WO2021087614A1 - Système microfluidique numérique intégré à gouttelettes permettant la création, le mélange, l'incubation et le tri de gouttelettes à la demande - Google Patents

Système microfluidique numérique intégré à gouttelettes permettant la création, le mélange, l'incubation et le tri de gouttelettes à la demande Download PDF

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WO2021087614A1
WO2021087614A1 PCT/CA2020/051506 CA2020051506W WO2021087614A1 WO 2021087614 A1 WO2021087614 A1 WO 2021087614A1 CA 2020051506 W CA2020051506 W CA 2020051506W WO 2021087614 A1 WO2021087614 A1 WO 2021087614A1
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droplet
droplets
fluid
microfluidic device
electrodes
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Steve SHIH
Fatemeh AHMADI
Kenza SAMLALI
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Valorbec, Société en commandite
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502715—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F31/00—Mixers with shaking, oscillating, or vibrating mechanisms
    • B01F31/65—Mixers with shaking, oscillating, or vibrating mechanisms the materials to be mixed being directly submitted to a pulsating movement, e.g. by means of an oscillating piston or air column
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/302—Micromixers the materials to be mixed flowing in the form of droplets
    • B01F33/3021—Micromixers the materials to be mixed flowing in the form of droplets the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/3031—Micromixers using electro-hydrodynamic [EHD] or electro-kinetic [EKI] phenomena to mix or move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502784—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/01—Drops
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0673—Handling of plugs of fluid surrounded by immiscible fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0424—Dielectrophoretic forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0415—Moving fluids with specific forces or mechanical means specific forces electrical forces, e.g. electrokinetic
    • B01L2400/0427—Electrowetting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/38—Diluting, dispersing or mixing samples

