Method and system for enhancing electromagnetic radiation signals from extracellular vesicles

Abstract

Systems, methods, and devices are described herein for use in detecting and / or monitoring a target extracellular vesicle ("EV"), for example, in detecting and / or monitoring the treatment of a disease, such as cancer, in a subject. The system can include a nanoplasmonic array of nanostructures arranged to form a periodic array on a substrate, each nanostructure can include a nanopillar, a spacer layer, for example, coated on a metal layer, and a plurality of metal nanoparticles coupled to each of the nanopillars. The nanoplasmonic array can amplify a specific wavelength of electromagnetic radiation and can be used to capture and image the target EV.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 391,999, filed Jul. 25, 2022, and U.S. Utility Patent Application No. 17 / 966,621, filed Oct. 14, 2022. The entire contents and disclosures of these applications are incorporated by reference into the disclosure of this application.

[0002] Description of Research Funded by the Federal Government This invention was made with government support under Grant Nos. R00CA201248 and R21CA217662 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field The present invention relates to a method for enhancing the signal of extracellular vesicles with electromagnetic radiation, for example optical, and more particularly to a small number of extracellular vesicles.

Background Art

[0004] Background Extracellular vesicles (EVs) present new possibilities as circulating biomarkers, especially for cancer, cardiovascular diseases, neurodegenerative diseases, and infectious diseases. These cell - derived phospholipid vesicles are abundant in various body fluids (e.g., blood, cerebrospinal fluid, urine, and saliva). More importantly, they carry various biomolecules (lipids, proteins, and genetic materials) originating from their parent cells, which can be utilized as a minimally invasive means to probe the molecular state of their cell origin.

[0005] In further leveraging the potential of EVs and accelerating their clinical applications, an important yet unresolved issue is to develop sensitive and robust standardized assays capable of determining the composition and molecular profile of EVs in clinical samples. However, their unique sizes (50 - 1000 nm) pose technical challenges in conventional analytical methods and often result in variable findings. Flow cytometry, for example, often underestimates the number of EVs; small EVs (i.e., typically exosomes less than 200 nm) may be missed due to their weak light scattering, or groups of vesicles may be counted as a single event. Conventional methods, especially for protein analysis (e.g., Western blotting, enzyme-linked immunosorbent assay / ELISA), consume large amounts of samples and involve extensive time-consuming processing steps, making them impractical for clinical scenarios. Therefore, the development of new molecular platforms for EVs is a crucial requirement for interpreting EVs as clinically relevant biomarkers.

Summary of the Invention

Problems to be Solved by the Invention

[0006] Overview The present disclosure relates to signal amplification strategies for boosting electromagnetic radiation signals, such as optical signals, generated from limited amounts of biomolecules present in individual EVs. In particular, strong optical resonances of metal nanostructures, such as gold or silver nanostructures, can be used to boost optical signals and significantly amplify optical signals emitted from EVs, such as fluorescence signals. Since plasmon enhancement is an inherently available signal amplification method, in certain embodiments, this inherently available method is combined with other chemical amplification strategies (e.g., branched DNA barcoding) to further improve sensitivity. The present disclosure relates to methods and systems for enhancing the optical signals of EVs.

[0007] The present disclosure also relates to a method of fabricating on a wafer scale a plasmonic substrate that can be used to amplify the signal of fluorescently labeled EVs for multi-channel single EV analysis without the need for additional chemical or enzymatic signal amplification processes. The multiplexed single EV molecule profiling method provides a better understanding of EV heterogeneity with higher EV detection sensitivity and accuracy than conventional methods. These methods also enable the discrimination of individual EVs (i.e., EVs from, for example, tumor, breast, or brain cells, or immune system cells) based on their cell origin.

Means for Solving the Problems

[0008] According to a first aspect, the present disclosure is a method of enhancing an electromagnetic radiation signal, such as an optical signal, from a target EV on a substrate, comprising: obtaining a nanoplasmonic array comprising a substrate, a plurality of nanostructures, and one or more affinity ligands immobilized on or adjacent to the nanostructures, wherein the affinity ligands bind, e.g., specifically bind, to the EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; flowing a liquid sample over the nanoplasmonic array at a flow rate that allows the EVs in the liquid sample, if present, to bind to the affinity ligands, thereby capturing the EVs on the nanoplasmonic array; labeling the target EVs among the EVs captured on the nanoplasmonic array with one or more different reporter groups; exposing the labeled target EVs captured on the nanoplasmonic array to a first electromagnetic radiation, thereby causing the target EVs and / or the reporter groups on the target EVs to emit, scatter, or reflect the first electromagnetic radiation or a second electromagnetic radiation as an electromagnetic radiation signal; and receiving all or a portion of the electromagnetic radiation signal, wherein the nanostructures within the nanoplasmonic array are arranged and dimensioned to amplify the electromagnetic radiation signal, thereby enhancing the electromagnetic radiation signal from the target EV on the substrate.

[0009] In some embodiments of these methods, one or more affinity ligands non-specifically bind to at least one surface marker on the EV and / or at least one intravesicular marker inside the EV, and the reporter group binds to a capture agent, such as an antibody, that specifically binds to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV, or one or more affinity ligands specifically bind to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV, and the reporter group binds to a capture agent that specifically or non-specifically binds to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV.

[0010] In certain embodiments, a plurality of nanostructures are arranged to form a periodic array of nanostructures on a substrate, and the periodic array of nanostructures is arranged and dimensioned to amplify an electromagnetic radiation signal emitted, scattered, or reflected by EVs bound to the nanostructures and / or EVs bound to the substrate near the nanostructures, or to amplify an electromagnetic radiation signal emitted, scattered, or reflected by a reporter group attached to the EV.

[0011] In various embodiments, the electromagnetic radiation signal can be or can include a fluorescence signal, a Raman signal, or darkfield scattering, and the method can further include obtaining an image of the amplified electromagnetic radiation signal, such as an optical signal.

[0012] In another aspect, the present disclosure is a method for detecting or monitoring EVs, such as cancer-derived EVs, and / or the use of the nanoplasmonic arrays described herein for detecting or monitoring EVs, such as cancer-derived EVs, wherein the liquid sample used in the methods described herein is from a subject, the reporter group is bound to a capture agent that binds, e.g., specifically binds, to a tumor-derived target EV, and the method further includes analyzing an acquired image to detect whether the liquid sample contains or includes tumor-derived target EVs, thereby detecting or monitoring, e.g., EVs from cancer or cancer tissue in a subject, or the origin of the EVs, e.g., the cancer or cancer tissue of the subject.

[0013] In certain embodiments, these methods and uses can further include discriminating EVs by size and discarding any EVs or other components larger than 1 micron; selecting target EVs from the EVs identified based on being positive for a target EV marker to generate selected target EVs; identifying selected target EVs as being of a particular organ or tissue origin based on being positive for an organ or tissue specific marker to generate specific selected target EVs; and analyzing individual specifically selected target EVs based on tetraspanin biomarkers on the surface of the specific target EVs, based on intracellular biomarkers within the specific target EVs, or based on both tetraspanin biomarkers and intracellular biomarkers.

[0014] In the methods and uses described herein, the reporter group can be, can include, or can consist of a first fluorescent label, and the reporter group can be, can include, or can consist of an antibody that specifically binds to a biomarker on the surface of the target EV. For example, in some embodiments, the antibody can be, can include, or can consist of at least two different types of antibodies, a first type of antibody that binds to EpCAM and a second type of antibody that binds to HER2. In other embodiments, the antibody can be, can include, or can consist of at least four different types of antibodies, a first type of antibody that binds to MUC1, a second type of antibody that binds to EGFR, a third type of antibody that binds to EpCAM, and a fourth type of antibody that binds to HER2.

[0015] In some embodiments, the methods and uses further include labeling a target EV comprising a first fluorescent label with a second fluorescent label different from the first fluorescent label. In some embodiments of the methods described herein, the cancer can be or can include breast cancer.

[0016] In another aspect, the present disclosure provides a method for detecting EVs, such as cancer-derived EVs, in a sample from a subject, and the use of the nanoplasmonic array described herein in a method for detecting EVs, such as cancer-derived EVs, in a sample from a subject, the method and use comprising collecting a biological sample from the subject; isolating EVs from the biological sample; capturing the EVs on a nanoplasmonic array, the nanoplasmonic array comprising a substrate, a plurality of nanostructures, and one or more affinity ligands immobilized on or adjacent to the nanostructures, the affinity ligands binding, e.g., specifically binding, to the EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling target EVs among the captured EVs with a plurality of different fluorescent reporter groups; performing multi-channel fluorescence imaging to form an image; and analyzing the image to detect cancer in the subject.

[0017] In these methods and uses, in certain embodiments, the plurality of nanostructures can be arranged to form a periodic array of nanostructures on the substrate, the periodic array of nanostructures being arranged and dimensioned to amplify a fluorescence signal emitted, scattered, or reflected by EVs bound to the nanostructures and / or EVs bound to the substrate near the nanostructures, or to amplify a fluorescence signal emitted, scattered, or reflected by reporter groups attached to the EVs.

[0018] In some embodiments, immunolabeling is performed using different antibodies that bind to different reporter groups and specifically bind to different biomarkers on the surface of the target EVs. For example, the antibodies can be, can include, or can consist of at least two different types of antibodies, where the first type of antibody binds to EpCAM and the second type of antibody binds to HER2. In other embodiments, the antibodies can be, can include, or can consist of at least four different types of antibodies, where the first type of antibody binds to MUC1, the second type of antibody binds to EGFR, the third type of antibody binds to EpCAM, and the fourth type of antibody binds to HER2. In some embodiments, the cancer is breast cancer.

[0019] In another aspect, the present disclosure provides a method of detecting individual target EVs, such as EVs associated with cancer diagnosis or cancer treatment monitoring, as well as the use of the nanoplasmonic array described herein for detecting individual target EVs associated with cancer diagnosis or cancer treatment monitoring, the method comprising: providing a biological sample from a subject containing one or more EVs; capturing one or more EVs on a nanoplasmonic array, the nanoplasmonic array comprising a substrate, a plurality of nanostructures, and one or more affinity ligands immobilized on or adjacent to the nanostructures, the affinity ligands binding, e.g., specifically binding, to the EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling the target EVs among the captured EVs with a fluorescent conjugate biomolecule; performing multi-channel fluorescence imaging to form an image; and analyzing the image, e.g., to detect the origin of the EVs, e.g., to detect cancer in a subject or to monitor cancer treatment in a subject undergoing cancer treatment.

[0020] In these methods and uses, the fluorescent conjugate biomolecule can be, can include, or can consist of at least three fluorescent molecules conjugated to at least three cancer-related biomolecules. For example, in certain embodiments, the fluorescent conjugate biomolecule can be, can include, or can consist of a fluorescent conjugate wheat germ agglutinin.

[0021] In some embodiments, the nanoplasmon array is configured to enhance the fluorescent signal from the fluorescent conjugate biomolecule by at least 2-fold as compared to the fluorescent signal from an image taken using a glass substrate instead of the nanoplasmon array.

[0022] In another aspect, the present disclosure provides a method, such as a method for diagnosing or monitoring the treatment of cancer, and the use of the nanoplasmon array described herein for diagnosing or monitoring the treatment of cancer, comprising 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 to a first EV-specific antibody and labeling with a first fluorescent conjugate biomolecule; contacting at least a second portion of the biological sample with a surface conjugated to a second EV-specific antibody and labeling with a second fluorescent conjugate biomolecule; contacting at least a third portion of the biological sample with a surface conjugated to the second EV-specific antibody and labeling with a third fluorescent conjugate biomolecule; performing multi-channel fluorescence imaging to form an image; and analyzing the image, for example, to detect the origin of the EVs and / or to detect cancer in the subject or monitor cancer treatment in a subject undergoing cancer treatment.

[0023] In these methods and uses, the surface can be, can include, or can consist of a nanoplasmonic array, which includes, or consists of, a substrate, a plurality of nanostructures, and one or more affinity ligands immobilized on or adjacent to the nanostructures, and the affinity ligand binds, e.g., specifically binds, to an EV to bind the EV to the nanostructure or to a substrate adjacent to the nanostructure.

[0024] In some embodiments, the nanoplasmonic array is configured to enhance the fluorescence signal in an image by at least 2-fold as compared to a fluorescence signal from an image taken using a glass substrate instead of the nanoplasmonic array.

[0025] In another aspect, the present disclosure provides a nanoplasmonic array for detecting a target EV, the array including: a substrate; a plurality of nanostructures arranged to form a periodic array of nanostructures on the substrate, the periodic array of nanostructures being arranged and dimensioned to amplify, near the nanostructures, one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by an EV bound to the nanostructure and / or an EV bound to a substrate near the nanostructure, or to amplify one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by a reporter group attached to the EV; and one or more affinity ligands immobilized on or adjacent to the nanostructures, the one or more affinity ligands selectively binding to the target EV to bind the target EV to the nanostructure or to a substrate adjacent to the nanostructure.

[0026] In various embodiments, the optical signal can be one or more of a fluorescence signal, a Raman signal, or darkfield scattering.

[0027] In certain embodiments, the nanostructures can be, can include, or can consist of a plurality of nanoholes formed within a metal film disposed within or on a substrate and arranged in an array. In other embodiments, the nanostructures can be, can include, or can consist of a plurality of nanoholes, nanowells, nanorods, nanodisks, nanopillars, nanogrooves, or any combination thereof, disposed in an array on the upper surface of a substrate. The nanostructures can be, for example, gold nanoparticles (NP: nanoparticle) coupled to a gold nanopillar structure (NP on nanopillar, or "NPOP").

[0028] In some embodiments, each of the nanostructures has a maximum size of about 30 - 400 nm, such as a diameter, width, or length. For example, the nanostructures can be nanorods or nanosquares having dimensions of a length of about 50 - about 300 nm, a width of about 20 - about 300 nm, and a height of about 20 - about 300 nm, or the nanostructures can be nanodisks having dimensions of a diameter of about 50 - about 200 nm and a height of about 20 - about 300 nm. Each of the nanopillars can have a maximum size of about 30 - about 500 nm.

[0029] In certain embodiments, the periodic array of nanostructures has a periodicity of about 400 - 800 nm or 400 - 2000 nm between the nanostructures. In certain embodiments, the nanostructures are nanopillars having dimensions of a diameter of about 20 - about 500 nm and a height of about 20 - about 300 nm and having a density of about 300 - about 750 nanopillars / μm on a substrate (e.g., Si, glass, indium tin oxide / ITO, polyethylene terephthalate (PET) polymer substrate). 2

[0030] ​In some embodiments, the affinity ligand binds to a capture agent, such as an antibody, and 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 at least one intravesicular marker within the target EV.

[0031] In some embodiments, the nanoplasmonic array further comprises, or consists of, a metal film disposed on the upper surface of a substrate, the metal film comprising a plurality of nanoholes penetrating the metal film with a periodicity selected to amplify one or more specific wavelengths of electromagnetic radiation, a periodicity of about 400 - 800 nm between the nanoholes, the metal film comprising a plurality of affinity ligands immobilized on or adjacent to the nanoholes, the plurality of affinity ligands selectively binding to markers on the surface of the target EV. The metal film can be, or can comprise, for example, a noble metal, a transition metal, an alkali metal, or any combination thereof. In various embodiments, either or both of the nanostructure and the metal film are, comprise, or consist of gold, silver, aluminum, or platinum.

[0032] In another aspect, the present disclosure is a method for detecting target EVs in a liquid sample and the use of the nanoplasmonic arrays described herein for detecting target EVs, the method comprising or consisting of: obtaining a nanoplasmonic array as described or claimed herein; flowing a liquid sample over the nanoplasmonic array at a flow rate that enables EVs in the liquid sample, if present, to bind to an affinity ligand, thereby capturing the EVs on the nanoplasmonic array; labeling the target EVs captured on the nanoplasmonic array with one or more reporter groups; projecting electromagnetic radiation of a first electromagnetic radiation of one or more specific wavelengths onto the labeled target EVs captured on the nanoplasmonic array, wherein the electromagnetic radiation of one or more specific wavelengths is selected such that the reporter groups 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 nanoplasmonic 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.

