WO2024026314A1 - Méthodes et systèmes d'amélioration de signaux de rayonnement électromagnétique à partir de vésicules extracellulaires - Google Patents

Méthodes et systèmes d'amélioration de signaux de rayonnement électromagnétique à partir de vésicules extracellulaires Download PDF

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WO2024026314A1
WO2024026314A1 PCT/US2023/070948 US2023070948W WO2024026314A1 WO 2024026314 A1 WO2024026314 A1 WO 2024026314A1 US 2023070948 W US2023070948 W US 2023070948W WO 2024026314 A1 WO2024026314 A1 WO 2024026314A1
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evs
nanostructures
antibodies
bind
nano
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PCT/US2023/070948
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English (en)
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Hyungsoon Im
Ralph Weissleder
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The General Hospital Corporation
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Publication of WO2024026314A1 publication Critical patent/WO2024026314A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G01N15/0205—Investigating particle size or size distribution by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/55—Specular reflectivity
    • G01N21/552—Attenuated total reflection
    • G01N21/553—Attenuated total reflection and using surface plasmons
    • G01N21/554—Attenuated total reflection and using surface plasmons detecting the surface plasmon resonance of nanostructured metals, e.g. localised surface plasmon resonance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/648—Specially adapted constructive features of fluorimeters using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering
    • G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons

Definitions

  • This invention relates to methods of enhancing electromagnetic radiation, e.g., optical, signals of extracellular vesicles, and more particularly to a small number of extracellular vesicles.
  • Extracellular vesicles present new opportunities as circulating biomarkers for cancers, cardiovascular, neurodegenerative, and infectious diseases, among others.
  • These cell-derived phospholipid vesicles are abundantly present in various bodily fluids (e.g., blood, cerebrospinal fluid, urine, and saliva). More importantly, they carry a variety of biomolecules (lipids, proteins, and genetic materials) originating from their parental cells, which can be harnessed as a minimally invasive means to probe the molecular status of their cellular origins.
  • the present disclosure relates to signal amplification strategies to boost electromagnetic radiation signals, e.g., optical signals, generated from limited amounts of biomolecules present in individual EVs.
  • electromagnetic radiation signals e.g., optical signals
  • the strong optical resonance of metallic nanostructure e.g., gold or silver nanostructures
  • the plasmon enhancement is an intrinsic signal amplification method, in certain embodiments, this intrinsic method is combined with other chemical amplification strategies (e.g., branched DNA barcodes) to improve the sensitivity even further.
  • the present disclosure relates to methods and systems for enhancing optical signals of EVs.
  • the present disclosure also relates to methods of using plasmonic substrates that can be fabricated on a wafer-scale to amplify fluorescent- labeled EVs’ signals for multichannel single EV analysis, without the need for additional chemical or enzymatic signal amplification processes.
  • the multiplexed single EV molecular profiling methods provide a better understanding of the heterogeneity of EVs with higher EV detection sensitivity and accuracy than past methods. These methods also enable the discrimination of individual EVs based on their cellular origins (i.e., EVs from, for example, tumor, breast, or brain cells, or from immune system cells).
  • the present disclosure provides methods of enhancing an electromagnetic radiation signal, e.g., an optical signal, from a target EV on a substrate, the method including or consisting of: obtaining a nano-plasmonic array including, a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands bind, e.g., specifically bind, to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; flowing a liquid sample over the nano-plasmonic array at a flow rate that enables EVs in the liquid sample, if any, to bind to the affinity ligands thus capturing the EVs on the nano-plasmonic array; labeling target EVs among EVs captured on the nano- plasmonic array with one or more different reporter groups; exposing the labeled target EVs captured on the nano-plasmonic array to a first electromagnetic radiation thereby causing the target
  • the one or more affinity ligands bind non-specifically to at least one surface marker on the EVs and/or to at least one intravesicular marker inside the EVs
  • the reporter groups are bound to capture agents, such as antibodies, that specifically bind to at least one surface marker on the target EVs and/or to at least one intravesicular marker inside the target EVs, or the one or more affinity ligands specifically bind to at least one surface marker on the target EVs and/or to at least one intravesicular marker inside the target EVs
  • the reporter groups are bound to capture agents that bind to at least one surface marker on the target EVs and/or to at least one intravesicular marker inside the target EVs, either specifically or non- specifically.
