Microfluidic device

Abstract

Microfluidic devices are disclosed. The microfluidic device includes a microscopic feature disposed in a loading conduit thereof, the microscopic feature configured to separate impurities from a fluid flowing from the loading conduit to a sample compartment. The loading conduit includes a filtration section including the microscopic feature and a narrowed section in fluid communication with the filtration section. The filter section has a larger cross-sectional area than the cross-sectional area of the narrowed section, thereby increasing the filtration capability of the loading conduit of the microfluidic device.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 549,048, filed February 2, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to microfluidic devices. More specifically, this disclosure relates to microfluidic devices for bioanalytical assays. Even more specifically, this disclosure relates to microfluidic devices including features for sample filtration. Background Technology

[0004] Microfluidic devices handle small-scale fluids. Typically, microfluidic devices operate at the sub-millimeter level and handle microliters, nanoliters, or even smaller volumes of fluid.

[0005] Microfluidic devices can be used to process samples for a variety of bioanalytical assays that typically require high accuracy in terms of sample size and / or concentration. However, many currently available microfluidic devices suffer from drawbacks, including contamination / impurities, air trapping, operational difficulties, and high manufacturing complexity, resulting in suboptimal performance when using these devices.

[0006] Overall, while microfluidic devices exist for bioanalytical testing, the field still needs improvement due to at least the aforementioned drawbacks of these devices. Utility Model Content

[0007] Solutions have been found to at least some of the aforementioned problems associated with microfluidic devices, particularly those used in bioanalytical assays. These solutions reside in the loading conduit of the microfluidic device, which includes microscopic features configured to separate impurities from the fluid loaded into the microfluidic device, thereby reducing the level of impurities / contaminants in the loaded liquid sample. This can help limit physical and / or chemical interferences caused by these impurities during bioanalytical assays, thus improving the accuracy of test results. For example, the microfluidic device of this disclosure can be used in digital PCR (dPCR) assays, which generate low-intensity signals from each sample partition. Therefore, interference caused by impurities in the sample fluid can have a significant negative impact on the accuracy of test results. The microfluidic device disclosed herein implements microscopic features (e.g., column arrays) in the sample loading conduit to facilitate the filtration of the sample fluid, which ultimately improves the performance of dPCR assays.

[0008] Furthermore, the loading conduit of the microfluidic device disclosed herein includes various sections with different cross-sectional areas, thereby further increasing the filtration capacity of the loading conduit and reducing impurities loaded into the sample compartment. For example, the loading conduit may include a filtration section and a narrow section in fluid communication with the filtration section, the filtration section including microscopic features for separating impurities from the fluid flowing therethrough. The cross-sectional area of ​​the filtration section may be larger than that of the narrow section, such that impurities including large particles cannot flow into the narrow section, thereby separating impurities from the fluid.

[0009] Therefore, the microfluidic device disclosed herein provides technical achievements for at least some of the problems associated with the aforementioned currently available microfluidic devices.

[0010] Some embodiments relate to microfluidic devices. In some aspects, the microfluidic device includes: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of micro-features disposed within the loading conduit and configured to separate impurities from fluid flowing from the loading conduit to the sample compartments. The loading conduit includes a filtration section and a narrow section in fluid communication with the filtration section. The filtration section has a larger cross-sectional area than the narrow section.

[0011] Some embodiments relate to microfluidic devices. In some aspects, the microfluidic device includes: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of columns disposed within the loading conduit and configured to separate impurities from fluid flowing from the loading conduit to the sample compartments. The loading conduit includes a first section, a filtering section, and a narrowing section. The first section, the filtering section, and the narrowing section have different cross-sectional areas and are configured to enhance the filtering capacity of the loading conduit. The plurality of columns are disposed within the filtering section of the loading conduit.

[0012] Some embodiments involve a method of loading a liquid comprising a biological sample into a microfluidic device disclosed herein. In some aspects, the method includes aspirating a liquid mixture to allow it to flow through a loading conduit into a sample compartment. The liquid in the sample compartment is substantially free of impurities having a minimum size greater than 10 micrometers.

[0013] Some embodiments relate to methods for processing biological samples. In some aspects, the method includes: applying multiple pressure pulses to a liquid comprising the biological sample, causing at least some of the liquid to flow through a loading conduit of the microfluidic device disclosed herein into a sample compartment; and performing PCR amplification by thermally cycling the liquid within the sample compartment.

[0014] The following includes definitions of various terms and phrases used throughout this specification.

[0015] The terms “about” or “approximately” are defined as those understood by one of ordinary skill in the art. In one non-limiting embodiment, these terms are defined as less than 10%, preferably less than 5%, more preferably less than 1%, and most preferably less than 0.5%.

[0016] The term “substantially” and its variants are defined as the range that includes within 10%, within 5%, within 1%, or within 0.5%.

[0017] A “chamber” is a structure that enables the deposition of non-sample fluids, fluids containing samples (such as biological samples), or solutions or reagents containing samples in a microfluidic device. Examples of structures that enable sample deposition and digitization include pores, chambers, and microchambers.

[0018] A "conduit" is a structure that enables the movement of a sample fluid or a non-sample fluid. Examples of structures that enable fluid movement include conduits, channels, microconduits, microchannels, siphon conduits, siphon channels, and siphon orifices.

[0019] The term "depth" used in reference to the microfluidic devices discussed in this specification generally refers to the distance from the bottom of the conduit, siphon or conduit, chamber or microchamber to the top of the sidewall of the conduit, siphon or conduit, chamber or microchamber, or the breathable membrane or film covering the conduit, siphon or conduit, chamber or microchamber.

[0020] "Fluid" generally refers to either a liquid or a gas. Fluids do not maintain a definite shape and flow or move such that the fluid particles undergo continuous surface area changes over an observable timeframe to fill the container in which they are placed. Therefore, fluids can have any suitable viscosity that allows for movement. If two or more fluids are present, a person skilled in the art can independently choose each fluid in substantially any fluid (liquid, gas, etc.).