[0033] In the method disclosed herein, the number of target EVs in the liquid sample can be less than 1000. In certain embodiments, the nanostructures comprise a plurality of nanoholes that penetrate a substrate or a metal film placed on a substrate, or the nanostructures comprise or consist of a plurality of nanorods, nanodisks, nanopillars, nanogrooves, or any combination thereof disposed on the upper surface of the substrate.

[0034] In some embodiments, the methods and uses described herein further comprise, or consist of, identifying EVs by size and discarding large (e.g., larger than 1 micron) components; selecting target EVs from the identified EVs based on positivity for a target EV marker; selecting target EVs as being of a specific organ or tissue origin based on positivity for an organ or tissue specific marker to generate specific target EVs; and analyzing individual specific target EVs based on extracellular biomarkers on the surface of the specific target EVs and / or based on intracellular biomarkers within the specific target EVs.

[0035] In yet another aspect, the disclosure provides a system for detecting target EVs in a liquid sample, such as a nanoplasmon array system, the system comprising a nanoplasmon array as described and claimed herein; a sample control unit comprising a pump; at least one fluid channel configured to flow a liquid sample over the nanoplasmon array at a flow rate controlled by the pump, enabling EVs in the liquid sample, if present, to bind to an affinity ligand, thereby capturing the EVs on the nanoplasmon array; at least one capture agent, such as an antibody, configured to bind to a reporter group and label the target EVs captured on the nanoplasmon array with one or more reporter groups; an imaging unit comprising a light source configured to project electromagnetic radiation onto the labeled target EVs captured on the nanoplasmon array; and an electromagnetic radiation detector, such as a camera or a CCD, configured to receive electromagnetic radiation emitted, scattered, or reflected by the target EVs captured on the nanoplasmon array or by the reporter groups on the labeled target EVs and capture an image of the labeled target EVs, wherein the electromagnetic radiation detector has one or more wavelengths amplified by the periodic array of nanostructures for electromagnetic radiation emitted, scattered, or reflected by scattered light reflected from the reporter groups.

[0036] In some embodiments of these systems, the nanostructures are arranged within an array and include or consist of a plurality of nanoholes formed within a metal film placed within or on a substrate. In other embodiments, the nanostructures include or consist of a plurality of nanorods, nanodisks, or nanogrooves arranged within an array on the upper surface of a substrate.

[0037] In some embodiments, the affinity ligand binds to a capture agent, which is configured to bind to at least one surface marker on the target EV. In other embodiments, the affinity ligand is configured to bind to at least one surface marker on the target EV and / or at least one intracellular marker within the target EV.

[0038] In some embodiments, a method is used to detect tumor-derived EVs using a QUAD marker signature (mucin 1, cell surface-bound (MUC1), epidermal growth factor receptor (EGFR), epithelial cellular adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)), for example, EVs derived from breast tumors. These methods can include, for example, treatment monitoring during chemotherapy to change a series of treatments during ongoing treatment using markers HER2 and EpCAM.

[0039] In another aspect, the present disclosure provides a three-dimensional nanoplasmon array of nanostructures, the array comprising or consisting of: a substrate; a plurality of nanostructures arranged to form a periodic array of nanostructures on the substrate, each nanostructure comprising a nanopillar having one end attached to the substrate; a metal layer coated on each of the plurality of nanopillars; a spacer layer coated on the metal layer on each of the plurality of nanopillars; a plurality of metal nanoparticles coupled to each of the nanopillars via the spacer layer; and one or more affinity ligands fixed on the nanostructure and / or fixed on the substrate adjacent to the nanostructure, the affinity ligands binding to a target extracellular vesicle (EV) to bind the target EV to the nanostructure or to the substrate adjacent to the nanostructure, and comprising or consisting of one or more affinity ligands.

[0040] In different embodiments, in these nanoplasmon arrays, the one or more affinity ligands are antibodies that bind to biomarkers on the surface of the target EV. In other embodiments, the antibody comprises at least two different types of antibodies, the first type of antibody binds to EpCAM, and the second type of antibody binds to HER2. In other embodiments, the antibody comprises at least four different types of antibodies, the first type of antibody binds to MUC1, the second type of antibody binds to EGFR, the third type of antibody binds to EpCAM, and the fourth type of antibody binds to HER2.

[0041] In some embodiments, the affinity ligand comprises or consists of a linker group, such as thiol-PEG-COOH. In some embodiments, the metal of the metal layer comprises or consists of gold. In various embodiments, the metal nanoparticles comprise or consist of gold. In some embodiments, the spacer layer comprises or consists of a self-assembled monolayer (SAM) of 1H,1H,2H,2H-perfluorodecanethiol (PFDT). In certain embodiments, the substrate comprises or consists of polyethylene terephthalate (PET).

[0042] Certain terms used herein are collected here. Unless otherwise specified or implied by context, the following terms and phrases include the meanings set forth below to assist in the description of particular embodiments.

[0043] As used herein, the terms "comprising" or "comprises" are used with respect to compositions, methods, and each of their components, which, while useful in one embodiment, allow for the addition of unspecified elements, whether useful or not.

[0044] The singular terms "a", "an", and "the" include plural referents unless the 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 non-limiting examples.

[0045] As used herein, the term "periodicity" refers to the recurrence or repetition of nanostructures at regular intervals due to their positioning on a substrate and / or within an array. Thus, as used herein, the term "periodic" refers to a regular predetermined pattern of nanostructures relative to one another, such as a lattice or other repeating unit configuration. A random distribution of nanostructures is not a periodic pattern.

[0046] As used herein, "surface plasmon resonance (SPR)" refers to the physical phenomenon in which incident light stimulates collective resonant electron oscillations at the interface between a flat metal surface, particularly between a negative permittivity material and a positive permittivity material stimulated by the incident light.

[0047] The term "localized surface plasmon resonance (LSPR)" refers to the surface plasmon resonance of nanostructures on the order of nanometers in size, such as metal nanoparticles. The oscillating electrons produce a strong electromagnetic field in the (non-conductive) surrounding medium near the surface of the metal.

[0048] As used herein, the terms "surface plasmon", "surface plasmon polariton", and "plasmon" refer to the collective oscillations of free electrons at a plasmonic surface, such as a metal. These oscillations give rise to self-standing surface electromagnetic waves that propagate in a direction parallel to the metal / dielectric (or metal / vacuum) interface. Since the waves are at the boundary between the metal and the external medium (e.g., air or water), these oscillations are very sensitive to any change in refractive index at this boundary, such as the adsorption of a molecular target, e.g., an EV, onto the metal surface. Furthermore, the electromagnetic field intensity decays exponentially from the metal surface into the surrounding environment (e.g., vacuum or dielectric). The maximum value of the electromagnetic field intensity can be found at the metal / dielectric or metal / vacuum interface.

[0049] As used herein, the term "sample" means any biological or other fluid that may contain one or more extracellular vesicles (e.g., exosomes). Such biological fluids include, but are not limited to, fluids derived from or containing cells, organisms (bacteria, viruses), lysed cells or organisms, cell 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 tissue or tumor, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymph fluid, tears, pleural effusion, nipple aspirate, breast milk, external sections of skin, respiratory, intestinal, and urogenital tracts, and prostatic fluid. Samples can be viral or bacterial samples, samples obtained from environmental sources such as contaminated water, air samples, or soil samples, as well as food industry samples.

[0050] A "biological sample" is derived from or obtained from a living organism. The organism can be the whole organism or can be cells or organs grown in culture. In one embodiment, a "biological sample" also refers to cells or cell populations or an amount of tissue or body fluid from a subject. Most often, the sample is removed from the 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" contains cells from a subject, but the term can also refer to acellular biological materials such as the acellular fractions of blood, saliva, or urine. In one embodiment, the biological sample is from the excision of a primary, secondary, or metastatic tumor, e.g., breast, ovarian, pancreatic, biliary, colorectal, glioblastoma, lung tumor, or a cell block from a pleural effusion, a bronchoscopic biopsy, or a core needle biopsy. Additionally, fine needle aspiration biological samples are also useful. In one embodiment, the biological sample includes primary ascites cells.

[0051] Biological samples also include explants and primary and / or transformed cell cultures derived from patient tissues. Biological samples can be provided by removing a sample of cells from a subject, but can also be achieved by using previously isolated cells or cell extracts (e.g., isolated by another person, at another time, and / or for another purpose). Archived tissues such as tissues with a treatment history or outcome history may be used. Biological samples include, but are not limited to, tissue biopsies, scrapings (e.g., buccal scrapings), whole blood or other body fluids such as plasma, serum, urine, saliva, cell cultures, urine, ascites, pleural effusion, bronchoalveolar lavage fluid (BALF), or cerebrospinal fluid.

[0052] Samples analyzed by the systems and methods described herein may be processed prior to analysis, for example, by purification or concentration of the EVs contained therein.

[0053] As used herein, "extracellular vesicle" ("EV") refers to a naturally occurring or synthetic vesicle that contains an internal cavity. EVs contain a lipid bilayer membrane that encloses the contents of the inner cavity. EVs can include, but are not limited to, exosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, liposomes, vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, matrix vesicles, or multivesicular bodies. EVs have dimensions up to about 10 microns, but typically are about 1000 nm or less.

[0054] Exosomes and microvesicles are types of EVs that can be shed by eukaryotic cells or bud off from the plasma membrane outside the cell. These membrane-bound vesicles are of heterogeneous size and have diameters in the range of about 10 nm to about 5000 nm. The methods and compositions described herein are equally applicable to microvesicles of any size.

[0055] In some of the literature, the term "exosome" also refers to a protein complex containing exoribonucleases involved in mRNA degradation and the processing of small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), and ribosomal RNA (rRNA). Such protein complexes do not have a membrane and are not "microvesicles" or "exosomes", and thus are not EVs as the term is used herein.

[0056] As used herein, the terms "patient" and "subject" are used interchangeably to refer to a human or an animal, such as a vertebrate, such as a mammal. Examples of mammals include, but are not limited to, primates, rodents, livestock, or game animals. Primates include chimpanzees, monkeys, and macaques, such as rhesus monkeys. Rodents include mice, rats, rabbits, and hamsters. Livestock and game animals include cows, horses, pigs, deer, buffalo, felines, such as house cats, canines, such as dogs, foxes, avians, such as chickens, ducks, and ostriches, and fish, such as salmon, bass, and trout. A patient or subject can be any subset of the above, such as including 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, such as a primate, such as a human. The subject can be male or female. Further, the subject can be at any stage of development, such as an embryo, fetus, infant, child, pre-adolescent, adolescent, young adult, mature adult, and elderly adult. A female subject can be pregnant or not pregnant.

[0057] In one embodiment, the subject can be a patient or a subject in a clinical setting. The subject can have or be suspected of having or at risk of developing a disease or disorder, or can already be diagnosed as having a disease or disorder. The subject can be a patient undergoing treatment.

[0058] As used herein, "capture agent" generally refers to any agent having specific binding to EVs (e.g., exosomes) or target EVs. The binding can be to markers present on all EVs, such as biomarkers, or to a subset of target EVs. Typically, the capture agent specifically binds to biomarkers (referred to herein as extracellular markers) that are fully or partially present on the outer surface of the EVs. However, in some embodiments, the capture agent specifically binds to markers (referred to herein as intracellular markers) present inside the EVs. The capture agent is immobilized on the surface (e.g., the sensing area) of the plasmon nanostructure that contacts the sample. Examples of capture agents include, but are not limited to, nucleic acids, oligonucleotides, peptides, polypeptides, aptamers, antigens, polyclonal antibodies, monoclonal antibodies, single-chain antibodies (scFv), antibody fragments, F(ab) fragments, F(ab’)2 fragments, Fv fragments, small organic molecules, polymers, compounds from combinatorial chemistry libraries, inorganic molecules, or any combination thereof.

[0059] The "nucleic acids" described herein can be RNA or DNA, can be single-stranded or double-stranded, and can be, for example, nucleic acids encoding a protein of interest, polynucleotides, oligonucleotides, nucleic acid analogs such as 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 such as, but not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc.

[0060] As used herein, the term DNA is defined as deoxyribonucleic acid. The term "polynucleotide" is used interchangeably herein with "nucleic acid" to refer to a polymer of nucleosides. Typically, a polynucleotide consists of nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine) that are found naturally in DNA or RNA and are linked by phosphodiester bonds. However, the term encompasses molecules containing nucleosides or nucleoside analogs that contain chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules can be used for specific applications.

[0061] As used herein, the term "polypeptide" refers to a polymer of amino acids. The terms "protein" and "polypeptide" are used interchangeably herein. A peptide is a relatively short polypeptide, typically about 2 to 60 amino acids in length. Polypeptides used herein typically contain amino acids such as the 20 L-amino acids 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 can be modified, for example, by the addition of chemical entities such as carbohydrate groups, phosphate groups, fatty acid groups, linkers for conjugation, functionalization, etc. Polypeptides having non-polypeptide moieties associated covalently or non-covalently are still considered "polypeptides". Examples of modifications include glycosylation and palmitoylation. Polypeptides can be purified from natural sources, produced using recombinant DNA technology, or synthesized by chemical means such as conventional solid-phase peptide synthesis. An "antigen" is defined herein as a substance that induces an immune response. An antigenic determinant is called an epitope, and an epitope in the context of a carrier molecule (optionally can be part of the same molecule, for example, botulinum neurotoxin A is a single molecule but has three different epitopes. Usually, antigens are foreign to the animals in which they produce an immune reaction.

[0062] As used herein, "antibody" 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 a single VL or VH antibody domain), Fv fragments including single-chain Fv fragments (scFv), Fab fragments, and F(ab’)2 fragments. Methods for constructing such antibody molecules are well known in the art. As used herein, the term "antibody" refers to an intact immunoglobulin, or a monoclonal or polyclonal antigen-binding fragment having an Fc (crystallizable fragment) region or an 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 fragment" includes, 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 containing at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide. The terms Fab, Fc, pFc’, F(ab’)2, and Fv are used in their standard immunological meanings (see, e.g., Klein, Immunology (John Wiley, New York, N.Y., 1982); Clark, W.R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Ritt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford).

[0063] As used herein, the term "reporter group" refers to a composition that can produce or enhance a detectable optical signal indicating the presence of a 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'-bipyridine, and R6G; and nanoparticles made of metals such as gold, semiconductors, plastics, polymers, and glass. Another example of a reporter group is the QUAD marker (mucin 1, cell surface-bound (MUC1), epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)).

[0064] As used herein, the term "label" refers to a composition that can produce or enhance a detectable signal indicating the presence of a target in a sample.

[0065] As used herein, the term "marker" or "biomarker" refers to a molecule that can bind to a capture agent for detecting EVs and associates with EVs. A marker can be any component of an EV that can be recognized by a capture agent. Examples of markers include, but are not limited to, proteins, or nucleic acids, or components of the lipid bilayer that constitutes the membrane of an EV. Useful markers include receptors (e.g., extracellular) and channel components. A marker can be either an extracellular marker or an intracellular 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 common to all EVs in a sample is referred to herein as a pan-EV marker.

[0066] As used herein, the term "QUAD biomarker" refers to MUC1, EpCAM, HER2, and EGFR.

[0067] As used herein, when one element is "fixed" to another element, the two elements are directly connected via a bond, such as an ionic bond, a covalent bond, a polar bond, or a hydrogen bond. When two elements are "bonded" to each other, the two elements are directly or indirectly connected via a bond or via other elements such as a linker group, e.g., PEG, or an affinity ligand described herein.

