Plasmon-enhanced biosensing of extracellular vesicles

Abstract

The present disclosure provides new assay configurations for molecular analysis of extracellular vesicles (EVs) for early cancer detection, treatment monitoring, and minimum residual disease analysis. The new methods use nanoplasmonic sensing platforms to significantly enhance EV fluorescence signals, obviating the need for supplementary chemical or enzymatic amplification processes, and enabling analysis of individual EVs. The new assay configurations leverage molecular specificity through antibodies and enable multi-color imaging to provide sensitive detection and multi-dimensional characterization.

Description

[0001] PLASMON-ENHANCED BIOSENSING OF EXTRACELLULAR VESICLES

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 543,190, filed on October 9, 2023. The entire contents of the foregoing are hereby incorporated by reference in their entireties. TECHNICAL FIELD

[0004] This invention relates to improved methods and systems for enhancing analysis of individual extracellular vesicles (EVs) using plasmonic sensor chip technology.

[0005] BACKGROUND

[0006] Various types of cancers are a leading cause of death worldwide. For example, breast cancer is the most common cancer among women worldwide, with more than 300,000 new cases annually in the United States. Despite advances in our understanding of molecular biology and targeted therapies, the 5-year survival rate for metastatic breast cancer is disappointingly low at below 30%. The failure is mainly due to acquired treatment resistance, which is common in all breast cancer subtypes. Making clinical decisions of continuing with the current therapy or pivoting will significantly improve patient care, minimizing ineffective treatments.

[0007] Molecular analysis of extracellular vesicles (EVs) represents anew paradigm for early cancer detection and treatment monitoring through liquid biopsies. In particular, EV analysis is important to understand the heterogeneity of EVs, identify the cellular origins of individual EVs, and detect key biomarkers in the subpopulations.

[0008] SUMMARY

[0009] The present disclosure relates to the use of various plasmonic resonance substrate devices and assay configurations to detect and analyze single EVs for different diseases. The new assay methods use a low-cost plasmonic substrate that can be fabricated on a wafer scale to amplify fluorescence signals from even single EVs without the need for additional chemical or enzymatic signal amplification processes. Different diseases are associated with different subpopulations of EVs and different biomarkers to be analyzed. For example, when a total EV count and quantification of EVs from multiple cellular origins are of interest, all EVs can be captured and analyzed for multiple biomarkers. In another example, if rare target EVs need to be detected, such as tumor-derived EVs from small tumor volumes for early cancer detection, only tumor-derived EVs can be captured based on tumor-specific capture antibodies. Thus, the new devices and methods can be used, for example, for cancer diagnosis and treatment monitoring, as well as for minimal residual disease monitoring after treatment has ended.

[0010] In a first aspect, the present disclosure provides methods for analyzing individual EVs utilizing plasmonic technology . The methods include or consist essentially of (a) separating EVs from a biological sample, e.g., using a size-based technique; (b) fluorescently labeling the EVs; (c) immobilizing the EVs on a plasmonic substrate containing nanostructures and functional linkers that bind to all types of EVs; (d) adding a plurality of different types of antibodies, wherein each type of antibody specifically binds to EVs from one type of cell, wherein each ty pe of antibody is bound to a specific type of reporter group, e.g., a fluorophore, and wherein the types of reporter groups are different, e.g., different colored fluorophores, to enable distinction between different EVs from different types of cells; and (e) employing multi-color imaging to detect individual types of EVs, e.g., a subpopulation of EVs of interest, immobilized on the substrate, thereby- enhancing EV detection accuracy and enabling multi-dimensional analysis.

[0011] In these methods, the plurality of different types of antibodies can be selected based on the characteristics of the EVs under investigation. The EVs can be immobilized on the plasmonic substrate by, for example, any one or more of (a) direct physisorption, (b) a molecular adhesive layer with affinity to large molecules, (c) a charged layer, or (d) covalent bonding with a linker layer. For example, the linker layer can include any one or more of (a) short carbon chains, (b) polyethylene glycol, or (c) other polymers. In other implementations, the EVs are immobilized by functionalization of the substrate, e.g., using SH-PEG-COOH with EDC / NHS activation.

[0012] In other implementations, the immobilization is mediated by, but not limited to, functionalization with amines, thiols, trans-Cyclooctene, or streptavidin (for biotinylated EVs).

[0013] The methods can further include selecting a type of antibody that specifically binds to EVs derived from a tumor cell to label tumor-derived EVs, or selecting a type of antibody that specifically binds to EVs derived from a specific type of tumor cells to label a specific type of tumor-derived EVs. In these methods, the type of antibody can include, but is not limited to, antibodies that specifically bind to p-gly coprotein (PgP) or survivin to profile drug resistance monitoring. In certain embodiments, the tumor is a solid tumor, e.g., breast cancer

[0014] In some implementations, the methods further include selecting a type of antibody that specifically binds to EVs derived from a specific type of diseased cell to label disease cell-derived EVs, e.g., from cells related to Alzheimer’s Disease or fibrosis.

[0015] In some implementations, the methods described herein are used when a subpopulation of EVs is present at or above about 0.5 percent to about 1.0 percent, e.g., at or above 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 percent, of a total number of EVs in the biological sample.

[0016] In another aspect, the disclosure provides methods for analyzing individual target EVs utilizing plasmonic technology . These methods include or consist essentially of (a) separating EVs from a biological sample, e.g., using a size-based technique; (b) fluorescently labeling the EVs; (c) immobilizing one type of target EV on a plasmonic substrate containing nanostructures and binding groups that specifically bind only to EVs derived from one type of cell; (d) adding one or more different types of antibodies, wherein each type of antibody specifically binds to a specific type of biomarker on the surface of the target EVs, wherein each type of antibody is bound to a specific type of reporter group, and wherein the types of reporter groups, e.g., fluorophores, are different, e.g., different colored fluorophores, to enable distinction between different biomarkers on the surface of the target EVs; and (e) employing multi-color imaging to detect individual types of biomarkers on the surface of the target EVs immobilized on the substrate, thereby enhancing EV detection accuracy and enabling multi-dimensional analysis.

[0017] In some implementations, the target EVs are immobilized on the plasmonic substrate by binding groups comprising one or more different types of monoclonal or polyclonal antibodies, wherein the monoclonal or polyclonal antibodies specifically bind to one or more surface biomarkers on the target EVs.

[0018] In some implementations, the methods can further include selecting a type of antibody that specifically binds to target EVs derived from a tumor cell to label tumor- derived target EVs, e.g., all tumor-derived target EVs. In certain implementations, the methods can further include selecting a type of antibody that specifically binds to target EVs derived from a specific type of tumor cell to label a specific type of tumor-derived target EVs.

[0019] In some implementations, the type of antibody includes, but is not limited to, antibodies that specifically bind to p-gly coprotein (PgP) or survivin to profile drug resistance monitoring.

[0020] In any of these methods, the tumor can be a solid tumor, e.g., breast cancer.

[0021] In other implementations, the methods can further include selecting a type of antibody that specifically binds to target EVs derived from a specific type of diseased cell to label disease cell-derived target EVs, e.g., wherein the disease is Alzheimer’s Disease or fibrosis.

[0022] In any of these methods for analyzing individual target EVs utilizing plasmonic technology, the methods are generally used for a subpopulation of target EVs present at or below about 0.5 percent to about 1.0 percent, e.g., at or below 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 percent, of a total number of EVs in the biological sample.

[0023] In certain embodiments, the antibodies include one or more, or all four, of a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, and a fourth type of antibody that specifically binds to HER2.

[0024] In some embodiments, the binding groups include one or more different types of antibodies immobilized on the surface of the substrate, wherein the at least two different types of antibodies are selected from a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, and a fourth type of antibody that specifically binds to HERZ. For example, all of the first, second, third, and fourth antibodies can be immobilized on the surface of the substrate.

[0025] In any of these methods described herein, the substrate can be a nanoparticles on pillars (NPOP) structure, e.g., that comprises gold nanoparticles bound to gold nanopillars. In some embodiments, the substrate is functionalized by using a citrate solution, e.g., a sodium citrate solution.

[0026] Any of these methods can further include obtaining image data for the captured EVs; and analyzing the captured EVs based on the image data. In another aspect, the present disclosure provides substrates for capturing target extracellular vesicles (EVs) in a liquid sample. These substrates include a nanoparticle on pillar (NPOP) structure functionalized with a citrate solution, and at least two different types of antibodies immobilized on the surface of the substrate, wherein the at least two different types of antibodies are selected from a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, a fourth type of antibody that specifically binds to HER2, a fifth type of antibody that specifically binds to p-glycoprotein (PgP), and a sixth type of antibody that specifically binds to survivin.

[0027] In some implementations, the at least two different types of antibodies include all four of the first, second, third, and fourth types of antibodies.

[0028] In some implementations, the at least two different types of antibodies include fifth and sixth antibodies for profiling drug resistant monitoring.

[0029] In certain implementations, the NPOP structure includes an array of gold nanorods arranged at a distance between neighboring nanorods of about 2500 nm to about 3500 nm, and wherein the nanorods have a length of about 70 nm to about 90 nm, a width of about 20 to about 30 nm, and a height of about 15 to about 25 nm.

[0030] In some embodiments, the at least two different types of antibodies are bound to the substrate using a solution having an antibody concentration of: about 0.35 to about 0.45 pg / ml for the first type of antibody, about 1.5 to about 2.5 pg / ml for the second type of antibody, about 0.06 to about 0. 10 pg / ml for the third type of antibody, and / or about 1.5 to about 2.5 pg / ml for the fourth type of antibody.

[0031] In another aspect, this disclosure provides methods of detecting or monitoring cancer, wherein the liquid sample used in the methods described herein is from a subject, wherein the reporter groups are bound to capture agents that specifically bind to tumor- derived target EVs, and wherein the methods further comprise analyzing the obtained image to detect whether the liquid sample comprises rare EVs, e g., tumor-derived target EVs, thereby detecting or monitoring cancer in the subject.

[0032] In certain implementations, these methods can further include identifying EVs by size and discarding any EVs or other components larger than one micron; selecting target EVs from the identified EVs based on positivity for target EV markers to generate selected target EVs; specifying selected target EVs as originating from specific organs or tissues by positivity for organ- or tissue-specific markers to generate specific, selected target EVs; and analyzing individual specific, selected target EVs based on tetraspanin biomarkers on the surface of the specific target EVs, based on intravesicular biomarkers within the specific target EVs, or based on both tetraspanin and intravesicular biomarkers.

[0033] In the methods described herein, the reporter groups can be, include, or consist of, a first fluorescent label, and the reporter groups can be, include, or consist of antibodies that specifically bind to a biomarker on the surface of the target EVs. For example, in some embodiments, the antibodies can be, include, or consist of at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2. In other embodiments, the antibodies can be, include, or consist of at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth ty pe bind to HER2.

[0034] In some embodiments, the methods further include labeling target EVs that include the first fluorescent label with a second fluorescent label that is different from the first fluorescent label. In some embodiments of the methods described herein, the cancer can be or include breast cancer.

[0035] In another aspect, this disclosure provides methods of detecting cancer in a subject, the methods including collecting a biological sample from the subject; isolating EVs from the biological sample; capturing EVs on a nano-plasmonic array, wherein the nano-plasmonic array includes, a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands specifically bind to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling target EVs among the captured EVs with multiple different fluorescent reporter groups; conducting multichannel fluorescence imaging to form an image; and analyzing the image to detect cancer in the subject.

[0036] In these methods, the plurality of nanostructures can be arranged, in certain embodiments, to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify fluorescent signals emitted, scattered, or reflected by EVs bound to the nanostructures and / or EVs bound to the substrate near the nanostructures, or to amplify fluorescent signals emitted, scattered, or reflected by reporter groups attached to the EVs. In some implementations, the immunolabeling is carried out with different antibodies that are bound to different reporter groups and that specifically bind to different biomarkers on the surface of the target EVs. For example, herein the antibodies can be, include, or consist of at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2. In other embodiments, the antibodies can be, include, or consist of at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth type bind to HER2. In some embodiments, the cancer is breast cancer.

[0037] In another aspect, the disclosure provides methods of detecting individual target EVs relevant to cancer diagnosis or cancer treatment monitoring, the methods including: providing a biological sample from a subject containing one or more EVs; capturing the one or more EVs on a nano-plasmonic array, wherein the nano-plasmonic array includes a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands specifically bind to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling target EVs among the captured EVs with fluorescence-conjugated biomolecules; conducting multi-channel fluorescence imaging to form an image; and analyzing the image to detect cancer in a subject or monitor a cancer treatment in a subject undergoing the cancer treatment.

[0038] In these methods, the fluorescence-conjugated biomolecules can be, include, or consist of at least three fluorescent molecules conjugated to at least three cancer- associated biomolecules. For example, in certain embodiments, the fluorescence- conjugated biomolecules can be, include, or consist of fluorescence-conjugated wheat germ agglutinin.

[0039] In some embodiments, the nano-plasmonic array is configured to enhance a fluoresce signal from the fluorescence-conjugated biomolecules at least two-fold compared to a fluorescence signal from an image taken using a glass substrate instead of the nano-plasmonic array.

