a main channel at a droplet extrusion region (e.g., for introducing droplets of a sample into the main channel), a detection region within or coincident with all or a portion of the main channel or droplet extrusion region, and a detector associated with the detection region. In certain embodiments the device may have two or more droplet extrusion regions. For example, embodiments are provided in which the analysis unit has a first inlet region in communication with the main channel at a first droplet extrusion region, a second inlet region in communication with the main channel at a second droplet extrusion region (for example, downstream from the first droplet extrusion region), and so forth. [0193] In some embodiments, a microfluidic device described herein is configured for a droplet generation frequency of at least about 10 Hz, 20 Hz, 30 Hz, 80 Hz, 100 Hz, 120 Hz, 150 Hz, 200 Hz, 300 Hz, 350 Hz, 400 Hz, 450 Hz, 500 Hz, 600 Hz, 1000 Hz, or higher.

[0194] Sorter embodiments of the device also have a discrimination region or branch point in communication with the main channel and with branch channels, and a flow control responsive to the detector. There may be a plurality of detection regions and detectors, working independently or together, e.g., to analyze one or more properties of a sample or encapsulation. The branch channels may each lead to an outlet region and to a well or reservoir. There may also be a plurality of inlet regions, each of which introduces droplets of a different sample (e.g., of cells, of virions or of molecules such as molecules of an enzyme or a substrate) into the main channel. Each of the one or more inlet regions may also communicate with a well or reservoir. [0195] As each droplet passes into the detection region, it is examined for a predetermined characteristic or activity (i.e., using the detector) and a corresponding signal is produced, for example indicating that “yes” the characteristic or activity is present, or “no” it is not. The signal may correspond to a characteristic qualitatively or quantitatively. That is, the amount of the signal can be measured and can correspond to the degree to which a characteristic or activity is present. For example, the strength of the signal may indicate the size of a molecule, or the potency or amount of an enzyme expressed by a cell, or a positive or negative reaction such as binding or hybridization of one molecule to another, a chemical reaction of a substrate catalyzed by an enzyme, or the activation or inhibition of an enzyme, or any other type of response. In response to the signal, data can be collected and/or a flow control can be activated to divert a droplet into one branch channel or another. Thus, samples within a droplet at a discrimination region can be sorted into an appropriate branch channel according to a signal produced by the corresponding examination at a detection region. In some embodiments, optical detection of molecular, cellular, viral, or other sample characteristics is used, for example directly or by use of a reporter associated with a characteristic chosen for sorting. However, other detection techniques may also be employed.

[0196] A variety of channels for sample flow and mixing can be microfabricated on a single chip and can be positioned at any location on the chip as the detection and discrimination or sorting points, e.g., for kinetic studies. A plurality of analysis units of the disclosure may be combined in one device. Microfabrication applied according to the disclosure eliminates the dead time occurring in conventional gel electrophoresis or flow cytometric kinetic studies, and achieves a better time-resolution. Furthermore, linear arrays of channels on a single chip, i.e., a multiplex system, can simultaneously detect and sort a sample by using an array of photo multiplier tubes (PMT) for parallel analysis of different channels. This arrangement can be used to improve throughput or for successive sample enrichment and can be adapted to provide a very high throughput to the microfluidic devices that exceeds the capacity permitted by conventional flow sorters. Circulation systems can be used in cooperation with these and other features of the disclosure. Microfluidic pumps and valves are one way of controlling fluid and sample flow. See, for example, U.S. patent application Ser. No. 60/186,856.

[0197] Microfabrication permits other technologies to be integrated or combined with flow cytometry on a single chip, such as PCR, moving cells using optical tweezer/cell trapping , transformation of cells by electroporation, pTAS, and DNA hybridization. Detectors and/or light filters that are used to detect viral (or cell) characteristics of the reporters can also be fabricated directly on the chip.

[0198] A device of the disclosure can be microfabricated with a sample solution reservoir or well at the inlet region, which is typically in fluid communication with an inlet channel. A reservoir may facilitate introduction of molecules or cells into the device and into the sample inlet channel of each analysis unit. An inlet region may have an opening such as in the floor of the microfabricated chip, to permit entry of the sample into the device. The inlet region may also contain a connector adapted to receive a suitable piece of tubing, such as liquid chromatography or HPLC tubing, through which a sample may be supplied. Such an arrangement facilitates introducing the sample solution under positive pressure to achieve a desired pressure at the droplet extrusion region.

[0199] A device of the disclosure may have an additional inlet region, in direct communication with the main channel at a location upstream of the droplet extrusion region, through which a pressurized stream or “flow” of a fluid is introduced into the main channel. In some embodiments, this fluid is one which is not miscible with the solvent or fluid of the sample. For example, in some embodiments, the fluid is a non-polar solvent, such as decane (e.g., tetradecane or hexadecane), and the sample (e.g., of cells, virions or molecules) is dissolved or suspended in an aqueous solution so that aqueous droplets of the sample are introduced into the pressurized stream of non-polar solvent at the droplet extrusion region.

[0200] The droplet extrusion or droplet formation region may also comprise two microfluidic channels carrying immiscible carrier fluid that are introduced on opposite sides of a main microfluidic channel. In some embodiments, the two microfluidic channels are substantially collinear. In some embodiments, such a junction resembles and X-shape. In some embodiments, the main microfluidic channel contains the sample or assay fluid. [0201] The main channel in turn communicates with two or more branch channels at another junction or “branch point”, forming, for example, a T-shape or a Y-shape. Other shapes and channel geometries may be used as desired. In sorting embodiments, the region at or surrounding the junction can also be referred to as a discrimination region or a sorting region. [0202] A detection region may be within, communicating with, or coincident with a portion of the main channel at or downstream of the droplet extrusion region. The discrimination region may be located immediately downstream of the detection region, or it may be separated by a suitable distance. It will be appreciated that the channels may have any suitable shape or crosssection (for example, tubular or grooved), and can be arranged in any suitable manner so long as flow can be directed from inlet to outlet and from one channel into another.

[0203] The channels of the disclosure may be microfabricated, for example by etching a silicon chip using conventional photolithography techniques or using a micromachining technology called “soft lithography”. These and other microfabrication methods may be used to provide inexpensive miniaturized devices, and in the case of soft lithography, can provide robust devices having beneficial properties such as improved flexibility, stability, and mechanical strength. When optical detection is employed, the devices provided herein may also provide minimal light scatter from molecule or cell (including virion) suspension and chamber material. In some embodiments, devices provided herein are relatively inexpensive and easy to set up.

[0204] A microfabricated device of the disclosure may be fabricated from a silicon microchip or silicon elastomer, printed, or embossed into a polymer such as Cyclic Olefin Polymer (COP), Cyclic Olefin Copolymer (COC), glass, plastic, or other materials (e.g., any material mentioned anywhere herein for fabrication of any miniaturized device including droplet microfluidic platforms, arrays, or any other suitable platform). In some embodiments, the dimensions of the chip are those of typical microchips, ranging between about 0.5 cm to about 5 cm per side and about 1 micron to about 1 cm in thickness. The device may contain at least one analysis unit having a main channel with a droplet extrusion region and a coincident detection region. The device may also contain at least one inlet region (which may contain an inlet channel) and one or more outlet regions (which may have fluid communication with a branch channel in each region). In a sorting embodiment, at least one detection region cooperates with at least one discrimination region to divert flow via a detector-originated signal. It shall be appreciated that the “regions” and “channels” are in fluid communication with each other and therefore may overlap, i.e., there may be no clear boundary where a region or channel begins or ends. A microfabricated device can be transparent and can be covered with a material having transparent properties, such as a glass coverslip, to permit detection of a reporter, for example, by an optical device such as an optical microscope.

[0205] The dimensions of the detection region are influenced by the nature of the sample under study and, in particular, by the size of the molecules or cells (including virions) under study. For example, viruses can have a diameter from about 20 nm to about 500 nm, although some extremely large viruses may reach lengths of about 2000 nm (i.e., as large or larger than some bacterial cells). By contrast, biological cells are typically many times larger. For example, mammalian cells can have a diameter of about 1 to 50 microns, more typically 10 to 30 microns, although some mammalian cells (e.g., fat cells) can be larger than 120 microns. Plant cells are generally 10 to 100 microns.

[0206] To prevent sample (e.g., cells, virions and other particles or molecules) or other material from adhering to the sides of the channels, the channels (and coverslip, if used) may have a coating which minimizes adhesion. Such a coating may be intrinsic to the material from which the device is manufactured, or it may be applied after the structural aspects of the channels have been microfabricated. “TEFLON” is an example of a coating that has suitable surface properties. Alternatively, the channels may be coated with a surfactant. Example surfactants may comprise sorbitan-based carboxylic acid esters (e.g., the “Span” surfactants, Fluka Chemika), including sorbitan monolaurate (Span20), sorbitan monopalmitate (Spa n 40), sorbitan monostearate (Span60) and sorbitan monooleate (Span80). Other non-limiting examples of non-ionic surfactants which may be used include polyoxy ethylenated alkylphenols (for example, nonyl-, p-dodecyl-, and dinonylphenols), poly oxy ethylenated straight chain alcohols, polyoxy ethylenated polyoxypropylene glycols, poly oxy ethylenated mercaptans, long chain carboxylic acid esters (for example, glyceryl and polyglyceryl esters of natural fatty acids, propylene glycol, sorbitol, polyoxyethylenated sorbitol esters, polyoxyethylene glycol esters, etc.) and alkanolamines (e.g., diethanolamine-fatty acid condensates and isopropanolamine-fatty acid condensates). In addition, ionic surfactants such as sodium dodecyl sulfate (SDS) may also be used.

[0207] A silicon substrate containing the microfabricated flow channels and other components may be covered and sealed, including with a transparent cover, e.g., thin glass or quartz, although other clear or opaque cover materials may be used. When external radiation sources or detectors are employed, the detection region may be covered with a clear cover material to allow optical access to the cells. For example, anodic bonding to a “PYREX” cover slip can be accomplished by washing both components in an aqueous H2SO4/H2O2 bath, rinsing in water, and then, for example, heating to about 350° C. while applying a voltage of 450V. [0208] In some examples, dielectrophoresis may produce dielectric objects, which may have no net charge, but have regions that are positively or negatively charged in relation to each other. Alternating or in addition, non-homogeneous electric fields in the presence of encapsulations, including droplets, and/or particles, such as cells or virions, cause the encapsulations and/or particles to become electrically polarized and thus to experience di electrophoretic forces. Depending on the dielectric polarizability of the particles and the suspending medium, dielectric particles will move either toward the regions of high field strength or low field strength. For example, the polarizability of living cells and virions depends on their composition, morphology, and phenotype and is highly dependent on the frequency of the applied electrical field. Thus, cells and virions of different types and in different physiological states generally possess distinctly different dielectric properties, which may provide a basis for cell separation, e.g., by differential di electrophoretic forces. Likewise, the polarizability of encapsulations, including droplets, also depends upon their size, shape and composition. For example, droplets that contain salts can be polarized. Individual manipulation of single encapsulations requires field differences (inhomogeneities) with dimensions close to the encapsulations.

[0209] In some examples, manipulation of droplets, particles, beads, or cells may depend on permittivity (a dielectric property) of the encapsulations and/or particles with the suspending medium. Thus, polymer particles, living cells and virions show negative dielectrophoresis at high-field frequencies in water. For example, dielectrophoretic forces experienced by a latex sphere in a 0.5 MV/m field (10V for a 20-micron electrode gap) in water are predicted to be about 0.2 piconewtons (pN) for a 3.4 micron latex sphere to 15 pN for a 15 micron latex sphere. These values are mostly greater than the hydrodynamic forces experienced by the sphere in a stream (about 0.3 pN for a 3.4 micron sphere and 1.5 pN for a 15 micron sphere). Therefore, manipulation of individual cells or particles can be accomplished in a streaming fluid, such as in a cell sorter device, using dielectrophoresis. Using conventional semiconductor technologies, electrodes can be microfabricated onto a substrate to control the force fields in a microfabricated sorting device of the disclosure. Dielectrophoresis is particularly suitable for moving objects that are electrical conductors. AC current may be used to prevent permanent alignment of ions. Megahertz frequencies are suitable to provide a net alignment, attractive force, and motion over relatively long distances.

[0210] Radiation pressure can also be used in the disclosure to deflect and move objects, e.g. encapsulations, droplets, and particles (molecules, cells, virions, etc.) contained therein, with focused beams of light such as lasers. Flow can also be obtained and controlled by providing a pressure differential or gradient between one or more channels of a device or in a method of the disclosure.

[0211] In some embodiments, molecules, cells, virions, beads, or droplets containing molecules, cells, beads, or virions can be moved by direct mechanical switching, e.g., with on- off valves or by squeezing the channels. Pressure control may also be used, for example, by raising or lowering an output well to change the pressure inside the channels on the chip. Different switching and flow control mechanisms can be combined on one chip or in one device and can work independently or together as desired.

[0212] Detection and discrimination for sorting can be accomplished in a variety of ways. The detector can be any device or method for interrogating a molecule, a cell or a virion as it passes through the detection region. Typically, molecules, cells or virions (or droplets containing such particles) are to be analyzed or sorted according to a predetermined characteristic that is directly or indirectly detectable, and the detector is selected or adapted to detect that characteristic. One detector is an optical detector, such as a microscope, which may be coupled with a computer and/or other image processing or enhancement devices to process images or information produced by the microscope using known techniques. For example, molecules can be analyzed and/or sorted by size or molecular weight. Enzymes can be analyzed and/or sorted by the extent to which they catalyze chemical reaction of a substrate (conversely, substrate can be analyzed and/or sorted by the level of chemical reactivity catalyzed by an enzyme). Cells and virions can be sorted according to whether they contain or produce a particular protein, by using an optical detector to examine each cell or virion for an optical indication of the presence or amount of that protein. The protein may itself be detectable, for example by a characteristic fluorescence, or it may be labeled or associated with a reporter that produces a detectable signal when the desired protein is present or is present in at least a threshold amount. There is no limit to the kind or number of characteristics that can be identified or measured using the techniques of the disclosure, which include without limitation surface characteristics of the cell or virion and intracellular characteristics, provided only that the characteristic or characteristics of interest for sorting can be sufficiently identified and detected or measured to distinguish cells having the desired characteristic(s) from those which do not. For example, any label or reporter as described herein can be used as the basis for analyzing and/or sorting molecules or cells (including virions), i.e., detecting molecules or cells to be collected.

[0213] In some embodiments, the samples (or encapsulations containing them) are analyzed and/or separated based on the intensity of a signal from an optically detectable reporter bound to or associated with them as they pass through a detection window or “detection region” in the device. In some embodiments, the samples are analyzed and/or separated based on the intensity of a signal from a detectable reporter. Molecules or cells or virions having an amount or level of the reporter at a selected threshold or within a selected range are diverted into a predetermined outlet or branch channel of the device. The reporter signal may be collected by a microscope and measured by a photo multiplier tube (PMT). A computer digitizes the PMT signal and controls the flow via valve action or electro-osmotic potentials. Alternatively, the signal can be recorded or quantified as a measure of the reporter and/or its corresponding characteristic or marker, e.g., for the purpose of evaluation and without necessarily proceeding to sort the molecules or cells.

[0214] In one embodiment, the chip is mounted on an inverted optical microscope. Fluorescence produced by a reporter is excited using a laser beam focused on molecules (e.g., DNA, protein, enzyme or substrate) or cells passing through a detection region. Fluorescent reporters include, e.g., rhodamine, fluorescein, Texas red, Cy 3, Cy 5, phycobiliprotein, green fluorescent protein (GFP), YOYO-1 and PicoGreen, to name a few. In molecular fingerprinting applications, the reporter labels are optionally fluorescently labeled single nucleotides, such as fluorescein-dNTP, rhodamine-dNTP, Cy3-dNTP, etc.; where dNTP represents dATP, dTTP, dUTP or dCTP. The reporter can also be chemically modified single nucleotides, such as biotin-dNTP. In other embodiments, the reporter can be fluorescently or chemically labeled amino acids or antibodies (which bind to a particular antigen, or fragment thereof, when expressed or displayed by a cell or virus).

[0215] Thus, in one aspect of the disclosure, the device can analyze and/or sort cells or virions based on the level of expression of selected cell markers, such as cell surface markers, which have a detectable reporter bound thereto, in a manner similar to that currently employed using fluorescence-activated cell sorting (FACS) machines. Proteins or other characteristics within a cell, and which do not necessarily appear on the cell surface, can also be identified, and used as a basis for sorting. In another aspect of the disclosure, the device can determine the size or molecular weight of molecules such as polynucleotides or polypeptides (including enzymes and other proteins) or fragments thereof passing through the detection region. Alternatively, the device can determine the presence or degree of some other characteristic indicated by a reporter. If desired, the cells, virions or molecules can be sorted based on this analysis. The sorted cells, virions or molecules can be collected from the outlet channels and used as needed. [0216] To detect a reporter or determine whether a molecule, cell or virion has a desired characteristic, the detection region may include an apparatus for stimulating a reporter for that characteristic to emit measurable light energy, e.g., a light source such as a laser, laser diode, high-intensity lamp, (e.g., mercury lamp), and the like. In embodiments where a lamp is used, the channels may be shielded from light in all regions except the detection region. In embodiments where a laser is used, the laser can be set to scan across a set of detection regions from different analysis units. In addition, laser diodes may be microfabricated into the same chip that contains the analysis units. Alternatively, laser diodes may be incorporated into a second chip (i.e., a laser diode chip) that is placed adjacent to the microfabricated analysis or sorter chip such that the laser light from the diodes shines on the detection region(s).

[0217] In some embodiments, an integrated semiconductor laser and/or an integrated photodiode detector are included on the silicon wafer in the vicinity of the detection region. This design provides the advantages of compactness and a shorter optical path for exciting and/or emitted radiation, thus minimizing distortion.

[0218] Sorting schemes can be accomplished in a variety of ways. According to the disclosure, molecules (such as DNA, protein, enzyme, or substrate) or particles (i.e., cells, including virions) are sorted dynamically in a flow stream of microscopic dimensions based on the detection or measurement of a characteristic, marker or reporter that is associated with the molecules or particles. More specifically, encapsulations of a solution (for example an aqueous solution or buffer), containing a sample of molecules, cells or virions, are introduced through a droplet extrusion region into a stream of fluid (for example, a non-polar fluid such as decane or other oil) in the main channel. The individual droplet encapsulations are then analyzed and/or sorted in the flow stream, thereby sorting the molecules, cells, virions, or scaffolds contained within the droplets.

[0219] The flow stream in the main channel may be continuous and may be stopped and started, reversed, or changed in speed. Prior to sorting, a liquid that does not contain samples molecules, cells, virions, or beads can be introduced into a sample inlet region (such as an inlet well or channel) and directed through the droplet extrusion region, e.g., by capillary action, to hydrate and prepare the device for use or to space out droplets. Likewise, buffer or oil can also be introduced into a main inlet region that communicates directly with the main channel to purge the device (e.g., or “dead” air) and prepare it for use. If desired, the pressure can be adjusted or equalized, for example, by adding buffer or oil to an outlet region.

[0220] The pressure at the droplet extrusion region can also be regulated by adjusting the pressure on the main and sample inlets, for example, with pressurized syringes feeding into those inlets. By controlling the pressure difference between the oil and water sources at the droplet extrusion region, the size and periodicity of the droplets generated may be regulated. Alternatively, a valve may be placed at or coincident to either the droplet extrusion region or the sample inlet connected thereto to control the flow of solution into the droplet extrusion region, thereby controlling the size and periodicity of the droplets. Periodicity and droplet volume may also depend on channel diameter, the viscosity of the fluids, and shear pressure. [0221] The droplet forming liquid is typically an aqueous buffer solution, such as ultrapure water (e.g., 18 mega-ohm resistivity, obtained, for example by column chromatography), 10 mM Tris HC1 and 1 mM EDTA (TE) buffer, phosphate buffer saline (PBS) or acetate buffer. Any liquid or buffer that is physiologically compatible with the population of molecules, cells or virions to be analyzed and/or sorted can be used. The fluid passing through the main channel and in which the droplets are formed is preferably one that is not miscible with the droplet forming fluid. In some embodiments, the fluid passing through the main channel is a non-polar solvent, for example decane (e.g., tetradecane or hexadecane), a fluorinated oil, or another oil. [0222] The fluids used in the disclosure may contain additives, such as agents which reduce surface tensions (surfactants). Exemplary surfactants may comprise Tween, Span, fluorinated oils, and other agents that are soluble in oil relative to water. Surfactants may aid in controlling or optimizing droplet size, flow and uniformity, for example by reducing the shear force needed to extrude or inject droplets into an intersecting channel. This may affect droplet volume and periodicity, or the rate or frequency at which droplets break off into an intersecting channel, as well as droplet stability. In some cases, surfactants may stabilize the droplets and avoid droplet elongation or merging or formation of large slugs or continuous aqueous films on channel surfaces.

[0223] Channels of the disclosure may be formed from silicon elastomer (e.g., RTV), urethane compositions, of from silicon-urethane composites such as those available from Polymer Technology Group (Berkeley, Calif.), e.g., PurSil™ and CarboSil™. Materials of chips may in some examples comprise glass, plastic, COC, CPC or other materials. The channels may also be coated with additives or agents, such as surfactants, TEFLON, or fluorinated oils such as octadecafluoroctane (98%, Aldrich) or fluorononane. TEFLON is particularly suitable for silicon elastomer (RTV) channels, which are hydrophobic and advantageously do not absorb water, but they may tend to swell when exposed to an oil phase. Swelling may alter channel dimensions and shape, and may even close off channels, or may affect the integrity of the chip, for example by stressing the seal between the elastomer and a coverslip. Urethane substrates do not tend to swell in oil but are hydrophilic, they may undesirably absorb water, and tend to use higher operating pressures. Hydrophobic coatings may be used to reduce or eliminate water absorption. Absorption or swelling issues may also be addressed by altering or optimizing pressure or droplet frequency (e.g. increasing periodicity to reduce absorption). RTV-urethane hybrids may be used to combine the hydrophobic properties of silicon with the hydrophilic properties of urethane.

[0224] Embodiments of the disclosure are also provided in which there are two or more droplet formation regions introducing droplets of samples into the main channel. For example, a first droplet extrusion region may introduce droplets of a first sample into a flow of fluid (e.g., oil) in the main channel and a second droplet extrusion region may introduce droplets of a second sample into the flow of fluid in main channel, and so forth. Optionally, the second droplet extrusion region is downstream from the first droplet extrusion region (e.g., about 30 pm). In one embodiment, the fluids introduced into the two or more different droplet extrusion regions comprise the same fluid or the same type of fluid (e.g., different aqueous solutions). For example, in one embodiment droplets of an aqueous solution containing an enzyme are introduced into the main channel at the first droplet extrusion region and droplets of aqueous solution containing a substrate for the enzyme are introduced into the main channel at the second droplet extrusion region. The introduction of droplets through the different extrusion regions may be controlled, e.g., so that the droplets combine (allowing, for example, the enzyme to catalyze a chemical reaction of the substrate). Alternatively, the droplets introduced at the different droplet extrusion regions may be droplets of different fluids which may be compatible or incompatible. For example, the different droplets may be different aqueous solutions, or droplets introduced at a first droplet extrusion region may be droplets of one fluid (e.g., an aqueous solution) whereas droplets introduced at a second droplet extrusion region may be another fluid (e.g., alcohol or oil).

[0225] The concentration (i.e., number) of scaffolds, molecules, cells, or bead density in a droplet can influence sorting efficiently and therefore may be optimized. In particular, the sample concentration should be dilute enough that most of the droplets contain no more than a singles scaffold, molecule, cell or , with only a small statistical chance that a droplet will contain two or more molecules, cells or bead. In some embodiments, the sample concentration should be such that a single cell is encapsulated with a single scaffold. This is to ensure that for the large majority of measurements, the level of reporter measured in each droplet as it passes through the detection region corresponds to a single molecule, cell or bead and not to two or more molecules, cells or beads. Additionally, ensuring that a single cell or bead is encapsulated with only a single encoded effector scaffold ensures that positive “hits” are correctly correlated with the correct effectors.

[0226] The parameters which govern this relationship are the volume of the droplets and the concentration of molecules, cells or beads in the sample solution. The probability that a droplet will contain two or more scaffolds, molecules, cells, or virions (P=2) can be expressed as P ^2=l-{ l+[bead]*V}xe -[bead]xV where “[bead]” is the concentration of molecules, cells or beads in units of number of molecules, cells or beads per cubic micron (pm3), and V is the volume of the droplet in units of pm3. It will be appreciated that P^2 can be minimized by decreasing the concentration of scaffolds, molecules, cells or beads in the sample solution. However, decreasing the concentration of molecules, cells or beads in the sample solution also results in an increased volume of solution processed through the device and can result in longer run times. Accordingly, it is desirable to minimize to presence of multiple molecules, cells or beads in the droplets (thereby increasing the accuracy of the sorting) and to reduce the volume of sample, thereby permitting a sorted sample in a reasonable time in a reasonable volume containing an acceptable concentration of molecules, cells or beads. The maximum tolerable P^2 depends on the desired “purity” of the sorted sample. The “purity” in this case refers to the fraction of sorted molecules, cells or beads that possess a desired characteristic (e.g., display a particular antigen, are in a specified size range or are a particular type of molecule, cell or bead). The purity of the sorted sample is inversely proportional to P^2. For example, in applications where high purity is not needed or desired a relatively high may be acceptable. For most applications, maintaining P^2 at or below about 0.1, or at or below about 0.01, provides satisfactory results.

[0227] A sample solution containing a mixture or population of molecule, cells or beads in a suitable carrier fluid (such as a liquid or buffer described above) is supplied to the sample inlet region, and droplets of the sample solution are introduced, at the droplet extrusion region, into the flow passing through the main channel. The force and direction of flow can be controlled by any desired method for controlling flow, for example, by a pressure differential, by valve action or by electro-osmotic flow (e.g., produced by electrodes at inlet and outlet channels). This permits the movement of the cells into one or more desired branch channels or outlet regions.

