Devices and methods for partitioning samples for digital assays

Abstract

Provided are devices, systems, and methods for partitioning a sample. Embodiments of the device includes a microfluidic cartridge for partitioning a sample in a fixed array, and includes a hole array for capillary filling, an enclosed chamber with a fixed geometry, a single input channel to the chamber, and at least one output channel. Methods for partitioning a sample into a hole assay includes, for example, providing the microfluidic device, filling the chamber and the hole array with an immiscible non-aqueous fluid, introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes, introducing a second non-aqueous fluid into the chamber to fill a first volume and a second volume without displacing the aqueous sample in the holes, and performing a digital assay after introducing the aqueous sample.

Description

MELIO.009WO PATENT DEVICES AND METHODS FOR PARTITIONING SAMPLES FOR DIGITAL ASSAYS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 598,379, filed on November 13, 2023, which is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure is related to devices, systems, and methods for the partitioning samples in a digital assay. More particularly, the devices, systems, and methods include loading a sequence of a water-immiscible fluid and an aqueous sample into a closed microfluidic chamber that contains an array of fixed partitions into which the aqueous sample is divided, which is promoted by the immiscible fluid. In some cases, the aqueous sample contains particles such as beads, or cells such as bacteria, micro-organisms, eukaryotic cells, or fungal cells. BACKGROUND

[0003] Biological analysis methods, especially genetic or nucleic acid analysis methods, in a digital format have been found to have multiple advantages. In a digital format, a sample is divided in a large number of partitions, in each of which an analysis or an assay is performed. The main advantages of digital assays are the possibility of absolute quantification, without the need for standard curves, and the reduction of background interferences due to the partitioning. SUMMARY

[0004] Embodiments provided herein relate to devices, systems, and methods for partitioning samples in a digital assay.

[0005] Some embodiments provided herein relate to devices, kits, and methods for partitioning a sample. Some embodiments provided herein relate to microfluidic cartridges for partitioning a sample in a fixed array. In some embodiments, the cartridges include a hole array for capillary filling. In some embodiments, each of a plurality of the holes in the hole array includes an open-ended first end and an open-ended second end. In some embodiments, the cartridgesinclude an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened-ended first end and a second volume below the open-ended second end, wherein the first volume and the second volume are fluidly connected by the holes of the array and wherein the second volume is capable of being filled only through the holes of the array. In some embodiments, the cartridges include an input channel to the enclosed chamber fluidically connected with the first volume. In some embodiments, the cartridges include at least one output channel fluidically connected with the first volume and the second volume. In some embodiments, the hole array includes at least 1000 holes. In some embodiments, the input channel is configured to flow fluid into the first volume and wherein the second volume is configured to receive fluid from the holes in the array. In some embodiments, the side-to-side width of each hole is between 1 and 300 microns. In some embodiments, the shape of each hole is a regular polygon. In some embodiments, a total surface area of the holes is at least 25% of the total surface area of the hole array. In some embodiments, the total surface area of the holes is about 50% of the total surface area of the hole array. In some embodiments, an inner surface of each hole is hydrophilic. In some embodiments, each hole is configured to retain an aqueous sample by capillary forces. In some embodiments, an inner surface of each hole is sufficiently hydrophilic that the holes will fill by capillarity when exposed to the aqueous sample. In some embodiments, the hole array includes at least 5,000 holes. In some embodiments, the hole array includes at least 10,000 holes. In some embodiments, a surface of the hole array in contact with the first volume includes silicon or a polymer. In some embodiments, the input channel is not connected with the second volume.

[0006] Some embodiments provided herein relate to kits. In some embodiments, the kits includes any of the microfluidic cartridges described herein and a non-aqueous immiscible fluid. In some embodiments, the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than an aqueous fluid. In some embodiments, the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil. In some embodiments, the non-aqueous immiscible fluid is FC-40 or HFE 7500. In some embodiments, the non-aqueous immiscible fluid is contained within the microfluidic cartridge. In some embodiments, each hole of the hole array, the first volume, and the second volume are filled with the non-aqueous immiscible fluid.

[0007] Some embodiments provided herein relate to methods for partitioning a sample into a hole array. In some embodiments, the methods include providing any of the microfluidiccartridges described herein or any of the kits described herein. In some embodiments, the methods include introducing the enclosed chamber and the hole array with an immiscible non-aqueous fluid. In some embodiments, the methods include introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes.

[0008] Some embodiments provided herein relate to additional methods of partitioning a sample into a hole array. In some embodiments, the methods include providing a microfluidic device. In some embodiments, the device includes a hole array for capillary filling. In some embodiments, each hole includes an open-ended first end and an open-ended second end In some embodiments, the device includes an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened-ended first end and a second volume below the open-ended second end. In some embodiments, the methods include filling the chamber and the hole array with an immiscible non-aqueous fluid. In some embodiments, the methods include introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes. In some embodiments, the hole array includes at least 1000 holes. In some embodiments, the methods further includes introducing a second non-aqueous fluid into the chamber to fill the first volume and the second volume without displacing the aqueous sample in the holes. In some embodiments, the aqueous sample includes particles, beads, cells, microorganisms, bacterial cells, eukaryotic cells, or fungal cells. In some embodiments, the aqueous sample has a volume that is less than the total volume of all of the holes of the array. In some embodiments, the microfluidic cartridge includes a single input channel is configured to flow fluid into the first volume and wherein the second volume is configured to receive fluid from the holes in the array. In some embodiments, the second volume is configured to receive fluid only from the holes in the array. In some embodiments, an inner surface of the holes is hydrophilic and wherein the holes fill by capillarity when exposed to the aqueous sample. In some embodiments, the array includes at least 5,000 holes. In some embodiments, the array includes at least 10,000 holes. In some embodiments, the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than the aqueous fluid. In some embodiments, the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil. In some embodiments, the non-aqueous immiscible fluid is FC-40 or HFE 7500. In someembodiments, the immiscible non-aqueous fluid includes a surfactant. In some embodiments, the methods further include performing a digital assay after introducing the aqueous sample. In some embodiments, the methods further include performing an assay to detect the presence of, identify, or quantify one or more nucleic acids included in the aqueous sample. In some embodiments, the digital assay or assay includes PCR amplification. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] In order to describe the manner in which the above-recited and other advantages and features of the present disclosure can be obtained, a more particular description will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments and are not therefore to be considered to be limiting of its scope, the disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:

[0010] Figure 1A illustrates an embodiment of an aqueous sample in the process of being removed in the upwards direction and the surface tension force between the aqueous sample, the second immiscible fluid, and the wall of the partition.

[0011] Figure 1B illustrates an embodiment of an aqueous sample in the process of being removed in the downwards direction and the surface tension force between the aqueous sample, the second immiscible fluid, and the wall of the partition.

[0012] Figure 2 illustrates an embodiment of a cross-section of a microfluidic chamber designed to automate the filling of a capillary hole array.

[0013] Figure 3 illustrates an embodiment of cells attached to silicon surfaces outside of the wells.

[0014] Figure 4 illustrates an embodiment of a top view of a microfluidic cartridge for filling a silicon hole array, using an input on one side only, split into three branches.

[0015] Figure 5 illustrates an embodiment of cross-section of a microfluidic chamber with a single input used for filling of a capillary hole array.

[0016] Figures 6A-6G illustrate an embodiment of an experimental set up describing the filling of a cartridge with a fluorinated oil.

[0017] Figures 7A-7D illustrate an embodiment of a video at the time when an oil droplet is placed on the top of the silicon chip with the holes using a pipette tip.

[0018] Figures 8A-8D illustrate an embodiment of a video at the time when the sample is getting filled into the holes by piercing the top non-aqueous layer using a fine pipette tip.

[0019] Figures 9A-9B illustrate an embodiment of a QuantStudio™ silicon chip with hydrophobic surface filled with fluorescent aqueous solution from the bottom left corner.

