Microfluidic devices and methods for processing biological samples - Patents.com

Abstract

A microfluidic device for processing a sample is disclosed, comprising an inlet port fluidly coupled to a closed system. The closed system comprises a plurality of linear loading conduits, a plurality of termination chambers, and a plurality of microchambers for receiving the sample. Also disclosed is a method for drawing a sample volume into a microchamber of the microfluidic device. One method includes applying a plurality of pressure pulses to contents within a microfluidic assembly, the plurality of pressure pulses including a first pressure applied for a first time interval and a second pressure applied for a second time interval, whereby a volume of the sample is drawn into the plurality of microchambers of the microfluidic device. These and other embodiments of microfluidic devices and methods are disclosed.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of U.S. Provisional Application Nos. 63 / 244,234 and 63 / 244,235, both filed on September 14, 2021. To the extent permitted by the applicable jurisdiction, the entire contents of these applications are incorporated herein by reference. [Background technology]

[0002] Microfluidic devices handle fluids on a small scale: typically, they operate on the sub-millimeter scale and handle microliters, nanoliters, or even smaller amounts of fluid.

[0003] One application of microfluidic structures is digital polymerase chain reaction (dPCR). dPCR involves diluting a nucleic acid sample into no more than one nucleic acid template in each chamber of a microfluidic structure that provides an array of multiple chambers, and running a PCR reaction across the entire array. The target nucleic acid is quantified by counting the chambers that successfully PCR-amplify the template and applying Poisson statistics to the results. Unlike the popular quantitative real-time PCR (qPCR), which quantifies the template by comparing the PCR amplification rate of an unknown sample to that of a set of known qPCR standards, dPCR has been proven to exhibit higher sensitivity, higher accuracy, and higher reproducibility.

[0004] For genomic researchers and clinicians, dPCR is particularly powerful in detecting rare variants, quantifying copy number variations, and quantifying next-generation sequencing libraries. Potential use in clinical settings for liquid biopsies with cell-free DNA and viral load quantification further enhances the value of dPCR technology. Existing dPCR solutions have used elastomeric valve arrays, silicon through-hole approaches, and microfluidic encapsulation of droplets. Despite the growing number of available dPCR platforms, dPCR has been at a disadvantage when compared to older qPCR techniques that rely on counting the number of PCR amplification cycles. A combination of throughput, ease of use, performance, and cost are the main barriers to dPCR's greater market adoption. Summary of the Invention

[0005] In microfluidic devices, the main fouling mechanism is air or air bubbles trapped inside the microstructure. This can be particularly problematic when thermoplastic materials are used to create microfluidic structures, as thermoplastics have very low gas permeability. To avoid fouling from trapped air, microfluidic structures either use simple straight or branched conduit designs with thermoplastic materials, or use highly gas permeable materials such as elastomers to fabricate the devices. However, simple designs limit the possible functionality of the microfluidic device, and elastomeric materials are difficult and expensive to manufacture, especially on a large scale.

[0006] Therefore, cost-effective designs that can be used in manufacturing microfluidic devices that can be effectively used for dPCR, such as microfluidic devices and structures that use thermoplastic materials, are desirable. In addition, because thermoplastic materials have low gas permeability, improved methods for reducing fouling in thermoplastic microfluidic devices by removing trapped air prior to performing the dPCR process on a sample are desirable.

[0007] The present disclosure describes microfluidic devices for sample processing and / or analysis. The microfluidic devices of the present disclosure may be formed from polymeric materials, such as thermoplastic materials, and may include a gas-permeable film that allows for pressurized outgassing (degassing) while functioning as a gas barrier when pressure is released. The use of thermoplastic materials to form the microfluidic devices allows for the use of inexpensive and highly scalable injection molding processes. The gas-permeable film provides the ability to outgas via pressurization and may avoid fouling issues that may be present in some microfluidic structures that do not include a gas-permeable film.

[0008] An embodiment of the invention disclosed herein includes a microfluidic device for processing a sample, the microfluidic device comprising an inlet port fluidly coupled to a closed system. The closed system comprises, or consists essentially of, or consists of a plurality of linear loading conduits, a plurality of end chambers, and a plurality of microchambers for receiving a sample. An end chamber of the plurality of end chambers is fluidly coupled to a linear loading conduit of the plurality of linear loading conduits. A plurality of microchambers of the plurality of microchambers are fluidly coupled to a linear loading conduit of the plurality of linear loading conduits. For a first plurality of microchambers and a first end chamber fluidly coupled to the same adjacent linear loading conduit, the volume of the first end chamber is equal to or less than the total volume of the first plurality of microchambers. The closed system is not provided with a waste reservoir larger than the end chamber. The inlet, the loading conduit, the microchambers, and the end chamber may all be in fluid communication with each other, and may optionally not be in fluid communication with another conduit or chamber.

[0009] Some embodiments of the present invention disclose a microfluidic device configured to process a sample, comprising an inlet port and a plurality of dead-end microfluidic assemblies, each of which is fluidically coupled to the inlet port. Each of the dead-end microfluidic assemblies comprises a linear loading conduit, a terminal chamber fluidically coupled to the linear loading conduit, and a plurality of microchambers for receiving a sample fluidically coupled to and proximate the linear loading conduit, the volume of the terminal chamber being equal to or less than the total volume of the plurality of microchambers. In some embodiments, at least one (e.g., all) of the microchambers are dead-ends. The dead-end microchambers optionally do not allow for sample fluid or other liquid exchange outside of the loading conduit, for example, via a siphon conduit fluidically connecting the microchamber to the loading conduit.

[0010] An embodiment of the invention disclosed herein also includes a microfluidic device for processing a sample, the device including an inlet port fluidly coupled to an array of loading conduits and sample-receiving microchambers via a first microconduit portion, and a wide conduit portion fluidly coupled between the inlet port and the first microconduit portion. The first microconduit portion has a depth less than a depth of the wide conduit portion. The first microconduit portion includes a linear path. The wide conduit portion includes a path including at least one of a linear path or a non-linear path oriented differently than the linear path of the first microconduit portion.

[0011] Another embodiment of the invention disclosed herein includes a microfluidic device for processing a biological sample, the microfluidic device comprising an inlet port and a plurality of loading conduits, each fluidly coupled to the inlet port and a plurality of microchambers. This embodiment includes a plurality of siphon conduits, each siphon conduit fluidly coupling the loading conduit to a microchamber. The microchamber includes a first side that substantially faces the loading conduit and a second side that in some embodiments does not substantially face the loading conduit, e.g., is not parallel to the loading conduit. In some embodiments, the second side does not face an adjacent microchamber, and the siphon conduit fluidly couples the loading conduit to the microchamber through the second side. In some embodiments, the siphon conduit has a non-linear (e.g., curved) shape to connect the loading conduit to the second side of the microchamber. Optionally, the orifice of the siphon conduit in the loading conduit is located upstream of the portion of the loading conduit closest to the microchamber.

[0012] Some embodiments of the invention discussed herein disclose a microfluidic device for processing a biological sample, the microfluidic device comprising an inlet port and a plurality of loading conduits, each loading conduit being fluidly coupled to the inlet port and a plurality of microchambers, one microchamber of the plurality of microchambers comprising a substantially rectangular three-dimensional shape including four substantially rectangular sidewalls, two adjacent sidewalls being joined by a curved corner.

[0013] A method of processing a biological sample in a microfluidic device comprising a plurality of microfluidic assemblies is disclosed. Each microfluidic assembly is configured to process a sample using a digital PCR process. Each of the microfluidic assemblies comprises an inlet, a loading conduit fluidly coupled to the inlet at a first end, one or more dead-end microchambers configured to receive a sample, and a siphon conduit fluidly coupled to the microchambers and the loading conduit. The method includes applying a plurality of pressure pulses to contents within the microfluidic assemblies. In one embodiment, applying the plurality of pressure pulses includes alternating between applying a first pressure for a first time interval and applying a second pressure for a second time interval, whereby a volume of the sample is drawn into the plurality of microchambers of the microfluidic device.

[0014] Methods of loading a biological sample into a microfluidic device for processing the biological sample are also disclosed. In some embodiments, the microfluidic device comprises an inlet, one or more loading conduits fluidly coupled to the inlet at a first end, a plurality of microchambers for receiving and / or digitizing the biological sample, and a plurality of siphon conduits fluidly coupling the plurality of microchambers to the one or more loading conduits. The microfluidic device further comprises a gas-permeable film forming a surface of the one or more loading conduits, the microchambers, and the siphon conduits.

[0015] In embodiments of these methods, a plurality of pressure pulses are applied to the fluid contents of the microfluidic device. The plurality of pressure pulses includes alternating peaks having a first pressure applied for a first time interval and valleys including a second pressure applied for a second time interval. After the series of pressure pulses are applied, air bubbles present in the plurality of microchambers are forced through the gas permeable film and a volume of reagent including the biological sample is drawn into the microchambers of the microfluidic device.