[0068] "Affinity ligand" is defined herein as a molecule that is either directly attached or fixed to a molecular spacer or a substrate or nanostructure and can also be directly attached to a capture agent. In some embodiments, the affinity ligand can be a capture agent. In other words, the affinity ligand physically links together a molecular spacer (or substrate or nanostructure) and a capture agent (or molecular spacer). In one embodiment, the affinity ligand is the first member of a specific binding pair. In such an embodiment, the capture agent can be the second member of the specific binding pair. Examples of such specific binding pairs include, but are not limited to, antigens, antibodies, haptens, oligonucleotides, polynucleotides, avidin, streptavidin, hormones, receptors, lectins, carbohydrates, IgG, protein A, and nucleic acid binding proteins. Affinity ligands can include, but are not limited to, nucleic acids, oligonucleotides, peptides, polypeptides, antigens, polyclonal antibodies, monoclonal antibodies, single-chain antibodies (scFv), antibody fragments, F(ab) fragments, F(ab')2 fragments, Fv fragments, organic small molecules, polymers, compounds from combinatorial chemistry libraries, inorganic molecules, or any combination thereof.

[0069] Examples of specific binding pairs include antigen - antibody, hapten - antibody or antibody - antibody pairs, complementary oligonucleotides or polynucleotides, avidin - biotin, streptavidin - biotin, hormone - receptor, ligand - receptor, lectin - carbohydrate, IgG - protein A, nucleic acid - nucleic acid binding protein, and nucleic acid - anti - nucleic acid antibody.

[0070] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, wherein a first entity binds to a second target entity with a higher specificity and affinity than it binds to a third non - target entity. In some embodiments, specific binding can refer to an affinity of a first entity for a second target entity that is at least 10 - fold greater than the affinity for a non - target entity. A reagent specific for a given target is a reagent that exhibits specific binding to that target under the conditions of the assay utilized. In certain embodiments, specific binding is indicated by a dissociation constant of ≦10 -8 M, ≦10 -9 M, ≦10 -10 M, or lower order.

[0071] Polyethylene glycol (PEG) is referred to herein as a possible component of a nanoplasmon array and is used as a molecular spacer. Various forms and combinations of PEG are envisioned for use as such a spacer. Polyethylene glycol (PEG) is a polyether compound with many uses from industrial manufacturing to medicine. The structure of PEG is (note the repeating element in parentheses): H-(O - CH2 - CH2) nIt is -OH. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE) depending on its molecular weight. PEG, PEO or POE refers to oligomers or polymers of ethylene oxide. Although the three names are chemically synonymous, when used in this specification, PEG refers to oligomers and polymers with a molecular mass of less than 20,000 g / mol, PEO refers to polymers with a molecular mass of more than 20,000 g / mol, and POE refers to polymers with any molecular mass. PEG and PEO are liquids or low melting point solids depending on their molecular weights. Different forms of PEG are also available depending on the initiator used in the polymerization process, and the most common initiator is monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated as mPEG. Low molecular weight PEG is also available as a purer oligomer called monodisperse, uniform, or discrete. Branched PEG has 3 to 10 PEG chains originating from a central core group. Star PEG has 10 to 100 PEG chains originating from a central core group. Comb PEG has multiple PEG chains usually grafted to a polymer backbone.

[0072] As used herein, "long-chain polyethylene glycol (PEG)" or "long PEG" is defined as a PEG polymer having a molecular weight of 750 Da or more.

[0073] As used herein, "short-chain PEG" or "short PEG" is defined as a PEG polymer having a molecular weight of 500 Da or less.

[0074] As used herein, "expression level" refers to the number of mRNA molecules and / or polypeptide molecules encoded by a gene of interest present in a cell or sample.

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Methods and materials for use in the present disclosure are described herein. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.

[0076] Details of one or more embodiments of the invention are set forth in the accompanying drawings, description, and claims. Other features, objects, and advantages of the invention will become apparent from the description, drawings, and claims.

Brief Description of the Drawings

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[0078] Like reference numerals in the various drawings indicate like elements. **Detailed Description** The present disclosure relates to systems, methods, and devices for detecting target extracellular vesicles (EVs). EVs can be, but are not limited to, exosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, liposomes, vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, matrix vesicles, or multivesicular bodies. EVs have multiple surface biomarkers that can be used as indicators for monitoring or diagnosing specific diseases, such as, among others, cancer, cardiovascular disease, neurodegenerative diseases, and infectious diseases. In particular, the new systems and methods can be used to detect and diagnose Alzheimer's disease and other neurodegenerative diseases, as well as to detect viruses, bacteria, and / or parasites by analyzing immune cells containing materials from infectious agents, for example.

[0079] However, their inherent sizes (50 - 1000 nm) pose technical challenges in conventional analytical methods and often result in variable findings. For example, EVs have dimensions up to about 10 microns, but typically are about 1000 nm or less, about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 240 nm or less, about 230 nm or less, about 220 nm or less, about 210 nm or less, about 200 nm or less, about 190 nm or less, about 180 nm or less, about 170 nm or less, about 160 nm or less, about 150 nm or less, about 140 nm or less, about 130 nm or less, about 120 nm or less, about 110 nm or less, about 100 nm or less, about 90 nm or less, about 80 nm or less, about 70 nm or less, about 60 nm or less, about 50 nm or less, about 40 nm or less, about 30 nm or less, about 20 nm or less, or about 10 nm or less.

[0080] Furthermore, EVs often provide weak detection signals, especially when the EV sample does not contain a sufficient number of EVs or when the abundance of proteins and / or intra - vesicular markers is low, which can make it difficult to perform sensitive and robust standardized assays for determining the composition and molecular profile of EVs in clinical samples.

[0081] The present disclosure provides solutions to these problems and enables the targeting of single EVs by amplifying individual optical signals to achieve accurate and precise multiplexed analysis of target EVs. By analyzing single EVs, the unique molecular profiles of cell - specific EVs can be revealed, thereby further facilitating the clinical use of EVs for constructing comprehensive EV "atlases" for different biological parameters (e.g., cell origin, cell state).

[0082] The nanoplasmonic system of the present disclosure enables multiplexed single EV analysis of target membranes and intracellular markers with improved sensitivity. Specifically, optical signals, such as fluorescence, are amplified using plasmonic metal nanostructures to provide high-sensitivity multi-channel EV biomarker profiling. The enhancement can be achieved, for example, by using a substrate having a periodic array of nanostructures such as nanoholes, nanorods, nanodisks, nanowells, nanosquares, nanopillars, nanogrooves, or any suitable periodic or aperiodic metal nanostructure. A copper or aluminum film or substrate can be used for UV illumination, and silver and gold can be used for visible wavelength illumination. Generally, the substrate is a non-metallic non-conductive substrate such as glass or plastic when used under a metal film, although metals, metal oxides, and semiconductors can also be used as substrates.

[0083] For example, a periodic array of Au nanoholes supports surface plasmon resonance extended to a long distance (about 100 nm) suitable for EVs. Furthermore, the resonance wavelength can be adjusted by tuning the periodicity and size of the nanoholes. The same can be done with nanostructures in the form of nanorods or nanodisks. In one embodiment, nanoplasmonic extracellular vesicle analysis (nPLEX-FL) using the enhanced fluorescence detection described herein provides a simple and robust signal amplification strategy that improves detection sensitivity and achieves multiplexed EV analysis, along with similar methods using other optical signals.

[0084] Preparation of Nanoplasmonic Arrays As used herein, a nanoplasmon array includes a substrate, a plurality of nanostructures on or within the substrate, and a plurality of affinity ligands immobilized on or adjacent to the nanostructures. Different surface chemistries (conjugates to the affinity ligands) can be used for the metals used to fabricate the nanostructures and the substrate (e.g., glass) in order to selectively immobilize the affinity ligands onto the nanostructures, such as on the surface of nanorods or nanodisks, within nanogrooves, and on the walls within or adjacent to 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.

[0085] In some embodiments, the plurality of affinity ligands are immobilized on or adjacent to the nanostructures, and the plurality of affinity ligands bind, e.g., specifically bind, to EVs or target EVs via a capture agent. Different types of affinity ligands can be used for the nanoplasmon array based on the corresponding EV preparation. For example, among high-affinity binding pairs, the substrate of the nanoplasmon array can include a biotin-binding protein (e.g., avidin) as an affinity ligand attached to the substrate, and then the EV or target EV needs to include the corresponding biotin as a capture agent to be captured by the nanoplasmon array. In some embodiments, the substrate includes a semiconductor, a non-conductor, a plastic, or any suitable transparent substrate. A method for attaching the corresponding capture agent to the EV is described below.

[0086] The inventors have developed an advanced nanoplasmonic EV sensing platform for single EV analysis using a new nanoplasmonic sensing platform for single EV detection. The system, called NEXT (Nanostructure-based Extracellular Vesicle Technology), includes an array of metals such as gold, silver, copper, or aluminum, nanostructures such as nanoholes, nanorods, nanodisks, or nanopillars, with dimensions, for example, less than 200 nm, which can be occupied by a single EV. For example, gold nanorods have high sensitivity up to single molecule detection and the possibility of precise adjustment of the resonance wavelength by adjusting the dimensions of the nanorods. The array of nanostructures can be fabricated reproducibly by advanced imprinting and deposition processes using standard nanoimprint lithography techniques.

[0087] The capture of individual EVs on each nanostructure, such as a nanorod, induces a spectral shift; these shifts from the nanorod array are simultaneously detected by dark-field imaging. Extensive validation studies were performed against benchmarks 1) single EV detection sensitivity; 2) specificity for capturing target EV subpopulations; and 3) robustness and reproducibility.

[0088] A high-density array of nanostructures such as nanorods of a metal such as gold (e.g., 10 5 arrays / cm 2) can be fabricated using a new nanoimprint lithography method that can pattern gold nanorod arrays at the wafer scale by a simple imprint and gold deposition process (Figs. 10A - 10D). This technique utilizes a reusable silicon mold having a nanopattern that is transferred to a target substrate coated with a thin resist layer (Figs. 10A - 10B). After imprinting, gold is deposited on the patterned area; subsequent removal of the resist leaves a nanorod array on the glass substrate (Fig. 10C). The inventors have previously fabricated various metal nanostructures at high density using known fabrication methods (ACS Nano 2011, 5, 7555 - 7564, 2011; Anal. Chem., 84, 6031 - 6039, 2012). Using these methods, with typical nanofabrication facilities, 10 chips per fabrication cycle can be produced in 4 hours or 100 chips in 1 week (10 cycles × 10 chips / cycle), which is a chip production rate sufficient for subsequent biological experiments. This mass production process is simple, reproducible, and scalable. Nanostructures can also be patterned with high precision using e - beam lithography.

[0089] Periodic nano - holes are fabricated by patterning a thin (50 - 200 nm thick) gold film on a substrate. The nano - holes can be patterned directly by focused ion beam milling or via lithography and metal etching. Deep ultraviolet (DUV) lithography is used to create a 200 - nm periodic circular pattern on a resist spin - coated on the gold film. Further, using the resist as an etching mask, the underlying gold film is etched by reactive ion etching or ion milling. Removal of the resist reveals the gold nano - hole pattern fabricated in the gold film.

[0090] The array chip is designed by comprehensive three-dimensional calculation. In one example, it is found that the nanorod dimensions of 80 nm (length) × 30 nm (width) × 20 nm (height) achieve the maximum sensitivity for 100 nm EV (average diameter) detection, and the dimensions and sensitivity of the array sensor are experimentally tested. When the size of the gold nanorods is less than 100 nm, it is also possible to capture a single EV on each nanorod. A gold nanorod array with a 3-μm spacing between nanorods enables a uniform distribution of EVs on the nanorod array; signals from individual EVs are clearly resolved using a 10× or higher objective lens. The total number of nanorods in the chip can be easily extended using the nanoimprint mold size. For example, a MicroSys, Digilab Inc. Microarray Spotter can be used to selectively functionalize the chip with affinity ligands. Using the spotter, 0.1 μL of solution is transferred from a 96-well plate and spotted onto an area designed with good reproducibility (variation less than 5%). Temperature and humidity are controlled inside the spotter chamber for consistent sample spotting and incubation conditions.

[0091] Metals, such as gold, and nanostructures, such as nanorods, exhibit a characteristic dark-field light-scattering peak at the resonance wavelength. Binding of EVs to the nanorod surface increases the local refractive index and red-shifts the peak wavelength. The spectral shift (i.e., EV binding) can also be detected by measuring the change in light intensity at a fixed wavelength, and there is an excellent correlation between the spectral and intensity measurements. The intensity measurement method can be used for high-throughput parallel signal readout from the entire array within the field of view. This technique is much faster than the sequential spectral measurements used in past systems and methods. Dark-field imaging is also compatible with epi-fluorescence measurements for molecular EV profiling in the same setup.

[0092] EVs, such as tumor-derived EVs, can be captured on nanorods and measured by the number of nanorods showing intensity changes induced by EV binding to the nanorod surface. Computational calculations using finite-difference time-domain (FDTD) solutions indicate that a single 100 nm EV binding induces a shift exceeding 10 nm, and that a shift large enough can be easily detected by dark-field intensity measurements. The signal also correlates with the size of the captured EVs, facilitating EV size measurement. Standard-sized nanospheres can be used for calibration, and the results can be compared with those obtained by a nanoparticle tracking analysis (NTA) system. In combination with molecular profiling, size information can be used to identify EV subtypes (e.g., exosomes vs. microvesicles).

[0093] By using dark-field imaging, the readout signal from the entire array can be measured simultaneously. Intensity measurements provide a much higher throughput in the readout of large arrays than spectral measurements. A temperature controller can be implemented to stabilize the light source temperature and / or increase the number of signal averages to avoid signal drift or fluctuations due to changes in the light source temperature and / or reduce background noise.

[0094] Figures 11A to 11D show an example of a nanoplasmon array with nanorods for plasmon-enhanced single EV sensing technology (NEXT). Figure 11A is a schematic diagram of a NEXT chip sensor consisting of a gold nanorod array fabricated within a grid. The small surface area of the nanorods enables single EV binding on each nanorod. Figure 11B is a scanning electron microscope (SEM) image of nanospheres captured on the gold nanorods (scale bar: 50 nm). Figure 11C is an SEM of a nanorod array fabricated by electron beam lithography, although such a nanoarray can also be fabricated using nanoimprint lithography (scale bar: 500 nm). Figure 11D is an SEM showing the binding of a single nanosphere mimicking an EV on the nanorod (scale bar: 200 nm). The captured EVs are labeled with immunofluorescent probes for high-throughput multi-channel analysis using plasmon-enhanced fluorescence detection. The captured EVs are evenly distributed according to the distance between the nanorods. This improves the accuracy of the analysis.

[0095] The fabrication procedure for making the nanopillars is shown in FIGS. 11Ea - g, which shows a new 3D plasmonic nanostructure composed of spherical gold nanoparticles (AuNPs) of 3D Au nanopillars (NPOP) with a 1-nm-thick uniform spacer layer between the AuNP and the nanopillar. This method is based on enhancing the surface diffusion of adsorbed Au atoms (i.e., adatoms) on a low-energy surface. The Au adatoms diffuse to the defect sites of the underlying film and form clustered atoms (i.e., AuNPs). This process enables the direct and selective formation of spherical Au nanoparticles by a simple Au deposition process without high-temperature annealing. The AuNP density can be highly increased by introducing more nucleation sites on the surface of the 3D rough Au nanopillars.

[0096] The 3D NPOP structure provides high-density hot spots and large effective volume molecular binding sites for high-sensitivity surface-enhanced Raman spectroscopy (SERS) and plasmon-enhanced fluorescence (PEF). The inventors demonstrate a much improved detection sensitivity for both SERS and PEF sensing applications. The fabrication method developed here is inexpensive, simple, reproducible, and applicable to scaled-up chip production.

[0097] Figure 11E-a shows a schematic diagram of the fabrication procedure of the 3D NPOP substrate. First, a polyethylene terephthalate (PET) substrate was etched using Ar plasma to form polymer nanopyramids. A 100-nm-thick Au film was deposited on the nanopyramids by either conventional sputtering or thermal evaporation to form high-density Au-coated nanopyramids. As a spacer layer, a self-assembled monolayer (SAM) of 1H,1H,2H,2H-perfluorodecanethiol (PFDT) was deposited on the Au nanopyramids. Finally, another layer of Au was deposited on the PFDT-coated Au nanopyramids (see, for example, Park et al., Advanced Functional Materials, 2019, 29(43):1904257, and US Patent Application Publication No. US 2019 / 0331605. These are hereby incorporated by reference in their entirety, including all figures and references cited).

