Integrated droplet sorter module for microfluidic devices

Abstract

A bidirectional microfluidic sorting device can sort, store, analyze, and re-sort aqueous microdroplets to characterize cell interactions and harvest cells with desired properties, such as highly effective immune cells. Sorting is provided by symmetrically configured forward and reverse sorting junctions configured to dielectrophoretically sort droplets based on their individual fluorescence. Unique features of the sorting device include the presence of a microdroplet docking array within the device, and a design that allows for reversible fluid flow, such that microdroplets can be sorted, then docked in the array, followed by reverse flow sorting again prior to collection from the device.

Description

[0001] TITLE

[0002] Integrated Droplet Sorter Module for Microfluidic Devices

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the priority of U.S. Provisional Appl. No. 63 / 657,753 filed 7 June 2024, the entirety of which is incorporated herein by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under Grant Numbers 5R01 GM127714-04 and 1 R33CA223908-01 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] BACKGROUND

[0008] Available microfluidic devices are capable of manipulating, analyzing, and recollecting single cells based on their functional characteristics. The use of docking arrays makes possible the simultaneous analysis of hundreds or even thousands of single cells or isolated groups of interacting single cells. There is a need to sort microdroplets containing these cells so as to divert, collect, and further study or culture selected cells from such microfluidic devices.

[0009] SUMMARY

[0010] The present technology provides a sorting junction for use with aqueous microdroplets suspended in a carrier oil. The microdroplets contain one or more single biological cells, which are capable of interacting within the aqueous microdroplet. The microdroplets also contain a fluorescent indicator whose emission is related to a functional property of one or more cells within the microdroplet, and is used to sort individual droplets. Unique features of the sorting device include the presence of a microdroplet docking array within the device, and a design that allows for reversible fluid flow, such that microdroplets can be sorted, then docked in the array, followed by reverse flow sorting again prior to collection from the device. The sorting device can be integrated into another microfluidic device or configured as a standalone device.

[0011] The technology can be further summarized with the following listing of features.

[0012] 1 . A sorting module for sorting aqueous microdroplets in an oil stream in a microfluidic device, the module comprising:

[0013] (i) an first inlet for a suspension of pre-formed aqueous microdroplets in oil, the aqueous microdroplets each comprising one or more individual cells suspended in an aqueous medium;

[0014] (ii) a second inlet for oil used to create a diluted suspension comprising said aqueous microdroplets and said oil;

[0015] (iii) channels fluidically coupling the first and second inlets to a microdroplet stream forming junction;

[0016] (iv) a bidirectional sorting junction capable of sorting said aqueous microdroplets in both a forward flow direction and a reverse flow direction, comprising a forward sorting junction, a reverse sorting junction, a connector channel fluidically coupled at one end to the forward sorting junction and at another end to the reverse sorting junction, a forward pair of electrodes, consisting of a ground electrode and a forward voltage electrode and configured to apply an electric field at the forward sorting junction, a reverse pair of electrodes, consisting of a ground electrode and a reverse voltage electrode and configured to apply an electric field at the reverse sorting junction, a forward input channel, fluidically coupled at one end to said stream forming junction and at another end to the connecting channel at the reverse sorting junction, one or more forward sorting channels fluidically coupled at said forward sorting junction to said connector channel, a reverse input channel, fluidically coupled at one end to the forward sorting junction, two or more reverse sorting channels fluidically coupled to the connector channel at the reverse sorting junction;

[0017] (v) a forward fluorescence detection zone, comprising an area for receiving a laser spot and an area for fluorescence emission detection;

[0018] (vi) a reverse fluorescence detection zone, comprising an area for receiving a laser spot and an area for fluorescence emission detection;

[0019] (vii) a docking array, comprising a plurality of docking positions for said aqueous microdroplets, the docking array fluidically coupled at a first end to said reverse input channel, and fluidically coupled at a second end to a docking array perfusion inlet;

[0020] (viii) a forward waste outlet, fluidically coupled to one of said one or more forward sorting channels;

[0021] (ix) a reverse waste outlet, fluidically coupled to one of said two or more reverse sorting channels; and

[0022] (x) a droplet collection outlet, fluidically coupled to one of said two or more reverse sorting channels.

