WO2024011134A1

WO2024011134A1 - Systems, devices and methods for multiplexed analysis

Info

Publication number: WO2024011134A1
Application number: PCT/US2023/069651
Authority: WO
Inventors: Keith WAWRZENIAK; Rui Zheng; Zheng XING
Original assignee: IsoPlexis Corporation
Priority date: 2022-07-05
Filing date: 2023-07-05
Publication date: 2024-01-11

Abstract

Embodiments of the current disclosure are directed to systems, methods and apparatus for the multiplexed analysis of biological material. In some embodiments, the apparatus may comprise an assembly including a baffle including a plurality of first openings, a substrate, and a membrane.

Description

SYSTEMS, DEVICES AND METHODS FOR MULTIPLEXED ANALYSIS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application No. 63/358,358, filed July 5, 2022, the contents of which are herein incorporated by reference in its entirety.

BACKGROUND

[0002] Multiplexed analysis of biological components of biological samples, either as single cells, cell populations, cell lysates, cell culture supernatants, or bodily fluids, is of great utility in the areas of basic research, diagnostics, and therapeutics. Robust, user-friendly, and more economical technologies to facilitate said multiplexed analyses remain of great need to the medical and research communities.

[0003] Automated devices, systems, and methods that improve accuracy, sensitivity, and reliability while reducing complexity of the overall device and/or system would hugely benefit the medical and research community by facilitating the discovery of novel therapeutics and the ability to directly monitor patients undergoing treatments, including chemotherapies, immunotherapies, cellular therapies, and the like.

BRIEF SUMMARY OF AT LEAST SOME OF THE EMBODIMENTS

[0004] Embodiments of the present disclosure are directed to methods, systems and devices for the multiplexed analysis of components, including proteins, antibodies, sugars, lipids, nucleic acids, and/or metabolites, in a biological sample. In particular embodiments, the methods, systems, and devices utilize fluid flow patterns along the z-axis and one or both of the x-axis and y-axis of a device. This flow pattern can provide for the simultaneous washing/assaying of a plurality of sample chambers using a single flow of wash/assay solution while ensuring better separation between neighboring sample chambers and washing out sample trapped in openings used for sample loading, which can reduce cross-contamination between neighboring wells and reduce "flare-ups" emanating from trapped sample subsequently entering a chamber.

[0005] In one aspect, microfluidic devices are provided. The microfluidic devices can comprise a body having a flow path, the flow path comprising a first fluidic structure; a second fluidic structure; and a first bridge disposed within the body or on a first side of the body and configured to fluidically couple the first fluidic structure and the second fluidic structure; wherein each of the first fluidic structure and the second fluidic structure comprises: (i) a first opening and a second opening, both the first opening and the second opening at a first side of the body, and (ii) a sample chamber disposed within the body or on a second side of the body, and the sample chamber is configured to fluidically couple with the first opening and the second opening; and wherein the first bridge is configured to fluidically couple the second opening of the first fluidic structure with the first opening of the second fluidic structure.

[0006] In another aspect, methods for analyzing a biological sample are provided. The methods can comprise loading a first biological sample into a first fluidic structure of a microfluidic device of the present disclosure via a first opening of the first fluidic structure, wherein a sample chamber surface of a sample chamber of the first fluidic structure comprises a capture agent; incubating the first biological sample within the sample chamber of the first fluidic structure, thereby allowing the first biological sample to interact with the capture agent, and detecting an interaction between components of the first biological sample and the capture agent. The first biological sample can be a biological fluid, such as serum, plasma, lymph, cerebrospinal fluid, and/or any other fluid comprising a mixture of biological molecules.

[0007] In another aspect, systems for analyzing a biological sample are provided. The systems can comprise a microfluidic device of the present disclosure and an instrument for operating the microfluidic device to perform a method of the present disclosure.

[0008] In another aspect, microfluidic devices configured for multiplexed sample analysis are provided. In some embodiments, the multiplexed microfluidic device comprises a body having a flow path, the flow path having: a first sample chamber disposed within the body or on a first side of the body; a second sample chamber disposed within the body or on the first side of the body; and a fluidic bridge structure disposed to fluidically couple the first sample chamber with the second sample chamber, the fluidic bridge structure having: a first z- channel having a first end disposed to fluidically couple with the first sample chamber, a second z-channel having a first end disposed to fluidically couple with the second sample chamber, and a bridge disposed to fluidically couple a second end of the first z-channel with a second end of the second z-channel. In some embodiments, the multiplexed microfluidic device further comprises: a first opening at a second side of the body disposed to fluidically couple with the first sample chamber for loading a biological sample (e.g., a first sample) therein; and/or a second opening at the second side of the body disposed to fluidically couple with the second sample chamber, optionally via the second end of the second z-channel, for loading a biological sample (e.g., a second sample) into the second chamber.

[0009] In some embodiments, the multiplexed microfluidic device is a multiplex assay device configured for multiplexed analysis of biological material, the device including: (a) a baffle comprising (i) a plurality of repeating fluidic structures, each of the plurality of repeating fluidic structures comprising a sealable first opening on a first side of the baffle, a sealable second opening on the first side of the baffle, a sample chamber disposed within the baffle or on a second side of the baffle, wherein the first side of the baffle is separated from the second side of the baffle by a thickness defining a z-axis of the baffle, a first channel extending along the z-axis from the sealable first opening to the sample chamber, and a second channel extending along the z-axis from the sealable second opening to the sample chamber, wherein the sealable first opening and the sealable second opening are in fluid communication via the first channel, the sample chamber, and the second channel; (ii) at least one connecting channel on the first side of the baffle, disposed between and in fluid communication with the sealable second opening of one of the plurality of repeating fluidic structures and the sealable first opening of another of the plurality of repeating fluidic structures; (iii) an input opening configured for receiving a flow and conducting the flow to the plurality of repeating fluidic structures; and (iv) an output opening configured for exhausting the flow from the plurality of repeating fluidic structures, and (b) a cover configured to cover and seal the plurality of sealable first and second openings on the first side of the baffle. In some embodiments, the cover is further configured to cover and seal that at least one connecting channel on the first side of the baffle.

[0010] According to some embodiments, a method of preparing a plurality of samples for analysis is provided which includes (a) loading each of the plurality of samples into different sample chambers of the multiplex assay device of any one of the above-described embodiments, via a corresponding one of the sealable first openings, and (b) applying the cover to the first side of the baffle, wherein the cover covers and seals the plurality of sealable first and second openings, without covering the input opening and the output opening. In some embodiments, the cover further covers and seals the at least one connecting channel.

[0011] In some embodiments, a multiplex assay device configured for multiplexed analysis of biological material is provided, the device including: a baffle comprising a plurality of first openings on a first side of the baffle, a plurality of second openings on the first side of the baffle, and a plurality of bridges on the first side of the baffle, wherein the first side of the baffle is separated from a second side of the baffle by a thickness defining a z-axis of the baffle, and wherein each one of the plurality of bridges fluidly couples corresponding ones of the plurality of second openings and the plurality of first openings; and a membrane comprising a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to respective ones of the plurality of first openings and the plurality of second openings; a cover; and a substrate, wherein corresponding ones of the plurality of first openings and the plurality of second openings are fluidly couplable via one of the plurality of elongated slots when the baffle, the membrane, and the substrate are assembled, with the membrane interposed between the baffle and the substrate, wherein a volume defined by the second side of the baffle, the substrate, and one of the plurality of elongated slots constitutes a sample chamber.

[0012] Some embodiments, such as those set out above (as well as other disclosed herein), may also include one and/or another of (and in some embodiments, a plurality of, in some embodiments, a majority of, in some embodiments, substantially all of, and in some embodiments, if not mutually exclusive, all of) the following features, structures, functionalities, steps, and clarifications, leading to yet further embodiments: the membrane comprises a first side and a second side, the first side configured to contact the baffle and the second side configured to contact the substrate; the membrane comprises a first end and a second end, an axis therebetween being an x-axis of the membrane, and wherein each of the plurality of elongated slots extends from a first end of the membrane towards a second end of the membrane, generally along the x-axis of the membrane; the membrane comprises a third end and a fourth end, an axis therebetween defining a y-axis of the membrane; a first subset of the plurality of elongated slots includes at least two elongated slots arranged parallel to one another, and, optionally, linearly arranged along the x-axis of the membrane; the membrane comprises silicone or a polymer such as, for example, polypropylene; the membrane comprises a polypropylene film coated on one or both sides with a silicone adhesive; the at least two elongated slots of the first subset of the plurality of elongated slots are spaced apart by a predetermined distance; the baffle further comprises a first end and a second end, an axis therebetween being an x-axis of the baffle, and wherein a first subset of the plurality of first openings and, optionally, a corresponding first subset of the plurality of second openings, are spaced apart along the x-axis of the baffle; the first subset of the plurality of first openings and the corresponding first subset of the plurality of second openings are spaced apart by the predetermined distance; the multiplex assay device further comprises a holder; the baffle is couplable to the holder; the baffle and holder are couplable such that the substrate and the membrane are arranged therebetween; the multiplex assay device further comprises a cover configured to cover the plurality of first openings and the plurality of second openings; the cover is configured to cover the plurality of first openings, and, optionally, the plurality of second openings, after a biological material sample has been pipetted into at least one of the plurality of first openings; the plurality of first openings is arranged in a plurality of rows along the x-axis of the baffle; the plurality of second openings is arranged in a plurality of rows along the x- axis of the baffle; the baffle comprises a third end and a fourth end, an axis therebetween defining a y-axis of the baffle; each of the plurality of first openings includes identifiable indicia; each of the plurality of first openings and each of the plurality of second openings extends from the first side of the baffle to the second side of the baffle along the z-axis of the baffle; the multiplex assay device further comprises at least one input opening; the at least one input opening is configured for receiving a flow; the flow path being configured to include (i.e., pass through) a plurality of sample chambers (e.g., a first subset or all of the sample chambers); the multiplex assay device further comprises at least one output opening; the at least one output opening is configured for exhausting the flow; the flow path being configured to include (i.e., pass through) a plurality of sample chambers (e.g., a first subset or all of the sample chambers); the multiplex assay device further comprises at least one flexible seal; the multiplex assay device further comprises a pair of flexible seals, one each for sealing engagement of a pipette tip (or other flow delivery/exhausting device) with the at least one input opening and the at least one output opening; the multiplex assay device further comprises a respective opening or recess for receiving a respective flexible seal; the multiplex assay device further comprises a coded label for identifying the multiplex assay device; the holder includes an opening (or transparent window) so as to image the substrate; each one of the plurality of bridges fluidly couples an opening of a first row of the plurality of second openings and a corresponding opening of a second row of the plurality of first openings; when the cover, the baffle, the membrane, and the substrate are assembled, a plurality of sample chambers are formed and a plurality of passages are formed between the at least one input opening and the at least one output opening via the plurality of first openings of the baffle, the plurality of sample chambers, the plurality of bridges, and the plurality of second openings of the baffle so as to establish a three dimensional channel between the at least one input opening and the at least one output opening.

[0013] In some embodiments, a multiplex assay device configured for multiplexed analysis of biological material is provided, and includes a baffle comprising a plurality of first openings arranged in a plurality of rows on a first side of the baffle, each of the plurality of first openings including identifiable indicia and extending from a first side of the baffle to a second side of the baffle, a plurality of second openings arranged in a plurality of rows on the first side of the baffle corresponding to the plurality of rows of the plurality of first openings, each of the plurality of rows of the plurality of second openings extending from the first side of the baffle to the second side of the baffle, a plurality of bridges arranged on the first side of the baffle, each one of the plurality of bridges fluidly coupling a second opening of a first row of the plurality of second openings and a corresponding first opening of a second row of the plurality of first openings, at least one input opening arranged on the baffle (optionally extending from the first side of the baffle to the second side of the baffle) configured for receiving a flow, and at least one output opening arranged on the baffle (optionally extending from the first side of the baffle to the second side of the baffle) configured for exhausting the flow, a substrate, a membrane having a first face and a second face, the membrane further configured with a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to corresponding ones of the plurality of first openings and the plurality of second openings when the baffle, the membrane, and the substrate are assembled with the membrane interposed between the baffle and the substrate, a volume defined by the second side of the baffle, the substrate, and each one of the plurality of elongated slots being a sample chamber, a cover configured to cover the plurality of first openings, and, optionally, the plurality of second openings, after a biological material sample has been pipetted into at least one of the plurality of first openings, a holder, and a pair of flexible seals, one each provided for the at least one input opening and the at least one output opening, wherein capture agents are disposed on a surface of the substrate and are contactable with biological material in a fluid within the sample chamber, wherein the baffle is couplable with the holder such that the substrate and membrane are arranged therebetween, wherein the holder includes an opening (or transparent window) so as to image the substrate, wherein each channel of the membrane is positioned below at least one first opening of each row of first openings, such that a sample loaded into a respective first opening proliferates along at least a portion of the channel to interact with capture agents of the substrate, and wherein a plurality of passages connect the at least one input opening to the at least one output opening, the plurality of passages including a first passage connecting the at least one input opening to one of a first row of the plurality of first openings, a second passage connecting the at least one output opening to one of a last row of the plurality of second openings, and a plurality of supporting passages connecting the first passage, the plurality of first openings, each sample chamber, the plurality of second openings, the plurality of bridges, and the second passage.

[0014] Some embodiments, such as those set out above (as well as other disclosed herein), may also include one and/or another of (and in some embodiments, a plurality of, in some embodiments, a majority of, in some embodiments, substantially all of, and in some embodiments, if not mutually exclusive, all of) the following features, structures, functionalities, steps, and clarifications, leading to yet further embodiments: the multiplex assay device further comprises a fluidics system in fluid communication with each of the at least one input opening arranged on the baffle and the at least one output opening arranged on the baffle and being configured to flow one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening.

[0015] According to some embodiments, a multiplex assay system configured for multiplexed analysis of biological material is provided which includes a receiving area configured to receiving a plurality of multiplex assay devices of any one of the above-described embodiments, a fluorescing device configured to expose the substrate and corresponding sample chambers to the fluorescing light, and an imager configured to image the substrate and sample chambers upon exposing the substrate and sample chambers to the fluorescing light.

[0016] Some embodiments, such as those set out above (as well as other disclosed herein), may also include one and/or another of (and in some embodiments, a plurality of, in some embodiments, a majority of, in some embodiments, substantially all of, and in some embodiments, if not mutually exclusive, all of) the following features, structures, functionalities, steps, and clarifications, leading to yet further embodiments: the system further comprises one selected from the group consisting of a graphical user interface (GUI), an electronic reader, and one or more processors configured with computer instructions operational thereon to cause the system to perform a plurality of steps of a method, wherein the one or more processors interface with a fluidics system in fluid communication with each of the at least one input opening arranged on the baffle and the at least one output opening arranged on the baffle to flow one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening; the GUI is configured to at least one of display information, display an output from the system, and/or receive input from a user; the electronic reader is configured to receive or otherwise obtain a code from each of the multiplex assay devices.

[0017] According to some embodiments, a method for multiplexed analysis of biological material using the multiplex assay system of any one of the above-described embodiments is provided and includes identifying each multiplex assay device of the multiplex assay system via reading of a code of a respective multiplex assay device, confirming proper application of the cover over the plurality of first openings and the plurality of second openings on the first side of the baffle of each identified multiplex assay device, incubating each multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents disposed on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, activating the fluorescing device, imaging the substrate from the opening in the holder upon exposure of the substrate to the fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image.

[0018] According to some embodiments, a multiplex assay system configured for multiplexed analysis of biological material is provided, where the system includes a receiving area configured to receiving a plurality of multiplex assay devices of any of the above-described embodiments, a graphical user interface configured to display information, output information from the system, and/or receive input from a user, a fluorescing device configured to expose the opening of a holder of each multiplex assay device to fluorescing light, an imager configured to image the substrate and corresponding elongated slots of the membrane upon the substrate being exposed to the fluorescing light, an electronic reader configured to receive or otherwise obtain a code from each of the multiplex assay devices, one or more processors configured with computer instructions operational thereon to cause the system to perform a method, comprising identifying each multiplex assay device via reading of a code of a respective multiplex assay device, confirming proper application of the cover over the plurality of first openings and the plurality of the second openings on the first side of the baffle of each multiplex assay device, incubating each multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents contained on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, activating the fluorescing device, imaging the substrate from the opening in the holder upon exposure of the substrate to the fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image.

