RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No.: 63/188,219 filed May 13, 2021, and entitled “TECHNIQUES FOR IMPROVED MICROFLUIDICS FLOW PROFILE," the entire contents of which is incorporated by reference herein.

BACKGROUND

[0002] Detection and identification of bacterial and viral pathogens present in cell containing solutions (e.g., blood, urine, CSF), mammalian cell culture, CHO cell matrix, CAR-T drug product, CAR-T specimen, CAR-NK drug product, body fluids, apheresis samples or samples related to immunotherapy), protein containing solutions (e.g., for pharmaceuticals during manufacturing, drug product, drug substance), analyte extraction from microbiome samples, water, sterile fluids and other fluids is possible by employing isolation on cultural media and metabolic fingerprinting methods. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassay and nucleic acid-based assays are now widely accepted techniques, providing more sensitive and specific detection and quantification of bacteria and microorganisms.

[0003] Dielectrophoresis (DEP) relates to a force in an electric field gradient on objects having dielectric moments. DEP has shown promise for particle separation, but has not yet been applied in clinical settings nor pharmaceutical quality assurance settings nor immunotherapy. DEP uses a natural or induced dipole to cause a net force on a particle in a region having an electric field gradient. The force depends on the Clausius-Mossotti factor associated with particle.

SUMMARY

[0004] Aspects of the technology described herein relate to improving uniformity of fluid flow through a microfluidic passage.

[0005] In some embodiments, a device is provided. The device comprises a body having a first internal wall and a second internal wall opposite the first internal wall, the first and second

1 internal walls defining a first microfluidic passage. The first microfluidic passage has an inlet arranged to receive a fluid into the first microfluidic passage, the inlet having a first cross- sectional area, a first region adjacent to the inlet, and a second region adjacent to the first region and having a second cross-sectional area larger than the first cross-sectional area, wherein the first internal wall has a smooth surface between the first region and the second region. The device further comprises first electrode configured to, when active, generate a dielectrophoretic force on a particle within the second region.

[0006] In one aspect, the device further comprises a first pillar arranged in the first region or the second region. In one aspect, the first pillar is positioned along a midpoint between the first internal wall and the second internal wall. In one aspect, the first pillar is cylindrical. In one aspect, the first pillar is configured to partially obstruct a flow of the fluid.

[0007] In one aspect, the first microfluidic passage further has a third region adjacent to the second region and having a third cross-sectional area that decreases with distance from the second region. In one aspect, at least one of the first internal wall or the second internal wall has a smooth surface between the second region and the third region.

[0008] In one aspect, the first microfluidic passage further has an outlet arranged to discharge the fluid from the first microfluidic passage, the outlet having a third cross-sectional area smaller than the second cross-sectional area.

[0009] In one aspect, a surface of the first internal wall within the second region is substantially parallel to a surface of the second internal wall within the second region.

[0010] In one aspect, the body defines a third internal wall, wherein the third internal wall is adjacent to the first internal wall and the second internal wall, and the first electrode is disposed on the third internal wall. In one aspect, the body defines a fourth internal wall opposite the third internal wall, wherein the fourth third internal wall is adjacent to the first internal wall and the second internal wall. In one aspect, the body defines an opening through the fourth internal wall, wherein the opening is arranged to pass fluid into the inlet. In one aspect, a distance between the third internal wall and the fourth internal wall is between 10pm and lOOpm.

[0011] In one aspect, a distance between the first internal wall and the second internal wall across the second region is between 500 pm and 50,000 pm.

[0012] In one aspect, a distance between the first internal wall and the second internal wall across the inlet is between 100 pm and 3,000 pm.

[0013] In one aspect, a ratio of the second cross-sectional area to the first cross-sectional area is between 1 : 1 and 30:1.

2 [0014] In one aspect, any curve in the first internal wall between the first region and the second region has a radius of at least 100 pm.

[0015] In one aspect, the first electrode is one of a plurality of electrodes arranged in a two- dimensional array.

[0016] In one aspect, the first electrode is configured to generate the di electrophoretic force on particles in the fluid such that the dielectrophoretic force captures at least a portion of the particles on a surface of the first electrode.

[0017] In one aspect, a uniformity of the second linear velocity across the second region is within +/- 20%.

[0018] In one aspect, the body further defines at least a second microfluidic passage, and the first microfluidic passage and the second microfluidic passage are configured to receive respective samples in parallel.

[0019] In one aspect, the device further comprises a first partition arranged in the second region, wherein the first partition is oriented along a direction substantially parallel to the first internal wall and the second internal wall.

[0020] In some embodiments, a device is provided. The device comprises a first layer having at least a first internal wall and a second internal wall opposite the first internal wall, the first internal wall and second internal wall defining a microfluidic passage. The microfluidic passage has an inlet arranged to receive a fluid, the inlet having a first width between the first internal wall and the second internal wall in a first direction perpendicular to a direction of flow of the fluid through the microfluidic passage, a widening region arranged to receive the fluid via the inlet, and a capture region arranged to receive the fluid via the widening region, the capture region having a second width between the first internal wall and the second internal wall in the first direction, wherein the first internal wall has a smooth surface between the widening region and the capture region. The device further comprises a first electrode configured to, when active, generate a dielectrophoretic force on a particle within the second region.

[0021] In one aspect, the first layer comprises a polymer.

[0022] In one aspect, the device further comprises a substrate layer, wherein the first electrode is disposed on a first surface of the substrate layer, the first layer is disposed on the substrate layer, and the substrate layer forms a third internal wall of the microfluidic passage. In one aspect, the substrate layer comprises silicon.

[0023] In one aspect, the device further comprises a second layer positioned on a first surface of the first layer, the second layer forming a third internal wall of the microfluidic passage. In one aspect, the second layer comprises a polymer. In one aspect, the second layer

3 defines an opening through third internal wall, wherein the opening is arranged to pass fluid into the inlet.

[0024] In one aspect, the device further comprises the first layer includes a first pillar arranged in the widening region or the capture region.

[0025] In one aspect, the device further comprises the first layer includes a first partition arranged in the capture region, wherein the first partition is oriented along a direction substantially parallel to the first internal wall and the second internal wall.

[0026] In some embodiments, a device is provided. The device comprises a body having a first internal wall and a second internal wall opposite the first internal wall, the first and second internal walls defining a first microfluidic passage. The first microfluidic passage has an inlet arranged to receive a fluid into the first microfluidic passage, the inlet having a first cross- sectional area, a first region adjacent to the inlet, and a second region adjacent to the first region and having a second cross-sectional area larger than the first cross-sectional area. The device further comprises a first electrode disposed adjacent to the second region, the first electrode configured to, when active, generate a dielectrophoretic force on a particle within the second region, a pump coupled to the body and configured to pump the fluid through the microfluidic passage, a signal generator electrically connected to the first electrode and configured to generate an AC voltage to drive the first electrode to produce an electric field within the second region, and a controller configured to control the signal generator to generate the AC voltage having frequency and amplitude characteristics such that when produced, the electric field captures on a surface of the first electrode, a target particle species in the fluid as the fluid traverses the microfluidic passage, and control the signal generator to alter generation of the AC voltage to release the target biological particle species.

[0027] In one aspect, at least one of the first internal wall or the second internal wall has a smooth surface between the first region and the second region.

[0028] In some embodiments, a device is provided. The device comprises a body defining a first microfluidic passage. The first microfluidic passage has an inlet arranged to receive a fluid into the first microfluidic passage, a capture region arranged to receive the fluid from the inlet, at least one electrode configured to generate, when active, a dielectrophoretic force on a particle within the capture region, and means for causing a substantially uniform flow velocity of the fluid in the capture region.

