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WO2016064755A2 - Modulation rapide de la composition de gouttelettes par des microvannes à membrane - Google Patents

Modulation rapide de la composition de gouttelettes par des microvannes à membrane Download PDF

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Publication number
WO2016064755A2
WO2016064755A2 PCT/US2015/056269 US2015056269W WO2016064755A2 WO 2016064755 A2 WO2016064755 A2 WO 2016064755A2 US 2015056269 W US2015056269 W US 2015056269W WO 2016064755 A2 WO2016064755 A2 WO 2016064755A2
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WIPO (PCT)
Prior art keywords
inlet
slmv
valve
inlet channel
droplet
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Ceased
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PCT/US2015/056269
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WO2016064755A3 (fr
Inventor
Adam R. Abate
Christopher Ochs
Chaitanya KANTAK
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University of California
University of California Berkeley
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University of California
University of California Berkeley
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Publication of WO2016064755A2 publication Critical patent/WO2016064755A2/fr
Publication of WO2016064755A3 publication Critical patent/WO2016064755A3/fr
Anticipated expiration legal-status Critical
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F23/00—Mixing according to the phases to be mixed, e.g. dispersing or emulsifying
    • B01F23/40—Mixing liquids with liquids; Emulsifying
    • B01F23/41—Emulsifying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F25/00—Flow mixers; Mixers for falling materials, e.g. solid particles
    • B01F25/40—Static mixers
    • B01F25/42—Static mixers in which the mixing is affected by moving the components jointly in changing directions, e.g. in tubes provided with baffles or obstructions
    • B01F25/43—Mixing tubes, e.g. wherein the material is moved in a radial or partly reversed direction
    • B01F25/433—Mixing tubes wherein the shape of the tube influences the mixing, e.g. mixing tubes with varying cross-section or provided with inwardly extending profiles
    • B01F25/4331—Mixers with bended, curved, coiled, wounded mixing tubes or comprising elements for bending the flow
    • 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
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00—Subject matter not provided for in other groups of this subclass
    • F16K99/0001—Microvalves
    • F16K99/0003—Constructional types of microvalves; Details of the cutting-off member
    • F16K99/0015—Diaphragm or membrane valves
    • 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
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F16—ENGINEERING ELEMENTS AND UNITS; GENERAL MEASURES FOR PRODUCING AND MAINTAINING EFFECTIVE FUNCTIONING OF MACHINES OR INSTALLATIONS; THERMAL INSULATION IN GENERAL
    • F16K—VALVES; TAPS; COCKS; ACTUATING-FLOATS; DEVICES FOR VENTING OR AERATING
    • F16K99/00—Subject matter not provided for in other groups of this subclass
    • F16K2099/0082—Microvalves adapted for a particular use
    • F16K2099/0084—Chemistry or biology, e.g. "lab-on-a-chip" technology

Definitions

  • Microfluidic valves have found use in technologies such as gene sequencing, single cell analysis, and structural and synthetic biology.
  • Various techniques have been developed to pump, switch, and isolate fluids in microfluidic channel networks via on-chip or off-chip control, including solenoids, various types of injectors, metal screws and pin-valves, single-layer and multilayer membrane valves.
  • MLMV multilayer membrane valves
  • These valves utilize a rounded geometry and vertical deflection of an elastic membrane to completely seal channels and achieve reliable, repeatable on-off actuation.
  • fabricating microfluidic devices with these valves requires specialized photoresists and a mask aligner; moreover minor errors in fabrication or alignment of control and flow layers can significantly impact valve performance.
  • SLMV Single-layer membrane valves
  • pressurized control channels deflect the elastic side wall of the flow channel, enabling modulation of flow rate.
  • small “gutters” remain open at the corners of the channels even when the valve is fully actuated.
  • these valves have been primarily relegated to use as sieve valves for high throughput particle and cell sorting.
  • aspects of the disclosure include a method of modulating droplet composition using a microfluidic device.
  • the method includes: (a) actuating a first valve, e.g., a membrane valve, in contact with a first inlet channel of the microfluidic device to provide a first modulated flow stream of a first liquid in a first inlet channel of the microfluidic device; (b) actuating a second valve, e.g., membrane valve, in contact with a second inlet channel of the microfluidic device to provide a second modulated flow stream of a second liquid in a second inlet channel of the microfluidic device; (c) receiving at a droplet generator of the microfluidic device the first and second modulated flow streams; and (d) forming via the droplet generator of the microfluidic device a first droplet comprising a volume of the first liquid and a volume of the second liquid, which volumes are determined by steps (a) and (b).
  • a first valve e.g.,
  • Figure 1 provides images and schematics illustrating various valve types in open (top) and closed state (bottom) showing membrane deflection.
  • Single layer membrane valves (A) and multilayer valves with rectangular channel geometry (B) achieve partial sealing.
  • Multilayer membrane valves with rounded flow channels achieve full sealing (C).
  • Figure 2 illustrates a schematic of a pincer-valve modulated microfluidic mixer and droplet generator, (A). Valve efficiency is visualized by comparing IR dye area fractions in the mixing region in the open (B) and closed (C) states.
  • Figure 3 shows a quantitative assessment of valve efficiency via measurement of droplet fluorescence.
  • A ON-OFF liquid control using a commercial off-chip solenoid valve as compared to
  • B actuation of on-chip pincer valves.
  • C Gradual control of fluorescein concentration (black squares) as a function of actuation pressure (open circles).
  • Figure 4 shows the results of combinatorial mixing in a five-channel device using pincer valves. Encapsulation of three dyes arranged by decreasing fluorescein concentration, (A). Agarose gel analysis of PCR-amplified Golden Gate Assembly products from combinatorial synthesis in a microfluidic mixer, (B). The left four lanes after a 1 kb ladder represent assembly product amplified with the correct primer pair; the final four lanes are misprimed PCRs (control).
  • FIG. 5 illustrates a schematic of a multi-inlet microfluidic mixer device (100), depicted in full view (A) and in an expanded view of one section of the device (B). Depicted are components of the device including an immiscible fluid (e.g., oil) inlet (101); collection chamber (102); a T-junction (103) leading to droplet maker or waste reservoir; a resistor (104); an enzyme and/or wash buffer inlet (105); an inlet (106) (e.g., a nucleic acid (e.g., DNA) inlet); and pincer valve (107).
  • an immiscible fluid e.g., oil
  • collection chamber 102
  • T-junction (103) leading to droplet maker or waste reservoir
  • a resistor 104
  • an enzyme and/or wash buffer inlet 105
  • an inlet (106) e.g., a nucleic acid (e.g., DNA) inlet
  • pincer valve 107
  • An exemplary workflow in which the device may find use is as follows: 1) open valves for desired input, e.g., nucleic acid (e.g., DNA fragments), open droplet maker valve and emulsify with enzymes; 2) close inlets (e.g., nucleic acid (e.g., DNA) inlets), switch to waste outlet, flush main channel, open valves for new combination; and 3) repeat until library is complete, collect emulsified library and incubate.
  • desired input e.g., nucleic acid (e.g., DNA fragments)
  • close inlets e.g., nucleic acid (e.g., DNA) inlets
  • Figure 6 illustrates a schematic of a droplet sorter and merger device
  • gas injection point 201
  • spacer immiscible fluid e.g., oil
  • moat e.g., moat
  • spacer immiscible fluid e.g., oil
  • Figure 7 illustrates a schematic of one section of the droplet sorter and merger device (200) in an expanded view. Depicted are components of the device including merger electrode (206), sorter electrode (208), bubble injector (207), drop re-injection point (209) and collection and waste channels (210).
  • An exemplary workflow in which the device may find use is as follows: 1) re-inject fiuorescently encoded droplets containing input, e.g., nucleic acid (e.g., DNA) fragments, and space with immiscible fiuid (e.g., oil); 2) select desired input, e.g., nucleic acid (e.g., DNA), droplets in sorter and deflect into outer channel; 3) group desired input, e.g., nucleic acid (e.g., DNA), combinations by injecting air bubbles (group size, e.g. 5-10 drops, flanked by air droplet to prevent merger with next group); and 4) merge groups of drops to achieve assembly, e.g., nucleic acid (e.g., DNA) assembly, collect emulsified library and incubate.
  • input e.g., nucleic acid (e.g., DNA) fragments, and space with immiscible fiuid (e.g.,
  • Figure 8 illustrates a schematic of a droplet library merger device
  • Figure 9 illustrates a schematic of one section of the droplet library merger device (300) in an expanded view. Depicted are components of the device including collection chamber (302), merger electrode (303), enzyme (e.g., GGA enzyme) inlet (304) and immiscible fluid (e.g., oil) inlets (305).
  • An exemplary workflow in which the device may find use is as follows: 1) inject emulsified gene circuit libraries of type A and B (GGA specific overhangs to allow only 1 : 1 combination); 2) group picoliter drops 1 : 1 with a large enzyme drop and merge (electrode ON); and 3) collect merged combinatorial library and incubate.
  • FIG. 10 illustrates a schematic of a multi-inlet micro fluidic combinatorial droplet generator device (400). Depicted are components of the device including miscible fluid (e.g., aqueous fluid) injectors with flowrate control (401), co- flow droplet generator (402), collection chamber for droplets or downstream device (403) and immiscible fluid (e.g., oil) inlet (404).