[0228] A “forward” sorting algorithm, according to the disclosure, includes embodiments where droplets from a droplet extrusion region flow through the device to a predetermined branch or outlet channel (which can be called a “waste channel”), until the level of measurable reporter of a molecule, cell or bead within a droplet is above a pre-set threshold. At that time, the flow is diverted to deliver the droplet (and the scaffold, molecule, cell, and/or bead contained therein) to another channel. For example, in an electro-osmotic embodiment, where switching is virtually instantaneous and throughput is limited by the highest voltage, the voltages are temporarily changed to divert the chosen droplet to another predetermined outlet channel (which can be called a “collection channel”). Sorting, including synchronizing detection of a reporter and diversion of the flow, can be controlled by various methods including computer or microprocessor control. Different algorithms for sorting in the microfluidic device can be implemented by different computer programs, such as programs used in conventional FACS devices. For example, a programmable card can be used to control switching, such as a Lab PC 1200 Card, available from National Instruments, Austin, Tex. Algorithms as sorting procedures can be programmed using C++, LAB VIEW, FLOWJO, or any suitable software.

[0229] A “reversible” sorting algorithm can be used in place of a “forward” mode, for example in embodiments where switching speed may be limited. For example, a pressure- switched scheme can be used instead of electro-osmotic flow and does not require high voltages and may be more robust for longer runs. However, mechanical constraints may cause the fluid switching speed to become rate-limiting. In a pressure-switched scheme the flow is stopped when a molecule or cell or virion of interest is detected within a droplet. By the time the flow stops, the droplet containing the molecule, cell or virion may be past the junction or branch point and be part of the way down the waste channel. In this situation, a reversible embodiment can be used. The system can be run backwards at a slower (switchable) speed (e.g., from waste to inlet), and the droplet is then switched to a different branch or collection channel. At that point, a potentially mis-sorted droplet (and the molecule, cell or virion therein) is “saved”, and the device can again be run at high speed in the forward direction. This “reversible” sorting method is not possible with standard FACS machines. FACS machines mostly sort aerosol droplets which cannot be reversed back to the chamber, in order to be redirected. The aerosol droplet sorters are virtually irreversible. Reversible sorting is particularly useful for identifying molecules, cells or virions that are rare (e.g., in molecular evolution and cancer cytological identification) or few in number, which may be misdirected due to a margin of error inherent to any fluidic device. The reversible nature of the device of the disclosure permits a reduction in this possible error.

[0230] In addition, a “reversible” sorting method permits multiple time course measurements of a molecule, cell or virion contained within a single droplet. This allows for observations or measurements of the same molecule, cell or virion at different times, because the flow reverses the cell back into the detection window again before redirecting the cell into a different channel. Thus, measurements can be compared or confirmed, and changes in properties over time can be examined, for example in kinetic studies.

[0231] When trying to separate scaffolds, molecules, cells or virions in a sample at a very low ratio to the total number of scaffolds, molecules, cells or virions, a sorting algorithm can be implemented that is not limited by the intrinsic switching speed of the device. Consequently, the droplets flow at the highest possible static (non-switching) speed from the inlet channel to the waste channel. Unwanted droplets (i.e., containing unwanted molecules, cells or virions) can be directed into the waste channel at the highest speed possible, and when a droplet containing a desired molecule, cell or virion is detected, the flow can be slowed down and then reversed, to direct the droplet back into the detection region, from where it can be redirected (i.e., to accomplish efficient switching). Hence the droplets (and the molecules, cells or virions contained therein) can flow at the highest possible static speed.

[0232] Provided herein are methods for controlling for variables such as temperature, pH and concentration. This may be accomplished by converging two aqueous streams to form droplets, where, for example, the first aqueous stream would contain 2X the concentration of component “A” desired in the droplet and the second aqueous stream would contain 2X the concentration of component “B” desired in the droplet, thus when the streams merge they would form a IX solution of both “A” and “B”. Different ratios of aqueous streams converging with different concentrations of reagents may also be sued to reach desired final concentrations of samples, scaffolds, and/or reagents. The concentrations in droplets are controlled by knowing what the concentrations are of components in each aqueous stream. This concept can be applied to pH, salt, concentration, etc. For temperature control a transparent stage may be used to heat the chip to a desired temperature.

[0233] Both the fluid comprising the droplets and the fluid carrying the droplets (i.e., the aqueous and non-polar fluids) may have a relatively low Reynolds Number, for example 10-2. The Reynolds Number represents an inverse relationship between the density and velocity of a fluid and its viscosity in a channel of given length. More viscous, less dense, slower moving fluids over a shorter distance will have a lower Reynolds Number, and are easier to divert, stop, start, or reverse without turbulence. Because of the small sizes and slow velocities, microfabricated fluid systems are often in a low Reynolds number regime (Re«l). In this regime, inertial effects, which cause turbulence and secondary flows, are negligible; viscous effects dominate the dynamics. These conditions are advantageous for sorting and are provided by microfabricated devices of the disclosure. Accordingly, the microfabricated devices of the disclosure are optionally operated at a low or very low Reynold's number.

[0234] In one aspect provided herein is a microfluidic device designed for droplet based encoded library screening. In some embodiments, the device comprises a first microfluidic channel comprising an aqueous fluid. In some embodiments, the device comprises a second microfluidic channel comprising a fluid immiscible with the aqueous stream. In some embodiments, the device comprises a junction at which the first microfluidic channel is in fluid communication with the second microfluidic channel. In some embodiments, the junction of the first and second microfluidic channels defines a device plane. In some embodiments, the junction is configured to form droplets of the aqueous fluid within the fluid from the second microfluidic channel. In some embodiments, the second microfluidic channel is configured to continue past the junction thereby defining an assay flow path. In some embodiments, the fluid from the second microfluidic channel with the droplets therein moves past the junction in a third microfluidic channel that defines an assay flow path. The assay flow path may also be called an incubation region. In some embodiments, the device comprises a cleavage region for cleaving effectors from scaffolds disposed within the assay flow path. In some embodiments, the device comprises a detection region. In some embodiments, the device comprises a sorting region. In some embodiments, the device comprises a stimulation region.

[0235] In some embodiments, the device comprises a third microfluidic channel. The third microfluidic channel may be in fluidic communication with the first microfluidic channel upstream of the junction of the first and second microfluidic channels. This third microfluidic channel may be used to mix an additional aqueous fluid with the first aqueous fluid prior to droplet formation, thus allowing the mixing of different sets of reagents shortly before the droplets are formed.

[0236] The junction of the first and second microfluidic channels is configured to create aqueous droplets encapsulated in the immiscible fluid of the second microfluidic channel. This junction may be of any configuration. In some embodiments, the junction is a T-junction. In some embodiments, the junction is at an oblique angle. In some embodiments, the junction further comprises a supplementary microfluidic channel. In some embodiments, the supplementary microfluidic channel comprises a second fluid immiscible with the aqueous stream. In some embodiments, the second fluid immiscible with the aqueous stream is the same as the fluid immiscible with the aqueous stream from the second microfluidic channel. In some embodiments, the second fluid immiscible with the aqueous stream is different from the fluid immiscible with the aqueous stream from the second microfluidic channel. In some embodiments, the second microfluidic channel and the supplementary microfluidic channel are positioned on opposite sides of the first microfluidic channel. In some embodiments, the second microfluidic channel and the supplementary microfluidic channel are configured to add their respective fluids immiscible with the aqueous stream simultaneously.

[0237] After the junction, the flow path of the second microfluidic channel may continue along the same trajectory for a least a short distance. After droplet formation, the channel downstream of the junction forms an assay flow path. The assay flow path is the path of the microfluidic channel where the screening assay is performed in the droplet. As the droplet continues along this assay flow path, additional unit operations can be performed on the droplet in sequences that allow an assay with a detectable readout to occur within the droplet. In some embodiments, the assay flow path comprises a cleavage region. In some embodiments, the assay flow path comprises a detection region. In some embodiments, the assay flow path comprises a sorting region. In some embodiments, the assay flow path comprises a stimulation region. In some embodiments, the assay flow path comprises a sorting region. In some embodiments, the assay flow path comprises a pico-inj ection region.

[0238] The assay flow path may be in any shape. In some embodiments, the assay flow path acts as an incubation region, allowing the assay to be performed over a desired length of time. In some embodiments, the assay flow path comprises a serpentine path region. The serpentine path region may contain a plurality of curves or turns. Such a pathway allows for an extended flow path to able to be embedded on a device of a small size. Additionally, the curves of the flow path may be used to orient various detectors, stimulators, sorters, or other components in a manner that minimizes background signal, crosstalk, or bleed through of various inputs into the droplets as they travel along the path. In some embodiments, this is accomplished by orienting the various inputs of unit operations along the curves or turns of the serpentine path. This minimizes the amount of the input that can travel along the flow path. For example, configuring a light source to input the light at a location along a curve or turn of the flow path minimizes the light that will travel along the path and reach droplets not the target of the emission.

[0239] The serpentine path region can be any length of the microfluidic device and can comprise any number of curves or turns. In some examples, the serpentine flow path may comprise at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000 or more curves or loops. The loops of the microfluidic device may allow for sample incubation for a set period of time. The incubation time corresponding to each loop can be measured and be known at the time of the screen. The loops may allow for performing dynamic kinetic assays.

[0240] In some embodiments, the chambers are configured such that the droplets formed on the microfluidic device have substantially the same residence time travelling through the device. In some embodiments, the microfluidic device is configured such that the droplets form on the device have substantially the same residence time travelling through the device. In some embodiments, this is measured by the dispersion ratio. The dispersion ratio is calculated according to the following formula: 60/Tavg; wherein T _av g is the average residence time of a droplet travelling through the device and <5 is the standard deviation of residence time of droplets travelling through the device. In some embodiments, the device has a dispersion ratio of at most about 10%, about 8%, about 6%, about 5%, about 4%, about 3%, about 2%, or about 1%. In some embodiments, the device has a dispersion ratio of at most about 10%. In some embodiments, the device has a dispersion ratio of at most about 8%. In some embodiments, the device has a dispersion ratio of at most about 6%. In some embodiments, the device has a dispersion ratio of at most about 5%. In some embodiments, the device has a dispersion ratio of at most about 4%. In some embodiments, the device has a dispersion ratio of at most about 3%. In some embodiments, the device has a dispersion ratio of at most about 2%. In some embodiments, the device has a dispersion ratio of at most about 1%.

[0241] In some embodiments, the device further comprises one or more collection chambers. In some embodiments, the one or more collection chambers are configured to receive a subset of the plurality of droplets passing through the assay flow path. In some embodiments, the collection chambers are configured to incubate the subset for an extended period of time. In some embodiments, the collection chambers are configured to lengthen the residence time for the subset of plurality of droplets.

[0242] In some embodiments, the device further comprises one or more shunts positioned along the flow path of the device. A shunt may be positioned at any location of the device. The shunt may be used for a variety of purposes. In some embodiments, a shunt is used to insert additional immiscible carrier fluid into the microfluidic channel in order to affect droplet spacing. In some embodiments, a shunt is used to divert droplets or carrier fluid off of the microfluidic device. In some embodiments, a shunt is used in initiation of the device. In some embodiments, a shunt is used in equilibration of the device. In some embodiments, the device is equilibrated [0243] In some embodiments, the assay flow path comprises a first shunt. In some embodiments, the first shunt is positioned in an upstream area of the assay flow path. In some embodiments, the first shunt is positioned upstream of the serpentine area of the assay flow path. In some embodiments, the first shunt is positioned upstream of the one or more chambers. In some embodiments, the first shunt is opened during an equilibration phase of using the device. In some embodiments, carrier fluid is run through the device in a reverse direction from normal operation during an equilibration stage of the device and allowed to exit the device through the first shunt. In some embodiments, aqueous droplets are simultaneously introduced into the microfluidic device upstream of the first shunt and allowed to exit the device through the first shunt. In some embodiments, the shunt is closed once pressures of input fluids on the device have been adjusted to desired levels in order to run the system as desired (e.g., flow rates, pressures, droplet size, droplet spacing, etc.).

[0244] In some embodiments, the first shunt configured to allow droplets to bypass at least a portion of the assay flow path. In some embodiments, an alternate flow path is coupled to the first shunt. The alternate flow path can have any property and can be used to affect the assay flow path in any manner. For example, the alternate flow path can be used to change the incubation time or residence time of droplets on the microfluidic device, add an additional reagent steam (e.g., a droplet merging junction or pico-inj ection site), or to incubate droplets off the device entirely.

[0245] The cleavage region may comprise a mechanism for liberating an effector that is linked to a bead by a cleavable linker. In some embodiments, the cleavage region comprises a pico-inj ection site or droplet merging site to introduce reagents to cleave the effector from a scaffold. In some embodiments, the cleavage region comprises a light source configured to cleave effectors from scaffolds disposed within the assay flow path. In some embodiments, the light source is a source of UV light. In some embodiments, the light source is a waveguide. In some embodiments, the light source is a fiberoptic cable. In some embodiments, the light source is a light source configured to cleave effectors from scaffolds disposed within the assay flow path. In some embodiments, the light source is configured to have an optical axis substantially parallel with the device plane. In some embodiments, the light source illuminates a passing droplet at a curve in the assay flow path. In some embodiments, the light source is configured to have an optical axis substantially perpendicular to the device plane. In some embodiments, the light source is aligned with the microfluidic channel of the cleavage region by pillars mounted on the device. In some embodiments, the light source is configured to emit light over an area covering multiple portions of the microfluidic channel passing through the cleavage region. In some embodiments, the cleavage region comprises a serpentine flow path. [0246] The cleavage region can be at any point along the microfluidic device depending upon the needs of the assay being employed on the device. In some embodiments, the cleavage region is upstream of the detection region, the sorting region, and the stimulation region. In some embodiments, the cleavage region is upstream of the detection region. In some embodiments, the cleavage region is upstream of the sorting region. In some embodiments, the cleavage region is upstream of the stimulation region. In some embodiments, the cleavage region is upstream of the detection region and the sorting region.

[0247] In some embodiments, the device comprises an additional inlet and outlet positioned on the microfluidic channel upstream and downstream of the cleavage region. In some embodiments, the inlet and outlets are positioned immediately before and immediately after the cleavage region.

[0248] In some embodiments, these inlets and outlets are configured to allow for a calibration of the cleavage region. The calibration allows for control over device-to-device variability in how much light the samples passing through the cleavage region are exposed to. Such variability can come from small changes to a variety of parameters of the device, including the coupling of the light source to the device. Variability in exposure intensity time and duration can lead to variability in amount of compound released from beads, which can cause errors in ultimate screening assay readouts.

[0249] In some embodiments, the inlets and outlets are used for the calibration procedure. In some embodiments, the calibration procedure comprises flowing a solution comprising a fluorescent dye through the cleavage region. FIG. 5D provides an exemplary depiction of the cleavage region for a microfluidic device described herein, wherein the calibration inlet and UV waveguide for exposing the encapsulations (e.g., droplets) to light are shown. In some embodiments, the calibrant channel is filled with UV-sensitive fluorophore to measure the UV intensity in the cleavage region. In some embodiments, the UV waveguide directs light from a UV LED coupled fiberoptic into a confined area. In some embodiments, the UV LED power is then set, based on a calibrant dye being measured.

[0250] In some embodiments, encoded effector-fluorophore beads are introduced into encapsulations (e.g., droplets) using a microfluidic device as described herein. FIG. 5A provides an exemplary solution of beads with an encoded effector modified with a fluorophore, wherein the solution can comprise a library of beads (One Bead One Compound (OBOC) library). As shown in FIG. 5B, the effector-fluorophore may be connected by a photo- cleavable, or pro-photo-cleavable linker. In some embodiments, the encoded-fluorophore beads are introduced into droplets at approximately 200 pL in volume. In some embodiments, the droplets are introduced into the cleavage region and exposed to the UV light. As shown in FIG. 5B, when exposed to the UV light, the effector is liberated (i.e. cleaving the photocleavable linker), such that the effector is released from the bead (FIG. 5C). The droplets then continue to flow through the microfluidic device, as described herein, until reaching an “interrogation region” of the microfluidic device, wherein the droplets are subject to laser excitation (e.g., confocal laser excitation at the detection region shown in FIG. 2B). The laser/LED shown in FIG. 2B excites the released effector-fluorophore. The emission from the encoded effector-fluorophore is then collected by PMT detectors shown in FIG. 2B and sent to a computer program connected to the sorting electrode shown in FIG. 2B.

[0251] The concentration of the released effector in the compartment (droplet) may depend on the intensity of the UV light. A higher, intensity, power, exposure time, or exposure energy of the stimulus (in this case UV light) can increase the concentration of the effector (in this case fluorophore) that is cleaved from the bead upon breaking the bond between the effector and the bead (cleaving the cleavable linker). Increasing the UV LED power increases the exposure, thereby facilitating the control of the final concentration of released effector- fluorophore. This feature can be used to perform dose-response drug screening, wherein varying concentrations of effectors may be screened against targets. The exposure energy of the stimulus may be used to control the compound dose.

[0252] The detection region is configured with a detector capable of detecting any desired readout of an assay to be performed on the device. In some embodiments, the detection region comprises a fluorometer. In some embodiments, the fluorometer comprises a photomultiplier tube (PMT) detector, a light source, an excitation filter and an emission filter. In some embodiments, the fluorometer is configured to have an optical axis substantially parallel to the device plane. In some embodiments, the fluorometer is configured to have an optical axis substantially perpendicular to the device plane. In some embodiments, the fluorometer illuminates a passing droplet at a curve in the assay flow path. In some embodiments, the detection region comprises confocal detection and laser scanning. In some embodiments, the detection region comprises a confocal laser scanning device.

[0253] FIG. 2B provides an exemplary schematic of encapsulation detection via confocal laser scanning. In some embodiments, the detection region comprises laser scanning. In some embodiments, the detection region comprises fluorescence. In some embodiments, the detection region comprises any combination of detection means described herein. The system may further comprise a brightfield, phase-contrast, and/or fluorescent microscope. The fluorescence excitation source can be used to generate a laser light which can expose a region (laser/LED source exposing the detection region shown on the microfluidic device shown in FIG. 2B)

[0254] In some embodiments, the detection region comprises an objective or fiber for emitting an excitation light into the detection region. In some embodiments, the detection region comprises an objective, fiber, or charged coupled device configured to collect emission from the detection region. In some embodiments, a single objective is configured to direct excitation and collect emission from the detection region. In some embodiments, the objective configured to collect emission from the detection region (which may be the same as the excitation objective) is an inverted objective lens. In some embodiments, the objective configured to collect emission from the detection region (which may be the same as the excitation objective) is configured to collect, collimate, and direct the emitted light through optical fibers. In some embodiments, the optical fibers are coupled to a detector configured to quantify the emission. In some embodiments, the detector configured to quantify the emission is a photomultiplier tube (PMT), charged coupled device, or photodiode. In some embodiments, the detection system may comprise a Field Programmable Gate Array (FPGA) sensor configured to detect the signals and send them to the computer system.

[0255] In some embodiments, the detection region is capable of being moved on the chip (across various regions of the microfluidic device/chip). For example, the laser may be aligned onto any region on the microfluidic chip, in the detection region or beyond the detection region. This may facilitate measuring signals from droplets in any location on the chip. In some embodiments, the detection region comprises an excitation light source that is not coupled to the device. In some embodiments, the detection region comprises an objective that is not coupled to the device. In some embodiments, having a light source or detector for the detection region not coupled to the device allows for the system to be adjusted based on assay need. For example, the system can be adjusted to increase or decrease the time between detection and sorting. Additionally, the system can be adjusted so that a single light source may be used for calibration and initialization of the device prior to performing a screening assay on the device. [0256] In some embodiments, the detection region is configured to detect two or more wavelengths of fluorescence. This allows for the detection of the abundance of a plurality of fluorescent probes. In some embodiments, the droplet being assayed may comprise a control fluorophore and an assay fluorophore. The assay fluorophore gives a readout of the assay, e.g., a positive or negative result of the assay. The control fluorophore, if present, may be detected and quantified. In some embodiments, the control fluorophore is placed into the aqueous fluid of the first microfluidic channel at a known concentration. When the droplet comprising the aqueous fluid of the first microfluidic channel reaches the detection region, the amount of control fluorophore fluorescence detected can be used to quantify the size of the droplet. This can be used to normalize the results of the assay fluorophore readout. In some embodiments, the detection region is configured to measure two or more assay fluorophores.

[0257] In some embodiments, the device comprises a single detection region. In some embodiments, the detection region is downstream of the cleavage region. In some embodiments, the detection region is downstream of the stimulation region. In some embodiments, the detection region is upstream of the sorting region.

[0258] In some embodiments, the device comprises multiple detection regions. When the device comprises multiple detection regions, they may be placed anywhere on the device. In some embodiments, the detection region is configured to be in communication with another region. For example, the detection region may be in communication with the sorting region to allow sorting to occur based on the detection of a signal. In some embodiments, a detection region is configured to be in communication with a pico-injector. When a detection region is in communication with a pico-injector, reagents or other assay components can be selectively added only when certain conditions are met, such as the presence or absence of a signal.

Stimulation region in microfluidic devices and arrays

[0259] Provided herein are miniaturized systems comprising a stimulation region or device. Alternatively, a system comprising a stimulation device or elements (e.g., electrodes, actuators, and other tools) may be used in conjunction with any screening system of the present disclosure.

[0260] A screening system of the present disclosure (e.g., a microfluidic device or arraybased system) may comprise a stimulation region. The stimulation region may comprise one or more actuators for stimulating a sample (e.g., stimulating an ion channel in a cell of a sample). Any method of stimulating an ion channel may be employed by the actuators when the device is configured to perform an ion channel modulation assay. The stimulation region may comprise one or more actuators which may be used for stimulating a sample (e.g., an ion channel). The one or more actuators for stimulating the sample (e.g., ion channel) may comprise at least one light source, at least one electrode, or at least one pico-inj ection site equipped with a chemical capable of stimulating a sample (e.g., an ion channel toxin). In some examples, the one or more actuators may comprise at least one light source. In some examples, the one or more actuators may comprise at least one electrode. In some examples, the one or more actuators may comprise an injection site for a stimulating chemical (e.g., an ion channel toxin). The stimulation region and features may be used to stimulate a sample in a droplet or a well.

[0261] In some embodiments, the one or more actuators comprises at least one electrode. Any type of electrode capable of delivering an electromagnetic current to the encapsulation may be employed. In some embodiments, the electrode lies along a wall of the assay flow path and delivers an electric field to the passing stream. In some embodiments, the electric field is pulsed to match the frequency at which droplets pass the electrode.

[0262] In some examples, the stimulation region (e.g., the one or more actuators may comprise a pair of electrodes. The stimulation region may be implemented in any screening system in the present disclosure. In case the screening system is a droplet microfluidic device, the pair of electrodes may be placed on opposite walls of the assay flow path. For example, when a droplet passes the pair of electrodes the droplet contacts the electrodes, thereby allowing a current to flow through the droplet. Alternatively, a pair of electrodes may be implemented in a compartment other than a droplet, such as a static compartment (e.g., a well of a microarray). The stimulation region may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 pairs or more pairs of electrodes. In some examples, the screening system may comprise from 1 to 20 pairs of electrodes.

[0263] Any number of actuators may be employed in the screening system. In some cases, the device may comprise from 1 to 20 actuators. In some examples, the screening system may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more actuators. [0264] In some cases, multiple stimulation regions may be used. In some cases, each well of a microarray may comprise a pair of electrodes capable of stimulating the sample. An example microfluidic device may comprise one or more stimulation regions. Stimulation regions in a microfluidic device may be distributed in any orientation throughout the microfluidic device. In some cases, the stimulation region may be downstream of the cleavage region. In some examples, the stimulation region may be upstream of the detection region. In some examples, the stimulation region is upstream of the sorting region.

[0265] The microfluidic device used for screening according to the methods of the present disclosure may comprise a sorting region. Any suitable sorting method may be used. The sorting region may be in communication with the detection region. The sorting region may comprise a sorting apparatus that sorts the droplets based on any sorting rule defined on the system (e.g., the detection of the presence, absence, or level of a signal detected by the detection region). [0266] The sorting region may comprise a sorting electrode. The sorting electro may comprise an electrophoresis electrode. In some examples, the sorting region may comprise a valve configured for sorting. The sorting region may comprise a deflectable membrane configured for sorting. The sorting region may comprise an acoustic wave generator configured for sorting. The sorting region may comprise an inlet for fluid configured to guide a passing droplet down a sorted path.

[0267] Control of flow of fluids through the device may be accomplished in any manner. In preferred embodiments, the flow of fluids is controlled by a device capable of delivering fluid through the device for a prolonged period of time and/or in a continuous fashion (e.g., a pneumatic pump or a peristaltic pump). Such pumps have several advantages over other pumps, such as syringe pumps, including the ability to run the system for a prolonged period of time at constant pressure, thus allowing for continuous feed of material through the device and control over residence time of droplets travelling through the device. In some examples, the flow of fluids is controlled by a continuous pump. In some embodiments, the flow of fluids is controlled by a pneumatic pump. In some embodiments, the fluids are delivered to the device from a reservoir of fluid off of the device. This allows the device to draw a much larger amount of fluid than would be possible from an on-device reservoir. Any of the sample fluids, immiscible fluids, spacing oil, focusing oil, or other fluid delivered onto the chip can be delivered in this manner. In some cases, the pumps may be capable of running fluids through the system for at least 10, 20, 30, 40, 50, 60 minutes (min) or longer. In some cases, the pumps may be capable of running fluids through the system for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 15, 16, 20, 24, 30, 40, 50, 60, 70, 80, 90, 100 hours (hr), or longer. The system may be capable of automation and over-night screening. In some cases, screens may be performed automatically without a human operator over-night for a prolonged period of time.