[0020] Figure 9C illustrates an embodiment of a chip with hydrophobic surface where the trailing immiscible oil pushes sample ahead.

[0021] Figure 9D illustrates an embodiment of a chip with hydrophobic surface where almost all the wells have been filled with sample and no more aqueous solution is left above the wells.

[0022] Figure 9E illustrates an embodiment of a bottom view of a chip with hydrophobic surface after filling the wells.

[0023] Figure 10A illustrates a histogram of intensity distribution of the filled silicon device imaged from the top.

[0024] Figure 10B illustrates a histogram of intensity distribution of the same filled silicon device imaged from the bottom.

[0025] Figures 11A-11E illustrate an embodiment of a silicon device with hydrophilic surfaces filled with fluorescent aqueous solution from the bottom left corner after filling with immiscible oil.

[0026] Figure 12 illustrates a histogram of intensity distribution of the filled silicon device.

[0027] Figure 13 illustrates an embodiment of a cartridge design used for the filling experiment.

[0028] Figures 14A-14E illustrate an embodiment of a QuantStudio™ silicon chip with a single input channel filled with fluorescent aqueous solution from the bottom left corner after filling with immiscible oil.

[0029] Figure 15 illustrates a histogram of intensity distribution of the filled QuantStudio™ silicon chip with a single input channel.

[0030] Figures 16A-16D illustrate an embodiment of a QuantStudio™ silicon filled from the bottom left corner with pure FC-40 as the immiscible fluid,

[0031] Figure 17 illustrates a histogram of intensity distribution of the filled QuantStudio™ silicon chip with pure FC-40.

[0032] Figure 18 illustrates a silicon chip filled with a fluorescent solution containing fluorescent E. coli cells. On the top left, an image of the entire chip, and on the top right, an enlargement that shows the individual cells more clearly. On the bottom left, the partitioned sections of the chip are shown which were analyzed individually for presence of particles outside the partitions. Bottom right panel shows the same experiment conducted with a QuantStudio™ silicon chip. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety herein. In case of conflict, the present specification, including definitions, will control. Other features and advantages will be apparent from the following detailed description and figures, and from the claims.

[0034] For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0035] It is noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the,” and any singular use of any word, include plural referents unless expressly and unequivocally limited to one referent. As used herein, the term “include” and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0036] As used herein, the term “biological sample” or “sample” has its ordinary meaning as understood in light of the specification, and refers to a material, substance, or solution that includes one or more biological molecules, chemicals, components, and / or compounds (e.g., a nucleic acid, DNA molecule, or RNA molecule) of interest to a user, manufacturer, or distributor of the various embodiments described or implied herein. A sample may include, but is not limited to, one or more of a DNA sequence (including cell-free DNA), an RNA sequence, a gene, an oligonucleotide, an amino acid sequence, a protein, a biomarker, a microorganism, or a cell (e.g., circulating tumor cell), or any other suitable target biomolecule. As used herein, the term “sample solution” has its ordinary meaning as understood in light of the specification, and refers to a liquid or fluid that includes at least one sample.

[0037] As used herein, the term “biological analysis” has its ordinary meaning as understood in light of the specification, and refers to the analysis of a biological substance in order to ascertain its influence on living organisms. A biological analysis is a scientific approach that combines analytical tools and biological content in one place to enhance deeper and broader understanding of biological relationships and processes to experimental observations and translation of that understanding to hypothesis. Biological analysis is typically done on mRNA, miRNA, protein, SNP, metabolites or any other biomolecule by performing basic statistics such as background correction, normalization, p-value, and so on.

[0038] As used herein, the term “capillary force” has its ordinary meaning as understood in light of the specification, and refers to a force required by a liquid flowing in a narrow space without the assistance of, or even in opposition to, any external forces like gravity. Capillary force acts at fluid-air-solid interfaces to minimize the surface energy of the interface. It is very vital for moving the liquid phases in the upward direction; the movement of liquid phase within the porous space is regulated by forces of adhesion, cohesion, and surface tension.

[0039] As used herein, the term “capillary pressure” has its ordinary meaning as understood in light of the specification, and refers to the pressure between two immiscible fluids in a thin tube, resulting from the interactions of forces between the fluids and solid walls of the tube. For example, if a small tube in water is overlaid by oil, water will rise up into the tube due to capillary pressure.

[0040] As used herein, the term “microfluidics” has its ordinary meaning as understood in light of the specification, and refers to the science which studies the behavior of fluids throughmicro-channels, and the technology of manufacturing microminiaturized devices containing chambers and tunnels through which fluids flow or are confined.

[0041] As used herein, the term “array” has its ordinary meaning as understood in light of the specification, and refers to an arrangement of distinct entities geometrically arranged on a surface. For example, an array may include an array of holes, or an arrangement of holes placed into a substrate or surface.

[0042] As used herein, the term “hydrophobic” has its ordinary meaning as understood in light of the specification, and refers to repellency of water.

[0043] As used herein, the term “hydrophilic” has its ordinary meaning as understood in light of the specification, and refers to having a strong affinity for water.

[0044] As used herein, the term “non-aqueous” has its ordinary meaning as understood in light of the specification, and refers to having characteristics of a liquid other than water.

[0045] As used herein, the term “immiscible” has its ordinary meaning as understood in light of the specification, and refers to a characteristic of a fluid being incapable of mixing or attaining homogeneity.

[0046] Without being bound by theory, the embodiments described herein relate to fluid flow in a microfluid device, and the filling of small holes by capillary forces. The forces involved in the fluid flow include pressure forces, viscosity, inertia, surface tension or capillary forces, and / or gravity. Due to the small dimensions of the channels and the holes in the hole array, capillary forces are expected to be strong, and gravity is expected to be less important. The operations of the embodiments described here are thus expected to be independent of any particular orientation. All terms referring to a given orientation such as “top”, “bottom”, “up”, “upper”, “bottom” or “lower” are to be understood to be used for convenience only, and are not intended to be limiting to a particular orientation of the device.

[0047] Polymerase chain reaction (PCR) may include a thermal cycling process, in which cycles of heating and cooling are used to provide repeated cycles of nucleic acid melting and enzymatic replication of nucleic acids. A number of PCR methods use thermal cycling involving alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps may be used first to physically separate nucleic acids, such as separating the two strands in a nucleic acid double helix, at a high temperature in a process called melting. At a lower temperature, each strand is then used as the template in synthesis by thepolymerase to selectively amplify a target nucleic acid during an annealing phase and extension phases. Example polymerases include heat-stable polymerase such as, for example, Taq polymerase. The selectivity of PCR results from the use of primers that are complementary to nucleic acid regions targeted for amplification under specific thermal cycling conditions. Primers (short nucleic acid fragments) containing sequences complementary to the target region along with a polymerase, are used to enable selective and repeated amplification.

[0048] Digital PCR (dPCR) is a refinement of conventional polymerase chain reaction (PCR) methods which can be used to directly quantify and clonally amplify nucleic acids (including DNA, cDNA, methylated DNA, RNA, or the like). One difference between dPCR and traditional PCR lays in the method of measuring nucleic acids amounts. In dPCR, a sample is separated into a large number of individual sample volumes or portions and respective PCR reactions are carried out in each sample portion individually. This separation allows for sensitive measurement of very small amounts of a nucleic acid. dPCR has been demonstrated as useful for studying variations in gene sequences, such as copy number variation or point mutations.

[0049] In dPCR, a sample is partitioned so that individual nucleic acid molecules to be assessed within the sample are localized and concentrated within many separate regions. While the starting number of copies of a molecule is proportional to the number of amplification cycles in conventional PCR, dPCR does not depend on determining a number of amplification cycles to determine the initial sample amount. Instead, the initial sample is partitioned into a large number of relatively small sample portions containing one copy, or approximately one copy, or no copy of the nucleic acid template or target. As a result, each partitioned sample portion may be characterized as a “0” or “1” for containing at least one of a type of target nucleic acid molecule, resulting in a negative (“0”) or positive (“1”) PCR reaction, respectively. The partitioning of the sample in this way may use Poisson statistics to provide an estimate of molecules in the initial sample. However, the accuracy of this estimate varies, depending on the number of “0” and “1” produced.

