CLAIM OF PRIORITY

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63/283,082, titled “METHODS AND APPARATUSES FOR SCALING UP CELL-FREE SYNTHESIS REACTIONS,” filed on November 24, 2021, which is herein incorporated by reference in its entirety.

BACKGROUND

[0002] In many enzymatic cell-free synthesis reactions, product yields drop as reaction volumes increase. For example, the specific yield of amplified DNA drops as the size of a polymerase chain reaction (PCR) increases. This is also true in cell-free protein expression reactions where the specific yield decreases dramatically with increased reaction volumes. Increasing the surface-to-volume ratio of cell-free enzymatic reactions can result in higher productivities. For example, the yield of PCR reactions is improved when the reaction is dispersed in small hydrophobic enclosures (e.g. emulsions). Thin-film reactions have also been validated as a viable solution for scaling up cell-free enzymatic reactions. For cell- free protein expression, thin-film reactions are particularly effective when the reaction’s surface interfaces with a hydrophobic material or with a gas such as air.

[0003] Current thin-film reaction formats are not ideal for sample mixing and collection. It may be particularly challenging to keep film thickness consistent to enable sufficient gas exchange (e.g. of oxygen), while providing strength and support. It would be beneficial to resolve these shortcomings in order to expand the applicability of the thin-film reaction format.

SUMMARY OF THE DISCLOSURE

[0004] The methods and apparatuses (e.g., systems, devices, etc.) described herein may generally relate to the production of biomolecular products using cell-free systems. For example, described herein are thin-film reaction cartridges that can be used with centrifugal or pneumatic systems for sample loading and collection. In some examples, the cartridges may include a semi- permeable membrane enabling gas exchange, while minimizing evaporation and the risk of contamination.

[0005] For example, described herein are cartridges for cell-free enzymatic reaction. In some examples, the cartridge comprises: an inlet; a narrow ant thin reaction chamber in fluid communication with the inlet, the narrow ant thin reaction chamber having a first major surface that is formed of a gas-exchange hydrophobic membrane and is separated from a second major surface by a thickness, t. wherein the first major surface has a height, h, and a width, w, wherein either or both the height and width of the first major surface are more than five times the thickness.

[0006] For example, a cartridge for cell-free enzymatic reaction may include: an inlet; a narrow ant thin reaction chamber in fluid communication with the inlet, the narrow ant thin reaction chamber having a first major surface and a second major surface that are opposite from each other and separated by a thickness, t, wherein the first and second major surfaces each comprise a gas-exchange hydrophobic membrane, further wherein the first major surface has a height, h, and a width, w, wherein either or both the height and width of the first major surface are more than five times the thickness.

[0007] In some examples a cartridge for cell-free enzymatic reaction includes: an inlet; and a narrow ant thin reaction chamber in fluid communication with the inlet, the narrow ant thin reaction chamber having a first major surface and a second major surface that are opposite from each other and separated by a thickness, t, wherein the first and second major surfaces each comprise a gas-exchange hydrophobic membrane, further wherein the first major surface has a height, h, and a width, w, wherein either or both the height and width of the first major surface are more than five times the thickness, further wherein cartridge does not include an outlet, but the cartridge is configured so that extraction occurs at least partially through the first gasexchange hydrophobic membrane acting as a pressure-responsive valve.

[0008] The second major surface may comprise a second gas-exchange hydrophobic membrane. The first and second major surfaces of the narrow ant thin reaction chambers may comprise hydrophobic membranes that comprise a semi-permeable gas-exchange material. Either the first or second gas-exchange hydrophobic membranes may be made at least in part from a fluoropolymer. For example, the fluoropolymer may be one or both of: PTFE (Polytetrafluoroethylene (PTFE) and Fluorinated ethylene propylene (FEP). In some examples the fluoropolymer comprises 12.5 micron FEP.

[0009] In some examples the cartridge includes an outlet. Alternatively and potentially advantageously, the narrow ant thin reaction chamber does not include an outlet; for example, the cartridge may be configured so that extraction occurs at least partially through the first gasexchange hydrophobic membrane acting as a pressure-responsive valve.

[0010] In any of these cartridges, the inlet may comprise a slot having an enlarged key-hole opening configured for positioning and aligning a pipette tip. In any of these cartridges, the narrow ant thin reaction chamber may comprise a porous fill material.

[0011] In some examples, the outlet includes a connector configured to connect to a source of positive or negative air pressure. [0012] Any of these devices may include an enclosure configured to seal over the cartridge for controlling or maintaining humidity.

[0013] Also described herein are methods of using and methods of making any of these apparatuses (e.g., cartridges). For example, a method for cell-free enzymatic production of a molecule within a cartridge may include: reacting components for cell-free enzymatic production of the molecule within a reaction chamber of the cartridge, wherein the reaction chamber is substantially narrow ant thin and includes at least one major surface comprising a hydrophobic, gas-exchange membrane that is configured to increase surface-to-volume ratio within the reaction chamber during synthesis; and applying a force greater than a minimum threshold to extract the molecule out of the reaction chamber, where the molecule is held within an aqueous environment within the reaction chamber until the applied force exceeds the minimum force.