[0021] As used herein, the term "biological sample" means a sample or solution containing any type of biochemical or component and / or any target molecule of interest to a user, manufacturer, or distributor of the various embodiments of this application described or implied herein, as well as any sample or solution containing related chemicals or compounds for performing bioassays, experiments, or tests. These biochemicals, components, or target molecules may include, but are not limited to, DNA sequences (including cell-free DNA), RNA sequences, genes, oligonucleotides, molecules, proteins, biomarkers, cells (e.g., circulating tumor cells), or any other suitable target biomolecule. A biological sample may include one or more of the following: at least one target nucleic acid sequence, at least one primer, at least one buffer, at least one nucleotide, at least one enzyme, at least one detergent, at least one blocking agent, or at least one dye, labeler, and / or detector suitable for detecting a target or reference nucleic acid sequence. In various embodiments, such biological components may be used in conjunction with one or more PCR methods and systems for applications such as fetal diagnostics, multiplex dPCR, viral detection and quantitative standards, genotyping, sequencing, experiments or protocols, sequencing validation, mutation detection, detection of genetically modified organisms, detection of rare alleles, and / or copy number changes.

[0022] "Microfluidic" generally refers to a device, structure, article, region, system, or chip comprising at least one catheter and optionally multiple siphon orifices or catheters, as well as an array of chambers or microchambers. For example, a catheter may have a cross-sectional dimension of less than or equal to about 1 mm, less than or equal to about 750 micrometers, less than or equal to about 500 micrometers, less than or equal to about 250 micrometers, less than or equal to about 100 micrometers, or smaller. A catheter or siphon catheter or orifice may have a cross-sectional dimension of less than or equal to about 50 micrometers, less than or equal to about 10 micrometers, or smaller.

[0023] As used in this specification and / or claims, the term "minimum size" means the minimum size of an object, particle, or any other individual impurity unit contained in a fluid, including overall height, overall width, overall length, and overall diameter.

[0024] The term “cross-sectional area” for each section of the loading conduit (e.g., loading pad, first section, filter section, or narrow section) disclosed throughout this specification is defined as a cross-sectional surface tangential to the intended flow direction of the fluid in the loading conduit.

[0025] The terms “suppress” or “reduce” or “prevent” or “avoid” or any variations thereof, when used in the claims and / or specification, include any measurable reduction or complete suppression to achieve the desired result.

[0026] The term “effective” as used in this specification and / or claims means sufficient to achieve the desired, expected, or anticipated result.

[0027] When used in the claims or description with the terms “comprising,” “including,” “containing,” or “having,” the use of the word “a” or “an” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more.”

[0028] The words “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include)”), or “containing” (and any form of containing, such as “contains” and “contain)”) are inclusive or open-ended and do not exclude additional, unlisted elements or steps of the process.

[0029] The apparatus, sample plate and / or device disclosed herein may “include” specific components, compositions, etc. disclosed throughout the specification, “consistently constitute” or “made up of” them.

[0030] Other objects, features, and advantages of the embodiments will become apparent from the following figures, detailed description, and examples. However, it should be understood that while the figures, detailed description, and examples indicate particular embodiments, they are given by way of illustration only and are not intended to be limiting. Furthermore, changes and modifications in spirit and scope are expected to become apparent to those skilled in the art from this detailed description. In another embodiment, features from a particular embodiment may be combined with features from other embodiments. For example, a feature from one embodiment may be combined with features from any of the other embodiments. In another embodiment, additional features may be added to the specific embodiments described herein. Attached Figure Description

[0031] For a more complete understanding, please refer to the following description in conjunction with the accompanying drawings, in which:

[0032] Figure 1 This is a schematic diagram of a microfluidic device according to the embodiments disclosed in this specification;

[0033] Figure 2AThis is a schematic top view of the filter section connected to the first section and narrow section of the loading conduit, as shown in the embodiment disclosed in the specification.

[0034] Figure 2B It is a schematic diagram of the front view of the cross-sectional area of ​​the first section connected to the filter section of the loading conduit according to the embodiment disclosed in the specification;

[0035] Figure 3A It is a schematic diagram of a sample plate based on the implementation scheme disclosed in the instruction manual;

[0036] Figure 3B It is a schematic diagram of a glass slide according to the embodiment disclosed in the specification, which is removably fixed to the frame of the sample plate;

[0037] Figure 4 Various configurations of the microstructure in the filtration section of a loading conduit for a microfluidic device according to embodiments disclosed in the specification are shown. Detailed Implementation

[0038] Currently available microfluidic devices for bioanalytical testing have several drawbacks, including contamination and air trapping, difficulty in use, and high production costs. The embodiments disclosed in this specification provide solutions to at least some of these problems. The solution is based on a microfluidic device comprising: a loading conduit in fluid communication with multiple sample compartments; and multiple micro-features disposed within the loading conduit and configured to separate impurities from the fluid flowing from the loading conduit to the sample compartments. Therefore, the microfluidic device is configured to reduce the chance of contamination in the sample compartments caused by impurities, thereby improving the accuracy of bioanalytical test results using the microfluidic device. Furthermore, the loading conduit may include multiple segments with different cross-sectional areas to increase the filtration capacity of the loading conduit, thereby further ensuring the accuracy of the bioanalytical test.

[0039] These and other non-limiting aspects of this application are discussed in more detail in the following sections.

[0040] A. Microfluidic devices

[0041] Microfluidic devices are typically designed to contain and / or process biological samples for bioanalytical assays. For conventional microfluidic devices used in bioanalytical assays, particularly digital polymerase chain reaction (dPCR) assays, contamination from impurities (e.g., solid particles, fibers, reagent precipitates, sample matrices, etc.) can significantly negatively impact the accuracy of test results due to the small sample compartment size and limited signal intensity of each sample compartment during assays. Currently available microfluidic devices may be limited in achieving integrated filtration and / or insufficient filtration capacity. The microfluidic devices disclosed in this specification implement microscopic features, such as columns, within the loading conduit to filter and separate impurities from the liquid containing the biological sample, thereby improving the purity of the sample loaded in the sample compartment and the accuracy of the test results.

