Cross-Reference

[0001] This application claims the benefit of United States Provisional Patent Application No. 62/932,897, filed on November 8, 2019, and United States Provisional Patent Application No. 63/080,788, filed on September 20, 2020. These documents are hereby incorporated by reference in their entirety.

Technical Field

[0002] The embodiments disclosed herein relate to microfluidic devices, systems, and methods, and more specifically, to devices, systems, and methods for integrating a droplet-digital microfluidic system for on-demand droplet creation, mixing, incubation, and sorting.

Background

[0003] Droplet microfluidics involves monodisperse aqueous droplets in sub nano- (or pico-) liter volumes that are generated by a pressure-driven flow in a continuous oil phase where droplets are typically analyzed and manipulated at very high rates (> 1000 droplets per second). The use of droplet microfluidic technology has enabled a wide variety of applications, specifically in the area of high-throughput chemistry and biology ^{1- 4} , and in particular high throughput screening and single cell analysis. This two-phase microfluidic format can undergo a number of different fluidic operations - droplet generation, encapsulation, mixing, and sorting. Sorting is in particular an important operation that allows selection of subpopulation of cells, DNA, and biomolecules in the droplets. ^5-7 A variety of sorting methods have been shown in literature using dielectrophoresis, magnetic, thermal, or acoustic methods. ^8-11 Each of these have their own advantages in terms of speed, reliability and ease of implementation. However, typical sorting methods are usually based only on binary sorting - i.e. sorting droplets that are based on two levels of output - which can limit the range of detecting rare events and to sort based on different constituents in the droplet (e.g., multiple concentrations of an additive).

[0004] There is an alternative type of microfluidics that enables on-demand droplet control called digital microfluidics. ¹² ’ ¹³ This platform allows manipulation of discrete droplets by electrostatic forces on an array of electrodes coated with an insulating dielectric. One of the main advantages of digital microfluidics (DMF) is it facilitates precise control over many different reagents simultaneously and independently. This has enabled DMF to be a well-suited platform to carry out many different types of applications, namely, cell-based assays, ¹⁴ ’ ¹⁵ synthetic biology, ¹⁶ ’ ¹⁷ and point-of-care diagnostics ¹⁸ ’ ¹⁹ Most of these types of applications are configured in a two-plate format, in which droplets are manipulated between a top and bottom substrate bearing a ground and driving electrodes respectively. There is another digital microfluidic configuration in which droplets are actuated on a single substrate with co-planar configuration of electrodes. Although in this configuration, droplets lack the capacity to dispense, this format does allow better mixing which is useful in applications carrying out chemical reactions. ²⁰ ’ ²¹ Likewise, it may be useful to couple single-plate DMF with microchannels as a chemical pre-processing unit without the need for pre-column reactions since DMF can rapidly mix different analytes in seconds and separated using the channels. ²² _’ ²³ The integrating DMF with other microfluidic paradigms combines advantages of both systems while minimizing the disadvantages of the individual systems.

Summary

[0005] Microfluidic devices are described herein. In accordance with a broad aspect, a microfluidic device comprising a first layer including a plurality of electrodes, a second layer disposed on top of the first layer, the second layer including a dielectric patterned over the plurality of electrodes, and a third layer disposed on top of the second layer is described herein. The third layer includes a droplet generator for generating droplets of fluid, a first inlet for receiving the droplets of fluid from the droplet generator, and a microchannel for carrying the droplets of fluid in solution from the first inlet towards an outlet. Actuation of one or more of the plurality of electrodes controls movement of the droplets of fluid from the first inlet towards the outlet. [0006] In accordance with another broad aspect, a microfluidic device comprising a first layer including a plurality of electrodes, a second layer disposed on top of the first layer, the second layer including a dielectric patterned over the plurality of electrodes, and a third layer disposed on top of the second layer is disclosed herein. The third layer includes a droplet generator for generating droplets of a first fluid in a second fluid, the droplet generator including a droplet generating channel for carrying the first fluid towards a main channel housing the second fluid. Actuation of the one or more of the plurality of electrodes controls movement of the first fluid in the droplet generating channel towards the main channel and generation of droplets of the first fluid in the main channel.

[0007] In accordance with another broad aspect, a method of controlling movement of droplets in a microchannel is described herein. The method includes generating one or more droplets of fluid at a droplet generator, directing the one or more droplets of fluid into the microchannel from the droplet generator and actuating one or more of a plurality of electrodes positioned below the microchannel to control movement of the droplets of fluid through the microchannel, such as but not limited to sorting them, mixing them, merging them, and storing them for incubation.

[0008] In accordance with another broad aspect, a method of mixing droplets in a microfluidic device is described herein. The method includes generating a first droplet, receiving the first droplet in a microchannel, directing the first droplet into a mixing region of the microchannel by actuating one or more electrodes positioned beneath the microchannel and mixing the first droplet with a second droplet in the mixing region.

[0009] In accordance with another broad aspect, a method of sorting droplets in a microchannel is described herein. The method includes generating one or more droplets of fluid at a droplet generator; directing the one or more droplets of fluid into the microchannel from the droplet generator; detecting the one or more droplets, optionally using an optical detector; and sorting the one or more droplets into two or more sorting microchannels by actuating one or more of a plurality of electrodes positioned below the microchannel and/or the two or more sorting microchannels. [0010] In accordance with another broad aspect, a use of a microfluidic device, method or system disclosed herein for analyzing and/or detecting one or more droplet constituents is described herein.

[0011] In accordance with another broad aspect, a use of a microfluidic device, method or system disclosed herein in an assay is described herein. The assay may be one of: an enzymatic assay, a single cell viability assay, a selection assay for directed evolution, a toxicity assay, a single cell drug inhibition assay, an assay for gene editing, an assay for single cell transfection, a single cell sorting assay, an isoclonal selection assay, an assay for delivery of chemicals, materials and/or drugs to single cells, an assay for analysis of cell products such as but not limited to antibodies, an incubation assay, and/or a microscopy-based assay.

[0012] These and other features and advantages of the present application will become apparent from the following detailed description taken together with the accompanying drawings. However, it should be understood that the detailed description and the specific examples, while indicating preferred embodiments of the application, are given by way of illustration only, since various changes and modifications within the spirit and scope of the application will become apparent to those skilled in the art from this detailed description.

Brief Description of the Drawings

[0013] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0014] Fig. 1a is an exploded view of the integrated droplet-digital microfluidic (ID2M) microfluidic device in accordance with an embodiment described herein. The bottom layer is the DMF configuration which is covered with a dielectric SU-8 layer ~ 7 pm thickness. The channel layer, in this embodiment, is 300 pm wide and 110-120 pm high and was fabricated on top of this layer. A PDMS slab of thickness ~ 5 mm was bonded to seal the channel layer.

[0015] Fig. 1 b is a schematic of the ID2M microfluidic device depicting the operations of the device, namely droplet dispensing (using T-junction and flow focusing), droplet mixing, droplet incubation, droplet detection, and droplet n-ary sorting. Reference number 102 shows the main channel on the device in which droplets are transported from one region to another. Mixing area contains sinking channels to reduce the oil flow rate.

[0016] Fig. 2a is a series of top view images depicting the droplet operations on an ID2M microfluidic device. Frames i-iii illustrate droplet generation from flow-focusing and on-demand (T-junction) techniques, and Frames iv-vi show subsequent merging and mixing of droplets. Frame vii shows droplet incubation (for incubating cells and other constituents) and Frames viii and ix show droplet sorting in four different channels.