[0023] 2. The sorting module of feature 1 , wherein the forward fluorescence detection zone is disposed in a portion of the forward input channel and the reverse fluorescence detection zone is disposed in a portion of the reverse input channel.

[0024] 3. The sorting module of feature 1 , wherein the forward and reverse fluorescence detection zones are fused to a single fluorescence detection zone, and wherein the single fluorescence detection zone is disposed within the connecting channel.

[0025] 4. The sorting module of any of features 1 -3, wherein a single ground electrode is used as the ground electrode in both the forward electrode pair and the reverse electrode pair.

[0026] 5. The sorting module of any of features 1 -3, wherein separate ground electrodes are used as the ground electrodes in the forward electrode pair and the reverse electrode pair.

[0027] 6. The sorting module of any of the preceding features, wherein both the forward and reverse electrode pairs are configured such that an AC voltage applied across either electrode pair forms a dielectrophoretic field that attracts aqueous microdroplets into a channel near the electrode pair across which the AC voltage is applied.

[0028] 7. The sorting module of any of the preceding features, wherein the docking array comprises 100 or more or 1000 or more docking sites for aqueous microdroplets.

[0029] 8. The sorting module of any of the preceding features, wherein the docking array has a width that increases from a first end coupled to the reverse input channel to a second end coupled to the docking array perfusion inlet.

[0030] 9. The sorting module of any of the preceding features, wherein the first inlet comprises a microdroplet collection chamber having a width that decreases as microdroplets flow towards the microdroplet stream forming junction.

[0031] 10. The sorting module of any of the preceding features that is integrated with a microfluidic device that forms said aqueous microdroplets using one or more cell suspensions and delivers them to the droplet inlet of the sorting module.

[0032] 11 . The sorting module of feature 10, wherein the microfluidic device further receives sorted aqueous microdroplets from the sorter module and subsequently provides one or more of analysis, incubation, merging, sorting, or harvesting of said sorted aqueous microdroplets.

[0033] 12. The sorting module of any of features 1-9, wherein the sorting module is configured as a standalone device.

[0034] 12a. The sorting module of any of features 1-12, further comprising a second docking array which is fluidically coupled to a reverse sorting channel.

[0035] 12b. The sorting module of any of features 1-12a, wherein the sorting module includes or does not include a merging junction.

[0036] 13. A system comprising; the sorting module of any of the preceding features; a light source for providing light for fluorescence excitation of one or more indicators of a state of cells contained in aqueous microdroplets within the sorting module; and a detector for receiving and quantifying fluorescence emission from the one or more indicators.

[0037] 14. The system of feature 13, wherein the light source is a laser and the detector is a photomultiplier tube.

[0038] 15. The system of feature 14, further comprising: a control module configured for controlling the light source, receiving and quantifying a signal from the detector, and sending said AC voltage to one or more of said pairs of electrodes in response to a said quantified signal that exceeds a predetermined threshold value for sorting microdroplets.

[0039] 16. The system of feature 14 or feature 15, further comprising: one or more pumps, vacuum sources, and / or valves used to control direction and magnitude of flow of a suspension of aqueous microdroplets through the sorting device.

[0040] 17. The system of any of features 14-16, further comprising a fluorescence microscope and / or computer for programming the control module and / or data acquisition, storage, and analysis.

[0041] 18. A method of sorting a suspension of aqueous microdroplets, the method comprising:

[0042] (a) providing the sorting device of any of features 1-12b or the system of any of features 13-17 and a suspension of aqueous microdroplets comprising cells and a fluorescent indicator;

[0043] (b) loading the suspension of aqueous microdroplets into the droplet inlet of the sorting device;

[0044] (c) loading oil into the oil inlet of the sorting device and forming a diluted aqueous microdroplet stream in the forward input channel;

[0045] (d) perfusing the diluted aqueous microdroplet stream through the forward input channel and the connecting channel to the forward sorting junction;

[0046] (e) determining a fluorescence emission of said fluorescent indicator for each aqueous microdroplet using the forward fluorescence detection zone;