  • the plurality of nanostructures are arranged to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify the electromagnetic radiation signals emitted, scattered, or reflected by EVs bound to the nanostructures and/or EVs bound to the substrate near the nanostructures, or to amplify the electromagnetic radiation signals emitted, scattered, or reflected by reporter groups attached to the EVs.
  • the electromagnetic radiation signal can be or include a fluorescent signal, a Raman signal, or dark-field scattering, and the methods can further include obtaining an image of the amplified electromagnetic radiation signal, e.g., optical signal.
  • this disclosure provides methods of detecting or monitoring EVs, e.g. cancer-derived EVs, and/or the use of the nano-plasmonic arrays described herein for detecting or monitoring EVs, e.g., cancer-derived EVs, wherein the liquid sample used in the methods described herein is from a subject, wherein the reporter groups are bound to capture agents that bind, e.g., specifically bind, to tumor-derived target EVs, and wherein the methods further include analyzing the obtained image(s) to detect whether the liquid sample contains or includes tumor-derived target EVs, thereby detecting or monitoring EVs, or the origin of the EVs, e.g. from a cancer or cancer tissue, e.g., in the subject.
  • the liquid sample used in the methods described herein is from a subject
  • the reporter groups are bound to capture agents that bind, e.g., specifically bind, to tumor-derived target EVs
  • the methods further include analyzing the obtained image(s) to detect whether
  • these methods and uses can further include identifying EVs by size and discarding any EVs or other components larger than one micron; selecting target EVs from the identified EVs based on positivity for target EV markers to generate selected target EVs; specifying selected target EVs as originating from specific organs or tissues by positivity for organ- or tissue-specific markers to generate specific, selected target EVs; and analyzing individual specific, selected target EVs based on tetraspanin biomarkers on the surface of the specific target EVs, based on intravesicular biomarkers within the specific target EVs, or based on both tetraspanin and intravesicular biomarkers.
  • the reporter groups can be, include, or consist of, a first fluorescent label
  • the reporter groups can be, include, or consist of antibodies that specifically bind to a biomarker on the surface of the target EVs.
  • the antibodies can be, include, or consist of at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2.
  • the antibodies can be, include, or consist of at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth type bind to HER2.
  • the methods and uses further include labeling target EVs that include the first fluorescent label with a second fluorescent label that is different from the first fluorescent label.
  • the cancer can be or include breast cancer.
  • this disclosure provides methods of detecting EVs, e.g., cancer- derived EVs, in a sample from a subject and uses of the nano-plasmonic arrays described herein in methods of detecting EVs, e.g., cancer-derived EVs, e.g., in a sample from a subject, the methods and uses including collecting a biological sample from the subject; isolating EVs from the biological sample; capturing EVs on a nano-plasmonic array, wherein the nano-plasmonic array includes, a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands bind, e.g., specifically bind, to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling target EVs among the captured EVs with multiple different fluorescent reporter groups; conducting multichannel fluorescence imaging to form an image; and analyzing the image
  • the plurality of nanostructures can be arranged, in certain embodiments, to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify fluorescent signals emitted, scattered, or reflected by EVs bound to the nanostructures and/or EVs bound to the substrate near the nanostructures, or to amplify fluorescent signals emitted, scattered, or reflected by reporter groups attached to the EVs.
  • the immunolabeling is carried out with different antibodies that are bound to different reporter groups and that specifically bind to different biomarkers on the surface of the target EVs.
  • the antibodies can be, include, or consist of at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2.
  • the antibodies can be, include, or consist of at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth type bind to HER2.
  • the cancer is breast cancer.
  • the disclosure provides methods of detecting individual target EVs, e.g., EVs relevant to cancer diagnosis or cancer treatment monitoring, and uses of the nano-plasmonic arrays described herein for detecting individual target EVs relevant to cancer diagnosis or cancer treatment monitoring, the methods and uses including: providing a biological sample from a subject containing one or more EVs; capturing the one or more EVs on a nano-plasmonic array, wherein the nano-plasmonic array includes a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands bind, e.g., specifically bind, to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures; immunolabeling target EVs among the captured EVs with fluorescence- conjugated biomolecules; conducting multi-channel fluorescence imaging to form an image; and analyzing the image, e.g., to detect an origin of the EVs
  • the fluorescence-conjugated biomolecules can be, include, or consist of at least three fluorescent molecules conjugated to at least three cancer-associated biomolecules.
