Methods and systems for nucleic acid analysis and quantification

Abstract

To provide a system for analyzing nucleic acid molecules.SOLUTION: A system comprises: a support unit configured to accept a device comprising a plurality of compartments, one compartment of the plurality of compartments being configured to permit gas flow from one compartment to an environment external to the compartment through the at least one gas permeable barrier, and the support unit being configured to contain a nucleic acid sample comprising nucleic acid molecules; a thermal unit in thermal communication with one compartment, the thermal unit being configured to subject the nucleic acid sample to heating; a detector operably coupled to one compartment; and one or more computer processors operably coupled to the thermal unit and the detector.SELECTED DRAWING: Figure 22A

Description

Technical Field

[0001] Cross-reference This invention claims the benefit of U.S. Provisional Patent Application No. 62 / 423,601, filed on November 17, 2016, which is hereby incorporated by reference in its entirety.

[0002] <Statement of Government Rights in the Patent> This invention was made with government support under Small Business Innovation Research Program grant numbers 1R43OD023028-01 and 1R43HG009640 awarded by the National Institutes of Health. The U.S. government has certain rights in this invention.

Background Art

[0003] A microfluidic device is a device that includes a structure for handling fluids on a small scale. Typically, a microfluidic device operates on the sub-millimeter scale and handles micro-liters, nano-liters, or even smaller amounts of fluid. In a microfluidic device, the main fouling mechanism is air trapped inside the micro-structure, i.e., air bubbles. This can be particularly problematic when using thermoplastic materials to fabricate microfluidic structures because the gas permeability of thermoplastics is very low.

[0004] To avoid fouling by trapped air, previous microfluidic structures have used either a simple linear channel design or a branched channel design using thermoplastic materials, or have manufactured the device using a material with a high gas permeability such as an elastomer. However, simple designs limit the potential functionality of microfluidic devices, and elastomer materials are difficult and expensive to manufacture, especially on a large scale.

[0005] One use of the microfluidic structure is in digital polymerase chain reaction (dPCR). dPCR dilutes a nucleic acid sample to a nucleic acid template of 1 or less in each section of a microfluidic structure that provides an array of many sections, and performs a PCR reaction across the array. By counting the sections in which the nucleic acid template has been successfully PCR amplified and applying Poisson statistics to the results, the target nucleic acid is quantified. Different from general quantitative real-time PCR (qPCR), in which the template is quantified by comparing the ratio of PCR amplification of an unknown sample to the ratio of a set of known qPCR standards, dPCR has been found to exhibit higher sensitivity, higher accuracy, and greater reproducibility.

[0006] For genome researchers and clinicians, dPCR is particularly powerful in the detection of rare mutations, the quantification of copy number polymorphisms, and the quantification of next-generation sequencing libraries. Potential uses in a clinical setting for liquid cytology using the quantification of cell-free DNA and viral loads further enhance the value of the dPCR technology. Existing dPCR solutions have used elastomeric valve arrays, silicon through-hole approaches, and microfluidic encapsulation of droplets in oil. Despite the increasing number of available dPCR platforms, dPCR has been at a disadvantage when compared to older qPCR technologies that rely on counting the number of PCR amplification cycles. The combination of throughput, ease of use, performance, and cost are the main barriers to adoption in the dPCR market. Summary of the Invention

[0007] Methods and systems are provided herein that can serve to amplify and quantify nucleic acids and to detect the presence or absence of targets, such as pathogens (e.g., bacteria), for example, actual or suspected targets. The present disclosure provides methods, systems, and devices that can enable sample preparation, sample amplification, and sample analysis through the use of digital polymerase chain reaction (dPCR). Thereby, nucleic acids can be amplified and quantified at low cost and with low complexity compared to other systems and methods.

[0008] The methods and systems of the present disclosure can use high-resolution melting (HRM) analysis to detect nucleic acid (e.g., deoxyribonucleic acid, DNA) sequence variants. The HRM methods of the present disclosure can generate sequence-dependent melting curves using single nucleotide resolution that are all completed by seamless integration using nucleic acid amplification such as polymerase chain reaction (PCR), optionally without a post-amplification processing step. In conjunction with broad-range PCR, the HRM methods and systems of the present disclosure can greatly expand the breadth of sequence variants that can be identified.

[0009] Various limitations associated with currently available melting curve approaches are recognized herein. Conventional bulk PCR / HRM assay designs can also be limited in addressing some important needs in genetic and epigenetic analysis. One important limitation is improper sensitivity due to interference from PCR inhibitors or excessive background human DNA. Another limiting need is the inability to analyze multiple sequence variants in a heterogeneous population in detail and accurately quantify each variant.

[0010] The need to determine the absolute amount of rare allelic variants and the relative allelic ratios is recognized herein. In infectious diseases, accurate detection and identification of low pathogen loads, as well as quantitative separation of mixed microbial populations that distinguish infection from colonization or contamination, further emphasizes the importance of quantitatively discriminating variants co-existing in a sample.

[0011] The present disclosure provides a digital HRM analysis platform that integrates HRM analysis and absolute quantification in digital polymerase chain reaction (dPCR). This can provide a detailed analysis at the single-molecule level and in a high-throughput manner.

[0012] In one aspect, the present disclosure provides a method for analyzing a plurality of nucleic acid molecules, including providing a device that includes a plurality of compartments, where at least a subset of the plurality of compartments includes a plurality of nucleic acid molecules, and each compartment of at least a subset of the plurality of compartments is configured to allow gas flow from at least a subset of the plurality of compartments to an environment external to at least a subset of the plurality of compartments through at least one barrier that separates at least a subset of the plurality of compartments from the external environment; collecting signals from at least a subset of the plurality of compartments while exposing at least a subset of the plurality of compartments to a controlled heat; and processing the collected signals to yield data indicative of a melting point of at least a subset of the plurality of nucleic acid molecules in at least a subset of the plurality of compartments.

[0013] In some embodiments, the method further comprises performing a nucleic acid amplification reaction on a nucleic acid sample under conditions sufficient to provide a plurality of nucleic acid molecules as amplification products of the nucleic acid sample, before providing a device comprising the plurality of nucleic acid molecules. In some embodiments, the method further comprises filling at least a subset of a plurality of compartments with a plurality of nucleic acid samples, before performing the nucleic acid amplification reaction. In some embodiments, the nucleic acid amplification reaction is performed in at least a subset of the plurality of compartments. In some embodiments, performing a nucleic acid amplification reaction on a nucleic acid sample comprises amplifying at least a portion of an internally transcribed spacer region of at least a subset of the nucleic acid molecules of the nucleic acid sample. In some embodiments, the nucleic acid amplification reaction uses one or more reagents selected from the group consisting of primers, deoxyribonucleotides, buffers, cofactors, intercalating dyes, and polymerases. In some embodiments, the one or more reagents comprise a fluorophore or a fluorescent label. In some embodiments, the method further comprises contacting at least a subset of the nucleic acid molecules of the nucleic acid sample with an intercalating dye, before performing the nucleic acid amplification reaction on the nucleic acid sample.

[0014] In some embodiments, while at least a subset of the plurality of compartments is exposed to controlled heat, collection of signals from at least a subset of the plurality of compartments is performed at multiple time points.

[0015] In some embodiments, while at least a subset of the plurality of compartments is exposed to controlled heat, collection of signals from at least a subset of the plurality of compartments comprises imaging at least a subset of the plurality of compartments to collect the signals.

[0016] In some embodiments, processing the collected signals comprises using the signals to generate signal-versus-temperature data for at least a subset of the plurality of nucleic acid molecules in at least a subset of the plurality of compartments.

[0017] In some embodiments, the plurality of nucleic acid molecules are derived from a sample that contains or is suspected of containing a pathogen. In some embodiments, the pathogen is at least one bacterium. In some embodiments, the at least one bacterium is selected from the group consisting of Bacillus anthracis, Bacillus cereus, Bacillus halodurans, Bacillus mycoides, Bacillus polymyxa, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus xylosus, Propionibacterium acnes, Enterococcus faecalis, Actinobacteria, Alphaproteobacteria, Bacteroidetes, Betaproteobacteria, Chlamydia, Epsilonproteobacteria, Firmicutes, Gammaproteobacteria, Spirochaetales, and Tenericutes. In some embodiments, the method further comprises isolating or extracting the plurality of nucleic acid molecules or a subset thereof from at least one bacterium prior to providing a device that includes the plurality of nucleic acid molecules. In some embodiments, the method further comprises using data indicative of a melting point to determine the presence or absence of a pathogen in at least a subset of the plurality of compartments.

[0018] In some embodiments, the sample from which the plurality of nucleic acid molecules are derived is a biological sample. In some embodiments, the biological sample includes a body fluid selected from the group consisting of blood, urine, semen, mucus, and saliva. In other embodiments, the sample from which the plurality of nucleic acid molecules are derived is an environmental sample.

[0019] In some embodiments, providing the device further comprises filling the plurality of compartments with the plurality of nucleic acid molecules, wherein during filling, gas in at least a subset of the plurality of compartments is subject to a flow from at least a subset of the plurality of compartments to an environment external to at least a subset of the plurality of compartments.

[0020] In some embodiments, the barrier comprises a polymeric material. In some embodiments, the polymeric material is a thermoplastic material. In some embodiments, the barrier is at least partially permeable to gas under a pressure differential applied across the barrier. In some embodiments, the barrier is substantially optically transparent. In some embodiments, the barrier has a thickness of from about 50 μm to about 200 μm.

[0021] In some embodiments, the device includes at least one inlet, at least one outlet, and at least one microchannel including a plurality of suction openings, wherein each of at least a subset of the plurality of compartments is in fluid communication with the at least one microchannel by the plurality of suction openings.

[0022] In some embodiments, the plurality of compartments includes from about 1,000 to about 20,000 compartments.

[0023] In some embodiments, the plurality of nucleic acid molecules are deoxyribonucleic acid molecules. In other embodiments, the plurality of nucleic acid molecules are ribonucleic acid molecules.

[0024] In another aspect, the present disclosure provides a method for analyzing a plurality of nucleic acid molecules, the method comprising: providing a device including a plurality of compartments, wherein at least a subset of the plurality of compartments includes a plurality of nucleic acid molecules, and wherein each compartment of at least a subset of the plurality of compartments is configured to allow gas flow from at least a subset of the plurality of compartments to an external environment of at least a subset of the plurality of compartments through at least one barrier separating at least a subset of the plurality of compartments from the external environment; exposing at least a subset of the plurality of compartments to conditions sufficient to perform a nucleic acid amplification reaction using the plurality of nucleic acid molecules to generate amplification products from at least a subset of the plurality of nucleic acid molecules; collecting signals from at least a subset of the plurality of compartments over a plurality of time points while exposing at least a subset of the plurality of compartments to the conditions; and processing the signals to determine the number of nucleic acid molecules in at least a subset of the plurality of compartments.

[0025] In some embodiments, subjecting at least a subset of the plurality of compartments to conditions sufficient to perform a nucleic acid amplification reaction includes performing a thermal cycle, and collecting a signal includes collecting more than once per thermal cycle a signal from each of at least a subset of the plurality of compartments. In some embodiments, the thermal cycle includes a denaturation step, an extension step, and an annealing step. In some embodiments, the thermal cycle is performed using a flat block thermal cycler.

[0026] In some embodiments, the nucleic acid amplification reaction uses one or more reagents selected from the group consisting of primers, deoxyribonucleotides, buffers, cofactors, intercalating dyes, and polymerases. In some embodiments, the one or more reagents include a fluorophore or a fluorescent label. In some embodiments, collecting a signal from at least a subset of the plurality of compartments over a plurality of time points includes imaging at least a subset of the plurality of compartments to collect the signal. In some embodiments, at least a subset of the plurality of compartments are imaged simultaneously. In some embodiments, the imaging is performed using a detector that detects fluorescence emission at two or more wavelengths. In some embodiments, processing the signal to determine the number of nucleic acid molecules in at least a subset of the plurality of compartments includes determining the light intensity of each of at least a subset of the plurality of compartments, where the light intensity is proportional to the amount of amplification product in each of at least a subset of the plurality of compartments.

[0027] In some embodiments, providing a device that includes a plurality of nucleic acid molecules further includes loading the plurality of nucleic acid molecules into the plurality of compartments, where during loading, gas in at least a subset of the plurality of compartments is subject to a flow from at least a subset of the plurality of compartments to an environment external to at least a subset of the plurality of compartments.

[0028] In some embodiments, the barrier comprises a polymeric material. In some embodiments, the polymeric material is a thermoplastic material. In some embodiments, the barrier is at least partially permeable to gas under a pressure differential applied across the barrier. In some embodiments, the barrier is substantially optically transparent. In some embodiments, the barrier has a thickness of from about 50 μm to about 200 μm.

[0029] In some embodiments, the device includes at least one inlet, at least one outlet, and at least one microchannel including a plurality of suction openings, wherein each of at least a subset of the plurality of compartments is in fluid communication with the at least one microchannel by the plurality of suction openings.

[0030] In some embodiments, the plurality of compartments includes from about 1,000 to about 20,000 compartments.

[0031] In some embodiments, the plurality of nucleic acid molecules are deoxyribonucleic acid molecules. In other embodiments, the plurality of nucleic acid molecules are ribonucleic acid molecules.

[0032] In a further aspect, the present disclosure provides a system for analyzing a plurality of nucleic acid molecules, the system comprising: a support unit configured to receive a device comprising a plurality of compartments, wherein each compartment of at least a subset of the plurality of compartments is configured to allow gas flow from at least a subset of the plurality of compartments to an external environment of at least a subset of the plurality of compartments through at least one barrier that separates at least a subset of the plurality of compartments from the external environment; a detector configured to collect signals from at least a subset of the plurality of compartments over a plurality of time points; and one or more computer processors operably coupled to the detector, wherein the one or more computer processors are: (i) exposing at least a subset of the plurality of compartments to conditions sufficient to perform a nucleic acid amplification reaction using a plurality of nucleic acid molecules to generate amplification products from at least a subset of the plurality of nucleic acid molecules; (ii) receiving signals collected by the detector from at least a subset of the plurality of compartments over a plurality of time points while the at least a subset of the plurality of compartments is being exposed to the conditions in (i); and (iii) processing the signals to determine the number of nucleic acid molecules in at least a subset of the plurality of compartments, including one or more computer processors programmed individually or collectively.

[0033] In some embodiments, the system further comprises a fluid flow device configured to direct a plurality of nucleic acid molecules into the plurality of compartments. In some embodiments, the one or more computer processors are programmed individually or collectively to direct the fluid flow device to fill the plurality of compartments with the plurality of nucleic acid molecules.

[0034] In another aspect, the present disclosure provides a system for analyzing a plurality of nucleic acid molecules, the system comprising: a support unit configured to receive a device including a plurality of compartments, wherein each compartment of at least a subset of the plurality of compartments is configured to allow gas flow from at least a subset of the plurality of compartments to an external environment of at least a subset of the plurality of compartments through at least one barrier that separates at least a subset of the plurality of compartments from the external environment; a heating unit configured to expose at least a subset of the plurality of compartments to controlled heating; a detector configured to collect signals from at least a subset of the plurality of compartments; and one or more computer processors operatively coupled to the heating unit and the detector, wherein the one or more computer processors are: (i) directed to the heating unit to expose at least a subset of the plurality of compartments to controlled heating; (ii) receive signals collected from at least a subset of the plurality of compartments by the detector while at least a subset of the plurality of compartments is exposed to controlled heating; and (iii) programmed, either individually or collectively, to process the signals collected in (ii) to provide data indicative of the melting points of at least a subset of the plurality of nucleic acid molecules in at least a subset of the plurality of compartments.

[0035] In some embodiments, the system further includes a fluid flow device configured to direct a plurality of nucleic acid molecules into the plurality of compartments. In some embodiments, the one or more computer processors are programmed, either individually or collectively, to direct the fluid flow device to fill the plurality of compartments with the plurality of nucleic acid molecules.

[0036] Another aspect of the present disclosure provides a non-transitory computer-readable medium including machine-executable code that, when executed by one or more computer processors, performs any of the methods described above or elsewhere in this specification.

[0037] Other aspects of the disclosure provide a system including one or more computer processors and computer memory coupled thereto. The computer memory includes machine-executable code that, when executed by the one or more computer processors, performs either the above method or any of the methods described elsewhere in this specification.

[0038] Further aspects and advantages of the disclosure will become apparent to those of ordinary skill in the art from the following detailed description, which, by way of example, shows and describes only exemplary embodiments of the disclosure. As will be understood from the following description, the disclosure may have other and different embodiments and its details may be modified in various obvious respects all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not as restrictive.

