Microfluidic array for sample digitization

Abstract

To provide systems, methods and devices for processing a biological sample.SOLUTION: The device may be a microfluidic device comprising a fluid flow path and a chamber. The fluid flow path may comprise a channel and an inlet port and no outlet port. The inlet port may be configured to direct a biological sample to the channel. The channel may be in fluid communication with the chamber. The chamber may be configured to receive a portion of the biological sample from the channel and retain the biological sample during processing.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] cross reference This invention claims the benefit of U.S. Provisional Patent Application No. 62 / 77,616, filed December 10, 2018, which is incorporated by reference herein in its entirety.

[0002] STATEMENT OF GOVERNMENT RIGHTS This invention was made with Government support under Small Business Innovation Research Grant No. 1R43CA221597-01A1 awarded by the National Cancer Institute. The United States Government has certain rights in the invention. [Background technology]

[0003] A microfluidic device is a device that includes structures that handle fluids on a small scale. Typically, microfluidic devices operate on the sub-millimeter scale and handle microliter, nanoliter, or smaller volumes of fluids. One application of microfluidic structures is in digital polymerase chain reaction (dPCR). For example, a microfluidic structure with multiple partitions can be used to partition a nucleic acid sample for dPCR. In dPCR, a nucleic acid sample can be diluted so that there is no more than one nucleic acid template in a partition, and a PCR reaction can be performed in each partition. The target nucleic acid is quantified by counting the partitions in which the nucleic acid template was successfully PCR amplified and applying Poisson statistics to the results.

[0004] For genomic researchers and clinicians, dPCR is particularly powerful in detecting rare mutations, quantifying copy number variations, and quantifying next-generation sequencing libraries. Potential applications in clinical settings for liquid cytology with cell-free DNA and viral load quantification further increase the value of dPCR technology. Summary of the Invention

[0005] Methods and devices are provided herein that can be useful for biological sample analysis, for example, amplifying and quantifying nucleic acid. The present disclosure provides methods, systems, and devices that can facilitate sample preparation, sample amplification, and sample analysis by using dPCR. Samples can be digitized or analyzed with little or no waste (e.g., 0 or about 0 sample dead volume). This allows sample analysis, for example, nucleic acid amplification and quantification, to be possible at low cost and low complexity compared to other systems and methods.

[0006] In one aspect, the present disclosure provides a microfluidic device for processing a biological sample, the microfluidic device comprising: a fluid flow path including a channel and an inlet port, where the fluid flow path does not include an outlet port, and where the inlet port is configured to direct a solution including the biological sample to the channel; and a chamber in fluid communication with the channel, where the chamber is configured to receive at least a portion of the solution from the channel and retain at least a portion of the solution during processing.

[0007] In some embodiments, the microfluidic device includes a plurality of chambers in fluid communication with a channel, where the plurality of chambers includes a chamber. In some embodiments, the channel includes a first end and a second end, and where the first end and the second end are connected to a single inlet port. In some embodiments, the fluid flow path is annular. In some embodiments, the channel includes a first end and a second end, and where the first end is connected to an inlet port and the second end is connected to a different inlet port.

[0008] In some embodiments, the chamber is configured to allow for pressurized gas release. In some embodiments, the chamber includes a film or membrane that allows for pressurized gas release. In some embodiments, the film or membrane is a polymeric film or membrane. In some embodiments, the polymeric film or membrane does not include an elastomer. In some embodiments, the film or membrane has a thickness of less than about 100 μm. In some embodiments, the thickness is less than about 50 μm. In some embodiments, the film or membrane is substantially impermeable to liquids.

[0009] In some embodiments, the fluid flow path or chamber does not include a valve. In some embodiments, the volume of the chamber is about 250 picoliters or less. In some embodiments, the volume of the chamber is about 500 picoliters or less. In some embodiments, the chamber has a cross-sectional dimension of about 250 μm or less. In some embodiments, the chamber has a depth of about 250 μm or less. In some embodiments, the microfluidic device further includes a siphon opening disposed between the channel and the chamber, where the siphon opening is configured to provide fluid communication between the channel and the chamber.

[0010] In another aspect, the disclosure provides a method for processing a biological sample, the method comprising: providing a device comprising: (i) a fluid flow path including a channel and an inlet port, the fluid flow path not including an outlet port; and (ii) a chamber in fluid communication with the channel; directing a solution including the biological sample from the inlet port to the channel; and directing at least a portion of the solution from the channel to the chamber, wherein the chamber retains at least a portion of the solution during processing.

[0011] In some embodiments, the device includes a plurality of chambers in fluid communication with the channel, and wherein the plurality of chambers includes the chamber. In some embodiments, the method further includes applying a single pressure differential to the inlet port to direct the solution from the inlet port to the channel and from the channel to the chamber. In some embodiments, the single pressure differential allows for pressurized outgassing of gas in the chamber.

[0012] In some embodiments, the method further comprises applying a first pressure differential to the inlet port to direct the solution from the inlet port to the channel. In some embodiments, the method further comprises applying a second pressure differential to the inlet port to direct the solution from the channel to the chamber. In some embodiments, the second pressure differential is greater than the first pressure differential. In some embodiments, the second pressure differential enables pressurized outgassing of gas in the chamber. In some embodiments, the chamber comprises a film or membrane, and where the film or membrane enables pressurized outgassing of gas in the chamber.

[0013] In some embodiments, the volume of the solution is equal to or less than the volume of the chamber. In some embodiments, the device distributes the solution containing the biological sample into the chamber such that no residual solution remains in the channel. In some embodiments, the method further comprises providing an immiscible fluid to the inlet port and directing the immiscible fluid into the channel. In some embodiments, the volume of the immiscible fluid is greater than the volume of the channel. In some embodiments, the biological sample is a nucleic acid molecule. In some embodiments, the method further comprises amplifying the nucleic acid molecule by thermal cycling the chamber. In some embodiments, the method further comprises controlling the temperature of the channel or chamber. In some embodiments, the method further comprises sensing one or more components of the biological sample or a reaction involving one or more components of the biological sample in the chamber. In some embodiments, sensing one or more components of the biological sample or the reaction comprises imaging the chamber.

[0014] In another aspect, the disclosure provides a system for processing a biological sample, the system including: a device including: (i) a fluid flow path including a channel and an inlet port, where the fluid flow path does not include an outlet port, and where the inlet port is configured to direct a solution including the biological sample into the channel; and (ii) a chamber in fluid communication with the channel, where the chamber is configured to receive at least a portion of the solution from the channel and to hold at least a portion of the solution during processing; a holder configured to receive and hold the device during processing; and a fluid flow module fluidly coupled to the inlet port and configured to provide a pressure differential to a subject, the fluid flow module configured to provide a pressure differential to (i) cause the solution to flow from the inlet port to the channel, and (ii) cause at least a portion of the solution to flow from the channel to the chamber.

[0015] In some embodiments, the device includes a plurality of chambers in fluid communication with the channel, and wherein the plurality of chambers comprises a chamber. In some embodiments, the chamber of the device is configured to allow pressurized outgassing of gas in the chamber when the fluid flow module applies a pressure differential to the inlet port. In some embodiments, the chamber includes a film or membrane configured to allow said pressurized outgassing.

[0016] In some embodiments, the system includes one or more computer processors operably coupled to the fluid flow module, where the one or more computer processors instruct the fluid flow module to provide a pressure differential when the fluid flow module is fluidly coupled to the inlet port, thereby causing the solution to flow from the inlet port into the channel and directing at least a portion of the solution from the channel into the chamber. In some embodiments, the system further includes a thermal module in thermal communication with the chamber, where the thermal module is configured to control a temperature of the chamber during processing. In some embodiments, the system further includes a sensing module in communication with the chamber, the sensing module configured to sense contents of the chamber during processing. In some embodiments, the sensing module is an optical module in optical communication with the chamber. In some embodiments, the optical module is configured to image the chamber.

[0017] In another aspect, the present disclosure provides a system for processing a biological sample, the system including: a holder configured to hold a device, the device including (i) a fluid flow path including a channel and an inlet port, but not an outlet port, and (ii) the chamber in fluid communication with the channel; and one or more computer processors configured to be operatively coupled to the device when the device is held by the holder, wherein the one or more computer processors are individually or collectively programmed to: (i) direct a solution including the biological sample from the inlet port to the channel, and (ii) direct at least a portion of the solution from the channel to the chamber, where the chamber retains at least the portion of the solution during the processing.

[0018] In some embodiments, the system further includes a fluid flow module operably coupled to the one or more computer processors, wherein the fluid flow module is configured to be operably coupled to the device when the device is held by the holder, and wherein the one or more computer processors are programmed to instruct the fluid flow module to direct the solution from the inlet port to the channel.

[0019] In some embodiments, the system further includes a thermal module in thermal communication with the chamber when the device is held by the holder, wherein the thermal module is configured to control a temperature of the chamber during the processing.

[0020] In some embodiments, the system further includes a detection module configured to communicate with the chamber when the device is held by the holder, wherein the detection module is configured to detect contents of the chamber during the processing. In some embodiments, the sensing module is an optical module in optical communication with the chamber. In some embodiments, the optical module is configured to image the chamber.

[0021] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which only exemplary embodiments of the present disclosure are shown and described. As will be seen from the following description, the present disclosure may be in other and different embodiments, each of whose details can be modified in various obvious respects, all without departing from the present disclosure. Thus, the drawings and description are to be regarded as illustrative in nature, and not restrictive.