[0098] Figure 11E-b shows a photograph of wafer-scale (90 cm 2 area) chip fabrication. Figures 11E-c and 11E-d are scanning electron images of the nanopyramids formed on the PET substrate and the 3D NPOP structure formed by depositing 20 nm of Au on the PFDT-coated nanopyramids (scale bar, 200 nm).

[0099] Figure 11E-e shows a transmission electron image of the 3D NPOP structure. The inset shows the size distribution of the AuNPs on the nanopyramids.

[0100] Figure 11E-f is a high-resolution transmission electron image showing an enlarged image of the white dashed box in (e). The thickness of the PFDT layer between the AuNP and the nanopillars is about 1 nm.

[0101] Figure 11E-g shows a finite element method (FEM) simulation of the 3D NPOP structure shown in the dashed box of 11E-e.

[0102] Isolation and Preparation of EVs A biological sample can be obtained from, for example, a human or other subject, and the cells can be cultured in a culture medium such as Dulbecco's modified Eagle's medium (DMEM, Cellgro). The medium can be supplemented with serum, such as 10% fetal bovine serum, and antibiotics, such as penicillin and / or streptomycin, and maintained under 5% CO2 (see, for example, Min et al., Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles, Advanced Biosystems, 2020, 4, 200003. DOI: 10.1002 / adbi.202000003. This document is hereby incorporated by reference in its entirety, including all figures and references cited).

[0103] EVs can be isolated using both standard ultracentrifugation (UC) and size-exclusion chromatography (SEC) methods. Further, EVs are isolated from the medium for the following process. In the case of UC, the filtrate is concentrated at, for example, 100,000×g for 1 hour. After removing the supernatant, the EV pellet is washed, for example, with PBS and centrifuged again, for example, at 100,000×g for 1 hour. The EV pellet is resuspended in a buffer or serum, such as PBS. In the case of SEC, the filtrate is loaded onto a filter (e.g., MWCO = 10 kDa) and centrifuged at, for example, 3500×g for 30 minutes at 4°C. After concentration, the volume is adjusted to 1 mL, for example, using PBS.

[0104] EVs can be selected using different biomarkers and their respective affinity binding pairs, as well as the manufacturer's instructions.

[0105] EV Labeling and Analysis Protocol EV analysis is performed based on the nPLEX-FL protocol described herein, which involves using multiple fluorescent labels, Raman signals, and dark-field scattering signals to detect target EVs for EV analysis. For fluorescence detection, EVs are labeled with a fluorescent probe conjugated to an affinity ligand. For Raman detection, molecules on the surface membrane or inside the EV can be directly detected, or the EVs are labeled with a Raman probe conjugated to an affinity ligand. In dark-field scattering detection, the scattering signal from the EVs can be directly detected without any label. The nanostructures of the nanoplasmon array are labeled with an affinity ligand that binds to the EV, e.g., specifically binds to the EV or binds to a capture agent that specifically binds to the EV, and then the substrate is exposed to the biological sample for a time sufficient to ensure that the substrate binds to a sufficient number of EVs.

[0106] In one embodiment, biotinylated EVs are captured on nanostructures coated with neutravidin, and subsequently fixed and permeabilized in a fixation / permeabilization solution. Surface passivation can be achieved by placing the surface in a blocking solution (Superblock PBS, Thermo Fisher) for 20 minutes (regardless of the presence or absence of EVs). This step is important to minimize unwanted non-specific binding. The captured EVs are stained in a two-step indirect labeling: first with a primary antibody and then with a compatible secondary antibody. Sufficient washing is performed between steps.

[0107] EVs are labeled with a capture agent such as streptavidin. Finally, the labeled EVs are attached to the nanostructures with a mounting solution via the capture agent and covered with a glass coverslip. Antibodies that can be used in the present disclosure are listed in Table 1 below. The primary antibody is used to specifically bind to specific biomarkers on the surface of EVs, and the secondary antibody is used to specifically bind to the primary antibody. Furthermore, the secondary antibody is conjugated with a reporter group used in image processing, such as a fluorescent probe, or a capture agent such as streptavidin. The assay buffer can be, for example, BD perm / wash buffer solution (BD Biosciences).

[0108] [Table 1]

[0109] Based on specific uses, any other antibody can also be used in the present disclosure. Considering different biomarkers of EVs, the corresponding antibodies can be selected. EV biomarkers related to different diseases and purposes are listed in Table 2 below.

[0110] [Table 2]

[0111] In some embodiments of the present disclosure, image processing of the captured EVs is performed using image analysis software such as ImageJ® and CellProfiler®. The streptavidin imaging channel is used to identify the location of the captured EVs and define the region of interest as a mask. For each molecular target (e.g., a protein on or inside the EV membrane), corresponding fluorescence images from the target molecule are aligned (aligning slices within the stack) using ImageJ® plugins. At each mask position, the average pixel intensity is obtained. The signal is corrected by subtracting the background signal surrounding the mask.

[0112] In some embodiments, the QUAD biomarker can be used, at least in some embodiments, in a multiplex analysis of surface markers on EVs to detect the presence of tumor cells in a sample by quantifying the levels of four biomarkers, e.g., MUC1, EGFR, EpCAM, and HER2, for diagnosing cancers, e.g., cancers of epithelial origin. Of the individual biomarkers investigated, MUC-1, HER2, EGFR, and EpCAM provide the highest diagnostic accuracy, but by combining these four markers, a very high level of accuracy superior to conventional clinical analysis has been established. See, e.g., U.S. Patent No. 10,712,343, which is incorporated herein by reference in its entirety. However, additional or alternative biomarkers may be used in the multiplex analysis, and two or three or four or five or six or more different biomarkers can be investigated.

[0113] In some embodiments, the tumor is a tumor of the pancreas, lung, breast, prostate, kidney, stomach, esophagus, bladder, endometrium, cervix, bile duct, thyroid, ovary, brain, head and neck, oral cavity, melanoma, liver, or colon.

[0114] A method for diagnosing or detecting the presence of cancer in a subject is described herein. The method includes obtaining a sample from the subject, isolating EVs from the sample, and assessing the presence and / or level of MUC-1, HER2, EGFR, and EpCAM in the EVs, and comparing the presence and / or level to one or more references, such as normal levels of MUC-1, HER2, EGFR, and EpCAM, such as a control reference representing the levels in a non-afflicted subject, and / or a disease reference representing the levels of the QUAD biomarker in a subject having cancer. In some embodiments, the method includes determining a value, such as a normalized expression value, for each of the biomarkers (MUC-1, HER2, EGFR, and EpCAM), and calculating a test score from the sum of the levels. The score can then be compared to a reference score, and the presence of a test score above (e.g., greater than or simply exceeding) the reference score indicates the presence of cancer in the subject. See, e.g., U.S. Patent No. 10,712,343.

[0115] The method can also be used, for example, to monitor a patient to determine, for example, whether a treatment has been effective, or whether the subject is experiencing a recurrence, or whether treatment resistance has emerged. In these embodiments, the method includes detecting the presence and / or level of a biomarker in EVs obtained from the subject over time, such as in a first or baseline sample and then in one or more subsequent EV samples, such as over a period of one week or several weeks or one month or several months. For some biomarkers, a decrease over time in the presence or level of the biomarker in the EVs, such as a decrease in the biomarker value, indicates improvement of the disease, such as that a treatment administered during the intervening time is effective in causing a decrease in the number of cancerous cells or tumor mass. The QUAD biomarker can be used in this analysis, although additional or alternative biomarkers can be used in a multiplex analysis of surface markers on the EVs.

[0116] The absence of a change in the presence or level of a biomarker, e.g., no change in the QUAD biomarker value of an EV, indicates that there is no change in the disease, e.g., that an intervening treatment was not effective in causing a decrease in the number of cancerous cells or tumor mass (however, in particularly aggressive diseases, the treatment may have been effective in halting progression, which can be a treatment goal).

[0117] The presence or level of a biomarker for an EV, e.g., a decrease over time in the QUAD biomarker value, a subsequent period of no change, or an increase in the presence or level of a biomarker, e.g., an increase in the quad biomarker value, indicates that an intervening treatment has lost effectiveness and may indicate the presence of the emergence of resistance to the treatment.

[0118] An increase over time in the presence or level of a biomarker, e.g., an increase in the QUAD biomarker value, indicates that the disease is progressing, e.g., that there has been an increase in the number of cancerous cells and / or tumor mass. Such an increase may also indicate a poor prognosis, e.g., an increased likelihood of death.

[0119] In some embodiments, the method includes the use of an antibody or an antigenic fragment or oligonucleotide thereof that specifically binds to a biomarker (i.e., does not substantially bind to other molecules). As used herein, the term "antibody" 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 that retain the ability to bind to an antigen. The antibody can be a polyclonal antibody, monoclonal antibody, recombinant antibody, chimeric antibody, deimmunized antibody or humanized antibody, fully human antibody, non-human antibody (e.g., mouse) or single-chain antibody. In some embodiments, the antibody has effector functions and can fix complement. In some embodiments, the antibody has a reduced or no ability to bind to an Fe receptor. For example, the antibody can be an isotype or subtype, fragment or other variant that does not support binding to an Fe receptor, e.g., has a mutagenized or deleted Fe receptor-binding region.

[0120] Methods for making antibodies and their fragments are known in the art. See, for example, U.S. Patent No. 10,712,343 and the citations therein.

[0121] Methods of Use New methods and nanoplasmon arrays can be used to analyze a single EV in multiple scenarios. For example, tumor-derived EVs contain protein and RNA markers that reflect primary tumor cells, and the new nanoplasmon array sensors can rapidly and sensitively detect tumor EVs directly from clinical samples. Thus, EV analysis offers compelling clinical potential for diagnosing cancer and monitoring long-term tumor responses to treatment.

[0122] The sensitive single-EV detection platform described herein significantly improves the understanding of EV biology, enables rapid and reliable screening of EVs from clinical specimens, and allows for the analysis of subtle phenotypic changes during treatment. Importantly, this will help the field understand how EVs align well with their primary tumor counterparts and whether EV counts and / or molecular profiles provide additional insights into cancer progression or treatment response. In the long term, successful achievement of high-throughput EV profiling in blood will open the way to other clinically relevant screening studies (e.g., EVs in other body fluids and cancer types). This results in more accessible tools to significantly accelerate the clinical adoption of EV analysis as a routine screening test for cancer care in the clinical setting. The single-EV detection platform described herein enables the identification of individual EVs derived from tumors or specific organs and the detection of specific target molecules on or inside the EVs from a target subpopulation that would otherwise be diluted or undetected by EVs from non-target origins. Molecular profiling of EVs from target-specific tumors or organs can indicate the molecular state of the originating cells.

[0123] Figure 20 shows the overall workflow of the plasmon-enhanced EV assay. First, EVs are isolated from human specimens (e.g., blood or other biological fluids) or cell culture supernatants.

[0124] The isolated EVs are captured on a plasmonic substrate having nanostructures, such as gold nanostructures (e.g., nanopillars, nanodisks, nanorods, nanoholes, nanoslits, nanoparticles, random structures), either by covalent bonding / linkage or by affinity ligands. In the former case, the surface of the plasmonic substrate (made of gold, silver, copper, aluminum, platinum, or combinations thereof) is functionalized with a linker (e.g., PEG or a carbon chain having a functional group) or a biomolecule adhesion layer to capture all EVs on the surface via non-specific interactions. In the latter case, a marker-positive target EV subpopulation is captured on the surface via an affinity ligand / capture agent (e.g., antibody, aptamer, peptide, nucleic acid). The first case is the capture of all EVs. The second case is the capture of target EVs.

[0125] Next, the captured EVs are immunolabeled with a fluorophore for marker analysis. Markers include, but are not limited to, surface proteins, intracellular proteins, lipids, and nucleic acids. The ligand used to capture EVs on the substrate can also be used for marker labeling. For example, the QUAD markers (MUC1, HER2, EGFR, and EpCAM) can be used. In the first case, only the target EVs among all EVs are labeled using a target EV-specific capture agent conjugated to a reporter group. The capture agent must specifically bind to a marker found only on target EVs, not on all EVs. In the second case, all EVs bound to the nanostructures and / or the substrate are target EVs and thus can all be labeled with a reporter group, and thus the reporter group can bind to a capture agent that binds to any EV (since only target EVs are present on the substrate). Of course, a target EV-specific reporter group can be used in both cases. The ultimate goal is to label only the target EVs with a reporter group.

[0126] The labeled target EVs are imaged, for example, by a fluorescence microscope. As shown in FIG. 20, images from multiple fluorescence channels can be acquired for multiplex analysis of EV markers at the single EV level. The images are then analyzed to detect the total number of EVs and their concentration, marker-positive EVs (EVs positive for CD63 / CD81 / CD9 (Pan-CD) or tumor-derived EVs), co-localization between channels, marker expression levels, correlation with clinical outcomes, etc.

[0127] FIG. 21A is a schematic diagram showing EV capture on a gold nanopillar array. The gold nanopillars can be decorated with gold nanoparticles to increase plasmon enhancement. The diameter of the gold nanopillars can be 20 - 500 nm, and the height can be 20 - 5000 nm. The diameter of the decorated nanoparticles can be 10 - 100 nm. The schematic diagram shows the case of non-specific EV capture by PEG-COOH after activation of carboxylation groups by EDC / NHS. Alternatively, antibodies can be immobilized on the nanopillar surface to capture marker-specific EVs. The captured EVs are labeled with fluorophore-conjugated antibodies.

[0128] Numerical simulation Furthermore, numerical simulation can be applied to calculate the resonance peak wavelength, determine the selection of labels for maximum optical signal amplification, and optimize the dimensions and materials of the nanostructures. The electrodynamics calculations can be performed using the finite-difference time-domain (FDTD) method. The electric field distribution irradiates a plane wave polarized in the x direction along the z direction. A 2 nm mesh size is used for a volume of 0.3×0.3×0.2 μm located at the center of the nanoholes. Periodic boundary conditions are imposed along the x and y directions, and a perfectly matched layer is used in the z direction. A z-polarized dipole source is used for the radiative decay rate simulation. The position of the dipole is set to x = 100 nm, y = 0 nm, and z = 6 nm so as to be located at the edge of the nanoholes of the nanoplasmon array and 6 nm above the Au surface. 3 is used.

[0129] Furthermore, statistical analysis and data plotting can be performed using GraphPad Prism 7. Group differences are tested using an unpaired t-test. All tests are two-sided, and P-values less than 0.05 are considered statistically significant.

[0130] General method Figures 1A - 1G show an example of a nanoplasmonic array for multiplexed single EV analysis. Figure 1A shows the procedure steps of multiplexed single EV analysis starting from the capture of EVs, e.g., target EVs, labeling of the captured target EVs, imaging of the labeled target EVs, and analysis of the images of the target EVs. EVs are captured on the nanohole surface and immunostained with fluorescent detection probes, and then the labeled target EVs are imaged in different fluorescent channels and their intensities are analyzed. For example, EVs are captured on the Au nanohole surface via an affinity ligand (e.g., capturing biotinylated EVs on an avidin-coated Au nanohole surface). Then, target EVs, which can be some or all of the captured EVs, are immunostained with fluorescently labeled antibodies in different color channels (typically 3 - 4 colors). Depending on the absorption and emission spectra of the fluorophore, the fluorescence signal is amplified by surface plasmon resonance (SPR) excited by the underlying Au nanohole structure.

[0131] Figure 1B shows a scanning electron micrograph of the periodic nanoholes in the nanoplasmonic array. The diameter of the nanohole is about 200 nm and the periodicity is 500 nm. The scale bar in Figure 1B is 1 μm. The nanostructure in Figure 1B is optimized as an SPR substrate, and the substrate is a 100-nm-thick Au film.

[0132] Figure 1C shows a finite-difference time-domain simulation showing the enhanced electromagnetic field confined to the nanohole surface of the nanoplasmon array. The strong field is responsible for the plasmon-enhanced fluorescence signal. The periodic nanohole imparting to the chip surface concentrates an electromagnetic field with a maximum electric field strength up to 300 times. The resonant field extends up to 110 nm in the z direction, which mainly covers small EVs (e.g., exosomes having an average diameter of 100 nm). In addition to the local near-field, fluorescence emission can be further enhanced by the interaction between proximal fluorophores within the resonance range and the Au nanostructures.