[0047] (f) sorting the aqueous microdroplets at the forward sorting junction based on the fluorescence emission, whereby an AC voltage is applied to the forward pair of electrodes when the fluorescence emission of an aqueous microdroplet exceeds a pre-determined value, the AC voltage producing a dielectric field that attracts the aqueous microdroplet into the reverse input channel, and whereby when the fluorescence emission of an aqueous microdroplet does not exceed the predetermined value the aqueous microdroplet proceeds into a forward sorting channel, optionally leading to a forward waste outlet;

[0048] (g) perfusing the sorted aqueous microdroplets in the reverse input channel into the docking array; (h) subjecting the docked aqueous microdroplets to observation, analysis, and / or treatment with an active agent or additional fluorescent indicator supplied through the docking array perfusion inlet;

[0049] (i) perfusing aqueous microdroplets from the docking array, through the reverse input channel and the connection channel to the reverse sorting junction;

[0050] (j) determining a fluorescence emission for each aqueous microdroplet using the reverse fluorescence detection zone;

[0051] (k) sorting the aqueous microdroplets at the reverse sorting junction based on their fluorescence emission, whereby an AC voltage is applied to the reverse pair of electrodes when the fluorescence emission of an aqueous microdroplet exceeds a pre-determined value, the AC voltage producing a dielectric field that attracts the aqueous microdroplet into a first reverse sorting channel, and whereby when the fluorescence emission of an aqueous microdroplet does not exceed the predetermined value the aqueous microdroplet proceeds into a second reverse sorting channel.

[0052] 19. The method of feature 18, wherein the first reverse sorting channel is fluidically coupled with the droplet collection outlet, and the second reverse sorting channel is fluidically coupled with the reverse waste outlet.

[0053] 20. The method of feature 18 or feature 19, further comprising collecting aqueous microdroplets from the droplet collection outlet.

[0054] 21 . The method of feature 20, further comprising releasing cells from the collected aqueous microdroplets.

[0055] 22. The method of feature 21 , further comprising analyzing the collected cells.

[0056] 23. The method of feature 22, wherein said analyzing comprises sequencing genomic DNA, RNA, and / or mtDNA from the collected cells.

[0057] 24. The method of feature 22 or 23, wherein said analyzing comprises performing proteomic analysis on proteins and / or peptides from the collected cells.

[0058] 25. The method of any of features 21-24, further comprising maintaining or expanding the collected cells in culture.

[0059] 26. The method of any of features 18-24, further comprising repeating the method of feature 18 using the collected microdroplets from step (k), or newly formed aqueous microdroplets comprising cells harvested therefrom, as the aqueous microdroplets provided in step (a) of the repetition of the method of feature 18.

[0060] 27. The method of any of features 18-26, further comprising: (aO) preparing the suspension of aqueous microdroplets using a device for forming aqueous microdroplets in oil from one or more aqueous cell suspensions and an oil.

[0061] 28. The method of any of features 18-27, wherein aqueous microdroplets are sorted twice without removing them from the sorting device.

[0062] 29. The method of any of features 18-28, wherein two or more functional assays are performed on cells within the aqueous microdroplets without removing the aqueous microdroplets from the sorting device.

[0063] As used herein, the expressions “aqueous microdroplet”, “microdroplet”, and “droplet” are synonymous.

[0064] BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Fig. 1 is a schematic representation of an embodiment of a sorting device of the present technology. The representation depicts the two-dimensional top view of the device layout. The forward flow direction is from left to right through sorting junction 1 12, and the reverse flow direction is from right to left.

[0066] Fig. 2 is a schematic representation of an embodiment of a sorting device of the present technology. The representation is a three dimensional view from above the device. The forward flow direction is from right to left across the sorting junction, and the reverse flow direction is from left to right.

[0067] DETAILED DESCRIPTION

[0068] The present technology provides devices, systems, and methods for sorting single cells and small groups of single cells co-encapsulated in aqueous microdroplets in an oil stream based on cell behaviorwithin the microdroplets. The technology allows separation of cell populations based on their function and interactions, and performing additional downstream analysis of the cells, including genomic and proteomic characterization. Sorting of microdroplets is based on fluorescent indicators, whose detection and quantification at the level of individual microdroplets provides a way to detect internal droplet functional changes with high accuracy and throughput.

