Microfluidic devices and methods for producing droplets for droplet-based assays

Abstract

Disclosed herein microfluidic devices for producing a plurality of sample-containing droplets for droplet-based assays. In addition, disclosed herein are methods for producing sample-containing droplets and to methods for carrying out droplet-based assays using the microfluidic devices described herein. The devices and methods allow for the low cost production of sample-containing droplets without the needs for pumps, valves, and the like, as well as the amplification and detection of samples containing a nucleic acid sequence of interest within the same microfluidic device where the droplets are formed.

Description

[0001] MICROFLUIDIC DEVICES AND METHODS FOR PRODUCING DROPLETS FOR DROPLET-BASED ASSAYS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of Finnish Patent Application No. 20235993, filed September 6, 2023, the entirety of which is incorporated by reference herein.

[0004] FIELD

[0005] The present invention is directed to microfluidic devices, and in particular to microfluidic devices for producing a plurality of sample-containing droplets for droplet-based assays. The present invention is also directed to methods for producing such droplets and to methods for carrying out a droplet-based assay using the microfluidic devices described herein.

[0006] BACKGROUND

[0007] A microfluidic device is a miniaturized system used to manipulate and analyze very small volumes of fluid, typically in the microliter or nanoliter range. Typically also, microfluidic devices are made from materials, such as glass or silicon, and contain a network of channels, chambers, valves, and pumps that allow the controlled movement and manipulation of fluids. The channels and chambers are generally only a few micrometers in size (e.g., width and depth), and the devices are often fabricated using microfabrication techniques, such as photolithography, soft lithography, or 3-D printing.

[0008] Microfluidic devices have a wide range of applications in fields such as chemistry, biology, medicine, and engineering. For example, they may be utilized for chemical synthesis, drug discovery, microparticle synthesis, DNA analysis, cell culture, and point-of-care diagnostics. Their small size and high precision make them well-suited for tasks that require precise control over small volumes of fluids, as well as for applications that require portability, disposability, and rapid analysis.

[0009] In point-of-care diagnostics, one method undergoing increased usage is ddPCR. ddPCR (droplet digital polymerase chain reaction) is a method used for the identification and / or quantification of nucleic acids (DNA or RNA) in a sample. The ddPCR technique is a modification of traditional PCR techniques. Advantageously, ddPCR enables the measurement of absolute nucleic acid quantities with higher precision, sensitivity, and accuracy, especially for low copy number nucleic acid molecules. To explain further, in ddPCR, a sample may be partitioned into thousands of tiny droplets, wherein each droplet serves as a miniature reaction vessel. Each droplet then undergoes PCR amplification to generate a large number of copies of a DNA or RNA molecule of interest, if even a single template is present within a droplet. After amplification, each droplet may be analyzed separately to determine the presence or absence of the DNA or RNA molecule of interest, and the number of copies of the molecule of interest may be quantified by counting the number of positive droplets. Since each droplet is analyzed individually, ddPCR provides a highly precise measurement of nucleic acid quantity, with lower variability and higher sensitivity than traditional PCR techniques. The technique is particularly useful for detecting rare mutations and gene fusions, DNA copy number variations, for detecting small changes in gene expression levels, or the like.

[0010] As mentioned, a key element of droplet-based assays, such as ddPCR, is the partitioning of a sample into thousands of tiny droplets. Typically, droplet formation is done by mixing of an aqueous phase containing the biological sample to be analyzed with an oil phase. Known techniques for preparing such droplets on a microscale level require pumps, valves, and the like. To ensure consistent and monodisperse droplet formation, the pumps of these systems provide accurate and controlled flow rates for the oil phase and the aqueous phase. This prevents the use of such systems in settings where limited resources are available. In addition, these systems may require intensive maintenance to ensure reliability, sterility, and consistent results over long-term usage. Long tubing lines between the pressure source and the droplet forming microfluidic device often lead to large dead volumes and loss of valuable sample, analyte or drug materials. Moreover, apparatus for generating these droplets are typically discrete units from the apparatus used to analyze the sample. Accordingly, known techniques are limited, require additional components, and / or require transfer of fluids from one apparatus to another, thereby adding costs, additional processing steps and time, and the risk of sample loss.

[0011] In view of the above, it would be desirable to provide a microfluidic device which is capable of generating a plurality of monodisperse sample droplets in situ and which also, in some embodiments, allows for immediate amplification and detection of the presence or absence of analyte(s) of interest, e.g., one or more DNA or RNA molecules of interest, within a single microfluidic device. SUMMARY

[0012] Aspects of the present invention overcomes the shortcoming of the prior art by disclosing novel and inventive microfluidic devices for producing a plurality of sample-containing droplets for droplet-based assays. In addition, disclosed herein are processes for producing sample-containing droplets and to methods for carrying out droplet-based assays using the microfluidic devices described herein. The devices and methods disclosed herein allow for the low cost production of monodisperse sample-containing droplets within a microfluidic device without the need for pumps, valves, separate components, and the like, as well as for the amplification and detection of samples comprising a nucleic acid sequence of interest within the same microfluidic device where the droplets are formed. Further, aspects of the present invention allow for use of the same microfluidic device for the generation of different sized droplets.

[0013] In accordance with one aspect of the present invention, there is disclosed a microfluidic device for providing a plurality of droplets for a droplet-based assay comprising: a body comprising:

[0014] (i) a plurality of first channels comprising a first fluid traveling there through and one or more second channels comprising a second fluid traveling there through, wherein the plurality of first channels join together to form a combined flow channel having a combined first fluid traveling there through;

[0015] (ii) a plurality of first inlets in fluid communication with the plurality of first channels for input of the first fluid to the plurality of first channels;

[0016] (iii) one or more second inlets in fluid communication with the one or more second channels for input of the second fluid to the one or more second channels; and

[0017] (iv) a droplet generation zone configured to generate an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase of the first fluid from at least the combined first fluid flow and the second fluid flow.

[0018] In accordance with another aspect of the present invention, there is disclosed a micro fluidic device for providing a plurality of droplets for a droplet-based assay comprising: a body comprising:

[0019] (i) a plurality of first inlets in fluid communication with a plurality of corresponding oil-containing channels formed in the body for delivery of oil to the plurality of corresponding oil-containing channels, and one or more second inlets in fluid communication with one or more corresponding sample-containing channels formed in the body for delivery of an aqueous biological sample-containing fluid to the one or more sample-containing channels, wherein the plurality of corresponding oil-containing channels join together to form a combined oil flow channel having a combined oil flow;

[0020] (ii) a droplet generation zone defined by the combined oil flow channel and the one or more sample-containing channels, wherein the droplet generation zone is configured to generate an emulsion from at least the combined oil flow and the aqueous biological samplecontaining fluid, the emulsion comprising a dispersed phase comprising a plurality of droplets of the aqueous biological sample-containing fluid dispersed in a continuous phase comprising the oil; and

[0021] (iii) optionally, a droplet collection chamber in fluid communication with the droplet generation zone for receipt of the emulsion comprising the plurality of the droplets generated in the droplet generation zone; and

[0022] (iv) an outlet downstream from at least one of the droplet collection chamber or the droplet generation zone.

[0023] In accordance with another aspect of the present invention, there is disclosed a process for forming droplets for a droplet-based assay comprising:

[0024] (i) providing a microfluidic device comprising a body, the body comprising a plurality of first inlets in fluid communication with a plurality of first channels formed in the body and one or more second inlets in fluid communication with one or more second channels formed in the body;

[0025] (ii) inputting a first fluid through the plurality of first inlets to the plurality of first channels and inputting a second fluid through the one or more second inlets to the one or more second channels, wherein the plurality of first channels join together to form a combined flow channel having a combined first fluid traveling there through; and

[0026] (iii) forming an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase comprising the first fluid from at least the combined first fluid and the second fluid.