[0133] Figure 1D shows images of fluorescent nanospheres (using Cy5, 200 nm) on glass and the nanoplasmonic substrate of the present invention. The scale bar in Figure 1D is 10 μm. The plasmon-enhanced fluorescence by the Au nanohole structure of the nPLEX chip using fluorescent nanospheres (Cy5, 200 nm) is tested in comparison with the glass substrate. Also, Figure 1E shows an example of a histogram of pixel intensities of the glass substrate and the present nanoplasmonic substrate. This shows that the fluorescence intensity of individual nanospheres is significantly higher on the nPLEX-FL substrate (two-sided t-test, p < 0.0001). Figure 1F shows the average fluorescence intensity of fluorescent nanospheres on glass and the nanoplasmonic substrate. The average fluorescence intensity increases by 18 times, and the signal-to-noise ratio (given by the signal divided by 3 times the standard deviation of the blank) increases 20 times from 17.7 (glass) to 358 (nPLEX-FL). There is no significant difference in the coefficient of variation given by the ratio of the standard deviation to the mean for the fluorescence intensity between glass (36.2%) and the nPLEX-FL substrate (33.6%), indicating that signal amplification does not increase intensity variation.

[0134] Figure 1G shows a finite-difference time-domain simulation showing the enhanced electromagnetic field around the nanohole. Scanning electron microscopy shows EVs captured by the functionalized Au nanohole chip. The dotted circle represents the enhanced electromagnetic field distribution around the nanohole.

[0135] Dual-mode imaging device Figure 2A shows an example of an optical system (NEXT readout system) for a nanoplasmon array for multiplexed single EV analysis that integrates dark field imaging with multi-channel fluorescence imaging. Figure 2A shows an example of an upright microscope setup 10 for dark field (transmission) and epi-fluorescence dual mode imaging. A charge-coupled device (CCD) 20 of the optical setup is used to capture electromagnetic radiation emitted, scattered, or reflected by reporter groups (e.g., fluorescent antibodies) on labeled target EVs captured on the nanoplasmon array. In some embodiments, the upright microscope setup 10 includes a filter set 22 for processing / filtering electromagnetic radiation, a microscope stage 26 on which a substrate having a nanoplasmon array is placed under the objective lens 24, and a camera, such as a CCD 20, for capturing the radiation processed by the filter set 22. The system also includes a dark field condenser 28 disposed below the stage 26, a primary light source 30 (e.g., an LED light source) arranged to irradiate the microscope stage 26 from above, and a secondary light source 32 arranged to irradiate the microscope stage 26 from below through the dark field condenser 28. In some embodiments, dark field scattering imaging is used to irradiate the nanoplasmon array from below and capture electromagnetic radiation emitted, scattered, or reflected from the nanoplasmon array through the objective lens 24.

[0136] In some examples, the second light source 32 and the dark field condenser 28 of the dark field scattering imaging system can be placed above the objective lens 24 to irradiate the microscope stage 26 from above.

[0137] In this system, the location of captured EVs or nano-sized particles can be detected by dark field imaging, and their marker positivity can be detected by fluorescence imaging. In this case, the dark field scattering signal can also be amplified by the underlying plasmonic substrate.

[0138] Figure 2B shows an example of a dark-field scattering image of the nanostructure array. The inset shows an enlarged image of the nanostructure array. In some embodiments, the nanostructure array may be a nanorod array, and each nanorod is separated by 2 μm.

Example

[0139] The present disclosure is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example

[0140] Example 1 - Characterization of the nanoplasmon array system The inventors investigated the plasmon enhancement of EVs. The inventors captured biotinylated EVs on glass and nPLEX-FL substrates, and subsequently labeled the captured EVs with streptavidin-conjugated dyes (Cy5, Figure 3E and AF488).

[0141] The nPLEX-FL chip was prepared using the above lithography method. The chip was incubated overnight at room temperature with thiolated biotin polyethylene glycol (PEG) (10×10 -3 m, PG2-BNTH-1k, Nanocs). After washing with PBS, an equimolar mixture of streptavidin molecules conjugated to either AlexaFluor488, Cy3, Cy5, or Cy5.5 (Biolegend) was incubated for 10 minutes. The concentration of each fluorescent dye was diluted to 2.5 μg mL-1, except for AlexaFluor488-conjugated streptavidin (25 μg mL-1 in PBS) due to a weak fluorescence signal compared to the other channels.

[0142] The inventors used a polyphenol-protein-based bioadhesive layer to capture the same amount of EVs on different substrates (glass and Au) and investigated the fluorescence intensity and detectable EV counts. When measuring the average signal enhancement factor for the fluorescence intensity after background correction, it was 1.54 for AF488 and 8.60 for Cy5 (Figure 3F). The overall signal enhancement of the captured EVs was not as prominent as that of the streptavidin monolayer coating, probably due to the difference in thickness between the EVs and the streptavidin monolayer (the electromagnetic field is stronger near the surface) (see Figures 3C and 3F). Nevertheless, the inventors were able to detect a significantly larger number of Cy5-labeled EVs on the nPLEX-FL chip compared to the glass substrate, showing higher sensitivity achieved by plasmon-enhanced signal amplification (Figure 3G).

[0143] The inventors also observed comparable average pixel intensities and EV counts for AF488-labeled EVs on both the nanohole chip and glass (see Figure 3H). This indicates that plasmon enhancement on the Cy5 dye reveals EVs with weak fluorescence signals that would otherwise not be detected without signal enhancement (glass substrate) or would be detected with weak enhancement (AF488). Therefore, the inventors assign low-abundance or important EV markers to the Cy5 channel in subsequent validation studies for maximum signal enhancement.

[0144] Specifically, Figure 3A is a series of fluorescence images of a nanoplasmon array / chip coated with four-color fluorophore-conjugated streptavidin (AF488, Cy3, Cy5, and Cy5.5). The scale bar in Figure 3A is 20 μm. The nanohole array was fabricated in a square area of 100×100 μm 2 in size, highlighted by the white dashed box, for example, the white dashed box shown in the fluorescence image using AF488.

[0145] Figure 3B shows the cross-sectional intensity profile along the gray horizontal dashed line in Figure 3A. Using a molecular monolayer, plasmon enhancement in different fluorescence channels was tested. The Au nanohole surface was functionalized with a thiolated biotin polyethylene glycol derivative (thiol-PEG-biotin), and fluorophore-conjugated streptavidin molecules were immobilized on the biotinylated Au surface. To prevent fluorescence quenching by the underlying Au substrate, the Au surface was functionalized with thiol-PEG-biotin (1 kDa, 6 - 8 nm) and neutravidin (60 kDa, 4 - 5 nm) to obtain an adhesion layer with a thickness of 10 - 13 nm. Figures 3A and 3B show strong signal enhancement in the nanohole diffraction grating (highlighted by the white dashed box) of a square area with a size of 100×100 μm 2 compared to the flat Au area (outside the square, Figure 3B).

[0146] Figure 3C shows an example of the enhancement factor (EF) of fluorescence intensity in different fluorescence channels. The signal enhancement is most dominant in the Cy5 channel, and the EF of the fluorescence intensity in the nanohole area is 23 times that in the flat Au area.

[0147] Figure 3D shows an example of the plasmon-supported light transmission spectrum through a nanohole array with the absorption / emission spectra of fluorophores superimposed. The intensities of Cy5.5 and Cy3 also increase by 17 times and 9 times, respectively, when the AF488 signal increases by 3 times. These EFs in different channels can be explained by the spectral overlap between the plasmon-supported light transmission through the nanohole and the absorption / emission spectra of the fluorophores. The light transmission peak was measured at 667 nm, which overlaps most with the Cy5 absorption (649 nm) and emission (666 nm) peaks, followed by Cy5.5 and Cy3.

[0148] Figures 3E to 3I show examples of plasmon enhancement for EVs. Figure 3E shows that biotinylated EVs are captured on a glass and nanoplasmon array substrate, and subsequently the captured EVs are labeled with streptavidin-conjugated dye. The captured EVs are labeled with Cy5-conjugated streptavidin and then imaged. The scale bar in Figure 3E is 10 μm. Figure 3H shows biotinylated EVs captured on a device surface coated with an L-3,4-dihydroxyphenylalanine (L-DOPA)-based bioadhesive layer. The captured EVs are labeled with AF488-conjugated streptavidin and then imaged. Figure 3I shows a comparison of the average fluorescence intensity, and Figure 3J shows the number of captured EVs in Figure 3H in the region of interest (ROI) between the nanohole chip and the glass substrate. An L-DOPA-based bioadhesive layer is used to capture the same density of EVs on different substrates (glass and Au), and the fluorescence intensity and detectable EV count are investigated.

[0149] Furthermore, Figure 3F shows an example of a histogram of the pixel intensity of the captured EVs in Figure 3E. When the average signal enhancement factor for the fluorescence intensity after background correction was measured, it was 1.54 for AF488 and 8.60 for Cy5. The overall enhancement is not significant compared to the streptavidin monolayer coating in Figure 3C, probably due to the strongest local electromagnetic field near the surface shown in Figure 3C.

[0150] Figure 3G shows the number of detected EVs in Figure 3E between the nanohole chip and the glass substrate. The fluorescence intensity was normalized by the background signal defined by the sum of the average fluorescence intensity and three times the standard deviation in the absence of EVs. It was detected that the number of Cy5-labeled EVs on the nPLEX-FL chip was orders of magnitude higher compared to the glass substrate, indicating higher sensitivity achieved by plasmon-enhanced signal amplification. In contrast, equivalent average pixel intensities and EV counts were observed for AF488-labeled EVs on both the nanohole chip and the glass. The observed difference is that plasmon-induced signal amplification of Cy5 reveals smaller EVs with weak signals that are not detected on the glass substrate. Therefore, due to the maximum spectral overlap with plasmon resonance, the Cy5 dye was selected to label low-abundance intracellular markers.

Example

[0151] Example 2 - Single EV Measurement We applied the nPLEX-FL technology to demonstrate its feasibility in multiplexed single EV analysis. We used glioblastoma cell lines for the tests: Gli36-WT and Gli36-EGFRvIII (which overexpresses human EGFRvIII). EGFR and EGFRvIII are target biomarkers for glioblastoma because amplification of EGFR and its variant (EGFRvIII) frequently occurs in glioblastoma. The presence of protein markers including 1) a combination of ubiquitous EV tetraspanins named CD-pan (CD9, CD63, and CD81); 2) GAPDH; 3) EGFR; and 4) EGFRvIII was examined by nPLEX-FL and benchmarked against Western blotting analysis as a standard method (see Figures 6A and 6B).

[0152] EVs were isolated from conditioned cell culture media. Nanoparticle tracking analysis showed that the isolated EVs used in this study had a size distribution in the range of 50 - 200 nm and an average diameter of 100 nm, which was also confirmed by transmission electron micrographs. The isolated EVs were biotinylated and diluted in pure buffer (1 - 10×10 8 EV mL-1 phosphate-buffered saline (PBS)) and captured on a neutravidin-coated gold nanohole surface. The captured EVs were immunolabeled against membrane (i.e., CD63, EGFR) and / or intracellular markers (i.e., GAPDH) and imaged under a fluorescence microscope. Since most EVs are smaller than the diffraction limit, the average blob size of the vesicles detected in the fluorescence images was approximately 500 nm (8 pixels, pixel size 63 nm). Single EVs generated a detectable fluorescence signal, which was confirmed by scanning electron micrographs. Some doublet EVs showed higher intensity in the streptavidin channel. Particles imaged larger than 1 μm (or 16 pixels) were considered large aggregates and excluded from our analysis.

[0153] The inventors selected well-established EV markers for proof-of-concept demonstration of EV profiling and subpopulation sorting based on marker signals. Considering fluorescence signal enhancement, the inventors assigned 1) the green dye (AF488) to markers with high abundance / easy detection and 2) the far-red dye (Cy5) to markers with low abundance / difficult detection. Figure 4A shows a representative nPLEX-FL image of biotinylated EVs labeled against CD-pan (AF488), streptavidin (Cy3), and GAPDH (Cy5). The inventors selected GAPDH as a representative intracellular marker, which is commonly used as a control in many other quantitative methods (e.g., Western blotting, qPCR). The inventors varied the EV concentration and counted the number of captured EVs. Line scans (Figure 4B) showed high signal-to-noise ratios and signal inhomogeneities for the selected markers on individual vesicles. The inventors then analyzed the raw intensity data for EV marker profiling. For a given marker, the inventors identified two subpopulations (marker-positive and marker-negative) that could be separated by an intensity cutoff (mean + 2 × standard deviation of the negative control). Approximately 40% of the captured streptavidin-positive vesicles were CD-pan positive, and the proportion of GAPDH-expressing CD-pan-positive EVs was 25% (Figure 3C). The false-positive rates in the control samples (without EVs) were negligible for both streptavidin staining (<1%) and antibody staining (<0.2%). Based on the negative control data, the inventors set the threshold for positivity at 1%.

[0154] In particular, Figure 4A shows that EVs from the Gli36-WT cell line are biotinylated and captured on the nanohole surface. Individual EVs are detected by staining with fluorescent Cy3-streptavidin (upper left). For molecular profiling, EVs are labeled with fluorescent antibodies against transmembrane EV markers (CD63) and intracellular markers (GAPDH). Multiple EV markers are selected to detect and classify single EVs based on marker expression levels. The AF488 dye is assigned to markers with high abundance / detectability, and Cy5 is assigned to markers with low abundance / detectability. Figure 4A shows a representative nPLEX-FL image of Gli36-WT-derived EVs labeled against CD63 (AF488) and GAPDH (Cy5). GAPDH is selected as a representative intracellular marker, which is commonly used as a control for many other quantitative methods (e.g., western blotting, qPCR).

[0155] Figure 4B shows a line scan demonstrating high signal-to-noise for the selected markers in this example. The grey shading emphasizes the EV positions. The line scan shows high signal-to-noise and heterogeneity for the selected markers on individual vesicles.

[0156] Figure 4C shows EV subtyping. Next, the raw intensity data shown in Figure 4B was analyzed for marker expression and EV subtyping. For a given marker, the inventors identified two subpopulations that could be separated by a 100 intensity cutoff: marker-positive and marker-negative. Approximately half of the captured vesicles had CD63 (46%), and of the CD63+ EVs, the fraction expressing GAPDH was 58%. A strong overlap (>95%) between GAPDH+ EVs and CD63+ EVs was confirmed.

[0157] The results show that a higher percentage of Cy5-GAPDH+ EVs on the nanohole chip is observed compared to other substrates, which may be due to plasmon-derived signal amplification in the Cy5 red channel.

Example

[0158] Example 3 - Demonstration of Tumor Diagnosis Possibility Figures 5A - 5E show an example of the measurement of tumor markers of captured EVs to demonstrate the tumor diagnosis possibility of the new system and method. EVs from three different cell lines (Gil36 - WT, Gli36 - EGFRvIII, MCF7) were biotinylated, captured on the nanohole array surface, and labeled with fluorescent antibodies against CD - pan marker panel (CD9, CD63, and CD81) and tumor markers including EGFR in Figure 5A and EGFRvIII in Figure 5B. The spots of the dotted - circle indicate tumor - marker - positive EVs in Figures 5A and 5B.

[0159] Figure 5C shows the Western blot analysis of EGFR expression in Gli36 - WT, Gli36 - EGFRvIII, and MCF - 7 cell lines. MCF - 7 cells served as a negative control for EGFR expression. Blotting antibodies against GAPDH were used for loading control.

[0160] The bar graphs in Figures 5D and 5E show EV subtyping. Ratio (%) = EV CD-pan+標的 + / EV CD-pan+ . As shown in Figure 5D, a significant proportion of Gli36 - WT EVs were positive for EGFR (54%), while a small proportion of Gli36 - EGFRvIII were positive for EGFR (7%). As shown in Figure 5E, more than 10% of Gli36 - EGFRvIII vesicles were positive for EGFRvIII, while Gli36 - WT and MCF7 showed EGFRvIII - positive ratios (<1%) below the threshold of statistical significance. Negative controls were prepared with the same procedure without EV incubation.