[0069] The present technology extends the capabilities of existing sorting devices with the addition of a droplet docking array for incubation and enrichment with agents of interest, such as cytokines, for example. The technology provides a sorting device that allows reperfusion of previously sorted droplets through the sorter screening junction for repeated single cell functional analysis, including analysis of immune cell and target cell interactions with evaluation of killing kinetics, and separation of cell populations based on function. A droplet merging junction can be added to the device as an optional module and provides additional applications.

[0070] The addition of a docking array to a microfluidic device for analyzing single cells in microdroplets provides advantages over generating droplets for direct off chip incubation and modulation without a docking array. The droplet microenvironments of encapsulated single cells can experience physical impact when being generated and transported off chip. This can result in droplet merging and potential death for internal encapsulated cells. Docking droplets on chip provides minimal physical impact for droplets, maintaining their internal microenvironment integrity. The array also provides a method of perfusing cell-containing microdroplets and enriching in cytokines or therapeutic agents to the docked droplets. The system can use perfusion in both forward and reverse flow directions, because the sorting junction includes additional symmetrical internal fluid circuits that can be utilized for droplet sorting of previously sorted droplets following initial docking and enrichment. This enables analysis of internal cell kinetics and behaviors based on two assays in place of only the one possible in previous droplet sorters.

[0071] The following are examples of procedures that can be carried out with the present sorting devices and systems including them. Droplets with encapsulated cells can be generated on device if a droplet formation junction is included in the device, or can be formed in a separate module or device coupled to the device. The droplets can be perfused within the device at a fixed flow rate. The droplets can be assessed at a sorting junction using an external laser / PMT system that detects variances in internal droplet fluorescence signal based on cellular state using an encapsulated fluorescent dye. Droplet and cell populations can be sorted and then separated using an electromagnetic field generated by a circuit activated by a pre-determined level of fluorescence detected by the laser / PMT system. The sorted droplet population can be retained on chip in an integrated docking array for evaluation by light microscopy, including fluorescence microscopy, to monitor cell interactions, changes in cell morphology, and killing of target cells by immune cells. For example, if the fluorescent indicator is an indicator of apoptosis, such as CellEvent Caspase 3 / 7 apoptosis dye (Invitrogen), droplets containing killed target cells, and therefore also containing an effective NK cell or killer T cell, can be sorted and isolated. The docked droplets and their contained cells can be treated with active agents such as cytokines or other therapeutic agents to test the ability of those active agents to modify cellular function, for example, to enhance the ability to kill target cells or to directly kill target cells or to render them more susceptible to other cells or other agents. Moreover, docked droplets can be perfused in the reverse direction and re-sorted, either using the same or different criteria compared to a previous sort. For example, a different fluorescent indicator can be used in a second or subsequent sort, which is sensitive to different aspects of cellular function. Alternatively, different sorting criteria, such as fluorescence threshold or PMT setting, or excitation or emission wavelength, can be used to render a sorting process more effective or to select out a different population of cells.