[0027] In accordance with yet another aspect of the present invention, there is disclosed a method of performing a droplet-based assay comprising:

[0028] (i) forming the emulsion according to a method as described herein, wherein the dispersed phase comprises a biological sample comprising at least one nucleic acid having a nucleic acid sequence of interest;

[0029] (ii) collecting the emulsion comprising the plurality of droplets within a droplet collection chamber of the microfluidic device; (iii) within the droplet collection chamber, amplifying the at least one nucleic acid comprising the nucleic acid sequence of interest within the plurality of droplets; and

[0030] (iv) within droplet collection chamber, detecting the presence of or a property of the droplets comprising the nucleic acid sequence of interest and / or removing the plurality of droplets from the droplet collection chamber and sequencing the nucleic acid sequence of interest.

[0031] In accordance with yet another aspect of the present invention, there is disclosed a process for forming droplets for a droplet-based assay comprising: providing a microfluidic device comprising a body, the body comprising a plurality of first inlets in fluid communication with a plurality of first channels formed in the body and one or more second inlets in fluid communication with one or more second channels formed in the body; inputting a first fluid through the plurality of first inlets to the plurality of first channels and inputting a second fluid through the one or more second inlets to the one or more second channels, wherein the plurality of first channels join together to form a combined flow channel having a combined first fluid traveling there through; and forming an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase comprising the first fluid from at least the combined first fluid and the second fluid, wherein the process further comprises varying a property, e.g., size and / or shape, of the formed droplets of the second fluid by blocking input to one or more of the first inlets or second inlets with one or more plugs.

[0032] In accordance with another aspect of the present invention, there is provided a micro fluidic device for providing a plurality of droplets for a droplet-based assay comprising: a body comprising: a plurality of first channels comprising a first fluid traveling there through and one or more second channels comprising a second fluid traveling there through, wherein the plurality of first channels join together to form a combined flow channel having a combined first fluid traveling there through; a plurality of first inlets in fluid communication with the plurality of first channels for input of the first fluid to the plurality of first channels; one or more second inlets in fluid communication with the one or more second channels for input of the second fluid to the one or more second channels; a droplet generation zone configured to generate an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase of the first fluid from at least the combined first fluid and the second fluid flow. one or more plugs disposed within one or more of the first inlets or the second inlets, wherein the plug is effective to vary a property of the formed droplets of the second fluid by blocking input to one or more of the first inlets or second inlets.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 illustrates a microfluidic device in accordance with one aspect of the present invention.

[0035] FIG. 2 illustrates a microfluidic device in accordance with another aspect of the present invention.

[0036] FIG. 3 illustrates a microfluidic device in accordance with yet another aspect of the present invention.

[0037] FIG. 4 illustrates a microfluidic device in accordance with yet another aspect of the present invention.

[0038] FIG. 5 illustrates a microfluidic device in accordance with yet another aspect of the present invention.

[0039] FIG. 6 illustrates a microfluidic device in accordance with yet another aspect of the present invention.

[0040] FIG. 7 illustrates the introduction of fluids from a multichannel pipette in accordance with another aspect of the present invention.

[0041] FIG. 8 illustrates a microfluidic device utilizing plug(s) in accordance with yet another aspect of the present invention.

[0042] FIG. 9 illustrates a microfluidic device utilizing plug(s) in accordance with yet another aspect of the present invention.

[0043] DETAIEED DESCRIPTION

[0044] In one aspect, the present disclosure relates to microfluidic devices for generating droplets, wherein the structure of the microfluidic device provides numerous advantages for droplet-based assays, such as ddPCR, and the digital isothermal amplification of nucleic acids, particularly in limited resource settings. This disclosure further discloses devices and methods for performing various methods of digital nucleic acid amplification in a droplet-based assay without the need for complex, non-portable, and expensive pumping and valving systems. Moreover, the microfluidic devices described herein are disposable and inexpensive to manufacture, thereby providing ease-of-use and sterility in experimental or diagnostic set-ups. Still further, aspects of the present invention allow for the use of a single microfluidic device for the generation of droplets of various sizes by varying the number of inputs and channels used, for example.

[0045] In some embodiments, the microfluidic devices are manually-powered microfluidic devices. As used herein, the term “manually-powered” refers to the provision of a force required to facilitate the movement of any fluids inside any of the disclosed microfluidic devices, wherein the force may be provided directly by hand or by the use of a hand-operated tool suitable for applying said force. In some embodiments, the hand-operated tool may be a handheld pipette, a handheld multichannel pipette, a syringe, or a roller. When provided, the handheld multichannel pipette is a handheld pipette having at least 2 channels for dispensing a liquid. In certain embodiments, the multichannel pipette has less than 25 channels, for example 24, 12, 8 or 4 channels. In other embodiments, the force required to facilitate the movement of any fluids inside any of the disclosed microfluidic devices is provided by an automated device, such as an electronic pipette or electronic multichannel pipette.

[0046] As used herein, the terms “droplet” or “droplets” refer to any droplet(s) in an emulsion comprising a dispersed phase within a continuous phase, wherein the dispersed phase is an aqueous solution and the continuous phase is oil or wherein the dispersed phase is oil and the continuous phase is an aqueous solution. In certain embodiments, the continuous phase is oil and the dispersed phase comprises an aqueous solution comprising a biological sample. In certain embodiments, the biological sample comprises at least one nucleic acid. In some embodiments, the aqueous solution also or instead comprises reagents that facilitate a nucleic acid amplification reaction. Additionally, in certain embodiments, the aqueous solution may comprise barcoded beads.

[0047] As used herein, the term “fluid” refers to a flowable liquid material.

[0048] As used herein, the term “fluid communication” means that that a fluid may travel directly or indirectly from one component to another component. Thus, A in fluid communication with B means that a fluid may travel directly or indirectly (such as through another component) from A to B.

[0049] As used herein, the term “oil” refers to any liquid or mixture of liquids that are immiscible with water. In an embodiment, the oil comprises a member selected from the group consisting of silicone oil, mineral oil, hydrocarbon oil, fluorocarbon oil, vegetable oil, and combinations thereof. In particular embodiments, the oil comprises a silicone oil. In certain embodiments, the oil comprises a fluorocarbon oil. In certain embodiments, the oil may comprise any other additional components or additives, such as at least one surfactant, reagent, preservative, particles, combinations thereof, or any other suitable material.

[0050] As used herein, the term “surfactant” refers to a surface-active agent capable of reducing the surface tension of a liquid to which it is added and / or the interfacial tension with another phase. In an embodiment, the surfactant comprises both a hydrophilic portion and a hydrophobic portion, which may collectively confer a dual hydrophilic-hydrophobic character on the surfactant. In an embodiment, the surfactant may comprise a fluorosurfactant or a fluorinated surfactant.

[0051] As used herein, the term “nucleic acid material” refers to any material comprising one or more nucleic acids, such as DNA and / or RNA or fragments of DNA and / or RNA. In addition, the term “nucleic acid material” may refer to material occurring in nature or may refer to synthetic materials. In some embodiments, the nucleic acid material may be modified DNA and / or modified RNA. In addition, the nucleic acid material may be single-stranded or doublestranded. In certain embodiments, the nucleic acid material may by connected to other molecules, such as proteins or beads.

[0052] As used herein, the terms “polymerase chain reaction” or “PCR” refer to a reaction for the in vitro amplification of specific nucleic acid sequences by the simultaneous primer extension of complementary strands of a nucleic acid sequence. In addition, PCR refers to a reaction for making multiple copies or replicates of a nucleic acid material of interest flanked by primer binding sites. Such reactions comprise one or more repetitions of the following steps: (i) denaturing the nucleic acid of interest; (ii) annealing primers to primer binding sites; and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Typically, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors that are well-known to those of ordinary skill in the art

[0053] As used herein, the terms “PCR reagent” or “PCR reaction mixture” are given their ordinary meaning in the art and may include one or more PCR reagents. In an embodiment, the PCR reagent or PCR reaction mixture comprises one or more of a polymerase enzyme (e.g., Taq Polymerase), template DNA, primers, deoxynucleotide triphosphate (dNTP), MgCh, cofactors, and physiological buffers.