[0019] According to some embodiments, a multiplex assay method for multiplexed analysis of biological material is provided and includes loading one or more biological samples into one or more of a plurality of first openings of the first side of the baffle of the multiplex assay device of any of the above-described embodiments, covering the first side of the baffle with a cover, placing the multiplex assay device within a processing system, identifying, via the processing system, the multiplex assay device via reading of a code of the multiplex assay device, confirming proper application of cover over the plurality of first openings, incubating the multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents contained on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, capturing an image of at least one of the substrate and elongated slots of the membrane upon exposure of the substrate to fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image.

[0020] Some embodiments, such as those set out above (as well as other disclosed herein), may also include one and/or another of (and in some embodiments, a plurality of, in some embodiments, a majority of, in some embodiments, substantially all of, and in some embodiments, if not mutually exclusive, all of) the following features, structures, functionalities, steps, and clarifications, leading to yet further embodiments: the plurality of samples comprises biological materials and the different sample chambers comprise capture agents; the method further comprises washing out of the different sample chambers any of the biological materials that are not bound to the capture agents by flowing a wash solution into the input opening, through each of the plurality of repeating fluidic structures in series, and out the output opening; the method further comprises labeling at least some of the biological materials bound to the capture agents in the sample chambers by flowing a labeling agent into the input opening, and through each of the plurality of repeating fluidic structures in series; the method further comprises washing out of the sample chambers any of the labeling agents that are not bound to the biological materials by flowing a wash solution into the input opening, through each of the plurality of repeating fluidic structures in series, and out the output opening.

[0021] In some embodiments, a system for multiplexed analysis of biological material is provided and includes a multiplex assay system, including a baffle comprising at least one input opening on a first side of the baffle, at least one output opening on the first side of the baffle, a plurality of first openings on the first side of the baffle, a plurality of second openings on the first side of the baffle, and a plurality of bridges on the first side of the baffle, wherein the first side of the baffle is separated from a second side of the baffle by a thickness defining a z-axis of the baffle, and wherein each one of the plurality of bridges fluidly couples corresponding ones of the plurality of second openings and the plurality of first openings, and a membrane comprising a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to respective ones of the plurality of first openings and the plurality of second openings, a cover; and a substrate, wherein corresponding ones of the plurality of first openings and the plurality of second openings are fluidly couplable via one of the plurality of elongated slots when the baffle, the membrane, and the substrate are assembled, a volume defined by the second side of the baffle, the substrate, and the one of the plurality of elongated slots being a sample chamber, and wherein the at least one input opening, the plurality of first openings, the elongated slots, the plurality of second openings, the plurality of bridges, and the at least one output opening are fluidly connected and form a plurality of passages, and a processor configured to actuate a fluidic system to flow one or more reagents through the plurality of passages via the at least one input opening, activating a fluorescing device to excite fluorophores of one of the one or more reagents, the fluorophores being bound to biological material captured by capture agents within sample chambers, the capture agents being bound to the substrate and instructing acquisition of images of the substrate.

[0022] These and other embodiments, objects and advantages of the embodiments, will become even more apparent with reference to the detailed description which follows, as well any associated figures corresponding thereto, a brief description of which is set out below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1A is an expanded view of a z-flow multiplex assay device (Z-MAD), according to exemplary embodiments of the present disclosure.

[0024] FIG. IB is an expanded view of a Z-MAD, according to exemplary embodiments of the present disclosure.

[0025] FIG. 1C is a cross-sectional view of a state of the Z-MAD, showing a top view of a body being a unitary structure having the baffle and the membrane respectively on opposite sides thereof.

[0026] FIG. ID is a cross-sectional view of a state of the Z-MAD, showing a bottom view of a body being a unitary structure having the baffle and the membrane respectively on opposite sides thereof.

[0027] FIG. 2 is an expanded view of a baffle, a membrane, and a substrate of a Z-MAD, according to exemplary embodiments of the present disclosure.

[0028] FIG. 3A is a via of a side of a baffle of a Z-MAD, the baffle comprising a plurality of first openings, according to exemplary embodiments of the present disclosure. [0029] FIG. 3B is a magnified view of a side of a baffle of a Z-MAD, the baffle comprising a plurality of first openings, according to exemplary embodiments of the present disclosure.

[0030] FIG. 3C illustrates a single pair of first opening and second opening coupling via a bridge.

[0031] FIG. 3D shows a perspective view of a row of row of adjacent ones of the plurality of first openings and the plurality of second openings.

[0032] FIG. 4 is a view of a side of a baffle with a membrane of the Z-MAD coupled thereto, according to exemplary embodiments of the present disclosure.

[0033] FIG. 5A is a view of a state of a Z-MAD, wherein a baffle and a membrane of the Z- MAD are coupled and before fluid is flowed into and/or through the Z-MAD, according to exemplary embodiments of the present disclosure.

[0034] FIG. 5B is a view of a state of the Z-MAD, wherein a baffle and a membrane of the Z- MAD are coupled and samples are added to the Z-MAD, according to exemplary embodiments of the present disclosure.

[0035] FIG. 5C is a view of a state of the Z-MAD, wherein a baffle and a membrane of the Z- MAD are coupled and a wash fluid is flowed through the Z-MAD, according to exemplary embodiments of the present disclosure.

[0036] FIG. 6A is a cross-sectional view of a state of the Z-MAD, wherein a baffle, a membrane, and a substrate are coupled and a fluid is flowed through the Z-MAD, according to exemplary embodiments of the present disclosure.

[0037] FIG. 6B is a cross-sectional view of a state of the Z-MAD, wherein a baffle, a membrane, and a substrate are coupled and a fluid is flowed through the Z-MAD, according to exemplary embodiments of the present disclosure.

[0038] FIG. 7 is a flow diagram of a method of use of the Z-MAD, according to exemplary embodiments of the present disclosure.

[0039] FIG. 8A is a schematic description of components of a Z-MAD for use with the method of use of the Z-MAD of FIG. 7, according to exemplary embodiments of the present disclosure.

[0040] FIG. 8B is a schematic description of components of a Z-MAD for use with the method of use of the Z-MAD of FIG. 7, according to exemplary embodiments of the present disclosure.

[0041] FIG. 8C is a schematic description of components of a Z-MAD for use with the method of use of the Z-MAD of FIG. 7, according to exemplary embodiments of the present disclosure.

[0042] FIG. 9 is a block diagram for a Z-MAD configured for multiplexed analysis of biological materials using one or more Z-MADs in a system, according to exemplary embodiments of the present disclosure. [0043] FIG. 10 shows graphs plotting RFU and concentration and showing a 4-Parameter Logistic Regression (4PL) curve.

[0044] FIG. 11A shows a bar chart demonstrating RFU values of high and low signal controls.

[0045] FIG. 11B shows a bar chart demonstrating concentration (pg/ml) of high and low signal controls.

[0046] FIG. 12 shows bar charts demonstrating multiplexed analyses of the secretomes of nine samples.

DETAILED DESCRIPTION

[0047] Some of the embodiments described herein include microfluidic devices, systems, computer readable media, and methods for multiplexed analysis of biological samples. In one example, one side of a microfluidic device includes sample chambers arranged in an array along an x-axis (or length) and a y-axis (or width) of the microfluidic device. Another side of the microfluidic device includes openings for providing the biological samples to the sample chambers via a z-channel along a z-axis (or depth or thickness) of the microfluidic device. Biological samples can be pipetted via the openings such that each of the sample chambers are separately loaded.

[0048] The sample chambers can also be part of a larger flow path that flows from an input opening of the microfluidic device and to an output opening of the microfluidic device for facilitating flow of wash buffers, reagents, or other fluids through the entire flow path having multiple sample chambers. For example, the z-channels that allow for separately loading each of the sample chambers can also form part of a fluidic structure that facilitates flow from one sample chamber to another via a bridge and another z-channel. If a fluid should be flowed through the entire flow path, then the fluid is provided to a first sample chamber which is elongated along the x-axis, flows up a z-channel along the z-axis and to a bridge which then flows along the x-axis again before flowing down the next z-channel to another sample chamber.

[0049] In some cases, this series of flows can repeat in a lane of sample chambers until the last sample chamber of the lane. At that point, the bridge is rotated relative to the other bridges of the lane of sample chambers such that the flow is in the y-direction for a short distance to provide the flow to the first sample chamber of an adjacent lane of sample chambers. Then the series of flows repeats (but in the opposite direction) before reaching the end of the adjacent lane. This allows for the flow path to have a serpentine flow "turning" at the ends of the lanes.

[0050] Thus, biological samples may be easily loaded into each of the sample chambers. However, because of the arrangement of the z-channels and bridges, the biological samples of each of the sample chambers are fluidically separated from each other if the pipetted volume of the biological sample is appropriate for the volume of the sample chamber. Any excess biological sample may flow into a z-channel, but the biological samples would still be relatively isolated from each other from sample chamber to sample chamber. This isolation of the sample chambers reduces cross-contamination of biological samples, while also allowing for a fluid path through all the sample chambers of the microfluidic device for when a fluid should be provided to the entire flow path of the microfluidic device.

Definitions

[0051] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0052] The terms "can" and "may" are used interchangeably in the present disclosure, and indicate that the referred to element, component, structure, function, functionality, objective, advantage, operation, step, process, apparatus, system, device, result, or clarification, has the ability to be used, included, or produced, or otherwise stand for the proposition indicated in the statement for which the term is used (or referred to) for a particular embodiment(s).

[0053] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0054] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0055] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0056] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "composed of," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0057] Although the terms "first" and "second" may be used herein to describe various features/elements (including steps), these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0058] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word "about" or "approximately," even if the term does not expressly appear. The phrase "about" or "approximately" may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/- 0.1% of the stated value (or range of values), +/- 1% of the stated value (or range of values), +/- 2% of the stated value (or range of values), +/- 5% of the stated value (or range of values), +/- 10% of the stated value (or range of values), etc. Any numerical values given herein should also be understood to include about or approximately that value, unless the context indicates otherwise. For example, if the value "10" is disclosed, then "about 10" is also disclosed. Any numerical range recited herein is intended to include all sub-ranges subsumed therein. It is also understood that when a value is disclosed that "less than or equal to" the value, "greater than or equal to the value" and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value "X" is disclosed the "less than or equal to X" as well as "greater than or equal to X" (e.g., where X is a numerical value) is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point "15" are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0059] As used herein, "substantially" means sufficient to work for the intended purpose. The term "substantially" thus allows for minor, insignificant variations from an absolute or perfect state, dimension, measurement, result, or the like such as would be expected by a person of ordinary skill in the field but that do not appreciably affect overall performance. When used with respect to numerical values or parameters or characteristics that can be expressed as numerical values, "substantially" means within ten percent.

[0060] As used herein, a "microfluidic device" is a type of fluidic device having a microfluidic circuit that contains at least one circuit element configured to hold a volume of fluid of less than about 100 microliters (uL), e.g., less than 90, 80, 70, 60, 50, 40, 30, 20, 10 uL. A microfluidic device may comprise a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, or more). In certain embodiments, one or more (e.g., all) of the at least one circuit elements is configured to hold a volume of fluid of between 0.1 uL and 10 mL, between 0.1 uL and 5 mL, between 0.1 uL and 4 mL, between 0.1 uL and 3 mL, between 0.1 uL and 2 mL, between 0.1 uL and 1 mL, between 1 uL and 1 mL, between 5 uL and 1 mL, between 10 uL and 1 mL, between 20 uL and 1 mL, between 30 uL and 1 mL, between 40 uL and 1 mL, between 50 uL and 1 mL, between 100 uL and 1 mL, between 200 uL and 1 mL, between 10 uLand 500 uL, between 50 uL and 500 uL, between 100 uL and 500 uL, or between 200 uL and 500 uL.

[0061] As used herein, extending "along an x-axis" refers to a structure, opening, channel, slot, flow, or the like extending along an x-axis of a structure and/or device of the disclosure. In some embodiments, the extending along an x-axis can further comprise extending along a y-axis and/or a z-axis. For example, a structure, opening, channel, slot, flow, or the like can extend along an x-axis while also extending across the y-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the x-axis) and/or the z-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the x-axis). In some embodiments, extending along the x-axis is not perpendicular relative to one or more of the y-axis, the z-axis, and the y-z plane. In some embodiments, extending along the x-axis is perpendicular relative to one or more of the y-axis, the z-axis, and the y-z plane. [0062] As used herein, extending "along a y-axis" refers to a structure, opening, channel, slot, flow, or the like extending along a y-axis of a structure and/or device of the disclosure. In some embodiments, the extending along a y-axis can further comprise extending along an x- axis and/or a z-axis. For example, a structure, opening, channel, slot, flow, or the like can extend along a y-axis while also extending across the x-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the y-axis) and/or the z-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the y-axis). In some embodiments, extending along the y-axis is not perpendicular relative to one or more of the x-axis, the z-axis, and the x-z plane. In some embodiments, extending along the y-axis is perpendicular relative to one or more of the x-axis, the z-axis, and the x-z plane.

[0063] As used herein, extending "along a z-axis" refers to a structure, opening, channel, slot, flow, or the like extending along a z-axis of a structure and/or device of the disclosure. In some embodiments, the extending along a z-axis can further comprise extending along an x- axis and/or a y-axis. For example, a structure, opening, channel, slot, flow, or the like can extend along a z-axis while also extending across the x-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the z-axis) and/or the y-axis (e.g. +/- 45 degrees, +/- 30 degrees, or +/- 15 degrees relative to the z-axis). In some embodiments, extending along the z-axis is not perpendicular relative to one or more of the x-axis, the y-axis, and the x-y plane. In some embodiments, extending along the z-axis is perpendicular relative to one or more of the x-axis, the y-axis, and the x-y plane.

[0064] Referring now to the Drawings, a multiplex assay device will be described according to embodiments of the present disclosure.

[0065] FIG. 1A and FIG. IB illustrate exploded perspective views of a microfluidic device, which can be referred to as a z-flow multiplex assay device (Z-MAD), according to an exemplary embodiment of the present disclosure. In embodiments, the Z-MAD 100 comprises a baffle 101 (which can also be referred to as a baffle, a plate, and the like), a membrane 102, and a substrate 103. In embodiments, the Z-MAD 100 of FIG. 1A and FIG. IB further comprises a cover 109, a holder 110 (which can be referred to as a frame), at least one flexible seal 108a, 108b, and a screw 117. The assembly of the baffle 101 and the membrane 102 forms a body 100a of the microfluidic device. The body 100a has a flow path comprising at least two fluidic structures and a bridge configured to fluidically couple the two fluidic structures. The flow path will be described in more detail below. In some embodiments like the one shown in FIG 1A and FIG. IB, the baffle 101 and the membrane 102 can be separate structures. For example, the baffle 101 and the membrane 102 are manufactured separately (and, in one example, be made of different materials) and then assembled together. While in other embodiments, the body of the microfluidic device is a unitary structure having the baffle 101 and the membrane 102 respectively on opposite sides thereof (FIG. 1C and FIG. ID). For example, the baffle 101 and the membrane 102 can be manufactured as part of the same body (e.g., manufactured by injection molding, additive manufacturing, etc.). As shown in FIG. ID, the feature of the membrane 102 is disposed on bottom side of the body 100a while the baffle 101 being (corresponding to) the top side of the body 100a.

[0066] In some embodiments, the Z-MAD is configured for multiplexed analysis of one or more biological samples.