[0050] Systems and methods for performing dPCR are described in the art, including, for example, in US 2017 / 0088879 and US 2016 / 0310949, each of which is incorporated by reference herein in its entirety. These references describe the use of dPCR on nucleic acids, such as DNA. However, the methods and systems described therein may be used in the context of the present application for the use of cells and single-cell analysis. For example, in some embodiments,a digital amplification technique is performed. For example, the digital amplification technique may include a dPCR assay, process, experiment, or test. In some embodiments, a sample or reaction solution is segregated, distributed, or divided, into a plurality of sample reaction volumes or reaction sites associated with a reaction device, fluidic device, sample holder, or other such device. The plurality of sample reaction volumes may include a first plurality of the sample reaction volumes each containing a cell and a second plurality of the sample reaction volumes each containing no cells. The plurality of sample reaction volumes or reaction sites are subjected to an amplification assay using, for example, at least a primer and probe or indicator dye. In some embodiments, the amplification assay is configured to amplify a target nucleic acid from the cell. During the dPCR assay, an indicator of the target present in any of the plurality of sample reaction volumes may be detected or measured. Similar to other types of PCR, dPCR may progress by exposing the partitioned sample reaction volumes, which contain reagents for amplification, to an amplification assay designed to amplify the target nucleic acid. For example, thermal cycling may be performed such that the template nucleic acid is amplified within the reaction volumes that include an initial one, or approximately one, copy of the template nucleic acid molecule.

[0051] There are multiple methods for partitioning a sample for a digital assay. One category of partitioning methods uses fixed partitions whose location in space is known beforehand. The use of fixed partitions has several advantages. First, each partition is separated in space and does not touch the other partitions, unlike droplets which are free to touch each other. This reduces the risk of exchange or interactions between the partitions. Second, detection is improved because the location of each sample is known, and as much time as needed can be used for detection. Third, the use of fixed partitions in known locations enables the possibility of selectively recovering certain partitions for further analysis.

[0052] Multiple methods of creating fixed partitions exist. One type of method uses chambers or wells connected by one or more flow channels that are filled with the sample, followed by flowing in an immiscible fluid to perform the separation into distinct partitions. Another method uses open holes, or wells open on two sides, that are filled by capillary forces, or surface tension forces. In all cases, after creating the partitions, it is useful to isolate them from each other, and protect them from evaporation and the outside environment by using one or more protective layers. These layers could be composed of an immiscible liquid, such as an oil, mineral oil, silicone oil, fluorinated liquids such as FC-40 or HFE 7500, or water-immiscible esters. Other possibleprotective layers that could be applied are solid, such as polymers, including thermoplastic polymer films, polydimethylsiloxane, other types of rubber films or septa, metal coated polymer films, and so forth.

[0053] An array of wells that are filled by capillary action can take the form of holes through the entire thickness of a sheet of a given material, and in which the inner surface of the holes is sufficiently hydrophilic that it will fill by capillarity when exposed to the aqueous sample. Once filled, the aqueous sample is retained by capillary forces. For digital assays, it is usually desirable to divide a sample into a large number of partitions, typically at least 1000 partitions, and most commonly 10,000 or more partitions. For example, in some embodiments, a device may include at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, or at least 20000 partitions, or an amount within a range defined by any two of the aforementioned values, or an amount greater than any of the aforementioned values. In general, the capillary pressure that causes the holes in the array to fill spontaneously with an aqueous solution are inversely proportional to the dimension of the hole. Figures 1A and 1B show an aqueous sample in the process of filling a capillary while pushing out the fluid that was there previously, which may be air, or a protective fluid such as an immiscible liquid as described above. The capillary forces and pressures in the case of a cylindrical partition of height h and radius r can be calculated as follows.

[0054] Since the surface is hydrophilic, the contact angle is usually below 90 degrees, and then the surface tension force is directed downwards. The surface tension force per unit length at the interface between the aqueous sample, the second immiscible fluid, and the wall of the partition is given by , and is parallel to the interface, with an angle of relative to the axis of the cylinder. By integrating over the perimeter of the circular interface, the total force along theaxis is given by:2 (1)

[0055] This force divided by the area of the partition is the capillary pressure givenby:

[0056] This is the familiar Young-Laplace equation, and it gives the capillary pressure that drives the spontaneous filling of a hole. The parameter .is the surface tension of the interface between the aqueous filling solution and the solution that was there previously, which could be air, a gas, or a water-immiscible liquid. Similarly, the contact angle is the angle at the surface of the interface between the aqueous filling solution and the displaced fluid. The surface tension of an interface of an aqueous solution with air is usually in the range of 50 to 80 mN / m (depending on the surfactants present), and the surface tension between an aqueous solution and a fluorinated fluid such as HFE-7500 is around 26 mN / m. The contact angles depend on many factors, including the properties of the surface, the nature of the two fluids, and the presence of surfactants in the aqueous solution and in the non-aqueous fluid.

[0057] When a hole has been filled by an aqueous fluid, the geometry at the exit will tend to inhibit further expansion. The direction of the force projected on the axis of the hole changes to an upward force at the exit due to the expansion at the exit. If the exit edge of the hole is sharp, the capillary pressure becomes (see Figure 1B): 2(3)

[0058] The capillary pressure relative to the axis of the hole now becomes negative, meaning that it stops further downward flow. However, depending on the actual sharpness of the exit, and the contact angle, it is still possible for the liquid to expand laterally on the bottom surface, because Figure 1B shows that the surface tension force has a horizontal component. The equations above can be generalized to the case of non-cylindrical cross-sections.

[0059] The array of wells that are filled by capillary action can take the form of holes through the entire thickness of a sheet of a material. The material used for the sheet with holes can be selected from a wide variety of options, but is often a heat-conducting hydrophilic material such as silicon or a metal. In the case of metals, many fabrication methods are available, including computer numerical control (CNC) machining, electrical discharge machining, chemical etching, water jet cutting, or laser cutting. In the case of silicon, the etching methods available are wet chemical etching, or one of many types of plasma etching. In both cases, the etching pattern can be defined by a form of photolithography, which has the advantage of being able to define dense patterns of small holes, with typical resolutions in the order of one micrometer. Silicon etchingcan be isotropic or anisotropic. To create dense arrays or small holes, anisotropic etching methods may be used, since they allow the creation of high aspect ratio holes. The most commonly used silicon anisotropic etching method is a form of plasma etching known as Deep Reactive Ion Etching (DRIE), or the Bosch Process, or Deep Silicon Etching. Hole arrays in silicon are commercially available for digital PCR, such as the QuantStudio™ (Thermo-Fisher), and have also been described in multiple publications.

[0060] It is important to fill a high fraction of the available aqueous sample in the partitions, without wasting an excessive amount. In the case of chambers connected by channels, the wasted volume consists of the volume in the fluidic connections between chambers that is removed by the separating fluid, such as a water-immiscible fluid or air. In the case of arrays of holes filled by capillary action, the sample solution must have a volume close to, or equal to, that of the total volume of all the holes. In some cases, such as in the QuantStudio™, this is done manually with a specific application tool. However, this has the disadvantage of requiring a high degree of skill by the user, and typically not all of the wells are filled. After the wells are filled, an immiscible separating fluid is usually introduced on both sides of the holes. For example, Gao et al. filled a hole array having S-shaped flow channels at a high filling rate, but demonstrated large volumes of sample loss. Gao, Biomicrofluidics, 14, 2020.