[0039] <Incorporation by reference> All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Brief Description of the Drawings

[0040] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments of the invention, and to the accompanying drawings (or “FIGURES” herein).

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[0041] Although various embodiments of the present invention are shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will occur to those skilled in the art without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be utilized.

[0042] As used herein, the terms "amplify" and "amplifying" are used interchangeably and generally refer to generating one or more copies or "amplification products" of a nucleic acid. For example, such amplification may use polymerase chain reaction (PCR) or isothermal amplification.

[0043] As used herein, the term "nucleic acid" generally refers to a polymeric form of nucleotides of any length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, or 1000 nucleotides), either deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may contain one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. Nucleotides may contain A, C, G, T, or U, or variants thereof. Nucleotides may contain any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunit that is specific for one of the more complementary A, C, G, T, or U, or that is complementary to a purine (i.e., A or G, or variants thereof) or a pyrimidine (i.e., C, T, or U, or variants thereof). In some examples, nucleic acids may be single-stranded or double-stranded, and in some cases, nucleic acid molecules may be circular. Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids may include gene or gene fragment coding or non-coding regions, loci defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, cDNA, recombinant nucleic acids, branched nucleic acids, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acids may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.

[0044] As used herein, the terms "polymerase chain reaction reagent" or "PCR reagent" are used interchangeably and generally refer to a composition containing the reagents necessary to complete a nucleic acid amplification reaction (e.g., DNA amplification). Non-limiting examples of such reagents include a primer set or priming site (e.g., a nick) having specificity for a target nucleic acid, a polymerase, an appropriate buffer, cofactors (e.g., divalent and monovalent cations), dNTPs, and other enzymes. PCR reagents may also include probes, indicators, and molecules containing probes and indicators.

[0045] As used herein, the term "probe" generally refers to a molecule containing a detectable moiety, the presence or absence of which can be used to detect the presence or absence of an amplification product. Non-limiting examples of detectable moieties can include radiolabels, stable isotope labels, fluorescent labels, chemiluminescent labels, enzyme labels, colorimetric labels, or any combination thereof.

[0046] As used herein, the term "extension" generally refers to the incorporation of nucleotides into a nucleic acid in a template directed fashion. Extension may occur with the aid of an enzyme. For example, extension may occur with the aid of a polymerase. Conditions under which extension can occur include an "extension temperature", which generally refers to the temperature at which extension is achieved, and an "extension time", which generally refers to the amount of time allotted for extension to occur.

[0047] As used herein, the term "indicator molecule" generally refers to a molecule containing a detectable moiety, the presence or absence of which can be used to indicate sample dispensing. Non-limiting examples of detectable moieties can include radiolabels, stable isotope labels, fluorescent labels, chemiluminescent labels, enzyme labels, colorimetric labels, or any combination thereof.

[0048] As used herein, the term "sample" generally refers to a sample that contains or is suspected of containing nucleic acid molecules, as used in the specification. Nucleic acid molecules can be present in or derived from, for example, samples of cells such as bacteria or in a living body. For example, the sample may be a biological sample containing one or more nucleic acid molecules. A biological sample can be obtained (e.g., extracted or isolated) from or can include one or more components selected from the group consisting of blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears. A biological sample can be a liquid sample or a tissue sample (e.g., a skin sample). The sample can be obtained from a cell-free body fluid such as whole blood. A sample from a cell-free body fluid can contain cell-free DNA and / or cell-free RNA. The sample can include circulating tumor cells. The sample is obtained from a subject, and the analysis of the nucleic acids contained therein is used for diagnostic purposes. Alternatively, the sample can be an environmental sample (e.g., soil, waste, ambient air, etc.), an industrial sample (e.g., a sample from any industrial process), and a food sample (e.g., dairy products, vegetable products, and meat products).

[0049] As used herein, the term "fluid" generally refers to a liquid or a gas. A liquid cannot maintain a defined shape and flows during an observable time frame to fill the container into which it is placed. Thus, a fluid can have an appropriate viscosity that allows it to flow. If more than one fluid is present, each fluid can be selected essentially independently of any fluid (liquid, gas, etc.) by one of ordinary skill in the art.

[0050] As used herein, the term "dispense" generally refers to dividing or dispersing into parts and sharing. For example, a dispensed sample is a sample isolated from other samples. Examples of structures that enable dispensing of samples include wells and microchambers.

[0051] As used herein, the term "microfluidic" generally refers to a chip, area, device, article, or system that includes at least one microchannel, a plurality of suction openings, and an array of microchambers. The microchannel can have a cross-sectional dimension of about 10 millimeters (mm) or less, about 5 mm or less, about 4 mm or less, about 3 mm or less, about 2 mm or less, about 1.5 mm or less, about 1 mm or less, about 750 micrometers (μm) or less, about 500 μm or less, about 250 μm or less, about 100 μm or less, or smaller.

[0052] As used herein, the term "depth" generally refers to the measured distance from the bottom of a microchannel, suction opening, or compartment (e.g., microchamber) to the thin film that caps the microchannel, the plurality of suction openings, and the array of compartments (e.g., microchambers).

[0053] As used herein, the terms "cross-section" or "cross-sectional" are used interchangeably and generally can refer to the dimension or area of a microchannel or suction opening that is substantially perpendicular to the longitudinal dimension of the feature.

[0054] The present disclosure includes microfluidic devices formed from polymeric materials such as thermoplastic materials, and includes the use of systems that incorporate thin films to allow pressurized gas release while functioning as a gas barrier when the pressure is released. The use of thermoplastic plastics to form microfluidic structures enables the use of inexpensive and highly scalable injection molding processes, and the thin films can provide the ability to release by pressurization and avoid fouling problems present in some microfluidic structures that do not incorporate such thin films.

[0055] One use for this structure is in a microfluidic design that incorporates an array of dead-end microchambers, connected by microchannels and formed, for example, from a thermoplastic. This design can be used, for example, in digital PCR (dPCR) or quantitative dPCR (qPCR) applications to dispense reagents to the array of microchambers, thereby for quantifying nucleic acids or for analyzing the amount and properties of nucleic acids dispensed in the array of microchambers in high-resolution melting (HRM) analysis.

[0056] <Microfluidic device for analyzing a sample> In one aspect, the present disclosure provides a method of using a system that includes a microfluidic device for analyzing and / or processing a sample. The device can include microchannels connected to an inlet and an outlet. The microfluidic device can also include a plurality of microchambers and a plurality of suction openings. The plurality of microchambers can be connected to the microchannels by the plurality of suction openings. The microfluidic device can include a thermoplastic thin film that caps and seals (e.g., hermetically seals) the microchannels, the microchambers, and the suction openings. The thermoplastic thin film can be at least partially gas permeable when a pressure differential is applied across the thermoplastic thin film.

[0057] A and B of FIG. 1 show examples of a microfluidic structure according to certain embodiments of the present disclosure. A of FIG. 1 shows an exemplary microfluidic device as viewed from above. The microfluidic device includes a microchannel (110) having an inlet (120) and an outlet (130). The microchannel is connected to a plurality of suction openings (101B)-(109B). The plurality of suction openings connect the microchannel to a plurality of microchambers (101A)-(109A). B of FIG. 1 shows a cross-sectional view of a single microchamber along the dashed line marked A-A'. The single microchamber (101A) is connected to the microchannel (110) by a suction opening (101B). The microfluidic device body (140) may be formed from a rigid plastic material (e.g., a thermoplastic material). The micro-structure of the microfluidic device may be capped and sealed by a thin film (150). The thin film (150) may be non-gas permeable when a small pressure difference is applied across the film and gas permeable when a large pressure difference is applied across the film. This may enable gas release through the thin film (150) when pressure is applied to the internal structure of the microfluidic device. Alternatively, gas release may occur when a vacuum is applied outside the microfluidic device.

[0058] The gas permeability of the thin film can be caused by high pressure. The pressure-induced gas-permeable thin film covers an array of microchambers or a subset thereof, and the microchannels and suction openings may be covered by a non-gas-permeable film. Alternatively, the pressure-induced gas-permeable thin film covers an array of microchambers or a subset thereof, and the suction openings, and the microchannels may be covered by a non-gas-permeable film. Alternatively, the pressure-induced gas-permeable thin film may cover an array of microchambers or a collective portion thereof, the suction openings, and the microchannels. The thickness of the thin film may be about 500 micrometers (μm) or less, about 250 μm or less, about 200 μm or less, about 150 μm or less, about 100 μm or less, about 75 μm or less, about 50 μm or less, about 25 μm or less, or thinner. The thickness of the thin film can be from about 0.1 μm to about 200 μm, or from about 0.5 μm to about 150 μm. For example, the thickness of the thin film can be from about 50 μm to about 200 μm. In some embodiments, the thickness of the thin film can be from about 100 μm to about 200 μm. For example, the thickness of the thin film is from about 100 μm to about 150 μm. In one example, the thin film is approximately 100 μm thick. The thickness of the film can be selected, inter alia, by the manufacturability of the thin film, the air permeability of the thin film, the volume of each compartment from which gas is released, the available pressure, and / or the desired time to complete the suction process.

[0059] A microfluidic device may include a single array of microchambers. Alternatively, a microfluidic device may include a plurality of arrays of microchambers, and each array of microchambers is isolated from other arrays. The arrays of microchambers may be arranged in a row, in a grid configuration, in an alternating pattern, or in any other configuration. A microfluidic device may have at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 40, at least about 50, or even more arrays of microchambers. The arrays of microchambers may be identical. A microfluidic device may include a plurality of arrays of non-identical microchambers. The arrays of microchambers may all have the same external dimensions (i.e., the length and width of the array of microchambers encompassing all features of the array of microchambers), or the arrays of microchambers may have various external dimensions.

[0060] The array of microchambers may have a width of up to about 100 mm, about 75 mm, about 50 mm, about 40 mm, about 30 mm, about 20 mm, about 10 mm, about 8 mm, about 6 mm, about 4 mm, about 2 mm, about 1 mm, or less. The array of microchambers may have a length of up to about 50 mm, about 40 mm, about 30 mm, about 20 mm, about 10 mm, about 8 mm, about 6 mm, about 4 mm, about 2 mm, 1 mm, or less. The width may be from about 1 mm to 100 mm, or from 10 mm to 50 mm. The length may be from about 1 mm to 50 mm, or from 5 mm to 20 mm.

[0061] In some embodiments, the array of microchambers can have a width of about 100 mm and a length of about 40 mm. In some embodiments, the array of microchambers can have a width of about 80 mm and a length of about 30 mm. In some embodiments, the array of microchambers can have a width of about 60 mm and a length of about 25 mm. In some embodiments, the array of microchambers can have a width of about 40 mm and a length of about 15 mm. In some embodiments, the array of microchambers can have a width of about 30 mm and a length of about 10 mm. In some embodiments, the array of microchambers can have a width of about 20 mm and a length of about 8 mm. In some embodiments, the array of microchambers can have a width of about 10 mm and a length of about 4 mm. The external dimensions may be determined by the total number of desired microchambers, the dimensions of each microchamber, and the minimum distance between each microchamber with respect to manufacturability.

[0062] The microchannels can be substantially parallel or substantially perpendicular to the long dimension of the microfluidic device. Alternatively, the microchannels may be neither substantially parallel nor substantially perpendicular to the long dimension of the microfluidic device. The angle between the microchannels and the long dimension of the microfluidic device can be at least about 5°, at least about 10°, at least about 15°, at least about 20°, at least about 30°, at least about 40°, at least about 50°, at least about 60°, at least about 70°, at least about 90°, at least about 100°, at least about 110°, at least about 120°, at least about 130°, at least about 140°, at least about 150°, at least about 160°, or at least about 170°. The microchannels can be a single long channel. The microchannels can have bends, curves, or angles. The microchannels can have a length dimension of 100 mm or less, about 75 mm or less, about 50 mm or less, about 40 mm or less, about 30 mm or less, about 20 mm or less, about 10 mm or less, about 8 mm or less, about 6 mm or less, about 4 mm or less, about 2 mm or less, or less. The length of the microchannels can be limited by the external length or width of the microfluidic device. The microchannels can have a depth of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, or less. The microchannels can have a cross-sectional dimension (e.g., width) of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 75 μm or less, about 50 μm or less, about 40 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, or less.

[0063] In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 100 μm in width and approximately 10 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 80 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 60 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 40 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 20 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 10 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 80 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 60 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 40 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 20 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the microchannel may be approximately 10 μm in width and approximately 10 μm in depth. The cross-sectional shape of the microchannel is not limited, and can be any suitable cross-sectional shape including, but not limited to, circular, elliptical, triangular, square, or rectangular. The cross-sectional area of the microchannel can be constant along the length of the microchannel. Alternatively, or additionally, the cross-sectional area of the microchannel can vary along the length of the microchannel.The cross-sectional area of the microchannel may vary between about 50% and 150%, about 60% and 125%, about 70% and 120%, about 80% and 115%, about 90% and 110%, about 95% and 100%, or about 98% and 102%. The cross-sectional area of the microchannel is about 10,000 square micrometers (μm²). 2 or less, about 7,500 μm 2 or less, about 5,000 μm 2 or less, about 2,500 μm 2 or less, about 1,000 μm 2 or less, about 750 μm 2 or less, about 500 μm 2 or less, about 400 μm 2 or less, about 300 μm 2 or less, about 200 μm 2 or less, about 100 μm 2 or less, or even less than that may be acceptable.

[0064] The microchannel may have a single inlet and a single outlet. Alternatively, the microchannel may have multiple inlets, multiple outlets, or multiple inlets and multiple outlets. The inlet and the outlet may have the same diameter, or they may have different diameters. The inlet and the outlet may have a diameter of about 2.5 millimeters (mm) or less, about 2 mm or less, about 1.5 mm or less, about 1 mm or less, about 0.5 mm or less, or less than that.

[0065] The array of microchambers may have at least about 1,000 microchambers, at least about 5,000 microchambers, at least about 10,000 microchambers, at least about 20,000 microchambers, at least about 30,000 microchambers, at least about 40,000 microchambers, at least about 50,000 microchambers, at least about 100,000 microchambers, or more microchambers. In some examples, the microfluidic device can have from about 10,000 to about 30,000 microchambers. In some examples, the microfluidic device can have from about 15,000 to about 25,000 microchambers. The microchambers can be cylindrical, hemispherical, or a combination of cylindrical and hemispherical. The microchambers may have a diameter of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 30 μm or less, about 15 μm or less, or less. The depth of the microchambers may be about 500 μm or less, about 250 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 30 μm or less, about 15 μm or less, or less. In some examples, the microchambers can have a diameter of about 30 μm and a depth of about 100 μm. In some examples, the microchambers can have a diameter of about 35 μm and a depth of about 80 μm. In some examples, the microchambers can have a diameter of about 40 μm and a depth of about 70 μm. In some examples, the microchambers can have a diameter of about 50 μm and a depth of about 60 μm. In some examples, the microchambers can have a diameter of about 60 μm and a depth of about 40 μm. In some examples, the microchambers can have a diameter of about 80 μm and a depth of about 35 μm. In some examples, the microchambers can have a diameter of about 100 μm and a depth of about 30 μm. The microchambers and microchannels can have the same depth. Alternatively, the microchambers and microchannels can have different depths.

[0066] The length of the suction opening may be constant. Alternatively, the length of the suction opening may vary. The suction opening may have a length dimension of about 150 μm or less, about 100 μm or less, about 50 μm or less, about 25 μm or less, about 10 μm or less, about 5 μm or less, or less than that. The depth of the suction opening may be about 50 μm or less, about 25 μm or less, about 10 μm or less, about 5 μm or less, or less than that. The suction opening may have a cross-sectional width of about 50 μm or less, about 40 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, about 5 μm or less, or less than that.

[0067] In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 30 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 10 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 50 μm in width and approximately 5 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 40 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 30 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 20 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 10 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 5 μm in width and approximately 50 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 40 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 30 μm in width and approximately 30 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 20 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 10 μm in width and approximately 10 μm in depth. In some examples, the dimensions of the cross-section of the suction opening can be approximately 5 μm in width and approximately 5 μm in depth. The cross-sectional shape of the suction opening is not limited, and can be any suitable cross-sectional shape including, but not limited to, circular, elliptical, triangular, square, or rectangular. The cross-sectional area of the suction opening can be constant along the length of the suction opening. Alternatively, or additionally, the cross-sectional area of the suction opening may vary along the length of the suction opening. The cross-sectional area of the suction opening may be larger at the connection to the microchannel than the cross-sectional area of the suction opening at the connection to the microchamber. Alternatively, the cross-sectional area of the suction opening at the connection to the microchamber may be larger than the cross-sectional area of the suction opening at the connection to the microchannel.The cross-sectional area of the suction opening may vary between about 50% and 150%, about 60% and 125%, about 70% and 120%, about 80% and 115%, about 90% and 110%, about 95% and 100%, or about 98% and 102%. The cross-sectional area of the suction opening is about 2,500 μm. 2 Hereinafter, about 1,000 μm 2 Hereinafter, about 750 μm 2 Hereinafter, about 500 μm 2 Hereinafter, about 250 μm 2 Hereinafter, about 100 μm 2 Hereinafter, about 75 μm 2 Hereinafter, about 50 μm 2 Hereinafter, about 25 μm 2 Hereinafter, or less than that may also be acceptable. The cross-sectional area of the suction opening at the connection to the microchannel may be less than or equal to the cross-sectional area of the microchannel. The cross-sectional area of the suction opening at the connection to the microchannel may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, about 5% or less, about 1% or less, or about 0.5% or less of the cross-sectional area of the microchannel.