[0067] In some embodiments, this biological sample is any fluid that contains a plurality of proteins, peptides, polysaccharides, saccharides, lipids, fatty acids, metabolites, nucleic acids, and/or particles comprising any of the foregoing biological molecules, such as lipid nanoparticles, vesicles, or viral particles. In some embodiments, the plurality of proteins, peptides, metabolites and/or nucleic acids are derived from a plurality of cells, a single cell, a cell lysate, or a cell culture supernatant. In some embodiments, the biological sample is a bodily fluid, e.g., obtained from an animal (e.g., an experimental animal, which may be a invertebrate, vertebrate, mammal, monkey, or primate) or a human (e.g., a human patient). In some embodiments, the bodily fluid is blood, serum, lymph, cerebrospinal fluid, pleural effusion, peritoneal fluid, saliva, tears, urine, semen and/or a fluid which has accumulated within a bodily cavity. In some embodiments, the metabolite is a small molecule. In some embodiments, the metabolite is glucose, glutamine, or lactate.

[0068] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the DNA is autosomal DNA, chromosomal DNA, cDNA, exosome DNA, single stranded DNA, or double stranded DNA. In some embodiments, the RNA is mRNA, rRNA, tRNA, snRNA, regulatory RNA, microRNA, exosome RNA, or double stranded RNA. In some embodiments, the RNA is an mRNA. In some embodiments, the RNA is a guide RNA from a CRISPR-Cas system.

[0069] In some embodiments, the single cell is an immune cell. In some embodiments, the plurality of cells is a homogenous cell population comprising a single cell type. In some embodiments, the plurality of cells is a heterogeneous cell population comprising more than one cell type.

[0070] In some embodiments, the single cell immune cell is a T-lymphocyte, a B-lymphocyte, a natural killer (NK) cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a basophil. In certain embodiments, the T-lymphocyte comprises a naive T-lymphocyte, an activated T- lymphocyte, an effector T- lymphocyte, a helper T-lymphocyte, a cytotoxic T-lymphocyte, a gamma-delta T-lymphocyte, a regulatory T-lymphocyte, a memory T-lymphocyte, or a memory stem T-lymphocyte. In some embodiments, the T-lymphocyte expresses a non- naturally occurring antigen receptor. In certain embodiments, the T-lymphocyte expresses a Chimeric Antigen Receptor (CAR).

[0071] In some embodiments, the heterogeneous cell population comprises one or more immune cells, where the one or more immune cells can comprise a T-lymphocyte, a B- lymphocyte, a natural killer (NK) cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a basophil. In certain embodiments, the T-lymphocyte comprises a naive T-lymphocyte, an activated T-lymphocyte, an effector T- lymphocyte, a helper T-lymphocyte, a cytotoxic T- lymphocyte, a gamma-delta T-lymphocyte, a regulatory T-lymphocyte, a memory T- lymphocyte, or a memory stem T-lymphocyte. In some embodiments, the T-lymphocyte expresses a non-naturally occurring antigen receptor. In certain embodiments, the T- lymphocyte expresses a Chimeric Antigen Receptor (CAR).

[0072] In some embodiments, the heterogeneous cell population comprises one or more immune cells, where the one or more immune cells can comprise a T-lymphocyte, a B- lymphocyte, a natural killer (NK) cell, a macrophage, a neutrophil, a mast cell, an eosinophil, or a basophil. In some embodiments, the B-lymphocyte comprises a plasmablast, a plasma cell, a memory B-lymphocyte, a regulatory B cell, a follicular B cell, or a marginal zone B cell.

[0073] In some embodiments, the cell culture supernatant is a media in which a cell (or a plurality of cells) was cultured. In some embodiments, during the culturing, the cell (or plurality of cells) is allowed or induced to produce one or more biological molecules of interest into the cell culture supernatant. The biological molecule(s) of interest can include a protein, a peptide, a polysaccharide, a mono- or di-saccharide, a lipid, a fatty acid, a metabolite, a nucleic acid, and/or any combination thereof, such as a glycosylated protein, a lipoprotein, riboprotein, or the like. The biological molecule(s) of interest can include particles, such as a viral particle, a vesicle, or a lipid nanoparticle. In some embodiments, the viral particle (can be also referred to as virion or capsid) comprises a full viral particle and/or an empty viral particle. A full viral particle (can be also referred to as virion or capsid) means a complete virus particle comprising a vector encapsulated within a capsid protein coat/shell. An empty viral particle means a capsid protein shell in the form of a viral particle but lacking a vector encapsulated therewithin. The virus of the viral particle can be an Adeno-associated virus (AAV), including but not limited to AAV1, AAV 2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAVll.

[0074] In certain embodiments, the biological molecule can be an antibody or a cytokine. The term "antibody" includes intact antibodies and binding fragments thereof. Typically, fragments compete with the intact antibody from which they were derived for specific binding to the target. Such fragments can include separate heavy chains, light chains Fab, Fab', F(a b')₂, F(ab)c, Dabs, nanobodies, and Fv. The cytokine can comprise, but not limited to, CCL-11, GM-CSF, Gran B, IFN-g, IL-10, IL-12, IL-13, IL-15, IL-17AJL-17F, IL-lb, IL-2, IL-21, IL-22, IL-4, IL-5, IL-6, IL-7, IL-8, IL-19, IP-10, MCP-1, MCP-4, MIP-lalpha, MIP-lbeta, perforin, RANTES, TGFbetal, TNF-alpha, TNF-beta, sCD137, and sCD4.

Body, Baffle, and Fluidic Structures

[0075] In embodiments, the body 100a comprises a plurality of first openings 106 and a plurality of second openings 107 disposed at a first side of the body 100a. As used herein in the specification and in the claims, the phrase "at a first side" of the body refers that the opening can be on a surface of the first side or be embedded within the body and proximal to the first side of the body such that it is closer to the first side than a second side of the body opposite from the first side. In some embodiments, the plurality of first openings 106 and the plurality of second openings 107 are all on the first side of the body 100a. In other embodiments, the plurality of first openings 106 are on the first side of the body 100a, while the plurality of second openings 107 are embedded within the body 100a and proximal to the first side of the body 100a. In embodiments, the plurality of first openings 106 and the plurality of second openings 107 of the baffle 101 may be arranged in a plurality of rows, as shown in FIG. 1A.

[0076] The first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body. The z-axis of the body as described herein indicates the thickness of the body. The body can also have an x-axis indicating the length of the body and a y-axis indicating the width of the body (e.g., as depicted in FIG. 6A and FIG. 6B). For convenience, the body, the baffle, the membrane, the substrate, and the microfluidic device described herein share the same x-axis, y-axis, and z-axis.

[0077] In the embodiment shown in the FIG. 1A and FIG. IB, the first side of the body 100a is the first baffle surface of the baffle 101, therefore, the plurality of first openings 106 and the plurality of second openings 107 are on the first baffle surface of the baffle 101. The plurality of first openings 106 and the plurality of second openings 107 may extend from a first side of the body 100a at least partially through the body 100a along a z-axis thereof. As shown in FIG. 1A and FIG. IB, in an exemplary embodiment the plurality of first openings 106 and the plurality of second openings 107 extend completely through the body 100a along the z-axis thereof from the first side of the body 100a to a second side of the body 100a. In other embodiments, the plurality of first openings 106 and the plurality of second openings 107 are captive openings such that they do not extend completely through the body 100a along the z-axis thereof. In such embodiments, the Z-MAD 100 is formed substantially from a unitary body.

[0078] In embodiments, the first z-channel is configured to fluidically couple the first opening 106 to a sample chamber, and the second z-channel is configured to fluidically couple the second opening 107 to the sample chamber. In this way, respective ones of the plurality of first openings 106 and the plurality of second openings 107 are configured to be fluidly coupled by the sample chamber. The sample chamber can be disposed within the body 100a or on a second side of the body 100a. Respective ones of the plurality of first openings 106 and the plurality of second openings 107 together with the sample chamber form a fluidic structure where a biological sample can be introduced, maintained, and/or assayed. For example, biological samples can be introduced into the plurality of first openings 106 and flow in the z-axis direction via the first z-channels to the corresponding sample chambers at the second side of the body. Because the volume of biological samples introduced into the plurality of first openings 106 is small, most of the sample would be retained in the sample chambers, and any excess sample beyond the volume of the sample chambers would then flow again in the z-axis direction and up the second z-channel back towards the first side of the body. However, due to the small amount of sample introduced, the flow of the sample would stop in the second z-channel or a negligible amount may spill over into the next chamber through the bridge and next z-channel. Thus, much or most of the biological sample would be retained within the sample chambers and maintained and/or assayed as described herein.

[0079] As described later, to facilitate flow throughout the entire flow path, fluid may be introduced into one of the sample chambers and flow in the x-axis direction via the elongated sample chambers and in the z-axis and x-axis directions via the z-channels and bridges. In some embodiments, a serpentine path may be formed by having the flow travel for a short distance in the y-axis direction via bridges at the end of a lane of sample chambers.

[0080] In some embodiments, the baffle 101 may have a thickness of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 millimeters (mm), or that falls within a range defined by two of the foregoing endpoints, e.g., between 1 mm and 10 mm, between 2 mm and 10 mm, between 3 mm and 10 mm, between 4 mm and 9 mm, between 4 mm and 8 mm, between 4 mm and 7 mm, between 4 mm and 6 mm, between 1 mm and 8 mm, between 2 mm and 7 mm, between 3 mm and 6 mm, between 2 mm and 5 mm, between 2 mm and 4 mm, etc. In some embodiments, the baffle 101 may be formed of a material having desirable optical properties. For instance, the material may be one having low fluorescence and/or autofluorescence. In some embodiments, the surface of the material has desirable hydrophilic properties that facilitate sample loading into the microfluidic device (e.g., into the z-channel). In some embodiments, the wettability of the surface of the material is characterized by a contact angle between 45° and 135° for a water droplet contacting the surface. In certain embodiments, the material may be a thermoplastic such as a poly(methyl methacrylate) (PMMA) or other similar polymers having desired hydrophilic properties. In some embodiments, the material may be a thermoset, such as polyester (PET) or poly(cyclohexylsilsesquioxane). In some embodiments, the material may be polydimethylsiloxane (PDMS). In some embodiments, the material can be a metal such as aluminum, anodized aluminum, and stainless steel, glass, ceramic, and the like. In some embodiments, the material is not a metal (e.g., not aluminum, anodized aluminum, stainless steel, etc.). In some embodiments, the baffle has a dark or black color that absorbs at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of incident light. In some embodiments, the baffle 101 can be manufactured by CNC (Computer Numerical Control) machining, injection molding, or additive manufacturing. The flatness of a surface of the baffle might be within 100, 80, 60, 50, 40, 20, or 10 urn.

[0081] In some embodiments, the first side of the baffle 101 comprises about 1 to about 1,000 openings. In some embodiments, the first side of the baffle 101 comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 openings or any number in between, or within a range defined by any two of the foregoing endpoints, of openings. In some embodiments, the first side of the baffle 101 comprises about 20 openings.

[0082] In some embodiments, the first side of the baffle 101 comprises about 1 to about 1,000 first openings. In some embodiments, the first side of the baffle 101 comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 first openings or any number in between, or range therein, of first openings. In some embodiments, the first side of the baffle 101 comprises about 20 first openings.

[0083] In some embodiments, the first side of the baffle 101 comprises about 1 to about 1,000 second openings. In some embodiments, the first side of the baffle 101 comprises about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 120, 140, 160, 180, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 second openings or any number in between, or within a range defined by any two of the foregoing endpoints, of second openings. In some embodiments, the first side of the baffle 101 comprises about 20 second openings. In some embodiments, the number of first openings and the number of second openings is equal. In embodiments, the at least one input opening 104, the at least one output opening 105, the plurality of first openings 106, and the plurality of second openings 107 may be referred to as apertures. The number of apertures per side of the baffle 101 may be between about 5 and about 1,000. In embodiments, the number of apertures per side of the baffle 101 may be between about 5 and about 1,000, about 10 and about 500, about 15 and about 100, about 20 and about 75, about 25 and about 50, or about 30 and about 45. In embodiments, the number of apertures per side of the baffle 101 may be greater than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, or 900. In an example, the number of apertures per side of the baffle 101 may be about 40. In certain embodiments, some or all of the first openings 106 may be chamfered.

Flow path and Bridge

[0084] The plurality of first openings 106 and the plurality of second openings 107 of the baffle 101 are fluidly coupled at the first side of the baffle 101 via a bridge or fluidic coupling formed at the first side of the baffle 101. The bridge can be disposed within the body 100a or on a first side of the body. In the example of FIG. 1A, the bridge is depicted as a depression on the first side of baffle 101. However, all or part of the bridge can also be within baffle 101. The bridge is configured to fluidically couple two fluidic structures. For example, a bridge is configured to couple a second opening of one fluidic structure with a first opening of another fluidic structure such that different sample chambers can be fluidically coupled with each other. In certain embodiments, the bridge is configured to be the only fluidic communication between any two fluidic structures of the microfluidic device. In certain embodiments, the bridge is configured to fluidically couple a second z-channel of one fluidic structure with a first z-channel of another fluidic structure. The bridge, and the remaining ones of a plurality of bridges, will be described in detail with reference to FIG. 3B. [0085] A flow path of the microfluidic device can comprise at least two fluidic structures and a bridge configured to fluidically couple the at least two fluidic structures. In certain embodiments, the flow path comprise (i) a first fluidic structure, including a first opening, a first z-channel, a sample chamber, a second z-channel, and a second opening thereof, (ii) a second fluidic structure, including a first opening, a first z-channel, a sample chamber, a second z-channel, and a second opening thereof, and (iii) a bridge configured to fluidically couple the second opening of the first fluidic structure and the first opening of the second fluidic structure.

[0086] In some embodiments, the first opening 106 of any fluidic structure of the present disclosure is configured to introduce a fluidic medium (e.g., a biological sample or a reagent) into the sample chamber of the fluidic structure. For example, a pipette can be maneuvered such that the tip is close to or within the first opening 106 and used to dispense a sample to a sample chamber via the first opening 106 and the corresponding z-channel fluidically coupling the first opening 106 with the sample chamber. The second opening 107, on the other hand, is configured to providing fluidic communication between adjacent fluidic structures via the bridge coupling therebetween. As described later herein, the fluidic communication between adjacent fluidic structures via the bridge coupling allows for formation of a serpentine fluidic path through all the sample chambers of the microfluidic device between the input opening 104 and the output opening 105 for wash buffers, capture agents, etc. as described later herein. The size of the first opening 106 and the size of the second opening 107 is not limited. Nevertheless, in preferred embodiments, the diameter of the opening is about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3 mm, or a range therein (e.g., any range defined by two of the foregoing endpoints).

[0087] In certain embodiments, the first opening is chamfered (or beveled) to correspond to a shape of a pipette tip so that a user, while pipetting a sample into a microfluidic device of the present invention, can insert the pipette tip into the first opening 106 for a better loading experience. For example, a sample may be loaded faster as the pipette tip is guided into the first opening 106. Additionally, less of the sample may be wasted due to incorrect positioning of the pipette. FIG. 3C illustrates a pair of first opening 130a and second opening 131a coupling via a bridge 113a, and FIG. 3D shows a perspective view of a row of row of adjacent ones of the plurality of first openings and the plurality of second openings. A chamfered structure 132a around the first opening 130a can be seen, which is configured to guide a pipette tip.

[0088] In certain embodiments, the first opening has an outer diameter of about 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, 1.5, 2, 2.5, 3 mm, or a range therein (e.g., any range defined by two of the foregoing endpoints, and an inner diameter of about 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9 mm, or a range therein (e.g., any range defined by two of the foregoing endpoints, with a 30, 35, 40, 45, 50, 55, 60, 65, 70 degree, or a range therein (e.g., any range defined by two of the foregoing endpoints, of chamfer. Thus, the diameter of the first opening tapers from a larger size to a smaller size in the z-axis direction towards a sample chamber. In some embodiments, the first z-channel extending from the first opening to the sample chamber can be tapered to correspond to a shape of a pipette tip.

[0089] In some embodiments, the diameter of the first opening is different from the diameter of the second opening. In some embodiments, the diameter of the first opening is larger than the diameter of the second opening. The difference in size between the first opening and the second opening allows a user to easily distinguish one from another so that they can load the fluidic medium through the first opening instead of the second opening. However, as previously discussed, in some embodiments the second opening may not be exposed at the surface and instead is embedded within the body.