[0014] As mentioned above, a hydrophobic, gas-exchange membrane may be a semi- permeable gas-exchange material. The hydrophobic, gas-exchange membrane may be made at least in part from a fluoropolymer. For example, the fluoropolymer may be selected from FEP or PTFE or a combination of both. In some examples extraction occurs through the hydrophobic, gas-exchange membrane acting as a pressure-responsive valve. Any of these methods may include extracting the molecule through the hydrophobic gas-exchange membrane acting as a pressure-responsive valve. For example, the extraction may occur through a hydrophobic porous membrane acting as a pressure-sensitive valve. In some examples the extraction occurs through a hydrophobic semi-permeable gas-exchange membrane. In other examples, the extraction may occur through a separate hydrophobic porous frit acting as a pres sure- sensitive valve.

[0015] The enzymatic reaction may be a cell-free protein expression reaction. One or more additional containers may be used for controlling or maintaining humidity.

[0016] All of the methods and apparatuses described herein, in any combination, are herein contemplated and can be used to achieve the benefits as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] A better understanding of the features and advantages of the methods and apparatuses described herein will be obtained by reference to the following detailed description that sets forth illustrative embodiments, and the accompanying drawings of which:

[0018] FIG. 1 illustrates an image of a protein (deGFP, a truncated version of the green fluorescent protein), expressed in three cell-free protein expression reaction volumes carried out in 1.5 mL polypropylene tubes and analyzed using a 12% SDS-PAGE Coomassie-stained gel. [0019] FIG. 2 is an example of specific yield comparisons among cell-free protein expression reactions carried out in 1.5 mL polypropylene tube at various reaction volumes. [0020] FIGS. 3 A and 3B schematically illustrate two conventional thin-film reaction formats used for cell-free reactions.

[0021] FIG. 4 is a graph showing an example of cell-free protein expression partial yield recovery by running a scaled-up thin film reaction atop a petri dish.

[0022] FIGS. 5A and 5B show two examples of deGFP expression in 150 pL and 500 pL oxygen-limited cell-free protein expression reactions, and the resulting color gradients caused by variation in oxygen availability.

[0023] FIGS. 6 A and 6B show an example of a thin-film reaction cartridge with two impermeable layers sandwiching the reaction contents, and a connector for extracting the contents using pneumatic means. A hydrophobic frit is used to contain the reaction during synthesis, and to enable sample extraction with adjustment of pressure differential. FIG. 6B shows a partial section through the cartridge of FIG. 6A.

[0024] FIG. 7 is a graph showing examples of low specific yields for reactions requiring exchange of gases carried out in thin-film reaction formats sandwiched between impermeable layers as compared to 15 pL reactions carried out in 1.5 mL polypropylene tubes.

[0025] FIG. 8 demonstrates various cartridges made to test specific yields of reactions carried out in resin-air, resin-membrane and membrane-membrane formats.

[0026] FIG. 9 is a graph showing specific yields of 50 pL reactions carried out in resin-air, resin-membrane and membrane-membrane formats compared to specific yield of 15 pL reactions carried out in 1.5 mL polypropylene tubes.

[0027] FIG. 10 shows an example of a membrane-based thin-film reaction cartridge designed to enable sample collection using centrifugal force (e.g. as a spin column) or using a pneumatic system (e.g. a vacuum manifold or a dedicated device).

[0028] FIG. 11 shows an example of a membrane-based thin-film reaction cartridge designed to enable sample collection using centrifugal force (e.g. as a spin column) or using a pneumatic system. FIG. 11 shows the cartridge attached to a pneumatic device for sample extraction.

[0029] FIG. 12 shows an example of a membrane-based thin-film reaction cartridge, where the membrane can act as a pressure-responsive valve.

[0030] FIG. 13 illustrates one example of a method of protein purification within membranebased cartridges.

[0031] FIG. 14 is a graph showing the results of an example purification run using a membrane-based cartridge starting from a cell-free reaction mixture.

[0032] FIG. 15 schematically illustrates one example of a method of cell-free synthesis within a membrane-based cartridge and collection of the resulting mixture. [0033] FIG. 16 schematically illustrates a number of features that may be included in any of the cartridges described herein.

[0034] FIG. 17 schematically illustrates additional features that may be included in a cartridge as described herein.

[0035] FIG. 18A shows examples of cartridges made with PTFE (Polytetrafluoroethylene) and FEP (Fluorinated ethylene propylene) and also visualizes the samples from resulting cell- free deGFP expression reactions in each cartridge.

[0036] FIG. 18B shows an example cartridge within an incub ation/collection tube.

[0037] FIG. 19 is a graph of the specific yields of 500 pL cell-free reactions carried out in a variety of conditions and cartridge formats.

[0038] FIG. 20 shows a specific yield comparison between a 15 pL reaction carried out in a 1.5 mL polypropylene tube and one carried out in a thin-film membrane-based cartridge as described herein. The resulting mixtures were analyzed on a 12% SDS-PAGE Coomassie- stained gel.

[0039] FIG. 21 is a graph showing consistent specific yields across various reaction volumes inside membrane-based cartridges as described herein. Specific yields of 100, 200 and 300 pL reactions were compared with those of 15 pL reactions carried out in a 1.5 mL polypropylene tubes.