[0016] Additional 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 illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details can be modified in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. [Brief description of the drawings]

[0017] [Figure 1] 1 illustrates a microfluidic device according to one embodiment of the present invention. [Figure 2-1] 1 illustrates a microfluidic device processing unit according to one embodiment of the present invention. [Figure 2-2] 1 illustrates a microfluidic device processing unit according to one embodiment of the present invention. [Diagram 3] FIG. 2 shows a bottom perspective view of a portion of a microfluidic device processing unit according to one embodiment of the present invention. [Figure 4] FIG. 2 shows a bottom view of a portion of a microfluidic device processing unit according to one embodiment of the present invention. [Diagram 5] FIG. 5 shows a side view of a portion of a microfluidic device processing unit as shown in FIG. 4 along the axis labeled "AB." [Figure 6A] FIG. 2 shows a bottom view of a portion of a microfluidic device processing unit according to one embodiment of the present invention. [Figure 6B] FIG. 6B shows a side view of a portion of the microfluidic device processing unit shown in FIG. 6A along the axis labeled "AB." [Figure 7] 1 illustrates a method for manufacturing an embodiment of the present invention. [Figure 8] FIG. 1 shows a block diagram of an exemplary machine that can be used with one or more embodiments of the present invention to perform a digital PCR process for a biological sample. [Figure 9]FIG. 1 shows a schematic diagram of a device for sample processing and / or analysis, together with a pneumatic unit for use in providing fluid loading control of a microfluidic device. [Figure 10] FIG. 1 illustrates an exemplary sample digitization process of one or more embodiments of the present invention. [Figure 11] 1 shows a graph depicting a pressure pulsing process of one or more embodiments of the present invention. [Figure 12] 9 illustrates an exemplary digital PCR laboratory workflow that may be performed by the machine illustrated in FIG. 8 according to one or more embodiments of the invention described herein. [Figure 13] 1 illustrates a digital PCR process for use in accordance with one or more embodiments of the present invention.

[0018] Although the invention has been described with reference to the above-mentioned drawing figures, the drawing figures are intended to be illustrative and other embodiments are consistent with the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention describes a microfluidic device that provides a microfluidic structure formed from a thermoplastic injection molding process, incorporating a semi-permeable membrane that is selectively permeable to air but not to sample liquid to allow pressurized gas release while functioning as a gas barrier when pressure is released. Using thermoplastics to form the microfluidic structure allows for the use of an inexpensive and scalable injection molding process, while the membrane provides the ability to release gas upon pressurization, avoiding the fouling issues that some microfluidic structures do not include. The microfluidic device structural design incorporates an array of dead-end microchambers connected by linear loading conduits (microconduits) and siphon conduits and formed from thermoplastic. The present invention embodiments incorporate a functional design optimized for manufacturability that can be used in digital PCR applications to deposit samples within the array of microchambers, which can then be used to quantify nucleic acids in digital PCR (dPCR).

[0020] Various embodiments will now be described in more detail below with reference to the accompanying drawings, which form a part of this specification and which show for the purpose of illustrating specific examples in which the embodiments can be practiced. However, this specification may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this specification will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. In particular, this specification may be embodied as a method or device. Thus, any of the various embodiments herein may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment combining software and hardware aspects. Thus, the following specification should not be construed in a limiting sense.

[0021] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0022] FIG. 1 shows a microfluidic device slide 1000 according to an embodiment of the present invention for processing samples. Multiple slides can be joined to an automation compatible plate frame by welding. The plate frame can be a standard format plate frame with a single inlet well, as known in the art. Other suitable methods can also be used to join multiple slides together. In one embodiment of the present invention, the single slide 1000 shown in FIG. 1 is a four-unit array and includes multiple processing units. For example, the slide 1000 in FIG. 1 includes four processing units 101, 102, 103, and 104. In other embodiments, the slide 1000 may include fewer or more than four processing units. For example, the slide 1000 can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 processing units. 1 include respective inlet ports, i.e., inlet port 111 for device 101, port 112 for device 102, port 113 for device 103, and port 104 for device 104. Devices 101-104 may include a single inlet port or multiple inlet ports in various embodiments of the invention. Other embodiments may include one, two, or more inlet ports.

[0023] Methods used in embodiments of the invention may include applying a single or multiple pressure differentials to an inlet port to direct a solution from the inlet port to a conduit, e.g., loading conduits 121-124 as shown in FIG. 1 for devices 101-104, respectively. Alternatively, or in addition, a device may include multiple inlet ports and a pressure differential may be applied to the multiple inlet ports. An inlet of a device (e.g., a microfluidic device) may be fluidly coupled to a fluidic control module, such as an air pump, vacuum source, or compressor. The fluidic control module may provide a positive or negative pressure to the inlet. The fluidic control module may apply a pressure differential to fill the device with a sample and deposit (e.g., digitize) the sample in a chamber or microchamber. Alternatively, or in addition, a sample may be deposited in multiple chambers or microchambers as described elsewhere herein. In one or more embodiments of the invention, the filling and deposition of a sample may be performed without the use of valves between the chambers or microchambers to load the sample into a chamber of a microchamber. For example, filling of a conduit may be performed by applying one or more pressure differentials between the sample in the inlet port and the conduit. The pressure differential(s) may be achieved by pressurizing the sample or by continuously applying a vacuum to the conduits and / or chambers or microchambers for one or more specified durations. Filling of the chambers and deposition of the sample-containing solution may be performed by applying a pressure differential between the conduits and the chambers for a specified duration. This may be achieved by pressurizing the conduits through an inlet port or by applying a vacuum to the chambers. The sample-containing solution may enter the chambers such that each chamber contains a portion of the sample.

[0024] 2 includes a diagram of the exemplary microfluidic device processing unit 101 of FIG. 1, according to one embodiment of the present invention. In this embodiment, the microfluidic device processing unit 101 comprises a closed system 201 fluidly coupled to an inlet port 111. Apart from the inlet port 111, there are no other inlets through which fluids can enter the closed system 201, and there are no outlets or outlet ports through which liquids can exit the closed system 201. The inlet port 111 is fluidly coupled to the closed system 201 via a loading conduit 121 comprising a wide conduit portion 203, which is coupled at a first end to the input port 111 and at a second end to a step-down conduit portion (e.g., a first microconduit portion) 204. The step-down conduit portion 204 is fluidly coupled at one end to the wide conduit portion 203 and at the other end to a loading conduit network portion 205. The loading conduit network section 205 is fluidly coupled to the step-down conduit section 204, the wide conduit section 203, and the inlet port 111 using a closure system 201. In one embodiment of the invention, the loading conduit network section 205 incorporates a "splitter" design, splitting from the step-down conduit section 204 into 2, 4, 8, 16, 32, then 64 conduits with equal fluidic resistance (having similar conduit depth and width as the step-down conduit section 204) to distribute the reagents evenly. In one embodiment of the invention, the splitter design may utilize curved paths or turns with radii of curvature similar to those described below.

[0025] The closed system 201 comprises a plurality of linear loading conduits 206, which are connected to the loading conduit network portion 205 via a fluid connection or coupling to each separate linear loading conduit 206. Each linear loading conduit 206 is fluidly coupled to a terminal chamber 207 at one end and a plurality of microchambers 208. In one embodiment of the invention, the terminal chamber 207 may comprise a receptacle or reservoir for waste or potential "overfill" of excess fluids, reagents, or samples, etc., that may cause reagents to crosstalk during the dPCR process if not properly routed into the terminal chamber 207. With the exception of the plurality of terminal chambers 207 and the plurality of microchambers 208, in some embodiments of the invention, the closed system 201 does not include other reservoirs for waste and / or fluid receptacles. Thus, each linear loading conduit, along with its adjacent microchambers and terminal chamber, may comprise a microchamber assembly or may be a dead end.

[0026] In some embodiments, the number of microchambers 208 in the closed system 201 is between 10,000 and 30,000. In one embodiment of the present invention, the multiple microchambers 208 fluidly coupled to the same adjacent loading conduit are arranged in two rows, one row of microchambers positioned on each side of the linear loading conduit 206. Each linear loading conduit 206 is also fluidly coupled to an input port 207. Other possible arrangements of microchambers within the microconduit may also be used within embodiments of the present invention. For example, the configurations, systems, methods, devices and systems disclosed in U.S. Pat. No. 9,845,499 to Hung et al., which is incorporated herein by reference in its entirety, U.S. Patent Application Publication No. 2019 / 0264260 to Zayac et al., which is incorporated herein by reference in its entirety, and U.S. Patent Application Publication No. 2020 / 0384471 to Lin et al., which is incorporated herein by reference in its entirety, may all be used within embodiments of the present invention.