[0054] As used herein, the term “isothermal amplification” refers to methods of nucleic acid amplification, wherein one or more nucleic acid sequences of interest are amplified at a constant temperature. In contrast to isothermal amplification, in traditional PCR-based methods, the reaction temperature is optimized for each step to facilitate amplification of the nucleic acid sequence of interest. Several different methods are known for isothermal amplification. In an embodiment, the isothermal amplification method is selected from the group consisting of LAMP (Loop-Mediated Isothermal Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), TRC (Transcription Reverse-transcription Concerted reaction), RPA (Recombinase Polymerase Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), HDA (Helicasedependent Amplification), RCA (Rolling Circle Amplification), MDA (Multiple Displacement Amplification), SIBA (Strand Invasion Based Amplification), and WGA (Whole Genome Amplification). The reagents required for performing said isothermal amplification methods are known and readily available for a person skilled in the art.

[0055] In certain embodiments, the isothermal amplification is multiplexed. As used herein, the term “multiplex,” “multiplexed,” or “multiplexed PCR,” or the like refers to a PCR method, wherein multiple nucleic acid sequences of interest (or a single nucleic sequence of interest and one or more reference sequences) are simultaneously amplified in the same reaction mixture. In other words, in certain embodiments, several different sequences may be amplified simultaneously. In some embodiments, distinct sets of primers are employed for each sequence being amplified. In some embodiments, the number of nucleic acid sequences of interest in a multiplex PCR is in the range of from 2 to 50, or from 2 to 40, or from 2 to 30.

[0056] As used herein, the term “nucleic acid sequence of interest” refers to one or more nucleic acid molecules comprising one or more sequences that the subject assay is configured to detect. In an embodiment, the detection steps may comprise amplification steps and imaging steps, wherein nucleic acid amplification only occurs in droplets comprising the nucleic acid sequence of interest. The sequence of interest may be of any length and have any sequence. In one embodiment, the nucleic acid sequence of interest comprises a sequence of a microbe, such as a bacterium, a virus, or a yeast. In other embodiments, the nucleic acid sequence of interest may comprise a nucleic acid sequence of a subject, such as a mammal. The nucleic acid sequence of interest may be a region of a gene or a chromosome. In certain embodiments, the sequence is specific or unique for the genotype or the organism to be detected. In some embodiments, the sequence of interest allows identification of an organism. For example, the sequence of interest may be a sequence specific to a particular strain of bacteria or a particular serotype, isolate or clade of a virus. In an embodiment, the nucleic acid sequence of interest is indicative of the presence of a pathogen. In certain embodiments, the pathogen is one that can be detected by PCR. In some embodiments, the nucleic acid sequence of interest may be a sequence of a viral pathogen. In some embodiments, the nucleic acid sequence of interest allows identification of a viral pathogen. Viral pathogens may include, for example, influenza viruses, SARS viruses, herpes viruses, hepatitis viruses, norovirus and adenoviruses. In certain embodiments, the nucleic acid sequence of interest is specific for SARS-CoV-2 or a variant thereof. In other embodiments, the nucleic acid sequence of interest may be a sequence of a bacterial pathogen. In some embodiments, the nucleic acid sequence of interest comprises one or more single nucleotide polymorphism (SNP). In some embodiments, the nucleic acid sequence of interest is a gene or sequence with a variable number of copies in the genome. In some embodiments, the nucleic acid sequence of interest is a fusion gene.

[0057] In some embodiments, the nucleic acid sequence of interest may be amplified for preparative purposes, for example, Next Generation Sequencing (NGS) library preparation, or for other preparation purposes, such as for phage display, ribosome display, or yeast display library preparation. In certain embodiments, the preparation comprises single-cell sequencing library preparation.

[0058] As used herein, the term “library” refers to a pool of nucleic acid material derived from a sample. Typically, a library is a collection of nucleic acid fragments. A library may be transferred and propagated in a population of microorganisms through the process of molecular cloning. A library comprises many different nucleic acid sequences of interest. In some embodiments, the library comprises a single-cell sequencing library. The term “single-cell sequencing library” refers to a library prepared from a single-cell. Single-cell sequencing can be used to detect the genome, transcriptome and other multi-omics of single cells. Single-cell sequencing provides a single-cell genome or transcriptome, which may be used, for example, for studying cell population differences and cellular evolutionary relationships.

[0059] Now referring to the figures, FIG. 1 illustrates a micro fluidic device 10 (hereinafter device 10 for ease of reference) in accordance with an aspect of the present invention for providing a plurality of droplets for a droplet-based assay. The device 10 comprises a body 12 having a plurality of first channels 14 (hereinafter first channels 14), a plurality of first inlets 16 (first inlets 16) in fluid communication with the first channels 14, one or more second channels 18 (second channels 18), and one or more second inlets 20 (second inlets 20) in fluid communication with the second channels 18. The device 10 is configured such that a first fluid is input into the first inlets 16 and travels through the first channels 14 and a second fluid is input into the second inlets 20 and travels through the second channels 18. As shown, at one or more locations, the first channels 14 join together to form a combined flow channel 22 having a combined first fluid traveling there through.

[0060] In addition to the first and second channels and inlets, the body 12 further comprises a droplet generation zone 24 configured to generate an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase of the first fluid. In certain embodiments, the droplet generation zone 24 is structured to provide droplet formation by any suitable passive droplet formation technique.

[0061] In one embodiment, the droplet generation zone 24 is defined by an intersection 26 of the combined flow channel 22 and the second channels 18. In an embodiment, the droplet generation zone 24 comprises one or more T-junctions as shown at intersection 26. In this way, the droplets are formed according to a cross-flow technique, wherein the flow of the continuous phase (e.g., through combined flow channel 22) and the flow of the dispersed phase channel(s), e.g., through second channel(s) 18, are contacted perpendicular to each other.

[0062] In other embodiments, as shown in FIG. 2, the droplet generation zone 24 is structurally configured to generate the plurality of droplets by flow-focusing the combined first fluid and second fluid from their respective channels 22, 18. In flow focusing, the continuous phase (combined (first) fluid flow) is introduced into at least two channels 22 that surround at least one channel 18 of the dispersed phase (second fluid). The fluids within the channels 22, 18 are combined and forced through a geometric constraint, such as an aperture of reduced diameter, thereby forcing the formation of microfluidic droplets by a geometrical constraint.

[0063] Alternatively, the droplet generation zone 24 may comprise any other suitable structural configuration suitable for forming an emulsion comprising a dispersed phase of the second fluid within a continuous phase of the first fluid, such as modification of the above configurations. One example of such a suitable structural configuration or modification may include, for example, non-perpendicular angle(s) of contact of the combined first flow channel 22 and the second channels 18 at the intersection 26.