[0061] There remains a need to develop methods that are (1) automated, and not dependent on user skill, (2) fill a large fraction of the available wells in the array, (3) do not waste a large portion of the sample, (4) do not consume large volumes of water-immiscible partitioning fluids, and (5) can be filled without leaving air pockets or bubbles that could interfere with subsequent thermal cycling.

[0062] To achieve a more reproducible method to fill the array of holes, the present disclosure relates to enclosed fluidic systems that allow for flow on both sides of the array. In some embodiments, the hole array, typically a silicon die with holes etched all the way through using a form of anisotropic plasma etching, is mounted in an enclosed microfluidic chamber. In some embodiments, the volume of the chamber around the array is small enough that an aqueous sample with a small volume can be introduced and brought into contact with all the holes in the array. The chamber must also allow the water-immiscible sealing fluid to contact the hole array on both sides to ensure that the partitions remain separated. That means there must be two open volumes, oneon each side of the hole array, with one or more fluidic connections on each side. Figure 2 depicts embodiments of a cross-section of a microfluidic chamber designed to fill a hole array.

[0063] An aqueous sample with a volume close to that of all the wells combined is introduced to the chamber, and flows over one of the outer surfaces of the capillary hole array. As it flows over the surface, the wells fill spontaneously with the aqueous sample due to surface tension forces. By the time the flow has covered the entire surface, the sample will be exhausted, and all of it, or almost all of it, has been introduced into the partitions. Capillary forces will then retain the samples in the partitions. An immiscible partitioning fluid can then be flowed along both sides of the hole array to flush out any remaining aqueous solution, and to ensure complete separation between the partitions.

[0064] The microfluidic chamber used to fill the wells in the capillary hole array can be fabricated in a microfluidic cartridge in which the hole array is mounted, and also provides all needed channels and connections. Microfluidic cartridges or systems are typically fabricated in polymers, because a variety of low cost and high-precision methods exist to shape the polymers. Examples of fabrication techniques include, for example, molding, injection molding, casting, CNC machining, laser cutting, or 3D printing. In some embodiments, clear thermoplastic polymers are used because they allow observation of the hole array, and optical detection of assays performed in the partitions.

[0065] In some embodiments, the use of a chamber combined with a precise flow system to control volumes and the flow rates of the solutions that enter the chamber allow the adjustment of the loading conditions to achieve essentially complete utilization of the sample. In some embodiments, this can be achieved by using a volume of aqueous sample that is slightly below that of the total volume of all the holes in the array. This will lead to the filling of less than the total number of partitions, but all of the sample will be consumed, leading to higher assay sensitivities by avoiding the waste on any samples. The loss of some of the partitions can be mitigated by making a slightly higher number of them than is required for the sensitivity of a digital assay.

[0066] However, there are a number of difficulties to be overcome in designing a fluidic system that introduces a sample into an array of partitions in a chamber. First, the polymers materials typically used in microfluidic fabrication all have surfaces that are hydrophobic to some degree. This makes filling a chamber without leaving some air on the edges difficult, and the fillingprocess can be irreproducible since it depends on the precise contact angles of the aqueous sample on all surfaces that it contacts. Filling is especially difficult when the chamber has sharp corners because air pockets tend to be trapped there. Hole arrays made of silicon usually have a square or rectangular shape due to the fabrication method employed, and the resulting corners are especially hard to fill completely.

[0067] Several methods are available to improve the filling of chambers and corners, but each of these adds their own difficulties and disadvantages. One is to treat the inner surface of the chamber to reduce the contact angle for filling with aqueous solutions. However, this makes fabrication more difficult and expensive, and some surface treatment methods such as oxygen plasmas or corona discharge only last for a limited time. Another method is to use phase guides, as is described in several publications. This helps the flow reach corners, but it also introduces additional edges and corners where part of the sample may be trapped unintentionally. A third method is to split the sample stream into multiple streams, and to fill from several corners at once. However, this increases the amount of sample that can be lost at the input ports, and makes the filling process more complex and less reproducible. It is therefore desirable to find an improved method of sample filling that avoids these disadvantages.

[0068] Another difficulty is that when filling a solution containing particles or cells, there is a possibility that the cells or particles could become attached to the outer surfaces of the array, and not in the inside of the holes. For instance, almost all of a cell-containing sample will eventually go into the holes, but during the distribution process cells or particles could adhere to the outer surface, and stay there even after the partitioning, and after the immiscible partitioning fluid is introduced. For instance, some types of cells have the capability of attaching themselves to surfaces (see Figure 3). In Figure 3, a QuantStudio™ (Thermo Fisher) chip was used, which has a hydrophobic surface outside the through holes and is hydrophilic inside. First, a sample containing fluorescent E. coli cells in the Thermo Fisher recommended Master Mix composition was loaded onto the chip using the recommended method, followed by the QuantStudio’s partitioning fluid. In the left panel of Figure 3, 15,000 cells were loaded in a 15 L reaction volume with the image taken using a fluorescence microscope with 10x zoom. The E. coli cell stuck to the outer surface is marked with a circle. In the right panel of Figure 3, 1500 cells were loaded in a 15 L reaction volume with images taken using a fluorescence microscope 4x zoom to show a wider field of view. The E. coli cells stuck to the outer surface are marked with circles. The presence ofcells outside of the fixed partitions could cause mistaken results in digital assays that rely on the separation of the partitions.

[0069] The present disclosure is based, in part, on the development of improved methods for partitioning an aqueous sample in a capillary hole array that overcomes these disadvantages. The methods include initially filling the chamber where the capillary hole array is located with a non-aqueous immiscible fluid with a lower surface tension and / or contact angle than for aqueous fluids. The ease, speed, and reliability of filling depends on the quantity , where is the surface tension of the non-aqueous fluid / air interface, and is the contact angle of the non-aqueous fluid with the material of the chamber walls. The speed by which channels fill depends on that quantity (see for instance Ichikawa et al., Journal of Colloid and Interface Science, 280 (2004) 155–164). One example of a suitable non-aqueous fluid would be the same fluid that is subsequently used as a separating fluid after the aqueous sample fill. Often this will be a fluorinated fluid such as a perfluorinated hydrocarbon (FC-40 for example), or a hydrofluoroether (HFE 7500 for example). Several other categories of suitable fluids would be mineral oil, liquid hydrocarbons, liquid esters, or silicone oils. In the case of hydrofluoroethers such as HFE 7500, the surface tension is in the order of 16 mN / m, well below the range of 50 to 80 mN / m typical for aqueous solutions. However, the contact angles will also be significantly lower, and that will drive significant and rapid spreading of the fluorinated liquid on surfaces such as silicon or polymers. The contact angle for HFE-7500 on a silicon surface is only 2.5 degrees, for instance (Wu et al.,Ind. Eng. Chem. Res. 2023, 62, 14735 14742), and is also very low on both hydrophilic andhydrophobic polymer resins (Mannel et al. (2021) Advanced Materials Technologies, 6. 10.1002 / admt.202100094). This results in a smooth and reliable initial fill, without leaving air gaps, and which also fills the partitioning holes and the entire space above and below the silicon chip with the partitioning holes. It is found that this filling consistently occurs without leaving any air voids or bubbles, unlike what was observed when the initial filling used an aqueous fluid.