[0040] FIG. 22 shows an example of a modified open format system that replaces the impermeable layer with a semi-permeable gas exchange membrane. This example also demonstrates the use of a snap lid to prevent excessive evaporation.

[0041] FIG. 23 shows an example of a hydrophobic semi-permeable gas exchange membrane that may be used to construct a multi-well plate capable of gas exchange as described herein.

[0042] FIG. 24 schematically illustrates an example of a cartridge as described herein.

[0043] FIGS. 25A-25C schematically illustrate an example of a cartridge as described herein. FIG. 25A shows a front view, FIG. 25B shows a side view and FIG. 25C shows a top view.

[0044] FIGS. 26A-26C schematically illustrate an example of a cartridge as described herein that does not include a discrete outlet. FIG. 26A shows a front view, FIG. 26B shows a side view and FIG. 26C shows a top view.

[0045] FIGS. 27A-27C schematically illustrate an example of a cartridge as described herein that includes a discrete outlet. FIG. 27A shows a front view, FIG. 27B shows a side view and FIG. 27C shows a top view. DETAILED DESCRIPTION

[0046] The methods and apparatuses described herein may allow scaling up enzymatic cell- free reactions and may include devices and apparatuses for liquid handling and collection of samples in thin-film reactions during expression and purification. In many enzymatic cell-free synthesis reactions, increasing reaction volume is often accompanied by a decrease in specific yield. For example, DNA yields drop as the size of polymerase chain reaction (PCR) mixtures increase. This is also true in cell-free protein expression reactions where the specific yield decreases dramatically with increased reaction volumes. FIG. 1 demonstrates an example of the reduction in yield when scaling up. In FIG. 1, an example protein (e.g., deGFP, a truncated version of the green fluorescent protein) was expressed in three cell-free protein expression reactions carried out in 1.5 mL polypropylene tubes, and the resulting mixtures were analyzed using a 12% SDS-PAGE Coomassie- stained gel. Band intensities for deGFP decreased as the reaction volumes increased.

[0047] The graph shown in FIG. 2 demonstrates the reduction in yield of mature deGFP as reaction volumes increase. The protein deGFP was expressed in cell-free protein expression reactions with increasing volumes inside 1.5mL polypropylene tubes and the resulting mixtures were analyzed for fluorescence (excitation: 488nm, emission: 507nm). A reduction in fluorescence intensity was observed with increased reaction volumes indicating a lower amount of mature deGFP produced in the cell-free enzymatic reactions. Although this is a well-known problem, it has proven difficult to address in a cost-effective and easy to use manner.

[0048] Increasing the surface-to-volume ratio of many cell-free enzymatic reactions can positively affect synthesis yield of target molecules within the reactions. This may be achieved, for example, using a thin-film reaction format. FIGS. 3A-3B illustrate examples of conventional thin-film reaction formats. FIG. 3A shows an example of a simple form (e.g., an “open format”), in which a reaction 302 is placed atop an impermeable membrane 301 (e.g. a plastic petri dish) and is exposed to surrounding gases (e.g. air) 303. Alternatively, as shown in FIG. 3B, the reaction 302 is sandwiched between two impermeable layers (the “closed format”) 301, 301’. The open format enables gas exchange, as may be required by some enzymatic cell-free reactions (e.g., cell-free protein expression systems relying on oxidative phosphorylation or synthesis of fluorescent proteins such as GFP requiring oxygen for maturation). However, this format is prone to contamination due to its open nature and can suffer from high evaporation rates. The open format also lends itself to pooling of samples in irregular forms, which can make the results inconsistent. The closed format, on the other hand, enables tight control over surface- to-volume ratios, but is unable to provide for sufficient exchange of gases. From a usability perspective, conventional thin-film formats are not well-suited for sample collection and handling and may result in significant sample loss due to evaporation or adherence to the impermeable layer.

[0049] For many enzymatic cell-free reactions, surface interactions may improve when the interface is a hydrophobic surface or a gas. For example, polymerase chain reactions (PCR) may improve in yield when dispersed in small hydrophobic enclosures that prevent DNA template adsorption. This may also be the case in cell-free protein synthesis reactions, where setting up the reaction atop a hydrophobic surface such as a petri dish can help recover at least some of the yield loss resulting from scale-up. The graph shown in FIG. 4 illustrates an improvement in cell- free deGFP expression yield for a reaction carried out on a petri dish in a humidity chamber (container). As shown, cell-free synthesis on the petri dish significantly increases the yield of mature deGFP compared to reactions carried out in 1.5 mL polypropylene tubes at similar volumes (e.g., compare with FIG. 2).