Example microfluidic device

[0268] An example microfluidic device for performing the methods of the present disclosure is shown in FIG. 4. The exemplary microfluidic device contains a first inlet 201. The first inlet 201 is configured to accept an aqueous fluid, such as an aqueous assay reagent. The exemplary microfluidic device also contains a second inlet 202. In this example, the second inlet 202 is configured to accept another aqueous fluid. This may be the same or different as the aqueous fluid added to the first inlet 201. The second inlet 202 may be configured to accept beads as provided herein, or the first inlet 201 may be so configured. In other examples of a microfluidic device, there may only be a single inlet stream. The exemplary microfluidic device shown in FIG. 4 further comprises a third inlet 203 for carrier fluid (e.g., an oil immiscible with an aqueous fluid) in fluid connection with a droplet formation junction or extrusion junction 204. The inlet 203 in this example is connected to the droplet formation junction 204 by two channels, each reaching an aqueous stream channel at the same point on opposite sides of the aqueous stream channel. The droplet formation junction 204 comprises a microfluidic channel that continues down the flow path towards cleavage region 206. The cleavage region may also be referred to as the exposure region. Near cleavage region or exposure region 206 is a fiberoptic waveguide 205 configured to deliver light (e.g., UV light) into the microfluidic channel of the cleavage region 206 for any reason (e.g., cleave a cleavable linker, polymerize a hydrogel, or otherwise stimulate the compartment for any intended purpose in the present disclosure) The fiberoptic waveguide 205 may be embedded in the plane of the device such that the light emitted enters the microfluidic channel of cleavage region/exposure region 206 from the device plane. The device also comprises an inlet for calibration fluid 207a in fluid connection with the cleavage region 106 and an outlet for calibration fluid 207b. The inlet for calibration fluid 207a is configured to receive and deliver to the cleavage region 206 a fluid configured to normalize photon exposure within the cleavage region. After passing through the cleavage region 206, the calibration fluid exits through the outlet for calibration fluid 207b. The cleavage region 206 is in fluid communication via a microfluidic channel to an incubation region 209. In the example of FIG. 4, the incubation region 209 contains a series of widened and deep chambers, each chamber connected to the next chamber in series by a microfluidic channel. The configuration of these chambers affects the flow rate and residence time of the droplets formed at droplet formation region 204 through the device as well as a dispersion ratio of the incubation times of the droplets through the channel. In some embodiments, the chambers are configured to prevent trapping of droplets as they pass through incubation region 209. Such configuration of the chambers is particularly important when using a carrier fluid that is denser than the aqueous droplets (e.g. 3-ethoxyperfluoro(2-methylhexane)). In some embodiments, this configuration is achieved by configuring the chambers and connecting channels to have only small difference in channel height between the chambers and the connecting channels. In some embodiments, the height of the chamber is about 80 pm and the height of the connecting channel is about 50 pm. As an additional design feature to aid in prevention of trapping of bubbles within the device, the height of the flow path does not change between the width of the chamber has been narrowed as the droplet approaches the connecting channel, thus facilitating the smooth transition of droplets from chamber to chamber without trapping. Configured on either end of incubation region 209 are bypass shunts 208a and 208b. The bypass shunts 208a and 208b are configured to allow a fluid coupled to the shunt to flow in or out of the main microfluidic channel. If fluid is diverted out of the main microfluidic channel at bypass shunt 208a, the material will not pass through incubation region 209. Positioned downstream of incubation region 209 is inlet for carrier fluid 210. Inlet for carrier fluid 210 is in fluid communication with the main microfluidic channel of the device and is configured to deliver additional immiscible carrier fluid into the main microfluidic channel to space droplets as desired. Also in fluid communication with the main microfluidic channel is inlet for carrier fluid 211, which is configured to deliver droplet focusing oil into the main microfluidic channel. Downstream of inlets for carrier fluid 210 and 211 is detection position 216. The detection position 216 indicates the point on the device that the desired signal from the assay being run on the chip is detected. The detection position 216 may be based on an alignment of an objective or fiber that directs an excitation light at the sample passing detection position 216 and an additional objective or fiber coupled to a detector configured to detect an emission from detection position 216. Alternatively, the objective for the excitation light may be configured to also collect the emission. In some embodiments, the excitation source is reflected from detection position 216 through an inverted objective lens, where the emission is collected, columnated, and directed through optical fibers for quantification by a photomultiplier tube (PMT) or other detector. In some embodiments, the objective or fiber aligned at detection position 216 is not coupled to the device. When not coupled to the device, the detector or emission objective or fiber can be moved to adjust the detection positions 216 on the device in order to adjust the time between detection and sorting. When not coupled to the device, the detector or emission objective may also be moved for use in calibration of the device or initiation of the device, thus allowing a single light source to be used for multiple functions. Downstream of inlets for carrier fluid 210 and 211 and detection position 216 is discrimination junction electrode 212. The discrimination junction electrode 212 may be a dielectrophoresis electrode configured to propel droplets down outlet 214 if the droplet is determined to display a signal with a predefined criteria (e.g., above or below a given threshold, such as a threshold defined for hits or a subpopulation selected for post-processing) or to outlet/waste path 215 if the droplet is determined to lack a signal fitting into the predefined criterial/threshold according to any method described anywhere herein. The discrimination junction electrode 212 is connected to a discrimination junction ground circuit, which is connected to the device at circuit connection point 213a. In some cases, an Optical Glue is displayed within the fiberoptic waveguide. In some embodiments, the Optical Glue helps to minimize scattering of the light from the fiberoptic wave guide. [0269] In some embodiments, for any microfluidic device described herein (e.g., FIG. 2B or FIG. 4), beads may be provided with an aqueous fluid via an inlet (e.g., inlet 201, inlet 203, or inlet 218). In some embodiments, the beads are suspended in the aqueous fluid, and provided from a bead source. In some embodiments, a tubing or other channel (“bead tubing”) provides fluidic communication between the bead source and a microfluidic device described herein. In some embodiments, said bead tubing comprises a rigid material. In some embodiments, a vibration motor or other vibration generating device is configured vibrate the bead tubing so as to maintain the beads as being suspended within the bead tubing (e.g., suspended within an aqueous fluid), and/or help maintain the beads as being spaced apart from each other. For example, without such vibration, in instances, the beads may settle along the walls of the bead tubing. In some instances, the beads may also or alternatively agglomerate together thereby forming “clumps” of beads. In some instances, such “clumps” of beads lead to ineffectual screening of assays and/or readings from signal measurement. In some instances, such settling of beads along the walls of a bead tubing and/or formation of “clumps” of beads results in a reduced amount or lack of beads that enter a microfluidic chip. In some embodiments, a vibration motor or other vibration generating device is configured vibrate the bead tubing so as to prevent or reduce the beads from settling within the bead tubing (for example, settling along the walls of the bead tubing).

[0270] In some embodiments, the vibration motor is configured to deliver high frequency vibration and/or low power (i.e., low amplitude of vibration of frequency). In some embodiments, the vibration frequency provided is optimize so as to prevent or reduce beads settling within the bead tubing, but also to prevent or reduce such vibration cascading to the flow profile of the beads within the bead tubing. In some embodiments, the vibration motor provides a vibration at a frequency of about 100Hz to about 200 Hz. In some embodiments, the vibration motor provides a vibration at a frequency of about 50Hz to about 300 Hz, or of about 25 Hz to about 500 Hz. In some embodiments, the vibration motor is coupled to the bead tubing. In some embodiments, the bead tubing passes through a channel within the vibration motor. In some embodiments, the vibration motor comprises a haptic motor.

[0271] In some embodiments, beads settling out in the bead tubing (for example along the walls of the bead tubing) results in an accumulation of beads within the bead tubing and may prevent beads from being disposed on the microfluidic chip. In some embodiments, such bead settling within a bead tubing is identified based on the detection and/or sorting region of the microfluidic chip, wherein no beads are detected with the corresponding measurements. In some embodiments, a feedback controller is provided and configured to modify the operation mode of a vibration motor based on no beads being detected. For example, if no beads are detected by the microfluidic device (for example at the detection and/or sorting regions), a feedback controller (in communication with such detection or sorting region) sends a signal to the vibration motor to turn on, or adjust the vibration frequency, so as to “unsettle” the beads within the bead tubing. In some embodiments, upon detecting beads (for e.g., by the detection region or sorting region), the feedback controller may turn off the vibration motor or revert the vibration frequency to a predetermined value.

[0272] The droplet formation junction 204 comprises a microfluidic channel that continues down the flow path towards cleavage region 206. Near cleavage region 206 is a UV waveguide 205 configured to deliver light into the microfluidic channel of the cleavage region 206. In some embodiments, the UV waveguide is a fiberoptic wave guide. The UV waveguide 205 is embedded in the plane of the device such that the light emitted enters the microfluidic channel of cleavage region 206 from the device plane. In some embodiments, the UV waveguide comprises a parabolic lens at an end closest to the cleavage region. In some embodiments, the parabolic lens is configured to columnate light inside the cleavage region. In some embodiments, the parabolic lens, or a curved lens, minimizes the tendency for the light from the UV waveguide to be scattered. In some embodiments, the cleavage region is exposed to UV light projected normal to the circuit plane, exposing a defined area to UV where the compound is cleaved. In some embodiments, an Optical Glue 217 is provided with the UV waveguide. In some embodiments, the Optical Glue 217 helps to minimize light being scattered by UV waveguide. Also, near cleavage region 206 may be a pillar (not shown) configured to fix a fiberoptic manifold which can be configured to emit light from above the plane of the device into the microfluidic channel of cleavage region 206.

[0273] The device also comprises an inlet for calibration fluid 207a in fluid connection with the cleavage region 206 and an outlet for calibration fluid 207b. The inlet for calibration fluid 207a is configured to receive and deliver to the cleavage region 206 a fluid configured to normalize photon exposure within the cleavage region. In some embodiments, the cleavage region 206 comprises a serpentine flow path. After passing through the cleavage region 206, the calibration fluid exits through the outlet for calibration fluid 207b. The cleavage region 206 is in fluid communication via a microfluidic channel to an incubation region 209. In some embodiments, the chambers are configured to prevent trapping of droplets as they pass through incubation region 209. Such configuration of the chambers is particularly important when using a carrier fluid that is denser than the aqueous droplets (e.g., 3-ethoxyperfluoro(2- methylhexane)). In some embodiments, the height of the chamber is about 30 pm to about 1,000 pm. In some embodiments, collection chambers 219 are optionally provided with this exemplary microfluidic device. Configured on either end of incubation region 209 are bypass shunts 208a and 208b. The bypass shunts 208a and 208b are configured to allow a fluid coupled to the shunt to flow in or out of the main microfluidic channel. If fluid is diverted out of the main microfluidic channel at bypass shunt 208a, the material will not pass through the incubation region 209. Positioned downstream of incubation region 209 is inlet for carrier fluid 210. Inlet for carrier fluid 210 is in fluid communication with the main microfluidic channel of the device and is configured to deliver additional immiscible carrier fluid into the main microfluidic channel in order to space droplets as desired. Also in fluid communication with the main microfluidic channel is inlet for carrier fluid 211, which is configured to deliver droplet focusing oil into the main microfluidic channel. In some embodiments, downstream of inlets for carrier fluid 210 and 211 is detection position 216. The detection position 216 indicates the point on the device that the desired signal from the assay being run on the chip is detected. The detection position 216 may be based on an alignment of an objective or fiber that directs an excitation light at the sample passing detection position 216 and an additional objective or fiber coupled to a detector configured to detect an emission from detection position 216. Alternatively, the objective for the excitation light may be configured to also collect the emission. In some embodiments, the excitation source is reflected from detection position 216 through an inverted objective lens, where the emission is collected, columnated, and directed through optical fibers for quantification by a photomultiplier tube or other detector. In some embodiments, the objective or fiber aligned at detection position 216 is not coupled to the device. When not coupled to the device, the detector or emission objective or fiber can be moved to adjust the detection positions 216 on the device to adjust the time between detection and sorting. When not coupled to the device, the detector or emission objective may also be moved for use in calibration of the device or initiation of the device, thus allowing a single light source to be used for multiple functions. Downstream of inlets for carrier fluid 210 and 211 and detection position 216 is discrimination junction electrode 212. The discrimination junction electrode 212 may be a dielectrophoresis electrode configured to propel droplets down outlet 214 if the droplet is determined to display a desired signal or to outlet 215 if the droplet is determined to lack a desired signal. The discrimination junction electrode 212 is connected to a discrimination junction ground circuit, which is connected to the device at circuit connection points 213a and 213b. Light exposure system

[0274] In some embodiments, for any miniaturized system described herein (e.g., microfluidic device, miniaturized array, or other system), an exposure light source may be provided or obtained, in addition to or alternative to the aforementioned UV waveguide. The light source may be top/down or bottom/up. Alternatively or in addition, the light source may have any orientation or angle. In some embodiments, the additional light source is a UV light source. In some embodiments, the light source is configured to provide light to the cleavage region or exposure region for a miniaturized system (e.g., microfluidic device or array) described herein. FIG. 7A provides an exemplary depiction of a light source 702 positioned over a cleavage region 704 for a microfluidic device 701. Any miniaturized system described herein may be used instead of the microfluidic device. The light source may have any position relative to the miniaturized screening device/system (above, below, side, angled).

[0275] With continued reference to FIG. 7A, in some embodiments, the light source is configured to provide light to the cleavage region in a downward (top/down) manner, upward manner (bottom/up), or at an angle. In some embodiments, the light source is configured to provide light perpendicular to a plane of the miniaturized device. In some embodiments, the light source is configured to provide light so as to reduce and/or minimize light scatter (e.g., by providing a more contained space of light being provided compared to other systems of the UV wave guide inserted inside the chip). In other examples, the direction of light might be angled with respect to the plane of the miniaturized device.

[0276] FIG. 7B provides another depiction of the light source 702 placed over a stage 710 for a miniaturized system described herein, wherein the light source is positioned over the location where the cleavage region of the miniaturized device would be situated. In some cases, the orientation of the light source may be bottom/up, from the side, or at any intended angle based on the application. The light source may be used for any application described anywhere herein.

[0277] In some embodiments, the miniaturized device is configured to be immobilized on the stage. In some embodiment, the light source may comprise a frame 712 that is configured to be mounted on top of the stage or at another location relative to the miniaturized device. In some cases, the light source may not touch the stage. In some embodiments, the frame 712 can be taped to the walls 714 of the stage. In some embodiments, the frame 712 comprises a 3- point adjustable height interface.

[0278] With reference to FIG. 7A, in some embodiments, the light source comprises a collimating lens 708. In some embodiments, the collimating lens comprises an SubMiniature version A (SMA) interface for SMA optical cable. In some embodiments, collimating lens comprises a 12mm diameter. In some embodiments, the collimating lens comprises an Edmunds 12 mm diameter lens. In some embodiments, the light source comprises a masking aperture 706 located in a portion 707 of the light source. In some embodiments, the shape of the aperture 706 reflects the area of light exposure in the cleavage region. In some embodiments, the light source comprises a sheet that will be mounted below the stage 710 to act as a light dump.

[0279] FIG. 7C provides a cutaway depiction of the light source, wherein reference character 716 comprises a right-angle UV optimized prism mirror. In some embodiments, the prism mirror comprises an Edmunds 10mm x 10mm right angle UV optimized prism mirror. As depicted in FIG. 7C, in some embodiments, light 709 is provided and reflected by a rightangle prism mirror 716, to pass through the aperture 706 and towards 711 the cleavage region. In some embodiments, the light source is configured to provide a greater light exposure to larger cleavage regions. In some applications, the light source may comprise or be a UV light source. Applications of UV exposure in the present disclosure may comprise effector cleavage, selective polymerization and light patterning, and hydrogel bead polymerization. All the methods and systems may be used for any other suitable application.

[0280] In some embodiments, a detector is provided to measure the light intensity exposed to the cleavage region or any other target location (exposure region or exposed region). In some embodiments, the detector measures light intensity in real-time. In some embodiments, the measured light intensity is output to a user interface and/or a controller. In some embodiments, the detector is provided below the cleavage region, wherein it receives light provided by the light source as described herein. In some embodiments, the detector and the user interface and/or controller provide a feedback system to maintain a light intensity provided to the exposed region according to a predetermined light intensity range. In some embodiments, the predetermined light intensity range correspond to a calibrated light intensity range. In some embodiments, the user interface and/or controller is configured to adjust the light intensity provided by the light source so as to maintain a light intensity provided to the exposed region according to a predetermined light intensity range (e.g., a calibrated light intensity range). In some embodiments, the calibrated light intensity range corresponds to an empirically generated calibration curve of material release (e.g., encoded effector, etc.) from scaffolds (e.g., beads) within an encapsulation (e.g., droplet), as described herein. In some embodiments, the user interface is configured to alert a user to adjust the light intensity of the light source. In some embodiments, light intensity variations occur due to a plurality of factors, such as variation in material of the light source, material of the microfluidic chip, etc. Similar examples may apply when the light source is used to expose the system for other applications.

Primer amplification method

[0281] Provided herein is a method for amplifying a primer to maximize cellular nucleic acid capture. In some screening methods provided herein, nucleic acid contents of cells are transferred to the nucleic acid encodings of various effectors. The nucleic acid encodings are sometimes linked to scaffolds, such as beads. However, a library of beads may comprise individual beads that may have dramatically different levels of nucleic acids encodings on the beads. Consequently, such beads are unable to attach significant levels of cellular nucleic acids, or other beads are able to attach substantially more levels of cellular nucleic acids. Such discrepancies make it difficult to determine if the cellular nucleic acid level differences are due to the differential effects of various effectors, or if there were simply less capture sites available to gather the cellular nucleic acids. Therefore, a method of producing additional primers to label the cellular nucleic acids with the nucleic acid encoding would have substantial benefits. [0282] In one aspect, provided herein is a method for amplifying a primer to maximize cellular nucleic acid capture. In some embodiments, the primer is a copy of a nucleic acid encoding (encoded nucleic acid primer). In some embodiments, the method comprises encapsulating a nucleic acid encoded scaffold with one or more cells, an amplification mix, and a nicking enzyme. In some embodiments, the nicking enzyme targets a specific nucleotide sequence. As described herein, a nucleic acid encoded scaffold is bound to one or more nucleic acid encodings. In some embodiments, the one or more nucleic acid encodings comprise a specific nucleotide sequence. In some embodiments, the cell is lysed to release one or more cellular nucleic acids. In some embodiments, the nucleic acid encoding is nicked with the nicking enzyme, thereby creating an encoded nucleic acid primer. In some embodiments, nicking refers to a single strand of an encoding being displaced. In some embodiments, the nicking enzyme targets a specific site in the nucleic acid encoding. In some embodiments, the specific site comprises the specific nucleotide sequence. In some embodiments, nicking the nucleic acid encoding creates an encoded nucleic acid primer. In some embodiments, the encoded nucleic acid primer is amplified. In some embodiments, the encoded nucleic acid primer is amplified via interaction between the nicking site and the amplification mix. In some embodiments, a released cellular nucleic acid is labeled with an encoded nucleic acid primer.

[0283] In some embodiments, amplifying the encoded nucleic acid primer comprises first creating a copy of the nucleic acid encoding, which is extended from the nicking site, followed by nicking the nucleic acid encoding copy. In some embodiments, amplifying the encoded nucleic acid primer comprises simultaneously 1) creating a copy of the nucleic acid encoding, which extends from the nicking site, and 2) displacing the nucleic acid encoding copy.

[0284] In some embodiments, the amplification mix comprises an amplification enzyme. In some embodiments, the amplification enzyme enables for the creation of a nucleic acid encoding copy, and then the subsequent nicking. In some embodiments, the nicking enzyme enables the nicking of the copy of the nucleic acid encoding copy. In some embodiments, the amplification enzyme enables for a copy of the nucleic acid encoding to be simultaneously created and displaced. In some embodiments, the amplification enzyme is a polymerase. In some embodiments, the creation of nucleic acid encoding copies and nicking, or the simultaneous creation and displacement of the nucleic acid encoding copies, repeats to generate a population of single stranded nucleic acid encodings that serve as a primer (encoded nucleic acid primer) for labeling cellular nucleic acids. In some embodiments, the encoded nucleic acid primers are generated isothermally.

[0285] In some embodiments, each encoded nucleic acid primer comprises a capture site that prescribes a target cellular nucleic acid to label a specific released cellular nucleic acid. In some embodiments, the target nucleic acid is a target mRNA. In some embodiments, the target mRNA encodes a protein of interest. In some embodiments, the nicking enzyme enables an increase in target mRNA capture and labeling with the nucleic acid encoding. In some embodiments, the target mRNA capture is increased by at least 10%, 25%, 50%, 100%, or 200%.

[0286] In some embodiments, a plurality of cellular nucleic acids are labeled with an respective encoded nucleic acid primer. In some embodiments, the nucleic acid encoded scaffold comprises a bead, and the encoded nucleic acid primer comprises a unique bead barcode and an effector encoding.

[0287] FIG. 3 illustrates an exemplary method for amplifying a primer to maximize cellular nucleic acid capture, as described herein. As shown in FIG. 3, in step 1, a nucleic acid encoded scaffold is shown with the nucleic acid encoding bound thereto, wherein a plurality of cellular encodings (e.g., nucleic acid) are also shown to have been released from a lysed cell. In some embodiments, the nucleic acid encoded scaffold and cellular encodings are provided within an encapsulation. The nicking site is identified on the nucleic acid encoding, along with a capture site. In some embodiments, the nicking site corresponds to a specific nucleotide sequence in the nucleic acid encoding. As shown in step 2, the nucleic acid encoding is nicked at the nicking site. As shown, in some embodiments, nicking herein refers to a single strand of the encoding being displaced from the nucleic acid encoded scaffold. As shown in steps 3-4 of FIG. 3, an amplification enzyme may interact with the nicking site, thereby creating a new copy of the nucleic acid encoding (step 4), while the previously nicked nucleic acid encoding copy (encoded nucleic acid primer) is unbound and moves within the encapsulation, such that the encoded nucleic acid primer may interact with a released cellular encoding (e.g., cellular nucleic acid), as shown in step 5. In some embodiments, the encoded nucleic acid primer labels the cellular encoding. In some embodiments, the capture site of the encoded nucleic acid primer prescribes a targeted cellular nucleic acid. In some embodiments, an enzyme enables such labeling. As shown in step 6, the encoded cell encoding is labeled with the encoded nucleic acid primer, while a created copy of the nucleic acid encoding is displaced from the scaffold, wherein the process returns to step 3.

[0288] The cell may be lysed in order to release the desired nucleic acids and to make the desired nucleic acids available for amplification. In some embodiments, the encapsulation further comprises a cell lysis buffer. In some embodiments, the lysis buffer is added by picoinjection. In some embodiments, the lysis buffer comprises a salt. In some embodiments, the lysis buffer comprises a detergent. In some embodiments, the detergent is SDS, Triton, or Tween. In some embodiments, the lysis buffer comprises a chemical which causes cell lysis. In some embodiments, cell lysis buffer is added to the encapsulation. In some embodiments, the cell lysis buffer is added to the encapsulation by pico-inj ection.

[0289] In some embodiments, an amplification mix is used to amplify nucleic acid encodings to create additional primers for labeling cellular nucleic acids of interest in a screen. In some embodiments, the amplification mix is an isothermal amplification mix. In some embodiments, the isothermal amplification mix comprises reagents for loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicasedependent amplification (HAD), recombinase polymerase amplification (RPA), rolling circle replication (RCA), or nicking enzyme amplification reaction (NEAR). In some embodiments, the encapsulation further comprises reagents for isothermal amplification of the target nucleic acid. In some embodiments, the method comprises adding reagents for isothermal amplification to the encapsulation. In some embodiments, the reagents for isothermal amplification are targeted to the specific nucleic acid sequence. In some embodiments, the amplification mix comprises a nicking enzyme. In some embodiments, the amplification mix comprises a nicking-enzyme amplification mixture. In some embodiments, the nicking enzyme is an endonuclease. In some embodiments, the nicking enzyme is a restriction enzyme. In some embodiments, the amplification mix comprises a reverse transcriptase. In some embodiments, the amplification mix comprises an amplification enzyme. In some embodiments, the amplification enzyme comprises a polymerase.

[0290] In some embodiments, the specific nucleotide sequence of interest can be amplified within the encapsulation. In some embodiments, the method comprises amplifying the cellular nucleic acid comprising the specific nucleotide sequence to produce amplified cellular nucleic acids. In some embodiments, amplifying the cellular nucleic acids is accomplished by PCR. In some embodiments, amplifying the cellular nucleic acids is accomplished by isothermal amplification. In some embodiments, cellular nucleic acids comprising the specific nucleotide sequence are amplified. In some embodiments, the amplified cellular nucleic acid is barcoded with the nucleic acid encoding the scaffold.

[0291] Any type of scaffold may be utilized in this method. In some embodiments, the scaffold acts as a solid support and keeps the nucleic acid encoding the scaffold linked in space to the scaffold. In some embodiments, the scaffold is a structure with a plurality of attachment points that allow linkage of one or more molecules. In some embodiments, the nucleic acid encoding the scaffold is bound to the scaffold. In some embodiments, the scaffold is a solid support. In some embodiments, the scaffold is a bead, a fiber, nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valent molecular assembly.

[0292] In some embodiments, the scaffold is a bead. In some embodiments, the bead is a polymer bead, a glass bead, a metal bead, or a magnetic bead. In some embodiments, the bead is a polymer bead. In some embodiments, the bead is a glass bead. In some embodiments, the bead is a metal bead. In some embodiments, the bead is a magnetic bead.