[0027] In some embodiments of the invention, the volume of the termination chamber is greater than the volume of the microchamber 208. In one embodiment of the invention, the volume of the termination chamber is at least about four times the volume of the microchamber. In one embodiment of the invention, the volume of the termination chamber is less than or equal to 10% of the total combined volume of the multiple microchambers fluidly coupled to the same proximate linear loading conduit 206. In other embodiments of the invention, the volume of the termination chamber is at least about five times, ten times, fifteen times, twenty times, or more, the volume of the microchamber. In some embodiments of the invention, the volume of the termination chamber is less than or equal to about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200% or more of the total combined volume of the multiple microchambers fluidly coupled to the linear loading conduit 206. In some embodiments of the invention, the combined volume of all end chambers is about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200% or more of the total combined volume of the plurality of microchambers fluidly coupled to the linear loading conduit 206. In other embodiments, the inlet port 111 is fluidly coupled to a sample reservoir and the volume of the end chambers is about 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200% or more of the total combined volume of the plurality of microchambers fluidly coupled to the linear loading conduit 206.

[0028] 3 shows a bottom perspective view of a portion of a microfluidic device processing unit according to an embodiment of the invention, showing a portion of two linear loading conduits 206. In one embodiment of the invention, the linear loading conduit 206 has a depth of about 10 microns. In one embodiment of the invention, the linear loading conduit 206 is fluidly coupled to a plurality of microchambers 208. Each microchamber 208 is fluidly coupled to the linear loading conduit 206 via a siphon conduit 302. In some embodiments of the invention, the siphon conduit 302 has a depth of at least about 10 microns and the microchambers 208 have a depth of at least about 100 microns.

[0029] FIG. 4 shows a bottom view of a portion of a microfluidic device processing unit according to an embodiment of the invention, showing a portion of a linear loading conduit 208 having four microchambers 208 arranged in two rows of two microchambers along the linear loading conduit portion 206. The microchambers 208 are connected to the linear loading conduit 206 by a siphon conduit 302. In some embodiments of the invention, the microchamber 208 is a substantially rectangular three-dimensional shape, as shown in FIGS. 3 and 4. In one embodiment of the invention, the microchamber comprises four substantially rectangular side walls, four side edges, four top (or bottom) edges, and a substantially rectangular top (or bottom) wall or base, with each side wall connected to two other side walls by a side edge, and the top (or bottom) wall connected to each side wall by four top (or bottom) edges. In some embodiments of the invention, each edge of the four side edges comprises an arc of at least about 90 degrees. In one embodiment of the invention, the arc comprises a radius of at least about 10 microns. In an injection molding process, having sharp edges or corners of 90 degrees may be physically prohibited. Adding a radius of curvature to the 90 degree corners of the rectangular microchambers aids in the release of the injection mold. Prior art microfluidic devices known in the art generally utilize cylindrical shaped microchambers, which are easier to manufacture using an inexpensive and highly scalable injection molding process. However, in dPCR applications, it is highly desirable to increase the sensitivity of the dPCR results by increasing the total volume analyzed, so microchambers with rectangular shapes are preferred over cylindrical shaped microchambers, as rectangular microchambers have a larger volume than cylindrical shapes with similar dimensions. In one embodiment of the present invention, rectangular microchambers 208 with a radius of curvature on each side edge of at least about 10 microns significantly improves the manufacturability of microfluidic devices using injection molding processes, while still increasing the total analysis volume per microchamber over prior art microfluidic structures.

[0030] On the one hand, maximizing the use of space by fitting as many microchambers as possible on a microfluidic device is optimal for the dPCR process, but current limitations of thermoplastic injection molding make it difficult to create tall, thin plastic walls, and therefore, for manufacturability purposes, some spacing between microchambers may be required to ensure structural integrity. Manufacturability may also be aided by positioning the microchambers equidistant from each other in a row adjacent to a linear loading conduit. The aspect ratio used in some embodiments of the invention may be defined as the height of the microchamber divided by the distance between two microchambers in a row or the distance between rows. Figures 3 and 4 also show microfluidic structures in some embodiments of the invention having an aspect ratio of at least about 3. Thus, in one embodiment of the invention, if the depth (or height) of the microchambers is 105 microns, the minimum distance between two microchambers is at least about 3. In another embodiment of the invention, the ratio of the depth of the microchambers to the minimum distance between two microchambers is at least about 5. In other embodiments of the invention, aspect ratios of at least about 3, 5, or more may be utilized.

[0031] In some embodiments of the invention, a siphon conduit 302 as shown in Figures 3 and 4 comprises part of the path to the microchambers. It is desirable to maximize the path distance between the microchambers, for example, to avoid crosstalk of dPCR reagents. In addition, reducing the presence of siphon conduits between the microchambers and adding a radius of curvature around both the base and opening of the microchambers also aids in the evacuation of injection molded structures, since sharp corners are more difficult to demold. Thus, a siphon conduit comprising a non-linear path may be included in an embodiment of the invention. In some embodiments of the invention, the non-linear path comprises at least one of an arc of at least about 90 degrees, a 90 degree angle, an acute angle, an obtuse angle, a curve, multiple curves, or multiple angles. In one embodiment of the invention, the non-linear path comprises an arc having a radius substantially equal to about 90 degrees and at least about 10 microns.

[0032] Additionally, from a manufacturability perspective, adding siphon conduits and placing them between microchambers may increase the difficulty of mold filling (similar to how limitations of the injection molding process may make it more difficult to create tall thin walls). Thus, longer siphon conduits of substantially equal length and placed at substantially equal intervals between microchambers may be considered optimal in terms of avoiding PCR reagent crosstalk. In one embodiment of the present invention, multiple siphon conduits may be placed at substantially equal distances from adjacent siphon conduits on the linear loading conduit.

[0033] FIG. 5 shows a side view of a portion of a microfluidic device processing unit as shown in FIG. 4 along the axis labeled "AB". A thermoplastic microfluidic structure including microchambers 208 is shown with the base wall facing "top" and a siphon conduit 302 positioned near the "bottom" and fluidically coupled to the microchambers 208 and linear loading conduit 206. A thin film 501 covers all of the microfluidic structure from the "bottom" labeled in FIG. 5. The "top" and "bottom" designations in FIG. 5 reflect its positioning on a standard plate frame, where the plate containing the processing unit including the microfluidic structure is flipped upside down and then laser welded to the frame with the reservoir. In one embodiment of the present invention, the thickness of the microfluidic structure in FIG. 5 from top to bottom (without the film) is about 1.5 mm. A thin film 501 is used to cover the surface of the microfluidic structure. The thin film is gas impermeable at low pressure, but allows gas release through the thin film when pressure is applied, and is therefore at least partially gas permeable under pressure. In some embodiments of the present invention, the gas-permeable film is not gas-permeable at atmospheric pressure, but is gas-permeable at pressures higher than atmospheric pressure. In some embodiments, the thin film is gas-permeable but not liquid-permeable at one or more selected pressures above atmospheric pressure. In some embodiments of the present invention, the thin film is about 80 microns thick and is composed of a cyclic olefin polymer. One suitable thin film used in embodiments of the present invention is the semi-gas-permeable film TOPAS® COC6013. In other embodiments of the present invention, semi-gas-permeable films having thicknesses of 60, 70, 80, 90, 100 microns or any range within these thicknesses can be used.

[0034] Figure 6A shows a top view of a portion of a microfluidic device processing unit showing the inlet port 111. The inlet port 111 comprises an inlet 601 and a landing pad 602. Figure 6A also shows the wide conduit portion 203 fluidly coupled to the inlet port 111 and the step-down conduit portion 204 fluidly coupled to the wide conduit portion 203. Figure 6B illustrates a side view of a portion of a microfluidic device processing unit depicting the inlet port 111, the wide conduit portion 204, and the step-down conduit portion 204 as shown in Figure 6A along the axis labeled "AB". A thin film 501 is used to cover the surface of the structure shown in Figures 6A and 6B.

[0035] Various features shown in Figures 6A and 6B are included in embodiments of the present invention to improve manufacturability during the thermoplastic injection molding process. For injection molding, a mold tool is created to receive the molten plastic. An insert, which can typically be made from nickel, has inverted microfluidic features including microchambers, microconduits, and landing pads that are "inserted" into the mold tool. An "insert pin" is part of the mold tool opposite the insert. When the mold closes, the insert pin hits the landing pad and the molten plastic moves into the mold, transferring the microfluidic features onto the insert. When the mold cools, the plastic forms the microfluidic device and is removed from the mold.

