Devices for assessing interactions and uses thereof

Abstract

Described here are devices and systems that allow for scalable on-chip screening of bioagents, chemicals, or combinatorial cellular libraries and use thereof, as well as methods of molding the devices. Bioagents or chemical samples are prepared to generate droplets. The devices described herein comprise arrays of microwells capable of receiving one or more individual droplets. The droplets are then transferred and pooled to be loaded to an MCSD described herein. Further provided herein are automated systems for high throughput screening comprising various components, such as a device described herein, a loading apparatus, one or more robot arms configured for automated loading and merging of droplets and automated transferring samples, an automated incubator and a controller. Also described here are methods for screening using the devices and systems described herein.

Description

Atty. Docket No. 202421-703601PCT PATENT APPLICATION DEVICES FOR ASSESSING INTERACTIONS AND USES THEREOF CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 788,468 filed April 14, 2025, which is incorporated by reference herein in their entirety.BACKGROUND

[0002] Biological cells exist in nature as part of ecosystems. Assembling many combinations of bioagents, chemicals, or bioagents-chemicals is logistically complex and difficult to achieve. Thus, there is a need for efficient and traceable systems for investigating combinatorial complexity of interactions between bioagents and / or chemicals.BRIEF SUMMARY

[0003] Provided herein are A method of screening, comprising: (a) providing a device, a pool of droplets comprising bioagents and / or chemicals, and one or more robotic arms, wherein the device comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents and / or chemicals; (b) mounting the device to a first robotic arm;(c) loading the one or more droplets in the pool to the device via an automated process that controls the first robotic arm to: (i) submerge or partially submerge the device in a bath unit configured to retain a pooled fluid that is immiscible with the droplets through the first robotic arm, wherein the device is tilted relative to the surface of the bath unit during the submerging step; (ii) add the droplets to the pooled; and (iii) move the device vertically relative to the surface of the pooled fluid such that one or more droplets are loaded to the device, thereby allowing the droplets to enter the device; (d) removing the device from the first robotic arm; (e) mounting the device to a second robotic arm;(f) moving the device to an imaging station using the second robotic arm;(g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the bioagents or chemicals in each droplet; (h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of bioagents and / or chemicals; (i) measuring optical signals of the combinations after the merging, wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms.

[0004] Provided herein are methods of screening for combinations of live cells with bioagents or chemicals, comprising: (a) providing a device, one or more robotic arms, and a pool of droplets comprising: (i) a population of live cells and (ii) bioagents and / or chemicals, wherein the deviceAtty. Docket No. 202421-703601comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets from the pool of droplets; (b) mounting the device to a first robotic arm; (c) loading the one or more droplets from the pool of droplets to the device by an automated process; (d) removing the device from the first robotic arm; (e) mounting the device to a second robotic arm; (f) moving the device to an imaging station using the second robotic arm; (g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the population of the live cells, bioagents or chemicals in each droplet; (h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of the population of the live cells, bioagents and / or chemicals; (i) measuring optical signals from the combinations after the merging, wherein the signals from combinations of the population of the live cells with the bioagents and / or chemicals indicate changes of activities of the population of live cells in presence of the bioagents and / or chemicals, wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms.

[0005] Provided herein are systems comprising: a micropatterned combinatorial screening device (MCSD) comprising an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; and a loading apparatus comprising: a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets; a holding unit for carrying the MCSD, wherein the holding unit is tilted at an angle relative to the surface of the pooled fluid.

[0006] Provided herein are methods of loading droplets for producing massively parallel combinations of bioagents or chemicals, comprising: (a) providing a micropatterned combinatorial screening device (MCSD), wherein the MCSD comprises: an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; (b) submerging or partially submerging the MCSD in a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets, wherein the array of microwells is tilted relative to the surface of the bath unit during the submerging step; (c) adding the droplets to the pooled fluid; and (d) moving the MCSD vertically relative to the surface of the pooled fluid such that the one or more droplets are loaded to the MCSD, thereby allowing the droplets to enter the MCSD to generate massively parallel combinations of cells or chemicals.

[0007] Provided herein are methods for label-free quantification of growth of a bioagent, the method comprising: providing a sample containing a bioagent in a growth medium to an array of microwells in a micropatterned combinatorial screening device (MCSD), wherein each microwell is capable of receiving one or more droplets; obtaining a plurality of label-free optical measurements from the sample over a predetermined time using the MCSD; generating data fromAtty. Docket No. 202421-703601the plurality of label-free optical measurements, wherein the data comprises optical density measurements, light transmittance measurements, entropy measurement, or phase contrast measurements; processing the data using at least one of the following: a deep neural network machine learning (ML) algorithm, wherein the deep neural network is trained to correlate one or more optical signal patterns with bacterial growth parameters; optical density measurement over time; light transmittance measurement over time; and entropy measurement over time; and determining at least one quantitative value relevant to the bacterial growth.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0009] FIGURE 1 is a flowchart showing a general workflow starting with upstream processes, including sample preparation and device design and manufacturing, followed by generation of droplets, transfer and pool of droplets, loading of droplets to device, sealing of device, and merging of droplets, ended with incubation and image data processing.

[0010] FIGURES 2A-2G are illustrations showing each step of an exemplary assay according to an embodiment of this disclosure including sample preparation (FIG. 2A), droplet generation (FIG. 2B), droplets loading (FIG. 2C), device sealing (FIG. 2D), pre-merge automated imaging (FIG. 2E), droplets imaging before merging (FIG.2F), droplet merging and droplets imaging after merging (FIG.2G).

[0011] FIGURES 3A-3E show illustrations of microwells and arrays of triangular posts in micropatterned combinatorial screening devices (MCSDs). FIG.3A illustrates several exemplary arrangements of microwells in an MCSD. FIG.3B is an illustration of an array of triangular posts with a zoom in showing a droplet situated within a group of six posts. FIG.3C is an illustration of an array of triangular posts showing unmerged droplets forming microwells of various sizes, 1 to 7, and having different shapes, circle, oval, triangle, trapezoid, parallelogram, and hexagon. FIG.3D is an illustration of an array of triangular posts showing repeats of microwells of the same size, having two or three droplets in a single microwell. FIG.3E is an illustration of single microwells having oval, triangle, and hexagon shapes with posts integrated into the edge of the microwells (top) and / or adhered to the central area of the microwells (middle and bottom).

[0012] FIGURES 4A-4D show embodiments of a mold. FIG. 4A, is a 3D-rendering of an exploded view of a mold, according to an embodiment of this disclosure. The mold comprises aAtty. Docket No. 202421-703601top piece and a back plate, such that the mold is configured to fit a wafer between the top piece layer and the back plate. FIG.4B, is a 3D-rendering of an exploded view of the mold illustrated in FIG. 4A. FIG. 4C at an alternate angle, is a 3D-rendering of an assembled view of a mold, according to another embodiment of this disclosure. The mold comprises a top piece, a mold material-constraining layer, and a back plate, such that the mold is configured to fit a wafer between the mold material-constraining layer and the back plate. FIG.4D, is a 3D-rendering of an exploded view of the mold illustrated in FIG.4C.

[0013] FIGURES 5A-5B are illustrations of a photolithography mask with fiducial marks. FIG.5A, is an illustration of a photolithography mask with fiducial marks and a wafer flat mark, according to an embodiment of this disclosure. FIG. 5B, is an expanded view of a fiducial mark for a k=2 mask, according to an embodiment of this disclosure.

[0014] FIGURE 6 is an image showing a close view of a droplet generator according to an embodiment of this disclosure.

[0015] FIGURES 7A-7H show embodiments of an MCSD in a holding unit. FIG. 7A is a side view image of an MCSD in a holding unit with a pipette and a tube filled with pooled droplets FIG. 7B, is a top view image of an MCSD in a holding unit. FIG. 7C, is an exploded side view image of an MCSD in a holding unit. FIG. 7D, is a side view image of an MCSD in a loading apparatus. FIG.7E, is an angled top view image of an MCSD in a holding unit. FIG.7F, is a zoom side view of a portion of an MCSD in a holding unit. A spring is shown in between two plates, and it is compressible by a wingnut tightening to a screw. FIG. 7G is a photograph perspective view of an exemplary holding unit. A spring is shown in between two plates, and it is compressible by a wingnut tightening to a screw. FIG. 7H is a photograph perspective view of an exemplary holding unit. A nylon spacer is shown in between two plates, and it is held in place by a wingnut tightening to a screw.

[0016] FIGURES. 8A-8B are photographs of an exemplary automated MCSD droplet loading apparatus. FIG.8A is a photograph showing the MCSD being suctioned to a robotic arm suspended over a tray of oil. FIG. 8B showing the automatic control of the MCSD through the robotic arm.

[0017] FIGURES 9A-9C are illustrations showing the top view (FIG. 9A), cut-away side view (FIG. 9B), and cut-away front view (FIG. 9C) of a bath unit according to an embodiment of this disclosure.

[0018] FIGURES 10A-10B are photographs showing the top view (FIG. 10A) and the side view (FIG. 10B) of an exemplary loading apparatus containing a holding unit, a bath unit, a robotic arm according to an embodiment of this disclosure.Atty. Docket No. 202421-703601

[0019] FIGURES 11A-11C are photographs illustrating an MCSD with a loading apparatus according to an embodiment of this disclosure. FIG. 11A shows mounting an MCSD to the holding unit. FIG. 11B and FIG. 11C shows the controlling of the MCSD via a robotic arm.

[0020] FIGURES 12A-12B show embodiments of a carrier. FIG. 12A, is a 3D-rendering of a carrier, according to an embodiment of this disclosure. FIG. 12B, is a photograph of a carrier, according to an embodiment of this disclosure.

[0021] FIGURE 13, is a 3D-rendering of an anaerobic carrier, according to an embodiment of this disclosure.

[0022] FIGURES 14A-14D show the configurations of an anaerobic carrier. FIG. 14A is a photograph of an anaerobic carrier, according to an embodiment of this disclosure FIG. 14B is a photograph of the anaerobic carrier of FIG. 14A with an inserted MCSD, according to an embodiment of this disclosure. FIG. 14C, is a photograph of the anaerobic carrier of FIG. 14B with a gasket and a lid, according to an embodiment of this disclosure. FIG. 14D, is a photograph of the anaerobic carrier of FIG. 14C, wherein the lid is clamped to the anaerobic carrier using a plurality of clips, i.e., 6 clips, to maintain a seal, according to an embodiment of this disclosure.

[0023] FIGURES 15A-15B are time-dependent spectra showing real-time monitored GFP signal change of E. coli-GFP (strain ai9) as compared to E. coli WT (strain ai9) under both aerobic (FIG.15A, hole poked) and anaerobic (FIG. 15B, sealed) conditions. The Y-axis represents the GFP signal intensity and the X-axis corresponds to time (hour).

[0024] FIGURE 16 is a plot showing E. coli GFP (strain ail2) maturation on an MCSD after imaging in anaerobic carrier as compared to E. coli WT (strain ai9). The Y-axis represents GFP fluorescence, whereas the X-axis corresponds to time (hours) after merging.

[0025] FIGURES 17A-17B are time-dependent UV-visible spectra (FIG. 17A) and fluorescence spectra (FIG. 17B) of E.co / z-UnaG without IPTG induction, but with bilirubin (br) addition at inoculation (br @ Oh), when IPTG was added for induction at inoculation with bilirubin addition (IPTG @ Oh, br @ Oh) , when IPTG was added for induction after 4 hours culturing and bilirubin was added after 18 hours culturing (IPTG @ 4h, br @ 18 h), and when IPTG was added for induction after 4 hours culturing and bilirubin was added at inoculation (IPTG @ 4h, br @ Oh). In FIG. 17A, the Y-axis represents optical density (OD) at 600 nm wavelength, whereas the X-axis corresponds to time (hour). In FIG. 17B, the Y-axis represents fluorescence intensity, whereas the X-axis corresponds to time (hour).

[0026] FIGURES 18A-18D are images of E.coli-UnaG cells without IPTG induction and bilirubin (br) addition (FIG. 18A), when IPTG was added for induction at inoculation without bilirubin addition (FIG. 18B), when IPTG was added for induction after 4 hours culturing and bilirubin wasAtty. Docket No. 202421-703601added after 18 hours culturing (FIG. 18C), and when 18 IPTG was added for induction after 4 hours culturing and bilirubin was added at inoculation (FIG. 18D). Images were taken after 26 hours culturing. The top and bottom images in each figure show the fluorescence and the morphology of live A. / z-UnaG cells, respectively.

[0027] FIGURES 19A-19B are time-dependent UV-visible spectra (FIG. 19A) and fluorescence spectra (FIG. 19B) of A. crz / z- Pepper when IPTG was added for induction at inoculation and HBC was either added at inoculation or at 24 h.In FIG. 19A, the Y-axis represents optical density (OD) at 600 nm wavelength, whereas the X-axis corresponds to time (hour). In FIG. 19B, the Y-axis represents fluorescence intensity, whereas the X-axis corresponds to time (hour).

[0028] FIGURES 20A-20C are images of live A. co / z- Pepper 28 h cultures without IPTG induction (FIG. 20A), with IPTG added at 0 h and HBC added at 0 h (FIG. 20B) and with IPTG added at 0 h and HBC added at 24 h (FIG. 20C). The top and the bottom images show the fluorescence and the morphology of live A". / z- Pepper, respectively.

[0029] FIGURES 21A-21F are images and plots showing an exemplary label-free assay for assessing the growth of microbes. FIG. 21A is an image showing optical observation of microbes in individual microwells of MCSD. FIG. 21B is an image showing a label-free C. acnes growth assay in an exemplary MCSD. FIG.21C and FIG.21E are zoomed-in views of the two microwells that are boxed in FIG. 21B. The zoomed-in view of the top microwell is shown in FIG. 21C. The zoomed-in view of the bottom microwell is shown in FIG.21E. FIG.21D and FIG.21F are plots showing the distribution of measured light transmittance values within the droplet area for microwells of FIG. 21C and FIG. 21E, respectively, as captured by a quantification model.

[0030] FIGURES 22A-22C are images showing optical observation of growth of C. albicans (FIG. 22A), S. aureus (FIG. 22B), and S. hominis (FIG. 22C) in individual microwells of an exemplary MCSD over time.

[0031] FIGURE 22D is a plot showing entropy-based growth of C. albicans, S. aureus, and S. hominis over time assayed with an exemplary MCSD. Y-axis represents the growth as determined by entropy measurements over a Laplacian of Gaussians (“LoG”) transformed image. X-axis represents time (hours).

[0032] FIGURE 23 shows S. aureus growth over time that is used as model training data.

[0033] FIGURE 24 is a plot showing the effect sizes of an exemplary target (P. aeruginosa) as determined using an MCSD versus a plate reader. Y-axis represents the effect sizes determined using a plate reader. X-axis represents the effect sizes as determined using an MCSD.Atty. Docket No. 202421-703601

[0034] FIGURES 25A-25B are images showing mammalian cells Jurkat E6-1 (JE6-1; ATCC TIB-152; human T lymphoblasts, FIG. 25A) and THP-1 (ATCC TIB-202; human monocytes, FIG. 25B) in droplets ready to be loaded onto an MCSD.

[0035] FIGURE 26 is an illustration of an automated system for droplet generation, loading, and sealing.

[0036] FIGURE 27 is an illustration of a robotic arm in the automated system which comprises a custom end of arm tool for picking up MCSDs.

[0037] FIGURE 28 is an illustration of the MCSD sealing portion of the system, provided in four perspectives.

[0038] FIGURE 29 is an illustration of the automated system in FIG. 26 with an additional robotic arm fitted with an electronic pipette for aspirating and dispensing of droplets.DETAILED DESCRIPTION

[0039] Provided herein are devices, systems, and methods for automated screening of randomly generated bioagents and / or chemicals via massively parallel assessment of combinatorial assemblages of droplet-encapsulated fluid volumes in a precise and efficient manner that requires minimal amount of manual labor. Briefly, further described are (1) systems; (2) automation applications; and (3) bioagents and chemicals.

[0040] A general workflow describing development of devices and assay methods is shown in FIG. 1. Briefly, micropattemed combinatorial screening devices (“MCSDs”) having an array of microwells for receiving sample inputs are designed and manufactured to incorporate fiducial marks to aid in downstream alignment of microscopy images. Bioagents or chemical samples are prepared to generate droplets. The droplets are then transferred and pooled to be loaded to an MCSD described herein. The MCSD is sealed after being loaded with droplets. After sealing, the MCSD is imaged for identification of droplet groupings. The droplet groupings in each microwell are then merged by a coalescing mechanism to generate combinations of bioagents and / or chemicals. The performance of the combinations is measured after incubation and time point scans.

[0041] Further provided herein are automated systems for high throughput screening comprising an MCSD, a loading apparatus, one or more robot arms configured for automated loading and merging of droplets and automated transferring samples, an automated incubator and a controller. The methods and devices provided herein for automation requires minimal human handling. The MCSD has been optimized so it comprises lower variation in thickness. Further provided herein are methods of making MCSDs with improvements to photomask design and mold design. Without bound to any theory, performing high-throughput screening with the automated systems reducesAtty. Docket No. 202421-703601cost and time (e.g., imaging time) and improves experimental consistency and reproducibility, especially when dealing with large-scale dataset.

[0042] The devices and systems provided herein can be used for screenings, such as aerobic screenings, anaerobic screenings, and examining interactions between chemicals and / or bioagents. Further provided herein are methods and systems of using the same with various cell types. In some embodiments, provided herein are methods and systems of using the same with anaerobic bacteria and yeast.

[0043] An exemplary method for high throughput screening using a system described herein is illustrated in FIGs. 2A-2G. As a first step, sample inputs are prepared and transferred to an optical plate. Optionally, a robotic arm carries the optical plate and transfers the optical plate to an imaging station to test the optical densities of the samples. Optionally, the content in each droplet is labeled with color barcode (FIG. 2A). The samples are loaded to droplet cartridges together with oil either manually or by using a programmed standard liquid handling robot. A droplet generator is used to convert the sample inputs to droplets. The size of droplets can be controlled by changing the flow rates of the oil and sample. Once each sample is converted to droplets, the droplets are collected in a single tube as a droplet pool. Optionally, the sample is converted to droplets using a droplet generator (FIG. 6). Droplets are then loaded to MCSDs in a loading apparatus comprising various units, such as a holding unit, a robotic arm, and a bath unit. In an exemplary embodiment, a micropatterned combinatorial screening device (MCSD) described herein is inserted into a holding unit attached to a robotic arm which positions the MCSD suspended over a pool of oil. The holding unit is tilted at an upward angle relative to the pool. The robotic arm lowers the MCSD down until the lower side of the MCSD is fully immersed in the oil while the higher side is still upward above the oil. The droplet pool is dispensed via pipette into oil right along the MCSD-oil interface (FIG.2C). As the angled MCSD moves up slowly under the control of the robotic arm, the droplet-air interface moves down relative to the MCSD. As droplets encounter new unfilled microwells, they rapidly enter and fill the unfilled microwells. After microwells of the MCSD are filled up with the droplets, the MCSD is taken off from the holding unit and sealed with a film (FIG. 2D). The sealed MCSD is then moved from the loading apparatus to a plate stacker (FIG. 2E, middle). The plate stacker can hold up to 14 MCSDs for large scale screening. A second robotic arm takes one MCSD at a time to a nearby imaging station for an initial scan of microwells under a microscope (“premerge image”) (FIG. 2E, left; FIG. 2F). After pre-merge image, the robotic arm moves the MCSD from the microscope to a merging station which contains a merging mechanism means, such as a corona treater (FIG. 2G). The corona treater generates an electric field, causing the droplets to spontaneously merge. After merging, the robotic arm moves the MCSD to a nearby incubatorAtty. Docket No. 202421-703601(FIG. 2E, right) for storage with controlled temperature until it is taken out for an eventual time point scan in the imaging station.Definitions

[0044] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included, unless the context clearly dictates otherwise.

[0045] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting of any embodiment. 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. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0046] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof.

[0047] The term “robotic arm” used herein refers to an electromechanical transport device or element that transports sample containers (e.g., an MCSD as described here) in various directions. In one embodiment, a robotic arm is controlled by a processor to transport sample containers to various stations (e.g., imaging station, stacker, or incubator). In another embodiment, a robotic arm refers to a motor of a loading apparatus and is connected with a holding unit to automate the vertical movement of a sample container (e.g., an MCSD as described herein) that is held by the holding unit.