[0068] The suction opening may be substantially perpendicular to the microchannel. Alternatively, the suction opening may not be substantially perpendicular to the microchannel. The angle between the suction opening and the microchannel may be at least about 5°, at least about 10°, at least about 15°, at least about 20°, at least about 30°, at least about 40°, at least about 50°, at least about 60°, at least about 70°, at least about 90°, at least about 100°, at least about 110°, at least about 120°, at least about 130°, at least about 140°, at least about 150°, at least about 160°, or at least about 170°.

[0069] The microchambers can be configured in various patterns. A and B of FIG. 2 illustrate typical patterns of the configuration of the microchambers, the suction openings, and the microchannels. A number of microchannels can be used, or a single microchannel can be used. In some embodiments, the microchannel may include a group of subchannels. The group of subchannels can be connected by one or more cross channels. In some of these embodiments, the subchannels can be substantially parallel to each other such that an array of microchambers forms a lattice of microchambers. A of FIG. 2 illustrates an embodiment in which parallel subchannels (230) and one or more cross channels (220) are used to form a lattice of microchambers.

[0070] The microchambers are configured to form a hexagonal lattice of microchambers, with curvilinear or angled subchannels connecting the microchambers. The hexagonal lattice of microchambers can also be formed and connected by a single microchannel, such as by a microchannel forming a meandering pattern (240) across the microfluidic device. B of FIG. 2 illustrates an embodiment in which a single microchannel with a meandering pattern forms a hexagonal lattice of microchambers. Another example of microchambers arranged in a meandering structure is shown in FIG. 12B.

[0071] The length of the sub-channel can be constant. Alternatively, the length of the sub-channel can vary. The sub-channel can have a length dimension of 100 mm or less, about 75 mm or less, about 50 mm or less, about 40 mm or less, about 30 mm or less, about 20 mm or less, about 10 mm or less, about 8 mm or less, about 6 mm or less, about 4 mm or less, about 2 mm or less, or less than that. The length of the sub-channel can be limited by the length or width outside the microfluidic device. The sub-channel may have the same cross-sectional dimensions as the micro-channel. Alternatively, the sub-channel may have different cross-sectional dimensions from the micro-channel. The sub-channel may have the same depth as the micro-channel and different cross-sectional dimensions. Alternatively, the sub-channel may have the same cross-sectional dimensions as the micro-channel and a different depth. For example, the sub-channel can have a depth of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 30 μm or less, about 15 μm or less, or less than that. The sub-channel can have a cross-sectional width of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 75 μm or less, about 50 μm or less, about 40 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, or less than that.

[0072] In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 100 μm in width and approximately 10 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 80 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 60 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 40 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 20 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 10 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 80 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 60 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 40 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 20 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-section of the sub-channel may be approximately 10 μm in width and approximately 10 μm in depth. The cross-sectional shape of the sub-channel is not limited, but can be any suitable cross-sectional shape including circular, elliptical, triangular, square, or rectangular. The cross-sectional shape of the sub-channel can be different from the cross-sectional shape of the micro-channel. The cross-sectional shape of the sub-channel can be the same as the cross-sectional shape of the micro-channel. The cross-sectional area of the sub-channel can be constant along the length of the sub-channel. Alternatively, or additionally, the cross-sectional area of the sub-channel can vary along the length of the micro-channel.The cross-sectional area of the sub-channel may vary between about 50% and 150%, about 60% and 125%, about 70% and 120%, about 80% and 115%, about 90% and 110%, about 95% and 100%, or about 98% and 102%. The cross-sectional area of the sub-channel is about 10,000 μm. 2 Hereinafter, about 7,500 μm 2 Hereinafter, about 5,000 μm 2 Hereinafter, about 2,500 μm 2 Hereinafter, about 1,000 μm 2 Hereinafter, about 750 μm 2 Hereinafter, about 500 μm 2 Hereinafter, about 400 μm 2 Hereinafter, about 300 μm 2 Hereinafter, about 200 μm 2 Hereinafter, about 100 μm 2 Hereinafter, or may be less than that. The cross-sectional area of the sub-channel may be the same as the cross-sectional area of the micro-channel. The cross-sectional area of the sub-channel may be less than or equal to the area of the cross-sectional area of the micro-channel. For example, the cross-sectional area of the sub-channel may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 20% or less, or less than that of the micro-channel cross-sectional area.

[0073] The length of the cross-channel can be constant. Alternatively, the length of the cross-channel can vary. The cross-channel may have a length dimension of about 100 mm or less, about 75 mm or less, about 50 mm or less, about 40 mm or less, about 30 mm or less, about 20 mm or less, about 10 mm or less, about 8 mm or less, about 6 mm or less, about 4 mm or less, about 2 mm or less, or less than that. The length of the cross-channel can be limited by the length or width outside the microfluidic device. The cross-channel may have the same cross-sectional dimensions as the microchannel. Alternatively, the cross-channel may have different cross-sectional dimensions from the microchannel. The cross-channel may have the same depth as the microchannel and different cross-sectional dimensions. Alternatively, the cross-channel may have the same cross-sectional dimensions as the microchannel and a different depth. For example, the cross-channel may have a depth of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 80 μm or less, about 60 μm or less, about 30 μm or less, about 15 μm or less, or less than that. The cross-channel may have a cross-sectional width of about 500 μm or less, about 250 μm or less, about 100 μm or less, about 75 μm or less, about 50 μm or less, about 40 μm or less, about 30 μm or less, about 20 μm or less, about 10 μm or less, or less than that.

[0074] In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 100 μm in width and approximately 10 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 80 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 60 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 40 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 20 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 10 μm in width and approximately 100 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 80 μm in width and approximately 80 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 60 μm in width and approximately 60 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 40 μm in width and approximately 40 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 20 μm in width and approximately 20 μm in depth. In some examples, the dimensions of the cross-channel cross-section may be approximately 10 μm in width and approximately 10 μm in depth.

[0075] The cross-sectional shape of the cross-channel is not limited and can be any suitable cross-sectional shape including circular, elliptical, triangular, square, or rectangular. The cross-sectional shape of the cross-channel may be different from the cross-sectional shape of the microchannel. The cross-sectional shape of the cross-channel may be the same as the cross-sectional shape of the microchannel. The cross-sectional area of the cross-channel may be constant within the length of the cross-channel. Alternatively, or additionally, the cross-sectional area of the cross-channel may vary within the length of the microchannel. The cross-sectional area of the cross-channel may vary between about 50% and 150%, about 60% and 125%, about 70% and 120%, about 80% and 115%, about 90% and 110%, about 95% and 100%, or about 98% and 102%. The cross-sectional area of the cross-channel may be about 10,000μm 2 or less, about 7,500μm 2 or less, about 5,000μm 2 or less, about 2,500μm 2 or less, about 1,000μm 2 or less, about 750μm 2 or less, about 500μm 2 or less, about 400μm 2 or less, about 300μm 2 or less, about 200μm 2 or less, about 100μm 2 or less, or even less than that. The cross-sectional area of the cross-channel may be the same as the cross-sectional area of the microchannel. Alternatively, the cross-sectional area of the cross-channel may be smaller than the area of the cross-sectional area of the microchannel. The cross-sectional area of the cross-channel may be about 98% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 20% or less, or even less than that of the cross-sectional area of the microchannel.

[0076] <Method for manufacturing a microfluidic device> Microfluidic devices useful in the methods and systems of the present disclosure can be manufactured by any useful method. For example, the manufacture of the device can include injection molding a thermoplastic to create a microfluidic structure. The microfluidic structure can include microchannels, a plurality of microchambers, and a plurality of suction openings. The plurality of microchambers can be connected to the microchannels by the plurality of suction openings. The microchannels can include an inlet and an outlet. A thermoplastic thin film may be applied to cap the microfluidic structure. The thermoplastic thin film may be at least partially gas permeable when a pressure differential is applied across the thermoplastic thin film.

[0077] The thermoplastic thin film may be formed by injection molding. The thermoplastic thin film may be applied to the microfluidic structure by thermal bonding. Alternatively, or additionally, the thin film may be applied by chemical bonding. The thermoplastic thin film can be formed as part of and during the injection molding process to form the microfluidic device.

[0078] The body and the thin film of the microfluidic device may contain the same material. Alternatively, the body and the thin film of the microfluidic device may contain different materials. The body and the thin film of the microfluidic device may contain a thermoplastic. Examples of thermoplastics include, but are not limited to, cycloolefin polymers, acrylics, acrylonitrile butadiene styrene, nylon, polylactic acid, polybenzimidazole, polycarbonate, polyethersulfone, polyetheretherketone, polyetherimide, polyethylene, polyphenylene oxide, polyphenylene sulfide, polypropylene, polystyrene, polyvinyl chloride, polytetrafluoroethylene, polyester, polyurethane, or any derivatives thereof. The microfluidic device may contain a homopolymer, a copolymer, or a combination thereof. The microfluidic device may be formed from a non-elastic material. Alternatively, or additionally, the microfluidic device may be formed from an elastic material.

[0079] Both the thermoplastic and the thin film may be composed of a cycloolefin polymer. One suitable thermoplastic is Zeonor 1430R (Zeon Chemical, Japan), and one suitable thin film is Zeonox 1060R (Zeon Chemical, Japan). The thin film may include a material that is non-gas permeable at low pressure and at least partially gas permeable under pressure.

[0080] The inlets and outlets of the microfluidic device of the present disclosure may be formed by mechanical drilling. Alternatively, the inlets and outlets are formed by melting, dissolving, or etching the thermoplastic.

[0081] FIG. 4 illustrates a method of manufacturing a device useful for the processing and / or analysis of samples. In FIG. 4, an injection molding process (401) is used to form a microfluidic structure. The microfluidic structure includes an array of microchambers connected to at least one microchannel via a suction opening, as shown in A and B of FIG. 1. The microfluidic structure is capped by a thin film. In the capping process, the openings on at least one side of the micro-structure are covered to close and seal the micro-structure. Capping can be performed by a process (402) of applying a thin film to the injection-molded microfluidic structure. Alternatively, capping can be performed by forming a thin film as part of the injection molding process (401).

[0082] As another example, although described in the context of a micro-structure formed by injection molding, microfluidic devices formed by other microfabrication techniques can also benefit from the use of such thin thermoplastic films to allow gas release as described above. Such techniques include microfabrication, microlithography, and hot embossing, as well as other microfabrication techniques.

[0083] The devices of the present disclosure may be disposable devices (e.g., designed for single use such as the analysis and / or processing of a single sample) or reusable devices (e.g., designed for multiple uses such as the analysis and / or processing of multiple samples). The selection of materials as inclusions in the device may reflect whether the device is used one or more times. For example, a disposable device may include materials that are not more expensive than reusable devices. Similarly, the manufacturing process can be adjusted according to the use of the device. For example, the fabrication process for a consumable device may produce less waste and / or include fewer or less expensive steps. Reusable devices can be washable and / or sterilizable to facilitate the analysis and / or processing of multiple samples using the same device. For example, a reusable device may include materials that can withstand high temperatures suitable for sterilization. A disposable device may or may not include such materials.

[0084] <Method for analyzing a sample> In one aspect, the present disclosure provides a method for using a microfluidic device to analyze a sample, such as a nucleic acid molecule. The nucleic acid molecule can be in or derived from a sample that contains or is suspected of containing a pathogen, such as a bacterium. The method can include providing a microfluidic device as described herein. The device can include microchannels. The microchannels can include microchannels connected to an inlet and an outlet. The microfluidic device may further include a plurality of microchambers connected to the microchannels by a plurality of suction openings. The microfluidic device can be sealed by a thin film (e.g., a thermoplastic thin film) disposed adjacent to the surface of the microfluidic device such that the thin film caps the microchannels, the plurality of microchambers, and the plurality of suction openings. Reagents and / or samples may also be added to the inlet or outlet. The microfluidic device can be filled by providing a first pressure difference between the reagent and / or sample and the microfluidic device and causing the reagent and / or sample to flow into the microfluidic device. A second pressure difference can be applied between the microchannels and the plurality of microchambers to move the reagent and / or sample into the plurality of microchambers and distribute the reagent and / or sample into the microchambers by passing gas in the plurality of microchambers through the thin film. The second pressure difference may be greater than the first pressure difference. A third pressure difference between the inlet and the outlet can be applied to introduce the fluid into the microchannels without introducing the fluid into the microchambers. The third pressure difference may be less than the second pressure difference. The reagent can be added before, after, or simultaneously with the sample. The reagent can also be provided in one or more compartments of the device by another method. For example, the reagent can be placed within one or more compartments before covering the one or more compartments with the thin film.

[0085] The inlet and outlet of the device can be in fluid communication with a pneumatic pump or a vacuum system. The pneumatic pump or vacuum system may either be a component of the system of the present disclosure or be separate therefrom. Filling and dispensing of the sample and / or reagent can be performed by applying a pressure difference across various features of the microfluidic device. Filling and dispensing of the sample and / or reagent can be performed without using valves between the microchambers and the microchannels to isolate the sample and / or reagent. For example, filling of the microchannel can be accomplished by applying a pressure difference between the sample and / or reagent to be filled and the microchannel. This pressure difference can be achieved by pressurizing the sample and / or reagent or by applying a vacuum to the microchannel. Filling of the microchamber can be performed by applying a pressure difference between the microchannel and the microchamber. This can be achieved by pressurizing the microchannel or by applying a vacuum to the microchamber. Dispensing of the sample and / or reagent can be performed by applying a pressure difference between the fluid and the microchannel. This pressure difference can be achieved by pressurizing the fluid or by applying a vacuum to the microchannel.

[0086] The membrane can have various permeability characteristics under various applied pressure differences. For example, the membrane may be non-gas permeable at a first pressure difference and a third pressure difference (e.g., low pressure) that can be a lesser degree of pressure difference. The membrane may be at least partially gas permeable at a second pressure difference (e.g., high pressure) that can be a greater degree of pressure difference. The first pressure difference and the third pressure difference may be the same or different. The first pressure difference can be the pressure difference between the reagent and the microfluidic device at the inlet or outlet. During filling of the microfluidic device, the pressure of the reagent may be higher than the pressure of the microfluidic device. During filling of the microfluidic device, the pressure difference (e.g., low pressure) between the reagent and the microfluidic device may be about 8 pounds per square inch (psi) or less, about 6 psi or less, about 4 psi or less, about 2 psi or less, about 1 psi or less, or less than that. In some examples, during filling of the microfluidic device, the pressure difference between the reagent and the microfluidic device may be from about 1 psi to about 8 psi. In some examples, during filling of the microfluidic device, the pressure difference between the reagent and the microfluidic device may be from about 1 psi to about 6 psi. In some examples, during filling of the microfluidic device, the pressure difference between the reagent and the microfluidic device may be from about 1 psi to about 4 psi. The microfluidic device may also be filled by applying a pressure difference between the reagent and the microfluidic device for a time of about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute or less, or less than that.

[0087] The filled microfluidic device can have a sample or one or more reagents in a microchannel, a suction opening, a microchamber, or any combination thereof. Backfilling of the sample or one or more reagents into the microchamber can occur when filling the microfluidic device or can occur while applying a second pressure difference. The second pressure difference (e.g., high pressure) can correspond to the pressure difference between the microchannel and the plurality of microchambers. While applying the second pressure difference, a first fluid in the high-pressure domain can push a second fluid in the lower-pressure domain out of the microfluidic device through a thin film. The first and second fluids can include a liquid or a gas. The liquid can include an aqueous mixture or an oily mixture. The second pressure difference can be achieved by pressurizing the microchannel. Alternatively, or additionally, the second pressure difference can be achieved by applying a vacuum to the microchamber. While applying the second pressure difference, the sample and / or reagent in the microchannel can flow into the microchamber. Further, while applying the second pressure difference, the gas trapped within the suction opening, microchamber, and microchannel can be released through the thin film. During backfilling of the microchamber and gas release, the pressure difference between the microchamber and the microchannel can be about 6 psi or more, about 8 psi or more, about 10 psi or more, about 12 psi or more, about 14 psi or more, about 16 psi or more, about 18 psi or more, about 20 psi or more, or greater. In some examples, during backfilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 20 psi. In some examples, during backfilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 18 psi. In some examples, during backfilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 16 psi. In some examples, during backfilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 14 psi.In some examples, during refilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 12 psi. In some examples, during refilling of the microchamber, the pressure difference between the microchamber and the microchannel is from about 8 psi to about 10 psi. The microchamber can be refilled and gas released by applying the pressure difference for about 5 minutes or more, about 10 minutes or more, about 15 minutes or more, about 20 minutes or more, about 25 minutes or more, about 30 minutes or more, or longer.