[0036] In some embodiments of the invention, the inlet port 111 includes angled sidewalls that may be formed using a mask during the thermoplastic injection molding process to prevent the formation of undercuts after repeated pin impacts for the inlet 601, theoretically increasing insert life and allowing for smoother part ejection by reducing sticking. Additionally, in some embodiments of the invention, the landing pad 602 includes a wider diameter than the inlet 601, thereby better accommodating pin impacts without damaging the edges of the landing pad. The landing pad 602 is a feature used during the microfluidic injection molding process and refers to a circular area for receiving an "insert pin" that forms a through hole in the plastic device. The insert pin physically "hits" the landing pad with every shot, degrading the pad. The landing pad 602 may also include sloped sidewalls with a draft angle. Without the draft angle, it may be difficult to remove the thermoplastic part from the mold. A landing pad with a draft angle aids in easier demolding. Thicker landing pads and landing pads with larger "pads" or diameters can improve the life of the insert and also provide greater tolerance for variations in the position of the insert pin. The draft angle (slope from straight vertical) may be present at about 5 degrees in the embodiments of the invention described herein, but may range anywhere from at least about 2 degrees to at least about 5 degrees, and more. Anything less than 2 degrees may present challenges to release.

[0037] The wide conduit section 203, as well as the microconduit splitter network and the multiple linear loading conduits, are shown in FIG. 6A as having an approximately 90 degree curve or turn before redirecting into the narrower step-down conduit section 204, having a depth of approximately 10 microns. In one embodiment of the invention, the 90 degree turn in the wide conduit section includes a 90 degree arc having a radius of curvature of at least approximately 10 microns, 25 microns, 50 microns, 100 microns, or more. In other embodiments of the invention, other angle turns, e.g., acute angles, obtuse angles, curves, and / or multiple curves and angles, may be used in the wide conduit section 204. In the embodiments of the invention disclosed herein, the inclusion of a 90 degree turn in the wider conduit before the step-down prevents shrinkage of the narrower loading conduit in the area of ​​the sharp bend as a result of natural part shrinkage as the plastic cools after the injection molding process. Such shrinkage at the turn can cause the narrower conduit to clog and thus deform during ejection. During the injection molding process, if a microconduit with a depth and / or width of 10 microns has a 90 degree turn, the conduit may shrink to a dimension of 6-8 microns in either direction, thereby causing potential conduit blockage issues.

[0038] FIG. 7 illustrates a manufacturing method used in an embodiment of the present invention. In FIG. 7, an injection molding process 701 is used to form a microfluidic structure or device. The microfluidic device includes an array of microchambers connected to at least one loading conduit and / or microconduit via a siphon conduit or siphon opening as shown in FIGS. 3 and 4. The surface of the microfluidic structure is covered by a semi-gas permeable film. In a covering process, openings in at least one surface of the microfluidic structure are covered to completely encapsulate the microstructure including the inlet port, the loading conduit, the microconduit, the siphon conduit, the microchamber, and the termination chamber. In some embodiments of the present invention, the covering is performed by a process 702 of applying a thin film to the injection molded microfluidic structure.

[0039] In one aspect, the present disclosure provides an apparatus for analyzing a nucleic acid sample using a microfluidic device. The apparatus may include a transfer stage configured to hold one or more microfluidic devices. The microfluidic device may include a microconduit with an inlet and an outlet, a plurality of microchambers connected to the microconduit by a plurality of siphon openings, and a thin film forming a surface of the microfluidic device. The apparatus may include a pneumatic module in fluid communication with the microfluidic device. The pneumatic module may load reagents into the microfluidic device and deposit reagents into the microchambers. The apparatus may include a thermal module in thermal communication with the plurality of microchambers. The thermal module may control the temperature of the microchambers and thermal cycle the microchambers. The apparatus may include an optical module capable of imaging the plurality of microchambers. The apparatus may also include a computer processor coupled to the transfer stage, the pneumatic module, the thermal module, and the optical module. The computer processor can be programmed to (i) direct the pneumatic module to load reagents into the microfluidic device and deposit the reagents into the multiple microchambers, (ii) direct the thermal module to thermal cycle the multiple microchambers, and (iii) direct the optical module to image the multiple microchambers.

[0040] The transfer stage may be configured to load microfluidic devices, hold microfluidic devices, and load microfluidic devices. The transfer stage may be stationary in one or more coordinates. Alternatively, or in addition, the transfer stage may be capable of moving in an X-direction, a Y-direction, a Z-direction, or any combination thereof. The transfer stage may be capable of holding a single microfluidic device. Alternatively, or in addition, the transfer stage may be capable of holding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, or more microfluidic devices.

[0041] The pneumatic module may be configured to be in fluid communication with the inlets and outlets of the microfluidic device. The pneumatic module may have multiple connection points that can connect to multiple inlets and multiple outlets. The pneumatic module may be capable of filling and / or backfilling a single array of microchambers at a time or multiple arrays of microchambers in parallel. The pneumatic module may further comprise a vacuum module. The pneumatic module may provide increased pressure to the microfluidic device or may provide a vacuum to the microfluidic device.

[0042] A thermal module can be configured in thermal communication with the microchambers of the microfluidic device. The thermal module can be configured to control the temperature of a single array of microchambers or to control the temperature of multiple arrays of microchambers. The thermal control module can implement the same thermal program across all arrays of microchambers or can implement different thermal programs for different arrays of microchambers.

[0043] The optical module may be configured to emit and detect light at multiple wavelengths. The emission wavelengths may correspond to the excitation wavelengths of the indicator and amplifier probes used. The emitted light may include wavelengths having a maximum intensity of about 450 nm, 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or any combination thereof. The detected light may include wavelengths having a maximum intensity of about 500 nm, 525 nm, 550 nm, 575 nm, 600 nm, 625 nm, 650 nm, 675 nm, 700 nm, or any combination thereof. The optical module may be configured to emit light at one, two, three, four, or more wavelengths. The optical module may be configured to detect light at one, two, three, four, or more wavelengths. A wavelength of the emitted light may correspond to an excitation wavelength of the indicator molecule. Another wavelength of the emitted light may correspond to an excitation wavelength of the amplifier probe. One detected wavelength of light may correspond to the emission wavelength of an indicator molecule. Another detected wavelength of light may correspond to an amplifier probe used to detect a reaction in the microchamber. The optical module may be configured to image a section of the array of microchambers. Alternatively, or in addition, the optical module may image the entire array of microchambers in a single image.

[0044] FIG. 8 shows a machine 800 for performing a digital PCR process, described below in FIG. 12. The machine 800 includes a pneumatics and pump module 801, which includes a pump and a manifold, and can be moved in the Z direction and is operable to perform the application of pressure as described in FIG. 10 and FIG. 11. The application of pressure to air refers to air already present in the microfluidic device at atmospheric pressure, which is then compressed as pressure is applied to push in fluid (e.g., sample fluid covered with non-sample fluid). The machine 800 also includes a thermal module 802, such as a flat block thermal cycler, for thermally cycling the microfluidic device, thereby performing a polymerase chain reaction. The machine 800 further includes an optical module 803, such as an epi-fluorescence optical module, that can optically determine which microchambers in the microfluidic device were successful in performing a PCR reaction. The optical module 803 can provide this information to a processor 804, which uses Poisson statistics to convert the raw counts of successful microchambers to nucleic acid concentrations. A transfer stage 805 may be used to move a given microfluidic device between the various modules to handle multiple microfluidic devices simultaneously. The above microfluidic devices, combined with incorporating this functionality into a single machine, reduce the cost, workflow complexity, and space requirements of dPCR over other implementations of dPCR.

[0045] FIG. 9 shows a schematic diagram of a slide 900 on a frame 905 along with a pneumatic unit 901 for use in providing device fluid control for fluids introduced to four different sample processing units (as shown and described above, but not shown separately in FIG. 9) on the slide 900. Fluids held in reservoirs 906 are introduced to the sample processing units through inlets (not shown separately in FIG. 9) of the processing units. As described herein, a series of pressure pulses including low and high pressures applied to the reservoirs 906 loads the sample solutions / reagents into microchambers (not shown separately) in the processing units. The pneumatic units 901 control the application of these pressures, and each unit 901 includes an electronic pressure regulator 902 and at least one valve 903. The units 901 interface with the frame 905 via a suitable mechanism, such as an O-ring, to transmit pressure to the reservoirs 906. More or fewer valves may be incorporated (e.g., depending on the number of reservoirs and / or whether separate pressure control for each reservoir is desired). The frame 905 and / or slide 900 can include mechanical keys to aid in orientation and alignment, such as tabs or other visual features (such as alignment marks, not shown).

[0046] Figure 10 illustrates an exemplary sample digitization process 1010 of one or more embodiments of the invention described herein. In step 1010 of Figure 10, a fluid or solution containing a sample may be loaded into an inlet port of a microfluidic device or processing unit. The microfluidic device may comprise an inlet port, a loading conduit, and multiple chambers or microchambers, as described above. The fluid loaded into the microfluidic device in step 1010 may include a fluid or solution containing a sample, or may include a fluid or solution not containing a sample, or may include both sample and non-sample fluids.