[0040] In another aspect, the present disclosure provides methods of cancer diagnosis or treatment monitoring, the methods including: providing a biological sample from a subject containing one or more EVs; contacting at least a first portion of the biological sample with a surface conjugated with a first EV-specific antibody and labeling with a first fluorescence-conjugated biomolecule; contacting at least a second portion of the biological sample with a surface conjugated with a second EV-specific antibody and labeling with a second fluorescence-conjugated biomolecule; contacting at least a third portion of the biological sample with a surface conjugated with a second EV-specific antibody and labeling with a third fluorescence-conjugated biomolecule; conducting multi-channel fluorescence imaging to form an image; and analyzing the image to detect cancer in a subject or monitor a cancer treatment in a subject undergoing the cancer treatment.

[0041] In these methods, the surface can be, include, or consist of a nano-plasmonic array, wherein the nano-plasmonic array includes or consists or a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands specifically bind to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures.

[0042] In some embodiments, the nano-plasmonic arrays are configured to enhance a fluoresce signal in the image at least two-fold compared to a fluorescence signal from an image taken using a glass substrate instead of the nano-plasmonic array.

[0043] In other aspects, the present disclosure provides nano-plasmonic arrays for detecting target EVs, the arrays including or consisting of a substrate; a plurality of nanostructures arranged to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by EVs bound to the nanostructures and / or EVs bound to the substrate near the nanostructures, or to amplify one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by reporter groups attached to the EVs; and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands selectively bind to target EVs to bind the target EVs to the nanostructures or to the substrate adjacent to the nanostructures.

[0044] In various embodiments, the optical signal can be one or more of a fluorescent signal, a Raman signal, or dark-field scattering.

[0045] In some embodiments, each of the nanostructures has a maximum size, e.g., diameter, width, or length, of about 30 to 400 nm. For example, the nanostructures can be nanorods or nanosquares and have dimensions of about 50 to about 300 nm in length, about 20 to about 300 nm in width, and about 20 to about 300 nm in height, or the nanostructures can be nanodisks and have dimensions of about 50 to about 200 nm in diameter and about 20 to about 300 nm in height. Each of the nanopillars can have a maximum size of about 30 to about 500 nm.

[0046] In certain embodiments, the periodic array of nanostructures has a periodicity of about 400 to 800 nm or 400 to 2000 nm between nanostructures. In certain embodiments, the nanostructures are nanopillars having a dimension of about 20 to about 500 nm in diameter and about 20 to about 300 nm in height with a density of about 300 to about 750 nanopillars per pm2on a substrate (e.g., Si, glass, indium tin oxide / ITO, polyethylene terephthalate (PET) polymer substrate).

[0047] In certain other embodiments, the periodic array of nanostructures has a periodicity' of about 2000 nm to 4000 nm or 2500 nm to 3500 nm or of about 2800 nm to 3200 nm about 3000 nm between nanostructures. In certain embodiments, the nanostructures are nanopillars having a dimension of about 60 to about 100 nm or about 70 to about 90 nm or about 75 to about 85 nm in length; about 20 to about 50 nm or about 20 to about 30 mm or about 25 to 35 nm in width; and about 10 to about 30 nm or about 15 to about 25 nm or about 20 nm in height.

[0048] In some implementations, the affinity ligands bind to a capture agent, e.g., an antibody, wherein the capture agent is configured to bind to at least one surface marker on the target EV. In certain embodiments, the affinity ligand is configured to bind to at least one surface marker on the target EV and / or to at least one intravesicular marker inside the target EV.

[0049] In some implementations, the nano-plasmonic arrays further include or consist of a metal film disposed on a top surface of the substrate, wherein the metal film comprises a plurality of nanoholes that penetrate the metal film in a periodicity selected to amplify one or more specific wavelengths of electromagnetic radiation, wherein the periodicity of about 400 to 800 nm between nanoholes, wherein the metal film comprises a plurality of affinity ligands fixed on or adjacent to the nanoholes, and wherein the plurality of affinity ligands selectively bind to markers on surfaces of the target EVs. The metal film can be or include, for example, a noble metal, a transition metal, an alkali metal, or any combination thereof. In various implementations, either or both the nanostructures and the metal film are, or include, or consist of gold, silver, aluminum, or platinum.

[0050] In another aspect, the disclosure includes methods for detecting target EVs in a liquid biological sample, the methods including or consisting of obtaining a nano- plasmonic array as described or claimed herein; flowing a liquid sample over the nano- plasmonic array at a flow rate that enables the EVs in the liquid sample, if any, to bind to the affinity ligands thus capturing the EVs on the nano-plasmonic array; labeling target EVs captured on the nano-plasmonic array with one or more reporter groups; projecting a first electromagnetic radiation at one or more specific wavelengths onto the labeled target EVs captured on the nano-plasmonic array, wherein the electromagnetic radiation at the one or more specific wavelengths is selected to cause the reporter groups to emit, scatter, or reflect the first electromagnetic radiation or a second electromagnetic radiation; receiving the first or second electromagnetic radiation emitted, scattered, or reflected by the reporter groups, wherein the nano-plasmonic array of nanostructures is arranged and dimensioned to amplify the first or second electromagnetic radiation emitted, scattered, or reflected by the reporter groups; and capturing an image of the amplified first or second electromagnetic radiation emitted, scattered, or reflected by the reporter groups.

[0051] In the methods disclosed herein, the number of the target EVs may be less than 1,000.

[0052] In certain embodiments, the nanostructures include a plurality of nanoholes that penetrate the substrate or a metal film disposed on the substrate, or the nanostructures include or consist of a plurality of nanorods, nanodisks, nanopillars, nanogrooves, or any combination thereof, arranged on a top surface of the substrate.

[0053] In some embodiments the methods as described herein further include or consist of identifying EVs by size and discarding large components, e.g., larger than one micron; selecting target EVs from the identified EVs based on positivity for target EV markers; selecting target EVs as originating from specific organs or tissues by positivity for organ- or tissue-specific markers to generate specific target EVs; and analyzing individual specific target EVs based on extravesicular biomarkers on the surface of the specific target EVs and / or based on intravesicular biomarkers within the specific target EVs.

[0054] In some embodiments, the methods are used to detect tumor-derived EVs using a QUAD marker signature (Mucin 1, cell surface associated (MUC1), Epidermal Growth Factor Receptor (EGFR), epithelial cellular adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)), e.g., EVs derived from breast tumors. These methods can include treatment monitoring during chemotherapy to change a course of therapy during ongoing therapy, e.g., using markers HER2 and EpCAM.

[0055] Certain terms employed with this document are collected here. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims.

[0056] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not.

[0057] The singular terms “a,” “an,” and “the” comprise plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation, “e.g.” is used herein to indicate a non-limiting example.

[0058] The term “periodicity,” as used herein, refers to a recurrence or repetition of a nanostructure at regular intervals by their positioning on the nanostructure. The term “periodic” as used herein therefore refers to the regular predefined pattern of nanostructures with respect to each other, such as a lattice or other repeating unit configuration. A random distribution of nanostructures is a periodic pattern.

[0059] “Surface plasmon resonance (SPR),” as used herein, refers to the physical phenomenon in which incident light stimulates collective resonant electron oscillations at planar metal surfaces, and in particular at interfaces between negative and positive permittivity materials stimulated by incident light.

[0060] The term “localized surface plasmon resonance (LSPR)” refers to surface plasmon resonance of nanometer-sized structures, such as a metallic nanoparticle. The oscillating electrons produce strong electromagnetic fields in the (non-conducting) ambient medium near the surface of the metal. As used herein, “surface plasmons,” “surface plasmon polaritons,” or “plasmons” refer to the collective oscillations of free electrons at plasmonic surfaces, such as metals. These oscillations result in self-sustaining, surface electromagnetic waves that propagate in a direction parallel to the metal / di electric (or metal / vacuum) interface. Because the wave is on the boundary of a metal and the external medium (air or water for example), these oscillations are very sensitive to any refractive index change of this boundary, such as, for example, the adsorption of a molecular target, such as an EV, to the metal surface. Additionally, the electromagnetic field strength decays exponentially from the metal surface to the surrounding environment (e.g., vacuum or dielectric). A maximum value of the electromagnetic field strength can be found at the metal / di electric or metal / vacuum interface.

[0061] As used herein, the term “sample” means any biological or other fluids that may contain one or more extracellular vesicles (e.g., exosomes). Such biological fluids include, without limitation, fluids derived from or containing cells, organisms (bacteria, viruses), lysed cells or organisms, cellular extracts, nuclear extracts, components of cells or organisms, extracellular fluid, media in which cells or organisms are cultured in vitro, blood, plasma, serum, gastrointestinal secretions, ascites, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, pleural fluid, nipple aspirates, breast milk, external sections of the skin, respiratory, intestinal, and genitourinary tracts, and prostatic fluid. A sample can be a viral or bacterial sample, a sample obtained from an environmental source, such as a body of polluted water, an air sample, or a soil sample, as well as a food industry sample.

[0062] A “biological sample” is derived or obtained from a living organism. The organism can be a whole organism or can be cells or organs grown in culture. In one embodiment, a “biological sample” also refers to a cell or population of cells or a quantity of tissue or fluid from a subject. Most often, a sample has been removed from a subject, but the term “biological sample” can also refer to cells or tissue analyzed in vivo i.e., without removal from the subject. Often, a “biological sample” will contain cells from a subject, but the term can also refer to non-cellular biological material, such as non- cellular fractions of blood, saliva, or urine. In one embodiment, a biological sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary, secondary, or metastatic tumor, e.g., breast, ovarian, pancreatic, biliary tract, colorectal, glioblastoma, lung tumors or a cell block from pleural fluid. In addition, fine needle aspirate biological samples are also useful. In one embodiment, a biological sample includes primary ascites cells.

[0063] Biological samples also include explants and primary and / or transformed cell cultures derived from patient tissues. A biological sample can be provided by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells or cellular extracts (e.g., isolated by another person, at another time, and / or for another purpose). Archival tissues, such as those having treatment or outcome history may also be used. Biological samples include, but are not limited to, tissue biopsies, scrapes (e.g., buccal scrapes), whole blood or other bodily fluids, such as plasma, serum, urine, saliva, cell culture, urine, ascites, pleural fluid, Broncho Alveolar Lavage Fluid (BALF), or cerebrospinal fluid.

[0064] The samples analyzed by the compositions and methods described herein may have been processed for purification or enrichment of EVs contained therein.

[0065] As used herein, an “extracellular vesicle” (“EV”) refers to a naturally occurring or synthetic vesicle that includes a cavity inside. The EVs comprise a lipid bilayer membrane enclosing contents of the internal cavity. An EV can include, but is not limited to, an ectosome, a microvesicle, a microparticle, an exosome, an oncosome, an apoptotic body, a liposome, a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, a matrix vesicle, or a multivesicular body. An EV has a dimension of up to about 10 microns, but are typically about 1000 nm or less.

[0066] Exosomes and microvesicles are types of EVs, and can be shed by eukaryotic cells, or budded off of the plasma membrane, to the exterior of the cell. These membrane-bound vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 5000 nm. The methods and compositions described herein are equally applicable for microvesicles of all sizes.

[0067] In some of the literature, the term “exosome” also refers to protein complexes containing exoribonucleases that are involved in mRNA degradation and the processing of small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and ribosomal RNAs (rRNA). Such protein complexes do not have membranes and are not “microvesicles” or “exosomes,” and thus are not EVs, as those terms are used here in. As used herein, the term “patient” and “subject” are used interchangeably to refer to a human or animal, such as a vertebrate, e.g., a mammal. Examples of mammals include, without limitation, primates, rodents, domestic animals, or game animals. Primates include chimpanzees, monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, rabbits, and hamsters. Domestic and game animals include cows, horses, pigs, deer, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, avian species, e.g., chicken, duck, and ostrich, and fish, e.g., trout, bass, and salmon. Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates, or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. A subject can be male or female. Additionally, a subject can be any stage of development, e.g., embryo, fetus, infant, child, pre-adolescent, adolescent, young adult, mature adult, and elderly adult. The female subject can be pregnant or not.

[0068] In one embodiment, the subject can be a patient or a subject in a clinical setting. The subject can be suspected of, or at risk for, having or developing a disease or disorder, or may have already been diagnosed as having a disease or disorder. The subject may be a patient undergoing treatment.

[0069] As used herein, a “capture agent” refers to any agent having specific binding for EVs generally (e.g., an exosome) or target EVs. Binding may be to a marker, e.g., biomarker, which is present on all EVs, or to a subset of target EVs. Typically the capture agent specifically binds to a biomarker fully or partially present on the external surface of the EVs (referred herein as an extravesicular marker), although in some embodiments, the capture agent specifically binds to a marker that is present on the intenor of the EV (referred herein as an intravesicular marker). The capture agent is immobilized on the surface of a plasmonic nanostructure that is contacted to the sample (e.g., the sensing area). Examples of capture agents include, without limitation, nucleic acids, oligonucleotides, peptides, polypeptides, aptamers, antigens, polyclonal antibodies, monoclonal antibodies, single chain antibodies (scFv), antibody portions, F(ab) fragments, F(ab')2 fragments, Fv fragments, small organic molecules, polymers, compounds from a combinatorial chemical library, inorganic molecule, or any combination thereof. A “nucleic acid,” as described herein, can be RNA or DNA, and can be single or double stranded, and can be, for example, a nucleic acid encoding a protein of interest, a polynucleotide, an oligonucleotide, a nucleic acid analogue, for example peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc. Nucleic acid sequences include, for example, but are not limited to, nucleic acid sequences that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.

[0070] As used herein, the term DNA is defined as deoxynbonucleic acid. The term “polynucleotide” is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides. Typically a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxy adenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine) joined by phosphodiester bonds. However the term encompasses molecules including nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules can be used for certain applications.