[0293] Beads for use in the systems and methods as described herein can be any size. In some embodiments, the beads are at most 10 nm, at most 100 nm, at most 1 pm, at most 10 pm, or at most 100 pm in diameter. In some embodiments, the beads are at least 10 nm, at least 100 nm, at least 1 pm, at least 10 pm, or at least 100 pm in diameter. In some embodiments, the beads are about 10 pm to about 100 pm in diameter.

[0294] The scaffolds may comprise effectors attached to the scaffold. In some embodiments, the effectors are attached to the scaffold by the cleavable linkers described herein. In some embodiments, the cleavable linker is cleaved by electromagnetic radiation, an enzyme, chemical reagent, heat, pH adjustment, sound or electrochemical reactivity. In some embodiments, the cleavable linker is cleaved from the scaffold using electromagnetic radiation. In some embodiments, the amount of effector cleaved is controlled by the intensity or duration of exposure to electromagnetic radiation. In some embodiments, the cleavable linker is cleaved using a cleavage reagent. In some embodiments, the amount of effector cleaved is controlled by the concentration of the cleavage reagent in the encapsulation. In some embodiments, the effector is cleaved from the scaffold using an enzyme. In some embodiments, the enzyme is a protease, a nuclease, or a hydrolase. In some embodiments, the rate of effector cleavage is controlled by the amount of enzyme in the encapsulation.

[0295] In some embodiments, the encoded nucleic acid primers amplified in the present methods are utilized to detect and quantify the amount of a target nucleic acid in the one or more cells being screened with an effector utilizing the nucleic acid encoded scaffold. In some embodiments, the encoded nucleic acid primer hybridizes with a target nucleic acid.

[0296] In some embodiments, the specific nucleotide sequence acts as an amplification primer with the target nucleic acid. In some embodiments, the target nucleic acid is barcoded with the nucleic acid encoding the scaffold using the specific nucleotide sequence. In some embodiments, the target nucleic acid is barcoded with the nucleic acid encoding the scaffold using the specific nucleotide sequence which has been extended with the nucleic acid encoding the scaffold.

[0297] The target nucleic acid can by any type of nucleic acid from a cell. In some embodiments, the target nucleic acid is a target mRNA. In some embodiments, the target mRNA encodes a protein of interest. In some embodiments, the target nucleic acid comprises a plurality of target mRNAs. In some embodiments, barcoding the plurality of target mRNAs creates an expression fingerprint of the cell treated with an effector. In some embodiments, the target nucleic acid is genomic DNA. In some embodiments, the target nucleic acid is mitochondrial DNA.

[0298] The methods provided herein increase target nucleic acid capture and labeling with the nucleic acid encoding the scaffold. In some embodiments, target nucleic acid capture is increased by at least 10%, 25%, 50%, 100%, or 200% compared to a method without the nicking enzyme that targets the specific nucleotide sequence. In some embodiments, target nucleic acid labeling is increased by at least 10%, 25%, 50%, 100%, or 200% compared to a method without the nicking enzyme that targets the specific nucleotide sequence. In some embodiments, target nucleic acid capture is increased by at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold compared to a method without the nicking enzyme that targets the specific nucleotide sequence. In some embodiments, target nucleic acid barcoding is increased by at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold compared to a method without the nicking enzyme that targets the specific nucleotide sequence. [0299] In some embodiments, labeling the cellular nucleic acids with encoded nucleic acid primers, as described herein, comprises barcoding the cellular nucleic acids. The encapsulation can further comprise barcoding reagents. In some embodiments, the encapsulation further comprises barcoding reagents. In some embodiments, the encapsulation further comprises barcoding reagents to effectuate the barcoding of the cellular nucleic acids with the encoded nucleic acid primers. In some embodiments, the encapsulation further comprises barcoding reagents to effectuate the barcoding of the nucleic acid encoding the scaffold with amplified nucleic acids.

[0300] The barcoding reagents can be any set of reagents that allow the j oining of different nucleic acids. In some embodiments, the barcoding reagents comprise an enzyme or chemical cross-linking reagent. In some embodiments, the enzyme is a polymerase, a ligase, a restriction enzyme, or a recombinase. In some embodiments, the enzyme is a polymerase. In some embodiments, the additional reagents comprise a chemical cross-linking reagent. In some embodiments, the chemical cross-linking reagent is psoralen.

[0301] The amplification of primers described herein can be performed at any time. In some embodiments, the above methods can be performed at the same time as an effector screen. In some embodiments, the cell is being screened against the effector. In some embodiments, an effector screen occurs concomitantly with the primer amplification method. In some embodiments, the primer amplification method described herein occurs prior to an effector screen. In some embodiments, the method is used to prepare the nucleic acid encoded scaffold for a screen. In some embodiments, the cell is used to prepare the nucleic acid encoded scaffold for a screen.

Effector load normalization method

[0302] Provided herein are methods of measuring effector loading onto scaffolds and libraries of scaffolds. Generally, when a library of encoded effectors bound to scaffolds is prepared, the final concentration of effectors bound to the scaffolds varies considerably among individual scaffolds. This is due to differences in yield of each synthesis step of the effector built onto the scaffold. Consequently, when ultimately used in a screen, different samples may receive different dosages of effectors. This can skew the results of the screen, as low potency, high abundance effectors may drown out the signal of higher potency, low abundance effectors. Thus, a method of determining effector loading onto scaffolds in a library can help avoid this skewing of results.

[0303] Provided herein are methods of measuring effector loading on scaffolds. In some embodiments, the method comprises (a) attaching an effector subunit to effector attachment sites on a plurality of scaffolds. In some embodiments, the method comprises (b) attaching a detectable label to any remaining free effector attachment sites on the plurality of scaffolds after the step of attaching an effector subunit. In some embodiments, the method comprises (c) removing a subset of scaffolds from the plurality. In some embodiments, the method comprises (d) measuring the amount of detectable label attached to the subset of scaffolds to determine the amount of effector subunits successfully attached to the effector attachment sites. In some embodiments, the method comprises (e) optionally activating the attached effector subunits to create new effector attachment sites. In some embodiments, the listed steps are repeated until a desired effector is assembled. In some embodiments, the scaffold further comprises a nucleic acid encoding the effector. In some embodiments, the method further comprises attaching nucleic acid encoding subunits to the scaffold corresponding to the effector subunits as the effector subunits are added to the scaffold. In some embodiments, there is no activating step after the last effector subunit is attached.

[0304] In some embodiments, each effector subunit attached to the scaffold is independently an amino acid, a small molecule fragment, a nucleotide, or a compound. In some embodiments, each effector subunit attached to the scaffold is an amino acid. In some embodiments, each effector subunit attached to the scaffold is a compound. In some embodiments, each effector subunit attached to the scaffold is a small molecule fragment. In some embodiments, each effector subunit attached to the scaffold is a nucleotide.

[0305] The effector attachment sites may have any group capable of performing a chemical reaction. In some embodiments, the effector attachment sites comprise reactive functionalities. In some embodiments, the effector attachment sites comprise amino groups, carboxylate groups, alcohol groups, phenol groups, alkyne groups, aldehyde groups, or ketone groups. In some embodiments, the effector attachment sites comprise amino or carboxylate groups. In some embodiments, the effector attachment sites comprise biorthogonal or CLICK chemistry reactive groups.

[0306] The encoding subunits can comprise functional groups that may react with the reactive functionalities on the effector attachment site. In some embodiments, the encoding subunits form a covalent bond with the reactive functionalities. In some embodiments, the effector subunits comprise reactive groups complementary to the effector attachment sites.

[0307] The detectable labels, in some embodiments, comprise functional groups that may react with the reactive functionalities on the effector attachment site. In some embodiments, the detectable labels form a covalent bond with the reactive functionalities. In some embodiments, the detectable labels comprise reactive groups complementary to the effector attachment sites.

[0308] The detectable label may any label that can produce a signal that can be detected and quantified. In some embodiments, the detectable label is a fluorophore.

[0309] In some embodiments, there is a yield associated with each effector attachment step. In some embodiments, the yield is measured a percentage of free effector attachment sites after the step of attaching an effector subunit. In some embodiments, at most 10%, at most 20%, at most 30%, at most 40%, or at most 50% of the effector attachment sites are free after the step of attaching the effector subunit.

[0310] A subset of beads may be removed to quantify the loading at each step of the synthesis of the desired effector. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 1%, no more than 2%, no more than 3%, no more than 5%, or no more than 10% of the remaining scaffolds. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 1% of the remaining scaffolds. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 2% of the remaining scaffolds. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 3% of the remaining scaffolds. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 5% of the remaining scaffolds. In some embodiments, removing a subset of the plurality of scaffolds comprises removing no more than 10% of the remaining scaffolds.

[0311] In some embodiments, wherein measuring the amount of detectable label attached to the subset of scaffolds to determine the amount of effector subunits successfully attached to the effector attachment sites comprises comparing the measurement of the detectable label to the measurement of detectable label on a scaffold without any effector subunits attached. In some embodiments, the amount of effector subunits successfully attached to the subset of scaffolds is expressed as a percentage of total attachment sites occupied by the effector subunits.

[0312] To begin a new step of attaching effector subunits, a previously attached effector subunit may need to be activated. In some embodiments, activation reveals the presence of a new effector attachment site. In some embodiments, optionally activating the attached effector subunits to create a new effector attachment site comprises removing a protecting group from the attached effector subunit. In some embodiments, the protecting group is an amino protecting group, a carboxylate protecting group, an alcohol protecting group, a phenol protecting group, an alkyne protecting group, an aldehyde protecting group, or a ketone protecting group. In some embodiments, the protecting group is an amino protecting group. In some embodiments, the amino protecting group is 9-fluorenylmethyloxcarbonyl (Fmoc), tertbutyloxycarbonyl (BOC), carbobenzyl oxy (Cbz), benzyl (Bz), tosyl (Ts) or tri chloroethyl chloroformate (Troc). In some embodiments, the protecting group is a carboxylate protecting group. In some embodiments, the carboxylate protecting group is a methyl ester, a benzyl ester, a tert-butyl ester, a 2,6-disubstituted phenolic ester, a silyl ester, or an orthoester. In some embodiments, the protecting group is an alcohol protecting group. In some embodiments, the protecting group is a phenol protecting group. In some embodiments, the protecting group is an alkyne protecting group. In some embodiments, the protecting group is an aldehyde protecting group. In some embodiments, the protecting group is a ketone protecting group.

[0313] The new effector attachment site can be any suitable reactive functionality. In some embodiments, the new effector attachment site is the same functionality as the previous effector attachment site. In some embodiments, the new effector attachment site is a different functionality from the previous effector attachment site.

[0314] The desired effectors can be synthesized using any number of steps and use any number of effector subunits. In some embodiments, steps (a)-(e) are repeated at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 10, or at least 20 times. In some embodiments, the desired effector is comprised of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 10, or at least 20 subunits.

[0315] Any type of scaffold may be used with the methods and systems provided herein. In some embodiments, the scaffold is a bead, a fiber, a nanofibrous scaffold, a molecular cage, a dendrimer, or a multi-valent molecular assembly. In some embodiments, the scaffold is a bead. In some embodiments, the bead is a polymer bead, a glass bead, a metal bead, or a magnetic bead. In some embodiments, the bead is a polymer bead. In some embodiments, the bead is a glass bead. In some embodiments, the bead is a metal bead. In some embodiments, the bead is a magnetic bead.

[0316] The beads utilized in the methods provided herein may be made of any material. In some embodiments, the bead is a polymer bead. In some embodiments, the bead comprises a polystyrene core. In some embodiments, the beads are derivatized with polyethylene glycol. In some embodiments, the beads are grafted with polyethylene glycol. In some embodiments, the polyethylene glycol contains reactive groups for the attachment of other functionalities, such as effectors or encodings. In some embodiments, the reactive group is an amino or carboxylate group. In some embodiments, the reactive group is at the terminal end of the polyethylene glycol chain. In some embodiments, the bead is a TentaGel® bead.

[0317] The polyethylene glycol (PEG) attached to the beads may be any size. In some embodiments, the PEG is up to 20 kDa. In some embodiments, the PEG is up to 5 kDa. In some embodiments, the PEG is about 3 kDa. In some embodiments, the PEG is about 2 to 3 kDa.

[0318] In some embodiments, the PEG group is attached to the bead by an alkyl linkage. In some embodiments, the PEG group is attached to a polystyrene bead by an alkyl linkage. In some embodiments, the bead is a TentaGel® M resin.

[0319] In some embodiments, the bead comprises a PEG attached to a bead through an alkyl linkage and the bead comprises two bifunction species. In some embodiments, the beads comprise surface modification on the outer surface of the beads that are orthogonally protected to reactive sites in the internal section of the beads. In some embodiments the beads comprise both cleavable and non-cleavable ligands. In some embodiments, the bead is a TentaGel® B resin.

[0320] Beads for use in the systems and methods as described herein can be any size. In some embodiments, the beads are at most 10 nm, at most 100 nm, at most 1 pm, at most 10 pm, or at most 100 pm in diameter. In some embodiments, the beads are at least 10 nm, at least 100 nm, at least 1 pm, at least 10 pm, or at least 100 pm in diameter. In some embodiments, the beads are about 10 pm to about 100 pm in diameter.

[0321] Nucleic acids encoding the effector are utilized in the described method. The nucleic acids encoding the effector may be bound to the scaffold as a pre-synthesized nucleic acid, synthesized concomitantly with the effector, or synthesized on the scaffold prior to synthesis of the effector. In some embodiments, a nucleic acid encoding the effector is attached to the scaffold. In some embodiments, the method further comprises attaching nucleic acid encoding subunits to the scaffold corresponding to the effector subunits as the effector subunits are added to the scaffold.

[0322] The methods described herein are especially useful when applied to libraries of effectors on scaffolds. In some embodiments, libraries of effectors are synthesized in parallel. In some embodiments, libraries of effectors are synthesized in individual wells. In some embodiments, libraries of effectors are synthesized using high-throughput synthesis techniques. In some embodiments, a library of effector loaded scaffolds are synthesized concurrently. The library of effector loaded scaffolds can be any size. In some embodiments, the library comprises at least 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , or 10 ¹⁶ effector loaded scaffolds. In some embodiments, each effector loaded scaffold comprises a unique effector. In some embodiments, some effector loaded scaffolds are repeated in the library.

[0323] In some embodiments, subsets of beads from an effector attachment step from the library are pooled prior to detection of the detectable label. In some embodiments, subsets of beads from all scaffolds in the library are pooled together. In some embodiments, a portion of the subset of beads from the scaffolds in the library are pooled together.

[0324] The pooled subsets of beads are placed into encapsulations for further analysis. An encapsulation refers to the formation of a compartment within a larger system. In some embodiments, the encapsulation is a droplet, an emulsion, a macrowell, a microwell, a bubble, or a microfluidic confinement. In some embodiments, a majority of the encapsulations comprise a single scaffold.

[0325] In some embodiments, the encapsulation is a droplet. In some embodiments, the droplet is at most 1 picoliter, at most 10 picoliters, at most 100 picoliters, at most 1 nanoliter, at most 10 nanoliters, at most 100 nanoliters, or at most 1 microliter in volume. In some embodiments, the droplet is at least 1 picoliter, at least 10 picoliters, at least 100 picoliters, at least 1 nanoliter, at least 10 nanoliters, at least 100 nanoliters, or at least 1 microliter in volume. In some embodiments, the droplet is between about 200 picoliters and about 10 nanoliters.

[0326] In some embodiments, the droplet is an aqueous droplet in a larger body of oil. In some embodiments, the droplets are placed in an oil emulsion. In some embodiments, the oil comprises a silicone oil, a fluorosilicone oil, a hydrocarbon oil, a mineral oil, a paraffin oil, a halogenated oil, or any combination thereof. In some embodiments, the oil comprises a silicone oil. In some embodiments, the oil comprises a fluorosilicone oil. In some embodiments, the oil comprises a hydrocarbon oil. In some embodiments, the oil comprises a mineral oil. In some embodiments, the oil comprises a paraffin oil. In some embodiments, the oil comprises a halogenated oil.

[0327] After the scaffolds are placed into encapsulations, the level of fluorophore bound to the scaffolds may be assessed. In some embodiments, scaffolds from the subset of scaffolds are binned according to the amount of detectable label detected. In some embodiments, each bin comprises a unique range of detectable label detected. In some embodiments, the bins correspond to 0-25%, 25-50%, 50-75%, and 75-100% loading of detectable label detected compared to scaffolds where no effector subunit was loaded. In some embodiments, the bins correspond to 0-20%, 20-40%, 40-60%, 60-80%, and 80-100% loading of detectable label detected compared to scaffolds where no effector subunit was loaded. In some embodiments, the bins correspond to 0-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, and 90-100% loading of detectable label detected compared to scaffolds where no effector subunit was loaded. Any combination of bins is acceptable to use with the methods and systems provided herein.

The bins may then be sequenced to reveal which effectors had particular yields in the attachment step. In some embodiments, the method further comprises the step of sequencing encoding nucleic acids or encoding nucleic acid subunits of the pools to reveal which effector subunits correspond to a particular yield in a step of attaching effector subunits to effector attachment sites. In some embodiments, the sequencing step is performed each time steps (a)- (e) are repeated. In some embodiments, yields of each step (a)-(e) for each unique scaffold are collected to create a dataset which reveals the loading of the complete desired effector on each scaffold. In some embodiments, yields of attachment of each encoder subunit for each unique scaffold are collected to create a dataset which reveals the loading of the complete desired effector on each scaffold. In some embodiments, the loading of desired effector on each unique scaffold is calculated.

Methods and systems for sample screening in miniaturized arrays

[0328] In some embodiments, the methods and systems of the present disclosure may comprise providing, obtaining, and/or utilizing array-based platforms. Array-based platforms with a solid support in the bottom may be particularly advantageous for seeding cells (e.g., adherent cells) and/or screening them. For many cell lines, it may be more suitable to perform the screens under conditions in which the cells are adhered to a solid surface/support. The reason may be that seeding cells on a solid support may better mimic the natural state of the cells (e.g., conditions in vivo). In some cases, the effect(s) of a compound or effector may be tested on a predefined or specific target of any kind according to the information provided elsewhere herein. Alternatively, in some cases, the overall effects of a compound on a sample or a population of cells may be mapped without directly assessing a predefined/ specific target. [0329] A variety of array platforms with any shape, size, geometry, and materials can be used for performing the methods of the present disclosure. In an example, the bottom substrate for the plurality of the partitions may be glass. An example glass microscopy cover slip may comprise a standard microscope glass slide. An example size of such microscope slide can be 75mm x 25mm x 1mm. In an example, a Globe Scientific 1324 Glass Microscope Slide was used as the bottom substrate for the array device. The surface of the glass slide was treated with silane. A plurality of wells was then fabricated on the silanized glass slide according to the methods and examples described elsewhere herein. [0330] Wells of a well-based platform and/or compartments of an array -based platform can be of any shape (circle, square, hexagon, or other shapes). Wells may also be referred to as miniaturized wells, microwells, nanowells, and picowells. In a particular example, the diameter of the fabricated wells may be from about 200 to about 300 micrometers/microns (pm). The height or depth of the well walls (e.g., the thickness of the material in which the wells are imprinted) may be from about 50 to about 200 microns. In some examples, the spacing between the wells may be from about 50 to about 100 microns. In some examples, the total internal volume of the wells may be from about 20 picoliters to about 100 nanoliters.

[0331] In a particular example, the well diameter may be about 200 pm and the well height (thickness of the well wall) may be about 50 pm. Accordingly, the internal volume of the well may be about 1.57 nanoliters (nL). In another example, the well diameter may be about 200 pm and the well height may be about 100 pm. Accordingly, the internal volume of the well may be about 3.14 nL. In another example, the well diameter may be about 200 pm and the well height may be about 200 pm. Accordingly, the internal volume of the well may be about 6.28 nL. In other examples, the diameter of the well may be about 300 pm, and the height of the well may be about 50 pm, 100 pm, or 200 pm. Accordingly, the internal volume of the well may be about 3.53 nL, 7.065 nL, or 14.13 nL, respectively.

[0332] In some cases, the diameter of the well may be at least 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400 pm, or above. In some cases, the diameter of the well may be at most about 1 cm, 500 pm, 400 pm, 300 pm, 200 pm, 150 pm, 100 pm, or smaller. The thickness or height of the well wall may be at least about 10, 20, 30, 40, 50, 100, 150, 200, 250, 300 pm or greater. In some cases, the height of the well wall may be at most about 500 pm, 400 pm, 300 pm, 200 pm, 100 pm, 50 pm, 30 pm, 20 pm or smaller. The volume of the well may be at least about 0.1, 0.2, 0.3 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 2, 2.2, 2.5, 3, 4, 5, 6, 7, 8, 10, 12, 14, 16, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500 nL, or greater. In some cases, the internal volume of the well may be at most about 900 nL, 800 nL, 700 nL, 600 nL, 500 nL, 400 nL, 300 nL, 200 nL, 100 nL, 50 nL, 30 nL, 20 nL, 10 nL, 5 nL, or less.

[0333] In some examples, the plurality of compartments may comprise at least 2 compartments. In some cases, the plurality of compartments may comprise from about 10 compartments to about ten million compartments or more. In some cases, the plurality of compartments may comprise at least 2, 10, 20, 30, 40, 50, 60, 100, 200, 300, 400, 500, 1000, 5000, 10,000, 20,000, 40,000, 60,000, 100,000, 1000,000, 10,000,000, 100,000,000 or more compartments. [0334] FIGs. 8A- 8C provide, respectively, view from the side, view from the top, and three-dimensional view of an exemplary miniaturized array for sample screening. A plurality of compartments 803 are built on a solid surface 801. The cells 802 are adhered to the bottom of the wells and the walls 803 are substantially non-adherent and impenetrable to the cells. The cells are seeded and grown on the bottom of the wells in a monolayer. In this example, the micro-array is open at the top (e.g., without a solid cap at the top and not sandwiched between two pieces of solid support/glass). Materials can be freely introduced in and out of the partitions/wells using a variety of techniques such as directly pouring, pipetting, robotic handling, flowing through tubes, and beyond. The solid substrate in the bottom of the compartments or wells can be made of any material and it can be the same as or different from the walls of the compartments. In some cases, the bottom substrate may be glass or plastic. The walls may be made of any material. In some cases, the wall material may be a hydrogel or polymer.

Well materials

[0335] The compartments or wells of a miniaturized well-based platform can be made of any suitable material. In some examples, the material of the wells may be preferred to be substantially impenetrable to liquids such as cell media, oil (e.g., fluorinated oil), and effectors of the present disclosure. In case of using a porous material for fabricating the wells, the mesh size can be small enough to prevent material diffusion therein, or such material transfer may be otherwise substantially blocked. In some cases, the wells (e.g., microwells) seek to isolate individual populations of cells and any compounds the effects of which on the cells are to be examined. As such, the walls of individual wells may be substantially impermeable to any material that can convolute the dosing of one cell population with another and such mass transfer is preferred to be substantially blocked and/or prevented.

[0336] In some examples, array platform/device material may be resistant to extracellular protein adsorption and/or cell growth. In some examples, the methods and systems of the present disclosure may be used to screen ion channels in cells in the array-based system. In some cases, the cells may be able to transmit ion channel signals to adjoining cells. Thus, if one population of cells is connected to another population of cells across two wells, it is possible that the signal from one cell population may trigger signals in the other. If the array platform material is resistant to ECM protein adsorption and cell growth, this may minimize the chance for cells growing between two wells.

[0337] In some examples, preferred well wall materials may be malleable into a predetermined shape or feature at the correct resolution (e.g., a high resolution). In some cases, array platform (e.g., microarray) material may be substantially transparent or highly transparent to light at given wavelengths, such as ambient light, UV, visible, Near-infrared (NIR), Short Wave Infrared (SWIR) or other light wavelengths which may be suitable for the purposes of the present disclosure. In some cases, the material may be substantially transparent to a light with a wavelength in the range between 300 nm to 2500 nm. Substantially transparent may be for example, at least about 70%, 80%, 90%, 92%, 95%, 97%, 98%, 99%, or above 99% transparent. This characteristic may help avoid light scattering or light absorption by the array material, which may affect the sample inside the array such as by stimulating the sample and/or signal capture from the samples or the cells therein. In some cases, the material of the compartments may have low autofluorescence.

[0338] The materials of the well walls and the bottom substrate can be the same or different. In a particular example, the wells were made of a polymerized hydrogel, using polyethylene glycol diacrylate (PEGDA) with a molecular weight of about 250 kDa as the monomer making up the polymerized hydrogel. The photoinitiator used for gelling/curing/solidifying the hydrogel was 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone mixed with the base in a 95% to 5% v/v ratio. The surface of the glass bottom was treated with silane. The surface of the wells were treated with extracellular matrix material (ECM) or a cell adhesive material such as vitronectin, fibronectin, or matrigel. In another example, the material of the wells was a mix of 250 kDa PEGDA monomer with 750 kDa PEGDA in monomer at a defined ratio. The ratio of the monomers, the concentrations of the monomers in the pre-polymer mixture, the molecular weights of each monomer in the pre-polymer mix, and the concentration of the photoinitiator, among other factors can be used to control and tune the mesh size of the polymerized hydrogel. In some cases, the pre-polymer mix may further comprise a spacer. The material used as the spacer, its chemical structure, molecular weight, additional chemical and physical properties, and the concentration of the spacer in the pre-polymer mix can also be used to control and tune the chemical properties of the resulting polymerized hydrogel. In some examples, any Zwitterionic polymers such as (poly(phosphorylcholine), poly(carboxybetaine), poly(sulfobetaine), poly(trimethylamine A-oxide) could be used to fabricate the wells. Another example for well material may comprise a Cyclic Olefin Polymer (COP) or Cyclic Olefin Copolymer (COC). Other examples for well material comprise glass and plastic. In such examples both the bottom and walls of the wells could be made of the same material (e.g., glass or plastic).