[0088] Samples and / or reagents can be dispensed by removing excess sample and / or reagent from the microchannel. Removing excess sample and / or reagent from the microchannel can prevent the reagent and / or sample in one microchamber from diffusing through the suction opening into the microchannel and other microchambers. Excess sample and / or reagent in the microchannel can be removed by introducing fluid at the inlet or outlet of the microchannel. The pressure of the fluid can be higher than the pressure of the microchannel, thereby creating a pressure difference between the fluid and the microchannel. The fluid can be oxygen, nitrogen, carbon dioxide, air, a noble gas, or any combination thereof. During dispensing of the sample, the pressure difference between the fluid and the microchannel can be about 8 psi or less, about 6 psi or less, about 4 psi or less, about 2 psi or less, about 1 psi or less, or less. In some examples, during dispensing of the sample and / or reagent, the pressure difference between the fluid and the microchannel is from about 1 psi to about 8 psi. In some examples, during dispensing of the sample and / or reagent, the pressure difference between the fluid and the microchannel is from about 1 psi to about 6 psi. In some examples, during dispensing of the sample and / or reagent, the pressure difference between the fluid and the microchannel is from about 1 psi to about 4 psi. Samples and / or reagents can be dispensed by applying a pressure difference between the fluid and the microchannel for about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute or less, or less.

[0089] A - D in FIG. 3 illustrate a method for using the microfluidic device shown in A of FIG. 1. In A of FIG. 3, a low pressure is applied to the reagent via a pneumatic pump (300) at the inlet (120), and the reagent is pushed into the microchannel (110), thereby filling the microchamber through the suction opening. The pressure causes the reagent to flow through the microchannel and thereby flow into the microchamber through the suction opening. During this time, gas bubbles such as air bubbles (301) may remain in the microchamber, the suction opening, or the microchannel. The filling by applying a low pressure can be continued until the microchamber, the suction opening, and the microchannel are substantially filled with the reagent. The reagent may be a reagent used in a polymerase chain reaction. The reagent can be diluted so that there is only one PCR template in the reagent per microchamber of the microfluidic device. For example, each compartment of at least a subset of the plurality of compartments of the device can contain at most one nucleic acid molecule. In some examples, each compartment of a subset of the plurality of compartments of the device can contain only one nucleic acid molecule.

[0090] In B of FIG. 3, the pneumatic pump (300) is connected to both the inlet (120) and the outlet (130), and a high pressure is applied. The high pressure is transmitted through the reagent and applied to gas bubbles such as air bubbles (301). Under the influence of the high pressure, the thin film (150) becomes gas permeable, and the air bubbles (301) can be released through the thin film (150). By applying this high pressure, the microchamber, the suction opening, and the microchannel are substantially free of gas bubbles, thereby avoiding fouling.

[0091] In C of FIG. 3, the fluid is reintroduced at the inlet (120) by applying a low pressure to the gas via the pneumatic pump (300). The pneumatic pressure may not be sufficient to allow the gas to escape through the thin film, or may not be high enough to suck up the gas bubbles and push them into the openings and microchambers. Instead, the gas can remove the reagent from the microchannel while leaving the isolated reagent in each microchamber and the suction opening. The gas may be air. Alternatively, the gas may be an inert gas such as nitrogen, carbon dioxide, or a noble gas. Such a gas can be used to avoid reactions between the reagent and the component gases of air.

[0092] D of FIG. 3 illustrates the state of the system after the low pressure is applied in C of FIG. 3. After the low pressure gas is applied, the reagent can be removed from the microchannel while the microchambers and the suction openings may remain filled with the reagent. The reagent can remain stationary within the microchamber due to the capillary force and high surface tension generated by the suction opening. The capillary force and high surface tension can prevent the reagent from flowing into the microchannel and minimize the evaporation of the reagent. A process similar to that described for A-3 to D of FIG. 3 can be used to distribute the sample within the device.

[0093] The distribution of the sample can be confirmed by the presence of an indicator in the reagent. The indicator can include a molecule containing a detectable moiety. The detectable moiety can include a radionuclide, a fluorescent label, a chemiluminescent label, an enzyme label, a colorimetric label, or any combination thereof. Non-limiting examples of radionuclides include 3H, 14C, 22Na, 32P, 33P, 35S, 42K, 45Ca, 59Fe, 123I, 124I, 125I, 131I, or 203Hg. Non-limiting examples of fluorescent labels include fluorescent proteins, optically active dyes (e.g., fluorescent dyes), organometallic fluorophores, or any combination thereof. Non-limiting examples of chemiluminescent labels include enzymes of the luciferase class such as Renilla luciferase, Gaussia luciferase, Cypridina luciferase, and firefly luciferase. Non-limiting examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, or other well-known labels.

[0094] The indicator molecule may be a fluorescent molecule. The fluorescent molecule may include a fluorescent protein, a fluorescent dye, and an organometallic fluorophore. The indicator molecule may be a protein fluorophore. The protein fluorophore may include a green fluorescent protein (GFP, a fluorescent protein that fluoresces in the green region of the spectrum, typically emitting light with a wavelength of 500-550 nanometers), a cyan fluorescent protein (CFP, a fluorescent protein that fluoresces in the cyan region of the spectrum, typically emitting light with a wavelength of 450-500 nanometers), and a red fluorescent protein (RFP, a fluorescent protein that fluoresces in the red region of the spectrum, typically emitting light with a wavelength of 600-650 nanometers).Non-limiting examples of protein fluorophores include mutants and spectral variants of AcGFP, AcGFP1, AmCyan, AmCyan1, AQ143, AsRed2, Azami Green, Azurite, BFP, Cerulean, CFP, CGFP, Citrine, copGFP, CyPet, dKeima-Tandem, DsRed, dsRed-Express, DsRed-Monomer, DsRed2, dTomato, dTomato-Tandem, EBFP, EBFP2, ECFP, EGFP, Emerald, EosFP, EYFP, GFP, HcRed-Tandem, HcRed1, JRed, Katuska, Kusabira Orange, Kusabira Orange2, mApple, mBanana, mCerulean, mCFP, mCherry, mCitrine, mECFP, mEmerald, mGrape1, mGrape2, mHoneydew, Midori-Ishi Cyan, mKeima, mKO, mOrange, mOrange2, mPlum, mRaspberry, mRFP1, mRuby, mStrawberry, mTagBFP, mTangerine, mTeal, mTomato, mTurquoise, mWasabi, PhiYFP, ReAsH, Sapphire, Superfolder GFP, T-Sapphire, TagCFP, TagGFP, TagRFP, TagRFP-T, TagYFP, tdTomato, Topaz, TurboGFP, Venus, YFP, YPet, ZsGreen, and ZsYellow1.

[0095] The indicator molecule can be a fluorescent dye. Non-limiting examples of fluorescent dyes are SYBR green; SYBR blue; DAPI; propidium iodine; Hoeste; SYBR gold; ethidium bromide; acridine; proflavine; acridine orange; acriflavine; fluorcoumanin; ellipticine; daunomycin; chloroquine, distamycin D; chromomycin; homidium; mitomycin; ruthenium polypyridyl; anthramycin; phenanthridine and acridine; propidium iodine; hexidium iodide; dihydroethidium; ethidium monoazide; ACMA; Hoechst 33258; Hoechst 33342; Hoechst 34580; DAPI; acridine orange; 7-AAD; actinomycin D; LDS751; hydroxystilbamidine; SYTOX Blue; SYTOX Green; SYTOX Orange; POPO-1; POPO-3; YOYO-1; YOYO-3; TOTO-1; TOTO-3; JOJO-1; LOLO-1; BOBO-1; BOBO-3; PO-PRO-1; PO-PRO-3; BO-PRO-1; BO-PRO-3; TO-PRO-1; TO-PRO-3; TO-PRO-5; JO-PRO-1; LO-PRO-1; YO-PRO-1; YO-PRO-3; PicoGreen; OliGreen; RiboGreen; SYBR Gold; SYBR Green I; SYBR Green II; SYBR DX; SYTO-40, SYTO-41, SYTO-42, SYTO-43, SYTO-44, and SYTO-45 (blue); SYTO-13, SYTO-16, SYTO-24, SYTO-21, SYTO-23, SYTO-12, SYTO-11, SYTO-20, SYTO-22, SYTO-15, SYTO-14, and SYTO-25 (green); SYTO-81, SYTO-80, SYTO-82, SYTO-83, SYTO-84, and SYTO-85 (orange); SYTO-64, SYTO-17, SYTO-59, SYTO-61, SYTO-62, SYTO-60, and SYTO-63 (red); fluorescein; fluorescein isothiocyanate (FITC);Tetramethylrhodamine isothiocyanate (TRITC); Rhodamine; Tetramethylrhodamine; R-phycoerythrin; Cy-2; Cy-3; Cy-3.5; Cy-5; Cy5.5;; Cy-7; Texas Red; Phar-Red; Allophycocyanin (APC); Sybr Green I; Sybr Green II; Sybr Gold; CellTracker Green; 7-AAD; Ethidium homodimer I; Ethidium homodimer II; Ethidium homodimer III; Umbelliferone; Eosin; Green fluorescent protein; Erythrosin; Coumarin; Methylcoumarin; Pyrene; Malachite green; Stilbene; Lucifer yellow; Cascade blue; Dichlorotriazinylamine fluorescein; Dansyl chloride; Fluorescent lanthanide complexes such as those containing europium and terbium, Carboxy·tetrachloro·fluorescein; 5 and / or)6-Carboxy·fluorescein (FAM); 5-(or 6-)Iodoacetamide fluorescein; 5-{[2(and 3)-5-(Acetylmercapto)-succinyl]amino}fluorescein (SAMSA-fluorescein); Lissamine rhodamine B sulfonyl chloride; 5 and / or 6 Carboxyrhodamine (ROX); 7-Amino-methyl-coumarin; 7-Amino-4-methylcoumarin-3-acetic acid (AMCA); BODIPY fluorophore; 8-Methoxypyrene-1;3;6-trisulfonic acid trisodium salt; 3;6-Disulfonate-4-amino-naphthalimide; Phycobilin protein; AlexaFluor350,405,430,488,532,546,555,568,594,610,633,635,647,660,680,700,750, and 790 dyes; DyLight350,405,488,550,594,633,650,680,755, and 800 dyes; and other fluorophores.;

[0096] The indicator molecule can be an organometallic fluorophore. Non-limiting examples of organometallic fluorophores include lanthanide ion chelates, and non-limiting examples of lanthanide ion chelates include tris(dibenzoylmethane)mono(1,10-phenanthroline)europium(III), tris(dibenzoylmethane)mono(5-amino-1,10-phenanthroline)europium(III), and Lumi4-Tb cryptate.

[0097] The signal can be collected (e.g., an image is taken) from a microfluidic device or a subset of its compartments (e.g., microchambers). Collection of the signal can include taking an image of the device or a subset of its compartments. The signal (e.g., image) can be collected simultaneously from a single microchamber, an array of microchambers, or multiple arrays of microchambers. The signal can be collected through the body of the microfluidic device, through a thin film of the microfluidic device, or both. The body of the microfluidic device may be substantially optically transparent. Alternatively, the body of the microfluidic device may be substantially optically opaque. Similarly, the thin film may be substantially optically transparent. Alternatively, the body of the microfluidic device may be substantially optically opaque.

[0098] Signals can be collected from a microfluidic device or a subset of its compartments at any useful time and at any useful frequency. For example, a signal (e.g., an image) may be collected before filling the microfluidic device with a reagent or sample. Signals may also be collected while filling the microfluidic device with a reagent or sample. Alternatively, or additionally, signals may be collected after filling the microfluidic device with a reagent or sample. For example, a signal may be collected to confirm the dispensing of a reagent or sample. Signals may be collected during a reaction (e.g., a nucleic acid amplification reaction) to monitor the product of the reaction (e.g., an amplification product). Similarly, signals may be collected during controlled heating of the device or a subset of its compartments (e.g., during high-resolution melting analysis). Signals may be collected at predetermined intervals, such as at specific times. Alternatively, or additionally, videos may be taken from the microfluidic device or a subset of its compartments. The predetermined intervals may include collecting signals (e.g., taking images) at least every 300 seconds, at least every 240 seconds, at least every 180 seconds, at least every 120 seconds, at least every 90 seconds, at least every 60 seconds, at least every 30 seconds, at least every 15 seconds, at least every 10 seconds, at least every 5 seconds, at least every 4 seconds, at least every 3 seconds, at least every 2 seconds, at least every 1 second, or more frequently during the reaction. As described herein, signals may further be collected in response to an instruction from a processor.

[0099] The methods described herein related to the use of microfluidic devices can include the amplification of multiple nucleic acid molecules from a sample. The microfluidic device can be filled with one or more amplification reagents such as nucleic acid molecules, components necessary for the amplification reaction (e.g., primers, polymerase, and deoxyribonucleotides), indicator molecules, and amplification probes. As described herein, the amplification reaction can include thermal cycling of multiple microchambers or a subset thereof. Detection of nucleic acid amplification can be performed by collecting signals (e.g., imaging) from multiple microchambers or a subset thereof of the microfluidic device. Nucleic acid molecules can be quantified by counting the microchambers in which the nucleic acid molecules were successfully amplified and applying Poisson statistics. The amount of nucleic acid molecules can also be quantified by processing signals collected at various time points throughout the amplification reaction. For example, during each thermal cycle (e.g., each amplification cycle) of the nucleic acid amplification reaction, one or more signals can be collected, and the signals can be used to determine the amplification rate, similar to, for example, real-time or quantitative polymerase chain reaction (real-time PCR or qPCR). Nucleic acid amplification and quantification can be performed in a single integrated unit, for example, within a predetermined compartment or a subset of multiple compartments of the device.

[0100] Various nucleic acid amplification reactions can be used to amplify nucleic acid molecules in a sample to generate amplification products. The amplification of a nucleic acid target may be linear, exponential, or a combination thereof. Non-limiting examples of nucleic acid amplification methods include primer extension, polymerase chain reaction, reverse transcription, isothermal amplification, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, and multiple displacement amplification. The amplification products of an amplification reaction can be DNA or RNA. For a sample containing DNA molecules, any DNA amplification method can be used. Examples of DNA amplification methods include, but are not limited to, PCR, real-time PCR, assembly PCR, asymmetric PCR, digital PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, overlap extension PCR, thermal asymmetric interlaced PCR, touchdown PCR, and ligase chain reaction. DNA amplification may be linear, exponential, or any combination thereof. As described herein, DNA amplification can also be achieved using digital PCR (dPCR), real-time quantitative PCR (qPCR), or quantitative digital PCR (qdPCR).

[0101] The reagents required for nucleic acid amplification may include a polymerase enzyme, a reverse primer, a forward primer, and an amplification probe. Examples of polymerase enzymes include, without limitation, nucleic acid polymerases, transcriptases, or ligases (i.e., enzymes that catalyze the formation of bonds). The polymerase enzyme may be of natural origin or synthetic. Examples of polymerases are DNA polymerase and RNA polymerase, thermostable polymerases, wild-type polymerases, modified polymerases, Escherichia coli DNA polymerase I, T7 DNA polymerase, bacteriophage T4 DNA polymerase Φ29 (phi29) DNA polymerase, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pwo polymerase, VENT polymerase, DEEPVENT polymerase, Ex-Taq polymerase, LA-Taw polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tca polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Tbr polymerase, Tfl polymerase, PfuTubo polymerase, Pyrobest polymerase, KOD polymerase, Bst polymerase, Sac polymerase, the Klenow fragment polymerase having 3'5' exonuclease activity, and variants, modified products, and derivatives thereof. For hot start polymerases, a denaturation step at a temperature of about 92°C to 95°C for about 2 minutes to 10 minutes may be required.