[0090] In embodiments that the microfluidic device comprises an array of fluidic structures, the flow path can comprise the plurality of fluidic structures and a plurality of bridges, wherein any one of the plurality of bridges is configured to fluidically couple two fluidic structures of the array via the second opening and the first opening thereof.

[0091] In some embodiments, two or more fluidic structures of the array are arranged in a lane of the flow path. In certain embodiments, the array of the fluidic structures is arranged into more than one lane spreading out over the body of the microfluidic device. In some embodiments, each lane of the extends along with the x-axis (or length) of the microfluidic device and is substantially parallel to one another and spaced apart from each other along the y-axis (or width) of the microfluidic device.

[0092] The fluidic devices of each lane of the flow path are configured to fluidically couple with at least one fluidic device of the same lane via a first bridge, which is as the bridge described above. In some embodiments, a fluidic structure of one lane of the flow path can be configured to fluidically couple with another fluidic structure of another lane via a second bridge. The second bridge is along an axis different than an axis of the first bridge. For example, the first bridge can be along the x-axis of the microfluidic device thereby directing a flow along the x-direction for a lane, and the second bridge can be along the y-axis of the microfluidic device thereby directing the flow along the y-direction to another lane.

[0093] In some embodiments, the flow path is serpentine, comprising (i) at least two fluidic structures and (ii) a first bridge and/or a second bridge. In certain embodiments, the flow path being serpentine comprises an array of fluidic structures, a first bridge, and a second bridge as shown in FIG. 5A and FIG. 5C. A flow is allowed to flow through each fluidic structure in the same lane via the first bridges and directed or turned into an adjacent lane via the second bridge. In some embodiments, the second bridge is configured to fluidically couple a second opening of a fluidic structure of a lane of the flow path with a first opening of a fluidic structure of an adjacent lane. In other embodiments, the second bridge is configured to fluidically couple a first opening of a fluidic structure of a lane of the flow path with a first opening of a fluidic structure of an adjacent lane. Yet in other embodiments, the second bridge is configured to f luidica lly couple a second opening of a fluidic structure of a lane of the flow path with a second opening of a fluidic structure of an adjacent lane.

[0094] For example, in FIG. 5B, fluid may be provided to the microfluidic device via input opening 104. The fluidic structures, bridges, etc. described herein construct a flow path through the microfluidic device to transport the fluid introduced via input opening 104 to output opening 105 such that the fluid flows through each of the sample chambers, z- channels, and bridges. Thus, the fluid flows through a first chamber, up a z-channel, through a bridge, and then back down another z-channel to a second sample chamber. This flow path repeats (i.e., from the second sample chamber up a z-channel, through a bridge, and then back down another z-channel to a third sample chamber) such that the flow path in the lane is along the x-axis (or length) due to the lengths of the sample chambers and the bridges, and along the z-axis due to the lengths of the z-channels.

[0095] At the end of a lane, the bridge is rotated with respect to the other bridges of the lane such that the bridge allows for the fluid to flow in the y-direction rather than the x-direction. Thus, at the end of the lane, the fluid flows from the last sample chamber of the lane up a z- channel, to a bridge which flows in the y-direction towards an adjacent lane, and then back down a z-channel to the first sample chamber within the adjacent lane as depicted by the arrows in FIG. 5B. By facilitating the flow via sample chambers, z-channels, and bridges, most of the length of the flow path can be along the x-axis, with some of the flow within the z-axis due to the z-channels, and the smallest length of the flow along the y-axis to allow for transitions to different lanes.

[0096] The example of FIG. 5B depicts a specific arrangement of sample chambers, bridges, and z-channels such that the flow path is longer in the x-axis than the y-axis. However, the arrangement may be different, for example, to have the flow path longer in the y-axis than the x-axis by changing the orientations of the bridges.

[0097] Moreover, the example of FIG. 5 depicts serpentine turns of the flow path. However, the microfluidic device may have separate input openings and output openings for each lane without having a serpentine flow path.

[0098] In another example, a single input opening may fluidically couple with each of the lanes, and a single output opening may fluidically couple with each of the lanes without having a serpentine flow path. A single, serpentine flow path allows for a single path for the fluid flow, while having multiple different paths increases the complexity of manufacturing and instrument design. Additionally, with multiple different paths, the fluidic resistance may differ from path-to-path and, therefore, the fluid flow may not be even due to the flow following the path of least fluidic resistance.

[0099] In embodiments, the body 100a comprises, on the first baffle surface of the baffle 101, at least one input opening 104 and at least one output opening 105. The at least one input opening 104 and the at least one output opening 105 may be in fluid communication to a flow path. In certain embodiments, the input opening 104 is configured for receiving a flow and conducting the flow to the flow path. In certain embodiments, the output opening 105 is configured for exhausting a flow from the flow path. The flow path, in some embodiments, comprises the plurality of first openings 106, the plurality of second openings 107, the plurality of bridges, a plurality of sample chambers defined in part by a plurality of elongated slots within the membrane 102, which will be described in detail below, a first passage 118, and a second passage 119.

[0100] The first passage 118 is configured to fluidica lly couple the input opening 104 with at least one of the plurality of fluidic structures. In some embodiments, the first passage 118 is configured to fluidically couple with a first opening 106 of one of the plurality of fluidic structures at the first z-channel thereof. The second passage 119 is configured to fluidically couple the output opening 105 with at least one of the plurality of fluidic structures. In some embodiments, the second passage 119 is configured to fluidically couple with a second opening 107 of one of the plurality of fluidic structures at the second z-channel thereof.

[0101] In embodiments, the baffle 101 further comprises at least one seal, which can be a flexible seal made of flexible materials. For instance, the baffle 101 may comprise a first flexible seal 108a arranged at a first end of the baffle 101 and configured to engage the at least one input opening 104 and a second flexible seal 108b arranged at a second end of the baffle 101 and configured to engage the at least one output opening 105. To this end, the first flexible seal 108a may comprise a first flexible seal opening 115a and the second flexible seal 108b may comprise a second flexible seal opening 115b, wherein each is configured to align with and provide access to the at least one input opening 104 and the at least one output opening 105. In some embodiments, each flexible seal has adhesive on one side thereof. As shown in FIG. 1A, in certain embodiments, a respective opening or recess for receiving a respective flexible seal is provided within the baffle 101 or another component of the Z-MAD 100.

[0102] In embodiments, the at least one flexible seal of the Z-MAD 100 comprises silicone rubber with an adhesive on a surface contacting the first side of the baffle 101. In some embodiments, the at least one flexible seal may be any thermoplastic elastomer that is sufficiently flexible to form a seal.

[0103] In embodiments, the baffle 101 further comprises a threaded hole 116 configured to receive screw 117. In certain embodiments, the screw 117 provides a magnetically responsive component to the Z-MAD 100, which may be used to engage a magnet in an analysis instrument to assist with, for example, positioning the Z-MAD within the analysis instrument. However, components other than a screw may be used to engage with the magnet.

Membrane and cover

[0104] In embodiments, the membrane 102 comprises a plurality of elongated slots 170, each of the plurality of elongated slots 170 being configured as a channel extending substantially from a first end of the membrane 102 toward a second end of the membrane (along the x- axis of the membrane 102). As shown in FIG. 1A and FIG. IB, a plurality of rows of elongated slots are disposed within the membrane 102. In some embodiments, the elongated slot is associated with the sample chamber, for example, the elongated slot defines the elongated shape of the sample chamber having a longitudinal axis along the x-axis of the microfluidic device.

[0105] In some embodiments, the membrane 102 includes a first side for positioning adjacent the baffle 101 and a second side to overlay the substrate 103 such that capture agents disposed on a first substrate surface 103' of the substrate 103 are accessible within each of the plurality of elongated slots 170. In some embodiments, when the baffle 101, the channel membrane 102, and the substrate 103 are assembled, a respective one of the plurality of elongated slots 170 of the membrane 102 corresponding with a surface of the first substrate surface 103' of the substrate 103 and a surface of the second baffle surface of baffle 101 define the sample chamber, which, as described above, is configured to fluidically couple with a first opening and a second opening forming a fluidic structure.

[0106] In some embodiments, a first subset of the plurality of elongated slots includes at least two elongated slots linearly arranged along the x-axis of the membrane. In some embodiments, the linearly arranged elongated slots are spaced apart by a predetermined distance. The size of the elongated slot is not limited, but in some embodiments, the elongated slot has a length of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 mm, or a range therebetween, or 5 to 20, 5 to 18, 5 to 15, 7 to 20, 7 to 18, or 7 to 15 mm. In some embodiments, the elongated slot has a width of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5 mm, or a range therebetween, or 0.5 to 2.5, 1 to 2.5, 1 to 2 mm. The elongated slots linearly arranged along the x-axis of the membrane allow for one lane, as previously described. Another lane is formed by other elongated slots also linearly arranged along the x-axis of the membrane but spaced apart from another lane along the y-axis. Thus, each of the elongated slots are spaced apart to form an array or grid at different non-overlapping positions along the x-axis and y-axis.

[0107] In embodiments, the membrane 102 may comprise silicone or a polymer such as polypropylene. In certain embodiments, the membrane 102 comprises a polypropylene film coated on one or both sides with a silicone adhesive. In some embodiments, the membrane 102 may be a transparent polypropylene film comprising an adhesive on both sides thereof. The adhesive can comprise, but not limited to, a silicone adhesive or an acrylic adhesive. In some embodiments, the membrane 102 includes at least one of low autofluorescence, compatibility with biological samples and reagents, low outgassing, an operating range of at least between -20°C (Celsius) and 40°C, and a total thickness of about 10, 20, 50, 100, 120, 140, 160, 180, 200, 220, 250, 300, 350, 400, 450, 500, 550, 600 micrometers or microns (urn), or any range therein (e.g., any range defined by two of the foregoing endpoints). In some embodiments, the thickness is 10 to 600, 20 to 600, 50 to 500, 50 to 450, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 500, 100 to 450, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 220, 120 to 400, 120 to 350, 120 to 300, 120 to 250, 120 to 220, 160 to 400, 160 to 350, 160 to 300, 160 to 250, 160 to 220, 180 to 400, 180 to 350, 180 to 300, 180 to 250, 180 to 220, 180 to 400, 180 to 350, 180 to 300, 180 to 250, 180 to 220 um.

[0108] In some embodiments, the elongated slots of the membrane can be manufactured by using laser to cut the polypropylene film with laser, but the present disclosure is not so limited to manufacturing via laser. For example, in other embodiments, the elongated slots may be stamped or die cut. In some embodiments, each of the plurality of elongated slots can have the same shape; while in some embodiments, one of the plurality of elongated slots can have a shape different from another one of the plurality of elongated slots, for example, in width, length, or both of the slot. Alternatively, the shapes of the elongated slot can vary in width to have different widths along the length, and each of the elongated slots need not vary in the same manner along their lengths.

[0109] In embodiments, the cover 109 may be a film, laminate, or other material suitable for covering and thereby sealing at least one or each of the plurality of first openings 106 and the plurality of second openings 107 of the baffle 101. The cover 109 may also be configured to cover and thereby seal each of the plurality of bridges and first openings 106 and the second openings 107 such that the flow path easily conducts the fluid through the bridge and the flow path is free from contamination via any exposed openings. That is, in the case where the bridge is a depression along on the surface of body 101, the cover 109 forms the top covering of the bridge such that the flow is constrained within the bridge and flows into the next z- channel. In an example, the cover 109 may be an adhesive film. To aid in imaging of the sample chamber and/or the substrate 103, the cover 109 may be opaque.

Substrate and capture agents

[0110] In embodiments, the substrate 103 comprises a first substrate surface 103' and a second substrate surface 103". The first substrate surface 103' of the substrate 103 may comprise a plurality of immobilized capture agents, each immobilized capture agent capable of specifically binding to one of a plurality of cellular components. To this end, the immobilized capture agents may be arranged uniformly, the immobilized capture agents may be attached to the first substrate surface 103' in a repeatable pattern, and/or each repetition of the capture agent pattern may align with a sample chamber. In some embodiments, the substrate 103 is made of a transparent material. In some embodiments, the substrate 103 is made of silica or a mixture that is predominantly silica. For example, the substrate 103 can be made of a glass. In some embodiments, the substrate 103 is coated with molecules suitable for immobilizing the capture agents. For example, the substrate 103 can be coated with poly-L Lysine (PLL). However, the present disclosure is not so limited. Other molecules and linking mechanisms, such as biotin-streptavidin linkages or covalent linkages (e.g., formed by "click chemistry") can also be employed for immobilizing the capture agents on a surface of the substrate 103. [0111] In embodiments, the capture agent can be a peptide, a protein, an oligonucleotide, or a combination thereof. In embodiments, preferred capture agents include antibodies. However, capture agents may include any entity that specifically binds to a target of interest in the biological sample. In some embodiments, the target is a protein, nucleic acid, metabolite, or viral particle. Detection of the binding between capture agents and targets may be achieved using a reporter molecule having a detectable entity. In certain embodiments, preferred detectable entities include antibodies or other agents that specifically bind the target. The detectable entity may comprise a detectable label. In some embodiments, the target is directly labeled with a detectable label and, therefore, the target itself is the detectable entity. Detectable labels may include, but are not limited to, fluorescent labels and chemiluminescent labels.

[0112] In embodiments, the first substrate surface 103' of the substrate 103 may comprise more than one kind of capture agents, for example two kinds of capture regents. In embodiments, the first substrate surface 103' of the substrate 103 may comprise between 3 and 80 different capture agents, 6 and 80 different capture agents, 12 and 80 different capture agents, 16 and 80 different capture agents, or 24 and 80 different capture agents, thereby allowing for the detection of between 3 and 80 different cellular components 6 and 80 different cellular components, 12 and 80 different cellular components, 16 and 80 different cellular components, or 24 and 80 different cellular components (for example), but may include greater than 10 different capture agents, thereby allowing for the detection of greater than 10 different cellular components, or may comprise greater than 42 different capture agents, thereby allowing for the detection of greater than 42 different cellular components, or may comprise greater than 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or any number in between of different capture agents, thereby allowing for the detection of greater than 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or any number in between of different cellular components. In embodiments, each of the different capture agents may be present within each sample chamber.

[0113] In embodiments, the capture agents may be antibodies. In some embodiments, the capture agents may be specific to cytokines and components of or stimulators of the immune system. In some embodiments, the effector cytokines are selected from the group consisting of CCL-11, GM-CSF, Gran B, IFN-g, IL-10, IL-12, IL-13, IL-15, IL-17AJL-17F, IL-lb, IL-2, IL-21, IL- 22, IL-4, IL-5, IL-6, IL-7, IL-8, IL-19, IP-10, MCP-1, MCP-4, MIP-lalpha, MIP-lbeta, perforin, RANTES, TGFbetal, TNF-alpha, TNF-beta, sCD137, and sCD40L.

[0114] In embodiments, the capture agents may be proteins. In some embodiments, the protein capture agents may be configured to capture antibodies present in the biological sample.

Sample chamber, holder, and assembly [0115] Prior to use, the above components may be assembled. In embodiments, the Z-MAD 100 may include the baffle 101 and the cover 109, features of the membrane 102, the substrate 103, and the holder 110 being incorporated within a single body construction of the baffle 101. The holder 110 is configured to receive the body 100a. To this end, assembly includes, after introduction of biological sample via the plurality of first openings 106, covering and sealing the plurality of first openings 106, the plurality of second openings 107, and the plurality of bridges. In some embodiments, the Z-MAD 100 comprises, separately or in select combinations, the components described above. To this end, assembly includes positioning the membrane 102 between the baffle 101 and the substrate 103. When coupled together, the baffle 101, the plurality of elongated slots of the membrane 102, and the substrate 103 define a plurality of sample chambers therebetween. The volume of each sample chamber is defined by the second side of the baffle 101, a thickness of the membrane 102, and the first substrate surface 103' of the substrate 103.