[0029] In some embodiments, a microfluidic device is provided. The microfluidic device comprises at least one microfluidic channel for receiving a sample. The at least one microfluidic channel is disposed in a body of the microfluidic device and comprises at least one electrode for

4 generating at least one dielectrophoretic force that acts on the sample, a first end comprising an inlet for receiving the sample, wherein the first end comprises at least one rounded edge, a second end comprising an outlet, wherein the body is disposed between the first and second ends, and one or more pillars disposed at one or more of the first and second ends.

[0030] In some aspects, the first end is tapered.

[0031] 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. In particular, all combinations of claimed subject matter appearing at the end of this disclosure are contemplated as being part of the inventive subject matter disclosed herein.

BRIEF DESCRIPTION OF DRAWINGS

[0032] Various non-limiting embodiments of the technology will be described with reference to the following figures. It should be appreciated that the figures are not necessarily drawn to scale.

[0033] FIG. 1 schematically illustrates a system for detection and quantification of live and dead bacteria in a sample, according to some embodiments;

[0034] FIG. 2 illustrates a microfluidic system for detection and quantification of live and dead bacteria in a sample in a sample, according to some embodiments;

[0035] FIG. 3 illustrates a static system for detection and quantification of live and dead bacteria in a sample, according to some embodiments;

[0036] FIG. 4 illustrates a first example implementation of a microfluidic device, according to some embodiments;

[0037] FIG. 5 shows a first view of a distribution of linear velocity of a fluid in the first example microfluidic device, according to some embodiments;

[0038] FIG. 6 shows a second view of the distribution of linear velocity of the fluid in the first example microfluidic device, according to some embodiments;

[0039] FIG. 7 shows various cross sections of the fluid in the first example microfluidic device at which flow profiles are measured, according to some embodiments;

[0040] FIG. 8 shows a graph illustrating the flow profiles at the cross sections of the first example microfluidic device illustrated in FIG. 7, according to some embodiments;

[0041] FIG. 9 illustrates a second example implementation of a microfluidic device, according to some embodiments;

5 [0042] FIG. 10 shows a first view of a distribution of linear velocity of a fluid in the second example microfluidic device, according to some embodiments;

[0043] FIG. 11 shows a second view of the distribution of linear velocity of the fluid in the second example microfluidic device, according to some embodiments;

[0044] FIG. 12 shows various cross sections of the fluid in the second example microfluidic device at which flow profiles are measured, according to some embodiments;

[0045] FIG. 13 shows a graph illustrating the flow profiles at the cross sections of the second example microfluidic device illustrated in FIG. 12, according to some embodiments; [0046] FIG. 14 illustrates a third example implementation of a microfluidic device, according to some embodiments;

[0047] FIG. 15 illustrates electrical components of the third example implementation of a microfluidic device, according to some embodiments;

[0048] FIG. 16 shows a first view of a distribution of linear velocity of a fluid in the third example microfluidic device, according to some embodiments;

[0049] FIG. 17 shows a second view of the distribution of linear velocity of the fluid in the third example microfluidic device, according to some embodiments;

[0050] FIG. 18 shows various cross sections of the fluid in the third example microfluidic device at which flow profiles are measured, according to some embodiments;

[0051] FIG. 19 shows a graph illustrating the flow profiles at the cross sections of the third example microfluidic device illustrated in FIG. 18, according to some embodiments; and [0052] FIG. 20 illustrates a fourth example implementation of a microfluidic device, according to some embodiments.

DETAILED DESCRIPTION

[0053] Aspects of the technology described herein relate to improving a fluid flow profile of a microfluidic device. In particular, the technology described herein provides techniques for detection and/or quantification of particles in a fluid sample using a microfluidic system comprising one or more electrodes configured to generate an electric field that exerts a dielectrophoretic force on particles in a fluid sample. Such particles may include, for example and without limitation, microorganisms, bacteria, yeast, mold, viruses, cells, T cells, NK cells, etc. Improving the flow profile of the sample fluid flowing through the microfluidic device can, among other benefits, increase the capture efficiency of the electrodes and enhance system performance.

6 [0054] The microfluidic device may have one or more microfluidic passages. The fluid sample may be pumped or otherwise passed through the microfluidic passage(s). A time-varying (AC) electric signal supplied to the electrode(s) may generate a dielectrophoretic force on particles within the sample. The microfluidic system may, by controlling a frequency and/or amplitude of the electrical signal, capture some of the particles from the sample such that they are held on or near the electrode(s) even as the sample fluid continues to flow through the microfluidic passage. Once captured, the sample particles may be detected, imaged, quantified, and/or otherwise measured. The microfluidic system may release some or all of the captured particles by modifying or turning off the electrical signal.

[0055] The capture efficiency refers to the proportion of sample particles that the microfluidic device is able to capture from flowing sample fluid. The capture efficiency relates to the force of the flow and the magnitude of the dielectrophoretic force. Thus, for a given dielectrophoretic force, the microfluidic device may capture particles from fluid moving up to a certain flow rate, above which capture efficiency decreases. Increasing the strength of the electric field and thus the dielectrophoretic force for a given electrode configuration may require increasing the amplitude of the electrical signal, which decreases the energy efficiency of the system, and may result in heating of the sample and/or electrodes, risking damage to each. Although a microfluidic system could increase capture efficiency by slowing a rate of flow through the microfluidic device, doing so may harm performance of the system in other ways; for example, by decreasing throughput, allowing sample particles and/or other residues to persist in a capture region after release, and/or reducing the system’s ability to distinguish sample species through selective capture and/or release.

[0056] In some cases, the geometry of a microfluidic passage may lead to an uneven flow profile of fluid flowing through various regions of the microfluidic device. For example, the microfluidic passage may widen to reduce a flow velocity of the sample fluid, and increase a surface area over which an electrode(s) can interact with sample particles. The microfluidic passage may receive a sample fluid at an inlet having a first cross-sectional area. The microfluidic passage may widen to a capture region having a larger cross-sectional area than the inlet. The capture region may be an area of the microfluidic passage containing and/or adjacent to the electrode(s), and from which sample particles are captured from the fluid sample via the dielectrophoretic force. The microfluidic passage may then narrow to an outlet having a smaller cross-sectional area than the capture region. Such a microfluidic passage configuration may result in an average flow velocity in the capture region that is lower than the flow velocity at the inlet; however, reducing flow velocity in this manner may result in an uneven flow profile (e.g.,

7 flow velocities that vary across the cross section of the microfluidic passage). Thus, some regions of the microfluidic device may experience higher flow velocities, which may reduce an ability of the electrode(s) adjacent to that region to capture sample particles out of the flow, lowering the overall capture efficiency of the microfluidic device.

[0057] Presented herein are techniques and structures for improving the capture efficiency of a microfluidic device by creating conditions for more uniform flow profile of a sample fluid across a capture region of a microfluidic passage. The techniques and structures may reduce the effect of, or eliminate dead and/or slow flow zones, eddies, and/or mixing that may cause sample particles to have different flow profiles. By creating a more uniform flow profile, the microfluidic system may reduce a range of flow velocities between different portions of the capture region of the microfluidic device. The overall flow rate may thus be kept relatively high, enabling higher throughput of the microfluidic system, without creating regions of poor local capture efficiency. In addition, the amplitude of the electrical signal may be kept lower while still providing for effective particle capture across a larger portion of the capture region, thus improving the energy efficiency of the system and preventing potentially problematic heating of the electrode(s) and/or sample. The increased capture efficiency may reduce false negatives (e.g., resulting from failing to capture and detect particles) when a sample is passed through the microfluidic passage. The more uniform flow profile may also allow for more effective flushing of the microfluidic passage to remove sample particles and/or other residues.