[0064] In some embodiments, the emulsion formed in the droplet generation zone 24 comprises a water-in-oil emulsion. To provide a water-in-oil emulsion, the first fluid comprises oil and the second fluid may comprise an aqueous liquid, which may be an aqueous biological samplecontaining liquid. In other embodiments, the emulsion may be an oil-in-water emulsion. To provide an oil-in-water emulsion, the first fluid may comprise an aqueous fluid and the second fluid may comprise oil, which may be an oil comprising a biological sample therein. The biological sample may comprise any suitable biological sample comprising an amount of nucleic acid material. In some embodiments, at least one nucleic acid is obtained or derived from the biological sample. In some embodiments, the aqueous liquid or the oil comprising a biological sample may be referred to as a “biological sample-containing fluid.” As used herein, the term “biological sample-containing fluid” refers to a fluid comprising at least one nucleic acid. In some embodiments, the biological sample-containing fluid is produced by extracting nucleic acids from a biological sample. The sample may be, for example, an environmental sample, a reference sample or a clinical sample. A clinical sample may be, for example, a sample obtained from a subject suspected of having, or having a disease. A clinical sample may comprise, for example, saliva, sputum, blood, plasma, serum, urine, or a stool sample. In other embodiments, the sample may comprise a tissue sample, a biopsy sample, or a cell sample. In some embodiments, the sample may be derived from a human or an animal, such as a domestic animal. In some embodiments, the sample may be obtained from water, such as contaminated water, soil, food, sewage, or other waste sources.

[0065] The body 12 of the micro fluidic device 10 may be formed from any suitable material(s), such as glass, silica, paper, or polymers, such as polydimethylsiloxane (PDMS), poly(methyl methacrylate (PMMA), thermoset polyester (TE), polystyrene (PS) or poly(ethylene glycol) diacrylate (PEGDA), photoresist SU-8 or combinations thereof. In some embodiments, the body of the micro fluidic device 10 may be formed from a deformable polymer having a degree of elasticity. In some embodiments, the polymer comprises polydimethylsiloxane (PDMS). In some embodiments, the micro fluidic device 10 may be formed from polydimethylsiloxane (PDMS). PDMS is a porous, flexible and transparent material and therefore well-suited for the device disclosed herein. PDMS is a particularly suitable material for the device when a multichannel pipette, for example, is used to input the fluids to the device 10, e.g., first fluid and second fluid, since the PDMS will deform about the tips of the micropipette. Alternatively, the body 12 of the micro fluidic may be formed from a rigid material, such as an inert and rigid polymer material.

[0066] The inlets 16, 20 may be provided in any suitable arrangement on the device corresponding to the desired number of channels 14, 18. In addition, the inlets 16, 20 may have any suitable size shape, such as a circular shape. Further, the inlets 16, 20 may be spaced apart from one another by any suitable distance. In certain embodiments, the inlets 16, 20 are spaced apart from one another by a distance of 9mm, 18mm, 27mm, or any distance divisible by 9mm so as to be compatible with standard multichannel pipettes. In certain embodiments, the inlets 16, 20 may be reinforced by a rigid material so as to accommodate tips of a multichannel pipette, particularly when using polymers that do not have sufficient elasticity for the body of the device 10.

[0067] The first channels 14 and the second channels 18 may be of any suitable dimension and shape. In some embodiments, the plurality of first channels 14 and the one or more second channels 18 each have of a width and depth of 1 pm to 500 pm, for example, from 100 pm to 300 pm. In some embodiments, the width of the channels 14, 18 may be from 60 pm to 100 pm. The height of the channels 14, 18 may be from about 1 pm to about 500 pm, for example, from about 80 pm to about 200 pm, such as about 100 pm to 150 pm. Typically, channel width and height are kept constant throughout the device 10.

[0068] The device 10 may be of any suitable size and shape, including thickness. In an embodiment, the thickness of the micro fluidic device 10 is from 1-5 mm, and in certain embodiments from 2-3 mm. It is appreciated that it is desirable that the thickness of the device 10 as a whole and the material on top (over) the channels 14, 18 be sufficient to ensure tight sealing of the input device, e.g., pipette tips into the first and second inlets 16, 20. In addition, it is appreciated that if the device 10 is too thin, there is an increasing likelihood that air will be trapped in the device 10.

[0069] Referring again to FIG. 1, in certain embodiments, the body 12 of the device 10 further comprises a droplet collection chamber 28 (collection chamber 28 or chamber 28) in fluid communication with the droplet generation zone 24 via pathway 29, for example, for receipt of the emulsion comprising the plurality of the droplets generated in the droplet generation zone 24. The droplet collection chamber 28 may comprise any suitable structure for collecting droplets formed in the micro fluidic device 10. The structure of the collection chamber should avoid trapping air bubbles inside the collection chamber 28 as the air bubbles decrease the available space for collecting the generated droplets. Furthermore, any air pockets inside the droplet collection chamber 28 may interfere with the detection of the droplets inside the collection chamber, or with the reaction conditions, for example for a PCR reaction, inside the droplet collection chamber 28. In addition, the droplet collection chamber 28 may comprise any suitable shape.

[0070] In one embodiment, the chamber 28 may have a diamond shape as shown in Figure 1. When a large volume of droplets are desired or expected, a diamond-shaped chamber 28 may have the advantage of holding more droplets per unit area relative to other shapes. In other embodiments, the chamber 28 may be in the shape of a meandering fluid pathway as shown in FIG. 4. In certain embodiments, the meandering pathway is configured to fill available space within the device 10. In some embodiments, the meandering pathway comprises a curved configuration, wherein a length of the chamber 28 is at least twice that of a width of the chamber 28. Alternatively, the chamber 28 may comprise any other suitable shape, such as a rectangular shape or a rhomboid shape.

[0071] In certain embodiments, the droplet collection chamber 28 has a depth which allows only a single layer of the droplets to be maintained within the droplet collection chamber 28. In this way, the droplet collection chamber 28 facilitates the imaging of the droplets within the chamber 28. In this way, any undesired overlap of the droplets is avoided during the imaging step. In a specific embodiment, the depth of the collection chamber is I um - 500pm, such as 1pm - 300pm, for example, 200 - 300 pm.

[0072] Downstream from the chamber 28, the device 10 further comprises an outlet 30 in fluid communication with the chamber 28. The outlet 30 allows for the flow of the first and second liquids through the first and second channels 14, 18, respectively. In some embodiments, the outlet 30 may be in fluid communication with an external device or apparatus for any materials, e.g., the droplets, from the device 10. The external device or apparatus may comprise any device or apparatus suitable for the further collection and / or analysis of materials exiting the outlet 30 of the device 10.

[0073] In some embodiments, as shown in FIG. 5, where the formed droplets are intended to be collected in an external device (other than device 10), the device 10 need not include the droplet collection chamber 28. In this instance, the outlet 30 may directly receive the emulsion comprising droplets from pathway 29.

[0074] In accordance with another aspect of the present invention, the chamber 28 may advantageously be utilized for the amplification of any nucleic acid material in the droplets and / or for the measurement of a property of the droplets, e.g., detection of one or more analytes of interest. In some embodiments, any materials necessary for amplification and / or measurement of the subject material are input upstream of the chamber 28 and may be delivered along with any fluid(s) traveling through the first channels 14, combined first fluid channel 22, second channels 18, and / or any additional channels provided.

[0075] In some embodiments, as shown in FIG. 6, the device 10 may further comprise one or more third channels 32 (third channels 32) and one or more third inlets 34 (third inlets 34) in fluid communication with the third channels 32. The device 10 is configured such that a third fluid is input into the third inputs 34 and travels through the channels 32. The third fluid may comprise any suitable material for the enhancement of, e.g., amplification, and / or measurement of a property of the droplets and / or emulsion. In some embodiments, the third fluid may comprise an aqueous reagent-containing fluid. The third fluid may thus allow for additional reagents, agents, additives or other components to be added to the formed emulsion and / or droplets.

[0076] It is appreciated that the ratio of the different inputs and / or channels as described herein may each be a result-effective variable for the formation of the droplets in the droplet generation zone 24.

[0077] The number of first channels, second channels, third channels (when provided), or any additional channels (when provided) may be any suitable value for generating droplets of a desired size. In some embodiments, the number of channels matches the number of inputs provided in the device, although it is understood that the present invention is not so limited, such as when a single inlet feeds more than one channel. In addition, in some embodiments, the number of inputs on the device 10 matches the number of channels or tips provided by the device, e.g., multichannel pipette, used to deliver the selected materials to the device 10, although again it is understood that the present invention is not limited. In some embodiments, the device 10 may include a total of 4, 8, 12, 24, or more channels, e.g., channels 14, 18.