[0071] The term “polypeptide” as used herein refers to a polymer of amino acids. The terms “protein” and “polypeptide” are used interchangeably herein. A peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and / or amino acid analogs known in the art can be used. One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc. A polypeptide that has a non-polypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide.” Examples of modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc. An “antigen” is defined herein as a substance inducing an immune response. The antigenic determinant group is termed an epitope, and the epitope in the context of a carrier molecule (that can optionally be part of the same molecule, for example, botulism neurotoxin A, a single molecule, has three different epitopes. Usually antigens are foreign to the animal in which they produce immune reactions.

[0072] As used herein, “antibodies” can include polyclonal and monoclonal antibodies and antigen-binding derivatives, or portions or fragments thereof. Well-known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab')2 fragment. Methods for the construction of such antibody molecules are well known in the art. As used herein, the term “antibody” refers to an intact immunoglobulin or to a monoclonal or polyclonal antigen-binding fragment with the Fc (crystallizable fragment) region or FcRn binding fragment of the Fc region. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. “Antigen-binding fragments” include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc', F(ab’) 2 and Fv are employed with standard immunological meanings (see, e.g., Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W. R. (1986) The Experimental Foundations of Modem Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford).

[0073] The term “reporter group,” as used herein, refers to a composition capable of producing or enhancing a detectable optical signal indicative of the presence of the target in a sample. Examples of reporter groups include fluorescent molecules, such as fluorescein isothiocyanate (FITC), tetramethylrhodamine (TRITC), Alexa Fluor® 488, Cy3, Cy5, Cy5.5, and Cy7; small molecules for Raman signals, such as benzenethiol, 4,4’-bipyndine, and R6G; and nanoparticles made of metal, semiconductor, plastic, polymer, and glass. Another example of reporter groups is a QUAD marker (Mucin 1, cell surface associated (MUC1), Epidermal Growth Factor Receptor (EGFR), epithelial cellular adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)), The terms “label,” as used herein, refer to a composition capable of producing or enhancing a detectable signal indicative of the presence of the target in a sample. “Labeling” is referred herein as a primary' antibody conjugated with a biomarker, or a secondary antibody conjugated with a primary antibody.

[0074] As used herein, the term “marker” or “biomarker” refers to a molecule that is associated with an EV, and can bind to a capture agent for detecting the EV. A marker can be any components of an EV that can be recognized by a capture agent. Examples of markers include, without limitation, proteins, or nucleic acids or a component of the lipid bilayer that makes up the membrane of the EV. Useful markers include receptors (e.g., extracellular) and channel components. A marker can be either an extravesicular or an intravesicular marker, as defined herein. A marker can be present on all EVs in a sample, or on a subset of EVs in a sample. A marker that is common to all EVs in a sample is referred to herein as a pan-EV marker.

[0075] The term “QUAD biomarkers” as used herein refers to MUC1, EpCAM, HERZ, and EGFR, which can all be labeled with reporter groups, such as fluorescent markers.

[0076] As used herein, when one element is “fixed” to another, the two elements are directly connected via bonds, e.g., ionic, covalent, polar, or hydrogen bonds. When two elements are “bound” to each other, the two elements are directly or indirectly connected, via bonds, or via other elements, such as linker groups, e.g., PEG, or affinity ligands as described herein.

[0077] An “affinity ligand” is defined herein as a molecule that is directly attached or fixed to a molecular spacer or to a substrate or nanostructure, and also can be directly attached to a capture agent or a molecular spacer. In some embodiments, an affinity ligand can be a capture agent. Stated another way, an affinity ligand physically links a molecular spacer (or substrate or nanostructure) and a capture agent (or molecular spacer) together. In one embodiment, the affinity ligand is a first member of a specific binding pair. In such an embodiment, the capture agents may be the second member of the specific binding pair. Examples of such specific binding pairs include, without limitation, antigens, antibodies, haptens, oligonucleotides, polynucleotides, avidin, streptavidin, hormones, receptors, lectins, carbohydrates, IgG, protein A, and nucleic acid binding proteins. An affinity ligand can include, but is not limited to, a nucleic acid, oligonucleotide, peptide, polypeptide, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), an antibody portion, F(ab) fragment, F(ab')2fragment, Fv fragment, small organic molecule, polymer, compounds from a combinatorial chemical library, inorganic molecule, or any combination thereof.

[0078] Examples of specific binding pairs include antigen-antibody, hapten-antibody or antibody-antibody pairs, complementary oligonucleotides or polynucleotides, avidinbiotin, streptavidin-biotin, hormone-receptor, ligand-receptors, lectin-carbohydrate, IgG- protein A, nucleic acid-nucleic acid binding protein, and nucleic acid-anti-nucleic acid antibody.

[0079] As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and / or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity that is at least 10 times greater than the affinity for the third non-target entity. A reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized. In certain embodiments, specific binding is indicated by a dissociation constant on the order of <10sM. <109M. <1010M or below.

[0080] Polyethylene glycol (PEG) is referred to herein as a possible component of the nano-plasmonic array and is used as a molecular spacer. A variety of forms and combinations of PEG are envisioned for use as such spacers. Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. The structure of PEG is (note the repeated element in parentheses): H — (0 — CH2-CH2)n-OH. PEG is also known as polyethylene oxide (PEG) or polyoxyethylene (POE), depending on its molecular weight. PEG, PEG, or POE refers to an oligomer or polymer of ethylene oxide. The three names are chemically synonymous, but as used herein, PEG refers to oligomers and polymers with a molecular mass below 20,000 g / mol, PEG refers to polymers with a molecular mass above 20,000 g / mol, and POE refers to a polymer of any molecular mass. PEG and PEO are liquids or low-melting solids, depending on their molecular weights. Different forms of PEG are also available, depending on the initiator used for the polymerization process — the most common initiator is a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG. Lower-molecular-weight PEGs are also available as purer oligomers, referred to as monodisperse, uniform, or discrete. Branched PEGs have three to ten PEG chains emanating from a central core group. Star PEGs have 10 to 100 PEG chains emanating from a central core group. Comb PEGs have multiple PEG chains normally grafted onto a polymer backbone.

[0081] A “long-chain polyethylene glycol (PEG)” or “long PEG” is defined herein as a PEG polymer having a molecular weight equal to or higher than 750 Da.

[0082] A “short-chain PEG” or “short PEG” is defined herein as a PEG polymer having a molecular weight equal to or less than 500 Da.

[0083] As used herein, “expression level” refers to the number of mRNA molecules and / or polypeptide molecules encoded by a gene of interest that are present in a cell or sample.

[0084] The plasmon-enhanced sensing methods using the new assay configurations and tests described herein enable sensitive, multiplexed EV analysis through simple assay procedures, and the multiplexed single EV analysis described herein significantly improves the detection accuracy for early cancer detection, treatment monitoring, and MRD monitoring.

[0085] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordin arv skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0086] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

[0087] DESCRIPTION OF DRAWINGS

[0088] FIG. 1 is a schematic illustration of an assay configuration designed to capture all EVs on a plasmonic resonance chip and label them with various combinations of different antibodies that bind selectively to different types of biomarkers on the surfaces of the EVs from different ty pes of cells. FIG. 2 is a schematic illustration of an assay configuration designed to detect a single subpopulation of EVs, e.g.. tumor-derived EVs, on a plasmonic resonance chip and interrogate drug resistant biomarkers within a tumor-derived EV subpopulation.

[0089] FIG. 3 is a schematic illustration of an assay configuration designed to capture a specific subpopulation of marker-positive EVs using a capture antibody, in this example EVs from a specific type of tumor, and label them with different antibodies that bind to additional biomarkers.

[0090] FIG. 4 is a schematic illustration of an assay configuration designed to detect various samples using a single plasmonic resonance chip.

[0091] FIG. 5 A is a highly magnified image of a “nanoparticle on pillar” (NPOP) plasmonic resonance chip substrate.

[0092] FIG. 5B is a bar graph showing mean fluorescence intensities streptavidin conjugated withAF488, Cy3, AF647, and Cy7 when they are immobilized on NPOP and plain Au substrates. Fold changes were shown on the top of the bar graphs.

[0093] FIG. 5C is a schematic illustration overview of multi-channel single EV analysis. An NPOP substrate was functionalized by SH-PEG-COOH (1.0 kDa), and the carboxylate functional groups were activated using EDC / NHS to capture AF555-labeled EVs. Alternatively, antibodies can be immobilized on the substrate surface to capture marker-specific EV subpopulations. Next, the captured EVs were fluorescently labeled by marker-specific antibodies in different fluorescence channels. Finally, the multi-color labeled EVs were imaged and analyzed. Scale bar, 10 pm.

[0094] FIGs. 6A-H are a series of images and graphs that illustrate plasmon-enhanced EV detection on NPOP substrates. EVs were fluorescently labeled by the same amount of AF488, AF555, AF647, and Cy7 fluorophores, and the differentially diluted EVs were mounted onto the adhesive molecule-treated substrates. A. Representative images of fluorescently labeled EVs on glass or NPOP substrate. Scale bar, 10 pm. B-D. Plots for comparison of EV intensities in different dilution factors on glass or NPOP substrates. AF555 co-localized EV signals with AF488 (B), AF647 (C), and Cy7 (D) were analyzed for intensity analysis. E. Bar graph for quantitative analysis of signal enhancement. The value of fold changes for each fluorescence was indicated on the top. Error bars are shown as mean ±SD from triplicate measurements. F-H. Scatter plots for individual EV fluorescence intensities labeled by AF555 and AF488 (F), AF647 (G), and Cy7 (H). FIGs. 7A-B are heatmaps that demonstrate results of single EV analysis on NPOP substrates. These heatmaps show fluorescence bleed-through to the other channel by an optical system with multi-band pass filter (A) and single cube filters (B).

[0095] FIG. 7C is a series of representative images for EV capture by functionalization with MUA (1.7 nm), SH-PEG-COOH (0.3 kDa, 2.8 nm), and SH-PEG-COOH (1.0 kDa, 7 nm) with or without EDC / NHS activation. Scale bars, 15 pm.

[0096] FIG. 7D is a bar graph of EV counting showed SH-PEG-COOH showed the least non-specific EV binding, ns, not significant; ****P < 0.0001 compared with the mock treatment, as assessed by two-way ANOVA with Bonferroni's multiple comparisons test. Error bars are shown as mean ±SD from the ten different images.

[0097] FIGs. 7E and 7F are a series of bar graphs that show comparisons of antibody immobilization method of physisorption or PEG methods. Captured AF555 (EV, 7E) counts and co-localized signal with AF647 / AF555 (HER2 / EV, 7F).

[0098] FIGs. 7G-J area a series of bar graphs that show selection of antibody concentrations against QUAD breast cancer markers, including MUC1 (G), HER2 (H), EpCAM (I), and EGFR (J). Bar graphs indicate the co-locahzation percentages of each antibody and EV signal. Lines indicate the signal-to-noise ratio in marker-positive and negative EV sets. The antibody concentration showed the highest value of signal-to-noise ratio was selected as the optimal concentration of each antibody. Error bars are shown as mean ±SD from triplicate measurements.

[0099] FIG. 8Ais a series of representative images of molecular profiling of single tumor cell-derived EVs for Quad markers (MUC1, HER2, EGFR, and EpCAM) in EVs derived from four different breast cancer cell lines. SKBR3, ERPR- / HER2+; MCF7, ERPR+ / HER2-; BT474, ERPR+ / HER2+; MDA-MB-231, ERPR- / HER2-. The min-max intensity values of each channel were shown in green (AF555) or red (AF647). Scale bar, 10 pm.

[0100] FIG. 8B is a series of bar graphs that show the percentages of co-localization for the EVs and Quad markers. Error bars are shown as mean ±SD from the four different images.

[0101] FIG. 8C is a pair of heat maps that show the differential abundances of Quad markers in four different tumor cell line-derived EVs, as assessed by single EV analysis (top) and originated cell lines, as assessed by flow cytometry (bottom). FIG. 8D is a dot-plot graph for a Pearson correlation coefficient that reveals that the multi-channel single EV analysis showed similar molecular patterns to the flow cytometry (r = 0.9656, P < 0.0001). The percentage of colocalization from the multichannel single EV analysis and flow cytometry were indicated in y- and x-axis, respectively. The solid blue line indicates the best linear fit. The two dashed lines indicate the 95% confidence interval.

[0102] FIG. 9Ais a series of representative images of multiplexed single EV detection and analysis on an NPOP substrate for pan-CDs (CD9, CD63, and CD81; AF488 dye), Quad marker (MUC1, HER2, EGFR, and EpCAM; AF647 dye), and additional HER2 (DL755 dye) profiling in EVs (AF555 dye) derived from four different breast cancer cell lines and normal HS371T cells. No EV sample was used as a negative control. The min- max intensity values of each channel were indicated at the bottom of the first row of images. Scale bar, 10 pm.

[0103] FIGs. 9B-D are a series of scatter plots for individual EV fluorescence intensities for pan-CDs (FIG. 9B, AF488 / AF555), Quad (FIG. 9C, AF647 / AF555), and HER2 (FIG. 9D, DL755 / AF555). The dashed lines indicate the intensity threshold value, which is mean + 3 times the standard deviation of no EV control to determine the marker positive signal. The pan-CDs, Quad, and HER2 -positive signals were shown in green-, red-, and violet-colored dots, respectively.