[0058] The techniques and structures described herein may include, for example, a microfluidic passage configured with a one or more smooth internal walls (e.g., without sharp inward or outward facing comers, cavities, and/or protrusions) between an inlet region and a capture region. In some implementations, the microfluidic passage may include one or more structural features such as pillars configured to reduce local fluid flow velocity in portions of the capture region where the flow velocity would otherwise be high relative to the average flow velocity in the microfluidic passage. In some implementations, one or more partitions may divide part or all of the capture region into two or more passages. By dividing the capture region into multiple passages, a more linear and/or uniform fluid flow through the capture region may be achieved. These and other features described herein may be used in combination as described below and illustrated in the accompanying drawings.

[0059] FIG. 1 illustrates an example system for detecting bacteria in a sample, in accordance with some embodiments. As shown in FIG. 1, the system 100 comprises a microfluidic device 104 in communication with a computing device 110.

8 [0060] The microfluidic device 104 may be any suitable device, examples of which are provided herein, in particular, with respect to FIG. 2. In some embodiments, microfluidic device 104 comprises a microfluidic chip having one or more passages (e.g., microfluidic channels or chambers) through which a fluid sample 102 is provided for analysis. Although the term “microfluidic passage” or simply “microfluidic channel” is used herein to describe a passage through which fluid flows through microfluidic device 104, it should be appreciated that a fluid passage having any suitable dimensions may be used as said passage or channel, and embodiments are not limited in this respect. Microfluidic device 104 may comprise a single channel or multiple channels configured to receive a single sample 102 (e.g., to perform different analyses on the sample) or multiple channels configured to receive different samples for analysis. In embodiments having multiple channels, the microfluidic device may be configured to process the single sample or multiple samples in parallel (e.g., at the same or substantially the same time).

[0061] As described herein, sample 102 may include any fluid containing bacteria or other microorganisms of interest. In some embodiments, the sample comprises a biological fluid such as saliva, urine, blood, water, any other fluid such as an environmental sample or potentially contaminated fluid, protein matrices, mammalian cell culture, bacterial culture, growth media, active pharmaceutical ingredients, enzyme products, or substances used in biomanufacturing, immunotherapy drugs, CAR-T drugs, CAR-NK drugs etc.

[0062] As shown, microfluidic device 104 includes at least one electrode 106. The at least one electrode 106 may be configured to receive one or more voltages to generate positive and/or negative dielectrophoresis (DEP) force(s) that act on a sample arranged proximate to the at least one electrode. In some embodiments, the at least one electrode 106 may be configured to receive one or more voltages (e.g., one or more AC voltages) to generate at least one dielectrophoresis force that acts on the sample. The at least one DEP force may cause certain components of the sample to move relative to (e.g., be attracted to or repulsed from) a surface of the at least one electrode 106. For example, in the absence of an electric field, bacteria and other components of the sample 102 may move freely relative to the surface of the electrode. In the presence of the electric field at least some components (e.g., bacteria) in the sample may be attracted to the electrode surface.

[0063] The microfluidic device 104 uses dielectrophoresis for purposes of separating viral particles from other components of a sample. Dielectrophoresis uses a natural or induced dipole to cause a net force on a particle in a region having an electric field gradient.

F = 2ne _m R ³ Re[CM(o ) VE ² (r, w)]

9 [0064] This force depends on the Clausius-Mossotti factor CM (OJ) defined by

[0065] where e° is the complex permittivity, e° =

[0066] The small size of bacteria presents an obstacle to optical observation and quantification of bacteria in the sample. The inventors have recognized that activation of the at least one electrode 106 results in an electric field that may be used to selectively trap bacteria on the surface of the electrode(s). When used with an optical detection system, capturing bacteria on the surface of the electrode(s) may prevent the bacteria from moving in and out of focus of the optical device to enable real-time bacteria detection and quantification, a process referred to herein as “on-chip quantification.”

[0067] The electric field used to capture the bacteria concentrates the bacteria, which enables imaging with fluorescence microcopy or another optical detection technique. Accordingly, bacterial capture using the techniques described herein allows for detection and quantification of bacteria at significantly lower limits compared to some conventional methods, such as the plate count method (PCM). The ability to detect and/or quantify bacteria in a sample, even in small amounts, may be useful in applications including, but not limited to, sterility testing, bioburden testing, challenge test, biomanufacturing, gene therapy, analysis of patient samples, vaccine development and/or biothreat detection.

[0068] For example, the at least one DEP force acting on the sample may cause bacteria (or certain bacteria) to separate from other components of the sample (e.g., via positive DEP). Bacteria in the sample may be attracted to the surface of the at least one electrode 106 allowing for enhanced detection and/or quantification, despite the small size and/or small amount of the bacteria in the sample. Although, microfluidic device 104 is illustrated as having a single electrode, it should be understood that in some embodiments, microfluidic device 104 comprises multiple electrodes arranged in any suitable configuration.

[0069] System 100 may further comprise a computing device 110 configured to control one or more aspects of microfluidic device 104. For example, computing device 110 may be configured to direct the sample 102 into a channel of the microfluidic device. In some embodiments, computing device 110 is configured to control the at least one electrode 106 to generate the at least one DEP force acting on the sample 102. In some embodiments, computing device 110 may cause one or more components of the microfluidic system (e.g., an optical device) to perform one or more of detection or quantification of the bacteria or other

10 microorganisms in the sample. Non-limiting examples of a computing device 110 that may be used in accordance with some embodiments are further described herein.

[0070] FIG. 2 illustrates an example system 200 for detecting the presence of bacteria in a sample, in accordance with some embodiments. System 200 includes microfluidic device 208 (e.g., a microfluidic chip) that includes one or more electrodes for generating DEP forces that act on a sample 204 provided as input to the system. Sample 204 may contain bacteria for which detection and/or quantification may be performed. The sample 204 may optionally undergo preparation prior to analysis. The system 200 may be flexible with respect to sample 204 volume; for example, the system 200 may analyze sample sizes from a few hundredths of a milliliter to several liters. The one or more electrodes may be arranged in any suitable configuration within the microfluidic device 208. For instance, in embodiments that include multiple electrodes, the electrodes may be arranged in one-dimension along the flow direction of the fluid, perpendicular to fluid flow direction or on a diagonal relative to the fluid flow direction. In some embodiments, a multidimensional (e.g., 2-dimensional, 3-dimensional) array of electrodes may be used. For instance, a dense array of electrodes arranged both along the direction of fluid flow and perpendicular to the direction of fluid flow may be used.

[0071] As shown in FIG. 2, a flow system 202 is provided. The flow system 202 may provide a solution for transporting the sample 204 to the microfluidic device 208. A first pump 206 may be used to pump the solution and the sample 204 to the microfluidic device 208 at a predetermined flow rate. First pump 206 may be of any suitable type. In some embodiments, first pump 206 is omitted and sample 204 is manually loaded (e.g., using a pipette) as input to one or more channels of microfluidic device 208.

[0072] Microfluidic device 208 is configured to receive sample 204 for processing. Microfluidic device 208 may include one or more passages through which the sample 204 flows. The one or more passages may include at least one electrode formed therein or adjacent thereto. For instance, the at least one electrode may be formed within a passage. The at least one electrode, when activated, is configured to generate an electric field that acts on the sample 204 as it flows through the one or more passages. An electrical system 212 (e.g., a signal generator or controller) is configured to provide one or more voltages to the at least one electrode of the microfluidic device 208 to tune the properties of the electric field for capture of a particular microorganism or microorganisms of interest. Further aspects of the electrical system 212, including example protocols for operating the microfluidic device 208 are provided herein.