[0048] The term “high throughput” refers to a capability of a system or method to enable quick performance of a very large number of experiments in parallel or in series. An example of a “highAtty. Docket No. 202421-703601throughput” system includes automation equipment with cell biology techniques to prepare, incubate, and / or conduct a large number of chemical, genetic, pharmacological, optical, and / or imaging analyses to screen one or more biological entities for at least one of a metabolite, an enzyme, a protein, a nucleic acid, a phenotype, a mutation, a metabolic pathway, a gene, an adaptation, and a capability, as discussed herein. According to some embodiments, “high throughput” refers to simultaneous or near simultaneous experiments on a scale ranging from at least about 96 experiments to at least about 10,000,000 experiments.Systems

[0049] Provided here are systems comprising a micropatterned combinatorial screening device (“MCSDs”) comprising arrays of microwells, wherein each microwell is capable of receiving one or more droplets and a loading apparatus for loading droplets to the device. In some embodiments, a system described herein further comprises a plurality of droplets deposited in each of the microwells (e.g., microwells on the MCSD), wherein each droplet comprises at least one type of bioagents or chemical compound or combinations of bioagents and / or chemical compounds. In some embodiments, devices and / or apparatus provided herein can form a module that is combined with other modules on a single interconnected machine. In some embodiments, a system described herein further comprises a plurality of modules or stations for processing the MCSD loaded with droplets.

[0050] Micropatterned combinatorial screening devices (“MCSDs”)

[0051] Provided here are systems comprising a micropatterned combinatorial screening device (“MCSDs”) comprising arrays of microwells, wherein each microwell is capable of receiving one or more individual droplets, and wherein the array comprises a variation in thickness across of less than about 0.5 mm. In some embodiments, each microwell of the array of microwells is approximately 80 pm to approximately 180 pm or more in diameter, approximately 80 pm to approximately 170 pm or more in diameter, approximately 125 pm to 165 pm in diameter, optionally approximately 130 pm to 160 pm in diameter, optionally approximately 135 pm to 155 pm in diameter, optionally approximately 140 pm to 150 pm in diameter, optionally approximately 145 pm to 150 pm in diameter, optionally approximately 148 pm in diameter, optionally precisely 148.2 pm in diameter. In some embodiments, each microwell of the array of microwells is approximately 110 pm to 120 pm deep. In some embodiments, each microwell of the array of microwells is approximately: 100 pm, 101 pm, 102 pm, 103 pm, 104 pm, 105 pm, 106 pm, 107 pm, 108 pm, 109 pm, 110 pm, 111 pm, 112 pm, 113 pm, 114 pm, 115 pm, 116 pm, 117 pm, 118 pm, 119 pm, 120 pm, 121 pm, 122 pm, 123 pm, 124 pm, 125 pm, 126 pm, 127 pm, 128 pm, 129Atty. Docket No. 202421-703601m, 130 pm, 131 pm, 132 pm, 133 pm, 134 pm, 135 pm, 136 pm, 137 pm, 138 pm, 139 pm, 140 pm, 141 pm, 142 pm, 143 pm, 144 pm, 145 pm, 146 pm, 147 pm, 148 pm, 149 pm, or 150 pm deep.

[0052] In some embodiments, an MCSD described herein comprises arrays of a given microwell geometry whose shape dictates the number (k) of droplets that can be grouped within the given microwell. In some embodiments, k is 1, 2, 3, 4, 5, 6, 7, or more. A k=2, k=3, or k=7 MCSD refers to an MCSD with each microwell configured to group 2, 3, or 7 droplets. FIG. 3A shows three different geometric shapes of microwells when k is 2, 3, or 7. In some embodiments, the choice of k depends on the goals of an assay condition using the device described herein.

[0053] In some embodiments, MCSDs described herein may further comprise posts to direct droplet loading into microwells. In some embodiments, the posts are integrated into the bottom of the microwells. An exemplary triangular post arrangement is shown in FIG. 3B, with carved microwells in FIG. 3C, and arrays of microwells in FIG. 3D. In this exemplary post arrangement the triangle edges are 40 pm as noted in FIG. 3B, and spacing between microwells is about 50 pm as noted in FIG. 3D. In some embodiments, three or more posts within a microwell encircle each droplet filled in a microwell, optionally six posts encircle each droplet in a microwell, optionally the posts are triangular, square, round, oval, rectangular, cylindrical or hemispherical, optionally each post is approximately 10 pm to 100 pm wide, optionally approximately 40 pm wide. Optionally, the posts are of a sufficient size to enhance loading of each of the majority of the microwells with one and only one droplet, as compared to a screening platform lacking said posts. In some embodiments, the posts are triangular. In some embodiments, in a triangular post arrangement, the triangle edges are from about 10 pm to about 100 pm. In some embodiments, in a triangular post arrangement, the triangle edges are about 10 pm, about 20 pm, about 30 pm, about 40 pm, about 50 pm, about 60 pm, about 70 pm, about 80 pm, about 90 pm, or about 100 pm. In some embodiments, the spacing between microwells is from about 5 pm to about 100 pm. In some embodiments, the spacing between microwells is about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, about 50 pm, about 55 pm, about 60 pm, about 65 pm, about 70 pm, about 75 pm, about 80 pm, about 85 pm, about 90 pm, about 95 pm, or about 100 pm. In some embodiments, one or more posts are positioned adjacent to the side of the microwells such that the posts are integrated into the sides of the microwells described herein. Exemplary embodiments showing microwells having posts integrated into the walls are illustrated in FIG. 3E. The top configuration illustrates a microwell in a k=2 MCSD having two posts integrated into the walls. The middle configuration illustrates a microwell in a k=3 MCSD having three posts integrated into the walls and one post centered in the microwell.Atty. Docket No. 202421-703601The botom configuration illustrates a microwell in a k=7 MCSD having six posts integrated into the walls and six posts sitting inside the central area of the microwell. In some embodiments, each microwell is configured to support up to 19 droplets. In some embodiments, each microwell is configured to support 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or more droplets.

[0054] In some embodiments, the MCSD is fabricated using hot embossing, molding of elastomers, injection molding, LIGA, soft lithography, silicon fabrication, or related thin film processing techniques. In some embodiments, due to the hydrophobic nature of some polymers, such as PDMS, which absorbs some proteins and may inhibit certain biological processes, a passivating agent is added to the MCSD. Exemplary passivating agents for inclusion include, but are not limited to, silanes, parylene, n-Dodecyl-b-D-maltoside (DDM), Tween-20, polyethylene glycol (PEG), albumin, and collagen. In some embodiments, the passivating agent comprises parylene C, a poly-monochloro para-xylylene, produced from dimer material, with one chlorine group per repeat unit on its main-chain phenyl ring.

[0055] In some embodiments, an MCSD described herein is generated through use of a photolithography mask, e.g., a photomask, and wafer design that allow for robust image analysis and improved consistency of resulting MCSD production. The photomask comprises an opaque plate with transparent areas that allow light to shine through in a defined pattern, such as, for example, corresponding to a microwell array. The photomask may be used during photolithography to control the pattern formed in a photoresist such that the pattern is transferred to a wafer to produce the plurality of MCSDs from the wafer. Specifically, the photomask is placed over the photoresist and UV light is shone through the photomask to crosslink the photoresist, such that un-crosslinked photoresist is washed away to leave the cross-linked photoresist in a defined pattern. In some embodiments, the wafer is made of silicon or glass. In some embodiments, the wafer comprises a 6-inch diameter. In some embodiments, the wafer comprises a missing section to create a wafer flat. In some embodiments, the photoresist is a positive photoresist or a negative photoresist, affecting whether the photoresist is crosslinked during exposure to UV light. In some embodiments, the negative photoresist is an epoxy-based negative photoresist, such as, for example, SU-8 photoresist. A thickness of the photoresist determines a height of the features. In some embodiments, the photomask comprises a plurality of fiducial marks. The fiducial marks may be used by image processing algorithms to align images of a given MCSD across time points. In some embodiments, the aligning may be performed manually or using automation systems. When performed manually, the fiducial marks reduce a time and increases ease for users to align the image perfectly between timepoints, as larger positional shifts of the MCSD can be accounted for. When performed with an automation system, the fiducial marks may be used to ensure correctAtty. Docket No. 202421-703601alignment between time points, regardless of whether a user can check on the alignment. In some embodiments, the photomask comprises a region on the MCSD comprising a plurality of k=l microwells. The region of k=l microwells allows individual sample inputs to be measured outside of any combinatorial context (e.g., k>l). In some embodiments, k=x, where x is the number of droplets that fill a given microwell. In some embodiments, k=l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the fiducial mark comprises a plurality of microwells in a specific pattern throughout the microwell field. In some embodiments, the photomask comprises a wafer flat mark. The wafer flat mark may allow for more accurate alignment of the photomask and wafer when the aligning is performed manually, which therefore allows the features in the middle of the MCSD to be evenly spaced from the MCSD’s edges.

[0056] In some embodiments, an MCSD described herein is generated through the use of a mold, wherein the mold comprises a plurality of layers for holding the wafer, the wafer comprising each of the plurality of microwells, and containing a mold material until curing of the mold material. In some embodiments, the mold material comprises a polymer. A height of the features based on the thickness of the photoresist determines a height of the features in the mold material. In some embodiments, the mold material comprises one or more of polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polycarbonate, styrene-ethylene-butylene-styrene (SEBS) and poly(methyl methacrylate) (PMMA). In some embodiments, the plurality of layers comprises a top piece and a black plate, wherein the top piece also serves as a mold materialconstraining layer. An exemplary embodiment according to this description is illustrated in FIGS.4A and 4B. In some embodiments, the plurality of layers comprises a top piece, a back plate, and a mold material-constraining layer between the top piece and the back plate. An exemplary embodiment according to this description is illustrated in FIGS.4C and 4D. In some embodiments, each of the plurality of layers comprises one or more layers made from acrylic or aluminum. The plurality of layers may be arranged such that the wafer is positioned between the mold materialconstraining layer and the back plate. In some embodiments, the back plate comprises a pocket configured to fit a shape of the wafer such that the wafer sits in the pocket of the back plate. In some embodiments, the mold material-constraining layer may be cut to sit on top of the wafer and align to a flat edge of the wafer such that the mold material-constraining layer is centered around the features that have been transferred to the wafer. In some embodiments, the pocket comprises a depth corresponding to a thickness of the wafer. In some embodiments, the depth is 2 mm. In some embodiments, the mold material-constraining layer is cut using laser cutting. The mold materialconstraining layer centering allows more even formation of the mold material, allowing for better loading of the MCSD due to more consistent droplet movement. Specifically, the mold designAtty. Docket No. 202421-703601allows for less mold material to seep between the wafer and the mold material-constraining layer, thus allowing more even, e.g., uniform, thickness of the MCSD. In some embodiments, the mold allows for the variation in thickness of the array to be less than about 0.5 mm. In some embodiments, the mold allows for the variation in thickness of the array to be less than about 0.4 mm. In some embodiments, the mold allows for the variation in thickness of the array to be less than about 0.3 mm. In some embodiments, the mold allows for the variation in thickness of the array to be about: 0.5 mm, 0.4 mm, 0.3 mm, or 0.2 mm. The mold material-constraining layer center also reduces imaging time, as fewer focus images are required with a reduced variation in z-height (e.g., depth) of the MCSD.Droplets

[0057] Provided herein are systems where a plurality of droplets is generated for analysis and loaded to an MCSD described herein when in fluidic contact with the MCSD. The term “droplets” described herein refer to small globules or drops composed of a first liquid in the body of a second liquid. The first and second fluids are immiscible with each other. In some embodiments, each droplet described herein encapsulates one or more chemicals or bioagents or a combination of both. The fluidic droplets may each be substantially the same shape and / or size. The shape and / or size can be determined, for example, by measuring the average diameter or other characteristic dimension of the droplets. The “average diameter” of a plurality or series of droplets is the arithmetic average of the average diameters of each of the droplets.

[0058] In some embodiments, a system described herein comprises a plurality of droplets deposited in each of the microwells of an MCSD described herein. In some embodiments, droplets are deposited in an MCSD together to generate massively parallel combinations of chemicals and / or bioagents. In some embodiments, each droplet comprises at least one type of bioagent or chemical compound. In some embodiments, processes and / or methods using the MCSDs described herein incorporate a combination of at least one biological cell and at least one chemical compound or mixture in each merged microwell. In some embodiments, an MCSD comprises droplets comprising at least two different types of biological cells. In some embodiments, an MCSD comprises single or merged droplets each comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more different types of biological cells.

[0059] In some embodiments, a majority of the individual droplets are of size approximately 120 pm to 150 pm in diameter, optionally 125 pm to 145 pm in diameter, optionally 130 pm to 140 pm in diameter. Further provided herein are encapsulated in droplets comprising a single type of biological cell material, or a molecular entity, or a mixture of either. In some embodiments, an MCSD described herein carries 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 200, 250, 500, 1,000 or more droplets.Atty. Docket No. 202421-703601In some embodiments, an MCSD carries more than 1,000, more than 2,000, more than 3,000, more than 4,000, more than 5,000, more than 6,000, more than 7,000, more than 8,000, more than 9,000, more than 10,000, more than 20,000, more than 30,000, more than 40,000, more than 50,000, more than 60,000, more than 70,000, more than 80,000, more than 90,000, more than 100,000, more than 110,000, more than 120,000, more than 130,000, more than 140,000, more than 150,000, more than 160,000, more than 170,000, more than 180,000, more than 190,000, more than 200,000, more than 210,000, more than 220,000, more than 230,000, more than 240,000, more than 250,000, more than 260,000, more than 270,000, more than 280,000, more than 290,000, more than 300,000, more than 310,000, more than 320,000, more than 330,000, more than 340,000, more than 350,000, more than 360,000, more than 370,000, more than 280,000, more than 390,000, more than 400,000, more than 410,000, more than 420,000, more than 430,000, more than 440,000, more than 450,000, more than 460,000, more than 470,000, more than 480,000, more than 490,000, more than 500,000, more than 510,000, more than 520,000, more than 530,000, more than 540,000, more than 550,000, more than 560,000, more than 570,000, more than 580,000, more than 590,000, more than 600,000, more than 610,000, more than 620,000, more than 630,000, more than 640,000, more than 650,000, more than 660,000, more than 670,000, more than 680,000, more than 690,000, more than 700,000, more than 710,000, more than 720,000, more than 730,000, more than 740,000, more than 750,000, more than 760,000, more than 770,000, more than 780,000, more than 790,000, more than 800,000, more than 810,000, more than 820,000, more than 830,000, more than 840,000, more than 850,000, more than 860,000, more than 870,000, more than 880,000, more than 890,000, more than 900,000, more than 910,000, more than 920,000, more than 930,000, more than 940,000, more than 950,000, more than 960,000, more than 970,000, more than 980,000, more than 990,000, or more than 1,000,000 droplets. In some embodiments, an MCSD carries up to 100,000, up to 110,000, up to 120,000, up to 130,000, up to 140,000, up to 150,000, up to 160,000, up to 170,000, up to 180,000, up to 190,000, up to 200,000, up to 210,000, up to 220,000, up to 230,000, up to 240,000, up to 250,000, up to 260,000, up to 270,000, up to 280,000, up to 290,000, up to 300,000, up to 310,000, up to 320,000, up to 330,000, up to 340,000, up to 350,000, up to 360,000, up to 370,000, up to 280,000, up to 390,000, up to 400,000, up to 410,000, up to 420,000, up to 430,000, up to 440,000, up to 450,000, up to 460,000, up to 470,000, up to 480,000, up to 490,000, up to 500,000, up to 510,000, up to 520,000, up to 530,000, up to 540,000, up to 550,000, up to 560,000, up to 570,000, up to 580,000, up to 590,000, up to 600,000, up to 610,000, up to 620,000, up to 630,000, up to 640,000, up to 650,000, up to 660,000, up to 670,000, up to 680,000, up to 690,000, up to 700,000, up to 710,000, up to 720,000, up to 730,000, up to 740,000, up to 750,000, up to 760,000, up to 770,000, up to 780,000, up to 790,000, up to 800,000, up to 810,000, up to 820,000, up to 830,000, up toAtty. Docket No. 202421-703601840,000, up to 850,000, up to 860,000, up to 870,000, up to 880,000, up to 890,000, up to 900,000, up to 910,000, up to 920,000, up to 930,000, up to 940,000, up to 950,000, up to 960,000, up to 970,000, up to 980,000, up to 990,000, or up to 1,000,000 droplets.

[0060] In some embodiments, the identity and position of the droplets within microwells is determined. In some embodiments, determination is a measurement of one or more fluorescent agents, proportions of which can be varied depending upon the contents of the particular droplet, allowing for determination of a ratio of such agents within a selected droplet. In some embodiments, droplets are distinguished based on luminescent and / or fluorescent dye content. Exemplary droplet identity fluorescent dyes for including are, but not limited to, Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, and / or Alexa Fluor 647. Such dyes can be employed in some embodiments to avoid wavelength overlap with optical assay fluorescent dyes. In some embodiments, luminescence and / or fluorescence-based assay signals are employed for optical assays of the microwell contents. Exemplary agents for such assays include, but are not limited to, green fluorescent protein (GFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), and resorufin.

[0061] In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on morphological traits of cells. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on growth of cells. In some embodiments, the growth of cells within a droplet is measured by measuring the density of cells. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on biological processes including but not limited to germination, sporulation, filamentation, and / or hyphal formation. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on cell clumping, aggregation, or diffuseness. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on cell motility, cell shapes, cellular group, or colony shapes. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on metabolic activity or state of cells. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on presence and level of specific metabolites produced by cells. In some embodiments, individual droplets and / or merged droplets of the MCSDs are screened based on presence / absence and / or levels of gene expression in cells. In further embodiments, individual droplets and / or merged droplets of the MCSDs are optically screened. The optical screening can include measurement of optical interference, luminescence, fluorescence, and / or brightfield. Exemplary agents for producing a detectable signal include, without limitation, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 536, Alexa Fluor 546, AlexaAtty. Docket No. 202421-703601Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, Alexa Fluor 790, RGF, CFP, GFP, YFP, mCherry, tdTomato, Venus, mNeonGreen and / or resorufin fluorescence. In some embodiments, where the fluorescence of one or more agents such Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 and / or Alexa Fluor 647, is measured, a ratio or ratios of Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594 and / or Alexa Fluor 647 identifies the single type of a chemical or a bioagent present in the individual droplet. In some embodiments, droplets are tagged for identification. In some embodiments, droplets containing discrete microbes are prepared and loaded onto an MCSD described herein, and microwells of the MCSD are designed to receive one or more droplets per microwell, as described elsewhere herein.

[0062] In some embodiments, a system described herein further comprises a droplet generator for converting samples to droplets. The term “droplet generator cartridge” used herein refers to a cartridge comprising arrays of input wells for depositing liquid sample to be converted to droplets. In some embodiments, a droplet generator delivers air pressure to the input wells through a custom manifold and controlled by a pressure controller to convert liquid sample inputs to droplets (FIG.2B). In some embodiments, droplets are produced on a Bio-Rad QX200 Droplet Generator. In some embodiments, droplets are produced on a customized droplet generator. An exemplary droplet generator module is illustrated in FIG. 6. As shown in FIG. 6, the droplet generator processes four droplet generator cartridges in parallel. The size of droplets can be controlled by changing the air pressure applied to the input wells, which in turns changes the flow rates of the oil and sample. Once each sample is converted to droplets, the droplets are collected in a single tube as a droplet pool.

[0063] In some embodiments, a system described herein further comprises a coalescing mechanism for merging at least two droplets in a microwell to form a single droplet. Droplets loaded into microwells are assessed and subjected to merging. Merging can occur by a mechanism sufficient to coalesce two or more droplets into a single droplet. Exemplary mechanisms for inclusion include, without limitation, electrocoalescence, thermal coalescence, acoustic coalescence, vortexing, or changes in surfactant concentration. In some embodiments, the droplets are merged by electrocoalescence. In some embodiments, the droplets are merged by a corona treater. A “corona treater” described herein is a module that produces an electric field. In some embodiments, the droplets are merging by rubbing the MCSD with a Kimwipe. After merging, individual droplets within each microwell are merged into a single merged droplet. In some embodiments, a corona treater is BD-20AC Laboratory Corona Treater from Electro-Technic Products.Atty. Docket No. 202421-703601

[0064] In some embodiments, across the array, individual droplets in k = 2-50 microwells, optionally in k = 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 microwells, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 droplets are merged into single merged droplets. In some embodiments, each single merged droplet harbors one or more types of bioagents and / or chemicals or mixtures. In one embodiment, at least one of the single merged droplets harbor a combination of at least one fungus and at least one bacteria. Optionally, a plurality of the single merged droplets harbor a combination of at least one fungus and at least one bacteria. Optionally, a majority of the single merged droplets harbor a combination of at least one fungus and at least one bacteria.