[0104] FIGs. 10A-D area a series of graphs that show the comparison of the mean fluorescence intensities fluorophore-conjugated streptavidin with AF488 (FIG. 10A), Cy3 (FIG. 10B), AF647 (FIG. 10C), and Cy7 (FIG. 10D) between the NPOP and plain Au substrates in different concentrations. Bar graphs for quantitative analysis of signal enhancements at 5 pg / ml of streptavidin are shown in the right. * denotes P < 0.01 in the Mann-Whitney test. Error bars are shown as mean ±SD from the six different images.

[0105] FIG. 11 A is a series of representative images of fluorescently labeled EVs (AF488, AF555, AF647, and Cy7) on glass and NPOP substrate that show the min-max intensity values of each channel indicated in the bottom of the first row images as a comparison of fluorescence signal enhancement. Scale bar, 10 pm.

[0106] FIGs. 11B-E are a series of histograms of detected EV counts based on fluorescence intensities for AF488 (FIG. 11B), AF555 (FIG. 11C), AF647 (FIG. HD), and Cy7 (FIG. HE). FIG. 12 is a bar graph overlapped with a graph that together show the results of rat-originated HER2 antibody concentration on the x-axis and the percent co-localization on the y-axis. The bar graphs indicate the co-localization percentages of HER2 antibody and EV signal. The solid line indicates the signal-to-noise ratio in the HER2-positive and HER2 -negative EV sets. The antibody concentration that showed the highest value of signal-to-noise ratio was selected as the best concentration of HER2 antibody. Error bars are shown as mean ±SD from quadruplicate measurements.

[0107] FIG. 13 A is an image of the results of a Western blotting analysis for the tetraspanins (CD9, CD63, and CD81) in EV lysates from five different cell lines including a benign cell line and four different breast cancer cell lines.

[0108] FIG. 13B is a series of graphs and images showing the size distributions and concentrations of cell line-derived EVs, as measured by nanoparticle tracking analysis. TEM images for each EVs were shown in the box. Scale bar. 100 nm.

[0109] FIGs. 14A-C are a senes of graphs showing changes of HER2 expression in the differential mixture of SKBR3 (HER2+) and MCR7 (HER2-) EVs. FIG. 14A shows EV counts (AF555). FIG. 14B shows counts and FIG. 14C shows co-localization percentages of HER2 (AF647) and EV (AF555) signal. Error bars are shown as mean ±SD from the four different images.

[0110] FIG. 14D is a series of three scatter plots for individual fluorescence intensities of EV(AF555) and HER2 (AF647) signal of MCF7 EV (left; MCF7:SKBR3=100:0, middle; MCF7:SKBR3=57. 1:42.9, right; MCF7:SKBR3=0: 100). The dashed lines indicate the intensity threshold value, which is mean + 3 times of standard deviation of no EV control to determine the marker-positive signal. HER2 -positive signals were shown in red colored dots (above dashed line) vs black dots (below dashed line).

[0111] FIGs. 15A-D are a series of bar graphs that show EV detection and marker profiling on NPOP substrates. These bar graphs show the EV counts (FIG. 15A, AF555) and the percentages of marker-positive EVs for pan-CDs (FIG. 15B, AF488 / AF555), QUAD (FIG. 15C, AF647 / AF555), and HER2 (FIG. 15D, DL755 / AF555). Error bars are shown as mean ±SD from the four different images.

[0112] FIGs. 16A-C is a series of bar graphs showing the results of profiling of cell-line- derived EVs spiked in normal human plasma. EVs from SKBR3 and MCF7 breast cancer cell lines were spiked in normal human plasma samples, and their marker signals were compared in the naive and spiked-in samples. A negative control includes ano-EV sample. The bar graphs in FIGs. 16A-C show marker-positive EV counts that were analyzed for spiked-in samples (blue bars, left side) and the same concentration of cell line EVs (red bars, right side). The results show the assays accurately detect markerpositive EVs for Cdmix (16A), QUAD (16B), and HER2 (16C) from plasma samples.

[0113] FIGs. 16D-I are a series of 2D scatter plots that show the marker-positive EVs. All EVs were labeled to be detected in the AF 555 channel, while respective markers were detected in AF488 for CDmix (a mixture of antibodies against CD63, CD81, and CD9, which are ubiquitously present in EVs) in AF647 channel for QUAD and DL755 channel in HER2. The plots show similar marker-positive rates for SKBR3- and MCF-7 EVs (16E, 16G, and 161) when they are spiked in plasma from ahealthy control (16D, 16F, and 16H).

[0114] FIGs. 17A-17D are a series of bar graphs that show the results of analysis of changes of tumor-derived EVs and p-gly coprotein (PgP) / survivin-positive tumor-derived EV counts before and after receiving paclitaxel treatment. EVs from two plasma samples obtained before and after paclitaxel neoadjuvant therapy were processed and the difference was analyzed. Green (dark) bars below zero indicate patients who showed good responses to paclitaxel, and red (lighter) bars above zero indicate patients who did not respond well to paclitaxel.

[0115] FIGs. 18A-18C are a pair of bar graphs, and a heat map, respectively, that show the results of the detection of tumor-derived EVs in human plasma samples.

[0116] DETAILED DESCRIPTION

[0117] The present disclosure relates to new assay configurations for detecting single EVs, or single types of EVs to provide a new sensing platform for EVs, which have emerged as promising circulating biomarkers in liquid biopsies for early cancer detection, treatment monitoring, and MRD analysis. These nano-sized vesicles are actively shed by tumor cells, carry a trove of biomolecules reflecting the molecular status of the originating tumor, and are abundantly present in easily accessible bio-fluids for liquid biopsy analysis, making them attractive new biomarkers. However, a major technical challenge in molecular EV analysis is the need for a sensitive, robust, and reproducible EV detection method, particularly for EV proteins that often require large sample amounts and extensive processing in conventional assays. The present new assay configurations provide a solution to these problems.

[0118] For example, the new methods can be used investigate treatment responses for cancer therapy, such as anti-HER2 therapy in breast cancer, e.g., trastuzumab deruxtecan (ENHERTU®), an anti-HER2 antibody-drug conjugate (ADC) for HER2-low breast cancer patients. While the treatment is effective, the resistance and side effects of toxic payload exist. The new methods disclosed herein enable monitoring of the cancer’s response and / or resistance to the drug in individual patients, and will greatly improve patient care with the drug treatment and pave new opportunities for other ADCs under trials.

[0119] The tumor-derived EVs (tEVs) contain protein and nucleic acid cargos, representing a molecular “snapshot” of tumors. The large abundance and ubiquitous presence of tEVs in bodily fluids have suggested the potential use of EVs as liquid biopsy biomarkers, because this analysis is minimally invasive and thus provides an easy way for repeated sampling using simple blood tests. Thus tEVs have particular utility for longitudinal disease monitoring and early detection of relapse, because the amount and molecular profiles of tEVs correlate with tumor burden and treatment efficacy.

[0120] Because non-tumor cells also shed EVs into circulation, the characterization of single EVs is critical to detect target-specific EVs, tumor-specific molecular biomarkers, and their temporal changes. However, single EV analysis is technically challenging due to EVs’ small sizes (i.e., fast movement) and surface areas (i.e., weak signals). The use of plasmonic nanostructures as described herein significantly amplifies EVs’ fluorescence signals. The enhancement is achieved by simply using a nanoplasmonic substrate instead of conventional glass or other plain substrates. Plasmon-enhanced fluorescence imaging enables single EV analyses with significantly improved sensitivity. As described herein, the new assay configurations provide highly sensitive and accurate single EV analysis. In addition, the examples described herein demonstrate the technology’s performance for profiling tumor-derived EVs across various breast cancer cell lines to monitor temporal changes upon drug treatments in both sensitive and resistant cell types.

[0121] EV Detection Assays This disclosure provides three different assay configurations that can be used for detecting and analyzing different types of EVs in different detection scenarios.

[0122] Assay Configuration 1 - Capturing all EVs and Labeling with Different Antibodies

[0123] As shown in FIG. 1, the first configuration is useful for detecting and analyzing all EVs in a given biological sample, e.g., that contains different types of EVs, shown in this figures as EV#1, EV#2, etc. By using different antibodies that bind selectively to the different types of EVs, this assay can be used to quantify a total EV count (by counting all antibodies), and relative counts for various subpopulations / types of the different EVs, by detecting the different types of antibodies, e.g., by using a different reporter group, e.g., different fluorescent label for the different types of antibodies. As shown in FIG. 1, antibody! selectively binds to any EV that is in the first subpopulation, EV#1. Antibody 1 selectively binds to any EV that is in the second subpopulation, EV#2, and so on.

[0124] The EVs can be bound to the substrate using any know methods, e g., the EVs can be captured directly on the substrate or using a linker layer immobilized on the substrate. The linkers can be short carbon chains, polyethylene glycol, and / or other polymers. These linkers are non-selective, in that they bind EVs of all subtypes to the substrate.

[0125] This assay configuration can be used when a target subpopulation is present at or above a certain percentage (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%), and this assay configuration is also useful when one needs to analyze EVs from different cellular origins. Examples include the analysis of EVs from tumors and different types of immune cells. This assay configuration is also useful for analyzing EVs for non-tumorous diseases, such as Alzheimer’s disease, fibrosis, other forms of dementia, lung fibrosis, tuberculosis, and liver cirrhosis. For example, for Alzheimer’s Disease, this assay can analyze EVs from different cell types in the brain (i.e., astrocytes, neurons, and microglia). For fibrosis, this assay can be used to analyze EVs from different cell types in the lung (e.g., epithelial, endothelial, fibroblast, and immune cells).

[0126] This multichannel, multiplexed analysis enables the profiling of disease- associated biomarkers in each subpopulation. Assay Configuration 2 - Capturing All EVs and Analyzing a Specific Subpopulation of EVs

[0127] The assay configuration shown in FIG. 2 illustrates the detection of a specific subpopulation or type of EVs, such as tumor-derived EVs (shown in FIG. 2 as “EV tumoral”), and subsequent profiling of drug-resistant biomarkers in only that specific type of EV, such as tumor-derived EVs.

[0128] In this assay configuration, there are still multiple different subpopulations of EVs bound to the substrate, as in the first assay configuration, but here, only a specific subpopulation of EVs is targeted with a first type of antibody, e.g., a QUAD antibody, as described herein. Thereafter, additional antibodies such as PgP or survivin antibodies, are used to analyze the drug resistance within the tumor-derived EV subpopulations. Additional antibodies can be used.

[0129] Like Configuration 1, Configuration 2 can be used when a target subpopulation, or subpopulation of interest, is not rare, present at or above a certain percentage (e.g., 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%).

[0130] Assay Configuration 3 - Capturing a Specific Subpopulation of EVs and Labeling for Biomarkers

[0131] As shown in FIG. 3, this third configuration is useful for detecting and analyzing a specific subpopulation of EVs recognized by capture antibodies that selectively bind to the particular subpopulation of EVs. This is different than in the first and second configurations, in which all EVs are first bound to the NPOP substrate.

[0132] The capture antibodies can be monoclonal antibodies for a single biomarker, e.g., target protein, on the surface of the specific subpopulation of EVs or a mixture of antibodies for multiple targets within the same subpopulation of EVs. This assay configuration enables one to quantify specific, marker-positive EV counts and to then detect additional biomarkers on the captured EVs.

[0133] Other affinity ligands (e.g., aptamers, DNA / RNA linkers) can be used instead of antibodies as capture affinity ligands. The capture affinity ligands can be immobilized directly on the NPOP substrate or via a linker layer immobilized on the substrate. The linker can also include short carbon chains, polyethylene glycol, and other polymers. This third configuration is useful when a target subpopulation is rare, e.g., tumor- derived EVs, such as subpopulations that are present at below a certain low percentage of total EVs in the biological sample, e.g., at or below 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.95, or 1.0%. Thus, one can use this configuration when one needs to analyze rare target EVs in the presence of a large number of non-target EVs. Examples include the analysis of EVs from tumors and different types of immune cells. This third configuration is also useful for analyzing EVs from small numbers of cells. For cancers, one can use this assay configuration to detect small, early-stage cancers.

[0134] In other implementations, the multichannel, multiplexed analysis enables the profiling of disease-associated biomarkers in the EV subpopulation. For example, it can be used to quantify and monitor tumor-derived EV counts and drug-resistant biomarkers within the tumor-derived EVs.

[0135] In some implementations, one can use one, two, or all three different configurations, in one or more different orders, to provide a highly selective and precise analysis of different subpopulations of EVs within larger collections of multiple different types of EVs within a given biological sample. It is particularly important to analyze even very scant rare tumor-derived EVs in clinical samples. For example, it is expected that the rate of tumor-derived EVs in plasma samples could be lower than 0.1% of total EVs. The rate could be even lower for early-stage cancer when the tumor size is small. In this case, the assays described in FIGs. 1 and 2, based on capturing all EVs in a given sample, may not have sufficient sensitivity to detect those rare tumor-derived EVs. In the assay configuration shown in FIG. 3, the target tumor-derived EVs are enriched by antibodies, e.g., QUAD capture antibodies, improving the statistical power of quantifying the tumor-derived EVs in clinical samples.