[0017] Fig. 2b is a graph showing droplet size as a function of oil flow rate at a constant water flow rate (0.0005 pL/s) using flow-focusing (hydrodynamic) and T-junction (on-demand) configurations. Each point represents eight droplets sampled. The error bars represent one standard deviation.

[0018] Fig. 3a is a close-up view of the detection region on the ID2M device.

[0019] Fig. 3b is a series of images of droplets containing fluorescein at four different concentrations (0.125, 0.25, 0.5, and 1 mM) being sorted into a respective channel.

[0020] Fig. 3c is a graph showing a time series during a sort showing the fluorescence signal (blue) for four concentrations of fluorescein and for droplets with only diluent (i.e. no fluorescein, yellow). Each droplet containing fluorescein is sorted by their threshold fluorescence intensity values (dashed lines).

[0021] Fig. 3d is a calibration curve showing the fluorescence as a function of fluorescein concentration. These average fluorescence values were used to create the threshold values for sorting. Error bars are ± 1 standard deviation. [0022] Fig. 4a is a graph showing optical density (OD) measurements as a function of ionic liquid (IL) concentrations for wild-type and two mutant yeast cells after 48 h incubation and at 30 °C.

[0023] Fig. 4b is a graph showing growth curves for the wild-type and mutant yeast cells in 100 mM ionic liquid.

[0024] Fig. 4c is a series of images of wild-type and mutant yeast cells cultured in incubation regions on the device for 48 h, confirming the differences between two cell lines. Cells are highlighted (circled regions) inside the droplet.

[0025] Fig. 4d is a graph containing raw data collected directly from the spectrometer showing the differences between the absorbance signals of droplets containing mutant and wild-type yeast.

[0026] Fig. 5 is a series of images showing on-demand droplet generation with T- junction configuration. Frame 1 shows a water flow 0.0005 [pL/s] with initialization of the electrodes. Frames 2-3 show actuation sequences to drag the fluid to the main channel and Frame 4 shows the required sequence actuation to break-off a droplet. A constant oil flow rate of 0.01 [pL/s] was maintained during this procedure.

[0027] Fig. 6 is a graph showing cell viability of yeast BY4741 strain as a function of different EMS treatment time. Cell viability was calculated by counting colonies growing on SD media plates after 48 h incubation at 30 °C.

[0028] Fig. 7 is a schematic illustrating the CAD model design for simulating the sink channel in COMSOL Multiphysics V5.2. For simplification, only the mixing and sinking channels with the following inlet and outlets of the system were modeled.

[0029] Fig. 8a, Fig. 8b, Fig. 8c and Fig. 8d are images showing finger-like structures on the boundary of the SU-85 negative photoresist layer. Fig. 8a shows cracks distributed in the resist layer fabricated with a straight edge mask, Fig. 8b and 8c show 10X and 20X images, respectively, of the same layer fabricated with mask design with fingers, and Fig. 8d shows the final mask design with finger-like boundaries. [0030] Fig. 9 is an image showing COMSOL simulation of the oil flow velocity in the mixing area, indicating a visible decrease in its velocity. Red arrows indicate the flow direction of the velocity field.

[0031] Fig. 10 is a schematic of the ID2M work flow for screening of a yeast mutant library for ionic liquid resistance based on growth (i.e. absorbance). All steps (except generating the mutant library) were conducted on the ID2M system.

[0032] Fig. 11 is a graph showing the time course (absorbance vs. time) plot for only oil phase (i.e. no droplets).

[0033] Fig. 12 is an image showing the MATLAB GUI interface used to automate the droplet operations which contains a region showing the electrode design (1), the real time view of the device (2), the voltage and frequency control for the droplet actuations (3), and the creation of user-defined droplet sequences that are preprogrammed (4).

[0034] Fig. 13 is a schematic of the ID2M automation setup and shows the connectivity of all the different components used in this system.

[0035] The skilled person in the art will understand that the drawings, further described below, are for illustration purposes only. The drawings are not intended to limit the scope of the applicant's teachings in any way. Also, it will be appreciated that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further aspects and features of the example embodiments described herein will appear from the following description taken together with the accompanying drawings.

Detailed Description

[0036] Various systems and methods are described below to provide an example of at least one embodiment of the claimed subject matter. No embodiment described below limits any claimed subject matter and any claimed subject matter may cover systems and methods that differ from those described below. The claimed subject matter are not limited to systems and methods having all of the features of any one system and method described below or to features common to multiple or all of the systems and methods described below. Subject matter that may be claimed may reside in any combination or sub-combination of the elements or process steps disclosed in any part of this document including its claims and figures. Accordingly, it will be appreciated by a person skilled in the art that a system or method disclosed in accordance with the teachings herein may embody any one or more of the features contained herein and that the features may be used in any particular combination or sub-combination that is physically feasible and realizable for its intended purpose.

[0037] Furthermore, it is possible that a system or method described below is not an embodiment of any claimed subject matter. Any subject matter that is disclosed in a system or method described herein that is not claimed in this document may be the subject matter of another protective instrument, for example, a continuing patent application, and the applicant(s), inventor(s) and/or owner(s) do not intend to abandon, disclaim, or dedicate to the public any such invention by its disclosure in this document.

[0038] It will also be appreciated that for simplicity and clarity of illustration, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or analogous elements. In addition, numerous specific details are set forth in order to provide a thorough understanding of the example embodiments described herein. However, it will be understood by those of ordinary skill in the art that the example embodiments described herein may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the example embodiments described herein. Also, the description is not to be considered as limiting the scope of the example embodiments described herein.

[0039] It should be noted that terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the result is not significantly changed. These terms of degree should be construed as including a deviation of the modified term, such as 1 %, 2%, 5%, or 10%, for example, if this deviation would not negate the meaning of the term it modifies.

[0040] Furthermore, the recitation of any numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about" which means a variation up to a certain amount of the number to which reference is being made, such as 1 %, 2%, 5%, or 10%, for example, if the end result is not significantly changed.

[0041] It should be noted that the term “coupled” used herein indicates that two elements can be directly coupled to one another or coupled to one another through one or more intermediate elements.

[0042] It should also be noted that, as used herein, the wording “and/or” is intended to represent an inclusive - or. That is, “X and/or Y” is intended to mean X or Y or both, for example. As a further example, “X, Y, and/or Z” is intended to mean X or Y or Z or any combination thereof.

[0043] The inventors have combined the use of single-plate DMF and droplet-in- channel microfluidics. While others have used digital microfluidics and combined it with other microfluidic paradigms, ¹⁷ _’ ^22-28 DMF in most of these studies was integrated with microchannels and used to control bulk fluid flow or for pre-separation of chemical reactions. The physical phenomenon behind the integration of electrowetting with microfluidics to control the size and frequency of drop formation and the binary sorting of droplets has been discussed ^26-28 however the presently disclosed device and method include advantages over such previously described methods, ^26-28 including the integration of on-demand droplet generation with n-ary sorting (as opposed to binary ²⁹ ) on the same device (which is called integrated digital-droplet microfluidic-ID2M). Furthermore, the presently disclosed device provides improvements for operations for typical droplet- based microfluidic assays.