[0070] In certain embodiments, a filling experiment was performed on a QuantStudio silicon chip (10 X 10 mm, such as at least 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000 or 25,000 hexagonal holes or within a range defined by any two of the aforementioned value with a side-to-side dimension of 52 micrometers). The chip was mounted in a fluidic cartridge as shown in Figure 4. In some embodiments, the cartridge is made of a clear thermoplastic polymer and has channels on both the upper and lower surfaces. In someembodiments, the top channels are shown as solid lines, and the bottom channels as dotted lines. In some embodiments, the silicon chip is mounted with adhesive in the square pocket. There is a single input connection to the top surface of the silicon, split into three branches that go to three of the corners. The advantage of a single input or inlet is that it simplifies the design of the microfluidic cartridge, and it reduces the potential dead volume when loading it. In some embodiments, there are two outlets, one at the top and one at the bottom, that are combined to a single outlet waste reservoir and outlet channel. Figure 5 shows the schematic cross-section of this type of cartridge. In some embodiments, an initial fill of this cartridge was performed with a fluorinated oil, namely the QuantStudio Fluid. The oil was driven by a volumetric pump at such as a typical rate of at least 1.0 μl / s, 1.1 μl / s, 1.2 μl / s, 1.3 μl / s, 1.4 μl / s, 1.5 μl / s, 1.6 μl / s, 1.7 μl / s, 1.8 μl / s, 1.9 μl / s or 2.0 μl / s or a typical rate within a range defined by any two of the aforementioned values. As shown in Figure 6A, the oil starts filling the cartridge from the top left corner. Figure 6B shows that subsequently, the fill also starts from the two neighboring corners, until the top of the cartridge is nearly completely filled as seen in Figure 6C. Figure 6D shows that after the top is filled, the oil breaks through to the bottom by going through the holes in the silicon. As shown in Figures 6E and 6F, the oil continues to fill the bottom. As shown in Figure 6G, the oil continues to fill until the cartridge is completely filled and there is no air present.

[0071] After this initial fill with a fluorinated oil, an aqueous sample was introduced without any air gap between the fluids. This can be done using a variety of methods, either using a microfluidic chamber with a defined volume, or with a sample injection valve such as an Idex MXT 715-000. The volume of this aqueous sample is carefully chosen to be close to that of the total volume of all the partitions. In some embodiments, the same volumetric pump as was used for the initial oil fill may be used for this step, but at a lower flow rate, at least 0.1 μl / s, 0.2 μl / s, 0.3 μl / s, 0.4 μl / s, 0.5 μl / s, 0.6 μl / s, 0.7 μl / s, 0.8 μl / s, 0.9 μl / s, or 1.0 μl / s or a rate within a range defined by any two of the aforementioned value. The aqueous sample flows into the chamber, displaces a portion of the initial non-aqueous filling fluid, and comes into contact with the holes that define the partitions. When an aqueous solution is in contact with the opening of a partition, it will spontaneously displace the non-aqueous fluid that was there initially due to the hydrophilicity of the inner surface of the openings, which causes a capillary filling force as described earlier.

[0072] In some embodiments, the displacement phenomenon is observed both in enclosed microfluidic chambers and in manual experiments in an open chamber. In some embodiments, the non-aqueous fluid is applied on the silicon chip with the hole array in an open chamber, and the fluid fills the openings and the space below them. As shown in Figures 7A-7D, an oil droplet is placed on the top of the silicon chip with the holes using a pipette tip. The non- aqueous fluid droplet remains on the surface for less than 1 second and then is spontaneously transferred to the bottom of the silicon chip via the through holes. When enough volume of non- aqueous fluid is applied, the silicon chip becomes completely covered with the fluid both on top and on the bottom. When aqueous samples are subsequently applied to the surface by piercing the top non-aqueous fluid layer with a fine pipette tip, they enter the array and displace the non- aqueous fluid that was there initially. Figures 8A-8D show that the sample is filled into the holes by piercing the top non-aqueous fluid layer using a fine pipette tip. Figure 8A shows that a sample droplet is placed above the non-aqueous fluid. In Figure 8C the sample doesn’t get partitioned. Figure 8B shows that when the aqueous sample is placed below the non-aqueous fluid using a fine pipette tip, the sample is then spontaneously partitioned by filling the holes and displacing the fluid that was there previously, as seen in Figure 8D.

[0073] In another embodiment, a fluorescent aqueous solution is used in an enclosed chamber, such as ROX, to allow filled wells to be detected. In some embodiments, the chamber and the silicon chip are initially completely filled with a fluorinated oil such as QuantStudio™ oil or FC-40. Figure 6 shows this initial oil fill. A solution of 5:1 diluted ROX in pHusion™ buffer was used as a sample in the experiment. The sample was loaded into a QuantStudio™ silicon chip, mounted into a chamber with a single sample inlet split into three channels that lead to three of the corners of the silicon chip as shown in Figure 4. The QuantStudio™ silicon chip has upper and lower surfaces that are hydrophobic, as opposed to the insides of the holes that are hydrophilic. The aqueous sample is followed by more QuantStudio™ oil to spread the aqueous sample entirely into the holes. As shown in Figures 9A-9E, 96% of the wells are filled with the fluorescent sample solution. Figures 9A and 9B shows that the fluorescent aqueous solution spreads from the bottom left corner, and fills the wells below as it moves along. Figure 9C shows the trailing immiscible oil pushes the sample ahead. In Figure 9D, almost all the wells have been filled and there is no more aqueous solution above the wells. Figure 9E shows the image from the bottom after the fill is complete. Figures 10A and 10B shows the histogram of the intensity distribution of the wellsand the total number of filled wells for the top and bottom views, respectively. It is seen that more filled wells can be counted from the bottom since that is a view unobstructed by the four fluidic vias in the corners seen in the top view.

[0074] In another embodiment, a fill was performed on a custom designed silicon device with similar size holes and hydrophilic surfaces. The silicon device had dimensions of 10 by 10 mm and was 300 micrometers thick. In some embodiments, the device is fabricated by oxidizing the surface, photolithographically patterning the oxide, and then plasma etching all the way through the wafer. The die has at least 21,350, 21,355, 21,360, 21,365, 21,370, 21,375, 21,380, 21,385, or 21,390 hexagonal holes or within a range defined by any two of the aforementioned value, with 52 micrometers side-to-side, and separated by 20 micrometer walls. After the etching, the photoresist and the oxide were stripped, leading to a surface of bare silicon covered with a native oxide. The surface is hydrophilic, both inside the wells and on the outer flat surfaces. The chamber was first filled with immiscible oil. The results of filling the chip with a fluorescent aqueous solution are shown in Figures 11A-11E and Figure 12. A good fill was possible in the absence of any hydrophobic treatments on the outer surfaces. As a result, 92% of the wells are filled with the sample solution.

[0075] In another embodiment, a QuantStudio (™) silicon chip was mounted in a cartridge with a single input channel that is not split, and leads directly to one of the corners of the silicon chip. The cartridge design used was described herein as Figure 13. The method of filling the cartridge is as described herein. The results of filling the chip with a single input channel are shown in Figures 14A-14E and Figure 15. 97% of the wells are filled indicating a good fill using a single input channel.

[0076] In some embodiments, the methods of filling the silicon chip includes filling with pure FC-40 as the immiscible fluid. FC-40 is a fully fluorinated fluorocarbon oil without any additives or surfactants. The cartridge design used was described herein as Figure 4. The results of filling the chip with FC-40 are shown in Figures 16A-16D and Figure 17. A 96% fill was obtained, indicating that the presence of additives or surfactants in the fluorinated oil is not required for a good fill.

[0077] In some embodiments, the methods described herein include sample solutions with high concentrations of particles or cells (about a billion cells per ml) and using two different fluorescence wavelengths, one for the aqueous solution, and a different one for the particles. Insome embodiments, the location of all particles were confirmed to be in the aqueous partitions after loading using the two different wavelengths. An image in Figure 18 shows a custom silicon array chip loaded using methods described herein with a fluorescent ROX solution containing cells that were fluorescent at green wavelengths. The QuantStudio™ oil was used for the initial fill, and for the oil that fills the chamber as a partitioning medium after the sample. The top left side panel of Figure 18 shows a large field of view displaying the entire chip with cells visible as bright dots. The top right side panel shows an enlarged view of the chip. In some embodiments, all of the cells are present in the aqueous partitions, instead of the wall region between the partitions. In some embodiments, the chip is manually analyzed by zooming into different sections of the chip. Although the concentration of the cells were 1000 times larger than in methods describes herein (as shown in Figure 3), no cells were observed to be outside the partitions as seen in the bottom left panel. The absence of cells outside the partitions confirms that by initially filling the silicon with fluorinated oil before the sample, it is possible to partition a sample containing particles or cells or microorganisms while ensuring that none of the particles, cells, or microorganisms are retained in a location outside the aqueous partitions, even at very high concentrations in the range of a billion cells per ml. The custom silicon used in the method is hydrophilic in nature both on the inside and outside the partitions.