[0136] FIG. 4 is a schematic view of illustrates an assay configuration designed to detect various samples using a single plasmonic resonance chip, in which the different shades of gray represent different colors of different fluorescent reporter groups. Different configurations with various antibody combinations are made as an array in a single chip. Each colored dot represents different antibody combinations or assay configurations, and thus, in this example shown in FIG. 4, there are 18 different assay configurations arranged and detected on a single chip. In some embodiments of the new detection assay configurations, QUAD biomarkers can be used for multiplexed analysis of surface markers on EVs, e.g., for methods for diagnosing cancer, e.g., cancer of epithelial origin, by detecting the presence of tumor cells in a sample, based (at least in some embodiments) on the quantification of levels of four biomarkers, e g., MUC1, EGFR, EpCAM, and HER2. Of individual biomarkers investigated, MUC-1, HER2, EGFR, and EpCAM provide the highest diagnostic accuracy, but combining these four markers established a very high level of accuracy that is superior to conventional clinical analysis. See, e.g., US Patent No. 10,712,343, which is incorporated herein by reference in its entirety. However, additional or alternative biomarkers can also be used in the multiplexed analysis.

[0137] In some embodiments, the tumor is a pancreas, lung, breast, prostate, kidney, stomach, esophagus, bladder, endometrial, cervix, biliary, thyroid, ovary, brain, head and neck, oral, melanoma, liver, or colon tumor.

[0138] Described herein are methods for diagnosing or detecting the presence of a cancer in a subject. The methods include obtaining a sample from a subject, isolating EVs from the sample, and evaluating the presence and / or level of MUC-1, HER2, EGFR, and EpCAM in the EVs, and comparing the presence and / or level with one or more references, e.g., a control reference that represents a normal level of MUC-1, HER2, EGFR, and EpCAM, e.g., a level in an unaffected subject, and / or a disease reference that represents a level of the QUAD biomarkers in a subject having cancer, using the assay configurations described herein.

[0139] In some embodiments, the methods using the new array configurations include determining a value, e.g., a normalized expression value, for each of the biomarkers (MUC-1, HER2, EGFR, and EpCAM), and calculating a test score from the sum of each of the levels. This score can then be compared to a reference score, wherein the presence of a test score above (e.g., equal to or above, or simply above) the reference score indicates the presence of cancer in the subject. See, e.g., US Patent No. 10,712,343.

[0140] The methods also can be used to monitor a patient, e.g., to determine whether a treatment has been effective, or whether a subject is experiencing a recurrence, or whether treatment resistance is emerging. In these embodiments, the methods include detecting the presence and / or level of biomarkers in EVs obtained from a subject over time, e.g., in a first or baseline sample, and then in one or more subsequent EV samples, e.g., over a period of one or more weeks or months, using the assay configurations described herein. For some biomarkers, a decrease over time in the presence or levels of the biomarkers, e.g., a decrease in the biomarker value, in the EVs indicates an improvement in the disease, e.g., that a treatment administered in the intervening time is effective to cause a decrease in a number of cancerous cells or tumor burden. While the QUAD biomarkers may be used in this analysis, additional or alternative biomarkers may also be used in the multiplexed analysis of surface markers on EVs.

[0141] No change in the presence or levels of the biomarkers, e.g., no change in the QUAD biomarker value of the EVs, indicates no change in disease, e.g., that any intervening treatment was not effective to cause a decrease in a number of cancerous cells or tumor burden (though in particularly aggressive disease the treatment may have been effective to halt progression, which may be a treatment goal).

[0142] A decrease over time in the presence or levels of the biomarkers, e.g., in the QUAD biomarker value, for the EVs, followed by a period of no change or an increase in the presence or levels of the biomarkers, e.g., in the quad biomarker value, indicates that any intervening treatment has lost effectiveness, and may indicate the presence of emerging resistance to the treatment.

[0143] An increase over time in the presence or levels of the biomarkers, e.g., an increase in the QUAD biomarker value, indicates that the disease is progressing, e.g., there has been an increase in a number of cancerous cells and / or tumor burden. Such an increase may also be indicative of a poor prognosis, e.g., an increased likelihood of mortality.

[0144] In some embodiments, the methods include the use of antibodies or antigen fragments thereof, or oligonucleotides, that specifically bind (i.e., do not bind substantially to other molecules) to the biomarkers. The term "antibody" as used herein refers to an immunoglobulin molecule or an antigen-binding portion thereof. Examples of antigen-binding portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments, which retain the ability to bind antigen. The antibody can be polyclonal, monoclonal, recombinant, chimeric, de-immunized or humanized, fully human, nonhuman, (e.g., murine), or single chain antibody. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind to an Fe receptor. For example, the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fe receptor, e.g., it has a mutagenized or deleted Fe receptor binding region. Antibodies that can be used in the assay configurations descnbed herein are described in further detail below.

[0145] Methods for making antibodies and fragments thereof are known in the art. See, e.g., US Patent No. 10,712,343 and the citations therein.

[0146] Characteristics ofNPOP Substrates for Plasmon-Enhanced Single EV Analysis

[0147] The primary objective of using NPOP substrates is to amplify fluorescent signals to detect relatively rare EVs, e.g., cancer cell-derived EVs, with various surface markers at the single EV level. The NPOP substrate provides a wide plasmonic hotspot in a 3D structure of gold nanopillars with AuNPs (FIG. 5 A), and is described in PCT Application Nos. WO2021 / 211756, entitled “Methods and Systems of Enhancing Optical Signals of Extracellular Vesicles,” WO2024 / 026314, entitled, “Methods and Systems of Enhancing Electromagnetic Radiation Signals from Extracellular Vesicles,” and WO2023 / 220377, entitled “Physisorption of Antibodies on Films,” which are incorporated herein by reference in their entireties, including the figures.

[0148] The nano-plasmonic arrays, e.g., NPOP substrates, can be made as follows. In general, the nano-plasmonic arrays used herein include a substrate, a plurality of nanostructures on or in the substrate, and a plurality of affinity ligands fixed on or adjacent to the nanostructures. Different surface chemistries (conjugates to affinity ligands) can be used for the metals used to make the nanostructures and the substrates (e.g., glass) to selectively fix the affinity ligands to the nanostructures, e.g., on the surface of nanorods or nanodisks, within nanogrooves, and on walls within the nanoholes or on the substrate or metal film adjacent to the nanoholes. The plurality of nanostructures are arranged to form a periodic array of nanostructures on the substrate, and the periodic array of nanostructures is arranged and dimensioned to amplify one or more specific wavelengths of electromagnetic radiation.

[0149] In some embodiments, the plurality of affinity ligands is fixed on or adjacent to the nanostructures, and the plurality of affinity ligands specifically bind to EVs or target EVs via a capture agent. Different types of affinity ligands can be used in the nano- plasmonic arrays based on a corresponding EV preparation. For example, among high affinity binding pairs, the substrate of the nano-plasmonic arrays can include a biotinbinding protein (e.g., avidin) as the affinity ligands attached on the substrate, then the EVs or target EVs are required to comprise a corresponding biotin as the capture agent to be captured by the nano-plasmonic arrays. In some embodiments, the substrate comprises semiconductors, non-conductors, plastics, or any suitable transparent substrates. Methods for attaching a corresponding capture agent to EVs are described below.

[0150] The systems include arrays of metal, e.g., gold, silver, copper, or aluminum, nanostructures, e.g., nanoholes, nanorods, nanodisks, or nanopillars, e.g., in sub-200 nm dimensions, which can be occupied by single EVs. For example, gold nanorods have a high sensitivity down to single molecule detection and precise tunability of resonant wavelengths by adjusting nanorod dimensions. The arrays of nanostructures can be made using standard nanoimprint lithography techniques with good reproducibility through advanced imprinting and deposition processes.

[0151] The capture of individual EVs on each nanostructure, e.g., nanorod, induces a spectral shift; those shifts from nanorod arrays will be simultaneously detected by darkfield imaging. Extensive validation studies are performed to benchmark 1) single EV detection sensitivity; 2) specificity for capture a target EV subpopulation; and 3) robustness and reproducibility.

[0152] Dense arrays (e.g., 105array per cm2) of metal, e.g., gold, nanostructures, e.g., nanorods, can be made using a new nanoimprint lithography method that can pattern gold nanorod arrays in a wafer-scale through simple imprinting and gold deposition processes. The technique utilizes a reusable silicon mold with nano-patterns that are transferred to a target substrate coated with a thin resist layer. After imprinting, gold is deposited onto the patterned area; subsequent removal of the resist will leave nanorod arrays on a glass substrate. This can be done using a known fabrication method (ACS Nano 2011, 5, 7555- 7564, 2011 and Anal. Chem, 84, 6031-6039, 2012).

[0153] In other implementations, periodic nanoholes are made by patterning a thin (50 to 200 nm thick) gold film on a substrate. Nanoholes can be directly patterned by focused ion-beam milling or through lithography and metal etching. Deep ultraviolet (DUV) lithography is used to make 200 nm periodic circular patterns on a resist spun-coated on the gold film. Furthermore, the underlying gold film is etched by reactive ion etching or ion milling using the resist as an etch mask. Resist removal reveals gold nanohole patterns made in the gold film.

[0154] Array chips are designed through comprehensive three-dimensional computational calculations, and we found in one example that the nanorod dimension of 80 nm (length) x 30 nm (width) x 20 nm (height) achieved maximum sensitivity for 100-nm EV (mean diameter) detection, and array sensor dimensions and sensitivities are experimentally tested. The sub-100 nm dimension of gold nanorods also allows single EV capture on each nanorod. Gold nanorod arrays with 3 pm separation between nanorods allow even distribution of EVs on the nanorod arrays; signals from individual EVs are clearly resolved using a 10x or higher objective. The total number of nanorods in a chip is readily scalable with a nanoimprint mold size. For example, one can use a microarray spotter (MicroSys™, Digilab Inc.) to functionalize the chip selectively with affinity ligands. With the spotter, 0.1 pL solutions are transferred from a 96-well plate and spotted on designed areas with good reproducibility (< 5% variation). Temperature and humidity are controlled inside the spotter chamber for consistent sample spotting and incubation conditions.

[0155] In certain implementations, the periodic array of nanostructures has a periodicity of about 400 to 800 nm or 400 to 2000 nm between nanostructures. In certain embodiments, the nanostructures are nanopillars having a dimension of about 20 to about 500 nm in diameter and about 20 to about 300 nm in height with a density of about 300 to about 750 nanopillars per pm2on a substrate (e.g., Si, glass, indium tin oxide / ITO, polyethylene terephthalate (PET) polymer substrate).

[0156] In certain other implementations, the periodic array of nanostructures has a periodicity' of about 2000 nm to 4000 nm or 2500 nm to 3500 nm or of about 2800 nm to 3200 nm, or about 3000 nm between nanostructures. In certain embodiments, the nanostructures are nanopillars having a dimension of about 60 to about 100 nm or about 70 to about 90 nm or about 75 to about 85 nm in length; about 20 to about 50 nm or about 20 to about 30 mm or about 25 to 35 nm in width; and about 10 to about 30 nm or about 15 to about 25 nm or about 20 nm in height.

[0157] A metal, e g., gold, nanostructure, e.g., nanorod, exhibits a unique dark-field light scattering peak at a resonant wavelength. EV binding to the nanorod surface increases a local refractive index, red-shifting the peak wavelength. The spectral shift (i.e. EV binding) can also be detected by measuring a light intensity change at a fixed wavelength, and there is an excellent correlation between spectral and intensity measurements. The intensity measurement method can be used for high throughput parallel signal reading from entire arrays in a field-of-view. This approach is much faster than sequential spectral measurements used in past systems and methods. The dark-field imaging is also compatible with epifluorescence measurements for molecular EV profiling in the same setup.

[0158] EVs, e.g., tumor-derived EVs, can be captured on nanorods and measured by the number of nanorods exhibiting intensity changes induced by EV binding to the nanorod surface. Calculation using a finite difference time-domain (FDTD) solution shows that single 100-nm EV binding induces more than a 10 nm shift, a sufficiently large shift readily detected by dark-field intensity measurements. The signal also correlates with the size of captured EVs, facilitating EV size measurements. Once can use size standard nanospheres for calibration and compare the results with those obtained by a nanoparticle tracking analysis (NT A) system. Combined with molecular profiling, the size information can be used to identify EV subtypes (e.g. exosomes vs microvesicles).

[0159] By employing dark-field imaging, readout signals from the entire arrays can be measured simultaneously. The intensity measurements provide much higher throughput in readouts of vast arrays than spectral measurements. To avoid signal drift or fluctuation due to changes in light source temperature, one can implement temperature controllers to stabilize the light source temperature and / or increase the number of signal averages to reduce background noises.

[0160] A fabrication procedure for making nanopillars is based on enhanced surface diffusion of adsorbed Au atoms (i.e., adatoms) on a low-energy surface; the Au adatoms diffuse into defective sites of the underlying film and form clustered atoms (i.e., AuNPs). This process enables the direct and selective formation of spherical Au nanoparticles through a simple Au deposition process without high-temperature annealing. The AuNPs density can be highly increased by introducing more nucleation sites on the 3D rough Au nanopillar surface.

[0161] The 3D NPOP structures provide high-density hotspots and large effective volumes of molecular binding sites for highly sensitive surface-enhanced Raman spectroscopy (SERS) and plasmon-enhanced fluorescence (PEF). First, a polyethylene terephthalate (PET) substrate is etched with Ar plasma to form polymer nanopillars. A 100 nm-thick Au film is then deposited onto the nanopillars by either conventional sputtering or thermal evaporation to form high-density Au-coated nanopillars. As a space layer, a self-assembled monolayer (SAM) of H. / H.2H.2H-perfluorodecanethiol (PFDT) can be vapor-deposited onto the Au nanopillars. Finally, another layer of Au is deposited onto the PFDT-coated Au nanopillars (see, e.g., Park et al., Advanced Functional Materials, 2019, 29(43): 1904257, and US Patent Application Publication No. US 2019 / 0331605, which are incorporated herein by reference in their entireties, including all figures and reference citations).