  • miscible fluid e.g., aqueous fluid
  • injectors with flowrate control 401
  • co- flow droplet generator 402
  • collection chamber for droplets or downstream device 403
  • immiscible fluid e.g., oil
  • the device may be used to create droplets with variable contents or can be coupled with other system such as a merger, sorter, and/or picoinjector.
  • a plurality of such combinatorial droplet generator devices may be used in a microfluidic device to generate synthetic molecule, e.g., nucleic acid (e.g., DNA), libraries as well as a plurality of inlets.
  • An exemplary workflow in which the device may find use is as follows: 1) apply pressure waveforms of continuous nature (e.g., sine/triangular/ saw-tooth, etc.) independently and simultaneously to the inlets of the device thereby controlling the flow-rates at every inlet with the specific waveform.
  • the combinations of different waveforms varying in frequency, amplitude and baseline cover the concentration space of various components needed for making a synthetic molecule, e.g., nucleic acid (e.g., DNA) library.
  • Non- continuous nature pressure waveform can be applied to the inlets of the device; 2) encoding the droplets with different fluorescent dyes to trace the amount of every reagent present in the droplet; and 3) generate droplets at high frequency using, e.g., a pressure/flow-rate based/ pneumatic/piezo based technique at the focusing geometry while scanning the concentration space.
  • the device may be utilized before a droplet-merger geometry to create further variability within the droplets.
  • Figure 11 illustrates schematics of two multi-inlet micro fluidic combinatorial droplet generator devices (A and B) (400) which include additional resistors (405) as compared to the device of Figure 10.
  • Figure 12 illustrates three aspects of a methodology for scanning concentration space to create a droplet library: microfluidic device, pressure actuated flow-rate control for miscible fluid, e.g., aqueous fluid, inlets, and bar-coding of droplets using dyes and readout by laser based detection (dropometer).
  • microfluidic device pressure actuated flow-rate control for miscible fluid, e.g., aqueous fluid, inlets
  • Figure 13 illustrates continuous functions (e.g., sine functions) with a period of 10, 100 and 1000 seconds for flow-rates controlled using a pressure source.
  • the figure on right shows the fraction of every component in a droplet.
  • the lower figure shows the sinusoidal nature of the concentration scan.
  • Figure 14 illustrates the 3D nature of parameter scanning for the multiplication factor of 10 and 4.
  • the denser nature of the 10X scan (A) is clearly evident in comparison to the 4X scan (B).
  • Figure 15 shows a comparison of a 3D scan for a sine (A) and a triangular function (B).
  • Figure 16 shows controlling of pressure/flow rates using sinusoidal or triangular functions or using a discontinuous step function.
  • a and B show ability of the pressure controller to independently vary frequency, amplitude and baseline of the pressure pulses simultaneously:
  • (C) shows the ability of pressure controller to control flow rates in discontinuous fashion.
  • the pressure pumps and three flow sensors were connected with the microf uidic device. The flow rates were stringently controlled for three input channels (B) in the increments of 0.25 ⁇ 1/ ⁇ .
  • the plotted graph shows measured values by three flow sensors (Fl, F2, F3) against time.
  • Figure 17 illustrates schematics of 3 versions of a microfluidic encapsulator and shows a picture of a polydimethylsiloxane (PDMS) device (A).
  • PDMS polydimethylsiloxane
  • Figure 18 illustrates the results of experiments with (i) dyes, (ii) model reagents, and (iii) (transcription-translation (TX-TL)) components using the encapsulator.
  • D Injection of fluorescein and Dylight 405 with period of 4 seconds.
  • Figure 19 illustrates the injection of IR dye, distilled water, polyethylene glycol (PEG) and bovine serum albumin (BSA) from four inlets of a device (A), stable laminar flows in the device (B), and control of the flow rates using the pressure controller (C).
  • PEG polyethylene glycol
  • BSA bovine serum albumin
  • Figure 20 illustrates injection of four different solutions into a device
  • A energy buffer, E. coli extract, DNA and diluting solution
  • B stability of four laminar streams at flow focusing junction
  • C flow rates of extract and buffer that were kept constant
  • flow rates of DNA and diluting solution that were varied in a oppositely phase sinusoidal fashion
  • D variability of deGFP expression within droplets observed at 20X and 2X
  • Figure 21 illustrates a schematic of components of a biological circuit of for maximizing expression of a protein of interest.
  • a primer refers to one or more primers, i.e., a single primer and multiple primers.
  • claims can be drafted to exclude any element, e.g., any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
  • each step may be performed after a predetermined amount of time has elapsed between steps, as desired.
  • the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more and including 5 hours or more.
  • he time between performing each step may be from 1 second to 10 seconds, from 10 seconds to 30 seconds, from 30 seconds to 60 seconds, from 60 seconds to 5 minutes, from 5 minutes to 10 minutes, from 10 minutes to 30 minutes, from 30 minutes to 60 minutes, or from 60 minutes to 5 hours or more.
  • each subsequent step is performed immediately after completion of the previous step.
  • a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.
  • mixture refers to a combination of elements, e.g., capture agents or analytes, that are interspersed and not in any particular order.
  • a mixture is homogeneous and not spatially separable into its different constituents.
  • examples of mixtures of elements include a number of different elements that are dissolved in the same aqueous solution, or a number of different elements attached to a solid support at random or in no particular order in which the different elements are not specially distinct. In other words, a mixture is not addressable.
  • an array of capture agents as is commonly known in the art and described below, is not a mixture of capture agents because the species of capture agents are spatially distinct and the array is addressable.
  • Isolated or “purified” general refers to isolation of a substance
  • a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample.
  • a "channel,” as used herein, means a feature on or in an article
  • the channel can have any cross-sectional shape (circular, oval, triangular, irregular, square or rectangular, or the like) and can be covered or uncovered. In embodiments where it is completely covered, at least one portion of the channel can have a cross-section that is completely enclosed, or the entire channel may be completely enclosed along its entire length with the exception of its inlet(s) and outlet(s).
  • a channel may also have an aspect ratio (length to average cross sectional dimension) of at least 2: 1, more typically at least 3: 1, 5: 1 , or 10: 1 or more.
  • An open channel generally will include characteristics that facilitate control over fluid transport, e.g., structural characteristics (an elongated indentation) and/or physical or chemical characteristics (hydrophobicity vs. hydrophilicity) or other characteristics that can exert a force (e.g., a containing force) on a fluid.
  • the fluid within the channel may partially or completely fill the channel.
  • the fluid may be held within the channel, for example, using surface tension (e.g., a concave or convex meniscus).
  • the channel may be of any size, for example, having a largest dimension perpendicular to fluid flow of less than about 5 or 2 millimeters, or less than about 1 millimeter, or less than about 500 microns, less than about 200 microns, less than about 100 microns, or less than about 50 or 25 microns.
  • the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. Larger channels, tubes, etc. can be used in a microfluidic device for a variety of purposes, e.g., to store fluids in bulk and to deliver fluids to components of the disclosed device.
  • the following examples are intended to illustrate certain embodiments of the present invention, but are not to be construed as limiting and do not exemplify the full scope of the invention.
  • Microfluidic refers to a device, apparatus or system including at least one fluid channel having a cross-sectional dimension of less than 1 mm, and a ratio of length to largest cross-sectional dimension of at least 3: 1.
  • a "microfluidic channel,” as used herein, is a channel meeting these criteria. In some cases, all of the channels of a microfluidic system are microfluidic channels.
  • the "cross-sectional dimension” (e.g., a diameter) or "cross-sectional area" of the channel is measured perpendicular to the direction of fluid flow.
  • Many fluid channels in systems described herein have maximum cross-sectional dimensions less than 2 mm, and in some cases, less than 1 mm, e.g., from 2 mm to 1 mm or from 1 mm to 0.5 mm.
  • all fluid channels containing embodiments described herein are microfluidic or have a largest cross sectional dimension of no more than 2 mm or 1 mm e.g., from 2 mm to 1 mm or from 1 mm to 0.5 mm.
  • the maximum cross-sectional dimension of the channel(s) containing embodiments described herein are less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns.
  • the maximum cross-sectional dimension of the channel(s) containing embodiments described herein are from 500 microns to 200 microns, from 200 microns to 100 microns, from 100 microns to 50 microns, or from 50 microns to 25 microns.
  • the dimensions of the channel may be chosen such that fluid is able to freely flow through the article or substrate.
  • the dimensions of the channel may also be chosen, for example, to allow a certain volumetric or linear flow rate of fluid in the channel.
  • the number of channels and the shape of the channels can be varied by any method known to those of ordinary skill in the art. In some cases, more than one channel or capillary may be used.
  • modulate encompasses a flow stream which is either modified by the action of one or more valves (e.g., where one or more valves have been actuated to reduce the cross-sectional area of a flow channel, e.g., an inlet channel, of the flow stream) or else not modified by the action of one or more valves (e.g., where one or more valves capable of being actuated to reduce the cross-sectional area of a flow channel of the flow stream have not been so actuated).
  • microfluidic devices of interest for practicing the subject methods are described first in greater detail. Next, methods for modulating droplet composition using a microfluidic device are reviewed. Methods of combining two or more fluids for droplet formation using a microfluidic device are also described.