[0078] In some embodiments, the methods described herein are repeated with QuantStudio™ silicon chip having a hydrophobic surface outside the partitions and hydrophilic inside. Similar results were obtained as shown in the bottom right panel in Figure 18 indicating that using the oil before filling the sample ensures that particles are located inside the silicon partitions, regardless of the surface treatment to adjust the hydrophobicity of the silicon surface.

[0079] Some embodiments provided herein relate to the following enumerated alternatives.

[0080] 1. A microfluidic cartridge for partitioning a sample in a fixed array, comprising: a hole array for capillary filling, wherein each of a plurality of the holes in the hole array comprises an open-ended first end and an open-ended second end; an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened- ended first end and a second volume below the open-ended second end, wherein the first volume and the second volume are fluidly connected by the holes of the array and wherein the second volume is capable of being filled only through the holes of the array; an input channel to theenclosed chamber fluidically connected with the first volume; and at least one output channel fluidically connected with the first volume and the second volume.

[0081] 2. The microfluidic cartridge of alternative 1, wherein the hole array comprises at least 1000 holes.

[0082] 3. The microfluidic cartridge of any one of alternatives 1-2, wherein the input channel is configured to flow fluid into the first volume and wherein the second volume is configured to receive fluid from the holes in the array.

[0083] 4. The microfluidic cartridge of any one of alternatives 1-3, wherein the side- to-side width of each hole is between 1 and 300 microns.

[0084] 5. The microfluidic cartridge of any one of alternatives 1-4, wherein the shape of each hole is a regular polygon.

[0085] 6. The microfluidic cartridge of any one of alternatives 1-5, wherein a total surface area of the holes is at least 25% of the total surface area of the hole array.

[0086] 7. The microfluidic cartridge of alternative 6, wherein the total surface area of the holes is about 50% of the total surface area of the hole array.

[0087] 8. The microfluidic cartridge of any one of alternatives 1-7, wherein an inner surface of each hole is hydrophilic.

[0088] 9. The microfluidic cartridge of any one of alternatives 1-8, wherein each hole is configured to retain an aqueous sample by capillary forces.

[0089] 10. The microfluidic cartridge of alternative 9, wherein an inner surface of each hole is sufficiently hydrophilic that the holes will fill by capillarity when exposed to the aqueous sample.

[0090] 11. The microfluidic cartridge of any one of alternatives 1-10, wherein the hole array comprises at least 5,000 holes.

[0091] 12. The microfluidic cartridge of any one of alternatives 1-11, wherein the hole array comprises at least 10,000 holes.

[0092] 13. The microfluidic cartridge of any one of alternatives 1-12, wherein a surface of the hole array in contact with the first volume comprises silicon or a polymer.

[0093] 14. The microfluidic cartridge of any one of alternatives 1-13, wherein the input channel is not connected with the second volume.

[0094] 15. A kit comprising a microfluidic cartridge of any one of alternatives 1-14 and a non-aqueous immiscible fluid.

[0095] 16. The kit of alternative 15, wherein the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than an aqueous fluid.

[0096] 17. The kit of any one of alternatives 15-16, wherein the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil.

[0097] 18. The kit of any one of alternatives 15-17, wherein the non-aqueous immiscible fluid is FC-40 or HFE 7500.

[0098] 19. The kit of any one of alternatives 15-18, wherein the non-aqueous immiscible fluid is contained within the microfluidic cartridge.

[0099] 20. The kit of alternative 19, wherein each hole of the hole array, the first volume, and the second volume are filled with the non-aqueous immiscible fluid.

[0100] 21. A method for partitioning a sample into a hole assay, comprising: providing the microfluidic cartridge of any one of alternatives 1-14 or the kit of any one of alternatives 15- 20; introducing the enclosed chamber and the hole array with an immiscible non-aqueous fluid; introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes.

[0101] 22. A method for partitioning a sample into a hole array, comprising: providing a microfluidic device, wherein the device comprises: a hole array for capillary filling, wherein each hole comprises an open-ended first end and an open-ended second end; and an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened-ended first end and a second volume below the open-ended second end; filling the chamber and the hole array with an immiscible non-aqueous fluid; and introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes.

[0102] 23. The method of alternative 22, wherein the hole array comprises at least 1000 holes.

[0103] 24. The method of any one of alternatives 21-23, further comprising introducing a second non-aqueous fluid into the chamber to fill the first volume and the second volume without displacing the aqueous sample in the holes.

[0104] 25. The method of any one of alternatives 21-24, wherein the aqueous sample comprises particles, beads, cells, microorganisms, bacterial cells, eukaryotic cells, or fungal cells.

[0105] 26. The method of any one of alternatives 21-25, wherein the aqueous sample has a volume that is less than the total volume of all of the holes of the array.

[0106] 27. The method of any one of alternatives 21-26, wherein the microfluidic cartridge comprises a single input channel is configured to flow fluid into the first volume and wherein the bottom volume is configured to receive fluid from the holes in the array.

[0107] 28. The method of alternatives 26, wherein the second volume is configured to receive fluid only from the holes in the array.

[0108] 29. The method of any one of alternatives 21-28, wherein an inner surface of the holes is hydrophilic and wherein the holes fill by capillarity when exposed to the aqueous sample.

[0109] 30. The method of any one of alternatives 21-29, wherein the array comprises at least 5,000 holes.

[0110] 31. The method of any one of alternatives 21-30, wherein the array comprises at least 10,000 holes.

[0111] 32. The method of any one of alternatives 21-31, wherein the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than the aqueous fluid.

[0112] 33. The method of any one of alternatives 21-32, wherein the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil.

[0113] 34. The method of any one of alternatives 21-33, wherein the non-aqueous immiscible fluid is FC-40 or HFE 7500.

[0114] 35. The method of any one of alternatives 21-34, wherein the immiscible non- aqueous fluid comprises a surfactant.

[0115] 36. The method of any one of alternatives 21-35, further comprising performing a digital assay after introducing the aqueous sample.

[0116] 37. The method of any one of alternatives 21-35, further comprising performing an assay to detect the presence of, identify, or quantify one or more nucleic acids comprised in the aqueous sample.

[0117] 38. The method of alternative 36 or 37, wherein the digital assay or assay comprises PCR amplification. EXAMPLES

[0118] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the disclosure, as it is described herein above and in the claims. EXAMPLE 1

[0119] The following example demonstrates filling a through-hole array with non- aqueous liquid followed by sample introduction in an enclosed microfluidic chamber.

[0120] A filling experiment was performed on a 10 X 10 mm QuantStudio 3D silicon chip (ThermoFisher) with approximately 20,000 hexagonal holes with a side-to-side dimension of 52 micrometers. This chip was mounted in a fluidic cartridge as shown in Figure 4. The cartridge is made of a clear thermoplastic polymer, and has channels on both the upper and lower surfaces. There is a single input connection to the top surface of the silicon, split into three branches that go to three of the corners.

[0121] An initial fill of this cartridge was performed with a fluorinated oil (QuantStudio™ 3D Digital PCR Immersion Fluid, from ThermoFisher). The oil was driven by a volumetric pump at a typical rate of 1.6 μl / s. The results are shown in Figures 6A-6G. As shown in Figure 6A, the oil starts filling the cartridge from the top left corner. Subsequently, the fill also starts from the two neighboring corners (Figure 6B), until the top of the cartridge is nearly completely filled (Figure 6C). After the top is filled, the oil breaks through to the bottom by going through the holes in the silicon (shown in Figure 6D) and continues to fill the bottom (Figures 6E and 6F) until the cartridge is completely filled and there is no more air present (Figure 6G).