[0072] An embodiment of a method for use with the system includes the following steps. (1 ) Encapsulation or co-encapsulation and incubation of one or more single cells using a separate droplet generation device. (2) Perfusion of droplets from the larger bottom port of the device giving ample room for droplets to spread out once they have entered the device. (3) Droplets perfuse towards a perpendicular carrier oil channel that propels droplets toward the sorting junction. (4) Droplets perfuse to the laser spot area where they are excited by the laser spot which can be adjusted to accommodate a variety of wavelengths, with the resulting emission recorded by an external photomultiplier tube (PMT). (5) The PMT registers the emission fluorescence as a voltage and if the voltage surpasses set thresholds and registers as a positive droplet, then the circuit is activated. Otherwise negative droplets will continue to flow towards the bottom waste channel which has a lower overall flow resistance than the top channel. (6) The circuit can be activated with an AC voltage of 1 kV and 12 kHz, which creates a corresponding di-electrophoretic field that pulls droplets towards the channel closest to the electrodes (e.g., upper channel). (7) Droplets can flow towards the docking array and become entrapped within that area by geometry towards the exit port. (8) Cytokines or other therapies can be gently perfused through the docking array where they will interact with and can be absorbed by the droplets and are incubated together. For example, the cytokines or other agents can be dissolved or suspended in the carrier oil, optionally as a microemulsion. (9) Carrier oil can be used to flow droplets back towards the sorting junction where they are once again assessed for internal fluorescence signal. (10) Droplets can be collected from the waste and collection ports for further, off-chip analysis. Referring now to the embodiment of a sorter device 100 of the present technology depicted in Fig. 1 , a pre-formed suspension of aqueous microdroplets in oil is injected at droplet inlet 101. The droplets migrate up the narrowing chamber towards a microchannel and filter 102, then perfuse toward microdroplet stream forming junction 117. At the stream forming junction, carrier oil added through oil inlet 103 mixes with the microdroplet suspension thereby diluting it out in a controlled manner; this has the effect of setting the time between drops arriving at the sorter junction to a desired range, to allow time for fluorescence detection, signal processing, and activation of the electrodes at time intervals required for droplet sorting. The diluted microdroplets perfuse in a “forward” direction (left to right in Fig. 1 ) down forward input channel 122 to forward fluorescence detection zone 113, where laser spot 1 15 (laser is off-chip) illuminates the droplets for fluorescence excitation and PMT 116 (PMT is off chip but can receive light, for example, via a fiber optic cable or other mechanism) detects fluorescence emission, providing an output voltage to an off-chip control unit (not shown). The control unit compares the PMT output to a predetermined threshold. If the threshold is met or exceeded, a suitable AC voltage is applied across ground electrode 108 and forward high voltage electrode 111 , resulting in the formation of a dielectric field that attracts the above threshold microdroplet to be drawn toward the electrodes by dielectrophoresis, resulting in sorting of the droplet into reverse input channel 123. Sub-threshold droplets proceed towards forward waste outlet 106. Above threshold droplets proceed into docking array 105, moving into docking sites (not shown) into the broadening end of the array. Docked droplets can optionally be perfused with one or more desired agents through docking array perfusion inlet 104. After an incubation period for interaction with perfused agents, or for observation by microscopy, during which droplets containing cel Is of further interest can be identified, direction of perfusion through the device can be reversed, upon which droplets will migrate back towards the narrow end of the array as depicted in 119, finally re-emerging into reverse sorting channel 123. During reverse sorting, droplets pass through reverse fluorescence detection zone 114, first through laser spot 115 and then through fluorescence emission detection area 116. A PMT produces a signal that represents the intensity of fluorescence emission, and if the signal meets or exceeds a threshold, an AC voltage is applied between ground electrode 108 and reverse high voltage electrode 110, thereby attracting the corresponding droplet to the nearest reverse sorting channel, leading to droplet collection outlet 108. Sub- threshold fluorescence leads to the droplet continuing toward reverse waste outlet 107. Note that the geometry of forward sorting junction 120 and reverse sorting junction 121 is configured such that absent an applied dielectrophoretic field a droplet will continue towards a default sorting channel (see arrows).

[0073] Fig. 2 illustrates a similar embodiment to that shown in Fig. 1 , but represented in three dimensions and as a mirror image architecture compared to the device of Fig. 1.

[0074] The sorting device can be fabricated using standard materials and techniques for the production of microfluidic devices. For example, a mask or mold can be prepared and the microfluidics portion of the device formed by casting PDMS material, which is then removed and bonded to a glass microscope slide or other inert substrate. Vertical shafts can be formed to provide fluid inlets and outlets. The device can be perfused through syringe pumps or other pumps to establish suitable flow rates for carrier oil, droplet suspension, and other reagents. Transparency is provided for microscopy at docking arrays and forfluorescence excitation and emission. In addition to pumps, valves and vacuum sources also can be used to regulate flow direction and rate as desired.

