Multiplexed immunoprofiling of single extracellular vesicles

Abstract

Methods, kits, and systems for analyzing a sample comprising extracellular vesicles. In various examples, a method comprises use of a fluidic device for the retention and discrete presentation of single extracellular vesicles for imaging-based analyses. In various examples, a method comprises use of a plurality of capture agents that are each linked to a different oligonucleotide and a corresponding plurality of labeled nucleic acid probes, where each of the labeled nucleic acid probes specifically hybridizes with one or more or each of the oligonucleotides of the capture agents, and sub-sets of the capture agents are detected by imaging-based means using iterative cycles using corresponding sub-sets of the labeled nucleic acid probes. In various examples, kits and systems are provided for carrying out a method of the disclosure.

Description

MULTIPLEXED IMMUNOPROFILING OF SINGLE EXTRACELLULAR VESICLESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 672,699, filed July 17, 2024 and titled “Multiplexed Immunoprofiling of Single Extracellular Vesicles”; the contents of the above-identified applications are hereby fully incorporated herein by reference in their entirety.TECHNICAL FIELD

[0002] The subject matter disclosed herein is generally directed to, inter alia, the analysis of the protein expression profiles of single extracellular vesicles.BACKGROUND OF THE DISCLOSURE

[0003] Extracellular vesicles (EVs) are lipid bilayer vesicles approximately 30 nm to 1 ,000 nm diameter that are produced by all cells of single-cellular and multicellular organisms. Because EVs can be readily found in mammalian biofluids at abundant levels (e.g., > ~109EVs / mL in human blood), and because EVs carry both nucleic acids and proteins that are characteristic of the cells from which the EVs derive, there is growing interest in using EVs as diagnostics for so-called liquid biopsies. Further, EVs are being exploited as therapeutic agents, as EVs can elicit biological effects or can deliver either intrinsic or exogenous payloads.

[0004] EV protein expression is inherently heterogeneous and varies widely among EV subpopulations, thus pooled analyses of EVs by conventional antibody-based methods (e.g., Western blotting, ELISA) are unable to detect low-expressed EV proteins and to characterize rare EV subpopulations. Consequently, there remains a need for methods, devices, and compositions for single EV analyses.

[0005] Current-generation methods for analyzing single EVs include, but are not limited to, super-resolution microscopy nano-flow cytometry. Both super-resolution microscopy and nano-flow cytometry require expensive, dedicated instruments. Nano-flow cytometry lacks the necessary sensitivity for detecting low- abundance, EV-expressed proteins, The lack of sensitivity is likely due to very short dwell time as EVs labeled with fluorophore-conjugated antibodies quickly pass through the nano-flow cytometer’s active sensing volume. Superresolution microscopy is generally limited to the analysis of only a few thousand single EVs (which is statistically insufficient for rare EV-expressed proteins) and often relies on someform of affinity-based, surface capture of EVs (which biases which EV subpopulations are subsequently analyzed). Moreover, neither super-resolution microscopy nor nano-flow Cytometry have the scalability that would support multiplexed immunoprofiling of tens-of- thousands or even hundreds-of-thousands single EVs.

[0006] The multiplexed detection of both intravesicular and surface proteins on individual EVs allow cell-type specific, EV protein profiles (e.g., immunoprofiling or immunoprofiles) which would be useful as diagnostics, prognostics, and QA / QC tools for therapeutically-purposed EVs.

[0007] Highly multiplexed protein detection has been previously described in the context of cells and tissue sections, where oligonucleotide-conjugated antibodies and reporter oligonucleotides (which hybridize to the oligonucleotides of the conjugated antibodies) are used to detect the presence and / or absence of antibody-labeled proteins on the fixed cells and / or fixed tissue sections. See, e.g., Nolan et al. “Highly Multiplexed Fluorescent Imaging” (U.S. Pat. No. 10,370,698). It is noteworthy that repeated cycles of detection as taught by Nolan et al. requires the use of a denaturant (e.g., chaotropic agent) at high concentration levels in order to remove (i.e., dehybridize) the reporter oligonucleotides between cycles. In various examples, chaotropic agents such as dimethyl sulfoxide, formamide, urea, or guanidinium are specified at high concentrations (i.e., incubations with 70% v / v to 90% v / v formamide or 70% v / v to 90% v / v dimethyl sulfoxide).SUMMARY OF THE DISCLOSURE

[0008] The present disclosure is directed to, inter alia, multiplexed immunoprofiles of single EVs.

[0009] In an aspect, the present disclosure provides methods for analyzing a sample (e.g., a biological sample, an analytical sample, or the like) (such as, for example, methods for performing a multiplexed single extracellular vesicle (EV) analyses). In various examples, a method comprises use of a device (e.g., a fluidic device). In various examples, a method comprises disposition of an EV sample onto a first layer of a device, thereby forming an analysis sample. In various examples, a method further comprises use of devices comprising a first layer, a second layer with fluidic channels, a third layer, an optional fourth layer, various openings and vias, and functionalized surfaces, as described herein. In various examples, analysis samples are formed by disposition of one or more pluralit(ies) of EVs onto a first layer of at least one device, and such samples are used for the one or more labeling and imaging methods disclosed herein so that the presence and / or abundance of theEVs’ components (e.g., EV biomarkers) can be detected and quantitated. In various examples, one or more sample(s) are used to perform a single EV immunoprofiling analysis as described herein.

[0010] In a further aspect, the present disclosure provides uses of devices (e.g., fluidic devices or the like). In various examples, a surface or surfaces of a device’s first layer is / are functionalized with molecules that are configured to promote or enable cross-linking EVs within the fluidic devices. Such cross-linking may be desirable in one or more method(s) described herein. Given the nature of such functionalization, however, introduction and flow of fluids into the fluidic devices is disrupted without the use of one or more mitigation(s). Therefore, in various examples, devices with mitigations for maintaining the desired fluid flow patterns are provided. Such mitigations may comprise devices comprising one or more functionalized surface(s) and / or one or more optional fourth layer(s).

[0011] In a further aspect, the present disclosure provides methods for analyzing a biological sample comprising at least one plurality of EVs (such as, for example, methods for performing a multiplexed single extracellular vesicle (EV) analyses). In various examples, a method comprises a multiplexed, immunoprofiling single EV analysis of one or more pluralit(ies) of EVs. In various examples, one or more EV sample(s) are disposed within one device or across multiple devices, thereby forming one or more corresponding analysis sample(s) that are analyzed by methods disclosed herein. In various examples, a method comprises use of one or more pluralit(ies) of capture agents, where each plurality is linked to a oligonucleotide (e.g., a distinct oligonucleotide). In various examples, a method comprises use of one or more corresponding pluralit(ies) of labeled nucleic acid probes to detect the presence and / or the absence of the one or more pluralit(ies) of capture agents, where a labeled nucleic acid probe specifically hybridizes with an oligonucleotide of a capture agent. In various examples, a method comprises use of capture agents comprising one or more pluralit(ies) of antibodies that selectively recognize one or more epitope(s) of EV-expressed components (e.g., EV-expressed protein biomarkers or the like). In various examples, a sample is labeled with the capture agents sequentially or en masse, and sub-sets of the capture agents are detected (e.g., the presence or absence) using iterative hybridization / label removal or inactivation cycles using corresponding sub-sets of the labeled nucleic acid probes. In various examples, a method further comprises use of labeled nucleic acid probes that are conjugated to one or more fluorophore(s), such that distinct fluorophores can be detected (e.g., presence or absence) by an imaging -based method or methods (e.g., during one or moreor all of the cycles). In various examples, a location of the EVs of the samples is detected by various labeling and / or imaging methods as described herein.

[0012] In a further aspect, the present disclosure provides kits. In various examples, kits comprise devices (e.g., fluidic devices), reagents (e.g., capture agents and / or labeled nucleic acid probes), buffers, instructions for practicing disclosed methods, or the like, or any combination thereof. In various examples, one or more kit(s) comprise(s) one or more device(s), one or more reagent(s), one or more buffer(s), one or more instruction(s), and the like, and any combination thereof. In various examples, a kit is configured to carry out a method of the present disclosure.

[0013] In a further aspect, the present disclosure provides systems. In various examples, a system comprises one or more system(s) that are configured for analyzing a biological sample. In various examples, a system is configured to carry out a method of the present disclosure. In various examples, a system comprises one or more or all of the following: an optical imager (e.g., a microscope), one or more device(s) (e.g., a fluidic device), one or more autosampler(s), one or more controller(s), one or more pump(s), one or more droplet generator(s), one or more processor(s), one or more image analysis module(s), one or more statistical model(s), one or more computer-readable medium(s) comprising instructions that, when executed by the one or more computer and / or processor, result in the analysis of a sample, or the like, or any combination thereof.

[0014] In a further aspect, methods, kits, and systems described herein find general use in a wide variety of applications for analyzing a wide variety of EV samples. Methods may be useful for generating multiplexed, single EV immunoprofiles. Methods may have many biomedical applications in a variety of fields of basic biology, phenotypic screening, high throughput screening, drug discovery, and the like. Further, methods may be useful for a number of clinical applications, such as, for example, diagnostics, prognostics, disease stratification, treatment efficacy monitoring, personalized medicine, clinical trials, companion diagnostics or drug-accompanying tests, and the like..

[0015] These and other aspects, objects, features, and advantages of the examples will become apparent to those having ordinary skill in the art upon consideration of the following figures and detailed description of examples.BRIEF DESCRIPTION OF THE FIGURES

[0016] For a fuller understanding of the nature and objects of the disclosure, reference should be made to the following detailed description taken in conjunction with the accompanying figures.

[0017] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present disclosure’s teachings in any way.

[0018] FIG. 1 shows A) a photograph of an example of a fluidic device used in methods provided in the present disclosure; and B) shows a rendering of an example of a fluidic device of the present disclosure (similar to the fluidic device example shown in A)).

[0019] FIG. 2 shows A) a photograph of another example of a fluidic device used in methods of the present disclosure; and B) shows a rendering of another example of a fluidic device of the present disclosure (similar to the fluidic device example shown in A)).

[0020] FIG. 3 shows A) an example of a use of a disclosed fluidic device to count single EVs via epifluorescent microscopy and analysis by a representative analysis module; B) shows analysis of single EVs by two orthogonal methods as a comparison; and C) to F) show another example of a use of a disclosed fluidic device to count single EVs with representative capture agents and universal EV stains as described herein.

[0021] FIG. 4 shows in A) to G) various steps or processes of a method of the present disclosure.

[0022] FIG. 5 shows an example of a multiplex, cell-of-origin single EV immunoprofile analysis of a method of the present disclosure.

[0023] FIG. 6 shows A) the liquid “spread-out” problem described in the present disclosure; and B) shows an example of a fluidic device of the present disclosure where a mitigation as described herein is applied to the fluidic device of B) to resolve the liquid "spread-out" problem.

[0024] FIG. 7 shows a rendering of an example of a fluidic device of the present disclosure, where an optional fourth layer of such a device is specified to resolve the liquid “spread-out” problem described herein.

[0025] FIG. 8 shows A) shows the liquid “spread-out” problem described in the present disclosure; and B) shows an example of a fluidic device of the present disclosure where a mitigation (i.e., an optional fourth layer) as described herein is applied to the fluidic device of B) to resolve the liquid "spread-out" problem.

[0026] FIG. 9 shows A) an effect of prolonged exposure to a chemical denaturant solution of the present disclosure on the second layer of a representative fluidic device; and B) shows the lack of an effect of similar prolonged exposure to an aqueous solution on the second layer of a representative fluidic device.

[0027] FIG. 10 shows in A) to D) the effects of various concentrations and / or exposure times of a chemical denaturant solution, in the presence and / or absence of a fixative agent, on retention of single EVs by examples of fluidic devices of the present disclosure.DETAILED DESCRIPTION OF THE DISCLOSURE

[0028] Although claimed subject matter will be described in terms of various examples, and various examples including examples that do not provide all of the benefits and features set forth herein, are also within the scope of this disclosure. For example, various structural, logical, process step, and electronic changes may be made without departing from the scope of the disclosure.

[0029] The headings provided herein are not limitations of the various aspects or examples of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0030] Throughout the present disclosure, all cited publications and patents are cited to disclose and describe the methods and / or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and / or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present disclosure.

[0031] Throughout the present disclosure, unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2nd edition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4th edition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2nd edition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2nd edition (2011).

[0032] Definitions of common terms and techniques in chemistry and organic chemistry can be found in e.g., Smith. Organic Synthesis, published by Academic Press. 2016; Tinoco et al. Physical Chemistry, 5th edition (2013) published by Pearson; Brown et al., Chemistry, The Central Science 14th ed. (2017), published by Pearson, Clayden et al., Organic Chemistry, 2nd ed. 2012, published by Oxford University Press; Carey and Sunberg, Advanced Organic Chemistry, Part A: Structure and Mechanisms, 5th ed. 2008, published by Springer; Carey and Sunberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, 5th ed. 2010, published by Springer, and Vollhardt and Schore, Organic Chemistry, Structure and Function; 8th ed. (2018) published by W.H. Freeman.

[0033] Throughout the present disclosure, where a range is expressed, a further aspect includes from the one particular value and / or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independentlybe included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure (e.g. the phrase “x to y” includes the range from ‘x ’to ‘y ’as well as the range greater than ‘x ’and less than ‘y’ ). The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less ’and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z ’as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater ’should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z ’as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x ’to ‘y’”, where ‘x ’and ‘y ’are numerical values, includes “about ‘x ’to about ‘y’.”

[0034] Throughout the present disclosure, ratios, concentrations, amounts, and other numerical data can be expressed in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0035] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub-ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

[0036] As used herein, unless otherwise indicated, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0037] As used herein, unless otherwise indicated, "about," "approximately," “substantially,” and / or the like, when used in connection with a measurable variable such as, for example, a parameter, an amount, a temporal duration, and / or the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by, e.g., given data set, art accepted standard, and / or with, e.g., a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as, for example, variations of + / - 10% or less, + / -5% or less, + / -!% or less, and + / -0.1 % or less of and from the specified value, insofar such variations are appropriate to perform in the examples of the present disclosure.

[0038] As used herein, unless otherwise indicated, the terms “about,” “approximate,” “at or about,” and “substantially” can further mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and / or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error, and / or the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0039] The term “optional” or “optionally” is used to mean that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0040] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0041] As used herein, unless otherwise indicated, the terms an “extracellular vesicle” or an “EV” refer to lipid bilayer vesicles produced by cells from single-cellular and multicellular organisms. The skilled artisan will recognize that EVs are also known by other terms such as, for example, exosomes, ectosomes, microsomes, microvesicles, apoptotic bodies, or the like. EVs are substantially about 30 nm (nm = nanometer(s)) to 1,000 nm indiameter. EVs may be borne by or may derive from any of the sources, samples, biologic entities, and organisms described below. In various examples, EVs carry components (e.g., EV biomarkers or the like) that are characteristic of the cells from which the EVs are derived. In various examples, the presence and / or absence of such components (e.g., EV biomarkers or the like) are capable of being or are detected by a method disclosed herein.

[0042] As used herein, unless otherwise indicated, an “EV source” refers to a biological sample or an environmental sample, in which EVs are borne and from which EVs can be isolated, separated, purified, or otherwise derived. A “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from any biologic entity or any organism. In various examples, the biological sample does not comprise whole cells, tissue samples, nor tissue sections obtained from a biologic entity or organism. The biological sample can contain (or be derived from) a “biofluid.” The term “biofluid” refers to a biological sample that is not a solid or at least contains a mix of solid and non-solid components, and further can comprise a bodily fluid. In various examples, a bodily fluid comprises any non-solid excretion, secretion, or other fluid present in an organism, or any combination thereof, including, for example: amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g., plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit, or the like, or any combination thereof. Biological samples include, but are not limited to, biofluids, cell cultures, and cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by venous puncture, or other collecting or sampling procedures. An alternative EV source may be an environmental sample (fluid, solid, semi-solid, and mixed) obtained from an environment (e.g., water source, soil, air, and / or the like), and may further comprise a culture of an environmental sample.

[0043] EVs can be isolated from an EV source by a variety of methods. An “EV purification” refers to any process whereby EVs are isolated, separated, purified, or the like, or otherwise derived from an EV source (e.g. a biological sample or an environmental sample). EV purification processes include, as non-limiting examples: filtration; centrifugation; differential ultracentrifugation; gradient ultracentrifugation; chromatography (e.g., gel-filtration, size-exclusion, and / or ion-exchange); affinity / ligand purification; antibody affinity purification; volume-exclusion precipitation; electrophoretic mobility-assisted purification; and the like. One or more EV purification process(es) may be employed and resultant EV preparations may have any range of purity and yield. Throughout the present disclosure, EV preparations produced by such EV purification processes are referred to as an “EV sample,” where such EV samples correspond to one or more pluralit(ies) of EVs.

[0044] As used herein, unless otherwise indicated, the terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate (e.g., a mammal, such as, for example, a human or the like). Non-limiting examples of mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed. A “subject” of diagnosis or treatment is a plant or animal, including, but not limited to, a human. Non-human animals subject to diagnosis or treatment include, for example, livestock, pets, and the like.

[0045] As used herein, unless otherwise indicated, the term “EV biomarker” refers to any component of an EV that can be indicated by binding to a capture agent. EV biomarkers include, as non-limiting examples: oligosaccharides; polysaccharides; proteoglycans; metabolites; lipids; proteins; nucleic acids (e.g., mRNA, microRNA, DNA, nucleosomes); and the like. An EV biomarker can also be indicated by immunological means (e.g., an antibody capture agent that is linked to an oligonucleotide, such as, for example, a distinct oligonucleotide). In various examples, the capture agent binds to a component (e.g., an EV biomarker) that is present within, on, or associated with an EV. A series (e.g., a panel) of such indicated EV biomarkers by immunological means may be referred to as “immunoprofiling” or as an “immunoprofile.” As used herein, unless otherwise indicated, the term “cell-of-origin EV biomarker” or “CofO EV biomarker” refers to any one or more component(s) (e.g., EV biomarker(s) or the like) that is indicated by capture agent binding which identifies if a plurality of EVs predominantly originates from one particular cell-type of a tissue. For example, antibody binding (e.g., capture agent binding) directed against certain EV-expressed proteins (e.g. EV biomarkers) may be used as one or more CofO EV biomarker(s) to indicate if a plurality of EVs is derived from a certain cell-type of a particular tissue, or to differentiate if one plurality of EVs predominantly represents a unique subpopulation originating from a particular cell type of a tissue. Non-limiting examples of CofO EV biomarkers are described throughout the present disclosure. Many CofO EV biomarkers are known or can be discovered and established, such as, for example, those identified by single cell proteomics (see, e.g.. The Human Proteome Tissue Atlas athttps: / / www.proteinatlas.org). It is generally assumed that if a particular cell-type predominantly expresses a protein, then EVs derived from that cell type will also express the same protein, where the protein is used to characterized or identified that particular cell-type, thus can be used as a CofO EV biomarker to characterize or to identify EVs originating from that particular cell-type.

[0046] As used herein, unless otherwise indicated, the term “multiplexing” refers to using more than one plurality of capture agents (and the corresponding number of labeled nucleic acid probe pluralities) for the simultaneous or sequential detection (e.g., the presence and / or the absence) and measurement of multiple EV biomarkers. Such a series (e.g., a panel) of detected EV biomarkers by immunological means may further be referred to as “multiplexed immunoprofiling” or as an “multiplexed immunoprofile.”

[0047] As used herein, unless otherwise indicated, the terms “antibody” and “immunoglobulin” are used interchangeably and are well understood by the skilled artisan. Those terms refer to a protein consisting of comprising, consisting essentially of, or consisting of one or more polypeptide(s) that specifically binds an antigen. One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and comprises, consists essentially of, of consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. The recognized immunoglobulin polypeptides include, but are not limited to, the kappa and lambda light chains and the alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or equivalents in other species. Full-length immunoglobulin “light chains” (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin “heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[0048] The terms “antibodies” and “immunoglobulin” herein include, but are not limited to, antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigens and / or epitopes and include, as non-limiting examples: Fab; Fv; scFv; Fd fragments; chimeric antibodies; humanized antibodies; minibodies; single-chain antibodies; fusion proteins comprising an antigen-binding portion of an antibody and a nonantibody protein; and the like. Also encompassed by the term are Fab', Fv, F(ab')2, and orother antibody fragments that retain specific binding to antigens and / or epitopes, and the like. Antibodies also may be in the form of monoclonal antibodies or in the form of polyclonal antibodies. Antibodies may exist in a variety of other forms including, for example, Fv, Fab, (Fab')2, as well as bi-functional (e.g., bi-specific) hybrid antibodies.

[0049] The term “specific binding” herein refers to the ability of a capture agent to preferentially bind to a particular analyte that is present in a mixture of different analytes. In various examples, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, where the concentration ratio between desirable to undesirable analytes present in a sample spans about more than about 10, more than about 100, or more than about 1,000 orders-of-magnitude difference.

[0050] In various examples, the affinity between a capture agent and analyte when they are specifically bound in a capture agent / analyte complex is characterized by a KD (e.g., dissociation constant) of about less than about 10"6M to less than about 10"12M, including all values and ranges therebetween, or less than about 1013M or lower.

[0051] As used herein, unless otherwise indicated, a “plurality” contains at least about 2 members. In certain cases, a plurality may have at least about 2, at least about 5, at least about 10, at least about 100, at least about 1000, at least about 10,000, at least about 100,000, at least about 106, at least about 107, at least about 108, or at least about 109or more members, including all integer values and ranges therebetween.

[0052] As used herein, unless otherwise indicated, the term “labeling” refers to binding one or more reagent(s) of the present disclosure to one or more component(s) of a sample. For example, a capture agent may be used to label any components of a sample (e.g., an antibody is used to label an epitope of an EV biomarker). Such labeling allows the presence and / or abundance of the components (e.g., EV biomarkers) to be determined by measuring the presence and / or abundance of the capture agent. In various examples, the measuring of the presence and / or absence of components (e.g., EV biomarkers) may be useful for detecting (e.g., the presence or the absence) a disease, measuring the progress of disease, the effects of treatment, or for measuring a process of interest. The term “labeling” refers to one or more method(s) for producing a labeled sample in which any necessary steps / processes are performed in any convenient order, as long as the required labeled sample is produced. For example, in various examples and as will be exemplified below, the capture agent may be linked to an oligonucleotide (e.g., an oligonucleotide) prior to binding of the antibody to the sample.