  • a multi-inlet microfluidic device including one or more integrated valves, e.g., membrane valves, e.g., one or more single-layer membrane valves (SLMV) and/or one or more multilayer membrane valves (MLMV).
  • integrated membrane valves of interest include those which are capable of modulating a microfluidic channel to substantially reduce a cross-sectional area of the channel thereby reducing its flow stream.
  • SLMVs of interest include pincer valves.
  • a pincer valve of the disclosure is a SLMV that is capable of modulating a microfluidic channel to substantially reduce a cross-sectional area of the channel thereby reducing its flow stream.
  • substantially reduced is meant that the cross-sectional area of the channel is reduced by 90% or more, such as by 92% or more, by 93% or more, 94%> or more, 95% or more, 96%> or more, 97% or more, 98% or more, 99% or more, or 100%.
  • the pincer valves may operate to modulate a flow stream in microfluidic channels that have rectangular cross-sections.
  • the pincer valves may modulate a microfluidic channel by applying sufficient pressure to substantially reduce a cross-sectional area of the channel, thereby modulating the flow stream.
  • the device may be composed of materials that provide for modulation of the microf uidic channel to achieve a desired reduction in cross-sectional area of the channel upon action of the pincer valve.
  • Figure 2 illustrates the operation of a pincer-valve modulated flow stream of a microfluidic device in the open (B) and closed (C) states.
  • any convenient methods may be adapted to make the subject integrated membrane valves, e.g., pincer valves, such as those methods described by Abate et al., "Valves And Other Flow Control In Fluidic Systems Including Microfluidic Systems” WO2009139898, the disclosure of which is herein incorporated by reference in its entirety.
  • An integrated membrane valve may include one or more control channels.
  • a control channel may have a length of greater than or equal to about 10 microns, greater than or equal to about 50 microns, greater than or equal to about 100 microns, greater than or equal to about 250 microns, greater than or equal to about 500 microns, greater than or equal to about 1 millimeter, greater than or equal to about 2 millimeters, greater than or equal to about 5 millimeters, or greater than or equal to about 1 centimeter.
  • the length of the control channel may be less than about 5 cm, for example. In some embodiments, the length of the control channel is the same as or less than the length of channel section which the control channel modulates.
  • the control channel may be substantially parallel to the channel section which it modulates, or there may be varying distances between portions of the controlled channel and portions of the channel section.
  • the width of the control channel can also vary. A greater width of the control channel may allow the control channel to have a higher pressure which can facilitate actuation in some cases.
  • the width of a control channel may be, for example, greater than or equal to about 10 microns, greater than or equal to about 25 microns, greater than or equal to about 50 microns, greater than or equal to about 100 microns, greater than or equal to about 250 microns, or greater than or equal to about 500 microns. In some instances, the width of the control channel is less than about 2 cm. The width of the control channel may be less than the length of the control channel.
  • the width of a channel section to be deformed by a valve may also influence the operation of the valve.
  • a smaller width of the channel section may be easier to deform upon actuation of a valve since there is less area to deform.
  • the length of the channel section deformed by the control channel is dependent upon the length of the control channel.
  • a control channel to cause deformation and/or deflection of a membrane, e.g., a membrane separating the control channel from a flow channel which the control channel modulates, may depend in part on the width of the membrane. Generally, a membrane having a smaller width may allow the application of less force and/or pressure to the control channel in order to deform the membrane.
  • an average width of a membrane positioned between a control channel and a channel section may be, for example, less than or equal to about 500 microns, less than or equal to about 250 microns, less than or equal to about 100 microns, less than or equal to about 75 microns, less than or equal to about 50 microns, less than or equal to about 25 microns, less than or equal to about 15 microns, or less than or equal to about 10 microns.
  • the width of the membrane may be greater than about 1 micron, for example. In some cases the membrane has an average width of about 10 microns to about 15 microns, from about 5 microns to about 25 microns, or from about 10 microns to 50 microns.
  • the elastic modulus (e.g., Young's modulus) of the membrane can also be varied in a microfiuidic system by, for example, using different materials to form the membrane and/or different amounts of crosslinker, which can change the stiffness of the material.
  • a membrane having less crosslinker may result in a relatively softer material and a higher elastic modulus. This higher elastic modulus may allow easier deformation of the membrane, and therefore less force and/or pressure can be applied to the membrane in order to cause deformation and/or deflection.
  • the Young's modulus of a membrane is from about 250 kPa to about 4,000 kPa. In certain embodiments, the Young's modulus of the membrane is from about 500 kPa to about 3,000 kPa, or from about 1,000 kPa to about 3,000 kPa.
  • the Young's modulus may be measured by, for example, applying a stress to a material and measuring the strain response, e.g., as described in more detail in X. Q. Brown, K. Ookawa, and J. Y. Wong, Biomaterials 26, 3123 (2005).
  • a channel section having a larger aspect ratio can result in more complete closure of the channel section compared to a channel section having a relatively lower aspect ratio (e.g., while applying the same amount of force and/or pressure from a valve).
  • the aspect ratio of a channel section may be, for example, at least about 1 : 1 , at least about 2: 1 , at least about 3 : 1 , at least about 5 : 1 , at least about 10: 1 , or at least about 20: 1.
  • the aspect ratio of a channel section may be less than about 50: 1 , for example.
  • the height of a channel section may be, for example, greater than or equal to about 10 microns, greater than or equal to about 25 microns, greater than or equal to about 50 microns, greater than or equal to about 100 microns, greater than or equal to about 250 microns, or greater than or equal to about 500 microns. In some instances, the height of a channel section is less than about 2 cm.
  • microchannels and/or microchambers comprising the devices described herein have a characteristic dimension (e.g. height or width or diameter) ranging from about 10 nm, or 100 nm, or 1 ⁇ up to about 500 ⁇ . In various embodiments the characteristic dimension ranges from about 1 , 5, 10, 15, 20, 25, 35, 50, or 100 ⁇ up to about 150, 200, 250, 300, or 400 ⁇ . In certain embodiments the characteristic dimension ranges from about 20, 40, or about 50 ⁇ up to about 100, 125, 150, 175 or 200 ⁇ .
  • a characteristic dimension e.g. height or width or diameter
  • the wall thickness between adjacent channels ranges from about 0.1 ⁇ to about 50 ⁇ , or about 1 ⁇ to about 50 ⁇ , more typically from about 5 ⁇ to about 40 ⁇ . In certain embodiments the wall thickness between adjacent channels ranges from about 5 ⁇ to about 10, 15, 20, or 25 ⁇ .
  • a device includes: two or more inlet channels fluidically connected to two or more fluid reservoirs; two or more valves, e.g., single layer membrane valves (SLMV) and/or multilayer membrane valves (MLMV) operably connected to, e.g., in contact with, the two or more inlet channels; and a droplet generator fluidically connected to the two or more inlet channels; wherein each SLMV is capable of reducing a cross-sectional area of the inlet channel with which it is in contact by 90% or more upon actuation thereof.
  • SLMV single layer membrane valves
  • MLMV multilayer membrane valves
  • each of the two or more SLMV and/or MLMV is capable of reducing a cross-sectional area of the inlet channel with which it is in contact by 90% or more, such as by 91 > or more, 92%> or more, 93 %> or more, 94%> or more, 95% or more, 96%> or more, 97%> or more, 98%> or more, 99%> or more, or 100%.
  • the device includes a multi-inlet microfluidic mixer that includes two or more inlet channels fluidically connected to two or more fluid reservoirs, such as 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, or even 1000 or more inlet channels fluidically connected to fluid reservoirs.
  • a multi-inlet microfluidic mixer that includes two or more inlet channels fluidically connected to two or more fluid reservoirs, such as 5 or more, 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, 100 or more, 200 or more, 500 or more, or even 1000 or more inlet channels fluidically connected to fluid reservoirs.
  • the device includes a multi-inlet microfluidic mixer that includes two or more inlet channels fluidically connected to two or more fluid reservoirs, such as from 5 to 10, from 10 to 20, from 20 to 30, from 30 to 40, from 40 to 50, from 50 to 60, from 60 to 70, from 70 to 80, from 80 to 90, from 90 to 100, from 100 to 200, 200 to 500, or 500 to 1000 or more inlet channels fluidically connected to fluid reservoirs.
  • the multi-inlet microfluidic mixer device may include any convenient components suitable for carrying out the functions of the device.
  • Figure 5 shows a multi -inlet microfluidic mixer device (100) having multiple inlet chambers (106) and channels (108) controlled by MLMVs (107) and fluidically connected to a collection chamber (102).
  • the two or more inlet channels connected to two or more fluid reservoirs can be positioned in series along a flow channel of a microfluidic device.
  • the two or more inlet channels connected to two or more fluid reservoirs can also be positioned flanking a flow channel of a microfluidic device as shown in Figure 5.
  • first and second inlet channels connected to first and second fluid reservoirs, respectively can be positioned on opposing sides of a flow channel of a microfluidic device as an inlet channel pair.
  • each MLMV 107 is configured to selectively reduce a cross-sectional area of inlet channel (108) of inlet chamber (106), e.g., by 90% or more upon actuation thereof, e.g., by 91% or more, 92%o or more, 93% or more, 94%> or more, 95% or more, 96%> or more, 97% or more, 98% or more, 99% or more, or 100%.