[0075] The sorting device can be configured as a module of a larger microfluidic device, such as one including a droplet formation junction to supply the suspension of aqueous microdroplets in oil, or to receive droplets leaving the sorting device through waste ports or droplet collection ports, for other uses. The host device optionally can contain one or more droplet merging junctions, so that harvested droplets from the sorting device can be merged with other droplets containing additional cells or reagents. Cells can be harvested from collected droplets and either maintained or expanded in cell culture or used directly for analysis, including extraction of materials for nucleic acid analysis including sequencing (DNA, RNA, or mtDNA) or proteomic analysis (proteins and peptides). Harvested cells also can be expanded, optionally genetically modified, and administered to a patient, including the cell donor, for use in immunotherapy of cancer or infection.

[0076] The novel and unusual features of the present technology include the following.

[0077] 1. The technology provides a method to sort single cells or groups of cells encapsulated in aqueous microdroplets in oil, based on internal kinetics of cell interactions or other cell behaviors, including cell functions linked to the presence or absence of molecular components incubated with the cells on chip (e.g., in a droplet docking array). Cells can be selected, enriched, or purified according to selected cell characteristics, and additional sorting runs can be carried out on a single microfluidic device.

[0078] 2. The technology can be used with many cellular assays and types of enrichment; it is not limited to assays including only a single incubation orsingle sorting process.

[0079] 3. This technology allows performing dual assay analysis of two separate cellular function variables to better understand and identify cellular events, including rare cellular events.

[0080] Advantages of the present technology include the following.

[0081] 1. Increasing complexity of on-chip processing reduces human interaction, decreases overall likelihood of failure, and increases ease of use.

[0082] 2. Multiple variables can be assessed in one experiment, compared with previous sorters which could only sort based on one variable per experiment.

[0083] 3. There is a decreased likelihood of inadvertent droplet merging or cell death compared to off-chip manipulation of droplets and interaction with droplets.

[0084] 4. Incubation with cytokines and other agents allows modulation of internal droplet microenvironments, which are typically inaccessible once droplets are on-chip.

[0085] Uses of the present technology include the following.

[0086] 1. Assessing the efficacy of encapsulated immune cell serial killers with coencapsulated tumor cells by enriching with additional tumor cell targets.

[0087] 2. Modeling of resistant tumor cells by testing therapies against various tumor cell types that have been sorted based on resistance markers.

[0088] 3. Assessment of immunotherapy efficiency via perfusion towards sorted cells in a docking array.

[0089] 4. Assessment of chemotherapy efficiency via perfusion of sorted cells in a docking array.

[0090] 5. 3D droplet matrix crosslinking (cell spheroids) and functional assessment by incorporating a hydrogel matrix into the droplets and crosslinking via docking array perfusion.

[0091] As used herein, "consisting essentially of" allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term "comprising", particularly in a listing of components of a composition or elements of a device, constitutes inclusion of alternative embodiments in which “comprising” is replaced with "consisting essentially of" or "consisting of".

[0092] While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

[0093] References

[0094] Sabhachandani, P., Motwani, V., Cohen, N., Sarkar, S., Torchilin, V., & Konry, T. (2016). Generation and functional assessment of 3D multicellular spheroids in droplet based microfluidics platform. Lab on a Chip, 16(3), 497-505. doi.org / 10.1039 / C5LC01 139F

[0095] Zhong, R., Sullivan, M., llpreti, N., Chen, R., De Ganzo, A., Yang, K., Yang, S., Jin, K., He, Y., Li, K., Xia, J., Ma, Z., Lee, L. P., Konry, T., & Huang, T. J. (2023). Cellular immunity analysis by a modular acoustofluidic platform: CIAMAP. Science Advances, 9(51), eadj9964. doi.org / 10.1126 / sciadv.adj9964

[0096] Sullivan, M. R., White, R. P., Dashnamoorthy Ravi, Kanetkar, N., Fridman, I. B., Ekenseair, A., Evens, A. M., & Konry, T. (2024). Characterizing influence of rCHOP treatment on diffuse large B-cell lymphoma microenvironment through in vitro microfluidic spheroid model. Cell Death & Disease, 15(1), 1-9. doi.org / 10.1038 / S41419-023-06299-6