[0053] As used herein, unless otherwise indicated, the term “a range of imaging modalities for imaging-based analyses’" includes, but is not limited to, a number of forms of microscopy. For example, bright-field and / or wide-field epifluorescence optical microscopy may be used and are well-known to the skilled artisan. Other non-limiting examples include two-photon microscopy, light sheet microscopy, total internal reflectance microscopy, confocal microscopy, as well as various forms of super-resolution microscopy (e.g., see U. J. Birk, Super-Resolution Microscopy: A Practical Guide, published by John Wiley & Sons, 2017) and the like. Forms of super-resolution microscopy include, as non-limiting examples: near-field optical random mapping (NORM) microscopy; structured illumination microscopy (SIM); reversible Saturable Optical Fluorescence Transitions (RESOLFT) microscopy; stimulated emission depletion (STED) microscopy; stochastic optical reconstruction microscopy (STORM); photo activated localization microscopy (PALM); and the like. Another example includes hyperspectral optical microscopy which collects a broad range of light wavelengths, such that the spectrum across the range of light wavelengths is obtained for each pixel in the image (e.g., see Grahn and Geladi, Techniques and Applications of Hyperspectral Image Analysis, published by John Wiley & Sons, 2007). A sensor such as, for example, a camera with an array of sensor pixels records the spectra. In hyperspectral imaging, the recorded spectra have fine wavelength resolution and cover a wide range of wavelengths. Hyperspectral imaging effectively measures continuous spectral bands, as opposed to multiband imaging which measures spaced spectral bands (the latter is the usual case for fluorescence optical microscopy which relies on specific bandpass emission, excitation, and dichroic mirrors). Any of these non-limiting examples of optical modalities may be used for the one or more reading step(s) / process(es) described in various examples, where distinct labels and / or universal / pan-EV stains as described below are imaged at such reading steps / processes.

[0054] As further used herein, the range of imaging modalities for imaging-based analyses also includes, but is not limited to, additional forms of microscopy that may be used to locate or image single EVs (e.g., the position of single EVs present on samples). Such optical modalities may be referred to as “light-scatter microscopy” since they generally rely on light scattering and / or reflectance caused by differences in refractive indices between the EV and the material on which the EV has been disposed. One non-limiting example of such optical modalities includes dark-field microscopy, where any unscattered light is excluded from the image (e.g., see U. Icha, Dark Field Microscopy: The practical handbook, published by Ulrike Icha, 2022). Consequently, the field around the specimen (e.g., a first layer lackingEVs at various positions) remains dark since there is no specimen to scatter the light, while the specimen (e.g., EVs disposed onto a first layer) scatters the light, resulting in the EVs being readily visible in the image. Dark-field optical microscopy can be used to locate the position of EVs of the analysis sample. Another non-limiting example includes confocal reflectance microscopy, which relies on scatter and reflectance of light by EVs from their lipid membranes due to different refractive indices compared to the surrounding aqueous medium. A pinhole aperture in the optical path is needed to eliminate the out-of- focus light and to increase the minor contrast of any scattered and / or reflected light. Any of these nonlimiting examples of optical modalities may be used for the one or more reading step(s) / process(es) described in various examples, where the position of single EVs as described below is imaged at such reading steps / processes. In various examples, light-scatter microscopy is used to image the position of single EVs in order to derive a count of the number of single EVs in the resultant image.

[0055] As used herein, unless otherwise indicated, the term “analysis sample” refers to a first layer of a device (e.g., a fluidic device) onto which one or more EV sample(s) are disposed. Such first layers may be comprised of a substantially flat material such as, for example, glass, ceramic, organic polymer membrane, inorganic membrane, or the like, onto which an EV sample is disposed such that the EVs are retained on at least a portion of the first layer of a fluidic device. Throughout the present disclosure, as will be apparent, the analysis sample may be synonymously referred to as “a sample” or as “the sample.” In various examples, the first layer may further be a functionalized first layer, where the material of the first layer is functionalized with moieties that promote EV capture and retention after cross-linking. As will also be apparent, the fluidic devices described below may comprise one or more analysis sample(s) after EV disposition.

[0056] As used herein, unless otherwise indicated, “fixative reagents” refer to reactive molecules that are used to cross-link EVs to functionalized first layers and / or to cross-link capture agents to EVs. In various examples, the captured EVs are cross-linked to a functionalized first layer and / or the capture agents cross-linked to EVs using any number of fixative reagents, where “fixative reagents” may include, as non-limiting examples: formalin; methanol; paraformaldehyde; methanol: acetic acid; glutaraldehyde; ethyl-3- (3dimethylaminopropyl) carbodiimide; bis(succinimidyl)suberate, bis(succinimidyl)polyethylene glycol; and the like.

[0057] As used herein, unless otherwise indicated, the term “single EV analysis” refers to the counting of individual EVs and / or the counting of the binding pattern of capture agentsand probes on individual EVs, as described by the one or more labeling and optical imaging method(s) of the present disclosure. One or more or each EV(s) within a plurality of EVs (e.g., an EV sample) can be thus analyzed by the one or more method(s) of the present disclosure. Various examples rely upon the disposition of discretely isolated, individual EVs onto a first layer (e.g., forming an analysis sample), and subsequently analyzing such samples by the labeling and imaging methods described herein. For purposes of this disclosure, “discretely isolated, individual EVs” refers to disposition of EVs onto a first layer such that one or more or each EV(s) is in its monomeric form is substantially separated upon disposition from one or more other or each other EV(s) about a multiple of the lateral optical resolution of the pixels of the sensor of the imaging system being used to image the one or more EV(s) (i.e., at about less than one-time, at about at least one-time, at least about two- times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs). Lateral optical resolution in the image is defined as the distance by which two specimens are separated in order to distinguish them as separate from one another in an image, which is equal to the radius (r) of the smallest point source in the image (theoretically defined as the first minimum of the Airy disk); see, e.g., Waters et al. J Cell Biol, 185 (7): 1135-1148). This radius (r) can be calculated as: r = 0.61 x l x NA , where Z is the wavelength of emission light and NA is the numerical aperture of the objective lens in the imaging system. Because in certain cases the images will be collected digitally using a sensor, such as, for example, a camera with an array of pixels or the like, the resolution of a digital image will depend on the physical size of the sensor’s pixels. Given the size of EVs relative to the typical pixel size of an imaging system sensor and that EV may be, for example, smaller than, equal to, or larger than the typical pixel size of an imaging system sensor there may be none, one, two, or three or more EV(s) per pixel of an imaging system sensor. Thus, due to the relative size of an EV to the size of an imaging system's sensor pixels, the distance separating the discretely isolated, individual EVs may be expressed in one or more multiple(s) of the pixel's lateral optical resolution. In various examples, the methods described herein for preparation of EVs and the disposition methods for forming analysis samples comprise forming one or more analysis sample(s) where about at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more of the EVs are disposed as substantially discretely isolated and individual EVs. In various examples, the discretely isolated, individual EVs comprise the disposition of EVs onto a first layer wherein the EVs are substantially separated (or separated) at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times thelateral optical resolution of the imaging system being used to image the EVs. In various examples, the discretely isolated, individual EVs are configured to be imaged at about less than one-time, at about at least one-time, at least about two-times, or at least about three- times the lateral optical resolution of the imaging system being used to image the EVs. In various examples, one or more method(s) comprise(s) imaging the discretely isolated, individual EVs that are substantially separated (or separated) at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs.

[0058] In various examples, the discretely isolated, individual EVs comprise the disposition of EVs onto a first layer, where the EVs are substantially separated (or separated) at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs. In various examples, the discretely isolated, individual EVs are configured to be imaged at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs. In various examples, one or more method(s) comprise(s) imaging the discretely isolated, individual EVs that are substantially separated (or separated) at about less than one-time, at about at least one-time, at least about two-times, or at least about three- times the lateral optical resolution of the imaging system being used to image the EVs.

[0059] As used herein, unless otherwise indicated, the term “Laplace pressure” refers to the pressure difference between the inside and the outside of a curved surface (e.g., a liquid droplet or the like) that forms the boundary between two fluid regions. The pressure difference is caused by the surface tension of the interface between liquid and gas, between two immiscible liquids, or between droplets of different radii. The Laplace pressure cane be defined by the Young-Laplace equation which is given as: Ap = -2 • y • Ri , where Ap is the pressure within the droplet, y is the surface tension of the fluid, and Ri is the radius of the droplet. Accordingly, larger droplets have lower pressure, while smaller droplets have higher pressure, and this difference in pressures can be used to direct flow within a device from the higher pressure (e.g., smaller droplet) point to the lower pressure (e.g., larger droplet) point. In various examples, flow within a device (e.g., fluidic device) of the present disclosure is Laplace pressure-driven flow.

[0060] As used herein, unless otherwise indicated, the term “complementary component” is used to refer to an epitope for an antibody or aptamer. Specifically, if the capture agent isan antibody or aptamer, then the complementary component for the capture agent is the epitope of an EV biomarker in the sample to which the antibody binds.

[0061] The term “epitope” as used herein refers to a small chemical group on the antigen molecule that is bound to by an antibody or aptamer. An antigen can have one or more epitope(s). In many cases, an epitope is roughly five amino acids or sugars in size. One skilled in the art understands that generally the overall three-dimensional structure or the specific linear sequence of the molecule can be the main criterion of antigenic specificity.

[0062] As used herein, unless otherwise indicated, the term “incubating” refers to maintaining a sample under conditions (which conditions include, but are not limited to, a period of time, a temperature, appropriate buffers, and the like, and any combination thereof) for carrying out the methods of the present disclosure.

[0063] As used herein, unless otherwise indicated, the term “capture agent” refers to an agent that can specifically bind to complementary components (e.g., EV biomarkers) in a sample. Non- limiting examples of capture agents include antibodies, aptamers, and the like. If antibodies or aptamers are used, in many cases they may bind to protein epitopes.

[0064] As used herein, unless otherwise indicated, the term “capture agent that is linked to an oligonucleotide” refers to a capture agent (e.g., antibody or aptamer) that is linked to an oligonucleotide (e.g., a distinct oligonucleotide) (such as, for example, by one or more of the linking method(s) described herein).

[0065] As used herein, unless otherwise indicated, the term “removing”, in the context of removing the labels and / or the probes that are associated with (e.g., hybridized to the oligonucleotides linked to the capture agents that are labeling a sample) refers to any method for physically separating the labels and / or probes from a sample. The labels and / or the probes can be removed from the sample by denaturation or by cleaving a linkage in the probe or a linker that attaches the label to the probe, for example, where the removal method used leaves the unhybridized oligonucleotides that are attached to the other antibodies intact and free to hybridize to the labeled probes used in the next cycle.

[0066] As used herein, unless otherwise indicated, the term “inactivating”, in the context of inactivating a label, refers to chemically modifying a label so that it no longer produces a detectable signal. Photobleaching or quenching are two ways to inactivate a label, although other ways are known.

[0067] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer comprising nucleotides (e.g., deoxyribonucleotides, ribonucleotides or a combination thereof), may be produced enzymatically or synthetically, may be PNA (asdescribed in U.S. Pat. No. 5,948,902 and the references cited therein), and can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids (e.g., can participate in Watson-Crick base pairing interactions). Naturally-occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. A locked nucleic acid (LNA), often referred to as an inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid”, or “UNA”, is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G' residue and a C' residue, where these residues correspond to non-naturally occurring forms, e.g., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in U.S. Application. No. 20050233340, which is incorporated by reference herein for disclosure of UNA.

[0068] As used herein, unless otherwise indicated, the term “oligonucleotide” refers to a multimer of nucleotide lengths define herein. Any oligonucleotide used herein may be composed of G, A, T and C, or bases that are capable of base pairing reliably with a complementary nucleotide. 7-deaza-adenine, 7-deaza-guanine, adenine, guanine, cytosine, thymine, uracil, 2-deaza-2-thio-guanosine, 2-thio-7-deaza- guanosine, 2-thio-adenine, 2-thio- 7-deaza-adenine, isoguanine, 7-deaza-guanine, 5,6-dihydrouridine, 5,6-dihydrothymine, xanthine, 7-deaza-xanthine, hypoxanthine, 7-deaza-xanthine, 2,6 diamino-7-deaza purine, 5- methyl-cytosine, 5-propynyl-uridine, 5-propynyl-cytidine, 2-thio-thymine or 2-thio-uridine are examples of such bases, although many others are known. As noted above, an oligonucleotide may be an LNA, a PNA, a UNA, or an morpholino oligomer, for example. The oligonucleotides used herein may contain natural or non-natural nucleotides or linkages.

[0069] As used herein, unless otherwise indicated, the term “reading” in the context of reading a fluorescent signal, refers to obtaining an image by scanning or by microscopy or the like, where the image shows the pattern of fluorescence as well as the intensity offluorescence in a field of view. Such reading may be accomplished, for example, by epifluorescent, hyperspectral, super-resolution microscopy, or the like, or any combination thereof. Reading may further refer to imaging the location of single EVs by light-scatter microscopy.

[0070] As used herein, unless otherwise indicated, the term “signal generated by”, in the context of reading refers to a signal produced or emitted by n EV upon imaging. In various examples, the generated signal is light scattering by an EV upon dark-field or confocal reflectance imaging. In various examples, the generated signal is fluorescence emitted directly from a fluorophore (e.g., from a universal stain or from a label nucleic acid probe) (where the distinct label is a fluorophore), a signal that is emitted indirectly via energy transfer to another fluorophore (e.g., by FRET), where a distinctly labeled nucleic acid probe bears the fluorophore. In various examples, the method is implemented using a molecular inversion probe with a donor fluorophore at one end and an acceptor fluorophore at the other for fluorescence energy transfer (FRET). When the probe is free in solution the two fluorophores are far apart. When they are brought into proximity by hybridization of the probe with the capture agent’s oligonucleotide, the two fluorophores are immediately adjacent to each other.

[0071] Other definitions of terms may appear throughout the specification.

[0072] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular examples described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular examples only and is not intended to be limiting.

[0073] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are described in association with the examples presented herein.

[0074] As will be apparent to those of skill in the art upon reading this disclosure, individual examples described and illustrated herein have discrete components and features that may be readily separated from or combined with the features of any of the other several examples without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0075] In an aspect, methods for analyzing a sample (e.g., a biological sample, an analysis sample or the like) of the present disclosure are described. In various examples, a method comprises use of a device (e.g., a fluidic device or the like). In various examples, amethod comprises disposition of an EV sample onto a first layer of a device, thereby forming an analysis sample. In various examples, a method further comprises use of devices comprising a first layer, a second layer with fluidic channels, a third layer, an optional fourth layer, various openings and vias, and functionalized surfaces, as described herein. In various examples, analysis samples are formed by disposition of one or more pluralit(ies) of EVs onto a first layer of at least one device, and such samples are used for the one or more labeling and imaging method(s) disclosed herein so that the presence and / or abundance and / or absence of the EV’s components (e.g., EV biomarker(s) or the like) can be detected and quantitated. In various examples, one or more sample(s) are used to perform a single EV immunoprofiling analysis as described herein.

[0076] In a further aspect, the uses of devices (e.g., fluidic devices) of the present disclosure are described. In various examples, various surface(s) (e.g., a surface or surfaces) of a device’s first layer is / are functionalized with molecules that are configured to promote or enable cross-linking EVs within the fluidic devices. Such cross-linking may be desirable in one or more method(s) described herein. Given the nature of such functionalization, however, introduction and flow of fluids into the fluidic devices is disrupted without the use of one or more mitigation(s). Therefore, in various examples, devices with mitigations for maintaining the desired fluid flow patterns are provided. Such mitigations may comprise devices comprising one or more functionalized surface(s) and / or one or more optional fourth layer(s).

[0077] Various means can be used to add or remove EV samples and / or other solutions to and from fluidic devices. In various examples, a method comprises use of centrifugation, filtration (in either normal or tangential modes), diafiltration, cross-flow filtration, absorption, affinity capture, electrophoretic transfer, positive pressure, negative pressure, La Place pressure, suction, gas pressurization, vacuum, aspiration, pipetting, hydrostatic pressure, droplet addition, or the like, or any combination thereof, for disposing EV samples onto a first layer of a fluidic device. Similar means may be used more generally in various examples for adding or withdrawing other solutions to or from fluidic devices (e.g., adding or withdrawing one or more capture agent solution(s), labeled nucleic probe solution, reagent solution, wash solution, buffer solution, blocking solution, or any combination thereof). In various examples, one or more pump(s) (e.g., vacuum pump(s), peristaltic pump(s), piezo pump(s), syringe pump(s), or the like) or droplet generator(s) (e.g., acoustic droplet generator(s), piezo droplet generator(s), or the like) is / are used to dispose the EV samples or for the addition / withdrawal of other solutions. In various examples, the disposition or the addition / withdrawal is manual (e.g., where the user controls a hand-held pipet) or byprogrammed liquid handling instrumentation. Any of the foregoing methods may be used in various examples for forming analysis samples where the EVs are disposed onto the first layer of a fluidic device as isolated, individual EVs as described herein.

[0078] The fluidic device may be comprised of a number of specified layers. In various examples, a method comprises use of a fluidic device comprising an optically transparent first layer, a second layer (e.g., an interstitial second layer), and an optically transparent third layer. In various examples, the second layer fluidically seals the first layer and the third layer, such that the sealing results in the first layer being disposed over at least a portion of the second layer. In various examples, the fluidic sealing results in the first layer being disposed over the entirety of the second layer. In various examples, the first layer and the third layer independently comprise one or more optional via(s) as described below.

[0079] The properties of the first and the third layers may be specified to endow imaging compatibility. The term “optically transparent” refers to any first layer or any third layer where the layer’s composition and other physical properties endows compatibility of such layers with the one or more imaging modalit(ies) described herein. Such optically transparent first and / or third layers independently comprise, as non-limiting examples: glass (e.g., quartz, soda lime, borosilicate, Willow® glass); polymers (e.g., nitrocellulose, polyether sulfone, polyvinylidene fluoride, cyclic olefin co-polymer, polydimethylsiloxane, acrylic, polycarbonate); ceramic or inorganic membrane (e.g., aluminum oxide, porous silicon, nanoporous silicon nitride); and the like. In various examples, the first layer and the third layer comprise the same material, while in various examples, the first layer and the third layer independently comprise at least two different materials. In various examples, the first layer and the third layer both comprise glass. In various examples, the first layer is nanoporous silicon nitride membrane (e.g., as disclosed in U.S. Pat. No. 9,789,239), while the third layer is a combination of a glass or a combination of at least two different polymeric materials. In various examples, the thickness of any first layer comprises a thickness from about 100 nm to about 500 m, including all 0.1 nm values and ranges therebetween. In various examples, the thickness of any third layer comprises a thickness of about 100 pm to about 2 mm, including all 0.1 nm values and ranges therebetween. In various examples, the first layer is nanoporous silicon nitride and the fluidic device is configured as described in U.S. Pat. No. 1 1,959,841 (incorporated herein by reference for its disclosure of fluidic devices).

[0080] The interstitial second layer may form fluidic channels. In various examples, the method further comprises use of a fluidic device comprising a second layer with one or more channel(s) of defined length, width, and / or height. In various examples, each channelindependently comprises a first opening and a second opening at each of the channel’s distal ends. The thickness of the second layer may be specified in order to define the height of such channels; e.g., the thickness of the second layer corresponds to the height of the channels. In various examples, the channels have a width of about 0.5 mm to about 5 mm, including all 0.01 mm values and ranges therebetween. In various examples, channels have a length of about 5 mm to about 20 mm, including all 0.01 mm values and ranges therebetween. In various examples, channels have a height of about 0.05 mm to about 1 mm, including all 0.01 mm values and ranges therebetween. In various examples, the number of channels formed within a second layer determines the number of channels of a fluidic device; (e.g., a fluidic device with at least one channel, at least two channels; at least three channels; at least four channels; at least five channels; at least six channels; or more than six channels).

[0081] A channel of a fluidic device or each of the channels of a fluidic device defines or define independently isolated instances of the first layer. In various examples, the method further comprises use of fluidic devices where the channels of the second layer independently define one or more portion(s) (e.g., instances or the like) of the first layer and there may be one or more such instance(s) within one fluidic device. Such instances correspond to independently isolated channels, where each channel comprises its own isolated portion of the first layer. The disposition of one or more EV sample(s) onto such instances of the first layer thus allows formation of one or more analysis sample(s) within one fluidic device. Accordingly, the fluidic devices may have at least one analysis sample, at least two analysis samples, at least three analysis samples, at least four analysis samples, at least five analysis samples, at least six analysis samples, or more than six analysis samples.

[0082] A second layer may comprise a variety of materials. In various examples, a method comprises use of a fluidic device where the second layer comprises a double-sided, pressure-sensitive adhesive (PSA)-coated tape (e.g., 3M F9460PC VHB Adhesive Transfer Tape and the like) or the like. Similar or and / or equivalent double-sided PSA tapes are well- known to the skilled artisan and widely available from various commercial vendors and may be suitable for the purposes disclosed herein. In various examples, a method comprises use of a fluidic device where the second layer comprises an inorganic material such as, for example, a non-metal, a metal, or a ceramic (e.g., a disposed layer of silicon, silicon dioxide, silicon nitride, aluminum oxide, titanium dioxide, aluminum, titanium, chromium, carbon in various forms, or the like, or any combination thereof), or the like, or any combination thereof. In various examples, a method comprises use of a fluidic device where the second layer comprises a polymer (e.g., polyamide, polydimethylsiloxane, polylactic acid, cyclic olefin co-polymer, other co-polymers, block co-polymers, a photoresist such as, for example, SU-8, any adhesive, polyetherimide, polytetrafluoroethylene, or the like, or any combination thereof) or the like. Various well-known means for disposition of such examples of second layer materials could be used and will be apparent given the nature and composition of the second layer, including, as non-limiting examples: low-pressure chemical vapor deposition; plasma-enhanced chemical vapor deposition; bonding; annealing; spin-coating; flash evaporation, e-beam evaporation; atomic layer deposition; or the like, or any combination thereof. The second layer material may be disposed onto a surface of a first layer or onto a surface of a third layer by any one of these methods. Of course, other methods will be apparent to the skilled artisan and these examples are listed for illustrative purposes only.