[0339] The bottom surface and/or walls may be made of any material. In some cases, glass, plastic, silicon, silica, polymer, polydimethylsiloxane (PDMS). In some examples, the bottom of the compartments/wells (e.g., the solid surface in the bottom of the platform) may be treated with a cell adhesive material to render the surface adhesive to cells and/or ECM. This will improve the surface properties for cell seeding. Any suitable cell adhesive material or chemical can be used. In some examples, the cell adhesive may be selected from the group consisting of Vitronectin, Matrigel, Fibronectin, Laminin, Poly-Lysine, and Extra-cellular Matrix (ECM) Protein, a Cell Adhesion Polymer (e.g., a synthetic cell adhesion polymer).

[0340] In some cases, the well walls may be made of a solid, semi-solid, partially porous material. The hydrogel may be at least partially hydrophobic. Such material may comprise or be polymer, plastic, silica, hydrogel, or any combination thereof. In some examples, the material of the walls may comprise any level of wettability or hydrophilicity. In some cases, the material may be hydrophobic. In some examples, the well walls may be made of a material (e.g., hydrogel) with a molecular weight of from about 10 to about 2000 daltons.

[0341] The well walls comprise a top surface. In some cases, the top surface of the well walls may be or may be rendered substantially non-adherent to cells. In some examples, the well walls may be made of a material that by itself is substantially non-adherent to cells. In some examples, the material of the wells may comprise or be glass through-holes or plastics (e.g., Cyclic Olefin Copolymer COC). In case of using COC, it can be made such that only the bottom surface of the well interior is tissue-culture treated (plasma treated) while the surfaces of the well walls (e.g., a material such as COC) have inherent low-specific protein and cell binding properties. In some examples, rendering the well top surface non-adherent to cells may be facilitated through surface treatment or coating. The top surface of the well walls may be coated with a coating material. Some examples of the coating materials coating the top surface(s) of the well walls may comprise PEG-DA, PDMS-Pluronic F127, PDMS-PDMS- PEG-BCP, PDMS-501W, fluoropolymers such as Novec 1720/1702/1700/2202 and Cytop, and zwitterionic polymers (including poly(phosphorylcholine), poly(carboxybetaine), poly(sulfobetaine), poly(trimethylamine A-oxide). In other examples, materials such as organogels and/or fluorogels may be used coat the well surfaces.

[0342] FIG. 9 schematically illustrates an exemplary workflow of a method for surface treatment of a miniaturized array platform to facilitate cell seeding or cell adhesion. In this example, The miniaturized array platform 900 comprised a plurality of circular wells 901 fabricated on a solid surface 902. The bottom surface of the platform was made of glass (e.g., a glass microscopy cover slip). The well walls/exteriors 903 were made of a hydrogel material (PEGDA). Prior to fabricating the wells on the glass slide, the surface of the glass slide was treated with silane. A PDMS mold was used to fabricate the hydrogel wells on the glass slide according to methods described elsewhere herein. After fabricating the array platform, a series of surface treatments were applied on the internal and external well surfaces to modify the surface properties, such as properties with respect to cell and extracellular matrix (ECM) adhesion to render the bottoms of the wells substantially adhesive to cell and the exteriors of the wells substantially non-adhesive to cells and ECM. Initially, water was used as a blocking liquid to fill in the wells and prevent the entry of coating material. The wells were then capped with an oil immiscible with water (e.g., a standard microfluidics fluorinated oil such as Novec HFE7500 by 3M). A hydrophobic surface treatment capable of rendering the top surfaces non- adhesive to cells and ECM (e.g., fluorosilane polymer diluted in a solvent (Novec 1720, 3M)) was added to the capping oil to treat the well exteriors. Such chemical was substantially hydrophobic, insoluble in water, low in surface tension, and configured to keep out dirt, dust, debris, cells, and particles away from the surfaces. Water inside the wells prevented the entry of the non-adhesive hydrophobic surface treatment material into the interior of the wells. The hydrophobic surface coating was evaporated over time to form a thin, transparent, permanent coating on the well exteriors (shown in light green). Next, the water was removed from the wells (e.g., aspirated), and a charged surface treatment (e.g., poly-D Lysine) was applied on the entire micro-array. The hydrophobic treatment prevented the charged surface treatment to substantially affect the well exteriors. The well interiors were modified by the charged surface treatment to render the well interiors substantially adhesive to cells and ECM or prime the surface for the addition of another surface treatment layer comprising a cell adhesive material such as Vitronectin. Next, Vitronectin or another cell adhesive material was applied on the entire array. The hydrophobic treatment on the top surfaces facilitated denaturing the cell adhesive material. The cell adhesive material rendered the solid surface of the bottom of the wells adhesive to cells and ECM.

[0343] FIG. 10 provides an example image of cells seeded in a miniaturized array. Adherent cells were seeded in the bottom of each well. The bottom of the well comprised a surface treatment which facilitated or enhanced cell adhesion. Surface treatments (e.g., as described elsewhere herein) facilitated improving surface properties for cell seeding and growth. The walls and tops of the wells were treated with a material which minimized cell adhesion and cell penetration. An assay was performed on the cells. The cells seeded in the miniaturized array platform were used in combination with encoded effectors, assays, detection systems, and other methods and systems described anywhere herein.

[0344] FIG. 11A shows an exemplary flow cell 1100 comprising a plurality of compartments 1101. A plurality of compartments (wells) 1101 were immobilized or built on a solid surface (e.g., glass) in the bottom of the flow cell 1100. The flow cell was fabricated to facilitate flow introduction into the compartments (e.g., wells). The top of the flow cell comprised a solid seal 1102 with one or more (e.g., two) openings 1103 to facilitate fluid flow. FIG. 11B shows four flow cells similar to flow cell 1100 immobilized on a solid support 1104. Each flow cell 1100 has an inlet tube 1105 inserted into an inlet port 1106 and an outlet tube 1107 inserted into an outlet port 1108 to facilitate fluid flow into and out of the wells 1101.

[0345] A solution comprising cell culture media and cells can be introduced into the flow cell through an opening incorporated in the solid seal on top of the flow cell using various techniques such as pipetting, robotic handling, and beyond. An assay can be performed in each compartment/well. The assay may comprise using a scaffold, wherein the scaffold comprises an effector covalently bound to the scaffold via a cleavable linker and a barcode (e.g., nucleic acid or optical barcode) on/in the scaffold which corresponds to and identifies the effector, as described anywhere herein. The cleavable linker can be cleaved by application of a stimulus to release the effector into the compartment to act on the cell. Assay activity can be measured by measuring a signal from each compartment. Based on the measured signal, the compartments that demonstrate a predefined/given effect can be marked as having an effect (positive or hit) or not having an effect (negative or non-hit), for example, using a computer program or software in communication with the system through a computer.

[0346] After the assay is completed, post-processing may be performed on the hits. In some cases, post-processing may comprise selective polymerization as described elsewhere herein. For example, a polymerizable monomer can be introduced into the flow cell. A digital mask and digital mirror device can be used to selectively polymerize select wells which were identified as non-hits to block liquid entry thereto. The hit compartments can remain substantially open to fluid flow. Liquid can be introduced into hit wells to extract the contents of the well (e.g., the hit scaffold to cleave the barcode from the scaffold, to add a secondary barcode to the barcode on the scaffold in order to mark or tag it as a hit, or to sequence the barcode of the scaffold inside the compartment and elucidate the identity or structure of the hit effector (e.g., compound or small molecule).

[0347] Any suitable system may be used to select wells and block liquid flow thereto. The system may comprise robotics, laser printing, 3D printing, or any combination thereof.

Methods of hit identification and separation in miniaturized platforms

[0348] The methods of the present disclosure may comprise identifying hits. A hit may be an effector that has or is suspected of having an effect on a sample, a target, or a cell. For example, effectors that are found to be active against an intended target (e.g., based on a threshold of a signal detected from the assay in presence of such effector in the compartment after release) may be referred to as “hits”. A scaffold on which a hit is identified may be referred to as a hit scaffold or positive scaffold. For example, a hit may be an effector for which activity against a target has found to be surpassing or below a threshold (e.g., predefined threshold or a hit identification condition defined during or after data acquisition/aggregation for the signal measured from the respective partition).

[0349] Activity or effect may be any effect described anywhere herein. A compartment or partition which contains at least one hit may be referred to as a positive compartment. In some cases, the positive compartment may further comprise one or more non-hits. A negative compartment may be a compartment that does not contain a hit. Whether or not the hit has a real effect, or it has been detected by mistake or because of an error, deficiency, artifact, by chance, due to an interruption in the screen, or any other reason during the screen (e.g., a false positive) can be determined by downstream validation assays. Hits may comprise validated/real hits and false positives. A validated hit has a real effect on the target, sample, or the cell. A non-validated hit is a false positive. In some cases, hits may comprise or be high-interest events of unknown veracity. In some cases, hits may not be treated as bona-fide until validated in replicate tests afterwards.

[0350] Upon performing an assay in the screening platform and/or once the signals are measured form the compartments, further processing may be performed to elucidate the identity of the hits. Such processing may comprise sorting the hit scaffolds. Hit identification and/or processing can be performed in various ways. Examples may comprise sorting and collecting the positive compartments/partitions (e.g., sort droplet, well, raft, or any other compartment such as by physically separating them in space or adding a membrane or barrier between them to partition them from one another), sorting and collecting the scaffolds/bead found/identified in the positive partitions (e.g., a partition containing at least one hit) with or without sorting the partitions themselves, for examples, extracting the hit scaffolds from the positive partitions and moving them to a separate container, or separating and sorting the barcodes of the hit scaffolds with or without sorting the scaffolds and partitions themselves. Separating the barcode from the scaffold may comprise breaking or cleaving the bond between the barcode and the scaffold via a stimulus. In some cases, cleaving the barcode from the bead may be performed or catalyzed by an enzyme.

[0351] In some examples, provided herein are methods for bead sorting, recovery, and barcode decoding to elucidate the identity of hits. An example workflow may comprise providing or obtaining a plurality of compartments (e.g., a plurality of droplets in a droplet microfluidic platform or a plurality of wells of a well array on a solid support), introducing a plurality of scaffolds (e.g., beads) and a plurality of cells into the compartments such that a subset of the compartments each include at least one cell and at least one scaffold, the scaffold comprising a barcode and an effector attached thereto, wherein the barcode is corresponding to and identifying the effector.

[0352] In some cases, a plurality of cells may be pre-seeded in the plurality of the compartments, and a plurality of scaffolds may be introduced into the plurality of compartments to enter the compartments and interact with the pre-seeded cells. A compartment may contain any number of cells and any number of scaffolds. The cells and the scaffolds may be introduced into the plurality of compartments in any order. For example, the scaffolds may be introduced into the compartments before or after the cells. In some cases, the cells may be suspension cells floating in the compartments and not necessarily pre-seeded in the compartments or adhered thereto.

[0353] The effector may be attached to the scaffold (e.g., bead) via a cleavable linker (e.g., photocleavable linker) and be releasable from the scaffold via a stimulus (e.g., UV light) further described elsewhere herein. The barcode may also be attached to the scaffold with a cleavable bond which may be cleaved via a stimulus (e.g., a chemical). In some cases, the barcode may be attached to the scaffold via a covalent bond that is not cleavable.

[0354] The compartment may further comprise assay reagents such as probes, reporters, and/or other materials for performing an intended assay in the compartment to measure the activity of the effector against the target (e.g., in or on the cell in some cases). A signal can be measured from each compartment which may be a measure of the activity of the effector against/on the target. The compartments may be labeled as either a positive compartment or negative compartment (e.g., based on the intensity of the signal). The positive compartments may be referred to as hit compartment (e.g., a compartment containing one or more hits (i.e., hit effector or hit scaffold). Compartments comprising a scaffold which has an intended effect on the assay or the target, based on the intensity of the signal, may be referred to as a positive compartment (a compartment bearing at least one hit). In some cases, a positive compartment may comprise more than one scaffold. For a compartment to be called as a positive compartment, the presence of one positive scaffold/effector (a hit) is sufficient, and the additional scaffolds in the compartment may not necessarily all have an effect on the target or be hits. A compartment marked as a negative compartment, which is a compartment not bearing a hit, may be sorted into a separate stream or space from the positive compartments.

Separating the compartments in space [0355] In some cases, the separation in space may be performed by partitioning, such as by a membrane or a blocking material which substantially blocks flow access to negative compartments, leaving the positive compartments substantially open to liquid flow. Alternatively, in some cases, the membrane or blocking material may substantially block flow access to positive compartments and leave the negative compartments substantially open to liquid flow.

[0356] An effect of an effector on a sample or cell may comprise a variety of effects, changes, and/or conditions. An effect may comprise the inhibition or agonism of a chemical or molecular moiety (e.g., a protein, an enzyme, a nucleic acid molecule) in the sample or the cell. For example, an effector may decrease an activity of such moiety in the sample (e.g., an inhibitor). For example, an inhibitor may be an enzyme inhibitor. In some cases, the effector may increase the activity of a moiety in the sample or the cell or catalyze it, in which case it may be called an agonist. In some examples, an effector may cause a change in a morphology of a sample or cell, cause redistribution of one or more moieties inside the sample or the cell, lead to aggregation/clustering, change of phase, or dissolutions of the moieties inside the sample or the cell. In some cases, such aggregation or clustering may manifest as a redistribution of signal intensity (fluorescence redistribution) inside the sample, the cell, and/or the compartment. For example, signals may be measured across an entire compartment, and the localization of signal intensity inside a single compartment may change over time as a result of aggregation of one or more chemical/molecular moieties. The methods of the present disclosure may comprise providing and utilizing high-content screening. High-content screening may comprise high-content signal detection/imaging of samples and cellular assays to detect and identify the effects. In some examples, the methods of the present disclosure may comprise performing high-content screening (HCS), high-content analysis (HCA), and/or cellomics. Such methods may comprise identifying substances such as small molecules, peptides, or RNA that alter the phenotype of a cell in an intended or targeted manner.

[0357] Upon the completion of the screen, the method may further comprise hit identification and sorting. In some examples, sample screening is performed in a droplet microfluidic platform described anywhere herein which may in some cases, comprise a sorting device in a droplet microfluidic platform comprising a sorting device/j unction, the negative compartments (e.g., encapsulations or droplets not containing hits) may follow a default flow path (e.g., a waste stream). A waveform pulse generator may generate a pulse to deflect the positive droplets into a hit stream, thereby separating the hits from the non-hits into different containers and/or separating the hits and non-hits in space, for example, into separate containers which can be later be separately processed. Alternatively or in addition, the negative compartments (e.g., non-hits) may be pulsed (e.g. using a pulse generator or any other kind of sorting device) into a separate stream, while the hits follow a default path. Such sorting may be performed according to any other method described anywhere herein.

[0358] Any rule may be defined for sample sorting or compartment sorting on the system (e.g., using a software) to separate and sort a sub-population of the compartments into separate containers (or otherwise partition them in space) based on a property measured in such compartment, beyond the definitions of positive and negative binary classification. Any number of sub-populations may be defined (e.g., on the system software based on the detected signal) and sorted (e.g., using a pulse generator or any other device mentioned in the disclosure) into any number of containers or bins for various applications and/or to meet various end goals. In some cases, a plurality of sub-populations may be defined and separated into a plurality of containers. Each sub-population may have one or more unique set of properties or characteristics which may have been identified before or during the screening by any kind of measurement, such as the assay signal generated through the screening system and detected by the system detectors described anywhere herein. The partitions (e.g., droplets) can be coalesced or temporarily destabilized to extract the beads from the partition (during the screen, after the screen, on the microfluidic device, or after collection from the device), the barcode on the beads in each container can be read (e.g., sequenced) to determine the effectors categorized and/or sorted as hits, non-hits, or based on other classification schemes.

[0359] In some examples, the present disclosure provides a screening method comprising providing or obtaining a plurality of compartments. In some cases, the compartments may be wells of a miniaturized array platform. In some cases, a first subset of the plurality of compartments may each comprise an effector bound to a scaffold and a barcode for identifying the effector, according to the bead-bound encoded effector libraries provided anywhere herein. The method may further comprise performing an assay in the plurality of compartments, thereby generating a signal indicative of the activity of the assay or an effect of the effector on the assay in each compartment of the plurality of compartments. The method may further comprise processing a second subset of the plurality of compartments based on the signal to substantially block liquid flow thereto, thereby yielding a third subset of the plurality of compartments which remain substantially open to liquid entry. This may be followed by flowing a liquid into the plurality of compartments and may be allowed to enter the third subset of the plurality of compartments such as to collect or sort the scaffold, the effector or the barcode, or to elucidate the identity or structure of the effector. The elucidating of the identity of the effector may comprise reading or decoding the barcode, such as measuring a property of the barcode. In some examples, the barcode may be a nucleic acid molecule (e.g., a DNA), and decoding may comprise sequencing. Sequencing may be performed inside the compartments or outside the compartments, after collecting the scaffolds or barcodes.

[0360] In some examples, the methods of the present disclosure (e.g., screening, compartment selection, and compartment processing) may be performed in a miniaturized platform comprising a plurality of compartments. The compartments may be according to any compartments provided anywhere herein. In some cases, the plurality of compartments may be a plurality of arrays or wells immobilized on a solid substrate.

[0361] Compartment processing may comprise selecting the second subset of compartments based on the signal. The signal may be measured in the compartments through an assay. In some cases, an assay may be performed to test the effect of a plurality of effectors on a one or more targets. The effectors screened may be covalently bound to a solid scaffold (e.g., a bead or a particle) via a cleavable linker. The scaffold may further comprise a barcode corresponding to and identifying the effector. In some cases, the scaffold may comprise or be a particle comprising a diameter of at least about 1 micrometer (pm), 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 10 pm, 15 pm, 20 pm, or other dimensions provided for particles (e.g., beads) anywhere herein. In some cases, the scaffolds may be substantially homogeneous or monodisperse in size.

[0362] In some examples, the barcode on the scaffolds may comprise or be a nucleic acid molecule, a DNA, an RNA, a peptide, a molecular weight barcode, or a peptide nucleic acid (PNA). The compartment may further comprise a reagent for detecting the signal.

[0363] Upon performing a screen in a miniaturized array, the compartments may be marked as positive compartments or negative compartments, such as by using a software program which is in communication with the screening platform/device (e.g., well array) via a computer. The hits and non-hits (positive compartments and negative compartments) can further be sorted or partitioned from one another, in some cases, by selective disruption of fluidic contact and/or separation in space. In some cases, such methods may be performed in miniaturized arrays or other compartmentalized platforms. The methods may comprise processing the compartments. In some examples, processing the compartments may comprise selective polymerization of the compartments (e.g., wells).

Selective polymerization

[0364] In some examples, compartment selection and processing may comprise providing a polymerizable monomer or hydrogel in the plurality of compartments, for example, after performing a screen in the plurality of compartments. In some cases, compartment processing (e.g., during or after sample screening) may comprise selectively polymerizing the polymerizable monomer in the second subset of the plurality of compartments by providing a stimulus thereto. The stimulus may comprise an energy, a chemical, or both. In some cases, the stimulating energy may comprise at least one of: electrical energy, electromagnetic energy, light, or heat.

[0365] In some cases, the polymerizable monomer or hydrogel may be photopolymerizable upon exposure to light. Light exposure or otherwise exposing the selected set of compartments to a stimulus may comprise exposing a pattern of energy wave (e.g., light) to a subset of the plurality of compartments which are selected to be polymerized. This may facilitate selectively polymerizing the selected subset of compartments (e.g., the second subset of the plurality of compartments). The pattern of light for selective light exposure and selective polymerization may be defined based on the signal. In some cases, the pattern of light may be defined using a digital mask, a digital mirror device (DMD), or both.

[0366] The methods may comprise screening a sample. In some cases, the compartment further comprises one or more cells (e.g., a cell or a plurality of cells) therein. The cell(s) may be adhered to the compartment (e.g., the bottom surface of a well). The methods and systems may comprise methods and systems for cell seeding, cell culture, and cell screening.

[0367] In some examples, the present disclosure provides a screening system comprising a miniaturized device. The miniaturized device may comprise a plurality of compartments. A first subset of the plurality of compartments may each comprise an effector bound to a scaffold and a barcode for identifying the effector. The screening system may further comprise a selector or selection device. In some cases, the selection device may comprise an illumination system. The selection device and/or the illumination system may comprise a light source, a digital mask, a digital mirror device, a signal detection device, a signal processing device, a computer system, hardware, and software, which may work individually or in concert to generate and apply a mask on the compartmentalized platform and expose the selected wells to the pattern of light, so as to stimulate the polymerization of the polymerizable monomer and perform the selective polymerization method.

[0368] In some examples, the selector may comprise a light source or illumination system, a microoptoelectromechanical system, or both. The microoptoelectromechanical system may comprise or be a digital micromirror device which may be connected to a computer system and a microscope-based system. The light source may comprise a UV light source. [0369] The system may comprise a blocking medium for selectively blocking flow into a plurality of compartments of the system. The system may comprise a flowing state configured to allow for liquid flow to the plurality of compartments or a selected subset thereof and a blocking state configured to block liquid flow to the plurality of compartments or a selected subset thereof. The system may comprise a detector configured to generate and/or detect a signal indicative of activity or structure of the effector screened in the plurality of compartments. The selector may be configured to activate the blocking state of the blocking medium in proximity of each compartment of a second subset of the plurality of compartments, based on the signal.

[0370] In some examples, the present disclosure provides a screening system comprising a miniaturized device or chip comprising a plurality of compartments. A first subset of the plurality of compartments may each comprise an effector bound to a scaffold and a barcode for identifying the effector. The system may further comprise a detector configured to detect and/or generate a signal indicative of activity or structure of the effector. The system may further comprise a selector configured to block liquid flow to a second subset of the plurality of compartments based on the signal, thereby yielding a third subset of the plurality of compartments that are substantially open to liquid entry. The system may further comprise a fluidic module or injection device configured to flow a liquid into a compartment of the plurality of compartments, such as the third subset of the plurality of compartments.

[0371] The liquid entered the third subset of the compartments may be configured to perform one or more tasks or serve one or more purposes as detailed anywhere herein. In some cases, the liquid may collect or sort the effector or the barcode. The injected liquid may comprise a reagent to facilitate the elucidation of the identity or structure of the effector in the selected compartments (e.g., positive compartments), such as a liquid used for decoding the barcode.

[0372] In some cases, the selector may comprise a light source and a microelectromechanical system. The fluidic module may be configured to provide a polymerizable monomer or hydrogel to the plurality of compartments. In some examples, the fluidics module (e.g., injection device) may inject a liquid comprising a reagent to be used for any purpose according to the methods provided elsewhere herein. In some examples, the selector may be configured to provide a stimulus to the polymerizable monomer or hydrogel located in proximity of the plurality of compartments.

[0373] The methods and systems provided herein may comprise providing a stimulus for performing the selective polymerization method. In some cases, the stimulus may be selected from the group consisting of an energy, electrical energy, electromagnetic energy, light, heat, and a chemical. The selective polymerization method may comprise providing and using a polymerizable monomer or hydrogel which may be photopolymerizable upon exposure to light. The selector in the system may be configured to expose a pattern of light to the plurality of compartments to selectively polymerize the second subset of the plurality of compartments. In some cases, the pattern of light is defined based on the signal. In some cases, the pattern of light may be defined using a digital mask, a digital mirror device (DMD), or both.

[0374] In some examples, the present disclosure provides a kit comprising a miniaturized device comprising a plurality of compartments, a library of different/unique effectors, such that each unique effector is bound to a bead, and the bead further comprises a barcode corresponding to and for identifying the effector. The kit may further comprise providing a blocking medium comprising a polymerizable monomer. The blocking medium may be configured to block liquid flow to the plurality of compartments upon exposure of the blocking medium to a stimulus according to any stimulus described elsewhere herein.

[0375] An example workflow for separating positive from negative compartments or separating the compartments based on other classification schemes according to the information provided anywhere herein can comprise performing the selective polymerization method. Selective polymerization may comprise introducing a polymerizable/cross-linkable monomer into the plurality of compartments/partitions (wells), selecting a subset of the compartments based on a predefined property or characteristic (e.g., positive/hit compartment vs. negative/non-hit compartment), and following a set of instructions to polymerize/cross-link the polymerizable monomer in the selected compartments without substantially polymerizing the monomer solution in the non-selected compartments. Selecting a subset of the compartments based on a predefined property may be performed based on a signal measured from each compartment. Performing this method may separate/isolate the selected compartments from the rest of the compartments in space, sort them, or partition them from one another. Separation may comprise separation by fluidic contact, separation in space, sorting one population from the other or a plurality of populations from each another in multiple containers or by adding a barrier between them to block material exchange among them. In some cases, this can be considered a method of binary classification followed by physical separation/partitioning of hit vs. non-hit compartments. Alternatively or in addition, this may be a non-binary categorization or classification of a plurality of sub-populations based on their properties or the signals measured from them through a screen or assay. [0376] The methods may comprise flowing a polymerizable monomer into the compartments (e.g., wells) followed by selective polymerization of the monomer solutions inside a subset of the compartments (e.g., wells) identified to have a given characteristics based on the detected assay signal, for example, identified to be negative or not comprising a hit. Such polymerization may at least partially block liquid flow entry into the negative compartments. In some cases, fluid flow into the compartment may be substantially blocked. For example, the barrier substantially blocking fluid flow may comprise or be a solidified or crosslinked hydrogel material which may comprise pores capable of mass transport in it. As such, the blockage of fluid flow may depend on the porosity of the polymerized material. In some cases, the blockage of fluid flow may be complete. In some cases, the blockage of fluid flow may be impeded but not complete (e.g., in some cases, fluid flow may be substantially blocked. In some examples, the goal of selective polymerization may be to substantially block access to negative compartments while the positive compartments /scaffolds (e.g., a compartment containing at least one hit) remain substantially accessible to future fluid flow. As such, the hits can be accessed in the positive compartments and either collected from the compartments or further processed inside or outside the compartments, for example, to elucidate the structure of the hit effectors found in the positive compartments. Alternatively, similar techniques may be performed to selectively polymerize the monomer mix inside the positive compartments and block liquid entry thereto. Fluid can then enter the negative wells to perform a process on the non-hit scaffolds or separate/sort them.