Definitions

  • 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.
  • 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).
  • DMF digital microfluidics
  • Microfluidic devices are described herein.
  • 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.
  • 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.
  • 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.
  • a method of controlling movement of droplets in a microchannel 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.
  • a method of mixing droplets in a microfluidic device 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.
  • a method of sorting droplets in a microchannel 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.
  • a use of a microfluidic device, method or system disclosed herein for analyzing and/or detecting one or more droplet constituents 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Fig. 3a is a close-up view of the detection region on the ID2M device.
  • 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.
  • 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).
  • 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.
  • 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.
  • Fig. 4b is a graph showing growth curves for the wild-type and mutant yeast cells in 100 mM ionic liquid.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • Fig. 8d shows the final mask design with finger-like boundaries.
  • 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.
  • 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.
  • Fig. 11 is a graph showing the time course (absorbance vs. time) plot for only oil phase (i.e. no droplets).
  • 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).
  • Fig. 13 is a schematic of the ID2M automation setup and shows the connectivity of all the different components used in this system.
  • 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.
  • Coupled indicates that two elements can be directly coupled to one another or coupled to one another through one or more intermediate elements.
  • 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.
  • 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.
  • 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).
  • a pressure-driven device e.g. droplets move through the device along a pressure gradient
  • droplet motion in the device can also be electrode-driven (such as but not limited to being driven by dielectrophoresis or electrostatics, for example).
  • the microchannel is positioned above one or more of the plurality of electrodes of the first layer.
  • 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.
  • the droplet generator is a T-junction droplet generator.
  • the droplet generator is a flow focusing droplet generator.
  • 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 requireaki 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).
  • the microchannel includes a droplet mixing region.
  • the droplet mixing region includes contains sinking microchannels to reduce an oil flow rate.
  • 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.
  • 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.
  • 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
  • the microchannel includes an incubation region.
  • the incubation region includes at least one trap extending away from the microchannel.
  • the microchannel includes a sorting region.
  • the sorting region includes two or more sorting microchannels configured to receive droplets from the microchannel.
  • the sorting region includes 2 sorting microchannels.
  • the sorting region includes 3 sorting microchannels.
  • the sorting region includes 4 sorting microchannels.
  • the sorting region includes 5 sorting microchannels.
  • the sorting region includes 6 sorting microchannels.
  • the first and second layers are fabricated on top of each other by standard photolithography.
  • the third layer is made of polydimethylsiloxane (PDMS).
  • the device includes a fourth layer disposed or laminated on top of the third layer to seal the third layer.
  • the fourth layer is made of PDMS.
  • the electrodes are co-planar electrodes.
  • the microfluidic device further comprises a fluorescence and/or absorbance detector.
  • the first layer is an electrode layer including a plurality of electrodes that are co-planar and placed under the channels.
  • the second layer is a dielectric material that acts as a capacitor.
  • the third layer is made of a transparent material.
  • the third layer may be made of one of: polydimethylsiloxane
  • PDMS photoresist
  • PMMA poly (methyl methacrylate)
  • plastic a polymer, silicon, glass, or a combination thereof.
  • the first fluid and the second fluid are immiscible.
  • the first fluid is immiscible in oil.
  • 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.
  • PBS phosphate buffer saline
  • surfactants such as Triton or Pluronics, dyes and stains, enzymes, proteins, RNA, DNA, transfection reagents, viral particles.
  • the second fluid is an oil
  • the droplet generating channel is positioned above one or more of the plurality of electrodes of the first layer.
  • the main channel is positioned above one or more of the plurality of electrodes of the first layer.
  • the droplet generator is a T-shaped droplet generator.
  • the droplet generator is a flow focusing droplet generator.
  • the device includes at least two droplet generators including at least one T-shaped droplet generator and at least one flow focusing droplet generator.
  • 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.
  • 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.
  • the second fluid is moved through the main channel by pressure driven flow.
  • the main channel includes a droplet mixing region.
  • the droplet mixing region includes one or more sinking microchannels to control the oil flow rate of the second fluid.
  • 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.
  • the main channel includes an incubation region.
  • droplets are trapped on-demand by actuating one or plurality of the electrodes.
  • 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.
  • 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.
  • 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.
  • DEP dielectrophoresis
  • two or more droplets can be merged inside the trap channel, such as by actuating electrodes.
  • the microchannel includes a sorting region.
  • the sorting region includes two or more sorting microchannels configured to receive droplets from the microchannel.
  • the first and second layers are fabricated on top of each other by standard photolithography.
  • the device further includes a fluorescence and/or absorbance detector
  • the device further includes an automation system for actuating the electrodes.
  • the device includes a syringe pump system to control the flow rates of fluids in the device.
  • detecting the one or more droplets is completed using a detector.
  • 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.
  • 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.
  • 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.
  • generating the one or more droplets of fluid includes generating the one or more droplets of fluid by a T-junction droplet generator.
  • generating the one or more droplets of fluid includes generating the T one or more droplets of fluid by a flow focusing droplet generator.
  • 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.
  • 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.
  • 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.
  • the method further comprises mixing the one or more droplets with a second droplet to obtain a droplet mixture.
  • 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.
  • the method further comprises incubating the one or more droplets.
  • the droplet may contain constituents such as one or more cells, such as for example yeast cells.
  • the method further comprises directing the one or more droplets into a detection region.
  • the detection region includes an optical detection area comprising one or more optical fibers placed perpendicular to the microchannel.
  • 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.
  • 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.
  • I2M Integrated droplet-digital microfluidic
  • 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).
  • AquapelTM 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).
  • 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.
  • 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.
  • SPB sodium phosphate buffer
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • channels were treated with AquapelTMfor ⁇ 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).
  • 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.
  • 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.
  • 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.
  • droplets were directed to the traps actuating the designated electrodes.
  • droplets pass through a detection region which were further sorted by actuation of the electrodes.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • PBS phosphate buffered saline
  • SPB sodium phosphate buffer
  • EMS ethyl methanesulfonate
  • 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.
  • 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.
  • 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.
  • 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.
  • Droplet manipulation was controlled by a GUI ( Figure 12) generated in a MATLAB (Mathworks, Natlick, MA) which controlled an chicken 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).
  • 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
  • ID2M 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.
  • FIG. 1 a 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
  • 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.
  • 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.
  • Electrode shape and design is important to ensure high-fidelity droplet movement on the device (Figure 1 b).
  • a one electrode design on the bottom plate with alternating ground and driving potentials was followed. 27 ’ 41
  • 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.
  • ground electrode or grounding line
  • 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).
  • APTES (3- aminopropyl)triethoxysilane 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.
  • 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.
  • a side channel i.e. a channel branching out of the main channel
  • the co-planar electrodes i.e. grounding and potential electrodes on the same plane
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • feedstocks e.g., lignin
  • ILs ionic liquid
  • a major disadvantage with typical 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.
  • 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.
  • the wild-type cells exhibited virtually no detectable growth in ionic liquid conditions.
  • 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.
  • FIG. 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).
  • 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).
  • 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.
  • IDM integrated droplet-digital microfluidic
  • 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.

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Abstract

La présente invention concerne des dispositifs, des systèmes et des procédés microfluidiques. Plus particulièrement, les dispositifs microfluidiques comprennent une première couche, comportant une pluralité d'électrodes ; une deuxième couche disposée sur la première couche, la deuxième couche comportant un diélectrique modelé sur la pluralité d'électrodes ; et une troisième couche, disposée au-dessus de la deuxième couche. La troisième couche comporte un générateur de gouttelettes permettant de générer des gouttelettes d'un premier fluide dans un second fluide, le générateur de gouttelettes comportant un canal de production de gouttelettes permettant le transport du premier fluide vers un canal principal logeant le second fluide. L'activation desdites électrodes de la pluralité d'électrodes commande le mouvement du premier fluide dans le canal de production de gouttelettes vers le canal principal et la production de gouttelettes du premier fluide dans le canal principal.
PCT/CA2020/051506 2019-11-08 2020-11-06 Système microfluidique numérique intégré à gouttelettes permettant la création, le mélange, l'incubation et le tri de gouttelettes à la demande WO2021087614A1 (fr)

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