[0073] An optical system 210 may be provided to facilitate analysis of the sample 204 by performing on-chip quantification. For example, the optical system 210 may comprise one or

11 more optical sensors (e.g., a red-green-blue camera) for viewing and/or imaging the sample. The optical sensor(s) may provide for enhanced detection and/or quantification of the bacteria and/or the other components of the sample 204 relative to detection and quantification techniques that require separate culturing of captured bacteria or an effluent sample from the device. Any suitable optical detector may be used. In some embodiments, the optical sensor(s) comprises a digital camera. In some embodiments, the optical sensor(s) comprises electronic sensors including CMOS compatible technology. In some embodiments, the optical sensor(s) comprise fiber optics. However, any suitable optical sensor(s) may be used. In some embodiments, bacteria in the sample are stained (e.g., with a fluorescent dye) and the optical system 210 is configured to perform microscopy (e.g., fluorescence microscopy) of captured stained bacteria.

In some embodiments, optical system 210 is configured to capture one or more images (e.g., color images) of the at least one electrode while the sample is flowing through the microfluidic device 208. In some embodiments, the detector comprises nanowire and/or nanoribbon sensors. In some embodiments, the field of view of the optical system 210 at a particular magnification is insufficient to capture the entire surface of the one or more electrodes. In such embodiments, the optical system 210 may be configured to capture multiple partially overlapping images that collectively cover the entire surface of the one or more electrodes. The multiple captured images may then be analyzed to detect and/or quantify the bacteria in the sample.

[0074] System 200 also includes computer 230 configured to control an operation of optical system 210 and/or to receive images from optical system 210 and to perform processing on the received images (e.g., to count a number of bacteria trapped by the microfluidic device 208). In some embodiments, the received images are analyzed to determine the number of bacteria captured by the at least one electrode. For instance, bacteria may be identified in the received images as spots (e.g., fluorescent spots) located on the edges of the electrodes. In this way a captured target bacterial species may be differentiated from other components in the sample that are not captured and may appear as floating above the at least one electrode or located between electrodes.

[0075] After the sample 204 is processed by the microfluidic device 208 and/or optical system 210 to capture and/or quantify bacteria on the electrode(s), the sample 204 may be removed from the microfluidic device 208. For example, a second pump 216 may be provided for pumping the sample 204 out of the microfluidic device 208. The second pump 216 may be of any suitable type. In some embodiments, system 200 comprises a flow sensor 214 for measuring a flow rate at which the sample 204 is removed from the microfluidic device 208. The flow

12 sensor 214 and the second pump 216 may be in communication to control a flow rate at which the sample 204 is removed from the microfluidic device 208.

[0076] As described herein, system 200 may be used for separating bacteria from other components (or for separating certain bacteria from other bacteria) in sample 204. System 200 comprises a waste region 218 arranged to receive other components of the sample 204 which have been separated from the bacteria by the microfluidic device 208 and subsequently removed from the sample 204, for example, using the second pump 216. In the description below, analysis of the fluid collected in waste region 218 may be referred to as analysis of the “effluent sample.” System 200 may further include effluent region 220 for receiving a purified version of sample 204 containing substantially only target bacteria that were captured using microfluidic device 208.

[0077] In some embodiments, an amount of time needed to process a sample using system 200 is substantially less than an amount of time required to process a sample using a conventional PCM sample processing system. As shown in FIG. 2, processing a sample using system 200 may include at least three steps. In step 250, a sample is provided as input to microfluidic device 208 and bacteria are captured from the sample in the presence of an applied electric field. In step 260, automated on-chip quantification is performed, for example, using an optical system and computer 230 to analyze one or more images recorded by optical system 210. In step 270, further analysis may be performed on samples in the waste region 218 and/or effluent region 220, as desired. In sum, the entire process for detecting and/or quantifying bacteria in a sample using system 200 may take on the order of minutes or an hour to a few hours, which is substantially faster than the multiple days (e.g., 1 to 14 days) typically required to process samples using PCM.

[0078] In some embodiments, rather than pumping sample 204 through one or more passages through which the sample flows, sample 204 may be manually provided as input to microfluidic device 208 for analysis. For instance, one or more droplets of sample 204 may be provided as input to microfluidic device 208 using a pipette or other suitable technique. In such embodiments, the sample is analyzed in a “static” condition rather than in a condition in which bacteria are captured by the at least one electrode as the sample flows past the electrode(s) (e.g., as in the case of system 200 as shown in FIG. 2). FIG. 3 illustrates a system 300 for detecting bacteria in a sample, according to some embodiments. As shown, system 300 may include many of the same components as system 200. The system 300 may, however, omit certain components of the system 200, such as the first pump 206, which are not needed when the sample is manually provided as input to the microfluidic device.

13 [0079] Microfluidic systems used in accordance with some embodiments of the present technology provide a precise and rapid system for qualitative and/or quantitative differentiation between live and dead bacteria and other organisms (e.g., spores). This differentiation may be based on measurements obtained from a single organism (e.g., bacteria, virus, fungi, yeast, etc.) or from a mix of organisms. Such measurements may provide information useful in quality assurance, product sterility and biomanufacturing processes, among others. For example, engineered tissues and organs require high quality and sterility assurance before they are implanted in patients. Similar to drugs with a short shelf life, immunotherapy drugs, engineered tissues require careful handling, are prone to contamination, and have a short shelf life before they must be implanted into patients.

[0080] Additionally, recent studies suggest that environmental contamination plays a significant role in Hospital Acquired Infections (HAIs) and in the unrecognized transmission of nosocomial pathogens during outbreaks, as well as ongoing sporadic transmission. Several pathogens can persist in the environment for extended periods and serve as vehicles of transmission and dissemination in the hospital setting. Cross-transmission of these pathogens can occur via hands of healthcare workers, who become contaminated directly from patient contact or indirectly by touching contaminated environmental surfaces. Less commonly, a patient could become colonized by direct contact with a contaminated environmental surface. Rapidly detecting the presence of live microorganisms may be important to verify whether water treatment, environmental surface treatment or sterilization of pharmaceutical substances or other sterile equipment was effective. If the treatment was successful, all microorganisms present in the sample should be dead.

[0081] Existing techniques for detecting bacteria in fluid samples (e.g., water and other fluids) may be inefficient in several ways including, but not limited to, their inability to detect low levels of contaminant and/or their inability to culture certain types of microorganisms. In some instances, existing detection methods may take days to provide results. While faster methods such as quantitative polymerase chain reaction (qPCR) can reduce the response time to a few hours, such methods require complex sample preparation, high costs, have limited portability, and cannot be used for process streamlining.

[0082] The techniques described herein may be implemented using a microfluidic system (e.g., the microfluidic systems described in FIGS. 1 through 3). For example, as described above, the microfluidic system may control particle motion in a fluid by dielectrophoresis (DEP), which describes the motion of all particles in a non-uniform electric field gradient. Using DEP, bacteria and other cells can be captured on a surface of one or more electrodes used to

14 generate an electric field having particular characteristics. The capture can be universal, capturing all particles within a range of sizes, or selective for a singular particle type, depending on the tuning of the electric field applied. The electrode(s) of the microfluidic system may be specially designed to maximize bacterial response to the electric field. An automated method to measure the bacterial response that allows for bacterial fingerprinting. The inventors have recognized and appreciated that live bacteria strains and dead bacteria strains can be distinguished based on their bacterial fingerprint in the presence of an electric field.

Accordingly, bacterial viability may be detected from a mix of strains using their unique fingerprints. In some embodiments, the strains may be unlabeled. In some embodiments, the techniques may be automated. In some embodiments, the techniques may be performed rapidly (e.g., results obtained in 30 minutes or less).

[0083] Various example implementations of a microfluidic device configured for improved flow distribution are described herein. The described implementations are not intended to be limiting, and one or more features described with reference to one implementation may be combined with one or more features described with reference to a different implementation. A first example implementation is described with reference to FIGS. 4-8 and includes a microfluidic passage with sharp comers. A second example implementation is described with reference to FIGS. 9-13 and includes a microfluidic passage with smooth internal walls. A third example implementation is described with reference to FIGS. 14-19 and includes a microfluidic passage having one or more structural features such as pillars configured to reduce local fluid flow velocity around the one or more structural features. A fourth example implementation is described with reference to FIG. 20 and includes a microfluidic passage with a distribution of pillars configured to further increase flow profile uniformity.