[0162] Isolation and Preparation of EVs

[0163] Biological samples are obtained, e.g., from a human or other subject, and cells can be cultured in culture media, such as Dulbecco’s modified Eagle’s medium (DMEM, Cellgro). Media can be supplemented with serum, e.g., 10% Fetal Bovine Serum, antibiotics, e.g., penicillin and / or streptomycin, and kept under 5% CO2 (see, e.g., Min et al., Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles, Advanced Biosystems, 2020, 4, 200003. DOI: 10.1002 / adbi.202000003, which is incorporated herein by reference in its entirety, including all figures and reference citations).

[0164] EVs can be isolated using both standard ultracentrifugation (UC) and sizeexclusion chromatography (SEC) methods. Furthermore, EVs are isolated from the medium for the next process. For UC, the filtrates are concentrated, e.g., by 100,000 x g for 1 hour. After the supernatant is removed, the EV pellet is washed, e.g., with PBS and centrifuged again, e.g., at 100,000 x g for 1 hour. The EV pellet is resuspended in buffer or serum, e.g., in PBS. For SEC, the filtrates are loaded onto filters, e.g., MWCO = 10 kDa, and centrifuged, e.g., at 3500 g for 30 minutes at 4 °C. After concentration, the volume is adjusted, e.g., to 1 mL with PBS.

[0165] EVs can be selected using different biomarkers and respective affinity binding pairs and their manufactures directions.

[0166] EV Labeling and Analysis Protocols The EV analysis is performed based on the protocols described herein, which includes using multiple fluorescent labels, Raman signals, and dark-field scattering signals to detect target EVs for EV analysis. For fluorescence detection, EVs are labeled by fluorescence probes conjugated with affinity ligands. For Raman detection, molecules on the surface membrane or inside of EVs can be directly detected or EVs are labeled by Raman probes conjugated with affinity ligands. For dark-field scattering detection, scattering signals from EVs can be directly detected without any labeling. The nanostructures of the nano-plasmonic arrays are labeled with affinity ligands that specifically bind to EVs or are bound to capture agents that specifically bind to EVs, and then the substrate is exposed to a biological sample for a sufficient time to ensure that the substrate is bound to a sufficient number of EVs.

[0167] In one embodiment, biotinylated EVs are captured on neutravi din-coated nanostructures, followed by EV fixation and permeabilization in a fix / perm solution. Surface passivation can be achieved by placing the surface (with or without EVs) in a blocking solution (Superblock™ PBS, Thermo Fisher) for 20 minutes. This step is important to minimize undesired nonspecific binding. The captured EVs are stained via two-step indirect labeling: first with primary antibodies then with compatible secondary antibodies. Thorough washing is done between steps.

[0168] The EVs are labeled with capture agents, such as streptavidin. Finally, the labeled EVs are attached to the nanostructures via the capture agents with a mounting solution and covered with a glass coverslip. Antibodies that can be used in the present disclosure are listed in Table 1 below. Primary antibodies are used to specifically bind to a specific biomarker on the surface of the EVs, and secondary antibodies are used to specifically bind to the primary antibodies. Furthermore, the secondary antibodies are conjugated with a reporter group, e.g., a fluorescent probe, to be used in image processing, or a capture agent such as streptavidin. The assay buffer can be, for example, a BD perm / wash buffer solution (BD Biosciences).

[0169] Table 1

[0170] List of Antibodies

[0171] Dilution Dilution Dilution

[0172] Antibody Vendor Cat # factor factor factor (Flow (Western (NPOP) cytometry) blotting)

[0173] Primary-rabbit

[0174] CD9 Invitrogen PA5- 1:12500

[0175] 85955

[0176] CD63 Proteintech 25682-1- 1:12500

[0177] AP

[0178] CD81 Cell Signaling 52892S 1:500

[0179] Primary-mouse

[0180] IgG 1 isotype control Invitrogen 14-4714- 1:500

[0181] 85 lgG2b isotype Biolegend 400301 1:500 control

[0182] CD9 Biolegend 312102 1:1000

[0183] CD63 Ancell 215-820 1:1000

[0184] CD81 Santa Cruz sc-166029 1:500

[0185] MUC1 Invitrogen MAI- 1:1000 1:2500

[0186] 06503

[0187] HER2 Biolegend 324402 1:500 1:250

[0188] EGFR Abeam ab30 1:1000 1:500

[0189] EpCAM Abeam ab85987 1:1000 1:12500

[0190] Primary-rat

[0191] HER2 Bio-rad MCA1788 1:2500

[0192] T

[0193] Secondary

[0194] Anti-rabbit IgG- Invitrogen 4412S 1:2000 1:2000

[0195] Alexa Fluor 488

[0196] Anti-mouse IgG- Invitrogen A-11001

[0197] Alexa Fluor 488

[0198] Anti-mouse IgG- Invitrogen A-21235 1:2000

[0199] Alexa Fluor 647 Anti-rat IgG-DyLight Cell Signaling SAS- 1:1000

[0200] 755 10023

[0201] Anti-mouse IgG-HRP Cell Signaling 7076S 1:2500

[0202] Other antibodies can also be used in the present disclosure based on the specific use. Considering different biomarkers of EVs, a corresponding antibody can be selected. EV biomarkers associated with different diseases and purposes are listed in Table 2, below.

[0203] Table 2. EV Biomarkers

[0204] Other biomarkers than those listed in Table 2 can, of course, also be used in the context of the methods and assay configurations described herein.

[0205] EXAMPLES

[0206] The disclosure is further described in the following examples, which do not limit the scope of the invention described in the claims. Materials and Methods

[0207] The following materials and methods were used in the examples described herein.

[0208] Single EV Analysis of Plasma-Derived EVs (Antibody-Capturing Method)

[0209] The following steps are used in the single EV analysis methods described herein.

[0210] EV Isolation: 250 pl of human plasma samples were centrifuged at 15,000 X g for 5 minutes to remove the cryoprecipitates. The supernatants were loaded onto the dual-mode size exclusion chromatography column which was packed with 2 mL of fractogel and 10 mL of CL4B sepharose. Fractions 5 and 6 of 1 ml fractions were used for EV isolation. The isolates were concentrated by using 100k MWCO centnfugal filter and adjusted the final volume as 25 pl.

[0211] EV Labeling: 20 pL of EVs were mixed with 0.8 pL of TFP-AF555 dye [mixture of 27.5 mM of Azido-dPEG®i2-TFP ester and 25 mM of AFDye 555 DBCO in an equal volume] and incubated for 1 hour at room temperature (RT) in a dark condition. Tetrafluorophenyl (TFP) ester chemistry is typically used to attach fluorophores to the primary amines of biomolecules.

[0212] Functionalization: NPOP substrate was functionalized by incubating in 100 mM of sodium citrate solution for 1 hour at RT. The substrate was washed with water and dried with nitrogen gas.

[0213] Antibody immobilization: Antibodies were diluted in 10 mM phosphate- buffered saline (PBS) and treated on the surface for 1 hour. After washing out the nonbounded antibodies with PBS three times.

[0214] Blocking: NPOP substrates were incubated with 10% goat serum in PBS for blocking for 20 minutes at room temperature.

[0215] EV loading: The AF555-labeled EVs in 1% goat serum in PBS were loaded onto the substrate and incubated for 1 hour in a dark condition. The non-bounded EVs were washed out with PBS three times.

[0216] Fix and Permeabilization: For antibody immunostaining, the bounded EVs were fixed and permeabilized by 4% paraformaldehyde and IX Perm / Wash buffer in PBS for 10 minutes, followed by washing with PBS three times.

[0217] Blocking: No treatment of blocking solution due to the prior blocking step. Primary antibody incubation: Antibodies were diluted in 2% goat serum in phosphate-buffered saline with Tween 20 (PBST) and incubated for 30 minutes at room temperature. The non-bounded antibodies were washed out by PBST washing three times.

[0218] Secondary antibody incubation: Antibodies were diluted in 2% goat serum in PBST and incubated for 30 minutes at room temperature. The non-bounded antibodies were washed out by PBST washing three times.

[0219] Mounting: After removing PBST, the NPOP substrate was mounted with ProLong™ gold mounting solution and microscopically imaged.

[0220] Cell Culture

[0221] The human breast cancer cell lines, including SKBR3, MCF7, BT474, MDA-MB- 231 cells, and normal breast cell, HS371T, were purchased from American Type Culture Collection (ATCC) and cultured at 37 °C in 5% CO2. SKBR3 and BT474 cells were grown in RPMI-1640 (Hy clone), and MCF7, MDA-MB-231, and HS371T cells were cultured in DMEM (Cyclone). All complete media contained 10% fetal bovine serum (FBS, ThermoFisher Scientific), 100 U / mL penicillin, and 100 pg / mL streptomycin (Millipore Sigma).

[0222] Flow Cytometric Analysis

[0223] All cells were collected and washed with phosphate-buffered saline (PBS) three times, followed by fixation using 4% paraformaldehyde. Next, the cells were incubated with blocking solution [0.5% bovine serum albumin (BSA, Millipore Sigma) in PBS] for 30 min at RT on aHulaMixer (ThermoFisher Scientific). The cells were then incubated with non-fluorescent-conjugated antibodies against Cluster of Differentiation 63 (CD63), Mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), epithelial cellular adhesion molecule (EpCAM) for 1 hour at RT on a HulaMixer®. The mouse IgGl and IgG2b antibodies were used as an isotype control. For fluorescence labeling, the cells were further incubated with Alexa Fluor (AF) 488-conjugated anti-mouse antibody for 30 minutes at RT on a HulaMixer®. The fluorescence signals were obtained with CytoFLEX® flow cytometer (Beckman Coulter) and the data were analyzed using FlowJo™ software version 10. Detailed information of antibodies, including vendor, catalog number, and dilution factor, are described in Table 1, above.

[0224] Isolation, Characterization, and Fluorescent Labeling of EVs

[0225] EVs were isolated as previously reported (Advanced Biosystems, 2020, 4(12):2000003).

[0226] Briefly, the cells were grown unto 80-90% confluence in a complete medium and washed twice with PBS to remove floating cells and the vesicles from FBS. The cells were then incubated in a conditioned medium supplemented with 1% Exosome-depleted FBS, 100 U / mL penicillin, and 100 pg / mL streptomycin for 48 hours. Next, the size exclusion chromatography (SEC) columns were prepared by packing 10 mL Sepharose CL-4B (Cytiva) at least one day before performing SEC. After 48 hours of incubation, the conditioned media were collected and filtered with a cell strainer, followed by centrifugation at 300 x g for 10 min at 4 °C to remove cells and debris. The supernatant was then collected and filtered through a 0.8 pm membrane filter (Millipore Sigma) and spun at 3,500 x g for 30 minutes at 4°C with Centricon® Plus-70 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma). Next, the concentrates were loaded onto the SEC column, and collected the fractions of 4 and 5 of 1 mL. Finally, the fractions were concentrated with the Amicon® Ultra-2 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma) at 3,500 x g for 30 minutes at 4°C and stored until use at -80°C.

[0227] For EV characterization, Western blot analysis was conducted using standard techniques. EVs from breast cancer and normal cells were lysed with RIPA lysis buffer (Cell Signaling Technology), and measured the concentration with a BCA protein assay kit (ThermoFisher Scientific). Five hundred ng of EV lysates were loaded onto the SDS- poly acrylamide gel and transferred to the nitrocellulose membrane. The membrane was probed with anti-CD9, anti-CD63, and anti-CD81. Detailed information of antibodies, including vendor, catalog number, and dilution factor, are described in Table 1. Next, the chemiluminescence signals were detected with an Azure 280 imaging system (Azure Biosystems). In addition, the size and number of EVs were determined by nanoparticle tracking analysis using Nanosight™ LM10 (Malvern Panalytical). EVs were labeled with AF555 dye as previously reported with minor modifications (Adv. Sci., 2022 Apr, 8(16): eabm3453; Nat Commun., 2023, 14: 1239; Adv. Sci., 2023, 2205148).

[0228] Briefly, 3 pL of 300 ng of EVs in PBS were mixed with 2 pL of 100 mM sodium bicarbonate (Millipore Sigma) and 0.2 pL of TFP-AF555 dye (mixture of 0.22 pM of Azido-dPEG®i2-TFP ester (Quanta Biodesign) and 0.2 pM of AFDye 555 DBCO (Click Chemistry Tools) in equal volumes) at RT for 1 hour in a dark condition. The labeled EVs were diluted with PBS in an appropriate concentration before loading on the substrates.