[0078] In some embodiments, a single device 10 may be used to form a plurality of different droplets sizes by blocking use of one or more inputs (and thus channels) in the device 10. For example, if a device 10 having 24 inputs is provided leading to first channels 14, second channels, and / or third channels as described herein, the user may wish to block one or more of the inputs of the device with a plug 40 (see FIGS. 8-9) so as to change the total number of channels utilized in the device and / or a ratio of different channels, e.g., first channels 14 and second channels 18, to one another. This may, in turn, provided droplets having different sizes, shapes, distances between each other in the flow and other desired droplet properties. Further advantageously, the use of one or more plugs 40 described herein allows a single device 10 to be utilized for the formation of different droplets using the same device 10 by varying the number of channels utilized and relative ratios of different channels in a single device vs. the expense of providing multiple devices with different numbers of channels.

[0079] To demonstrate, FIG. 8 illustrates a first example of the use of a plug 40 in accordance with an aspect of the present invention. In the embodiment shown, the device 10 includes four first channels 14 and four second channels 18. Accordingly, absent the use of the plug 40, the ratio of first channels to second channels would be 1 : 1. As shown, if a plug 40 is utilized to block the input to one of the second channels 18, the ratio of second channels 18 (for input of a dispersed phase, for example) to first channels 14 (for input of a continuous phase) is modified to 3:4. In this way, droplets having different properties, e.g., shape and / or size, such as reduced droplet size, may be produced. In the embodiment shown in FIG. 8, the plug 10 may be used, for example, to produce water-in-oil droplets by using only seven out of eight tips of an 8-channel pipette (e.g., one inlet to a second channel 14 plugged with plug 40; three tips deliver an aqueous fluid to inlets leading to second channels 18; and four tips deliver an oil phase to inlets leading to first channels 14).

[0080] FIG. 9 illustrates the same device 10 as in FIG. 8, but instead with three plugs 40 in use blocking three of the four second channels 18. This changes the ratio of second channels 18 to first channels from 1 : 1 to 1 :4, thereby forming smaller droplets relative to the embodiment of FIG. 8. In the embodiment shown in FIG. 9, the plug 10 may be used, for example, to produce water-in-oil droplets by using only five out of eight tips of an 8-channel pipette (e.g., three inlets to second channels 14 are plugged with plugs 40; one tip delivers an aqueous fluid to inlets leading to second channel(s) 18; and four tips deliver an oil phase to inlets leading to first channels 14).

[0081] In addition to the above-mentioned benefits of using a plug 40, the plug 40 may also prevent the unintended introduction of fluid and / or air into the device 10 that may undesirably affect droplet formation within the device. Further, the plug 40 may be of any suitable material, such as an inert polymeric material, and may be sized and shaped so as to form an air-tight seal in the plug. The plug 40 can also comprise or be pieces of tapes or other adhesives for sealing and blocking the desired inlets. In some embodiments, the plug(s) are removeable with the use of a suitable instrument, tool, or the like.

[0082] Similar to the number of each type of channel and the total number of channels, the ratio of the number of first channels 14, second channels 18, to further channels, e.g., third channels 32, may be any suitable value. In this way, the droplet size, shape, or other propert(ies) of the droplets may be modified by alternating the ratio of particular channels to one another. In an embodiment, for example, the size, shape, droplet distances to each other in the flow and / or other properties of the formed droplets may be affected by changing the ratio of first channels 14 to the number of second channels 18. In some embodiments, the ratio of the number of the first channels 14 to the number second channels 18 may be from 1 : 15 to 15 : 1 , for example 1 : 1 to 7:1. A configuration showing a total of eight (8) first channels 14 and second channels 18 is shown in FIG. 3, although it is understood that the present invention is not so limited.

[0083] In certain embodiments, the ratio of the number of the first channels 14 to the number of second channels 18 may be from 1 :3 to 3 : 1 as was shown in FIG. 1. In some embodiments, the ratio of the number of the first channels 14 to the number of second channels 18 may be, for example, 1 :1 or 1 :3. Typically, the number of first channels 14 is at least greater than the number of second channels 18 since the first fluid (aqueous solution or oil) traveling through the first channels 18 becomes the continuous phase in which the dispersed phase (second fluid from second channels 18) is dispersed. It is appreciated that the ratio of the number of first inlets 16 to the number of second inlets 20 may similarly be the same ratio as the number of first channels 14 to the number of second channels 18. In some embodiments, the ratio of inlets is different than the ratio of channels, such as, again, when a single inlet feeds more than one channel.

[0084] To explain the use of the device 10 in a single cell application, cells and beads / lysis buffer may be inputted to the device 10 from separate inlets (e.g., second inlets 20 and third inlets 34 to second channels 18 and third channels 32, respectively). In such embodiments, the channels 18, 32 may merge together to provide a combined fluid flow channel 35 just before the droplet generation zone 24, e.g., a T-junction, where droplet formation takes place when intersecting with the combined first fluid traveling through the combined flow channel 22. In this way, the second fluid and third fluid will be mixed within the same droplet such that the droplets each include cells, and beads / lysis buffer, for example.

[0085] In particular embodiments, the aqueous reagent-containing fluid may comprise a member selected from the group consisting of bar-coded beads, lysis buffer, and combinations thereof. Bar-coded beads and lysis buffer may be used, for example, in applications related to singlecell analysis, such as single-cell sequencing. In single-cell analysis, it is desirable that cells are first separated, then lysed, and finally the nucleic acid sequences therein are amplified. Providing a third fluid may allow the control of the sequence of these events.

[0086] Another aspect of the present disclosure is a process for forming droplets for a dropletbased assay. The process comprises a first step of providing a micro fluidic device 10 as described herein. In addition, the process further comprises inputting the first fluid through the first inlets 16 to the first channels 14 and inputting a second fluid through the second inlets 20 to the second channels 18. In the process, as was shown in FIG. 1, the first channels 14 join together to form a combined flow channel 22 having a combined first fluid traveling there through. In some embodiments, additional fluids, e.g., a third fluid, may be input into additional inputs, e.g., third inlets 34, and travel through third channels 32 as described herein.

[0087] The fluids described herein may be input into the micro fluidic device 10 by any suitable method or device that provides a pressure effective to push the flow of the desired fluids (e.g., first, second, and / or third fluids) through the microfluidic device 10. In a particular embodiment, the first fluid, the second fluid, and / or further fluids (e.g., third fluid when present) may be manually inputted into the device 10. In this way, the process may be performed without the use of external pumps, valves, or the like. In some embodiments, the inputting of the desired fluids to the microfluidic device may be performed manually by a human user. In certain embodiments, the first and second fluids may be manually inputted into the first channels 14 and the second channels 18 (and third channels 32 when present) from a multichannel pipette 36 having tips 38 as shown in FIG. 7. In this way, in one aspect, the present invention provides a process where no external pumping power is needed. This may result in a substantial reduction in time, costs, materials and process steps, and simplifies the droplet formation process for droplet-based assays. As such, the processes described herein may also be performed in any laboratory using standard laboratory equipment. Further, due to the design of the device 10, a wide range of applications may be more easily performed with the process. These applications include for example single-cell analytics, droplet digital PCR (ddPCR), and drug nanoparticle research.