[0044] Accordingly, in an aspect, there is provided a microfluidic device comprising a first layer including a plurality of electrodes, a second layer disposed on top of the first layer, the second layer including a dielectric patterned over the plurality of electrodes, and a third layer disposed on top of the second layer is described herein. The third layer includes a droplet generator for generating droplets of fluid, a first inlet for receiving the droplets of fluid from the droplet generator, and a microchannel for carrying the droplets of fluid in solution from the first inlet towards an outlet. Actuation of one or more of the plurality of electrodes controls movement of the droplets of fluid from the first inlet towards the outlet.

[0045] In some embodiments, the microfluidic device is a pressure-driven device (e.g. droplets move through the device along a pressure gradient) and droplet motion in the device can also be electrode-driven (such as but not limited to being driven by dielectrophoresis or electrostatics, for example).

[0046] In some embodiments, the microchannel is positioned above one or more of the plurality of electrodes of the first layer.

[0047] In some embodiments, the droplet generator is positioned over one or more electrodes of the plurality of electrodes of the first layer and is configured to generate a droplet based on coordinated actuation of the one or more electrodes.

[0048] In some embodiments, the droplet generator is a T-junction droplet generator.

[0049] In some embodiments, the droplet generator is a flow focusing droplet generator.

[0050] An improvement provided in the device disclosed herein relates to on- demand droplet mixing enabling control and creation of different concentration of droplets. Typical droplet-in-channel techniques have depended on fusion ³⁰ or picoinjection ³¹ methods for mixing but these techniques only allow one reagent addition to an existing droplet and require exquisite control over flow rates, timing, and fluidic resistance. The presently disclosed integrated device can create a range of different concentrations with multiple additions of reagent droplets by application of an electric potential without any consideration for other parameters (e.g., timing).

[0051] In some embodiments, the microchannel includes a droplet mixing region.

[0052] In some embodiments, the droplet mixing region includes contains sinking microchannels to reduce an oil flow rate. Herein, for example, the second fluid may be an oil such as but not limited to a fluorinated oil such as for example a hydrofluoroether (3M) or Fluorinert FC series (3M), an oil including one or more hydrocarbon(s), or other organic solvents with or without surfactants. [0053] Another improvement provided in the presently disclosed device includes areas for trapping and incubation of droplets in which droplets can be individually trapped and incubated for > 48 h. To date, this operation has not been shown on such a device and does not require delay lines ³² · ³³ or on- and off-chip reservoirs for incubation ³⁴ · ³⁵

[0054] In some embodiments, the microchannel includes an incubation region.

[0055] In some embodiments, the incubation region includes at least one trap extending away from the microchannel.

[0056] In some embodiments, the microchannel includes a sorting region.

[0057] In some embodiments, the sorting region includes two or more sorting microchannels configured to receive droplets from the microchannel. For example, the sorting region includes 2 sorting microchannels. For example, the sorting region includes 3 sorting microchannels. For example, the sorting region includes 4 sorting microchannels. For example, the sorting region includes 5 sorting microchannels. For example, the sorting region includes 6 sorting microchannels.

[0058] In some embodiments, the first and second layers are fabricated on top of each other by standard photolithography.

[0059] In some embodiments, the third layer is made of polydimethylsiloxane (PDMS).

[0060] In some embodiments, the device includes a fourth layer disposed or laminated on top of the third layer to seal the third layer.

[0061] In some embodiments, the fourth layer is made of PDMS.

[0062] In some embodiments, the electrodes are co-planar electrodes.

[0063] In some embodiments, the microfluidic device further comprises a fluorescence and/or absorbance detector.

[0064] In some embodiments, the first layer is an electrode layer including a plurality of electrodes that are co-planar and placed under the channels.

[0065] In some embodiments, the second layer is a dielectric material that acts as a capacitor. [0066] In some embodiments, the third layer is made of a transparent material.

[0067] For example, the third layer may be made of one of: polydimethylsiloxane

(PDMS), a photoresist, poly (methyl methacrylate) (PMMA), a plastic, a polymer, silicon, glass, or a combination thereof.

[0068] In some embodiments, the first fluid and the second fluid are immiscible.

[0069] In some embodiments, the first fluid is immiscible in oil. For example, the first fluid may be an aqueous fluid or an aqueous phase including but not limited to fluids such as water, a cell culture media, a buffered solution such as but not limited to phosphate buffer saline (PBS), sometimes containing other chemical or biological compounds such as but not limited to agarose or other gelling agents, surfactants such as Triton or Pluronics, dyes and stains, enzymes, proteins, RNA, DNA, transfection reagents, viral particles.

[0070] In some embodiments, the second fluid is an oil.

[0071] In some embodiments, the droplet generating channel is positioned above one or more of the plurality of electrodes of the first layer.

[0072] In some embodiments, the main channel is positioned above one or more of the plurality of electrodes of the first layer.

[0073] In some embodiments, the droplet generator is a T-shaped droplet generator.

[0074] In some embodiments, the droplet generator is a flow focusing droplet generator.

[0075] In some embodiments, the device includes at least two droplet generators including at least one T-shaped droplet generator and at least one flow focusing droplet generator.

[0076] In some embodiments, the droplet generator is an on-demand droplet generator where droplet generation is controlled by a combination of hydrodynamic flow and actuation of plurality of the electrodes. [0077] In some embodiments, a volume of the droplets is controlled by changing a flow rate of the second fluid in the main channel and actuating a plurality of electrodes to move the first fluid in the droplet generating channel.

[0078] In some embodiments, the second fluid is moved through the main channel by pressure driven flow.

[0079] In some embodiments, the main channel includes a droplet mixing region.

[0080] In some embodiments, the droplet mixing region includes one or more sinking microchannels to control the oil flow rate of the second fluid.

[0081] In some embodiments, the one or more sinking microchannels is a serpentine channel having a number of turns, the sinking microchannel providing an increased flow length to increase a flow resistance in the sinking channel to be higher than a flow resistance in the main channel.

[0082] In some embodiments, the main channel includes an incubation region.

[0083] The microfluidic device of claim 19, wherein the incubation region includes at least one trap extending away from the microchannel.

[0084] In some embodiments, droplets are trapped on-demand by actuating one or plurality of the electrodes.

[0085] In some embodiments, the trapped droplet contains biological material, the biological material including one or more bacterial cells, human cells, mammalian cells, yeast cells, algae cells, plant cells, insect cells or fungal cells, DNA, RNA, proteins, dead cells, barcodes, nucleotides, antibodies, beads or the like.

[0086] In some embodiments, the trapped droplet can individually be addressed via actuating one or plurality of electrodes, the droplet and its constituents can be trapped using electrodes, the droplet and its constituents can be released using electrodes, and the droplet and its constituents can be maintained in the trap for as long as required using the electrodes or the hydrodynamic flow or a combination thereof. [0087] In some embodiments, actuation of the one or more of the plurality of electrodes controls movement of a droplet from the second fluid when the one or more electrodes operate in a dielectrophoresis (DEP) mode or an electrowetting mode.

[0088] In some embodiments, two or more droplets can be merged inside the trap channel, such as by actuating electrodes.

[0089] In some embodiments, the microchannel includes a sorting region.

[0090] In some embodiments, the sorting region includes two or more sorting microchannels configured to receive droplets from the microchannel.

[0091] In some embodiments, the first and second layers are fabricated on top of each other by standard photolithography.