[0161] To further test the diagnostic possibility for clinical applications, the inventors used approximately 10 from Gli36 - WT and Gli36 - EGFRvIII cell lines 10Individual EVs were spiked into 1 mL of human plasma sample. EVs were isolated from the spiked plasma sample using a size exclusion column (Izon column), biotinylated, and then loaded onto the chip (1 - 5 μL). The captured EVs were labeled against CD-pan (AF488), streptavidin (Cy3), and EGFR or EGFRvIII (Cy5). The inventors implemented a decision tree algorithm using a nested gating strategy for classifying the EV population based on EGFR and EGFRvIII signals (Figure 6A). Briefly, particles labeled with Cy3-conjugated streptavidin were first detected and pre-screened by size exclusion (<1 μm) to exclude large aggregates from the analysis. Among the particles positive for streptavidin, the inventors defined EVs positive for CD-pan markers (CD9, CD63, and CD81). The pre-screened EVs were then sub-gated with the target glioblastoma markers of EGFR or EGFRvIII. The inventors performed an output analysis of the Mann-Whitney test using two independent groups (EV positive and negative) and calculated the required EV sample size (n>100) considering a statistical output of 0.9 and an effect size of 0.43. Considering the EV surface coverage of 0.1 - 0.5 EV μm -2 the minimum area required is approximately 200 - 1000 μm 2 However, the inventors used fluorescence images (n = 4) at the full field of view (FOV: 120 μm × 100 μm) and sampled thousands of vesicles per measurement to ensure statistical significance and robust analysis.

[0162] Figures 6B and 6C show the biomarker distribution analysis at a single EV level. The inventors plotted bivariate histograms from 3-channel fluorescence images in a 120 μm × 100 μm FOV. On average, the inventors detected approximately 4200 particles positive for streptavidin in a single image (minimum = 3604, maximum = 5057 EVs, Figure 6D). The inventors observed a positivity of 10 - 15% of streptavidin-positive particles for the CD-pan marker (Figure 6E). The lower proportion of CD-pan+ streptavidin+ particles in plasma samples compared to pure buffer (PBS) may be due to the presence of lipoproteins and plasma protein aggregates in human plasma. For marker profiling, the detected EVs positive for CD-pan were screened for target markers of EGFR and EGFRvIII. In plasma samples spiked with EVs from Gli36-WT and Gli36-EGFRvIII cell lines, approximately 10 - 20% of the detected EVs were positive for EGFR in both samples (Figure 6F). However, approximately 10% of the EVs were positive for EGFRvIII only in plasma samples with Gli36-EGFRvIII EVs, while the other samples with Gli36-WT EVs showed a positive EV ratio of less than 1%, which is below the threshold (Figure 6G). Equivalent biomarker positivity for EGFR and EGFRvIII was observed between plasma samples and pure buffer samples.

[0163] These results indicate that EGFRvIII mutant protein can be detected using glioblastoma EVs.

Example

[0164] Example 4 - Characterization of EVs Isolated from Tumor Cell Lines The nPLEX-FL technology was extended to demonstrate its feasibility in multiplexed single EV analysis. Glioblastoma (GBM) cell lines were used for the study: Gli36-WT and Gli36-EGFRvIII (a clone of Gli36EV that is positive for the EGFRvIII mutation). EVs were collected from conditioned cell culture media, membrane-filtered, biotinylated, immobilized on the surface of a nanohole array chip, and immunolabeled against membrane (i.e., CD63, EGFR) and / or intracellular markers (i.e., GAPDH). The isolated EVs used in this study had an average diameter of 100 nm and a size distribution ranging from 50 to 200 nm, and high purity was determined by Western blotting for ubiquitous EV protein markers (CD9, CD63, and CD81, Figure 7B). Avidin-functionalized Au chips showed high specificity for biotinylated EV capture, which was confirmed by electron microscopy.

[0165] Specifically, Figure 7A shows the size distribution graphs of Gli36-WT and Gli36-EGFRvIII EVs obtained by nanoparticle tracking analysis (NTA). Figure 7B shows Western blot measurements of Gli36-WT and Gli36-EGFRvIII EVs to determine the pan-CD marker expression levels (CD9 / CD63 / CD81) in bulk.

[0166] The results demonstrate that multiplexed single EV analysis is feasible using the nanoplasmonic array devices and methods disclosed herein.

Example

[0167] Example 5 - Characterization of EVs with Varying Concentrations Figure 8A shows EVs from the OVCA429 cell line that were biotinylated and captured on a nanoplasmon array device. EVs were collected from conditioned cell culture media, membrane filtered, biotinylated, and immobilized on the surface of a nanohole array chip. The captured EVs were labeled with a pan-CD marker that is a combination of CD9, CD63, and CD81 (AF488) and EGFR (Cy5). The EVs were artificially color-coded for visual aid. The scale bar in Figure 8A is 10 μm.

[0168] The three bar graphs in Figure 8B show various EV concentrations (a four-fold difference) and the number of captured EVs. Regardless of the EV concentration, approximately half of the CD-pan+ EVs expressed EGFR. The inventors discriminated individual vesicles by staining the EVs with Cy3-streptavidin.

[0169] These results indicate that the devices and methods disclosed herein can be used for single-vesicle analysis even when the EV concentration varies.

Example

[0170] Example 6 - Negative Control to Demonstrate Test Sensitivity Figures 9A - 9B show negative controls to demonstrate the test sensitivity and specificity of captured EVs. EVs from the MCF7 cell line were collected from conditioned cell culture media, membrane filtered, biotinylated, and immobilized on the surface of a nanohole array chip. The EVs were labeled with a CDpan marker that is a combination of CD63 / CD81 / CD9 (AF488) and EGFR (shown in Figure 9A) or EGFRvIII (Cy5) (shown in Figure 9B).

[0171] Negative controls (e.g., no EVs) were prepared in the same procedure without EV incubation. The results in Figures 9A - 9B show the statistical significance of the present nanoplasmon array sensor system for detecting target EVs.

Example

[0172] Example 7 - Optical Property Evaluation of Nanorod Arrays Figures 12A to 12B show examples of the optical property evaluation of the nanorod sensor array described in this specification.

[0173] The graph in Figure 12A shows the results of finite-difference time-domain (FDTD) simulations showing the optical resonance peaks for different-sized nanorods having lengths of 40, 60, 80, 100, and 120 nm. Optical tuning is important to maximize the fluorescence signal enhancement due to the surface plasmon resonance of the nanorods. The nanoplasmon array can have a specific size / dimension for each of the nanorods based on the size of the target EV and / or other requirements (e.g., to detect the specific wavelength of SPR) in the analysis process.

[0174] The graph in Figure 12B shows the experimental results of the spectral shift and intensity change of dark-field scattering as the surface refractive index increases from 1.33 to 1.45. Different mixing ratios of water and ethanol mixtures were prepared and applied to the nanorod array to vary the refractive index from 1.33 to 1.45. The spectral shift was measured and plotted against the surface refractive index. The spectral shift was calculated using the peak wavelength at a surface refractive index of 1.33 as a reference. The binding of EVs to the nanorods increases the surface refractive index and shifts the resonance peak. The EV binding event can be detected by measuring either the spectral shift or the change in scattered light intensity.

Example

[0175] Example 8 - Optical Resonance of Nanorods vs. Nanodisks Figure 13A is a side view of an EV captured on a nanorod. Figure 13B is a top view of a nanodisk in the array. Figure 13C is a scanning electron microscope image of the top surface of an EV on the nanostructure.

[0176] FIG. 13D is a graph showing the scattering intensity (a.u.) of nanodisks having different diameters of 40, 60, 80, 100, 120, 140, 160, 180, or 200 nm calculated by FDTD simulation. As shown, the scattering intensity increases with the diameter of the nanodisk, and the wavelength of the peak scattering intensity also increases with the diameter of the nanodisk.

[0177] FIG. 13E is a graph showing the peak shift (nm) of nanodisks having different diameters from 40 to 200 nm. As shown, the peak shift is highest for nanodisks having a diameter of 40 nm, decreases sharply at diameters from 60 nm to 80 nm, and then continues to decrease as the diameter increases until it levels off at about 180 nm.

[0178] FIG. 13F is a top view of a nanorod having a length L and a width of 30 nm. FIG. 13G is a graph showing the scattering intensity (a.u) of nanorods having different lengths of 40, 60, 80, 100, 120, 140, 160, 180, and 200 nm. As shown, the peak scattering intensity increases with the wavelength and the length of the nanorod.

[0179] FIG. 13H is a graph showing the peak shift (nm) of nanorods having different lengths from 40 to 120 nm. As shown, the peak shift decreases with the length of the nanorod.

[0180] The nanoplasmon array can be designed to have nanostructures with nanorods or nanodisks of specific sizes / dimensions based on the size of the target EV and other requirements for detecting the target EV (e.g., for detecting the specific wavelength of SPR). Any shape similar to the nanorod or nanodisk can also be used as a plasmonic nanostructure to amplify the fluorescence and dark-field signals.

Example

[0181] Example 9 - Finite Difference Time Domain (FDTD) Simulation Figures 14A to 14C show FDTD simulations showing the spectral shift of dark-field scattering upon EV binding to nanodisks at different locations and distances to the substrate.

[0182] Figure 14A is a diagram of Scenario 1 of the first EV binding location and its detected peak wavelength, together with the corresponding graph. Figure 14B is a diagram of Scenario 2 of the second EV binding location and its detected peak wavelength, together with a microscopic image showing the electromagnetic wave. Figure 14C is a diagram of Scenario 3 of the third EV binding location and its detected peak wavelength, together with a microscopic image showing the electromagnetic wave concentrated on the nanodisk surface. The results show that single EV binding to the nanodisk surface in various binding scenarios can be detected by measuring the spectral shift of the dark-field scattering peak wavelength.

Example

[0183] Example 10 - Real-time EV Binding Experiment The real-time binding of EVs to nanodisks was analyzed using the systems and methods described herein.

[0184] Figures 15A to 15D show an example of EV binding detection by measuring the change in dark-field scattering intensity in real time. Time line (1) is before EV binding to the nanodisk at the center of the array (the central circle) as shown in Figure 15A. Time line (2) is after EV binding to the nanodisk at the center of the array (the central circle) as shown in Figure 15B.

[0185] The graph in Figure 15C shows real-time measurement values indicating a sharp intensity change at time point (2) upon EV binding to the central nanodisk. In particular, Figure 15C shows that the intensity increases within 100 seconds when the EV binds to the nanodisk (the central circle), as the signal difference shown in Figures 15A and 15B.

[0186] The graph of FIG. 15D shows a real-time measurement that does not show two sharp intensity changes indicating that it does not bind to any of the nanodisks in the array. In particular, FIG. 15D shows the change over time in the dark-field scattering intensity of control nanodisks without affinity ligands (control 1 and control 2 in FIGS. 15A and 15B). There is no EV binding, and thus no intensity change is measured. The vertical dashed lines (1) and (2) in FIGS. 15C and 15D indicate the time points at which FIGS. 15A and 15B were taken.

Example

[0187] Example 11 - Spectral Shift of Dark-Field Scattering of Nanodisks Using the above methods and nanodisk arrays, the spectral shift of dark-field scattering caused by the nanodisks was analyzed. FIGS. 16A - 16C show the results of a method by which EV binding can be detected by measuring the spectral shift of dark-field scattering by the nanodisks. EV binding was confirmed by overlay with the EV fluorescence image. EVs were labeled with a specific fluorescent probe to localize the nanodisks that captured the EVs.

[0188] FIGS. 16A - 16C show an overlaid image of the fluorescence channel of the EVs and the dark-field scattering of the nanodisks, indicating that the EVs are captured on the nanodisks (circles in the image). The dark-field scattering spectra before and after EV binding on these nanodisks are shown on the right, demonstrating that EV binding to the nanodisk surface induces a spectral shift.

[0189] The image of FIG. 16A shows the central nanodisk showing a fluorescence signal indicating EV binding. The attached graph of the single nanodisk spectrum shows a slight spectral shift in the normalized scattering before or after EV binding, and no shift means complete overlap.

[0190] The image of Fig. 16B shows a central nanodisk with a change in fluorescence indicating EV binding. The attached graph of the single nanodisk spectrum shows a significant rightward (increase in peak wavelength) shift in the normalized scattering after EV binding.

[0191] The image of Fig. 16C shows a nanodisk with a fluorescence signal indicating EV binding. The attached graph of the single nanodisk spectrum shows no significant shift in the normalized scattering before or after EV binding, and the lack of shift implies complete overlap.

Example

[0192] Example 12 - Plasmon Enhancement of Dark-Field and Fluorescence Signals Using Nanodisks of Different Diameters As shown in Figs. 17 and 18A - 18D, the nanoplasmon array can be designed to have nanodisks with corresponding diameters specific to the respective dark-field / fluorescence labeling for detecting target EVs. A monolayer of fluorescent molecules was formed on top of the nanodisk array, and the fluorescence signals on the nanodisks and the substrate were measured.

[0193] Fig. 17 shows the plasmon enhancement of dark-field and three different fluorescence signals (TRITC, Cy5, and Cy5.5) for nanodisks with different diameters of 80, 100, 120, 140, 160, 180, and 200 nm.

[0194] Figs. 18A - 18D are graphs showing the plasmon enhancement of dark-field and fluorescence signals for different-sized nanodisks. Fig. 18A is a graph of the plasmon intensity for dark-field measurements corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200 nm, and as shown, the level increases with diameter up to about 180 nm and then slightly decreases at 200 nm.

[0195] Figure 18B is a graph of the plasmon intensity of TRITC corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200 nm. As shown, the level increases rapidly with the diameter up to 120 nm, then decreases rapidly up to 140 nm, decreases slightly up to 180 nm, and increases again up to 200 nm.

[0196] Figure 18C is a graph of the plasmon intensity of Cy5 corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200. As shown, the level decreases slightly from 80 to 100 nm, then increases rapidly with the diameter up to 120 nm, and decreases up to 200 nm.

[0197] Figure 18D is a graph of the plasmon intensity of Cy5.5 corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200 nm. As shown, the level remains the same from 80 to 100 nm, increases rapidly with the diameter from 100 to 140 nm, then decreases rapidly up to 180 nm, and decreases slightly up to 200 nm.

[0198] TRICT and Cy5 showed maximum intensity when coated on 120 nm (diameter) nanodisks, while Cy5.5 showed maximum intensity on 140 nm diameter nanodisks.

Example

[0199] Isolation and Preparation of Example 13 - EV EVs were isolated from cell cultures of Gli36-WT (ATCC), Gli36-EGFRvIII (generated from Gli36-WT by lentiviral transduction), and MCF-7 cells (ATCC) grown in DMEM (Cellgro), and OVCA429 cells (ATCC) cultured in RPMI-1640 medium (Cellgro). The medium was supplemented with 10% fetal bovine serum (FBS, Thermo Fisher), 100 U / mL penicillin, and 100 μg / mL streptomycin (Cellgro) at 37 °C in 5% CO2. Additionally, the cell lines were tested and found to be free of mycoplasma contamination (MycoAlert™ Mycoplasma Detection Kit, Lonza).

[0200] For EV isolation and biotinylation, EVs were incubated for 48 h in DMEM containing 1% exosome-depleted FBS (Thermo Fisher) prior to EV collection. The conditioned medium was collected and centrifuged, for example, at 300×g for 5 min, and then the supernatant was filtered through, for example, a 0.2-μm membrane filter (Millipore Sigma).

[0201] EVs were isolated using both standard ultracentrifugation (UC) and size exclusion chromatography (SEC) methods: (i) for UC, the filtrate was concentrated by centrifugation at 100,000×g for 1 h. After removing the supernatant, the EV pellet was washed with buffer or saline, for example, PBS, and centrifuged at 100,000×g for 1 h. The EV pellet was resuspended in PBS, and (ii) for SEC, the filtrate was loaded onto a centrifugal filter (Centricon® Plus-70 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma)) and centrifuged at 3,500×g for 30 min at low temperature, for example, 4 °C.