[0088] In accordance with an aspect of the present invention, the devices and processes described herein enable the formation of emulsions comprising droplets by a single manual thumb move by the user (press of multichannel pipette). In this way, a user can control the ratio(s) of multiple channels, e.g., water:oil channels, and flexibly change the flowrates of the respective channels by controlling the number of inlets used, e.g., active water vs oil inlets or number of pipette tips used to input the desired materials or combine the flows of multiple channels. Although the manual push by a user's hand may be less controllable, due to, for example, pressure fluctuation, manually pushing the materials to the device 10 still produces monodisperse droplets because any fluctuations are transferred equally to all active channels. As such, the ratio of phases, e.g., water:oil, in the droplet generation zone 24 will remain the same throughout the push.

[0089] After inputting into the device 10, the respective fluids, e.g., first, second, and / or third fluids, travel through the device 10 to the droplet generation zone 24. In an embodiment, the fluids are inputted into the device at the same or approximately the same pressure (+ / - 10%) as one another. As noted above, the first fluid channels 14 join to together to form a combined first fluid channel 22. In an embodiment, the first fluids in the plurality of channels 14 combine and produce a combined first fluid having a greater flow rate than the flow rate of the second fluid, which aids in proper droplet formation Accordingly, when the combined first fluid and the second fluid, for example, combine at the droplet generation zone 24, the second fluid becomes an emulsion comprising a dispersed phase comprising droplets within a continuous phase of the first fluid.

[0090] As described above, in one embodiment, the first fluid is oil and the second fluid is an aqueous solution to form a water-in-oil emulsion. In other embodiments, the first fluid comprises an aqueous solution and the second fluid is oil to form an oil-in-water emulsion. In either instance, in some embodiments, the dispersed phase comprising droplets comprises a biological sample having at least one nucleic acid having a nucleic acid sequence of interest therein.

[0091] The size of the droplets formed by the dispersed phase can be controlled by controlling the flow rate of the continuous phase and the flow rate of the dispersed phase. In general, the higher ratio of the flow rate of the continuous phase to the flow rate of the dispersed phase, the smaller the generated droplets are. In one embodiment, a flow rate ratio of from 1 :3 to 1 :15, such as 1 :3, 1 :7, 1 :11, or 1 :15, of the dispersed to the continuous phase, respectively, results in an average droplet diameter of 110 pm when using approximately a channel width of 100 pm and a height of 150 pm.

[0092] In certain embodiments, the process may further comprise, prior to inputting any of the fluids described herein into their respective channels, pre-filling one or more of the channels 14, 18, and / or 32 (when present) with the first fluid that forms the continuous phase of the emulsion. In a particular embodiment, the process further comprises, prior inputting the first fluid through the plurality of first inlets 16 to the plurality of first channels 14 and inputting the second fluid through the one or more second inlets 20 to the one or more second channels 18, pre-filling the plurality of first channels 14 and the one or more second channels 18 with the first fluid. In an embodiment, the first fluid is oil in the case of forming a water-in-oil emulsion. The present inventors have found that so doing may improve the monodispersity of the formed droplets when desired. In some embodiments, the process may comprise plugging one or more of the inlets of the device, e.g., first inlets 16 or second inlets 20, with a plug 40 to prevent introduction of air and / or fluid to the inlets, e.g., first inlets 16 or second inlets 20 as described above.

[0093] In accordance with another aspect of the present invention, there is provided a method of performing a droplet-based assay. First, the method comprises forming an emulsion comprising a dispersed phase comprising droplets in a continuous phase as described herein, wherein the dispersed phase comprises a biological sample comprising at least one nucleic acid sequence of interest.

[0094] In some embodiments, the method performing a droplet-based assay further comprises collecting the formed emulsion comprising the plurality of droplets within the droplet collection chamber 28 of the microfluidic device 10. In certain embodiments, the droplets in the chamber 28 do not need to be removed for any further processing, e.g., amplification and / or analysis as described herein. Once the droplets are collected within chamber 28, the process for performing a dropletbased assay may further comprise, amplifying the at least one nucleic acid comprising the nucleic acid sequence of interest within the plurality of droplets in the chamber 28 of the device 10. In some embodiments, the amplification may comprise polymerase chain reaction (PCR) amplification or isothermal amplification. In particular embodiments, the isothermal amplification may be selected from the group consisting of LAMP (Loop-Mediated Isothermal Amplification), NASBA (Nucleic Acid Sequence-Based Amplification), TRC (Transcription Reverse-transcription Concerted reaction), RPA (Recombinase Polymerase Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), or HDA (Helicase-dependent Amplification). RCA (Rolling Circle Amplification), MDA (Multiple Displacement Amplification), SIBA (Strand Invasion Based Amplification), and WGA (Whole Genome Amplification).

[0095] In some embodiments, the amplifying step may comprise droplet digital PCR (ddPCR). In some embodiments, at least one of the fluids, e.g., second fluid, may comprise or further comprise a PCR reaction mixture. The PCR reaction mixture may comprise a reagent for detecting amplified DNA, for example, an intercalating dye. In certain embodiments, the intercalating dye is a fluorescent dye. The intercalating dye may be connected to a factor capable of specifically binding to the nucleic acid sequence of interest. In some embodiments, the intercalating dye may be connected to a primer that has a complimentary region with the nucleic acid sequence of interest. In certain embodiments, a primer that has a complimentary region with the nucleic acid sequence of interest may comprise a fluorescent label. In certain embodiments, two or more primers binding to two or more different nucleic acid sequence of interest may be utilized. In some embodiments, each primer may have a different fluorescent dye.

[0096] Further, in some embodiments, the amplifying step may comprise droplet digital isothermal amplification. In such embodiments, at least one of the fluids, e.g., the second fluid, may further comprise an isothermal amplification reaction mixture. The isothermal amplification reaction mixture may comprise a reagent for detecting amplified DNA, for example, an intercalating dye as explained above. In certain embodiments, the intercalacting dye is a fluorescent dye.

[0097] Once the amplifying of the at least one nucleic acid comprising the nucleic acid sequence of interest within the plurality of droplets in the chamber 28 of the device 10, the process for performing a droplet-based assay may further comprise: (i) within the chamber 28, detecting the presence of or a property of the droplets comprising the nucleic acid sequence of interest within the chamber 28; and / or (ii) removing the plurality of droplets from the chamber 28 and sequencing the nucleic acid sequence of interest.

[0098] In an embodiment, the detecting of a presence of or a property of the droplets may be done by measuring an appropriate signal provided by the droplets. The signal may be measured using available methods for the detection of nucleic acids. In some embodiments, the reagents for amplification may comprise a fluorescent dye capable of binding to the nucleic acid material or nucleic acid sequence of interest. The detection may then be performed by any available method for detecting fluorescence, such as fluorescence microscopy or fluorescence spectroscopy. In other embodiments, the detecting step may be performed by imaging technologies, such as microscopic techniques, for example, by fluorescent microscopy. In still other embodiments, the plurality of droplets may be removed from the droplet collection chamber and sequencing of the nucleic acid sequence of interest may be performed.

[0099] In accordance with yet another aspect of the present invention, the dispersed phase comprising droplets may comprise cells, for example, cancer cells, B-cells, or bacterial cells. The emulsion be formed from the first fluid, second fluid, and a third fluid as described herein. In this embodiment, the third fluid may comprise a lysis buffer and / or bar-coded beads. In some embodiments, the third fluid is contacted with the cells in the emulsion. In certain embodiments, each droplet has one cell and one bar-coded bead. As used herein, the term “bar- coded” refers to a unique label that allows differentiating between nucleic acid materials derived from different cells. The bar-code may be, for example, a unique nucleic acid sequence. In bar-coded beads, the bar-code is typically connected to a bead, such as magnetic beads, acrylamide beads, or hydroxylated methacrylic polymer beads.

[0100] In some embodiments, bar-coded beads are biocompatible. The aim with using such bar- coded beads is to label the nucleic acid material contained in the cell so that it may be differentiated from nucleic acid material derived from another cell. In some embodiments, the cells are subjected to a lysis step before or during the amplifying step. In certain embodiments, the cells are brought into contact with the bar-coded beads prior to the lysis. In some embodiments, the bar-coded beads may identify individual cells from which the nucleic acid sequence of interest originates.