[0042] refer to Figure 1 The diagram shows a schematic of a microfluidic device 100. The microfluidic device 100 includes a loading conduit 110 in fluid communication with a plurality of sample compartments 208. In some embodiments, the loading conduit 110 includes a loading pad 101, a first section 102, a filtering section 103, and a narrow section 104.

[0043] According to some embodiments, the loading pad 101 is configured as an inlet for receiving fluid (e.g., a liquid containing a biological sample). The cross-sectional area of ​​the loading pad 101 may be 0.47 mm. 2 up to 0.83mm 2 The range and all values ​​and ranges within it, including the following range: 0.47mm 2 up to 0.50mm 2 0.50mm 2 Up to 0.53mm 2 0.53mm 2 up to 0.56mm 2 0.56mm 2 up to 0.59mm 2 0.59mm 2 Up to 0.62mm 2 0.62mm 2 Up to 0.65mm 2 0.65mm 2 Up to 0.68mm 2 0.68mm 2 Up to 0.71mm 2 0.71mm 2 Up to 0.74mm 2 0.74mm 2 Up to 0.77mm 2 0.77mm 2 Up to 0.80mm 2 and 0.80mm2 up to 0.83mm 2 In some embodiments, the outlet of loading pad 101 is in fluid communication with the first section 102. Loading pad 101 has a larger cross-sectional area than the first section 102. The cross-sectional area of ​​the first section 102 is approximately 0.010 mm². 2 Up to 0.020mm 2 and all values ​​and ranges therein, including the following range: 0.010 mm 2 Up to 0.011mm 2 0.011mm 2 Up to 0.012mm 2 0.012mm 2 Up to 0.013mm 2 0.013mm 2 Up to 0.014mm 2 0.014mm 2 Up to 0.015mm 2 0.015mm 2 Up to 0.016mm 2 0.016mm 2 Up to 0.017mm 2 0.017mm 2 Up to 0.018mm 2 0.018mm 2 Up to 0.019mm 2 and 0.019mm 2 Up to 0.020mm 2 In some embodiments, the ratio of the cross-sectional area of ​​the loading pad 101 to the cross-sectional area of ​​the first segment 102 may be in the range of 29.3 to 83 and for all ranges and values ​​therebetween, including the following ranges: 29.3 to 30, 30 to 33, 33 to 36, 36 to 39, 39 to 42, 42 to 45, 45 to 48, 48 to 51, 51 to 54, 54 to 57, 57 to 60, 60 to 63, 63 to 66, 66 to 69, 69 to 72, 72 to 75, 75 to 78, 78 to 82, and 82 to 83.

[0044] In some implementations, the outlet of the first section 102 is in fluid communication with the inlet of the filtration section 103. In some aspects, the outlet of the first section 102 and the inlet of the filtration section 103 form a curved boundary (e.g., Figure 2A (As shown). Compared to a straight boundary, a curved boundary is constructed to increase the filtration capacity of the loading conduit 110 by increasing the cross-sectional area of ​​the loading conduit 110. In some embodiments, the depth of the first segment 102 (e.g.) Figure 2BThe d1 shown is approximately 80 micrometers to 120 micrometers and all values ​​therein, including the following ranges: 80 micrometers to 84 micrometers, 84 micrometers to 88 micrometers, 88 micrometers to 92 micrometers, 92 micrometers to 96 micrometers, 96 micrometers to 100 micrometers, 100 micrometers to 104 micrometers, 104 micrometers to 108 micrometers, 108 micrometers to 112 micrometers, 112 micrometers to 116 micrometers, and 116 micrometers to 120 micrometers. The depth of filter section 103 (e.g., Figure 2B The d shown f The range and values ​​are approximately 10 micrometers to 20 micrometers and therebetween, including the following ranges: 10 micrometers to 12 micrometers, 12 micrometers to 14 micrometers, 14 micrometers to 16 micrometers, 16 micrometers to 18 micrometers, and 18 micrometers to 20 micrometers. The transition between the depth of the first section 102 and the depth of the filtration section 103 is sloping (e.g., ...). Figure 2B As shown in S1), to prevent fluid trapping. In some embodiments, the first segment 102 has a length of about 0.7 mm to 1.5 mm (e.g., S1). Figure 2A and Figure 2B (as shown in the x-direction) and a width of approximately 0.10 mm to 0.14 mm (e.g.) Figure 2A and Figure 2B (as shown in the y-direction). The filter section 103 may have a length of approximately 0.2 mm to 0.25 mm (e.g., in the y-direction). Figure 2A and Figure 2B (as shown in the x-direction) and a width of approximately 0.10 mm to 0.14 mm (e.g.) Figure 2A and Figure 2B y direction shown).

[0045] In some embodiments, multiple micro-features are disposed within the filtration section 103 of the loading conduit 110. The micro-features are configured to separate impurities from the fluid flowing from the loading conduit 110 to the sample compartment 208. Exemplary micro-features may include columns, parallel channels, debris-deflecting columns, and any combination thereof. Exemplary impurities may include particles, fibers, reagent precipitates, sample matrices, and any combination thereof. In some cases, impurities may have a minimum size of 10 micrometers to 50 micrometers and values ​​thereof, including the following ranges: 10 micrometers to 15 micrometers, 15 micrometers to 20 micrometers, 20 micrometers to 25 micrometers, 25 micrometers to 30 micrometers, 30 micrometers to 35 micrometers, 35 micrometers to 40 micrometers, 40 micrometers to 45 micrometers, and 45 micrometers to 50 micrometers.

[0046] In some embodiments, the microscopic features include column 105. In some aspects, column 105 is positioned near the inlet of narrow section 104. According to embodiments, column 105 is further configured to limit emulsion formation and / or air retention as fluid flows toward narrow section 104 through filtration section 103. Each column in column 105 may have a cross-sectional area (a cross-sectional area tangent to the height of the column) that is substantially circular, triangular, rectangular, square, polygonal, or any combination thereof.