[0092] In some embodiments, the electrodes are co-planar electrodes. The electrodes may be made of any conductive material. The electrodes may be covered with a dielectric material with a specific (i.e. selected) permittivity. The electrodes may be positioned under the fluidic channels (but are not limited to this position). The electrodes can have a coplanar orientation (but are not limited to this orientation). To control fluid droplets, a potential may be sent to the electrodes, with the aim to generate an electric field, at least on top of the dielectric, between the active and grounded electrodes.

[0093] In some embodiments, the device further includes a fluorescence and/or absorbance detector

[0094] In some embodiments, the device further includes an automation system for actuating the electrodes.

[0095] In some embodiments, the device includes a syringe pump system to control the flow rates of fluids in the device.

[0096] In some embodiments, detecting the one or more droplets is completed using a detector.

[0097] In some embodiments, the detector is one of an optical fiber, one or more photo sensors, one or more photomultiplier tubes, a microscope or the like. [0098] In some embodiments, sorting the one or more droplets into two or more groups is based on their solution or their constituents.

[0099] As shown in the Examples provided herein, the presently disclosed device and method was shown to be useful in various applications such as in a biological study (instead of manipulation of water and oil ²⁶²⁸ ) that examines mutant and wild-type yeast cells under ionic liquid conditions which may for example be useful for applications related to biofuel production. More specifically, the presently disclosed platform has been validated as a robust on-demand screening system by sorting fluorescein droplets of different concentration with an efficiency of ~ 96 %. The utility of the system is further demonstrated by culturing and sorting tolerant yeast mutants and wild-type yeast cells in ionic liquid based on their growth profiles. This platform for both droplet and digital microfluidics may the potential to be used for screening different conditions on-chip and for applications like directed evolution.

[0100] Accordingly, another aspect disclosed herein relates to a method of controlling movement of droplets in a microchannel. The method includes generating one or more droplets of fluid at a droplet generator, directing the one or more droplets of fluid into the microchannel from the droplet generator and actuating one or more of a plurality of electrodes positioned below the microchannel to control movement of the droplets of fluid through the microchannel.

[0101] In some embodiments, generating the one or more droplets of fluid includes generating the one or more droplets of fluid by a T-junction droplet generator.

[0102] In some embodiments, generating the one or more droplets of fluid includes generating the T one or more droplets of fluid by a flow focusing droplet generator.

[0103] In some embodiments, directing the one or more droplets of fluid into the microchannel includes actuating one or more of a plurality of electrodes positioned below an inlet of the microchannel to control movement of the droplets of fluid from the droplet generator into the microchannel.

[0104] A further aspect relates to a method of mixing droplets inside a channel by using a combination of pressure driven flow and electrode induced droplet movement. The method includes generating a first droplet either by pressure driven flow only or by additionally using electrodes, receiving the first droplet in a microchannel, directing the first droplet into a mixing region of the microchannel by actuating one or more electrodes positioned beneath the microchannel, merging the first droplet with a second droplet in the mixing region, and mixing the droplet by actuating electrodes and moving the merged.

[0105] Yet another aspect relates to a method of sorting droplets in a microchannel. The method includes generating one or more droplets of fluid at a droplet generator; directing the one or more droplets of fluid into the microchannel from the droplet generator; detecting the one or more droplets, optionally using an optical detector; and sorting the one or more droplets into two or more sorting microchannels by actuating one or more of a plurality of electrodes positioned below the microchannel and/or the two or more sorting microchannels.

[0106] In some embodiments, the method further comprises mixing the one or more droplets with a second droplet to obtain a droplet mixture. For example, a first droplet is directed into a mixing region of the microchannel by actuating one or more electrodes positioned beneath the microchannel and mixing the first droplet with a second droplet in the mixing region.

[0107] In some embodiments, the method further comprises incubating the one or more droplets. For example, the droplet may contain constituents such as one or more cells, such as for example yeast cells.

[0108] In some embodiments, the method further comprises directing the one or more droplets into a detection region. For example, the detection region includes an optical detection area comprising one or more optical fibers placed perpendicular to the microchannel.

[0109] For example, the use of the microfluidic device, method or system herein described for analyzing and/or detecting one or more droplet constituents comprises using fluorescence and/or absorbance for analyzing and/or detecting the one or more droplet constituents. [0110] The presently disclosed device may provide improvements in the field of digital and droplet microfluidics as this can possibly enable more control for droplet microfluidic devices while increase droplet throughput for digital microfluidic devices.

Examples: Integrated droplet-digital microfluidic (ID2M) device and method of making thereof

MATERIALS AND METHODS

Reagents and Materials

[0111] 1-ethyl-3-methylimidazolium acetate > 95 % (HPLC grade), ethyl methanesulfonate, sodium thiosulfate, sodium hydroxide (lab grade), fluorescein (free acid) dye content 95%, yeast nitrogen base without amino acids and with ammonium sulfate, bovine serum albumin (lyophilized powder) > 96 %, and a-D-glucose anhydrous 96% were purchased from Sigma (Oakville, ON Canada), unless specified otherwise. L- leucine, L-histidine, L-methionine, and uracil were purchased from Bio Basic Canada Inc. Yeast BY4741 strain (genotype: MATa his3A1 leu2A0 met15A0 ura3A0) was generously was used. 3M Novec HFE7500 engineering fluid was purchased from M.G. Chemicals (Burlington, ON Canada). Aquapel™ was purchased from Aquapel.ca (Lachute, QC Canada). 20 g of 5% wt of fluoro- surfactant dissolved in HFE7500 was purchased from Ran Biotechnologies (Beverly, MA). Sodium phosphate monobasic and sodium phosphate dibasic (Anhydrous, ASC grade) were purchased from BioShop (Burlington, ON).

[0112] Photolithography reagents and supplies included chromium coated with S1811 photoresist on glass slides from Telic (Valencia, CA), MF-321 positive photoresist developer from Rohm and Haas (Marlborough, MA), CR-4 chromium etchant from OM Group (Cleveland, OH), and AZ-300T photoresist stripper from AZ Electronic Materials (Somerville, NJ). Polylactic acid (PLA) material for 3D printing was purchased from 3Dshop (Mississauga, ON, Canada). Poly(dimethylsiloxane) (PDMS -- Sylgard 194) was purchased from Krayden Inc. (Westminster, CO). SU8 photoresist and developer were purchased from Microchem (Westborough, MA). De-ionized (Dl) water had a resistivity of 18 MW·ah at 25 °C.

[0113] A 100 mM sodium phosphate buffer (SPB) was prepared by mixing 5.77 mL of 1 M Na ₂ HP0 ₄ and 4.23 mL of 1 M NaH ₂ P0 ₄ Solutions (pH 7.0). 5 g of sodium thiosulfate salt was added to deionized water to produce a 5 % (w/v) sodium thiosulfate (STS) solution.

[0114] Fluorescein solutions (0.5 mM) was prepared by adding 1.66 mg of fluorescein powder (332.3 g/mol) to 10 mL 1 M NaOH solution that was made by adding 0.4 g NaOH to 10 mL Dl water.