[0047] High pressure may be applied to compress the air in the microfluidic network, including the loading conduit, the linear loading conduit, the siphon conduit, the microchamber, and the end chamber, which draws the sample fluid into the microfluidic network. The amount of fluid drawn in should be approximately equal to the air compressed according to the ideal gas law. Since the volume of the loading conduit (having a depth of at least about 10 microns and a width of at least about 10 microns) is smaller than the volume of the microchamber (having a depth of at least about 100 microns), all the loading conduits should be filled with sample fluid during this operation, which means that most of the compressed air stays in the microchamber and the end chamber. The compressed air continues to escape through the thin film, drawing more sample fluid into the network of microconduits, loading conduits, siphon conduits, and into the microchamber. The non-sample fluid overlaid on top of the sample fluid is later drawn into the microfluidic network, while the sample fluid continues to replace the space occupied by the air, and the air continues to escape through the film.

[0048] In step 1010, a series of one or more pressure pulses may be applied to the inlet port of the microfluidic device. The pressure pulses may include applying a high pressure for a first predetermined period (e.g., a short time interval) immediately followed by a low pressure for a second predetermined period (e.g., a short time interval). In one embodiment of the invention, the pulses start initially (when no fluid is present in the array because the material is hydrophobic) for 1 minute (6 cycles of 75 psi / 10 psi for 5 seconds / 5 seconds). In other words, a high pressure pulse can be applied at 75 psi for a short time interval, e.g., 5 seconds, followed immediately by a low pressure pulse at 10 psi for a short time interval, e.g., 5 seconds, followed immediately by a high pressure pulse again at 75 psi for 5 seconds. Thus, a high pressure pulse followed by a low pressure pulse can be repeatedly applied at successive 5 second time intervals for 6 cycles. In other embodiments of the invention, a greater or lesser number of cycles can be contemplated, e.g., 5, 10, 12, 20, or more cycles. There can be different high or low pressure pulses, for example low pressure pulses, for example less than or greater than 10 psi for low pressure pulses, or greater than or less than 75 psi for high pressure pulses.

[0049] In step 1020, after the series of pressure pulses have been applied, a solution or fluid containing the sample may be loaded into one or more of the microchambers. To fully digitize the sample fluid into the microchambers, high pressure (e.g., 75 psi) may be applied for a predetermined period of time (e.g., in one embodiment of the invention, a longer time interval (e.g., 24 minutes)) while the sample fluid is present in the microchambers. The reason for step 1020 is that some small amount of air (e.g., air bubbles) may still be trapped in the microchambers. Thus, applying continuous pressure at 75 psi for 24 minutes allows the microfluidic device to continue outgassing, most likely through both dissolution of air into the sample and slow outgassing when the air is no longer in contact with the surface of the film. Finally, in some embodiments of the invention, a lower equilibrium pressure (e.g., 50 psi) may be applied for a predetermined period of time (e.g., 5 minutes) before the start of the dPCR process. In one embodiment of the invention, the reason for lowering to 50 psi for 5 minutes is that the pressure is held at 50 psi during dPCR. If the closed system is not equilibrated prior to the start of PCR, where the temperature is heated to 96 degrees Celsius, any residual air in the closed system will expand and push the sample out and potentially cause crosstalk.

[0050] To reduce crosstalk between the deposited samples, the conduits or microconduits fluidly coupled to the microchambers may be substantially or completely free of sample fluid following step 1020. To achieve this, in one embodiment, the volume of sample fluid injected into the inlet port of the microfluidic device is less than or substantially less than the total volume of the chamber or microchambers of the microfluidic device. For example, in one embodiment, the total volume of all microchambers in the processing unit is about 11 microliters, but only 9 microliters of sample fluid may be loaded into the processing unit. In other embodiments, the total volume of sample fluid is less than the total combined volume of the microchambers of the microfluidic device, for example, the volume of sample fluid loaded into the microfluidic device is less than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% of the total combined volume. In some embodiments, a non-sample fluid different from the sample fluid may also be loaded into the inlet port, as described in step 1010 discussed above. Following step 1020, the non-sample fluid may therefore be primarily present in the bottom of the conduits and / or microconduits and microchambers (held in place by surface tension), and the sample fluid is primarily present in the chambers and microchambers. In one embodiment of the invention, the sample fluid in one chamber may not be in substantial fluid communication with the sample fluid located in another chamber after step 1020.

[0051] FIG. 11 shows a graph 1100 illustrating the pressure pulsing process of one or more embodiments of the present invention described above, with pressure (PSI) plotted on the y-axis and time (seconds) plotted on the x-axis.

[0052] In some cases, alternative processes or methods to those described in connection with Figures 10 and 11 above may be employed to digitize a sample. For example, a single pressure difference may be used to deliver a sample solution (e.g., containing nucleic acid molecules of interest) to a conduit, and the same pressure difference may be used to continue to digitize a chamber having the solution (e.g., delivering the solution from the conduit to the chamber). Furthermore, the single pressure difference may be high enough to allow pressurized degassing or degassing of the conduit and / or chamber. Alternatively, or in addition, the pressure difference for delivering the solution containing the sample to the conduit may be a first pressure difference. The pressure difference for delivering the solution from the conduit to the chamber may be a second pressure difference. The first and second pressure differences may be the same (e.g., equal) or different. In one example, the second pressure difference may be 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 high enough to allow pressurized degassing or degassing of the conduit or chamber. In some cases, a third pressure difference may be used to allow pressurized degassing or degassing of the conduit and / or chamber. Pressurized degassing or degassing of the conduit or chamber 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 chamber and / or conduit through the film or membrane to the environment outside the chamber and / or conduit.

[0053] FIG. 12 shows an exemplary digital PCR process that can be performed by the machine shown in FIG. 8 using one or more embodiments of the invention described herein. The digitalization process can be easily integrated into a typical laboratory workflow for nucleic acid assays, as shown in FIG. 12. The biological sample preparation in step 1210 (sample preparation step) can be performed similarly to other PCR-based workflows known in the art, including nucleic acid isolation and mixing of the biological sample with a master mix and primers / probes. The master mix is ​​a mixture containing precursors and enzymes used as components in RT-PCR techniques in molecular biology. Such a mixture known in the art can include at least a mixture of dNTPs, MgCl2, adding Taq polymerase, pH buffer, and mixing in nuclease-free water. The microfluidic device plate shown in step 1220 (plate loading step) is loaded as described herein with respect to Figures 10 and 11 (e.g., pipetting of sample fluid mixture followed by pipetting of oil overlay) and then placed into an instrument similar to that shown in Figure 8 as described herein, which integrates pneumatic loading and digitization of reagents, thermal cycling, and data / image acquisition (sample digitization+PCR+image acquisition steps). The acquired data of the PCR reaction can then be analyzed by software downstream to provide results such as the concentration of a target gene in a biological sample.

[0054] FIG. 13 illustrates a digital PCR process used in accordance with one or more embodiments of the present invention described above. In step 1301, reagents (fluids) containing a sample are digitized in a microchamber of a microfluidic device. In step 1302, the reagents are thermal cycled to run a PCR reaction on the digitized reagents in the microchambers. This step can be performed, for example, using a flat block thermal cycler as described above with respect to FIG. 8. In step 1303, image acquisition is performed to determine which microchambers successfully ran a PCR reaction. Image acquisition can be performed, for example, using a three-color probe detection unit. In step 1304, Poisson statistics are applied to the count of microchambers determined in step 1303 to convert the raw number of positive chambers into a quantifiable nucleic acid concentration.

[0055] term Terms used herein with reference to the embodiments of the invention disclosed herein are to be given their ordinary meaning by those of ordinary skill in the art, unless otherwise indicated explicitly or by context.

[0056] "Chamber" refers to a structure that allows for the deposition of non-sample fluids, sample-containing fluids such as biological samples, or sample-containing solutions or reagents within a microfluidic device. Examples of structures that allow for sample deposition and digitization include wells, chambers, and microchambers.

[0057] "Conduit" refers to a structure that allows a path for the movement of a sample fluid or a non-sample fluid. Examples of structures that allow a path for fluid movement include conduits, passages, microconduits, micropassages, siphon conduits, siphon passages, and siphon openings.

[0058] "Depth" as used with respect to the microfluidic devices discussed herein generally refers to the distance measured from the bottom of a conduit, siphon opening or conduit, chamber, or microchamber to the top of the sidewall of the conduit, siphon opening or conduit, chamber, or microchamber, or to a gas permeable film or membrane covering the conduit, siphon opening or conduit, chamber, or microchamber.

[0059] The terms "digitized" or "digitalization" may be used interchangeably and generally refer to a sample that has been distributed into one or more microchambers. A digitized sample may or may not be in fluid communication with another digitized sample. A digitized sample may not exchange materials (e.g., reagents, analytes) or interact with another digitized sample. Examples of structures that allow for digitization of a sample include wells, chambers, and microchambers.