[0116] In some embodiments, the volume of each sample chamber is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 microliters (uL), or any range therein (e.g., any range defined by two of the foregoing endpoints). In certain embodiments, the volume of each sample chamber is 1 to 20 uL, 2 to 19 uL, 3 to 18 uL, 4 to 17 uL, 5 to 16 uL, 5 to 15 uL, 5 to 14 uL, 5 to 13 uL, 5 to 12 uL, 5 to 11 uL, 5 to 10 uL, 6 to 16 uL, 6 to 15 uL, 6 to 14 uL, 6 to 13 uL, 6 to 12 uL, 6 to 11 uL, or 6 to 10 uL.

[0117] In embodiments, a biological sample volume applied to the plurality of first openings, and subsequently to the plurality of sample chambers, is between 10 nanoliters (nl_) and 100 microliters (uL). In certain embodiments, the volume of each sample chamber is designed to accommodate a biological sample having a volume of 0.01, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 uL, or any range therein (e.g., any range defined by two of the foregoing endpoints), or 0.1 to 20, 0.5 to 20, 1 to 20, 0.1 to 19, 0.5 to 19, 1 to 19, 2 to 19, 0.1 to 15, 0.5 to 15, 1 to 15, 2 to 15, 3 to 15, 3 to 18, 4 to 17, 5 to 16, 5 to 15, 5 to 14, 5 to 13, 5 to 12, 5 to 11, 5 to 10, 6 to 16, 6 to 15, 6 to 14, 6 to 13, 6 to 12, 6 to 11, or 6 to 10 uL. In some embodiments, the volume of each sample chamber is designed to accommodate a biological sample having a volume of smaller than 15, 14, 13, 12, 11, 10, 9.5, 9. 8.5, 8, 7.5, 7, 6.5, 6, 5.5, or 5 uL.

[0118] The baffle 101, the membrane 102, and the substrate 103 may secured within the holder 110. Snap features 111 of the holder 110, or similar features configured to couple components, may be configured to engage flanges of the baffle 101, such as flange 112, in order to retain the components in the holder 110. In some embodiments, when the holder 110 is coupled with the body 100a (e.g., coupled with the baffle 101 the membrane 102, and the substrate 103), the holder 110 does not block either or both of the first opening 106 and the second opening 107 on the first side of the body 100a.

[0119] In embodiments, the holder 110 comprises a window 110' through which the substrate 103 and at least one of the sample chambers can be visible, interrogated, imaged, and the like. Though not shown in the Drawings, in some embodiments, the holder 110 may further comprise a coded label for identifying the Z-MAD when used independently or within a system of Z-MADs.

[0120] In embodiments, the holder 110 may be formed of a material having desirable optical properties. For instance, the material may be one having low fluorescence and/or autofluorescence. In certain embodiments, the material may be a thermoplastic such as a poly(methyl methacrylate), a thermoset, a metal such as aluminum, anodized aluminum, and stainless steel, glass, ceramic, and the like. In an example, the material may be polydimethylsiloxane (PDMS).

[0121] In embodiments, the Z-MAD 100 of FIG. 1A and FIG. IB comprises a number of components that may be formed independently or integrally according to manufacturing capabilities. For instance, the baffle 101, the membrane 102, the substrate 103, and the holder 110 may be integrally formed.

[0122] After introducing the biological sample via the plurality of first openings 106 of the baffle 101, the cover 109 may be positioned over the first side of the baffle 101 (excluding the at least one input opening 104 and the at least one output opening 105).

[0123] With reference now to FIG. 2, a perspective view of a baffle 101, a membrane 102, and a substrate 103 of a Z-MAD are shown. As alluded above, a first side of the baffle 101 includes at least one row of a plurality of first openings, at least one row of a plurality of second openings, at least one input opening 104, and at least one output opening 105. As in FIG. 2, the at least one row of the plurality of first openings may include a first row 106a, a second row 106b, a third row 106c, and a fourth row 106d. The at least one row of the plurality of second openings may include a first row 107a, a second row 107b, a third row 107c, and a fourth row 107d.

[0124] A plurality of bridges may fluidly couple adjacent ones of the plurality of first openings and the plurality of second openings on the first side of the baffle 101. For instance, bridge 113 may fluidly couple aligned ones of the first row 106a of the plurality of first openings and the second row 107b of the plurality of second openings. A plurality of elongated slots 170 of the membrane 102 may, when coupled together with the baffle 101 and the substrate 103, form a sample chamber that fluidly couples corresponding ones of the plurality of first openings and the plurality of second openings at a second side of the baffle 101. For instance, sample chamber 170' fluidly couples aligned ones of the third row 107c of the plurality of second openings and the third row 106c of the plurality of first openings. By forming these fluid communications within and about the baffle 101 (with or without the presence of a cover), it can be appreciated how fluid may travel in three-dimensions between the at least one input opening 104 and the at least one output opening 105.

[0125] FIG. 2 will be similarly described with reference to FIG. 3A and FIG. 3B. [0126] FIG. 3A shows a top view of the first side of the baffle 101. The first side of the baffle

101 comprises a plurality of first openings, a plurality of second openings, at least one input opening 104, at least one output opening 105, and a plurality of bridges. It can be appreciated that the arrangement and labeling of the apertures of the first side of the baffle 101 in FIG. 3A and FIG. 3B is opposite that of previous Drawings. This is intentionally done to demonstrate the flexibility of the system to be used in more than one orientation.

[0127] In embodiments, and with reference to FIG. 3A, the first side of the baffle 101 comprises at least a first row 120 of the plurality of first openings, a second row 121 of the plurality of first openings, a third row 122 of the plurality of first openings, and a fourth row 123 of the plurality of first openings. Additionally, the first side of the baffle 101 comprises a first row 125 of the plurality of second openings, a second row 126 of the plurality of second openings, a third row 127 of the plurality of second openings, and a fourth row 128 of the plurality of second openings. Adjacent the fourth row 128 of the plurality of second openings, the first side of the baffle 101 may comprise an opening 129 configured to prevent samples from moving toward the at least one input opening 104. In other words, the opening 129 provides a barrier between chambers closest to the at least one input opening 104. Similarly, adjacent the first row of the plurality of first openings, the first side of the baffle 101 may comprise an opening 124 configured to prevent samples from moving toward the at least one output opening 105. FIG. 3B is a detailed view of the area outlined with dashed lines in FIG. 3A.

[0128] FIG. 3B illustrates a single row of adjacent ones of the plurality of first openings and the plurality of second openings. The single row includes first openings 130a, 130b, 130c, 130d, 130e and second openings 131a, 131b, 131c, 131d, 131e. The first openings 130a, 130b, 130c, 130d, 130e and the second openings 131a, 131b, 131c, 131d, 131e are fluidly coupled, respectively, by bridges 113a, 113b, 113c, 113d, 113e.

[0129] With reference now to FIG. 4, a view of a second side of a baffle 101 and a membrane

102 coupled thereto is shown.

[0130] In embodiments, and in view of FIG. 3A, rows 120, 121, 122, 123 of first openings and rows 125, 126, 127, 128 of second openings extend through the baffle 101 from the first side to the second side. Accordingly, FIG. 4 shows a first row 120 of the plurality of first openings, a second row 121 of the plurality of first openings, a third row 122 of the plurality of first openings, and a fourth row 123 of the plurality of first openings. Additionally, FIG. 4 shows a first row 125 of the plurality of second openings, a second row 126 of the plurality of second openings, a third row 127 of the plurality of second openings, and a fourth row 128 of the plurality of second openings. When the baffle 101 is coupled with the membrane 102 and the substrate (not shown), sample chambers are formed therebetween and aligned with corresponding ones of the plurality of first openings and the plurality of second openings. For instance, a first row 140 of sample chambers can be formed between corresponding ones of the first row 120 of the plurality of first openings and the first row 125 of the plurality of second openings. Similarly, a second row 141 of sample chambers, a third row 142 of sample chambers, and a fourth row 143 of sample chambers may be formed between corresponding ones of the plurality of first openings and the plurality of second openings, as shown in FIG. 4. Each of the sample chambers comprises a volume defined by the second side of the baffle 101, the membrane 102, and the first substrate surface of the substrate (not shown).

[0131] Referring now to FIG. 5A through FIG. 5C, and in view of the description of the previous Drawings, during use, use of the Z-MAD 100 will be described.

[0132] In embodiments, a number of wash steps may be performed at a variety of different times. The wash step may include flowing, by a fluidic system optionally controlled by a processor circuit, washing fluid through the plurality of passages of the Z-MAD 100, the plurality of passages including the at least one input opening 104 of the baffle 101, the plurality of bridges of the baffle 101, the plurality of first openings of the baffle 101, the plurality of second openings of the baffle 101, the sample chambers formed by the assembly, the first passage 119, and the second passage 118.

[0133] In embodiments, a fluid comprising capture agents may be flowed, by the fluidic system, through the plurality of passages of the Z-MAD 100 in order to immobilize the capture agents within sample chambers of the Z-MAD 100.

[0134] In embodiments, biological samples may be loaded into the sample chambers via the plurality of first openings on the first side of the baffle 101. In other words, the biological samples are injected into rows 120, 121, 122, 123 of the plurality of first openings and permitted to fill corresponding rows 140, 141, 142, 142 of sample chambers in the directions indicated by the black arrows shown in FIG. 5B. At the time of biological sample loading, the biological sample in each sample chamber is isolated from the biological samples in the other sample chambers.

[0135] Following sample loading, the cover may be placed on the first side of the baffle 101 in order to cover and seal the plurality of first openings, the plurality of second openings, and the plurality of bridges therebetween. The Z-MAD 100 may then be positioned within an instrument operably engaged with the fluidics system to control fluid and reagent flow within the Z-MAD 100. The fluidics system may be in fluid communication with the plurality of passages of the Z-MAD 100 via the at least one flexible seal. One or more reagent and/or wash solutions can then be flowed into the Z-MAD 100 via the at least one input opening 104, through the plurality of passages, and out of the Z-MAD 100 via the at least one output opening 105. Such a fluid flow path can be visually described by the serpentine flow path indicated by the black arrows in FIG. 5C.

[0136] FIG. 5C includes a dashed line associated with an 'A' indicating a portion of the image from which the cross-sectional schematics of FIG. 6A and FIG. 6B are obtained. As shown in FIG. 6A and FIG. 6B, fluid flow within the plurality of passages of the Z-MAD 100, and particularly between the sample chambers in the Z-MAD 100, utilizes the "z-channels" of the plurality of first openings and the plurality of second openings and the plurality of bridges to traverse between rows (i.e., lanes) of sample chambers, between columns of sample chambers, and between rows of first openings and second openings. As in FIG. 6A and FIG. 6B, the Z-MAD 100 may include a baffle 101, a holder 110, a membrane 102, a substrate 103, a cover 109, and at least one flexible seal.

[0137] For convenience, fluids flowing along a length of the baffle 101 are referred to as flowing along the "x-axis" (as denoted in FIG. 6A), and fluids flowing "up" or "down" through the baffle 101 via the z-channels are referred to as flowing along the "z-axis" (as denoted in FIG. 6A). As previously discussed, fluids can also flow along the y-axis to allow for a serpentine turn.

[0138] In embodiments, and as shown by the white arrows in FIG. 6A and FIG. 6B, fluid flow traverses the plurality of passages by traveling in both the x-axis, the y-axis (as denoted in FIG. 6B), and the z-axis of the baffle 101. For instance, after being introduced into the Z-MAD 100 via the at least one input opening 104, fluid flows within the Z-MAD 100 as follows: (1) from the first side of the baffle 101 to the second side of the baffle 101 via z-channel 144; (2) along the x-axis through a first sample chamber 152; (3) "up" along the z-axis via z-channel 145; (4) along the x-axis through a first bridge 156; (5) "down" along the z-axis via z-channel 146; (6) along the x-axis through a second sample chamber 153; (7) "up" along the z-axis via z-channel 147; (8) along the x-axis through a second bridge 157; (9) "down" along the z-axis via z- channel 148; (10) along the x-axis through a third sample chamber 154; (11) "up" along the z- axis via z-channel 149; (12) along the x-axis through a third bridge 158; (13) "down" along the z-axis via z-channel 150; (14) along the x-axis through a fourth sample chamber 155; and (15) "up" along the z-axis via z-channel 151.

Methods

[0139] According to embodiments, use of the assembled Z-MAD 100 comprises loading one or more biological samples into the plurality of first openings 106 of the baffle 101. Each opening of the plurality of first openings 106 may be fluidly coupled with a sample chamber defined in part by one of the plurality of elongated slots 170 of the membrane 102. Loading the one or more biological samples includes injecting the biological sample, using for example a pipette, into one or more of the plurality of first openings 106 and allowing the biological sample to flow into and fill a corresponding sample chamber. After sample loading, the cover 109 may be placed on the first side of the baffle 101 to cover and seal the plurality of first openings 106, the plurality of second openings 107, and the plurality of bridges. The at least one input opening 104 and the at least one output opening 105 may then be used to deliver reagents and/or washes to the sample chambers. To further illustrate the use of the Z-MAD 100, FIG. 5A through FIG. 5C provides a semi-transparent top view of the baffle 101 with the membrane 102 disposed between the second side of the baffle 101 and the substrate 103 (not shown). FIG. 6A and FIG. 6B illustrate cross-sectional views of the assembled Z-MAD 100. Such Drawings will be described in more detail below. [0140] With reference now to FIG. 7, a method of using a Z-MAD of the present disclosure is described. It should be appreciated that the method of FIG. 7 can be performed manually, can be partially automated, or can be fully automated. To this end, the method of FIG. 7 can be performed at least partially by one or more processors configured to interact with the Z- MAD. A schematic of such an interaction is shown and described in FIG. 9. For instance, flowing of fluids and reagents may be controlled by a processor(s) in conjunction with a fluidic system, imaging may be controlled by a processor(s) in conjunction with an imaging system (or imager) (e.g., a camera), and reading may be controlled by a processor(s) in conjunction with an electronic reader. The biological samples pipetted into the sample chambers can be performed manually or performed by an instrument controlled by a processor(s). In one example, the biological samples may be pipetted manually, but the flowing of fluids and reagents through the entire flow path may be controlled by the processor(s), or vice versa. Each of the processes of method 700, including experimental planning to result analysis and visualization can be controlled by processor(s) and a user via a graphical user interface of a computing device.

[0141] At process 701 of method 700, a Z-MAD system is provided. As shown in FIG. 8A through FIG. 8C, the Z-MAD may be provided as one of a plurality of systems. For instance, as in FIG. 8A, the Z-MAD may be provided as a plurality of individual components, including a baffle, a substrate, a membrane, a cover, a holder, and at least one flexible seal, amongst other ancillary components described herein. In another instance, as in FIG. 8B, the Z-MAD may be provided as a plurality of individual components, including a baffle, a substrate, a membrane, and a cover, amongst other ancillary components described herein, but wherein a holder and the at least one flexible seal is not included. In another instance, as in FIG. 8C, the Z-MAD may be provided as an individual integrated baffle, wherein one or more of the plurality of components described above as being included within the Z-MAD may formed in a unitary body. In the event the Z-MAD of FIG. 8A and/or FIG. 8B is provided, the Z-MAD may be provided in an assembled form, in a partially assembled form (cover not assembled), or in a disassembled form.

[0142] Method 700 is described below in view of a fully assembled Z-MAD having previously been doped with capture agents specifically designed for particular analytes (and the like) of the biological sample to be introduced.

[0143] At step 702 of method 700, biological sample can be added to the sample chambers via the plurality of first openings. After adding the biological sample, the Z-MAD may be incubated for a predetermined period of time to allow the biological sample to travel the first openings, enter the sample chambers, and interact with the capture agents.

[0144] At step 703 of method 700, a wash via the plurality of passages may be performed. In embodiments, the wash fluid may be phosphate buffered saline, double distilled water, and the like. [0145] At step 704 of method 700, detection antibodies may be flowed through the plurality of passages of the Z-MAD. When flowing through the sample chambers of the Z-MAD, the detection antibodies may bind to analytes captured by the capture agents immobilized on the substrate and within the sample chambers. In some embodiments, the detection antibodies may be fluorescent antibodies configured for imaging and/or reading.