[0202] After concentration, the volume was adjusted to 1 mL using PBS. SEC was performed with modifications. Briefly, a 10 mL syringe (BD Biosciences) equipped with a nylon mesh (Millipore Sigma) having a pore size of 20 μm at the bottom was prepared and filled with 10 mL of Sepharose CL-4B (GE healthcare). The concentrate was loaded on top, and 1 mL of six fractions were collected under a constant gravity flow by adding PBS to the top of the column. Fractions 4 and 5 were used for EV isolation. These were loaded onto an Amicon Ultra-2 Centrifugal Filter (MWCO = 10 kDa, Millipore Sigma) and centrifuged at 3,500 × g for 30 minutes at 4 °C. The isolated EVs were stored at -80 °C until further measurement.

[0203] The isolated EVs were resuspended in buffer or saline, such as PBS, and incubated with a capture agent, such as EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher), for a sufficient time at room temperature, such as 30 minutes. For example, a 20-fold molar excess of sulfo-NHS-biotin relative to the EV protein was used in a 0.5 mL volume. Four to six biotins were incorporated per molecule. Then, excess biotin was removed using an Exosome Spin Column, MW3000 (Thermo Fisher) according to the kit instructions. The prepared EVs were filtered using a 0.22 μm centrifugal filter (Ultrafree®, Millipore).

Example

[0204] Example 14 - Plasmon-Enhanced EV Detection on NPOP Substrates This example compares an NPOP (nanoparticle-on-nanopillar) substrate with a glass substrate for EV detection. The inventors fluorescently labeled EVs with AF555 or AF647 dyes and applied the same aliquot to the substrates. Since the substrates were made of different materials, the inventors coated the substrate surfaces with a cell adhesion layer (Corning® CELL-TAK™) to ensure the same surface chemistry.

[0205] The TFP dye was prepared as follows. First, the inventors prepared 27.5 mM azido-dPEG®-TFP ester (Quanta Biodesign) using anhydrous DMSO (Sigma). 12 Next, the inventors prepared 25 mM AFdye DBCO using anhydrous DMSO (Sigma), and then an equal volume of azido-dPEG®-TFP ester and AFdye DBCO were mixed with a HulaMixer® (ThermoFisher Scientific) at room temperature for 2 hours. 12 The EVs were labeled as follows. First, the inventors mixed 0.2 μL of TFP-AF555 or AF647 dye, 3 μg of 300 ng SKBR3 EVs in PBS, and 2 μg of 100 mM sodium bicarbonate, and incubated for 1 hour at room temperature under dark conditions. Next, the inventors loaded the labeled EVs onto a Zeba™ Micro Spin Desalting Column (ThermoFisher Scientific) to remove unbound TFP dye, centrifuged at 1,500 × g for 2 minutes, and collected the filtrate. Then, the inventors reloaded the filtrate onto a Zeba™ Micro Spin Desalting Column, centrifuged at 1,500 × g for 2 minutes, and collected the filtrate.

[0206]

[0207] ​The substrate and EV samples were prepared as follows. The inventors first cleaned the substrate, incubated the glass in absolute ethanol for 5 minutes, and then washed it with water. The NPOP was washed with water and the water was removed with nitrogen gas. The inventors attached PDMS (4 mm wells, 2 mm thickness) to the substrate for EV attachment. The inventors prepared a cell and tissue adhesive solution (CELL-TAK™, Corning) to attach EVs onto the substrate without surface chemistry. Then, the inventors mixed 30 μg of Cell-Tak, 10 mM sodium bicarbonate, and 17 mN sodium hydroxide, added the resulting mixture to the wells, and incubated for 30 minutes for surface absorption. Next, the inventors washed the substrate three times with water, diluted and added the fluorescently labeled EVs with PBS, incubated for 30 minutes at dilutions of 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, and washed the unbound EVs three times with PBS. Then, the inventors removed the PDMS wells, added Prolong Gold™ anti-fade mounting solution (ThermoFisher Scientific) onto the substrate, and mounted the coverslip.

[0208] Images were taken and analyzed as follows. The inventors acquired three images of the EV signal by upright fluorescence microscopy (Zeiss) with a 5-second exposure time for each channel (AF555 and AF647) using a 40x objective lens. The inventors converted the raw czi files to Tiff image files, loaded the images into ImageJ® Fiji, and subtracted the background signal. The inventors analyzed the EV number and intensity for each channel using the ComDet Plugin with fixed parameters (approximate particle size: 4.00 pixels, intensity threshold (SD): around 3.00). The inventors used Prism software (GraphPad) to describe the differences in EV count, EV intensity, and mean intensity of AF555 or AF647-labeled EVs at different dilution factors.

[0209] The results are shown in FIGS. 19A - 19D, indicating that more EVs can be detected when EVs are captured on the NPOP substrate rather than on the glass substrate. This was due to signal enhancement by the plasmonic substrate that amplifies the fluorescence signal of EVs. The overall signal was amplified, and a larger number of EVs were detected for both the AF555 channel and the AF647 channel. Enhancement can occur for dyes having an excitation wavelength longer than 530 nm.

[0210] FIG. 19A shows fluorescence images of AF555 or AF647 labeled EVs on a glass substrate or an NPOP substrate. The same amount of labeled EVs with different dilution factors was mounted on the adhesive molecule treated substrates. Scale bar, 50 μm.

[0211] FIG. 19B shows a pair of graphs for comparing the EV count numbers at different dilution factors on a glass substrate (black line) or an NPOP substrate (lighter line). The NPOP substrate detected a larger number of EVs for both AF555 labeled EVs and AF647 labeled EVs. Left panel, AF555 labeled EVs; right panel, AF647 labeled EVs. Error bars represent mean ± SD.

[0212] FIG. 19C is a pair of histogram plots for comparing the EV intensities (1:1600 dilution) on glass (black graph) and NPOP substrates (lighter line). The intensity of the detected EVs was higher on the NPOP substrate. Left panel, AF555 labeled EVs; right panel, AF647 labeled EVs.

[0213] FIG. 19D is a bar graph for the quantitative analysis of signal enhancement. As evaluated by two - way ANOVA with Bonferroni's multiple comparison test, the NPOP substrate enhanced the signal intensity by 2 - fold (AF555) and 9 - fold (AF647) compared to the glass substrate (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). Error bars are shown as mean ± SD from three different images.

[0214] These results show a significant advantage of the NPOP substrate over the glass substrate for EV detection.

Example

[0215] Example 15 - Comparison of Different Linkers for Capturing EVs on NPOP Substrate The inventors tested different thiol linker groups on the NPOP substrate. The linker groups have two functions. First, when their carboxylated groups are activated by EDC / NHS, the linkers function as affinity ligands and provide binding sites for EVs through covalent bonding between the activated functional groups on the linker and the EVs. Second, the linker groups serve as an immobilization layer (simulated) to minimize non-specific molecular binding. In this test, the inventors showed that thiol-PEG-COOH (1.0 kDa) showed a greater difference between desirable (EDC / NHS) EV capture and undesirable (simulated) EV capture than the other tested linkers.

[0216] The inventors labeled EVs by mixing 0.2 μg of TFP-AF555 dye, 3 μg of 300 ng of EVs in PBS, and 2 μg of 100 mM sodium bicarbonate and incubating for 1 hour at room temperature under dark conditions. The inventors loaded the labeled EVs onto a Zeba™ Micro Spin Desalting Column (ThermoFisher Scientific) to remove unbound TFP dye, centrifuged at 1,500 × g for 2 minutes, and collected the filtrate. The inventors reloaded the filtrate onto a Zeba™ Micro Spin Desalting Column, centrifuged at 1,500 × g for 2 minutes, and collected the filtrate.

[0217] The inventors prepared NPOP substrates and captured EVs by washing the substrates with water. The inventors then removed the water with nitrogen gas and placed the substrates in a humid chamber. The inventors functionalized the NPOP substrates with COOH molecules of different lengths. For mercaptoundecanoic acid (MUA), the inventors mixed 10 mM 11-mercaptoundecanoic acid (Sigma) and 1-octanethiol (Sigma) in absolute ethanol, added the mixture onto the NPOP substrates, and incubated for 2 hours at room temperature. Then, they washed with ethanol and water. For SH-PEG-COOH (0.4 kDa), the inventors prepared 0.25 mM SH-PEG-COOH (0.4 kDa, Nanocs) in water, added the mixture to the NPOP substrates, incubated for 4 hours at room temperature, and washed with water. For SH-PEG-COOH (1.0 kDa), the inventors prepared 0.25 mM SH-PEG-COOH (1.0 kDa, Nanocs) in water, added the mixture to the NPOP substrates, incubated for 4 hours at room temperature, and washed with water.

[0218] The inventors then treated the NPOP substrates with a mixture of 0.1 M MES (pH 6.0) for mock treatment and 50 mM EDC (ThermoFisher Scientific) and 125 mM sulfo-NHS (ThermoFisher Scientific) in 0.1 M MES (pH 6.0) for 7 minutes to capture EVs by covalent bonding. The inventors then washed the NPOP substrates with MES and PBS, incubated AF555-labeled EVs for 30 minutes at room temperature in the dark, washed away unbound EVs three times with PBS, added Prolong Gold™ anti-fade mounting solution (ThermoFisher scientific) onto the substrates, and mounted a coverslip.

[0219] Images were taken and analyzed as follows. The inventors acquired 10 images of EV signals using upright fluorescence microscopy (Zeiss) with a 40x objective lens and an exposure time of 5 seconds for the AF555 channel. The inventors converted the raw czi files to Tiff image files, loaded the images into ImageJ (registered trademark) Fiji, subtracted the background signal, and analyzed the EV number and intensity for each channel using the ComDet (registered trademark) Plugin with fixed parameters (approximate particle size: 4.00 pixels; intensity threshold (SD): around 3.00). The inventors used Prism software (GraphPad) to describe the difference in EV counts for AF555-labeled EVs of different lengths.

[0220] The results are shown in FIGS. 21B and 21C. FIG. 21B is a series of representative images of EV capture by functionalization with MUA (length 1.7 nm), SH-PEG-COOH (0.3 kDa, length 2.8 nm), and SH-PEG-COOH (1.0 kDa, length 7 nm) with or without EDC / NHS activation. Scale bar, 50 μm.

[0221] FIG. 21C is a bar graph of EV counts showing that SH-PEG-COOH showed the most non-specific EV binding (ns, not significant; ****P < 0.0001 compared to mock treatment when evaluated by two-way ANOVA with Bonferroni's multiple comparison test). Error bars are shown as mean ± SD from 10 different images. EDC / NHS activation appears to provide the most significant advantage with the 1.0 kD linker and no benefit with the MUA linker. Overall, the SH-PEG-COOH linker was better than the MUA linker.

[0222] FIG. 21D is a series of representative images of EVs (green) and markers (IgG or HER2, red) labeled with different colors. To evaluate differential marker expression, the percentage of co-localization (marker / EV) was analyzed. Co-localized signals are indicated by white arrows. Scale bar, 10 μm.

[0223] The results indicate that different linkers can provide excellent results.

Example

[0224] Example 16 - Multi - channel detection for molecular profiling of tumor - derived EVs using QUAD markers by single - EV analysis on an NPOP substrate This example examines the molecular profiling of tumor - derived EVs from four different breast cancer cell lines. These cell lines represent the four major subtypes of breast cancer based on their HER2, ER, and PR expression levels. The inventors tested EVs derived from the cell lines with their QUAD markers (MUC1, HER2, EGFR, and EpCAM) and compared their levels in the EVs and their parental cells. The results show that each cell line and their EVs exhibit different positive levels for these QUAD markers, but the overall molecular profiling pattern is similar between the EVs and their originating cells, supporting the use of EVs as surrogate markers for tumors.

[0225] The inventors prepared NPOP and captured EVs as follows. The inventors washed the substrate with water, removed the water with nitrogen gas, and placed the NPOP in a humid chamber. The inventors functionalized the NPOP substrate with 0.25 mM SH - PEG - COOH (1.0 kDa, Nanocs) and 0.75 mM SH - mPEG (0.35 kDa, Nanocs) in water for 4 hours at room temperature. Then, the inventors washed the NPOP substrate with water and treated it with a mixture of 50 mM EDC (ThermoFisher Scientific) and 125 mM sulfo - NHS (ThermoFisher Scientific) in 0.1 M MES (pH 6.0) for 7 minutes to capture EVs by covalent bonding. Then, the inventors incubated the EVs at room temperature for 30 minutes in the dark to generate SKBR3EV, MCF7EV, BT474EV, and MDA - MB - 231EV, and washed this three times with PBS.

[0226] The inventors immunostained the QUAD marker and labeled EVs as follows. The inventors fixed and permeabilized the EVs with 4% paraformaldehyde (Electron Microscopy Sciences) and 1× Perm / Wash Buffer (BD) for 10 minutes at room temperature, washed three times with PBS, and blocked the EVs and the NPOP substrate with 100% SuperBlock™ (PBS) Blocking Buffer (ThermoFisher Scientific) for 30 minutes at room temperature. The inventors then added primary antibodies against MUC1, HER2, EGFR, and EpCAM in 10% SuperBlock™ (PBS) blocking buffer in PBS and incubated for 30 minutes at room temperature (the following concentrations: 0.08 μg / mL mouse IgG1 isotype control (Invitrogen, 14-4714-85); 0.4 μg / mL mouse IgG1 isotype control (Invitrogen, 14-4714-85); 2.0 μg / mL mouse IgG1 isotype control (Invitrogen, 14-4714-85); and 0.08 μg / mL mouse IgG2b isotype control (Biolegend, 400301); 0.4 μg / mL mouse IgG1 MUC1 (Invitrogen, MA1-06503); 2.0 μg / mL mouse IgG1 HER2 (Biolegend, 324402); 0.08 μg / mL mouse IgG2b EGFR (Abcam, ab30); and 0.08 μg / mL mouse IgG1 EpCAM (Abcam, ab85987). The NPOP substrate was then washed three times with PBST.

[0227] Next, the inventors added goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 647 (ThermoFisher Scientific) in 10% SuperBlock™ (PBS) blocking buffer in PBS, incubated for 20 minutes at room temperature, washed the NPOP substrate three times with PBST, added 0.25 μg / mL of wheat germ agglutinin, Alexa Fluor™ 555 conjugate (ThermoFisher Scientific) for 20 minutes at room temperature in the dark, washed the NPOP substrate three times with PBST, added Prolong Gold™ antifade mounting solution (ThermoFisher scientific) onto the substrate, and mounted a coverslip.

[0228] Images were taken and analyzed as follows. The inventors acquired four images of EV signals (AF555) and antibody signals (AF647) using upright fluorescence microscopy (Zeiss) with a 40× objective lens and an exposure time of 5 seconds. The inventors converted the raw czi files to Tiff image files, loaded the images using ImageJ™ Fiji, and subtracted the background signal. Next, the inventors analyzed the number of EVs and antibody signals and their co-localization using the ComDet® Plugin with fixed parameters (approximate particle size: 4.00 pixels; intensity threshold (SD): around 3.00). The inventors described the co-localization percentage (co-localized EVs / total EVs), heatmap, and correlation plot using Prism™ software (GraphPad).