[0050] For cell-free protein expression reactions not requiring exchange of gases such as oxygen, it is also possible to sandwich the reaction between two impermeable layers to increase the surface area in contact with a hydrophobic surface, similar to FIG. 3B. While this set up can provide good control over surface-to-volume ratios, it is ill-suited for reactions requiring exchange of gases such as cell-free protein expression reactions relying on oxidative phosphorylation or for synthesis of fluorescent proteins requiring oxygen for maturation. FIGS. 5A-5B demonstrate cell-free expression of deGFP at larger scales (150 pL, FIG. 5A, and 500 pL, FIG. 5B). In FIGS. 5A-5B, the cell-free reactions resulted in a color gradient that indicates that the reactions may be oxygen-limited where the reaction components are placed further away from the gas interface; the color from deGFP is concentrated at the surface 510 of the fluid within the tube, rather than deeper into the fluid volume 511, indicating either lower expression yield or lower deGFP maturation levels.

[0051] Conventional thin-film formats (e.g. cell-free protein expression atop a petri dish) also results in excessive evaporation, sample loss due to surface adhesion and inconsistent yields due to irregular pooling of the reaction mixture. As such, alternate methods and apparatuses are needed to overcome these challenges.

[0052] The cartridges described herein are configured to enable extraction via centrifugation and/or pressure differential at very high efficiency. FIG. 6A illustrates one such example, including a reaction chamber 615 formed as part of a hydrophobic body 607 that is sandwiched between two hydrophobic acrylic membranes. The device includes a hydrophobic frit 609 at the end to prevent unintentional loss of fluid and/or evaporation, as well as a connector 611 (e.g., Luer lock connector). Two versions of this type of cartridge were built, setting the distance between the two hydrophobic walls at 0.75 mm and 1 mm. In some examples, the cartridges may contain a porous material with a known liquid entry pressure to enable containment of the sample during the reaction and further collection of the sample by adjusting centrifugal force or pressure differential. The exemplary device shown in FIG. 6A allowed for efficient sample collection, however it was not optimized for synthesis reactions requiring oxygen, such as cell- free synthesis of mature deGFP. 250 pL cell-free protein expression reactions with template DNA encoding for deGFP were used to test this. FIG. 7 shows the fluorescence measurement results indicating that the specific yield of mature deGFP made in this form factor is significantly lower than that of those carried out in 15 pL reactions in 1.5 mL polypropylene tubes. Thus in some examples a cartridge as described herein may include an air (e.g., oxygen) permeable surface for one or both sides of a relatively narrow ant thin reaction chamber. In general, as used herein a narrow and thin reaction chamber may be substantially flat. For example, a narrow and thin reaction chamber may be configured so that the distance between the opposing major sides of the reaction chamber are separated by a distance (thickness, t) that is much, much less than the height or width. For example, the spacing between the opposing major sides may be smaller than 0.5 mm, 1 mm, 1.5 mm, 2 mm, 2.5 mm, 3 mm, 4 mm, 5 mm, 10 mm, 20 mm, etc.

[0053] In any of these examples of cartridges described herein the reaction chambers may be shaped as generally flat structures, forming the narrow and thin reaction chamber having the gas- permeable (e.g., air-permeable) porous hydrophobic membrane on one or more sides. The reaction chambers may be square, rectangular, etc. with the two major sides (e.g., formed at least in part by one or more gas-permeable (e.g., air-permeable) porous hydrophobic membranes) approximately parallel from each other. In some examples the reaction chamber(s) may have curved walls, including cylindrical walls (e.g., tubes). In some examples the reaction chamber(s) may be bent or curved. More than one reaction chamber(s) or more than one sections of reaction chambers may be included as part of a cartridge. For example multiple reaction chamber may be arranged as a stack, with air gaps between them.

[0054] For example, FIG. 8 illustrates different versions of small-scale cartridges configured to contain, e.g., 50-60 pL of cell-free reactions. These cartridges may include a relatively flattened profile, in which all, or a portion, of one or both sides is formed of a gas-permeable (e.g., air-permeable) porous hydrophobic membrane forming the reaction chamber. The apparatus may include an inlet and an outlet. In some examples the outlet and/or inlet of the cartridge may be plugged (e.g., with silicone tubing 831) to prevent accidental drainage and to reduce or prevent evaporation. In any of these examples, a hydrophobic permeable or semi- permeable gas exchange membrane may be used to provide the necessary surface area enabling significant gas exchange. The use of such membranes may also protect the sample from contamination and can also limit the rate of evaporation during and after the reaction. For example, FIG. 8 illustrates examples in which PTFE semi-permeable gas exchange membranes provide a hydrophobic interface while enabling gas exchange.

[0055] As shown in FIG. 8, experiments with three thin-film cartridge designs were tested against 15 pL and 50 pL reactions carried out in a 1.5 mL polypropylene tubes. These designs included: resin-air, resin-membrane and membrane-membrane. The resin was made out of a hydrophobic polymer material. The membranes were made out of porous PTFE material and were attached to the cartridges using an adhesive (e.g., adhesive layer). The total reaction size in each cartridge of these examples was 50 pL (as described herein, larger or smaller sizes may be used). FIG. 9 summarizes the results for one such experiment using these prototype cartridge devices. After controlling for evaporation, all three set-ups showed at least some yield recovery when compared to reactions carried out in 1.5 mL polypropylene tubes. However, the membrane-based cartridges were significantly easier to handle due to a lower risk of spillage and contamination. They also demonstrated reduced evaporation rates, while providing sufficient gas exchange for maturation of deGFP.