[0022] Incorporation by Reference All publications, patents, and patent applications mentioned in the specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference. In the event that any publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material. [Brief description of the drawings]

[0023] 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 present invention can be obtained by reference to the following detailed description that sets forth embodiments, in which the principles of the invention are utilized, and the accompanying drawings (or "Figures" herein) of which:

[0024] [Figure 1A] 1A to 1F generally illustrate examples of microfluidic devices and methods for filling the microfluidic devices, with FIG. 1A generally illustrating loading a sample and an immiscible fluid into a microfluidic device. [Figure 1B] 1A to 1F generally illustrate a microfluidic device and a method for filling the microfluidic device, and FIG. 1B generally illustrates pressurizing the microfluidic device to load a sample into the channels. [Figure 1C] 1A to 1F generally illustrate a microfluidic device and a method for filling the microfluidic device, and FIG. 1C generally illustrates continued pressurization to vent the fluid flow path and continue filling the channel with sample. [Figure 1D] 1A to 1F generally illustrate a microfluidic device and a method for filling the microfluidic device, and FIG. 1D generally illustrates partial digitization of a sample into a chamber, filling of oil into a channel, and movement of air. [Figure 1E]1A to 1F illustrate generally a microfluidic device and a method for filling the microfluidic device, and FIG. 1E illustrates generally the further digitization and air movement. [Figure 1F] 1A to 1F illustrate generally a microfluidic device and a method for filling the microfluidic device, and FIG. 1F illustrates generally the complete digitization of a sample. [Figure 2A] 2A to 2E show example images of sample digitization in a microfluidic device, with FIG. 2A showing an example of a microfluidic device. [Figure 2B] 2A-2E show example images of sample digitization in a microfluidic device, and FIG. 2B shows an example of pressurized loading of a sample into a microfluidic device. [Figure 2C] 2A-2E show example images of sample digitization in a microfluidic device, and FIG. 2C shows an example of a sample and immiscible fluid filling a channel. [Figure 2D] 2A to 2E show example images of sample digitization in a microfluidic device, and FIG. 2D shows an example of partial filling of the sample into a chamber. [Figure 2E] 2A to 2E show example images of sample digitization in a microfluidic device, with FIG. 2E showing an example of complete digitization of a sample. [Diagram 3] 1 illustrates diagrammatically an example of a method for digitization of a sample. [Figure 4] 1 illustrates generally an example of a method for digital polymerase chain reaction (dPCR). [Diagram 5] 1 illustrates generally an example of a system for digitizing and analyzing a sample. [Figure 6] 1 illustrates a computer system that is programmed or configured to perform the methods provided herein. [Figure 7A] 7A and 7B show a microfluidic device containing multiple slides, with each slide containing multiple processing units. [Figure 7B] 7A and 7B show a microfluidic device containing multiple slides, with each slide containing multiple processing units. [Figure 8] A microscope image of a single processing unit is shown. [Figure 9A] 9A through 9D show four different points in time during the digitization process. [Figure 9B] 9A through 9D show four different points in time during the digitization process. [Figure 9C] 9A through 9D show four different points in time during the digitization process. [Figure 9D] 9A through 9D show four different points in time during the digitization process. [Figure 10] 1 illustrates a laboratory workflow integrating the reagent digitization process described herein. [Figure 11] An example screenshot of the image analysis software is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] While various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous modifications, changes, and substitutions may occur to those skilled in the art without departing from the invention. It is understood that various alternatives to the embodiments of the invention described herein may be utilized.

[0026] As used herein, the terms "amplification" and "amplifying" are used interchangeably and generally to refer to producing one or more copies of a nucleic acid or "amplification product." For example, such amplification may be using polymerase chain reaction (PCR) or isothermal amplification.

[0027] As used herein, the term "nucleic acid" generally refers to a biological macromolecule that includes nucleic acid subunits (e.g., nucleotides), either deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 500, or 1000 nucleotides). A nucleic acid may include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (TO), and uracil (U), or variants thereof. A nucleotide may include A, C, G, T, or U, or variants thereof. A nucleotide may include 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 subunits specific to one of the more complementary A, C, G, T, or U, or complementary to a purine (i.e., A or G, or variants thereof) or pyrimidine (i.e., C, T, or U, or variants thereof). In some examples, the nucleic acid may be single-stranded or double-stranded, and in some cases, the nucleic acid molecule is circular. Non-limiting examples of nucleic acids include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), including DNA and RNA. Nucleic acids may include coding or non-coding regions of a gene or gene fragment, 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. A nucleic acid may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.

[0028] As used herein, the terms "polymerase chain reaction reagents" or "PCR reagents" are used interchangeably and generally to refer to compositions that contain reagents for accomplishing a nucleic acid amplification reaction (e.g., DNA amplification), non-limiting examples of which include a primer set or priming site (e.g., a nick) with 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 that contain probes and indicators.

[0029] As used herein, the term "probe" generally refers to a molecule that includes a detectable moiety, and the presence or absence of the probe may be used to detect the presence or absence of an amplification product. Non-limiting examples of detectable moieties may include a radiolabel, a stable isotope label, a fluorescent label, a chemiluminescent label, an enzyme label, a colorimetric label, or any combination thereof.

[0030] As used herein, the term "extension" refers to the incorporation of nucleotides into a nucleic acid, typically in a template directed fashion. Extension may also occur with the assistance of an enzyme. For example, extension may occur with the assistance of a polymerase. Conditions under which extension may occur include "extension temperature," which generally refers to the temperature at which extension is achieved, and "extension duration," which generally refers to the amount of time allotted for extension to occur.

[0031] As used herein, the term "indicator molecule" generally refers to a molecule that includes a detectable moiety, and the presence or absence of the indicator molecule may be used to indicate the partitioning of a sample. Non-limiting examples of detectable moieties may include a radiolabel, a stable isotope label, a fluorescent label, a chemiluminescent label, an enzyme label, a colorimetric label, or any combination thereof.

[0032] The term "sample", as used herein, generally refers to a sample that contains or is suspected of containing a nucleic acid molecule. For example, a sample can be a biological sample that contains one or more nucleic acid molecules. A biological sample can be obtained (e.g., extracted or separated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal discharge, saliva, stool, and tears. A biological sample can be a liquid tissue or tissue sample (e.g., a skin sample). In some examples, a sample is obtained from acellular bodily fluids such as whole blood. In such instances, a sample can include cell-free DNA and / or cell-free RNA. In some examples, a sample can include circulating tumor cells. In some embodiments, a sample is 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). A sample can be processed prior to loading into a microfluidic device. For example, a sample can be processed to lyse cells, purify nucleic acid molecules, and / or include reagents.

[0033] As used herein, the term "fluid" generally refers to a liquid or a gas. A liquid will not be able to maintain a set shape and will flow within an observable time frame, filling a container in which it is placed. Thus, a fluid may have an appropriate viscosity that allows it to flow. When more than one fluid is present, each fluid may be independently selected by one of skill in the art from essentially any fluid (liquid, gas, etc.).

[0034] As used herein, the term "partition" generally refers to a division or distribution into multiple portions, or to a share. For example, a partitioned sample is a sample that is isolated from other samples. Examples of structures that allow for the distribution of samples include wells and chambers.

[0035] As used herein, the terms "digitized" or "digitization" may be used interchangeably and generally refer to a sample that has been distributed into one or more compartments. A digitized sample may or may not be in fluid communication with another digitized sample. A digitized sample may not interact with or exchange materials (e.g., reagents, analytes, etc.) with other digitized samples.

[0036] As used herein, the term "microfluidic" generally refers to a chip, area, device, article, or system that includes at least one channel, a plurality of wicking openings, and an array of chambers. The channels may have cross-sectional dimensions 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 less.

[0037] As used herein, the term "depth" generally refers to the distance measured from the bottom of a channel, wicking opening, or chamber to the membrane that caps the array of channels, wicking openings, and chambers.

[0038] As used herein, the terms "cross-section" and "cross-sectional" may be used interchangeably and typically refer to a dimension or area of ​​a channel or wicking opening that is substantially perpendicular to the external long dimension.

[0039] As used herein, the terms "pressurized gas release" or "pressurized venting" may be used interchangeably and generally refer to the removal or venting of gas (e.g., air, nitrogen, oxygen, etc.) from a channel or chamber of a device (e.g., a microfluidic device) to an environment outside the channel or chamber by application of a pressure differential. The pressure differential may be applied between the channel or chamber and the environment outside the channel or chamber. The pressure differential may be provided by the action of a pressure source on one or more inlets of the device or by the action of a vacuum source on one or more surfaces of the device. Pressurized gas release or pressurized venting may be enabled through a film or membrane that encases one or more sides of the channel or chamber.

[0040] Whenever the terms "at least," "greater than," or "greater than or equal to" precede a first number in a series of two or more numerical values, the terms "at least," "greater than," or "greater than or equal to" apply to each and every number in the series. For example, 1 or more, 2, or 3 is equivalent to 1 or more, 2 or more, or 3 or more.

[0041] Whenever the terms "slightly," "less than," or "or less than" precede a first number in a series of two or more numerical values, the terms "slightly," "less than," or "or less than" apply to each and every number in the series. For example, 3 or less, 2, or 1 is equivalent to 3 or less, 2 or less, or 1 or less.

[0042] The present disclosure provides microfluidic devices for the processing and / or analysis of samples. The microfluidic devices of the present disclosure may be formed from polymeric materials (e.g., thermoplastics) and may include thin films that allow for pressurized degassing while serving as a gas barrier when pressure is released. The microfluidic devices may be chips or cartridges. The microfluidic devices of the present disclosure may be single-use and / or disposable devices. Alternatively, the microfluidic devices may be multi-use devices. The use of polymers (e.g., thermoplastics) to form the microfluidic structures may allow for the use of low-cost, highly scalable injection molding methods, yet the thin films provide the ability to degas via pressurization while avoiding fouling issues that may exist in some microfluidic structures that do not incorporate such thin films.

[0043] For example, because microfluidic devices operate at the sub-millimeter scale and handle microliter, nanoliter, or smaller amounts of fluid, the primary fouling mechanism may be air trapped inside the microstructure, i.e., air bubbles. This may be particularly problematic when using polymeric materials such as thermoplastics to fabricate microfluidic structures, because thermoplastics have very low gas permeability. To avoid fouling from trapped air, other microfluidic structures use either simple straight channel designs or simple branched channel designs in thermoplastic materials, or else use materials with high gas permeability, such as elastomers, to fabricate the devices. However, the simple designs limit the possible functions of the microfluidic devices, and elastomeric materials are difficult and expensive to manufacture, especially at scale.

[0044] One use for this structure is in a microfluidic design incorporating an array of dead-end chambers formed from thermoplastics and connected by channels. This design may be used in the detection and analysis of biological analytes. For example, the microfluidic design may be used to distribute reagents into an array of chambers (e.g., multiple chambers) in digital polymerase chain reaction (dPCR) applications, and thereby to quantify nucleic acids in dPCR.

[0045] Microfluidic devices for analyzing biological samples In an aspect, the present disclosure provides a device (e.g., a microfluidic device) for processing a biological sample. The device (e.g., a microfluidic device) may include one unit including a fluid flow path and a chamber. The device may include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, or more units. The fluid flow path may include a channel and an inlet port. The fluid flow path may not include an outlet port. The inlet port may be in fluid communication with the channel. The inlet port can be configured to direct a biological sample to the channel. The chamber can be in fluid communication with the channel. The chamber can be configured to receive at least a portion of the solution from the channel and to retain a portion of the solution during processing.