[0060] The term "fluid" generally refers to a liquid or gas. A fluid does not maintain a defined shape, but flows or moves such that particles of the fluid undergo continuous changes in area during an observable time frame to fill the container in which it is placed. Thus, a fluid may have any suitable viscosity capable of allowing movement. When more than one fluid is present, each fluid may be independently selected from among essentially any fluid (liquid, gas, etc.) by one of ordinary skill in the art.

[0061] "Microfluidic" generally refers to a device, structure, article, region, system, or chip that includes at least one conduit, optionally a plurality of siphon openings or conduits, and an array of chambers or microchambers. For example, the conduits may have cross-sectional dimensions of about 1 millimeter or less, about 750 microns or less, about 500 microns or less, about 250 microns or less, about 100 microns or less, or less. The conduits or siphon conduits or openings may have cross-sectional dimensions of about 50 microns or less, about 10 microns or less, or less.

[0062] The terms "pressurized venting" or "pressurized degassing" may be used interchangeably and generally refer to the removal or evacuating of one or more gases (e.g., air, nitrogen, oxygen, carbon dioxide, etc.) from a conduit, opening, or chamber of a device, such as a microfluidic device, to an environment external to the chamber, conduit, or opening through the application of a pressure differential. The pressure differential may be applied between the conduit or chamber and the environment external to the conduit or chamber. The pressure differential may be provided by application of a pressure source or pressure to one or more inlets to the device, or a vacuum source to one or more surfaces of the device. Pressurized venting or degassing may be enabled through a gas-permeable film, membrane, or membrane covering one or more sides of the conduit, chamber, or opening.

[0063] A "sample" as used herein generally refers to any sample that contains or is suspected of containing a nucleic acid molecule. For example, a sample may be a biological sample that contains one or more nucleic acid molecules. A biological sample may be obtained (e.g., extracted or isolated) from or may contain blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretion, sputum, stool, and tears. A biological sample may be a fluid sample or a tissue sample (e.g., skin). A sample may be obtained from an acellular body fluid and may contain cell-free DNA or cell-free RNA. A sample may contain tumor cells. In some embodiments, a sample may include an environmental sample (e.g., soil, waste, water, ambient air, etc.), an industrial sample (e.g., a sample from any industrial process), or a food sample (e.g., dairy products, vegetable products, and meat products). A sample may be processed before loading into a microfluidic device. For example, a sample may be processed to lyse cells, purify nucleic acid molecules, and / or contain reagents.

[0064] Further embodiments Embodiment 1A. A microfluidic device configured to process a sample, comprising an inlet port and a closure system fluidically coupled to the inlet port, the closure system comprising a plurality of linear loading conduits, a plurality of termination chambers, one of the plurality of termination chambers being fluidically coupled to a linear loading conduit of the plurality of linear loading conduits, and a plurality of microchambers for receiving a sample, one of the plurality of microchambers being fluidically coupled to a linear loading conduit of the plurality of linear loading conduits, wherein for a first plurality of microchambers and a first termination chamber fluidically coupled to the same adjacent linear loading conduit, a volume of the first termination chamber is less than or equal to a total volume of the first plurality of microchambers, and further wherein a waste reservoir larger than any one of the termination chambers is excluded from the closure system.

[0065] Embodiment 2A. The microfluidic device of embodiment 1A, further comprising a plurality of siphon conduits, one of the plurality of siphon conduits being fluidly coupled between the linear loading conduit and the microchamber.

[0066] Embodiment 3A. The microfluidic device of embodiment 1A, wherein the volume of the termination chamber is greater than the volume of one of the microchambers.

[0067] Embodiment 4A. The microfluidic device of embodiment 3A, wherein the volume of the termination chamber is at least about four times the volume of the microchamber.

[0068] Embodiment 5A. The microfluidic device of embodiment 2A, wherein the first dimension of the linear loading conduit and the first dimension of the siphon conduit are less than about 10 microns.

[0069] Embodiment 6A. The microfluidic device of embodiment 1A, wherein a microchamber of the plurality of microchambers has a first dimension of at least about 100 microns.

[0070] Embodiment 7A. The microfluidic device of embodiment 1A, further comprising a thin film applied to the microfluidic device, the thin film forming a surface of the closed system.

[0071] Embodiment 8A. The microfluidic device of embodiment 7A, wherein the surface formed by the thin film provides an exterior surface of a plurality of microchambers.

[0072] Embodiment 9A. The microfluidic device of embodiment 8A, wherein the surface formed by the thin film provides an outer surface of a plurality of linear loading conduits.

[0073] Embodiment 10A. A microfluidic device as described in embodiment 9A, wherein the surface formed by the thin film provides an exterior surface of a plurality of end chambers.

[0074] Embodiment 11A. The microfluidic device of any of embodiments 7A to 10A, wherein the thin film has a thickness of about 70 to 90 microns.

[0075] Embodiment 12A. A microfluidic device according to any of embodiments 7A to 10A, wherein the thin film has a thickness of about 80 microns.

[0076] Embodiment 13A. The microfluidic device of any of embodiments 7A to 12A, wherein the thin film comprises a gas-permeable thermoplastic material.

[0077] Embodiment 14A. The microfluidic device of embodiment 13A, wherein the gas permeable thermoplastic material is not permeable to the sample.

[0078] Embodiment 15A. The microfluidic device of any of Embodiments 13A to 14A, wherein the gas permeable thermoplastic material comprises a cyclic olefin copolymer.

[0079] Embodiment 16A. The microfluidic device of any of Embodiments 1A to 15A, wherein the volume of the first terminal chamber is fifty percent (50%) or less of the total volume of the first plurality of microchambers.

[0080] Embodiment 17A. The microfluidic device of any of Embodiments 1A to 15A, wherein the volume of the first terminal chamber is twenty-five percent (25%) or less of the total volume of the first plurality of microchambers.

[0081] Embodiment 18A. The microfluidic device of any of Embodiments 1A to 15A, wherein the volume of the first terminal chamber is no more than ten percent (10%) of the total volume of the first plurality of microchambers.

[0082] Embodiment 19A. The microfluidic device of any of Embodiments 1A to 18A, comprising an injection molded thermoplastic material.

[0083] Embodiment 1B. A microfluidic device configured to process a sample, comprising an inlet port and a plurality of dead-end microfluidic assemblies, each of the dead-end microfluidic assemblies fluidly coupled to the inlet port, each of the dead-end microfluidic assemblies comprising a linear loading conduit, a termination chamber fluidly coupled to the linear loading conduit, and a plurality of microchambers fluidly coupled to the linear loading conduit and configured to receive a sample proximate to the linear loading conduit, wherein a volume of the termination chamber is less than or equal to a combined volume of the plurality of microchambers.

[0084] Embodiment 2B. The microfluidic device of embodiment 1B, wherein each of the dead-end microfluidic assemblies further comprises a plurality of siphon conduits, and a siphon conduit of the plurality of siphon conduits is fluidly coupled between the linear loading conduit and a microchamber of the plurality of microchambers.

[0085] Embodiment 3B. The microfluidic device of embodiment 1B, wherein the volume of the termination chamber is greater than the volume of one of the microchambers.

[0086] Embodiment 4B. The microfluidic device of embodiment 3B, wherein the volume of the end chamber is at least about four times the volume of the microchamber.

[0087] Embodiment 5B. The microfluidic device of embodiment 2B, wherein the first dimension of the linear loading conduit and the first dimension of the siphon conduit are each less than about 10 microns.

[0088] Embodiment 6B. The microfluidic device of embodiment 1B, wherein a microchamber of the plurality of microchambers has a first dimension of at least about 100 microns.

[0089] Embodiment 7B. The microfluidic device of embodiment 1B, further comprising a thin film applied to the microfluidic device, the thin film forming a surface of the microfluidic device.

[0090] Embodiment 8B. The microfluidic device of embodiment 7B, wherein the surface formed by the thin film provides an exterior surface of a plurality of microchambers.

[0091] Embodiment 9B. The microfluidic device of embodiment 8B, wherein the surface formed by the thin film further provides an outer surface of the linear loading conduit.

[0092] Embodiment 10B. The microfluidic device of embodiment 9B, wherein the surface formed by the thin film further provides an exterior surface of a termination chamber.

[0093] Embodiment 11B. A microfluidic device according to any of embodiments 7B-10B, wherein the thin film has a thickness of about 70-90 microns.

[0094] Embodiment 12B. The microfluidic device of any of embodiments 7B-10B, wherein the thin film has a thickness of about 80 microns.

[0095] Embodiment 13B. The microfluidic device of any of Embodiments 7B-12B, wherein the membrane comprises a gas-permeable thermoplastic material.

[0096] Embodiment 14B. The microfluidic device of embodiment 13B, wherein the gas permeable thermoplastic material is not permeable to the sample.

[0097] Embodiment 15B. The microfluidic device of any of Embodiments 13B-14B, wherein the gas permeable thermoplastic material comprises a cyclic olefin copolymer.