Claims

CLAIMSWhat is claimed is1 . A sorting module for sorting aqueous microdroplets in an oil stream in a microfluidic device, the module comprising:(i) an first inlet for a suspension of pre-formed aqueous microdroplets in oil, the aqueous microdroplets each comprising one or more individual cells suspended in an aqueous medium;(ii) a second inlet for oil used to create a diluted suspension comprising said aqueous microdroplets and said oil;(iii) channels fluidically coupling the first and second inlets to a microdroplet stream forming junction;(iv) a bidirectional sorting junction capable of sorting said aqueous microdroplets in both a forward flow direction and a reverse flow direction, comprising a forward sorting junction, a reverse sorting junction, a connector channel fluidically coupled at one end to the forward sorting junction and at another end to the reverse sorting junction, a forward pair of electrodes, consisting of a ground electrode and a forward voltage electrode and configured to apply an electric field at the forward sorting junction, a reverse pair of electrodes, consisting of a ground electrode and a reverse voltage electrode and configured to apply an electric field at the reverse sorting junction, a forward input channel, fluidically coupled at one end to said stream forming junction and at another end to the connecting channel at the reverse sorting junction, one or more forward sorting channels fluidically coupled at said forward sorting junction to said connector channel, a reverse input channel, fluidically coupled at one end to the forward sorting junction, two or more reverse sorting channels fluidically coupled to the connector channel at the reverse sorting junction;(v) a forward fluorescence detection zone, comprising an area for receiving a laser spot and an area for fluorescence emission detection;(vi) a reverse fluorescence detection zone, comprising an area for receiving a laser spot and an area for fluorescence emission detection;(vii) a docking array, comprising a plurality of docking positions for said aqueous microdroplets, the docking array fluidically coupled at a first end to said reverse input channel, and fluidically coupled at a second end to a docking array perfusion inlet;(viii) a forward waste outlet, fluidically coupled to one of said one or more forward sorting channels;(ix) a reverse waste outlet, fluidically coupled to one of said two or more reverse sorting channels; and(x) a droplet collection outlet, fluidically coupled to one of said two or more reverse sorting channels.

2. The sorting module of claim 1 , wherein the forward fluorescence detection zone is disposed in a portion of the forward input channel and the reverse fluorescence detection zone is disposed in a portion of the reverse input channel.

3. The sorting module of claim 1 , wherein the forward and reverse fluorescence detection zones are fused to a single fluorescence detection zone, and wherein the single fluorescence detection zone is disposed within the connecting channel.

4. The sorting module of claim 1 , wherein a single ground electrode is used as the ground electrode in both the forward electrode pair and the reverse electrode pair.

5. The sorting module of claim 1 , wherein separate ground electrodes are used as the ground electrodes in the forward electrode pair and the reverse electrode pair.

6. The sorting module of claim 1 , wherein both the forward and reverse electrode pairs are configured such that an AC voltage applied across either electrode pair forms a dielectrophoretic field that attracts aqueous microdroplets into a channel near the electrode pair across which the AC voltage is applied.

7. The sorting module of claim 1 , wherein the docking array comprises 100 or more or 1000 or more docking sites for aqueous microdroplets.

8. The sorting module of claim 1 , wherein the docking array has a width that increases from a first end coupled to the reverse input channel to a second end coupled to the docking array perfusion inlet. of the prec9. The sorting module of claim 1 , wherein the first inlet comprises a microdroplet collection chamber having a width that decreases as microdroplets flow towards the microdroplet stream forming junction.

10. The sorting module of claim 1 that is integrated with a microfluidic device that forms said aqueous microdroplets using one or more cell suspensions and delivers them to the droplet inlet of the sorting module.11 . The sorting module of claim 10, wherein the microfluidic device further receives sorted aqueous microdroplets from the sorter module and subsequently provides one or more of analysis, incubation, merging, sorting, or harvesting of said sorted aqueous microdroplets.

12. The sorting module of claim 1 , wherein the sorting module is configured as a standalone device.

13. A system comprising; the sorting module of claim 1 ; a light source for providing light for fluorescence excitation of one or more indicators of a state of cells contained in aqueous microdroplets within the sorting module; and a detector for receiving and quantifying fluorescence emission from the one or more indicators.