[0046] The fluid flow path may include one channel or multiple channels. The fluid flow path may include at least 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40, 50, or more channels. The channels may each be fluidically isolated from one another. Alternatively, or additionally, multiple channels may be in fluid communication with one another. The channel may include a first end and a second end. The first end and the second end may be connected to a single inlet port. A channel with a first end and a second end connected to a single inlet port may be in a circular and / or looped configuration such that fluid entering the channel through the inlet port may be simultaneously directed through the first end and the second end of the channel. Alternatively, the first end and the second end may be connected to different inlet ports. The fluid flow path and / or chamber may not include a valve to stop or slow fluid flow or to isolate the chambers.

[0047] The device may include a long dimension and a short dimension. The long dimension may be about 20 centimeters (cm) or less, about 15 cm or less, about 10 cm or less, about 8 cm or less, about 6 cm or less, about 5 cm or less, about 4 cm or less, about 3 cm or less, about 2 cm or less, about 1 cm or less, or less. The short dimension of the device may be about 10 cm or less, about 8 cm or less, about 6 cm or less, about 5 cm or less, about 4 cm or less, about 3 cm or less, about 2 cm or less, about 1 cm or less, about 0.5 cm or less, or less. In one example, the dimensions of the device (e.g., a microfluidic device) are about 7.5 cm by 2.5 cm. The channel may be substantially parallel to the long dimension of the microfluidic device. Alternatively, or additionally, the channel may be substantially perpendicular to the long dimension of the microfluidic device (e.g., parallel to the short dimension of the device). Alternatively or additionally, the channel may be neither substantially parallel nor substantially perpendicular to the long dimension of the microfluidic device. The angle between the channel and the long dimension of the microfluidic device may be at least about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 30 degrees, about 40 degrees, about 50 degrees, about 60 degrees, about 70 degrees, or about 90 degrees. In one example, the channel is a single long channel. Alternatively or additionally, the channel may have a bend, curve, or corner. The channel may have a long dimension of about 100 millimeters (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 channel may be limited by the external length or width of the microfluidic device. The channels may have a depth of 500 micrometers (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 channels may have a cross-sectional dimension (e.g., width or diameter) 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.

[0048] In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 80 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 60 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 40 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 20 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 100 μm wide and about 10 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 80 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 60 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 40 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 20 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 10 μm wide and about 100 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 80 μm wide and about 80 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 60 μm wide and about 60 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 40 μm wide and about 40 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 20 μm wide and about 20 μm deep. In some examples, the cross-sectional dimensions of the channel may be about 10 μm wide and about 10 μm deep.

[0049] The cross-sectional shape of the channel may be any suitable cross-sectional shape, including, but not limited to, circular, elliptical, triangular, square, or rectangular. The cross-sectional area of ​​the channel may be constant along the length of the channel. Alternatively, or additionally, the cross-sectional area of ​​the channel may vary along the length of the channel. The cross-sectional area of ​​the channel may range from about 50% to 150%, about 60% to 125%, about 70% to 120%, about 80% to 115%, about 90% to 110%, about 95% to 100%, or about 98% to 102%. The cross-sectional area of ​​the channel may be about 10,000 square micrometers (μm 2 ) or less, approximately 7,500μm 2Below, approximately 5,000μm 2 Below, approximately 2,500μm 2 Below, approximately 1,000μm 2 Below, approximately 750μm 2 , about 500μm 2 Below, approximately 400μm 2 Below, approximately 300μm 2 Below, approximately 200μm 2 Below, approximately 100μm 2 It may be equal to or less than.

[0050] The channel may have a single inlet or multiple inlets. The inlet(s) may have the same or different diameters. The inlet(s) 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 smaller.

[0051] The device may include multiple chambers. The multiple chambers may be an array of chambers. The device may include a single array of chambers, or multiple arrays of chambers, with each array fluidically isolated from the other arrays. The arrays of chambers may be arranged in a line, in a grid configuration, in an alternating pattern, or in any other configuration. A microfluidic device may have 1, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, or more chamber arrays. The arrays of chambers may be identical or different (e.g., have different numbers or configurations of chambers). The arrays of chambers may all have the same external dimensions (i.e., the length and width of the array of chambers that encloses all portions of the array of chambers) or the arrays of chambers may have different external dimensions. The array of chambers may have a width 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, about 1 mm or less, or smaller. The array of chambers may have a major dimension of 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, about 1 mm or less, or smaller. The width may be about 1 mm to 100 mm, or 10 mm to 50 mm. The length may be about 1 mm to 50 mm, or 5 mm to 20 mm.

[0052] The array of chambers may have about 1,000 or more chambers, about 5,000 chambers, about 10,000 chambers, about 20,000 chambers, about 30,000 chambers, about 40,000 chambers, about 50,000 chambers, about 100,000 chambers, or more. In one example, the microfluidic device may have about 10,000 to 30,000 chambers. In another example, the microfluidic device may have about 15,000 to 25,000 chambers. The chambers may be cylindrical, hemispherical, or a combination of cylindrical or hemispherical. Alternatively or additionally, the chamber may be cubic in shape. The chamber may have a cross-sectional dimension 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. In one example, the chamber has a cross-sectional dimension (e.g., diameter or side length) of about 250 μm or less. In another example, the chamber has a cross-sectional dimension (e.g., diameter or side length) of about 100 μm or less. In another example, the chamber has a cross-sectional dimension (e.g., diameter or side length) of about 50 μm or less.

[0053] The depth of the chamber 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 one example, the chamber may have a cross-sectional dimension of about 30 μm and a depth of about 100 μm. In another example, the chamber may have a cross-sectional dimension of about 35 μm and a depth of about 80 μm. In another example, the chamber may have a cross-sectional dimension of about 40 μm and a depth of about 70 μm. In another example, the chamber may have a cross-sectional dimension of about 50 μm and a depth of about 60 μm. In another example, the chamber may have a cross-sectional dimension of about 60 μm and a depth of about 40 μm. In another example, the chamber may have a cross-sectional dimension of about 80 μm and a depth of about 35 μm. In another example, the chamber may have a cross-sectional dimension of about 100 μm and a depth of about 30 μm. In another example, the chamber and the channel have the same depth. In an alternative embodiment, the chamber and the channel have different depths.

[0054] The chambers may have any volume. The chambers may have the same volume or the volumes may vary across the microfluidic device. The chambers may have a volume of about 1000 picoliters (pL) or less, about 900 pL or less, about 800 pL or less, about 700 pL or less, about 600 pL or less, about 500 pL or less, about 400 pL or less, about 300 pL or less, about 200 pL or less, about 100 pL or less, about 75 pL or less, about 50 pL or less, about 25 pL or less, or less than picoliters. The chamber may have a volume of about 25 pL to 50 pL, about 25 pL to 75 pL, about 25 pL to 100 pL, about 25 pL to 200 pL, about 25 pL to 300 pL, about 25 pL to 400 pL, about 25 pL to 500 pL, about 25 pL to 600 pL, about 25 pL to 700 pL, about 25 pL to 800 pL, about 25 pL to 900 pL, or about 25 pL to 1000 pL. In one example, the chamber has a volume of 250 pL or less. In another example, the chamber has a volume of about 150 pL or less.

[0055] The volume of the channel may be less than or equal to the total volume of the chamber. In one example, the volume of the channel is less than the total volume of the chamber. The volume of the channel may have a volume that is 95% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, or less than the total volume of the chamber.

[0056] The device may further include a wicking opening disposed between the channel and the chamber. The suction opening can be one of a plurality of suction openings connecting the channel to a plurality of chambers, where the suction opening is configured to provide fluid communication between the channel and the chamber. The length of the suction opening can be constant or different across the device (e.g., a microfluidic device). The suction opening can have a major dimension of about 150 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. The depth of the suction opening can be about 50 μm or less, about 25 μm or less, about 10 μm or less, about 5 μm or less, or less. The suction opening can have a cross-sectional dimension 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.

[0057] The cross-sectional shape of the suction opening may 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 may 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 channel than the cross-sectional area of ​​the suction opening at the connection to the chamber. Alternatively, the cross-sectional area of ​​the suction opening at the connection to the chamber may be larger than the cross-sectional area of ​​the suction opening at the connection to the channel. The cross-sectional area of ​​the suction opening may range from about 50% to about 150%, about 60% to about 125%, about 70% to about 120%, about 80% to about 115%, about 90% to about 110%, about 95% to about 100%, or about 98% to about 102%. The cross-sectional area of ​​the suction opening may be greater than about 2,500 μm 2 Below, approximately 1,000μm 2 Below, approximately 750μm 2 , about 500μm 2 Below, approximately 250μm 2 Below, approximately 100μm 2 Below, approximately 75μm 2 Below, approximately 50μm 2 Below, approximately 25μm 2The cross-sectional area of ​​the suction opening at the connection to the channel may be less than or equal to the cross-sectional area of ​​the channel. The cross-sectional area of ​​the suction opening at the connection to the channel may be less than or equal to about 98%, less than or equal to about 95%, less than or equal to about 90%, less than or equal to about 85%, less than or equal to about 80%, less than or equal to about 75%, less than or equal to about 70%, less than or equal to about 60%, less than or equal to about 50%, less than or equal to about 40%, less than or equal to about 30%, less than or equal to about 20%, less than or equal to about 10%, less than or equal to about 5%, less than or equal to about 1%, less than or equal to about 0.5%, or less than or equal to the cross-sectional area of ​​the channel. The suction opening may be substantially perpendicular to the channel. Alternatively or additionally, the suction opening is not substantially perpendicular to the channel. The angle between the suction opening and the channel may be at least about 5 degrees, about 10 degrees, about 15 degrees, about 20 degrees, about 30 degrees, about 40 degrees, about 50 degrees, about 60 degrees, about 70 degrees, or about 90 degrees.

[0058] The microfluidic device may be configured to allow pressurized gas release or venting of the channels, chambers, wicking openings, or any combination thereof. Pressurized gas release or venting may be provided by a film or membrane configured to allow pressurized gas release or venting. The film or membrane may be permeable to gas above a pressure threshold. The film or membrane may be impermeable (e.g., impermeable or substantially impermeable) to liquids such as, but not limited to, oil or other solvents. The channels, chambers, wicking openings, or any combination thereof may include a film or membrane. In one example, the chamber includes a gas-permeable film or membrane, and the channel and / or wicking opening do not include a gas-permeable film or membrane. In another example, the chamber and wicking opening include a gas-permeable film or membrane, and the channel does not include a gas-permeable film or membrane. In another example, the chamber, channel, and wicking opening include a gas-permeable film or membrane.