[0056] In some examples cartridges were designed to enable sample collection using centrifugal force (e.g. as a spin column or part of a spin column) or using pneumatic systems (e.g., through which positive or negative pressure may be applied). FIG. 10 illustrates one example of a cartridge apparatus configured for use with pneumatic pressure, including a flat reaction chamber having at least one side (though in some example both flat sides are) formed of a porous (or semi-porous) gas-exchange hydrophobic membrane membranes (e.g., a gasexchange, hydrophobic, porous membrane). In some examples, the apparatus (e.g., cartridge) may be configured to function either as spin columns or as cartridges capable of being connected to a pneumatic device (e.g. through a Luer connection), or both. In some examples, cartridges may include a hydrophobic porous material (UHMWPE or PTFE), e.g., membrane, that retains the reaction components in the cartridge during expression, while also permitting extraction of the contents using centrifugation or pneumatic force. For example, in some cases the inlet and/or outlet may include a hydrophobic frit that may retain the sample fluid until sufficient force (pneumatic force or centrifugation force) is applied.

[0057] In some examples, the cartridge may include one or more porous hydrophobic membrane that enables exchange of gases, e.g., air, while providing a hydrophobic surface in a thin-film reaction format (see e.g., FIG. 11). In any of these examples the cartridge may be housed in an enclosure (e.g. a tube, including a capped tube) to further reduce evaporation. In some examples the air flow within the enclosure may be controlled to facilitate further exchange of gases. For example, an inlet (e.g., hole, pinhole, etc.) may be included in the enclosure and/or the cap of the enclosure (e.g., tube) and/or an electronic pump may control the transfer of gases through the enclosure and/or the cartridge. Thus, some of the cartridges described herein may be configured to include or direct airflow, including airflow across the one or more hydrophobic, gas-exchange, membranes (e.g., porous or semi-porous membranes). In any of these examples, a humidification chamber (container) may be used as either part of the cartridge or a device to control the rate of evaporation from the cartridge. In FIG. 11, the device 1100 includes flat reaction chamber 1115 having one or more walls formed of a hydrophobic semi-permeable gas exchange material 1135. The base of the device includes an outlet including a frit containing a hydrophobic porous material 1109 and a connector shown connected to a pneumatic device 1133.

[0058] In some examples, such as those shown in FIG. 12, the cartridges may be configured such that the hydrophobic gas exchange membrane also acts as a pressure-responsive valve, which may remove the need or benefit for a different porous material for this purpose. This configuration may minimize sample loss and dead volume that may occur by using an additional frit. In the example shown in FIG. 12, an inlet may be included for the application of material into the flat reaction chamber, but a dedicated outlet (with or without a frit) is not included. Instead, pressure and/or force (centrifugal force) may be applied to drive reaction product, or fluid containing the reaction product, through the porous or semi-porous membrane. Alternatively or additionally, the reaction chamber may include one or more porous filling materials that may retain the fluid, reactant(s) and product(s) within the reaction chamber until pressure and/or force is applied. In some examples the bottom of the reaction chamber may include an opening.

[0059] In some examples purification may be done directly within the cartridges. For example, binding, wash and elution steps may be carried out within the expression cartridge, including within the reaction chamber. FIG. 13 illustrates one example of a method that includes purification using a cartridge as described herein. In FIG. 13 chromatography beads are used to purify a product. To illustrate this capability, cell-free expressed SxHistidine-tagged deGFP was formed 1301 within a cartridge inserted into a container (e.g., tube) as shown to the left in FIG. 13. Purification substrate (e.g., purification beads) were added 1303, and the product was purified in the cartridge, which is configured as a 15 mL spin column cartridge in this example. The purification substrate is retained within the flat reaction chamber by the gas-exchange, hydrophobic, semi-permeable membrane, though fluid may be pulled through the porous/semi- porous gas-exchange hydrophobic membrane, when force and/or pressure is applied, e.g., by centrifugation. The purification beads may include, for example, Nickel-NTA beads. After adding the purification substrate, the material may be mixed (by vortexing, inverting, shaking and/or pipetting), and all or some of the liquid may be removed by centrifugation, leaving the substrate with bound product (e.g., protein). The substrate material including the bound product was then washed 1305 with wash buffer, and mixed (e.g., by vortexing, inverting, shaking and/or pipetting). The wash fluid may be removed through the gas-exchange, hydrophobic, semi- permeable membrane by centrifugation (“flow through”). Finally, the protein product material may be eluted from the substrate by adding elution buffer and mixing 1307, to release the product material. The container (e.g., tube) may be swapped for a new tube to avoid impurities. Samples were collected during the flow-through, wash and elution steps and fluorescence was measured. FIG. 14 shows an example of fluorescence measurements during each step of a method such as that shown in FIG. 13, indicating successful purification of 6xHistidine-tagged deGFP within the cartridge format.