[0229] Functionalization and Immunofluorescence Staining

[0230] For comparison of surface functionalization, NPOP surface was functionalized by MUA, SH-PEG-COOH (0.4 kDa), and SH-PEG-COOH (1.0 kDa), respectively. Briefly, 10 mM of 11-Mercaptoundecanoic acid (Millipore Sigma) and 1 -Octanethiol (Millipore Sigma) were mixed in absolute ethanol and applied onto the NPOP substrate for 2 hours, followed by sequential washing with absolute ethanol and water. In addition for SH-PEG- COOH (0.4 and 1.0 kDa) treatment, 0.25 mM of each SH-PEG-COOH (Nanocs) prepared in water were applied onto the NPOP substrate for 4 hour and washed with water. Next, NPOP substrates were treated with 50 mM of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, ThermoFisher Scientific) and 125 mM of sulfo-N- hydroxysulfosuccinimide (NHS, ThermoFisher Scientific) mixture in 0.1M MES (pH 6.0) for 7 minutes. After activation, TFP-AF555-labeled EVs were loaded onto the NPOP substrate and incubated for 30 minutes. The substrates were then washed with PBS and the images were obtained by Zeiss upright automated epifluorescence microscope with a 40* (NA = 0.95) objective lens and a sCMOS camera (Hamamatsu).

[0231] For immunofluorescence staining of EVs, NPOP substrates were functionalized by treatment of 0.25 mM of SH-PEG-COOH (1.0 kDa, Nanocs) and 0.75 mM of SH- mPEG (0.35 kDa, Nanocs) mixture in water for 4 hr. NPOP substrates were then incubated in the mixture of 50 mM EDC (ThermoFisher Scientific) and 125 mM of sulfo- NHS (ThermoFisher Scientific) in 0. 1 M MES (pH 6.0) for 7 minutes to capture the EVs by covalent bonding. TFP-AF555-labeled EVs were loaded onto the NPOP substrate immediately and incubated for 30 minutes. Next, the EVs were fixed and permeabilized by 4% paraformaldehyde (Electron Microscopy Sciences) and IX Perm / Wash buffer (BD bioscience) in PBS for 10 minutes, followed by 30 minutes blocking with Superblock® (ThermoFisher Scientific). The EVs were then fluorescently labeled by sequential incubation with primary and secondary antibodies as indicated in Table 1.

[0232] Image Processing

[0233] Images were analyzed using ImageJ® and custom-built Jupyter™ Notebook code. Background intensity was subtracted using the rolling ball method (radius = 20), and the ImageJ Comdet® plugin was used to detect EV locations from the AF555 channel. 3x3 averaged pixel intensity was obtained from AF647 and DL755 channels. The intensity thresholds for AF 647 and DL755 channels were defined by IgG control experiments, and the threshold value was defined by mean + 3 x standard deviation.

[0234] For each molecular target (e.g., proteins on the membrane or inside of EVs), the corresponding fluorescent images from target molecules are aligned using ImageJ® plugins (Align slices in the stack). At each mask position, average pixel intensities are obtained. The signal is corrected by subtracting background signal surrounding the mask.

[0235] The following examples are illustrative, and not limiting.

[0236] Example 1 - Analysis of Signal Amplification of NPOP vs. Other Substrates

[0237] Using the NPOP substrate, we investigated whether the signals of various fluorophores in different channels are amplified. After immobilizing an equal amount of biotin on both plain gold and NPOP substrate, we induced the binding of streptavidin conjugated with AF488, Cy3, AF647, and Cy7 in a concentration gradient.

[0238] We observed a significant increase in the fluorescence signals of all channels on the NPOP substrate compared to the plain gold substrate, particularly in the AF647 (49.52 fold) and Cy7 (46.10 fold) channels, where the fluorescence signals were dramatically amplified compared with the plain Au substrate (FIG. 5B and FIGs. 10A-10D).

[0239] The strategy of single EV analysis using plasmon-enhanced NPOP substrate is shown in FIG. 5C. The conventional immunolabeling of EVs was applied in this system with brief modifications. For EV capture, the substrate was fictionalized with a mixture of carboxylated and methylated polyethylene glycol (PEG) as previously described (Advanced Science, 2023, 10 (8), 2205148; Nat. Biotechnol., 2014, 32:490-495). For plasma-derived EVs, antibodies against cancer markers were immobilized onto the NPOP substrate by the physisorption method to bind the EVs that have affinity to the antibodies as previously reported (ACS Appl Material Interfaces, 2022, 14:26548-26556) and WO2023 / 220377. Furthermore, the antibody concentration for the EV or cancer markers was further designed to reduce the false-positive signals.

[0240] Finally, the multiplexed and sensitive detection of protein markers at the single EV levels was achieved by NPOP substrate.

[0241] Next, we aimed to compare the signal enhancement of fluorescently labeled EVs in each channel between NPOP and glass substrates. EVs were labeled with AF488, AF555, AF647, and Cy7 TFP dyes and immobilized onto substrates using Cell-Tak, a bioadhesive molecule, under consistent binding conditions following serial dilution of the EV samples. Analysis of the fluorescence signals from EVs attached at specific concentrations revealed significant signal amplification in the AF555, AF647, and Cy7 channels (FIGs. 11A-11E). Moreover, since AF555 was assigned as the EV channel, an examination of co-localization with AF555-labeled EVs revealed a prominent increase in the number of EVs and signal intensity in the AF647 and Cy7 channels (FIG. 6A).

[0242] This observation was further substantiated through signal intensity analysis, which showed an 11.02X-fold increase in the AF647 signal and a 13. HX-fold increase in the Cy7 signal (FIGs. 6B-E). To provide a clearer visualization of these effects in a single EV analysis, scatter plotting was performed. This analysis showed a right shift in the case of AF488 / AF555 co-localized EVs due to signal enhancement by AF555 (FIG. 6F). Conversely, EVs exhibiting co-localization with AF647 and Cy7 showed both rightward and upward shifts across all channels, indicating signal enhancement on NPOP substrate compared with the glass substrate (FIGs. 6G and 6H).

[0243] Overall, these results suggest that the utilization of NPOP in single EV analysis enables the detection of a higher number of EVs that are relatively less abundant in plasma, thereby facilitating the sensitive detection of cancer cell-derived EVs. Based on these experimental performances, we employed the NPOP substrate in subsequent experiments.

[0244] Example 2 - Single EV Analysis on NPOP Substrates We next determined the best scenarios for the followings:

[0245] 1) confirmation of channel-to-channel bleed-through for fluorescence multichannel EV imaging;

[0246] 2) functionalization of EVs for direct binding to the NPOP substrate;

[0247] 3) immobilization methods for EV capture utilizing antibody specificity; and

[0248] 4) selection of best antibody concentration for EV antibody labeling.

[0249] First, to confirm channel-to-channel bleed-through, we examined the bleed- through from the multiband pass filter previously attached to the conventional fluorescence microscope. After labeling EVs with respective AF488, AF555, AF647, and Cy7, we observed the number of EVs in each channel within the same field of view. AF555-labeled EVs showed a bleed-through of 37.21% in the AF488 channel, AF647 EVs showed a bleed-through of 17.06% in the AF555 channel, and Cy7 EVs showed a bleed-through of 9.22% in the AF647 channel. Our observations revealed that EVs labeled with higher wavelength fluorophores exhibited bleed-through in the adjacent channel (FIG. 7A). To solve this issue, we replaced the optical system with single cube filters specific to each channel, resulting in a significant reduction in bleed-through artifacts (FIG. 7B).

[0250] Second, we pursued the functionalization of EVs for direct binding to the NPOP substrate. To achieve this, we employed different lengths of PEG linkers, including 11- mercaptoundecanoic acid (~1.7 nm), SH-PEG-COOH (0.4 kDa, -2.8 nm), and SH- PEGCOOH (1.0 kDa, 7 nm). Following the functionalization process, we activated the NPOP substrate using EDC / NHS and attached EVs onto the surface. We assessed the degree of non-specific binding of EVs in these functionalized substrates and observed a significant reduction in non-specific binding when utilizing the 1.0 kDa PEG linker (FIGs. 7C-7D).

[0251] Third, we selected an appropriate immobilization method for EV capture based on antibody specificity. We compared two methods: 1) Immobilization of antibodies using SH-PEG-COOH (1.0 kDa, 7 nm) through EDC / NHS activation, similar to EV binding, and 2) Direct physisorption of antibodies onto the substrate surface (Adv. Funct. Mater. 2019, 1904257), In this experiment, we prepared EV samples, including those with no EVs, normal plasma EVs, and normal plasma EVs spiked with SKBR3 EVs (HER2- positive). As the capture antibody, we employed a QUAD antibody, which consists of MUC1, HER2, EGFR, and EpCAM antibodies. Following AF555 labeling of EVs, we processed them on the antibody immobilized substrates and performed HER2 staining using conventional immunostaining.

[0252] Our results demonstrated that the QUAD-negative control EVs (no EV and normal plasma EVs) exhibited significant differences compared with the QUAD-positive EVs (SKBR3-spiked normal plasma EVs) only with the physisorption method (FIG. 7E) of assay configuration 3, even though the HER2 -positive signals from different channel showed significant differences with PEG and physisorption methods (FIG. 7F). These results indicated that the physisorption method using Configuration 3 reduces the nonspecific binding of EVs.

[0253] Fourth, we also selected the best antibody concentrations for EV antibody labeling, a crucial step in our study. Excessive antibody usage during EV staining can lead to nonspecific binding. To determine the appropriate antibody concentrations, we performed a serial dilution and prepared marker-positive or negative cell-derived EVs. By confirming the specificity of the antibodies for their respective markers, we calculated the signal -to-noise ratio for the signals observed in positive and negative EVs. The selected antibody concentrations were determined as follows: 0.4 pg / mL for MUC1, 2 pg / mL for HER2, 0.08 pg / mL for EpCAM, and 2 pg / mE for EGFR in the case of the QUAD antibody (FIGs. 7G-7J). Additionally, the rat HER2 antibody exhibited optimal binding at a concentration of 0.4 pg / mL (FIG. 12).

[0254] Furthermore, the bar graphs of FIGs. 16A-C show the results of profiling of cell- line-derived EVs spiked in normal human plasma. EVs from SKBR3 and MCF7 breast cancer cell lines were spiked in normal human plasma samples, and their marker signals were compared in the naive and spiked-in samples. A negative control includes a no-EV sample. The bar graphs in FIGs. 16A-C show marker-positive EV counts that were analyzed for spiked-in samples (blue bars, left side) and the same concentration of cell line EVs (red bars, right side). The results show the assays accurately detect markerpositive EVs for Cdmix (16A), QUAD (16B), and HER2 (16C) from plasma samples.

[0255] FIGs. 16D-I are a series of 2D scatter plots that show the marker-positive EVs. All EVs were labeled to be detected in the AF 555 channel, while respective markers were detected in AF488 for CDmix (a mixture of antibodies against CD63, CD81, and CD9, which are ubiquitously present in EVs) in AF647 channel for QUAD and DL755 channel in HER2. The plots show similar marker-positive rates for SKBR3- and MCF-7 EVs (16E, 16G, 161) when they are spiked in plasma from a healthy control (16D, 16F, 16H).

[0256] These results show that we obtained the same levels of signals even if those target tumor-derived EVs are spiked in normal plasma that contain many non-tumor EVs. Taken together these selected conditions were subsequently employed in our experiments.

[0257] Example 3 - Single EV Analysis of Breast Cancer Cell-Derived EVs

[0258] Next, we evaluated the molecular profiling capability of NPOP substrate for the QUAD (MUC1, HER2, EGFR, EpCAM) in breast cancer cell-derived EVs. Firstly, EVs derived from breast cancer cell lines, including SKBR3, MCF7, BT474, and MDA-MB- 231 cells (FIGs. 13A-13B), were then labeled with AF555 and attached to the functionalized substrate with SH-PEG-COOH linker using assay configuration 2. Subsequently, immunofluorescence staining was conducted using our selected concentrations against respective antibodies, followed by labeling with AF 647 dye (FIG. 8 A). The number of AF647-labeled EVs (marker channel) showing co-localization with the AF555 signal (EV channel) was analyzed and converted into colocalization percentage values. In SKBR3 EVs, markers exhibiting high co-localization were HER2 and EpCAM. In MCF7, MUC1 and EpCAM showed high co-localization, while in BT474, HER2 and EpCAM were high with moderate expression of MUC1, while in MDA-MB-231, EGFR exhibited only higher expression (FIG. 8B).

[0259] The heatmap of the upper panel in FIG. 8C describes the expression levels of each marker and EVs in terms of z-scores through NPOP substrate single EV analysis. For comparative analysis of marker expression, cellular expression in each cell was analyzed through flow cytometry and presented in the lower panel of the heatmap of FIG. 8C. Quantitative analysis of NPOP assay and cellular flow cytometry demonstrated a significant and strong correlation (FIG. 8D, Pearson correlation coefficient, r = 0.9656, P < 0.0001). These results indicate the high EV molecular profiling capability of NPOP substrate single EV analysis.

[0260] We next conducted multiplexed single EV analysis using the NPOP substrate through multi-color imaging. Similar to the previous experiment, breast cancer cell- derived EVs were labeled with AF555 dye and attached to the substrate. For immunofluorescence staining, CD mix (a rabbit-onginated antibody mixture of CD9, CD63, and CD81) was assigned to the AF488 channel, QUAD mix (a mouse-originated antibody mixture of MUC1, HER2, EGFR, and EpCAM) was assigned to the AF 647 channel, and HER2 (a rat-originated antibody) was assigned to the DL755 channel. The additional HER2 -level interrogation would be clinically important to assess the treatment efficacy of anti-HER2 antibody-drug conjugate, which was recently approved by the FDA for HER2-low breast cancer patients.