Device fabrication and operation

[0115] ID2M device masks were designed using AutoCAD 2016 and a transparent photomask was printed by CAD/Art Services Inc. (Bandon, OR). The ID2M microfluidic chip consisted of three layers: a digital microfluidic, dielectric, and channel layer (Figure 1a). As described previously, ¹⁶ ’ ³⁶ electrodes were patterned on a glass substrate with chromium and coated with positive photoresist S1811 , by UV exposure (5 s) on a Quintel Q-4000 mask aligner (Neutronix Quintel, Morgan Hill, CA). Exposed substrates were developed in Microposit MF-321 developer (2 min), rinsed with Dl water, and post-baked on a hot plate (115 °C, 1 min). Substrates were etched in chromium (CR-4) etchant (2 min). Remaining photoresist was stripped in AZ300T (2 min). DMF devices were rinsed by acetone, isopropanol (IPA), and Dl water. The device surface was treated with a plasma cleaner (Harrick Plasma PDC-001 , Ithaca, NY) for 2 min and then immediately spin-coated (Laurell, North Wales, PA) with 7 pm SU8- 5 photoresist (10 s, 500 rpm, 30 s 2000 rpm). SU-8 5 was soft-baked (1. 65 °C, 2 min, 2. 95°C, 5 min) and exposed to UV light (5 s) under the dielectric mask. Post-exposure bake (1. 65°C, 1 min, 2. 95 °C, 1 min) was followed by immersing in SU-8 developer (2 min). Substrates are rinsed with IPA and Dl water, a hard bake was performed in three steps (1. 65 °C, 2 min, 95 °C, 4 min, 3. 180 °C, 10 min). For the channel layer, devices were cleaned again with IPA and Dl water prior to plasma cleaning (2 min). Next, SU-8 2075 photoresist was immediately spin-coated (1. 10 s 500 rpm, 2. 30 s 2000 rpm) on the chip as a 110-120 pm third layer, and soft-baked (65 °C, 3 min; 95 °C, 9 min). Following UV exposure (15 s), devices were post-baked (1. 65 °C, 2 min- 2. 95°C, 7 min), developed in SU-8 developer (7 min) and rinsed with IPA and Dl water. The devices were hard-baked (1 . 65 °C, 2 min, 2. 95 °C, 4 min, 2. 180 °C, 10 min). The integrated microfluidic chip was bonded to a slab (60 mm x 30 mm) of ~ 0.5 mm thick PDMS (1 : 10 weight ratio, w/w curing agent to prepolymer, cured at 65 °C for 3 hours). Inlets and outlets were created using a 0.75 mm puncher (Biopsy Punch, Sklar, West Chester, PA). Before bonding, the PDMS slab was plasma-treated for ~1 min and exposed to (3-aminopropyl)triethoxysilane 99% in a desiccator for 30 min. PDMS was immediately bonded to the device and baked at 160 °C for 20 min. Before operation, channels were treated with Aquapel™for ~5 min and rinsed with HFE oil mixed with 0.75% fluorosurfactant. Syringes were prepared with the following fittings and tubing: 1/4-28 to 10- 32 PEEK adapter, (10-32) peek union assembly, finger tight micro ferrule 10-32 coned for 1/32" OD, and PEEK tubing (1/32” diameter) from IDEX Health & Science, LLC (Oak Harbor, WA). Gastight glass 500 pL-syringes were purchased from Hamilton (Reno, NV) and installed on the neMESYS system (Cetoni, Korbussen, DE).

[0116] Device operation comprised of five stages: droplet generation by a flow- focusing or T-junction configuration followed by droplet mixing, incubation, detection, and sorting. Droplet generation by flow-focusing was implemented by initializing the flow rates using the neMESYS for the aqueous and oil flow rates to 0.0005 [pL/s] and 0.01 [pL/s] respectively. For the T-junction configuration, droplets were created on-demand by four steps: (1 ) the aqueous flow was initialized at 0.0005 [pL/s], (2) when the aqueous flow reaches the sixth electrode, an AC voltage (15 kHz, 200 Vrms) was used to drive the flow to the T-junction, (3) two electrodes were sequentially actuated (i.e. electrodes are turned on and off) to drag the fluid to the main channel (shown in red; Figure 1 b) and (4) a ~ 30 nl_ droplet is formed by both intersecting the oil phase with flow rate of 0.01 [pL/s] and turning on electrodes in the T-junction and main channel as shown in Figure 5. After on- demand droplet generation, droplets were pressure-driven using the oil phase in the main channel and using actuation sequences to drive the droplet into the mixing region (15 kHz, 200 Vrms, under oil flowrate of 0.01 pL/s). Droplets were mixed by actuating underlying electrodes and the mixed droplet was actuated to the main channel. For incubation, droplets were directed to the traps actuating the designated electrodes. After incubation, droplets pass through a detection region which were further sorted by actuation of the electrodes. For droplet size calculations, images of the droplets were acquired and uploaded into ImageJ (National Institute of Health, USA). An imaging pipeline was created to calculate the droplet volume based on an ellipsoid volume formula given that the droplet height was set to 110 pm. ID2M microfluidic optical fiber detection interface

[0117] The optical fiber detection interface consists of a Flame spectrometer (Ocean Optics, Largo, FL), two bare fiber (100 pm core) with numerical aperture of 0.22, and a multi-channel LED light sources that contains four high-power (1 mW) LED modules: 470, 530, 590, 627 nm. Two optical fibers were inserted into two fabricated 300 pm channels that were perpendicular to the direction of the fluid flow (see Figure 1 b). One fiber was connected to the multi-channel LED source, while the other was connected to the Flame spectrometer. The fiber ends were polished carefully using the ocean optics termination kit and fitted with an SMA connector by the help of bare boots for guiding the bare fiber. The distance between the fiber and the channel is ~ 200 pm. All data were collected using the Ocean View spectroscopy software (Ocean Optics, Largo, FL) using the following settings: integration time 100 ms, boxcar smoothing width = 3, number of scans = 5, update rate = 1. Strip chart was enabled to collect data from a single wavelength (530 nm) and executed without stopping.

On-chip calibration curves - fluorescein measurement

[0118] A droplet containing fluorescein (1 mM each in 1 M NaOH buffer, pH 9) was generated using the flow-focusing configuration with fluorescein (0.0005 pL/s) and HFE oil (0.01 pL/s). A droplet of buffer or water (~ 30 nL) was generated using the on-demand T-junction configuration. The droplets were merged and mixed by actuation of underlying electrodes. The amount of buffer droplets added to one fluorescein droplet created four different concentrations: 1 , 0.5, 0.25, and 0.125 mM. After mixing droplets were detected by using our optical fiber setup, and sorted by actuating a sorting sequence for one of the four different on-demand sorting channels. Peak intensities were recorded for each concentration with time traces of the recorded signals. The standard deviation was calculated from 20 replicates.

EMS mutagenesis and generating ionic liquid resistant yeast strains

[0119] Before generating the mutant library, wild-type S. cerevisiae BY4741 yeast cells were stored on agar plates containing synthetic defined medium (6.8 yeast nitrogen base without amino acids, 20 g agar, 20 g 2% glucose, 20 g methionine, 20 g histidine, 20 g uracil, 120 g leucine) at 4°C. Wild-type yeast was grown in 50 mL of synthetic defined medium (30 °C, 200 rpm) for 48 hours. Aliquots of 2 ^c 10 ⁸ yeast cells (O.D. ~ 1 ) were transferred to four micro-centrifuge tubes corresponding to technical triplicate and one control sample. The cells were washed two times with phosphate buffered saline (PBS) and a single time with sodium phosphate buffer (SPB) (0.1 M- pH 7.0). After centrifugation, the pellets were re-suspended in 1.5 ml_ SPB. For mutagenesis, cells were exposed to ethyl methanesulfonate (EMS) according to Winston’s protocol. ³⁷ To generate a standard curve for viability after EMS mutagenesis, 15 ml_ Falcon tubes (corresponding to three different EMS treatment time) were filled with 1 ml_ SPB and 0.7 ml_ cell solution of each micro-centrifuge tube. 50 pL of EMS was added to three of the 15 ml_ falcon tubes in a biological safety cabinet. The control sample (i.e. wild-type cells) were kept without EMS addition. All tubes were incubated at 30°C on a shaker (200 rpm) for 30 min. Cells were exposed to EMS for 40, 50, 60, 75, and 90 min. Mutagenesis was stopped by adding 8 ml_ of 5 % (w/v) sterile sodium thiosulfate (STS) solution at each time point. Aliquots of each falcon tubes diluted in SD media were plated on solid SD media. Plates were incubated at 30 °C for 48 h. Cell viability was measured by comparing colony formation of each EMS time point and the wild-type cells (Figure 6).