[0083] The channels may be formed in the second layer by a variety of means. Well- known means for forming the channels into the disposed materials of the second layer can be used and will be apparent given the nature and composition of the second layer. In various examples where the second layer is a non-metal, a metal, a ceramic, or the like, or any combination thereof, a method includes photoresist patterning, followed by wet chemical etching or reactive ion etching, or the like. Alternatively, the channels may be formed upon disposition of the second layer; e.g., the material forming the second layer is disposed through a shadow mask or the like, where the pattern of the shadow mask corresponds to the desired pattern of the channels. In various examples where the second layer is formed from double-sided PSA tape or from a polymer or the like, the channels are formed by laser cutting, die stamping, numerical-controlled machining or cutting, embossing, soft lithography, pattern stenciling, or the like, or any combination thereof. Similar methods may be used to form vias in first layers, third layers, and fourth layers (as described below.) Of course, other methods will be apparent to the skilled artisan and these examples are listed for illustrative purposes only.

[0084] The second layer may be fluidically sealed to the first layer and the third layer by a variety of means. Well-known means for sealing the layers could be used and will be apparent given the nature and composition of the second layer. In various examples where the second layer is a pressure-sensitive adhesive (PSA)-coated, double-sided tape or the like, then activation of the PSA is used for fluidic sealing of the layers. In various examples where the second layer is a polymer or the like, then gluing, oxygen plasma or UV-ozone activation and bonding (with or without additional use of pressure) is used for fluidically sealing the layers. In various examples where the second layer is a non-metal, a metal, a ceramic, or the like, or any combination thereof, then plasma-enhanced thermal bonding (with or without pressure),sintering, thermal annealing, or laser-assisted sintering is used for fluidically sealing the layers. Similar methods may be used for sealing a third layer to a fourth layer (as described below). Of course, other methods will be apparent to the skilled artisan and these examples are listed for illustrative purposes only.

[0085] The layers of the fluidic device may be alternatively specified. In various examples, the method further comprises use of a fluidic device comprising any previously described first layer, an optional thin interstitial second layer, and an optically transparent third layer into which the channels have been formed. The term “thin” herein designates a second layer comprising a thickness of about 100 nm to about 5,000 nm, including all 0.1 nm values and ranges therebetween. In various examples, the second layer fluidically seals the first layer and the third layer, such that the sealing results in the first layer being disposed over at least a portion of the second layer, or the sealing may result in the first layer being disposed over the entirety of the second layer. In various examples, the third layer comprises channels formed down into the material comprising the third layer (e.g., the channels have been formed into the third layer in the z-direction by any of the patterning and etching methods disclosed herein). Accordingly, the depth of such channels defines the height of the channels as previously described. The pattern formed within the optional second layer would thus substantially correspond or correspond to the pattern formed in the third layer so that together the patterns define the channels. In some of these various examples, the first layer and third layer are in direct contact and the fluidic sealing (as previously described for second layers) is between the first layer and the third layer (since the second layer is optionally omitted).

[0086] The fluidic device may also have a fourth layer. In various examples, the method further comprises use of any of the previously described first layers, any of the previously described second layers, any of the previously described third layers, and a fourth layer with one or more via(s) as described below. In various examples, the fourth layer comprises glass, non- metallic, metallic, ceramic, or polymeric material (e.g., material comprising polymer, polymer, or the like), or the like, or any combination thereof. In various examples, the thickness of the fourth layer is from about 20 m to about 1,000 pm, including all 0.1 pm values and ranges therebetween. In various examples, the fourth layer is a chemical functionalization layer of about 1 Angstrom to about 100 Angstrom thickness, including all 0.1 Angstrom values and ranges therebetween.

[0087] The openings of the channels and the vias of the layers may be positionally aligned for fluidic access. In various examples, the openings of the one or more channel(s)correspond to one or more via(s) formed within a first layer, a third layer, and / or a fourth layer, such that liquids can be added or withdrawn from the channels at their distal ends through such vias. Any of the layers’ vias may have defined dimensions and shapes. In various examples, a method comprises use of a fluidic device with various combinations and numbers of vias; e.g., a fluidic device with one or more via(s) through the third layer only; a fluidic device with one or more via(s) through both the first layer and the third layer; or a fluidic device with one more vias in the first layer and the fourth layer. Accordingly, the vias may have any degree of iteration and / or combination as desired. In various examples, the openings of the channels and any via of any first layer, any third layer, or any optional fourth layer are registered or aligned in the z-direction of the fluidic device.

[0088] The dimensions and / or shapes of the openings and / or vias may be specified to allow for certain desired properties for the fluidic devices with functionalized first layers. In various examples, the method further comprises use of a fluidic device comprising a functionalized first layer. In various examples, the use of a functionalized first layer necessitates that the fluidic device incorporates an optional fourth layer in order to maintain desirable liquid flow properties. For example, assuming both surfaces of the first layer are similarly functionalized with a moiety for EV cross-linking, then such surfaces may present polar and / or charged moieties under the typical buffer conditions of the methods disclosed herein (e.g., -NHs+groups at pH 7 to pH 8). Under these conditions, introducing a liquid or a sample into the channels may consequently be disrupted by the functionalization because the liquid will preferentially “spread-out” onto the outer surface of the first layer and not within the channel defined in part by the opposite, bottom, or underside of the first layer. Various examples comprising an optional fourth layer with one or more via(s) thus solve this potential disruption problem as such vias can be defined in order to direct the liquid as desired into the channels. Alternatively, the fourth layer may correspond to a differentially functionalized first layer as described below in order to mitigate the liquid “spread-out” problem.

[0089] The functionalization of the first layer may be variously specified to promote cross-linking of the EVs to the functionalized first layer. In various examples, EVs captured onto a functionalized first layer is cross-linked using any number of fixative reagents as disclosed herein. In various examples, a functionalized first layer promotes such fixation as its surface moieties can react with the fixative agents. Such functionalized first layers may be necessitated by all the stringent washing steps / processes described in the present disclosure; e.g., the conditions for removing labeled probes would remove EVs as well if the EVs were not otherwise cross-linked to the first layer. During an EV disposition step / process, EVs maybe initially disposed onto all inner surfaces of the channels of the fluidic devices. However, as will become apparent, the EVs that remain after cross-linking and washing are those EVs cross-linked to the bottom or underside surface of the functionalized first layer (e.g., the surface of the functionalized first layer facing the inner channel). Thus, the EVs analyzed by the labeling and imaging methods of the present disclosure are those cross-linked to this bottom or underside surface of the functionalized first layer (e.g., the optical imager is focused at this surface during the reading steps / processes described below).

[0090] Non-limiting examples of means for the functionalization of first layers may include vapor-phase or solution-phase covalent reaction of any number of well-known chemistries to the first layer, such as, for example, aldehydes, silanes, anhydrides, epihalohydrins, carbenes, and the like, and any combination thereof, with terminal groups chosen to endow the resultant surface with one or more desired propert(ies). For example, a first layer (e.g., comprising glass, ceramic, polymeric membrane, or inorganic membrane) is functionalized with an amine -presenting terminal group silane so that amine-to-amine crosslinking reagents can be used. In various examples, the first layer comprises one or more form(s) of glass and the first layer is similarly functionalized on both of its surfaces or on at least two surfaces (e.g., planar surfaces or opposite surfaces, such as, for example, a top surface and a bottom surface, or the like) by (3 -aminopropyl) triethoxysilane (APTES) or the like. In various examples, the first layer comprises nanoporous silicon nitride and the first layer is similarly functionalized on both of its surfaces or at least two of its surfaces by APTES or the like. The resultant polar and charged surfaces (at about pH 7) would promote liquid “spread-out” as characterized by a sessile water droplet contact angle less than about 90°, thus may require the use of a fourth layer to mitigate the liquid “spread-out” problem as described below.

[0091] Fourth layers may be used to mitigate the liquid “spread-out” problem associated with functionalized first layers. The fourth layer may alternatively be a chemical functionalization or the like. In various examples, the first layer comprises one or more form(s) of glass and at least two surfaces (e.g., two opposite surfaces, such as, for example, top and bottom surfaces) of the first layer are differentially functionalized; e.g., where one surface of the first layer is functionalized with dichloromethylsilane (DCMS), dimethyldichlorosilan (DMDCS) hexamethyldisilazane (HDMS) or other similar resultant hydrophobic silane, while the opposite or opposing surface is functionalized with APTES. In various examples, the DCMS, DMDCS, HMDS or another hydrophobic silane or the like would correspond to an optional fourth layer. The resultant hydrophobic nature of DCMS,DMDCS, HMDS, or other silane, or the like functionalized glass (as characterized by a sessile water droplet contact angle greater than about 90°) would prevent the liquid “spread- out” problem previously describe and thus represents a novel means for mitigating the undesirable consequence of the glass ’other surface’s APTES functionalization. In various examples, the bottom or underside surface of the first layer functionalized with APTES would be oriented into the channel such that its resultant surface-presented amine groups would be readily cross-linked to EVs (e.g., using one or more fixative reagent(s) disclosed herein). In various examples, the first layer is not functionalized on both of its surfaces (e.g., opposing surfaces, such as, for example, a top surface and a bottom or underside surface) with a moiety that renders the surface hydrophobic (as characterized by a sessile water droplet contact angle of greater than about 90°).

[0092] The vias of a fourth layer can be specified to induce Laplace pressure-driven flow. In various examples, a method comprises use of a fluidic device comprising a functionalized first layer, any of the previously described second layers, any of the previously described third layers, and a fourth layer with one or more via(s) of specified dimensions, where the functionalized first layer has been similarly functionalized on both of its surfaces or at least two surfaces with a resultant hydrophilic moiety (e.g., a hydrophilic functionalized first layer). In various examples, the diameter of the fourth layer vias at one end of the fluidic device’s channels is greater than the diameter of the fourth layer vias at the other opposite, bottom, or underside surface or end of the fluid device A channels. In various examples, the ratio of fourth layer vias at each distal channel end is independently about 2: 1 to about 10:1, including all integer ratio values and ranges therebetween. In various examples, the one of more fourth layer vias comprises a diameter of about 50 pm to about 5 mm, including all 0.1 pm values and ranges therebetween. In various examples, Laplace pressure-driven flow would be directed from the smaller diameter vias toward the opposing distal ends of the channels with the larger diameter vias, thereby mitigating the liquid “spread-out” problem caused by the hydrophilic functionalized first layer.

[0093] Various examples of fluidic devices are described herein. In various examples, methods of the present disclosure dictate that the fluidic devices have particular features and properties. First, in various examples, where a functionalized first layer is present for effective retention of EVs via cross-linking, the fluidic device comprises a fourth layers described herein (e.g., to resolve the liquid “spread-out” problem). Second, repeated exposure to concentrated chemical denaturant solutions (described below) during the labeling and reading cycles (described below) will degrade the integrity of certain polymers and / or PSAtapes, if such polymers or PSA tapes are used as the second layer for assembly and sealing of devices. Consequently, various examples of the fluidic devices comprise a first layer and a third layer where each layer independently comprises a glass, a second layer comprising a metal, a non-metal, a ceramic, or the like, or any combination thereof, and a fourth layer comprising a glass, a metal, a non-metal, a polymer, a ceramic, or the like or any combination thereof, or a chemical functionalization, and where the first layer is functionalized on at least its bottom or underside surface (e.g., the surface of the first layer facing the inner channel). In various examples, the first layer is differentially functionalized; e.g., APTES functionalization on its bottom or underside surface and DCMS, DMDCS, HMDS or other resultant hydrophobic silane on its opposing surface; thus, forming a chemical functionalization fourth layer. In various examples, glass, metal, non-metal, ceramic, or the like, or any combination thereof of any such layers endows the devices with desirable and / or necessary chemical compatibility (e.g., resistance to concentrated chemical denaturants). In various examples, the fourth layer comprises a polymer or the like, and a chemically resistant polymer such as, for example, poly etherimide, polytetrafluoroethylene, or the like, or any combination thereof is used for the fourth layer. In various examples, the fluidic devices are assembled and sealed using plasma-enhanced thermal bonding (with or without pressure), sintering, thermal annealing, laser-assisted sintering, or the like, or any combination thereof. In various examples, these assembly and sealing methods also endow the devices with desirable and / or necessary chemical resistance properties. In various examples, the fluidic device does not comprise a functionalized first layer hydrophobically functionalized on both of its surfaces (e.g., opposite surfaces, such as, for example, a top surface and a bottom or underside surface). In various examples, the fluidic device does not comprise a second layer comprising PSA tape.

[0094] Fluidic devices may be fabricated as individual units or in batches. The fluidic devices may be formed as single units; (e.g., any of the layers are individually prepared and then assembled and fluidically sealed). Alternatively, multiple devices may be prepared in batches; e.g., any of the layers are prepared on tape rolls or as sheets or as wafers, then singulated into individual units following assembly and fluidic sealing. Roll-to-roll processes or other scaled handling (e.g., robotic or other automation) may be used to fabricate the fluidic devices. The functionalization, deposition, patterning, etching, and other preparation steps / processes may be carried out in any order of preference, with any degree of repetition and / or combination, as desired for any of the previously described device layers. Various means may be employed for singulating individual fluidic devices from tape rolls, sheets, orwafers; e.g., cutting, stamping, saw-dicing, laser-dicing, stealth-dicing, and the like, and any combination thereof. The size of the tape rolls, the sheets, or the wafers on which devices are fabricated may be of any size or variation, as well as the number of devices per tape roll, sheet, and wafer. Any of the foregoing methods may further be combined with any degree of iteration and repetition to fabricate the fluidic devices.

[0095] In a further aspect, methods for analyzing a biological sample comprising at least one plurality of EVs of the present disclosure are described. In various examples, a method comprises a multiplexed, immunoprofiling single EV analysis of one or more pluralit(ies) of EVs. In various examples, one or more EV sample(s) are disposed within one fluidic device or across multiple fluidic devices, thereby forming one or more corresponding analysis sample(s) that are analyzed by one or more method(s) disclosed herein. In various examples, a method comprises use of one or more pluralit(ies) of capture agents, where a plurality is or each plurality is independently linked to an oligonucleotide (e.g., a distinct oligonucleotide). In various examples, a method comprises use of one or more corresponding pluralit(ies) of labeled nucleic acid probes to detect the presence and / or absence of the one or more pluralit(ies) of capture agents. In various examples, a labeled nucleic acid probe or each of the labeled nucleic acid probes specifically hybridizes with an oligonucleotide of a capture agent. In various examples, a method comprises use of capture agents comprising one or more pluralit(ies) of antibodies that selectively recognize(s) one or more epitope(s) of EV- expressed components (e.g., EV-expressed protein biomarkers). In various examples, a sample is labeled with the capture agents sequentially or en masse, and sub-sets of the capture agents are detected (e.g., the absence and / or presence) using iterative hybridization / label removal or inactivation cycles using corresponding sub-sets of the labeled nucleic acid probes. In various examples, the method further comprises use of labeled nucleic acid probes that are conjugated to one or more fluorophore(s), such that distinct fluorophores can be detected (e.g., the present or absence) by imaging-based methods during one or more or each of the cycle(s).

[0096] EVs may be variously disposed onto fluidic devices as desired. In various examples, a method comprises disposition of the same EV sample into all the channels of a fluidic device; e.g. the resultant analysis samples would all be the same within one fluidic device. In various examples, one or more EV sample(s) are disposed into one or more channel(s) of one or more fluidic device(s); e.g., the resultant analysis samples can have any desired degree of similarity, difference, iteration, and / or repetition within one fluidic device or across multiple fluidic devices. In various examples, the EVs are cross-linked to thefunctionalized first layers of fluidic devices after disposition or are cross-linked concurrently to both the fluidic devices and to capture agents after EV sample disposition and capture agent addition. Such cross-linking may be done using any of the fixative reagents described herein.

[0097] Capture agents and labeled nucleic acid probes are used in the methods for indicating the presence and / or abundance of EV components (e.g., EV biomarkers). In various examples, the method further comprises use of one or more pluralit(ies) of capture agents and one or more pluralit(ies) of corresponding labeled nucleic acid probes, where each plurality of capture agent is each linked to its own distinguishing but different oligonucleotide, and where the term “corresponding” is intended to mean that the number of labeled nucleic acid probes is the same as the number of capture agents used. In various examples, each plurality of the labeled nucleic acid probes is complementary to and specifically hybridizes with only one particular plurality of capture agent’s linked oligonucleotides. As an illustrative example, if there are about 50 capture agents (from one particular plurality of capture agent) and each capture agent is linked to one oligonucleotide, then there are about 50 labeled nucleic acid probes, where each labeled nucleic acid probe is complementary to and specifically hybridizes with one oligonucleotide. As an illustrative example, all the capture agents within one particular plurality of capture agent are each linked to the same oligonucleotide, thus distinguishing that particular plurality, and the various pluralities of capture agents each has its own different oligonucleotide. The capture agents may label the components (e.g., EV biomarkers) of the one or more pluralit(ies) of EVs forming the analysis sample.

[0098] The number of capture agents and labeled nucleic acid probes used in the method can be specified as desired. In various examples, the method is performed using about 3 to about 100 or more capture agents and labeled nucleic acid probes, including all integer values and ranges therebetween. In various examples, the number of capture agents and labeled nucleic acid probes are at least about 3, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more than about 100 capture agents and labeled nucleic acid probes. In various examples, each capture agent would be one plurality of capture agent linked to its own distinguishing and different oligonucleotide, and one plurality of labeled nucleic acid probe in corresponding numbers would be used for each capture agent used. In various examples, the method further comprises labeling one or more sample(s) with one or more capture agent pluralit(ies) and any corresponding number of labeled nucleic acid probepluralities as desired. In various examples, one or more sample(s) are within one fluidic device or distributed across multiple fluidic devices and the one or more pluralit(ies) of capture agents (and corresponding labeled nucleic acid probes) are used to label these samples with any degree of similarity, difference, iteration, repetition, or the like, or any combination thereof as desired. In other words, any number or any combination of capture agents (and corresponding nucleic acid probes) can be added to one or more channel(s) within one fluidic device or across multiple fluidic devices. In various examples, at least one capture agent (e.g., a cell-of-origin EV biomarker capture agent as described herein) is used to label all the samples or any group or portion of the samples; e.g., at least one capture agent within the mixture of the one or more capture agent(s) is similarly added to at least two or more fluidic channels such that at least two samples are similarly labeled by the common or shared one or more capture agent(s). In various examples, the labeling involves contacting the sample (e.g., an EV sample disposed onto a first layer within the channel of a fluidic device) with the specified number of capture agents sequentially or en masse under conditions where the capture agents bind to complementary components within, on, or associated with the EVs (e.g., epitopes of EV biomarkers).

[0099] Distinct labels may be used to detect (e.g., the presence and / or absence) capture agent-labeled samples. In various examples, following cross-linking of the capture agents to the sample, a method comprises specifically hybridizing a sub-set of the labeled nucleic acid probes with the sample, where the probes in the sub-set are distinctly labeled, thereby forming a sub-set of distinctly labeled probe / oligonucleotide duplexes. “Sub-set” refers to a fraction or portion of the distinctly labeled probe / oligonucleotide duplexes formed out of all the possible labeled probe / oligonucleotide duplexes that could be formed (depending on the number and variety of capture agents linked to oligonucleotides that were used to label the sample). The term “distinctly labeled” means that the labels can be separately detected (e.g., the presence or absence) and distinguished upon imaging (e.g., reading), even if they are present within, on, or at the same EV. The number of distinct labels that can be separately distinguished is a function of the imaging modality used to detect (e.g., the presence and / or absence) the labels. For instance, given their characteristics as previously described, hyperspectral imaging theoretically can detect e.g., the presence and / or absence) and distinguish a larger number of distinct labels than conventional fluorescent imaging. Accordingly, in various examples, a method comprises forming a sub-set of about 2 to about 5 or more distinctly labeled probe / oligonucleotide duplexes. In various examples, a method comprises forming at least about 2, at least about 3, at least about 4, at least about 5, or morethan about 5 distinctly labeled probe / oligonucleotide duplexes. In various examples, the probe / oligonucleotide duplexes are read (e.g., imaged) with either hyperspectral imaging and / or fluorescence imaging, following formation of the duplexes. In various examples, a method comprises specifically hybridizing about 2, about 3, about 4, about 5, or more than about 5 of the labeled nucleic acid probes with one or more sample(s), thereby producing labeled probe / oligonucleotide duplexes that are linked to antibodies that are bound to components (e.g., EV biomarkers). The hybridization may be carried out for any desired time-course (e.g., from about 1 minute to about 5 minutes, including all second values and ranges therebetween) and / or at any desired temperature (e.g., from about 10 °C to about 50 °C, including all 0.1 °C values and ranges therebetween).

[0100] A variety of distinct labels may be used. In various examples, a method comprises use of distinct labels comprising a fluorophore, a pro-fluorophore, a secondary activatable fluorophore, a fluorescent protein, a visible stain, a polychromatic barcode, or an absorbance tag, or the like, or any combination thereof. In various examples, any of the foregoing labels are covalently conjugated to the nucleic acid probe. In various examples, a method comprises use of an oligonucleotide that delivers an enzyme that delivers a fluorophore or there is an enzymatic amplification of signal. In various examples, a method detects a signal generated by fluorescence resonance energy transfer (FRET) or the like. In various examples, the method uses a secondary nucleic acid amplification step / process, including hybridization chain reaction, branched DNA (bDNA) amplification, or the like. In various examples, a method comprises detection of tyramide signal amplification (TSA) reagents and fluorophores. In various examples, a method comprises detection of the distinct label by fluorescent optical microscopy, hyperspectral imaging microscopy, super-resolution microscopy, or the like, or any combination thereof. Suitable distinct labels include, as nonlimiting examples: Cascade Blue; Pacific Blue; fluorescein and its derivatives; Oregon Green; Texas Red; rhodamine and its derivatives; Cy3; Pacific Orange; Cy5; any of the Atto brand fluorophores; aany of the Alexa Fluor brand fluorophores; and the like, and any combination thereof. Other non-limiting examples of distinct labels can be found in, for instance, The Molecular Probes handbook. A guide to fluorescent probes and labeling technologies, 11th Edition, published by ThermoFisher Scientific.

[0101] In various examples, distinct labels have one or more distinguishable spectral properties). In various examples, a method comprises use of distinct labels that are specified so that they are spectrally distinguishable (e.g., independently detectable) from one another, meaning that the labels can be independently measured (e.g., when the labels are mixed in animage). In other words, the amounts of label present (e.g., the amount of fluorescence if the label is a fluorophore) for each of the labels are separately determinable, even when the labels are within, on, or at the same EV. Likewise, the signal generated by the universal or pan-EV stain described below are chosen so that it is also separately distinguishable during a reading (e.g., imaging) step or steps / process or processes. “Spectrally distinguishable” refers to, for example, that the distinct label has well-defined excitation, emission, and / or absorbance wavelength bands that can be qualitatively and / or quantitatively identified by the one or more optical modalit(ies) described herein.