[0146] At step 705 of method 700, a wash via the plurality of passages may be performed.

[0147] At step 706 of method 700, the sample chambers of the Z-MAD may be imaged and/or read by an imager and/or electronic reader.

[0148] Wash step(s) performed after sample loading, as a result of the z-flow pattern within the z-channels and the bridges therebetween, can provide better separation between neighboring chambers and wash out sample trapped in the z-channels after loading, which can reduce cross-contamination between neighboring wells and reduce "flare-ups" emanating from trapped sample subsequently entering a chamber.

System

[0149] Another aspect of the present disclosure provides a system for analyzing a biological sample, comprising the microfluidic device according to an embodiment of the present disclosure, and an instrument for operating the microfluidic device to perform the method according to an embodiment of the present disclosure.

[0150] With reference now to FIG. 9, and in view of the semi-automated or automated method of FIG. 7, in some embodiments, a multiplex assay system 950 configured for multiplexed analysis of biological material is provided. The multiplex assay system 950 includes at least one Z-MAD 900 in communication with one or more of a fluidic system 920, a fluorescing device 921, an imager 922, and an electronic reader 923. In embodiments, the fluorescing device 921 and the imager 922 may be separate or combined within a single imaging system. In embodiments, the imager 922 may be a fluorescence imager such as a fluorescence microscope, a fluorescence spectrophotometer, and the like. In embodiments, the electronic reader 923 may be an optical scanner configured to scan a barcode conveying an identity of a particular one of the at least one Z-MAD 900. In embodiments, the fluidics system 920, the fluorescing device 921, the imager 922, and the electronic reader 923 may interface with the at least one Z-MAD 900 and may be controlled by processor(s) 910 in order to perform the methods of the present disclosure on the Z-MAD 900. In embodiments, a user may interface with the system via a graphical user interface 904 on a computing device 914. The graphical user interface can be configured to at least one of (and preferably all of) display information, output information from the system 950, receive input from the user via the computing device 914, the fluorescing device 921, the fluorescing device 921 being configured to expose the opening of the holder of the at least one Z-MAD 900 to fluorescing light, the imager 922 configured to image the substrate and corresponding sample chambers upon the substrate being exposed to the fluorescing light, the electronic reader 923 configured to receive or otherwise obtain an identity code from each of the Z-MADs, and one or more processors 910 configured with computer instructions operational thereon to cause the system to perform the methods of the present disclosure. One of skill in the art will appreciate that the disclosed system, in some embodiments, includes structure to aid in providing, pumping, and exhausting various fluids/materials to the at least one Z-MAD, and may also include structure to aid in incubating materials within the at least one Z-MAD.

Exemplary Data and Results

Example 1: Chamber Isolation Test using Z-flow Multiplex Assay Device (Z-MAD)

[0151] 4.5 pL/chamber of either 1:200 ng/ml AF647-labelled rabbit anti-IgG and 1:200 ng/ml AF488-labelled mouse anti-IgG in lx cell lysis buffer was loaded into 20 chambers of a Z-flow multiplex assay device (Z-MAD) in the pattern shown in Table 1.

Table 1: Sample Loading Map of Z-MAD

[0152] The cover was applied to the Z-MAD and the Z-MAD was loaded in an IsoSpark™ instrument (IsoPlexis, Inc.). The rabbit anti-IgG and mouse anti-IgG was incubated in the chambers, which were coated with rabbit and mouse IgG antibodies against p-lkBa, p-PRAS40, Cleaved PARP, p-eif4E, p-STAT5, p-MEKl/2, Alpha Tubulin, p-p44-42 MAPK, p-S6 Ribosomal, p-STAT3, p-p90RSK, p-Rb, p-STATl, p-NF-kB p65, and p-MET. The IsoSpark™ instrument then performed a wash protocol, which flowed a wash buffer through the chambers of the Z-MAD in a serpentine pattern to remove unbound rabbit anti-IgG and mouse anti-IgG from the Z- MAD. The IsoSpark™ instrument then imaged the chambers to detect the remaining, bound rabbit anti-IgG and mouse anti-IgG.

[0153] Image analysis showed that there was no detectable cross-contamination between chambers, indicating that the z-channels effectively contained the AF647-labelled rabbit anti- IgG and inAF488-labelled mouse anti-IgG their intended chambers.

Example 2: Comparative Study Of Z-flow Multiplex Assay Device (Z-MAD)

[0154] 5 pL/ Chamber of 1500 pg/ml recombinant protein ("Recomb") with ELISA buffer was loaded into chambers of either a Z-MAD or multiplex assay device without z-channels (non-Z- MAD) in the pattern shown below in Table 2. The recombinant proteins included recombinant human GM-CSF, Granzyme B, IFN-y, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-15, ILI A, IP-10, MCP-1, MIP-la, MIP-ip, Perforin, SCD137, TNF-a, and TNF- .

Table 2: Sample Loading Map for Comparison Study

[0155] The cover was applied to the Z-MAD and non-Z-MAD and both were loaded in an IsoSpark™ instrument (IsoPlexis, Inc.). In the Z-MAD, each chamber was separated from the next chamber in series by two z-channels and a bridge. In the non-Z-MAD, each chamber was separated from the next chamber in series by an air gap. The Recomb were incubated in the chambers, which were coated with antibodies against human GM-CSF, Granzyme B, IFN-y, IL- 2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-la, MIP-1 , Perforin, sCD137, TNF-a, and TNF- . The IsoSpark™ instrument then performed a wash protocol, which flowed a wash buffer through the chambers of the Z-MAD and non-Z-MAD in a serpentine pattern to remove unbound Recomb. The serpentine flow path through chambers in the Z-MAD utilized flows in the x-, y-, and z-dimensions, whereas the flow path between chambers in the non-Z-MAD utilized only x- and y-dimensions. The IsoSpark™ instrument then performed a detection reagent protocol, which flowed fluorescently labeled detection antibodies through the chambers of the Z-MAD and non-Z-MAD in a serpentine pattern. Following incubation of the detection antibodies in the chambers, a further wash protocol was performed to remove unbound detection antibodies. The IsoSpark™ instrument then imaged the chambers of each device to detect the labeled Recomb. [0156] Image analysis showed that the Z-MAD had better-defined chambers with no detectable cross-contamination between chambers, as compared to the non-Z-MAD. The non-Z-MAD exhibited significant sample carryover between some chambers.

Example 3: 32 Sample Z-flow Multiplex Assay Device (Z-MAD)

[0157] 4 pL/chamber of either 1:200 ng/ml AF647-labelled rabbit anti-IgG and 1:200 ng/ml AF488-labelled mouse anti-IgG in RPMI cell culture media was loaded into 32 chambers of a Z-flow multiplex assay device (Z-MAD) in the pattern shown in Table 3.

Table 3: Sample Loading Map of Z-MAD for 32 Sample Study

[0158] The cover was applied to the Z-MAD and the Z-MAD was loaded in an IsoSpark™ instrument (IsoPlexis, Inc.). The rabbit anti-IgG and mouse anti-IgG was incubated in the chambers, which were coated with rabbit and mouse IgG antibodies against human GM-CSF, Granzyme B, IFN-y, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-15, IL-17A, IP-10, MCP-1, MIP-la, MIP-1|3, Perforin, sCD137, TNF-a, and TNF- . The IsoSpark™ instrument then performed a wash protocol, which flowed a wash buffer through the chambers of the Z-MAD in a serpentine pattern to remove unbound rabbit anti-IgG and mouse anti-IgG from the Z- MAD. The IsoSpark™ instrument then imaged the chambers to detect the remaining, bound rabbit anti-IgG and mouse anti-IgG. [0159] Image analysis showed that there was no detectable cross-contamination between chambers, indicating that the z-channels effectively contained the AF647-labelled rabbit anti- IgG and inAF488-labelled mouse anti-IgG their intended chambers.

[0160] Example 4: Detection of recombinant proteins for the generation of calibration curves and Detection and quantitation of native proteins from cellular supernatants

[0161] A recombinant protein calibrator blend of known concentration was serially diluted 5 times from 20000 pg/ml to 19.5 pg/ml using lx ELISA buffer. 7.5 pL/chamber of each calibrator (Call-Cal6), background (IX ELISA buffer), RPMI media, cell supernatant (Samples 1-9), and control samples (HuCtrl-1, HuCtrl-4) were loaded into 20 chambers of a Z-flow multiplex assay device (Z-MAD) in the pattern shown in Table 3.

Table 4: Sample Loading Map of Z-MAD

[0162] The cover tape was applied to the Z-MAD and the Z-MAD was loaded in an IsoSpark™ instrument (IsoPlexis, Inc.). The Z-MAD incubated for 1 hour on the IsoSpark™ instrument before a wash protocol initiated, which flowed a wash buffer through the chambers of the Z- MAD in a serpentine pattern to remove unbound proteins in all the wells. Biotinylated detection antibodies were then introduced to the Z-MAD that bind the proteins bound to the coated capture antibodies. After excess detection antibodies were washed away, streptavidin conjugated fluorophore was introduced to the Z-MAD. After washing away excess streptavidin conjugated fluorophore, the IsoSpark™ instrument imaged the chambers using an onboard excitation laser, microscope objective assembly, and associated CCD camera. [0163] Using the IsoSpeak® software, the calibrator points were plotted on a graph and a 4- Parameter Logistic Regression (4PL) curve fit was applied to interpolate unknown values (FIG. 10). A clear dose-response relationship was shown whereby as the concentration of recombinant protein increases so does the resulting signal intensity (RFU). The interpolated values here were used to translate RFU values from samples with unknown protein concentrations into pg/ml values.

[0164] HuCtrl-1 and HuCtrl-4 are control samples whereby the same recombinant proteins used for calibrators are spiked into RPMI media at high and low concentrations, respectively. These samples were used to confirm protein quantitation is possible in the same sample matrix as the cell supernatants loaded on the same Z-MAD. FIG. 11A and FIG. 11B demonstrate how the interpolated values from the calibration curve fitting can be used to translate RFU to pg/ml.

[0165] Cell supernatants were used to confirm the ability to detect proteins of interest secreted by cells cultured in RPMI cell culture media. The cells were collected several PBMC samples and cell lines and were exposed to a variety of stimulants for certain periods of time before the cell supernatant was collected. The cell supernatant sample RFUs were converted to pg/ml (FIG. 12) based on the same interpolated values generated from the calibration curves shown in FIG. 10. The results show, by using the microfluidic device and method according to embodiments of the present disclosure, different secretomes, as a result of cell types and stimulation conditions, were able to be multiplexed and observed.

ADDITIONAL REMARKS

[0166] While various inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be an example and that the actual parameters, dimensions, materials, and configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims, equivalents thereto, and any claims supported by the present disclosure, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, method, functionality, and step, described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, methods, and steps, if such features, systems, articles, materials, kits, methods, functionality, and steps, are not mutually inconsistent, is included within the inventive scope of the present disclosure. Embodiments disclosed herein may also be combined with one or more features, as well as complete systems, devices and/or methods, to yield yet other embodiments and inventions. Moreover, some embodiments, may be distinguishable from the prior art by specifically lacking one and/or another feature disclosed in the particular prior art reference(s); i.e., claims to some embodiments may be distinguishable from the prior art by including one or more negative limitations.

[0167] Also, various inventive concepts may be embodied as one or more methods, of which an example has been provided. The acts performed as part of the method may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different than illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.

[0168] Any and all references to publications or other documents, including but not limited to, patents, patent applications, articles, webpages, books, etc., presented anywhere in the present application, are herein incorporated by reference in their entirety. Moreover, all definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

NUMBERED EMBODIMENTS OF THE DISCLOSURE

[0169] Notwithstanding the appended claims, the disclosure sets forth the following numbered embodiments:

[0170] (1) A multiplex assay device configured for multiplexed analysis of biological material, the multiplex assay device comprising a baffle comprising a plurality of first openings on a first side of the baffle, a plurality of second openings on the first side of the baffle and a plurality of bridges on the first side of the baffle, wherein the first side of the baffle is separated from a second side of the baffle by a thickness defining a z-axis of the baffle, and wherein each one of the plurality of bridges fluidly couples corresponding ones of the plurality of second openings and the plurality of first openings, and a membrane comprising a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to respective ones of the plurality of first openings and the plurality of second openings, a cover; and a substrate, wherein corresponding ones of the plurality of first openings and the plurality of second openings are fluidly couplable via one of the plurality of elongated slots when the baffle, the membrane, and the substrate are assembled, a volume defined by the second side of the baffle, the substrate, and the one of the plurality of elongated slots being a sample chamber. [0171] (2) The multiplex assay device of (1), wherein the membrane comprises a first side and a second side, the first side configured to contact the baffle and the second side configured to contact the substrate.

[0172] (3) The multiplex assay device of either (1) or (2), wherein the membrane comprises a first end and a second end, an axis therebetween being an x-axis of the membrane, and wherein each of the plurality of elongated slots extends from a first end of the membrane towards a second end of the membrane along the x-axis of the membrane.

[0173] (4) The multiplex assay device of any one of (1) to (3), wherein the membrane comprises a third end and a fourth end, an axis therebetween defining a y-axis of the membrane.

[0174] (5) The multiplex assay device of any one of (1) to (4), wherein a first subset of the plurality of elongated slots includes at least two elongated slots linearly arranged along the x- axis of the membrane.

[0175] (6) The multiplex assay device of any one of (1) to (5), wherein the at least two elongated slots of the first subset of the plurality of elongated slots are spaced apart by a predetermined distance.

[0176] (7) The multiplex assay device of any one of (1) to (6), wherein the baffle further comprises a first end and a second end, an axis therebetween being an x-axis of the baffle, and wherein a first subset of the plurality of first openings and a corresponding first subset of the plurality of second openings are spaced apart along the x-axis of the baffle.

[0177] (8) The multiplex assay device of any one of (1) to (7), wherein the first subset of the plurality of first openings and the corresponding first subset of the plurality of second openings are spaced apart by the predetermined distance.

[0178] (9) The multiplex assay device of any one of (1) to (8), further comprising a holder.

[0179] (10) The multiplex assay device of any one of (1) to (9), wherein the baffle is couplable to the holder.

[0180] (11) The multiplex assay device of any one of (1) to (10), wherein the baffle and holder are couplable such that the substrate and the membrane are arranged therebetween.

[0181] (12) The multiplex assay device of any one of (1) to (11), further comprising a cover configured to cover the plurality of first openings and the plurality of second openings.

[0182] (13) The multiplex assay device of any one of (1) to (12), wherein the cover is configured to cover the plurality of first openings and the plurality of second openings after a biological material sample has been pipetted into at least one of the plurality of first openings.

[0183] (14) The multiplex assay device of any one of (1) to (13), wherein the plurality of first openings is arranged in a plurality of rows along the x-axis of the baffle. [0184] (15) The multiplex assay device of any one of (1) to (14), wherein the plurality of second openings is arranged in a plurality of rows along the x-axis of the baffle.

[0185] (16) The multiplex assay device of any one of (1) to (15), wherein the baffle comprises a third end and a fourth end, an axis therebetween defining a y-axis of the baffle.

[0186] (17) The multiplex assay device of any one of (1) to (16), wherein each of the plurality of first openings includes identifiable indicia.

[0187] (18) The multiplex assay device of any one of (1) to (17), wherein each of the plurality of first openings and each of the plurality of second openings extends from the first side of the baffle to the second side of the baffle along the z-axis of the baffle.

[0188] (19) The multiplex assay device of any one of (1) to (18), further comprising at least one input opening.

[0189] (20) The multiplex assay device of any one of (1) to (19), wherein the at least one input opening extends from the first side of the baffle to the second side of the baffle.

[0190] (21) The multiplex assay device of any one of (1) to (20), wherein the at least one input opening is configured for receiving a flow.

[0191] (22) The multiplex assay device of any one of (1) to (21), further comprising at least one output opening.