[0065] In some embodiments, at least one of the single merged droplets harbors a combination of at least one fungus and at least one chemical compound or mixture. In some embodiments, a plurality of the single merged droplets harbor a combination of at least one fungus and at least one chemical compound or mixture. In some embodiments, a majority of the single merged droplets harbor a combination of at least one fungus and at least one chemical compound or mixture.

[0066] In some embodiments, at least one of the single merged droplets harbors a combination of at least one bacteria and at least one chemical compound or mixture. In some embodiments, a plurality of the single merged droplets harbor a combination of at least one bacteria and at least one chemical compound or mixture. In some embodiments, a majority of the single merged droplets harbor a combination of at least one bacteria and at least one chemical compound or mixture. In an embodiment, at least one of the single merged droplets harbors a combination of at least one bacteria, at least one fungus and at least one chemical compound or mixture. In some embodiments, a plurality of the single merged droplets harbor a combination of at least one bacteria, at least one fungus and at least one chemical compound or mixture. In some embodiments, a majority of the single merged droplets harbor a combination of at least one bacteria, at least one fungus and at least one chemical compound or mixture.

[0067] In some embodiments, after merging, each microwell comprises at least two different types of microbes, three different types of microbes, four different types of microbes, at least five different types of microbes, optionally at least six different types of microbes, optionally at least seven different types of microbes, optionally at least eight different types of microbes, optionally at least nine different types of microbes, optionally at least ten different types of microbes, optionally at least eleven different types of microbes, optionally at least twelve different types of microbes, optionally at least thirteen different types of microbes, optionally at least fourteen different types of microbes, optionally at least fifteen different types of microbes, optionally at least sixteen different types of microbes, optionally at least seventeen different types of microbes,Atty. Docket No. 202421-703601optionally at least eighteen different types of microbes, or optionally at least nineteen different types of microbes.Loading apparatus

[0068] Provided herein are systems comprising an MCSD described herein and a loading apparatus. A “loading apparatus” described herein refers to a set of equipment designed for loading droplets to an MCSD described herein. In some embodiments, a loading apparatus comprises one or more units including but not limited to a bath unit, a holding unit, and a robotic arm. In some embodiments, a loading apparatus comprises a holding unit. A “holding unit” described herein refers to an equipment for carrying an MCSD described in place to assist loading of droplets.

[0069] In some embodiments, a holding unit described herein comprises a first scaffold plate and a second scaffold plate, wherein the MCSD sits on the top of the first scaffold plate with the microwells upside down; a plurality of solid compressible spacers that when inserted define a flow channel beneath the one or more arrays of microwells; one or more fasteners for securing the first scaffold plate and the second scaffold plate such that the two scaffold plates are secured against each spacer of the plurality of solid compressible spacers. In some embodiments, the compressible spacers are springs. In some embodiments, the compressible spacers are solid. In some embodiments, each of the plurality of solid compressible spacers are plastic. In some embodiments, each of the plurality of solid compressible spacers comprises rubber, polyurethane, polyethylene, or silicon. In some embodiments, the compressible spacers are nylon. An exemplary holding unit with an MCSD sitting on the top of the top scaffold plate is illustrated in FIG. 7A. FIGS. 7A-7H.In some embodiments, an exemplary MCSD holding unit is illustrated in side view in FIG. 7A.As can be seen, pooled droplets of cells are collected and deposited in a flow channel that interfaces with an MCSD that rests on a top scaffold plate described herein. Continuing with the exemplary MCSD of FIG. 7A, a top view can be seen in FIG. 7B showing an MCSD that rests on the top scaffold plate of a holding unit. From the exploded side view, FIG. 7C, an MCSD comprising a backing piece 711 and a body 709 is shown above a holding unit. The holding unit comprises two scaffold plates, i.e., a first scaffold plate 707 and a second scaffold plate 705, screws with wingnut fasteners and springs and a base 701. FIG. 7D shows a side view of the device with an MCSD resting on the first scaffold plate described in FIG. 7A. The same arrangement is shown in FIG.7E from an angled top view. Looking at a zoom of a fastener unit in FIG. 7F, a base 701 is shown onto which the second scaffold plate 705 and the first scaffold plate 707 of a holding unit are positioned, an MCSD is placed on the holding unit such that the backing piece 711 of the MCSD sits on the top of the first scaffold plate 707 and the body 709 of the MCSD extends over the frame of the first scaffold plate 707, a spring 713 is situated between the second scaffold plate 705Atty. Docket No. 202421-703601(optionally transparent) and the first scaffold plate 707, and a wingnut 715 and a socket head cap screw 703 fasten the second scaffold plate 705 and a first scaffold plate 707 in furtherance of creating a fluid path. FIG. 7G shows a photograph of a perspective view of the holding unit described in FIG. 7A, showing the socket head cap screw 703, the second scaffold plate 705, the first scaffold plate 707, the spring 713, and the wingnut 715. FIG. 7H shows a photograph of an additional embodiment of a holding unit described in FIG. 7G, though with the springs 713 replaced with solid spacers 714, such as, for example, nylon spacers.

[0070] As illustrated in FIG. 7A, a loading apparatus provided herein can comprise a flow path or flow space between an MCSD described herein and a base or other surface below and facing the MCSD (FIG.7A). When in a relaxed state, the flow space can be about 1 mm to 4 mm wide. Flow space distance can be maintained by a variety of mechanisms including repulsive magnetic force or physical resistance. In some embodiments, surfaces forming the flow path are pre-wetted with an injection of oil to fill the entirety of the flow space. Once injected, buoyant in the surrounding oil, the droplets are distributed around the flow space, optionally via tilting the loading apparatus. In some embodiments, the MCSD comprises a size of about 85 mm x about 120 mm. In some embodiments, the MCSD comprises a planar area of at least 250, 500, 750, 10000, 11000, 12000, 12500, 15000, 20000 or more mmA2. In some embodiments, a loading apparatus described herein comprises a bath unit or a flow channel.

[0071] In some embodiments, the loading of droplets is performed with pipettes, as exemplified in FIG. 7A. In some embodiments, the loading comprises filling the droplets into the flow space between an MCSD and the second scaffold plate such that the droplets randomly enter each of the microwells. In some embodiments, the flow space faces each of a feature side of each of the microwells. In some embodiments, the flow space may be controlled using a plurality of spacers, allowing for more even spacing between the second scaffold plate and the microwells such that movement of the droplets through the flow space is consistent. Due to the even thickness of the MCSDs, the spacers allow for adjustment of the spacing for changing droplet movement, thereby allowing further customization of droplet movement based on user requirements. Consistent droplet movement during the loading also leads to improved loading efficiency of each of the plurality of microwells. In some embodiments, each of the plurality of spacers is made of nylon.

[0072] In some embodiments, a system described herein comprises a MCSD described herein comprising an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; and a loading apparatus wherein the loading apparatus comprises: a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets; and a holding unit for carrying the device, wherein the holding unit is tilted at an angleAtty. Docket No. 202421-703601relative to the surface of the pooled fluid. In some embodiments, the holding unit is tilted at an angle of 5 degrees to 85 degrees relative to the surface of the pooled fluid. In some embodiments, the holding unit is tilted at an angle of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 degrees relative to the surface of the pooled fluid. In some embodiments, the holding unit is tilted at an angle of 10 degrees relative to the surface of the pooled fluid. In some embodiments, an MCSD described herein is removably attached to the holding unit. In some embodiments, an MCSD described herein is suctioned to the holding unit. An exemplary holding unit to which an MCSD is suctioned is illustrated in FIGS. 8A and 8B. As shown in FIGS. 8A and 8B, the holding unit is suctioned to the backing piece of an MCSD. In some embodiments, the backing piece comprises glass-like material sufficient to create an adherence by suction. In some embodiments, the backing piece comprises acrylic. In some embodiments, a holding unit comprises a mounting piece such that an MCSD described herein can be slid into the holding unit as illustrated in FIG.11 A. In some embodiments, the holding unit is titled at an angle relative to the surface of the pooled fluid.

[0073] In some embodiments, a loading apparatus described herein further comprises a robotic arm connected to the holding unit. In some embodiments, an MCSD described herein, after being mounted to the holding unit, can be moved vertically via control of the robotic arm (FIGS. 11B and 11C). After being mounted to the holding unit, the robotic arm positions the MCSD suspended over a pool of oil. The holding unit is tilted at an upward angle relative to the pool. The robotic arm lowers the MCSD down until the lower side of the MCSD is fully immersed in the oil while the higher side is still upward above the oil. The droplet pool is dispensed via pipette into oil right along the MCSD-oil interface (FIG.11C). As the angled MCSD moves up slowly under the control of the robotic arm, the droplet-air interface moves down relative to the MCSD. As droplets encounter new unfilled microwells, they rapidly enter and fill the unfilled microwells.

[0074] In some embodiments, a bath unit described herein is configured to retain a pooled fluid that is immiscible with the one or more droplets. In some embodiments, the pooled fluid is oil. In some embodiments, the pooled fluid is mineral oil. In some embodiments the pooled fluid is fluorinated oil. In some embodiments, a bath unit described herein comprises one or more ports in or adjacent to the bottom surface of the bath unit for controlling flow of the pooled fluid and one or more outlet for removing liquid in the bath unit. An exemplary configuration of a bath unit according to one of the embodiments is illustrated in FIGS. 9A-9C. As shown in FIG. 9A, pooled fluid can pass through the port in the bottom surface of the bath unit, while pooled fluid with unloaded droplets can drain from the outlet adjacent to the top of the bath unit. In some embodiments, the bath unit further comprises pumps, a reservoir, a waste container, or anyAtty. Docket No. 202421-703601combination thereof. An exemplary configuration of a system comprising an MCSD and a loading apparatus comprising a bath unit comprising an oil reservoir, a plurality of valves, and a plurality of pumps is illustrated in FIGS. 10A-10B. In some embodiments, as illustrated in FIGS. 11A-11C, the process for loading droplets into an MCSD described herein comprises: tilting the MCSD relative to the surface of a bath unit described herein; submerging or partially submerging the MCSD in a bath unit described herein, adding the droplets to the pooled fluid; and moving vertically the MCSD such that the one or more droplets are loaded to the MCSD, thereby allowing the droplets to enter the MCSD to generate massively parallel combinations of cells or chemicals. In some embodiments, the moving step is repeated until the droplets are loaded to more than 50% of the microwells. In some embodiments, the moving step is repeated until the droplets are loaded to more than 60%, more than 70%, more than 80%, more than 90%, or more than 90% of the microwells. In some embodiments, the moving step is repeated until the droplets are loaded to all of the microwells.

[0075] In some embodiments, a robot arm is able to pick up an MCSD from a stack using a suction cup as illustrated in FIG 27. The robot arm is then able to submerge the MCSD in the pooled oil in the bath unit illustrated in FIG 26. The robot arm can hold the MCSD at an angle relative to the oil surface and uses the machine vision camera illustrated in FIG 27 to adjust the position of the MCSD relative to the surface of the oil. In some embodiments, a second robot arm with an electronic pipette mounted to it aspirates droplets from a tube and dispenses them under the MCSD. In some embodiments, the MCSD is withdrawn from the pool of oil and transferred to the sealing device as illustrated in FIG 26 and FIG 28. In some embodiments a seal is placed on top of the bottom roller. The MCSD is positioned between the top and bottom rollers. The bottom roller is pushed up with the two actuators, pressing the seal against the roller. In some embodiments, the top roller is rotated by a motor connected via a belt. Friction between the top roller and the back of the MCSD causes the MCSD and the seal to be pushed through the sealer.Carrier

[0076] In some embodiments, a system described herein further comprises a carrier for assisting the automated handling of the MCSDs described herein. In some embodiments, as illustrated in FIGS. 12A, the carrier comprises a protrusion, such as a lip, on an inside of the carrier, such that when an MCSD is inserted into the carrier, a backing piece of the MCSD is supported by the protrusion. In some embodiments, dimensions of the carrier may conform to ANSI SLAS 4-2004 (R2012) microplate standards, with a length of 127.76 mm, width of 85.48 mm and height of 15 mm. The dimensions allow the carrier to be handled the same way as a microplate using automationAtty. Docket No. 202421-703601tools, such as, for example through robot arms and / or an automated incubator that is sized for standard, off-the-shelf grippers. Further, the dimensions allow standard-height plate storage stacks to be used in the incubator without further modification to handling of the MCSDs. Further, for example, the dimensions of the carrier allow the MCSD to be moved on and off a microscope and into the incubator through the automation tools.

[0077] In some embodiments, the carrier is an anaerobic carrier. The anaerobic carrier comprises the carrier described herein with modifications, wherein the modifications are configured to maintain anaerobic conditions for each of the systems. In some embodiments, the modifications allow for airtight sealing of the MCSD to the anaerobic carrier. In some embodiments, a first plate, comprising a glass material or glass-like material, is adhered to a bottom side of the anaerobic carrier and sealed to the anaerobic carrier with a sealant. The MCSD described herein may then be inserted into the anaerobic carrier above the first plate, and a second plate is further placed onto the anaerobic carrier above the MCSD as a lid. In some embodiments, a gasket is inserted between the anaerobic carrier and the second plate to ensure an airtight seal between the anaerobic carrier and the second plate. In some embodiments, the second plate is clamped to the anaerobic carrier, such as, for example, using clips, clasps, braces, brackets, or fasteners. In some embodiments, once the MCSD has been assembled in the anaerobic carrier and sealed, the assembly may be moved to the microscope while maintaining anaerobic conditions for imaging. In some embodiments, the anaerobic carrier may be fabricated through 3D printing or Computer Numerical Control (“CNC”) machining.Optical Imaging or Screening

[0078] In some embodiments, a system provided herein further comprises an imaging station for performing optical imaging or screening. In some embodiments, optical imaging or screening is performed using a camera located in the base. In some embodiments, the MCSD is relocated from the loading apparatus described herein to a different location for viewing by a camera. In some embodiments, the camera viewing includes a lamp. In some embodiments, the camera viewing includes a visible spectrum lamp. In some embodiments, the optical screening comprises measurement of luminescence and / or fluorescence, optionally autofluorescence, optionally in a label-free optical assay, optionally wherein the optical screening comprises measurement of GFP, YFP and / or resorufin fluorescence. In some embodiments, measurement of fluorescence comprises using a filter and a photomultiplier tube to detect emitted light. In some embodiments, the system for fluorescence measurement comprises a light source and filters for stimulating fluorescence andAtty. Docket No. 202421-703601a photomultiplier tube to detect light emitted by a sample using fiber optic cables to send and receive light.

[0079] In some embodiments, the optical screening comprises a label-free optical assay. The term “label-free assay” or “label-free approach” means droplets are assayed based upon analysis without fluorescence-based technologies. In some embodiments, with a label-free approach described herein, brightfield images are taken for the droplets containing cells to assay the growth and morphology of the cells. In some embodiments, where an MCSD is used for assaying the growth and morphology of the cells, brightfield images are taken over the time course for the droplets containing cells in an MCSD (e.g., FIG. 21A). For example, as shown FIG. 21A, in a label-free C. acnes growth assay conducted with an MCSD, brightfield images were captured at a certain time point. As the bacteria grew, the growth of C. acnes in each cell (e.g., FIG.21C and FIG. 21E, which are the zoomed-in views of the two microwells boxed in FIG. 21B) could be easily characterized by light transmittance as assessed by measuring pixel intensities within the images taken by the camera (FIG. 21D and FIG. 21F).

[0080] In some embodiments, a dye or stain is added to the droplets, wherein the dye or stain specifically binds a bioagent and fluoresces. In some embodiments, the dye or stain comprises a compound that is chemically modified, metabolized, or otherwise transformed by an enzyme or an organism, such that the transformation produces a detectable change in one or more fluorescent characteristics of the compound. In some embodiments, after the dye or stain is added to the droplets, fluorescent images are taken for the droplets containing cells to assay the growth and morphology of the cells. In some embodiments, where an MCSD is used for assaying the growth and morphology of the cells, fluorescent images are taken over the time course for the droplets containing cells in an MCSD.

[0081] In some embodiments, the growth of a bioagent is assessed by a computer vision assessment utilizing a ResNet-50 based Machine Learning (ML) algorithm that detects the growth of the bioagent. The ML model used for growth assessment is trained per target on a linear time course of images, which provide snapshots of different amounts of growth. These timepoints provide the units of measurement used throughout the analysis.

[0082] In some embodiments, the growth of a bioagent is assessed by classical image processing methods. In some embodiments, for example, a battery of metrics, such as image intensity and entropy, are calculated using classical image processing techniques. In some embodiments, the assessment comprises selecting the best image features to complement the ML-based growth quantification model as described herein. A separate linear ML model is trained on the data derived from all different analyses in order to optimally combine them into a “final growth score,” whichAtty. Docket No. 202421-703601is a quantitative value for the bioagent to be assessed. As exemplified in FIG. 22A, FIG. 22B, FIG. 22C, and FIG. 22D, brightfield images were taken for each bioagent containing cell (C. albicans in FIG.22A, S. aureus in FIG.22B, and S. hominis in FIG.22C) in an exemplary MCSD.An entropy-based growth metric shown in FIG. 22D, which accurately characterizes relative growth rates of phenotypically different targets.

[0083] In some embodiments, a system described herein comprises a fluorescence microscope which can be used for the screening of merged droplets in microwells. Both label and label-free approaches have been exemplified herein and are contemplated for further application. In exemplified embodiments, fluorescence microscopy was performed using a Nikon Ti2-E inverted fluorescence microscope with fluorescence excitation by a Lumencor Sola light emitting diode illuminator (100% power setting). Each signal from the samples within the microwells of an MCSD described herein (e.g., signal from a dye) was detected with a different excitation wavelength generated by a collection of filter cubes. In certain aspects, growth of complex microbial communities can be measured in massively parallel fashion across varying carbon sources. It is further contemplated that growth or other fluorescent readouts from the microwells could be monitored in the presence or absence of other agents, including probiotics, antibiotics and / or a wide range of small molecule drugs, peptides or other biologies, optionally in the presence or absence of varying other environmental conditions (e.g., temperature or other conditions). In certain embodiments, combination phenotypes that can be tracked via optical assays performed upon the MCSD include fluorescent protein expression and respiration-driven reduction of resazurin to the fluorescent product resorufin, among others. In some embodiments, MCSD combination phenotypes in the microwells of an MCSD described herein can be tracked via use of phase contrast microscopy as a readout, among other contemplated readouts.

[0084] In some embodiments, after the MCSD has been imaged one or more times and a certain amount of time has passed, the featured surface of the MCSD is cooled. In some embodiments, the MCSD is cooled after 1 minute to 10 minutes. In some embodiments, the MCSD is cooled after 10 minutes to 1 hour. In some embodiments, the MCSD is cooled after 1 hour to 24 hours. In some embodiments, the MCSD is cooled after 1 day to 7 days. Various methods can be used to cool the surface, including placing the surface of the MCSD against a flat surface, such as a block of metal, which has been cooled to a sufficient temperature to solidify the medium contained inside the droplets within the device. After sufficient time has passed at a suitable temperature, the adhesive seal on the surface of the MCSD is removed and the MCSD is immersed in a fluorinated oil for loading additional droplets.Atty. Docket No. 202421-703601

[0085] There are many benefits of using this method. In some embodiments, the unsealing and additional droplet loading process described herein allows for non-optical, spatially-encoded detection methods, which expands the potential applications of MCSD to measure other biological indicators which cannot easily be encoded as optical signals. In some embodiments, because the unsealing process allows the MCSD to preserve the spatial information about which test articles are present in which microwells, techniques such as spatial sequencing, spatial transcriptomics, imaging mass spectrometry and others can be applied to the unsealed MCSD contents to measure how the different combinations present in each well affect other phenotypes of interest with non-optical readouts.