[0098] Embodiment 16B. The microfluidic device of any of Embodiments 1B-15B, wherein the volume of the terminal chamber is fifty percent (50%) or less of the total volume of the plurality of microchambers.

[0099] Embodiment 17B. The microfluidic device of any of Embodiments 1B-15B, wherein the volume of the terminal chamber is twenty-five percent (25%) or less of the total volume of the plurality of microchambers.

[0100] Embodiment 18B. The microfluidic device of any of Embodiments 1B-15B, wherein the volume of the terminal chamber is ten percent (10%) or less of the total volume of the plurality of microchambers.

[0101] Embodiment 19B. The microfluidic device of any of Embodiments 1B-18B, further comprising an injection molded thermoplastic material.

[0102] Embodiment 1C. A microfluidic device configured to process a sample, comprising: an inlet port fluidly coupled to an array of loading conduits and sample-receiving microchambers via a first microconduit portion; and a wide conduit portion fluidly coupled between the inlet port and the first microconduit portion, the first microconduit portion having a depth less than a depth of the wide conduit portion, the first microconduit portion comprising a straight conduit, the wide conduit portion comprising a portion including at least one of straight conduits or non-straight conduits oriented differently than the straight conduits of the first microconduit portion.

[0103] Embodiment 2C. The microfluidic device of embodiment 1C, wherein the wide conduit portion has a depth of at least about 100 microns.

[0104] Embodiment 3C. The microfluidic device of embodiment 2C, wherein the depth of the first microconduit portion is about 10 microns or less.

[0105] Embodiment 4C. The microfluidic device of embodiment 1C, wherein the path of the first microconduit pathway comprises at least one of at least about a 90 degree curve, a 90 degree angle, an acute angle, an obtuse angle, a curve, multiple curves, or multiple angles.

[0106] Embodiment 5C. The microfluidic device of embodiment 1C, wherein the microfluidic device is a thermoplastic injection molded microfluidic device.

[0107] Embodiment 6C. A microfluidic device as described in embodiment 1C, further comprising a thin film in the form of a surface of the microfluidic device.

[0108] Embodiment 7C. A microfluidic device as described in embodiment 6C, wherein the thin film provides an outer surface of the sample-receiving microchamber.

[0109] Embodiment 8C. The microfluidic device of embodiment 7C, wherein the thin film in the form of a surface further provides an exterior surface of the loading conduit.

[0110] Embodiment 9C. A microfluidic device according to any of embodiments 6C to 8C, wherein the thin film has a thickness of about 70 to 90 microns.

[0111] Embodiment 10C. A microfluidic device according to any of embodiments 6C-8C, wherein the thin film has a thickness of about 80 microns.

[0112] Embodiment 11C. A microfluidic device according to any of embodiments 6C-10C, wherein the thin film comprises a gas-permeable thermoplastic material.

[0113] Embodiment 12C. A microfluidic device according to embodiment 13C, wherein the gas permeable thermoplastic material is not permeable to the sample.

[0114] Embodiment 13C. A microfluidic device according to any of embodiments 6C-12C, wherein the gas permeable thermoplastic material comprises a cyclic olefin copolymer.

[0115] Embodiment 14C. A microfluidic device according to any of embodiments 1C-13C, wherein at least a portion of the microfluidic device is substantially optically transparent.

[0116] Embodiment 15C. A microfluidic device according to any of embodiments 1C-14C, wherein the microfluidic device is one of a plurality of microfluidic devices that collectively form a sequential injection molded thermoplastic part.

[0117] Embodiment 16C. The microfluidic device of embodiment 15C, wherein the microfluidic device is not fluidically coupled to another microfluidic device of the plurality of microfluidic devices.

[0118] Embodiment 1D. A microfluidic device configured for processing a biological sample, comprising: an inlet port; a plurality of microchambers; a plurality of loading conduits each fluidly coupled to the inlet port and the plurality of microchambers; and a plurality of siphon conduits, each of the siphon conduits fluidly coupling the loading conduit to the microchamber, wherein a microchamber of the plurality of microchambers optionally comprises a first side substantially facing the loading conduit and a second side that does not face the loading conduit or substantially faces an adjacent microchamber of the plurality of microchambers, wherein the siphon conduit fluidly couples the loading conduit to the microchamber via the second side.

[0119] Embodiment 2D. The microfluidic device of embodiment 1D, wherein the siphon conduit comprises a curved conduit.

[0120] Embodiment 3D. The microfluidic device of embodiment 2D, wherein the curved path includes an approximately 90 degree turn.

[0121] Embodiment 4D. The microfluidic device of embodiment 2D, wherein the curved conduit comprises a turn having a radius of curvature of at least about 10 microns.

[0122] Embodiment 5D. A microfluidic device as described in embodiment 1D, wherein each of the plurality of siphon conduits is substantially equal in length.

[0123] Embodiment 6D. A microfluidic device as described in embodiment 1D, wherein each of the plurality of siphon conduits is disposed at a substantially equidistant position on the loading conduit.

[0124] Embodiment 7D. The microfluidic device of embodiment 1D, wherein the microfluidic device is a thermoplastic injection molded microfluidic device.

[0125] Embodiment 8D. A microfluidic device according to any of embodiments 1D-7D, further comprising a thin film applied in the form of a surface of the microfluidic device.

[0126] Embodiment 9D. The microfluidic device of embodiment 8D, wherein the thin film in the form of a surface provides an exterior surface of a plurality of microchambers.

[0127] Embodiment 10D. The microfluidic device of embodiment 9D, wherein the surface formed by the thin film further provides an outer surface of a plurality of loading conduits.

[0128] Embodiment 11D. A microfluidic device according to any of embodiments 8D-10D, wherein the thin film has a thickness of about 70-90 microns.

[0129] Embodiment 12D. A microfluidic device according to any of embodiments 8D-10D, wherein the thin film has a thickness of about 80 microns.

[0130] Embodiment 13D. The microfluidic device of any of embodiments 8D-12D, wherein the thin film comprises a gas-permeable thermoplastic material.

[0131] Embodiment 14D. The microfluidic device of embodiment 13D, wherein the gas permeable thermoplastic material is not permeable to the sample.

[0132] Embodiment 15D. The microfluidic device of any of embodiments 13D-14D, wherein the gas permeable thermoplastic material comprises a cyclic olefin copolymer.

[0133] Embodiment 16D. A microfluidic device according to any of embodiments 1D-15D, wherein at least a portion of the microfluidic device is substantially optically transparent.

[0134] Embodiment 17D. A microfluidic device according to any of embodiments 1D-15D, wherein the microfluidic device is one of a plurality of microfluidic devices that collectively form a sequential injection molded thermoplastic part.

[0135] Embodiment 18D. The microfluidic device of embodiment 17D, wherein the microfluidic device is not fluidically coupled to another microfluidic device of the plurality of microfluidic devices.

[0136] Embodiment 1E. A microfluidic device configured to process a biological sample, comprising: an inlet port; a plurality of microchambers; and a plurality of loading conduits, each fluidly coupled to the inlet port and the plurality of microchambers, wherein one microchamber of the plurality of microchambers comprises a substantially rectangular three-dimensional shape including four substantially rectangular sidewalls, and two adjacent sidewalls are joined by a curved corner.

[0137] Embodiment 2E A microfluidic device as described in embodiment 1E, wherein the curved corners have a radius of at least about 10 microns.

[0138] Embodiment 3E A microfluidic device as described in embodiment 1E, wherein each of the plurality of microchambers has a depth of at least about 100 microns.

[0139] Embodiment 4E A microfluidic device as described in embodiment 1E, wherein the ratio of the depth of the microchamber to the minimum distance between the microchamber and an adjacent microchamber of the plurality of microchambers is at least about 3:1.

[0140] Embodiment 5E A microfluidic device as described in embodiment 1E, wherein the ratio of the depth of the microchamber to the minimum distance between the microchamber and an adjacent microchamber of the plurality of microchambers is at least about 5:1.

[0141] Embodiment 1F. A method of loading a sample into a microfluidic device including a plurality of microfluidic assemblies each configured to process the sample, the method comprising applying a plurality of pressure pulses to contents within the microfluidic assemblies, the plurality of pressure pulses comprising a first pressure applied for a first time interval and a second pressure applied for a second time interval, each of the microfluidic assemblies comprising an inlet, a loading conduit fluidly coupled at a first end to the inlet, at least one dead-end microchamber configured to receive a sample, and a siphon conduit fluidly coupled to the microchamber and the loading conduit, wherein a volume of the sample is drawn into the plurality of microchambers of the microfluidic device.

[0142] Embodiment 2F. The method of embodiment 1F, wherein the volume of sample drawn into the microfluidic device is less than the total volume capacity of the microchamber.

[0143] Embodiment 3F. The method of embodiment 1F, wherein the first pressure is at least about 75 psi and the second pressure is at least about 10 psi.