[0122] After the initial fill with a fluorinated oil, an aqueous sample consisting of Phusion® High-Fidelity DNA Polymerase Buffer (ThermoFisher) at 1X concentration, and Biotium ROX passive Reference Dye at a final concentration of 5X (10:1 dilution of the 50X stock solution) was introduced without any air gap between the fluids. The same volumetric pump as was used for the initial oil fill was used for this step, but at a lower flow rate, in the range of 0.3 to 0.8 μl / s. In this step, the aqueous sample flowed into the chamber, displaced a portion of the initialnon-aqueous filling fluid, and came into contact with the holes that define the partitions. Introduction of the aqueous sample was also performed using a microfluidic chamber with a defined volume, or with a sample injection valve such as an Idex MXT 715-000. The volume of this aqueous sample was designed such that it was close to that of the total volume of all the partitions in the array. Upon introducing the aqueous sample, it was observed that when the aqueous sample came into contact with the opening of a partition on the array, it spontaneously displaced the non-aqueous fluid that was there initially. This is due to the hydrophilicity of the inner surface of the openings, which causes a capillary filling force. EXAMPLE 2

[0123] The following example demonstrates filling a through-hole array with non- aqueous liquid followed by sample introduction in an open chamber.

[0124] A similar assay to Example 1 was performed in an open chamber containing a silicon chip with the hole array. The non-aqueous fluid (a droplet of QuantStudio™ 3D Digital PCR Immersion Fluid) was applied on top of the silicon chip using a pipette tip. Figures 7A-7D shows screen grabs taken from a video showing that the fluid filled the openings and the space below them. The non-aqueous fluid droplet remains on the surface for less than 1 second and then was spontaneously transferred to the bottom of the silicon chip via the through holes. Upon application of the full volume of the non-aqueous fluid, the silicon chip became completely covered with the fluid both on top as well as on the bottom.

[0125] An aqueous sample (Phusion® High-Fidelity DNA Polymerase Buffer (ThermoFisher) at 1X concentration, and Biotium ROX passive Reference Dye at a final concentration of 5X) was applied to the surface by piercing the top non-aqueous fluid layer with a fine pipette tip. Similar to the displacement seen in Example 3, the sample entered the holes in the array and displaced the non-aqueous fluid that was there initially. Figures 8A-8D show the screen grabs from a video of the sample being filled into the holes by piercing the top non-aqueous fluid layer using a fine pipette tip, comparing the placement of the sample above and below the non- aqueous fluid. A sample droplet was placed above the non-aqueous fluid as shown in Figure 8A and the sample did not partition (Figure 8C). In contrast, as shown in Figure 8B, the sample was placed below the non-aqueous fluid using a fine pipette tip; the sample then spontaneously partitioned, filling the holes and displacing the fluid that was there previously (Figure 8D).EXAMPLE 3

[0126] The following example demonstrates filling a through-hole array with a single inlet split into 3 channels.

[0127] Filling experiments in an enclosed chamber typically used a fluorescent aqueous solution, such as ROX, to allow filled wells to be detected. In this Example 3 and all of the following examples, the chamber and the silicon chip itself were initially completely filled with a fluorinated oil such as QuantStudio™ 3D Digital PCR Immersion Fluid or FC-40. These initial oil fills were all similar to the one shown in Figure 6.

[0128] The sample in this experiment was a the same as in Example 1 (Phusion® High- Fidelity DNA Polymerase Buffer (ThermoFisher) at 1X concentration, and Biotium ROX passive Reference Dye at a final concentration of 5X). This sample was loaded into a QuantStudio™ silicon chip, mounted into a chamber with a single sample inlet split into three channels that lead to three of the corners of the silicon chip (design shown in Figure 4). The QuantStudio™ silicon chip has upper and lower surfaces that are hydrophobic and the insides of the holes are hydrophilic. The aqueous sample was applied as described in Example 1. This was followed by more QuantStudio™ oil (QuantStudio™ 3D Digital PCR Immersion Fluid) to spread the aqueous sample entirely into the holes. The process resulted in 96% of the wells being filled with the fluorescent sample solution as shown in Figures 9A-9E. As shown in Figures 9A and 9B, the fluorescent aqueous solution spread from the bottom left corner, and filled the wells below as it moved along the array. As shown in Figure 9C, the trailing immiscible oil pushed the sample ahead of it until almost all the wells were filled and there was no more aqueous solution above the wells (as shown in Figure 9D). Figure 9E shows the image from the bottom after the fill was completed. A histogram of the intensity distribution of the wells and the total number of filled wells is shown in Figure 10A (top view) and Figure 10B (bottom view). More filled wells can be counted from the bottom since that is a view unobstructed by the four fluidic vias in the corners seen in the top view. EXAMPLE 4

[0129] The following example demonstrates filling a through-hole array with hydrophilic surfaces.

[0130] A similar fill to Example 3 was performed on a custom-designed silicon device with holes of a similar size to the QuantStudio chip but having hydrophilic surfaces. This silicondevice had dimensions of 10 by 10 mm and was 300 micrometers thick. It was fabricated by oxidizing the surface, photolithographically patterning the oxide, and then plasma etching all the way through the wafer. Each die has 21,383 hexagonal holes 52 micrometers side-to-side, separated by 20 micrometer walls. After the etching, the photoresist and the oxide were stripped, leading to a surface of bare silicon covered with a native oxide, providing a hydrophilic surface both inside the wells and on the outer flat surfaces.

[0131] The chamber was first filled with QuantStudio™ 3D Digital PCR Immersion Fluid, followed by introduction of a fluorescent aqueous solution (same sample as in Example 1) as described in Example 3. The results of filling this chip are shown in Figures 11 and 12. Surprisingly, a good fill was achieved in the absence of any hydrophobic treatments on the outer surfaces of the holes. A 92% filling of the wells with the sample solution was achieved. EXAMPLE 5

[0132] The following example demonstrates filling a through-hole array with a single inlet split without a split into additional channels.

[0133] A QuantStudio™ silicon chip was mounted in a cartridge with a single input channel that leads directly to one of the corners of the silicon chip and does not split into further channels. This cartridge design is shown in Figure 13. The filling experiment described in Example 3 was repeated with this device. The results are shown in Figures 14A-14E and Figure 15. A 97% filling of the wells with sample was observed with this chip. EXAMPLE 6

[0134] The following example demonstrates filling a through-hole array with a fully fluorinated fluorocarbon oil without any additives or surfactants.

[0135] A similar filling experiment to Example 3 was performed using pure FC-40 as the immiscible non-aqueous fluid. FC-40 (available from 3M Corp.) is a fully fluorinated fluorocarbon oil without any additives or surfactants. The cartridge design used is shown in Figure 4. A 96% fill of sample was obtained, indicating that the presence of additives or surfactants in the fluorinated oil is not required for a good fill of the sample.EXAMPLE 7

[0136] The following example demonstrates filling a through-hole array with a concentrated sample.

[0137] Filling of the hole array was performed with sample solutions that contained very high concentration particles or cells (approximately one billion cells per ml). Two different fluorescence wavelengths, one for the aqueous solution, and a different one for the particles were employed for this analysis to image the location of the cells or particles after loading, and to confirm that all particles were located in the aqueous partitions. Figure 18 shows an image of the custom silicon array chip (the same type as described in Example 4) used for this analysis loaded by a fluorescent ROX solution described in Examples 1 and 3 that also contained cells that were fluorescent at green wavelengths. These cells were E. Coli cells modified to express GFP (green fluorescent protein). QuantStudio™ oil was used for the initial fill, and for the oil that fills the chamber as a partitioning medium after the sample was loaded. Figure 18, top left shows a large field of view displaying the entire chip with the cells visible as bright dots. Figure 18, top right shows a 4x zoomed in view of the chip. It can be seen that all the cells are present in the aqueous partitions, and not on the wall region between the partitions. The chip was manually analyzed by zooming into different sections of the chip and no cells were observed to be outside the partitions (Figure 18, bottom left). The initial filling experiment was done with the custom chip that has hydrophilic surfaces both inside and outside the partitions. The fill experiment was repeated with the QuantStudio™ silicon chip, which has a hydrophobic surface outside the partitions and hydrophilic inside. As shown in Figure 18 (bottom right), no cells were observed to be outside the partitions.