  • MLMV 107 is configured to selectively reduce a cross-sectional area of inlet channel (108) of inlet chamber (106), e.g., by from 90%> to 95% or from 95% to 100% upon actuation thereof.
  • Each MLMV shown in Figure 5 is operably connected to a channel with respect to which it is configured to selectively reduce the cross-sectional area thereby modulating and/or starting or stopping a flow stream.
  • the MLMVs of the embodiment depicted in Figure 5 are thus capable of providing for continuous modulation via modulated flow through one or more inlet channels and/or selective on/off switching between inlet channels to vary the component concentration of droplets generated via the device.
  • the device includes a multi-inlet microfluidic mixer (e.g., as described herein) that is configured to: 1) actuate, e.g., open, one or more valves (e.g., MLMVs or SLMV (e.g., pincer valves)) for desired inlets or reservoir chambers (e.g., an inlet for a nucleic acid (e.g., DNA) fragment of interest), actuate, e.g., open, a droplet maker valve and emulsify the flow stream with a reagent of interest (e.g., an enzyme).
  • a reagent of interest e.g., an enzyme
  • the multi-inlet microfluidic mixer is further configured to: 2) actuate, e.g., close, the inlets valves, switch to waste outlet, flush main channel, and actuate, e.g., open, valves for a new combination.
  • the multi-inlet microfluidic mixer is configured for repeated operations (e.g., as described herein) that provide a desired mixture of liquids in the collection chamber.
  • the mixture is a nucleic acid, e.g., DNA, library of interest, and the device is configured to collect, emulsify and incubate the mixture.
  • each of the two or more SLMV includes a pincer valve including first and second control channels, the first and second control channels each including a membrane facing the inlet channel with which the SLMV is in contact, which membrane is capable of being deflected into a flow path of the inlet channel with which the SLMV is in contact, upon actuation of the SLMV.
  • the membrane is capable of being deflected into a flow path of the inlet channel with which the SLMV is in contact such that an open flow rate of the inlet channel is reduced by 3000-fold or more, such as by 4000-fold or more, 5000-fold or more, 6000-fold or more, 7000-fold or more, 8000- fold or more, 9000-fold or more, 10,000 fold or more, or even more.
  • each SLMV is capable of being pressurized to 30 psi or more, such as to 40 psi or more, 50 psi or more, 60 psi or more, 65 psi or more, or even more, to deflect the membranes of the first and second control channels in the pincer valve of the SLMV into the inlet channel.
  • each SLMV is capable of being pressurized to from 30 psi to 40 psi, from 40 psi to 50 psi, from 50 psi to 60psi, or from 60 psi to 65 psi or more, to deflect the membranes of the first and second control channels in the pincer valve of the SLMV into the inlet channel.
  • each SLMV is connected to a pressure source, and one or more SLMV is pressurized to 30 psi or more, such as pressurized to 40 psi or more, 50 psi or more, 60 psi or more, 65 psi or more, or even more, thereby deflecting the membranes of the first and second control channels in the pincer valve of the SLMV into the inlet channel.
  • each SLMV is connected to a pressure source, and one or more SLMV is pressurized to from 30 psi to 40 psi, from 40 psi to 50 psi, from 50 psi to 60 psi, or from 60 psi to 65 psi or more, thereby deflecting the membranes of the first and second control channels in the pincer valve of the SLMV into the inlet channel.
  • valves e.g., solenoid valves, and/or non-valve -based implementations may be utilized to control fluid flow as appropriate.
  • a non-valve -based implementation is the use of pressurized inlets to modulate fluid flow. By modulating the pressure applied to one or more fluid inlets, the fluid flow from the one or more inlets may be modulated.
  • Pressure as applied to one or more membrane valves described herein, may be either positive or negative as appropriate under the circumstances.
  • negative pressure may be applied under suitable circumstances to a membrane valve to open the valve, while positive pressure may be applied under suitable circumstances to close the valve.
  • the device may be fabricated from any convenient materials.
  • the device is fabricated from an elastic polymer.
  • the device may include a membrane composed of a sufficiently flexible or elastic polymer such that the channels in the membrane may be substantially closed by the action of an actuator.
  • Polymers of interest include, but are not limited to, elastomers such as organosilicone polymers, e.g., polydimethylsiloxane. Any convenient polydimethylsiloxanes may find use in fabricating the device.
  • Exemplary polydimethylsiloxane polymers include those sold under the trademark Sylgard® by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186.
  • the device is fabricated from polydimethylsiloxane (PDMS) (6% cross linker) polymer.
  • the device may include multiple inlet channels for introducing multiple fluids into the device, where the flow of the fluids through each of the inlet channels may be controlled by an integrated membrane valve, e.g., a MLMV or a SLMV (e.g., a pincer valve).
  • the device may include multiple inlets channels, fluid reservoirs and integrated membrane valves, such as 2 or more, 5 or more, 10 or more, 20 or more, 40 or more, 100 or more, 200 or more, 500 or more inlets channels, fluid reservoirs and integrated membrane valves, e.g., MLMVs and/or SLMVs (e.g., pincer valves).
  • the device includes five or more inlet channels fluidically connected to five or more fluid reservoirs; and five or more integrated membrane valves, e.g., MLMVs and/or SLMVs (e.g., pincer valves), in contact with the five or more inlet channels; wherein each integrated membrane valve is capable of reducing a cross-sectional area of the inlet channel with which it is in contact by 90% or more upon actuation thereof.
  • integrated membrane valves e.g., MLMVs and/or SLMVs (e.g., pincer valves
  • the subject device includes a droplet sorter and/or droplet merger.
  • a droplet sorter and or merger may be configured to provide for the sorting and movement of droplets through desired microfluidic channels of the device.
  • a droplet sorter/merger may be configured to provide for the merging of droplets of interest, e.g., in a collection chamber.
  • the droplet sorter/merger device may include any convenient components suitable for carrying out the functions of the device.
  • Figures 6 and 7 shows a droplet sorter and/or merger device (200) including components such as an gas (e.g., air) injection point (201), a spacer immiscible fluid (e.g., oil) chamber for gas (e.g., air) bubbles (202), a moat (203), spacer immiscible fluid (e.g., oil) chambers (204) and a droplet re-injection point (205), a merger electrode (206), a sorter electrode (208), bubble injector (207), drop re-injection point (209) and collection and waste channels (210).
  • an gas e.g., air
  • spacer immiscible fluid e.g., oil
  • 202 spacer immiscible fluid
  • the device includes a droplet sorter/merger
  • droplet sorter/merger is further configured to: 2) select desired droplets (e.g., DNA- containing droplets) in the sorter and deflect them into an outer channel, e.g., via operation of an electrode.
  • desired droplets e.g., DNA- containing droplets
  • the droplet sorter/merger is further configured to: 3) group desired droplet combinations (e.g., desired DNA combinations) by injecting gas, e.g., air, bubbles (group size, e.g. 5-10 drops, flanked by gas, e.g., air, droplet to prevent merger with next group).
  • group desired droplet combinations e.g., desired DNA combinations
  • group size e.g. 5-10 drops
  • flanked by gas e.g., air
  • droplet sorter/merger is further configured to: 4) merge groups of droplets to achieve assembly of a mixture of interest (e.g., DNA assembly), to collect an emulsified mixture (e.g., DNA library) and incubate to any convenient time.
  • the subject device may include an electrode.
  • the device includes one or more electrodes for detecting cells or droplets and/or for controlling droplet combination and/or for controlling fluid flow. Any convenient electrodes and methods may be adapted for use in the subject devices.
  • the device is configured to provide for selection of a liquid flow path among two or more alternative flow paths via the use of electrophoretic forces and the selective use of electrodes.
  • the subject devices may include a resistor, capacitor and/or a diode.
  • the resistor may be utilized as a heat source.
  • the device may include a microfluidic resistor that is tunable.
  • the device includes a resistor that is configured to control the flow, e.g., the movement of bubbles or droplets through a microfluidic circuit of the subject device, e.g., by means of thermocapillary effects.
  • the device may include one or more droplet generators fluidically connected to the inlet channels of the device.
  • Suitable droplet generators which may be utilized in accordance with the present disclosure to form droplets from fluid streams modulated using the integrated membrane valves of the present disclosure may include any suitable droplet generators know in the art, e.g., the droplet generators described in PCT Publication No. WO 2014/028378, the disclosure of which is incorporated by reference herein in its entirety and for all purposes.
  • the device may include one or more components, such as any convenient components that find use in microfluidic devices, e.g., pumps, switches, solenoids, resistors, injectors, valves, reservoirs, where the components may be on- chip or off-chip.
  • Components of microfluidic devices of interest include those described by Abate et al, "Valves And Other Flow Control In Fluidic Systems Including Microfluidic Systems" WO 2009/139898; and those described by Chang et al. US20120258487 the disclosures of which are herein incorporated by reference in their entirety and for all purposes.
  • the components of the device described herein may have various configurations which may be chosen depending on the desired performance characteristics.
  • Figure 2 illustrates a schematic of an exemplary microfluidic device.
  • the device includes a resistor positioned upstream of the droplet generator.
  • the subject device includes a droplet library merger device.
  • the droplet library merger device may include any convenient components suitable for carrying out the functions of the device.