  • the fluorescence- conjugated biomolecules can be, include, or consist of fluorescence-conjugated wheat germ agglutinin.
  • the nano-plasmonic arrays are configured to enhance a fluoresce signal from the fluorescence-conjugated biomolecules at least two-fold compared to a fluorescence signal from an image taken using a glass substrate instead of the nano-plasmonic array.
  • the present disclosure provides methods, e.g., methods of cancer diagnosis or treatment monitoring, and uses of the nano-plasmonic arrays described herein for cancer diagnosis or treatment monitoring, the methods and uses including: providing a biological sample from a subject containing one or more EVs; contacting at least a first portion of the biological sample with a surface conjugated with a first EV-specific antibody and labeling with a first fluorescence-conjugated biomolecule; contacting at least a second portion of the biological sample with a surface conjugated with a second EV-specific antibody and labeling with a second fluorescence- conjugated biomolecule; contacting at least a third portion of the biological sample with a surface conjugated with a second EV-specific antibody and labeling with a third fluorescence-conjugated biomolecule; conducting multi-channel fluorescence imaging to form an image; and analyzing the image, e.g., to detect an origin of the EVs, and/or to detect cancer in a subject or monitor a cancer treatment in a
  • the surface can be, include, or consist of a nano- plasmonic array, wherein the nano-plasmonic array includes or consists or a substrate, a plurality of nanostructures, and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands bind, e.g., specifically bind, to EVs to bind the EVs to the nanostructures or to the substrate adjacent to the nanostructures.
  • the nano-plasmonic arrays are configured to enhance a fluoresce signal in the image at least two-fold compared to a fluorescence signal from an image taken using a glass substrate instead of the nano-plasmonic array.
  • the present disclosure provides nano-plasmonic arrays for detecting target EVs, the arrays including or consisting of a substrate; a plurality of nanostructures arranged to form a periodic array of nanostructures on the substrate, wherein the periodic array of nanostructures is arranged and dimensioned to amplify one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by EVs bound to the nanostructures and/or EVs bound to the substrate near the nanostructures, or to amplify one or more optical signals of electromagnetic radiation emitted, scattered, or reflected by reporter groups attached to the EVs; and one or more affinity ligands fixed on or adjacent to the nanostructures, wherein the affinity ligands selectively bind to target EVs to bind the target EVs to the nanostructures or to the substrate adjacent to the nanostructures.
  • the optical signal can be one or more of a fluorescent signal, a Raman signal, or dark-field scattering.
  • the nanostructures can be or include or consist of a plurality of nanoholes arranged in an array and formed in the substrate or in a metal film disposed on the substrate.
  • the nanostructures can be or include or consist of a plurality of nanoholes, nanowells, nanorods, nanodisks, nanopillars, nanogrooves, or any combinations thereof, arranged in an array on a top surface of the substrate.
  • the nanostructures can be, for example, gold nanoparticles (NPs) bound to gold nanopillars (NPs on nanopillars, or “NPOP”) structures.
  • each of the nanostructures has a maximum size, e.g., diameter, width, or length, of about 30 to 400 nm.
  • the nanostructures can be nanorods or nanosquares and have dimensions of about 50 to about 300 nm in length, about 20 to about 300 nm in width, and about 20 to about 300 nm in height, or the nanostructures can be nanodisks and have dimensions of about 50 to about 200 nm in diameter and about 20 to about 300 nm in height.
  • Each of the nanopillars can have a maximum size of about 30 to about 500 nm.
  • the periodic array of nanostructures has a periodicity of about 400 to 800 nm or 400 to 2000 nm between nanostructures.
  • the nanostructures are nanopillars having a dimension of about 20 to about 500 nm in diameter and about 20 to about 300 nm in height with a density of about 300 to about 750 nanopillars per pm 2 on a substrate (e.g., Si, glass, indium tin oxide/ITO, polyethylene terephthalate (PET) polymer substrate).