[0120] To generate 1 -ethyl-3-methylimidazolium acetate IL resistant cells, the mutagenesis is repeated for 60, 75, and 90 mins. Resulting aliquots were inoculated in 5 ml_ synthetic defined medium for 24 h at 30 °C on a shaker with 200 rpm. Next, the mutants were inoculated in 5 ml_ synthetic defined medium and 50, 75, or 100 mM 1 -ethyl- 3-methylimidazolium acetate IL and incubated for 24 h at 30 °C on a shaker with 200 rpm. 1 mL aliquots of each test tubes along with a wild-type sample were diluted 100 times with SD media and then were plated onto several solid SD plates containing 50, 75, or 100 mM IL. These plates were incubated for 4-6 days at 30 °C. Colonies were randomly selected from the plates and cultured in 5 mL SD media at 30 °C. After 24 h, the OD of the culture was measured and if the OD was greater than 0.3, samples were diluted and cultured in different ionic liquid conditions otherwise they were discarded. If selected, an aliquot (depending on IL concentration) from the 5 mL culture was added to the wells of a microwell plate to make up a final volume of 200 pL. In each well, the OD was measured every 20 min at 30 °C with shaking at 200 rpm for 48 hours using a Tecan Sunrise microplate reader (Tecan, Salzburg, Austria) with the following settings (measurement wavelength: 595 nm). Three replicates were measured for each condition.

N-ary sorting of yeast mutants library on ID2M device

[0121] For analyzing the effect of IL on wild-type and mutant yeast on chip, the two fastest growing IL tolerant mutants and wild-type yeast were cultured in SD without IL for 48 h. A 500 pL syringe was prepared with a cell suspension of 2 ^c 10 ⁵ cells/mL in SD media containing 1 % bovine serum albumin (BSA) and a syringe containing HFE oil with 2 % fluorinated surfactant. Both syringes were connected to the inlets of the device using PEEK tubing (1/32 inch diameter). Cell encapsulation was performed through flow focusing (using Poisson statistics) with flow rates of 0.0008 pL/s and 0.01 pL/s for cells and oil, respectively to generate a droplet with volume of ~ 35 nL. For the T-junction droplet generator, a syringe was filled with 200 mM IL and ~ 35 nL droplets were formed on demand. Droplets containing a single cell were actuated into the mixing region by sequentially applying ~ 200 V _P.P (15 kHz) to the electrodes. The droplet was merged with an on-demand generated droplet of IL and mixed by moving the droplet back-and-forth along the linear path. Upon mixing the droplet with a 200 mM IL, the mixed droplet of cells and IL (with a final concentration of 100 mM IL) was actuated to the main channel and was trapped into incubation slot using actuation. This process was repeated for three other incubation regions. After trapping all four droplets, the ID2M device was removed from the automation system and droplets were incubated for 48 h at 30 °C in a humidified chamber.

[0122] After incubation, droplets were actuated to the main channel and passed through the optical detection area where the two optical fibers were placed perpendicular to the main channel. According to the absorbance peaks differences, droplets were sorted into three groups using the three sorting channels. Any excess droplets in this procedure was actuated to the waste channel. During all droplet operation procedures (i.e. mixing, trapping, incubation, sorting) and when droplets were in the main channel, oil flow rates were maintained at 0.01 pL/s.

COMSOL simulation [0123] A simulation of the mixing area with the sinking channels was conducted using COMSOL Multiphysics V5.3 (COMSOL Inc., Cambridge, MA, USA). Parameters are shown in Table 1 and following assumptions were made for simplification: 1) Newtonian fluid, 2) no- slip boundary condition, and 3) incompressible flow. A single phase laminar flow using Navier Stokes model was selected as the physics of our stationary study with the assumption that our fluid is 3M™ Novec™ 7100 Engineered Fluid. Wall boundaries and inlet and outlet were defined as depicted in Figure 7. The inlet velocity of the fluid flow was initialized to 0.033 nvs ¹ .

Table 1. COMSOL simulation parameters used for modeling the sinking channels in the mixing area.

ID2M automation system

[0124] Droplet manipulation was controlled by a GUI (Figure 12) generated in a MATLAB (Mathworks, Natlick, MA) which controlled an Arduino Uno that interfaced to a control board consisting of a network of high-voltage relays (AQW216 Panasonic, Digikey, Winnipeg, MB). The control board delivered AC signals from a high-voltage amplifier (PZD-700A, Trek Inc., Lockport, NY) paired with a function generator (33201 A Agilent, Allied Electronics, Ottawa, ON) to initiate actuation sequences on the device (Figure 13). Additionally, the GUI controlled the neMESYS syringe pump and Flame spectrometer (Ocean Optics, Largo, FL). The ID2M microfluidic chip is mounted on a pogo pin-control board (104 pins) with a 3D printed base platform as previously reported ¹⁶ ’ ³⁶ and was placed on the stage of an inverted IX-73 Olympus microscope (Olympus Canada, Mississauga, ON). RESULTS AND DISCUSSION

Device characterization and optimization

[0125] A microfluidics architecture called ID2M has been developed. ID2M merges droplet microfluidics (useful for generating and sorting droplets) with digital microfluidics (useful for on-demand droplet manipulation and individual control of droplets). The ID2M device was formed by creating a single-plate DMF device (i.e. the ground and driving electrodes are co-planar) and fabricating a network of channels on top, with inlets and outlets for generating and sorting droplets respectively, and an area for droplet mixing. An exploded view (Figure 1 a) shows the digital microfluidic device as the bottom substrate with 104 patterned electrodes, the dielectric layer (substrate 1 and 2), the network of channels patterned in SU-8 photoresist, and a slab of PDMS with inlets and outlets (substrates 3 and 4). This multilayer integrated architecture facilitates pressure-based and on-demand droplet generation using flow focusing and T-junction configurations respectively, on-demand droplet mixing, on-demand droplet trapping and incubation, and on-demand droplet sorting. The combined multilayer architecture may provide a significant advance over other types droplet-to-digital methods which rely on two separate design configurations which can cause difficulties in moving the droplet from one platform to the other as reported previously. ^{17 2224}

[0126] Droplets in the main channel are moved by pressure flow and electrical potentials move droplets to the mixing, incubation, and sorting regions (i.e. away from the main channel) (Figure 1 b). A central feature of this design is that droplets in the main channel can be moved to the mixing area to merge with other droplets. For example, a droplet containing dilution buffer is generated on-demand via actuation from the T- junction, then actuated to the mixing area, and merged and mixed with other droplets in the main channel. This process can be repeated to create of a diluent series of droplets. After generating the diluent droplet, these droplets can be actuated to the main channel and can be incubated in the trap and sorted in one of the channels (after incubation) using electrostatic actuation. Typical droplet microfluidic systems use electrocoalescence ^{38 39} or picoinjection ³¹ ' ⁴⁰ techniques to sequentially add reagents to droplets at different times. Flowever, these techniques, as of yet, have not demonstrated the generation of a dilution series of droplets. In addition to generation of a diluent series of droplets, the droplets are capable of being sorted in four different channels. This allows for droplet samples to be sorted by multiple conditions based on a larger gradient, like multiple levels of fluorescence and absorbance, instead of typical binary sorters. This suggests that using a system (such as ID2M) can provide direct droplet control that enables generation of a droplet dilution series and droplet sorting in multiple fractions for droplet microfluidic systems.