  • Figures 8 and 9 shows a droplet library merger device (300), including droplet library inlets with filters (301), a collection chamber (302), a merger electrode (303), an reagent inlet (304) (e.g., an enzyme inlet) and immiscible fluid, e.g., oil, inlets (305).
  • the droplet library merger device is configured to: 1) inject into the device libraries of multiple types (e.g., emulsified gene circuit libraries of type A and B, such as libraries where GGA specific overhangs allow only a 1 : 1 combination).
  • the droplet library merger device is further configured to: 2) group picoliter droplets of interest (e.g., in a ratio of 1 : 1, as described above) with a reagent drop (e.g., a large enzyme drop) and merge (e.g., by action of electrode ON).
  • the droplet library merger device is further configured to: 3) collect a merged combinatorial library and incubate for any convenient time.
  • the subject device includes a multi-inlet microfluidic combinatorial droplet generator.
  • the multi-inlet microfluidic combinatorial droplet generator may include any convenient components suitable for carrying out the functions of the device.
  • Figures 10 and 11 show multi- inlet microfluidic combinatorial droplet generators (400), including components such as miscible fluid, e.g., aqueous fluid, injectors with flowrate control (401), co-flow droplet generator (402), collection chamber for droplets or downstream device (403), immiscible fluid, e.g., oil, inlet (404), and resistors (405).
  • the multi-inlet microfluidic combinatorial droplet generator device is configured to: apply pressure waveforms of continuous nature (e.g., sine, triangular, or saw-tooth waveforms, e.g., as described herein) independently and simultaneously to the inlets of the device thereby controlling the flow-rates at every inlet with the specific waveform.
  • pressure waveforms of continuous nature e.g., sine, triangular, or saw-tooth waveforms, e.g., as described herein
  • a variety of combinations of different waveforms may be utilized and may vary in frequency, amplitude and/or baseline to cover the desired concentration space for a variety of components of interest, e.g., components for making a DNA library.
  • non-continuous nature pressure waveform can be applied to the inlets of the device.
  • the multi-inlet microfluidic combinatorial droplet generator device is configured to: encode the droplets with different markers (e.g., fluorescent dyes) to trace the amount of every reagent present in a droplet.
  • the multi-inlet microfluidic combinatorial droplet generator device is configured to: generate droplets at high frequency using, e.g., a pressure, flow-rate based, pneumatic, and/or piezo-based technique at the focusing geometry, while scanning the concentration space. The device may be utilized before a droplet-merger geometry to create further variability within the droplets.
  • the subject devices may be configured to prepare a library of droplets that scans the concentration space with respect to one or more library components of interest.
  • the library may include multiple droplets each containing a different concentration of a component of interest.
  • the device is configured to scan concentration space utilizing pressure-based components and methods.
  • the device is configured to scan concentration space utilizing flow-rate based components and methods.
  • the device is configured to scan concentration space utilizing pneumatic-based components and methods.
  • the device is configured to scan concentration space utilizing piezo-based components and methods.
  • Figures 13-16 show methods of controlling of pressure and flowrates in the subject devices using sinusoidal or triangular functions or using a discontinuous step functions.
  • the methods and devices described herein use an oil/water system or an air/aqueous fluid system for droplet generation.
  • the systems need not be so limited.
  • any of a number of immiscible fluids including, but not limited to, carbon tetrachloride, chloroform, cyclohexane, 1 ,2-dichloroethane, dichloromethane, diethyl ether, dimethyl formamide, ethyl acetate, heptane, hexane, methyl-tert-butyl ether, pentane, toluene, 2,2,4-trimethylpentan, and the like may be utilized.
  • the first fluid and second fluid need not be immiscible in each other.
  • injected droplets can be kept separate from each other simply by adjusting flow rates in the microchannels and rate of droplet formation to form separated droplets.
  • sample solutions e.g., buffer solutions, cell culture solutions, etc. and carrier fluid (e.g., mineral oil with surfactant) are injected into the channels via inlet ports and/or tubings or channels and are driven by compressed air.
  • carrier fluid e.g., mineral oil with surfactant
  • the flow rates of solutions and oils can be controlled by pressure regulators.
  • the droplets are formed by shearing force at the T-junction of a microchannel where oils and solutions meet. It is understood that variables such as, droplet size, spacing, and transportation speed can be adjusted by fine tuning a variety of parameters/configurations of the device, such as the ratio between solution and oil flow rate. By opening different solution valves at different times, various droplet sequences can be generated and queue in the flow channel. Before two trains of droplets are mixed, a synchronization structure is utilized to ensure droplets arrive at mixing spot at the same time.
  • This approach is intended to be illustrative and not limiting. Any of a number of approaches can be used to convey the fluids, or mixtures of droplets, particles, cells, etc. along the channels of the subject devices described herein. Such approaches include, but are not limited to syringe pumps, peristaltic pumps, electrokinetic pumps, bubble pumps, air pressure driven pumps, and gravity-driven pumps.
  • FIG. 12 illustrates a schematic of a system configured for barcoded droplets.
  • a droplet may be barcoded using combinations of dyes in any convenient ratios.
  • the subject devices may be configured to encode droplets of interest by the addition of dyes to the droplets.
  • the presence, size and/or speed of droplets in microfluidic channels of the subject device can be detected by using a photomultiplier (PMT) sensor.
  • the presence, size and speed of droplets in microfluidic channels of the subject device can be detected by using a capacitive sensor.
  • PMT photomultiplier
  • Cross-contamination between the droplets can be eliminated by introducing a passivation layer between the sensing electrodes and droplets.
  • Coplanar electrodes can be used to form a capacitance through the microfluidic channel. The change in capacitance due to the presence of a droplet in the sensing area can be detected and used to determine the size and speed of the droplet.
  • a single pair of electrodes can be used to detect the presence of a droplet and the interdigital finger design can be used to detect the size and speed of the droplet.
  • the measured droplet information can be displayed through an interface in real-time.
  • chambers in the microfluidic device have reagents and reactants flowing through for an incubation period, during which a reaction of interest can be monitored (e.g., using fluorescence).
  • An automated stage mounted to a microscope with a CCD camera can be used to rapidly collect data and analyze each chamber or channel. Stage operation can be operated using any convenient devices with image data analysis.
  • the devices described herein can be configured to carry out standardized restriction enzyme assembly protocols. Such protocols include, but are not limited to BioBricksTM, BglBricks, and Golden Gate methods. In certain embodiments, sequence-independent overlap techniques, such as In-FusionTM, SLIC and Gibson isothermal assembly can be used for larger assemblies.
  • the device(s) described herein are configured to receive instructions or to receive software implementing instructions and/or to perform operations according to one or more assembly systems (e.g., gene assembly systems) described herein. Any convenient assembly methods can be readily adapted for use in the subject devices and methods described herein.
  • BioBrick approach standardizes the DNA assembly process, and facilitates automation and part re -use.
  • BioBrick assembly standards such as that originally developed at MIT (see, e.g., Shetty et al. (2008) J. Biol. Eng., 2: 5), as well as the UC Berkeley BglBrick standard (see, e.g., Anderson et al. (2010) J. Biol. Eng., 4: 1).
  • SLIC, or sequence and ligase independent cloning see, e.g., Li et al. (2007) Nature Meth., 4: 251-256, as its name implies, does not utilize restriction enzymes or ligase.
  • a DNA sequence fragment to be cloned into a destination vector is PCR amplified with oligos (oligonucleotides) whose 5' termini contain about 25 bp of sequence homology to the ends of the destination vector, linearized either by restriction digest or PCR amplification.
  • Gibson DNA assembly so named after the developer of the method (see, e.g., Gibson et al. (2009) Nature Meth., 6: 343-345), is analogous to SLIC, except that it uses a dedicated exonuclease (no dNTP addition step), and uses a ligase to seal the single stranded nicks.
  • the Golden-gate method see, e.g., Engler et al.
  • the Golden-gate method relies upon the use of type lis endonucleases, whose recognition sites are distal from their cut sites. Although there are several different type lis endonucleases to choose from, one example uses Bsal (equivalent to Eco31I) (the Golden-gate method only uses a single type lis endonuclease at time).
  • the subject device may be fabricated using any convenient methods and materials. There are a variety of formats, materials, and size scales for constructing the subject devices and various integrated fluidic systems.
  • the devices described herein are made of PDMS (or other polymers), fabricated using a technique called "soft lithography".
  • PDMS is a material of interest for a variety of reasons including, but not limited to: (i) low cost; (ii) optical transparency; (iii) ease of molding; (iv) elastomeric character; (v) surface chemistry of oxidized PDMS can be controlled using conventional siloxane chemistry; (vi) compatible with cell culture (non-toxic, gas permeable).
  • Soft lithographic rapid prototyping may be employed to fabricate the desired microfluidic channel systems.
  • Substrate materials of interest for use in the subject devices include, but are not limited to, transparent substrate such as polymers, plastics, glass, quartz, or other dielectric materials, nontransparent substrates including translucent or opaque plastics, silicon, metal, ceramic, and the like.
  • channel materials for use in the subject devices include, but are not limited to, flexible polymers such as PDMS, plastics, and the like, and nonflexible materials such as stiff plastics, glass, silicon, quartz, metals, and the like.
  • any convenient components of the device may be automated.
  • control of the integrated membrane valves of the device is automated.