[0086] In some embodiments, the unsealing and additional droplet loading process described herein allows the use of toxic or unstable reagents for detection which otherwise could not be used on MCSD. Some assays detect the abundance of target organisms or measure specific biological activities by using a chemical reagent which is toxic to the organisms of interest. For such assays the compound cannot be added early in the assay before the organisms have grown, as the chemical would suppress growth. Instead, the organisms must first be grown on MCSDs, and the chemical reagent introduced later. An example of this would be certain types of fungal stains which are absorbed into the fungal cell wall and can be toxic to the fungus being measuredin some embodiments, the unsealing and additional droplet loading process described herein allows use of more flexible assay protocols which can be designed for greater accuracy / resemblance to the real-world biological system of interest. In some embodiments, the unsealing and additional droplet loading process described herein allows the addition of environmental perturbations to the articles being tested and the ability to measure their response. In some embodiments, the unsealing and additional droplet loading process described herein allows the addition of more nutrients to microwells, allowing for longer MCSD experiments.Automation Applications

[0087] Provided herein is an automated system for automated handling and microscopy of the MCSDs disclosed herein. In some embodiments, the automated system comprises one or more robot arms, a device for merging the droplets, an automated incubator, one or more microscopes, and a controller in communication with each of the one or more robot arms, the device for merging the droplets, the automated incubator, and the microscope. In some embodiments, the automated system is controlled by a main software program executable on a computer configured to analyze one or more input signals acquired by one or more sensory devices associated with each of the robot arms, the automated incubator, and the microscope. In some embodiments, the main softwareAtty. Docket No. 202421-703601comprises a scheduling software. In some embodiments, the main software is configured to allow a user to input a number of MCSDs to be imaged and a number of timepoints to be taken by the microscope. In some embodiments, the main software is configured to control the robot arm to move one or more MCSDs from a storage location to the microscope and trigger the microscope to obtain an image. Therefrom, the robot arm may be configured to move the one or more MCSDs to the automated incubator so as to store the one or more MCSDs at a pre-determined temperature. Therefrom, the robot arm may be configured to grab another of the MCSDs from the storage location and repeat the aforementioned processes. In some embodiments, the robot arm may be configured to move one of the MCSDs to a device for merging the droplets, such as a corona treater, which is then triggered by the main software to merge the droplets. In some embodiments, after merging, the robot arm may move one of the MCSDs to the microscope for obtaining another image. In some embodiments, the robot arm may be configured to perform one or more of: unloading any of the one or more MCSDs from the automated incubator, moving it back to the microscope, obtaining another image, and moving the one or more MCSDs back to the automated incubator. This may be repeated for any of the one or more MCSDs and timepoints for which the processes are programmed by the user.

[0088] In some embodiments, the controller comprises a computer with programmable software that is designed to perform one or more of: autonomously controlling the robot arm to move each of the MCSDs as required; and autonomously control inputs and various parameters of each of the automated incubator and the microscope. In an embodiment, the robot arm may include a high-precision mechanical positioning system and a control unit. In an embodiment, the control unit is a dedicated computer and motor driver that takes commands from a master computer and controls the robot's position and motion. In some embodiments, the robot arm is programmable to actuate tools attached to an end of the robot arm. An emergency stop switch may be present in case of malfunction of the automated imaging system, allowing immediate cessation of the motion of the robot arm. The motion of the robot arm may also be immediately stopped through the controlling software, if desired. If the robot arm is stopped in either of these ways, it may retreat to its original home position. In an embodiment, the robot arm may be provided with capability to hold its present position, while allowing someone to manipulate its joints with ample force may also be included. In an embodiment, as an additional safety feature, the robot arm does not move as a result of gravitational forces. In an embodiment, the robot arm is a mobile robot capable of avoiding obstacles and path following so that the automated imaging system can autonomously move around a laboratory room or floor. In some embodiments, the main software program may provide a graphical user interface (GUI) through which a user may interact with the system. In anAtty. Docket No. 202421-703601embodiment, this entails use of the GUI to control what process the automated imaging system carries out, along with certain parameters to be selected to further describe the details of the process.

[0089] In some embodiments, an automated system described herein comprises an automated imaging system. In some embodiments, the automated imaging setup comprises a PreciseFlex 400 robot arm, a Cytomat 2C450 automated incubator, and a Nikon Ti2-E microscope. In some embodiments, the automated imaging system comprises a processor in communication with each of the robot arm, the automated incubator, and the microscope. In some embodiments, images of samples (e.g., droplets within the microwells of a MCSD) may be taken at 0 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, and / or 96 hours later, and at programmable intervals therebetween. In some embodiments, images of the samples may be taken at 1 day to 28 days later. In some embodiments, images of the samples may be taken at 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days later.

[0090] In some embodiments, the automated system may comprise at least one computer-readable storage medium in communication with the processor, having encoded thereon instructions that, when executed by the processor, causes the processor to perform one or more of the following steps: (a) allow user input for the number of MCSDs to image and the number of time points at which they would like each of the MCSDs to be imaged; (b) control the robot arm to move an MCSD loaded with the droplets described herein from a storage location to the microscope; (c) trigger the microscope to obtain an image of the first MCSD; (d) merge the droplets in the MCSD and move the MCSD after merging to a stacker; (e) repeat steps (b)-(d) for additional MCSDs such that up to 14 MCSDs are placed in a stacker; (f) move the first MCSD to the incubator using the robot arm and store the first MCSD at a predetermined temperature; (g) unload the first MCSD from the automated incubator using the robot arm; (h) trigger the microscope to obtain an image of the first MCSD after incubation; (i) move the first MCSD back to the automated incubator; and (j) repeat steps (f)-(i) for each of the additional MCSDs.

[0091] A computer herein refers to any device with a processor capable of executing logic or coded instructions, and could be a server, personal computer, set top box, smart phone, pad computer or media device, to name a few such devices. The computer may include one or more processing units (also referred to herein as CPUs), which interface with at least one computer bus. Also interfacing with computer bus are persistent storage medium / media, network interface, memory, e.g., random access memory (RAM), run-time transient memory, read only memory (ROM), etc., media diskAtty. Docket No. 202421-703601drive interface as an interface for a drive that can read and / or write to media including removable media such as floppy, CD-ROM, DVD, etc. media, display interface as interface for a monitor or other display device, keyboard interface as interface for a keyboard, pointing device interface as an interface for a mouse or other pointing device, and miscellaneous other interfaces not shown individually, such as parallel and serial port interfaces, a universal serial bus (USB) interface, and the like.

[0092] Memory interfaces with computer bus so as to provide information stored in memory to CPU during execution of software programs such as an operating system, application programs, device drivers, and software modules that comprise program code, and / or computer-executable process steps, incorporating functionality described herein, e.g., one or more of process flows described herein. CPU first loads computer-executable process steps from storage, e.g., memory, storage medium / media, removable media drive, and / or other storage device. CPU can then execute the stored process steps in order to execute the loaded computer-executable process steps. Stored data, e.g., data stored by a storage device, can be accessed by CPU during the execution of computer-executable process steps.

[0093] Persistent storage medium / media is a computer readable storage medium(s) that can be used to store software and data, e.g., an operating system and one or more application programs. In some embodiments, persistent storage medium / media can also be used to store device drivers, such as one or more of a digital camera driver, monitor driver, printer driver, scanner driver, or other device drivers, web pages, content files, playlists and other files. In some embodiments, persistent storage medium / media can further include program modules and data files used to implement one or more embodiments of the present disclosure.

[0094] For the purposes of this disclosure a computer readable medium stores computer data, where data can include computer program code that is executable by a computer, in machine readable form. By way of example, and not limitation, a computer readable medium may comprise computer readable storage media, for tangible or fixed storage of data, or communication media for transient interpretation of code-containing signals. Computer readable storage media, as used herein, refers to physical or tangible storage (as opposed to signals) and includes without limitation volatile and non-volatile, removable and non-removable media implemented in any method or technology for the tangible storage of information such as computer-readable instructions, data structures, program modules or other data. In some embodiments, a computer readable storage media includes, but is not limited to, RAM, ROM, EPROM, EEPROM, flash memory or other solid state memory technology, CD-ROM, DVD, or other optical storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other physical orAtty. Docket No. 202421-703601material medium which can be used to tangibly store the desired information or data or instructions and which can be accessed by a computer or processor.

[0095] For the purposes of this disclosure a module is a software, hardware, or firmware (or combinations thereof) system, process or functionality, or component thereof, that performs or facilitates the processes, features, and / or functions described herein (with or without human interaction or augmentation). In some embodiments, a module can include sub-modules. In some embodiments, software components of a module may be stored on a computer readable medium. In some embodiments, modules may be integral to one or more servers, or be loaded and executed by one or more servers. In some embodiments, one or more modules may be grouped into an engine or an application.

[0096] In some embodiments, assays performed by the automated system comprising automated droplet generation, pooling, loading and imaging as described herein provides for improved efficiency as compared to those performed by an otherwise equivalent system with manual droplet generation, pooling, loading and imaging. In some embodiments, an “improved efficiency” is evaluated by increased combinations of bioagents and / or chemicals tested on a single MCSD. In some embodiments, an “improved efficiency” is evaluated by increased maximum MCSDs screened per day. In some embodiments, an “improved efficiency” is evaluated by reduced full-time equivalent (FTE) required for screening per day. FTE is a workload / staffing unit. 1.0 FTE means one person working full time on the screening effort.Bioagents and chemicals

[0097] Provided herein are systems and uses thereof for analysis of interactions between chemicals and / or bioagents that are merged in the droplets described herein. Bioagent types for inclusion in such methods include prokaryotic or eukaryotic cells. Bioagent types for inclusion in such methods include bacteria, archaea, eukarya, plants, animal, or fungi cells. In some cases, a bioagent is a strict aerobic cell, a strict anaerobic cell, a facultative aerobic cell, a facultative anaerobic cell, an aerotol erant anaerobic cell, an anaerobic-tolerant aerobic cell, or a microaerophilic cell. In some cases, a bioagent can be used in a system that is in an anaerobic, an aerobic, or a microaerophilic environment. In some cases, a bioagent can be an isolated and / or purified bioagent. In some cases, a bioagent is genetically engineered to express a gene. For example, a bioagent can be engineered to express a fluorescent reporter such as GFP or luciferase. In some cases, a bioagent comprises promoters that drive expression of genes include, but are not limited to a metabolic gene, a virulence-related gene, a transcription and / or translation related gene (e.g., a transcription factor), a structural associated gene (e.g., a gene associated with the cell membrane), a cell signaling gene,Atty. Docket No. 202421-703601a quorum sensing gene, a replication associated gene, a protein processing gene, a protein folding gene, a secretion gene, a cell division gene, a biosynthesis gene, a cell wall gene, a cell membrane gene, an antibiotic resistance gene, or any combination thereof. Chemical types for inclusion in such methods include small molecules and macromolecules. In some embodiments, a chemical is a biological molecule. Biological molecules include, but are not limited to fatty acids, glucose, amino acids, nucleic acids, cholesterols, secondary metabolites, proteins, DNA, andRNA. In some embodiments, a chemical is a pharmaceutical compound. Pharmaceutical compounds include, but are not limited to antibiotics, anti-inflammatory compounds, antiepileptics, lipid regulators, antiseptics, antidepressants, anticancer agents, and personal care products.

[0098] In some embodiments, a cell described herein is grown for 20 min to about 120 hours or longer than 120 hours. In some cases, a cell is grown for 20 min to about 120 min, 1 hour to about 10 hours, 2 hours to about 12 hours, 6 hours to about 24 hours, 10 hours to about 50 hours, 24 hours to about 72 hours, 48 hours to about 100 hours, or 72 hours to about 120 hours. In some cases, a cell is grown for more than, or equal to about: 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, or 120 hours.

[0099] In some embodiments, a cell herein is grown for 1 day to about 40 days. In some embodiments, a cell herein is grown for 1 day to 10 days, 5 days to 15 days, 10 days to 20 days, 15 days to 25 days, 20 days to 30 days, 25 days to 35 days, or 30 days to 40 days. In some embodiments, a cell herein is grown for about: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 37 days, 38 days, 39 days, or 40 days.

[0100] In some embodiments, a cell herein is grown at a temperature of about 20°C to about 40°C. In some cases, a cell herein is grown at a temperature of 1°C to about 99°C, 10°C to about 20°C, about 15°C to about 30°C, about 20°C to about 40°C, about 30°C to about 60°C, about 40°C to about 70°C, or about 50°C to about 99°C,. In some cases, a cell is grown at a temperature of more than or equal to about: 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C,Atty. Docket No. 202421-70360128°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, or 100°C.

[0101] In some embodiments, a bioagent herein includes an isolated bacterial strain. Example bacterial strains for inclusion in a composition, device, or method described herein include Achromobacter spp, Acidaminococcus fermenlans. Acinetobacter calcoaceticus, Actinomyces spp, Actinomyces viscosus, Actinomyces naeslundii, Aeromonas spp, Aggregatibacter actinomycetemcomitans, Anaerobiospirillum spp, Alcaligenes faecalis, Arachnia propionica, Bacillus spp, Bacteroides spp, Bacteroides gingivalis, Bacteroides fragilis, Bacteroides intermedins, Bacteroides melaninogenicus, Bacteroides pneumosintes, Bacterionema matruchotii, Bifidobacterium spp, Buchnera aphidicola, Butyriviberio fibrosolvens, Campylobacter spp, Campylobacter coli, Campylobacter sputorum, Campylobacter upsaliensis, Capnocytophaga spp, Clostridium spp, Citrobacter freundii, Clostridium difficile, Clostridium sordellii, Corynebacterium spp, Eikenella corrodens, Enterobacter cloacae, Enterococcus spp, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Eubacterium spp, Flavobacterium spp, Fusobacterium spp, Fusobacterium nucleatum, Gordonia Bacterium spp, Haemophilus parainfluenzae, Haemophilus paraphrophilus, Lactobacillus spp, Leptotrichia buccalis, Methanobrevibacter smithii, Morganella morganii, Mycobacteria spp, Mycoplasma spp, Micrococcus spp, Mycoplasma spp, Mycobacterium chelonae, Neisseria spp, Neisseria sicca, Peptococcus spp, Peptostreptococcus spp, Plesiomonas shigelloides, Porphyromonas gingivalis, Propionibacterium spp, Propionibacterium acnes, Providencia spp, Pseudomonas aeruginosa, Ruminococcus bromii, Rothia dentocariosa, Ruminococcus spp, Sarcina spp, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus anginosus, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus sobrinus, Streptococcus viridans, Torulopsis glabrata, Treponema denticola, Treponema refringens, Veillonella spp, Vibrio spp, Vibrio sputorum, Wolinella succinogenes and Yersinia enter ocolitica. In certain embodiments, exemplary bacteria include Achromobacter spp. (e.g., Achromobacter denitrificans, Achromobacter xylosoxidans, Achromobacter ruhlandii); Actinomadura spp. (e.g., Actinomadura luteofluorescens, Actinomadura madurae, Actinomadura pelletieri, Actinomadura viridis); Agrobacterium spp. (e.g., Agrobacterium radiobacter, Agrobacterium luteum, Agrobacterium agile, Agrobacterium rubi); Arthrobacter spp. (e.g., Arthrobacter arilaitensis, Arthrobacter chlorophenolicus, Arthrobacter aurescens); Bacillus spp. (e.g., Bacillus cereus, Bacillus subtilis,Atty. Docket No. 202421-703601Bacillus coagulans, Bacillus psychrosaccharolyticus, Bacillus amyloliquefaciens, Bacillus lentus, Bacillus circulans, Bacillus firmus); Burkholderia spp. (e.g., Bur kholderia gladioli, Burkholderia plantarii, Burkholderia cepacia); Clostridium spp. (e.g., Clostridium orbiscindens, Clostridium formicaceticum) ; Escherichia coli; Ewingella spp. (e.g., Ewingella americana); Flavobacterium spp. (e.g., Flavobacterium flevense, Flavobacterium aquatile, Flavobacterium saccharophilum, Flavobacterium hydatis, Flavobacterium johnsoniae) ; Flexibacter spp. (e.g., Flexibacter flexilis, Flexibacter columnare); Herbaspirillum frisingense; Hyphomicrobium spp. (e.g., Hyphomicrobium aestuarii); Micromonospora spp. (e.g., Micromonospora rosaria, Micromonospora facile, Micromonospora zavarzinii, Micromonospora denitrificans) ; Mycobacterium spp. (e.g., Mycobacterium neoaurum) ; Nocardia spp. (e.g., Nocardia jiangxiensis, Nocardia miyunensis); Paenibacillus spp. (e.g., Paenibacillus macquariensis, Paenibacillus macerans, Paenibacillus alvei, Paenibacillus polymyxa, Paenibacillus chibensis); Pseudomonas spp. (e.g., Pseudomonas aeruginosa, Pseudomonas putida, Pseudomonas jluorescens, Pseudomonas citronellolis, Pseudomonas chlororaphis, Pseudomonas aurantiaca, Pseudomonas pseudopalustris, Pseudomonas palustris, Pseudomonas syringae, Pseudomonas veronii, Pseudomonas aurantiaca); Ralstonia spp. (e.g., Ralstonia solanacearum, Ralstonia pickettii, Ralstonia syzygii); Rhodococcus spp. (e.g., Rhodococcus erythropolis, Rhodococcus rhodochrous) ; Serratia spp. (e.g., Serratia marcescens, Serratia liquefaciens) ; Sphingomonas spp. (e.g., Sphingomonas echinoides, Sphingomonas leidyi, Sphingomonas wittichii); and / or Streptomyces spp. (e.g., Streptomyces lividans, Streptomyces coelicolor, Streptomyces tanashiensis, Streptomyces clavuligerus, Streptomyces griseus). In some instances, a bacteria for include in such methods include Staphylococcus capitis, Staphylococcus caprae, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus intermedins, Staphylococcus lugdunensis, Staphylococcus pasteuri, Staphylococcus saprophyticus, Staphylococcus warneri, Bacillus velezensis, Bacillus amyloliquefaciens, Bacillus cecembensis, Bacillus cereus, Bacillus sp. Lzh-5, Bacillus sp. KbaLl, Bacillus sp. SJ-10, Bacillus mediterraneensis, Bacillus mycoides, Bacillus nakamurai, Bacillus thuringiensis, Bacillus tropicus, Bacillus paranthracis, Bacillus vallismortis, Micrococcus aloeverae, Psychrobacillus sp. INOPOl, Paenibacillus lentimorbus, Paenibacillus elgii, Bacillus wiedmannii, Brevibacillus parabrevis, Dermacoccus nishinomiyaensis, Kocuria marina, Lysinibacillus boronitolerans, Lysinibacillus macroides, Lysinibacillus sp. FN11, Lysinibacillus sp. UBA5994, Lysinibacillus sp. PB300, Lysinibacillus sp. YS11, Lysinibacillus boronitolerans, Lysinibacillus fusiformis, Macrococcus goetzii, and Staphylococcus aureus. In some embodiments, bacteria provided herein are phototrophic bacteria (e.g., Prochlorococcus, Synechococcus) .Atty. Docket No. 202421-703601

[0102] In some embodiments, a bioagent herein includes fungi. Exemplary fungi for inclusion in the systems and methods described herein include Ascomycetes (e.g., Venturia, Aspergillus, Podosphaera, Erysiphe, Monilinia, Uncinula, Aureobasidium, Sclerophoma) ; Basidiomycetes (e.g., Hemileia, Rhizoctonia, Puccinia, Coniophora, Serpula, Poria, Uromyces, Gloeophyllum, Lentinus, Coriolus, Irpex); zygomycetes (e.g., Mucor) and fungi imperfecti (e.g., Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia, Penicillium, Geotrichum, and Zymoseptoria). In certain embodiments, representative but not comprehensive examples of fungi are Venturia, Aspergillus, Podosphaera, Erysiphe, Monilinia, Uncinula, Aureobasidium, Sclerophoma, Acremonium, Actinoplanes, Agaricus, Chrysosporium, Colletotrichum, Coprinus, Cryptococcus, Filibasidum, Humicola, Magnaporthe, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Phytophthora, Piromyces, Panerochaete, Pleurotus, Pythium, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Hemileia, Rhizoctonia, Puccinia, Coniophora, Serpula, Poria, Uromyces, Gloeophyllum, Lentinus, Coriolus, Irpex, Mucor, Botrytis, Helminthosporium, Rhynchosporium, Fusarium, Septoria, Cercospora, Alternaria, Pyricularia, Penicillium, Geotrichum, and / or Zymoseptoria. In some instances, a fungi for include in such methods include Cryptococcus neoformans, Candida auris, Aspergillus fumigatus, Candida albicans, Nakaseomyces glabrata (Candida glabrata), Histoplasma spp., eumycetoma causative agents, Mucorales, Fusarium species, Candida tropicalis, Candida parapsilosis, Scedosporium species, Lomentospora prolificans, Coccidioides species, Pichia kudriavzeveii (Candida krusei), Cryptococcus gattii, Talaromyces marneffei, Pneumocystis jirovecii, and Paracoccidioides species.