[0084] FIG. 4 illustrates a first example implementation of a microfluidic device 400, according to some embodiments. The microfluidic device 400 may have a body with internal walls 430A and 430B (e.g., inward-facing surfaces) that define a microfluidic passage having an inlet region 410, a widening region 420, a capture region 440, a narrowing region 450, and an outlet region 460. A sample fluid may enter the microfluidic passage via the inlet region 410, and exit via the outlet region 460. An electrode (or array of electrodes) 445 may generate a time- varying (AC) electric field within the capture region 440 that exerts a dielectrophoretic force on one or multiple types of sample particles within the sample fluid. Such particles may include, for example and without limitation, microorganisms, bacteria, yeast, mold, viruses, cells, T cells, NK cells, etc. The electrode(s) 445 may be disposed on a third inward-facing surface (e.g., defining a top or bottom of the microfluidic passage). In some implementations, the electrode(s)

15 445 may be disposed on or in other or additional internal walls of the microfluidic device 400. An electric signal may be provided to the electrodes 445 via one or more electrical contacts 447A and 447B. The resulting dielectrophoretic force may capture some sample particles for detection by, for example, the optical system 210.

[0085] In some implementations, the inlet region 410 may have a width 411 between 100 pm and 50,000 pm. In some implementations, the width 411 may be between 500 pm and 5,000 pm. In some implementations, the capture region 440 may have a width 441 of between 500 pm and 50,000 pm. In some implementations, the width 441 may be between 1,000 pm and 10,000 pm. In some implementations, a ratio of the capture region width 441 and the inlet region width 411 may be between 1:1 and 30:1. In some implementations, the ratio of the width 441 and the width 411 may be between 2: 1 and 10:1.

[0086] In some implementations, the microfluidic device 400 may be assembled from individual layers. Materials used for constructing each layer may be chosen based on desired properties; for example, electrical and/or thermal conductivity. In some implementations, a first layer (e.g., upon which the electrode(s) 445 may be disposed) may include silicon, which may act as an electrical insulator but a thermal conductor (e.g., to remove heat generated by the electrode(s) 445). In some implementations, other layers may comprise a polymer. The additional layers may include a spacer layer (e.g., which may define the internal walls 430A and 430B) and a top layer, which may cover the microfluidic passage. In some implementations, one or more polymer layers and/or components may be injection molded and assembled with, for example, a silicon substrate including the electrode(s) 445.

[0087] The inlet region 410 may be configured to receive a sample fluid (e.g., from an opening) and provide it to the capture region 440 where sample particles may interact with the electric fields generated by the electrode(s) 445. The capture region 440 may have a larger cross sectional area than the inlet region 410 such that a flow velocity of the sample fluid is lower than in the inlet region. In the widening region 420, the microfluidic passage may widen in one or more dimensions perpendicular to a direction of flow (e.g., a distance between the internal walls 430A and 430B may progressively increase as fluid travels from the inlet region 410 to the capture region 440). The widening region 420 may be configured to slow the flow velocity of the sample fluid and spread the flow over a larger surface area within the capture region 440 to increase interaction between sample particles and the electrode 445. The microfluidic passage may be defined by internal walls 430A and 430B, which may progressively increase in distance from each other between the inlet region 410 and the capture region 440. The internal walls

16 430A and 430B may, in some implementations, be parallel or substantially parallel in the capture region 440.

[0088] The outlet region 460 may be configured to funnel or otherwise convey sample fluid from the capture region 440 to an outlet. In the narrowing region 450, the microfluidic passage may progressively narrow in one or more dimensions perpendicular to a direction of flow. The flow velocity of the sample fluid may increase as the cross-sectional area of the microfluidic passage decreases towards the outlet region 460.

[0089] In some implementations, the inlet region 410 may be configured with a needle shape. In such implementations, the inlet region 410 may have a length-to-width ratio of 10:1 or higher; that is, a length of the inlet region 410 (e.g., measured in a direction of fluid flow) may be ten or more times as large as its width (e.g., measured between internal walls 430A and 430B). The needle shape may reduce or eliminate non-specific particle binding at the inlet interface. The needle shape may enhance microfluidic device connections to the remainder of the flow system providing a user-friendly interface and ease of use, in contrast to tubing based connections.

[0090] Table 1 lists capture efficiency of the microfluidic device 400 shown in FIG. 4 for various influent concentrations and flow rates. As shown, the capture efficiency may depend on flow rate and electric field settings (e.g., how strong the dielectrophoretic force is for particular particles being captured). Table 1 shows that the microfluidic device 400 has capture efficiency of 99.991% at a flow rate 480ul/min, 99.99% - 100% at a flow rate 240ul/min. Accordingly, at flow rates less than 480 uL/min, the capture efficiency is greater than or equal to 99.99%. Though not shown, flow rates as low as 10 uL/min were tested and showed capture efficiencies of at least 99.99%. As flow rates are increased, the capture efficiency is decreased, but still remains above 99.9% in experiments in which the flow rate was 960 uL/min or less.

17

Table 1: Capture efficiency of the first example implementation of a microfluidic device shown in FIG. 4 for various influent concentrations and flow rates

[0091] In the microfluidic device 400, internal walls 430A and 430B may have one or more comers 425A and 425B or other non-smooth transitions. The comers 425A and 425B may create dead or slow flow zones, eddy currents, lower mixing, and other effects that result in an uneven flow profile across a portion of the microfluidic passage. In other words, sample particles in different portions of the capture region 440 may have different flow velocities, which may contribute to non-uniform capture efficiencies across the electrode(s) 445.

[0092] FIG. 5 shows a first view of a distribution of linear velocity of a fluid in the microfluidic device 400, according to some embodiments. FIGS. 5 through 8 were generated using COMSOL Multiphysics simulation software developed by COMSOL Inc. of Stockholm, Sweden. FIG. 5 shows a distribution of the linear velocity of the fluid in a half height of the microfluidic channel of the microfluidic device 400. Threshold of the max value was set to 80 mm/s flow velocity.

[0093] FIG. 5 shows a relatively high flow velocity in the inlet region 410 slowing to a lower flow velocity in the capture region 440. FIG. 5 also reveals “dead zones” of reduced flow velocity in the vicinity of the comers 425 A and 425B. These dead zones create areas of lower flow velocity that persist across the capture region 440, especially along the walls of the microfluidic channel. As a result, sample particles in some portions of the capture region 440 (e.g., in the center of the microfluidic channel) may have a flow velocity high enough to overcome the dielectrophoretic force and evade capture, while sample fluid in other portions of

18 the capture region 440 (e.g., along the walls of the microfluidic channel) may not achieve a flow velocity high enough to remove sample particles and/or other residue after release of the dielectrophoretic force.

[0094] FIG. 6 shows an alternative view of the distribution of linear velocity of the fluid in the microfluidic device 400, according to some embodiments. The distribution is shown in a half height of the microfluidics channel. The velocity scale has been adjusted to further reveal dead zones 610A, 610B, 610C, and 610D in the vicinity of the comers 425A and 425B. Threshold of the max value was set to 28 mm/s and 18 mm/s to min value.

[0095] FIG. 7 indicates multiple cross sections through the microfluidic device 400 at which a flow profile of the fluid flowing through the cross section may be measured, according to some embodiments. The cross sections include a first cross section 720 across a middle portion of the widening region 420, a second cross section 730 at a boundary of the capture region 440, a third cross section 740 across a middle portion of the capture region 440, and a fourth cross section 750 across the narrowing region 450. The second cross section 730 may represent a boundary after which particles may begin to be effectively captured by the electrode(s). The fourth cross section 750 may represent a boundary after which particles are no longer effectively captured by the electrode(s). The resulting flow profiles measured through each of the cross sections indicated in FIG. 7 are shown in graph 800 of FIG. 8.