  • a substrate e.g., Si, glass, indium tin oxide/ITO, polyethylene terephthalate (PET) polymer substrate.
  • the affinity ligands bind to a capture agent, e.g., an antibody, wherein the capture agent is configured to bind to at least one surface marker on the target EV.
  • the affinity ligand is configured to bind to at least one surface marker on the target EV and/or to at least one intravesicular marker inside the target EV.
  • the nano-plasmonic arrays further include or consist of a metal film disposed on a top surface of the substrate, wherein the metal film comprises a plurality of nanoholes that penetrate the metal film in a periodicity selected to amplify one or more specific wavelengths of electromagnetic radiation, wherein the periodicity of about 400 to 800 nm between nanoholes, wherein the metal film comprises a plurality of affinity ligands fixed on or adjacent to the nanoholes, and wherein the plurality of affinity ligands selectively bind to markers on surfaces of the target EVs.
  • the metal film can be or include, for example, a noble metal, a transition metal, an alkali metal, or any combination thereof.
  • either or both the nanostructures and the metal film are, or include, or consist of gold, silver, aluminum, or platinum.
  • the disclosure includes methods for detecting target EVs in a liquid sample and uses of the nano-plasmonic arrays described herein for detecting target EVs, the methods and uses including or consisting of obtaining a nano-plasmonic array as described or claimed herein; flowing a liquid sample over the nano-plasmonic array at a flow rate that enables the EVs in the liquid sample, if any, to bind to the affinity ligands thus capturing the EVs on the nano-plasmonic array; labeling target EVs captured on the nano-plasmonic array with one or more reporter groups; projecting a first electromagnetic radiation at one or more specific wavelengths onto the labeled target EVs captured on the nano-plasmonic array, wherein the electromagnetic radiation at the one or more specific wavelengths is selected to cause the reporter groups to emit, scatter, or reflect the first electromagnetic radiation or a second electromagnetic radiation; receiving the first or second electromagnetic radiation emitted, scattered, or reflected by the reporter groups, wherein the nano-plasmonic array of nanostructures is arranged and dimensioned
  • the number of the target EVs in the liquid sample may be less than 1000.
  • the nanostructures include a plurality of nanoholes that penetrate the substrate or a metal film disposed on the substrate, or the nanostructures include or consist of a plurality of nanorods, nanodisks, nanopillars, nanogrooves, or any combination thereof, arranged on a top surface of the substrate.
  • the methods and uses as described herein further include or consist of identifying EVs by size and discarding large components, e.g., larger than one micron; selecting target EVs from the identified EVs based on positivity for target EV markers; selecting target EVs as originating from specific organs or tissues by positivity for organ- or tissue-specific markers to generate specific target EVs; and analyzing individual specific target EVs based on extravesicular biomarkers on the surface of the specific target EVs and/or based on intravesicular biomarkers within the specific target EVs.
  • the disclosure provides systems, e.g., nano-plasmonic array systems, for detecting target EVs in a liquid sample, the systems including or consisting of a nano-plasmonic array as described and claimed herein; a sample control unit comprising a pump; at least one fluidic channel configured to flow a liquid sample over the nano-plasmonic array at a flow rate controlled by the pump that enables the EVs in the liquid sample, if any, to bind to the affinity ligands, thus capturing the EVs on the nano-plasmonic array; and at least one capture agent, e.g., an antibody, bound to a reporter group to label target EVs captured on the nano-plasmonic array with one or more reporter groups; and an imaging unit comprising a light source configured to project electromagnetic radiation onto the labeled target EVs captured on the nano-plasmonic array; and an electromagnetic radiation detector, e.g., a camera or CCD, configured to receive electromagnetic radiation emitted, scattered, or reflected by the target EVs or
  • the nanostructures include or consist of a plurality of nanoholes arranged in an array and formed in the substrate or in a metal film disposed on the substrate. In other embodiments, the nanostructures include or consist of a plurality of nanorods, nanodisks, or nanogrooves arranged in an array on a top surface of the substrate.
  • the affinity ligands bind to a capture agent, wherein the capture agent 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 to at least one intravesicular marker inside the target EV.