[0047] According to some embodiments, columns 105 are arranged in at least one row. In some cases, columns 105 in the filtration section 103 are arranged in an interlaced pattern comprising at least three rows. In some aspects, columns 105 are positioned in an interlaced pattern to minimize the retention of stagnant fluid in the filtration section 103 and / or to promote a change in the direction of the fluid flow path to increase the chance of capturing impurities between columns 105. In some embodiments, each column in column 105 may have a substantially circular top surface and a substantially circular bottom surface. In some aspects, the bottom surface of each column in column 105 is larger than the top surface. The columns in column 105 may taper toward their top. According to embodiments, the top surface of each column in column 105 may have a diameter ranging from 0.018 mm to 0.022 mm and all ranges and values ​​therebetween, including the ranges of 0.018 mm to 0.019 mm, 0.019 mm to 0.020 mm, 0.020 mm to 0.021 mm, and 0.021 mm to 0.022 mm. The bottom surface of each column in column 105 may have a diameter ranging from 0.020 mm to 0.028 mm and all values ​​therebetween, including 0.020 mm to 0.021 mm, 0.021 mm to 0.022 mm, 0.022 mm to 0.023 mm, 0.023 mm to 0.024 mm, 0.024 mm to 0.025 mm, 0.025 mm to 0.026 mm, 0.026 mm to 0.027 mm, and 0.027 mm to 0.028 mm. The height of at least one column in column 105 may range from 0.012 mm to 0.016 mm and all values ​​therebetween, including the following ranges: 0.012 mm to 0.013 mm, 0.013 mm to 0.014 mm, 0.014 mm to 0.015 mm, and 0.015 mm to 0.016 mm. In some embodiments, column 105 comprises cyclic olefin polymers, cyclic olefin copolymers, or any combination thereof.

[0048] According to some implementation schemes, columns 105 are arranged in three rows, with the first row containing four columns, the second row containing three columns, and the third row containing four columns (e.g., ...). Figure 2AAs shown, rows are numbered from left to right. In some embodiments, columns 105 are positioned such that the minimum distance between two adjacent columns is approximately 6 to 10 micrometers and all ranges and values ​​in between, including the following ranges: 6 to 7 micrometers, 7 to 8 micrometers, 8 to 9 micrometers, and 9 to 10 micrometers. According to the embodiment, the column ( Figure 2A The columns 105a or 105b shown are positioned at each corner of the end of the filter section 103 that connects to the narrow section 104 (near the end of the narrow section 104). In some aspects, columns 105a and 105b are configured to further restrict emulsion formation in the fluid flowing through the filter section 103. In embodiments, the outlet of the filter section 103 is in fluid communication with the narrow section 104. In some embodiments, the cross-sectional area of ​​the filter section 103 is 0.0010 mm. 2 Up to 0.0025mm 2 The range and all ranges and values ​​within it, including the following range: 0.0010 mm 2 up to 0.0013mm 2 0.0013mm 2 Up to 0.0016mm 2 0.0016mm 2 Up to 0.0019mm 2 0.0019mm 2 Up to 0.0022mm 2 and 0.0022mm 2 Up to 0.0025mm 2 The cross-sectional area of ​​the narrow section 104 is 0.00025 mm. 2 up to 0.00046mm 2 The range and all ranges and values ​​within it, including the following range: 0.00025mm 2 up to 0.00028mm 2 0.00028mm 2 up to 0.00031mm 2 0.00031mm 2 up to 0.00034mm 2 0.00034mm 2 up to 0.00037mm 2 0.00037mm 2 up to 0.00040mm 2 0.00040mm 2 up to 0.00043mm 2 and 0.00043mm 2 up to 0.00046mm 2The ratio of the cross-sectional area of ​​the filtration section 103 to the cross-sectional area of ​​the narrow section 104 can be in the range of 4 to 5.4 and all ranges and values ​​therebetween. The narrow section 104 can have a depth of 12 micrometers to 16 micrometers and all ranges and values ​​therebetween, including the following ranges: 12 micrometers to 13 micrometers, 13 micrometers to 14 micrometers, 14 micrometers to 15 micrometers, and 15 micrometers to 16 micrometers. In some embodiments, the column 105 having a substantially circular top and bottom surface is configured to break down impurities into smaller impurities compared to columns having other shapes of top and bottom surfaces, thereby preventing larger impurities from clogging the loading conduit.

[0049] In some aspects, the sample compartment 208 of the microfluidic device 100 is configured as a partition to accommodate a biological sample for bioanalytical testing. The sample compartment 208 may include any structure that can divide the fluid into multiple partitions. Exemplary sample compartments may include microchambers, through-holes, micropores, recesses, and any combination thereof.

[0050] In some embodiments, the microfluidic device 100 is configured to process biological samples and / or reagents for polymerase chain reaction (PCR). In some aspects, the microfluidic device 100 is configured to contain biological samples and / or reagents for digital PCR (dPCR). Each sample compartment in the sample compartment 208 may have a volume of 100 picoliters to 1000 picoliters. In some respects, each sample compartment in sample compartment 208 has a volume of 477 picoliters to 583 picoliters and all values ​​and ranges therebetween, including the following ranges: 477 picoliters to 487 picoliters, 487 picoliters to 497 picoliters, 497 picoliters to 507 picoliters, 507 picoliters to 517 picoliters, 517 picoliters to 527 picoliters, 527 picoliters to 537 picoliters, 537 picoliters to 547 picoliters, 547 picoliters to 557 picoliters, 557 picoliters to 567 picoliters, 567 picoliters to 577 picoliters and 577 picoliters to 583 picoliters.

[0051] In some cases, sample compartment 208 comprises microcells having a depth of at least about 100 micrometers. The ratio of the depth of the microcell to the minimum distance between the microcell and adjacent microcells may be at least about 3:1 or at least about 5:1. In some aspects, the microcells 208 of the sample compartment comprise a substantially rectangular three-dimensional shape comprising four substantially rectangular sidewalls. Two adjacent sidewalls of the microcell may be connected by a curved corner. The curved corner may have a radius of at least about 10 micrometers. In embodiments, the microfluidic device 100 comprises at least 8,000 sample compartments or at least 10,000 sample compartments. In some cases, the microfluidic device 100 comprises at least 20,000 sample compartments. According to embodiments, the total volume of sample compartment 208 is greater than the volume of loading conduit 110.