[0060] In some examples, the cartridges may be configured for use as spin columns. FIG. 15 illustrates an example of a cartridge configured as a spin column and an example of a method for using such a cartridge. The spin-column format can further reduce evaporation during synthesis, since the reaction can be enclosed by simply capping the tube during and after the reaction. Using this format, only small amounts of evaporation were observed. Fluid 1540 (e.g., reaction components) may be injected or inserted into the flat reaction chamber of the cartridge 1500, and the cartridge may be inserted into the mouth of a tube 1504 forming the container. The lid of the container 1504 may be closed over the cartridge, securing it at the top of the tube, so that the flat reaction chamber 1515 is suspended above the bottom of the container, separated by a space 1540. Following centrifugation, fluid 1540’ passing through the cartridge may be collected at the bottom of the container.

[0061] In any of these examples a permeable or semi-permeable hydrophobic gas-exchange membrane may be used to form one or more walls of the reaction chamber. The flat reaction chamber may be configured as a reactors having a low reaction thickness to allow for facile exchange of gases. In some examples the spacing between the major (large) walls of the reaction chamber may have an approximately fixed separation, giving the reaction chamber a constant thickness that may be maintained throughout the length of the reaction chamber (in at least one dimension) to provide similar surface-to-volume ratios regardless of the reaction volume. In some examples liquid entry pressure of the hydrophobic retaining material may be specifically selected so that the reaction components are retained at low pressures/centrifugal forces and can be collected at higher pressures/centrifugal forces. For example, the dimensions and materials (including material thicknesses), and in particular the gas-exchange, hydrophobic, semi- permeable membrane, may be configured so that aqueous fluid is retained in the flat reaction chamber unless the force (e.g., pressure and/or force due to centrifugation) exceeds a minimum threshold, which may be set by the applied pressure and/or centrifugation (in centrifugal force, xg, e.g., 1000 xg, 2000 xg, 3000 xg, 4000 xg, 5000 xg, 6000 xg, 7000 xg, 8000 xg, etc.). FIG. 16 illustrates some such examples.

[0062] FIG. 16 (left) schematically illustrates an example of a section through a flat reaction chamber 1615 of a cartridge 1600 as described herein. In this example both major walls of the reaction chamber are formed of a semi-permeable gas-exchange membrane 1642 that are separated from each other by a narrow thickness, t, compared to the width, w, and length, I, of the reaction chamber 1615.

[0063] In some examples, cartridges may be configured to facilitate sample loading and mixing. For example, any of the cartridges described herein may include an inlet having a slot with an enlarged key-hole opening configured for positioning and aligning a pipette tip for sample loading so that all or the majority of the material to be inserted ends up in the flat reaction chamber (e.g., the thin/flat chamber forming the flat reaction chamber). Furthermore, any of these cartridges may include a reservoir at the top the cartridge to enable loading of larger amounts of material, e.g., buffer (e.g. for washing, purification, etc.) and to catch any sample overflow from the thin/flat reaction chamber during mixing and loading. For example, FIG. 17 illustrates examples of cartridges including some of these features. In any of these cartridges, the flat reaction chamber may be configured so that the ratio of the width, w, to the thickness, t, of the reaction chamber formed by the one or more gas-exchange, hydrophobic, porous/semi- porous membranes is 4 or greater, 5 or greater, 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 or greater, 11 or greater, 15 or greater, 20 or greater, 25 or greater, 30 or greater, etc. (e.g., the width may be 5 times greater than the thickness, 10 times greater than the thickness, 15 times greater than the thickness, 30 times greater than the thickness, etc.).

[0064] Any appropriate gas-exchange, hydrophobic, membrane (porous/semi-porous) may be used. For example, a porous gas exchange membrane may be made of a fluoropolymer. In some such examples, the fluoropolymer is a porous PTFE (Polytetrafluoroethylene) or FEP (Fluorinated Ethylene Propylene). FEP has particularly high selectivity for oxygen over water vapor. However, even at low thicknesses (e.g. 12.5 microns), the oxygen permeability can be lower than that of porous PTFE. FEP also has notable optical clarity, enabling the user to monitor the sample visually or through automated optical means. FIGS. 18A-B illustrate cartridges made with PTFE and FEP and also shows examples of samples of cell-free deGFP expression reactions carried out in each cartridge. For comparison, a 15 uL reaction volume performed in a tube (Eppendorf tube) is shown on the left. The use of an FEP membrane (e.g., a 12.5 micron FEP membrane) significantly reduced the rate of evaporation and was commensurate with a large recovery of specific yield. After controlling for evaporation, PTFE film outperformed the FEP film, likely due to higher gas exchange rates, though either material may be used. FIG. 18B shows an example of a cartridge within an enclosure configured to hold the cartridge at the top of the closed enclosure (e.g., tube).

[0065] To demonstrate the efficacy of some of the example cartridges described herein, 500 pL cell-free deGFP expression reactions were carried out in a variety of conditions and cartridge formats. In these experiments, the use of semi-permeable gas exchange membranes in the thin- film format significantly increased yield over other reaction set-ups. FIG. 19 demonstrates fluorescence measurements for these reactions after 6 hours of incubation. The resulting specific yield from one of the 500 pL cartridge runs was also compared with a sample from a 15 pL reaction carried out in a 1.5 mL polypropylene tube. Samples were analyzed on a 12% SDS- PAGE Coomassie- stained gel. Band intensities for deGFP were similar between the two reaction formats (see, e.g., FIG. 20). As shown, examples having a pair of porous (or semi-porous) gasexchange hydrophobic membrane membranes both formed of PTFE, even at large volume, had a very large yield, as compared to smaller volume (e.g., 500 pL in 1.5 mL tube) examples.