[0096] In the graph 800, a first line 820 illustrates a flow profile at the first cross section 720 (in the widening region 420). A second line 830 illustrates a flow profile at the second cross section 730 (at or near a boundary of the capture region 440). A third line 840 illustrates a flow profile at the third cross section 740 (across a middle portion of the capture region 440). A fourth line 850 illustrates a flow profile at the fourth cross section 750 (in the narrowing region 450).

[0097] The graph 800 shows a relatively uniform flow profile across a width of a middle portion the capture region 440, as shown by the third line 840. The second line 830, however reveals flow velocities that vary significantly across the boundary at the beginning of the capture region 440. The graph 800 thus demonstrates how the dead spots can cause an uneven flow profile across portions of the capture region 440, potentially resulting in reduced capture efficiency of the microfluidic device 400.

[0098] FIG. 9 illustrates a second example implementation of a microfluidic device 900, according to some embodiments. Similar to the microfluidic device 400, the microfluidic device 900 may have a body with internal walls 930A and 930B (e.g., inward-facing surfaces) that define a microfluidic passage having an inlet region 910, a widening region 920, a capture

19 region 940, a narrowing region 950, and an outlet region 960. A sample fluid may enter the microfluidic passage via an opening 905 leading to the inlet region 910, and exit via an opening 965 from an outlet region 960. The openings 905 and 965 may be defined in a top surface of the microfluidic device 900. An electrode (or array of electrodes) adjacent to the capture region 940 may generate a time-varying (AC) electric field within the capture region 940 that creates a dielectrophoretic force on sample particles within the sample fluid. An electric signal may be provided to the electrodes via one or more electrodes. The resulting dielectrophoretic force may capture some sample particles for detection by, for example, the optical system 210. Such particles may include, for example and without limitation, microorganisms, bacteria, yeast, mold, viruses, cells, T cells, NK cells, etc.

[0099] In some implementations, the inlet region 910 may have a width 911 between 100 pm and 30,000 pm. In some implementations, the width 911 may be between 500 pm and 5,000 pm. In some implementations, the capture region 940 may have a width 941 of between 500 pm and 50,000 pm. In some implementations, the width 941 may be between 1,000 pm and 10,000 pm. In some implementations, a ratio of the capture region width 941 and the inlet region width 911 may be between 1:1 and 30:1. In some implementations, the ratio of the width 941 and the width 911 may be between 2: 1 and 10:1.

[0100] The widening region 920 may be configured to slow the flow velocity of the sample fluid and spread the flow over a larger surface area within the capture region 940 to increase interaction between sample particles and the electrode. The microfluidic passage may be defined by the internal walls 930A and 930B, which may progressively increase in distance from each other between the inlet region 910 and the capture region 940. In the microfluidic device 900, the internal walls 930A and 930B have smooth surfaces (e.g., without sharp inward or outward facing comers, cavities, and/or protrusions) between the inlet region 910 and the capture region 940. The smooth internal walls 930A and 930B may reduce or eliminate dead and/or slow flow zones, and improve the uniformity of flow profile across the capture region 940. The more uniform flow profile may increase capture efficiency of the microfluidic device 900. A more uniform flow profile may also improve electrical efficiency by allowing for a lower amplitude electric field to be used to capture sample particles across a greater proportion of the capture region 940. The increased capture efficiency may reduce false negatives (e.g., resulting from failing to capture and detect particles). Reducing dead and/or slow flow zones may also provide for more effective flushing of the microfluidic passage and removing of residue. In some implementations, the smooth internal walls 930A and 930B may have one or more curves. In some implementations, any curve in the smooth internal walls 930A and 930B between the inlet

20 region 910 and the capture region 940 may have a radius 921 of at least 100 pm. In some implementations, the radius 921 may be at least 50 pm. In some implementations, the radius 921 may be at least 200 pm. In some implementations, the radius 921 may be at least 500 pm.

[0101] The outlet region 960 may be configured to funnel or otherwise convey sample fluid from the capture region 940 to an outlet. In the narrowing region 950, the microfluidic passage may progressively narrow in one or more dimensions perpendicular to a direction of flow. The flow velocity of the sample fluid may increase as the cross-sectional area of the microfluidic passage decreases towards the outlet region 960. In some implementations, one or both of the internal walls 930A and 930B of the microfluidic device 900 may have smooth surface between the capture region 940 and the narrowing region 950. In some implementations, the smooth surface may extend to the outlet region 960 as well.

[0102] The openings 905 and 965 may be defined in the top layer of the microfluidic device 900. Positioning the openings on the top surface of the microfluidic device (e.g., as shown in FIGS. 9 and/or 14) may improve manufacturability and reliability. For instance, it may be easier to create a reliable seal around an opening positioned on the top surface of the microfluidic device rather than on a side surface. For example, in a layered microfluidic device construction, a side surface of the microfluidic device may be irregular or have grooves or weaknesses between layers. It may be simpler from a manufacturing standpoint to create a smoother top surface on the microfluidic device. The smoother top surface may form a better seal with a tube, gasket, or ferule. In addition, openings positioned on the top surface of the microfluidic device may be made to arbitrary shape (including circular), while side openings may be constrained by the layered construction of the device to shapes (e.g., rectangular) that may be more difficult to interface with the tube or hose providing the sample fluid.

[0103] FIG. 10 shows a first view of a distribution of linear velocity of a fluid in the microfluidic device 900, according to some embodiments. FIG. 10 shows a distribution of the linear velocity of the fluid in a half height of the microfluidics channel of the microfluidic device 900. Threshold of the max value is set to 80 mm/s flow velocity. Similar to the distribution of flow velocity in in FIG. 5, the distribution of flow velocity in FIG. 10 shows a relatively high flow velocity in the inlet region 910 slowing to a lower flow velocity in the capture region 940; however, the distribution of flow velocity in FIG. 10 is more uniform than the distribution of flow velocity in FIG. 5 due, at least in part, to the smooth internal walls 930A and 930B, which reduce or eliminate dead and/or slow flow zones along the smooth internal walls 930A and 930B.

21 [0104] FIG. 11 shows an alternate view of the distribution of linear velocity of the fluid in the microfluidic device 900, according to some embodiments. The distribution is shown in a half height of the microfluidics channel. The velocity scale been adjusted to enhance flow non uniformities along the internal walls 930A and 930B. Threshold of the max value is set to 28 mm/s and 18 mm/s to min value. Although FIG. 11 reveals slow-flow zones 1110A, 1110B,

11 IOC, and 1110D along the internal walls 930A and 930B, the effect is reduced compared to the slow-flow zones of the microfluidic device 400, as shown in FIG. 6.

[0105] FIG. 12 shows multiple cross sections of the fluid in the microfluidic device 900, across which flow profiles may be measured, according to some embodiments. The cross sections include a first cross section 1220 across a middle portion of the widening region 920, a second cross section 1230 at a boundary of the capture region 940, a third cross section 1240 across a middle portion of the capture region 940, and a fourth cross section 1250 across the narrowing region 950. The second cross section 1230 may represent a boundary after which particles may begin to be effectively captured by the electrode(s). The fourth cross section 1250 may represent a boundary after which particles are no longer effectively captured by the electrode(s). The resulting flow profiles measured through each of the cross sections indicated in FIG. 12 are shown in graph 1300 of FIG. 13.