  • the subject devices and systems may include a controller which synchronizes the operations of the components of the subject device, e.g., valves, inlets, resistors, etc.
  • a computer program product is described including a computer usable medium having control logic stored therein.
  • the control logic when executed by the processor of the computer, causes the processor to perform functions described herein.
  • Any convenient controller devices may be utilized in the subject systems and devices.
  • the controller may be operably coupled to any convenient components of the subject devices, e.g., valves, inlets, resistors, etc and provide instructions for carrying out the functions of the system, such as the controlled movement of fluids through the microfluidic circuits of the subject devices (e.g., as described herein).
  • the system includes a processor operably coupled to a memory that includes instructions stored thereon for performing functions described herein.
  • Figure 12 shows a schematic of a flow-rate controlled device.
  • Memory may be any suitable device in which the processor can store and retrieve data, such as magnetic, optical, or solid state storage devices (including magnetic or optical disks or tape or RAM, or any other suitable device, either fixed or portable).
  • the processor may include a general purpose digital microprocessor suitably programmed from a computer readable medium carrying necessary program code. Programming can be provided remotely to processor through a communication channel, or previously saved in a computer program product such as memory or some other portable or fixed computer readable storage medium using any of those devices in connection with memory.
  • a magnetic or optical disk may carry the programming, and can be read by a disk writer/reader.
  • the subject systems also include programming, e.g., in the form of computer program products, computer- readable medium, algorithms, etc. for use in controlling the subject systems and practicing the methods as described herein.
  • aspects of the present disclosure include methods for modulating droplet composition using a microfluidic device (e.g., as described herein).
  • the method includes: (a) actuating a first valve, e.g., a membrane valve, in contact with a first inlet channel of the microfluidic device to provide a first modulated flow stream of a first liquid in a first inlet channel of the microfluidic device; (b) actuating a second valve, e.g., a membrane valve, in contact with a second inlet channel of the microfluidic device to provide a second modulated flow stream of a second liquid in a second inlet channel of the microfluidic device; (c) receiving at a droplet generator of the microfluidic device the first and second modulated flow streams; and (d) forming via the droplet generator of the microfluidic device a first droplet including a volume of the first liquid and a volume of the second liquid, which volumes are determined by steps (a) and (b).
  • a first valve e.g., a membrane valve
  • the method also includes: (e) actuating the first valve, e.g., membrane valve, to provide a third modulated flow stream of the first liquid in the first inlet channel of the microfluidic device; (f) actuating the second valve, e.g., membrane valve, to provide a fourth modulated flow stream of the second liquid in the second inlet channel of the microfluidic device; (g) receiving at the droplet generator of the microfluidic device the third and fourth modulated flow streams; and (h) forming via the droplet generator of the microfluidic device a second droplet including a volume of the first liquid and a volume of the second liquid, which volumes are determined by steps (e) and (f), wherein the volume of the first liquid and the volume of the second liquid in the second droplet are different than the volume of the first liquid and the volume of the second liquid in the first droplet.
  • first valve e.g., membrane valve
  • the first membrane valve and the second membrane valve are independently selected from a multilayer membrane valve (MLMV) and a single-layer membrane valve (SLMV).
  • MLMV multilayer membrane valve
  • SLMV single-layer membrane valve
  • the first membrane valve is capable of reducing a cross-sectional area of the first inlet channel by 90% or more (such as by 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96%) or more, 97% or more, 98% or more, 99% or more, or 100%) upon actuation thereof
  • the second membrane valve is capable of reducing a cross-sectional area of the second inlet channel by 90% or more (such as by 91% or more, 92% or more, 93%) or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) upon actuation thereof.
  • the first and second liquids include a biological construct.
  • the biological construct is a gene or a fragment thereof.
  • the first and second liquids each include a nucleic acid.
  • the method further includes introducing into the first or second droplet one or more reagents sufficient to enable one or more procedures selected from the group consisting of nucleic acid assembly, ligation, amplification (e.g., PCR), cloning, expression and cell transformation.
  • the one or more reagents includes a type II endonuclease (e.g., Golden Gate assembly method).
  • a type II endonuclease e.g., Golden Gate assembly method. Any convenient methods and reagents may be utilized in connection with reactions in the droplets, such as those described by Engler et al, "Generation of families of construct variants using golden gate shuffling", Methods Mol Biol. 2011;729: 167-81; and "A One Pot, One Step, Precision Cloning Method with High Throughput Capability", PLoS ONE 3(11): e3647, 2008.
  • the first liquid and the second liquid include a first and second detectable agent respectively. Any convenient detectable agents may be utilized in the subject methods.
  • the first and/or second detectable agent includes a dye (e.g., a fluorescent dye, e.g., a lanthanide dye).
  • a dye e.g., a fluorescent dye, e.g., a lanthanide dye.
  • a dye may be a colorimetric dye, a fluorescent dye (i.e., a fluorophore) or a luminescent dye.
  • Fluorophores of interest include but are not limited to, fluorescein, 6-FAM, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamme, carboxyrhodamme 6G, carboxyrhodol, carboxyrhodamme 110, Cascade Blue, Cascade Yellow, coumarin, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy-Chrome, phycoerythrin, PerCP (peridinin chlorophyll-a Protein), PerCP-Cy5.5, JOE (6-carboxy-4',5'-dichloro-2',7'- dimethoxyfluorescein), NED, ROX (5-(and-6)-carboxy-X-rhodamine), HEX, Lucifer Yellow
  • Exemplary lanthanide dyes include europium chelates, terbium chelates and samarium chelates.
  • Aspects of the invention include methods of combining two or more fluids for droplet formation using a micro fluidic device (e.g., as described herein).
  • the method includes: (a) modulating a flow stream in a first inlet channel of the microfluidic device using a first single layer membrane valve (SLMV) in contact with the first inlet channel to transfer a first volume of a first liquid to a droplet generator of the microfluidic device, wherein the modulating includes actuating the first SLMV, and wherein the first SLMV is capable of reducing a cross-sectional area of the first inlet channel by 90% or more upon actuation thereof; and (b) modulating a flow stream in a second inlet channel of the microfluidic device using a second SLMV in contact with the second inlet channel to transfer a second volume of a second liquid to the droplet generator of the microfluidic device, wherein the modulating includes actuating the second SLMV, and wherein the second SLMV is capable of reducing a cross-sectional area of the second inlet channel by 90% or more upon actuation thereof.
  • SLMV single layer membrane valve
  • the method includes actuating the first or second SLMV to reduce the cross-sectional area of the first or second inlet channel by 90%) or more (such as by 91%> or more, 92% or more, 93% or more, 94%> or more, 95%) or more, 96%> or more, 97% or more, 98%> or more, 99% or more, or 100%) respectively.
  • the first and second SLMV are capable of reducing a cross-sectional area of the first and second inlet channels respectively by 92% or more (e.g., 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%).
  • the method includes actuating the first or second SLMV to reduce the cross-sectional area of the first or second inlet channel by 92% or more respectively.
  • the first SLMV includes a first pincer valve including first and second control channels, the first and second control channels each including a membrane facing the first inlet channel, which membrane is capable of being deflected into a flow path of the first inlet channel upon actuation of the first SLMV; and wherein the second SLMV includes a second pincer valve including first and second control channels, the first and second control channels each including a membrane facing the second inlet channel, which membrane is capable of being deflected into a flow path of the second inlet channel upon actuation of the second SLMV.
  • modulating a flow stream in the first or second inlet channels includes applying pressure to the first or second SLMV respectively, which pressure is sufficient to deflect the membranes of the first and second control channels, in the first or second pincer valves, into the first or second inlet channels and reduce the open flow rate of the first or second inlet channels by 3000-fold or more, such as by 4000-fold or more, 5000-fold or more, 6000-fold or more, 7000-fold or more, 8000-fold or more, 9000-fold or more or even by 10,000 fold or more.
  • modulating a flow stream in the first or second inlet channels includes applying a pressure of 30 psi or more, such as 40 psi or more, 50 psi or more, 60psi or more, 65 psi or more, 70 psi or more, 75 psi or more, 70 psi or more, 85 psi or more, 90 psi or more, 95 psi or more, 100 psi or more, or even more, to the first or second SLMV respectively, which pressure is sufficient to deflect the membranes of the first and second control channels, in the first or second pincer valves, into the first or second inlet channels.
  • a pressure of 30 psi or more such as 40 psi or more, 50 psi or more, 60psi or more, 65 psi or more, 70 psi or more, 75 psi or more, 70 psi or more, 85 psi or more, 90 psi or more,
  • modulating a flow stream in the first or second inlet channels includes applying a pressure of from 30 psi to 40 psi, from 40 psi to 50 psi, from 50 psi to 60 psi, from 60 psi to 65 psi, from 65 psi to 70 psi, from 70 psi to 75 psi, from 75 psi to 80 psi, from 80 psi to 85 psi or more, from 90 psi to 95 psi, or from 95 psi to 100 psi or more, to the first or second SLMV respectively, which pressure is sufficient to deflect the membranes of the first and second control channels, in the first or second pincer valves, into the first or second inlet channels.
  • the method is performed using a microfluidic device fabricated from PDMS (6% cross linker) polymer.
  • the first and second volumes are picoliter (pL) volumes of fluid, e.g., a volume of less than 1 ⁇ , such as 500 pL or less, 100 pL or less, 50pL or less, or lOpL or less.