[0102] The nucleic acid amplification reaction may include an amplification probe. The amplification probe may be a sequence-specific oligonucleotide probe. The amplification probe may be optically active when hybridized to the amplification product. The amplification probe can only be detected as the nucleic acid amplification proceeds. The intensity of a signal (e.g., an optical signal) collected from a plurality of compartments containing nucleic acid molecules may be proportional to the amount of amplification product contained in the compartment. For example, the signal collected from a particular compartment may be proportional to the amount of amplification product in that particular compartment. The probe may be linked to any of the optically active detectable moieties (e.g., dyes) described herein and may further include a quencher capable of blocking the optical activity of the associated dye. Non-limiting examples of probes that may be useful as detectable moieties include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, Lion probes, locked nucleic acid probes, or molecular beacons. Non-limiting examples of quenchers that may be useful for blocking the optical activity of the probe include Black Hole Quencher (BHQ), Iowa Black FQ and RQ quenchers, or internal ZEN quenchers. Alternatively, or in addition, the probe or quencher may be any known probe useful in the context of the methods of the present disclosure.

[0103] The amplification probe may be a dual-labeled fluorescent probe. The dual-labeled probe may include a fluorescent reporter and a fluorescent quencher linked to a nucleic acid. The fluorescent reporter and the fluorescent quencher may be arranged in proximity to each other. The proximity of the fluorescent reporter and the fluorescent quencher may also block the optical activity of the fluorescent reporter. The dual-labeled probe can bind to the nucleic acid molecule to be amplified. During amplification, the fluorescent reporter and the fluorescent quencher may be cleaved by the exonuclease activity of the polymerase. Cleaving the fluorescent reporter and the fluorescent quencher from the amplification probe may restore the optical activity to the fluorescent reporter and enable detection. The dual-labeled fluorescent probe may include a 5' fluorescent reporter having a maximum excitation wavelength of about 450 nanometers (nm), 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or higher, and a maximum emission wavelength of about 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or higher. The dual-labeled fluorescent probe may also include a 3' fluorescent quencher. The fluorescent quencher may also quench the emission wavelength of fluorescence between about 380 nm to 550 nm, 390 nm to 625 nm, 470 nm to 560 nm, 480 nm to 580 nm, 550 nm to 650 nm, 550 nm to 750 nm, or 620 nm to 730 nm.

[0104] The nucleic acid amplification reaction carried out within the microchamber of the device can include thermal cycling of the microchamber of the microfluidic device, or a subset thereof. Thermal cycling can include controlling the temperature of the microfluidic device by applying heating or cooling to the microfluidic device. The heating method or cooling method can include resistive heating or resistive cooling, radiative heating or radiative cooling, conductive heating or conductive cooling, convective heating or convective cooling, or any combination thereof. The thermal cycle can include incubating the microchamber at a temperature high enough to denature nucleic acid molecules for a period of time, and then incubating the microchamber at an extension temperature for an extension period. The thermal cycle can also include incubating the microchamber at an annealing temperature for a period of time sufficient to anneal primers to the nucleic acid molecules. The denaturation temperature can vary, for example, depending on a particular nucleic acid sample, the reagents used, and the desired reaction conditions. The denaturation temperature can be from about 80°C to about 110°C. The denaturation temperature can be from about 85°C to about 105°C. The denaturation temperature can be from about 90°C to about 100°C. The denaturation temperature can be from about 90°C to about 98°C. The denaturation temperature can be from about 92°C to about 95°C. The denaturation temperature can be at least about 80°C, at least about 81°C, at least about 82°C, at least about 83°C, at least about 84°C, at least about 85°C, at least about 86°C, at least about 87°C, at least about 88°C, at least about 89°C, at least about 90°C, at least about 91°C, at least about 92°C, at least about 93°C, at least about 94°C, at least about 95°C, at least about 96°C, at least about 97°C, at least about 98°C, at least about 99°C, at least about 100°C, or higher.

[0105] The denaturation period may vary, for example, depending on a specific nucleic acid sample, the reagents used, and the desired reaction conditions. The denaturation period may be 300 seconds or less, 240 seconds or less, 180 seconds or less, 120 seconds or less, 90 seconds or less, 60 seconds or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, or 1 second or less. Alternatively, the denaturation period may be about 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0106] The extension temperature may vary, for example, depending on a specific nucleic acid sample, the reagents used, and the desired reaction conditions. The extension temperature can be from about 30°C to about 80°C. The extension temperature can be from about 35°C to about 75°C. The extension temperature can be from about 45°C to about 65°C. The extension temperature can be from about 55°C to about 65°C. The extension temperature can be from about 40°C to about 60°C. The extension temperature may be at least about 35°C, at least about 36°C, at least about 37°C, at least about 38°C, at least about 39°C, at least about 40°C, at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, at least about 45°C, at least about 46°C, at least about 47°C, at least about 48°C, at least about 49°C, at least about 50°C, at least about 51°C, at least about 52°C, at least about 53°C, at least about 54°C, at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, or at least about 80°C.

[0107] The extension time may vary depending on, for example, a specific nucleic acid sample, the reagents used, and the desired reaction conditions. The extension time may be about 300 seconds or less, 240 seconds or less, 180 seconds or less, 120 seconds or less, 90 seconds or less, 60 seconds or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, or 1 second or less. Alternatively, the extension time may be about 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0108] The annealing temperature may vary depending on, for example, a specific nucleic acid sample, the reagents used, and the desired reaction conditions. The annealing temperature can be from about 30°C to about 80°C. The annealing temperature can be from about 35°C to about 75°C. The annealing temperature can be from about 45°C to about 65°C. The annealing temperature can be from about 55°C to about 65°C. The annealing temperature can be from about 40°C to about 60°C. The annealing temperature can be at least about 35°C, at least about 36°C, at least about 37°C, at least about 38°C, at least about 39°C, at least about 40°C, at least about 41°C, at least about 42°C, at least about 43°C, at least about 44°C, at least about 45°C, at least about 46°C, at least about 47°C, at least about 48°C, at least about 49°C, at least about 50°C, at least about 51°C, at least about 52°C, at least about 53°C, at least about 54°C, at least about 55°C, at least about 56°C, at least about 57°C, at least about 58°C, at least about 59°C, at least about 60°C, at least about 61°C, at least about 62°C, at least about 63°C, at least about 64°C, at least about 65°C, at least about 66°C, at least about 67°C, at least about 68°C, at least about 69°C, at least about 70°C, at least about 71°C, at least about 72°C, at least about 73°C, at least about 74°C, at least about 75°C, at least about 76°C, at least about 77°C, at least about 78°C, at least about 79°C, or at least about 80°C.

[0109] The annealing time may vary depending on, for example, a specific nucleic acid sample, the reagents used, and the desired reaction conditions. The annealing period may be about 300 seconds or less, 240 seconds or less, 180 seconds or less, 120 seconds or less, 90 seconds or less, 60 seconds or less, 55 seconds or less, 50 seconds or less, 45 seconds or less, 40 seconds or less, 35 seconds or less, 30 seconds or less, 25 seconds or less, 20 seconds or less, 15 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, or even 1 second or less. Alternatively, the annealing period may be only about 120 seconds, 90 seconds, 60 seconds, 55 seconds, 50 seconds, 45 seconds, 40 seconds, 35 seconds, 30 seconds, 25 seconds, 20 seconds, 15 seconds, 10 seconds, 5 seconds, 2 seconds, or 1 second.

[0110] Nucleic acid amplification may include a number of cycles of thermal cycling (e.g., a number of amplification cycles). Any suitable number of cycles may be performed. The number of cycles performed may be more than about 5 cycles, more than about 10 cycles, more than about 15 cycles, more than about 20 cycles, more than about 30 cycles, more than about 40 cycles, more than about 50 cycles, more than about 60 cycles, more than about 70 cycles, more than about 80 cycles, more than about 90 cycles, more than about 100 cycles, or more. The number of cycles performed may depend on the number of cycles necessary to obtain a detectable amplification product. For example, the number of cycles necessary to detect nucleic acid amplification during PCR (e.g., dPCR, qPCR, or qdPCR) may be about 100 or less, about 90 or less, about 80 or less, about 70 or less, about 60 or less, about 50 or less, about 40 or less, about 30 or less, about 20 or less, about 15 or less, about 10 or less, about 5 or fewer cycles, or even less.

[0111] The time to achieve a detectable amount of amplification product may vary depending on, for example, a specific nucleic acid sample, the reagents used, the amplification reaction used, the number of amplification cycles used, and the desired reaction conditions. The time to achieve a detectable amount of amplification product may be about 120 minutes or less, 90 minutes or less, 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, or 5 minutes or less.

[0112] In some embodiments, the ramping rate (i.e., the rate at which the microchamber transitions from one temperature to another) is important for amplification. For example, the temperature and time at which an amplification reaction results in a detectable amount of amplification product may vary depending on the ramping rate. The ramping rate can affect the time, temperature, or both time and temperature used during amplification. The ramping rate may be constant between cycles or may vary between cycles. The ramping rate can be adjusted based on the sample being processed. For example, an optimal ramping rate can be selected to provide a robust and efficient amplification method.

[0113] Figure 5 illustrates a digital PCR process utilized with the microfluidic device described above. In step (501), the reagents are dispensed as shown in FIGS. 3A - 3D. In step (502), the reagents are subjected to thermal cycling to perform a PCR reaction on the reagents in the microchambers. This step may be performed, for example, using a flat block thermal cycler. In step (503), image acquisition is performed to determine which microchambers successfully performed the PCR reaction. Image acquisition may be performed, for example, using a three-color probe detection unit. In step (504), Poisson statistics are applied to the calculation of the microchambers determined in step (503) to convert the raw number of positive chambers to nucleic acid concentration.

[0114] A method for analyzing a plurality of nucleic acid molecules can include providing a device that includes a plurality of compartments as described herein. At least a subset of the plurality of compartments can contain a plurality of nucleic acid molecules (e.g., deoxyribonucleic acid or ribonucleic acid molecules). Each compartment of the subset of the plurality of compartments can be configured to allow gas flow from the compartment to the external environment through at least one barrier that separates the compartment from the external environment. Thereafter, the subset of the plurality of compartments can be exposed to conditions sufficient to perform a nucleic acid amplification reaction using the plurality of nucleic acid molecules to generate amplification products from at least a subset of the plurality of nucleic acid molecules. While the subset of the plurality of compartments is being exposed to these conditions, signals can be collected from the subset of the plurality of compartments over a plurality of time points. Thereafter, the signals collected from the plurality of compartments can be processed to determine the number of nucleic acid molecules in the subset of the plurality of compartments. Signal processing can be performed while the amplification reaction is in progress or after the amplification reaction is complete.

[0115] As described herein, exposing a subset of the plurality of compartments to conditions sufficient to perform a nucleic acid amplification reaction can include thermal cycling. The thermal cycling can include a denaturation phase, an extension phase, and an annealing phase and can include any useful combination of temperature and duration. Any useful number of thermal cycles can be performed. For example, if signals are being processed while the amplification reaction is in progress, a processor that controls the thermal cycling process can be programmed to reach a threshold after the thermal cycling is complete. Alternatively, a user can interact with a system that performs the amplification and signal collection processes and can choose to end the thermal cycling after a predetermined number of cycles. The thermal cycling can be performed using a flat block thermal cycler or any other useful temperature control device.

[0116] Collecting signals from a subset of multiple compartments can involve collecting more than one signal per compartment per thermal cycle. For example, signals may be collected during each annealing step, each extension step, each denaturation step, or any combination thereof. Alternatively, a system performing the method may be programmed to collect signals at a plurality of predetermined time points. These time points may be equidistant (e.g., every 5 seconds) or follow a predetermined pattern (e.g., every 5 seconds for the first 100 seconds, then every 20 seconds or any other useful pattern). As described herein, signal collection may include imaging. The detector may be configured to image all of a subset of multiple compartments of the device simultaneously. The detector for imaging can detect fluorescence emission at two or more wavelengths. Such a detector can measure nucleic acid amplification products corresponding to various starting nucleic acid molecules (e.g., templates). For example, a sample containing two different nucleic acid molecules can be exposed to two different primers, each of which contains a different detectable label (e.g., a dye or a fluorescent probe) and is specific for a different nucleic acid molecule. The various detectable labels can emit fluorescence signals at various wavelengths, each of which can be detected by the same detector. Determining the number of nucleic acid molecules in a subset of multiple compartments involves determining the light intensity of each compartment that is proportional to the amount of amplification product in that compartment.

[0117] As described herein, the nucleic acid amplification reaction includes one or more reagents. For example, reagents such as primers, deoxyribonucleotides, buffers, cofactors, intercalating dyes, and polymerases may be used. These reagents can be loaded into the device before, after, or simultaneously with the sample being loaded into the device. Multiple nucleic acid molecules may be loaded into multiple compartments of the device using controlled fluid flow (e.g., as described with respect to D of A-3 in FIG. 3). The gas in a subset of multiple compartments may be exposed to a flow from the compartment to the external environment. For example, as described herein, loading the device or a subset of its multiple compartments with a sample containing nucleic acid molecules can cause gas release through a barrier from the compartment.

[0118] Devices used in methods for analyzing nucleic acids can have any of the features described herein. The barrier of the device can comprise a polymeric material such as a thermoplastic material or can be a thin film. The barrier can be substantially optically transparent. The barrier can have a thickness of from about 50 μm to about 200 μm (e.g., about 50 μm, 100 μm, 150 μm, or 200 μm). The device can include at least one inlet, at least one outlet, and at least one microchannel including a plurality of suction openings. A subset of the plurality of compartments can be in fluid communication with the microchannel by the plurality of suction openings. The plurality of compartments can include from about 1,000 to about 20,000 compartments (e.g., at least about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 10,000, 15,000, or 20,000 compartments).

[0119] Figure 11 illustrates a quantitative digital PCR process utilized with the above-described microfluidic device. Panel A of FIG. 11 shows a display of a subset of compartments of a typical microfluidic device. In some compartments, no nucleic acid template is present; in other compartments, one or more templates are present. Panel B of FIG. 11 illustrates the amplification kinetics of samples in each compartment of a typical device. As illustrated in Panel B of FIG. 11, compartments with different numbers of nucleic acid templates present exhibit different amplification kinetics. Compartments without templates do not amplify. Otherwise, compartments amplify faster if they have more templates present compared to other compartments. Each vertical dashed line represents a single amplification cycle for a total of five amplification cycles. Although five cycles are illustrated, any number of cycles may be performed depending on the particular features of the method and / or the structure of the system. For example, when a wider range of templates potentially exist in any given compartment, more cycles may be required to provide absolute quantification, and thus the number of nucleic acid molecules that may potentially exist in any given compartment, the reagents used, and other reaction conditions can affect the number of cycles required. Panel C of FIG. 11 illustrates the results of the qdPCR process applied to the amplification kinetics shown in Panel B of FIG. 11. Specifically, FIG. 11C illustrates the number of nucleic acid templates calculated to be present in each compartment based on the amplification kinetics measured during the five PCR amplification cycles shown in FIG. 11B.

[0120] Figure 16 illustrates a method of performing qdPCR using the device described herein. In step (1601), one or more reagents are loaded into a plurality of compartments or a subset thereof of the device. Loading of the reagents and / or samples can be performed, for example, as described herein. Optionally, the reagents may be placed in the compartments or another portion of the device (e.g., using an automated mechanical process) prior to placing a thin film over the compartments (e.g., sealing the device). For example, reagent packets, blister packs, gels, or other such components may be placed in the compartments prior to sealing the device. As described herein, a sample containing a plurality of nucleic acid molecules may also be added.

[0121] In step (1602), the device may be loaded into a system for performing qPCR. For example, the device may be physically placed on a thermal unit such as a heating block and fixed in place by a pneumatic fixture, or placed in a slot, groove, or recess in the housing of the analysis system. Registration marks and / or mechanical keys can facilitate placement of the device. The device may be loaded into a mechanical loading unit that arranges devices for placement and analysis in a continuous sequence. In some cases, step (1602) is performed after the device is placed within the analysis system and before step (1601) and the reagents are loaded into the device. Such a system can include, as described herein, a fluid flow device for loading reagents into the device and / or other mechanical and fluid components including reservoirs, pumps, valves, and meters.

[0122] In step (1603), the nucleic acid amplification reaction is performed using a subset of the plurality of compartments of the device or a plurality of nucleic acids loaded into that subset. As described herein, the amplification reaction can include one or more thermal cycles. For example, a PCR amplification reaction can include a denaturation phase, an annealing phase, and an extension phase. The amplification cycle can last from about 60 to 180 seconds (e.g., 30 seconds at the denaturation temperature and 120 seconds at the annealing / extension temperature, for about 150 seconds). Additional steps and / or different durations may be used.