[0377] In some examples, a system is obtained or provided for performing the assay, hit identification, and selective polymerization. The system may comprise a platform such as a miniaturized device comprising the plurality of partitions/compartments, a computer or signal processing device, software, a microscope, an objective, a detector device, a light source, a digital mirror device, a digital mask, or any combinations thereof. Light can pass through a microscope stage through an objective to expose the plurality of wells or a selected subset thereof such as to polymerize predefined regions of the platform or the selected compartments/wells.

[0378] The polymerizable monomer used in the selective polymerization method may be any suitable material and it may be curable or cross-linkable upon application of a stimulus. The stimulus may comprise an energy, electrical energy, electromagnetic energy, light, heat, a change in PH, a reagent, and/or a chemical such as a catalyst. In some cases, the material used in selective polymerization may be photopolymerizable upon application of a light at a predefined wavelength and intensity. In some cases, the crosslinking light may be UV. The UV light may have any suitable power density. In a particular example, a power density of the UV light used for crosslinking the hydrogel may be from about 1 to about 2

[0379] In some examples, the wavelength of the light used for polymerization may be at least about 250 nm, 300 nm, 400 nm, or more. In some examples, the wavelength of the light may be from about 200 to about 500 nm. In some examples, the power density of the light (e.g., UV light) may be at least about 0.1 milliwatts (mW), 0.2 mW, 0.3 mW, 0.4 mW, 0.5 mW, or above. The exposure time may be at least about 1 second (s), 10 s, 30 s, 50 s, 1 minute(s) (min), 2 min, 3 min, 4 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, or more. The light wavelength, power density, and exposure time can be adjusted to reach the intended results based on the application.

Light patterning and selective light exposure

[0380] In some examples, the methods and systems of the present disclosure facilitate selective light patterning and selective light exposure on various regions on a substrate or a system (e.g., a plurality of compartments on a miniaturized screening platform such as a chip, microfluidic chip, miniaturized well array platform, and beyond) at the same or at varying light intensities and wavelengths, such as to facilitate/enable various applications.

[0381] In some examples, the methods and systems of the present disclosure comprise providing a scaffold comprising an effector covalently bound to the scaffold via a photocleavable linker (PCL) and releasable via photolytic cleavage of the PCL. The effector may be further encoded by an encoding or barcode (e.g., optical barcode or oligonucleotide barcode) which is corresponding to the effector and is configured to identify it. In some cases, the effector comprises a plurality of subunits (e.g., a number of building blocks making up a small molecule or polymer). The barcode may also comprise a plurality of subunits (e.g., an oligonucleotide barcode comprising multiple subunits or sequences ligated/covalently attached to one another), and the plurality of the subunits of the barcode correspond to and identify the plurality of the subunits of the effector. Alternatively or in addition, the barcode or a number of the subunits of the barcode may be corresponding to and identifying the scaffold (e.g., bead). The effector, barcode, and the scaffold may comprise various embodiments according to the descriptions provided anywhere herein. In some cases, the subunits of the barcode may record the sequential order of addition of each subunit of the effector, thereby containing a record of the order of synthetic steps used to create the effector.

[0382] In some examples, selective light patterning may be applied to expose a selected subset of compartments/wells in the compartmentalized/miniaturized system (e.g., array/microarray platform) to a given light intensity. The light intensity may affect or control the amount (e.g., concentration) of the released effector/compound in the compartment. For example, increasing the light intensity may release a greater amount or concentration/dose of the effector/compound and a lower light intensity may lead to a lower released concentration in the compartment. The concentration of the released effector inside each compartment can be controlled by the intensity of the light that is exposed to that compartment, or the percentage of the light that passes through a filter or digital mask to expose the compartment. This feature can be used to create a light gradient for exposing the compartments to energies (e., lights) at varying energy/power densities, thereby generating a concentration gradient for the released effector among the compartments. This can be used to perform a titration or dose-response study (e.g., testing a plurality of different concentrations of a released effector on the same sample) of the effector/compound across the compartments. This method can be used to screen any effector. In some examples, the effectors may be small molecules composed of a plurality of building blocks. For example, a small molecule may comprise at least 1, 2, 3, 4, or more building blocks (e.g., builtthrough 1, 2, 3, or 4 cycles in a split and pool combinatorial synthesis process). In other words, an effector/compound library may comprise 1, 2, 3, 4, or more cycles. In some cases, the number of cycles correspond to the number of building blocks and the number of synthesis steps during an example split and pool synthesis. In other examples, the effector may be a known compound or drug pre-synthesized on the scaffold (e.g., using methods other than modular attachment or split and pool synthesis of a plurality of subunits or building blocks). In some cases, an effector may be a single unit. In some cases, the technology may be used to screen known drugs, tool compounds, or FDA-approved drugs.

[0383] In some examples, a plurality of cells or a population of cells may be introduced into or seeded in an array presented herein. A response/signal may be measured from the compartments and/or the cells in the array, for example, generated by an assay performed in the wells of the array, and detected by a detector. A number of cells, a number of compartments, a number of regions on the platform, or any combination thereof may be selected to be exposed to light at a given intensity, energy, and/or power. The methods and systems provided herein can be used to selectively expose such cells to light at the intended intensity for any purpose. [0384] An example of a system and workflow for performing screening (e.g., cell screening in a miniaturized compartmentalized screening system such as a flow cell), perturbation analysis with encoded effector libraries, and/or selective light patterning is provided in FIG. 12. This figure shows an exemplary system and workflow for performing the methods of the present disclosure. This exemplary workflow comprises providing or obtaining a system comprising a digital mirror device (DMD) and a flow cell. A miniaturized compartmentalized screening device (e.g., a flow cell provided anywhere herein) is set up on the microscope stage. The flow cell comprises a plurality of compartments such as miniaturized wells. The miniaturized wells may comprise a biological sample, particles, and/or cells. The system further comprises a flow controller and a computer connected thereto for controlling flow rates and components of solutions inserted into the flow cell inlets. The solution may comprise assay materials, media, oil, polymers used for the selective polymerization described elsewhere herein, and any other solution described anywhere herein for performing the methods. A signal detector, camera, or signal processing device in the system detects a signal and/or captures one or more images of the wells or subsets thereof. The subsets can be defined by the user or identified automatically by the computer software. The workflow may be manual or automated. In some cases, the system may be operated by a person. Alternatively or in addition, the system may be operated by robots. The signal (e.g., images) can be processed manually and/or automatically by a user or any computer program to define a set of conditions for hit identification (e.g., defining criteria for marking certain compartments as positive). Alternatively or in addition, any sub-population beyond hit vs. non-hit may be defined. A digital mask can then be created (e.g., using the software on the computer or any other computer and computer program). The digital mask can be used to selectively expose certain wells to light to selectively polymerize the wells identified as hits using the digital mirror device (DMD), according to the methods for selective polymerization and light patterning.

[0385] An exemplary digital mask for selective light exposure is shown in FIG. 13A. This digital mask may be used with the system shown in FIG. 12 and other methods and systems described anywhere herein. FIG. 13B shows an exemplary digital mask overlayed on an exemplary miniaturized array for screening. The circles 1301 show the digital mask overlayed on a subset of wells to selectively expose those wells and their contents to light (e.g., UV light) in order to selectively polymerize the polymerizable monomer solutions in the exposed wells, thereby substantially blocking liquid flow thereto to perform selective polymerization, lightpatterning, and sample post-processing during or after a screen. Another subset of wells 1302 remain unexposed to UV light.

[0386] FIG. 14 shows an example of selectively polymerized miniaturized wells 1401 of the methods and systems of the present disclosure. The system comprises a plurality of circular wells on a solid surface. Cells were initially seeded in the majority or all of the wells. A subset of the wells contained at least one cell and at least one scaffold comprising an effector and a barcode identifying the effector. The scaffold/bead and the bead-bound encoded effector can be according to any embodiment thereof described anywhere herein. An assay was performed inside each well. A signal indicative of an effect of an effector (e.g., released from a scaffold) was measured from the wells. A threshold was defined for the signal which marked select wells as hit wells (i.e., wells in which the effector was determined to have a significant effect on the assay and/or the cells). Based on such determination, a digital mask (e.g., such as the digital mask shown in FIGs. 13A and 13B) was generated for selectively exposing the non-hit wells to a light capable of stimulating or initiating the cross-linking or polymerization of the polymerizable monomer (e.g., using the system shown in FIG. 12 including a computer and software program), thereby blocking liquid flow into the identified non-hit/negative wells. A liquid polymerizable monomer was introduced into the majority or all of the wells, after the completion of the assay screen. The digital mask and digital mirror device (DMD) were used to expose the non-hit wells to the stimulating light (e.g., UV) which initiated the crosslinking of the monomers. The polymerizable monomer in the non-hit wells were selectively polymerized by exposure to the light, thereby substantially blocking liquid flow thereto and substantially trapping and retaining the contents of the non-hit wells 1401, such that the contents of those wells would remain in place in presence of fluid flow. A liquid (e.g., oil and surfactant) was introduced into the system and allowed to enter the non-polymerized (hit) wells 1402 to collect their contents. The liquid was substantially impenetrable to non-hit wells 291 as those wells contained the cross-linked polymer facilitated through selective polymerization using the digital mask and digital mirror device. As such, the contents of the non-hit and nonpolymerized wells 1401 remained therein, while the contents of the hit wells 1402 well collected therefrom. This image shows the difference in appearance of selectively polymerized wells 1401 vs. non-polymerized wells 1402. Downstream assays enriched for active effectors. A subset of the hits were validated as active effectors. This demonstrated the efficacy of the described method.

[0387] FIG. 15 shows an example of selective light exposure and compound release from scaffolds in miniaturized wells. The system comprised a plurality of circular wells on a solid surface. A plurality of scaffolds (beads) comprising a fluorophore-bound (e.g., covalently attached) to the scaffold via a photocleavable (PCL) linker were introduced into the plurality of wells of the system. In this example, the effector was a fluorophore bound to the bead. The fluorophore-bound scaffolds were intended to characterize effector release from the scaffold upon exposure to light. Upon introduction of the scaffolds into the system, a subset of the wells remained empty, another subset of the microwells contained a single scaffold, another subset of the wells contained more than one scaffold (e.g., two or more scaffolds). A system similar

- I l l - to the schematic shown in FIG. 12 comprising a digital mask and digital mirror device were used to select a plurality of wells for exposure to light to release the fluorophores from those scaffolds in their respective wells, using a software program on a computer in communication with or as part of the system. In this control experiment, well selection was arbitrary. The selected wells were exposed to UV light through an objective below the solid surface on a microscope stage, the PCL was cleaved by UV exposure, and the fluorophore was released from the scaffold into the selected wells. Any other UV exposure system could be used (e.g., the illumination system or UV exposure system shown in FIGs. 7A-7C operated in a top-down and/or bottom-up position, or used to expose the system at any suitable angle). The selected wells demonstrated a measurable fluorescent light upon fluorescent microscopy. This example demonstrates the application of the digital mask and the digital mirror device for selectively exposing a subset of the wells to UV light and releasing compounds from the scaffolds into wells. Following the experiment, the system was monitored for a prolonged period to study the dynamics of fluorophore penetration into adjacent wells over time. The fluorophore was not found to penetrate or diffuse into adjacent wells by any significant degree over several hours. This demonstrated an application of the methods and systems of the present disclosure for selectively exposing a plurality of wells, and that the wells were successful in isolating the samples therein without their contents leaking across the wells over time and cross contaminating the compartmentalized samples.

[0388] FIG. 16 provides another demonstration of the selective polymerization method using a DMD. A custom digital mask was used to selectively polymerize a subset of the plurality of compartments of a miniaturized well array using a DMD and light patterning techniques disclosed in the present disclosure. Fluorescent beads were flown into the compartments and selected based on the fluorescent signals detected from them using the system detectors. Brightfield images and fluorescent images were then used to identify select wells and the bead spatially (i.e., based on location in space). Based on the images, a custom mask was generated to block off UV light from select wells 1601 and keep the beads therein intact. Once the mask was generated, the DMD automatically determined and polymerized the selected wells 1600 using the methods and systems of the present disclosure. Another subset of wells 1601 remained unpolymerized and open to liquid flow, as an example of separating compartments in space.

Isolation and sorting of sub-populations after a screen

[0389] The methods of the present disclosure comprise screening in arrays. The compartments (e.g., wells of an array) may contain cells, assay reagents, and a scaffold-bound encoded effector according to the descriptions provided elsewhere herein. An assay may be performed in at least a subset of the compartments (e.g., some or all of the compartments). The assayed compartments may be identified or marked as positive (a compartment containing a hit), negative (a compartment not containing a hit), or marked as another class/category. The hit may be an effector identified to have a defined effect on the cell(s) based on a signal measured from the assay. The assay and hit identification may be followed by the selective polymerization method. For example, after performing the assay and identifying the hits, a photopolymerizable hydrogel pre-polymer may be flowed into the plurality of compartments (array/microarray) to fill the compartments (e.g., a subset or all of the wells of the array platform, for example, non-discriminatively). The wells considered as negative (non-hits) may be selectively polymerized using the selective polymerization and light patterning methods and systems described anywhere herein (e.g., using a digital mirror device, a microscope-based system, a computer, and software). In some cases, such polymerization may substantially block liquid flow into and/or from the polymerized compartments, thereby effectively prevent interactions between the scaffold entrapped inside the compartment within the polymerized matrix, such that after flowing reagents or solutions into the device (into the plurality of compartments), they would not enter the selectively polymerized compartments. Remainders or residual amounts of hydrogel pre-polymer may be washed out of the array using any proper wash protocol (e.g., using a proper buffer).

[0390] The next steps may comprise performing a variety of downstream processes and techniques to either isolate or sort a sub-population of the compartments and/or the scaffolds therein based on their properties as determined by the signal (e.g., hit scaffolds), or elucidate the identity of the effectors on the scaffolds using the information available through the barcode, such as by measuring a property of the barcode (e.g., decoding or sequencing) inside the compartment or after collection of the scaffold and/or the barcode from the compartment. Examples of such techniques may comprise the emulsification method for scaffold sorting, barcode cleavage, selective barcode extension or barcode tagging, selective barcode extension, and/or selective barcode replication for hit elucidation.

Emulsification method for scaffold sorting

[0391] In some examples, following a screen and performing the selective polymerization method, liquid can be introduced into the system and allowed to enter the positive compartments without substantially penetrating the negative compartments. In some cases, such liquid may comprise an oil immiscible with the contents of the compartment (e.g., an aqueous solution comprising assay reagents and/or cells). The oil may collect the effector from a compartment which has remained substantially open to liquid flow after performing the selective polymerization method. This method may be herein referred to as “emulsification”.

[0392] In some examples, the liquid flown into the compartments for performing the emulsification method may comprise oil and surfactant. As the surfactant-containing oil flows into the compartments which are open to fluid entry (e.g., positive compartments), it can remove the aqueous solution and/or the scaffold from the compartment. This method can facilitate collecting and sorting the positive scaffolds, separating the positive scaffolds from the negative scaffolds, and preparing the positive scaffolds for barcode readout and/or decoding (e.g., sequencing analysis) to identify the positive effectors. In some examples, the combination of selective polymerization and emulsification allows for collecting the contents of the positive compartments (e.g., hit scaffolds) without collecting the contents of the negative compartments (non-hit scaffolds), thereby separating the hits and non-hits in space and/or sorting them. Alternatively or in addition, in some examples, ultra- sonication and/or bulk motion may be used to extract the scaffolds.

Barcode cleavage

[0393] The methods of the present disclosure may comprise barcode cleavage. Barcode cleavage may be performed on positive scaffolds after identifying them through a screen in a miniaturized system provided herein (e.g., droplet-based system or well array system). In some examples, after identifying hits (e.g., based on a property of the detected signal), the barcodes of the hits may be cleaved from the scaffolds (e.g., enzymatic cleavage) and collected for decoding (e.g., sequencing) outside the partitions/compartments. For example, a plurality of encoded effectors may be introduced into the plurality of the compartments containing cells, the assay may be performed in the plurality of compartments. A subset of the wells may be identified as positive (e.g., containing a hit). Selective polymerization may be performed to polymerize the hydrogel in negative compartments to substantially block liquid flow thereto. A solution comprising a cleavage reagent (e.g., cleavage enzymes) may be flown into the positive compartments to cleave the barcodes of the hits and collect them for further processing (e.g., downstream amplification and/or sequencing). Downstream amplification and/or sequencing may in some cases be performed inside the same compartments. In other examples, the cleaved barcodes may be collected and transferred outside the compartments for performing the downstream amplification and/or sequencing.

[0394] In some examples, the methods of the present disclosure comprise cleaving the barcode/encoding (e.g., nucleic acid encoding or DNA encoding/tag) from the scaffold. In some examples, the nucleic acid encoding (e.g., DNA) is cleaved via a cleavage catalyst or enzyme. A catalyst or enzyme may catalyze the cleavage reaction rate and/or facilitate the cleavage of the barcode, encoding, and/or tag from the scaffold. The selective polymerization method may facilitate controlling where (e.g., in which compartments) the barcodes are cleaved.

[0395] FIG. 17 provides an exemplary method for enzymatic cleavage and collection of DNA barcodes from beads. The exemplary method comprises selective enzymatic cleavage of DNA barcode from the beads such that the cleaved barcode could be sequenced inside the compartment or collected from the compartment for off-chip decoding and elucidation of the identity and/or structures of effectors identified as hits after performing a screen followed by selective polymerization of the non-hit compartments.

[0396] With reference to FIG. 17, Panel (a) shows a bead comprising a nucleic acid barcode. The bead may further comprise an effector that is encoded by the barcode. The encoded effector may be bound to the bead via a cleavable linker or be incorporated inside the bead. The effector may be releasable upon cleavage of the linker or releasable upon degradation of the bead. A stimulus may be provided to release the effector by cleaving the cleavable linker or by degrading/resolving the bead. The DNA barcode on the bead shown in the figure comprises a cleavage site/sequence (abc) in proximity to the DNA barcode connection site to the bead. The cleavage sequence can be recognized by a cleavage enzyme.

[0397] With reference to FIG. 17, panel (b) shows an exemplary screen and assay which can be performed inside the plurality of compartments followed by the selective polymerization method. The non-hit/negative compartments can be blocked using a polymerized hydrogel to substantially block liquid flow thereto (shown on the right, polymerized). With reference to panel (c), a cleavage mix can be supplied into the compartments identified as hit (shown on the left) with the polymerized matrix acting as a barrier against the entry of the cleavage enzyme solution into the non-hit compartments (shown on the right). The cleavage enzyme can nick/cut the DNA barcode at the cleavage site, thereby releasing the DNA barcodes from the bead into the solution in the compartment. Beads trapped in a cured photopolymer in the non-hit compartments may continue to have DNA tags bonded thereto (e.g., to their surface). The supernatant from the plurality of compartments can be collected from the system. Such supernatant may comprise the cleaved DNA barcodes which can then be amplified and decoded to elucidate the identity and structures of the hits.

Selective barcode extension or barcode tagging

[0398] The methods of the present disclosure may comprise selective extension and replication of barcode/encoding. In some examples, selective barcode extension may be performed after performing the assay, measuring the assay signal (e.g., a first signal), identifying the hits, and polymerizing the non-hit compartments to block liquid flow thereto. The selection may be performed in other ways. In some cases, categories other than hits and non-hits may be selected to be processed. In some cases, the non-hits may be selected to be processed. The selective barcode extension method may comprise flowing a solution into the selected compartments (e.g., microwells that have been selected based on an intended criteria or property of the signal in order to get processed) which is allowed to enter the selected compartments (e.g., hits) but be substantially impenetrable into the non-selected compartments (e.g., non-hits) due to the presence of the polymerized matrix as a result of the selective polymerization method.

[0399] The selective barcode extension method may comprise flowing a solution or extension mix into the plurality of compartments, such as to enter the selected compartments (positive/hit-containing wells) but not the non-selected compartments (e.g., negative wells or wells which do not contain a hit). The solution or extension mix may comprise one or more reagents. The reagents in the extension mix may comprise one or more proteins or enzymes, in some cases, a composition of a plurality of enzymes or a panel of enzymes. The one or more enzymes and/or reagents in the extension mix may initiate the extension of the nucleic acid barcode on the scaffolds contained in the selected/positive compartments (e.g., as identified based on the assay signal, for example in case the effector of the scaffold is identified as a hit based on the assay signal measured in the compartment). In some cases, the same method may be applied on any sub-population among the compartments, based on a measured property in the compartment (e.g., based on the signal). For example, in some cases, the selective polymerization method may be applied to block liquid flow into a defined population based on a criteria or threshold of the signal. In some cases, selective polymerization may be used to block liquid flow to positive compartments, and the contents of the negative compartments may be chosen for post-processing according to the methods presented herein.

[0400] The extension and/or replication can be performed using a variety of methods. In an example, a screen according to the methods of the present disclosure is performed in a well array using a DNA-encoded effector library. In such example, an effector is bound to a scaffold (e.g., a bead) via a cleavable linker or is otherwise provided inside the bead. The effector is releasable from the bead upon cleavage of the cleavable linker. Alternatively, the effector may be inside the bead and be releasable upon degradation of the bead. The effector is encoded with a DNA barcode present on the same bead which is corresponding to and identifying the structure of the effector. For example, the effector may comprise a plurality of subunits (e.g., building blocks of a small molecule), and a DNA barcode may comprise a plurality of sections/sequences that are corresponding to and identifying the subunits of the effector using a database generated throughout the synthesis of the on-bead DNA-encoded library. The synthesis may have been performed using split and pool synthesis. That is, both the DNA barcode and the effector may be incorporated inside or onto the bead or a combination of both, prior to introduction into any screening platform and the plurality of compartments. The beads and cells can be loaded into an array platform according to the methods and systems presented herein. An assay can be performed in the compartments (e.g., wells). The compartments may be identified as positive compartments (hits), negative compartments (non-hit containing compartments) or otherwise based on a property or threshold of the signal. In some cases, the non-hits may be selectively polymerized to block liquid flow thereto. The barcode extension method may be used to extend the barcodes of the identified hits in the positive compartments. [0401] The DNA barcode (e.g., a double-stranded/dsDNA barcode) extension method may comprise flowing a solution comprising one or more enzymes (e.g., a panel of enzymes) and reagents into the compartments to initiate the DNA barcode extension and replication. An example method of extension/replication of a DNA barcode may comprise flowing in an exonuclease to remove one strand of the DNA barcode (e.g., one strand of the dsDNA barcode), converting the dsDNA into a single stranded DNA (ssDNA). Due to the blocking effect of the polymerized wells, the enzyme will only be able to act on bead DNA tags within unpolymerized wells and will have minimal to no effect on the blocked/non-selected compartments and contents thereof. Next, a combination of primers, deoxyribonucleoside triphosphates (dNTPs), and polymerase enzymes may be flowed into the array. The primers may first bind to a specific corresponding sequence on the ssDNA barcode, providing a small section of dsDNA. This may provide a site where the DNA polymerase can bind to the DNA tag. The DNA polymerase can then begin to extend the primer, incorporating dNTPs in a manner that corresponds to the opposing parent DNA strand. After the extension process, the ssDNA can be converted back to dsDNA, where the newly built strand is only bound via annealing to its opposing strand and not through covalent linkage to the bead. Flowing a “strip” reagent such as NaOH may result in a disassociation between the newly built strand and the parent strand. This may release a copy of the DNA tag into the surrounding supernatant, thus providing a tag that can be recovered for decoding. The process of DNA polymerase extension and stripping can be repeated over and over again to increase the number of copies of the stripped extended DNA barcode inside the compartments of the system, leading to a concentration of DNA tags in solution that can be clearly measured over any background signal, for example, upon extraction from the plurality of compartments. Additionally, because wells sealed with photopolymerized hydrogel prevent interactions between any enzymes in the liquid flown into the system and the DNA barcodes/tags, only barcodes/tags from the selected compartments (e.g., scaffolds/beads considered as positive or hit containing) will be replicated and released into solution, which can be recovered from the well array.

[0402] An example method for performing DNA barcode/tag extension and replication may comprise initiating isothermal linear amplification. In some cases, an ensemble of reagents and/or enzymes may recognize a specific marker on the dsDNA barcode which may induce, insert, facilitate, or otherwise permit a priming event. Such event may extend the primer to create a copy of the barcode and repeat this process without iterative rounds of cycling temperature and/or reagents. In some cases, the extension and dissociation methods provided herein may allow for enrichment and/or repeated decoding of the encoded hit scaffolds.

[0403] FIG. 18 shows an Exemplary method for DNA barcode extension, stripping, and collection from positive (hit-containing) compartments following assay screening and selective polymerization by light patterning. A DNA-encoded effector library (e.g., a bead-bound library) comprising a plurality of beads, each bead comprising an effector bound to the surface of the bead via a cleavable linker or encapsulated inside a degradable bead according to the embodiments presented elsewhere herein such that the effector is releasable from the bead upon cleavage of the cleavable linker or degradation of the bead, and the effect of the effector on a target or biological sample can be assayed inside the compartment using the methods provided anywhere herein. The effector may be barcoded with a nucleic acid molecule such as a DNA barcode comprising a sequence unique to the structure of the effector according to the embodiments presented anywhere herein. The DNA barcode may further comprise a sequence unique to the bead itself corresponding to and for identifying and counting the bead (beadspecific barcode or sequence). In this example, the DNA barcode may further comprise a cleavage site/sequence. The cleavage site/sequence (abc) may be in proximity or adjacent to the site at which the DNA barcode is bound (e.g., covalently bound) to the surface of the bead. The encoded-effector beads may be loaded into a plurality of compartments in a miniaturized well array platform comprising a plurality of cells, as described anywhere herein (e.g., shown in FIG. 10) The cells may be seeded in the wells of the miniaturized well array before or during encoded effector bead library loading. A subset of the plurality of the compartments in the well array platform may comprise at least one cell or a plurality of cells and at least one encoded effector scaffold/bead. The compartments may contain any number of cells and/or any number of beads. A subset of the compartments may each contain at least one cell and at least one bead (e.g., one bead). A screen (e.g., assay screen) can be performed in the plurality of compartments to identify a subset of the compartments as containing hits (positive) and another subset as non-hits (negative or not containing a hit).