  • the first and second volumes are reproducible with an accuracy of ⁇ 500pL or less, such as ⁇ 300pL or less, ⁇ 100pL or less, ⁇ 50pL or less, ⁇ 30pL or less, or ⁇ 10pL or less.
  • the term "reproducible with an accuracy of refers to the standard deviation of at least 5 measurements related to volume (e.g., at least 10 volume measurements). Any convenient methods may be used to assess reproducibility of the volumes.
  • the method includes forming a droplet using the droplet generator of the microfluidic device, wherein the droplet includes a volume of the first liquid and a volume of the second liquid, which volumes are determined by steps (a) and (b).
  • the method includes transferring volumes of 5 or more liquids to the droplet generator of the microfluidic device via 5 or more inlet channels (e.g., 10 or more, 20 or more 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, or 500 or more inlet channels).
  • 5 or more inlet channels e.g., 10 or more, 20 or more 30 or more, 40 or more, 50 or more, 100 or more, 200 or more, or 500 or more inlet channels.
  • the first and second liquids include a biological construct.
  • the biological construct may be a gene or a fragment thereof.
  • the first and second liquids each include a nucleic acid.
  • the droplet may further include one or more reagents sufficient to enable one or more procedures selected from the group consisting of nucleic acid assembly, ligation, amplification (e.g., PCR), cloning, expression and cell transformation.
  • the one or more reagents includes a type II endonuclease (e.g., Golden Gate assembly method).
  • the first liquid and the second liquid include a first and second detectable agent (e.g., as described herein) respectively.
  • the first and/or second detectable agent includes a dye (e.g., a fluorescent dye, e.g., a lanthanide dye).
  • the methods and devices of the invention find use in a variety of applications.
  • Applications of interest include, but are not limited to numerous biological applications utilizing microfluidic droplets and screening applications.
  • the subject methods and devices may find use in any convenient applications where the ability to generate droplets with defined concentrations of reagents over a large dynamic range is desirable. For example, by loading different solutions into the five inlets and controllably modulating the relative ratios of the solutions encapsulated in droplets, it is possible to scan large concentration parameter spaces quickly.
  • Applications of interest for the disclosed devices and methods include the optimization of metabolic pathways or gene circuits requiring precise combinations of inducers, cofactors, DNA constructs, and other components (see e.g., M. Quach et al., Bioinformatics, 2007, 23, 3209-3216; and Y. Dharmadi et al., Biotechnology and Bioengineering, 2006, 94, 821-829).
  • Generating combinatorial libraries of droplets encapsulating different reagents is useful for a broad array of applications, including for creating spectrally encoded beads and performing synthetic biology screens (see e.g., R. E. Gerver et al, Lab Chip, 2012, 12, 4716-4723; and Y. Dharmadi et al., Biotechnology and Bioengineering, 2006, 94, 821-829).
  • Another example is building defined DNA constructs. The ability to modulate a DNA oligos concentration by 3000X is sufficient to dictate whether or not that oligo will integrate into the construct being created by Golden Gate assembly (GGA).
  • Other applications of interest include screening applications where large combinatorial spaces of reagents are of interest.
  • Applications for which the disclosed methods and devices are well suited include those requiring rapid screening of many different reagents and many or all possible combinations thereof. For example, screening for protein crystallization conditions, screening for desirable conditions in multi-parameter biological circuits (e.g., maximizing protein expression systems), antibiotic or drug screens, and screening of cell free extracts, etc., are of interest in connection with the disclosed methods and devices.
  • the subject methods and devices provide precise control over flow rate in microfluidic inlet channels enabling precise control of downstream droplet composition.
  • the disclosed pincer valves exhibit negligible leakiness in the fully closed state, allowing modulation of flow rate and downstream reagent concentration over three orders of magnitude.
  • the simplicity of these valves, the ease with which they can be integrated into microfluidic devices, and their reliability, robustness, and performance, provide for a range of biological applications, including for generating spectrally encoded droplets and beads, performing DNA synthesis, and screening large, combinatorial chemical and biological parameter spaces.
  • microfluidic devices are fabricated using standard photolithography.
  • PDMS replicas are cast by pouring PDMS at 16: 1 ratio of basexuring agent (Sylgard 184, Dow Chemical, MI, USA) and baked at 80 °C for 1 hour.
  • Fabricated channel widths are as follows: resistor (20 um), flow channels in valve area (30 ⁇ ), regular flow channels (60 ⁇ ), droplet generator nozzle (20 x 40 ⁇ ), PDMS membranes (20 ⁇ ).
  • access holes are punched with a 0.75 mm biopsy punch and PDMS-coated glass slides (10 ⁇ ) are bonded to the bottom of the devices using oxygen plasma treatment.
  • DNA fragments from lambda phage genome are as follows: A (250mer) forward: ACG TTG GTC TCA GCT TCG TTC CGT GCT GTC C (SEQ ID NO: l); A (250mer) reverse: GAT ATC TTT AAT GTG GGG CTG GTT GCC TCC T (SEQ ID NO:2); A' (500mer) forward: GGT ATG GTC TCA GCT TCG GCC CTT GTG ACT G (SEQ ID NO:3); A' (500mer) reverse: GGA ATT GAG GCC GTC CCC GTT GAC GCA CTC C (SEQ ID NO:4); B (350mer) forward: TTA GTG GTC TCA AAG CGC AGC TTG GCC TGA A (SEQ ID NO:5); B (350mer) reverse: GTT AAT TTC CCT TGC AGC GCT GGG CTT TGT A (SEQ ID NO:6); B' (700mer) forward: GGC TCG GTC TC AAA GCT C
  • SLMVs are used to modulate the concentrations of DNA oligos in droplets by over three orders of magnitude, sufficient to reliably control the sequences of the constructs assembled in each droplet.
  • the use of these valves to generate a library of droplets labelled with controlled combinations of dyes is demonstrated, a first step in the creation of spectrally-encoded particles (see e.g., R. E. Gerver et al, Lab Chip, 2012, 12, 4716- 4723).
  • Membrane valves utilize a pressurized chamber to deflect the flexible wall of a flow channel, increasing flow channel hydrodynamic resistance and thereby enabling regulation of flow rate.
  • Three membrane valve types for lab-on-a-chip applications are shown in Figure 1.
  • SLMV is the simplest to design and to fabricate, but may exhibit gutter flow (Fig. 1A), similar to the more complex MLMV based on rectangular channel geometries (Fig. IB).
  • Fig. 1A Similar to the more complex MLMV based on rectangular channel geometries
  • leaky gutter flow results from the use of rectangular flow channels that do not completely seal in the off state.
  • a rounded geometry can be implemented that
  • MLMV with rounded channels thus afford the best performance of the three valve types (in terms of leakiness) but are also the hardest to fabricate, requiring both the specialized photoresists to generate rounded channels and precision alignment of flow and control layers.
  • single-layer membrane valves are the simplest and the most robust, because they can be fabricated in the same layer as the flow channel, obviating the need for control and flow network alignment, which is an error prone step in the fabrication process.
  • complete sealing of the flow channel is not essential; rather, a marked change in flow rate between on and off states is sufficient.
  • a device was created that allows modulation of the flow rates of five liquids injected into a droplet generator (Fig. 2, Panel A).
  • the five liquids were pressurized to the same values and injected into the inlet ports of the micro fluidic device; since the inlet channels of each liquid are identical, their flow rates into the droplet generator are equal unless the valves are actuated.
  • the valves include a symmetric pincer geometry deflecting ⁇ 20 ⁇ membranes into their respective fluidic channels using pressures ranging from 0 to 67 psi. To facilitate membrane deflection, all devices were fabricated from "soft" PDMS (6% cross linker) and sealed at the bottom with a flexible PDMS layer on glass.
  • one of the aqueous solutions was dyed with 2 mg/mL IR dye 783 (central inlet) and water was injected into the other inlets (Fig. 2, Panel B).
  • IR dye 783 central inlet
  • the flow rate of this liquid was estimated and changes with actuation of the valve on the central channel before and after full valve actuation were observed (Fig. 2, Panel B and Fig. 2, Panel C, respectively).
  • the IR dye allowed qualitatively assessment of valve performance, but to quantify leakage as a function of valve actuation, this dye was replaced with a fluorescent dye (100 ⁇ fluorescein in PBS), and buffer (PBS) was injected into the other inlets; consequently, the droplets generated contained a volume of fluorescent dye proportional to the flow rate of the central inlet.
  • a fluorescent dye 100 ⁇ fluorescein in PBS
  • PBS buffer
  • aqueous solutions were pressurized with a custom manifold (12.7 psi) and a fluorinated oil containing 2 wt% of a biocompatible surfactant (RAN Technologies) was supplied at 600 ⁇ 71 ⁇ using a syringe pump.
  • RAN Technologies a biocompatible surfactant
  • pincer valves achieved a reduction in droplet fluorescence of >3,000X, significantly outperforming the solenoid valve; this corresponds to an estimated 92% constriction in cross-sectional area of the channel with actuation.
  • Pincer valves are analogue: the degree of deflection of the membrane, and thus the change in flow rate in the channel, is proportional to the pressure applied to the valve; this allows not just on-off control but also continuous modulation of flow rate (Fig. 3, Panel C).