[0052] According to some embodiments, the microfluidic device 100 also includes a plurality of termination chambers 307 in fluid communication with the loading conduit 110 and the sample compartment 208. In some embodiments, the termination chambers 307 are configured to contain overflow and / or residual volumes of liquid entering from the loading conduit 110 and / or the sample compartment 208. In embodiments, the loading conduit 110 may be connected to and in fluid communication with a plurality of branch conduits, such that incoming fluid can flow from the loading conduit 110 to the plurality of branch conduits. In some cases, the microfluidic device 100 also includes a plurality of siphon conduits, and the siphon conduits of the plurality of siphon conduits are fluidly coupled to the branch conduits of the plurality of branch conduits to the sample compartments in the plurality of sample compartments 208. In embodiments, the microfluidic device 100 is configured such that incoming fluid is received in the loading conduit 110 and flows sequentially through the branch conduits and siphon conduits into the sample compartments in the plurality of sample compartments 208.

[0053] B. Sample Plate

[0054] In the implementation scheme, sample plates are provided that improve sample quality and accuracy in bioanalytical tests compared to conventional sample plates. Sample plates may include, for example... Figure 1 , Figure 2A and Figure 2B The disclosed and illustrated microfluidic devices (e.g., microfluidic device 100).

[0055] In some implementations, such as Figure 3A As shown, the sample plate 300 includes a frame 311 configured to hold one or more glass slides 312 thereon. Figure 3B As shown, there are more than two of the above-discussed examples (e.g., such as...). Figure 1 The microfluidic devices (e.g., microfluidic devices 100, 100a, 100b, 100c, and 100d) shown in the microfluidic device 100 are disposed or formed on, and / or integrated therein, on, a glass slide 312. In some cases, the glass slide 312 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 microfluidic devices. In embodiments, the frame 311 may be substantially rectangular. The frame 311 may be configured to hold 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 glass slides (e.g., glass slide 312). According to embodiments, the frame 311 may include multiple ports (e.g., port 315). Each loading pad (e.g., Figure 1 The loading pad 101 shown and Figure 3BThe loading pads 101a-101d shown may correspond to and / or be in fluid communication with a port of frame 311. A port (e.g., port 315) may be configured to expel gas from a microfluidic device on a slide. A port (e.g., port 315) may be further configured to receive fluid (e.g., a biological sample) into a microfluidic device disposed on and / or within a slide (e.g., slide 312). According to some embodiments, multiple ports are disposed and / or formed on multiple strips (e.g., strip 316) of frame 311. According to some embodiments, each strip is positioned such that when one or more slides (e.g., slide 312) are secured to frame 311, each port is fitted onto a loading pad (e.g., loading pads 100 and 101a-101d) of the microfluidic device. In some embodiments, the strips (e.g., strip 316) may be disposed across the width or length of frame 311.

[0056] According to an embodiment, frame 311 may include a plurality of gaskets (e.g., gasket 314). Each gasket is configured to be removably secured above a port (e.g., port 315). In some aspects, each gasket includes a cap-like structure disposed above the port. In an embodiment, the gasket is configured to seal the port (e.g., port 316) relative to the external environment. In some embodiments, the gasket (e.g., gasket 314) is configured to be removably secured to a pressure manifold. In some embodiments, the pressure manifold is configured to apply pressure to the microfluidic device through the gasket. In some embodiments, the pressure manifold is further configured to load the microfluidic device through the gasket.

[0057] In some embodiments, sample plate 300 includes a membrane (e.g., membrane 313) applied over one or more microfluidic devices (e.g., microfluidic devices 100, 100a-100d), the membrane being configured to form a cover for sample compartment 208. The membrane may be further configured to form a cover for loading at least a portion of conduits 110, 110a-110d. In embodiments, the membrane (e.g., membrane 313) is impermeable at lower pressures but allows degassing through the membrane when pressure is applied, thus it is at least partially permeable under pressure. In some embodiments of this application, the permeable membrane is impermeable at atmospheric pressure but permeable at pressures above atmospheric pressure. In some embodiments, the membrane is permeable but not liquid-permeable at one or more selected pressures above atmospheric pressure. In some embodiments of this application, the membrane has a thickness of approximately 80 micrometers and is composed of a cyclic olefin polymer. A suitable membrane used in embodiments of this application is the semi-permeable membrane TOPAS. ®COC 6013. In other embodiments of this application, a semi-permeable membrane with a thickness of 60 micrometers, 70 micrometers, 80 micrometers, 90 micrometers, 100 micrometers, or any thickness within these ranges may be used.

[0058] C. Methods using microfluidic devices

[0059] In the embodiments, methods are provided for loading a liquid comprising a biological sample into the microfluidic device disclosed above (e.g., microfluidic devices 100, 100a-100d). The method of loading the liquid comprising the biological sample may include aspirating the liquid to allow it to flow through a loading conduit (e.g., loading conduit 110) into a sample compartment 208. The liquid in the sample compartment may be substantially free of impurities having a minimum size greater than 10 micrometers.

[0060] In this embodiment, the liquid includes reagents used for the PCR process. The liquid may also include oil, water, or any combination thereof. The oil may include silicone oil. According to this embodiment, high pressure can be applied to compress air in a microfluidic device comprising a loading conduit, a siphon conduit, a sample compartment (e.g., a microchamber), and a termination chamber, which draws liquid into the sample compartment. According to the ideal gas law, the amount of fluid drawn in should be approximately equal to the amount of compressed air. Since the volume of the loading conduit (having a depth of at least about 10 micrometers and a width of at least about 10 micrometers) is smaller than the volume of the sample compartment (having a depth of at least about 100 micrometers), under this action, all loading conduits should be filled with sample fluid, meaning that most of the compressed air will remain in the sample compartment and termination chamber. Compressed air will continue to escape through the membrane, drawing more fluid, including biological samples, into the loading conduit, the siphon conduit, and into the sample compartment (e.g., the microchamber). The sample fluid covering the top of the sample fluid, without sample fluid, will later be drawn into the microfluidic device, while the fluid including biological samples continues to displace the space occupied by air, and air continues to escape through the membrane.