[0123] In operation (1604), signals are collected from each of a subset of a plurality of sections of the device. For example, an image can be obtained from the sections and an optical signal can be measured. Imaging can be performed, for example, by moving the optical unit to scan the thermal unit, by moving the thermal unit to scan the optical unit, or by moving both the optical unit and the thermal unit to enable imaging of the device or a subset of its plurality of sections. Signals from the entire device can be collected at once (e.g., using one or more detectors such as one or more cameras, or using a single detector configured to collect signals from the entire device at once), or the signals can be collected at once from only a part of the device (e.g., corresponding to a subset of a plurality of sections of the device). In the former case, scanning of the thermal unit and / or a detector may not be required. The signals may be collected one or more times during amplification, for example, to provide an estimate of the amplification dynamics during each cycle. In some cases, the signals can be collected only once after each amplification cycle to determine the amount of amplification that occurred after the amplification cycle was completed. Collection of the signals can include imaging a subset of a plurality of sections of the device. Imaging can be performed using a fluorescent dye that intercalates with double-stranded nucleic acid, or using a quenched DNA probe that fluoresces only after reacting with a complementary sequence. In either case, imaging can be performed by illuminating the sections with an excitation light source suitable for the fluorescent probe, or by determining which sections fluoresce and the fluorescence intensity. In some cases, operations (1603) and (1604) can be performed in parallel using the signals collected (e.g., imaged) during a nucleic acid amplification reaction (e.g., a thermal cycle).

[0124] In step (1605), the amplification kinetics in each of a subset of the plurality of compartments of the device are determined based on the signals collected in step (1604). By determining the amplification rate, the original number of nucleic acid molecules contained in each of the subset of the plurality of compartments can be estimated. For example, in FIG. 11B, the amplification kinetics of an array of compartments are shown corresponding to five amplification cycles. The number of nucleic acid molecules corresponding to each compartment in the array is shown in FIG. 11C and can be determined, for example, by measuring the amount of fluorescence exceeding the background intensity and correlating it with a large number of nucleic acid molecules (e.g., templates). In such an example, compartments that originally contained more nucleic acid molecules amplify more rapidly and thus generate detectable fluorescence earlier. The earlier the time (e.g., amplification cycle) at which fluorescence becomes detectable, the more templates originally existed in the compartment. Thus, the number of nucleic acid molecules present can be determined by the number of cycles completed or the number of cycles remaining when the fluorescence first becomes detectable. Other methods for determining the amplification kinetics may also be applied. In each case, the final measurement is the number of templates originally present in each compartment based on the amount of signal collected, the timing at which the signal is detected, and / or the rate of increase in signal generation.

[0125] In step (1606), the total number of nucleic acid molecules present in a subset of the plurality of compartments of the device is determined by summing the number of nucleic acid molecules originally present in each of the subset of the plurality of compartments.

[0126] In step (1607), the device is unloaded from the system for performing qdPCR. Unloading the device (e.g., removing it) may include a reflection of the loading procedure of step (1601) or a different procedure. For example, a manually loaded device can be automatically unloaded by a mechanical loading device or by other means (e.g., by a vacuum pick-and-place system). Thereafter, the process can be repeated using another device.

[0127] <Method for Thermodynamic Analysis of Nucleic Acid Samples> The present disclosure provides a method for thermodynamic analysis of a sample. For example, the devices described herein can be used for high-resolution melting (HRM) analysis. A method for analyzing a plurality of nucleic acid molecules can include providing a device that includes a plurality of compartments as described herein. At least a subset of the plurality of compartments can contain a plurality of nucleic acid molecules (e.g., deoxyribonucleic acid or ribonucleic acid molecules). Each compartment of the subset of the plurality of compartments can be configured to allow gas flow from the compartment to the environment external to the compartment through at least one barrier that separates the compartment from the external environment. Subsequently, the subset of the plurality of compartments can be subjected to controlled heating. While the subset of the plurality of compartments is subjected to these conditions, signals can be collected from the subset of the plurality of compartments, for example, over a plurality of time points. Subsequently, the signals collected from the plurality of compartments can be processed to yield data indicative of the melting points of at least a subset of the plurality of nucleic acid molecules in the subset of the plurality of compartments. The signal processing can be performed while the controlled heating is in progress or after the controlled heating is completed.

[0128] The method can further include performing a nucleic acid amplification reaction on a nucleic acid sample (e.g., as described herein) under conditions sufficient to yield a plurality of nucleic acid molecules as amplification products of the nucleic acid sample. The amplification reaction can be performed in a subset of the plurality of compartments. For example, a sample containing nucleic acid molecules can be loaded into a subset of the plurality of compartments prior to performing the nucleic acid amplification reaction. Performing the amplification reaction can include heating a subset of the plurality of compartments using the same thermal unit (e.g., a heater) that is used to perform the controlled heating of the subset of the plurality of compartments. The amplification reaction can include one or more reagents such as one or more primers, deoxyribonucleotides, buffers, cofactors, intercalating dyes, and polymerases, or any combination thereof. The reagents can include a detectable label such as a fluorophore or a fluorescent label. In some examples, it can be useful to contact at least a subset of the nucleic acid molecules of the nucleic acid sample with an intercalating dye prior to performing the amplification reaction.

[0129] Controlled heating of a subset of the plurality of compartments of the device can be carried out at any useful rate over any useful temperature range. For example, the controlled heating can be carried out at a lower temperature of at least about 25°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, or about 95°C, or higher. The controlled heating can be carried out at a higher temperature of at least about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 91°C, about 92°C, about 93°C, about 94°C, about 95°C, about 96°C, about 97°C, about 98°C, about 99°C, or about 100°C, or higher. The temperature can be increased by any useful increment. For example, the temperature can be increased by at least about 0.01°C, about 0.05°C, about 0.1°C, about 0.2°C, about 0.3°C, about 0.4°C, about 0.5°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, or about 10°C, or higher. The controlled heating can also occur over temperature increments at unequal intervals. For example, the temperature can be increased by about 0.1°C (e.g., fine-grained measurements) over a range where significant melting of the nucleic acid molecule is expected and by about 1°C (e.g., coarse-grained measurements) over a range where significant melting of the nucleic acid molecule is not expected.Controlled heating may be carried out at any useful rate, such as at least about 0.0001 °C / second, about 0.002 °C / second, about 0.0003 °C / second, about 0.004 °C / second, 0.005 °C / second, about 0.0006 °C / second, about 0.0007 °C / second, about 0.0008 °C / second, about 0.0009 °C / second, about 0.001 °C / second, about 0.0002 °C / second, about 0.003 °C / second, about 0.0004 °C / second, about 0.0005 °C / second, about 0.006 °C / second, about 0.007 °C / second, about 0.008 °C / second, about 0.009 °C / second, about 0.01 °C / second, about 0.02 °C / second, about 0.03 °C / second, about 0.04 °C / second, about 0.05 °C / second, about 0.06 °C / second, about 0.07 °C / second, about 0.08 °C / second, about 0.09 °C / second, about 0.1 °C / second, about 0.2 °C / second, about 0.3 °C / second, 0.4 °C / second, about 0.5 °C / second, about 0.6 °C / second, about 0.7 °C / second, about 0.8 °C / second, about 0.9 °C / second, about 1 °C / second, about 2 °C / second, about 3 °C / second, about 4 °C / second, and about 5 °C / second, or more. A heat unit (e.g., a heater) that executes the controlled heating process can maintain a predetermined temperature over any useful period. For example, the predetermined temperature can be maintained for at least about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 70 seconds, about 80 seconds, about 90 seconds, about 100 seconds, about 110 seconds, about 120 seconds, about 130 seconds, about 140 seconds, about 150 seconds, about 160 seconds, about 170 seconds, about 180 seconds, about 190 seconds, about 200 seconds, about 210 seconds, about 220 seconds, about 230 seconds, about 240 seconds, about 250 seconds, or about 300 seconds, or longer.

[0130] Signals can be collected from a subset of multiple compartments at any desired time point. For example, signals can be collected at least once every about 1 second, about 2 seconds, about 3 seconds, about 4 seconds, about 5 seconds, about 6 seconds, about 7 seconds, about 8 seconds, about 9 seconds, about 10 seconds, about 20 seconds, about 30 seconds, about 45 seconds, about 60 seconds, about 70 seconds, about 80 seconds, about 90 seconds, about 100 seconds, about 110 seconds, about 120 seconds, about 130 seconds, about 140 seconds, about 150 seconds, about 160 seconds, about 170 seconds, about 180 seconds, about 190 seconds, about 200 seconds, about 210 seconds, about 220 seconds, about 230 seconds, about 240 seconds, about 250 seconds, about 300 seconds, or more often. Signals can be collected more than once per temperature interval. For example, signals can be collected at the end of a temperature interval, before increasing the temperature for the next temperature interval. As described herein, the collection of signals can include imaging. Processing the collected signals can include using signals that generate signal-to-temperature data for a subset of multiple nucleic acid molecules in a subset of multiple compartments.

[0131] The plurality of nucleic acid molecules analyzed by the method can be derived from a sample that contains or is suspected of containing a pathogen. The pathogen can be at least one bacterium. The bacterium is not limited, but can be selected from the group consisting of Bacillus anthracis, Bacillus cereus, Bacillus halodurans, Bacillus mycoides, Bacillus polymyxa, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus capitis, Staphylococcus caprae, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lentus, Staphylococcus lugdunensis, Staphylococcus saprophyticus, Staphylococcus xylosus, Propionibacterium acnes, Enterococcus faecalis, Actinobacteria, Alphaproteobacteria, Bacteroidetes, Betaproteobacteria, Chlamydia, Epsilonproteobacteria, Firmicutes, Gammaproteobacteria, Spirochaetales, and Tenericutes. The method can include a further processing step for isolating or extracting nucleic acid molecules from the bacterium. Performing a nucleic acid amplification reaction on a nucleic acid sample can include amplifying at least a part of the internal transcribed spacer region of a subset of the nucleic acid molecules of the nucleic acid sample. Alternatively, or additionally, amplification of ribosomal RNA (e.g., 16S) may occur.

[0132] The sample used in the method described herein can be a biological sample. The biological sample can include a body fluid selected from the group consisting of blood, urine, semen, mucus, saliva, and any combination thereof. Alternatively, as described herein, the sample can be an environmental sample.

[0133] The method can further include a step of filling a plurality of compartments of a device with a plurality of nucleic acid molecules, wherein during filling, gas in a subset of the plurality of compartments containing the plurality of nucleic acid molecules is subject to a flow from the subset of the plurality of compartments to the external environment.

[0134] A device used in a method for analyzing nucleic acid molecules can have any of the features described herein. The barrier of the device can comprise a polymeric material such as a thermoplastic material or can be a thin film. The barrier can be substantially optically transparent. The barrier can have a thickness of from about 50 μm to about 200 μm (e.g., about 50 μm, 100 μm, 150 μm, or 200 μm). The device can include at least one inlet, at least one outlet, and at least one microchannel including a plurality of suction openings. A subset of the plurality of compartments can be in fluid communication with the microchannel by way of the plurality of suction openings. The plurality of compartments can include from about 1,000 to about 20,000 compartments (e.g., about 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 10,000, 15,000, or 20,000 compartments).

[0135] Figures 22A-22B schematically illustrate high-resolution melting (HRM) analysis. Figure 22A illustrates the difference between digital HRM analysis and bulk HRM analysis. As shown in the upper panel, in digital HRM analysis, each compartment contains at most one target DNA molecule and the melting curves of various bacteria are resolved. The lower panel shows bulk HRM analysis where a single, non-differentiable melting curve is measured from a heterogeneous sample. Figure 22B shows HRM curves for a mixed sample containing a number of different bacterial species. Distinct helicity curves for Staphylococcus aureus, Enterococcus faecalis, and Propionibacterium acnes are shown in the upper panel and the lower panel shows their use for determining compartment occupancy using Poisson statistics.

[0136] E of A-23 in FIG. 23 shows HRM data for various bacterial species. FIG. 23A shows 16S and internal transcribed spacer (ITS) composite derivative HRM curves for 89 different bacteria. Notably, the melting curve corresponding to the ITS region of the bacterial nucleic acid molecule shows a wider temperature range and greater curve diversity than the melting curve corresponding to the 16S ribosomal RNA. FIG. 23B shows HRM curves for seven different species of the genus Bacillus. FIG. 23C shows HRM curves for nine different species of the genus Staphylococcus. FIG. 23D shows HRM curves for five different species of Streptococcus pneumoniae. FIG. 23E shows a heat map of ITS sequence homology for 153 different bacterial species organized by phylum.

[0137] A-B of FIG. 24 schematically illustrate HRM analysis. FIG. 24A illustrates the partitioning of DNA from a sample amplified in a bulk PCR reaction containing multiple human gDNA and HIV proviral DNA molecules. The sample is partitioned such that the HIV proviral DNA is not disrupted and there is an average of approximately 3 billion g base pairs of DNA per compartment. FIG. 24B shows a virtual HRM analysis for the compartments in which PCR occurs. Each panel shows the temperature-dependent fluorescence signal corresponding to various theoretical compartment populations. The leftmost panel illustrates the virtual signal for a compartment containing all five amplicons; the middle panel illustrates the virtual signal for a compartment containing only amplicons 1, 2, and 4; and the rightmost panel illustrates the virtual signal for a compartment containing only amplicons 2 and 5.

[0138] <System for analyzing a sample> In one aspect, the present disclosure provides a system for using a microfluidic device (e.g., as described herein) to analyze a plurality of nucleic acid molecules. The system can include a support unit configured to receive a device that includes a plurality of compartments. Each compartment of a subset of the plurality of compartments of the device can be configured to allow gas flow from the compartment to the external environment outside the compartment through at least one barrier that separates the subset of the plurality of compartments from the external environment. The system can also include a detector configured to collect signals from a subset of the plurality of compartments of the device over a plurality of time points. The system can also include one or more computer processors operably coupled to the detector. As described herein, the one or more computer processors can be programmed individually or collectively to expose a subset of the plurality of compartments to conditions sufficient to perform a nucleic acid amplification reaction using the plurality of nucleic acid molecules to generate amplification products from at least a subset of the plurality of nucleic acid molecules. While the amplification reaction is proceeding, the one or more computer processors can also be programmed to receive signals collected by the detector from a subset of the plurality of compartments over a plurality of time points. The signals can be collected and stored by the detector and sent to the processor at a predetermined time, or provided to the processor when the signals are collected. The one or more computer processors can be programmed to command the collection of signals from a subset of the plurality of compartments. Further, they can be programmed to process the collected signals to determine the number of nucleic acid molecules in a subset of the plurality of compartments. The system can further include a fluid flow device (e.g., a pneumatic unit) configured to direct the plurality of nucleic acid molecules into the plurality of compartments. The one or more computer processors can be programmed individually or collectively to direct the fluid flow device to fill the plurality of compartments with the plurality of nucleic acid molecules.

[0139] The present disclosure also provides a system for using a microfluidic device (e.g., as described herein) to analyze a sample that contains or is suspected of containing a plurality of nucleic acid molecules. Such an analysis can include a thermodynamic evaluation of the dissociation characteristics of nucleic acid molecules such as DNA in the sample. The thermodynamic evaluation can include determination of the melting point of the DNA and the binding strength of the individual strands of the DNA molecule.

[0140] As described above, the system can include a support unit and a detector. The system can further include a thermal unit configured to expose a subset of the plurality of compartments to controlled heating. The system can also include one or more computer processors operably coupled to the detector. The one or more computer processors can be programmed individually or collectively to direct the thermal unit to expose a subset of the plurality of compartments to controlled heating. The one or more computer processors can also be programmed to receive signals collected from the subset of the plurality of compartments by the detector while the subset of the plurality of compartments is being exposed to controlled heating. Further, they can be programmed to process the signals collected to yield data indicative of the melting points of the subset of the plurality of compartments. The system can further include a fluid flow device (e.g., a pneumatic unit) configured to direct the plurality of nucleic acid molecules to the plurality of compartments. The one or more computer processors can be programmed individually or collectively to direct the fluid flow device to fill the plurality of compartments with the plurality of nucleic acid molecules.

[0141] The system may include a support unit, such as a transfer stage, platform, slot, or groove, configured to hold one or more microfluidic devices. The microfluidic device may include a microchannel having an inlet and an outlet, a plurality of microchambers connected to the microchannel by a plurality of suction openings, and a thin film (e.g., a thermoplastic thin film) that caps or covers the microfluidic device. The apparatus may include a pneumatic unit in fluid communication with the microfluidic device. The pneumatic unit can fill the microfluidic device with a reagent and distribute the reagent into the microchambers. The system may include a thermal unit in thermal communication with the plurality of microchambers. The thermal unit can control the temperature of the microchambers and thermally cycle the microchambers. The system may include a detector for collecting signals from the microchambers of the device or a subset thereof. The detector may be an optical unit capable of imaging the plurality of microchambers. The system may also include one or more computer processors coupled to the support unit, the pneumatic unit, the thermal unit, and the detector (e.g., the optical unit). The one or more computer processors may be programmed to (i) instruct the pneumatic unit to fill the microfluidic device with a reagent and distribute the reagent into the plurality of microchambers, (ii) instruct the thermal unit to thermally cycle the plurality of microchambers, and (iii) instruct the detector (e.g., the optical unit) to collect signals (e.g., image) from the plurality of microchambers.