[0404] With continued reference to FIG. 18, panel (b) shows that the non -hit (negative) compartments (e.g., a compartment not containing a hit) can be selectively polymerized by light patterning according to the methods of the present disclosure such as to substantially block liquid flow to and/or from the non-hit (negative) compartment, while the hit- containing/positive compartments remain substantially open to liquid entry. One or more washing steps may be performed to clean residual liquid after performing the polymerization. [0405] With continued reference to FIG. 18, panel (c) shows that a solution may be added to (e.g., injected to or loaded into) the plurality of compartments and allowed to enter the positive wells while the polymerized matrix substantially blocks liquid entry into the negative compartments. The added solution may comprise or be a barcode extension solution/mix which may comprise a plurality of reagent components such as one or more enzymes (e.g., an enzyme panel), one or more primers, a buffer, and optionally, additional components. The added solution may comprise an extension enzyme such as a polymerase and/or an exonuclease. The added solution may further comprise one or more primers. The exonuclease may cut/cleave the DNA barcode at the cleavage site (abc), shown on the left. This converts the double-stranded DNA barcode into a single-stranded DNA which may remain attached (e.g., covalently attached) to the surface of the bead. The cleavage of the DNA barcode will not take place in the negative wells due to the presence of the polymerized matrix incorporated therein as a result of the selective polymerization method (e.g., performed by light patterning) according to the methods provided anywhere herein (shown on the right) since the extension mix will be substantially impenetrable into the negative wells due to the presence of the polymerized hydrogel.

[0406] With continued reference to FIG. 18, panel (c) shows that the added solution (e.g., extension mixture added to the positive compartments) may further comprise a polymerase enzyme. The polymerase may extend the single-stranded DNA attached to the surface of the bead which remains thereon after getting cleaved by the exonuclease, as described in panel (b) of FIG. 18, thereby creating a newly produced complementary DNA strand on the bead which was identified to initially contain a hit effector. The added solution may further comprise a stripping reagent configured to cleave the newly produced complementary strand from the bead. In some examples, such stripping reagent may comprise or be sodium hydroxide. This can thereby create a released complementary strand into the solution inside the positive compartment which forms the new composition of the contents of the compartment. The contents of the compartments can be sorted or collected out of the compartments for further processing. For example, the contents of the compartments which may contain one or more (e.g., multiple copies of) the newly formed complementary strand may be aspirated or collected using any suitable methods (a pipette, an aspirator, a robotic liquid handler, or beyond, manually or automatically in any suitable platform). The oligonucleotides (e.g., the newly produced complementary strand created and stripped according to the extension and cleaving methods described herein) can be decoded by sequencing. In some cases, the DNA barcode extension and stripping of the newly formed strand may be repeated more than once (e.g., through multiple cycles) to produce a measurable concentration of DNA for each identified/detected hit or to enhance the quality of the signal (e.g., signal to noise ratio) for such measurements. The compartments identified as not containing a hit (negative, shown on the right) comprise a polymerized hydrogel blocking liquid entry thereto. As such, the solution added to the plurality of compartments may be substantially impenetrable into the negative compartments. Therefore, the complementary DNAs will not be produced inside those wells. Once the solution is collected from the plurality of compartments, the sequences read from the collected solutions may elucidate the structure of effectors identified as hits through reading the sequence of the newly formed complementary strands formed in the positive compartments but not the negative compartments. That is, in some cases, minimal to no signal will be detected, or minimal to no sequences will be read which corresponds to non-hit effectors.

[0407] In some examples, once the hits and the positive compartments are identified through a screen in a compartmentalized platform, the negative compartments (e.g., wells) can be selectively exposed to a stimulation to intentionally damage the DNA barcodes on the beads in a selected subset of the compartments. Such intentional damage may be referred to as intentional ablation. Intentional ablation may be performed selectively. As such, ablation may comprise selective intentional ablation/damaging of the barcodes. Intentional selective ablation may be performed by selecting a subset of the compartments using the systems of the present disclosure, such as the selector device and illumination module. Ablation may comprise exposing a damaging wave of energy in any form (e.g., light, heat, chemical, or another type of condition which may intentionally cause damage). In some cases, ablation may be performed by light exposure, in which case it may be referred to as photoablation. For example, a subset of the compartments may be selected based on the properties of the contents of those compartments, as identified through a screen, for example, based on a property or intensity of an assay signal measured for such compartment. In some cases, the compartments selected for ablation (e.g., photoablation) may comprise or be the negative compartments (compartments not containing hits). In other examples, another subset of compartments may be selected to be subjected to photoablation of their barcodes. For example, after the assay is performed and hits are identified, the negative wells can be exposed to high intensity or high energy (e.g., low wavelength) UV to intentionally damage the barcodes of the non-hit/negative scaffolds. In such case, upon performing sequencing in the compartments, the final sequencing readouts will be from positive scaffolds and the barcodes corresponding to hit effectors only. Minimal to no sequencing readouts may be obtained from negative/non-hit scaffolds. Alternatively or in addition, any other appropriate method and/or energy may be used to ablate the barcodes of the negative compartments.

[0408] The methods presented herein may be applied on any sub-population defined based on the signal beyond the binary definitions of hits vs. non-hits. For example, any number of sub-populations may be defined and processed using any post-processing method described herein. The intended methods may be repeated for as many numbers of times as intended in various combinations and/or iterations. For example, a combination of methods may be applied, and each method may be repeated 1 time, 2 times, 3 times, 4 times, 5 times, 10 times, or more.

[0409] The methods and systems provided herein may facilitate decoding the barcode inside the compartments (e.g., droplet or well. For example, the compartment may be a well, a screen according to any method described anywhere herein may be performed. Some or all of the compartments (e.g., one or more wells) may be selected for their barcodes to be elucidated. In some cases, the barcodes may be collected from the compartments and sequenced after collection. In some examples, the sequencing may be performed inside the compartment. Sequencing inside the compartment (e.g., well) may be performed using a variety of techniques.

[0410] An example technique for decoding in a well may comprise sequencing by probehybridization and signal tracking. The barcode (e.g., a nucleic acid molecule) may be hybridized to a probe. A signal may be detected from the probe that is hybridized to the barcode. The signal may be tracked over time. The signal may be indicative of the sequence of the barcode. The signal may be any kind of signal. In some cases, the signal may be optical or fluorescent. The signal may be detected by any suitable detector (e.g., an optical detector, a sensor, a camera, a PMT, or any other suitable device). In an example, the signal may be detected by imaging. Images may be processed to quantify the signal. The quantity of the signal may elucidate the sequence of the barcode and thereby the structure of the effector which the barcode is encoding.

[0411] In some examples, the barcode may be an optical barcode, as in, a barcode with optical properties which can be excited to emit a light. Measuring the light emitted from the barcode may elucidate the structure of the effector that the barcode is encoding. Decoding may be performed in the screening compartment (e.g., a well or a droplet in which the assay is performed, and the effector may have interacted with the sample/target). The method may comprise optical barcode imaging and signal analysis. Signal analysis may comprise capturing images and processing them to quantify the signal. The quantity of the signal may elucidate the structure of the effector. This method may be performed without collecting the barcode and/or the scaffold from the array, and instead, decoding the barcode in the screening platform in real time. The same technique can be applied to a droplet compartment.

[0412] The methods may comprise encoding extension in a partitioned array by polymerase and elution by denaturation. Denaturation may comprise chemical denaturation, PH denaturation, heat denaturation, an/or any combination thereof. The method may comprise barcode amplification inside the compartments or after collecting the barcode from the compartments. Barcode amplification may comprise PCR (thermocycling amplification), RCA (generating a cyclic copy of the barcode and isothermally amplifying it), Linear amplification (isothermal T7 promoter extension), Linear amplification by strand-displacing polymerase extension and nicking enzyme.

[0413] The methods of the present disclosure may comprise partitioned decoding. An example of partitioned decoding may comprise aspiration of one or more selected wells into bins for PCR analysis. In some cases, the selection may be performed based on the signal detected from the wells. In some cases, partitioning may comprise light patterning and/or selective polymerization as detailed elsewhere herein. Selection of the wells may be performed by a variety of techniques detailed anywhere herein, in some examples using DMD directed UV exposure for crosslinking a hydrogel matrix in wells. In some examples, the selection and processing of the wells may be performed using selective UV-laser scanning of wells to crosslink the hydrogel matrix in the selected wells. In some examples, barcodes may be retrieved from a partitioned array (e.g., partitioned using selective polymerization or another method). In some cases, barcodes may be cleaved from the scaffold (e.g., bead). Barcode cleavage may comprise chemical cleavage, photochemical cleavage, enzymatic cleavage (e.g., using a cleavage enzyme such as protease or endonuclease). [0414] In some examples, the encoded effectors may be sequestered from the partitioned array (e.g., a partitioned array using the selective polymerization and light pattern methods described anywhere herein). In some cases, droplets may be formed in wells. The droplets formed in the wells may be sequestered to collect the scaffolds/beads. In some examples, the beads may be sequestered by flow (e.g., also referred to as “the emulsification method” elsewhere herein). In some examples, the scaffolds may be sequestered from the compartments using acoustic waves. Other examples, for sequestering or moving the scaffolds may comprise magnetic field, electric field, electromagnetic field, or other methods.

Applications and samples

Morphological changes in the sample

[0415] The signal from the sample may be a morphological or visual change in the sample which can be measured by imaging the encapsulation. In some embodiments, detecting the signal comprises recording images of the sample in the encapsulation. In some embodiments, detecting the signal comprises recording a series of images of the sample in the encapsulation. In some embodiments, detecting a signal comprises recording a series of images of samples in encapsulations and superimposing the series of images of the sample. In some embodiments, detecting a signal comprises detecting morphological or visual changes in the sample measured by recording a series of images of the encapsulation.

[0416] In some embodiments, morphology changes in a sample, such as one or more cells, can be detected by an imaging sensor, capturing trans illuminated light with a high-speed shutter, where composite video frames offers multiple full-cell images that can aid in shape determination. In some embodiments, morphology changes in a sample, such as one or more cells, can be detected by an imaging sensor, capturing trans illuminated light from a high frequency pulsed light source, increasing temporal resolution and sharpening the perimeter of the cell. In one manifestation, morphology changes can be detected by fluorescence emission from a cell traversing a laser-light sheet excitation region. In some embodiments, the emission is captured by Avalanche Photodiode (APD) or charged coupled detector (CCD), in a onedimensional array of pixels, binned by time, then restitched into a composite fluorescencemicroscopy image.

[0417] In some embodiments, detecting the signal comprises recording images of the sample, wherein the sample is a cell. In some embodiments, recording images of the cell provides information about cell morphology, mitotic stage, levels of expressed proteins, levels of cellular components, cell health, or combinations thereof. In some embodiments, the encapsulation comprises a detection agent. In some embodiments, the detection agent is an intercalation dye. In some embodiments, the intercalation dye is ethidium bromide, propidium iodide, crystal violet, a dUTP-conjugated probe, DAPI (4’,6-diamidino-2-phenylindole), 7- AAD (7-aminoactinomycin D), Hoechst 33258, Hoechst 33342, Hoechst 34580, combinations thereof, or derivatives thereof. In some embodiments, the detection agent highlights different regions of the cell. In some embodiments, the detection agent highlights a particular organelle. In some embodiments, the organelle is a mitochondrion, Golgi apparatus, endoplasmic reticulum, nucleus, ribosomes, cellular membrane, nucleolus, liposome, lipid vesicle, lysosome, or vacuole. In some embodiments, the organelle is a mitochondrion. In some embodiments, the organelle is the nucleus.

[0418] Samples of any type can be utilized with the methods and systems provided herein. In some embodiments, the sample is a biological sample. In some embodiments, the sample comprises one or more cells, one or more proteins, one or more enzymes, one or more nucleic acids, one or more cellular lysates, or one or more tissue extracts.

Cell types

[0419] In some embodiments, the sample is a cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is SH-SY5Y, Human neuroblastoma; Hep G2, Human Caucasian hepatocyte carcinoma; 293 (also known as HEK 293), Human Embryo Kidney; RAW 264.7, Mouse monocyte macrophage; HeLa, Human cervix epitheloid carcinoma; MRC-5 (PD 19), Human fetal lung; A2780, Human ovarian carcinoma; CACO-2, Human Caucasian colon adenocarcinoma; THP 1, Human monocytic leukemia; A549, Human Caucasian lung carcinoma; MRC-5 (PD 30), Human fetal lung; MCF7, Human Caucasian breast adenocarcinoma; SNL 76/7, Mouse SIM strain embryonic fibroblast; C2C12, Mouse C3H muscle myoblast; Jurkat E6.1, Human leukemic T cell lymphoblast; U937, Human Caucasian histiocytic lymphoma; L929, Mouse C3H/An connective tissue; 3T3 LI, Mouse Embryo; HL60, Human Caucasian promyelocytic leukaemia; PC- 12, Rat adrenal phaeochromocytoma; HT29, Human Caucasian colon adenocarcinoma; OE33, Human Caucasian oesophageal carcinoma; OE19, Human Caucasian oesophageal carcinoma; NIH 3T3, Mouse Swiss NIH embryo; MDA-MB-231, Human Caucasian breast adenocarcinoma; K562, Human Caucasian chronic myelogenous leukemia; U-87 MG, Human glioblastoma astrocytoma; MRC-5 (PD 25), Human fetal lung; A2780cis, Human ovarian carcinoma; B9, Mouse B cell hybridoma; CHO- Kl, Hamster Chinese ovary; MDCK, Canine Cocker Spaniel kidney; 1321N1, Human brain astrocytoma; A431, Human squamous carcinoma; ATDC5, Mouse 129 teratocarcinoma AT805 derived; RCC4 PLUS VECTOR ALONE, Renal cell carcinoma cell line RCC4 stably transfected with an empty expression vector, pcDNA3, conferring neomycin resistance.; HUVEC (S200-05n), Human Pre-screened Umbilical Vein Endothelial Cells (HUVEC); neonatal; Vero, Monkey African Green kidney; RCC4 PLUS VHL, Renal cell carcinoma cell line RCC4 stably transfected with pcDNA3-VHL; Fao, Rat hepatoma; J774A.1, Mouse BALB/c monocyte macrophage; MC3T3-E1, Mouse C57BL/6 calvaria; J774.2, Mouse BALB/c monocyte macrophage; PNT1 A, Human post pubertal prostate normal, immortalised with SV40; U-2 OS, Human Osteosarcoma; HCT 116, Human colon carcinoma; MA104, Monkey African Green kidney; BEAS-2B, Human bronchial epithelium, normal; NB2-11, Rat lymphoma; BHK 21 (clone 13), Hamster Syrian kidney; NSO, Mouse myeloma; Neuro 2a, Mouse Albino neuroblastoma; SP2/0-Agl4, Mouse x Mouse myeloma, non-producing; T47D, Human breast tumor; 1301, Human T-cell leukemia; MDCK-II, Canine Cocker Spaniel Kidney; PNT2, Human prostate normal, immortalized with SV40; PC-3, Human Caucasian prostate adenocarcinoma; TF1, Human erythroleukaemia; COS-7, Monkey African green kidney, SV40 transformed; MDCK, Canine Cocker Spaniel kidney; HUVEC (200-05n), Human Umbilical Vein Endothelial Cells (HUVEC); neonatal; NCLH322, Human Caucasian bronchioalveolar carcinoma; SK.N.SH, Human Caucasian neuroblastoma; LNCaP.FGC, Human Caucasian prostate carcinoma; OE21, Human Caucasian oesophageal squamous cell carcinoma; PSN1, Human pancreatic adenocarcinoma; ISHIKAWA, Human Asian endometrial adenocarcinoma; MFE-280, Human Caucasian endometrial adenocarcinoma; MG-63, Human osteosarcoma; RK 13, Rabbit kidney, BVDV negative; EoL-1 cell, Human eosinophilic leukemia; VCaP, Human Prostate Cancer Metastasis; tsA201, Human embryonal kidney, SV40 transformed; CHO, Hamster Chinese ovary; HT 1080, Human fibrosarcoma; PANC-1, Human Caucasian pancreas; Saos-2, Human primary osteogenic sarcoma; Fibroblast Growth Medium (116K-500), Fibroblast Growth Medium Kit; ND7/23, Mouse neuroblastoma x Rat neuron hybrid; SK-OV-3, Human Caucasian ovary adenocarcinoma; COV434, Human ovarian granulosa tumor; Hep 3B, Human hepatocyte carcinoma; Vero (WHO), Monkey African Green kidney; Nthy-ori 3-1, Human thyroid follicular epithelial; U373 MG (Uppsala), Human glioblastoma astrocytoma; A375, Human malignant melanoma; AGS, Human Caucasian gastric adenocarcinoma; CAKI 2, Human Caucasian kidney carcinoma; COLO 205, Human Caucasian colon adenocarcinoma; COR-L23, Human Caucasian lung large cell carcinoma; IMR 32, Human Caucasian neuroblastoma; QT 35, Quail Japanese fibrosarcoma; WI 38, Human Caucasian fetal lung; HMVII, Human vaginal malignant melanoma; HT55, Human colon carcinoma; TK6, Human lymphoblast, thymidine kinase heterozygote; SP2/0- AG14 (AC -FREE), Mouse x mouse hybridoma non-secreting, serum-free, animal component (AC) free; AR42J, or Rat exocrine pancreatic tumor, or any combination thereof

[0420] The sample may comprise a protein, a recombinant protein, a mutant protein, an enzyme, a mutant enzyme, a protease, a hydrolase, a kinase, a recombinase, a reductase, a dehydrogenase, an isomerase, a synthetase, an oxidoreductase, a transferase, a lyase, a ligase, or any mutant thereof. The sample may comprise any suitable number of cells from at least 1, 2, 3, 4, 5, 10, 100, 1000, 10000 or more cells seeded in each compartment.

Ion channel screen

[0421] The methods and systems of the present disclosure may be used to screen cells. In some cases, screens may be performed on ion channels in cells. For example, the target may be one or more ion channels in one or more cell types. The encoded effectors may be used to perturb ion channels and/or modulate their activity. The effect of the encoded effectors on ion channels may be tested using the screening systems present anywhere herein. In some cases, screening may be performed in an array-based system described anywhere herein, in cells seeded in the micro-array, such as any array and system shown in any one of figures FIG. 8A, FIG. 8B, FIG, 8C, FIG. 9, FIG. 10, FIG. 11B, FIG. 12 or other figures or sections in the present disclosure.

[0422] In some cases, ion channels may be endogenous to the cells. In other cases, ion channels may not be endogenous to the cells. Ion channels may be mutant ion channels, such as an ion channel comprising a mutation. In some cases, mutations may cause the ion channel to be sensitive to stimulation (e.g., optical stimulation). Alternatively, the ion channel may be sensitive to stimulation (e.g., optical stimulation) for another reason.

[0423] In some examples, the ion channels may be stimulated as part of performing the methods of the present disclosure. Various kinds of stimulation may be applied. Stimulation may comprise electrostimulation, optical stimulation, chemical stimulation, any combination thereof, or other kind of stimulations. The stimulation may comprise optical stimulation, electromagnetic radiation, UV-VIS, near-infrared radiation, UV radiation, stimulation with visible light, or any combination thereof. Any suitable light wavelength and intensity may be used to stimulate ion channels. Stimulation may be applied at any suitable frequency.

[0424] In some examples, electrostimulation is performed on the cells using one or more electrodes which may be embedded in or used in conjunction with a screening system of the present disclosure. The screening system used for ion channel screening with or without electrostimulation may be any screening system mentioned anywhere herein, such as a droplet microfluidic device (e.g., FIG. 4) or an array-based system (e.g., FIG. 8A, FIG. 8B, FIG, 8C, FIG. 9, FIG. 10, FIG. 11B, FIG. 12), or another suitable screening system.

[0425] The methods for ion channel screening may comprise for searching for an effector with an effect on an ion channel of a cell. The effector can comprise any therapeutic modality. In some cases, an effector may be a small molecule compound, a biologic, a gene, a protein, a peptide, or any other effector mentioned in the present disclosure. The effect may be inhibitory or agnostic. The effector may be an inhibitor or an agonist. The effector may increase or decrease the activity of the ion channel. The effector may be inert and not have an effect on the ion channel. Encoded effector libraries may be screened against cells to find effectors with a predetermined effect. In some cases, the ion channel may be a protein expressed by a cell.

[0426] One or more voltage sensors may be provided or obtained in a screening system of the present disclosure or as an add-on set of tools to be used in conjunction with the screening system. The cells may be provided in the compartments (e.g., droplet or well), the cells may be stimulated for ion channels to be activated, the voltage sensors may be used to detect a signal indicative of the activity of the ion channel. This method may be performed in presence and/or absence of an encoded effector which may be used to perturb the cell to modulate the activity of the ion channel. A library of encoded effector libraries can be screened against ion channels of the cells to identify effectors with the predetermined effect. The encoded effectors and screening methods and systems are described in detailed throughout the entire disclosure. Any encoded effector embodiment or screening system embodiment may be used for ion channel screening.

[0427] The set of voltage sensor probes may comprise any suitable probe. For example, the set of voltage sensor probes comprise a FRET pair, a voltage-sensitive oxonol, a fluorescent coumarin, a DiSBAC compound, a coumarin phospholipid, a DiSBAC compound, a coumarin phospholipid, a DiSBAC ₂ , DiSBAC ₄ , DiSBACe, CC1-DMPE, CC2-DMPE, a DiSBAC ₂ (3), DiSBAC ₂ (5), DiSBAC ₄ (3), DiSBAC ₄ (5), DiSBAC ₆ (3), DiSBAC ₆ (5), CC1-DMPE, CC2- DMPE, DiSBACe, CC2-DMPE or any combination or derivative thereof.

[0428] The ion channel screened using the methods and systems of the present disclosure may be any kind of ion channel. The ion channel may comprise or be a protein, sodium, calcium, chloride, proton, potassium ion channel protein, calcium ion channel protein, chloride ion channel protein, proton ion channel proteins, or other kind of protein. An ion channel protein may comprise or be a voltage gated ion channel protein. The voltage gated ion channel may comprise or be a protein, sodium, calcium, chloride, proton, potassium ion channel protein, calcium ion channel protein, chloride ion channel protein, proton ion channel protein. The ion channel protein may be endogenous to the cell, an exogeneous ion channel protein, incorporated into the cell through a vector, expressed in the cells (e.g., after being incorporated into the cell by a vector), a gene encoding the ion channel protein transiently transfected into the cell, an overexpressed protein, or other kind of ion channel.

[0429] The screening method comprises detecting a signal from at least one member of the set of voltage sensor probes. The signal may be electromagnetic radiation, luminescence, fluorescence, or another kind of signal. In some cases, the electromagnetic radiation may be emitted due to a FRET interaction. In an example, the signal may be an increase, decrease, or change in electromagnetic radiation as compared to a compartment without the encoded effector. In another example, the signal may be an increase, decrease, or change in electromagnetic radiation as compared to the compartment before the stimulation of the ion channel.

[0430] In some cases, an engineered cell line may contain a first ion channel, in addition to a second light-sensitive ion channel, where upon stimulation with light, the second ion channel opens, and indirectly causes the first ion channel to also open. In this way, an effector may be tested for an effect on the first ion channel, e.g., by directly stimulating a second ion channel, then measuring the effect of the effector on the first ion channel.

Condensate detection

[0431] In some examples, a change in the condition of the sample or target may be observed as a result of the progression of an assay, test, or experiment in presence or absence of an encoded effector. In some examples, the observed change may be a redistribution of the signal in space. For example, an assay may test phase condensation. A sample may go through a phase change during the course of an assay. The condition of the sample and/or a change thereof may comprise liquid-liquid phase separation (LLPS) or phase condensation. LLPS or phase condensation may result in formation of condensates in the sample. The condensates may be an indication of a biological condition or activity. In some cases, condensate formation may be a measure of protein-protein interactions (ppi) in a sample or in a cell in a sample. For example, an assay may measure one or more protein-protein interactions through manifestation of a phase condensation (formation of condensates) in the sample. In some examples, such assays may be performed to study protein-protein interaction networks or protein-nucleic acid interaction networks (e.g., protein-RNA interaction network). In some examples, stressed- induced biomolecular condensates, also referred to as “condensates” or “stress granules” may form during a screen, detected using the screening platforms presented herein. The terms “condensates” and “stress granules” (SG) may be used interchangeably. In some cases, the effects of members of an encoded effector library provided herein may be tested on stress granules, condensates, protein-protein interaction networks, and protein-RNA interaction networks. The methods and systems provided herein may facilitate drug discovery and drug development for protein-protein-interaction networks or protein-RNA networks.