[0061] In the step of aspirating a liquid containing a biological sample to flow through a loading catheter, a series of one or more pressure pulses may be applied to the loading pad of the microfluidic device. The pressure pulses may include applying a high pressure for a first predetermined time period (e.g., a short time interval), followed immediately by applying a low pressure for a second predetermined time period (e.g., a short time interval). In one embodiment of this application, the pulses are initiated for 1 minute (6 cycles, each cycle being 75 psi / 10 psi (5 seconds / 5 seconds)) at the beginning (since the material is hydrophobic). In other words, a higher pressure pulse at 75 psi may be applied for a short time interval (e.g., 5 seconds), followed immediately by a lower pressure pulse at 10 psi for a short time interval (e.g., 5 seconds), followed immediately by another higher pressure pulse at 75 psi for 5 seconds. Thus, in 6 cycles, higher and lower pressure pulses may be repeatedly applied sequentially within consecutive 5-second time intervals. In other embodiments of this application, higher or lower numbers of cycles may be considered, such as 5, 10, 12, 20, or more cycles. Different higher or lower pressure pulses can also be applied, for example, lower pressure pulses using less than or greater than 10 psi, or higher pressure pulses using greater than or less than 75 psi. According to some embodiments, high pressure can be continuously applied to the loading pad for an extended period of time (e.g., greater than 1 minute, or greater than 5 minutes, or sometimes greater than 20 minutes) to further degas the sample compartment (e.g., microchamber).

[0062] According to the implementation scheme, the microfluidic device in this method is used to prepare samples for digital PCR testing, and each sample compartment can hold zero, one, or more than one copy of a target molecule. The method may also include performing PCR amplification by thermally cycling the liquid within the sample compartment. The method also includes capturing images of the sample compartments of the microfluidic device and determining the number of sample compartments within which PCR amplification has been successfully achieved based on the images. Prior to thermal cycling, the liquid (sample partitions) in the sample compartments is substantially free of impurities with a minimum size greater than 10 micrometers due to the filtration capabilities provided by the loading conduits (microscopic features in the filtration sections, such as columns) and / or various cross-sectional area sizes of each section, thereby reducing the negative impact of impurities in the incoming sample liquid on the dPCR test results.

[0063] As part of this disclosure, specific examples are included below. These examples are for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art will readily recognize that parameters can be changed or modified to produce substantially the same results.

[0064] Example

[0065] (Efficiency of various structures with microscopic features)

[0066] Eight microstructural features in the filtration section of the test-loaded catheter (AE and control group, such as...) Figure 4 The filtration efficiency (as shown) was determined. Each test used a liquid containing 10-micron beads (fluorescing under a fluorescence microscope) at a concentration of 450 to 500 beads. The microfluidic device discussed above was loaded with these beads. The filtration sections and microchambers (sample compartments) were observed under a microscope to determine the filtration efficiency.

[0067] Beads were observed in the filtration sections of structures A, B, C, D, E, F, and G. No beads were observed in the filtration section of the control group. Additionally, air traps were observed in structures B and C. The emulsion risk, manufacturability risk, and functional risk of structures A, E, and G were further evaluated. Structure G (only one row of columns) was not selected due to its high functional risk caused by a lack of redundancy. Structure E includes a square column array as a micro-feature for filtration; however, the manufacturability risk of a square column array is high compared to circular columns. Therefore, structure A was selected. Structure A requires further improvement to achieve the final design of the filtration section of the loading conduit for the microfluidic device.

[0068] In the context of this specification, at least the following embodiments are described. Embodiment 1 is a microfluidic device comprising: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of micro-features disposed within the loading conduit and configured to separate impurities from fluid flowing from the loading conduit to the sample compartments, wherein the loading conduit includes a filtration section and a narrow section in fluid communication with the filtration section; wherein the filtration section has a larger cross-sectional area than the narrow section. Embodiment 2 is the microfluidic device according to Embodiment 1, wherein the loading conduit further includes a loading pad and a first section in fluid communication with an outlet of the loading pad, the loading pad being configured to receive the fluid therein, and wherein the loading pad has a larger cross-sectional area than the first section. Embodiment 3 is the microfluidic device according to Embodiment 2, wherein the cross-sectional area of ​​the loading pad is 0.47 mm. 2 up to 0.83mm 2 Within the range, and the cross-sectional area of ​​the first segment is within 0.01 mm. 2 Up to 0.016mm 2 Within the range. Embodiment 4 is a microfluidic device according to Embodiment 2 or 3, wherein the cross-sectional area of ​​the filtration section is within 0.0010 mm. 2 Up to 0.0025mm 2 Within the range, and the cross-sectional area of ​​the narrow section is within 0.00025 mm. 2 up to 0.00046mm 2Within the range. Embodiment 5 is a microfluidic device according to any one of embodiments 2 to 4, wherein the outlet of the first section is in fluid communication with the inlet of the filtration section. Embodiment 6 is a microfluidic device according to embodiment 5, wherein the outlet of the first section and the inlet of the filtration section form a curved boundary, the curved boundary being configured to increase the filtration capacity of the loading conduit. Embodiment 7 is a microfluidic device according to any one of embodiments 1 to 6, wherein the microscopic features include columns, debris deflecting columns, parallel channels, or combinations thereof. Embodiment 8 is a microfluidic device according to embodiment 7, wherein the columns are disposed in the filtration section near the inlet of the narrow section of the loading conduit. Embodiment 9 is a microfluidic device according to any one of embodiments 7 and 8, wherein the columns are further configured to restrict emulsion formation and air retention when the fluid flows through the filtration section. Embodiment 10 is a microfluidic device according to any one of embodiments 7 to 9, wherein each of the columns has a cross-sectional area in the shape of a circle, triangle, rectangle, square, polygon, or a combination thereof. Embodiment 11 is a microfluidic device according to any one of embodiments 7 to 10, wherein the columns in the filtration section are arranged in at least one row. Embodiment 12 is a microfluidic device according to any one of embodiments 7 to 11, wherein the columns in the filtration section are arranged in an interlaced pattern comprising at least three rows. Embodiment 13 is a microfluidic device according to any one of embodiments 7 to 12, wherein the columns are positioned such that the minimum distance between two adjacent columns is approximately 6 to 10 micrometers. Embodiment 14 is a microfluidic device according to any one of embodiments 7 to 13, wherein columns are disposed at each corner of the end of the filtration section connected to the narrow section. Embodiment 15 is a microfluidic device according to embodiment 14, wherein the columns at each corner of the end of the filtration section are configured to restrict emulsion formation of the liquid flowing through the filtration section. Embodiment 16 is a microfluidic device according to any one of embodiments 7 to 15, wherein each column of the filtration section has a circular cross-sectional area with a diameter of 20 to 24 micrometers. Embodiment 17 is a microfluidic device according to any one of embodiments 7 to 16, wherein the column comprises a cyclic olefin polymer, a cyclic olefin copolymer, or a combination thereof. Embodiment 18 is a microfluidic device according to any one of embodiments 1 to 17, wherein each sample compartment has a volume of approximately 477 picoliters to 583 picoliters. Embodiment 19 is a microfluidic device according to any one of embodiments 1 to 18, wherein the microfluidic device is configured to contain samples and reagents for polymerase chain reaction (PCR).Embodiment 20 is a microfluidic device according to Embodiment 19, wherein the microfluidic device is configured to contain samples and reagents for digital PCR (dPCR). Embodiment 21 is a microfluidic device according to Embodiment 20, wherein the loading conduit is in fluid communication with at least 10,000 sample compartments.