  • Another advantage of the pincer valves is that the volume displaced by the valve membrane under actuation is small, so that a relatively small fluid flux is required to equilibrate the pressure downstream of the valve; this allows accurate modulation of flow rate on a phase time scale, as illustrated by the reversibility of flow to actuation in points 4, 5, and 6 in Fig. 3, Panel C.
  • Example 3 Multi-inlet microfluidic combinatorial droplet generator
  • a multi-inlet microfluidic combinatorial droplet generator (e.g., as depicted in Figures 10 and 11) is used independently to create droplets with variable contents, or is coupled with other downstream microfluidic systems, such as droplet merger, a droplet sorter, a pico-injector or a double emulsion maker.
  • a plurality of such combinatorial droplet generators may be utilized in a microfluidic device.
  • the device includes a plurality of inlets for the droplet generator.
  • the flow focusing geometry can optionally be replaced with a T-junction for generating droplets.
  • Non-continuous nature pressure waveform can also be applied to the inlets of the device.
  • the device can be used prior to droplet-merger geometry to create further variability within the droplet.
  • Figure 11 (Panel A) The schematic represents a microfluidic device with four or more inlets which are merging at a common junction, (Panel B) other versions of the encapsulator
  • Figure 12 illustrates three methodologies that are used for scanning concentration space to create a droplet library: microfluidic device, pressure actuated flow-rate control for aqueous inlets, and bar-coding of droplets using dyes and readout by laser based detection (dropometer).
  • F4 C-(F1 + F2 + F3)
  • T3 10, 100, 1000 seconds respectively with 10 as the multiplication factor; in theory, Tl, T2 and T3 and multiplication factor can be any finite value).
  • the baseline values of CI, C2 and C3 ensure that scanned values of flow rates are positive for the duration of an entire experiment. This also indicates the presence of every component or reaction agent in a droplet. In Figure 13 on top right corner is shown the strength of an every inlet component in an individual droplet.
  • the first three sinusoidal inlets Fl, F2 and F3 can vary to the maximum value of 25% of total droplet volume.
  • the 3D function which is an additive function of sine waves, can scan parameter space as shown in Figure 13, bottom left within the limits of 25% of total drop volume for x, y and z axis.
  • the multiplication factor can determine the meshing density of the parameter space.
  • the multiplication factor of 10 could result in meshing the space in denser manner than in case of the factor of 4. Determination of meshing density can be chosen after due consideration of the following factors: (i) Application for which combinations are generated (ii) Total number of variables involved for a parameter space (iii) time limitations on the experiment.
  • Figure 14 3D nature of parameter scanning for the multiplication factor of 10 and 4. The denser nature of 10X scan is clearly evident in comparison to 4X scan.
  • the lower multiplication factor can yield faster scans. 4X scans can create approximate 4 reads of the fast solution for a read of the slower solution. This should be sufficient for most applications and should allow user to scan almost 8 solutions in total— for example, Is, 4s, 16s, 64s, 256s, 1024s, 4096s, 16384s - i.e., 8 solutions can be scanned at this resolution in ⁇ 5 hrs.
  • the sinusoidal nature of scanning functions can generate uneven scanning density within a parameter space.
  • the scanning density for the slowest input is higher in the extremities, which creates higher number of samples than in the middle portion of the concentration space. This issue is circumvented by scanning the parameter space using triangular waves wherein even scanning of concentration space is evident as shown in Figure 15.
  • the various functions that can be generated using a pressure controller are described herein.
  • Droplets can be generated with variable concentration for different solutions that a user wants to combine ahead of time, and then a droplet merger technique is used to merge combinations of these droplets. Each merged droplet gets a random assortment of the other droplet types, yielding unique concentrations within it. If the droplets are labeled, the concentration can be read out optically by the dropometer. This method of creating combinations has an advantage of higher speeds for screening the concentration space. If the droplets merger rate is ⁇ 1 kHz, the combination of the merger-combinatorial technique allows substantially larger screens.
  • a technique of flow control using pincer membrane valve can also be added to the microfluidic device to achieve high-speed concentration scanning.
  • the flow rates were stringently controlled for three input channels (figure b) in the increments of 0.25 ⁇ 1/ ⁇ .
  • the plotted graph shows measured values by three flow sensors (Fl , F2, F3) against time. (4) Triangular function to control pressure with period 1 and 2 seconds.
  • Example 5 Design and fabrication of microfluidic encapsulator
  • FIG. 17 Schematic of 3 versions microfluidic encapsulator and a picture of a PDMS device (A), (B) Generation of droplets encapsulating four inlet streams of water and dyes.
  • the device height is varied as 20, 35 or 50 ⁇ .
  • Version 3 of the device was finalized after three iterations of design-fabricate-test cycles. The devices were fabricated using one-step photolithography in a photopolymer, SU-8.
  • the master mold is used for replica molding in PDMS using soft lithography.
  • the device is bonded with a glass slide using oxygen plasma.
  • the device is treated with Aquapel for making the surface hydrophobic.
  • the pressure controller is used to control aqueous inlets, and oil inlet is operated using a syringe pump.
  • Figure 17(B) shows successful droplet generation using pressure controller based actuation of reservoirs. All values are in mbar for 4 aqueous inlets and flow rate of oil (HFE) is kept constant at 400 ⁇ /hr. Aqueous (IR 783 dye/water) microdroplets formed in HFE with 2% crytox-based surfactant can be seen to grow in size as the inlet pressure increases.
  • Figure 18 illustrates the results of experiments with (i) dyes (ii) model reagents (iii) (transcription-translation (TX-TL)) components using the encapsulator.
  • D Injection of fluorescein and Dylight 405 with period of 4 seconds.
  • Figure 9 Injection of IR dye, distilled water, PEG and BSA from four inlets (Panel A), stable laminar flows in the device (Panel B), controlling the flow rates using the pressure controller (Panel C).
  • TX-TL transcription-translation
  • an experiment was done using model reagents.
  • Four inlets were used: Polyethylene glycol (PEG) was used to mimic energy buffer; concentrated 40 mg/ml bovine serum albumin (BSA) was used to mimic highly viscous extract; IR dye and water were injected from remaining two inlets (Figure 9, Panel A).
  • the volume of each reservoir is a minimum 250 ⁇ in order to run the calibration step and actual scan experiment.
  • the inlets of PEG and BSA were kept constant at 1.5 ⁇ /min and 1.2 ⁇ /min respectively, and flow rates of IR dye and water were varied with oppositely phase sine wave, which varies between 0.5 to 2.5 ⁇ /min with a period of 120 seconds.
  • the four laminar streams were successfully established and calibration step was completed using Fluigent system. The implantation of sinusoidal nature of two streams was confirmed from the flow sensors (S range, 0-7 ⁇ /min) and was visually confirmed by high speed camera.
  • Inlet 1 Buffer (25% of drop volume, constant)
  • Inlet 2 Extract (33% of drop volume, constant)
  • Inlet 3 DNA in H20 ( deGFP on OR20RlPr, O. lnM-lOnM)
  • Inlet 1 Buffer + Extract ( Flowrate was kept constant, 55% of drop volume)
  • Inlet 2 DNA in H20 (deGFP on OR20RlPr, O. lnM-lOnM)
  • Example 7 Scanning of concentration space for (transcription-translation (TX- TL)) reaction for multi parameter biological circuits for maximizing protein expression
  • Feed Forward Loop (FFL) circuit is tested in a droplet system as described herein.
  • AraC and arabinose enhances TetR and deGFP production, whereas TetPv starts to inhibit deGFP production which initially gives a pulse of increase in deGFP which decreases as the reaction progresses.
  • Figure 21 depicts components of the reaction.
  • pi 1 psigma70-AraC; AmpR
  • pl2 pBAD-TetR; AmpR
  • pl4 pBAD-TetO-deGFP-ssrA; AmpR
  • Tube 1 (MM 0.915 274 pL 274 ul 6.23 16.41 Out of range for FR +p1 +p48+Ana) sensors or use syringe pump (perturbation ?)
  • Tube 3 (p 12) O.0OE9D-D 1.77- 8.85 pL 1.77- 3.S5 ul 0.D5-0.27 0.11 -D.53 FR sensor: ⁇
  • Tube 4 (nuclease-free 0.0211-D. 21.76 -633 pL 6.33-21.76 ul 0.19-0.65 0.38-1.31 FR Sensor: ⁇ water) D726
  • ch droplet contains 2-10 nM of ll, 2-10 nM of pl2, 4 nM of pl4, 2 nM of p43 ⁇ 4 TX-TL MM and 0.2% Arabirose
  • Crystallization of protein requires scanning of multi-dimensional chemical space which can have various parameters, such as (i) protein concentration;
  • DNA libraries are prepared using the methods and devices described herein.

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Abstract

La présente invention concerne un procédé pour moduler la composition de gouttelettes au moyen d'un dispositif microfluidique. L'invention concerne également un procédé pour combiner deux ou plusieurs fluides pour former des gouttelettes au moyen d'un dispositif microfluidique et des dispositifs pour mettre en oeuvre les procédés de l'invention.
PCT/US2015/056269 2014-10-20 2015-10-19 Modulation rapide de la composition de gouttelettes par des microvannes à membrane Ceased WO2016064755A2 (fr)

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