[0432] Provided herein are method and systems for detecting formation of ribonucleoprotein (RNP) granule assembly. In some cases, the methods and systems may comprise detecting and screening formation of stress granules (SG). A stress granule may be a dynamic and reversible cytoplasmic assembly formed in eukaryotic cells in response to cells. In some cases, SG formation may be detected in cells. In some cases, SG formation may be detected in cell-free samples. In some cases, SGs may form or assemble through liquid-liquid phase separation (LLPS) arising from interactions distributed unevenly across a core protein- RNA interaction network. In some examples, the central node of this network may comprise or be a moiety or molecule (e.g., G3BP1) which may function as a molecular switch which may trigger RNA-dependent LLPS in response to rise in intracellular free RNA concentrations. In some cases, increasing the levels of RNA in a sample may lead to SG formation or condensate formation. In some cases, G3BP1 may comprise one or more intrinsically disordered regions (IDRs) which may regulate its intrinsic propensity for LLPS. This propensity may be tuned by phosphorylation within the IDRs. Other factors affecting SG assembly may arise through positive or negative cooperativity by extrinsic G3BP1 -binding factors that strengthen or weaken, respectively, the core SG network. SGs may show up as condensates in screening and may be referred to as condensates.

[0433] In some examples, the effectors of the present disclosure may be capable of affecting SG networks, SG formation in living organisms, protein-interaction networks, protein-RNA interaction networks, molecular switches, IDRs, phosphorylation in G3BP1 or IDRs thereof, and/or positive or negative cooperativity by extrinsic G3BP1 -binding factors. SG networks, SG formation in living organisms, protein-interaction networks, protein-RNA interaction networks, molecular switches, IDRs, phosphorylation in G3BP1 or IDRs thereof, and/or positive or negative cooperativity by extrinsic G3BP1 -binding factors may be relevant in one or more pathological conditions or diseases and may be targets for drug discovery. The encoded effectors and screening platforms of the present disclosure may facilitate such drug discovery for the afore-mentioned targets.

[0434] In some cases, RNP granules assemble by liquid-liquid phase separation (LLPS), which may occur when protein-laden RNAs that are dispersed in the cytoplasm or nucleoplasm (soluble phase) coalesce into a concentrated state (condensed phase). In this condensed phase, the highly concentrated RNAs and RNA binding proteins (RBPs) may behave as a single organelle with liquid-like properties. The constituents of membranelles organelles may remain in dynamic equilibrium with the surrounding nucleoplasm or cytoplasm and may form transiently or persist indefinitely. Some RBPs, particularly those harboring low complexity domains (LCDs), undergo concentration dependent LLPS. In some examples, the methods and systems of the present disclosure may facilitate detection, screening, and perturbation of RNA- binding proteins (RBPs). The effectors of the present disclosure may be screened for effects on RBPs. The methods and systems of the present disclosure may facilitate drug screening, discovery, and development for affecting RBPs.

[0435] Condensates may show up as a change in signal locality, such as pixels, parts of pixels, or a plurality of pixels in an image which are brighter than the background. Fluorescence from the may aggregate, accumulate, or otherwise become brighter in the regions of the image where the condensates are formed. In some cases, this may also decrease the brightness of the background of the image. Therefore, a condensate may comprise a higher signal to background ratio compared to the areas of the image where condensates are not present. This effect may progress over time. More condensates may form in the sample/image over time. In some cases, a plurality of condensates may form in a sample over time. The plurality of condensates may comprise different sizes and intensities. In some examples, the number of condensates formed in a sample may be an indication of a condition of the sample. In some examples, the intensity of the condensates may be an indication of a condition of the sample. In some examples, a density of condensates (number of condensates per unit volume or per unit surface) may be measured, screened, and monitored over time. The properties of the condensates such as the number of condensates, size of condensates, intensity of the condensates, and other detectable properties of condensates may be used to assess a condition regarding the sample.

[0436] In some cases, an effect of an effector or effector library on a sample may be screened. In some cases, an effect of an effector on condensate formation may be screened. The assay may assay a protein-protein interaction in a sample or a cell. In a particular example, protein-protein interaction may be screened in a cell in presence and/or absence of an effector. The effector may be any effector mentioned anywhere herein. The effector may be an encoded effector described anywhere herein, for example, an encoded effector bound to a bead, such as shown in FIG. 1. The sample may be compartmentalized in any screening platform described herein. The screening platform may be a droplet-based platform, such as shown in FIG. 2B or FIG. 4. Alternatively, the screening platform may be a well array platform such as shown in FIG. 10, FIG. 11 A, FIG. 11B, FIG. 11, FIG. 12, FIGs. 14-16, or as described elsewhere An effector may be cleaved from a bead and released into the compartment and allowed to interact with a sample in presence of an assay capable of detecting protein-protein interactions in the sample which may comprise a cell. The protein-protein interaction may manifest as a change in fluorescence distribution and/or condensate formation. Fluorescence distribution or condensate formation may be monitored over time during assay incubation. Signals may be detected using any detector mentioned herein. The properties of the condensates may be an indication of the activity of the assay and the protein-protein interactions being tested. The changes in the signals over time may indicate the change in the condensates. The condition of the condensates and their various properties may be affected by an effector. The effect of the effector on condensate formation may be detected using the methods and systems provided herein. The condensates may be detected by imaging (e.g., fluorescence imaging).

[0437] In some examples, condensates may be detected in a droplet-based screening platform provided herein. In such case, the compartment/encapsulation is a droplet. A condensate may be a bright three-dimensional (3D) region (e.g., a sphere brighter than the droplet) inside the droplet. As a result of an assay, one or more condensates may be formed inside the droplets. The condensates in the droplets may be detected using any suitable detector described anywhere herein, such as a system comprising any combination of a camera (fluorescent imaging system), image processing device, signal processing device, optical train, using laser induced fluorescence (LIF) and PMTs. The settings, workflows, protocols, assay reagents, optical filters, and other conditions may be determined, set up, and integrated in each case to work properly individually and/or in concert toward the detection and screening goals (e.g., high-throughput screening of encoded effector libraries).

[0438] In case of condensate detection by imaging, images of compartments may be captured. A compartment may be a droplet in a microfluidic device. The droplet may be imaged. The condensates may be form during incubation in the microfluidic device and be present during the detection. In some cases, a droplet comprising an effector may be compared to a droplet not containing an effector. The number of condensates in presence and absence of effectors may be compared. In some cases, the intensity of the condensates under different conditions (e.g., presence of effector) may be compared. In some cases, the condensates may aggregate together to form larger and/or brighter aggregated condensates. In some cases, the formed condensates may aggregate on the beads of encoded effector libraries, thereby causing or increasing a fluorescence to be detected from the bead. Such appearance or increase in bead fluorescence may be an indication of condensate formation and/or activity of the assay. In each case, the effect of the effector on the activity of the biological event (e.g., protein-protein interaction in a sample and/or in a cell) may be assessed by observation and/or detection of the condition and properties of the condensates, however such properties manifest in the compartment (e.g., droplet or well). In some cases, an effector changes the properties of condensates over time, compared to a control sample not containing an effector. In some cases, the condensates may manifest or appear as small spikes on a droplet digital signal trace detected by a system presented herein.

[0439] An example demonstrating a condensate formation assay in a static compartment (well) is shown in FIGs. 19A and 19B. In these examples, assay components for phase condensation were mixed. Assay components may comprise a protein, RNA, and an enzyme. In this example, the sample contained G3BP1 stress granule assembly factor 1 (+Green Fluorescence Protein), RNA, and Caprinlwt (Caprinl wild type). Parameters considered for assay development and set-up may comprise temperature, ionic strength of the buffer (salts and PH), viscosity, crowding agents, detergents, mixing or turbulence in the sample, and sample incubation time, which can be adjusted to optimize assay conditions for screening. Upon mixing of the components and incubation, phase condensation and/or LLPS may occur and be detected over time. FIG. 19C shows a schematic illustrating the progression of the assay leading to Liquid-Liquid Phase Separation (LLPS). “SG” represents stress granule formation. The assay components (G3BP1 (+Green Fluorescence) Protein, RNA, and CaprinlWT) were added and incubated. In some cases, CaprinlWT may act in a catalysis capacity in the assay/reaction. Over time, the assay may progress, LLPS may occur in the compartment leading to the formation of small bubbles also referred to as condensates or stress granules. FIG. 19C schematically illustrates the increase in the number of condensates from right to left, marked with an arrow. The reaction rate is catalyzed by the addition of CaprinlWT.

[0440] With reference to FIG. 19A, a compartment comprising a sample containing SG components (as described with reference to FIG. 19C) was imaged over time using the methods and systems provided herein. The time span shown in this particular example was 30 minutes. The assay can progress for any intended duration, such as 1 min, 2 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 80 min, 90 min, 2 hr, 3 hr, 4 hr, 5 hr, or longer. The rate of the assay can be adjusted by adjusting assay reagents. The properties of the condensates as mentioned elsewhere herein can be monitored during a screen or after data acquisition. The number and/or the intensity of condensates may increase over time.

[0441] FIG. 19B shows a plurality of condensates formed in a compartment, illustrated as bright spots in an image 1900 captured via fluorescent microscopy performed on a cell-free and bead-free sample in absence of an encoded effector in a compartment (well). The image was processed to quantify the fluorescent signal detecting the condensates. A line scan is shown on image 2000 across which the intensity of the pixels of the image were measured. The intensity of the signal in the plurality of regions across the line scan are shown in plot 1901. X axis indicates the distance across the line scan. The Y axis is the intensity of the signal at each point. The background of the image comprises an intensity of about 80 (arbitrary units (AU). The high-intensity areas peak around 160 AU and 180 AU. The distance across the X axis is a measure of the size of the condensate. The intensity of the peaks is a measure of the brightness of each condensate. Graph 1901 indicated two signal peaks corresponding to the two condensates across the line scan in image 1900.

[0442] Various factors may affect condensate formation in an assay and its dynamics over time. Such factors may comprise temperature, PH, Ionic Strength of the assay buffer, buffer viscosity, crowding agents, detergents, mixing, and static or dynamic conditions of the compartment. For example, a well in an array-based system is static. A droplet may be dynamic. A droplet may move and shake as it travels through a microfluidic device. Such factors may increase mixing and turbulence or semi -turbulence inside the droplet. Such dynamic conditions may affect and alternate the assay conditions and may be accounted for and adjusted during assay development and assay optimization.

[0443] An example of condensate detection using a droplet-based platform is shown in FIG. 20. The droplet-based platform and workflow used in this example was similar to the system and workflow schematically illustrated in FIG. 2B. This example was performed with assay reagents in absence of beads. The droplets in this example did not contain beads. The signals detected are purely representative of the assay and condensate formation in droplets. The microfluidic device used was similar to the device shown in FIG. 4. Compartments or encapsulations in this example were droplets. FIG. 20 demonstrates the successful detection of condensates (stress granules (SG)) formed under dynamic flow conditions in a droplet microfluidic system. In the plots shown in FIG. 20, X axis represents time (min). Y axis represents digital signal measured using a PMT in the system. This PMT is arbitrarily marked as PMT1 on the system used. The signal was measured in Volts [V], The signal was detected using a Field Programmable Gate Array (FPGA) sensor. The FPGA sensor in this example was the detector, or a part of the detection system. Signals were recorded over time (Tl, T2, T3, T4, T5, and T6). These time points were measured across the loops of the incubation line (assay flow path) of the droplet-based microfluidic device shown in FIG. 4. The incubation time for each device region was known at the time of the experiment. As such, the progression of the assay and condensate formation in the droplets of a droplet microfluidic device and flow path were detected and monitored over time, across the device. This experiment was performed in absence of cells and in absence of beads.

[0444] In FIG. 20, The droplet trace 2001 in T1 shows a droplet in early regions/loops in the device at an early time point (approximately T1 ~ 0 min). Condensates are not present in droplet trace 2101 as they have not formed yet. This is because the assay has not progress yet (TO). After 2 minutes, another series of signals were captured (T2). Droplet trace 2002 detected in time point T2 shows an appearance of a small spike in the droplet trace, an early indication of a formation of a dim and small condensate. Droplet trace 2003 captured at T3 clearly indicates a spike demonstrating the formation of a condensate in the droplet. Droplet trace 2004 and the other droplet traces taken at time point T5 show additional examples of condensate formation and growth over time in the droplet microfluidic device. Multiple spikes in droplet trace 2004 indicates multiple condensates. In trace 2004, four spikes are observed. In other traces in the same plot, larger spikes are observed. Larger spikes indicate brighter condensates. More spikes detected on the same droplet trace indicate larger numbers of condensates per droplet. Droplet trace 2005 taken at time point T6 ~ 18 min also indicates spikes indicative of condensate formation in droplets. In some examples, the intensity of the spikes shown in this plot reach above PMT 1 = 0.05 V. Spikes of this intensity are absent in the time traces taken at Tl. This indicates that the condensates have formed over time and are a result of the progression of the assay indicative of an activity of the target. The biological activity in this example was a protein-RNA interaction. The encoded effector libraries provided herein can be integrated with this workflow and the effects thereof may be tested on the protein-RNA interaction networks using similar assays and methods and any screening platform provided anywhere in the present disclosure. This may facilitate drug discovery for protein-RNA interaction networks and protein-protein interaction (ppi) networks.

[0445] FIGs. 21A and 21B provide additional examples of condensate detection in a static miniaturized compartmentalized system. FIG. 21A shows a plurality of condensates (stress granules) formed in a static compartment. In some cases, the number of condensates may be counted as an indication of the activity of the assay over time. For example, the number (quantity) of the condensates may increase over time. FIG. 21B shows a plot presenting the relative frequency of condensates of various diameters. In some examples, the sample may be exposed to an effector. The effector may affect the number of condensates formed over time. For example, a greater number of condensates may be formed in absence of a compound, compared to in presence of the compound. That may indicate that the effector has successfully reduced the number of condensates formed, and therefore has been active against the target or the assay. The methods and systems of the present disclosure facilitate screening condensate detection in presence and absence of drug candidates such as using the bead-bound encoded effector libraries and the miniaturized screening platforms.

Definitions

[0446] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0447] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0448] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a sample” includes a plurality of samples, including mixtures thereof.

[0449] The terms “determining,” “measuring,” “detecting,” “evaluating,” “assessing,” “assaying,” and “analyzing” are often used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of’ can include determining the amount of something present in addition to determining whether it is present or absent depending on the context.

[0450] The term “zw vivo" is used to describe an event that takes place in a subject’s body. [0451] The term “ex vivo” is used to describe an event that takes place outside of a subj ect’ s body. An ex vivo assay is not performed on a subject. Rather, it is performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample is an “zzz vitro” assay.

[0452] The term “/// vitro” is used to describe an event that takes places contained in a container for holding laboratory reagent such that it is separated from the biological source from which the material is obtained. In vitro assays can encompass cell-based assays in which living or dead cells are employed. In vitro assays can also encompass a cell-free assay in which no intact cells are employed.

[0453] The term “hit” refers to an effector that has been screened against a sample and returned a positive result. The positive result may depend upon the nature of the screen being employed, but may include, without limitation, an indication of efficacy against a target being interrogated. In some cases, hits may be high-interest events of unknown veracity. In some cases, hits may not be treated as bona-fide until validated (e.g., in replicate tests) afterward. Downstream validation assays may be performed to validate the hits or identify them as false positives.

[0454] The term “screen” as used herein refers to performing an assay using a plurality of effectors in order to determine the effect various effectors have on a particular sample.

[0455] The term “sequencing” refers to determining the nucleotide sequence of a nucleic acid. Any suitable method for sequencing may be employed with the methods and systems provided herein. The sequencing may be accomplished by next generation sequencing. Next generation sequencing encompasses many kinds of sequencing such as pyrosequencing, sequencing-by-synthesis, single-molecule sequencing, second- generation sequencing, nanopore sequencing, sequencing by ligation, or sequencing by hybridization. Next-generation sequencing platforms are those commercially available from Illumina (RNA-Seq) and Helicos (Digital Gene Expression or "DGE"). Next generation sequencing methods include, but are not limited to those commercialized by: 1 ) 454/Roche Lifesciences including but not limited to the methods and apparatus described in Margulies et al., Nature (2005) 437:376-380 (2005); and US Patent Nos. 7,244,559; 7,335,762; 7,21 1,390; 7,244,567; 7,264,929; 7,323,305; 2) Helicos Biosciences Corporation (Cambridge, MA) as described in U.S. application Ser. No. 1 1/167046, and US Patent Nos. 7501245; 7491498; 7,276,720; and in U.S. Patent Application Publication Nos. US20090061439; US20080087826; US20060286566; US2006002471 1; US20060024678; US20080213770; and US20080103058; 3) Applied Biosystems (e.g. SOLiD sequencing); 4) Dover Systems (e.g., Polonator G.007 sequencing); 5) Illumina, Inc. as described in US Patent Nos. 5,750,341; 6,306,597; and 5,969,1 19; and 6) Pacific Biosciences as described in US Patent Nos. 7,462,452; 7,476,504; 7,405,281; 7,170,050; 7,462,468; 7,476,503; 7,315,019; 7,302,146; 7,313,308; and US Application Publication Nos. US20090029385; US20090068655; US20090024331; and US20080206764. Such methods and apparatuses are provided here by way of example and are not intended to be limiting.

[0456] The term “barcode” refers to a nucleic acid sequence that is unique to a particular system. The barcode may be unique to a particular method or to a particular effector. The nucleic acid encodings of the methods and systems provided herein are analogous to barcodes in that they are unique nucleic acid sequences that can be used to identify the structure of a given effector. The length of a barcode or nucleic acid encoding should be sufficient to differentiate between all the effectors in a given library.

[0457] The term “flow” means any movement of liquid or solid through a device or in a method of the disclosure, and encompasses without limitation any fluid stream, and any material moving with, within or against the stream, whether or not the material is carried by the stream. For example, the movement of molecules, cells or virions through a device or in a method of the disclosure, e.g. through channels of a microfluidic chip of the disclosure, comprises a flow. This is so, according to the disclosure, whether or not the molecules, cells or virions are carried by a stream of fluid also comprising a flow, or whether the molecules, cells or virions are caused to move by some other direct or indirect force or motivation, and whether or not the nature of any motivating force is known or understood. The application of any force may be used to provide a flow, including without limitation, pressure, capillary action, electro-osmosis, electrophoresis, dielectrophoresis, optical tweezers, and combinations thereof, without regard for any particular theory or mechanism of action, so long as molecules, cells or virions are directed for detection, measurement or sorting according to the disclosure.

[0458] An “inlet region” is an area of a microfabricated chip that receives molecules, cells or virions for detection measurement or sorting. The inlet region may contain an inlet channel, a well or reservoir, an opening, and other features which facilitate the entry of molecules, cells or virions into the device. A chip may contain more than one inlet region if desired. The inlet region is in fluid communication with the main channel and is upstream therefrom.

[0459] An “outlet region” is an area of a microfabricated chip that collects or dispenses molecules, cells or virions after detection, measurement or sorting. An outlet region is downstream from a discrimination region and may contain branch channels or outlet channels. A chip may contain more than one outlet region if desired.

[0460] An “analysis unit” is a microfabricated substrate, e.g., a microfabricated chip, having at least one inlet region, at least one main channel, at least one detection region and at least one outlet region. Sorting embodiments of the analysis unit include a discrimination region and/or a branch point, e.g., downstream of the detection region, that forms at least two branch channels and two outlet regions. A device according to the disclosure may comprise a plurality of analysis units.

[0461] A “main channel” is a channel of the chip of the disclosure which permits the flow of molecules, cells or virions past a detection region for detection (identification), measurement, or sorting. In a chip designed for sorting, the main channel also comprises a discrimination region. The detection and discrimination regions can be placed or fabricated into the main channel. The main channel is typically in fluid communication with an inlet channel or inlet region, which permits the flow of molecules, cells or virions into the main channel. The main channel is also typically in fluid communication with an outlet region and optionally with branch channels, each of which may have an outlet channel or waste channel. These channels permit the flow of cells out of the main channel.

[0462] A “detection region” is a location within the chip, typically within the main channel where molecules, cells or virions to be identified, measured or sorted on the basis of a predetermined characteristic. In an embodiment, molecules, cells or virions are examined one at a time, and the characteristic is detected or measured optically, for example, by testing for the presence or amount of a reporter. For example, the detection region is in communication with one or more microscopes, diodes, light stimulating devices, (e.g., lasers), photo multiplier tubes, and processors (e.g., computers and software), and combinations thereof, which cooperate to detect a signal representative of a characteristic, marker, or reporter, and to determine and direct the measurement or the sorting action at the discrimination region. In sorting embodiments, the detection region is in fluid communication with a discrimination region and is at, proximate to, or upstream of the discrimination region. [0463] A “carrier fluid,” “immiscible fluid,” or “immiscible carrier fluid” or similar term as used herein refers to a liquid in which a sample or assay liquid is incapable of mixing and allows formation of droplets of the sample or assay liquid within the carrier fluid. These terms are used interchangeable herein and are meant to encompass the same materials. Nonlimiting examples of such carrier fluids include silicon-based oils, silicone oils, hydrophobic oils (e.g. squalene, fluorinated oils, perfluorinated oils), or any fluid capable of encapsulating another desired liquid containing a sample to be analyzed.

[0464] An “extrusion region,” “droplet extrusion region,” or “droplet formation region” is a junction between an inlet region and the main channel of a chip of the disclosure, which permits the introduction of a pressurized fluid to the main channel at an angle perpendicular to the flow of fluid in the main channel. In some embodiments, the fluid introduced to the main channel through the extrusion region is “incompatible” (i.e., immiscible) with the fluid in the main channel so that droplets of the fluid introduced through the extrusion region are sheared off into the stream of fluid in the main channel.

[0465] A “discrimination region” or “branch point” is a junction of a channel where the flow of molecules, cells or virions can change direction to enter one or more other channels, e.g., a branch channel, depending on a signal received in connection with an examination in the detection region. Typically, a discrimination region is monitored and/or under the control of a detection region, and therefore a discrimination region may “correspond” to such detection region. The discrimination region is in communication with and is influenced by one or more sorting techniques or flow control systems, e.g., electric, electro-osmotic, (micro-) valve, etc. A flow control system can employ a variety of sorting techniques to change or direct the flow of molecules, cells or virions into a predetermined branch channel. [0466] A “branch channel” is a channel which is in communication with a discrimination region and a main channel. Typically, a branch channel receives molecules, cells or virions depending on the molecule, cell or virion characteristic of interest as detected by the detection region and sorted at the discrimination region. A branch channel may be in communication with other channels to permit additional sorting. Alternatively, a branch channel may also have an outlet region and/or terminate with a well or reservoir to allow collection or disposal of the molecules, cells or virions.

[0467] The term “forward sorting” or flow describes a one-direction flow of molecules, cells or virions, typically from an inlet region (upstream) to an outlet region (downstream), and in some instances without a change in direction, e.g., opposing the “forward” flow. In some embodiments, molecules, cells or virions travel forward in a linear fashion, i.e., in single file. A “forward” sorting algorithm consists of running molecules, cells or virions from the input channel to the waste channel, until a molecule, cell or virion is identified to have an optically detectable signal (e.g. fluorescence) that is above a pre-set threshold, at which point voltages are temporarily changed to electro-osmotically divert the molecule or to the collection channel. [0468] The term “reversible sorting” or flow describes a movement or flow that can change, i.e., reverse direction, for example, from a forward direction to an opposing backwards direction. Stated another way, reversible sorting permits a change in the direction of flow from a downstream to an upstream direction. This may be useful for more accurate sorting, for example, by allowing for confirmation of a sorting decision, selection of particular branch channel, or to correct an improperly selected channel.

[0469] Different “sorting algorithms” for sorting in the microfluidic device can be implemented by different programs, for example under the control of a personal computer. As an example, consider a pressure-switched scheme instead of electro-osmotic flow. Electro-osmotic switching is virtually instantaneous and throughput is limited by the highest voltage that can be applied to the sorter (which also affects the run time through ion depletion effects). A pressure switched-scheme does not require high voltages and is more robust for longer runs. However, mechanical compliance in the system is likely to cause the fluid switching speed to become rate-limiting with the “forward” sorting program. Since the fluid is at low Reynolds number and is completely reversible, when trying to separate rare molecules, cells or virions, one can implement a sorting algorithm that is not limited by the intrinsic switching speed of the device. The molecules, cells or virions flow at the highest possible static (non-switching) speed from the input to the waste. When an interesting molecule, cell or virion is detected, the flow is stopped. By the time the flow stops, the molecule, cell or virion may be past the junction and part way down the waste channel. The system is then run backwards at a slow (switchable) speed from waste to input, and the molecule, cell or virion is switched to the collection channel when it passes through the detection region. At that point, the molecule, cell or virion is “saved” and the device can be run at high speed in the forward direction again. Similarly, a device of the disclosure that is used for analysis, without sorting, can be run in reverse to re-read or verify the detection or analysis made for one or more molecules, cells or virions in the detection region. This “reversible” analysis or sorting method is not possible with standard gel electrophoresis technologies (for molecules) nor with conventional FACS machines (for cells). Reversible algorithms are particularly useful for collecting rare molecules, cells or virions or making multiple time course measurements of a molecule or single cell.

[0470] The term “emulsion” refers to a preparation of one liquid distributed in small globules (also referred to herein as drops or droplets) in the body of a second liquid. The first liquid, which is dispersed in globules, is referred to as the discontinuous phase, whereas the second liquid is referred to as the continuous phase or the dispersion medium. In one embodiment, the continuous phase is an aqueous solution and the discontinuous phase is a hydrophobic fluid, such as an oil (e.g., decane, tetradecane, or hexadecane). Such an emulsion is referred to here as an oil in water emulsion. In another embodiment, an emulsion may be a water in oil emulsion. In such an embodiment, the discontinuous phase is an aqueous solution and the continuous phase is a hydrophobic fluid such as an oil. The droplets or globules of oil in an oil in water emulsion are also referred to herein as “micelles”, whereas globules of water in a water in oil emulsion may be referred to as “reverse micelles”.

[0471] As used herein, the term “about” a number refers to that number plus or minus 10% of that number. The term “about” a range refers to that range minus 10% of its lowest value and plus 10% of its greatest value.

[0472] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.