[0261] For this purpose, negative control samples of No EV (PBS) were prepared for the EV and CD mix channels, and EVs derived from normal cells in breast tissue (HS371T) were prepared as a negative control for the QUAD channel. In the No EV sample, no signal was observed in any of the CD mix, EV, QUAD, or HER2 channels. In HS371T, a normal cell EV sample, signals were only observed in the CD mix and EV channels. Additionally, in breast cancer cell EVs, signals were observed in the CD mix, EV, and QUAD channels, while the HER2 signal was only detected in the HER2 -positive cells SKBR3 and BT474 EVs (FIG. 9A, and FIGs. 15A-15D).

[0262] When examining the co-localization of the signals in each channel with the EV channel (AF555), the amounts of marker expressions in EVs are shown in FIGs 14A-D. Furthermore, scatter plots were used to represent the signal intensity in the AF555 channel and the signal intensity in each channel. These allowed to group subpopulations of EVs to exhibit positivity for specific markers, even though they showed high heterogeneity within the same cell EV (FIGs. 9B-D). FIGs. 14A-C are a series of graphs showing changes of HER2 expression in the differential mixture of SKBR3 (HER2+) and MCR7 (HER2-) EVs. FIG. 14A shows EV counts (AF555). FIG. 14B shows counts and FIG. 14C shows co-localization percentages of HER2 (AF647) and EV (AF555) signal. Error bars are shown as mean ±SD from the four different images.

[0263] FIG. 14D is a series of three scatter plots for individual fluorescence intensities of EV(AF555) and HER2 (AF647) signal of MCF7 EV (left; MCF7:SKBR3=100:0, middle; MCF7:SKBR3=57.1:42.9, right; MCF7:SKBR3=0: 100). The dashed lines indicate the intensity threshold value, which is mean + 3 times of standard deviation of no EV control to determine the marker-positive signal. HER2 -positive signals were shown in red colored dots (above dashed line) vs black dots (below dashed line). FIGs. 15A-D are a series of bar graphs that show EV detection and marker profiling on NPOP substrates. These bar graphs show the EV counts (FIG. 15A, AF555) and the percentages of marker-positive EVs for pan-CDs (FIG. 15B, AF488 / AF555), QUAD (FIG. 15C, AF647 / AF555), and HER2 (FIG. 15D, DL755 / AF555). Error bars are shown as mean ±SD from the four different images. Basically, EV markers (CDmix) are detected from all cell lines. QUAD are detected in all except for HS371T. HER2 is only detected in HER2+ cell-derived EVs.

[0264] Through this analysis, we confirmed the multiplexing capability of NPOP substrate-based single EV analysis using multi-color imaging.

[0265] Example 4 - Paclitaxel Treatment Monitoring of Breast Cancer Patients

[0266] In a clinical study, we obtained plasma samples of 22 breast cancer patients who received neoadjuvant therapy with paclitaxel (a total of 44 samples). The two plasma samples were collected 1) before receiving the paclitaxel treatment (Tl) and 2) after finishing the paclitaxel treatment, but before surgery (T2). EVs were isolated from plasma samples and labeled by TFP-AF555. The sensor surface was functionalized with QUAD antibodies to capture tumor-derived EVs using the assay configuration 3. We labeled those captured tumor-derived EV subpopulations using antibodies against PgP / survivin, which are labeled and imaged in AF647.

[0267] We analyzed changes in tumor-derived EV counts (AF555) and PgP- or survivin- positive EV counts (AF647) within the tumor-derived EV subpopulations. Within this cohort, we identified an increase in co-colocalization counts (AF555 and AF647; QUAD mix and P-gp_Survivin mix) in T2 compared to Tl among 11 patients classified as poor responders. We classified responding and non-responding groups based on the changes in PgP / survivin-positive tumor-derived EV counts. If the drug-resistant marker-positive EV increased, we called these “non-responding” patients, and if the number decreased, we call them “responding” patients.

[0268] Conversely, the remaining 11 patients were classified as good responders due to reduced co-colocalized EVs in T2 compared to Tl. Among the 22 patients, and based on the clinical outcome based on pathological complete response, we correctly identified 21 out of 22 patients (a success rate of over 95%), and the results are shown in FIGs. 17A- 17D In particular, FIGs. 17A-17D show the results of analysis of changes of tumor- derived EVs and PgP / survivin-positive tumor-derived EV counts before and after receiving paclitaxel treatment. EVs from two plasma samples obtained before and after paclitaxel neoadjuvant therapy were processed and the difference was analyzed. Green (dark) bars below the horizontal zero line indicate patients who showed good responses to paclitaxel, and red (gray) bars above the zero line indicate patients who did not respond well to paclitaxel. FIG. 17A shows changes after treatment in QUAD-positive EV counts. FIG. 17B shows changes after treatment in PgP / survivin-positive EVs. FIG. 17C shows the changes after treatment in the QUAD-positive EVs as a percentage. FIG. 17D shows changes after treatment in PgP / survivin positive EVs as a percentage.

[0269] In this double-blind test, the new assay configuration 3 correctly identified 21 out of 22 patients (95.4%) for their responses to paclitaxel (one exception was Pl 7).

[0270] Example 5 - Clinical Studies with Biobank Samples

[0271] We obtained human plasma samples of breast cancer patients and healthy controls from the MGB biobank and analyzed the tumor-derived EV counts in the plasma samples. The tumor-derived EVs were defined based on the QUAD-positivity. The NPOP substrates were coated with QUAD capture antibodies using assay configuration 3, and TFP-labeled EVs were detected. The number of tumor-derived EV counts were defined by TFP-labeled EV counts captured by QUAD antibodies on the NPOP substrates.

[0272] EVs were isolated from plasma samples using dual -mode chromatography and stained for TFP-AF555. We conducted the analysis in a blind manner. In the first test, we had 6 samples (3 breast cancer and 3 normal), and we correctly identified 5 out of 6 samples (FIG. 18A).

[0273] In the second test, we had 8 samples from 4 patients (2 breast cancer and 2 normal) with duplicates. In this test, we correctly identified all test samples. FIG. 18C shows the results of the cluster analysis, in which the samples from the same patients were aligned next to each other (S8-S2; S7-S1; S4-S6; S3-S5). (FIG. 18B).

[0274] OTHER EMBODIMENTS

[0275] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the disclosure, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for analyzing individual extracellular vesicles (EVs) utilizing plasmonic technology, the method comprising:(a) separating EVs from a biological sample, e.g., using a size-based technique;(b) fluorescently labeling the EVs;(c) immobilizing the EVs on a plasmonic substrate containing nanostructures and functional linkers that bind to all types of EVs;(d) adding a plurality of different types of antibodies, wherein each type of antibody specifically binds to EVs from one type of cell, wherein each type of antibody is bound to a specific type of reporter group, e.g., a fluorophore, and wherein the types of reporter groups are different, e.g., different colored fluorophores, to enable distinction between different EVs from different types of cells; and(e) employing multi-color imaging to detect individual types of EVs immobilized on the substrate, thereby enhancing EV detection accuracy and enabling multidimensional analysis.

2. The method of claim 1, wherein the plurality of different types of antibodies are selected based on the characteristics of the EVs under investigation.

3. The method of claim 1 or claim 2, wherein the EVs are immobilized on the plasmonic substrate by any one or more of (a) direct physisorption, (b) a molecular adhesive layer with affinity to large molecules, (c) a charged layer, or (d) covalent bonding with a linker layer.

4. The method of claim 3, wherein the linker layer compnses any one or more of (a) short carbon chains, (b) polyethylene glycol, or (c) other polymers.

5. The method of any one of claims 1-4, wherein the EVs are immobilized by functionalization of the substrate.

6. The method of claim 5, wherein the immobilization is mediated by, but not limited to, functionalization using SH-PEG-COOH with EDC / NHS activation.

7. The method of claim 5, wherein the immobilization is mediated by, but not limited to, functionalization with amines, thiols, trans-Cyclooctene, or streptavidin (for biotinylated EVs).

8. The method of any one of claims 1-7, further comprising selecting a type of antibody that specifically binds to EVs derived from a tumor cell to label tumor-derived EVs9. The method of any one of claims 1-7, further comprising selecting a type of antibody that specifically binds to EVs derived from a specific type of tumor cells to label a specific type of tumor-derived EVs.

10. The method of claim 9, wherein the type of antibody includes, but is not limited to, antibodies that specifically bind to p-gly coprotein (PgP) or survivin to profile drug resistance monitoring.

11. The method of any one of claims 8-10, wherein the tumor is a solid tumor, e.g., breast cancer.

12. The method of any one of claims 1-7, further comprising selecting a type of antibody that specifically binds to EVs derived from a specific type of diseased cell to label disease cell-derived EVs.

13. The method of claim 12, wherein the disease is Alzheimer’s Disease or fibrosis.

14. The method of any one of claims 1-13, wherein the method is used when a target subpopulation of EVs is present at or above 0.5 percent or 1.0 percent of a total number of EVs in the biological sample.

15. A method for analyzing individual target extracellular vesicles (EVs) utilizing plasmonic technology, the method comprising:(a) separating EVs from a biological sample, e.g., using a size-based technique;(b) fluorescently labeling the EVs;(c) immobilizing one type of target EV on a plasmonic substrate containing nanostructures and binding groups that specifically bind only to EVs derived from one type of cell;(d) adding one or more different types of antibodies, wherein each type of antibody specifically binds to a specific type of biomarker on the surface of the target EVs, wherein each type of antibody is bound to a specific type of reporter group, and wherein the types of reporter groups, e.g., fluorophores, are different, e.g., different colored fluorophores, to enable distinction between different biomarkers on the surface of the target EVs; and(e) employing multi-color imaging to detect individual types of biomarkers on the surface of the target EVs immobilized on the substrate, thereby enhancing EV detection accuracy and enabling multi-dimensional analysis.

16. The method of claim 15, wherein the target EVs are immobilized on the plasmonic substrate by binding groups comprising one or more different types of monoclonal or polyclonal antibodies, wherein the monoclonal or polyclonal antibodies specifically bind to one or more surface biomarkers on the target EVs.

17. The method of claim 15 or claim 16, further comprising selecting a type of antibody that specifically binds to target EVs derived from a tumor cell to label tumor- derived target EVs.

18. The method of any one of claims 15-17, further comprising selecting a type of antibody that specifically binds to target EVs derived from a specific type of tumor cells to label a specific type of tumor-derived target EVs.

19. The method of claim 18, wherein the type of antibody includes, but is not limited to, antibodies that specifically bind to p-gly coprotein (PgP) or survivin to profile drug resistance monitoring.

20. The method of any one of claims 17-19, wherein the tumor is a solid tumor, e.g., breast cancer.

21. The method of claim 15 or claim 16, further comprising selecting a type of antibody that specifically binds to target EVs derived from a specific type of diseased cell to label disease cell-derived target EVs.

22. The method of claim 21, wherein the disease is Alzheimer’s Disease or fibrosis.

23. The method of any one of claims 15-22, wherein the method is used for a subpopulation of target EVs present at or below 0.5 percent or 1.0 percent of a total number of EVs in the biological sample.

24. The method of any one of claims 1-23, wherein the antibodies comprise one or more, or all four, of a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, and a fourth type of antibody that specifically binds to HER2.

25. The method of any one of claims 15-23, wherein the binding groups comprise one or more different types of antibodies immobilized on the surface of the substrate, wherein the at least two different types of antibodies are selected from a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, and a fourth type of antibody that specifically binds to HER2.

26. The method of claim 25, wherein the first, second, third, and fourth antibodies are immobilized on the surface of the substrate.

27. The method of any one of claims 1-26, wherein the substrate comprises a nanoparticles on pillars (NPOP) structure.

28. The method according to claim 27, wherein the NPOP structure comprises gold nanoparticles bound to gold nanopillars.

29. The method of claim 27 or claim 28, wherein the substrate is functionalized by using a citrate solution, e.g., a sodium citrate solution.

30. The method of any one of claims 1-29, further comprising: obtaining image data for the captured EVs; and analyzing the captured EVs based on the image data.

31. A substrate for capturing target extracellular vesicles (EV s) in a liquid sample, comprising: a nanoparticle on pillar (NPOP) structure functionalized with a citrate solution, and at least two different types of antibodies immobilized on the surface of the substrate, wherein the at least two different types of antibodies are selected from a first type of antibody that specifically binds to MUC1, a second type of antibody that specifically binds to EGFR, a third type of antibody that specifically binds to EpCAM, a fourth type of antibody that specifically binds to HER2, a fifth type of antibody that specifically binds to p-glycoprotein (PgP), and a sixth type of antibody that specifically binds to survivin.

32. The substrate of claim 31, wherein the at least two different types of antibodies comprise the first, second, third, and fourth types of antibodies.

33. The substrate of claim 31 or claim 32, wherein the at least two different types of antibodies comprise the fifth and sixth antibodies for profiling drug resistant monitoring.

34. The substrate of claim 31, claim 32, or claim 33, wherein the NPOP structure comprises an array of gold nanorods arranged at a distance between neighboring nanorods of about 2500 nm to about 3500 nm, and wherein the nanorods have a length of about 70 nm to about 90 nm, a width of about 20 to about 30 nm, and a height of about 15 to about 25 nm.

35. The substrate of claim 32, wherein the at least two different types of antibodies were bound to the substrate from a solution having an antibody concentration of: about 0.35 to about 0.45 pg / ml for the first type of antibody, about 1.5 to about 2.5 pg / ml for the second type of antibody, about 0.06 to about 0. 10 pg / ml for the third type of antibody, and / or about 1.5 to about 2.5 pg / ml for the fourth type of antibody.

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