[0103] Exemplary yeast, a type of fungi, for inclusion in the systems and methods described herein include Saccharomyces, Candida, Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula, Schizosaccharomyces and Yarrowia. Exemplary species include Saccharomyces cerevisiae, Saccharomyces pastorianus, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Candida kefyr, Candida laurentii, Cryptococcus neoformans, Hansenula anomala, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus var. Lactis, Pichia pastoris, Rhodotorula rubra, Schizosaccharomyces pombe, Leucosporidium frigidum, Saccharomyces telluris, Candida sloofft, Torulopsis, Trichosporon cutaneum, Dekkera intermedia, Candida blankii, Cryptococcus gattii, Rhodotorula mucilaginosa, Brettanomyces bruxellensis, Candida stellata, Torulaspora delbrueckii, Zygosaccharomyces bailii, Brettanomyces anomalus, Brettanomyces custersianus,Atty. Docket No. 202421-703601Brettanomyces naardenensis, Brettanomyces nanus, Dekkera bruxellensis, Dekkera anomala and Yarrowia lipolytica.

[0104] In some embodiments, a bioagent described herein comprises an algae cell. Exemplary algae cells for inclusion in the systems and methods described herein include Amphora, Bacillariophyceae, Dunaliella, Chlorella, Chlamydomonas, Phormidium, Cyanophyta (cyanobacteria), Nannochloropsis, Spirulina, and Ochromonas.

[0105] In some embodiments, a bioagent described herein comprises a plant cell. In some embodiments, a plant cell is from a monocotyledonous plant including but not limited to maize, rice, wheat, and setaria. In some embodiments, a plant cell is from a dicotyledonous plant including but not limited to cassava, potato, soybean, tomato, tobacco, alfalfa, Physcomitrella patens and Arabidopsis.

[0106] In some embodiments, a bioagent described herein comprises a mammalian cell. Exemplary mammalian cells for inclusion in the systems and methods described herein include epithelial cells, fibroblasts, keratinocytes, melanocytes, endothelial cells, muscle cells, hematopoietic cells, and / or mesenchymal stem cells. In some embodiments, the mammalian cells are primary cells. In some embodiments, the mammalian cells are primate cells. In some embodiments, the mammalian cells are non-primate mammalian cells. In some embodiments, the mammalian cells are non-human primate cells. In some embodiments, the mammalian cells are human cells. In some embodiments, the human cells comprise human T lymphoblasts (see, e.g., FIG. 25A) and / or human monocytes (see e.g., FIG. 25B).

[0107] Also provided herein are bacterial, plant fungal and mammalian cells that modulate the expression of a gene in a microorganism, or modulate the expression of a protein in a microorganism. In some cases, the microorganism can be a pathogenic microorganism. In some cases, the microorganism can be a bacterial, a fungal, or a mammalian cell. In some cases, a compound, such as a compound from a library of compounds modulates the expression of a gene in a microorganism, or modulates the expression of a protein in a microorganism. In some cases, the modulation is an increase of the gene expression or the protein expression. In some cases, the modulation is a decrease of the gene expression or the protein expression. In some cases, microorganisms herein provide for a reduction in the expression of a gene or protein from S. aureus or S. pyogenes. In some cases, microorganisms herein modulate the expression of a gene or protein from a mammalian cell. In some cases, microorganisms herein modulate the expression of a gene or protein from a fungal cell. Exemplary genes for modulation and suppression include, without limitation, a virulence gene, a metabolism gene, a transcription gene, a translation gene, a protein processing gene, a protein folding gene, a secretion gene, a cell division gene, a biosynthesis gene,Atty. Docket No. 202421-703601a cell wall gene, a cell membrane gene, an antibiotic resistance gene, and a protein coded by one of these genes. In some cases, the gene or protein is associated with growth, regulation (e.g., virulence regulation, metabolic regulation, or growth regulation), direct virulence such as a toxin, stress, or a metabolic state.

[0108] In some cases, the gene is gmk, agr,psma, sigB, saeR, and / or ccpA. In some cases, the protein is encoded by gmk, agr, psma, sigB, saeR, and / or ccpA. In some cases, the expression of a gene is measured by a promoter-reporter strain. For example, a promoter of a gene of interest such as, gmk, agr, psma, sigB, saeR, or ccpA can be fused to a fluorescent reporter such as GFP, or a luminescent reporter such as luciferase to measure the amount of gene expression. Exemplary genes for promoter-reporter strains are shown in Table 1.

[0109] In some cases, gene expression is used to assess constitutive metabolic function, quorum sensing, a toxin that damages host tissue, a stress response sigma factor, virulence regulation, and / or carbon catabolite repression. In some cases, gene expression is measured by a Northern blot, a Western blot, a promoter-reporter gene, a microarray, a PCR assay such as a reverse transcription polymerase chain reaction, a Serial Analysis of Gene Expression (SAGE), a next generation sequencing technique such as RNA Seq, or a combination of these methods. In some cases, the expression of a protein is measured. In some cases, protein expression is measured by a Western blot, an enzyme-linked immunoassay (ELISA), an SDS-PAGE gel, a Bicinchoninic acid (BCA) assay, or any assay that quantifies protein expression and / or amounts.

[0110] Also provided here are anaerobic bacterial strains modified to express a reporter system. In some embodiments, the reporter system is designed specifically for anaerobic screening. In some embodiments, the anaerobic bacterial strains are modified to express a fluorescent protein. In some embodiments, the fluorescent protein is ligand-inducible. In some embodiments, the fluorescent protein requires an exogenous ligand to fluoresce. In some embodiments, the anaerobic bacterial strains are modified to express unaG. UnaG belongs to the fatty-acid-binding protein family and is a unique ligand-inducible fluorescent protein. It binds unconjugated bilirubin non-covalently as a fluorogenic ligand and fluoresces in the absence of oxygen. In some embodiments, the anaerobic bacterial strains are modified to transcribe an aptamer. In some embodiments, the anaerobic bacterial strains are modified to transcribe a Pepper fluorescent RNA. Pepper fluorescent RNAs are fluorogenic aptamer tags. Pepper aptamer binds to a chromophore and fluoresces in the absence of oxygen. In some embodiment, a chromophore described herein is ((4-((2-hydroxyethyl)(methyl) amino)-benzylidene)-cyanophenyl-acetonitrile) (HBC).Table 1: Promoter-Reporter StainsAtty. Docket No. 202421-703601Species Promoter Protein Function Associated with Protein Genbank Genome Genbank Reference for gene ReferenceS', aureus gmk Constitutive Growth WP 000368227.1 GenBank: CP000253.1 metabolic Range: 1126551 to function 1126850S. aureus agr Quorum sensing Virulence ADF28639.1 GenBank ID:induction (regulatory) CP000253.1 Range:2093636 to 2093876 S. aureus psma Toxin that Virulence WP_014373781.1 GenBank ID:damages host (direct) CP000253.1 Range: tissue 412992 to 413258 S. aureus sigB Stress response Stress CAC6986262.1 GenBank ID:sigma factor (regulatory) CP000253.1 Range:2133482 to 2133781 S. aureus saeR Broad virulence Virulence WP_000149344.1 GenBank ID:regulation (regulatory) CP000253.1 Range:700936 to 701683 S. aureus ccpA Carbon catabolite Metabolic state WP 000219066.1 GenBank ID:repression CP000253.1 Range:1757336 to 1757835 C. albicans ECE1 Toxin that Virulence XP 711594.1 GenBank ID:damages host (direct) GCF 000182965.3; tissue Chromosome: 4;NC 032092.1 (726514..727329, complement) C. albicans TDH3 Constitutive Growth XP 719909.1 GenBank ID:metabolic GCF 000182965.3; function Chromosome: 3;NC 032091.1 (1574865..1575872) C. albicans RPS1 Constitutive Growth XP 721500.1 GenBank ID:metabolic GCF 000182965.3; function Chromosome: 1;NC 032089.1 (643226..643996) C. albicans SAP2 Secreted Aspartyl Virulence XP 711047.1 GenBank ID:protease (direct) GCF 000182965.3;Chromosome: R; NC 032096.1 (1692632..1693828) C. albicans SAP5 Secreted Aspartyl Virulence XP 719147.1 GenBank ID:protease (direct) GCF 000182965.3;Chromosome: 6; NC 032094.1 (635173..636429) C. albicans ALS3 Surface adhesion Virulence XP 710435.2 GenBank ID:protein (direct) GCF 000182965.3;Chromosome: R; NC 032096.1 (1532277..1535744, complement) s. TDH3 Constitutive Growth NP 011708.3 GenBank ID: cerevisiae metabolic GCA 000146045.2;function Chromosome: VII;NC 001139.9 (882812..883810,complement)Atty. Docket No. 202421-703601EXEMPLARY EMBODIMENTS

[0111] Provided herein are A method of screening, comprising: (a) providing a device, a pool of droplets comprising bioagents and / or chemicals, and one or more robotic arms, wherein the device comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents and / or chemicals; (b) mounting the device to a first robotic arm;(c) loading the one or more droplets in the pool to the device via an automated process that controls the first robotic arm to: (i) submerge or partially submerge the device in a bath unit configured to retain a pooled fluid that is immiscible with the droplets through the first robotic arm, wherein the device is tilted relative to the surface of the bath unit during the submerging step; (ii) add the droplets to the pooled; and (iii) move the device vertically relative to the surface of the pooled fluid such that one or more droplets are loaded to the device, thereby allowing the droplets to enter the device; (d) removing the device from the first robotic arm; (e) mounting the device to a second robotic arm;(f) moving the device to an imaging station using the second robotic arm;(g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the bioagents or chemicals in each droplet; (h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of bioagents and / or chemicals; (i) measuring optical signals of the combinations after the merging, wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms. Further provided herein are methods, wherein prior to step (b), the droplets are formed using a droplet generator. Further provided herein are methods, wherein after the droplets are formed, the droplets are aspirated and dispensed with an electronic pipette mounted to a third robotic arm. Further provided herein are methods, wherein the droplets comprise bioagents and chemicals, wherein the method assays growth of the bioagents in the presence of the chemicals. Further provided herein are methods, wherein the device is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein the device is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein the device is tilted at an angle of 10 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein prior to step (e), the device is sealed. Further provided herein are methods, wherein the device is sealed with a film or a glass. Further provided herein are methods, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof. Further provided herein are methods, wherein step (i) further comprises: (1) receiving input information from a user of the device to be imaged and a number of timepoints to be taken by an imaging apparatus; (2) controlling the second roboticAtty. Docket No. 202421-703601arm to move the device from a first location to the imaging apparatus to obtain first images; (3) controlling the second robotic arm to move the device to an automated incubator for storing the device; (4) unloading the device from the automated incubator and moving the device back to the imaging apparatus to obtain second images; (5) moving the device back to the automated incubator; and (6) repeating one or more of steps (1 )-(5) for the device according to the number of timepoints. Further provided herein are methods, wherein the device is positioned in a carrier, wherein the one or more robotic arms grip the carrier to move the device. Further provided herein are methods, wherein the droplets comprise a plurality of bioagents and / or chemical compounds. Further provided herein are methods, wherein the bioagents comprise prokaryotic cells. Further provided herein are methods, wherein the prokaryotic cells comprise bacteria. Further provided herein are methods, wherein the bacteria is an odor-causing bacteria. Further provided herein are methods, wherein the bioagents comprise eukaryotic cells. Further provided herein are methods, wherein the eukaryotic cells comprise fungi. Further provided herein are methods, wherein the fungi comprise filamentous fungi. Further provided herein are methods, wherein the fungi comprise yeast. Further provided herein are methods, wherein the eukaryotic cells comprise protists. Further provided herein are methods, wherein the protists comprise eukaryotic algae. Further provided herein are methods, wherein the eukaryotic cells comprise mammalian cells. Further provided herein are methods, wherein the bioagents are anaerobic microbes or anaerobic yeasts. Further provided herein are methods, wherein the methods further comprise detecting and determining a level of a signal. Further provided herein are methods, wherein the signal is from a morphology measurement. Further provided herein are methods, wherein the signal is from an entropy measurement. Further provided herein are methods, wherein the signal is from an overall optical interference. Further provided herein are methods, wherein the signal is from one or more reporters. Further provided herein are methods, wherein the one or more reporters are expressed in an anaerobic microbe. Further provided herein are methods, wherein the one or more reporters comprises a fluorescent protein. Further provided herein are methods, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen. Further provided herein are methods, wherein the fluorescent protein is a ligand-inducible fluorescent protein. Further provided herein are methods, wherein the ligand-inducible fluorescent protein is UnaG. Further provided herein are methods, wherein the one or more reporters comprise an RNA aptamer, wherein the RNA aptamer fluoresces when bound to a chromophore. Further provided herein are methods, wherein the methods further comprise unsealing the device and repeating step (c). Further provided herein are methods, wherein the device is cooled before the unsealing step. Further provided herein are methods, wherein the device is cooled at -70 °C. Further provided herein areAtty. Docket No. 202421-703601methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased number of combinations of bioagents and / or chemicals tested on a single device. Further provided herein are methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased maximum number of devices screened per day. Further provided herein are methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by a reduced full-time equivalent (FTE) required to perform an equal number of screenings per day.

[0112] Provided herein are methods of screening for combinations of live cells with bioagents or chemicals, comprising: (a) providing a device, one or more robotic arms, and a pool of droplets comprising: (i) a population of live cells and (ii) bioagents and / or chemicals, wherein the device comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets from the pool of droplets; (b) mounting the device to a first robotic arm; (c) loading the one or more droplets from the pool of droplets to the device by an automated process; (d) removing the device from the first robotic arm; (e) mounting the device to a second robotic arm; (f) moving the device to an imaging station using the second robotic arm; (g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the population of the live cells, bioagents or chemicals in each droplet; (h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of the population of the live cells, bioagents and / or chemicals; (i) measuring optical signals from the combinations after the merging, wherein the signals from combinations of the population of the live cells with the bioagents and / or chemicals indicate changes of activities of the population of live cells in presence of the bioagents and / or chemicals, wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms. Further provided herein are methods, wherein prior to step (b), the droplets are formed using a droplet generator. Further provided herein are methods, wherein after the droplets are formed, the droplets are aspirated and dispensed with an electronic pipette mounted to a third robotic arm. Further provided herein are methods, wherein the droplets comprise bioagents and / or chemicals, wherein the method assays growth of the bioagents in presence of the chemicals. Further provided herein are methods, wherein step (c) comprises: (1) submerging or partially submerging the deviceAtty. Docket No. 202421-703601in a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets through the first robotic arm, wherein the device is tilted relative to the surface of the bath unit during the submerging step; (2) adding droplets to the pooled liquid; and (3) moving the device vertically relative to the surface of the pooled fluid such that one or more droplets are loaded to the device, thereby allowing the droplets to enter the device. Further provided herein are methods, wherein the device is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein the device is tilted at an angle of from 5 degree to 30 degree relative to the surface of the pooled fluid. Further provided herein are methods, wherein the device is tilted at an angle of 10 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein prior to step (e), the device is sealed. Further provided herein are methods, wherein the device is sealed with a film or a glass. Further provided herein are methods, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof. Further provided herein are methods, wherein step (i) further comprises: (1) receiving input information from a user of the device to be imaged and a number of timepoints to be taken by an imaging apparatus; (2) controlling the second robotic arm to move the device from a first location to the imaging apparatus to obtain first images; (3) controlling the second robotic arm to move the device to an automated incubator for storing the device; (4) unloading the device from the automated incubator and moving the device back to the imaging apparatus to obtain second images; (5) moving the device back to the automated incubator; and (6) repeating one or more of steps ( 1 )-(5) for the device according to the number of timepoints. The method of any one of claims 42 to 53, wherein the device is positioned in a carrier, wherein the one or more robotic arms grab the carrier to move the device. Further provided herein are methods, wherein the droplets comprise a plurality of bioagents and / or chemical compounds. Further provided herein are methods, wherein the population of the live cells comprise prokaryotic cells. Further provided herein are methods, wherein the prokaryotic cells comprise bacteria. Further provided herein are methods, wherein the bacteria is an odor-causing bacteria. Further provided herein are methods, wherein the bioagents comprise eukaryotic cells. Further provided herein are methods, wherein the eukaryotic cells comprise fungi. Further provided herein are methods, wherein the fungi comprise filamentous fungi. Further provided herein are methods, wherein the fungi comprise yeast. Further provided herein are methods, wherein the eukaryotic cells comprise protists. Further provided herein are methods, wherein the protists comprise eukaryotic algae. Further provided herein are methods, wherein the eukaryotic cells comprise mammalian cells. Further provided herein are methods, wherein the bioagents are anaerobicAtty. Docket No. 202421-703601microbes or anaerobic yeasts. Further provided herein Further provided herein are methods, wherein the signal is from morphology measurement. Further provided herein are methods, wherein the signal is from entropy measurement. Further provided herein are methods, wherein the signal is from overall optical interference. Further provided herein are methods, wherein the signal is from one or more reporters. Further provided herein are methods, wherein the one or more reporters are expressed in an anaerobic microbe. Further provided herein are methods, wherein the one or more reporters comprises a fluorescent protein. Further provided herein are methods, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen. Further provided herein are methods, wherein the fluorescent protein is a ligandinducible fluorescent protein. Further provided herein are methods, wherein the ligand-inducible fluorescent protein is UnaG. Further provided herein are methods, wherein the one or more reporters comprise an RNA aptamer, wherein the RNA aptamer fluoresces when bound to a chromophore. Further provided herein are methods, wherein the method further comprise unsealing the device and repeating step (c). Further provided herein are methods, wherein the device is cooled before the unsealing step. Further provided herein are methods, wherein the device is cooled at -70 °C. Further provided herein are methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased number of combinations of bioagents and / or chemicals tested on a single device. Further provided herein are methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased maximum number of devices screened per day. Further provided herein are methods, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by a reduced full-time equivalent (FTE) required to perform an equal number of screenings per day.