[0102] The binding pattern of probes and capture agents on EVs, as well as the location of EVs themselves, can be imaged during a reading step / process. In various examples, after the sample has been washed to remove labeled nucleic acid probes that have not hybridized, the method further comprises reading the sample to obtain an image showing the binding pattern for at least a portion of the, substantially all the, or each sub-set(s) of probes hybridized to capture agents in a prior hybridization step or process. The reading (e.g., imaging) may be done using any of the appropriate optical modalities disclosed herein. The universal or pan-EV stain described below may also be imaged at one or more or each reading step(s) or process(es). Alternatively, the location of single EVs may be imaged by light-scatter microscopy at each reading step / processes. Accordingly, in various examples, a method comprises repeated reading steps / processes (within hybridization and removal / inactivation cycles) where the binding patterns of probes and capture agents, as well as the location of EVs, are imaged during one or more or each step(s) or process(s).

[0103] Probes can be removed or inactivated following hybridization and reading steps / processes. After reading the binding and hybridization pattern for a prior sub-set of probes, the method further comprises inactivating or removing the labels and / or the probes that are associated with the sample labeled by the capture agents linked to oligonucleotides. The inactivation and / or removal thus leaves the plurality of capture agents and their associated oligonucleotides still bound to the sample but in an unhybridized form. The labels that are associated with the sample may be removed or inactivated by a variety of methods including, in non-limiting examples: denaturation (in which case the label and the probe in its entirety may be released and can be washed away); by cleaving a linkage in the probe (in which case the label and part of the probe may be released and can be washed away); by cleaving both the probe and the oligonucleotide to which the probe is hybridized (to release a fragment that can be washed away); by cleaving the linkage between the probe and the label (in which case the label will be released and can be washed away); or by inactivating thelabel itself (e.g., by modifying or breaking bonds in the label, thereby preventing the label from producing a signal); or the like. In all of these removal methods, the unhybridized oligonucleotides that are attached to the other antibodies are intact and free to hybridize to the set of labeled probes used in the next cycle (e.g., subsequent successive sub-sets of probe hybridization).

[0104] The probes can be removed in several manners. In various examples, a method comprises removing the hybridized probes from the sample by denaturation, leaving the other capture agents (e.g., the capture agents that are not hybridized to a probe) and their associated oligonucleotides still bound to the sample. In various examples, a method comprises not removing the hybridized probes from the sample by denaturation, leaving the other capture agents (e.g., the capture agents that are not hybridized to a probe) and their associated oligonucleotides still bound to the sample. In various examples, a method comprises the removal of labels by cleaving at least one bond in the probes that are associated with the sample, or a linker that links the probes to the labels, thereby releasing the labels from the probes. This cleavage can be done enzymatically, chemically, via exposure to light, or the like, or any combination thereof. Alternatively, the labels can be inactivated by photobleaching or by chemically altering the label or the like or any combination thereof). In various examples the probes are not removed by displacement with a competitively binding oligonucleotide that would displace the probe (e.g., no removal by a displacement oligonucleotide).

[0105] After removal of the probes, the sample may be iteratively hybridized with additional sub-sets of different labeled probes. In various examples, following the removal of a first sub-set of probes, a method comprises addition and hybridization of a second sub-set of labeled probes (e.g., a second sub-set of about 2, about 3, about 4, or about 5 or more labeled probes, where the probes are each distinctly labeled). The sample then may be re-read to produce an image showing the binding pattern for one or more or each of the most recently hybridized sub-set(s) of probes. For example, a first sub-set of probes may be hybridized, read, and then removed, followed by similar steps / processes for a second, third, and successive sub-sets of labeled probes. In various examples, method comprises hybridizing a first sub-set of probes to a sample, reading the first sub-set’s binding pattern, and then removing the first sub-set, followed by additional cycles of hybridization, reading, and removal steps / processes on the same sample for a second, third, and successive sub-sets of labeled probes). After one or more or each reading step or process in these cycles, the probes may be removed from the sample (e.g., by denaturation or another method) and thehybridization and reading steps / processes may be repeated with a different sub-set of distinctly labeled probes.

[0106] In various examples, a method comprises repeating the hybridization, reading, label removal or inactivation steps / processes over multiple cycles on the same sample with a first, second, third, and successive sub-sets of distinctly labeled nucleic acid probes, where each repeat is followed by removal of the probes (e.g., by denaturation or another method), except for the final repeat, such that a plurality of images of the sample are produced, where each image corresponds to the binding pattern for a sub-set of labeled nucleic acid probes labeling capture agents that labeled any of the sample’s complimentary components (e.g. EV biomarkers or the like). The hybridization, reading, and / or probe removal / inactivation steps / processes can be repeated over multiple cycles until all of the probes have been analyzed. Further, the EVs may be optionally imaged by the universal / pan-EV stain or light- scatter imaging at one or more or each reading step(s) or process(es) of such cycles.

[0107] Following disposition of EVs onto a functionalized first layer and subsequent cross-linking, the cycles may be repeated to read the binding pattern of multiple capture agents and their corresponding probes, as well as the position of EVs on the sample, for as many cycles as desired. In various examples, the method further comprises: a) disposing at least one EV sample onto at least one first layer, thereby forming at least one analysis sample within at least one fluidic device; b) contacting the at least one analysis sample with one or more pluralit(ies) of capture agents; c) cross-linking the EVs to the first layer and the EVs to the one or more capture agent(s); d) hybridizing a first sub-set of labeled nucleic acid probes with the at least one analysis sample, where the probes in the first sub-set are distinctly labeled, thereby forming a first sub-set of labeled probe / oligonucleotide duplexes; e) reading the sample to obtain an image showing the binding pattern for at least a portion, substantially all, or each of the probes hybridized in the prior step; f) removing the first sub-set of probes from the at least one analysis sample, by denaturation or another method, leaving the plurality of capture agents and their associated oligonucleotides still bound to the at least one analysis sample; g) hybridizing a second sub-set of labeled nucleic acid probes with the at least one analysis sample, where the probes in the second sub-set are distinctly labeled, thereby forming a second sub-set of labeled probe / oligonucleotide duplexes;h) reading the sample to obtain an image showing the binding pattern for at least a portion, substantially all, or each of the probes in the second sub-set of probes; i) removing the second sub-set of probes from the at least one analysis sample, by denaturation or another method, leaving the plurality of capture agents and their associated oligonucleotides still bound to the at least one analysis sample; j) optionally, imaging the EVs at each reading step by a universal EV stain or by light- scatter imaging; and k) optionally, repeating steps / processes g) through j) over multiple cycles for a third subset and successive sub-sets of labeled nucleic acid probes.

[0108] In various examples, steps / processes g) through]) are repeated for as many cycles as desired; e.g., until the binding patterns for at least a portion, substantially all, or all capture agents have been read and imaged across one or more or all the samples (such as, for example, biological sample(s) and / or analysis sample(s)) being analyzed, where the number of probes corresponding to the number of capture agents used to label the samples. In various examples, the probes are optionally removed from the sample at the final cycle. In various examples, a method comprises repeating the hybridization, reading, and removal steps / processes over two cycles through about 20 or more cycles. In various examples, a method comprises repeating the hybridization, reading, and removal steps / processes over at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles, at least about 11 cycles, at least about 12 cycles, at least about 13 cycles, at least about 14 cycles, at least about 15 cycles, at least about 16 cycles, at least about 17 cycles, at least about 18 cycles, at least about 19 cycles, at least about 20 cycles, or more than about 20 cycles. With such numbers of cycles, multiplexing can be achieved so that multiplexed immunoprofiles of EV biomarkers can be generated.

[0109] A variety of chemical-based, enzyme-catalyzed or photo-induced cleavage methods may be alternatively used instead of denaturation. In various examples, a method comprises use of probes that contain a chemically or photo-cleavable linkage so that they can be fragmented by exposure to a chemical, light, or the like. In various examples, a method comprises use of a restriction enzyme or a double-stranded DNA-specific endonuclease to cleave the duplexes since they are double-stranded. In various examples, a method comprises use of a probe that contains a uracil (which may be cleaved by USER), or contains a hairpin that contains a mismatch, which can be cleaved using a mismatch- specific endonuclease. In various examples, after cleavage the melting temperature (Tm) of the fragment of the probethat contains the label may be insufficiently high to remain base paired with the oligonucleotide and, as such, the fragment will disassociate from the oligonucleotide. In various examples, a method comprises use of a probe and label that are connected by a photo- cleavable or chemically-cleavable linker or the like or any combination thereof. Cleavage of the linker(s) releases the label from the sample. In various examples, method comprises use of an RNA probe which can be degraded using an RNAse. In various examples, a method comprises use of a probe with an enzymatically cleavable linkage. For example, esters can be cleaved by an esterase and a glycan can be cleaved by a glycosidase. Alternatively, the label itself may be inactivated by modifying the label, for example, by photobleaching or by chemical quenching with hydrogen peroxide, or other means or any combination thereof.

[0110] In various examples, after reading the sample, a method comprises removing the probes hybridized in a previous step from the sample by denaturation (e.g., by un-annealing the labeled probes from the oligonucleotides and washing them away), leaving the capture agents and their associated oligonucleotides still bound to the sample. This step may be done using any suitable chemical denaturant (e.g., formamide, dimethylsulfoxide (DMSO), urea, guanidinium chloride, or the like), using ultrapure water (e.g., greater than or equal to about 18 MOhm resistivity water) with / without the addition of a divalent cation chelation agent (e.g. 0-10 mM ethylenediaminetriacetic acid (EDTA) or the like), using a toehold release strategy (e.g., see Kennedy-Darling, Chembiochem. 2014 15: 2353-2356), or by using heat, base, a topoisomerase, or a single-strand binding agent (e.g., SSBP). This step can also be achieved through hybridization of an oligonucleotide with a greater affinity (e.g., PNA-based oligonucleotide). In various examples, a method comprises removal of the probes by incubating the sample in ultrapure water, for a period from about 1 minute to about 5 minutes, including all second values and ranges therebetween. In various examples, a method comprises removal of the probes by incubating the sample in about 1 mM to about 10 mM EDTA solution, including all 0.1 mM values and ranges therebetween, for a period from about 1 minute to about 5 minutes, including all second values and ranges therebetween. In various examples, the EDTA is at least about 1 mM, at least about 5 mM, or at least about 10 mM. In various examples, a method comprises removal of the probes by incubating the sample in about 1% to about 10% v / v concentration formamide, including all 0.1% values and ranges therebetween, or in about 1% to about 10% v / v concentration DMSO, including all 0.1% values and ranges therebetween, for a period from about 1 minute to about 5 minutes, including all second values and ranges therebetween. In various examples, the formamide or the DMSO is at least about 1%, at least about 5%, or at least about 10% v / vconcentration. In any of the foregoing examples, the denaturant (i.e., ultrapure water, EDTA, formamide, or DMSO) is incubated for at least about 1 minute, at least about 2 minutes, at least about 3 minutes, at least about 4 minutes, or at least about 5 minutes. This denaturation step may be repeated, if desired or necessary, so that substantially all or all of the hybridized probes have been removed, and may be followed by a wash as desired. As would be apparent, this step is not implemented enzymatically (e.g., does not use an enzyme such as, for example, a DNase, endonuclease, nor restriction enzyme) so does not result in cleavage of any covalent bonds (e.g., in any of the probes or oligonucleotides), nor does any enzyme release the probe from the oligonucleotide (e.g., by a topoisomerase), and further does not remove any capture agents from the sample. With any of the foregoing, the strands of the probe / oligonucleotide duplexes are separated from one another (e.g., denatured) and the separated probes, which are now free in solution, can be washed away, leaving the capture agents and their associated oligonucleotides intact and in place.

[0111] It may be desirable that the denaturation step be specified for the chemical sensitivity of EVs. In various examples, the method does not comprise removal of the probes by incubating the sample in a chemical denaturant (e.g., urea, formamide, DMSO, or guanidinium, or the like), where the denaturant concentration is about greater than 10% v / v concentration, about greater than 15% v / v concentration, about greater than 20% v / v concentration, or about greater than 25% v / v concentration . In various examples, a chemical denaturant concentration is about less than or equal to about 10% v / v in the various examples of the methods disclosed herein. Without intending to be bound by any particular theory, it is considered higher concentrations of such denaturants will negatively affect the EVs (e.g., dissolve the EVs or the like), which would obviate the desired outcomes and intended purposes of the methods disclosed herein. It is considered that, given their nanoscale size and surface area, EVs are sensitive to dissolution by high concentrations of chemical denaturants (i.e., 70% to 90% v / v) that are required when similarly processing cells and / or tissue sections.

[0112] A cleavable linkage may be alternatively be used to remove the probes. If a cleavable linkage is used (e.g., in the probes or to connect the probes to the labels, it is desirable that the cleavable linker capable of being selectively cleaved using a stimulus (e.g., light or a change in its environment) without breakage of bonds in the oligonucleotides attached to the antibodies. In various examples, a method comprises use of a cleavable linkage that is a disulfide bond, which can be readily broken using a reducing agent (e.g., 0- mercaptoethanol, dithiothreitol, or the like), or the like, or any combination thereof. Other suitable cleavable linkers include, but are not limited to, base-cleavable sites such as, forexample, esters, particularly succinates (e.g., cleavable by, for example, ammonia or trimethylamine or the like), quaternary ammonium salts (e.g., cleavable by, for example, diisopropylamine or the like) and urethanes (e.g., cleavable by, for example, aqueous sodium hydroxide or the like); acid-cleavable sites such as, for example, benzyl alcohol derivatives (e.g., cleavable by, for example, trifluoroacetic acid or the like), teicoplanin aglycone (e.g., cleavable by, for example, trifluoroacetic acid followed by base or the like), acetals and thioacetals (e.g., cleavable by, for example, trifluoroacetic acid or the like), thioethers (e.g., cleavable by, for example, HF or cresol or the like) and sulfonyls (e.g., cleavable by, for example, trifluoromethane sulfonic acid, trifluoroacetic acid, thioanisole, or the like, or any combination thereof); nucleophile-cleavable sites such as, for example, phthalamide (e.g., cleavable by, for example, substituted hydrazines or the like), esters (e.g., cleavable by, for example, aluminum trichloride or the like); and Weinreb amide (e.g., cleavable by, for example, lithium aluminum hydride or the like); and other types of chemically cleavable sites, including, but not limited to, phosphorothioate (e.g., cleavable by, for example, silver or mercuric ions or the like) and diisopropyldialkoxysilyl (e.g., cleavable by, for example, fluoride ions and the like), and any combination thereof. Other cleavable bonds will be apparent to those skilled in the art or are described in the pertinent literature and texts (e.g., Brown (1997) Contemporary Organic Synthesis 4(3); 216-237). In various examples, a cleavable bond is cleaved by an enzyme or the like.

[0113] In various examples, a photocleavable (“PC’-) linker (e.g., a uv-cleavable linker) is employed. Suitable photocleavable linkers for use include, but are not limited to, orthonitrobenzyl-based linkers, phenacyl linkers, alkoxybenzoin linkers, chromium arene complex linkers, NpSSMpact linkers and pivaloylglycol linkers, e.g., as described in Guillier et al (Chem Rev. 2000 Jun. 14; 100(6):2091- 158), and the like any combination thereof. Nonlimiting examples of linking groups that may be employed in the subject methods are described in Guillier et al, supra and Olejnik et al. (Methods in Enzymology 1998 291 : 135- 154), and further described in U.S. Pat. No. 6,027,890 (incorporated herein by reference for its disclosure of linking groups); Olejnik et al. (Proc. Natl. Acad Sci, 92:7590-94); Ogata et al. (Anal. Chem. 2002 74:4702-4708); Bai et al. (Nucl. Acids Res. 2004 32:535-541); Zhao et al. (Anal. Chem. 2002 74:4259-4268); and Sanford et al. (Chem Mater. 1998 10: 1510-20), and are purchasable from Ambergen; NHS-PC-LC-Biotin) from Link Technologies or ThermoFisher Scientific).

[0114] The DNA sequences used may be selected in order to decrease (e.g., minimize) background staining. In various examples, the DNA sequences of the oligonucleotides linkedto the capture agents and of the nucleic acid probes used are selected in order to decrease (e.g., minimize) background staining, for example, either from non-specific adsorption, through binding to endogenous genomic sequences (RNA or DNA) or the like, or any combination thereof. Likewise, in various examples, a method comprises use of hybridization and / or washing buffers formulated to decrease (e.g., minimize) background staining either from non-specific adsorption or through binding to endogenous genomic sequences (RNA or DNA) or through binding to other reporter sequences. In various examples, sheared salmon sperm DNA and / or a combination of multiple pluralities of nucleic acid probes (lacking distinct labels) are incorporated into a blocking buffer used to pre-treat a sample prior to the step of adding capture agents to block non-specific binding of labeled nucleic acid probes in subsequent steps / processes. Alternatively, a molecular excess of the nucleic acid probes, lacking any distinguishing label, may be supplemented into a blocking buffer for similar purposes. Common blocking buffers suitable for the methods disclosed herein are well- known to the skilled artisan and may include, but are not limited to, buffering agents (e.g., Tris, HEPS, or the like), salts (e.g., NaCl or the like), polysorbates (e.g., Tween-20 or the like), detergents (e.g., Triton X-100, BR1DJ-35, or the like), and proteins (e.g., fish eye gelatin or the like), and the like, and any combination thereof.

[0115] The oligonucleotides and the probes may have defined lengths. In various examples, a method comprises use of nucleic acid probes that are about 8 to about 20 nucleotides in length (e.g., at least about 8, at least about 10, at least about 11, at least about 17, at least about 18, or at least about 20 nucleotides in length). In various examples, a method comprises use of probes that are as short as about 5 nucleotides in length to as long as about 150 nucleotides in length (e.g., about 5 nucleotides in length to about 100 nucleotides in length, including all integer values and ranges therebetween). In various examples, a probe comprises or exhibits a calculated melting temperature (Tm) of about 15 °C to about 70 °C, including all 0.1 °C values and ranges therebetween (e.g., about 20 °C to about 60 °C or about 35 °C to about 50 °C) such that the duplexes of the hybridization step have a Tm in the same range. In various examples, the Tm is calculated using the IDT oligoanalyzer program (available at IDTs website and described in Owczarzy et al., Nucleic Acids Res. 2008 36: W163-9) using default settings of about 50 mM Na+, about 250 nA oligonucleotide. It is well-known to the skilled artisan that the effective Tm of the various hybridized duplexes, as well as the unhybridized oligonucleotide counterparts, will be influenced by the presence and absence, for example of monovalent and / or divalent cations (e.g., K+, Na+, Mg2+, or the like), as well as chaotropic agents (e.g., urea, formamide, DMSO, guanidinium, or the like).Effectively lower Tm will be realized in the absence of monovalent and divalent cations (e.g., in ultrapure water + / - EDTA), thus the duplexes may be more easily denatured at lower incubation temperatures. Similarly, effectively lower Tm will be realized in the presence of chaotropic agents, thus the duplexes may be more easily denatured at lower incubation temperatures.

[0116] The sequence of the probes can be any sequence, although certain sequences may be specified in order to minimize background staining as previously described. In various examples, a method comprises use of probes that are Tm-matched, where the term “Tm- matched” refers to sequences that have melting temperatures that are within a defined range (e.g., less than about 15° C, less than about 10° C, or less than about 5° C). In various examples, a method comprises use of probes labeled at the 5' end, the 3' end, or anywhere in between, and / or probes that are specifically cleavable (e.g., may contain a cleavable linker). In various examples, a method comprises use of oligonucleotides that are at least about 5 nucleotides in length to about 40 nucleotides in length, including all integer values and ranges therebetween (e.g., at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, or at least about 40 nucleotides in length).

[0117] The sequence of the oligonucleotides may be specified as needed. In various examples, a method comprises use of oligonucleotides sequences (to which the capture agents are linked) that are the same length and are perfectly complementary to the labeled probes. In various examples, the oligonucleotides are linked to the capture agents by a linker that spaces the oligonucleotide from the capture agents. In various examples, a method comprises use of oligonucleotide sequences (to which the capture agents are linked) that are longer than the sequences of the labeled nucleic acid probes and otherwise identical to one other except for a sub-sequence that is complementary to a single labeled nucleic acid probe. In various examples, the extra sequence acts as a linker to space the oligonucleotides from the capture agents. Capture agents may be derivatized with oligonucleotides with any degree of derivation efficiency or extent (e.g., any degree of labeling), while oligonucleotides may be linked to capture agents using any convenient method (see, e.g., Gong et al., Bioconjugate Chem. 2016 27: 217-225 and Kazane et al. Proc Natl Acad Sci 2012 109: 3731-3736). A variety of labeling methods are available. In various examples, unique oligonucleotides are linked to the capture agents directly using any suitable chemical moiety on the capture agent (e.g., a cysteine residue or via an engineered site). In various examples, a method comprises use of capture agents where a common oligonucleotide is conjugated directly to all of the capture agent(s) using any suitable chemistry, and unique oligonucleotides may be linked tothe common oligonucleotides enzymatically (e.g., by ligation). In various examples, a method comprises use of capture agents where unique oligonucleotides are linked to the capture agents directly or indirectly via a non-covalent interaction (e.g., via a biotin / streptavidin or an equivalent thereof, via an aptamer or secondary antibody, or via a protein-protein interaction such as, for example, a leucine-zipper tag interaction or the like). In various examples, a method comprises use of other entities that can bind to one another in a specific manner, thereby substituting for the oligonucleotides and probes of the present disclosure (e.g., leucine zipper pairs, antigen / antibody pairs, or the like or any combination thereof).