[0192] (23) The multiplex assay device of any one of (1) to (22), wherein the at least one output opening extends from the first side of the baffle to the second side of the baffle.

[0193] (24) The multiplex assay device of any one of (1) to (23), wherein the at least one output opening is configured for exhausting a flow.

[0194] (25) The multiplex assay device of any one of (1) to (24), further comprising at least one flexible seal.

[0195] (26) The multiplex assay device of any one of (1) to (25), further comprising a pair of flexible seals, one each for sealing the at least one input opening and the at least one output opening.

[0196] (27) The multiplex assay device of any one of (1) to (26), further comprising a respective opening or recess for receiving a respective flexible seal.

[0197] (28) The multiplex assay device of any one of (1) to (27), further comprising a coded label for identifying the multiplex assay device.

[0198] (29) The multiplex assay device of any one of (1) to (28), wherein the holder includes an opening so as to image the substrate. [0199] (30) The multiplex assay device of any one of (1) to (29), wherein each one of the plurality of bridges fluidly couples an opening of a first row of the plurality of second openings and a corresponding opening of a second row of the plurality of first openings.

[0200] (31) The multiplex assay device of any one of (1) to (30), wherein, when the cover, the baffle, the membrane, and the substrate are assembled, a plurality of sample chambers are formed and a plurality of passages are formed between the at least one input opening and the at least one output opening via the plurality of first openings of the baffle, the plurality of sample chambers, the plurality of bridges, and the plurality of second openings of the baffle so as to establish a three dimensional channel between the at least one input opening and the at least one output opening.

[0201] (32) A multiplex assay device configured for multiplexed analysis of biological material, comprising baffle comprising a plurality of first openings arranged in a plurality of rows on a first side of the baffle, each of the plurality of first openings including identifiable indicia and extending from a first side of the baffle to a second side of the baffle, a plurality of second openings arranged in a plurality of rows on the first side of the baffle corresponding to the plurality of rows of the plurality of first openings, each of the plurality of rows of the plurality of second openings extending from the first side of the baffle to the second side of the baffle, a plurality of bridges arranged on the first side of the baffle, each one of the plurality of bridges fluidly coupling a second opening of a first row of the plurality of second openings and a corresponding first opening of a second row of the plurality of first openings, at least one input opening arranged on the baffle and extending from the first side of the baffle to the second side of the baffle and configured for receiving a flow, and at least one output opening arranged on the baffle and extending from the first side of the baffle to the second side of the baffle and configured for exhausting the flow, a substrate, a membrane having a first face and a second face, the membrane further configured with a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to corresponding ones of the plurality of first openings and the plurality of second openings when the baffle, the membrane, and the substrate are assembled, a volume defined by the second side of the baffle, the substrate, and the one of the plurality of elongated slots being a sample chamber, a cover configured to cover the plurality of first openings and the plurality of second openings after a biological material sample has been pipetted into at least one of the plurality of first openings, a holder, and a pair of flexible seals, one each provided for the at least one input opening and the at least one output opening, wherein capture agents are disposed on a surface of the substrate and are contactable with biological material in a fluid within the sample chamber, wherein the baffle is couplable with the holder such that the substrate and membrane are arranged therebetween, wherein the holder includes an opening so as to image the substrate, wherein each channel of the membrane is positioned below at least one first opening of each row of first openings, such that a sample loaded into a respective first opening proliferates along at least a portion of the channel to interact with capture agents of the substrate, and wherein a plurality of passages connect the at least one input opening to the at least one output opening, the plurality of passages including a first passage connecting the at least one input opening to one of a first row of the plurality of first openings, a second passage connecting the at least one output opening to one of a last row of the plurality of second openings, and a plurality of supporting passages connecting the first passage, the plurality of first openings, each sample chamber, the plurality of second openings, the plurality of bridges, and the second passage.

[0202] (33) The multiplex assay device of (32), further comprising a fluidics system in fluid communication with each of the at least one input opening arranged on the baffle and the at least one output opening arranged on the baffle and being configured to flow one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening.

[0203] (34) A multiplex assay system configured for multiplexed analysis of biological material, the multiplex assay system comprising a receiving area configured to receiving a plurality of multiplex assay devices of any one of (1) to (32), a fluorescing device configured to expose the substrate and corresponding elongated slots of the membrane to the fluorescing light, and an imager configured to image the substrate and corresponding elongated slots of the membrane upon the substrate and elongated slots being exposed to the fluorescing light.

[0204] (35) The system of (34), further comprising one selected from the group consisting of a graphical user interface (GUI), an electronic reader, and one or more processors configured with computer instructions operational thereon to cause the system to perform a plurality of steps of a method, wherein the one or more processors interface with a fluidics system in fluid communication with each of the at least one input opening arranged on the baffle and the at least one output opening arranged on the baffle to flow one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening.

[0205] (36) The multiplex assay system of (35), wherein the GUI is configured to at least one of display information, display an output from the system, and/or receive input from a user.

[0206] (37) The multiplex assay system of either of (35) or (36), wherein the electronic reader is configured to receive or otherwise obtain a code from each of the multiplex assay devices.

[0207] (38) A method for multiplexed analysis of biological material using the multiplex assay system of any one of (34) to (37), comprising identifying each multiplex assay device of the multiplex assay system via reading of a code of a respective multiplex assay device, confirming proper application of the cover over the plurality of first openings and the plurality of second openings on the first side of the baffle of each identified multiplex assay device, incubating each multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents disposed on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, activating the fluorescing device, imaging the substrate from the opening in the holder upon exposure of the substrate to the fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image.

[0208] (39) A multiplex assay system configured for multiplexed analysis of biological material, the system comprising a receiving area configured to receiving a plurality of multiplex assay devices of any of (1) to (34), a graphical user interface configured to display information, output information from the system, and/or receive input from a user, a fluorescing device configured to expose the opening of a holder of each multiplex assay device to fluorescing light, an imager configured to image the substrate and corresponding elongated slots of the membrane upon the substrate being exposed to the fluorescing light, an electronic reader configured to receive or otherwise obtain a code from each of the multiplex assay devices, one or more processors configured with computer instructions operational thereon to cause the system to perform a method, comprising identifying each multiplex assay device via reading of a code of a respective multiplex assay device, confirming proper application of the cover over the plurality of first openings and the plurality of the second openings on the first side of the baffle of each multiplex assay device, incubating each multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents contained on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, activating the fluorescing device, imaging the substrate from the opening in the holder upon exposure of the substrate to the fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image.

[0209] (40) A multiplex assay method for multiplexed analysis of biological material comprising loading one or more biological samples into one or more of a plurality of first openings of the first side of the baffle of the multiplex assay device of any of (1) to (39), covering the first side of the baffle with a cover, placing the multiplex assay device within a processing system, identifying, via the processing system, the multiplex assay device via reading of a code of the multiplex assay device, confirming proper application of cover over the plurality of first openings, incubating the multiplex assay device over a period of time such that one or more components of the biological samples loaded into the plurality of first openings bind to capture agents contained on the substrate, flowing one or more reagents through the plurality of passages connecting the at least one input opening to the at least one output opening, capturing an image of at least one of the substrate and elongated slots of the membrane via an opening in the multiplex assay device upon exposure of the substrate to fluorescing light, and generating one or more graphs, charts, and/or information based on the acquired image. [0210] (41) A multiplex assay device configured for multiplexed analysis of biological material, the multiplex assay device comprising (a) a baffle comprising (i) a plurality of repeating fluidic structures, each of the plurality of repeating fluidic structures comprising a sealable first opening on a first side of the baffle, a sealable second opening on the first side of the baffle, a sample chamber disposed within the baffle or on a second side of the baffle, wherein the first side of the baffle is separated from the second side of the baffle by a thickness defining a z-axis of the baffle, a first channel extending along the z-axis from the sealable first opening to the sample chamber, and a second channel extending along the z-axis from the sealable second opening to the sample chamber, wherein the sealable first opening and the sealable second opening are in fluid communication via the first channel, the sample chamber, and the second channel, (ii) at least one connecting channel on the first side of the baffle and disposed between and in fluid communication with the sealable second opening of one of the plurality of repeating fluidic structures and the sealable first opening of another of the plurality of repeating fluidic structures, (iii) an input opening configured for receiving a flow and conducting the flow to the plurality of repeating fluidic structures, and (iv) an output opening configured for exhausting the flow from the plurality of repeating fluidic structures, and (b) a cover configured to sea lingly cover the plurality of repeating fluidic structures and the at least one connecting channel on the first side of the baffle.

[0211] (42) A method of preparing a plurality of samples for analysis, the method comprising (a) loading each of the plurality of samples into different sample chambers of the multiplex assay device of (41) via a corresponding one of the sealable first openings, and (b) applying the cover to the first side of the baffle, wherein the cover sealingly covers the plurality of repeating fluidic structures and the at least one connecting channel, and wherein the cover does not cover the input opening and the output opening.

[0212] (43) The method of either (41) or (42), wherein the plurality of samples comprises biological materials and the different sample chambers comprise capture agents.

[0213] (44) The method of any one of (41) to (43), further comprising washing out of the different sample chambers any of the biological materials that are not bound to the capture agents by flowing a wash solution into the input opening, through each of the plurality of repeating fluidic structures in series, and out the output opening.

[0214] (45) The method of any one of (41) to (44), further comprising labeling at least some of the biological materials bound to the capture agents in the sample chambers by flowing a labeling agent into the input opening, and through each of the plurality of repeating fluidic structures in series.

[0215] (46) The method of any one of (41) to (45), further comprising washing out of the sample chambers any of the labeling agents that are not bound to the biological materials by flowing a wash solution into the input opening, through each of the plurality of repeating fluidic structures in series, and out the output opening. [0216] (47) A system for multiplexed analysis of biological material, comprising a multiplex assay system, comprising a baffle comprising at least one input opening on a first side of the baffle, at least one output opening on the first side of the baffle, a plurality of first openings on the first side of the baffle, a plurality of second openings on the first side of the baffle, and a plurality of bridges on the first side of the baffle, wherein the first side of the baffle is separated from a second side of the baffle by a thickness defining a z-axis of the baffle, and wherein each one of the plurality of bridges fluidly couples corresponding ones of the plurality of second openings and the plurality of first openings, and a membrane comprising a plurality of elongated slots, each one of the plurality of elongated slots being fluidly couplable to respective ones of the plurality of first openings and the plurality of second openings, a cover; and a substrate, wherein corresponding ones of the plurality of first openings and the plurality of second openings are fluidly couplable via one of the plurality of elongated slots when the baffle, the membrane, and the substrate are assembled, a volume defined by the second side of the baffle, the substrate, and the one of the plurality of elongated slots being a sample chamber, and wherein the at least one input opening, the plurality of first openings, the elongated slots, the plurality of second openings, the plurality of bridges, and the at least one output opening are fluidly connected and form a plurality of passages, and a processor configured to actuate a fluidic system to flow one or more reagents through the plurality of passages via the at least one input opening, activating a fluorescing device to excite fluorophores of one of the one or more reagents, the fluorophores being bound to biological material captured by capture agents within sample chambers, the capture agents being bound to the substrate and instructing acquisition of images of the substrate.

[0217] Additional embodiments are listed below.

[0218] Embodiment Al: A microfluidic device, comprising a body having a flow path, the flow path comprising: a first fluidic structure; a second fluidic structure; and a first bridge disposed within the body or on a first side of the body and configured to fluidically couple the first fluidic structure and the second fluidic structure; wherein each of the first fluidic structure and the second fluidic structure comprises: (i) a first opening and a second opening, and (ii) a sample chamber disposed within the body or on a second side of the body, the first opening of the first fluidic structure and the first opening of the second fluidic structure are disposed at a first side of the body, and each sample chamber is configured to fluidically couple with the first opening and the second opening; and the first bridge is configured to fluidically couple the second opening of the first fluidic structure with the first opening of the second fluidic structure.

[0219] Embodiment A2: The microfluidic device of Embodiment Al, wherein the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the first opening extends from the first side toward the second side of the body (e.g., along the z-axis) to define a first z-channel configured to fluidically couple the first opening with the sample chamber of the second fluidic structure. [0220] Embodiment A3: The microfluidic device of Embodiment Al or Embodiment A2, wherein the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the second opening extends toward the second side of the body (e.g., along the z-axis) to define a second z-channel configured to f luidica lly couple the second opening with the sample chamber of the first fluidic structure.

[0221] Embodiment A4: The microfluidic device of Embodiment A3, wherein the second opening is disposed at the first side of the body, and the second opening extends from the first side toward the second side of the body.

[0222] Embodiment A5: The microfluidic device of Embodiment A3 or Embodiment A4, wherein the first bridge is configured to f luidica lly couple the first z-channel and the second z-channel along an axis different than the z-axis.

[0223] Embodiment A6: The microfluidic device of any one of Embodiments Al to A5, wherein the first bridge is configured to be the only fluidic communication between the first fluidic structure and the second fluidic structure.

[0224] Embodiment A7: The microfluidic device of any one of Embodiments Al to A6, wherein the sample chamber is elongated.

[0225] Embodiment A8: The microfluidic device of any one of Embodiments Al to A7, wherein the body further comprises an input opening configured for receiving a flow and conducting the flow to the flow path.

[0226] Embodiment A9: The microfluidic device of Embodiment A8, further comprising a first flexible seal configured to seal the input opening.

[0227] Embodiment A10: The microfluidic device of any one of Embodiments Al to A9, wherein the body further comprises an output opening configured for exhausting a flow from the flow path.

[0228] Embodiment All: The microfluidic device of Embodiment A10, further comprising a second flexible seal configured to seal the output opening.

[0229] Embodiment A12: The microfluidic device of any one of Embodiments Al to All, wherein each sample chamber is disposed on the second side of the body.

[0230] Embodiment A13: The microfluidic device of Embodiment A12, wherein the body comprises a baffle and a membrane, the baffle comprises a first baffle surface corresponding to the first side of the body and a second baffle surface coupled with a first membrane surface of the membrane; and wherein the membrane comprises a second membrane surface corresponding to the second side of the body, and a first elongated slot and a second elongated slot, and the first elongated slot is associated with the sample chamber of the first fluidic structure, the second elongated slot is associated with the sample chamber of the second fluidic structure, and each of the first and second elongated slots is configured to fluidically couple with the first opening and the second opening of the corresponding first fluidic structure and the second fluidic structure.

[0231] Embodiment A14: The microfluidic device of Embodiment A13, wherein: for each of the first and second fluidic structures, the corresponding first opening is disposed at the first baffle surface of the baffle, extends to the second baffle surface of the baffle, and opens to the corresponding elongated slot of the membrane.

[0232] Embodiment A15: The microfluidic device of Embodiment A13 or Embodiment A14, wherein: for each of the first and second fluidic structures, the corresponding second opening extends to the second baffle surface of the baffle, and opens to the corresponding elongated slot of the membrane.

[0233] Embodiment A16: The microfluidic device of any one of Embodiments A13 to A15, further comprising a substrate coupled with the second membrane surface of the membrane, and wherein the substrate, the elongated slot of the membrane, and the second membrane surface of the baffle define the sample chamber.

[0234] Embodiment A17: The microfluidic device of Embodiment A16, wherein a surface of the substrate is coated with a capture agent, and when the substrate is assembled with the baffle and the membrane, the capture agent is accessible the sample chamber.

[0235] Embodiment A18: The microfluidic device of Embodiment A17, wherein the capture agent is a peptide, a protein, an oligonucleotide, or a combination thereof.

[0236] Embodiment A19: The method of Embodiment A17 or Embodiment A18, wherein the capture agent comprises a first capture agent and a second capture agent, and the first capture agent is different from the second capture agent.

[0237] Embodiment A20: The microfluidic device of any one of Embodiments A13 to A19, wherein both the first elongated slot and the second elongated slot extend alone a length of the membrane.

[0238] Embodiment A21: The microfluidic device of Embodiment A20, wherein the first elongated slot and the second elongated slot are linearly arranged and spaced apart by a predetermined distance.