[0113] Provided herein are systems comprising: a micropatterned combinatorial screening device (MCSD) comprising an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; and a loading apparatus comprising: a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets; a holding unit for carrying the MCSD, wherein the holding unit is tilted at an angle relative to the surface of the pooled fluid. Further provided herein are systems, wherein the systems furtherAtty. Docket No. 202421-703601comprise a plurality of droplets deposited in each of the microwells, wherein each droplet comprises at least one type of bioagents or chemicals. Further provided herein are systems, wherein the bioagents comprise prokaryotic cells. Further provided herein are systems, wherein the prokaryotic cells comprise bacteria. Further provided herein are systems, wherein the bioagents comprises eukaryotic cells. Further provided herein are systems, wherein the eukaryotic cells comprise fungi. Further provided herein are systems, wherein the fungi comprise filamentous fungi. Further provided herein are systems, wherein the fungi comprise yeast. Further provided herein are systems, wherein the eukaryotic cells comprise protists. Further provided herein are systems, wherein the protists comprise eukaryotic algae. Further provided herein are systems, wherein the eukaryotic cells comprise mammalian cells. Further provided herein are systems, wherein upon movement of the holding unit relative to the surface of the pooled fluid, the one or more droplets are loaded into the MCSD, thereby allowing at least one of the one or more droplets to enter in each of the microwells to form massively parallel combinations of bioagents or chemicals. Further provided herein are systems, wherein the holding unit comprises a mounting piece, wherein the MCSD is slid into the holding unit through the mounting piece. Further provided herein are systems, wherein the MCSD is suctioned to the holding unit. Further provided herein are systems, wherein the bath unit comprises one or more ports in or adjacent to the bottom surface of the bath unit for controlling flow of the pooled fluid and one or more outlet for removing liquid in the bath unit. Further provided herein are systems, wherein the systems further comprise multiple pumps, valves, a reservoir, a waste container, or any combination thereof. Further provided herein are systems, wherein the pooled fluid comprises an oil. Further provided herein are systems, wherein the holding unit is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid. Further provided herein are systems, wherein the holding unit is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid. Further provided herein are systems, wherein the holding unit is tilted at an angle of 10 degrees relative to the surface of the pooled fluid. Further provided herein are systems, wherein the systems further comprise a droplet generator, wherein the droplet generator comprises a pressure manifold and one or more input arrays, wherein each input array comprises multiple input wells. Further provided herein are systems, wherein the systems further comprise a droplet generator, wherein the droplet generator comprises four input arrays. Further provided herein are systems, wherein the loading apparatus further comprises one or more robotic arms connected to the holding unit, wherein the one or more robotic arms move the holding unit via an automated process. Further provided herein are systems, wherein the systems further comprise a carrier, wherein the carrier comprises a frame for placing the MCSD, thereby allowing the one or more robotic arms to grip the MCSD. FurtherAtty. Docket No. 202421-703601provided herein are systems, wherein the MCSD comprises a variation in thickness of less than about 0.5 mm. Further provided herein are systems, wherein the MCSD comprises a variation in thickness of less than about 0.4 mm. Further provided herein are systems, wherein the MCSD comprises a variation in thickness of less than about 0.3 mm. Further provided herein are systems, wherein the MCSD comprises one or more of polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC), cyclic olefin polymer (COP) polycarbonate, styrene-ethylene-butylene-styrene (SEBS), and poly(m ethyl acrylate) (PMMA). Further provided herein are systems, wherein each microwell is sized to capture at least one droplet, at least two droplets, at least three droplets, at least four droplets, at least five droplets, at least six droplets, at least seven droplets, at least eight droplets, at least nine droplets, at least ten droplets, at least eleven droplets, at least twelve droplets, at least thirteen droplets, at least fourteen droplets, at least fifteen droplets, at least sixteen droplets, at least seventeen droplets at least eighteen droplets, or at least nineteen droplets. Further provided herein are systems, wherein each microwell is sized to capture droplets of a same size or microwells are sized to capture droplets of a different size. Further provided herein are systems, wherein the systems further comprise a coalescing mechanism means for merging at least two droplets in a microwell to form a single droplet. Further provided herein are systems, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof. Further provided herein are systems, wherein the coalescing mechanism means is a corona treater, wherein the corona treater generates an electric field for merging at least two droplets in a microwell to form a single droplet. Further provided herein are systems, wherein each microwell comprises a plurality of posts. Further provided herein are systems, wherein the posts are rectangular, triangular, or circular in shape when viewed from above. Further provided herein are systems, wherein the plurality of posts are integrated into the sides of microwells and / or adhered to the bottom of the microwells. Further provided herein are systems, wherein the droplets comprise one or more reporters. Further provided herein are systems, wherein the one or more reporters are small molecule reporters. Further provided herein are systems, wherein the systems further comprise an anaerobic microbe strain. Further provided herein are systems, wherein the one or more reporters are expressed in the anaerobic microbe strain. Further provided herein are systems, wherein the one or more reporters comprises a fluorescent protein. Further provided herein are systems, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen. Further provided herein are systems, wherein the fluorescent protein is a ligand-inducible fluorescent protein. Further provided herein are systems, wherein the ligand-inducible fluorescent protein is UnaG. Further provided herein are systems, wherein the one or more reporters comprise an RNA aptamerAtty. Docket No. 202421-703601and wherein the RNA aptamer fluoresces when bound to a chromophore. Further provided herein are systems, wherein the RNA aptamer is a Pepper aptamer. Further provided herein are systems, wherein the system further comprise a controller, wherein the controller is in communication with each of the one or more robot arms.

[0114] Provided herein are methods of loading droplets for producing massively parallel combinations of bioagents or chemicals, comprising: (a) providing a micropatterned combinatorial screening device (MCSD), wherein the MCSD comprises: an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; (b) submerging or partially submerging the MCSD in a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets, wherein the array of microwells is tilted relative to the surface of the bath unit during the submerging step; (c) adding the droplets to the pooled fluid; and (d) moving the MCSD vertically relative to the surface of the pooled fluid such that the one or more droplets are loaded to the MCSD, thereby allowing the droplets to enter the MCSD to generate massively parallel combinations of cells or chemicals. Further provided herein are methods, wherein the bioagents comprise prokaryotic cells. Further provided herein are methods, wherein the prokaryotic cells comprise bacteria. Further provided herein are methods, wherein the bioagents comprises eukaryotic cells. Further provided herein are methods, wherein the eukaryotic cells comprise fungi. Further provided herein are methods, wherein the fungi comprise filamentous fungi. Further provided herein are methods, wherein the fungi comprise yeast. Further provided herein are methods, wherein the eukaryotic cells comprise protists. Further provided herein are methods, wherein the protists comprise eukaryotic algae. Further provided herein are methods, wherein the eukaryotic cells comprise mammalian cells. Further provided herein are methods, wherein the one or more droplets is added at a loading edge of the device. Further provided herein are methods, wherein the MCSD is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein the MCSD is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein the MCSD is tilted at an angle of 10 degrees relative to the surface of the pooled fluid. Further provided herein are methods, wherein step (d) comprises repeatedly moving the MCSD vertically until the droplets are loaded to more than 90% of the microwells. Further provided herein are methods, wherein before step (c), the droplets are generated in a droplet generator, wherein the droplet generator comprises a pressure manifold and one or more input arrays, wherein each input array comprises multiple input wells. Further provided herein are methods, wherein after the droplets are generated, the droplets are aspirated and dispensed with anAtty. Docket No. 202421-703601electronic pipette mounted to a third robotic arm. Further provided herein are methods, wherein the pooled fluid is an oil.

[0115] Provided herein are methods for label-free quantification of growth of a bioagent, the method comprising: providing a sample containing a bioagent in a growth medium to an array of microwells in a micropatterned combinatorial screening device (MCSD), wherein each microwell is capable of receiving one or more droplets; obtaining a plurality of label-free optical measurements from the sample over a predetermined time using the MCSD; generating data from the plurality of label-free optical measurements, wherein the data comprises optical density measurements, light transmittance measurements, entropy measurement, or phase contrast measurements; processing the data using at least one of the following: a deep neural network machine learning (ML) algorithm, wherein the deep neural network is trained to correlate one or more optical signal patterns with bacterial growth parameters; optical density measurement over time; light transmittance measurement over time; and entropy measurement over time; and determining at least one quantitative value relevant to the bacterial growth. Further provided herein are methods, wherein step (d) comprises processing the data consisting of the following: a deep neural network machine learning (ML) algorithm, wherein the deep neural network is trained to correlate one or more optical signal patterns with bacterial growth parameters; optical density measurement over time; light transmittance measurement over time; and entropy measurement over time. Further provided herein are methods wherein the at least one quantitative value is a median growth value of the bacteria. Further provided herein are methods wherein the bioagent comprises prokaryotic cells. Further provided herein are methods wherein the prokaryotic cells comprise bacteria. Further provided herein are methods wherein the bacteria is an odor-causing bacteria. Further provided herein are methods wherein the bioagent comprises eukaryotic cells. Further provided herein are methods wherein the eukaryotic cells comprise fungi. Further provided herein are methods wherein the fungi comprise filamentous fungi. Further provided herein are methods wherein the fungi comprise yeast. Further provided herein are methods wherein the eukaryotic cells comprise protists. Further provided herein are methods, wherein the protists comprise eukaryotic algae. Further provided herein are methods wherein the eukaryotic cells comprise mammalian cells.EXAMPLES EXAMPLE 1: Assay Methods

[0116] Described in this example is an exemplary assay method using a system described herein including the following steps:Atty. Docket No. 202421-703601

[0117] (1) Sample preparation. Overnight cultures of microbe samples are prepared as liquid samples and transferred to an optical plate. A robotic arm carries an optical plate and transfers the optical plate to a plate reader (BioTek Synergy Hl Multimode Reader) to test the optical densities of the liquid samples. Optionally, the content in each droplet is labeled with color barcode (FIG. 2A).

[0118] (2) Droplet generation. The samples are loaded to droplet cartridges together with oil by using programmed standard liquid handling robots (Cybio® Felix or Opentrons OT2). A customized droplet generator delivers air pressure to the input wells through a custom manifold and controlled by an Elveflow OBI pressure controller to convert the sample inputs to droplets (FIG. 2B) As illustrated in FIG. 6, the customized droplet generator can process four droplet cartridges in parallel. The size of droplets can be controlled by changing the flow rates of the oil and the liquid sample through changing the air pressure. Once each liquid sample is converted to droplets, the droplets are collected in a single tube as a droplet pool.

[0119] (3) Droplets loading. A micropatterned combinatorial screening device (“MCSD”) described herein was inserted into a holding unit attached to a robotic arm which positioned the MCSD suspended over a pool of oil. The holding unit was tilted at an upward angle relative to the pool. A robotic arm lowers the MCSD down until the lower side of the MCSD is fully immersed in oil while the higher side is still upward above oil (FIG. 2C). The droplet pool is dispensed via pipette into oil right along the MCSD-oil interface. As the angled MCSD moves up slowly under the control of the robotic arm, the droplet-air interface moves down the MCSD. As droplets encounter new unfilled microwells, they rapidly enter and fill the unfilled microwells.

[0120] (4) Device sealing. After microwells of the micropattemed combinatorial screening device are filled up with the droplets, the MCSD is taken off from the robotic arm and sealed with a film (FIG. 2D). As shown in FIG. 2D, an MCSD is sealed with a sealer. The sealer contains a roller, on which a film is paced. The roller has rotational degrees of freedom. A film seal is pressed up against an MCSD as it is pushed along the roller.

[0121] (5) Pre-merge image taking. The sealed MCSD is placed in a carrier, which is then placed on a plate stacker before imaging (FIG. 2E). The dimensions of the carrier conform to ANSI SLAS 4-2004 (R2012) microplate standards. The plate stacker can hold up to 14 MCSDs with carriers for large scale screening. A second robotic arm takes one MCSD at a time to a nearby imaging station for initial scans of microwells under a microscope (“pre-merge image”) (FIG.2F).

[0122] (6) Droplets merging. After pre-merge image taking, the robotic arm moves the MCSD from the microscope to a merging station which contains a corona treater (FIG. 2G). The corona treater generates an electric field, causing the droplets to spontaneously merge.Atty. Docket No. 202421-703601

[0123] (7) Treatment and measurement after merging. After merging, the robotic arm moves the MCSD to a nearby incubator for storage with controlled temperature until it is taken out for an eventual time point scan at the imaging station (FIG. 2G).EXAMPLE 2: MCSD Photomask / Wafer Example

[0124] Described in this example is a photomask for generating features of photoresist on the wafers, such that the black regions are opaque and the white are transparent for allowing UV-light to shine through and crosslink an SU-8 photoresist. As shown in FIG. 5A, The photomask comprises a top left fiducial mark, a bottom left fiducial mark, and a bottom right fiducial mark in a microwell field, according to some embodiments of this disclosure. Here, the region comprises 11,488 k=l microwells, and a spatially distinct section of k=l microwells is indicated on the left side. Further, the fiducial marks each comprise 36 k=l microwells arranged in a double cross pattern. FIG. 5B illustrates a close-up view of an exemplary fiducial mark, according to some embodiments of this disclosure, for a k=2 mask.EXAMPLE 3: Mold Examples

[0125] Described in this example are two molds according to some embodiments of this disclosure. As illustrated in FIGS. 4A-4B, the mold comprises an aluminum top plate and an aluminum back plate, such that the mold is configured to fit a wafer between the aluminum top plate and the back plate ().

[0126] FIGS. 4C-4D illustrate another exemplary mold according to some embodiments of this disclosure. The second mold comprises an aluminum top piece, an acrylic mold materialconstraining layer, and an aluminum back plate, such that the mold is configured to fit a wafer between the mold material-constraining layer and the back plate (FIGS. 4C-4D). The back plate comprises a pocket configured to fit the wafer such that the wafer sits in the pocket of the back plate. The mold material-constraining layer is laser cut to sit on top of the wafer and align to the flat edge of the wafer such that the mold material-constraining layer is centered around the features that have been transferred to the wafer. The depth of the pocket is 2 mm.EXAMPLE 4: Carrier for the MCSD

[0127] Described in this example a 3D-rendering and a photograph of an exemplary embodiment of the carrier with a micropatterned combinatorial screening device inserted. The carrier in FIGS. 12A-12B conforms to the ANSI SLAS 4-2004 (R2012) microplate standards, comprising a length of 127.76 mm, a width of 85.48 mm and a height of 15 mm. This allows theAtty. Docket No. 202421-703601carrier to be handled in the same way as a microplate with robot grippers and the automated incubator (i.e. the size that nearly all off-the-shelf laboratory grippers are standardized for). This also ensures that standard-height plate storage stacks can be used in the incubator without any modification. With the carrier, the MCSDs can now be moved on and off the microscope and into the incubator with the robot arm.EXAMPLE 5: Anaerobic or Microaerophilic Carrier Example

[0128] Described in this example is an anaerobic carrier for the MCSD according to some embodiments of this disclosure. The anaerobic carrier in FIG. 13 is fabricated from a stereolithography (SLA) 3D printer or a computer numerical control (CNC) machine, which provides a more airtight print than produced through a fused deposition modeling (FDM) 3D printer through the use of a resin that cures upon exposure to ultraviolet (UV light).

[0129] FIG. 14A is a photograph of an anaerobic carrier, FIG. 14B is a photograph of the anaerobic carrier of FIG. 14A with an inserted MCSD, FIG. 14C is a photograph of the anaerobic carrier of FIG. 14B with a gasket and a lid, and FIG. 14D is a photograph of the anaerobic carrier of FIG. 14C, wherein the lid is clamped to the anaerobic carrier using 6 clips to maintain a seal, according to an embodiment of this disclosure. The anaerobic carrier contains a 1.2 mm-thick glass that is glued in place and sealed to the anaerobic carrier using a silicone sealant. The lid is laser cut. The gasket comprises a 1 / 16” butyl rubber for ensuring the airtight seal. The second plate is clamped to the carrier using 6 clips.EXAMPLE 6: Additional Automated Loading Example

[0130] Described in this example is an automated loading system. The MCSD, suctioned to a robotic arm, was suspended over a tray of oil (FIG. 8A). Droplets were then added to the oil at the loading edge of the MCSD. As the MCSD, which is at an angle relative to the surface of the oil, is lifted (FIG.8B), the oil-device interface moves down the MCSD, carrying the droplets with it and loading them into microwells.EXAMPLE 7: Anaerobic Assay Strains for Anaerobic Screening System

[0131] To develop a GFP reporter system, strains of Escherichia coH. Bacteroides thetaiotaomicron, and Staphylococcus aureus were modified to express GFP. While GFP did not fluoresce without oxygen, the modified strains expressing GFP could be used as an endpoint assay after removing the reporter from an anaerobic environment. The modified strains were grown without oxygen, and GFP signal of the modified strains was tested in a microtiter plate that wasAtty. Docket No. 202421-703601sealed in anaerobic conditions. A hole was poked in some wells to allow aeration and other wells remained unsealed and fluorescence was measured in an aerobic environment (as shown FIGS.15A-15B for E. coli strains). The results show the GFP signal quickly became detectable and was stable under hole poked conditions (FIG. 15A) as compared to non-aerated cultures (FIG. 15B).

[0132] To understand the maturation of GFP signal upon exposure to oxygen, cultures of wild-type and GFP-labeled E. coli were grown anaerobically. Following normalization, they were loaded into an MSCD within an anaerobic chamber and imaged prior to merging while in an anaerobic carrier (labeled as timepoint -1 in FIG. 16). Following pre-merge imaging, the MSCD was removed from the anaerobic carrier, merged using a Kimwipe, and imaged at T=0. The MCSD was then incubated aerobically at room temperature and imaged every hour. The results show the fluorescent signal from the GFP-labeled E. coli rapidly mature between the anaerobic pre-merge timepoint and the initial post-merge T=0 timepoint with additional increase at 1 hr followed by a plateau in signal.

[0133] Two other anaerobic reporter systems were developed for the anaerobic screening system. A UnaG / Bilirubin system was developed by modifying strains of E. coli to express inducible UnaG. After IPTG induction, UnaG fluoresced in the presence of the fluorogenic ligand bilirubin (FIGS. 17A-17B) and the fluorescence in live cells was detectable under a microscope (FIGS. 18A-18D)

[0134] A Pepper system was developed by modifying the E. coli to transcribe Pepper, an RNA aptamer that fluoresces when bound to a chromophore, such as benzylidene-cyanophenyl (HBC). Pepper fluoresced in the presence of HBC. (FIGS. 19A-19B). The fluorescence in live cells expressing Pepper was also detectable under a microscope (FIGS. 20A-20C). In both cases, the fluorescence decreased over time. Though the fluorescence became much weaker, it was still detectable after 24 hours.EXAMPLE 8: Screening Isolated Bacterial Strains for Interactions with a Pathogenic Bacterial Species

[0135] Isolated bacterial species are screened in a system described herein to determine isolate(s) interaction with strains of S. aureus. Briefly, bacterial isolates and S. aureus strains are added to an MCSD that contains thousands of microwells. Strains are mixed with fluorescent agents with different excitation and / or emission wavelengths for identification and when mixed create a unique emission wavelength code that is read by an imaging device to determine input. The S. aureus strains are promoter / reporter strains that are used to screen for inhibition of gene expression. Decreased expression of the promoter / reporter strains when grown with bacterialAtty. Docket No. 202421-703601isolate(s) is indicated as a decrease in fluorescence as compared to the promoter / reporter strain grown alone. Increased expression of the promoter / reporter strains when grown with bacterial isolate(s) is indicated as an increase in fluorescence as compared to the promoter / reporter strain grown alone.

[0136] The S. aureus promoter / reporter strains contain a fluorescent reporter under the control of a promoter of a gene of interest. The promoter / reporter strains generated are as follows: Strain 1- agr promoter for quorum sensing induction, Strain 2- psma promoter for a toxin that damages host tissue, Strain 3- saeR promoter for virulence regulation, Strain 4- sigB promoter for the stress response sigma factor, Strain 5- ccpA promoter for metabolism (e.g., carbon catabolite repression), and strain 6- gmk promoter for constitutive metabolic function.EXAMPLE 9: Screening Isolated Bacterial Strains for Interactions with a Pathogenic Fungal Species

[0137] Isolated bacteria species are screened in a screening system described herein to determine isolate(s) interaction with a pathogenic fungal species, such as Candida albicans. Briefly, bacterial isolates and Candida albicans strains are added to an MCSD that contains thousands of microwells. Strains are mixed with fluorescent agents with different wavelengths for identification and when mixed create a unique wavelength code that is read by an imaging device to determine input. The Candida albicans strains are promoter / reporter strains that are used to screen for inhibition of gene expression. Decreased expression of the promoter / reporter strains when grown with bacterial isolate(s) is indicated as a decrease in fluorescence as compared to the promoter / reporter strain grown alone. Increased expression of the promoter / reporter strains when grown with bacterial isolate(s) is indicated as an increase in fluorescence as compared to the promoter / reporter strain grown alone.

[0138] The Candida albicans promoter / reporter strains contain a fluorescent reporter under the control of a promoter of a gene of interest. The promoter / reporter strains to be developed are as follows: Candida albicans Strain 1 - ECE1 promoter for detection of a toxin that damages host tissue, Candida albicans Strain 2 - TDH3 promoter for detection of constitutive metabolism function, Candida albicans Strain 3 - RPS1 2 promoter for detection of constitutive metabolism function, Candida albicans Strain 4 - SAP2 promoter for detection of secreted aspartyl protease, Candida albicans Strain 5 - SAP5 promoter for detection of secreted aspartyl protease, and Candida albicans Strain 6 - ALS3 promoter for detection of surface adhesion protein.Atty. Docket No. 202421-703601EXAMPLE 10: Automated System with Automated Droplet Generation, Loading, and Sealing

[0139] Described in this example is a set up providing for automated droplet generation, automated loading of droplets, and automated sealing of MCSD after loading. Specifically, loading and sealing of MCSDs are integrated into one automated set-up. MCSDs are picked up with a robot arm using a custom end of arm tool (holding unit) that has a suction cup (FIG. 26 and FIG. 27).The end of arm tool also has a machine vision camera that is used during the loading of droplets. It ensures the oil level is correctly aligned to the MCSD edge. This robot arm submerges the MCSD in the same oil bath from the original filing.