[0118] A reading step (e.g., imaging step or the like) or process (e.g., imaging process or the like) or each reading step (e.g., imaging step) produces an image of the sample showing the pattern of binding of a sub-set of probes and capture agents, where such binding patterns can indicate the presence and / or abundance of components (e.g., EV biomarkers). In various examples, a method comprises analyzing, comparing, overlaying, or the like, or any combination thereof of at least two or more of the images. In various examples, the method further comprises analyzing, comparing, overlaying, or the like, or any combination thereof any one or more image(s) in any desired combination and / or sequence with any one or more other image(s). In various examples, the method further comprises overlaying at least a portion, substantially all or all of the images to produce an image showing the pattern of binding of at least a portion, substantially all, or all the capture agents to the sample. In various examples, a method further comprises limiting the overlaying such that the resultant image shows where the binding pattern of any one or more capture agent(s) to the sample is aligned (e.g., colocated with or the like) the binding pattern of one or more particular capture agent(s). In various examples, the one or more particular capture agent(s) are a cell-of-origin (CofO) EV biomarker; e.g., the binding pattern for one or more EV biomarkers’ capture agent(s) are shown or only shown in the resultant overlaid image when they colocate with the binding pattern for one or more CofO EV biomarkers’ capture agents, indicating those EV biomarkers are colocated to the same EV, or the like. In various examples, the resultant overlaid images are further limited to show binding patterns of any capture agent that only colocate with the universal EV stain or the light-scatter EV location. The term “colocate with” refers to the analyzing, comparing, overlaying, or the like, or any combination thereof of images that shows where capture agent binding patterns are registered to the same EV and / or where the capture agent binding patterns are registered with the universal EV stain (or the light-scatter EV image) on the same EV. The term “registered” refers to superimposition of the signals generated by any of the foregoing when the images are overlaid. Thus,colocalization herein described allows the presence and / or abundance of EV biomarkers to be indicated on single EVs, thereby enabling single EV analyses as disclosed herein.

[0119] The position of single EVs on the sample can be identified by a variety of means. In various examples, a method comprises imaging single EVs that have been labeled with a universal or pan-EV stain or the like. The terms “universal” and “pan- EV” mean that all the EVs in an EV sample are commonly labeled with a distinctly identifiable moiety, where such moiety can be optionally imaged during any of the previously described reading (e.g., imaging) steps / processes. Such universal or pan-EV stains may include, as non-limiting examples: lipid bilayer-permeable and amine reactive dyes such as, for example, carboxyfluorescein diacetate succinimidyl ester(CMFDA-SE; available from Thermo Fisher); impermeable and amine-reactive V-hydroxysuccinimide (NHS) ester fhiorophore derivatives(e.g., Atto 390-NHS, Pacific Blue-NHS, AlexaFluor 488-NHS, Atto 550-NHS, and the like; available from Thermo Fisher or MilliporeSigma); lipid-soluble fluorogenic dyes such as, for example, MemGlow brand dyes(available from Cytoskeleton Inc.), Cell Mask brand dyes(available from Thermo Fisher), or ACOE brand dyes (available from Acoerela Inc.); impermeable and thiol-reactive (e.g., fluorescein-polyethyleneglycol- maleimide of varying polyethyleneglycol length) or amine-reactive N-hydroxy succinimide (NHS) ester fluorophore derivatives (e.g., Sulfo-Cy3-NHS, Atto 390-NHS, Pacific Blue- NHS, AlexaFluor 488-NHS, Atto 550-NHS, and the like; available from Thermo Fisher or MilliporeSigma); and the like; and any combination thereof. In various examples, a method comprises labeling an EV sample with the universal EV stain prior to, or after, contact with a fluidic device. Alternatively, a method comprises labeling an EV sample with a universal stain prior to any EV purification process. Other labeling means and universal stains are available. As will be apparent, the foregoing examples impart a fluorophore moiety to the EVs, such that single EVs can be identified using one or more form(s) of fluorescent microscopy, fluorescent imaging, or the like, or any combination thereof. In various examples, a method comprises use of light-scatter microscopy imaging or the like to locate single EVs on a sample or samples of the present disclosure.

[0120] Various image analysis modules may be used to analyze the plurality of binding pattern and EV images. Combinations of image analysis modules may be used. An image analysis module used may transform the signals from fluorophores or each fluorophore to produce a plurality of false color images. The image analysis module may overlay the plurality of false color images (e.g., superimpose the false colors at each pixel) to obtain a multiplexed false color image. Multiple images (e.g., unweighted or weighted; limited bycolocation with a CofO EV biomarker or the EV position from the universal stain or the lightscatter image) may be transformed into a single false color, so as to represent one or more particular combination(s) of binding patterns of specific capture agents to the same EV (e.g., to generate a multiplexed, EV biomarker immunoprofile of single EVs). False colors of any binding patterns may be assigned to specific capture agents or combinations of capture agents, based on manual input from the user. Likewise, a false color may be assigned to the EV from the universal EV stain or from the light-scatter image, and / or false colors may be assigned to specific capture agents or combinations of capture agents (e.g., when such binding patterns colocate with the universal EV stain or to the light-scatter EV image, based on manual input from the user). The intensity of assigned false colors may be adjusted by the user to represent certain single EV labeling patterns (e.g., intensities are thresholded to show only those single EVs with specific or combinations of labeling and / or colocation). An image analysis module may further be configured to count single EVs via the universal stain, the light-scatter image, any combination of binding patterns of capture agents, as well as any combination thereof. Such single EV counts may be exportable to a spreadsheet (e.g. csv, tsv, or .xlsx file) or other numerical manipulation file format suitable for subsequent analyses (e.g., Statistical and / or bioinformatic models). An image analysis module may further be configured to adjust, normalize, threshold, or the like, or any combination thereof the intensity, contrast of signal intensities, or false colors, or the like, or any combination thereof, to perform a convolution operation (such as, for example, blurring or sharpening of the intensities, false colors, or the like), or perform any other suitable operations to enhance the image. An image analysis module may perform any of the above operations to align pixels obtained from successive images, to blur, smooth, or the like, or any combination thereof intensities or false colors across pixels obtained from successive images. An image analysis module may further be configured to reconstruct one image from multiple images of the same imaging field (e.g., either from a series of images that are taken at small changes in the focal plane in the z-direction that are typical for confocal microscopy, or from a series of time- lapsed images at the same focal plane). An image analysis module may further be configured for deconvolution or extraction of particular signals from a spectrum comprising mixed signals (e.g., deconvolution or extraction of the emission maximal intensities from multiple distinct fluorophores in one spectral image obtained by hyperspectral imaging). Other means for image manipulation and image analysis module configurations are possible and will be apparent to the skilled artisan.

[0121] The resultant images produced by an image analysis module or modules may be viewed side-by-side or, in various examples, the images are superimposed or combined. In various examples, the images are in color, where the colors used in the images correspond to the capture agents used to form the imaged binding patterns and / or to the location of EVs identified by the universal stain or the light-scatter EV image.

[0122] Overlaid images and resultant EV biomarker colocation counts may be used to generate cell-of-origin, single EV biomarker immunoprofiles. In various examples, a method further comprises using the resultant EV counts from the image analysis module to generate a cell-of-origin EV biomarker immunoprofile or the like. In various examples, the capture agents are antibodies or the like, and the EV counts would be derived from overlaid images where the binding pattern for one or more EV biomarker capture agent(s) are shown in resultant overlaid images only when they substantially colocate or colocate with binding patterns for one or more particularly specified CofO EV biomarker capture agent(s) (indicating that the former EV biomarkers are colocated to the same EV as the one or more CofO EV biomarker(s)). In various examples, a method further comprises use of a statistical and / or bioinformatic analysis model for generating the cell-of-origin EV biomarker immunoprofile. Non-limiting examples of suitable statistical and / or bioinformatic analysis approaches include, but are not limited to, models based on logistic-regression, discriminant analysis, principal component analysis, clustering, random forests, or other machine learning algorithms, or the like, or any combination thereof. In various examples, such models compare the at least a portion, substantially all, or an entire population of EVs expressing the EV biomarkers in general to the population of EVs expressing the at least one CofO EV biomarker and the other EV biomarkers, in order to ascertain if the latter population represents EVs from the specific CofO cell type, where “represent” refers to a statistically significant difference is determined by the model. In various examples, a method further comprises using similar statistical and / or bioinformatic analysis models for generating one or more single EV biomarker immunoprofile(s) that are not based on any cell-of-origin limitations.

[0123] An image analysis module may be implemented in various examples. The image analysis method may be implemented on a computer or the like. In various examples, a general-purpose computer or the like is configured to a functional arrangement for the methods and programs disclosed herein. The hardware architecture of such a computer is well known by a person skilled in the art, and can comprise hardware components including, but not limited to, one or more processor(s) (CPU), a random-access memory (RAM), a read-only memory (ROM), an internal or external data storage medium (e.g., hard disk drive), or any combination thereof. A computer system may also comprise one or more graphic board(s) for processing and outputting graphical information to display means. The above components may be suitably interconnected via a bus inside the computer. The computer may further comprise suitable interfaces for communicating with general-purpose external components such as, for example, a monitor, a keyboard, a mouse, a network, or the like, or any combination thereof. In various examples, the computer is capable of parallel processing or is part of a network configured for parallel or distributive computing to increase the processing power for the present methods and programs or the like, or any combination thereof. In various examples, a program code read out from a storage medium is written into a memory provided in an expanded board inserted in a computer, or an expanded unit connected to the computer, and a CPU or the like provided in the expanded board or expanded unit can actually perform a part or all of the operations according to the instructions of the program code, so as to accomplish the functions described below. In various examples, a method is performed using a cloud computing system or the like. In various examples, data files and / or the programming are exported to a cloud computer or the like, which runs the program, and returns an output to the user.

[0124] In a further aspect, kits of the present disclosure are described. In various examples, a kit comprises one or more or all of the following: one or more device(s) (e.g., fluidic device(s) or the like), reagent(s) (e.g., capture agent(s), labeled nucleic acid probe(s), or the like, or any combination thereof), buffer(s), or instruction(s) for practicing the disclosed methods. In various examples, a kit comprises one or more device(s), one or more reagent(s), one or more buffer(s), one or more instruction(s), or the like, or any combination thereof.

[0125] A kit may provide devices for practicing the disclosed methods. In various examples, the one or more device(s) are any of the fluidic devices of the present disclosure.

[0126] A kit may provide reagents and / or buffers for practicing the disclosed methods. In various examples, a kit comprises one or more pluralit(ies) of nucleic acid probes as disclosed herein. One or more or each plurality of nucleic acid probe(s) may be labeled with a distinct label, or may not be labeled with any such label. The one or more pluralit(ies) of labeled or unlabeled nucleic acid probes may be in separate vessels or mixed in one or more vessel(s) to any level of (or a desired level) of combination. In various examples, a kit further comprises one or more pluralit(ies) of capture agents that may be linked to oligonucleotides as disclosed herein. In various examples, the oligonucleotides each independently comprise asequence that is complementary to the full-length sequence of one plurality of nucleic acid probe or the like. In various examples, the oligonucleotides are longer than the sequence length of its complimentary nucleic acid probe. The one or more pluralit(ies) of capture agent may be in separate vessels or mixed in one or more vessel(s) to any level of combination. In various examples, a kit further comprises a reagent or reagents and / or a buffer or buffers for conjugation of one or more oligonucleotide(s) of the present disclosure with a capture agent, which may be user-supplied capture agent. In various examples, the reagent(s) and / or buffer(s) of a kits are present in one container or in separate containers, while certain compatible reagents and / or buffers are recombined into a single container, as desired.

[0127] A kit may provide instructions for practicing the disclosed method. In various examples, the one or more instruction(s) comprise instructions for using the device(s), reagent(s), buffer(s), or the like, or any combination thereof of the kit to practice a disclosed method or methods; e.g., instructions for sample analysis. The one or more instruction(s) for practicing the disclosed methods may be recorded on a suitable recording medium or the like. For example, instructions may be printed on a substrate, such as, for example, paper, plastic, or the like, or any combination thereof. As such, the instruction(s) may be present in a kit as a package insert, in the labeling of any container, or any labeling of reagents and / or buffers, or any combination thereof. In various examples, the one or more instruction(s) are present as an electronic storage data file present on a suitable computer readable storage medium; e.g., CD- ROM, diskette, or the like, or the like or any combination thereof. In yet various examples, the one or more instruction(s) are not present in or with a kit, but means are provided for obtaining the one or more instruction(s) from a remote source; e.g., providing a web address or the like, where the instructions can be viewed and / or from which the instructions can be downloaded via the internet.

[0128] In a further aspect, systems of the present disclosure are described. In various examples, a system comprises one or more system(s) that are independently configured for analyzing a sample or samples. In various examples, a systems comprises an optical imager, one or more fluidic device(s), one or more autosampler(s), one or more controller(s), one or more pump(s), one or more droplet generator(s), one or more processor(s), and one or more computer-readable medium(s) independently comprising instructions that, e.g., when executed by one or more computer(s) and / or processor(s), result in the analysis of a sample.

[0129] The system can include various optical imagers. In various examples, an optical imager comprises one or more microscope(s) independently configured to carry out any of the methods and associated optical imaging modalities disclosed herein. In various examples,the microscope is configured to perform bright- field and wide-field epifluorescent microscopy. In various examples, the microscope is configured to perform confocal reflectance scattering and confocal fluorescent microscopy. In various examples, the microscope is configured to perform dark-field and hyperspectral imaging. In various examples, the microscope is configured to perform at least super-resolution microscopy or the like. In various examples, such microscopes would be used to analyze the one or more sample(s) produced by a labeling method or methods disclosed herein. The microscope may further comprise one or more light source(s), one or more camera(s) or detector(s), one or more objective lens(es), one or more condenser lens(es), one or more sample stage(s), one or more excitation, emission, dichroic mirror set(s), or the like, or any combination thereof. The microscope may interface with the one or more interface(s) described herein and / or in various examples, the optical imaging system may be controlled by one or more controller(s) described herein.

[0130] The system can include various fluidic devices and / or autosampler(s). In various examples, a system comprises a device or devices, which is / are independently a fluidic device or device(s) of the present disclosure. In various examples, a system comprises a fluidic device, where each cycle of analysis may involve delivery into and out of the fluidic device of up to three types of solutions: 1) labeled nucleic acid probe mixtures; 2) washing buffer; 3) chemical denaturant solutions, or any combination thereof. In various examples, for ease of use and reproducibility purposes, a system comprises a fluidic device configured for automated liquid handling of one or more or all of the solutions. In various examples, a system further comprises an autosampler programmed in line with a series of pumps or droplet generators controlling the automated liquid handling of a solution or solutions. In various examples, a system further comprises a programmed autosampler configured to deliver EV samples, fixative reagents, blocking buffers, or the like, or any combination thereof, with any degree of combination and / or repetition, to one or more fluidic device(s).

[0131] The system can include various controllers, processors, computer readable medium(s), or the like. The system may include a controller, processor and computer readable medium that are configured or adapted to control or operate one or more system component(s). In various examples, a system comprises a controller that is in communication with one or more component(s) of the system, and any such controller may be configured to control components of the system and / or execute one or more operation(s) or function(s) of the system. In various examples, a system comprises a processor and a computer-readable medium, which may include memory media and / or storage media. Applications and / oroperating systems embodied as computer-readable instructions on computer-readable memory can be executed by the processor to provide some or all of the functionalities described herein. In various examples, a system comprises an entire set of commands for automated completion of one or more analysis cycle(s) or for delivery one or more EV sample(s), fixative reagent(s), blocking buffers, or the like, or any combination thereof. Such commands may be written in any suitable computer programming language.

[0132] A system can include various interfaces. In various examples, a system comprises a user interface, such as, for example, a graphical user interface (GUI) or the like. In various examples, the user interface adapted and / or configured to receive input from a user, and to execute one or more of the method(s) as described herein. In various examples, a system comprises a GUI or the like that is configured to display data, information, or the like, or any combination thereof to a user.

[0133] In a further aspect, methods, kits, and systems described herein find general use in a wide variety of applications for analyzing a wide variety of EV samples. The methods may be useful for generating multiplexed, single EV immunoprofile(s). The methods may have many biomedical applications in a variety of fields of basic biology, phenotypic screening, high throughput screening, drug discovery, and the like. Further, the methods may be useful for a number of clinical applications, such as, for example, diagnostics, prognostics, disease stratification, treatment efficacy monitoring, personalized medicine, clinical trials, companion diagnostics or drug-accompanying tests, and the like, and any combination thereof.

[0134] In various examples, the EV source is a biological sample obtained from a subject or from a patient, from which EVs are purified by an EV purification and then disposed within a device as described herein, forming an analysis sample. Such samples may then be analyzed by a disclosed method or methods to generate one or more single EV, multiplex EV biomarker immunoprofile or a cell-of-origin EV biomarker immunoprofile(s) (e.g., where the capture agents are antibodies or the like). Non-invasively obtained biofluids such as, for example, venous-puncture blood draws, urine samples, or the like may be EV sources that can be analyzed by the present methods as “liquid biopsies” which offer advantages of repeatable sampling for longitudinal (time-course) studies and avoid invasive procedures such as, for example, surgery or deep tissue needle aspirations that are presently used for obtaining biopsy tissue samples. In various examples of the present disclosure, a biological sample does not comprise whole cells, tissue samples, nor tissue sections obtained from a biologic entity, nor is any analysis sample comprising disposed whole cells or tissue sections analyzed by any method of the present disclosure.

[0135] Cell-type-specific EVs may be identified by single EV, cell-of-origin EV biomarker immunoprofiles using the disclosed methods. In various examples, one or more panel(s) comprising one or more pluralit(ies) of capture agents (e.g., antibodies or the like) includes one or more capture agent(s) (e.g., antibodies or the like) that are indicative of one or more cell-of-origin EV biomarker(s) or the like. A panel or panels may provide immunoprofiles that are useful for identifying EVs derived from specific cell types within particular contexts. A non-limiting example of such a context is a disease stratification and / or treatment efficacy monitoring, where such immunoprofiling could ask to what extent epithelial cell-derived EVs, identified by expression of the EpCAM CofO EV biomarker, also express metabolic, angiogenic, wild-type or mutant cell cycle regulatory, and immune checkpoint biomarkers, from a carcinoma patient’s ’blood, where the patient is undergoing immunotherapy. As another non-limiting example, multiple, independent cell-of-origin EV biomarker immunoprofiles are generated for broader phenotypic screening; e.g., where each fluidic channel of a fluidic device or devices disclosed herein is independently dedicated to identifying EVs derived from a distinct cell type (e.g., epithelial, stromal, and multiple immune cell types and the like) and by asking to what extent one or more or each cell-type- specific EV population expresses cancer and immune checkpoint markers, activation markers, exhaustion markers, or the like, or any combination thereof, respectively.

[0136] Cell-of-origin (CofO) EV biomarkers for identifying EVs from cells of particular tissue types of interest are well-known and / or can be discovered and established. For example, EVs originating from cells of particular tissues of interest would be useful. Such tissues include, as non-limiting examples, tumor / cancer (sarcoma, carcinoma, melanoma, adenoma, etc.), healthy (e.g., non- neoplastic), skin, soft tissue, bone, breast, colon, liver, kidney, adrenal, gastrointestinal, pancreatic, gall bladder, salivary gland, cervical, ovary, uterus, testis, prostate, lung, thymus, thyroid, parathyroid, pituitary, glandular, ductal, brain, spinal cord, ocular, nerve, cardiac muscle, smooth muscle, and skeletal muscle, and the like, and any combination thereof. Particular CofO EV biomarkers may include, as non-limiting examples: CD3 for T cell-derived EVs, CD4 for helper T cell-derived EVs, CD8 for cytotoxic T cell-derived EVs; CD11c for dendritic cell-derived EVs; CD 14 for monocyte- derived EVs; CD19 and / or CD20 for B cell-derived EVs; CD31 and / or CD54 for endothelialcell derived EVs; CD56 for natural killer cell-derived EVs; CD68 for macrophage-derived EVs; CD71 for reticulocyte-derived EVs; HLA-DR (MHC II) for antigen-presenting cell- derived EVs; EpCAM for epithelial cell-derived EVs; GP100 for melanocyte-derived EVs; pan-collagen for fibroblast-derived EVs; podoplanin for podocyte-derived EVs; L1CAM forneuron-derived EVs; ALDH1L1 for astrocyte-derived EVs; GFAP for glial cell-derived EVs; and the like. EV biomarkers may serve both as CofO EV biomarkers and as other EV biomarkers, depending on the context and / or the image analysis procedure.

[0137] Two or more cell-of-origin (CofO) EV biomarkers may be combined for identifying particular cell type-derived EVs from particular tissues of interest. In various examples, at least about 2 (e.g., at least about 3, at least about 4, at least about 5 or more) CofO EV biomarkers are used to identify EVs deriving from certain cells of certain tissues. For instance, CD3 and CD8 may be used to identify EVs originating from T cells of the cytotoxic T cell linage, or CD3 and CD4 could be used to identify EVs originating from T cells of the helper T cell linage. In other examples, one or more CofO biomarker(s) for lung tissue are used in combination with one or more epithelial CofO biomarker(s) to identify EVs from lung epithelial cells; one or more CofO biomarker(s) for prostate tissue are used in combination with one or more epithelial CofO biomarker(s) to identify EVs from prostate epithelial cells; one or more CofO biomarker(s) for hepatic or pancreatic tissues or the like are be used in combination with one or more ductal CofO biomarker(s) to identify EVs from hepatic ductal or pancreatic ductal cells or the like, respectively; or one or more CofO biomarker(s) for brain tissue are used in combination with one or more endothelial CofO biomarker(s) to identify EVs from brain endothelial vasculature cells; or the like; or any combination thereof. Of course, other combinations of CofO tissue and cell type biomarkers are possible.

[0138] Capture agents may bind to CofO EV biomarkers and to other EV biomarkers (e.g., other than CofO EV biomarkers) and may include EV biomarkers that are indicative of a disease or of a pathophysiological condition or the like or any combination thereof. In various examples, such EV biomarkers are indicative of, for example: infections (e.g., by viruses, bacteria, fungi, or protozoa, or the like or any combination thereof); cancers; cardiovascular conditions; ischemia-reperfusion injuries; neurological conditions; renal conditions; auto-immune conditions; metabolic conditions; endocrinological conditions; urological conditions; reproductive conditions; musculoskeletal conditions; immunological conditions; hepatic conditions; gastroenterological conditions; pancreatic conditions; hematological conditions;; or the like; or any combination thereof.

[0139] As a non-limiting example where the disease may be a cancer, the EV biomarkers may include, for example, carcinoembryonic antigen (for identification of adenocarcinomas), cytokeratins (for identification of carcinomas but may also be expressed in some sarcomas), CD 15 and CD30 (for Hodgkin's disease), alpha fetoprotein (for yolk sac tumors andhepatocellular carcinoma), CD117 (for gastrointestinal stromal tumors), CD10 (for renal cell carcinoma and acute lymphoblastic leukemia), prostate specific antigen (for prostate cancer), estrogens and progesterone receptors (for breast tumor identification), CD20 (for identification of B-cell lymphomas), CD3 (for identification of T-cell lymphomas), mutant p53 (for identification of various tumors), and mutant KRAS (for identifying various carcinomas), and the like, and any combination thereof. Of course, other cancer EV biomarkers may be known, discovered, and established, and thus used in the methods disclosed herein.