[0142] The support unit may be configured to receive a microfluidic device, hold the microfluidic device, and output the microfluidic device. The support unit may be stationary at one or more coordinates. Alternatively, or additionally, the support unit may be movable in the X direction, Y direction, Z direction, or any combination thereof. The support unit may be capable of holding a single microfluidic device. Alternatively, or additionally, the transfer stage may be capable of holding at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more microfluidic devices.

[0143] The pneumatic unit may be configured to be in fluid communication with the inlets and outlets of the microfluidic device. The pneumatic unit may have a number of connection points connectable to a number of inlets and a number of outlets. The pneumatic unit may be able to fill, refill, and dispense a single array of microchambers, or a number of arrays of microchambers in succession. The pneumatic unit may further include a vacuum unit. The pneumatic unit may bring about a pressure increase or a vacuum in the microfluidic device.

[0144] The thermal unit may be configured to be in thermal communication with the microchambers of the microfluidic device. The thermal unit may be configured to control the temperature of a single array of microchambers, or to control the temperature of a number of arrays of microchambers. The thermal control unit may implement the same thermal program across all arrays of microchambers, or different thermal programs for different arrays of microchambers. The thermal unit may be configured to perform both thermal cycling and controlled heating. Alternatively, the system may include a plurality of thermal units, each of which is configured to perform a separate thermal process such as thermal cycling and controlled heating.

[0145] The detector may be configured to collect signals from all or a subset of a plurality of compartments of the device. For example, the detector can collect light, impedance, or any other useful signal type. The detector can be an optical unit. The optical unit may be configured to emit and detect light of multiple wavelengths. The emission wavelength may correspond to the excitation wavelength of the indicator and amplification probe used. The emitted light may include wavelengths having a maximum intensity of about 450 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or any combination thereof. The detected light may include wavelengths having a maximum intensity of about 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or any combination thereof. The optical unit can be configured to emit 1, 2, 3, or 4 or more wavelengths of light. The optical unit can be configured to detect 1, 2, 3, or 4 or more wavelengths of light. The emitted wavelength of light may correspond to the excitation wavelength of the indicator molecule. Another emitted wavelength of light may correspond to the excitation wavelength of the amplification probe. One detected wavelength of light may correspond to the emission wavelength of the indicator molecule. Another detected wavelength of light may correspond to the amplification probe used to detect the reaction within the microchamber. The optical unit can be configured to image a subset of the microchambers or a section of an array of microchambers. Alternatively, or additionally, the optical unit can image the entire array of microchambers in a single image.

[0146] Figure 6 illustrates a machine (600) for performing the process of Figure 5 in a single machine. The machine (600) includes a pneumatic unit (601), which includes a pump and a manifold and is movable in the Z direction and may be operable to apply pressure as described in Figure 3A - Figure 3D. The machine (600) also includes a thermal unit (602), such as a flat block thermal cycler, to thermally cycle the microfluidic device, thereby causing a polymerase chain reaction. The machine (600) further includes an optical unit (603), such as an epi - fluorescence optical unit, that can optically determine which microchambers in the microfluidic device are successfully performing the PCR reaction. The optical unit (603) supplies this information to a processor (604) that uses Poisson statistics to convert the raw count of successful microchambers to nucleic acid concentration. A support unit (605) (e.g., a transfer stage) can be used to move a given microfluidic device between various units and to handle multiple microfluidic devices simultaneously. Combined with incorporating this functionality into a single machine, the microfluidic device reduces the cost, workflow complexity, and space requirements related to dPCR compared to other implementations of dPCR.

[0147] Figure 12A illustrates a device according to one embodiment of the present disclosure. The device has eight independent reaction arrays, each of which can be independently filled with reagents. Each reaction array has 20,000 individual compartments. Figure 12B illustrates the configuration of a portion of the compartments within one reaction array of the device. This illustration shows the tortuous path of the filling conduit and a close - up view of the compartments to be filled stemming off from the filling conduit. Each circle represents an individual compartment that can be filled with a reagent containing zero, one, or more nucleic acid templates, which are individually amplified and analyzed as described herein.

[0148] Figure 13 illustrates a schematic of the device, along with a pneumatic unit used to provide fluid control to the device. Each port provides a fluid interface to one of eight (in this embodiment) reaction arrays on the device (1300). As described herein, a series of low and high pressures applied at the inlets and outlets of each reaction array fill the compartments within the reaction array with reagent. The pneumatic unit (1301) controls the application of these pressures and includes an electronic pressure regulator (1302), at least two valves (1303 and 1304). More valves can be incorporated (e.g., to provide separate filling of each reaction array). The device may include a mechanical key to assist with orientation and alignment (such as a tab (1305) at the top of the device) within the system of the present disclosure, or may include visual features such as registration marks (not shown).

[0149] Figure 14A illustrates a flat block thermal cycling device used in an embodiment of the present disclosure. The flat block thermal cycling device provides thermal control to enable PCR amplification cycles in accordance with an embodiment of the present disclosure. The flat block includes threaded holes (not shown) such that an air clamp can be directly attached to the flat block. This can be used to secure the device to the flat block, for example, for thermal cycling of samples and reagents contained within the device, as described herein.

[0150] Figure 4B illustrates the flat block thermal cycle unit (1401) of Figure 4A with an additional air clamp (1402), where the clamp is opened so that a device (1403), such as the device of Figure 12A, can be loaded into the thermal unit. In the illustration, the device (1403) is loaded into an integrated thermal unit / clamp system for PCR amplification cycles. After the device (1403) is loaded into the thermal unit / clamp system, air pressure can be applied via pneumatic drive to fix it with the air clamp (1402) and hold the device in place. The pneumatic drive may be integrated into the above pneumatic unit with respect to Figure 13 or may be a separate pneumatic system. In a system where the pneumatic unit is integrated, an empty device is placed in the thermal cycling device, and reagents can be filled while the device remains in a fixed position on the block. Filling of reagents and / or samples can also be performed using a separate pneumatic unit, for example, before a reagent / sample filling device is loaded into the system for amplification purposes.

[0151] Figure 15 illustrates a complete qdPCR system (150) used in an embodiment of the present disclosure. The system (1500) includes a thermal unit (1501), an optical unit (1502) (including components (1502A, 1502B, and 1502C)), a pneumatic unit (1503), and a mechanical unit (1504). Each unit cooperates with other units to perform the qdPCR process according to an embodiment of the present disclosure.

[0152] The thermal unit (1501) provides thermal cycling and / or controlled heating of a reagent and / or sample filling device, or a subset of its plurality of compartments. The thermal unit (1501) provides the ability to subject the device to thermal cycling and / or controlled heating. As described herein, the thermal cycle may include a denaturation stage, an annealing stage, and an extension stage. A single PCR amplification cycle can occur, for example, in about 60 to 120 seconds. Other temperature profiles may also be used in embodiments according to the present disclosure. For example, the thermal unit (1501) may be configured to heat the device to a holding temperature that enables storage of the product of the PCR amplification reaction without further change. As described herein, the thermal unit (1501) may also be configured to perform controlled heating (e.g., high-resolution melting analysis). The thermal unit (1501) may include components such as a temperature adjustment unit, a temperature probe, circuitry, and other useful components. The thermal unit (1501) can include a support unit for supporting the device and can incorporate an air clamp (1505) for fixing the device to the thermal unit (1501).

[0153] The optical unit (1502) provides imaging of the device or a subset of its plurality of compartments during, before, and / or after an amplification or controlled heating process. The optical unit (1502) may be configured to image each compartment at least once, for example, per amplification cycle. As described herein, each compartment may be imaged more frequently (e.g., twice per amplification cycle, or ten times per amplification cycle). The optical unit (1502) may also be configured to image each compartment at multiple time points during a controlled heating process.

[0154] The optical unit (1502) can image the device partially (e.g., imaging a 5x5 grid of compartments in each image), as a whole, or for each array (e.g., imaging all 20,000 compartments in a given array in a single image). If the optical unit (1502) can image the entire device in one image, it may not be necessary to move the optical unit (1502). For partial imaging, the optical unit (1502) can be configured to direct itself towards the device (e.g., using a mechanical key or registration marks) and then scan across the compartments of the device. Alternatively, the optical unit (1502) can be fixed in position and the thermal unit (1501) can be moved to enable the optical unit (1502) to image each compartment or group of compartments. The optical unit (1502) may be configured to orient itself using registration marks printed on the device. The optical unit (1502) can be oriented by the position of the thermal unit (1501) or by the positioning of the optical unit (1502), the thermal unit (1501), and any associated handling devices. As described herein, the optical unit (1502) includes a light source (1502A), an excitation filter (1502B), a dichroic mirror (1502C), an emission filter (1502D), a focusing lens (1502E), and an image sensor (1502F).

[0155] The pneumatic unit (1503) provides functionality for fluid handling and / or fixation of the device. The same pneumatic unit (1503) can provide both the fluid handling / reagent filling function and the air clamping function for the air clamp (1505) mounted on the thermal unit (1501) to the device. Alternatively, the pneumatic unit (1503) can simply provide the clamping function for the air clamp (1505) and a separate pneumatic unit (not shown) can provide reagent filling to the device.

[0156] The mechanical unit (1504) provides mechanical handling and movement of various components. In the illustrated embodiment, the mechanical unit (1504) provides the ability to scan the optical unit (1502) across the compartments of the device. In this embodiment, the mechanical unit (1504) moves the optical unit (1502) to the next imaging position to enable it to be imaged, and then repeats the process until all compartments have been imaged. This process can be repeated as many times as necessary, depending on the desired number of amplification cycles, the number of images per cycle, and / or the controlled heating process being targeted. The mechanical unit (1504) can move the thermal unit (1501) instead of the optical unit (1502). For example, the mechanical unit (1504) can move the thermal unit (1501) to the imaging position so that an image can be taken by the optical unit (1502), and then the thermal unit (1501) can be moved again to enable imaging of the new imaging position. This process may be repeated as needed. The mechanical unit (1504) can provide additional functions such as mechanical handling of the device to load it onto the thermal unit (1501) prior to the implementation of the qPCR process or to automatically unload it from the thermal unit (1501) after completion of the qPCR process.

[0157] The system of the present disclosure can include multiple examples of one or more thermal units (1501), optical units (1502), pneumatic units (1503), and mechanical units (1504), and can be incorporated into the same system to provide an automated system that can process multiple devices simultaneously or can sequentially perform various steps on various devices. Additionally, the system can incorporate a processor to perform analysis of the amplification kinetics detected by the optical unit (1502) or for other analysis functions as described above with respect to the present disclosure.

[0158] Figures 17A-17B show an exemplary system for the processing of nucleic acid molecules. Figure 17A shows an entire exemplary system for processing nucleic acid molecules, including a user interface (1701) coupled to a computer processor (1702), a thermal unit (1703), and a camera (1704). Figure 17B shows a close-up image illustrating the camera (1704), LED emitter / heat sink (1705), filter cube (1706), and shutter (1707) on the device (1708).

[0159] The scope of the present disclosure is not limited by the specific embodiments described herein. Indeed, various other embodiments and variations of the present disclosure will be apparent to those skilled in the art from the foregoing description and the accompanying drawings.

[0160] For example, while dPCR applications are also described in context, other microfluidic devices that may require a number of isolated microchambers filled with a liquid isolated via a gas or other fluid may benefit from the use of a thin thermoplastic membrane, where gas release can avoid gas fouling while also providing benefits with respect to manufacturability and cost. Apart from PCR, other nucleic acid amplification methods such as loop mediated isothermal amplification can be applied to perform digital detection of specific nucleic acid sequences in accordance with embodiments of the present disclosure. The microchambers can also be used to isolate a single cell, and the aspiration opening is designed to be close to the diameter of the cell to be isolated. If the aspiration opening is much smaller than the size of blood cells, the methods described herein can be used, for example, to separate the plasma from whole blood.

[0161] <Computer System for Analyzing Nucleic Acid Samples> The present disclosure provides a computer control system programmed to implement the methods of the present disclosure. FIG. 7 shows a computer system (701) that can be programmed or otherwise configured for processing and analyzing nucleic acid samples, including dispensing, amplifying, and detecting the samples. The computer system (701) can regulate various aspects of the methods and systems of the present disclosure. The computer system (701) may be a user's electronic device or a computer system located remotely with respect to the electronic device. The electronic device may be a mobile electronic device.

[0162] The computer system (701) includes a central processing unit (CPU, also referred to herein as "processor" and "computer processor") (705), which can be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (701) also includes a memory or storage location (710) (e.g., random access memory, read-only memory, flash memory), an electronic storage device (715) (e.g., hard disk), a communication interface (720) (e.g., network adapter) for communicating with one or more other systems, and peripheral devices (725) such as cache, other memory, data storage devices, and / or an electronic display adapter. The memory (710), storage device (715), interface (720), and peripheral devices (725) communicate with the CPU (705) through a communication bus (solid line) such as a motherboard. The storage device (715) may be a data storage device (or data repository) for storing data. The computer system (701) is operably coupled to a computer network ("network") (730) with the aid of the communication interface (720). The network (730) may be the Internet, the Internet and / or an extranet, or an intranet and / or an extranet capable of communicating with the Internet. The network (730) may in some cases be a telecommunications and / or data network. The network (730) can include one or more computer servers that enable distributed computing such as cloud computing. The network (730) may in some cases implement a peer-to-peer network that enables devices connected to the computer system (701) to operate as clients or servers with the aid of the computer system (701).

[0163] The CPU (705) can execute a series of machine-readable instructions that can be embodied in a program or software. Those instructions can be stored in a storage location such as the memory (710). Those instructions can be directed to the CPU (705) and continue to program or otherwise configure the CPU (705) to execute the method of the present disclosure. Examples of operations executed by the CPU (705) can include fetch, decode, execute, and write-back.

[0164] The CPU (705) can be part of a circuit such as an integrated circuit. One or more other components of the system (701) may be included in the circuit. In some cases, the circuit is an application-specific integrated circuit (ASIC).

[0165] The storage device (715) can store files such as drivers, libraries, and stored programs. The storage device (715) can store user data, such as user preferences, and user programs. The computer system (701) can include one or more additional data storage devices external to the computer system (701), such as being located on a remote server that communicates with the computer system (701) via an intranet or the Internet in some cases.

[0166] A computer system (701) can communicate with one or more remote computer systems through a network (730). For example, the computer system (701) can communicate with a remote computer system of a user (e.g., a service providing company). Examples of remote computer systems include personal computers (e.g., portable PCs), slates or tablet PCs (e.g., Apple® iPad®, Samsung® Galaxy Tab), phones, smartphones (e.g., Apple® iPhone®, Android-enabled devices, Blackberry®), or mobile information terminals. A user can access the computer system (701) via the network (730).

[0167] The methods as described herein can be implemented by machine (e.g., computer processor) executable code stored in an electronic storage device of a computer system (701), such as, for example, on a memory (710) or an electronic storage device (715). In some embodiments, the machine executable code or machine readable code can be provided in the form of software. During use, the code can be executed by a processor (705). Optionally, the code can be retrieved by the processor (705) from the storage device (715) and stored in the memory (710) for easy access. In some situations, the electronic storage device (715) can be eliminated and the machine executable instructions can be stored in the memory (710).

[0168] The code can be pre-compiled and configured for use with a machine having a processor suitable for executing the code, or can be compiled during runtime. The code can be provided in a programming language that can be selected to execute the code in a pre-compiled manner or in a just-in-time compiled manner.

[0169] In one aspect, the present disclosure provides a non-transitory computer-readable medium including machine-executable code that, when executed by one or more computer processors, implements a method for forming a microfluidic device to amplify and quantify a nucleic acid sample. The method includes: thermally injecting a thermoplastic to fabricate a microfluidic structure including at least one microchannel, a plurality of microchambers, and a plurality of suction openings, wherein the plurality of microchambers are connected to the at least one microchannel by the plurality of suction openings; forming at least one inlet and at least one outlet, wherein the at least one inlet and the at least one outlet are in fluid communication with at least the microchannel; and applying a thermoplastic thin film to cap the microfluidic structure, wherein the thermoplastic thin film is at least partially gas permeable to a pressure difference and is applied across the thermoplastic thin film.