[0059] The film or membrane can be a thin film. The film or membrane can be a polymer. The film can be a thermoplastic film or membrane. The film or membrane can be free of elastomeric materials. The gas permeable film or membrane can cover the fluid flow path, channel, chamber, or any combination thereof. In one example, the gas permeable film or membrane covers the chamber. In another example, the gas permeable film or membrane covers the chamber and the channel. The gas permeability of the film can be induced by high pressure. The thickness of the film can be 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 less. In one example, the film or membrane has a thickness of about 100 μm or less. In another example, the film or membrane has a thickness of about 50 μm or less. In another example, the film or membrane has a thickness of about 25 μm or less. The thickness of the film or membrane can be from about 0.1 μm to about 200 μm, from about 0.5 μm to about 150 μm, or from about 25 μm to about 100 μm. In one example, the thickness of the film or membrane is from about 25 μm to about 100 μm. The thickness of the film may be selected depending on the manufacturability of the film, the air permeability of the film, the volume of each chamber or compartment to be degassed, the available pressure, and / or the time to complete the digitization process.

[0060] The film or membrane may be configured to take advantage of different permeation characteristics under different applied pressures. For example, a thin film may be gas impermeable at a first pressure differential (e.g., low pressure) and at least partially gas permeable at a second pressure differential (e.g., high pressure). The first pressure differential (e.g., low pressure differential) may be about 8 pounds per square inch (psi) or less, 6 psi or less, 4 psi or less, 2 psi or less, 1 psi or less, or less. In one example, the film or membrane is substantially impermeable to gas at a pressure differential of less than 4 psi. The second pressure differential (e.g., high pressure differential) may be about 1 psi or more, about 2 psi or more, about 4 psi or more, 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 20 psi or more, or more. In one example, the film or membrane is substantially gas permeable at a pressure of 4 psi or more.

[0061] Method for Analysing a Biological Sample In another aspect, the disclosure provides a method for processing a biological sample. The method may include providing a device (e.g., a microfluidic device). The device may include a fluid flow path and a chamber. The fluid flow path may include a channel and an inlet port. The fluid flow path may not include an outlet port. The chamber may be in fluid communication with the channel. A solution including the biological sample may be directed from the inlet port to the channel. At least a portion of the solution may be directed from the channel to the chamber. The chamber may retain said portion of the sample during processing of the solution and the biological sample.

[0062] The device may include a chamber or multiple chambers. The device may have a single inlet port or multiple inlet ports. In one example, the device includes a single inlet port. In another example, the device includes two or more inlet ports. The device may be as described elsewhere herein.

[0063] The method may further include applying a single or multiple pressure differentials to the inlet ports to direct the solution from the inlet ports to the channels. Alternatively or additionally, the device may include multiple inlet ports and the pressure differential may be applied to the multiple inlet ports. The inlet of the device (e.g., a microfluidic device) may be in fluid communication with a fluid flow module, such as a pneumatic pump, a vacuum source, or a compressor. The fluid flow module may provide a positive or negative pressure to the inlet. The fluid flow module may apply a pressure differential to fill the device with the sample and distribute (e.g., digitize) the sample into the chambers. Alternatively or additionally, the sample may be distributed into multiple chambers as described elsewhere herein. The filling and distribution of the sample may be performed without using valves between the chambers and the channels to separate the samples. For example, filling of the channel may be performed by applying a pressure differential between the sample in the inlet port and the channel. This pressure differential may be achieved by pressurizing the sample or by applying a vacuum to the channel or chamber. Filling the chambers and distributing the solution may be performed by applying a pressure differential between the channels and the chambers. This may be accomplished by pressurizing the channels through an inlet port or by applying a vacuum to the chambers. The solution containing the sample may enter the chambers such that each chamber contains at least a portion of the solution.

[0064] In some cases, a single pressure difference may be used to deliver a solution containing a biological sample (including a molecular target of interest) to the channel, and the same pressure difference may be used to continue digitizing the chamber with the solution (i.e., delivering the solution from the channel to the chamber). Furthermore, the single pressure difference may be sufficiently high to allow pressurized outgassing or degassing of the channel and / or chamber. Alternatively or additionally, the pressure difference for delivering the solution containing the sample to the channel may be a first pressure difference. The pressure difference for delivering the solution from the channel to the chamber(s) may be a second pressure difference. The first pressure difference and the second pressure difference may be the same or different. In one example, the second pressure difference is greater than the first pressure difference. Alternatively, the second pressure difference may be less than the first pressure difference. The first pressure difference, the second pressure difference, or both may be sufficiently high to allow pressurized outgassing or degassing of the channel and / or chamber. In some cases, a third pressure difference may be used to allow pressurized outgassing or venting of the channels and / or chambers. Pressurized outgassing or venting of the channel or chamber(s) may be enabled by a film or membrane. For example, when a pressure threshold is reached, the film or membrane may allow gas to move from the chambers and / or channels, through the film or membrane, to the environment external to the chambers and / or channels.

[0065] The film or membrane may be configured to take advantage of different permeation characteristics under different applied pressures. For example, the film or membrane may be gas impermeable at a first pressure difference (e.g., low pressure) and gas permeable at a second pressure difference (e.g., high pressure). The first pressure difference and the second pressure difference may be the same or different. During filling of the microfluidic device, the pressure of the inlet port may be higher than the pressure of the channel, allowing the solution at the inlet port to enter the channel. The first pressure difference (e.g., low pressure difference) may 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 one example, the first pressure difference may be about 1 psi to 8 psi. In another example, the first pressure difference may be about 1 psi to 6 psi. In another example, the first pressure difference can be from about 1 psi to 4 psi. The chamber of the device can be filled by applying a second pressure difference between the inlet and the chamber. The second pressure difference can direct fluid from the channel into the chamber and gas from the channel and / or chamber to an environment external to the channel and / or chamber. The second pressure difference can be about 1 psi or more, about 2 psi or more, about 4 psi or more, 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 20 psi or more, or more. In one example, the second pressure difference is more than 4 psi. In one example, the second pressure difference is more than 8 psi. The microfluidic device can be filled and the sample dispensed by applying the first pressure difference, the second pressure difference, or a combination thereof for about 20 minutes or less, about 15 minutes, about 10 minutes, about 5 minutes, about 3 minutes, about 2 minutes, about 1 minute or less, or less.

[0066] The sample may be dispensed by removing excess sample from the channel by refilling the channel with a gas or fluid that is immiscible with the aqueous solution containing the biological sample. The immiscible fluid may be provided after providing the solution containing the sample, such that the solution enters the channel first, followed by the immiscible fluid. The immiscible fluid may be any fluid that does not mix with the aqueous fluid. The gas may be oxygen, nitrogen, carbon dioxide, air, noble gases, or any combination thereof. The immiscible fluid may be an oil or an organic solvent. For example, the immiscible fluid may be silicone oil, or other types of oil / organic solvents that have similar properties compared to silicone oil. Alternatively, removing the sample from the channel may prevent the reagent in one chamber from diffusing into the channel and into the other chamber through the wicking opening. The sample in the channel is removed from the channel by dispensing the sample into the chamber or by removing excess sample from the channel so that the sample does not remain in the channel.

[0067] Directing the solution from the channel to the chamber or chambers may distribute the sample. The device may allow for the partitioning of the sample into the chamber or digitization of the sample such that no residual solution remains in the channels and / or wicking openings (e.g., no or substantially no sample dead volume). The solution containing the sample may be dispensed such that there is zero sample dead volume (e.g., all sample and reagent inputs to the device are fluidically isolated within the chamber), which may prevent or reduce sample and reagent waste. Alternatively, or additionally, the sample may be dispensed by providing a sample volume smaller than the volume of the chamber. The volume of the channel may be less than the total volume of the chamber, so that all sample loaded into the channel is distributed to the chamber. The total volume of the solution containing the sample may be less than the total volume of the chamber. The volume of the solution may be 100%, 99%, 98%, 95%, 90%, 85%, 80%, or less of the total volume of the chamber. The solution may be added to the inlet port simultaneously with or prior to the gas or immiscible fluid being added to the inlet port. The volume of the gas or immiscible fluid may be equal to or greater than the volume of the channel, to fluidically separate the chambers. A small amount of the gas or immiscible fluid may enter the wicking opening or chamber.

[0068] 1A to 1F illustrate schematic examples of methods for filling a microfluidic device. FIG. 1A illustrates schematic loading of a sample and an immiscible fluid into a microfluidic device. The microfluidic device includes an input port (101), a channel (102), and a chamber (103). The channel and the chamber of the microfluidic device are filled with air (104). The sample (105) is introduced or injected into the input port (101). FIG. 1B illustrates schematic pressurizing the microfluidic device to load the sample (105) into the channel. In this example, the microfluidic device includes a single inlet port connected to both ends of the channel in a loop configuration. When pressure is applied, the sample (105) is introduced through both ends of the channel simultaneously. FIG. 1C illustrates schematic continued pressurization to vent the fluid flow path and continue loading the sample into the channel. As the sample (105) enters the chamber (103), a portion of the channel (102) fills with an immiscible fluid (106), such as oil or gas, which may be added simultaneously with the sample or sequentially (e.g., sample then immiscible fluid). As the sample (105) and immiscible fluid (106) fill the channels and chambers, air (104) is directed through the film or membrane and from the outside of the device. FIG. 1D illustrates a schematic of the partial digitization of the sample (105) into the chamber (103) and the continued filling of the channel (102) with the immiscible fluid (106). As the sample (105) enters the chamber (103), the air (104) in the chamber (103) is evacuated through the film or membrane. FIG. 1E illustrates diagrammatically further digitization and the movement of air (104). As the immiscible fluid (106) fills the channel from both ends, the sample is directed into the chamber (103) and the volume of the sample (105) in the channel is reduced. FIG. 1F illustrates diagrammatically the complete digitization of the sample (105), where the immiscible fluid (106) fills the entire channel (102) and the sample (105) is isolated in the chamber (103).In another example, the device has multiple inlet ports and a fluid immiscible with the sample is injected simultaneously into each port to fill the channels and chambers.

[0069] 2A-2E show example images of sample digitization in a microfluidic device. FIG. 2A shows a microfluidic device with two inlet ports. Sample and an immiscible fluid, in this example oil, are injected simultaneously into both inlet ports. FIG. 2B shows the filling of the microfluidic device with sample and oil by pressure. Both inlet ports are pressurized simultaneously to evenly direct the sample and oil into the channels of the device. FIG. 2C and FIG. 2D show the sample and oil gradually filling the channels and chambers of the device. FIG. 2E shows an example of the device after completion of digitization or sample partitioning within the device.