[0069] Embodiment 22 is a microfluidic device for processing biological samples. The microfluidic device includes: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of columns disposed within the loading conduit and configured to separate impurities from fluid flowing from the loading conduit to the sample compartments; wherein the loading conduit includes a first section, a filtration section, and a narrow section, and wherein the first section, the filtration section, and the narrow section have different cross-sectional areas and are configured to enhance the filtration capacity of the loading conduit. Embodiment 23 is a microfluidic device according to Embodiment 22, wherein the loading conduit further includes a loading pad configured to receive liquid into the loading conduit, and the liquid sequentially flows through the loading pad, the first section, the filtration section, and the narrow section to the sample compartment. Embodiment 24 is a microfluidic device according to Embodiment 23, wherein the cross-sectional area of ​​the loading pad is 0.47 mm². 2 up to 0.83mm 2 Within the range, and the cross-sectional area of ​​the first segment is within 0.010 mm. 2 Up to 0.016mm 2 Within the range. Embodiment 25 is a microfluidic device according to any one of embodiments 22 and 24, wherein the cross-sectional area of ​​the filtration section is within 0.0010 mm. 2 Up to 0.0025mm 2 Within the range, and the cross-sectional area of ​​the narrow section is within 0.00025 mm. 2 up to 0.00046mm 2Within the range. Embodiment 26 is a microfluidic device according to any one of embodiments 22 to 25, wherein the column is a cylinder having a cross-sectional diameter of 20 micrometers to 24 micrometers. Embodiment 27 is a microfluidic device according to any one of embodiments 22 to 26, wherein the columns are arranged in an interlaced pattern to form an array. Embodiment 28 is a microfluidic device according to any one of embodiments 22 to 27, wherein the columns are disposed in the filtration section. Embodiment 29 is a microfluidic device according to embodiment 28, wherein the columns are disposed at the end of the filtration section near the narrow section. Embodiment 30 is a microfluidic device according to embodiment 29, wherein the columns are disposed at each corner of the filtration section near the end of the narrow section and are configured to restrict emulsion formation. Embodiment 31 is a microfluidic device according to any one of embodiments 22 to 30, wherein the columns are arranged in an array comprising at least one row. Embodiment 32 is a microfluidic device according to embodiment 31, wherein the array comprises 3 rows, wherein the first row comprises 4 columns, the second row comprises 3 columns, and the third row comprises 4 columns. Embodiment 33 is a microfluidic device according to any one of embodiments 22 to 32, wherein the impurities include microparticles, fibers, reagent precipitates, sample matrices, or combinations thereof. Embodiment 34 is a microfluidic device according to any one of embodiments 22 to 33, wherein the impurities include microparticles having a minimum size of 10 micrometers to 50 micrometers. Embodiment 35 is a microfluidic device according to any one of embodiments 22 to 34, wherein the fluid includes oil, water, nucleic acids, or combinations thereof. Embodiment 36 is a microfluidic device according to any one of embodiments 22 to 35, wherein each sample compartment has a volume of 477 picoliters to 583 picoliters. Embodiment 37 is a microfluidic device according to any one of embodiments 22 to 36, wherein the microfluidic device includes at least 10,000 sample compartments. Embodiment 38 is a microfluidic device according to any one of embodiments 22 to 37, wherein the microfluidic device includes a plurality of termination chambers in fluid communication with the loading conduit and the sample compartments. Embodiment 39 is a microfluidic device according to any one of embodiments 22 to 38, wherein the termination chambers are configured to receive overflow or remaining volumes of the fluid. Embodiment 40 is a microfluidic device according to any one of embodiments 22 to 39, the microfluidic device further comprising a plurality of siphon conduits, one of the plurality of siphon conduits being in fluid communication with the loading conduit and the sample compartment. Embodiment 41 is a microfluidic device according to embodiment 40, wherein the siphon conduit is configured such that there is fluid communication between the loading conduit and the sample compartment.Embodiment 42 is a microfluidic device according to any one of embodiments 22 to 41, the microfluidic device further comprising a membrane applied to the microfluidic device, the membrane being configured to form a cover for the sample compartment. Embodiment 43 is a microfluidic device according to embodiment 42, wherein the membrane applied to the microfluidic device is further configured to form a cover for at least a portion of the loading conduit. Embodiment 44 is a microfluidic device according to any one of embodiments 42 and 43, wherein the membrane comprises a breathable material. Embodiment 45 is a microfluidic device according to any one of embodiments 22 to 44, wherein the ratio of the depth of the sample compartment to the minimum distance between two adjacent sample compartments is 3.96 to 3.09. Embodiment 46 is a microfluidic device according to any one of embodiments 22 to 45, wherein the microfluidic device is configured to process samples for polymerase chain reaction (PCR). Embodiment 47 is a microfluidic device according to any one of embodiments 22 to 45, wherein the microfluidic device is configured to process samples for digital PCR (dPCR).