[0066] In some examples, the cartridges enable linear scaling of reactions without significant change in specific yield. For example, no significant change in specific yield was observed between 100, 200 and 300 pL reactions carried out in cartridges designed as 15 mL tube spin columns (see FIG. 21).

[0067] Any of these examples may use a modified open format, replacing one or both impermeable layer with a semi-permeable gas exchange membrane. In some examples, a cap or lid (e.g. a snap lid) may be used to prevent excessive evaporation. In some examples, this format can also double as a purification column. One such example is demonstrated in FIG. 22.

[0068] Some examples may include a hydrophobic semi-permeable gas exchange membrane configured to be used to construct a multi- well plate capable of gas exchange. For example, FIG. 23 illustrates an example of this configuration. In this example, a multi-well plate body is shown forming a plurality of reaction chambers in which each chamber includes a gas-exchange (and semi-permeable) hydrophobic membrane. In some examples, the semi-permeable gas exchange membrane layer may be made of low-thickness (e.g. 12.5 micron) FEP film to enable high optical opacity, while allowing for oxygen permeation.

[0069] In some examples the cartridges described herein may be used for production of a protein through cell-free protein synthesis. In some examples, enzymes of transcription and translation (e.g. crude cell lysate) and the reaction buffers including energy sources and amino acids (e.g. 1.2 mM ATP, 0.85 mM GTP, 0.85 mM UTP, 0.85 mM CTP, 31 pg/mL Folinic Acid, 171 pg/mL tRNA, 0.4 mM Nicotinamide Adenine Dinucleotide (NAD), 0.27 mM Coenzyme A (CoA), 4 mM Oxalic Acid, 1.5 mM Spermidine, and 57 mM HEPES buffer, 10 mM Mg(Glu)i, 130 mM K(Glu), 3 mM of each of the 20 amino acids and 30 mM 3PGA) are combined with template DNA and pipetted into the cartridge. In some examples, the reactions are incubated for 1-16 hours at 16-37 C to carry out protein expression.

[0070] For example, cartridges may be configured to enable purification using chromatography within the reaction chamber after the desired biomolecule has been synthesized. For example, Ni-NTA beads may be used as the purification substrate for His-tagged proteins, while using a binding buffer (e.g. 5 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol), a wash buffer (e.g. 25 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol) and an elution buffer (e.g. 250 mM imidazole, 500 mM NaCl, 50 mM Trizma base pH 7.5, 0.5 mM TCEP, 5% glycerol).

[0071] In other examples, the cartridges may be used for in vitro transcription and/or purification of RNA from DNA templates. In one such example, T7 RNA polymerase (25 U/pL final concentration) is mixed with a transcription buffer, NTPs and inorganic pyrophosphatase (0.005 U/pL final concentration) and is placed inside the thin-fdm reaction chamber. In some examples, silica beads or a silica membranes may be used to capture the RNA post transcription. [0072] In yet other examples, the cartridges are used for in vitro DNA synthesis through a process such as polymerase chain reaction (PCR). In some such examples, DNA polymerase, a PCR buffer, a magnesium salt and dNTPs are placed inside the thin-film reaction chamber. In some examples, silica beads or a silica membrane may be used to capture the amplified DNA.

[0073] FIG. 24 shows an example of a cartridge 2400 similar to that shown in FIG. 8. In this example, the cartridge includes a body forming a substantially flat reaction chamber 2415 that includes at least one wall formed of a gas-permeable hydrophobic membrane 2442. The reaction chamber may be accessed by an inlet 2417 that may include a hydrophobic frit, or may be plugged or pluggable to prevent evaporation and/or fluid loss. In some examples the cartridge may also include an outlet 2417 that may include a hydrophobic frit, or may be plugged or pluggable.

[0074] FIGS. 25A-25C illustrate an example of another cartridge 2500 similar to that shown in FIG. 24 and FIG. 8. In this example, the cartridge 2500 also includes a reaction chamber 2515 formed, in this case, between a pair of porous (or semi-porous) gas-exchange hydrophobic membrane membranes 2542, 2542’. The reaction chamber is substantially flat, as the gasexchange hydrophobic membranes are separated from each other by a distance that is much smaller than the length and/or width of the gas-exchange hydrophobic membrane. In this example the inlet 2517 having a slot 2519 with an enlarged key -hole opening 2521 configured for positioning and aligning a pipette tip, as shown in FIG. 25C. The cartridge may also include an outlet 2542 that may include a frit 2509 (e.g., a hydrophobic frit) and/or a connector 2511 (such as a Luer lock connector). [0075] FIGS. 26A-26C illustrate another example of a cartridge 2600. In this example the cartridge may be configured for larger volume samples (e.g., between 50-500 pL, between 100- 500 pL, between 200-500 pL, etc.), and include an inlet 2617 with a slot 2619 having an enlarged key-hole opening 2621 configured for positioning and aligning a pipette tip. The inlet is fluidly connected to the flat reaction chamber 2615, that is formed of one (or a pair) of porous (or semi-porous) gas-exchange hydrophobic membrane membranes separated from each other by a very narrow thickness, t. Thus, the reaction chamber has a thickness, t, that is much smaller than the width, w, and length, I, of the reaction chamber. The thickness may be maintained by one or more spacers within and/or around the periphery of the reaction chamber. In some examples just one side of the reaction chamber is formed of the porous (or semi-porous) gasexchange hydrophobic membrane, while in some examples both major surface (sides) of the reaction chamber are formed of a porous (or semi-porous) gas-exchange hydrophobic membrane. As described above, the example cartridge shown in FIGS. 26A-26C may be used with the application of force, e.g., by centrifugation, positive pressure, etc. to drive the product material (e.g., protein, etc.) out of the reaction chamber in a controllable manner, e.g., by applying centrifugation above a threshold force value (e.g., greater than 5000 xg), without the need for a separate outlet.