14. The system of claim 13, wherein the light source is a laser and the detector is a photomultiplier tube.

15. The system of claim 14, further comprising: a control module configured for controlling the light source, receiving and quantifying a signal from the detector, and sending said AC voltage to one or more of said pairs of electrodes in response to a said quantified signal that exceeds a predetermined threshold value for sorting microdroplets.

16. The system of claim 14, further comprising:one or more pumps, vacuum sources, and / or valves used to control direction and magnitude of flow of a suspension of aqueous microdroplets through the sorting device.

17. The system of claim 14, further comprising a fluorescence microscope and / or computer for programming the control module and / or data acquisition, storage, and analysis.

18. A method of sorting a suspension of aqueous microdroplets, the method comprising:(a) providing the system of claim 17 and a suspension of aqueous microdroplets comprising cells and a fluorescent indicator;(b) loading the suspension of aqueous microdroplets into the droplet inlet of the sorting device;(c) loading oil into the oil inlet of the sorting device and forming a diluted aqueous microdroplet stream in the forward input channel;(d) perfusing the diluted aqueous microdroplet stream through the forward input channel and the connecting channel to the forward sorting junction;(e) determining a fluorescence emission of said fluorescent indicator for each aqueous microdroplet using the forward fluorescence detection zone;(f) sorting the aqueous microdroplets at the forward sorting junction based on the fluorescence emission, whereby an AC voltage is applied to the forward pair of electrodes when the fluorescence emission of an aqueous microdroplet exceeds a pre-determined value, the AC voltage producing a dielectric field that attracts the aqueous microdroplet into the reverse input channel, and whereby when the fluorescence emission of an aqueous microdroplet does not exceed the predetermined value the aqueous microdroplet proceeds into a forward sorting channel, optionally leading to a forward waste outlet;(g) perfusing the sorted aqueous microdroplets in the reverse input channel into the docking array;(h) subjecting the docked aqueous microdroplets to observation, analysis, and / or treatment with an active agent or additional fluorescent indicator supplied through the docking array perfusion inlet;(i) perfusing aqueous microdroplets from the docking array, through the reverse input channel and the connection channel to the reverse sorting junction;(j) determining a fluorescence emission for each aqueous microdroplet using the reverse fluorescence detection zone;(k) sorting the aqueous microdroplets at the reverse sorting junction based on their fluorescence emission, whereby an AC voltage is applied to the reverse pair of electrodes when the fluorescence emission of an aqueous microdroplet exceeds a pre-determined value, the AC voltage producing a dielectric field that attracts the aqueous microdroplet intoa first reverse sorting channel, and whereby when the fluorescence emission of an aqueous microdroplet does not exceed the predetermined value the aqueous microdroplet proceeds into a second reverse sorting channel.

19. The method of claim 18, wherein the first reverse sorting channel is fluidically coupled with the droplet collection outlet, and the second reverse sorting channel is fluidically coupled with the reverse waste outlet.

20. The method of claim 19, further comprising collecting aqueous microdroplets from the droplet collection outlet.21 . The method of claim 20, further comprising releasing cells from the collected aqueous microdroplets.

22. The method of claim 21 , further comprising analyzing the collected cells.

23. The method of claim 22, wherein said analyzing comprises sequencing genomic DNA, RNA, and / or mtDNA from the collected cells.

24. The method of claim 22, wherein said analyzing comprises performing proteomic analysis on proteins and / or peptides from the collected cells.

25. The method of claim 21 , further comprising maintaining or expanding the collected cells in culture.

26. The method of claim 18, further comprising repeating the method using the collected microdroplets from step (k), or newly formed aqueous microdroplets comprising cells harvested therefrom, as the aqueous microdroplets provided in step (a) of the repetition of the method.

27. The method of claim 18, further comprising:(aO) preparing the suspension of aqueous microdroplets using a device for forming aqueous microdroplets in oil from one or more aqueous cell suspensions and an oil.

28. The method of claim 18, wherein aqueous microdroplets are sorted twice without removing them from the sorting device.

29. The method of claim 18, wherein two or more functional assays are performed on cells within the aqueous microdroplets without removing the aqueous microdroplets from the sorting device.

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