[0140] The methods can be used to analyze EVs from a subject to determine, for example, whether the originating cells are normal or diseased or the like, or if such originating cells are responding to a treatment, or the like, or any combination thereof. In various examples, a method is employed for single EV analysis in order to determine the degree of disease or pathophysiological condition affecting the cell type of specific interest or the like. In various examples, a method is employed for generating multiple single EV analyses for EVs originating from multiple cell types in order to differentiate phenotypes of such multiple cells in the context of a disease or a condition for diagnosis, prognosis, disease stratification, treatment efficacy monitoring, or the like, or any combination thereof. The term “originating cell” refers to the cell or cells from which the EVs are derived, purified, and analyzed by the disclosed methods.

[0141] The disclosed methods find particular utility in examining EV samples using a plurality of antibodies as the one or more pluralit(ies) of capture agents. In various examples, each plurality of antibody labels and binds a particular EV biomarker (e.g., a protein or the like within, on, or associated with an EV or the like). One or more series of different antibodies (e.g., collectively a panel of antibodies) targeting such EV biomarkers may be used together in the disclosed methods (e.g., for disease diagnosis or prognosis). Some or all of the antibody labeling data generated from any such panel may be used for the purposes disclosed herein. Such panels may be referred to as multiplexed panels as understood within the context of the present disclosure. The one or more panel(s) may comprise any desired level of combination and thus have any desired level of multiplexing. In various examples, the multiplexing comprises about a 3-plex panel to about a 100-plex or more panel, including all integer values and ranges therebetween, depending on the number of different antibodies used). In various examples, the panel comprises a panel of at least about 3-plex, at least about 10-plex, at least about 20-plex, at least about 30-plex, at least about 40-plex, at least about50-plex, at least about 60-plex, at least about 70-plex, at least about 80-plex, at least about 90-plex, at least about 100-plex, or greater than about 100-plex.

[0142] In various examples, the disease of interest is cancer or the like and the panel comprising one or more pluralit(ies) of antibodies target multiple EV biomarkers which may include, in non- limiting examples:Acute leukemia panel: CD3, CD7, CD20, CD34, CD45, CD56, CD117, MPO, PAX-5, and TdT.Adenocarcinoma vs. mesothelioma panel: Pan-cytokeratin (Pan-CK), CEA, MOC-31 , BerEP4, TTF1, calretinin, and WT-1.Bladder vs. prostate carcinoma panel: CK7, CK20, PSA, CK 903, p63, EpCAM, EGFR, and MUC2.Breast cancer panel: ER, PR, Ki-67, and HER2.Burkitt vs. DLBC lymphoma panel: BCL-2, c-MYC, Ki-67.Carcinoma unknown primary site (female) panel: EpCAM, EGFR, MUC2, CK7, CK20, mammaglobin, ER, TTF1, CEA, CA19-9, S100, synaptophysin, and WT-1.Carcinoma unknown primary site (male) panel: EpCAM, EGFR, MLJC1, CK7, CK20, TTF1, PSA, CEA, CA19-9, S100, and synaptophysin.Gastrointestinal stromal tumor panel: APC, BRAF, HRAS, KIT, KRAS, NF1, NRAS, PDGFRA, PIK3CA, SETD2, and TP53 where each is either mutant or wild-type). Hepatoma I cholangio vs. metastatic hepatoma: HSA, HepPar 1, CDX2, CK7, CK20, CAM 5.2, TTF-1, and CEA.Hodgkin vs. non-Hodgkin lymphoma panel: BOB-1, BCL-6, CD3, CD10, CD15, CD20, CD30, CD45 LCA, CD79a, MUM1, OCT-2, PAX-5, and EBER ISH.Lung cancer panel: chromogranin A, synaptophysin, CK7, p63, TTF-1, EpCAM, EGFR, and MUC2.Lung vs. metastatic breast carcinoma panel: TTF1, mammaglobin, GCDFP-15 (BRST-2), ER, EGFR, MUC2, and EpCAM.Lymphoma phenotype panel: BCL-2, BCL-6, CD3, CD4, CD5, CD7, CD8, CD10, CD15, CD20, CD30, CD79a, CD138, cyclin DI, Ki67, MUM1, PAX5, TdT, and EBER ISH.Lymphoma vs. carcinoma panel: CD30, CD45, CD68, CD117, pan-CK, MPO, S100, synaptophysin, EGFR, MUC2, and EpCAM.Lymphoma vs. reactive hyperplasia panel: BCL-2, BCL-6, CD3, CD5, CD10, CD20, CD23, CD43, cyclin, Dl„ and Ki-67.• Melanoma vs. squamous cell carcinoma panel: GP100, EpCAM, EGFR, MUC2, CD68, Factor Xllla, CEA, S-100,HMB-45, MART-l / Melan-A, tyrosinase, and pan-CK.• Mismatch repair proteins panel: MLH1, MSH2, MSH6, and PMS2 (may be combined with any other cancer EV biomarker panel).• Neuroendocrine neoplasm panel: CD56, synaptophysin, chromogranin A, TTF-1, pan- CK, and CEA.• Plasma cell neoplasm panel: CD19, CD20, CD38, CD43, CD56, CD79a, CD138, cyclin DI , EMA, kappa, lambda, KI-67, and MUM1 .• Prostate vs. colon Carcinoma panel: EpCAM, EGFR, MUC2, CDX2, CK 20, CEA, CA19-9, PLAP keratin-7, and PSA.• Soft tissue tumor panel: pan-CK, SMA, desmin, S100, CD34, vimentin, and CD68.• T cell lymphoma panel: ALK1, CD2, CD3, CD4, CD5, CD7, CD8, CD10, CD20, CD21, CD30, CD56, TdT, and EBER ISH.• Pancreatic ductal adenocarcinoma panel: MUC1, MUC2, MUC4, MUC5AC, MUC6, DAS-1, PSCA, TSP-1, TSP-2, PLEC1, S100, STMN1, ZEB1, HOOK1, PTPN6, FBN1, CD73, CLDN18, TIMP1, EphA2, CYFRA21-1, LRG1, MSLN, HER2, GRP94, EpCAM, EGFR, p53mut, and KRASmut.• Ovarian cancer panel: EpCAM, CD24, VCAN, HE4, CA125, and TNC; or the like or any combination thereof.

[0143] In various examples, a condition of interest is myocardial infarction or the like and the panel(s) independently comprising one or more pluralit(ies) of antibodies or the like target multiple EV biomarkers or the like, which may include, in non-limiting examples,: Cardiac troponin, B-type natriuretic peptide, interleukin-6, agouti-related peptide, chemokine-motif ligand 1 , NF-kappa-B essential modulator, placental growth factor, platelet- derived growth factor, angiopoietin 1 , tissue transglutaminase, glyoxalase I, chemokine-motif ligand 17, chymotrypsin C, and proto-oncogene tyrosine-protein kinase SRC and the like any combination thereof.

[0144] In various examples, a condition of interest is ischemia-reperfusion injury or the like and the panel(s) independently comprising one or more pluralit(ies) of antibodies or the like target multiple EV biomarkers or the like, which may include, in non-limiting examples: CD8, CD14, CD2, CD326, CD142, CD31, CD62P, CD42a, CD44, and MCSP, and the like, and any combination thereof.

[0145] In various examples, a condition of interest is diabetic kidney disease or the like and the panel(s) independently comprise one or more pluralit(ies) of antibodies or the like configured to target one or more EV biomarker(s).

[0146] In various examples, the condition of interest is immune exhaustion or the like and the panel(s) independently comprising one or more pluralit(ies) of antibodies or the like target multiple EV biomarkers, which may be indicative of immunological inhibition, activation, or the like, or any combination thereof that are measured within particular patients. Such patients may include, as non-limiting examples, patients with cancer, autoimmune diseases, chronic infection, or immunotherapy (e.g., anti-immune checkpoint antibody therapy), or the like, or any combination thereof. In various examples, EV biomarkers indicative of immunological immunological inhibition , activation, or the like, or any combination thereof may be used to analyze an EV sample with or without any combined CofO EV biomarkers or the like. In various examples, one or more antibod(ies) or the like independently targeting one or more EV biomarker(s) for immunological activators, immunological inhibitors, or the like, or any combination thereof and one or more CofO EV biomarker(s) are used to analyze an EV sample or samples. In various examples, the presence and / or abundance of immunological inhibitors and activators on stromal, endothelial, epithelial, immunological cells, or the like, or any combination thereof are be analyzed. Immunological activation EV biomarkers may include, as non-limiting examples, CD127, CD25, CD27, CD40, CD44, CD80, GZMB, HLA-DR, ICOS, KI-67, and PD-12, and the like, and any combination thereof. Immunological inhibition EV biomarkers may include, as nonlimiting examples, 4-1BB, ARG1, B7-H3, CTLA4, GITR, IDO1, LAG3, OX40L, PD-1, PD- Ll, STING, TIM-3, VISTA, and the like, and any combination thereof.

[0147] In various examples, a method comprises obtaining the one or more pluralit(ies) of images as described above (e.g., an electronic form of which may have been retrieved and / or forwarded from a remote location or the like), and one or more user(s) may remotely analyze the one or more image(s) by one or more image analysis and / or statistical process(es) described herein. The conclusions or results of such analyses may be summarized in a report or electronic medical record or the like. Such reports, records, or the like may be retrieved or forwarded to a doctor, other medical professional, patient (from whom the EV sample was obtained), or the like, or any combination thereof to determine whether a patient has a disease or a condition or the like or any combination thereof or to report to a patient that they may have a disease or a condition or the like or any combination thereof. In various examples, one or more image(s), analys(es), report(s), record(s) or the like thereof are used as a diagnosticor the like to determine whether a patient or a subject has a disease or a condition or the like or any combination thereof. In various examples, one or more image(s), analys(es), report(s), record(s) or the like thereof are used as a diagnostic or the like are used to stage a disease, to stratify a disease, to differentiate a disease, or the like or any combination thereof. In various examples, the one or more image(s), analys(es), report(s), record(s), or the like thereof are used to monitor a patient’s response to a therapy or other intervention.

[0148] In various examples, data is forwarded to a “remote location”, where “remote location,” refers to a location other than the location at which the image is examined for example, a remote location could be another location (e.g., office, lab, etc.) in the same city, another location in a different city, another location in a different state, another location in a different country, etc. As such, when one item is indicated as being “remote” from another, the two items can be in the same room but separated, or at least in different rooms or different buildings, and can be at least one mile, ten miles, or at least one hundred miles apart. “Communicating” information refers to transmitting the data representing that information as electrical signals over a suitable communication channel (e.g., a private or public network). “Forwarding” an item refers to any means of getting that item from one location to the next, whether by physically transporting that item or otherwise (where that is possible) and nonlimiting examples include, at least in the case of data, physically transporting a medium carrying the data, communicating the data, or the like. Non-limiting examples of communicating media include radio or infra-red transmission channels as well as a network connection to another computer or networked device, and the internet or including email transmissions and information recorded on websites and the like.

[0149] The disclosed methods can be used to diagnose a patient with a disease or condition or the like. In various examples, the presence or absence of one or more EV biomarker(s) (e.g., indicated by analyzing the EV sample derived from the patient or the like) indicates that the patient has or does not have a particular disease or condition or the like. In various examples, a patient is diagnosed with a disease or condition or the like by comparing a sample from the patient with a sample from a healthy control. In various examples, a level of one or more EV biomarker(s) or the like, relative to the control, is measured. In various examples, a difference in the levels of the one or more EV biomarker(s) in a patient's sample relative to the control indicates that the patient has or does not have a disease or condition.

[0150] The disclosed methods can be used to determine a treatment plan for a patient. In various examples, the measured presence or absence of one or more EV biomarker(s) or the like indicates a patient is responsive or refractory or the like to a particular therapy. In variousexamples, a presence or absence of one or more EV biomarker(s) or the like indicates a disease is refractory or the like to a specific therapy, and an alternative therapy may be administered. In various examples, a patient is currently receiving the therapy and the presence or absence of one or more EV biomarker(s) or the like indicates that the therapy is no longer effective.

[0151] The disclosed methods can be used in a variety of fields of basic biology, phenotypic screening, high throughput screening, drug discovery, and the like. In various examples, a method comprises screening, discovery, or research applications or the like, or any combination thereof, which may include, as non-limiting examples, discovery of a drug target or targets (e.g., where the one or more EV biomarker(s) may be subsequently identified and then targeted for drug therapy); drug screening (e.g., where the one or more analyzed EV biomarker(s) is / are used to measure the effects of a drug or the like); determining drug susceptibility (e.g., where drug susceptibility is associated with the presence or absence of one or more EV biomarker(s) or the like); phenotypic screening (e.g., where it is desirable to measure the one or more EV subpopulation(s) deriving from one or more cell type(s)). In various examples, two or more different EV samples are compared using one or more of the above methods.

[0152] The methods can be used to compare one or more experimental sample(s) and one or more control sample(s); e.g., where the experimental samples of interest are compared to the control samples. In various examples, different EV samples are derived from pairs of different cells or the like, where experimental samples are derived from one or more experimental cell-type(s) of interest (e.g., abnormal cells or the like), while control samples may be derived from one or more normal or wild-type cell(s) or the like. Such pairs of different cells from which experimental and control EV samples may be derived include, in non-limiting examples, EVs isolated from: environmental samples; cells of a tissue biopsy or from a biofluid (e.g., from a patient with a disease or a condition); normal cells from the same tissue or biofluid (usually from the same patient); cells grown in tissue culture that are either primary cells or immortalized cells that may be infected with a pathogen, or treated with an exogenous stimuli (e.g., with environmental or chemical agents such as, for example, peptides, hormones, altered temperature, growth condition, physical stress, cellular transformation, etc.); normal cells (e.g., a cell that is otherwise identical to the experimental cell except that it is not immortal, infected, or treated, etc.); cells from a mammal with a disease or condition, a geriatric mammal, or a mammal exposed to a condition, and a cell from a mammal of the same species (e.g., of the same family or the like), that is healthy oryoung, including differentiated cells and non-differentiated cells from the same mammal (e.g., one cell being the progenitor of the other cell in a mammal); or the like, or any combination thereof. In various examples, cells from which experimental and control EV samples are derived further include EVs isolated from cells of different tissues (e.g., brain vs gastrointestinal) or from cells of different status (e.g., before and after a stimulus is applied on the same cell-type) or the like, or any combination thereof. In various examples, cells from which experimental and control EV samples are derived further include EVs isolated from cells that are susceptible to infection by a pathogen vs. cells that are resistant to infection by the pathogen. In various examples, cells from which experimental and control EV samples are derived further include EVs isolated from undifferentiated cells vs. differentiated cells.

[0153] The biologic entity from which EVs are sourced can vary. In the various examples, EV samples are derived from cells of any organism; e.g., from bacteria, yeast, plants, animals (such as, for example, fish, birds, reptiles, amphibians, and mammals and the like), and the like, and any combination thereof. In various examples, mammalian cells (e.g., cells from mice, rabbits, primates, or humans, or cultured derivatives thereof or the like or any combination thereof) are used.