[0076] FIGS. 27A-27C illustrate an example of a cartridge similar to that shown in FIG. 26A-26C that includes an outlet 2742 having both a connector 7211 and a hydrophobic frit 2709 for preventing fluid (e.g., reaction product) from eluting out of the reaction chamber until a minimum force is applied (e.g., by positive/negative pressure and/or centrifugation). As in FIGS. 26A-26C the cartridge 2700 shown in FIGS. 27A-27C includes an inlet 2717 (with a slot 2719 having an enlarged key-hole opening 2721 configured for positioning and aligning a pipette tip). The cartridge also includes an elongate and flat reaction chamber 2715 with one or both major sides formed of a porous (or semi-porous) gas-exchange hydrophobic membrane 2707. The major walls of the reaction chamber are separated from each other by a thickness, t, and the reaction chamber has a width, w, and a length, I.

[0077] As mentioned above, any of these cartridges may include a porous fill material within all or some of the reaction chamber. The porous fill material may have a known liquid entry pressure to enable containment of the sample during the reaction and further release and collection of the sample by adjusting centrifugal force or pressure differential. The porous fill material may be present throughout the length and volume of the reaction chamber of the cartridge or just at an upper and/or lower region, such as at or near the outlet.

[0078] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, it should be appreciated that all combinations of the foregoing concepts and additional concepts discussed in greater detail below (provided such concepts are not mutually inconsistent) are contemplated as being part of the inventive subject matter disclosed herein and may be used to achieve the benefits described herein.

[0079] A person of ordinary skill in the art will recognize that any process or method disclosed herein can be modified in many ways. The process parameters and sequence of the steps described and/or illustrated herein are given by way of example only and can be varied as desired. For example, while the steps illustrated and/or described herein may be shown or discussed in a particular order, these steps do not necessarily need to be performed in the order illustrated or discussed.

[0080] The various exemplary methods described and/or illustrated herein may also omit one or more of the steps described or illustrated herein or comprise additional steps in addition to those disclosed. Further, a step of any method as disclosed herein can be combined with any one or more steps of any other method as disclosed herein.

[0081] When a feature or element is herein referred to as being "on" another feature or element, it can be directly on the other feature or element or intervening features and/or elements may also be present. In contrast, when a feature or element is referred to as being "directly on" another feature or element, there are no intervening features or elements present. It will also be understood that, when a feature or element is referred to as being "connected", "attached" or "coupled" to another feature or element, it can be directly connected, attached or coupled to the other feature or element or intervening features or elements may be present. In contrast, when a feature or element is referred to as being "directly connected", "directly attached" or "directly coupled" to another feature or element, there are no intervening features or elements present. Although described or shown with respect to one embodiment, the features and elements so described or shown can apply to other embodiments. It will also be appreciated by those of skill in the art that references to a structure or feature that is disposed "adjacent" another feature may have portions that overlap or underlie the adjacent feature.

[0082] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" and/or "comprising," when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items and may be abbreviated as "/" .

[0083] Spatially relative terms, such as "under", "below", "lower", "over", "upper" and the like, may be used herein for ease of description to describe one element or feature's relationship to another element(s) or feature(s) as illustrated in the figures. It will be understood that the spatially relative terms are intended to encompass different orientations of the device in use or operation in addition to the orientation depicted in the figures. For example, if a device in the figures is inverted, elements described as "under”, or "beneath" other elements or features would then be oriented "over" the other elements or features. Thus, the exemplary term "under" can encompass both an orientation of over and under. The device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly. Similarly, the terms "upwardly", "downwardly", "vertical", "horizontal" and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

[0084] Although the terms “first” and “second” may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0085] In general, any of the apparatuses and methods described herein should be understood to be inclusive, but all or a sub-set of the components and/or steps may alternatively be exclusive and may be expressed as “consisting of’ or alternatively “consisting essentially of’ the various components, steps, sub-components or sub-steps.

[0086] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or “approximately,” even if the term does not expressly appear. The phrase “about” or “approximately” may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value " 10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point “15” are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0087] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0088] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the subject matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term “invention” merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.