[0170] In one aspect, the present disclosure provides a non-transitory computer-readable medium including machine-executable code that, when executed by one or more computer processors, implements a method for analyzing and quantifying a nucleic acid sample. The method includes: providing a microfluidic device including a plurality of microchambers; filling the microfluidic device with a sample and / or one or more reagents as described herein (e.g., using a pneumatic unit or fluid flow device and a series of pressure differences); heating a plurality of microchambers of the device or a subset thereof periodically (e.g., for an amplification reaction by thermal cycling) and / or with controlled intensification (e.g., for high-resolution melting or other thermodynamic analysis); collecting signals from the plurality of microchambers or subset thereof during an amplification reaction or controlled heating, or during or after the progression of these processes; processing the signals collected from the plurality of microchambers to determine the number of nucleic acid molecules in a subset of the plurality of compartments and / or to provide data indicative of melting points corresponding to the plurality of nucleic acid molecules or a subset thereof in a subset of the plurality of compartments, the method including one or more of these steps. One or more processors may also be programmed to implement a method of filling the microchambers of the device or a subset thereof with a sample and / or reagents.The method comprises: providing a microfluidic device comprising at least one microchannel, wherein the at least one microchannel comprises at least one inlet and at least one outlet, and wherein the microfluidic device further comprises a plurality of microchambers connected to the microchannel by a plurality of suction openings, and a thermoplastic thin film disposed adjacent to the surface of the microfluidic device so as to cap the microchannel, the plurality of microchambers, and the plurality of suction openings; providing a reagent at at least one inlet or at least one outlet; filling the microfluidic device by providing a first pressure difference between the sample and / or reagent and the microfluidic device, wherein the first pressure difference causes the sample and / or reagent to flow into the microfluidic device; applying a second pressure difference between the microchannel and the plurality of microchambers to move the sample and / or reagent into the plurality of microchambers and to cause the gas in the plurality of microchambers to pass through the thermoplastic thin film that caps or covers the plurality of microchambers, the plurality of suction openings, and the microchannel, wherein the second pressure difference is greater than the first pressure difference; and applying a third pressure difference between the at least one inlet and the at least one outlet to introduce fluid into the microchannel without introducing fluid into the microchambers, wherein the third pressure difference is less than the second pressure difference.

[0171] Aspects of the systems and methods provided herein, such as computer system (701), may be embodied in programming. Various aspects of the technology may typically be considered as a "product" or "article" in the form of machine (or processor) executable code and / or associated data held on or embodied in a type of machine-readable medium. Machine executable code can be stored in an electronic memory device such as a memory (e.g., read only memory, random access memory, flash memory) or a hard disk. A "storage" type medium can include various semiconductor memories, tape drives, disk drives, etc., which can provide non-transitory storage at any time with respect to software programming, tangible memories such as computers, processors, or any or all of their associated units or modules. All or part of the software is sometimes communicated via the Internet or various other communication networks. Such communication can enable, for example, the loading of software from one computer or processor to another, such as from a management server or host computer to an application server computer platform. Thus, another type of medium that can hold software elements includes optical, electrical, and electromagnetic waves such as those used via various air links and wired and optical terrestrial communication line networks between local devices. Physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered as media that hold software. As used herein, the term such as "readable medium" of a computer or machine refers to any medium involved in providing instructions to a processor for execution, unless limited to non-transitory tangible "storage" media.

[0172] Accordingly, machine-readable media such as computer-executable code may take many forms including, but not limited to, tangible storage media, carrier wave media, or physical transmission media. Non-volatile storage media includes, for example, optical disks or magnetic disks such as any computer(s) in which a database as shown in the drawings may be implemented, such as any of the storage devices in any computer. Volatile storage media includes dynamic memory such as the main memory of such a computer platform. Tangible transmission media includes coaxial cables; copper wires and fiber optics including wires that form part of a bus within a computer system. Carrier wave transmission media may take the form of electrical or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Thus, common forms of computer-readable media include, for example: floppy disks, flexible disks, hard disks, magnetic tape, other magnetic media, CD-ROM, DVD or DVD-ROM, other optical media, punch cards, paper tape, other physical storage media with patterns of holes, RAM, ROM, PROM and EPROM, FLASH-EPROM, other memory chips or cartridges, carrier waves carrying data or instructions, cables or links that transmit such carrier waves, or other media that a computer can read programming code and / or data from. Many of these forms of computer-readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0173] A computer system (701) includes, or is communicable with, an electronic display (735) that includes a user interface (UI) (740) for providing, for example, an epithelial tissue depth profile. Examples of UIs include, but are not limited to, graphical user interfaces (GUIs) and web-based user interfaces.

[0174] The methods and systems of the present disclosure can be implemented by one or more algorithms. The algorithms can be implemented by software when executed by a central processing unit (705). The algorithms can, for example, adjust the systems or methods of implementation provided herein.

[0175] Preferred embodiments of the present disclosure are shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the invention described herein. It should be understood that various alternatives to the embodiments of the invention described herein can be utilized in practicing the invention. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

[0176] Example 1: Demonstration of Reagent Dispensing Demonstrate the dispensing of reagents using a microfluidic device assembled using standard microscope slide dimensions. The total dimensions of the microfluidic device are 1 inch in width, 3 inches in length, and 0.6 inches in thickness. The device includes the design of four different microchamber arrays and a total of eight different microchamber arrays. Figure 8A shows an enlarged perspective view of an 8-unit device and one of the four array designs. The microfluidic device was molded from cycloolefin polymer (COP), Zeonor 790R (Zeon Chemicals, Japan), and sealed by thermally bonding a 100-μm COP thin film, Zeonox ZF14 (Zeon Chemicals, Japan). The enlarged microfluidic segment shown has a tortuous microchannel connected to the microchamber by a suction opening. The microchambers are in a grid configuration. The depth of the microchambers and microchannels is 40 μm, and the depth of the suction opening is 10 μm. Each isolated microfluidic segment has inlet and outlet channels. Before thermally bonding the membrane to the base of the microfluidic device, mechanically drill holes in the inlet and outlet channels. The diameter of the inlet and outlet channels is 1.6 mm.

[0177] Figure 8B shows fluorescence images of reagent filling, microchamber refilling, and dispensing. Before filling the microfluidic device, dispense 2 microliters (μL) of 4-kilodalton (kDa) fluorescein conjugated dextran (Sigma-Aldrich, St. Louis, MO) into the inlet. Then, bring the microfluidic device into contact with a pneumatic control device. The pneumatic control device fills the microchannels of the microfluidic device by applying a pressure of 4 psi to the inlet for 3 minutes. The microchambers are filled by pressurizing both the inlet and outlet to 10 psi for 20 minutes. Then, the reagent is dispensed by flowing air at 4 psi from the inlet of the microfluidic device, and the reagent is removed from the microchannels.

[0178] Figure 20A shows images corresponding to the differential viral loading of a series of devices. The panels include images of compartments containing 5, 0.5, 0.05, and 0.005 nucleic acid copies per compartment. These images were taken after thermal cycling and were imaged using a fluorescence imaging device for the FAM and ROX fluorophores. Figure 20B shows the corresponding Poisson analysis of the images of Figure 20A using Image J software and Image R software. Similarly, Figure 21 shows images corresponding to the differential filling of a series of devices. The panels include images of compartments containing 10, 1.0, 0.1, and 0.01 nucleic acid copies per compartment. The rightmost panel shows the density and fractional occupancy corresponding to each device.

[0179] Example 2: Workflow for a single instrument for dPCR Methods for the amplification and quantification of nucleic acids in a microfluidic device can be implemented with a single instrument. The instrument can enable reagent dispensing, thermal cycling, image acquisition, and data analysis. Figure 9 shows a prototype instrument capable of a single instrument workflow. The instrument is designed to accommodate up to four devices at a time and enable simultaneous image acquisition and thermal cycling. The instrument includes a pneumatic unit for dispensing reagents, a thermal unit for controlling temperature and performing thermal cycling, an optical unit for imaging, and a scanning unit. The optical unit has two fluorescence imaging functions and can detect fluorescence emissions of approximately 520 nm and 600 nm, corresponding to the emission wavelengths of the FAM and ROX fluorophores, respectively. The optical unit has a 25 mm × 25 mm field of view and a numerical aperture (NA) of 0.14.

[0180] The workflow of a single device can be tested using an established qPCR assay that utilizes TaqMan probes as reporters. Briefly, a nucleic acid sample is mixed with PCR reagents. The PCR reagents include a forward primer, a reverse primer, a TaqMan probe, and a ROX indicator. The sequence of the forward primer is 5’-GCC TCA ATA AAG CTT GCC TTG-3’. The sequence of the reverse primer is 5’-GGG GCG CAC TGC TAG AGA-3’. The sequence of the TaqMan probe is 5’-[FAM]-CCA GAG TCA CAC AAC AGA CGG GCA CA-[BHQ1]-3’. The nucleic acid sample and the PCR reagents are filled and dispensed into a microfluidic device after the above protocol. PCR amplification is performed by raising the temperature of the microchamber to 95°C and holding that temperature for 10 minutes, followed by lowering the temperature of the microchamber from 95°C to 59°C at a rate of 2.4°C per second, holding at 59°C for 1 minute, and then returning the temperature to 95°C for 40 cycles. A-D of Figure 10 show fluorescence images of samples containing approximately one copy of the nucleic acid template per compartment and compartments with zero copies of the nucleic acid template per compartment after PCR amplification (template-free control, i.e., NTC), and plots of the fluorescence intensity of samples containing approximately one nucleic acid copy per compartment and NTC compartments after PCR amplification. A of Figure 10 shows a fluorescence image of the dispensed sample without the nucleic acid template, where each gray dot represents a single microchamber containing the PCR reagents. The image is taken by exciting the ROX indicator within each microchamber using light of approximately 575 nm and imaging the emission spectrum with a maximum emission at 600 nm. B of Figure 10 shows the dispensed sample containing approximately one copy of the nucleic acid template per compartment after PCR amplification. After PCR amplification, the imaging shows microchambers containing the ROX indicator and microchambers containing both the ROX indicator and emission from the FAM probe. The FAM probe has a maximum excitation wavelength of approximately 495 nm and an emission wavelength of approximately 520 nm. Each individual microchamber contains the ROX indicator, the FAM probe, and the BHQ-1 quencher.Similar to A of FIG. 10, each gray dot represents a microchamber containing a dispensed sample without a nucleic acid template. The white dots represent microchambers containing successfully amplified nucleic acid samples. When PCR amplification is successful, the FAM fluorophore and the BHQ-1 quencher may be cleaved from the TaqMan probe, resulting in a detectable fluorescence signal. FIGS. 10C and 10D each show a two-dimensional scatter plot of the FAM fluorescence intensity as a function of the ROX fluorescence intensity for each microchamber of the dispensed and amplified microfluidic device. FIG. 10C shows a sample with zero nucleic acid template per compartment, resulting in a predominantly constant FAM fluorescence intensity over a variety of ROX fluorescence intensities. FIG. 10D shows a sample containing approximately one copy of the nucleic acid template per compartment, resulting in a FAM fluorescence intensity that varies as a function of the ROX fluorescence intensity due to the presence of the amplification signal within the compartment.

[0181] Example 3: qdPCR A known PCR reagent kit was used to prepare two reagent mixtures, and the first reagent mixture had 10 times the amount of target as the second reagent mixture. The reagents were filled into two adjacent units of the device and pneumatically adjusted using an off-the-shelf controller and a custom interface jig. The device (1708) was then removed and placed into the system of FIGS. 17A - 17B. The device (1708) was held on a standard flat block thermal cycler (1703) by a glass piece for good thermal contact. The device (1708) was thermally cycled 40 times using a standard 96 - 61°Cx40 PCR protocol. During the low temperature step (61°C) of each cycle (cycles 11 - 40), images were automatically taken using a camera (1704). The first row of images taken at cycle 20, the second row of images taken at cycle 30, and the third row of images taken at cycle 40 are shown in FIG. 18. The images in the upper row correspond to filling at a copy rate of 10 per compartment, and the lower row corresponds to 1 copy per compartment. A custom ImageJ plugin was used to extract mean intensity data for 53 selected points of the 10 - fold image set in each of the 30 - cycle images. These data are illustrated in FIG. 19 and are normalized against background variation.

[0182] Example 4: HRM Analysis Known reagent kits were used to prepare two reagent mixtures, with the first reagent mixture having 10 times the amount of target as the second reagent mixture. The reagents were filled into two adjacent units of the device and pneumatically adjusted using an off-the-shelf controller and custom interface jig. The device (1708) was then removed and placed into the system of FIGS. 17A - 17B. The device (1708) was held on a standard flat block thermal cycler (1703) by glass pieces for good thermal contact. The device (1708) was thermally cycled 40 times using a standard 96 - 61°Cx40 PCR protocol to complete the dPCR process. As the thermal unit (1703) raised the temperature from approximately 60°C to approximately 90°C at a rate of about 0.1°C / s, the camera (1704) continuously imaged the device every 5 seconds. The first row of images taken at approximately 70°C, the second row taken at approximately 80°C, and the third row taken at approximately 90°C are shown in FIG. 25. The images in the upper row correspond to fillings at a copy rate of 10 per compartment, and the lower row corresponds to 1 copy per compartment. A custom ImageJ plugin was used to extract the average intensity data for 54 selected points of the 10x image set. These data are illustrated in FIG. 26.

[0183] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the present invention be limited by the specific examples provided within this specification. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Those skilled in the art will envision many changes, variations, and substitutions without departing from the present invention. Furthermore, it will be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative ratios described herein, which depend on various conditions and variables. It must be understood that various alternatives of the embodiments of the present invention described herein may be utilized in the practice of the present invention. Accordingly, the present invention is contemplated to extend to any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. A method for quantifying a plurality of nucleic acid molecules, comprising: (a) distributing a sample containing the plurality of nucleic acid molecules into a plurality of compartments of a device using a fluid flow device, wherein the plurality of compartments are in the form of an array of dead-end microchambers; (b) subjecting the plurality of compartments containing the sample to thermal cycle conditions sufficient to perform a nucleic acid amplification reaction on the plurality of nucleic acid molecules, thereby amplifying the plurality of nucleic acid molecules to generate amplification products; (c) collecting more than one signal per thermal cycle over a plurality of time points from the sample contained in the plurality of compartments subjected to the conditions; (d) processing the signal to quantify the plurality of nucleic acid molecules. The method further comprises: The plurality of compartments are covered by a barrier, and the barrier is at least partially permeable to gas under a pressure difference applied across the barrier.

2. The method according to claim 1, wherein the compartments within the plurality of compartments have a diameter of about 500 micrometers (μm) or less.

3. The method according to claim 2, wherein the compartments have a depth of about 500 μm or less.

4. The method according to claim 1, wherein the plurality of compartments includes about 1000 to about 20000 compartments.

5. The method according to claim 1, wherein the thermal cycle is performed using a flat block thermal cycler.

6. The method according to claim 1, wherein subjecting the plurality of compartments containing the sample to the conditions and collecting the signal are performed in parallel.

7. The method according to claim 1, wherein subjecting the plurality of compartments containing the sample to the conditions and collecting the signal are performed sequentially.

8. The method according to claim 1, wherein processing the signal includes determining the amplification rate of the first nucleic acid molecule among the plurality of nucleic acid molecules over the plurality of time points.

9. The method according to claim 8, further comprising determining the number of the first nucleic acid molecules in a first compartment of the plurality of compartments using the amplification rate.

10. The method according to claim 9, wherein determining the number of the first nucleic acid molecules in the first compartment includes comparing the amplification rate of the first nucleic acid molecule in the first compartment with the amplification rate of the first nucleic acid molecule in another compartment of the plurality of compartments.

11. The method according to claim 1, wherein the signal is an optical signal. **Claim 12** The method according to claim 1, wherein collecting the signal includes imaging each of the plurality of compartments to collect the signal. **Claim 13** The method according to claim 1, wherein collecting the signal includes detecting fluorescence emission at two or more wavelengths using a detector. **Claim 14** The method according to claim 1, wherein processing the signal includes determining light intensities in compartments within the plurality of compartments over the plurality of time points. **Claim 15** The method according to claim 14, wherein processing the signal further includes determining an amplification rate of nucleic acid molecules within the plurality of nucleic acid molecules using the light intensities. **Claim 16** The method according to claim 1, wherein processing the signal includes simultaneously collecting the signal from each of the plurality of compartments. **Claim 17** The method according to claim 1, wherein dispensing the sample includes applying a pressure difference across the barrier using the fluid flow device and flowing gas within the plurality of compartments through the barrier to an external environment.

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