[0070] 3 illustrates generally an example of a method for digitization of a sample. A sample and an immiscible fluid may be provided at the inlet port(s) of a microfluidic device (301). The inlet port(s) can be pressurized to fill the channel with the sample and immiscible fluid (302). The inlet port can be further pressurized to load the sample into the chamber and fill the channel with the immiscible fluid to provide complete digitization of the sample (304).

[0071] Partitioning of the sample may be confirmed by the presence of an indicator in the reagent. The indicator may include a molecule that includes a detectable moiety. The detectable moiety may include a radioactive species, a fluorescent label, a chemiluminescent label, an enzymatic label, a colorimetric label, or any combination thereof. Non-limiting examples of radioactive species include: 3 H, 14 C. 22 Na, 32 P, 33 P, 35 S, 42 K, 45 Ca, 59 Fe, 123 I, 124 I, 125 I, 131I, or 203 Contains Hg. 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 Cypridina luciferase, Gaussia luciferase, Renilla luciferase, and Firefly luciferase. Non-limiting examples of enzyme labels include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase, glucose oxidase, or other types of labels.

[0072] The indicator microparticles can be fluorescent molecules. Fluorescent molecules may include fluorescent proteins, fluorescent dyes, and organometallic fluorophores. In some embodiments, the indicator molecules are protein fluorophores. Protein fluorophores may include green fluorescent protein.Non-limiting examples of protein fluorophores include AcGFP, AcGFP1, AmCyan, AmCyan1, AQ143, AsRed2, Azami, Cyan Fluorescent Proteins (CFPs, fluorescent proteins that fluoresce in the green region of the spectrum and emit light having a wavelength generally between 500-550 nanometers), Cyan Fluorescent Proteins (CFPs, fluorescent proteins that fluoresce in the green region of the spectrum and emit light having a wavelength generally between 450-500 nanometers), and Red Fluorescent Proteins (RFPs, fluorescent proteins that fluoresce in the red region of the spectrum and emit light having a wavelength generally between 600-650 nanometers). Green, Azurite, BFP, Cerulean, CFP, CGFP, Citrine, copGFP, CyPet, dKeima-Tandem, DsRed, dsRed-Express, DsRed-Monomer, DsRed 2, 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 Including GFP, T-Sapphire, TagCFP, TagGFP, TagRFP, TagRFP-T, TagYFP, tdTomato, Topaz, TurboGFP, Venus, YFP, YPet, ZsGreen, and ZsYellow1.

[0073] The indicator molecule can be a fluorescent dye. Non-limiting examples of fluorescent dyes include SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridine, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyl, anthramycin, phenanthridine and acridine, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and ethidium homodimer-2, ethidium monoazide, and 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, Cyber ​​Green I, Cyber ​​Green II, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (blue), SYTO-13, -16, -24, -21, -23, -12, -11, -20, -22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, -60, -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, ethidium bromide, umbelliferone, eosin, green fluorescent protein, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer yellow, cascade blue, dichlorotriazinylamine fluorescein fluorescein), dansyl chloride, fluorescent lanthanide complexes such as those containing europium and terbium, carboxytetrachlorofluorescein, 5 and / or 6-carboxyfluorescein (FAM), 5-(or 6-)iodoacetamidofluorescein, 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 and 7-amino No-4-methylcoumarin-3-acetic acid (AMCA), BODIPY fluorophores, 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt, 3,6-disulfonate-4-amino-naphthalimide, phycobiliproteins, AlexaFluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, 750, and 790 dyes, DyLight 350, 405, 488, 550, 594, 633, 650, 680, 800 dyes, or other fluorophores.

[0074] The indicator molecule can be an organometallic fluorophore. Non-limiting examples of organometallic fluorophores include lanthanide ion chelates, non-limiting examples of lanthanide ion chelates include tris(dibenzoylmethane)mono(1,10-phenanthroline)europium(lll), tris(dibenzoylmethane)mono(5-amino-1,10-phenanthroline)europium(lll), and Lumi4-Tb cryptate.

[0075] FIG. 4 illustrates a schematic example of a method for digital polymerase chain reaction (dPCR) using a microfluidic device. Samples and reagents may be dispensed (401) as shown in FIGS. 2A-2E. The samples and reagents are thermally cycled (402) to perform a PCR reaction in the reagents within the chambers. Thermal cycling may be performed using, for example, a flat block thermal cycler. Image acquisition (403) may be performed to determine which chambers successfully performed a PCR reaction. Image acquisition may be performed using, for example, a three-color probe detection unit. Poisson statistics may be applied to the chamber counts (404) to convert the raw number of positive chambers determined in (403) to nucleic acid concentrations.

[0076] The method may further include detecting one or more components of the solution, one or more components of the biological sample, or a reaction with one or more components of the biological sample. The detecting one or more components of the solution, one or more components of the biological sample, or a reaction may include imaging the chamber. An image of the microfluidic device may be taken. Images of a single chamber, an array of chambers, or multiple arrays of chambers may be taken simultaneously. The image may be taken through the body of the microfluidic device. The image may be taken through a film or membrane of the microfluidic device. In one example, the image is taken through both the body and the membrane of the microfluidic device. The body of the microfluidic device may be substantially optically transparent. Alternatively, the body of the microfluidic device may be substantially optically opaque. In one example, the film or membrane may be substantially optically transparent. The image may be taken prior to filling the microfluidic device with a sample. The image may also be taken after filling the microfluidic device with a sample. The image may be taken while filling the microfluidic device with a sample. The image may be taken to confirm sample distribution. The image may be taken during the reaction to monitor the products of the reaction. In one example, the products of the reaction include amplification products. The images may be taken at predetermined intervals. Alternatively or additionally, a video of the microfluidic device may be taken. The predetermined intervals may include taking images at least about every 300 seconds, 240 seconds, 180 seconds, 120 seconds, 90 seconds, 60 seconds, 30 seconds, 15 seconds, 10 seconds, 5 seconds, 4 seconds, 3 seconds, 2 seconds, 1 second, or more frequently during the reaction.

[0077] The biological sample may be any biological specimen, such as, but not limited to, a nucleic acid molecule, a protein, an enzyme, an antibody, or other biological molecule. For example, the biological sample may include one or more nucleic acid molecules. Processing the nucleic acid molecule may further include thermocycling the chamber(s) to amplify the nucleic acid molecule. The method may further include controlling the temperature of the channel or chamber(s). The method for using the microfluidic device may further include amplifying the nucleic acid sample. The microfluidic device may be filled with an amplification reagent including the nucleic acid molecule, components used in the amplification reaction, an indicator molecule, and an amplification probe. The amplification may be performed by thermocycling the multiple chambers. Detection of the nucleic acid amplification may be performed by imaging the chambers of the microfluidic device. The nucleic acid molecule may be quantified by counting the chambers in which the nucleic acid molecule was successfully amplified and applying Poisson statistics. In some embodiments, the amplification and quantification of the nucleic acid may be performed in a single integrated unit.

[0078] Various nucleic acid amplification reactions can be used to amplify nucleic acid molecules in a sample to generate an amplification product. Amplification of nucleic acid targets can 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. In some embodiments, the amplification product is DNA or RNA. For embodiments directed to DNA amplification, any method of DNA amplification can be utilized. Methods of DNA amplification 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. In some embodiments, the DNA amplification is linear, exponential, or any combination thereof. In some embodiments, the DNA amplification is achieved using digital PCR (dPCR).

[0079] Reagents used in nucleic acid amplification may include a polymerizing enzyme, a reverse primer, a forward primer, and an amplification probe. Examples of polymerizing enzymes include, but are not limited to, a nucleic acid polymerase, a transcriptase, or a ligase (i.e., an enzyme that catalyzes the formation of a bond). Polymerases can be naturally occurring or synthetic. Examples of polymerases include DNA polymerases and RNA polymerases, thermostable polymerases, wild-type polymerases, modified polymerases, E. 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 These include Taq polymerase, Tbr polymerase, Tfl polymerase, PfuTubo polymerase, Pyrobest polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment polymerase with 3'-5' exonuclease activity, and mutants, modified products, and derivatives thereof. For hot start polymerases, a denaturation cycle at a temperature of about 92°C to 95°C for about 2 minutes to 10 minutes may be used.

[0080] The amplification probe may be a sequence-specific oligonucleotide probe. The amplification probe may be optically active when hybridized with the amplification product. In some embodiments, the amplification probe is detectable only when nucleic acid amplification proceeds. The intensity of the optical signal may be proportional to the amount of amplification product. The probe may be conjugated to any of the optically active detectable moieties (e.g., dyes) described herein, and may also include a quencher that can block 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 quencher and Iowa Black RQ quencher, or internal ZEN quencher. Alternatively, or additionally, the probe or quencher may be any probe useful in the context of the disclosed methods.

[0081] The amplification probe is a dual-labeled fluorescent probe. The dual-labeled probe may include a fluorescent reporter and a fluorescent quencher bound to a nucleic acid. The fluorescent reporter and the fluorescent quencher may be located in close proximity to each other. The proximity of the fluorescent reporter and the fluorescent quencher may block the optical activity of the fluorescent reporter. The dual-labeled probe may bind to a 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. Upon cleavage of the fluorescent reporter and the fluorescent quencher from the amplification probe, the fluorescent reporter may regain its optical activity, allowing detection. The dual-labeled fluorescent probe may include a 5' fluorescent reporter with an excitation wavelength maximum of at least about 450 nanometers (nm), 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or higher, or an emission wavelength maximum 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 quench the fluorescence emission wavelength of 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.

[0082] Nucleic acid amplification may be performed by thermal cycling a chamber of a microfluidic device. Thermal cycling may include controlling the temperature of the microfluidic device by applying heating or cooling to the microfluidic device. The heating or cooling method may include resistive heating or cooling, radiative heating or cooling, conductive heating or cooling, convective heating or cooling, or any combination thereof. Thermal cycling may include a cycle of incubating the chamber at a temperature high enough to denature the nucleic acid molecules for a duration, followed by incubation of the chamber at the extension temperature for the duration of the extension. The denaturation temperature may vary depending, for example, on the particular nucleic acid sample, the reagents used, and the reaction conditions. The denaturation temperature may be about 80°C to 110°C, 85°C to about 105°C, 90°C to about 100°C, 90°C to about 98°C, 92°C to about 95°C. The denaturation temperature can be at least about 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C, 100°C, or higher.