[0127] Electrode shape and design is important to ensure high-fidelity droplet movement on the device (Figure 1 b). In initial electrode designs, a one electrode design on the bottom plate with alternating ground and driving potentials was followed. ²⁷ ’ ⁴¹ However, droplets in the main channel were not able to overcome the pressure generated from the oil flow rate and could not be actuated into the mixing, incubation, or different sorting regions. A coplanar electrode configuration (i.e. with adjacent ground and actuated electrodes on the same plane), as shown by some groups, ⁴²⁴⁴ showed optimal droplet manipulation. The introduction of a ground electrode (or grounding line) on the same plane may not generate the highest applied force as compared to other electrode designs, ⁴² but the selected design is easiest to fabricate and is capable of overcoming the applied pressure on the droplet in our system (oil flow rate of 0.005-0.05 pL/s).

[0128] The fabrication protocol for the ID2M devices needed to be optimized to ensure strong adhesion of the dielectric, channel, and PDMS layers during fabrication, and to allow droplets to be controlled by application of electric potentials in the mixing area. For the former challenge, it was found that introducing 300 pm spaced repeated finger-like structures on the boundary of the dielectric layer increases adhesion to the substrate (Figure 8). Layers that did not have these finger-like structures or if the repeated finger like structures are spaced far apart (> 500 pm), SU-85 tends to peel or crack easily. It is hypothesized these cracks are mostly made by internal stresses as high evaporation and heating/cooling rate in addition to temperature differences in different layers of SU-8 5 causes residual stresses in the layer. ⁴⁵ To increase the adhesion of the PDMS slab to the SU-8 layer, (3- aminopropyl)triethoxysilane (APTES) ⁴⁶ vapor deposition was used after plasma treatment of the PDMS, and the slab was exposed to the vapor of APTES in a desiccator for 30 min, forming aminosilane molecule on the surface of the PDMS. This surface favorably reacts with the epoxy group from the SU-8 surface which strengthens the bond between the PDMS and SU-8 layer.

[0129] To slow down the flow rate and to enable droplets to be actuated from the main channel to the mixing area, sinking channels were added in the mixing area. Multiple sink channels ⁴⁷ were added to create flow eddies from the main flow channel which allow the oil phase to have multiple flow paths (Figure 1 b and Figure 9). This reduction in oil flow rate enables droplets in the main channel to be actuated into the mixing channel. In initial designs, a side channel (i.e. a channel branching out of the main channel) was created with the co-planar electrodes (i.e. grounding and potential electrodes on the same plane); however, droplets were not capable of being moved by actuation from the main channel to the mixing area. Increasing of voltage ⁴⁸ was explored; however higher voltage tends to cause dielectric breakdown in the oil phase and cause droplet breakup which creates small satellite droplets. The sink channels are particularly important when a droplet is already in the mixing area since the droplet acts as a plug (i.e. increasing the hydrodynamic resistance). ⁴⁹ In one embodiment, the sinking channel(s) may include a serpentine channel for sinking the continuous flow fluid (e.g. the oil phase fluid). The serpentine channel may control the resistance of flow of the sinking channel based on the length of this channel. The number of turns is designed such that the resistance in sinking channel becomes much more than the resistance in the main channel to inhibit the oil phase from exiting the main channel and entering the sinking channel. Since the hydrodynamic resistance in the mixing channel is higher than the main channel when a droplet is present, the generated droplets favor flow in the main channel. Alternatively, having multiple sink channels creates multiple flow paths (i.e. reducing the resistance in the mixing channel), leading to mixing of the droplets in this area. Flaving a mixing area with a sinking channel provides for performing droplet operations in all directions using electrodes, not following pressure driven flow. For example, two droplets may be brought into the region, merged, and mixed together by moving the droplet repetitively within the area. [0130] An additional component for successful device operation was optimization of the configuration of the n-ary sorting channels. Initial testing was conducted with Y- shaped configuration, ⁵ ’ ⁵⁰ in which droplets are discriminated by two (or more ⁹ · ^{51 52} ) physical characteristics. However, the Y-channels have a tendency to create a stagnation zone (i.e. an area where the droplet faces an uncontrolled choice for an outlet) even with the additional bias of the electric potentials. The additional bias also creates an asymmetric presence of drops (creating different resistances) when it is expanded to more than two channels. ²⁹ Instead, a symmetrical T-channel that consists of four different sorting areas with similar resistances was designed. Pressure-driven droplets are detected using the optical interface and are biased directly to a channel by actuation. Rails ⁵³ or linear electrodes ⁵⁴ with the symmetric T-channels may be contemplated to reduce the footprint and to increase the number of sorting channels.

On-demand droplet generation, mixing, incubation, and sorting

[0131] The presently disclosed system enables integration of a variety of fluidic manipulations steps such as on-demand droplet generation, merging and mixing, and n- ary sorting. As shown in Figure 2a, droplets can be generated through flow-focusing geometry or by on-demand generation using T-junction (Frame i, ii, and iii), stored (Frame iv and v), merged and mixed (Frame vi), incubated (Frame vii), and sorted (Frame viii and ix). The device can generate droplets on-demand by using a T-junction configuration which combines the pressure of the continuous oil phase and electrostatic actuation of the aqueous flow. As shown in Figure 2b, the droplet volume generated by the T-junction can be tuned by only changing the oil flow rate (as opposed to tuning both aqueous and oil flows) ⁵⁵ ’ ⁵⁶ and using actuation to move the aqueous flow. This setup enables a wide range of volumes being generated (40-115 nl_) by tuning the oil flow between 0.001 and 0.06 pL/s. As a comparison, droplets were generated hydrodynamically by changing the oil flow rate (while keeping the aqueous flow rate constant) which resulted in minimal changes in the volume when increasing the oil flow rate > 0.01 pL/s. It is hypothesized that traditional systems for tuning droplet sizes is limited by the orifice size and the relative strength of interfacial tension and hydrodynamic shear forces, ³² which can be alleviated using on-demand droplet generation. In addition to on-demand droplet generation, mixing and sorting are particularly useful capabilities, as most droplet microfluidic systems are incapable of generating dilutions of droplets and sorting them into multiple channels. In the design disclosed herein, after droplet generation, droplets can be actuated to the mixing area and merged with another droplet (Figure 2a, frame iv-vi) and transferred to the main channel area for sorting and analysis (Figure 2a, frame vii-ix). To illustrate this, this method was used to generate calibration standards on this platform with sorting analysis.