[0140] As illustrated in FIG.29, a robot arm is installed, with an electronic pipette mounted to it for aspirating and dispensing the droplets. The pipette mounted to the second robot arm aspirates droplets from a tube and dispenses them underneath the MCSD. The first robot arm then moves through a similar sequence as before by raising and lowering the MCSD to load droplets into the wells.

[0141] Sealing is done by the device as illustrated in FIG.28. This device functions in the same way as the manual version in FIG. 2D. It includes two rollers. The top rod / roller is fixed in place and can be rotated by a motor via a belt. The bottom roller is fixed to two pneumatic actuators that raise and lower the bottom roller. There is a ramp that supports and aligns the seal to the roller. To seal an MCSD, a seal is placed on the loading ramp. The robot arm with the MCSD withdraws the MCSD from the oil bath and moves it between the two rollers. The bottom roller is pressed up by two pneumatic actuators to press the seal against the MCSD. The top roller is rotated by the motor. The friction between the top roller and the back side of the MCSD causes the MCSD and seal to be pushed forward. Once the MCSD is pushed all the way through, the actuators retract the bottom roller releasing the MCSD.EXAMPLE 11: Introduction of Organisms to a Community at Different Times to Test Their Effects

[0142] To examine how an established microbial community responds to the arrival of a new / foreign organism, an assay with MCSD is conducted by loading a variety of test organisms onto an MCSD at the beginning of the assay and allowing them to grow together. The assay process is the same as what is described in Example 1. After images are taken, the sealed MCSD is placed on a metal block which has been cooled to at least -70 °C and chilled for up to 5 minutes. The seal is then quickly pulled off the MCSD surface. The freezing process stabilizes the previously loaded and merged droplets in the microwells so that the unsealing process leaves the majority of themAtty. Docket No. 202421-703601undisturbed. The MCSD is then re-immersed in oil to gently thaw that MCSD. Additional droplets containing organisms which are new to the community are then loaded onto the MCSD and occupy open spaces in the microwells that are created after the merging step earlier in the process. This MCSD is then re-sealed and the newly added droplets are merged with the rest of the droplet contents, allowing the introduction of the “foreign organisms” for testing which communities prevent or permit its engraftment in the community.EXAMPLE 12: Detection of Specific Biological Activities with Toxic Chemical Reagent

[0143] Described herein is an example for testing the abundance and activities of a target organism Yarrowia lipolytica in presence of a fungal stain Calcofluor White. This fungal stain Calcofluor White can be absorbed into the fungal cell wall of the organism Yarrowia lipolytica and is toxic to the fungus being measured. Accordingly, the fungal stain Calcofluor White cannot be introduced at an early stage of the assay prior to growth of organism Yarrowia lipolytica, as early exposure to the stain would suppress fungal growth and interfere with the measurement. Instead, the organisms Yarrowia lipolytica must first be grown on the MCSD, and the fungal stain Calcofluor White is introduced later. The assay process is the same as what is described in Example 1. After images are taken, the sealed MCSD is placed on a metal block which has been cooled to at least -70 °C and chilled for up to 5 minutes. The seal is then quickly pulled off the MCSD surface. The freezing process stabilizes the previously loaded and merged droplets in the microwells so that the unsealing process leaves the majority of them undisturbed. The MCSD is then re-immersed in oil to gently thaw that chip. Additional droplets containing the fungal stain Calcofluor White are then loaded on to the MCSD and occupy open spaces in the microwells that are created after the merging step earlier in the process. This MCSD is then re-sealed and the newly added droplets are merged with the rest of the droplet contents, allowing the introduction of the fungal stains for testing.EXAMPLE 13. Screening with Nucleic Acid Probe-Based Detection with MCSD

[0144] The components of nucleic acid probe-based detection are usually degraded by live bacteria present in the droplets. To conduct screening with MCSD with nucleic acid probe-based detection, droplets containing live bacteria to be tested are introduced to MCSD, sealed, and incubated for a certain period of time. Then the sealed MCSD is placed on a metal block which has been cooled to at least -70 °C and chilled for up to 5 minutes. The seal is then quickly pulled off the MCSD surface. The freezing process stabilizes the previously loaded and merged droplets in the microwells so that the unsealing process leaves the majority of them undisturbed. The MCSDAtty. Docket No. 202421-703601is then re-immersed in oil to gently thaw that chip. Additional droplets containing the nucleic acid probes are then loaded on to the MCSD to occupy open spaces in the microwells that are created after the merging step earlier in the process. This MCSD is then re-sealed and the newly added droplets are merged with the rest of the droplet contents, allowing the introduction of the nucleic acid probe for detection.EXAMPLE 14: Screening of Supernatants from Isolated Bacterial Strains for Interactions with Microbes of Interest

[0145] A screen using the MCSD with an automated system described herein was designed to measure the effects of pairwise combinations of supernatants from a supernatant library on the growth of selected target microbes of interest (e.g., odor-causing microbes). The supernatant library was subsetted (more details below in this section) such that a set of ninety-six inputs (see “MCSD screen execution”) were loaded onto each MCSD, with 4,656 possible pairwise combinations. To assemble the pairwise combinations, “k=2” (2-droplet microwell) MCSDs were used, each containing 11,488 one-input microwells and 113,199 two-input microwells. We aimed to achieve ~20 replicates of each possible combination. Based on what was expected to be a conservative estimate that each MCSD would yield 45,000 usable microwells after all data filtering steps, we chose to run each set of inputs in triplicate - the same pool of input droplets was loaded onto three separate MCSDs to increase the overall replicate count.

[0146] The library was handled as two subsets: an AF-BHI library and AF-uTSB library, each containing 230 supernatants. Pairwise combinations across the two subsets were not sampled. Each subset library was divided into five “blocks” of supernatants, consisting of 46 supernatants each. Each MCSD sampled two blocks of supernatants, for a total of 92 supernatant inputs and three control inputs (more details below in this section). These inputs were combined with the target microbial strain in a 1:1 ratio, such that each input was diluted to 50% (v / v). Additionally, a non-inoculated control consisting of 50% volume assay medium combined with 50% volume supernatant library medium was included to serve as a contamination check.

[0147] Three inputs were included to serve as various types of “untreated” controls for the target: 1) A PBS control, which would provide no additional nutrients 2) An AF-BHI or AF-uTSB medium control, as appropriate for each respective subset library, which would provide additional nutrients to the target, and 3) A target supernatant control (generated by growing up the target microbial species in either AF-BHI or AF-uTSB, as appropriate to match the subset library; S. epidermidis, S. hominis, S. pyogenes, and C. acnes did not grow in AF-uTSB, so supernatants were generated from TSB cultures), which was hypothesized to not affect growth of the target strain,Atty. Docket No. 202421-703601but could most closely mimic the supernatant inputs. The PBS control was ultimately used for all analyses.Table 2. Microbial target strainsai number Species Strain Sourceail 5 Staphylococcus aureus TCH1516 ATCC BAA-1717ai 16 Candida albicans SC5314 ATCC MYA-2876ai82 Pseudomonas PA14 NR-50573aeruginosaail39 Cutibacterium acnes VPI 0389 ATCC 6919ail 56 Cutibacterium acnes VPI 4979 ATCC 11827ai203 Staphylococcus 1194 ATCC 700563epidermidisai207 Staphylococcus hominis MK 129 ATCC 27846ai225 Streptococcus pyogenes Bruno (CIP ATCC 19615104226)ai230 Corynebacterium M221 ATCC 49528striatumai235 Malassezia M609 ATCC 42756pachydermatis

[0148] Media and growth conditions for microbial targets

[0149] The strains used are shown in Table 2. S. aureus, S. epider midis, P. aeruginosa, S. pyogenes, S. hominis, and C. striatum were streaked on TSA and incubated at 37°C for 24 hours aerobically to isolate single colonies. C. albicans was streaked on TSA and incubated at 30°C for 24 hours to isolate single colonies. Single colonies were inoculated into TSB and grown overnight in a shaking incubator (250 RPM) at 30°C for 24 hours. Cultures were mixed by pipetting up and down and ODeoo was measured.Atty. Docket No. 202421-703601

[0150] C. acnes was streaked onto BRU agar and incubated at 37°C in an anaerobic chamber for 3 - 4 days. Single colonies were inoculated into reduced TSB + 0.5g / L cysteine and incubated at 37°C in the anaerobic chamber without shaking for 4 days. Cultures were mixed by pipetting up and down and ODeoo was measured.

[0151] M. pachydermatis was streaked onto YPD agar and incubated at 37°C for 2 - 3 days aerobically. Single colonies were inoculated into YPD and grown in a shaking incubator (250 RPM) at 37°C for 4 days. Cultures were mixed by pipetting up and down and ODeoo was measured.

[0152] MCSD manufacturing

[0153] A “k=2” MCSD, containing ~113,0002-droplet microwells and ~11,000 1-droplet microwells, were used for these experiments. Custom silicone wafer molds were created by photolithography (Custom Nanotech). The chips were fabricated by soft lithography from polydimethylsiloxane (PDMS; Dow Corning Sylgard) and coated with 1 ,5-pm parylene C (Kayaku Advanced Materials, formerly Paratronix).

[0154] Growth assay quantification development of targets on MCSD

[0155] Microbial targets were grown as specified in the “Media and growth conditions for microbial targets” section. ODeoo of . aeruginosa, S. hominis, C. striatum were normalized to 0.01 in TSB. ODeoo of C. acnes strains were normalized to 0.05 in TSB + 0.5g / L cysteine. ODeoo of S. epider midis, C. albicans, S. pyogenes were normalized to 0.1 in TSB. ODeoo of AT. pachydermatis was normalized to 0.1 in YPD and sonicated using a Branson 5800 Cleaner at maximum power for 10 minutes. For S. aureus, several suspensions normalized to ODeoo 0.02, 0.2, 1, 2.5, 5 were made. A media (TSB) control without S. aureus was also included. AlexaFluor fluorescent dye 488 (AF488) was added into suspensions of microbial targets and standardized to a final concentration of 0.5 pM. An Integra ViaFlo96 was used to pipette 3.5 pl of a “color code”, or a unique ratio of three AlexaFluor fluorescent dyes (AF555, AF594, and AF647) into each input in the plate. The color codes were standardized to a combined final concentration of 2.5 pM. The mixtures were pipetted up and down to mix. 30 pl of each input were transferred into the sample wells of BioRad DG8 droplet cartridges using an Analytik Jena CyBio FeliX, with the exception of inputs containing AT pachydermatis which were vigorously mixed by additional pipetting to break up cell clumps and manually transferred to droplet cartridges to minimize the amount of cells that would settle at the bottom of the plate. 50 pl of 2% 008-FluoroSurfactant (RAN Biotechnologies) in Novec 7500 oil (3M) was transferred to the oil wells of the DG8 cartridges. Targets and corresponding media controls were dropletized (1 nl) using a droplet generator consisting of a custom pressure manifold and pressure controller (Elveflow OBI) to generate droplets that were approximately 130 pm in diameter. Pressures of 320 mbar and 380 mbar were used for the oil andAtty. Docket No. 202421-703601sample channels respectively for blocks containing inputs in TSB-based media (all microbes with the exception of M. pachydermatis). Pressures of 275 mbar and 425 mbar were used for the oil and sample channels respectively for blocks containing inputs in YPD-based media (M. pachydermatis). Droplets were pooled to prepare a droplet suspension using an Opentrons liquid handling robot and loaded into a custom-built MCSD loading apparatus. Each microwell randomly sampled 2 droplets. All MCSDs were imaged using a Nikon Eclipse Ti2 at 2x magnification using 4 channels (brightfield, and fluorescent channels to capture AF555, AF594, and AF647) before droplets were merged. Droplets were then merged within their microwells via ~10 s of exposure to an alternating-current field (4.5 MHz, 10,000-45,000 volts; Electro-Technic Products corona treater) and imaged again (t = 0) at 4x magnification using 2 channels (brightfield and a fluorescent channel to capture AF488). The MCSDs containing P. aeruginosa, S. hominis, C. striatum, S. epidermidis, C. albicans, and S. pyogenes were incubated at 30°C and imaged at 4x magnification using 2 channels (brightfield and a fluorescent channel to capture AF488) every hour for 24 hours. MCSDs containing S. aureus were only imaged at t = 0 and the various known normalized densities prepared at the outset of the experiment were used for growth modeling. The MCSDs containing M. pachydermatis were incubated at 37°C and imaged every 3 hours for 72 hours. The MCSDs containing C. acnes were incubated at 37°C in an anaerobic chamber in a sealed plastic bag with damp paper towels to maintain humidity and imaged at hours 0, 12, 24, 32, 48, 56, 72, 80.

[0156] Microbial growth in droplets was quantified using a deep convolutional neural network trained on microwell images. The images contained droplets with either a known optical density (for S. aureus) or a defined growth time. The model uses a ResNet-50 backbone with a modified classification head that outputs a predicted mean and log variance. Thus, for new data, the model estimated either the optical density or the equivalent hours of growth under ideal conditions.

[0157] MCSD Screen Execution

[0158] Microbial targets were grown as specified in the “Media and growth conditions for microbial targets” section. ODeoo of each target except S. aureus were normalized to 2x the original densities mentioned in the above section “Growth assay quantification development of targets on MCSD workflow”. S. aureus was normalized to an ODeoo of 0.02. The media in which targets were normalized contained 2x the concentrations of solute (2x TSB, 2x TSB + Ig / L cysteine, and 2x YPD) containing 1 pM AF488. 96-well plates containing arrayed supernatants were thawed at 25°C for 10 minutes and centrifuged at 1000 RCF for 30 seconds prior to use. The appropriate microbial target was added to each well of the plate containing arrayed supernatants in a 1:1 (v / v) ratio. One well containing blank assay medium was not inoculated with microbes to serve as aAtty. Docket No. 202421-703601negative control. An Integra ViaFlo96 was used to ensure each input in the plate received 3.5 pl of a “color code”, or a unique ratio of three AlexaFluor fluorescent dyes (AF555, AF594, and AF647) to achieve a final concentration of 2.5 pM. The mixtures were pipetted up and down to mix. 30 pl of each input were transferred into the sample wells of Bio-Rad DG8 droplet cartridges using an Analytik Jena CyBio FeliX, with the exception of inputs containing M. pachydermatis which were vigorously mixed by additional pipetting to break up cell clumps and manually transferred to droplet cartridges to minimize the amount of cells that would settle at the bottom of the plate. 50 pl of 2% 008-FluoroSurfactant (RAN Biotechnologies) in Novec 7500 oil (3M) was transferred to the oil wells of the DG8 cartridges. Targets and corresponding media controls were dropletized (~1 nl) using a droplet generator consisting of a custom pressure manifold and pressure controller (Elveflow OBI) to generate droplets that were approximately 130 pm in diameter. Pressures of 320 mbar and 380 mbar were used for the oil and sample channels respectively for blocks containing inputs in TSB-based media (all microbes with the exception of M. pachydermatis). Pressures of 275 mbar and 425 mbar were used for the oil and sample channels respectively for blocks containing inputs in YPD-based media (M. pachydermatis). Droplets were pooled to prepare a droplet suspension using an Opentrons liquid handling robot. 475 pl of droplets from the pool were loaded into a custom-built MCSD loading apparatus. This was done 3 times per pool of droplets to increase the number of replicates obtained for each supernatant combination. Each k=2 microwell randomly sampled 2 droplets and each k=l microwell sampled 1 droplet.

[0159] All MCSDs were imaged using a Nikon Eclipse Ti2 microscope with a Teledyne Kinetix sCMOS camera. The microscope was controlled with custom python scripts that used Pycro-Manager and Micro-Manager. All MCSDs were imaged at 2x magnification using 4 channels (brightfield, and fluorescent channels to capture AF555, AF594, and AF647) at 126 tile positions. Droplets were then merged within their microwells via about 10 s of exposure to an alternating-current field (4.5 MHz, 10,000-45,000 volts; Electro-Technic Products corona treater). MCSDs were then incubated in specified conditions until they were imaged again. MCSDs containing S. aureus, P. aeruginosa, S. hominis, C. striatum, S. epidermidis, C. albicans, and S. pyogenes were incubated at 30°C. MCSDs containing M. pachydermatis were incubated at 37°C in a sealed plastic bag with damp paper towels to maintain humidity. MCSDs containing C. acnes were incubated at 37°C in an anaerobic chamber in a sealed plastic bag with damp paper towels to maintain humidity.

[0160] Merged droplets on MCSDs were imaged at 4x magnification using 2 channels (brightfield, and a fluorescent channel to capture AF488) at 360 tile positions to quantify microbial growth in each microwell. For S. aureus, C. albicans, P. aeruginosa, S. epidermidis, S. hominis,Atty. Docket No. 202421-703601C. striatum, and S. pyogenes, images were taken 10 and 16 hours post-merge. For M. pachydermatis, images were taken 48 hours post-merge. For C. acnes, images were taken 72 hours and 80 hours post-merge.

[0161] MCSD Screen Analysis

[0162] A montage of each MCSD was constructed from individual image tiles by calculating the offset between overlapping tiles edges and optimizing the correct overlap for each tile pair. Individual well boundaries were identified prior to droplet merging using a custom-trained convolutional neural network, and the contents of each droplet were identified based on the ratio of dyes in the three fluorescence channels. Each well was tracked to the corresponding location in the post-merging image using a series of elastic registration steps. The amount of microbial growth per well was then quantified by applying a deep convolutional neural network (see “Growth assay quantification development of targets on MCSD” above) to the image of each microwell. Briefly, a machine learning (“ML”) model used for growth assessment was trained per target on a linear time course of images, which provided snapshots of different amounts of growth (FIG. 23). These timepoints provide the units of measurement used throughout the analysis. “Media only” and “target only” controls set the normalization range for growth measurements. The “media only” controls provide the lower bound of the normalization. The median growth value of these wells is set by definition to an effect size of 0. Isolated target growth (the “target only” controls) provide the upper bound of normalization. The median growth value of these wells is set by definition to an effect size of 1, and it is generally equal to the saturation point of the time course. The ML model scored each well in the time course units it which was trained. Then those values were normalized to the control range. For example, in a single microwell of an MCSD, to evaluate the growth of a microbe (e.g., S. aureus), an effect size = 0.1 means in the presence of a combination of two supernatants, the culture got to 0.1 (i.e., in around 1.2 hours of what the culture achieved in isolation. An effect size = 1.2 means in the presence of a combination of two supernatants, the culture got to 1.2 (i.e. “14 hours”) of what the culture achieved in isolation, suggesting that the culture grew more than it could in the absence of the two supernatants. A comparison of the effect sizes determined by MCSD and plate reader is shown in FIG. 24 (correlation coefficient = 0.93), showing a strong consistency of the results as determined using MCSD.

[0163] Data was excluded from analysis if microwells contained more than one droplet for k=l or any number other than two droplets for k=2. Microwells with incomplete droplet merging, irregular merged droplet shapes, or abnormal droplet areas were also removed as part of data quality filtering.Atty. Docket No. 202421-703601

[0164] For each supernatant pair, a summary growth metric was calculated as the background- subtracted signal relative to background-subtracted target-only signal to generate growth scores for further downstream analysis.

[0165] This example demonstrated a high-throughput screening assay with the devices and methods described herein having technical advantages compared to conventional arrays for exploring and exploiting new bacterial networks or suppression of odor-causing bacteria on skin with high-volume datasets. With the automated droplet generation, pooling, loading and imaging, the efficiency in running the screening improved significantly. A comparison between screening performed with the automated system and a corresponding manual system is provided in Table 3. Table 3. MCSD screen performed with automated system vs. manual systemManual System Automated SystemGeneral Features Fully manual workflow Automated droplet generation,pooling, loading, and imaging Total Combinations Tested 5,424,888 33,599,731Max MCSDs Per 6 24Screening DayFull-Time Equivalents 3 1-1.5(FTEs) Per Screening Day

[0166] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Atty. Docket No. 202421-703601CLAIMS WHAT IS CLAIMED IS:

1. A method of screening, comprising:(a) providing a device, a pool of droplets comprising bioagents and / or chemicals, and one or more robotic arms,wherein the device comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents and / or chemicals;(b) mounting the device to a first robotic arm;(c) loading the one or more droplets in the pool to the device via an automated process that controls the first robotic arm to: (i) submerge or partially submerge the device in a bath unit configured to retain a pooled fluid that is immiscible with the droplets through the first robotic arm, wherein the device is tilted relative to the surface of the bath unit during the submerging step; (ii) add the droplets to the pooled; and (iii) move the device vertically relative to the surface of the pooled fluid such that one or more droplets are loaded to the device, thereby allowing the droplets to enter the device;(d) removing the device from the first robotic arm;(e) mounting the device to a second robotic arm;(f) moving the device to an imaging station using the second robotic arm;(g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the bioagents or chemicals in each droplet;(h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of bioagents and / or chemicals;(i) measuring optical signals of the combinations after the merging, wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms.