[0083] The duration for denaturation may vary depending, for example, on the particular nucleic acid sample, the reagents used, and the reaction conditions. The duration for denaturation 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 about 1 second or less.

[0084] The extension temperature may vary depending on, for example, the particular nucleic acid sample, the reagents used, and the reaction conditions. The extension temperature may be about 30°C to 80°C, 35°C to 75°C, 45°C to 65°C, 55°C to 65°C, 40°C to 60°C. The extension temperature can be at least about 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, or a temperature of about 80°C.

[0085] The extension time may vary depending on, for example, the specific nucleic acid sample, the reagents used, and the reaction conditions. In some embodiments, the duration for extension 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 about 1 second or less. In an alternative embodiment, the duration for extension 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 about 1 second. In one example, the duration of the extension reaction is about 10 seconds or less.

[0086] Nucleic acid amplification may include multiple cycles of thermal cycling. Any suitable number of cycles may be performed. The number of cycles may be greater than about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more. The number of cycles performed may depend on the number of cycles required to obtain detectable amplification products. For example, the number of cycles required to detect nucleic acid amplification during dPCR may be about 100 cycles or less, 90 cycles or less, 80 cycles or less, 70 cycles or less, 60 cycles or less, 50 cycles or less, 40 cycles or less, 30 cycles or less, 20 cycles or less, 15 cycles or less, 10 cycles or less, 5 cycles or less, or less. In one example, about 40 cycles or less are used, and the cycle time is about 20 minutes or less.

[0087] The time to achieve a detectable amount of amplification product may vary depending on the particular nucleic acid sample, the reagents used, the amplification reaction used, the number of amplification cycles used, and the reaction conditions. In some embodiments, 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 about 5 minutes or less. In one example, a detectable amount of amplification product may be reached in less than 20 minutes.

[0088] In some embodiments, the ramping rate (i.e., the rate at which the chamber transitions from one temperature to another) is important for amplification. For example, the temperature and time at which the amplification reaction results in a detectable amount of amplification product may vary depending on the ramping rate. The ramping rate may also affect the time, temperature, or both time and temperature used during amplification. In some embodiments, the ramping rate is constant between cycles. In some embodiments, the ramping rate varies between cycles. The ramping rate may also be adjusted based on the sample being processed. For example, an optimal ramping rate may be selected to provide a robust and efficient amplification method.

[0089] System for analyzing biological samples - Patent application In another aspect, the present disclosure may provide a system for processing a biological sample. The system may include a device (e.g., a microfluidic device) holder and a fluid flow channel. The device may include a fluid flow path and a chamber. The fluid flow path may include a channel and an inlet port. The fluid flow path may not include an outlet port. The inlet port may be configured to direct the biological sample into the channel. The chamber may be in fluid communication with the channel. The chamber may be configured to receive at least one of the solutions comprising the biological sample from the channel and to hold the solution during processing. The holder is configured to receive and hold the device during processing. The fluid flow module is fluidly coupled to the inlet port and configured to provide a pressure differential to flow the solution from the inlet port into the channel. Additionally, the fluid flow module is configured to provide a pressure differential to flow the solution from the channel into the chamber.

[0090] The holder may be a shelf, receptacle, or stage for housing the device. In one example, the holder is a transfer stage. The transfer stage may be configured to input, hold, and output a microfluidic device. The microfluidic device may be any device described elsewhere herein. The transfer stage may be stationary in one or more coordinates. Alternatively, or additionally, the transfer stage may be movable in the X-direction, Y-direction, Z-direction, or any combination thereof. The transfer stage may be capable of holding a single microfluidic device. Alternatively, or additionally, the transfer stage may be capable of holding at least two, three, four, five, six, seven, eight, nine, ten, or more microfluidic devices.

[0091] The fluid flow module may be a pneumatic module and / or a vacuum module. The fluid flow module may be configured to be in fluid communication with the inlet port(s) of the microfluidic device. The fluid flow module may have multiple connection points that can connect to multiple inlet ports. The fluid flow module may be capable of filling, refilling, and dispensing a single array of chambers at once, or multiple arrays of chambers in tandem. The fluid flow module may be a pneumatic module combined with a vacuum module. The fluid flow module may provide a pressure increase to the microfluidic device or may provide a vacuum to the microfluidic device.

[0092] The system may further include a thermal module. The thermal module may be configured to be in thermal communication with the chambers of the microfluidic device. The thermal module may be configured to control the temperature of a single array of chambers or to control the temperature of multiple arrays of chambers. Each array of chambers may be individually addressable by the thermal module. For example, the thermal module may implement the same thermal program across all chamber arrays or may implement different thermal programs in different chamber arrays. The thermal module may be configured to be in thermal communication with the microfluidic device and / or the chambers of the microfluidic device. The thermal module may heat or cool the microfluidic device. One or more surfaces of the microfluidic device may be in direct contact with the thermal module. Alternatively or additionally, a thermally conductive material may be disposed between the thermal module and the microfluidic device. The thermal module may maintain the temperature across the surface of the microfluidic device such that the deviation is about 2° C. or less, 1.5° C. or less, 1° C. or less, 0.9° C. or less, 0.8° C. or less, 0.7° C. or less, 0.6° C. or less, 0.5° C. or less, 0.4° C. or less, 0.3° C. or less, 0.2° C. or less, 0.1° C. or less, or less. The thermal module may maintain the temperature of the surface of the microfluidic device within plus or minus 0.5° C., 0.4° C., 0.3° C., 0.2° C., 0.1° C., 0.05° C., or less than a temperature setpoint.

[0093] The system may further include a detection module. The detection module may provide electronic or optical detection. In one example, the detection module is an optical module that provides optical detection. The optical module may be configured to emit and detect multiple wavelengths of light. The emission wavelengths may correspond to the excitation wavelengths of the indicator and amplifier probes used. The emission 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 detection 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 module may be configured to emit one, two, three, or more than four wavelengths of light. The optical module may be configured to detect one, two, three, or more wavelengths of light. One emitted wavelength of light may correspond to an excitation wavelength of an indicator molecule. Another emitted wavelength of light may correspond to an excitation wavelength of an amplifier probe. One detected wavelength of light may correspond to an emission wavelength of an indicator molecule. Another detected wavelength of light may correspond to an amplifier probe used to detect a reaction in the chamber. The optical module may be configured to image a section of the array of chambers. Alternatively, or additionally, the optical module may image the entire array of chambers in a single image. In one example, the optical module is configured to take a video of the device.

[0094] FIG. 5 illustrates a system (500) for carrying out the process of FIG. 4 in a single system. The system (500) includes a fluid flow module (501), which may include a pump, a vacuum, and a manifold, and may be operable to move in the Z direction and perform the application of pressure as described in FIGS. 1A-1F. The system (500) may also include a thermal module (502), such as a flat block thermal cycler, to thermally cycle the microfluidic device, thereby causing the polymerase chain reaction to occur. The system (500) further includes an optical module (503), such as an epi-fluorescent optical module, that may optically determine whether a chamber in the microfluidic device successfully performed a PCR reaction. The optical module (503) provides this information to a processor (504), which may use Poisson statistics to convert the raw counts of successful chambers to nucleic acid concentrations. The holder (505) may be used to move a given microfluidic device between the various modules and to handle multiple microfluidic devices simultaneously. The above microfluidic devices, combined with the incorporation of this functionality into a single machine, may reduce the cost, workflow complexity, and space requirements for dPCR over other implementations of dPCR.

[0095] The system may further include a robotic arm. The robotic arm may move, change, or arrange the position of the microfluidic device. Alternatively or additionally, the robotic arm may position or move other components of the system (e.g., the fluid flow module or the detection module). The detection module may include a camera (e.g., a complementary metal oxide semiconductor (CMOS) camera) and a filter cube. The filter cube may change or modify the wavelength of the excitation light and / or the wavelength of the light detected by the camera. The fluid flow module may include a manifold (e.g., a pneumatic manifold) and / or one or more pumps. The manifold may be in an upright position such that the manifold does not contact the microfluidic device. The upright position may be used when filling and / or imaging the microfluidic device. The manifold may be in a down position such that the manifold contacts the microfluidic device. The manifold may be used to fill the microfluidic device with fluids (e.g., samples and reagents). The manifold may apply pressure to the fluid flow device to hold the device in place during use and / or to prevent warping, bending, or other stresses. In one example, the manifold applies downward pressure and holds the microfluidic device against a thermal module.

[0096] The system may further include one or more computer processors. The one or more computer processors may be operably coupled to the fluid flow module, the holder, the thermal module, the sensing module, the robotic arm, or any combination thereof. In one example, the one or more computer processors are operably coupled to the fluid flow module. The one or more computer processors may be individually or collectively programmed to instruct the fluid flow module to provide a pressure differential to the inlet port when the fluid flow module is fluidly coupled to the inlet port to flow and thereby dispense the solution from the inlet port to the channel and / or from the channel to the chamber(s) through pressurized gas release of the chamber.

[0097] The scope of the present disclosure is not limited by the specific embodiments described herein. Indeed, other various embodiments and variations of the present disclosure in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings.

[0098] For example, although described in the context of dPCR applications, other microfluidic devices that may require multiple isolated chambers filled with liquids isolated via gas or other fluids may benefit from the use of thin thermoplastic membranes, allowing venting to avoid gas fouling while also providing benefits in terms of manufacturability and cost. Apart from PCR, other nucleic acid amplification methods such as loop mediated isothermal amplification may be applied to perform the digital detection of specific nucleic acid sequences of the embodiments of the present disclosure. The chambers may also be used to isolate single cells, with the suction opening designed to be close to the diameter of the cells to be isolated. In some embodiments, the embodiments of the present disclosure may be used to separate plasma from whole blood, when the suction opening is much smaller than the size of a blood cell.

[0099] The described systems may be used in conjunction with any of the devices (e.g., microfluidic devices) described elsewhere herein. In addition, the described systems may be used to practice any of the methods described elsewhere herein.

[0100] Computer Systems The present disclosure provides a computer system programmed to carry out the methods of the present disclosure. Figure 6 shows a computer device (601) programmed or otherwise configured for processing and analysis of biological samples (e.g., nucleic acid molecules). The computer device (601) can regulate various aspects of the systems and methods of the present disclosure, such as loading, digitizing, and analyzing biological samples. The computer system (601) is an electronic device of a user or a computer system, which is located remotely relative to the electronic device. The electronic device may be a mobile electronic device capable of or otherwise configured to monitor and control a biological analysis system.