[0239] Embodiment A22: The microfluidic device of any one of Embodiments Al to A21, wherein the first opening has a diameter that is different than a diameter of the second opening.

[0240] Embodiment A23: The microfluidic device of any one of Embodiments Al to A22, wherein the first opening includes a chamfer.

[0241] Embodiment A24: The microfluidic device of any one of Embodiments Al to A23, wherein the flow path comprises an array of fluidic structures comprising the first fluidic structure and the second fluidic structure, and each fluidic structure of the array is the same structure as the first fluidic structure or the second fluidic structure.

[0242] Embodiment A25: The microfluidic device of Embodiment A24, wherein the flow path further comprises a plurality of first bridges, each of the plurality of first bridges is configured to fluidically couple the second opening of one fluidic structure of the array and the first opening of another one of the fluidic structures of the array.

[0243] Embodiment A26: The microfluidic device of Embodiment A25, wherein one of the fluidic structures of the array is configured to fluidically couple with another one of the fluidic structures of the array via one of the plurality of first bridges.

[0244] Embodiment A27: The microfluidic device of any one of Embodiments A24 to A26, wherein two or more fluidic structures of the array are arranged in a lane of the flow path.

[0245] Embodiment A28: The microfluidic device of any one of Embodiments A24 to A27, wherein two or more fluidic structures of the array are arranged in a first lane of the flow path, and another two or more fluidic structures of the array are arranged in a second lane of the flow path.

[0246] Embodiment A29: The microfluidic device of any one of Embodiments Al to A28, wherein the flow path is serpentine.

[0247] Embodiment A30: The microfluidic device of Embodiment A29, wherein the flow path is serpentine, the first lane and the second lane of the flow path are configured to be fluidically coupled via a second bridge along an axis different than an axis of the first bridge, and the second bridge is configured to couple the second opening of one of the fluidic structures of at least one of the first lane and the second lane of the flow path with the first opening of another one of the fluidic structures of another one of the first lane and the second lane of the flow path.

[0248] Embodiment A31: The microfluidic device of any one of Embodiments Al to A30, wherein the sample chamber has a volume between 1 and 20 uL.

[0249] Embodiment A32: The microfluidic device of any one of Embodiments Al to A31, further comprising a holder configured to receive the body.

[0250] Embodiment A33: The microfluidic device of Embodiment A32, wherein when the holder is coupled with the body, the holder does not block either or both of the first opening and the second opening at the first side of the body.

[0251] Embodiment A34: The microfluidic device of Embodiment A32 or Embodiment A33, wherein the holder further comprises a window from which at least one of the sample chambers is visible from the second side of the body. [0252] Embodiment A35: The microfluidic device of any one of Embodiments Al to A34, further comprising a cover, configured to cover the first side of the body thereby sealing the first opening and the second opening.

[0253] Embodiment A36: The microfluidic device of any one of Embodiments Al to A35, wherein the first side of the body comprises an identifiable indicium corresponding to the first opening.

[0254] Embodiment A37: A method for analyzing a biological sample, comprising: loading a first biological sample into the first fluidic structure of the microfluidic device of any one of Embodiments Al to A36 via the first opening of the first fluidic structure, wherein a sample chamber surface of the sample chamber of the first fluidic structure comprises a capture agent; incubating the first biological sample within the first fluidic structure thereby allowing the first biological sample to interact with the capture agent, and detecting an interaction between the first biological sample and the capture agent.

[0255] Embodiment A38: The method of Embodiment A37, wherein the body comprises a baffle and a membrane, and the microfluidic device further comprises a substrate, the baffle comprises a first baffle surface corresponding to the first side of the body and a second baffle surface coupled with a first membrane surface of the membrane; wherein the membrane comprises a second membrane surface corresponding to the second side of the body, and an elongated slot, the elongate slot is associated with one of the sample chambers and is configured to fluidically couple with the first opening and the second opening, a surface of the substrate is coated with the capture agent whereby when the substrate is assembled with the baffle and the membrane, and the surface of the substrate is the surface of the sample chamber comprising the capture agent.

[0256] Embodiment A39: The method of Embodiment A37 or Embodiment A38, wherein the body further comprises an input opening configured for receiving a flow and conducting the flow to the flow path, and the method further comprises flushing the flow path by introducing a wash solution into the input opening.

[0257] Embodiment A40: The method of Embodiment A39, wherein flushing the flow path is performed before detecting the interaction between the first biological sample and the capture agent.

[0258] Embodiment A41: The method of any one of Embodiments A37 to A40, wherein detecting the interaction between the first biological sample and the capture agent comprises introducing a reporter molecule into the microfluidic device.

[0259] Embodiment A42: The method of Embodiment A41, wherein the reporter molecule is labeled with a detectable entity, and detecting the interaction between the first biological sample and the capture agent comprises detecting a signal associated with the detectable entity. [0260] Embodiment A43: The method of any one of Embodiments A37 to A42, further comprising loading a second biological sample into the second fluidic structure of the microfluidic device via the first opening of the second fluidic structure, wherein a sample chamber surface of the sample chamber of the second fluidic structure comprises the capture agent.

[0261] Embodiment A44: The method of Embodiment A43, further comprising detecting an interaction between the second biological sample and the capture agent within the second fluidic structure.

[0262] Embodiment A45: The method of any one of Embodiments A37 to A44, wherein the capture agent is a peptide, a protein, an oligonucleotide, or a combination thereof.

[0263] Embodiment A46: The method of any one of Embodiments A37 to A45, wherein the capture agent comprises a first capture agent and a second capture agent, and the first capture agent is different from the second capture agent.

[0264] Embodiment A47: The method of any one of Embodiments A37 to A46, wherein the flow path of the microfluidic device comprises an array of fluidic structures, each fluidic structure of the array is the same structure as the first fluidic structure; and the method further comprises loading one respective biological sample of a plurality of biological samples into one of the fluidic structures of the array via the first opening thereof.

[0265] Embodiment A48: The method of Embodiment A47, further comprising detecting an interaction between the respective biological sample with the capture agent within the respective one fluidic structure.

[0266] Embodiment A49: The method of any one of Embodiments A37 to A48, further comprising applying a cover membrane to the first side of the body thereby sealing the first opening and the second opening at the first side of the body.

[0267] Embodiment A50: A system for analyzing a biological sample, comprising the microfluidic device of any one of Embodiments Al to A36, and an instrument for operating the microfluidic device to perform the method of any one of Embodiments A37 to A49.

[0268] Embodiment A51: A microfluidic device comprising a body having a flow path, the flow path having: a first sample chamber disposed within the body or on a first side of the body; a second sample chamber disposed within the body or on the first side of the body; and a fluidic bridge structure disposed to fluidically couple the first sample chamber with the second sample chamber, the fluidic bridge structure having: a first z-channel having a first end disposed to fluidically couple with the first sample chamber, a second z-channel having a first end disposed to fluidically couple with the second sample chamber, a bridge disposed to fluidically couple with a second end of the first z-channel and a second end of the second z- channel, and a first opening at a second side of the body and disposed to fluidically couple with the second end of the second z-channel for loading biological sample into the second chamber.

[0269] Embodiment A52: The microfluidic device of Embodiment A51, wherein the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the first z-channel extends from the first sample chamber toward the second side of the body.

[0270] Embodiment A53: The microfluidic device of Embodiment A51 or Embodiment A52, wherein the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the second z-channel extends from the second sample chamber toward the second side of the body.

[0271] Embodiment A54: The microfluidic device of Embodiment A52 or Embodiment A53, wherein the bridge is disposed to fluidically couple with the second end of the first z-channel and the second end of the second z-channel along an axis different than the z-axis.

[0272] Embodiment A55: The microfluidic device of any one of Embodiments 1 to 4, wherein the fluidic bridge structure is configured to be the only fluidic communication between the first sample chamber and the second sample chamber.

[0273] Embodiment A56: The microfluidic device of any one of Embodiments Al to A5, wherein the first sample chamber and/or the second sample chamber are elongated.

[0274] Embodiment A57: The microfluidic device of any one of Embodiments Al to A6, wherein the body further comprises an input opening configured for receiving a flow and conducting the flow to the flow path.

[0275] Embodiment A58: The microfluidic device of any one of Embodiments Al to A8, wherein the body further comprises an output opening configured for exhausting a flow from the flow path.

[0276] Embodiment A59: The microfluidic device of any one of Embodiments Al to A8, wherein the first opening is chamfered.

[0277] Embodiment A60: The microfluidic device of any one of Embodiments Al to A9, wherein the flow path is serpentine.

Claims

What is claimed is:

1. A microfluidic device, comprising a body having a flow path, the flow path comprising: a first fluidic structure; a second fluidic structure; and a first bridge disposed within the body or on a first side of the body and configured to fluidically couple the first fluidic structure and the second fluidic structure; wherein: each of the first fluidic structure and the second fluidic structure comprises:

(i) a first opening and a second opening, and

(ii) a sample chamber disposed within the body or on a second side of the body, the first opening of the first fluidic structure and the first opening of the second fluidic structure are disposed at a first side of the body, and each sample chamber is configured to fluidically couple with the first opening and the second opening; and the first bridge is configured to fluidically couple the second opening of the first fluidic structure with the first opening of the second fluidic structure.

2. The microfluidic device of claim 1, wherein: the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the first opening extends from the first side toward the second side of the body to define a first z-channel configured to fluidically couple the first opening with the sample chamber of the second fluidic structure.

3. The microfluidic device of claim 1, wherein: the first side of the body is separated from the second side of the body by a thickness defining a z-axis of the body, and the second opening extends toward the second side of the body to define a second z- channel configured to fluidically couple the second opening with the sample chamber of the first fluidic structure. The microfluidic device of claim 3, wherein the second opening is disposed at the first side of the body, and the second opening extends from the first side toward the second side of the body. The microfluidic device of claim 3, wherein the first bridge is configured to fl uidica lly couple the first z-channel and the second z-channel along an axis different than the z- axis. The microfluidic device of claim 1, wherein the first bridge is configured to be the only fluidic communication between the first fluidic structure and the second fluidic structure. The microfluidic device of claim 1, wherein the body further comprises an input opening configured for receiving a flow and conducting the flow to the flow path. The microfluidic device of claim 1, wherein the body further comprises an output opening configured for exhausting a flow from the flow path. The microfluidic device of claim 1 to 8, wherein: each sample chamber is disposed on the second side of the body, the body comprises a baffle and a membrane, the baffle comprises a first baffle surface corresponding to the first side of the body and a second baffle surface coupled with a first membrane surface of the membrane, the membrane comprises a second membrane surface corresponding to the second side of the body, and a first elongated slot and a second elongated slot, and the first elongated slot is associated with the sample chamber of the first fluidic structure, the second elongated slot is associated with the sample chamber of the second fluidic structure, and each of the first and second elongated slots is configured to fl uidica I ly couple with the first opening and the second opening of the corresponding first fluidic structure and the second fluidic structure. The microfluidic device of claim 9, wherein: for each of the first and second fluidic structures, the corresponding first opening is disposed at the first baffle surface, extends to the second baffle surface, and opens to the corresponding elongated slot of the membrane, and the corresponding second opening baffle extends to the second baffle surface, and opens to the corresponding elongated slot of the membrane. The microfluidic device of claim 9, further comprising a substrate coupled with the second membrane surface, wherein the substrate, the elongated slot of the membrane, and the second baffle surface define the sample chamber. The microfluidic device of claim 11, wherein: a surface of the substrate is coated with a capture agent, and when the substrate is assembled with the baffle and the membrane, the capture agent is accessible the sample chamber. The microfluidic device of claim 12, wherein the capture agent is a peptide, a protein, an oligonucleotide, or a combination thereof. The microfluidic device of claim 12, wherein the capture agent comprises a first capture agent and a second capture agent, and the first capture agent is different from the second capture agent. The microfluidic device of claim 9, wherein both the first elongated slot and the second elongated slot extend along with a length of the membrane. The microfluidic device of claim 15, wherein the first elongated slot and the second elongated slot are linearly arranged and spaced apart by a predetermined distance. The microfluidic device of claim 1, wherein the first opening includes a chamfer. The microfluidic device of claim 1, wherein: the flow path comprises an array of fluidic structures comprising the first fluidic structure and the second fluidic structure, and each fluidic structure of the array is the same structure as the first fluidic structure or the second fluidic structure. The microfluidic device of claim 18, wherein: the flow path further comprises a plurality of first bridges, and each of the plurality of first bridges is configured to fluidically couple the second opening of one of the fluidic structures of the array and the first opening of another one of the fluidic structures of the array. The microfluidic device of claim 19, wherein one of the fluidic structures of the array is configured to fluidically couple with another one of the fluidic structures of the array via one of the plurality of first bridges. The microfluidic device of claim 18, wherein: two or more fluidic structures of the array are arranged in a first lane of the flow path, and another two or more fluidic structures of the array are arranged in a second lane of the flow path. The microfluidic device of claim 1, wherein the flow path is serpentine. The microfluidic device of claim 21, wherein: the flow path is serpentine, the first lane and the second lane of the flow path are configured to be fluidically coupled via a second bridge along an axis different than an axis of the first bridge, and the second bridge is configured to couple the second opening of one of the fluidic structures of at least one of the first lane and the second lane of the flow path with the first opening of another one of the fluidic structures of another one of the first lane and the second lane of the flow path. The microfluidic device of claim 1, wherein the sample chamber has a volume of between 1 and 20 uL. The microfluidic device of claim 1, further comprising a holder configured to receive the body. The microfluidic device of claim 25, wherein when the holder is coupled with the body, the holder does not block either or both of the first opening and the second opening at the first side of the body. The microfluidic device of claim 25, wherein the holder further comprises a window from which at least one of the sample chambers is visible from the second side of the body. The microfluidic device of claim 1, further comprising a cover, configured to cover the first side of the body thereby sealing the first opening and the second opening. A method for analyzing a biological sample, comprising: loading a first biological sample into the first fluidic structure of the microfluidic device of claim 1 via the first opening of the first fluidic structure, wherein a surface of the sample chamber of the first fluidic structure comprises a capture agent; incubating the first biological sample within the first fluidic structure thereby allowing the first biological sample to interact with the capture agent, and detecting an interaction between the first biological sample and the capture agent. The method of claim 29, wherein: the body comprises a baffle and a membrane, and the microfluidic device further comprises a substrate, the baffle comprises a first baffle surface corresponding to the first side of the body and a second baffle surface coupled with a first membrane surface of the membrane; the membrane comprises a second membrane surface corresponding to the second side of the body, and an elongated slot, the elongate slot is associated with one of the sample chambers and is configured to fluidically couple with the first opening and the second opening, a surface of the substrate is coated with the capture agent whereby when the substrate is assembled with the baffle and the membrane, and the surface of the substrate is the surface of the sample chamber comprising the capture agent. The method of claim 29, wherein: the body further comprises an input opening configured for receiving a flow and conducting the flow to the flow path, and the method further comprises flushing the flow path by introducing a wash solution into the input opening. The method of claim 31, wherein flushing the flow path is performed before detecting the interaction between the first biological sample and the capture agent. The method of claim 29, wherein detecting the interaction between the first biological sample and the capture agent comprises introducing a reporter molecule into the microfluidic device. The method of claim 29, further comprising loading a second biological sample into the second fluidic structure of the microfluidic device via the first opening of the second fluidic structure, wherein a sample chamber surface of the sample chamber of the second fluidic structure comprises the capture agent. The method of claim 34, further comprising detecting an interaction between the second biological sample and the capture agent within the second fluidic structure. The method of claim 29, wherein the capture agent comprises a first capture agent and a second capture agent, and the first capture agent is different from the second capture. The method of any one of claims 29 to 36, wherein: the flow path of the microfluidic device comprises an array of fluidic structures, each fluidic structure of the array is the same structure as the first fluidic structure; and the method further comprises loading one respective biological sample of a plurality of biological samples into one of the fluidic structures of the array via the first opening thereof. The method of claim 37, further comprising detecting an interaction between the respective biological sample with the capture agent within the respective fluidic structure.