[0106] In the graph 1300, a first line 1320 illustrates a flow profile at the first cross section 1220 (in the widening region 920). A second line 1330 illustrates a flow profile at the second cross section 1230 (at or near a boundary of the capture region 940). A third line 1340 illustrates a flow profile at the third cross section 1240 (across a middle portion of the capture region 940). A fourth line 1350 illustrates a flow profile at the fourth cross section 1250 (in the narrowing region 950).

[0107] The second line 1330 in the graph 1300 shows improved flow profile uniformity at the second cross section 1230 (representing the leading boundary of the capture region 940) relative to the second line 830 in the graph 800. In addition, a maximum flow velocity at the trailing boundary of the capture region 940 (e.g., at the fourth line 1350) has been reduced from 55 mm/s for the microfluidic device 400 to 30 mm/s for the microfluidic device 900. The graph 1300 thus demonstrates improved uniformity of flow profile across both a width and length of the capture region 940. The design of the microfluidic device 900 may therefore exhibit improved capture efficiency and/or electrical efficiency over the design of the microfluidic device 400.

[0108] FIG. 14 illustrates a third example implementation of a microfluidic device 1400, according to some embodiments. The microfluidic device 1400 may have a body with internal

22 walls 1430A and 1430B (e.g., inward-facing surfaces) that define a microfluidic passage having an inlet region 1410, a widening region 1420, a capture region 1440, a narrowing region 1450, and an outlet region 1460. A sample fluid may enter the microfluidic passage via an opening 1405 leading to the inlet region 1410, and exit via an opening 1465 from the outlet region 1460. The openings 1405 and 1465 may be defined in a top surface of the microfluidic device 1400. One or more electrodes may generate a time-varying (AC) electric field within the capture region 1440 to capture sample particles within the sample fluid via a di electrophoretic force. The optical system 210 may detect the captured particles. Such particles may include, for example and without limitation, microorganisms, bacteria, yeast, mold, viruses, cells, T cells, NK cells, etc.

[0109] In some implementations, the inlet region 1410 may have a width 1411 between 100 pm and 30,000 pm. In some implementations, the width 1411 may be between 500 pm and 5,000 pm. In some implementations, the capture region 1440 may have a width 1441 of between 500 pm and 50,000 pm. In some implementations, the width 1441 may be between 1,000 pm and 10,000 pm. In some implementations, a ratio of the capture region width 1441 and the inlet region width 1411 may be between 1:1 and 30:1. In some implementations, the ratio of the width 1441 and the width 1411 may be between 2:1 and 10:1.

[0110] Similar to the microfluidic device 900, the widening region 1420 of the microfluidic device 1400 may be configured to slow the flow velocity of the sample fluid and spread the flow over a larger surface area within the capture region 1440 to increase interaction between sample particles and the electrode. The microfluidic passage may be defined by internal walls 1430A and 1430B, which may progressively increase in distance from each other between the inlet region 1410 and the capture region 1440. The internal walls 1430A and 1430B of the microfluidic device 1400 have smooth surfaces (e.g., without sharp inward or outward facing comers, cavities, and/or protrusions) between the inlet region 1410 and the capture region 1440. The smooth internal walls 1430A and 1430B may reduce or eliminate dead and/or slow flow zones, thereby improving the uniformity of flow profile across the capture region 1440. In some implementations, the smooth internal walls 1430A and 1430B may have one or more curves. In some implementations, any curve in the smooth internal walls 1430A and 1430B between the inlet region 1410 and the capture region 1440 may have a radius 1421 of at least 100 pm. In some implementations, the radius 1421 may be at least 50 pm. In some implementations, the radius 1421 may be at least 200 pm. In some implementations, the radius 1421 may be at least 500 pm.

23 [0111] The microfluidic device 1400 may include one or more protrusions into the microfluidic passage such as one or more pillars 1425. The pillar(s) 1425 may further contribute to a more uniform flow profile across a width of the capture region 1440 my slowing fluid flowing through the center of the widening region 1430 and/or the capture region 1440 relative to fluid flowing in the periphery (e.g., along the walls of the microfluidic passage). The pillar(s) 1425 may be of various shapes including cylindrical, as shown in FIG. 14, polygonal, elongated, etc. FIGS. 16 through 19, described below, show results of simulations of flow through the microfluidic device 1400.

[0112] The microfluidic device 1400 may also include one or more partitions 1435A and 1435B (singularly or collectively “partition(s) 1435”), which may divide part or all of the capture region 1440 into two or more passages. The partition(s) 1435 may further reduce the size and/or effect of dead and slow-flow zones, and cause a more linear and/or uniform fluid flow through the passages. In addition, the partition(s) 1435 may provide structural support to the microfluidic device 1400 by, for example, maintaining a distance between top and bottom of the microfluidic channel in the presence of internal pressure (e.g., of fluid in the microfluidic channel) and/or external pressure (e.g., applied via seals at the openings 1405 and/or 1465, and or other mechanical sources). In some implementations, the one or more partitions 1435 may be elongated and extend across some or all of a length of the capture region 1440 and, in some implementations, extend beyond a length of the capture region 1440 (e.g., into the widening region 1420 and/or narrowing region 1450). In some implementations, one or more partitions 1435A and/or 1435B may be parallel or substantially parallel to the internal walls 1430A and/or 1430B.

[0113] The more uniform flow profile provided by the pillar(s) 1425 and/or the partitions 1435 may increase capture efficiency of the microfluidic device 1400. The electrical efficiency may also be improved by enabling a lower electric field to be used to capture sample particles across a greater proportion of the capture region 1440. The increased capture efficiency may reduce false negatives (e.g., resulting from failing to capture and detect particles). Reducing dead and/or slow flow zones may also provide for more effective flushing of the microfluidic passage and removing of residue.

[0114] FIG. 15 illustrates electrical components of the microfluidic device 1400, according to some embodiments. A time-varying (AC) electric signal may be provided, via one or more electrical contacts 1547A and 1547B, to an electrode (or array of electrodes) 1545 adjacent to the capture region 1440. The electric signal applied to the electrode(s) 1545 may create a time- varying (AC) electric field within the capture region 1440. The electric field may exert a

24 dielectrophoretic force on sample particles within the sample fluid. The dielectrophoretic force may capture some or all of the sample particles on or near the electrode(s) 1545. The resulting dielectrophoretic force may capture some sample particles for detection by, for example, the optical system 210.

[0115] FIG. 16 shows a first view of a distribution of linear velocity of a fluid in the microfluidic device 1400, according to some embodiments. FIG. 16 shows a distribution of the linear velocity of the fluid in a half height of the microfluidics channel of the microfluidic device 1400. Threshold of the max value was set to 80 mm/s flow velocity. Similar to the distribution of flow velocity in in FIGS. 5 and FIG. 10, the distribution of flow velocity in FIG. 16 shows a relatively high flow velocity in the inlet region 1410 slowing to a lower flow velocity in the capture region 1440. Similar to FIG. 10, the distribution of flow velocity in FIG. 16 shows reduced dead and/or slow flow zones along the smooth internal walls 1430A and 1430B. The pillar(s) 1425 may further contribute to a more uniform flow profile across a width of the capture region 1440 my slowing fluid flowing through the center of the widening region 1430 and/or the capture region 1440 relative to fluid flowing in the periphery (e.g., closer to the internal walls 1430A and 1430B). In addition, flow linearity is improved across the capture region 1440.

[0116] FIG. 17 shows an alternate view of the distribution of linear velocity of the fluid in the microfluidic device 1400, according to some embodiments. The distribution is shown in a half height of the microfluidics channel. The velocity scale has been adjusted to enhance flow non-uniformities along the internal walls 1430A and 1430B. Threshold of the max value was set to 28 mm/s and 18 mm/s to min value. FIG. 17 reveals less slowing of flow locally along the internal walls 1430A and 1430B relative to the flow profile for the microfluidic device 900 as shown in FIG. 11. For example, slow flow zones 1710A and 1710B are reduced for the microfluidic device 1400 relative to the slow flow zones shown in those areas for the microfluidic device 900.