[0101] The computer system (601) includes a central processing unit (CPU, herein "processor" and "computer processor") (605), which may be a single-core or multi-core processor, or multiple processors for parallel processing. The computer system (601) also includes memory or memory locations (610) (e.g., random access memory, read-only memory, flash memory), electronic storage devices (615) (e.g., hard disks), communication interfaces (620) (e.g., network adapters) for communicating with one or more other systems, and peripherals (625), such as cache, other memories, data storage devices, and / or electronic display adapters. The memory 610, storage devices 615, interfaces 620, and peripherals 625 communicate with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage unit (615) may be a data storage unit (or data repository) for storing data. The computer system (601) may be operatively coupled to a computer network ("network") (630) with the aid of a communication interface (620). The network (630) may be the Internet, an Internet and / or an extranet, or an intranet and / or an extranet in communication with the Internet. The network (630) may be a telecommunications and / or data network in some cases. The network (630) may include one or more computer servers that may enable distributed computing, such as cloud computing. The network (630) may, in some cases, with the aid of the computer system (601), implement a peer-to-peer network, which may enable devices coupled to the computer system (601) to act as clients or servers.

[0102] The CPU (605) can execute a series of machine-readable instructions, which may be embodied in a program or software. The instructions may be stored in a memory location, such as the memory (610). The instructions may be directed to the CPU (605), which may then program or configure the CPU (605) to implement the methods of the present disclosure. Examples of operations performed by the CPU (605) may include fetch, decode, execute, and writeback.

[0103] The CPU (605) may be part of a circuit, such as an integrated circuit. One or more other components of the system (601) may be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0104] The storage device (615) can store files such as drivers, libraries, and saved programs. The storage device (615) can store user data, such as user preferences and user programs. The computer system (601) may optionally include one or more additional data storage devices external to the computer system (601), such as located on a remote server in communication with the computer system (601) over an intranet or the Internet.

[0105] The computer system (601) can communicate with one or more remote computer systems through the network (630). For example, the computer system (601) can communicate with a remote computer system of a user (e.g., a laboratory technician, a scientist, a researcher, or a medical technician). Examples of remote computer systems include a personal computer (e.g., a portable PC), a slate or tablet PC (e.g., an Apple® iPad®, a Samsung® Galaxy Tab), a phone, a smartphone (e.g., an Apple® iPhone®, an Android®-enabled device, a Blackberry®), or a personal digital assistant. The user can access the computer system (601) through the network (630).

[0106] Methods as described herein may be implemented by machine (e.g., computer processor) executable code stored in an electronic storage location of the computer system (601), such as on memory (610) or electronic storage (615). Machine executable or machine readable code may be provided in the form of software. During use, the code may be executed by the processor (605). In some cases, the code may be retrieved from the electronic storage device (615) and stored in memory (610) for easy access by the processor (605). In some situations, the electronic storage device (615) may be omitted and the machine executable instructions are stored in memory (610).

[0107] The code may be precompiled and configured for use with a machine having a suitable processor to execute the code, or may be compiled during run-time. The code may be provided in a programming language that can be selected to enable the code to be executed in a precompiled or as-compiled fashion.

[0108] Aspects of the systems and methods provided herein, such as the computer system (601), may be integrated in programming. Various aspects of the technology may be thought of as a "product" or "article of manufacture" typically in the form of machine (or processor) executable code and / or associated data embodied in a type of machine-readable medium. The machine executable code may be stored in an electronic storage device, such as a memory (e.g., read-only memory, random access memory, flash memory) or a hard disk. A "storage" type medium may include any or all of the tangible memory of a computer, processor, etc., such as various semiconductor memories, tape drives, disk drives, etc., that may provide non-transitory storage at any time for software programming. All or a portion of the software may also be communicated from time to time over the Internet or various other communication networks. Such communication may, for example, enable loading of the software from one computer or processor to another, for example, from a management server or host computer to a computer platform of an application server. Thus, other types of media that may carry software elements include optical, electrical, and electromagnetic waves, such as those used over wired and optical land-line networks between local devices, and various air links. The physical elements that carry such waves, such as wired or wireless links, optical links, etc., may also be considered media bearing the software. As used herein, unless limited to non-transitory tangible "storage" media, terms such as computer or machine "readable medium" refer to any medium that participates in providing instructions to a processor for execution.

[0109] Thus, a machine-readable medium such as a computer executable code may take many forms, including but not limited to a tangible storage medium, a carrier wave medium, or a physical transmission medium. Non-volatile storage media include optical or magnetic disks, such as any of the storage devices in any computer(s), such as may be used to implement the databases, etc., shown in the figures. Volatile storage media include dynamic memory, such as the main memory of such a computer platform. Tangible transmission media include coaxial cables, copper wire, and fiber optics, including the wires that comprise a bus within a computer system. Carrier wave transmission media may take the form of electric 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 tapes, other magnetic media, CD-ROMs, DVDs or DVD-ROMs, other optical media, punch cards, paper tapes, other physical storage media with patterns of holes, RAM, ROM, PROMs and EPROMs, FLASH-EPROMs, other memory chips or cartridges, carrier waves that transport data or instructions, cables or links that transmit such carrier waves, or other media from which a computer may read programming code and / or data. 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.

[0110] The computer system (601) includes or is in communication with an electronic display device (635) that includes a user interface (UI) (640) for providing processing parameters, data analysis, and results of a biological assay or reaction (e.g., PCR). Examples of UIs include, without limitation, graphical user interfaces (GUI) and web-based user interfaces.

[0111] The methods and systems of the present disclosure may be implemented by one or more algorithms. The algorithms can be implemented as software when executed by the central processing unit (605). The algorithms can, for example, regulate or control the system or perform the methods provided herein (e.g., sample loading, thermal cycling, sensing, etc.). EXAMPLES

[0112] Example 1: Microfluidic device containing multiple processing units 7A and 7B show an example of a microfluidic device for processing a biological sample. The microfluidic device (701) includes multiple slides, for example, four slides as shown in FIG. 7B. The microfluidic device (701) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more slides. The multiple slides may be joined to an automation-compatible plate frame by welding. The plate frame may be a standard format plate frame including multiple single inlet wells as shown in FIG. 7B. Other suitable methods may also be used to join multiple slides together. A single slide (702) (a four-unit array with approximately 20,000 compartments per unit) includes multiple processing units, for example, four processing units as shown in FIG. 7A. Additionally, the slide (702) may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more processing units.

[0113] Further, a single processing unit includes approximately 20,000 chambers / compartments, and a single compartment / chamber (704) is shown in FIG. 7A. Also, each processing unit includes a channel as illustrated in FIGS. 1A-1F. Each processing unit includes a single inlet port as described herein. The single chamber / compartment (704) has a length of approximately 85 μm, a width of approximately 65 μm, and a height of approximately 100 μm. Furthermore, the single chamber / compartment (704) may include a microchannel (705), which has a depth of approximately 10 μm and a width of approximately 15 μm. As a result of the dimensions of each processing unit, for example, the processing unit (703) may include a total analysis volume of approximately 11.5 μL. Furthermore, each processing unit includes less than 10% dead volume.

[0114] Example 2: Loading of biological samples and reaction reagents into a microfluidic device Figure 8 shows a microscope image of a single processing unit (801). Figure 8 further includes a 650 μm scale bar shown in solid lines in the corners of the microscope image. As shown by the various configurations of the main channel (802), (803), and (804), the main channel (805) is configured to open into multiple microchannels configured to connect and fluidically communicate with one or more compartments / chambers.

[0115] For example, to fill a processing unit, a liquid combination of a biological sample and reaction reagents is first flowed into the processing unit, followed by an immiscible fluid, such as an inert silicone oil, that is configured to remove the combined liquid from the main channel and isolate individual compartments / chambers for PCR reactions, such as thermal cycling, and subsequent detection analysis.

[0116] Furthermore, to solve the problem of overfilling fluid, each processing unit may include one or more sacrificial chambers (806), which are configured to connect and fluidly communicate with the main channel (805). Each main channel may connect and fluidly communicate with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sacrificial chambers. The sacrificial chambers may be of different shapes and may contain different volumes. When a processing unit is overfilled with fluid and some residual liquid remains in the main channel, especially toward the end of the fluid flow, one or more neighboring compartments / chambers become in fluid communication with the overfilled processing unit. Furthermore, one or more target molecules contained in the overfilled fluid may bleed into adjacent compartments / chambers during the PCR amplification process. The one or more sacrificial chambers (806) located in each processing unit are configured to capture excess fluid and allow immiscible fluids to completely purge the main channel. As a result, the sacrificial chambers help accommodate slight displacements in the volume of the loaded combined fluids (e.g., biological sample and reagents) (e.g., slight displacements may be caused by pipetting errors). The sacrificial chambers (806) may also help isolate the PCR reactions in each compartment / chamber so that the signal generated from the PCR reactions can be accurately quantified.

[0117] Example 3: Digitization of a fluid combining a biological sample and a reaction reagent Figures 9A-9D show the fluid loading and digitization of combined biological samples and reaction reagents using a single inlet with an optional outlet. A pneumatic control device (not shown in Figures 9A-9D) is utilized, and a PCR reagent mix containing the common qPCR correction dye ROX is used to visualize the digitization process.

[0118] The digitization process can be accomplished using either a single pressure step or multiple pressure steps. For example, about 10 μL of reaction reagent (including target nucleic acid molecules from a biological sample) is filled (e.g., by pipetting) into the inlet well (706) (FIG. 7A) on the microfluidic device (701) to fill each processing unit of the microfluidic device (701) (FIG. 7A). In addition, about 7 μL of isolation buffer / silicone oil is filled (e.g., by pipetting) into the inlet well (706) on the microfluidic device (701). A positive pressure of about 50 PSI is then applied to the inlet well (706) using a pneumatic pressure control device for about 60 minutes or until completion of reagent digitization. The pressure of about 50 PSI may be gradually increased. The isolation buffer / silicone oil serves as an overlay liquid and is configured to rest on the PCR reagents during the filling process, which ensures that the PCR reagents enter the microfluidic array first, because the isolation buffer / silicone oil has a lower density than water. After the digitization process is complete, the compartments / chambers are fluidically isolated and, when activated, are capable of performing independent PCR.