[0101] EXAMPLES

[0102] Microfluidic device design

[0103] Microfluidic device designs were drawn using AutoCad 2020 software and printed as high-resolution polymer film photomasks (Micro litho). In one embodiment, the produced microfluidic devices included four inlets (three for oil and one for aqueous sample). An 8- channel pipette was used to make the droplets and to provide a user with the capability of influencing droplet size by altering the water-to-oil ratio. The inlets lead to a droplet generating T-junction from where the created droplets are brought into a larger collection chamber. Two types of collection chambers are utilized: a meandering shape (FIG. 4) and a diamond shape (e.g., FIG. 1). The channel width was 100 pm throughout the microfluidic device, except at the collection chamber. At the collection chamber, the diamond-shaped was approximately 8.8 mm tall and 26.5mm long, measured at the widest and longest portions of the chamber. The meander-shaped chamber was 100 mm tall and 1 mm wide.

[0104] A master for the microfluidic devices was made using SU-8 photolithography. The used recipe was following: SU-8 2075 (MicroChem) was spin-coated on top of a silicon wafer 1250 rpm for 30s following soft bake on a hotplate 65°C for 5min and 95°C for 15min, UV exposure (Cloe, UV-kub2) through the mask for 10 s and post exposure bake for 3.5 min at 65°C and 9 min at 95°C. The master was developed submerging it to SU-8 developer (MicroChem) for 15 min and washing it afterwards with isopropyl alcohol. The master was finalized with 15 min 120°C hard bake. The channel height used in all micro fluidic devices was measured by profilometry. (Bruker Dektak XT).

[0105] PDMS casting and device finalization

[0106] PDMS (SYLGARD 184, DOW) was mixed with 10:1 ratio of monomer :crosslinker. PDMS mixture was degassed and poured on top of the SU-8 master. PDMS curing took place in 65°C oven over 2 hours. After curing, PDMS was peeled of the master and chips were cut out. Inlet and outlet holes were punched at this stage. The microfluidic devices and glass slides were cleaned using tape, isopropyl alcohol and dried with compressed air. The microfluidic devices were bonded on the glass slides with oxygen plasma (plasma oven, Femto Science, 70ml / min oxygen) 60W power for 60s.

[0107] Monodispersity of the droplets

[0108] The processes and devices disclosed herein demonstrate that the produced manually- pressurized microfluidic devices can produce uniform and monodisperse droplets quickly and without forming significant reagent waste.

[0109] Two methods were tested for droplet generation in order to assess the monodispersity: (1) the droplets were either generated directly on an empty micro fluidic device or alternatively generated on a microfluidic device where the channels of the microfluidic device were prefilled with oil. The same oil was used in a prefilling step as in droplet generation. The droplets formed on an empty device created two-peaked profile where the droplets were around 180 pm or 250 gm in diameter, whereas the prefilled device created droplets which were approximately 178 gm in diameter. Therefore, it may be beneficial to prefill the device with oil and only then generate the droplets. Without wishing to be bound by theory, it is believed that prefilling the channels of the microfluidic device with oil standardizes the hydraulic resistance at various points of the droplet generation. Although this adds an extra step, the gain in monodispersity is significant.

[0110] Monodispersity of the droplets is a key component in ddPCR analytics. The effect of the flow rate (how fast the pipette was pressed) was further tested in order to determine any effect on the droplet size distribution. A slow flow rate (0.45 gl / s of oil and 0.15 gl / s of sample) was compared to a fast flow rate (0.9 gl / s of oil and 0.3 gl / s of sample). The ratio of the continuous to the discontinuous phase was 3:1, due to the design. The droplets were generated in a squeezing mode in both cases. The monodispersity of the droplets can be estimated using polydispersity index (PI), which is based on the size heterogeneity of a sample. Collected data showed that the press rate did not have a significant effect on either the average size of the droplets or the polydispersity index, although the droplets with the lower press rate were slightly larger. With high-pressure droplets, the average diameter was 190gm with a standard deviation of 28gm while, with low-pressure droplets, the average diameter was 199gm with a standard deviation of 28gm. We concluded that the produced manually-pressurized microfluidic devices are capable of producing droplets with intermediary monodispersity and that there may be an expected trade-off between monodispersity and lower component and / or instrumentation requirements. The monodispersity of the droplets can be somewhat improved by discarding the first created droplets since they had the tendency to be more polydisperse. The same phenomenon could be seen using commercial pumps as it takes a moment to establish a constant flow of equal sized droplets.

[0111] It is to be understood that the embodiments of the invention disclosed are not limited to the particular structures, process steps, or materials disclosed herein, but are extended to equivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0112] Reference throughout this specification to one embodiment or an embodiment means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Where reference is made to a numerical value using a term, such as, e.g., about or substantially, the exact numerical value is also disclosed.

[0113] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another but are to be considered as separate and autonomous representations of the present invention.

[0114] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of “a” or “an”, that is, a singular form, throughout this document does not exclude a plurality.

[0115] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as the examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0116] While the above description is illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, except as by the claims set forth below.

Claims

CLAIMS1. A micro fluidic device (10) for providing a plurality of droplets for a droplet-based assay comprising: a body comprising: a plurality of first channels (14) comprising a first fluid traveling there through and one or more second channels (18) comprising a second fluid traveling there through, wherein the plurality of first channels (14) join together to form a combined flow channel (22) having a combined first fluid traveling there through; a plurality of first inlets (16) in fluid communication with the plurality of first channels (14) for input of the first fluid to the plurality of first channels (14); one or more second inlets (20) in fluid communication with the one or more second channels (18) for input of the second fluid to the one or more second channels (18); a droplet generation zone (24) configured to generate an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase of the first fluid from at least the combined first fluid and the second fluid flow.

2. The microfluidic device (10) of claim 1, further comprising a droplet collection chamber (28) in fluid communication with the droplet generation zone (24) for receipt of the plurality of the droplets generated in the droplet generation zone (24).

3. The micro fluidic device (10) of claim 1 or 2, further comprising an outlet (10) in fluid communication with at least one of the droplet generation zone (24) or the droplet collection chamber (28).

4. The micro fluidic device (10) of any of the preceding claims, wherein the emulsion is a water-in-oil emulsion, and wherein the first fluid comprises oil and the second fluid comprises an aqueous biological sample-containing fluid.

5. The micro fluidic device (10) of any of the preceding claims, wherein the emulsion is an oil-in-water emulsion, and wherein the first fluid comprises an aqueous fluid and the second fluid comprises oil comprising a biological sample therein.

6. The micro fluidic device (10) of any one of the preceding claims, wherein the body (12) is formed from a polymer having a degree of elasticity.

7. The microfluidic device (10) of claim 6, wherein the polymer comprises poly dimethylsiloxane (PDMS).

8. The micro fluidic device (10) of any one of the preceding claims, wherein the ratio of the number of the first channels (14) to the number of second channels (18) is from 1 :15 to 15:1, for example 1 : 1 to 7 : 1.

9. The micro fluidic device (10) of any one of the preceding claims, wherein the droplet generation zone (24) is defined by an intersection of the combined flow channel (22) and the one or more second channels (18).

10. The micro fluidic device (10) of any one of the preceding claims, wherein the first fluid or the second fluid comprises a nucleic acid material therein.

11. The micro fluidic device (10) of any one of claims 2 to 10, wherein the droplet collection chamber (28) is further configured for the amplification of the nucleic acid material in the plurality of droplets and subsequent measurement of a signal from the plurality of droplets.

12. The micro fluidic device (10) of claim 11, wherein the amplification comprises polymerase chain reaction (PCR) amplification or isothermal amplification.