2. The method of claim 1, wherein prior to step (b), the droplets are formed using a droplet generator.

3. The method of claim 2, wherein after the droplets are formed, the droplets are aspirated and dispensed with an electronic pipette mounted to a third robotic arm.

4. The method of claim 1, wherein the droplets comprise bioagents and chemicals, wherein the method assays growth of the bioagents in the presence of the chemicals.

5. The method of claim 1, wherein the device is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid.Atty. Docket No. 202421-7036016. The method of claim 5, wherein the device is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid.

7. The method of claim 5, wherein the device is tilted at an angle of 10 degrees relative to the surface of the pooled fluid.

8. The method of claim 1, wherein prior to step (e), the device is sealed.

9. The method of claim 8, wherein the device is sealed with a film or a glass.

10. The method of claim 1, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof.

11. The method of any one of claims 1 to 10, wherein step (i) further comprises:(1) receiving input information from a user of the device to be imaged and a number of timepoints to be taken by an imaging apparatus;(2) controlling the second robotic arm to move the device from a first location to the imaging apparatus to obtain first images;(3) controlling the second robotic arm to move the device to an automated incubator for storing the device;(4) unloading the device from the automated incubator and moving the device back to the imaging apparatus to obtain second images;(5) moving the device back to the automated incubator; and(6) repeating one or more of steps ( 1 )-(5) for the device according to the number of timepoints.

12. The method of any one of claims 1 to 11, wherein the device is positioned in a carrier, wherein the one or more robotic arms grip the carrier to move the device.

13. The method of claim 1, wherein the droplets comprise a plurality of bioagents and / or chemical compounds.

14. The method of any one of claims 1 to 13, wherein the bioagents comprise prokaryotic cells.

15. The method of claim 14, wherein the prokaryotic cells comprise bacteria.

16. The method of claim 15, wherein the bacteria is an odor-causing bacteria.

17. The method of claim 1 to 13, wherein the bioagents comprise eukaryotic cells.

18. The method of claim 17, wherein the eukaryotic cells comprise fungi.

19. The method of claim 18, wherein the fungi comprise filamentous fungi.

20. The method of claim 18, wherein the fungi comprise yeast.

21. The method of claim 17, wherein the eukaryotic cells comprise protists.Atty. Docket No. 202421-70360122. The method of claim 21, wherein the protists comprise eukaryotic algae.

23. The method of claim 17, wherein the eukaryotic cells comprise mammalian cells.

24. The method of claim 13, wherein the bioagents are anaerobic microbes or anaerobic yeasts.

25. The method of claim 1, further comprising detecting and determining a level of a signal.

26. The method of claim 25, wherein the signal is from a morphology measurement.

27. The method of claim 25, wherein the signal is from an entropy measurement.

28. The method of claim 25, wherein the signal is from an overall optical interference.

29. The method of claim 25, wherein the signal is from one or more reporters.

30. The method of claim 29, wherein the one or more reporters are expressed in an anaerobic microbe.

31. The method of claim 30, wherein the one or more reporters comprises a fluorescent protein.

32. The method of claim 31, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen.

33. The method of claim 31, wherein the fluorescent protein is a ligand-inducible fluorescent protein.

34. The method of claim 33, wherein the ligand-inducible fluorescent protein is UnaG.

35. The method of claim 29, wherein the one or more reporters comprise an RNA aptamer, wherein the RNA aptamer fluoresces when bound to a chromophore.

36. The method of claim 8, further comprising unsealing the device and repeating step (c).

37. The method of claim 36, wherein the device is cooled before the unsealing step.

38. The method of claim 37, wherein the device is cooled at -70 °C.

39. The method of claim 1, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased number of combinations of bioagents and / or chemicals tested on a single device.

40. The method of claim 1, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual dropletAtty. Docket No. 202421-703601generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased maximum number of devices screened per day.

41. The method of claim 1, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by a reduced full-time equivalent (FTE) required to perform an equal number of screenings per day.

42. A method of screening for combinations of live cells with bioagents or chemicals, comprising:(a) providing a device, one or more robotic arms, and a pool of droplets comprising: (i) a population of live cells and (ii) bioagents and / or chemicals, wherein the device comprises an array of microwells, wherein each microwell is capable of receiving one or more droplets from the pool of droplets;(b) mounting the device to a first robotic arm;(c) loading the one or more droplets from the pool of droplets to the device by an automated process;(d) removing the device from the first robotic arm;(e) mounting the device to a second robotic arm;(f) moving the device to an imaging station using the second robotic arm;(g) measuring optical signals from each droplet in the microwells, wherein the measuring determines the identities of the population of the live cells, bioagents or chemicals in each droplet;(h) applying a coalescing mechanism to merge the droplets loaded to the microwells, thereby generating in parallel combinations of the population of the live cells, bioagents and / or chemicals;(i) measuring optical signals from the combinations after the merging, wherein the signals from combinations of the population of the live cells with the bioagents and / or chemicals indicate changes of activities of the population of live cells in presence of the bioagents and / or chemicals,wherein the steps (b), (c), (g) to (i) are performed by an automated process employing the one or more robotic arms.

43. The method of claim 42, wherein prior to step (b), the droplets are formed using a droplet generator.Atty. Docket No. 202421-70360144. The method of claim 43, wherein after the droplets are formed, the droplets are aspirated and dispensed with an electronic pipette mounted to a third robotic arm.

45. The method of claim 42, wherein the droplets comprise bioagents and / or chemicals, wherein the method assays growth of the bioagents in presence of the chemicals.

46. The method of claim 42, wherein step (c) comprises:(1) submerging or partially submerging the device in a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets through the first robotic arm, wherein the device is tilted relative to the surface of the bath unit during the submerging step;(2) adding droplets to the pooled liquid; and(3) moving the device vertically relative to the surface of the pooled fluid such that one or more droplets are loaded to the device, thereby allowing the droplets to enter the device.

47. The method of claim 46, wherein the device is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid.

48. The method of claim 46, wherein the device is tilted at an angle of from 5 degree to 30 degree relative to the surface of the pooled fluid.

49. The method of claim 46, wherein the device is tilted at an angle of 10 degrees relative to the surface of the pooled fluid.

50. The method of claim 42, wherein prior to step (e), the device is sealed.

51. The method of claim 50, wherein the device is sealed with a film or a glass.

52. The method of claim 42, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof.

53. The method of any one of claims 42 to 52, wherein step (i) further comprises:(1) receiving input information from a user of the device to be imaged and a number of timepoints to be taken by an imaging apparatus;(2) controlling the second robotic arm to move the device from a first location to the imaging apparatus to obtain first images;(3) controlling the second robotic arm to move the device to an automated incubator for storing the device;(4) unloading the device from the automated incubator and moving the device back to the imaging apparatus to obtain second images;(5) moving the device back to the automated incubator; andAtty. Docket No. 202421-703601(6) repeating one or more of steps ( 1 )-(5) for the device according to the number of timepoints.

54. The method of any one of claims 42 to 53, wherein the device is positioned in a carrier, wherein the one or more robotic arms grab the carrier to move the device.

55. The method of claim 42, wherein the droplets comprise a plurality of bioagents and / or chemical compounds.

56. The method of any one of claims 42 to 55, wherein the population of the live cells comprise prokaryotic cells.

57. The method of claim 56, wherein the prokaryotic cells comprise bacteria.

58. The method of claim 57, wherein the bacteria is an odor-causing bacteria.

59. The method of claim 42 to 55, wherein the bioagents comprise eukaryotic cells.

60. The method of claim 59, wherein the eukaryotic cells comprise fungi.

61. The method of claim 60, wherein the fungi comprise filamentous fungi.

62. The method of claim 60, wherein the fungi comprise yeast.

63. The method of claim 59, wherein the eukaryotic cells comprise protists.

64. The method of claim 63, wherein the protists comprise eukaryotic algae.

65. The method of claim 59, wherein the eukaryotic cells comprise mammalian cells.

66. The method of claim 42, wherein the bioagents are anaerobic microbes or anaerobic yeasts.

67. The method of claim 42, further comprising detecting and determining a level of a signal.

68. The method of claim 67, wherein the signal is from morphology measurement.

69. The method of claim 67, wherein the signal is from entropy measurement.

70. The method of claim 67, wherein the signal is from overall optical interference.

71. The method of claim 67, wherein the signal is from one or more reporters.

72. The method of claim 71, wherein the one or more reporters are expressed in an anaerobic microbe.

73. The method of claim 72, wherein the one or more reporters comprises a fluorescent protein.

74. The method of claim 73, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen.

75. The method of claim 73, wherein the fluorescent protein is a ligand-inducible fluorescent protein.Atty. Docket No. 202421-70360176. The method of claim 75, wherein the ligand-inducible fluorescent protein is UnaG.

77. The method of claim 71, wherein the one or more reporters comprise an RNA aptamer, wherein the RNA aptamer fluoresces when bound to a chromophore.

78. The method of claim 50, further comprising unsealing the device and repeating step (c).

79. The method of claim 78, wherein the device is cooled before the unsealing step.

80. The method of claim 79, wherein the device is cooled at -70 °C.

81. The method of claim 42, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased number of combinations of bioagents and / or chemicals tested on a single device.

82. The method of claim 42, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by an increased maximum number of devices screened per day.

83. The method of claim 42, wherein the method provides for improved efficiency as compared to the method performed by an otherwise equivalent system with manual droplet generation, manual pooling, and / or manual loading and imaging, wherein the improved efficiency is determined by a reduced full-time equivalent (FTE) required to perform an equal number of screenings per day.

84. A system comprising:a micropattemed combinatorial screening device (MCSD) comprising an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals; anda loading apparatus comprising:a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets;a holding unit for carrying the MCSD, wherein the holding unit is tilted at an angle relative to the surface of the pooled fluid.

85. The system of claim 84, further comprising a plurality of droplets deposited in each of the microwells, wherein each droplet comprises at least one type of bioagents or chemicals.Atty. Docket No. 202421-70360186. The system of claim 85, wherein the bioagents comprise prokaryotic cells.

87. The system of claim 86, wherein the prokaryotic cells comprise bacteria.

88. The system of claim 85, wherein the bioagents comprises eukaryotic cells.

89. The system of claim 88, wherein the eukaryotic cells comprise fungi.

90. The system of claim 89, wherein the fungi comprise filamentous fungi.

91. The system of claim 89, wherein the fungi comprise yeast.

92. The system of claim 88, wherein the eukaryotic cells comprise protists.

93. The system of claim 92, wherein the protists comprise eukaryotic algae.

94. The system of claim 88, wherein the eukaryotic cells comprise mammalian cells.

95. The system of claim 84, wherein upon movement of the holding unit relative to the surface of the pooled fluid, the one or more droplets are loaded into the MCSD, thereby allowing at least one of the one or more droplets to enter in each of the microwells to form massively parallel combinations of bioagents or chemicals.

96. The system of claim 84, wherein the holding unit comprises a mounting piece, wherein the MCSD is slid into the holding unit through the mounting piece.

97. The system of claim 84, wherein the MCSD is suctioned to the holding unit.

98. The system of claim 84, wherein the bath unit comprises one or more ports in or adjacent to the bottom surface of the bath unit for controlling flow of the pooled fluid and one or more outlet for removing liquid in the bath unit.

99. The system of claim 98, further comprising multiple pumps, valves, a reservoir, a waste container, or any combination thereof.

100. The system of claim 84, wherein the pooled fluid comprises an oil.

101. The system of claim 84, wherein the holding unit is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid.

102. The system of claim 101, wherein the holding unit is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid.

103. The system of claim 101, wherein the holding unit is tilted at an angle of 10 degrees relative to the surface of the pooled fluid.

104. The system of claim 84, further comprising a droplet generator, wherein the droplet generator comprises a pressure manifold and one or more input arrays, wherein each input array comprises multiple input wells.

105. The system of claim 104, further comprising a droplet generator, wherein the droplet generator comprises four input arrays.Atty. Docket No. 202421-703601106. The system of claim 84, wherein the loading apparatus further comprises one or more robotic arms connected to the holding unit, wherein the one or more robotic arms move the holding unit via an automated process.

107. The system of claim 106, further comprising a carrier, wherein the carrier comprises a frame for placing the MCSD, thereby allowing the one or more robotic arms to grip the MCSD.

108. The system of claim 84, wherein the MCSD comprises a variation in thickness of less than about 0.5 mm.

109. The system of claim 108, wherein the MCSD comprises a variation in thickness of less than about 0.4 mm.

110. The system of claim 109, wherein the MCSD comprises a variation in thickness of less than about 0.3 mm.

111. The system of claim 84, wherein the MCSD comprises one or more of polydimethylsiloxane (PDMS), cyclic olefin copolymer (COC), cyclic olefin polymer (COP) polycarbonate, styrene-ethylene-butylene-styrene (SEBS), and poly(m ethyl acrylate) (PMMA).

112. The system of any one of claims 84 to 111, wherein each microwell is sized to capture at least one droplet, at least two droplets, at least three droplets, at least four droplets, at least five droplets, at least six droplets, at least seven droplets, at least eight droplets, at least nine droplets, at least ten droplets, at least eleven droplets, at least twelve droplets, at least thirteen droplets, at least fourteen droplets, at least fifteen droplets, at least sixteen droplets, at least seventeen droplets at least eighteen droplets, or at least nineteen droplets.

113. The system of any one of claims 84 to 111, wherein each microwell is sized to capture droplets of a same size or microwells are sized to capture droplets of a different size.

114. The system of any of claims 84 to 113, further comprising a coalescing mechanism means for merging at least two droplets in a microwell to form a single droplet.

115. The system of claim 114, wherein the coalescing mechanism coalesces the droplets by electrocoalescence, thermal coalescence, acoustic coalescence, changes in surfactant concentration, or a combination thereof.

116. The system of claim 114, wherein the coalescing mechanism means is a corona treater, wherein the corona treater generates an electric field for merging at least two droplets in a microwell to form a single droplet.

117. The system of any one of claims 84 to 116, wherein each microwell comprises a plurality of posts.Atty. Docket No. 202421-703601118. The system of claim 117, wherein the posts are rectangular, triangular, or circular in shape when viewed from above.

119. The system of claim 117, wherein the plurality of posts are integrated into the sides of microwells and / or adhered to the bottom of the microwells.

120. The system of claim 85, wherein the droplets comprise one or more reporters.

121. The system of claim 120, wherein the one or more reporters are small molecule reporters.

122. The system of claim 121, further comprising an anaerobic microbe strain.

123. The system of claim 122, wherein the one or more reporters are expressed in the anaerobic microbe strain.

124. The system of claim 123, wherein the one or more reporters comprises a fluorescent protein.

125. The system of claim 124, wherein the fluorescent protein is GFP, RFP, CFP, YFP, mCherry, tdTomato, Venus, or mNeonGreen.

126. The system of claim 124, wherein the fluorescent protein is a ligand-inducible fluorescent protein.

127. The system of claim 126, wherein the ligand-inducible fluorescent protein is UnaG.

128. The system of claim 120, wherein the one or more reporters comprise an RNA aptamer and wherein the RNA aptamer fluoresces when bound to a chromophore.

129. The system of claim 128, wherein the RNA aptamer is a Pepper aptamer.

130. The system of any one of claims 84 to 129, further comprising a controller, wherein the controller is in communication with each of the one or more robot arms.

131. A method of loading droplets for producing massively parallel combinations of bioagents or chemicals, comprising:(a) providing a micropattemed combinatorial screening device (MCSD), wherein the MCSD comprises:an array of microwells, wherein each microwell is capable of receiving one or more droplets comprising bioagents or chemicals;(b) submerging or partially submerging the MCSD in a bath unit configured to retain a pooled fluid that is immiscible with the one or more droplets, wherein the array of microwells is tilted relative to the surface of the bath unit during the submerging step;(c) adding the droplets to the pooled fluid; andAtty. Docket No. 202421-703601(d) moving the MCSD vertically relative to the surface of the pooled fluid such that the one or more droplets are loaded to the MCSD, thereby allowing the droplets to enter the MCSD to generate massively parallel combinations of cells or chemicals.

132. The method of claim 131, wherein the bioagents comprise prokaryotic cells.

133. The method of claim 132, wherein the prokaryotic cells comprise bacteria.

134. The method of claim 131, wherein the bioagents comprises eukaryotic cells.

135. The method of claim 134, wherein the eukaryotic cells comprise fungi.

136. The method of claim 135, wherein the fungi comprise filamentous fungi.

137. The method of claim 135, wherein the fungi comprise yeast.

138. The method of claim 134, wherein the eukaryotic cells comprise protists.

139. The method of claim 138, wherein the protists comprise eukaryotic algae.

140. The method of claim 134, wherein the eukaryotic cells comprise mammalian cells.

141. The method of claim 131, wherein the one or more droplets is added at a loading edge of the device.

142. The method of claim 131, wherein the MCSD is tilted at an angle of from 5 degrees to 85 degrees relative to the surface of the pooled fluid.

143. The method of claim 142, wherein the MCSD is tilted at an angle of from 5 degrees to 30 degrees relative to the surface of the pooled fluid.

144. The method of claim 142, wherein the MCSD is tilted at an angle of 10 degrees relative to the surface of the pooled fluid.

145. The method of claim 131, wherein step (d) comprises repeatedly moving the MCSD vertically until the droplets are loaded to more than 90% of the microwells.

146. The method of claim 131, wherein before step (c), the droplets are generated in a droplet generator, wherein the droplet generator comprises a pressure manifold and one or more input arrays, wherein each input array comprises multiple input wells.

147. The method of claim 146, wherein after the droplets are generated, the droplets are aspirated and dispensed with an electronic pipette mounted to a third robotic arm.

148. The method of claim 131, wherein the pooled fluid is an oil.

149. A method for label-free quantification of growth of a bioagent, the method comprising:(a) providing a sample containing a bioagent in a growth medium to an array of microwells in a micropatterned combinatorial screening device (MCSD), wherein each microwell is capable of receiving one or more droplets;(b) obtaining a plurality of label -free optical measurements from the sample over a predetermined time using the MCSD;Atty. Docket No. 202421-703601(c) generating data from the plurality of label-free optical measurements, wherein the data comprises optical density measurements, light transmittance measurements, entropy measurement, or phase contrast measurements;(d) processing the data using at least one of the following:(i) a deep neural network machine learning (ML) algorithm, wherein the deep neural network is trained to correlate one or more optical signal patterns with bacterial growth parameters;(ii) optical density measurement over time;(iii) light transmittance measurement over time; and (iv) entropy measurement over time, and(e) determining at least one quantitative value relevant to the bacterial growth.

150. The method of claim 149, wherein step (d) comprises processing the data consisting of the following:(i) a deep neural network machine learning (ML) algorithm, wherein the deep neural network is trained to correlate one or more optical signal patterns with bacterial growth parameters;(ii) optical density measurement over time;(iii) light transmittance measurement over time; and(iv) entropy measurement over time.

151. The method of claim 149, wherein the at least one quantitative value is a median growth value of the bacteria.

152. The method of claim 149, wherein the bioagent comprises prokaryotic cells.

153. The system of claim 150, wherein the prokaryotic cells comprise bacteria.

154. The system of claim 153, wherein the bacteria is an odor-causing bacteria.

155. The system of claim 149, wherein the bioagent comprises eukaryotic cells.

156. The system of claim 155, wherein the eukaryotic cells comprise fungi.

157. The system of claim 156, wherein the fungi comprise filamentous fungi.

158. The system of claim 156, wherein the fungi comprise yeast.

159. The system of claim 155, wherein the eukaryotic cells comprise protists.

160. The system of claim 159, wherein the protists comprise eukaryotic algae.

161. The system of claim 155, wherein the eukaryotic cells comprise mammalian cells.

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