[0154] In the following Statements, various examples of the methods and structures of the present disclosure described:Statement 1. A method for performing a multiplexed single EV analysis, the method comprising the steps / processes of: a) disposing at least one EV sample (e.g., one or more EV samples comprising one or more pluralit(ies) of EVs) onto a first layer of at least one fluidic device, thereby forming at least one analysis sample within at least one fluidic device; b) contacting the at least one EV sample with one or more pluralit(ies) of capture agents; c) cross-linking the EVs (or at least a portion, substantially all, or all of the pluralit(ies) of EVs to the first layer and the at least a portion, substantially all, or all of the one or more pluralit(ies) of EVs) to the one or more pluralit(ies) of capture agent(s)to the first layer and the EVs to the one or more capture agent(s); d) hybridizing a first sub-set of labeled nucleic acid probes with the at least one EV sample, thereby forming a first sub-set of labeled probe / oligonucleotide duplexes (e.g., wherein the first sub-set of labeled probe / oligonucleotide duplexes form a binding pattern); e) reading the at least one EV sample to obtain an image showing the binding pattern for each of the probes hybridized in the prior step (or the binding pattern for each of thehybridized nucleic acid probes of the first sub-set of the labeled nucleic acid probes from d); f) removing the first sub-set of probes from the at least one EV sample, by denaturation or another method; g) hybridizing a second sub-set of labeled nucleic acid probes with the at least one EV sample, thereby forming a second sub-set of labeled probe / oligonucleotide duplexes; h) reading the at least one EV sample to obtain an image showing the binding pattern for each of the probes in the second sub-set of probes; i) removing the second sub-set of probes from the at least one EV sample (such as, for example, by denaturation or another method or the like); j) optionally, imaging the location of the EVs (or imaging a location of at least a portion, substantially all or all of the pluralit(ies) of EVs crosslinked to one or more pluralities of capture agent(s) and hybridized to a nucleic acid probe or probes) at each reading step by a universal EV stain or by light-scatter imaging; and k) optionally, repeating steps / processes g) through j) over multiple cycles for a third sub-set and successive sub-sets of labeled nucleic acid probes,(e.g., where the reading(s) (e.g., reading step(s) / process(es)) produce(s) images of the binding patterns of the sub-set(s) of capture agents and their corresponding labeled nucleic acid probes and the location of EVs (or at least a portion, substantially all or all of the pluralit(ies) of EVs crosslinked to one or more pluralities of capture agent(s) and hybridized to a nucleic acid probe or probes)).Statement 2. A method according to Statement 1, where the EV sample comprises at least one plurality of EVs derived by one or more EV purification process(es) from a biological sample or an environmental sample.Statement 3. A method according to Statements 1 or 2, where the analysis sample does not comprise any cells nor any tissue sections (a cell or cells or a tissue section or tissue sections).Statement 4. A method according to Statements 1-3, where the disposition of the EV sample comprises discretely isolated, individual EVs onto a first layer of a fluidic device, thereby forming the at least one analysis sample within at least one fluidic device (the disposing results in a disposition of the EV sample comprising discretely isolated, individual EVs on the first layer of the fluidic device and / or at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more of the EVs are discretely isolated and individual EVs).Statement 4A. A method according to Statements 1-4, where the EVs of the at least one analysis sample are substantially disposed as at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more discretely isolated and individual EVs.Statement 4B. A method according to Statements 1-4A, where the discretely isolated, individual EVs comprise the disposition of EVs onto a first layer wherein the EVs are substantially separated (or separated) at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs.Statement 4C. A method according to Statements 1-4B, where the discretely isolated, individual EVs are configured to be imaged at about less than one-time, at about at least onetime, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs.Statement 4D. A method according to Statements 1-4C, where the discretely isolated, individual EVs are imaged at about less than one-time, at about at least one-time, at least about two-times, or at least about three-times the lateral optical resolution of the imaging system being used to image the EVs.Statement 4E. A method according to Statements 1-4D, where two or more analysis samples are formed within one fluidic device or across multiple fluidic devices.Statement 5. A method according to Statements 1-4E, where the fluidic device comprises a first layer, a second layer, and a third layer, with one or more independent channel(s) and any combination of first layer vias and / or third layer vias.Statement 5 A. A method according to Statements 1-5, where the fluidic device further comprises a first layer, a thin second layer, and a third layer into which the one or more channel(s) of the fluidic device have been formed, with one or more independent channel(s) and any combination of first layer vias and / or third layer vias.Statement 6. A method according to Statements 1-5 A, where the fluidic device further comprises a functionalized first layer, a second layer, a third layer, and a fourth layer, with one or more independent channel(s) and any combination of first layer vias, third layer vias, fourth layer vias, or the like.Statement 7. A method according to Statements 1-6, where the fluidic device further comprises a first layer and a third layer where each layer independently comprises a glass, a second layer comprising a metal, a non-metal, or a ceramic, and a fourth layer comprising a glass, a metal, a non-metal, a polymer, a ceramic, or a chemical functionalization, and wherethe first layer is functionalized on at least one of its surfaces(e.g., the surface of the first layer facing the inner channel).Statement 7A. A method according to Statements 1-7, where the fluidic device further comprises a differentially functionalized first layer.Statement 7B. A method according to Statements 1-7A, where the functionalized first layer is APTES -functionalized on at least one of its surfaces (e.g., the surface of the first layer facing the inner channel).Statement 8. A method according to Statements 1 -7B, where the fourth layer comprises (or is) a chemical functionalization.Statement 8A. A method according to Statements 1-8, where the first layer is not hydrophobic on both of its surfaces (e.g., both a top surface and a bottom surface).Statement 9. A method according to Statements 1-8A, where the contact with the one or more pluralit(ies) of capture agents comprises sequential or en masse contact, and where the contact further comprises labeling two or more analysis samples within at least one fluidic device with any number of plurality of capture agents such that the two or more samples are labeled with any degree of similarity, difference, iteration, repetition, or the like, or any combination thereof as desired.Statement 10. A method according to Statements 1-9, where the contact with the one or more pluralit(ies) of capture agents further comprises labeling two or more analysis samples within at least one fluidic device with at least one common capture agent (e.g., a cell-of-origin EV biomarker capture agent as described herein).Statement 11. A method according to Statements 1-10, where the one or more pluralit(ies) of capture agents further comprises at least about 3, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more than about 100 capture agents.Statement 12. A method according to Statements 1-11, where each plurality of capture agent further comprises a plurality of one antibody that is linked to its own distinguishing and different oligonucleotide.Statement 13. A method according to Statements 1-12, where the oligonucleotides linked to capture agents have a sequence length of at least about 5, at least about 10, at least about 20, at least about 30, at least about 45, or more than about 45 nucleotides.Statement 14. A method according to Statements 1-13, where the EVs are cross-linked to the functionalized first layer and where the one or more pluralit(ies) of capture agents are crosslinked to the EVs by a fixative reagent.Statement 15. A method according to Statements 1-14, where the hybridizing of labeled nucleic acid probes comprises contacting the at least one analysis sample after cross-linking with one or more pluralit(ies) of labeled nucleic acid probes (or the hybridizing of one or more or all of the sub-set(s) of labeled nucleic acid probes comprises contacting at the least one analysis sample with one or more pluralit(ies) of labeled nucleic acid probes).Statement 16. A method according to Statements 1-15, where the hybridizing further comprises selectively hybridizing one plurality of labeled nucleic acid probe to one distinct plurality of oligonucleotide each linked to one plurality of capture agent (or one or more pluralit(ies) of labeled nucleic acid probes, wherein each of the pluralit(ies) of labeled nucleic acid probes is hybridized to a distinct plurality of oligonucleotides, wherein each oligonucleotide of the distinct plurality of oligonucleotides is linked to a capture agent). Statement 17. A method according to Statements 1-16, where the number of labeled nucleic acid probes hybridized to the at least one analysis sample corresponds to the number of capture agents of the at least one analysis sample (a number of labeled nucleic acid probes hybridized in the one or more or all sub-set(s) to the at least one analysis sample corresponds to a number of capture agents labeled in an individual sub-set).Statement 18. A method according to Statements 1-17, where the hybridizing further comprises contacting the at least one analysis sample with a sub-set comprising at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5 pluralities of labeled nucleic acid probes, thereby forming at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5 sub-sets of labeled probe / oligonucleotide duplexes.Statement 19. A method according to Statements 1-18, where the hybridizing is (e.g., of one or more sub-set(s) is / are) from about 1 minute to about 5 minutes, including all integer second values and ranges therebetween.Statement 20. A method according to Statements 1-19, where the labeled nucleic acid probe / oligonucleotide duplexes have a Tm from about 15 °C to about 75 °C, including all 0.1 °C values and ranges therebetween.Statement 21. A method according to Statements 1-20, where the labeled nucleic acid probes have a sequence length of at least about 5, at least about 10, at least about 20, at least about 30, at least about 45, or more than about 45 nucleotides.Statement 22. A method according to Statements 1-21, where the labeled nucleic acid probes are distinctly labeled with one or more fluorophore(s).Statement 23. A method according to Statements 1-22, where removing the probes comprises use of a chemical denaturant and where the removing is not by displacing the labeled nucleic acid probes using displacement oligonucleotides.Statement 24. A method according to Statements 1-23, where the chemical denaturant is either ultrapure water, about 1 mM to about 10 mM EDTA, including all 0.1 mM values and ranges therebetween, about 1% to about 10% v / v concentration formamide, including all 0.1% values and ranges therebetween, or about 1% to about 10% v / v concentration DMSO, including all 0. 1 % values and ranges therebetween (e.g., where the removing is an incubation for a period of about 1 minute to about 5 minutes, including all integer second values and ranges therebetween).Statement 25. A method according to Statements 1-24, where the sequences of the oligonucleotides to which the capture agents are linked are longer than sequences of the labeled nucleic acid probes, but otherwise identical to one other except for a sub-sequence that is complementary to a single labeled nucleic acid probe (or a sequence of an oligonucleotide to which a capture agent is linked is longer than a sequence of a labeled nucleic acid probe, but the sequence of the oligonucleotide to which the capture agent is linked is identical to the sequence of the labeled nucleic acid probe except for a sub-sequence of the oligonucleotide to which the capture agent is linked that is complementary to a single labeled nucleic acid probe).Statement 26. A method according to Statements 1-25, where the hybridizing, reading, and removing steps / processes (e.g., steps / processes g) through))) are repeated over at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles, at least about 11 cycles, at least about 12 cycles, at least about 13 cycles, at least about 14 cycles, at least about 15 cycles, at least about 16 cycles, at least about 17 cycles, at least about 18 cycles, at least about 19 cycles, at least about 20 cycles, or more than about 20 cycles.Statement 27. A method according to Statements 1-26, where the removing step is optionally omitted for a or the last cycle.Statement 28. A method according to Statements 1-27, where the method further comprises analyzing at least two of the images.Statement 29. A method according to Statements 1-28, where the analyzing further comprises counting the binding patterns of capture agents on single EVs and / or counting a or thenumber of single EVs by the universal stain EV location images or the light-scatter EV location images.Statement 30. A method according to Statements 1-29, where the analyzing of images comprises comparing or overlaying at least two of the images.Statement 31. A method according to Statements 1-30, where the analyzing of images further comprises comparing or overlaying all of the images to produce an image showing the pattern of binding of all of the capture agents and the locations of EVs.Statement 32. A method according to Statements 1-31 , where the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more other capture agent(s).Statement 33. A method according to Statements 1-32, where the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more cell-of-origin capture agent(s).Statement 34. A method according to Statements 1-33, where the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the one or more image(s) showing the location of EVs imaged by the universal stain or by light-scatter.Statement35. A method according to Statements 1-34, where the method further comprises performing an analysis using a statistical model on any combination of single EV binding pattern counts and / or on any single EV location counts.Statement 36. A method according to Statements 1-35, where reading of the binding patterns of capture agents and labeled nucleic acid probes, universal EV stains, or the like, or any combination thereof, are done by fluorescence microscopy, super-resolution microscopy, confocal microscopy, hyperspectral imaging, or any combination thereof.Statement 37. A method according to Statements 1-36, where the reading of the location of EVs is done by imaging any universal EV stain or by light-scatter microscopy.Statement 38. A method according to Statements 1-37, where the one or more pluralit(ies) of capture agents are aptamers.Statement 39. A method according to Statements 1-38, where the removing steps / processes result in the plurality of capture agents and their linked oligonucleotides still being bound to the at least one analysis sample (or one or more or all of the removing(s) results / result in atleast a portion, substantially all, or all of the crosslinked pluralit(ies) of EVs, the crosslinked pluralit(ies) of capture agents, and linked oligonucleotides of the capture agents remaining bound to the at least one analysis sample).Statement 40. A method according to Statements 1-39, where the removing steps / processes are alternatively done by chemical inactivation or by cleaving one or more bond(s).Statement 41. A method according to Statements 1-40, where the cleaving is done enzymatically, chemically, or via exposure to light, or the like.Statement 42. A kit comprising one or more of any fluidic device(s) of the present disclosure (or a kit comprising: one or more fluidic device(s); one or more pluralit(ies) of labeled nucleic acid probes; one or more pluralit(ies) of capture agents; one or more reagent(s) and / or one or more buffer(s); and one or more instruction set(s).Statement 43. A kit according to Statement 42, the kit further comprising one or more pluralit(ies) of nucleic acid probes as disclosed herein, where each plurality of nucleic acid probe is labeled with a distinct label (e.g., a fluorophore).Statement 44. A kit according to Statements 42 or 43, the kit further comprising one or more pluralit(ies) of nucleic acid probes as disclosed herein, where each plurality of nucleic acid probe is not labeled.Statement 45. A kit according to Statements 42-44, where the one or more pluralit(ies) of labeled or unlabeled nucleic acid probes are in separate vessels or mixed in one or more vessel(s) to any level of combination.Statement 46. A kit according to Statements 42-45, the kit further comprising one or more pluralit(ies) of capture agents that may be linked to oligonucleotides as disclosed herein, where the oligonucleotides each comprise a sequence that is complementary to the sequence of one plurality of nucleic acid probes of the kit, or further comprising one or more pluralit(ies) of capture agents linked to oligonucleotides, wherein the oligonucleotides each comprise a sequence that is complementary to a sequence of a plurality of nucleic acid probes of the kit.Statement 47. A kit according to Statements 42-46, where the oligonucleotides comprise a sequence longer than a or the nucleic acid probes of the kit or where the oligonucleotides comprise a sequence length shorter than the nucleic acid probes of the kit, or any combination thereof.Statement 48. A kit according to Statements 42-47, where the one or more pluralit(ies) of capture agents is / are in separate vessels or mixed in one or more vessel(s) (e.g., to any level of combination).Statement 49. A kit according to Statements 42-48, the kit further comprising one or more oligonucleotide(s), one or more reagent(s), one or more buffer(s), or any combination thereof (e.g., for a user to conjugate one or more oligonucleotide(s) of the present disclosure with a user-supplied capture agent).Statement 50. A kit according to Statements 42-49, where the nucleic acid probes comprise a cleavable linkage that is not a phosphodiester bond.Statement 51. A kit according to Statements 42-50, the kit further comprising one or more reagent(s) and / or one or more buffer(s) for carrying out a method of the present disclosure (such as for example, a method of any one of Statements 1-41).Statement 52. A kit according to Statements 42-51, where the one or more reagent(s) and / or the one or more buffer(s) are in one container or in separate containers.Statement 53. A kit according to Statements 42-52, the kit further comprising one or more instruction(s) for practicing a method or methods of the present disclosure (such as, for example, one or more methods of any one claims 1-41) and / or for using any fluidic devices, reagents, buffers, or the like, or any combination thereof of the present disclosure (or the one or more instruction set(s) specify a use of one or more or all of the fluidic device(s), one or more or all of the one or more pluralit(ies) of labeled nucleic acid probes, one or more one or more of the one or more pluralit(ies) of capture agents, one or more or all of the one or more buffer(s), or any combination thereof of the kit).Statement 54. A system configured to carry out a method for performing a multiplexed single EV analysis of the present disclosure (such as, for example, a method of any one of Statements 1-41) (e.g., a method for performing a multiplexed single EV analysis the analysis comprising the steps / processes of or a system configured to carry out one or more or all of the following: a) disposing at least one EV sample onto a first layer of at least one fluidic device, thereby forming at least one analysis sample within at least one fluidic device; b) contacting the at least one analysis sample with one or more pluralit(ies) of capture agents; c) cross-linking the EVs to the first layer and the EVs to the one or more capture agent(s); d) hybridizing a first sub-set of labeled nucleic acid probes with the at least one analysis sample, thereby forming a first sub-set of labeled probe / oligonucleotide duplexes; e) reading the at least one analysis sample to obtain an image showing the binding pattern for each of the probes hybridized in the prior step;f) removing the first sub-set of probes from the at least one analysis sample, by denaturation or another method; g) hybridizing a second sub-set of labeled nucleic acid probes with the at least one analysis sample, thereby forming a second sub-set of labeled probe / oligonucleotide duplexes; h) reading the at least one analysis sample to obtain an image showing the binding pattern for each of the probes in the second sub-set of probes; i) removing the second sub-set of probes from the at least one analysis sample, by denaturation or another method; j) optionally, imaging the location of the EVs at each reading step by a universal EV stain or by light- scatter imaging; and k) optionally, repeating steps / processes r) through u) over multiple cycles for a third sub-set and successive sub-sets of labeled nucleic acid probes; where the reading steps / processes produce images of the binding patterns of the sub-sets of capture agents and their corresponding labeled nucleic acid probes and the location of EVs). Statement 55. A system according to Statement 54, the system comprising one or more or all of the following: one or more fluidic device(s), one or more optical imager(s), one or more auto-sampler(s), one or more controller(s), one or more pump(s), one or more droplet generator(s), one or more processor(s), one or more computer-readable medium / media, one or more image analysis module(s), one or more statistical model(s), or the like, or any combination thereof.Statement 56. A system according to Statements 54 or 55, where the one or more computer- readable medium / media further comprises one or more instruction(s) that, when executed by the at least one processor, cause the at least one controller to carry out the analysis of the at least one analysis sample (or one or more or all of the one or more auto-sampler(s) further controls one or more or all of the one or more pump(s) and / or one or more or all of the one or more droplet generator(s) that dispenses one or more EV sample(s) at or into an opening or openings and / or vias of one or more or all of the one or more fluidic device(s)).Statement 57. A system according to Statements 54-56, where one or more or all of the one or more auto-sampler(s) further controls / control one or more or all of the one or more pump(s) and / or one or more or all of the one or more droplet generator(s) that dispenses one or more EV sample(s) at or into the openings and / or vias of one or more fluidic device(s) (or an opening or openings and / or vias of one or more or all of the one or more fluidic device(s)). Statement 58. A system according to Statements 54-57, where the auto-sampler further controls one or more pump(s) and / or one or more droplet generator(s) that add and / or removeone or more capture agent solution(s), one or more nucleic acid probe solution(s), one or more fixative reagent solution(s), one or more chemical denaturant solution(s), and one or more buffer solution(s), to and from the at least one fluidic device through one or more opening(s) and / or one or more via(s) in such devices.Statement 59. A system according to Statements 54-58, where the system is further configured to carry out the hybridizing, reading, and removing steps / processes (e.g., steps / processes g) through j)) over at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles, at least about 11 cycles, at least about 12 cycles, at least about 13 cycles, at least about 14 cycles, at least about 15 cycles, at least about 16 cycles, at least about 17 cycles, at least about 18 cycles, at least about 19 cycles, at least about 20 cycles, or more than about 20 cycles.Statement 60. A system according to Statements 54-59, where one or more or all of the image analysis module(s) is / are configured to analyze at least two of the images.Statement 61. A system according to Statements 54-60, where one or more or all of the image analysis module(s) is / are configured or further configured to count the binding pattern(s) of capture agents on single EVs and / or to count the number of single EVs by the universal stain EV location images or the light-scatter EV location images or the like or any combination thereof.Statement 62. A system according to Statements 54-61, where one or more or all of the image analysis module(s) is / are further configured to compare or overlay at least two of the images. Statement 63. A system according to Statements 54-62, where one or more or all of the image analysis module(s) is / are further configured to compare or overlay all of the images to produce an image showing the pattern of binding of all of the capture agents and the locations of EVs.Statement 64. A system according to Statements 54-63, where the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more other capture agent(s).Statement 65. A system according to Statements 54-64, where the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more cell-of-origin capture agent(s).Statement 66. A system according to Statements 54-65, where the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the one or more image(s) showing the location of EVs imaged by the universal stain or by light-scatter. Statement 67. A system according to Statements 54-66, where the analysis further comprises using a statistical model on any combination of single EV binding pattern counts and / or on any single EV location counts.Statement 68. A system according to Statements 54-67, where one or more or all of the at least one optical imager(s) performs / perform the reading of one or more (or the) binding pattem(s) of capture agents and labeled nucleic acid probes, universal EV stains, or the like, or any combination thereof.Statement 69. A system according to Statements 54-68, where one or more or all of the at least one optical imager(s) performs / perform the reading of a (or the) location of EVs by imaging any universal EV stain or by light-scatter microscopy or the like or any combination thereof.Statement 70. A system according to Statements 54-69, where the optical imager is a microscope configured to carry out any of the optical imaging modalities of the present disclosure (e.g., bright-field microscopy, wide-field epifluorescent microscopy, confocal reflectance scattering, confocal fluorescent microscopy, dark-field imaging, hyperspectral imaging, super-resolution microscopy, or the like, or any combination thereof).

[0155] The steps / processes of the methods described in the various examples disclosed herein are sufficient carry out a method of the present disclosure. Thus, in various examples, a method consists essentially of a combination of the steps / processes of the methods disclosed herein. In various other examples, a method consists of such steps / processes.

[0156] Now having described examples of the present disclosure, in general, the following Examples describe some additional examples of the present disclosure. While examples of the present disclosure are described in connection with the following Examples and the corresponding text and figures, there is no intent to limit the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of the present disclosure. The following Examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or nearatmospheric. Standard temperature and pressure are defined as about 20 °C to about 25 °C and 1 atmosphere, respectively.EXAMPLE 1

[0157] This example shows examples and uses of the fluidic devices used in the methods provided in the present disclosure, including representative capture agents and universal EV stains.

[0158] In FIG. 1: Panel A): A fluidic device example produced by laser cutting a glass coverslip to form openings (a.k.a., vias) within a first layer. The second layer was formed by knife cutting a double-sided, pressure-sensitive adhesive (PSA) tape that was 50 pm thick (3M F9460PC VHB Adhesive Transfer Tape). The third layer comprised a conventional 25 x 75 mm microscope slide. The PSA tape fhiidically sealed the three layers of the device. The channels within the second layer were 50 m H x 4 mm W x 12 mm L with approximately 3 pL volume. Panel B) A rendering of a fluidic device of the present disclosure, where the device has a first layer, an optional thin second layer, a third layer, and the first layer is disposed over the entirety of the second layer. Fluidic access to the channels of the second layer is provided by openings (e.g., vias) through the first layer.

[0159] In FIG. 2: Panel A): An example of a fluidic device of the present disclosure, where the device has a first layer, a second layer, a third layer, and the first layer is disposed over a portion of the second layer so that the distal ends of the channels are exposed for fluidic access. Panel B): A rendering of an example of a fluidic device of the present disclosure, similar to the device shown in FIG. 2A).

[0160] In FIG. 3: Panel A): Purified EVs that expressed a green fluorescent protein (GFP)-CD63 fusion protein were applied to a device of FIG. 1A), where the device’s first layer was hydrophobic on both its surfaces. Prior to addition, the concentration of these EVs was counted by nanoparticle tracking analysis (NTA). A log-dilution series of such EVs was applied across the channels of one device and a resultant representative fluorescent micrograph is shown. The EVs were counted by identifying GFP+ particles by epifluorescent microscopy and then counting them using NIH Image J / Fiji and the ComDet plug-in as the image analysis module. Panel B): Comparison of the GFP+ EVs counted by microscopy versus those counted by NTA. (note good linearity). Panel C): EVs from human plasma were isolated by size-exclusion chromatography. The EVs were then labeled with CMFDA-SE (a universal EV stain), rendering the EVs CFSE+. The EV samples were then loaded onto a device of FIG. 1 A) with a hydrophobic first layer. Next, the channels of the device wereblocked in an immunoassay blocking solution, then further labeled with a representative capture agent (either anti-human CD3 antibody or IgG isotype control antibody). Both antibodies were conjugated to AlexaFluor 546. Immunofluoresnce detection was then performed similar to that for imaging and counting GFP+ EVs. Approximately 2% EVs were CD3+ and < 0.05% background (isotype antibody-i- EVs). Panels D) to F): EVs were counterstained with CFSE and counts are expressed as CD3+ / CFSE+ or isotype control+ / CFSE+. A representative false-colored micrograph is shown with EVs CFSE+ (D) and CD3+ (E), then overlayed (F), arrows indicate colocalization events). Scale bars: 500 nm.EXAMPLE 2

[0161] This example shows conceptually some of the steps / processes comprising the method provided in the present disclosure, as well as an example of a multiplex, cell-of- origin single EV immunoprofile analysis.

[0162] In FIG. 4: The capture agents (e.g., antibodies linked to oligonucleotides) and labeled nucleic probes conjugated to fluorophores, along with EVs captured onto a surface (e.g., a first layer), are graphically depicted to illustrate in principle some of the labeling and reading steps / processes of the disclosed method.

[0163] The flow diagram depicts: A) disposition of universally stained EVs onto a surface (e.g., first layer of a fluidic device); B) binding and cross-linking oligonucleotide- linked antibodies (e.g., capture agents) to EVs; C) hybridizing a first sub-set of labeled nucleic acid probes to a first sub-set of antibodies, D) then imaging the EVs and the first subset of probes; E) removing by dehybridization the first sub-set of probes; and F) hybridizing a second sub-set of probes to a second sub-set of antibodies, G) imaging the EVs and the second sub-set of probes. Steps / Processes B-G can be repeated through several more iterations as desired. The strategy effectively allows the “re-use” of four fluorophores for single EV biomarker multiplexed analyses, where the “permanent” universal EV stain signal can be colocated with the binding patterns of capture agents and probes. Three symbols are shown only for illustrative purposes and are in no way intended to be limiting of the scope of the disclosure. This figure was created using Biorender.com.

[0164] A 55-plex, cell-of-origin, single EV analysis was carried out using the disclosed methods. A flowchart representation of the steps / processes of the analysis is shown in in FIG. 5.

[0165] To illustrate, one EV sample can be introduced to six channels of a representative fluidic device. In this case, each of the channels thus corresponds to six analysis samples.

[0166] Each channel is then labeled with one common cell-of-origin (CofO) EV biomarker capture agent (e.g., the same capture agent is repeated across each of the six channels). Each channel is further labeled with its own set of nine other EV biomarker capture agents en masse and subsequently all the components are cross-linked. The related workflow is graphically depicted.

[0167] Top row: EV sample labeled with universal EV stain to yield fluorescently blue EVs (represented by dotted line). The EV sample is then loaded into six channels, forming six analysis samples.

[0168] Middle row: The fluidic device’s six channels are represented by one oval. Each channel (e.g., analysis sample) is labeled with a shared cell-of-origin EV biomarker capture agent and its own set of nine other EV biomarker capture agents. Labeled nucleic acid probes would then be cycled in sub-sets of four probes per cycle. The locations of EVs and the capture agents ’binding patterns would be imaged at each cycle. Dashed line is used to represent the probe corresponding to the cell-of-origin EV biomarker capture agent, while cut circle, triangle, and diamond shapes are used to represent the probes corresponding to the other EV biomarker capture agents read during each cycle. Each of the EV s and the capture agent and probes will be imaged and image analysis modules will be used to count the EVs, binding patterns, and associated colocated signals.

[0169] Bottom row: a statistical model, such as, for example, logistical regression or other standard bioinformatic model, is then used to generate cell-of-origin-specific EV classifications based on biomarker colocation with CofO+ EVs.

[0170] Note: Ab 1 / 2 / 3 and similar represent biomarker- targeting antibody capture agents used within each channel and related fluorophore color. The figure is intended for illustrative purposes and is in no way intended to limit the scope of the disclosure.EXAMPLE 3

[0171] This example shows the liquid “spread-out” problem described in the present disclosure, which is mitigated by the various examples of the fluidic devices described herein.

[0172] In FIG. 6: Panel A): A fluidic device similar to that of FIG. 2A) was assembled. However, this device had an untreated first layer (e.g., the glass coverslip was used as received from the vendor). The water droplet contact angle for this first layer is shown in Table 1. Also shown is the liquid “spread-out” of a blue solution added to this device, where the solution spreads out on the upper surface of the first layer and not into the channeldefined in part by the opposite (e.g. bottom or underside) surface of the first layer. Panel B): A fluidic device similar to that of FIG. 6A) was assembled. However, this device had a hydrophobic functionalized first layer, unlike the first layer of FIG. 6A). The water droplet contact angle for this first layer is shown in Table 1. Also shown is the desired fluid flow pattern, where the blue solution enters the channels of the device. This example demonstrates a chemical functionalization approach (as a representative fourth layer of a device) to resolve the problem shown in FIG. 6A). Note that in both panels, the water droplet contact angles were measured on the bottom or underside surface of the first layer facing the inside of the channels and report hydrophilic and hydrophobic surfaces (respectively for FIGS. 6A) and 6B)).