13. The micro fluidic device (10) of any one of the preceding claims, further comprising one or more third channels (32) comprising a third fluid traveling there through and a plurality of third inlets (34) in fluid communication with the one or more third channels (32) for input of the third fluid to the one or more third channels (32), and wherein the third fluid comprises an aqueous reagent-containing fluid.

14. The micro fluidic device (10) of claim 13, wherein the aqueous reagent-containing fluid comprises a member selected from the group consisting of bar-coded beads, lysis buffer, and combinations thereof.T115. The micro fluidic device (10) of claim 13 or 14, wherein the one or more third channels intersect with the combined flow channel (22) or the one or more second channels (18) such that the emulsion is formed from the first, second, and third fluids.

16. The microfluidic device (10) of any of the preceding claims, further comprising a plug (40) disposed within one or more of the first inlets (16) or the second inlets (20).

17. A micro fluidic device (10) for providing a plurality of droplets for a droplet-based assay comprising: a body (12) comprising: a plurality of first inlets (16) in fluid communication with a plurality of corresponding oil-containing channels (14) formed in the body for delivery of oil to the plurality of corresponding oil-containing channels, and one or more second inlets (20) in fluid communication with one or more corresponding sample-containing channels (18) formed in the body for delivery of an aqueous biological sample-containing fluid to the one or more sample-containing channels (18), wherein the plurality of corresponding oil-containing channels (14) join together to form a combined oil flow channel having a combined oil flow; a droplet generation zone (24) configured to generate an emulsion from at least the combined oil flow and the aqueous biological sample-containing fluid, the emulsion comprising a dispersed phase comprising a plurality of droplets of the aqueous biological sample-containing fluid dispersed in a continuous phase comprising the oil; optionally, a droplet collection chamber (28) in fluid communication with the droplet generation zone (24) for receipt of the emulsion comprising the plurality of the droplets generated in the droplet generation zone (24); and an outlet (30) downstream from at least one of the droplet collection chamber (28) or the droplet generation zone (24).

18. A process for forming droplets for a droplet-based assay comprising: providing a micro fluidic device (10) comprising a body (12), the body (12) comprising a plurality of first inlets (16) in fluid communication with a plurality of first channels (14) formed in the body and one or more second inlets (20) in fluid communication with one or more second channels (18) formed in the body (12); inputting a first fluid through the plurality of first inlets (16) to the plurality of first channels (14) and inputting a second fluid through the one or more second inlets (20) to the one or more second channels (18), wherein the plurality of first channels (14) join together to form a combined flow channel (22) having a combined first fluid traveling there through; and forming an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase comprising the first fluid from at least the combined first fluid and the second fluid.

19. The process of claim 18, wherein the emulsion is a water-in-oil emulsion, and wherein the first fluid comprises oil and the second fluid comprises an aqueous biological samplecontaining fluid.

20. The process of any one of claims 18 to 19, wherein the ratio of the number of the first channels (14) to the number of the second channels (18) is from 1 :15 to 15:1, for example 1 :1 to 7:1.

21. The process of any one of claims 18 to 20, wherein the first fluid or the second fluid comprises a nucleic acid material therein.

22. The process of any one of claims 18 to 21, wherein the inputting of the first and second fluids to the micro fluidic device (10) is performed manually by a human user.

23. The process of claim 22, wherein the first and second fluids are manually inputted into the plurality of first channels (14) and the one or more second channels (18) from a handheld multichannel pipette, and wherein the handheld multichannel pipette provides a pressure effective to push the flow of the first and second fluids through the micro fluidic device (10).

24. The process of any one of claims 18 to 23, further comprising, prior to inputting the first fluid through the plurality of first inlets (16) to the plurality of first channels (14) and inputting the second fluid through the one or more second inlets (20) to the one or more second channels (18), pre-filling the plurality of first channels (18) and the one or more second channels (18) with the first fluid.

25. The process of any one of claims 18 to 24, further comprising plugging one or more of the first inlets (16) or the second inlets (20) with a plug (40).

26. A method of performing a droplet-based assay comprising: forming the emulsion according to any one of claims 18 to 25, wherein the dispersed phase comprises a biological sample comprising at least one nucleic acid having a nucleic acid sequence of interest; collecting the plurality of droplets within a droplet collection chamber (28) of the micro fluidic device (10); within the droplet collection chamber (28), amplifying the at least one nucleic acid comprising the nucleic acid sequence of interest within the plurality of droplets; and within droplet collection chamber (28), detecting the presence of or a property of the droplets comprising the nucleic acid sequence of interest and / or removing the plurality of droplets from the droplet collection chamber and sequencing the nucleic acid sequence of interest.

27. The method according to claim 25, wherein the amplifying step comprises droplet digital PCR (ddPCR), wherein the second fluid further comprises a PCR reaction mixture, and wherein the PCR reaction mixture comprises a reagent for detecting amplified DNA, for example, an intercalating dye.

28. The method according to claim 26 or 27, wherein the amplifying step comprises droplet digital isothermal amplification, wherein the second fluid further comprises an isothermal amplification reaction mixture, and wherein the isothermal amplification reaction mixture comprises a reagent for detecting amplified DNA, for example, an intercalating dye.

29. The method according to any one of the claims 26 to 28, wherein the intercalacting dye is a fluorescent dye and the detecting step is performed using fluorescent spectroscopy or fluorescent microscopy.

30. The method according to any of the claims 26 to 29, wherein the dispersed phase comprises cells, for example, cancer cells, B-cells, or bacterial cells.

31. The method according to claim 30, wherein the emulsion is formed from the first fluid, the second fluid, and a third fluid, wherein the third fluid comprises at least one of lysis buffer or bar-coded beads, and wherein cells are subjected to lysis before or during the amplifying step.

32. The method according to claim 29, wherein the bar-coded beads identify individual cells from which the nucleic acid sequence of interest originates.

33. A process for forming droplets for a droplet-based assay comprising: providing a micro fluidic device (10) comprising a body (12), the body (12) comprising a plurality of first inlets (16) in fluid communication with a plurality of first channels (14) formed in the body and one or more second inlets (20) in fluid communication with one or more second channels (18) formed in the body (12); inputting a first fluid through the plurality of first inlets (16) to the plurality of first channels (14) and inputting a second fluid through the one or more second inlets (20) to the one or more second channels (18), wherein the plurality of first channels (14) join together to form a combined flow channel (22) having a combined first fluid traveling there through; and forming an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase comprising the first fluid from at least the combined first fluid and the second fluid, wherein the process further comprises varying a property of the formed droplets of the second fluid by blocking input to one or more of the first inlets (16) or second inlets (20) with one or more plugs (40).

34. The process of claim 33, wherein the property is a size or a shape of the formed droplets.

35. A micro fluidic device (10) for providing a plurality of droplets for a droplet-based assay comprising: a body comprising: a plurality of first channels (14) comprising a first fluid traveling there through and one or more second channels (18) comprising a second fluid traveling there through, wherein the plurality of first channels (14) join together to form a combined flow channel (22) having a combined first fluid traveling there through; a plurality of first inlets (16) in fluid communication with the plurality of first channels (14) for input of the first fluid to the plurality of first channels (14); one or more second inlets (20) in fluid communication with the one or more second channels (18) for input of the second fluid to the one or more second channels (18); a droplet generation zone (24) configured to generate an emulsion comprising a dispersed phase comprising a plurality of droplets of the second fluid dispersed in a continuous phase of the first fluid from at least the combined first fluid and the second fluid flow. one or more plugs (40) disposed within one or more of the first inlets (16) or the second inlets (20), wherein the plug (40) is effective to vary a property of the formed droplets of the second fluid by blocking input to one or more of the first inlets (16) or second inlets (20).

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