[0117] FIG. 18 shows multiple cross sections of the fluid in the microfluidic device 1400 at which flow profiles may be measured, according to some embodiments. The cross sections include a first cross section 1820 across a middle portion of the widening region 1420, a second cross section 1830 at a boundary of the capture region 1440, a third cross section 1840 across a middle portion of the capture region 1440, and a fourth cross section 1850 across the narrowing region 1450. The second cross section 1830 may represent a boundary after which particles may begin to be effectively captured by the electrode(s). The fourth cross section 1850 may represent a boundary after which particles are no longer effectively captured by the electrode(s). The

25 resulting flow profiles measured through each of the cross sections indicated in FIG. 18 are shown in graph 1900 of FIG. 19.

[0118] In the graph 1900, a first line 1920 illustrates a flow profile at the first cross section 1820 (in the widening region 1420). A second line 1930 illustrates a flow profile at the second cross section 1830 (at or near a boundary of the capture region 1440). A third line 1940 illustrates a flow profile at the third cross section 1840 (across a middle portion of the capture region 1440). A fourth line 1950 illustrates a flow profile at the fourth cross section 1850 (in the narrowing region 1450).

[0119] The graph 1900 shows improved flow profile uniformity at both the second cross section 1830 (representing the leading boundary of the capture region 1440) and the fourth cross section 1850 (representing a trailing boundary of the capture region 1440) relative to the cross- sectional flow profiles shown in the graph 1300. In addition, a maximum flow velocity at the trailing boundary of the capture region 1440 (e.g., at the fourth line 1950) has been reduced from 55 mm/s for the microfluidic device 400 to less than 30 mm/s for the microfluidic device 1400. The graph 1900 thus demonstrates further improved uniformity of flow profile across both a width and length of the capture region 1440. The design of the microfluidic device 1400 may therefore exhibit improved capture efficiency and/or electrical efficiency relative to the design of the microfluidic device 400.

[0120] The inventors have recognized that it may be desirable for a microfluidic system to have unified particle velocity entering the electrode area of the microfluidic device.

Subsequently, particles in the sample are exposed to the electric field created by the electrodes, which exerts a dielectrophoretic force on particles in the sample, and bacteria are captured on/near the electrode edges. The capture efficiency may relate to the resultant force of fluid flow against the sample particles and the dielectrophoretic force. A more uniform and/or constant flow force across the electrode area can improve capture efficiency. Accordingly, the inventors have recognized the benefit of improving uniformity of flow velocity, and thus flow force, across the electrode area of a microfluidic device.

[0121] Table 2 below compares flow velocity ranges for the example microfluidic devices 400, 900, and 1400 described herein. Data was generated for an input flow of 80 mm/s.

26

Table 2: Comparison of flow velocity ranges for the three example microfluidic devices

[0122] FIG. 20 illustrates a fourth example implementation of a microfluidic device 2000, according to some embodiments. The microfluidic device 2000 is similar to the microfluidic device 1400; however, the microfluidic device 2000 may include a structure of pillars 1425 in place of some or all of the partitions 1435. A structure of pillars 1425 distributed in the widening region 2020, the capture region 2040, and/or the narrowing region 2050 may be configured to provide a flow profile with even more uniform linear velocity within even tighter variation. In addition, the pillars 1425 may add structural support to the microfluidic channel by, for example, maintaining a distance between top and bottom of the microfluidic channel in the presence of internal pressure (e.g., of fluid in the microfluidic channel) and/or external pressure (e.g., applied via seals at the inlet and/or outlet, and or other mechanical sources). The number and placement of the pillars 1425 shown in FIG. 20 is one example configuration, and other numbers and/or positions of pillars 1425 are possible.

[0123] Having thus described several aspects and embodiments of the technology set forth in the disclosure, it is to be appreciated that various alterations, modifications, and improvements will readily occur to those skilled in the art.

[0124] Such alterations, modifications, and improvements are intended to be within the spirit and scope of the technology described herein. For example, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the embodiments described herein. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described. In addition, any combination of two or more features, systems, articles, materials, kits, and/or methods described herein, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

27 [0125] The above-described embodiments can be implemented in any of numerous ways. One or more aspects and embodiments of the present disclosure involving the performance of processes or methods may utilize program instructions executable by a device (e.g., a computer, a processor, or other device) to perform, or control performance of, the processes or methods. In this respect, various inventive concepts may be embodied as a computer readable storage medium (or multiple computer readable storage media) (e.g., a computer memory, one or more floppy discs, compact discs, optical discs, magnetic tapes, flash memories, circuit configurations in Field Programmable Gate Arrays or other semiconductor devices, or other tangible computer storage medium) encoded with one or more programs that, when executed on one or more computers or other processors, perform methods that implement one or more of the various embodiments described above. The computer readable medium or media can be transportable, such that the program or programs stored thereon can be loaded onto one or more different computers or other processors to implement various ones of the aspects described above. In some embodiments, computer readable media may be non-transitory media.

[0126] The terms “program” or “software” are used herein in a generic sense to refer to any type of computer code or set of computer-executable instructions that can be employed to program a computer or other processor to implement various aspects as described above. Additionally, it should be appreciated that according to one aspect, one or more computer programs that when executed perform methods of the present disclosure need not reside on a single computer or processor but may be distributed in a modular fashion among a number of different computers or processors to implement various aspects of the present disclosure.

[0127] Computer-executable instructions may be in many forms, such as program modules, executed by one or more computers or other devices. Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Typically, the functionality of the program modules may be combined or distributed as desired in various embodiments.

[0128] Also, data structures may be stored in computer-readable media in any suitable form. For simplicity of illustration, data structures may be shown to have fields that are related through location in the data structure. Such relationships may likewise be achieved by assigning storage for the fields with locations in a computer-readable medium that convey relationship between the fields. However, any suitable mechanism may be used to establish a relationship between information in fields of a data structure, including through the use of pointers, tags or other mechanisms that establish relationship between data elements.

28 [0129] The above-described embodiments of the present technology can be implemented in any of numerous ways. For example, the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as a controller that controls the above-described function. A controller can be implemented in numerous ways, such as with dedicated hardware, or with general purpose hardware (e.g., one or more processor) that is programmed using microcode or software to perform the functions recited above and may be implemented in a combination of ways when the controller corresponds to multiple components of a system.

[0130] Further, it should be appreciated that a computer may be embodied in any of a number of forms, such as a rack-mounted computer, a desktop computer, a laptop computer, or a tablet computer, as non-limiting examples. Additionally, a computer may be embedded in a device not generally regarded as a computer but with suitable processing capabilities, including a Personal Digital Assistant (PDA), a smartphone or any other suitable portable or fixed electronic device.

[0131] Also, a computer may have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include printers or display screens for visual presentation of output and speakers or other sound generating devices for audible presentation of output. Examples of input devices that can be used for a user interface include keyboards, and pointing devices, such as mice, touch pads, and digitizing tablets. As another example, a computer may receive input information through speech recognition or in other audible formats.

[0132] Such computers may be interconnected by one or more networks in any suitable form, including a local area network or a wide area network, such as an enterprise network, and intelligent network (IN) or the Internet. Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks.

[0133] Also, as described, some aspects may be embodied as one or more methods. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

29 [0134] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0135] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” [0136] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as anon-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0137] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0138] Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having,"

30 “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

[0139] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.

[0140] The terms “substantially”, “approximately”, and “about” may be used to mean within ±20% of a target value in some embodiments, within ±10% of a target value in some embodiments, within ±5% of a target value in some embodiments, within ±2% of a target value in some embodiments. The terms “approximately” and “about” may include the target value. [0141] User of ordinal terms such as “first,” “second,” “third,” etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claim elements.

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