[0119] Furthermore, Figures 9A-D show four different time points during the digitization process. Figure 9A shows a first time point where the first reagent first enters the array. Figure 9B shows a second time point where the reagent has passed almost the entire array and the filling channels in the array are filled with reagent, but the microchambers are mostly empty. Figure 9C shows a third time point where the reagent has entered the microchambers, but the chambers are still in fluid communication at this time, and Figure 9D shows a fourth time point where the reagent has run out and oil has moved through the array, displacing all of the reagent in the main fluidic channels. The first row of Figures 9A-D demonstrates fluorescent images of the entire array at four time points in the progression as the reagent begins to fill the array, fills most of the main filling channels, enters the microchambers, and is replaced by silicone oil only in the main channel. The middle row of Figures 9A-D shows a schematic representation of the microchamber array at the four time points mentioned above. The last rows of Figures 9A to 9D show enlarged images of the time progression.

[0120] Example 4: Integration of the digitization process into the laboratory workflow The digitization process of the present invention can be easily integrated into a typical laboratory workflow for nucleic acid assays, as exemplified by FIG. 10. Biological sample preparation (sample preparation step) may be performed in other PCR-based workflows, including isolation of nucleic acids and combining Master Mix and primers / probes with the biological sample. The microfluidic device (701) is filled (fill plate step) as described herein (e.g., by pipetting the sample mixture followed by pipetting an oil overlay) and then placed into an instrument that integrates pneumatic filling / digitization of reagents, thermal cycling, and data / image acquisition (reagent dispensing+PCR+image acquisition step and analysis step). The acquired data of the PCR reaction can then be analyzed by downstream software to provide results such as the concentration of the target gene in the original biological sample.

[0121] Example 5: Image Analysis Figure 11 shows a screenshot of the user interface of the software that performs analysis on the acquired images. Figure 11 demonstrates actual results from a dPCR assay with four different indicators (indicated by four different fluorescent colors) using human genomic DNA as a sample. The analysis settings shown in panel (1101) are selectable by the user. Results from one processing unit (unit C3 in this case) out of 16 processing units on the microfluidic device (701) are shown in the form of a scatter plot in panel (1104) and in the form of concentration values ​​in panel (1103). The central image in panel (1102) is a composite image overlaid with positives from each of the four optical channels to detect the target gene. The fifth optical channel is used as a quality control channel.

[0122] While preferred embodiments of the present invention have been shown and described herein, 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 herein. 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. Many modifications, changes, and substitutions will occur to those skilled in the art 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 proportions set forth herein, which depend upon a variety of conditions and variables. It is to be understood that various alternatives to the embodiments of the present invention described herein may be utilized in the practice of the present invention. It is therefore contemplated that the present invention shall cover any such alternatives, modifications, variations, or equivalents. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of the claims and their equivalents are covered thereby.

Claims

1. 1. A microfluidic device for processing a biological sample, the microfluidic device comprising: a fluid flow path including a channel and an inlet port, wherein the fluid flow path does not include an outlet port, and wherein the inlet port is configured to direct a solution including the biological sample into the channel; a chamber in fluid communication with the channel, wherein the chamber is configured to receive at least a portion of the solution from the channel and to retain at least the portion of the solution during the processing.

2. The microfluidic device of claim 1 , further comprising a plurality of chambers in fluid communication with the channel, wherein the plurality of chambers includes the chamber.

3. 10. The microfluidic device of claim 1, wherein the channel comprises a first end and a second end, and wherein the first end and the second end are connected to a single inlet port.

4. The microfluidic device of claim 3 , wherein the fluid flow path is annular.

5. 2. The microfluidic device of claim 1, wherein the channel comprises a first end and a second end, and wherein the first end is connected to the inlet port and the second end is connected to a different inlet port.

6. The microfluidic device of claim 1 , wherein the chamber is configured to allow for pressurized gas release.

7. The microfluidic device of claim 6 , wherein the chamber includes a film or membrane that allows for the release of pressurized gas.

8. The microfluidic device of claim 7 , wherein the film or membrane is a polymer film or membrane.

9. The microfluidic device of claim 8 , wherein the polymeric film or membrane is elastomer-free.

10. The microfluidic device of claim 7 , wherein the film or membrane has a thickness of less than about 100 μm.

11. The microfluidic device of claim 10, wherein the thickness is less than about 50 μm.

12. The microfluidic device of claim 7 , wherein the film or membrane is substantially impermeable to liquids.

13. The microfluidic device of claim 1 , wherein the fluid flow paths or the chambers do not include valves.

14. The microfluidic device of claim 1 , wherein the chamber has a volume of about 500 picoliters or less.

15. The microfluidic device of claim 1 , wherein the chamber has a volume of about 250 picoliters or less.

16. The microfluidic device of claim 1 , wherein the chamber has a cross-sectional dimension of about 250 μm or less.

17. The microfluidic device of claim 1 , wherein the chamber has a depth of about 250 μm or less.

18. 10. The microfluidic device of claim 1, further comprising a siphon opening disposed between the channel and the chamber, wherein the siphon opening is configured to provide fluid communication between the channel and the chamber.

19. The microfluidic device of claim 1 , further comprising a sacrificial chamber in fluid communication with the channel.

20. 20. The microfluidic device of claim 19, wherein the sacrificial chamber is configured to hold an excess portion of a solution containing the biological sample.

21. 1. A method for processing a biological sample, comprising: (a) providing a device comprising: (i) a fluid flow path comprising a channel and an inlet port, the fluid flow path not including an outlet port; and (ii) a chamber in fluid communication with the channel; (b) directing a solution containing the biological sample into the channel from the inlet port; (c) directing at least a portion of the solution from the channel to the chamber, wherein the chamber retains at least the portion of the solution during the processing.

22. 22. The method of claim 21, wherein the device comprises a plurality of chambers in fluid communication with the channel, and wherein the plurality of chambers includes the chamber.

23. 22. The method of claim 21, further comprising applying a single pressure differential to the inlet port to direct the solution from the inlet port into the channel and from the channel into the chamber.

24. 24. The method of claim 23, wherein the single pressure differential allows for pressurized outgassing of gas in the chamber.

25. 22. The method of claim 21, further comprising applying a first pressure differential to the inlet port to direct the solution from the inlet port into the channel.

26. 26. The method of claim 25, further comprising applying a second pressure differential to the inlet port to direct the solution from the channel to the chamber.

27. 27. The method of claim 26, wherein the second pressure differential is greater than the first pressure differential.

28. 27. The method of claim 26, wherein the second pressure differential enables pressurized outgassing of gas in the chamber.

29. 30. The method of claim 24 or 28, wherein the chamber includes a film or membrane, wherein the film or membrane allows for pressurized outgassing of the gas in the chamber.

30. 22. The method of claim 21, wherein the volume of the solution is equal to or less than the volume of the chamber.

31. 31. The method of claim 30, wherein the device dispenses the solution containing the biological sample into the chamber such that no residual solution remains within the channel.

32. 22. The method of claim 21, further comprising the steps of providing an immiscible fluid to the inlet port and directing the immiscible fluid into the channel.

33. 33. The method of claim 32, wherein the volume of the immiscible fluid is greater than the volume of the channel.

34. 22. The method of claim 21, wherein the biological sample is a nucleic acid molecule.

35. 35. The method of claim 34, further comprising amplifying the nucleic acid molecule by thermocycling the chamber.

36. The method of claim 21 , further comprising controlling a temperature of the channel or the chamber.

37. 22. The method of claim 21, further comprising sensing one or more components of the biological sample or a reaction with the one or more components of the biological sample in the chamber.

38. 38. The method of claim 37, wherein sensing the one or more components or the reaction of the biological sample comprises imaging the chamber.

39. 1. A system for processing a biological sample, the system comprising: A device comprising: (i) a fluid flow path including a channel and an inlet port, wherein the fluid flow path does not include an outlet port, and wherein the inlet port is configured to direct a solution including the biological sample to the channel; and (ii) a chamber in fluid communication with the channel, wherein the chamber is configured to receive at least a portion of the solution from the channel and to retain at least the portion of the solution during the processing; a holder configured to receive or hold the device during the processing; a fluid flow module fluidly coupled to the inlet port and configured to (i) cause the solution to flow from the inlet port into the channel, and (ii) provide a pressure differential to cause at least a portion of the solution to flow from the channel to the chamber.

40. 40. The system of claim 39, wherein the device comprises a plurality of chambers in fluid communication with the channel, and wherein the plurality of chambers includes the chamber.

41. 40. The system of claim 39, wherein the chamber of the device is configured to allow pressurized outgassing of gas in the chamber when the fluid flow module applies the pressure differential to the inlet port.

42. 42. The system of claim 41, wherein the chamber includes a film or membrane configured to allow for the release of pressurized gas.

43. 40. The system of claim 39, further comprising one or more computer processors operably coupled to the fluid flow module, wherein the one or more computer processors are individually or collectively programmed to instruct the fluid flow module to provide a pressure differential when the fluid flow module is fluidly coupled to the inlet port, thereby causing the solution to flow from the inlet port into the channel and directing at least the portion of the solution from the channel to the chamber.

44. 40. The system of claim 39, further comprising a thermal module in thermal communication with the chamber, wherein the thermal module is configured to control a temperature of the chamber during the process.

45. 40. The system of claim 39, further comprising a sensing module in communication with the chamber, the sensing module configured to sense contents of the chamber during the processing.

46. 46. ​​The system of claim 45, wherein the sensing module is an optical module in optical communication with the chamber.

47. 47. The system of claim 46, wherein the optical module is configured to image the chamber.

48. 1. A system for processing a biological sample, the system comprising: A holder configured to hold a device, the device comprising: (i) a fluid flow path including a channel and an inlet port, but not including an outlet port; and (ii) a chamber in fluid communication with the channel; and one or more computer processors configured to be operatively coupled to the device when the device is held by the holder, wherein the one or more computer processors are individually or collectively programmed to: (i) direct a solution containing the biological sample from the inlet port to the channel, and (ii) direct at least a portion of the solution from the channel to the chamber, wherein the chamber retains at least a portion of the solution during the processing.

49. 49. The system of claim 48, further comprising a fluid flow module operably coupled to the one or more computer processors, wherein the fluid flow module is configured to be operably coupled to the device when the device is held by the holder, and wherein the one or more computer processors are programmed to instruct the fluid flow module to direct the solution from the inlet port to the channel.

50. 49. The system of claim 48, further comprising a thermal module configured to be in thermal communication with the chamber when the system is held by the holder, wherein the thermal module is configured to control a temperature of the chamber during the processing.

51. 49. The system of claim 48, further comprising a detection module configured to communicate with the chamber when the device is held in the holder, wherein the detection module is configured to detect contents of the chamber during the processing.

52. 52. The system of claim 51, wherein the sensing module is an optical module in optical communication.

53. 53. The system of claim 52, wherein the optical module is configured to image the chamber.

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