[0138] These observations were in contrast to a filling experiment performed without using the immiscible non-aqueous fluid (oil) loaded prior to the sample. Using the QuantStudio™ silicon chip, a sample containing fluorescent E. coli cells in the ThermoFisher recommended Master Mix composition was loaded onto the chip using the recommended method, followed by the QuantStudio’s partitioning fluid. The chip was loaded with 15000 cells in a 15 L reaction volume and images were taken using a fluorescence microscope with a 10x zoom. Figure 3 shows that some E. coli cells stuck to the outer surface; one such cell is marked with a circle. The filling experiment was repeated with a lower concentration of sample, 1500 cells in a 15 L reaction volume. Images were taken using a fluorescence microscope with a 4x zoom to show a wider fieldof view. As shown in Figure 3, the E. coli cells stuck to the outer surfaces; these cells are marked with circles. The presence of cells outside of the fixed partitions could cause mistaken results such as in digital assays that rely on the separation of the partitions.

[0139] The results indicate that by initially filling the array with immiscible non- aqueous fluid before the sample, allows partitioning of the sample containing particles or cells or microorganisms while ensuring that none of the particles, cells, or microorganisms are retained in a location outside the partitions. Moreover, the use of the immiscible non-aqueous fluid prior to sample loading provides this advantage even at very high concentrations of sample.

[0140] With respect to the use of plural and / or singular terms herein, those having skill in the art can translate from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity.

[0141] It will be understood by those of skill within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles "a" or "an" limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least oneof A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0142] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0143] Any of the features of an embodiment of the first through second aspects is applicable to all aspects and embodiments identified herein. Moreover, any of the features of an embodiment of the first through third aspects is independently combinable, partly or wholly with other embodiments described herein in any way, e.g., one, two, or three or more embodiments may be combinable in whole or in part. Further, any of the features of an embodiment of the first through third aspects may be made optional to other aspects or embodiments.

[0144] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises,” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0145] By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

Claims

WHAT IS CLAIMED IS:

1. A microfluidic cartridge for partitioning a sample in a fixed array, comprising: a hole array for capillary filling, wherein each of a plurality of the holes in the hole array comprises an open-ended first end and an open-ended second end; an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened-ended first end and a second volume below the open-ended second end, wherein the first volume and the second volume are fluidly connected by the holes of the array and wherein the second volume is capable of being filled only through the holes of the array; an input channel to the enclosed chamber fluidically connected with the first volume; and at least one output channel fluidically connected with the first volume and the second volume.

2. The microfluidic cartridge of claim 1, wherein the hole array comprises at least 1000 holes.

3. The microfluidic cartridge of claim 1, wherein the input channel is configured to flow fluid into the first volume and wherein the second volume is configured to receive fluid from the holes in the array.

4. The microfluidic cartridge of claim 1, wherein the side-to-side width of each hole is between 1 and 300 microns.

5. The microfluidic cartridge of claim 1, wherein the shape of each hole is a regular polygon.

6. The microfluidic cartridge of claim 1, wherein a total surface area of the holes is at least 25% of the total surface area of the hole array.

7. The microfluidic cartridge of claim 6, wherein the total surface area of the holes is about 50% of the total surface area of the hole array.

8. The microfluidic cartridge of claim 1, wherein an inner surface of each hole is hydrophilic.

9. The microfluidic cartridge of claim 1, wherein each hole is configured to retain an aqueous sample by capillary forces.

10. The microfluidic cartridge of claim 9, wherein an inner surface of each hole is sufficiently hydrophilic that the holes will fill by capillarity when exposed to the aqueous sample.

11. The microfluidic cartridge of claim 1, wherein the hole array comprises at least 5,000 holes.

12. The microfluidic cartridge of claim 1, wherein the hole array comprises at least 10,000 holes.

13. The microfluidic cartridge of claim 1, wherein a surface of the hole array in contact with the first volume comprises silicon or a polymer.

14. The microfluidic cartridge of claim 1, wherein the input channel is not connected with the second volume.

15. A kit comprising a microfluidic cartridge of claim 1 and a non-aqueous immiscible fluid.

16. The kit of claim 15, wherein the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than an aqueous fluid.

17. The kit of any one of claim 15, wherein the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil.

18. The kit of claim 15, wherein the non-aqueous immiscible fluid is FC-40 or HFE 7500.

19. The kit of claim 15, wherein the non-aqueous immiscible fluid is contained within the microfluidic cartridge.

20. The kit of claim 19, wherein each hole of the hole array, the first volume, and the second volume are filled with the non-aqueous immiscible fluid.

21. A method for partitioning a sample into a hole assay, comprising: providing the microfluidic cartridge of claim 1; introducing the enclosed chamber and the hole array with an immiscible non- aqueous fluid; introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes.

22. A method for partitioning a sample into a hole array, comprising: providing a microfluidic device, wherein the device comprises:a hole array for capillary filling, wherein each hole comprises an open- ended first end and an open-ended second end; and an enclosed chamber in which the hole array is mounted in a configuration to provide a first volume above the opened-ended first end and a second volume below the open-ended second end; filling the chamber and the hole array with an immiscible non-aqueous fluid; and introducing an aqueous sample in the holes of the array, whereby the aqueous sample is partitioned by spontaneously displacing the non-aqueous fluid in the holes.

23. The method of claim 22, wherein the hole array comprises at least 1000 holes.

24. The method of claim 22, further comprising introducing a second non-aqueous fluid into the chamber to fill the first volume and the second volume without displacing the aqueous sample in the holes.

25. The method of claim 22, wherein the aqueous sample comprises particles, beads, cells, microorganisms, bacterial cells, eukaryotic cells, or fungal cells.

26. The method of claim 22, wherein the aqueous sample has a volume that is less than the total volume of all of the holes of the array.

27. The method of claim 22, wherein the microfluidic cartridge comprises a single input channel is configured to flow fluid into the first volume and wherein the second volume is configured to receive fluid from the holes in the array.

28. The method of claim 26, wherein the second volume is configured to receive fluid only from the holes in the array.

29. The method of claim 22, wherein an inner surface of the holes is hydrophilic and wherein the holes fill by capillarity when exposed to the aqueous sample.

30. The method of claim 22, wherein the array comprises at least 5,000 holes.

31. The method of claim 22, wherein the array comprises at least 10,000 holes.

32. The method of claim 22, wherein the non-aqueous immiscible fluid has a lower surface tension and / or contact angle than the aqueous fluid.

33. The method of claim 22, wherein the non-aqueous immiscible fluid is a fluorinated fluid, a perfluorinated hydrocarbon, a hydrofluoroether, mineral oil, a liquid hydrocarbon, a liquid ester, or a silicone oil.

34. The method of claim 22, wherein the non-aqueous immiscible fluid is FC-40 or HFE 7500.

35. The method of claim 22, wherein the immiscible non-aqueous fluid comprises a surfactant.

36. The method of claim 22, further comprising performing a digital assay after introducing the aqueous sample.

37. The method of claim 22, further comprising performing an assay to detect the presence of, identify, or quantify one or more nucleic acids comprised in the aqueous sample.

38. The method of claim 36, wherein the digital assay or assay comprises PCR amplification.

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