[0144] Embodiment 4F. The method of embodiment 1F, wherein the first time interval and the second time interval are substantially equal.

[0145] Embodiment 5F. The method of embodiment 1F, wherein the first time interval and the second time interval are each at least about 2.5 seconds and at most about 10 seconds.

[0146] Embodiment 6F. The method of any of embodiments 1F-5F, wherein the microfluidic device further comprises a gas permeable film forming a surface of at least one of the loading conduit, the at least one microchamber, and the siphon conduit.

[0147] Embodiment 7F. The method of embodiment 6F, wherein the gas-permeable film is not gas-permeable at atmospheric pressure, but is gas-permeable at pressures greater than atmospheric pressure.

[0148] Embodiment 8F. The method of embodiment 6F, wherein applying a pressure pulse causes gas in the at least one microchamber to pass through a gas permeable film.

[0149] Embodiment 9F. The method of embodiment 6F, wherein the gas permeable film has a thickness of less than about 80 microns.

[0150] Embodiment 10F. The method of embodiment 6F, wherein the gas permeable film comprises a thermoplastic material.

[0151] Embodiment 11F. The method of embodiment 10F, wherein the thermoplastic material comprises a cyclic olefin copolymer.

[0152] Embodiment 12F. The method of embodiment 6F, wherein the gas permeable film is substantially transparent.

[0153] Embodiment 13F. The method of embodiment 6F, wherein the gas permeable film is configured to be substantially impermeable to liquids.

[0154] Embodiment 14F. The method of any of embodiments 1F-13F, wherein the method is performed using a single integrated machine.

[0155] Embodiment 15F. The method of any of embodiments 1F-14F, wherein the sample comprises polymerase chain reaction (PCR) reagents and nucleic acid molecules.

[0156] Embodiment 16F. A method for processing a biological sample, comprising the loading method of any one of embodiments 1F to 15F, and further comprising performing PCR amplification by thermal cycling the multiple microchambers.

[0157] Embodiment 17F. The method of embodiment 16F, further comprising acquiring an image of the plurality of microchambers.

[0158] Embodiment 18F. The method of embodiment 17F, further comprising counting the number of microchambers within an image of the plurality of microchambers in which PCR amplification was successfully achieved.

[0159] Embodiment 19F. The method of embodiment 18F, further comprising applying Poisson statistics to the number of the plurality of microchambers in which PCR amplification was successfully achieved to derive the nucleic acid concentration.

[0160] Embodiment 20F. The method of any of embodiments 1F-19F, further comprising at least one dead-end termination chamber fluidly coupled to the loading conduit and configured to receive an overfill of sample to reduce crosstalk.

[0161] Embodiment 21F. The method of any of embodiments 1F-20F, wherein the loading conduit further comprises a plurality of secondary conduits and a splitter conduit structure fluidly coupling at least one inlet to the plurality of secondary conduits.

[0162] Embodiment 22F. The method of embodiment 21F, wherein the plurality of secondary conduits comprises a plurality of linear secondary conduits, each of the plurality of linear secondary conduits being connected to the splitter conduit structure at a first secondary conduit end and connected to the termination chamber at a second secondary conduit end.

[0163] Embodiment 23F. A method of loading a sample into a microfluidic device configured to process the sample, the microfluidic device comprising an inlet, at least one loading conduit fluidly coupled to the inlet at a first end, a plurality of microchambers, and a plurality of siphon conduits fluidly coupling the plurality of microchambers to the at least one loading conduit, the method comprising applying a plurality of pressure pulses to a fluid content of the microfluidic device, the plurality of pressure pulses comprising alternating peaks comprising a first pressure applied for a first interval of time and a plurality of valleys comprising a second pressure applied for a second interval of time, thereby forcing gas in the plurality of microchambers through a gas permeable film, wherein a volume of reagent comprising the sample is drawn into the microchambers of the microfluidic device, the microfluidic device further comprising a gas permeable film forming a surface of the at least one loading conduit, the plurality of microchambers, and the plurality of siphon conduits.

[0164] Embodiment 24F. The method of embodiment 23F, wherein the volume of reagents, including the sample, drawn into the microfluidic device is less than the total volume capacity of the plurality of microchambers.

[0165] Embodiment 25F. The method of any of embodiments 23F-24F, further comprising drawing a volume of non-sample fluid into the microfluidic device.

[0166] Embodiment 26F. The method of any of embodiments 23F-25F, wherein the first pressure is at least about 75 psi and the second pressure is about 10 psi.

[0167] Embodiment 27F. The method of any of embodiments 23F-26F, wherein the first time interval and the second time interval are substantially equal.

[0168] Embodiment 28F. The method of any of embodiments 23F-26F, wherein the first time interval and the second time interval are each at least about 2.5 seconds and at most about 10 seconds.

[0169] Embodiment 29F. The method of any of embodiments 23F-28F, further comprising applying a third pressure to the fluid contents of the microfluidic device for a third time interval after the multiple pressure pulses are applied.

[0170] Embodiment 30F. The method of embodiment 29F, wherein the third pressure is at least about 50 psi.

[0171] Embodiment 31F. The method of any of embodiments 29F-30F, wherein the third time interval is at least about 5 minutes.

[0172] Embodiment 32F. The method of any of embodiments 23F-31F, wherein the gas permeable film has a thickness of less than about 80 microns.

[0173] Embodiment 33F. The method of any of embodiments 23F-32F, wherein the gas permeable film comprises a thermoplastic material.

[0174] Embodiment 34F. The method of embodiment 33F, wherein the thermoplastic material comprises a cyclic olefin copolymer.

[0175] Embodiment 35F. The method of any of embodiments 23F-34F, wherein the gas permeable film is substantially transparent.

[0176] Embodiment 36F. The method of any of embodiments 23F-35F, wherein the gas-permeable film is configured to be substantially impermeable to liquids.

Claims

1. 1. A microfluidic device configured to process a sample, comprising: an inlet port; a closure system fluidly coupled to the inlet port, the closure system comprising: a plurality of linear loading conduits; a plurality of termination chambers, one of the plurality of termination chambers being fluidly coupled to one of the plurality of linear loading conduits; a plurality of microchambers for receiving the sample, wherein a plurality of microchambers of the plurality of microchambers are fluidly coupled to a linear loading conduit of the plurality of linear loading conduits; 1. A microfluidic device comprising: a first plurality of microchambers and a first terminal chamber fluidly coupled to the same adjacent linear loading conduit; a volume of the first terminal chamber that is less than or equal to a total volume of the first plurality of microchambers; and a waste reservoir larger than any of the terminal chambers that is excluded from the closed system.

2. 10. The microfluidic device of claim 1, further comprising a plurality of siphon conduits, wherein one siphon conduit of the plurality of siphon conduits is fluidly coupled between the linear loading conduit and the microchamber.

3. The microfluidic device of claim 1 , wherein the volume of the termination chamber is greater than the volume of one of the microchambers.

4. The microfluidic device of claim 3 , wherein the volume of the termination chamber is at least about four times the volume of the microchamber.

5. 3. The microfluidic device of claim 2, wherein the first dimension of the linear loading conduit and the first dimension of the siphon conduit are less than about 10 microns.

6. 10. The microfluidic device of claim 1, wherein one microchamber of the plurality of microchambers has a first dimension of at least about 100 microns.

7. The microfluidic device of claim 1 , further comprising a membrane disposed on the microfluidic device, the membrane forming a surface of the closed system.

8. The microfluidic device of claim 7 , wherein the surface formed by the thin film provides an outer surface of the plurality of microchambers.

9. The microfluidic device of claim 8 , wherein the surface formed by the thin film provides an outer surface of the plurality of linear loading conduits.

10. The microfluidic device of claim 9 , wherein the surface formed by the thin film provides an outer surface of the plurality of termination chambers.

11. The microfluidic device of claim 7, wherein the thin film has a thickness of about 70 to 90 microns.

12. The microfluidic device of claim 7 , wherein the thin film has a thickness of about 80 microns.

13. The microfluidic device of claim 7 , wherein the membrane comprises a gas-permeable thermoplastic material.

14. The microfluidic device of claim 13 , wherein the gas-permeable thermoplastic material is impermeable to the sample.

15. The microfluidic device of claim 13 , wherein the gas-permeable thermoplastic material comprises a cyclic olefin copolymer.

16. The microfluidic device of any one of claims 1 to 15, wherein the volume of the first terminal chamber is less than or equal to fifty percent (50%) of the total volume of the first plurality of microchambers.

17. The microfluidic device of any one of claims 1 to 15, wherein the volume of the first terminal chamber is no more than twenty-five percent (25%) of the total volume of the first plurality of microchambers.

18. The microfluidic device of any one of claims 1 to 15, wherein the volume of the first terminal chamber is less than or equal to ten percent (10%) of the total volume of the first plurality of microchambers.

19. The microfluidic device of any one of claims 1 to 15, comprising an injection molded thermoplastic material.

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