[0070] Embodiment 48 is a method of loading a liquid comprising a biological sample into a microfluidic device according to any one of embodiments 22 to 47. The method includes: aspirating the liquid to allow it to flow through the loading conduit into the sample compartment; wherein the liquid in the sample compartment is substantially free of impurities having a size greater than 10 micrometers. Embodiment 49 is the method according to embodiment 48, wherein the liquid further comprises reagents for a PCR process. Embodiment 50 is the method according to embodiment 49, wherein the liquid further comprises oil, an aqueous solution, nucleic acid, or a combination thereof.

[0071] Embodiment 51 is a method for processing a biological sample. The method includes: applying multiple pressure pulses to a liquid mixture comprising the biological sample, causing at least some of the liquid mixture to flow through the loading conduit of the microfluidic device according to any one of embodiments 22 to 47 into the sample compartment; and performing PCR amplification by thermally cycling the liquid mixture in the sample compartment. Embodiment 52 is the method according to embodiment 51, wherein the liquid mixture in the sample compartment is substantially free of impurities having a minimum size greater than 10 micrometers. Embodiment 53 is the method according to any one of embodiments 51 and 52, further comprising capturing an image of the sample compartment of the microfluidic device. Embodiment 54 is the method according to embodiment 53, further comprising determining the number of sample compartments within which PCR amplification has been successfully performed based on the image of the sample compartment of the microfluidic device.

[0072] Although embodiments of the present application and their advantages have been described in detail, it should be understood that various changes, substitutions, and alterations can be made herein without departing from the spirit and scope of the embodiments as defined by the appended claims. Furthermore, the scope of this application is not intended to be limited to the specific embodiments of the systems, apparatus, machines, articles, compositions of matter, methods, and steps described in the specification. As will be readily apparent to those skilled in the art, based on the foregoing disclosure, currently existing or later-developed systems, apparatus, processes, machines, articles, compositions of matter, methods, or steps that perform substantially the same functions or achieve substantially the same results as the corresponding embodiments described herein can be utilized. Therefore, the appended claims are intended to include such processes, machines, manufactures, compositions of matter, methods, or steps within their scope.

Claims

1. A microfluidic device, characterized in that, The microfluidic device comprises: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of microfeatures disposed within the loading conduit configured to separate impurities from a fluid flowing from the loading conduit to the sample compartments; wherein the loading conduit comprises a filter section and a narrow section in fluid communication with the filter section; wherein the filter section has a larger cross-sectional area than the narrow section.

2. The microfluidic device of claim 1, wherein, The loading conduit further comprises a loading pad and a first section in fluid communication with an outlet of the loading pad, the loading pad configured to receive the fluid therein, and wherein the loading pad has a larger cross-sectional area than the first section.

3. The microfluidic device of claim 2, wherein, The cross-sectional area of the loading pad is in the range of 0.47mm 2 to 0.83mm 2 and the cross-sectional area of the first section is in the range of 0.01mm 2 to 0.016mm 2 .

4. The microfluidic device of claim 2, wherein, The cross-sectional area of the filter section is in the range of 0.0010 mm 2 to 0.0025 mm 2 and the cross-sectional area of the narrow section is in the range of 0.00025 mm 2 to 0.00046 mm 2 .

5. The microfluidic device of claim 2, wherein, An outlet of the first section is in fluid communication with an inlet of the filter section.

6. The microfluidic device of claim 5, wherein, The outlet of the first section and the inlet of the filter section form a curved boundary configured to increase a filtering capacity of the loading conduit.

7. The microfluidic device of any one of claims 1 to 6, wherein, The microfeatures comprise posts, chip diverting posts, parallel channels, or combinations thereof.

8. The microfluidic device of claim 7, wherein, The posts are disposed in the filter section proximate an inlet of the narrow section of the loading conduit.

9. The microfluidic device of claim 7, wherein, The posts are further configured to limit emulsion formation and air trapping as the fluid flows through the filter section.

10. The microfluidic device of claim 7, wherein, Each of the posts has a cross-sectional area in a shape of a circle, a triangle, a rectangle, a square, a polygon, or combinations thereof.

11. The microfluidic device of claim 7, wherein, The posts in the filter section are arranged in at least one row.

12. The microfluidic device of claim 7, wherein, The posts in the filter section are arranged in a staggered pattern comprising at least 3 rows.

13. The microfluidic device of claim 7, wherein, The posts are positioned such that a minimum distance between two adjacent posts is 6 microns to 10 microns.

14. The microfluidic device of claim 7, wherein, A post is disposed at each corner of an end of the filter section connected to the narrow section.

15. The microfluidic device of claim 14, wherein, The post at each corner of the end of the filter section is configured to limit emulsion formation of a liquid flowing through the filter section.

16. The microfluidic device of claim 7, wherein, Each post of the filter section has a circular cross-sectional area with a diameter of 20 microns to 24 microns.

17. The microfluidic device of any one of claims 1 to 6 and 8 to 16, wherein, Each of the sample compartments has a volume of 477 picoliters to 583 picoliters.

18. The microfluidic device of any one of claims 1 to 6 and 8 to 16, wherein, The microfluidic device is configured to house a sample and reagents for a polymerase chain reaction.

19. The microfluidic device of claim 18, wherein, The microfluidic device is configured to house a sample and reagents for a digital polymerase chain reaction.

20. The microfluidic device of claim 19, wherein, The loading conduit is in fluid communication with at least 10,000 sample compartments.

21. A microfluidic device for processing a biological sample, characterized in that, The microfluidic device comprises: a loading conduit in fluid communication with a plurality of sample compartments; and a plurality of posts disposed in the loading conduit configured to separate impurities from a fluid flowing from the loading conduit to the sample compartments; wherein the loading conduit comprises a first section, a filter section, and a narrow section, and wherein the first section, the filter section, and the narrow section have different cross-sectional areas configured to enhance a filtering capacity of the loading conduit.

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