[0173] Table 1. Water contact angle with various coverslip conditions.

[0174] In FIG. 7 : Shown is a rendering of a fluidic device with a first layer, a second layer, a third layer, and a fourth layer, where the first layer is disposed over a portion of the second layer so that the distal ends of the channels are exposed for fluidic access.

[0175] In FIG. 8: Panel A): A fluidic device with layers similar to those shown for the device in FIG. 2A) was assembled. However, the first layer of this device was oxidized by KOH treatment prior to assembly. Shown is the liquid “spread-out” of the blue solution added to this device, where the solution spreads out on the upper surface of the first layer and not into the channel defined in part by the opposite (e.g. bottom or underside) surface of the first layer. Panel B): A fluidic device similar to that of FIG. 8A) was assembled. However, an additional layer (e.g., a fourth layer) of 130 pm thick parafilm with vias was added to the device. The same oxidized first layer was used for its assembly. Shown is the desired fluid flow pattern, where the blue solution enters the channels of the device. This example demonstrates how a fourth layer of a device resolves the problem shown in FIG. 8A). Note that the water droplet contact angle was measured on the bottom or underside surface of the first layer facing the inside of the channels and reports a hydrophilic surface; same first layer used for both devices of FIGS. 8A) and 8B), Table 2.

[0176] Table 2. Water contact angle of 1st layer with and without fourth layer.With fourth layer § 14.226°

[0177] In FIG. 9: Panel A): A fluidic device with layers similar to those shown for the device in FIG. 2A) was assembled. The fluidic device was filled with a chemical denaturant solution (e.g., DMSO), thereby exposing the second layer comprising a PSA tape to a concentrated chemical denaturant during a prolonged (e.g., overnight) exposure. Shown is the device after such exposure, where the PSA tape is swollen and has taken on a cloudy and stretched appearance, implying that the tape has changed in composition. Panel B): A fluidic device similar to that of FIG. 9A) was assembled. The second layer was exposed to an aqueous solution without DMSO overnight. Shown is the comparison between the two conditions, where the aqueous solution does not cause tape swelling.EXAMPLE 4

[0178] This example shows the effects of a representative chemical denaturant on the retention of EVs by fluidic devices of the present disclosure.

[0179] In FIG. 10, CFSE-labeled EVs obtained from size-exclusion chromatography purification of human pooled plasma were used (similar to those described for FIG. 3). These CFSE-labeled EVs were loaded into fluidic devices similar to those shown in FIG.s 1 and 2, then treated and analyzed as described below. All fluidic devices used for data reported in FIG. 10 had an APTES-functionalized glass coverslip on both of its surfaces (i.e., the surfaces of the fluidic device’s second layer). It was assumed that if a chemical denaturant (i.e., DMSO) affects the integrity of EVs (e.g., by dissolution of the EVs), then the fluidic devices will retain fewer EVs as the presence and exposure time to the chemical denaturant (i.e., DMSO) are increased.

[0180] In Panel A), data for EV retention are reported as CFSE+ particle counts. Four fluidic devices were used for this example. After the initial loading and incubation steps, two devices received no fixative agent, while two were exposed to a fixative agent (BS3; 20 mins (mins = minutes) at room temperature (RT)), thereby cross-linking the EVs to the APTES layer. Following the fixative agent incubation, one of the no fixative agent fluidic devices andone of the BS3-treated fluidic devices were further exposed to 5% v / v DMSO (30 min at RT), while the other two fluidic devices received no DMSO. Finally, the number of CFSE+ EVs in all four fluidic devices was measured. Comparing the EV counts across the four conditions represented by the four differentially treated fluidic devices, the data show that the fixative agent (i.e., BS3) is necessary for the retention of EVs in the presence of a chemical denaturant (i.e., DMSO).

[0181] In Panel B), data for EV retention are reported as CFSE+ particle counts. After the initial loading and incubation steps, the devices were subsequently exposed to BS3 (20 mins at RT), then either PBS for 10 or 30 mins (as control), 20% v / v DMSO for 30 mins, or 75% v / v DMSO for 10 mins. Finally, the number of CFSE+ EVs in all fluidic devices was measured. For the two DMSO cases, there was approximately 40% lower EV particles observed compared to the PBS-treated control cases. These data suggest that chemical denaturant conditions (i.e., exposure to DMSO) at levels previously reported to be compatible with fixed cells and fixed tissue sections are incompatible with more chemically sensitive EVs (even after the EVs have been exposed to a fixative agent).

[0182] In Panel C), data for EV retention are reported as percentage of particle retention normalized to a control case. After the initial loading and incubation steps, the devices were subsequently exposed to BS3 (20 mins at RT), then incubated in no DMSO (PBS-only control) or increasing concentrations of DMSO (0.5%, 1%, 5%, 10%, 15%, 20%, or 30% v / v in PBS) for 10 mins at RT. Finally, the number of CFSE+ EVs in all fluidic devices was measured and the DMSO-treated cases were normalized to the PBS-only control case. The data show a marked loss of the CFSE+ EV particles at 20% and 30% v / v DMSO (i.e., about 30% loss), while the other cases show approximately 90% retention of the CFSE+ EV particles at the lower DMSO concentrations (i.e., about 10% loss). These data suggest EVs may tolerate exposure to solutions comprising less than 20% v / v chemical denaturant concentration.

[0183] In Panel D), data for EV retention are reported as CFSE+ particle counts. After the initial loading and incubation steps, the devices were subsequently exposed to BS3 (20 mins at RT), then incubated in no DMSO (water-only control) or increasing concentrations of DMSO (2%, 5%, or 10% v / v in water) for 10 mins at RT. Finally, the number of CFSE+ EVs in all fluidic devices was measured. The results show that about 90% of the CFSE EV particles were retained in the presence of a chemical denaturant (i.e., DMSO) from 2% to 10% v / v concentration, suggesting that EVs can tolerate this level of exposure to a chemical denaturant like DMSO.

[0184] Various modifications and variations of the described methods, kits, and systems of the present disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific Examples, it will be understood that it is capable of further modifications and that the disclosure as claimed should not be unduly limited to such specific Examples. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure. This application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure come within known customary practice within the art to which the disclosure pertains and may be applied to the essential features herein before set forth.

Claims

CLAIMS:

1. A method for performing a multiplexed single extracellular vesicle (EV) analysis, the method comprising: a) disposing at least one EV sample comprising one or more pluralit(ies) of EVs onto a first layer of at least one fluidic device, thereby forming at least one analysis sample within at least one fluidic device; b) contacting the at least one EV sample with one or more pluralit(ies) of capture agents; c) cross-linking at least a portion, substantially all, or all of the pluralit(ies) of EVs to the first layer and at least a portion, substantially all, or all of the one or more pluralit(ies) of EVs to the one or more pluralit(ies) of capture agent(s); d) hybridizing a first sub-set of labeled nucleic acid probes with the at least one EV sample, thereby forming a first sub-set of labeled probe / oligonucleotide duplexes, wherein the first sub-set of labeled probe / oligonucleotide duplexes form a binding pattern; e) reading the at least one EV sample to obtain an image showing the binding pattern for each of the hybridized nucleic acid probes of the first sub-set of the labeled nucleic acid probes from d); f) removing the first suh-set of probes from the at least one EV sample, by denaturation or by inactivation; g) hybridizing a second sub-set of labeled nucleic acid probes with the at least one EV sample, thereby forming a second sub-set of labeled probe / oligonucleotide duplexes; h) reading the at least one EV sample to obtain an image showing the binding pattern for each of the probes in the second sub-set of probes; i) removing the second sub-set of probes from the at least one EV sample, by denaturation or by inactivation; j) optionally, imaging a location of at least a portion, substantially all or all of the pluralit(ies) of EVs crosslinked to one or more pluralities of capture agent(s) and hybridized to a nucleic acid probe or probes at each reading step by a universal EV stain or by light-scatter imaging; k) optionally, repeating steps g) through j) over multiple cycles for a third sub-set and successive sub-sets of labeled nucleic acid probes; and wherein the reading(s) produce(s) images of the binding patterns of the sub-set(s) of capture agents and their corresponding labeled nucleic acid probes and the location of the at least aportion, substantially all or all of the pluralit(ies) of EVs crosslinked to one or more pluralities of capture agent(s) and hybridized to a nucleic acid probe or probes.

2. The method of claim 1 , wherein the EV sample comprises at least one plurality of EVs derived by one or more EV purification process(es) from a biological sample or an environmental sample.

3. The method of claim 1 , wherein the analysis sample does not comprise a cell or cells or a tissue section or tissue sections.

4. The method of claim 1 , wherein the disposing results in a disposition of the EV sample comprising discretely isolated, individual EVs on the first layer of the fluidic device and / or at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% or more of the EVs are discretely isolated and individual EVs.

5. The method of claim 1, wherein two or more analysis samples are formed within one fluidic device or wherein two or more analysis samples are formed across multiple fluidic devices.

6. The method of claim 1, wherein the fluidic device comprises a first layer, a second layer, and a third layer, with one or more independent channel(s) and any combination of first layer vias and / or third layer vias.

7. The method of claim 6, wherein the fluidic device further comprises a first layer, a thin second layer, and a third layer into which the one or more channels ) of the fluidic device have been formed, with one or more independent channel(s) and any combination of first layer vias and / or third layer vias.

8. The method of claim 6, wherein the fluidic device further comprises a functionalized first layer, a second layer, a third layer, and a fourth layer, with one or more independent channel(s) and any combination of first, third, and / or fourth layer vias.

9. The method of claim 8, wherein the fluidic device further comprises a functionalized first layer, a thin second layer, a third layer into which channels have been formed, and a fourthlayer, with one or more independent channel(s) and any combination of first, third, and / or fourth layer vias.

10. The method of claim 8, wherein the fluidic device further comprises a first layer and a third layer where each layer independently comprises a glass, a second layer comprising a metal, a non-metal, a ceramic, or any combination thereof, and a fourth layer comprising a glass, a metal, a non-metal, a polymer, a ceramic, or any combination thereof and wherein the first layer is hydrophilically functionalized on at least one of its surfaces.

11. The method of claim 10, wherein the fluidic device further comprises a differentially functionalized first layer.

12. The method of claim 10, wherein the functionalized first layer is APTES-functionalized on at least one of its surfaces.

13. The method of claim 10, wherein the fourth layer is a chemical functionalization.

14. The method of claim 1, wherein the contact with the one or more pluralit(ies) of capture agents comprises sequential or en masse contact, and wherein the contact further comprises labeling two or more analysis samples within any number of fluidic device with any number of plurality of capture agents such that the two or more samples are labeled with any degree of similarity, difference, iteration, and / or repetition as desired.

15. The method of claim 14, wherein the contact with the one or more pluralit(ies) of capture agents further comprises labeling two or more analysis samples within any number of fluidic devices with at least one cell-of-origin EV biomarker capture agent, as well as any number of other capture agents.

16. The method of claim 15, wherein the one or more plurality es) of capture agents further comprises at least about 3, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, or more than about 100 capture agents.

17. The method of claim 16, wherein each plurality of capture agent further comprises a plurality of one antibody that is linked to its own distinguishing and different oligonucleotide.

18. The method of claim 17, wherein the oligonucleotides linked to capture agents have a sequence length of at least about 5, at least about 10, at least about 20, at least about 30, at least about 45, or more than about 45 nucleotides.

19. The method of claim 1 , wherein the EVs are cross-linked to the functionalized first layer of the at least one fluidic device and wherein the one or more pluralit(ies) of capture agents are cross-linked to the EVs by a fixative reagent.

20. The method of claim 1, wherein the hybridizing of one or more or all of the sub-set(s) of labeled nucleic acid probes comprises contacting at the least one analysis sample with one or more pluralit(ies) of labeled nucleic acid probes.

21. The method of claim 20, wherein the hybridizing further comprises selectively hybridizing one or more pluralit(ies) of labeled nucleic acid probes, wherein each of the pluralit(ies) of labeled nucleic acid probes is hybridized to a distinct plurality of oligonucleotides, wherein each oligonucleotide of the distinct plurality of oligonucleotides is linked to a capture agent.

22. The method of claim 21 , wherein a number of labeled nucleic acid probes hybridized in the one or more or all sub-set(s) to the at least one analysis sample corresponds to a number of capture agents labeled in an individual sub-set.

23. The method of claim 20, wherein the hybridizing further comprises contacting the at least one analysis sample with a sub-set comprising at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5 pluralities of labeled nucleic acid probes, thereby forming at least about 2, at least about 3, at least about 4, at least about 5, or more than about 5 sub-sets of labeled probe / oligonucleotide duplexes.

24. The method of claim 1, wherein the hybridizing of one or more or all of the sub-set(s) is / are from about 1 minute to about 5 minutes.

25. The method of claim 1, wherein any of the labeled nucleic acid probe / oligonucleotide duplexes have a Tm from about 15 °C to about 75 °C.

26. The method of claim 20, wherein the labeled nucleic acid probes have a sequence length of at least about 5, at least about 10, at least about 20, at least about 30, at least about 45, or more than about 45 nucleotides.

27. The method of claim 20, wherein the labeled nucleic acid probes are distinctly labeled with one or more fluorophore(s).

28. The method of claim 1, wherein the removing by denaturation further comprises use of a chemical denaturant and wherein the removing is not by displacing the labeled nucleic acid probes using displacement oligonucleotides.

29. The method of claim 28, wherein the chemical denaturant is ultrapure water, about 1 mM to about 10 mM EDTA, about 1% to about 10% v / v concentration formamide, or about 1% to about 10% v / v concentration DMSO.

30. The method of claim 1, wherein a sequence of an oligonucleotide to which a capture agent is linked is longer than a sequence of a labeled nucleic acid probe, but the sequence of the oligonucleotide to which the capture agent is linked is identical to the sequence of the labeled nucleic acid probe except for a sub-sequence of the oligonucleotide to which the capture agent is linked that is complementary to a single labeled nucleic acid probe.

31. The method of claim 1, wherein the hybridizing, reading, and removing steps (e.g., steps g) through j)) are repeated over at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles, at least about 11 cycles, at least about 12 cycles, at least about 13 cycles, at least about 14 cycles, at least about 15 cycles, at least about 16 cycles, at least about 17 cycles, at least about 18 cycles, at least about 19 cycles, at least about 20 cycles, or more than about 20 cycles.

32. The method of claim 31, wherein the removing step is optionally omitted for a last cycle.

33. The method of claim 1, wherein the method further comprises analyzing at least two of the images.

34. The method of claim 33, wherein the analyzing further comprises counting the binding patterns of capture agents on single EVs and / or counting a number of single EVs by the universal stain EV location images or the light-scatter EV location images.

35. The method of claim 34, wherein the analyzing of images further comprises comparing or overlaying at least two of the images.

36. The method of claim 35, wherein the analyzing of images further comprises comparing or overlaying all of the images to produce an image showing the pattern of binding of all of the capture agents and the locations of EVs.

37. The method of claim 35, wherein the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more other capture agent(s).

38. The method of claim 35, wherein the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more cell-of-origin capture agent(s).

39. The method of claim 35, wherein the analyzing of images further comprises limiting the comparing or overlaying of images to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the one or more image(s) showing the location of EVs imaged by the universal stain or by light-scatter.

40. The method of claim 35, wherein the method further comprises performing an analysis using a statistical model on any combination of single EV binding pattern counts and / or on any single EV location counts.

41. The method of claim 1, wherein reading of any binding patterns of capture agents and labeled nucleic acid probes, and / or any universal EV stains, are done by fluorescence microscopy, super-resolution microscopy, confocal microscopy, hyperspectral imaging, or any combination thereof.

42. The method of claim 1, wherein the reading of any location of EVs is done by imaging any universal EV stain or by light-scatter microscopy.

43. The method of claim 1, wherein the one or more pluralit(ies) of capture agents are aptamers.

44. The method of claim 1, wherein one or more or all of the removing(s) results / result in at least a portion, substantially all, or all of the crosslinked pluralit(ies) of EVs, the crosslinked pluralit(ies) of capture agents, and linked oligonucleotides of the capture agents remaining bound to the at least one analysis sample.

45. The method of claim 1, wherein the removing by inactivation further comprises chemical inactivation or by cleaving one or more bond(s).

46. The method of claim 45, wherein the cleaving is done enzymatically, chemically, or via exposure to light.

47. A kit comprising: one or more fluidic device(s); one or more pluralit(ies) of labeled nucleic acid probes; one or more pluralit(ies) of capture agents; one or more reagent(s) and / or one or more buffer(s); and one or more instruction set(s).

48. The kit of claim 47, wherein each plurality of nucleic acid probe is labeled with a distinct label.

49. The kit of claim 47, wherein each plurality of nucleic acid probe is not labeled.

50. The kit of claim 47, wherein the one or more pluralit(ies) of labeled or unlabeled nucleic acid probes are in separate vessels or mixed in one or more vessel(s) to any level of combination.

51. The kit of claim 47, the kit further comprising one or more pluralit(ies) of capture agents linked to oligonucleotides, wherein the oligonucleotides each comprise a sequence that is complementary to a sequence of a plurality of nucleic acid probes of the kit.

52. The kit of claim 51, wherein the oligonucleotides comprise a sequence length longer than a sequence of a corresponding complimentary labeled nucleic acid probe of the kit.

53. The kit of claim 51, wherein the oligonucleotides comprise a sequence length shorter than a sequence of a corresponding complimentary labeled nucleic acid probe of the kit.

54. The kit of claim 51, wherein the one or more pluralit(ies) of capture agents is / are in separate vessels or mixed in one or more vessel(s).

55. The kit of claim 47, the kit optionally comprising one or more oligonucleotide(s), one or more reagent(s), one or more buffer(s) for a user to conjugate one or more oligonucleotide(s) with a user-supplied capture agent.

56. The kit of claim 47, wherein the nucleic acid probes comprise a cleavable linkage that is not a phosphodiester bond.

57. The kit of claim 47, wherein the one or more reagent(s) and / or one or more buffer(s) are used to carrying out a method for performing a multiplexed single extracellular vesicle (EV) analysis.

58. The kit of claim 47, wherein one or more or all of the one or more reagent(s) and / or one or more or all of the one or more buffer(s) are in a container or in separate containers.

59. The kit of claim 47, wherein the one or more instruction set(s) specify a use of one or more or all of the fluidic device(s), one or more or all of the one or more pluralit(ies) of labeled nucleic acid probes, one or more one or more of the one or more pluralit(ies) ofcapture agents, one or more or all of the one or more buffer(s), or any combination thereof of the kit.

60. The kit of claim 47, wherein the one or more instruction set(s) specify how to practice a method for performing a multiplexed single extracellular vesicle (EV) analysis.

61. A system configured to carry out the method for performing a multiplexed single extracellular vesicle (EV) analysis of claim 1 .

62. The system of claim 61, the system comprising one or more or all of: one or more fluidic device(s); one or more optical imager(s); one or more auto-sampler(s); one or more controller(s); one or more pump(s); one or more droplet generator(s); one or more processor(s); one or more computer-readable medium / media; one or more image analysis module(s); or one or more statistical model(s).

63. The system of claim 62, wherein the one or more computer-readable medium / media further comprises one or more instruction(s) that, when executed by at least one of the one or more processor(s), cause at least one of the one or more controller(s) to carry out the multiplexed single extracellular vesicle (EV) analysis of the at least one analysis sample.

64. The system of claim 63, wherein one or more or all of the one or more auto-sampler(s) further control(s) one or more or all of the one or more pump(s) and / or one or more or all of the one or more droplet generator(s) that dispenses one or more EV sample(s) at or into an opening or openings and / or vias of one or more or all of the one or more fluidic device(s).

65. The system of claim 63, wherein one or more or all of the auto-sampler(s) further controls / control one or more or all of the one or more pump(s) and / or one or more or all of the one or more droplet generator(s) that add and / or remove one or more capture agentsolution(s), one or more nucleic acid probe solution(s), one or more fixative reagent solution(s), one or more chemical denaturant solution(s), and one or more buffer solution(s), to and from the at least one fluidic device through one or more opening(s) and / or one or more via(s) of the at least one fluidic device.

66. The system of claim 62, wherein the system is further configured to carry out the hybridizing, reading, and removing steps over at least about 2 cycles, at least about 3 cycles, at least about 4 cycles, at least about 5 cycles, at least about 6 cycles, at least about 7 cycles, at least about 8 cycles, at least about 9 cycles, at least about 10 cycles, at least about 11 cycles, at least about 12 cycles, at least about 13 cycles, at least about 14 cycles, at least about 15 cycles, at least about 16 cycles, at least about 17 cycles, at least about 18 cycles, at least about 19 cycles, at least about 20 cycles, or more than about 20 cycles.

67. The system of claim 62, wherein one or more or all of the image analysis module(s) is / are configured to analyze at least two of the images.

68. The system of claim 67, wherein the one or more or all of the image analysis module(s) is / are further configured to count one or more binding pattern(s) of capture agents on single EVs and / or to count a number of single EVs by the universal stain EV location images or the light-scatter EV location images.

69. The system of claim 68, wherein one or more or all of the image analysis module(s) is / are further configured to compare or overlay at least two of the images.

70. The system of claim 69, wherein one or more or all of the image analysis module(s) is / are further configured to compare or overlay all of the images to produce an image showing the pattern of binding of all of the capture agents and the locations of EVs.

71. The system of claim 69, wherein the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more other capture agent(s).

12. The system of claim 69, wherein the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the binding patterns of one or more cell- of-origin capture agent(s).

73. The system of claim 69, wherein the comparing or overlaying of images is further limited to produce one or more image(s) showing any desired combination of binding patterns of one or more capture agent(s) only when colocated with the one or more image(s) showing the location of EVs imaged by the universal stain or by light-scatter.

74. The system of claim 69, wherein the analysis further comprises using a statistical model on any combination of single EV binding pattern counts and / or on any single EV location counts.

75. The system of claim 62, wherein one or more or all of the one or more optical imager(s) performs / perform the reading of the binding patterns of capture agents and labeled nucleic acid probes and / or universal EV stains.

76. The system of claim 62, wherein one or more or all of the one or more optical imager(s) performs / perform the reading of the location of EVs by imaging any universal EV stain or by light-scatter microscopy.

77. The system of claim 62, wherein the optical imager is a microscope configured to carry out bright-field microscopy, wide-field epifluorescent microscopy, confocal reflectance scattering and / or confocal fluorescent microscopy, dark-field imaging, hyperspectral imaging, super-resolution microscopy, or combination thereof.

Images