[0229] For flow cytometry, the inventors harvested the cultured cells by trypsin treatment, washed the cells three times by centrifugation, and resuspended the pellet in PBS. The inventors added 4% paraformaldehyde (Electron Microscopy Sciences) and 1× Perm / Wash Buffer (BD) and incubated for 10 minutes at room temperature. The inventors then washed the cells three times by centrifugation and resuspended the pellet in PBS. The inventors then added primary antibodies against MUC1, HER2, EGFR, and EpCAM in 10% SuperBlock™ (PBS) blocking buffer in PBS at the following concentrations (1 μg / mL mouse IgG1 isotype control (Invitrogen, 14-4714-85); 1 μg / mL mouse IgG2b isotype control (Biolegend, 400301); 1 μg / mL mouse IgG1 MUC1 (Invitrogen, MA1-06503); 1 μg / mL mouse IgG1 HER2 (Biolegend, 324402); 1 μg / mL mouse IgG2b EGFR (Abcam, ab30); and 1 μg / mL mouse IgG1 EpCAM (Abcam, ab85987)) and incubated for 1 hour at room temperature. The inventors then washed the cells three times by centrifugation and resuspended the pellet in PBS. The inventors added goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 488 (ThermoFisher Scientific) in 10% SuperBlock™ (PBS) blocking buffer in PBS and incubated for 30 minutes at room temperature. The inventors then washed the cells three times by centrifugation and resuspended the pellet in PBS. The inventors loaded the cells into a 96-well plate and read the signal intensity with a Cytoflex® Flow Cytometer (Beckman Coulter).

[0230] The inventors analyzed the differential expression of markers within cell lines using the median of the signals using Flowjo (registered trademark) software. The results are shown in FIGS. 22A to 22E, which are images demonstrating the molecular profiling of tumor cell-derived EVs for QUAD markers by single EV analysis on the NPOP substrate.

[0231] FIG. 22A is a series of representative images of multi-channel single EV analysis for QUAD marker (MUC1, HER2, EGFR, and EpCAM) profiling in EVs derived from four different breast cancer cell lines (SKBR3, ERPR- / HER2+; MCF7, ERPR+ / HER2-; BT474, ERPR+ / HER2+; MDA-MB-231, ERPR- / HER2-). Scale bar, 10 μm.

[0232] FIG. 22B is a bar graph of the percentage of co-localization for EVs and QUAD markers. Error bars are shown as mean ± SD from four different images.

[0233] FIGS. 22C to 22D are a pair of heat maps showing the differential abundance of QUAD markers in EVs derived from four different tumor cell lines evaluated by single EV analysis (21C) and in the originating cell lines evaluated by flow cytometry (21D).

[0234] FIG. 22E is a dot plot for the Pearson correlation coefficient, revealing that multi-channel single EV analysis showed a molecular pattern similar to that of flow cytometry (r = 0.8438, P < 0.0001). The percentage of co-localization from multi-channel single EV analysis and flow cytometry is shown on the y-axis and x-axis, respectively. The solid line indicates the best linear fit.

[0235] The results show that each cell line and their EVs have different positive levels for these QUAD markers, but the overall molecular profiling patterns are similar between EVs and their originating cells, supporting the use of EVs as surrogate markers for different tumors.

Example

[0236] Example 17 - Plasmon Enhancement on the Cy7 Channel Imaging of EVs in the Cy7 channel was difficult due to the weak fluorescence signals generated from the dye and the channel. However, by using plasmon enhancement, the inventors were able to significantly amplify the EV fluorescence signal when the EVs were captured on the NPOP substrate compared to other planar substrates (glass or gold). This resulted in higher EV detection sensitivity.

[0237] The method used in this example is the same as described in Example 14, except for the use of TFP - Cy7 instead of the TFP - AF dye. The results are shown in different graphs of FIGS. 23A - 23C.

[0238] FIG. 23A is a graph showing the EV count versus the dilution factor. The EV count was clearly highest over various dilution factors (upper line) compared to the gold and glass substrates (dark line where the EV count rapidly decreased from a dilution factor of about 90 on the left to a dilution factor of 1000 where the EV count was 0).

[0239] FIG. 23B is a histogram plot of the conversion of Cy7. Similarly, the large curve on the right representing the NPOP substrate showed a significantly higher EV count than glass or gold.

[0240] FIG. 23C is a bar graph showing that AF488 - CD63 and Cy7 - CD63 had the highest percentage of colonization compared to AF488 - IgG or Vy7 - IgG.

[0241] The results show that the NPOP substrate is superior to glass or gold substrates with respect to the total captured EVs, is much more sensitive, and can thus detect EVs at much lower concentrations than glass or gold substrates.

Example

[0242] Example 18 - CD63 Detection on EVs In this example, EVs were labeled with the TFP - AF555 dye and captured on the NPOP substrate. The captured EVs were then labeled with a CD63 primary antibody followed by a secondary antibody conjugated to AF647. An isotype IgG control was used as a negative control.

[0243] For EV labeling, the inventors mixed 0.4 μg of the TFP - AF555 dye, 6 μg of 2×10 8 MCF7 EVs in PBS, and 4 μg of 100 mM sodium bicarbonate and incubated for 1 hour at room temperature under dark conditions. The inventors then loaded the labeled EVs onto a Zeba™ Micro Spin Desalting Column (ThermoFisher Scientific) to remove unbound TFP dye, centrifuged at 1,500×g for 2 minutes, and collected the filtrate. The inventors then re - loaded the filtrate onto the Zeba™ Micro Spin Desalting Column, centrifuged at 1,500×g for 2 minutes, and collected the filtrate. The inventors then diluted the EVs in PBS to concentrations of 1×10 8 、2.5×10 7 、6.25×10 6 、1.56×10 6 EV / mL.

[0244] For NPOP preparation and EV capture, the inventors washed the substrate with water, removed the water with nitrogen gas, and placed it in a wet chamber. The inventors functionalized the NPOP substrate with 0.25 mM SH-PEG-COOH (1.0 kDa, Nanocs) and 0.75 mM SH-mPEG (0.35 kDa, Nanocs) in water for 4 hours at room temperature and then washed it with water. The inventors treated the NPOP substrate with a mixture of 50 mM EDC (ThermoFisher Scientific) and 125 mM sulfo-NHS (ThermoFisher Scientific) in 0.1 M MES (pH 6.0) for 7 minutes to capture EVs by covalent bonding. The inventors incubated serial diluted EVs at the following concentrations (1×10 8 EV / mL, 2.5×10 7 EV / mL, 6.25×10 6 EV / mL, 1.56×10 6 EV / mL) for 30 minutes at room temperature in the dark and washed it three times with PBS.

[0245] The inventors immunostained the QUAD marker and labeled EVs as follows. The inventors fixed and permeabilized the EVs with 4% paraformaldehyde (Electron Microscopy Sciences) and 1× Perm / Wash Buffer (BD) for 10 minutes at room temperature, washed them three times with PBS, and blocked the EVs and the NPOP substrate with 100% SuperBlock™ (PBS) Blocking Buffer (ThermoFisher Scientific) for 30 minutes at room temperature. Then, the inventors added primary antibodies against mouse IgG and CD63 in 10% SuperBlock™ (PBS) blocking buffer in PBS and incubated them for 30 minutes at room temperature (5 μg / mL mouse IgG1 isotype control (Invitrogen, 14-4714-85); 10 μg / mL mouse IgG1 CD63 (Ancell, 215-820)), washed the NPOP substrate three times with PBST, added goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 647 (ThermoFisher Scientific) in 10% SuperBlock™ (PBS) blocking buffer in PBS, and incubated them for 20 minutes at room temperature. Then, the inventors washed the NPOP substrate three times with PBST, added Prolong Gold™ antifade mounting solution (ThermoFisher scientific) to the substrate, and mounted the coverslip.

[0246] Images were taken and analyzed as follows. The inventors acquired four images of the EV signal (AF555) and the antibody signal (AF647) by upright fluorescence microscopy (Zeiss) with a 40× objective lens and an exposure time of 5 seconds. The inventors converted the raw czi files to Tiff image files, loaded the images into ImageJ™ Fiji, and subtracted the background signal. Then, the inventors analyzed the number of EVs and the antibody signal and their co-localization using the ComDet® Plugin with fixed parameters (approximate particle size: 4.00 pixels; intensity threshold (SD): around 3.00).

[0247] The inventors have shown in Figures 24A and 24B plots of EV and marker-positive EV counts in serial-diluted EVs generated by Prism software (GraphPad), showing similar numbers of EVs captured on two samples and decreasing with titration of EV concentration. However, as expected, the CD63+ EV counts are significantly higher than the IgG isotype (control), and the CD63-positive EV counts are proportional to the EV concentration applied to the surface.

[0248] Other embodiments Although this specification contains many details of specific embodiments, these should not be construed as limitations on the scope of any invention or of what may be claimed, but rather as descriptions of features that may be specific to particular embodiments of a particular invention. The particular features described herein in the context of separate embodiments may also be implemented in combination within a single embodiment. Conversely, the various features described in the context of a single embodiment may also be implemented separately in multiple embodiments or in any suitable sub-combination. Further, features may be described above as acting in certain combinations and initially claimed as such, but one or more features from a claimed combination may in some cases be excised from that combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination.

[0249] Particular embodiments of the subject matter have been described. Other embodiments, variations, and permutations of the described embodiments will be apparent to those skilled in the art and are within the following claims. Operations are shown in the drawings or claims in a particular order, but this should not be understood as requiring that such operations be performed in the particular order shown or sequentially to achieve desirable results (some operations may be optional). In certain circumstances, multitasking and parallel processing may be advantageous.

[0250] Furthermore, the separation and / or integration of the various system modules and components in the above-described embodiments should not be understood as requiring such separation and / or integration in all embodiments, and it should be understood that the described program components and systems can generally be integrated together into a single software product or packaged into multiple software products.

[0251] Accordingly, the above description of different embodiments and implementations does not define or limit the present disclosure. Other changes, substitutions, and modifications are possible without departing from the spirit and scope of the present disclosure.

Claims

1. A method for enhancing electromagnetic radiation signals from target extracellular vesicles (EVs) on a substrate, circuit board and Multiple nanostructures, One or more affinity ligands fixed on or adjacent to the nanostructure, wherein the affinity ligands bind to the EV and bind the EV to the nanostructure, or bind to the substrate adjacent to the nanostructure, To obtain a nanoplasmon array comprising, If EVs are present in the liquid sample, the EVs are captured on the nanoplasmon array by flowing the liquid sample onto the nanoplasmon array at a flow rate that allows them to bind to the affinity ligand. Labeling the target EVs among those captured on the nanoplasmon array with one or more different reporter groups, Exposing the labeled target EVs trapped on the nanoplasmon array to a first electromagnetic radiation, thereby causing the target EVs and / or reporter groups on the target EVs to emit, scatter, or reflect the first or second electromagnetic radiation as an electromagnetic radiation signal, and Receiving all or part of the aforementioned electromagnetic radiation signal, wherein the nanostructures within the nanoplasmon array are arranged and sized to amplify the electromagnetic radiation signal, thereby enhancing the electromagnetic radiation signal from the target EV on the substrate. Methods that include...

2. (i) The one or more affinity ligands nonspecifically bind to at least one surface marker on the EV and / or at least one intravesicular marker inside the EV, and the reporter group binds to a scavenger that specifically binds to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV, or (ii) The one or more affinity ligands specifically bind to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV, and the reporter group binds to a scavenger that specifically or nonspecificly binds to at least one surface marker on the target EV and / or at least one intravesicular marker inside the target EV. The method according to claim 1.

3. The method according to claim 1, wherein the plurality of nanostructures are arranged on the substrate to form a periodic array of nanostructures, and the periodic array of nanostructures is arranged and sized to amplify the electromagnetic radiation signal emitted, scattered, or reflected by EVs bonded to the nanostructures and / or EVs bonded to the substrate near the nanostructures, or to amplify the electromagnetic radiation signal emitted, scattered, or reflected by reporter groups attached to the EVs.

4. The method according to claim 1, wherein the electromagnetic radiation signal includes a fluorescence signal, a Raman signal, or dark-field scattering.

5. The method according to claim 1, further comprising obtaining an image of the amplified electromagnetic radiation signal.

6. The method according to claim 5, wherein the liquid sample is from a subject, the reporter group is bound to a capture agent that specifically binds to tumor-derived target EVs, and the method further comprises analyzing the acquired image to detect whether the liquid sample contains tumor-derived target EVs, thereby detecting or monitoring cancer in the subject.

7. Identify extravasation (EVs) by size and discard any EVs or other components larger than 1 micron. To generate a selected target EV, select a target EV from the identified EVs based on positivity for the target EV marker. To generate specifically selected target EVs, identify selected target EVs that originate from a specific organ or tissue by being positive for organ-specific or tissue-specific markers, and Analyzing individual specific selected target EVs based on a tetraspanin biomarker on the surface of the specific target EV, based on an intravesicular biomarker within the specific target EV, or based on both a tetraspanin biomarker and an intravesicular biomarker. The method according to claim 6, further comprising:

8. The method according to claim 6, wherein the reporter group includes a first fluorescent label.

9. The method according to claim 6, wherein the reporter group comprises an antibody that specifically binds to a biomarker on the surface of the target EV.

10. The method according to claim 9, wherein the antibody comprises at least two different types of antibodies, the first type of antibody binding to EpCAM and the second type of antibody binding to HER2.

11. The method according to claim 9, wherein the antibody comprises at least four different types of antibodies, the first type of antibody binding to MUC1, the second type of antibody binding to EGFR, the third type of antibody binding to EpCAM, and the fourth type of antibody binding to HER2.

12. The method according to claim 8, further comprising labeling the target EV containing the first fluorescent label with a second fluorescent label different from the first fluorescent label.

13. The method according to claim 6, wherein the cancer is breast cancer.

14. Isolating extracellular vesicles (EVs) from biological samples, The method involves trapping EVs on a nanoplasmon array, wherein the nanoplasmon array is circuit board and Multiple nanostructures, One or more affinity ligands fixed on or adjacent to the nanostructure, wherein the affinity ligands bind to the EV and bind the EV to the nanostructure, or bind to the substrate adjacent to the nanostructure, The system includes capturing EVs on a nanoplasmon array, The target EVs among the captured EVs are immunolabeled with multiple different fluorescent reporter groups. Performing multichannel fluorescence imaging to form an image, and To analyze the aforementioned images and determine the origin of the EVs in the biological sample, Methods that include...

15. The method according to claim 14, wherein the plurality of nanostructures are arranged on the substrate to form a periodic array of nanostructures, and the periodic array of nanostructures is arranged and sized to amplify fluorescence signals emitted, scattered, or reflected by EVs bonded to the nanostructures and / or EVs bonded to the substrate near the nanostructures, or to amplify fluorescence signals emitted, scattered, or reflected by reporter groups attached to the EVs.

16. The method according to claim 14, wherein the immunolabeling is performed using different antibodies that bind to different reporter groups and specifically bind to different biomarkers on the surface of the target EV.

17. The method according to claim 16, wherein the antibody comprises at least two different types of antibodies, the first type of antibody binding to EpCAM and the second type of antibody binding to HER2.

18. The method according to claim 16, wherein the antibody comprises at least four different types of antibodies, the first type of antibody binding to MUC1, the second type of antibody binding to EGFR, the third type of antibody binding to EpCAM, and the fourth type of antibody binding to HER2.

19. The method according to any one of claims 1 to 18, wherein the nanostructure contains gold.

20. The method involves capturing one or more extracellular vesicles (EVs) from a biological sample onto a nanoplasmon array, wherein the nanoplasmon array is circuit board and Multiple nanostructures, One or more affinity ligands fixed on or adjacent to the nanostructure, wherein the affinity ligands bind to the EV and bind the EV to the nanostructure, or bind to the substrate adjacent to the nanostructure, To be captured on a nanoplasmon array, The target EVs among the captured EVs are immunolabeled with a fluorescently conjugated biomolecule. Performing multichannel fluorescence imaging to form an image, and Analyzing the aforementioned images to determine whether each of the target EVs originates from cancer tissue, Methods that include...

21. The method according to claim 14, wherein the fluorescent conjugate biomolecule comprises at least three fluorescent molecules conjugated to at least three cancer-related biomolecules.

22. The method according to claim 20, wherein the fluorescent conjugate biomolecule comprises a fluorescent conjugate wheat germ agglutinin.

23. The method according to claim 20, wherein the fluorescent conjugate biomolecule comprises a fluorescent conjugate antibody that specifically binds to a biomarker on cancer cells.

24. The method according to claim 20, wherein the nanoplasmon array is configured to enhance the fluorescence signal from the fluorescent conjugate biomolecule by at least twofold compared to the fluorescence signal from an image taken using a glass substrate instead of the nanoplasmon array.

Images