[0132] Dilutions were formed by merging a droplet containing analyte (fluorescein) with a droplet of diluent (buffer). This merged droplet was mixed (by moving the merged droplet in a linear pattern - up-and-down - for several seconds ⁵⁷ ) producing a droplet with a 2x dilution of analyte. This droplet was analyzed by optical detection (Figure 3a) and sorted for further processing. Subsequent droplets of analyte with different concentrations (4x and 8x) followed a similar protocol except the droplet containing fluorescein was mixed with two, three, orfour droplets of diluent respectively (Figure 3b). Note that this type of process, which includes on- demand droplet generation and mixing to create different droplets of different concentration of analytes was only made possible with the integration of digital microfluidics. Such operations were not possible with typical droplet microfluidic platforms unless increasing the number of inlets and injectors or reinjecting droplets into the device. ⁴⁰ The devices used in this experiment were done in droplet-in-channels with minimal inlets, which allowed for a maximum 8x dilution of stock analyte. It is expected that more dilutions could be implemented or mixing different types of analytes could be implemented by using these devices.

[0133] Figure 3c summarizes the results from the dilution series experiment with fluorescein. The emitted fluorescence from the droplet was detected by the spectrometer which outputted arbitrary units proportional to the emitted fluorescence of the droplet. As shown in Figure 3c, the yellow curve depicts droplets that have minimal emitted fluorescence (i.e. droplets of diluent without fluorescein). The blue curve shows the fluorescence intensity for different concentrations of fluorescein. As expected, the highest fluorescein concentration (1 mM) showed the highest signal with a sorting threshold ~1900 arbitrary units and the lower fluorescein concentration (0.125 mM) showed the lowest signal with a threshold of ~ 700. A calibration curve (N = 10) was generated by plotting the ratio of analyte peak intensity as a function of analyte concentration (Figure 3d). The precision in each measurement (RSD = 3.2%, 4.6 %, 7.5%, and 10.7% for the stock, 2x, 4x, and 8x dilution, respectively) and the correlation coefficient (R ² = 0.99) demonstrates that the method is reproducible and linear. Furthermore, the sorting efficiency was measured by sorting positive-fluorescein (1 mM) vs. negative-fluorescein droplets and obtained ~96 % efficiency for positive (i.e. fluorescent) droplets which was found to be similar to other reported sorting efficiencies. ⁵⁸

ID2M application - effect of ionic liquid on yeast mutants

[0134] As an application of this work, the effects of ionic liquid (IL) on wild-type and mutant yeast cells were examined. Ionic liquid has been used as a promising pretreatment method for breaking down polysaccharides from typical feedstocks (e.g., lignin) for sustainable production of renewable biofuels. ⁵⁹ · ⁶⁰ Typically, there has been a wide range of available ILs that are suitable for effectively breaking down the required biomass. ^{61 62} However, a major disadvantage with typical ILs (especially imidazolium ILs) is their inherent microbial toxicity which can either arrest growth of microbial cells, like E.coli or S. cerevisiae, or inhibit biofuel-related enzymes which can reduce the overall yield of biofuel production. ⁶³ · ⁶⁴ Hence, there is interest in investigating the mechanisms of tolerance for microbes to different levels of IL.

[0135] Here, the effects of IL on wild-type and mutant yeast cells were compared and the ability to interrogate each cell type with different IL concentrations and to sort cells based on their growth differences was shown. It is believed that this is the first time that microbes have been cultured, mixed with ionic liquid and sorted based on multiple conditions (i.e. not binary). As a first step, a random mutant library (via ethyl methylsulfonate treatment) was created and their growth rates under IL conditions were verified (Figure 4a). Three types of yeast cells were chosen: wild-type and two best performing IL tolerant mutants and cultured them with and without 100 mM ionic liquid. As shown in Figure 4b, the mutant cells showed faster rates (~ 2.2 and ~ 2.3 cell per hour for mutant #1 and mutant #2, respectively) compared to the wild-type cells (~ 0 cell per hour) in ionic liquid. In fact, the wild-type cells exhibited virtually no detectable growth in ionic liquid conditions. When cultured without ionic liquid, the wild type cells showed faster rates than both mutant cells (~ 3.4 and 3.7 cell per hour for the mutants and ~ 3.8 cell per hour for the wild-type). The mechanisms of ionic liquid tolerance are still under debate, but it is hypothesized that the location of the mutations in the yeast are in areas that are related to efflux pumps (i.e. to bring IL in-and-out of the cells) ⁶⁵ and to transcriptional regulators that are related to stabilizing stress response ⁶⁶ More work is required to determine the genotype location of the mutations (i.e. single-cell sequencing) ⁶⁷ , but this experiment confirmed the capability of obtaining three different strains that may be used to show the utility of the device.

[0136] After selecting mutant phenotypes, mutant and wild-type yeast cells exposed to ionic liquid were sorted. Figure 10 shows the workflow for sorting yeast cells, starting with encapsulation of single cells in droplets (Poisson) and actuating them into the mixing channel. Droplets were mixed with a droplet of 200 mM IL, generated from the on-demand T-channel configuration. The droplet was incubated in one of the four trapping regions and after 24 h it was further analyzed by absorbance and sorted by their growth (i.e. cell number). Figure 4c shows droplets that contained wild-type and mutant-type yeast cells with 100 mM IL. Mutant- type cells showed significant difference in the cell density compared to wild-type cells which are matching the growth rate results. Droplet absorbance signals (Figure 4d) increased as the cell density increases while the signal for the oil phase remains constant (~0.04-0.07; see Figure 11 for oil signal). In practice, the absorbance of the droplet is greater than that of the oil at higher cell densities (> 20 cells) and similar to oil at low cell densities (< 5 cells). Indeed, sensitivity of the signals depend on fiber alignment and background lighting which in this case was measured to be < 0.5 %. It is proposed that improvement on the optical setup ⁶⁸ or device fabrication ⁶⁹ can increase the sensitivity of the design and expand the range of cell densities being observed. The presently disclosed method enables a wide variety of droplet operations that is typically not possible with droplet or digital microfluidic systems - encapsulation, mixing (to generate different ionic liquid concentrations), culture and incubation, and n- ary sorting. The presently disclosed method may be particularly useful for high-throughput applications that require a creation of different drug concentrations or clonal libraries and sorting them at multiple levels. CONCLUSION

[0137] An integrated droplet-digital microfluidic (ID2M) system that uses a combination of pressure- and electrical-based methods for the manipulation of droplets on chip has been developed. In this presently disclosed method, four enhanced fluidic operations were created. First, droplets are generated by on-demand T-junction droplet generators (along with traditional flow-focusing techniques) which could generate a wide range of droplet volumes by tuning only the oil flow rate. Secondly, droplets were actuated to a mixing region that enabled merging with other droplets to form a dilution series of droplets. Third, after mixing, droplets could be trapped and incubated for several days simply by activating electrodes to guide the droplet into incubation traps. Lastly, this design included four channels (i.e. n-ary) for sorting droplets that contained different concentrations or constituents using fluorescence or absorbance. The utility of this microfluidic device is demonstrated by studying the effects of ionic liquid on wild-type and mutant yeast cells. Using the four controlled fluidic steps, the cells could be sorted into different fractions based on absorbance that can be analyzed downstream.

[0138] While the applicant's teachings described herein are in conjunction with various embodiments for illustrative purposes, it is not intended that the applicant's teachings be limited to such embodiments as the embodiments described herein are intended to be examples. On the contrary, the applicant's teachings described and illustrated herein encompass various alternatives, modifications, and equivalents, without departing from the embodiments described herein, the general scope of which is defined in the appended claims.

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