  • the methods are used to detect tumor-derived EVs using a QUAD marker signature (Mucin 1, cell surface associated (MUC1), Epidermal Growth Factor Receptor (EGFR), epithelial cellular adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)), e.g., EVs derived from breast tumors. These methods can include treatment monitoring during chemotherapy to change a course of therapy during ongoing therapy, e.g., using markers HER2 and EpCAM.
  • MUC1 cell surface associated
  • EGFR Epidermal Growth Factor Receptor
  • EpCAM epithelial cellular adhesion molecule
  • HER2 human epidermal growth
  • the disclosure provides three-dimensional nano-plasmonic arrays of nanostructures, wherein the array includes or consists of a substrate; a plurality of nanostructures arranged to form a periodic array of nanostructures on the substrate, wherein each nanostructure includes or consists of a nanopillar attached at one end to a substrate; a metallic layer coated onto each of the plurality of nanopillars; a spacer layer coated onto the metallic layer on each of the plurality of nanopillars; a plurality of metal nanoparticles bound to each of the nanopillars via the spacer layer; and one or more affinity ligands fixed on the nanostructures and/or fixed on the substrate adjacent to the nanostructures, wherein the affinity ligands bind to target extracellular vesicles (EVs) to bind the target EVs to the nanostructures or to the substrate adjacent to the nanostructures.
  • EVs extracellular vesicles
  • the one or more affinity ligands are antibodies that bind to a biomarker on the surface of the target EVs.
  • the antibodies include at least two different types of antibodies, wherein antibodies of a first type bind to EpCAM and antibodies of a second type bind to HER2.
  • the antibodies include at least four different types of antibodies, wherein antibodies of a first type bind to MUC1, antibodies of a second type bind to EGFR, antibodies of a third type bind to EpCAM, and antibodies of a fourth type bind to HER2.
  • the affinity ligands include or consist of linker groups, e.g., thiol-PEG-COOH.
  • the metal of the metallic layer includes or consists of gold.
  • the metal nanoparticles include or consist of gold.
  • the spacer layer includes or consists of a self-assembled monolayer (SAM) of 1H, ///,2//,2//-perfluorodecanethiol (PFDT).
  • the substrate includes or consists of polyethylene terephthalate (PET).
  • compositions, methods, and respective component(s) thereof are used in reference to compositions, methods, and respective component(s) thereof, that are useful to an embodiment, yet open to the inclusion of unspecified elements, whether useful or not.
  • peripherality refers to a recurrence or repetition of a nanostructure at regular intervals by their positioning on a substrate and/or in an array.
  • the term “periodic” as used herein therefore refers to the regular predefined pattern of nanostructures with respect to each other, such as a lattice or other repeating unit configuration. A random distribution of nanostructures is a periodic pattern.
  • SPR Surface plasmon resonance
  • LSPR localized surface plasmon resonance
  • the terms “surface plasmons,” “surface plasmon polaritons,” and “plasmons” refer to the collective oscillations of free electrons at plasmonic surfaces, such as metals. These oscillations result in self-sustaining, surface electromagnetic waves that propagate in a direction parallel to the metal/di electric (or metal/vacuum) interface. Because the wave is on the boundary of a metal and the external medium (air or water, for example), these oscillations are very sensitive to any refractive index change of this boundary, such as, for example, the adsorption of a molecular target, such as an EV, to the metal surface. Additionally, the electromagnetic field strength decays exponentially from the metal surface to the surrounding environment (e.g., vacuum or dielectric). A maximum value of the electromagnetic field strength can be found at the metal/di electric or metal/vacuum interface.
  • sample means any biological or other fluids that may contain one or more extracellular vesicles (e.g., exosomes).
  • biological fluids include, without limitation, fluids derived from or containing cells, organisms (bacteria, viruses), lysed cells or organisms, cellular extracts, nuclear extracts, components of cells or organisms, extracellular fluid, media in which cells or organisms are cultured in vitro, blood, plasma, serum, gastrointestinal secretions, ascites, homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymphatic fluid, tears, pleural fluid, nipple aspirates, breast milk, external sections of the skin, respiratory, intestinal, and genitourinary tracts, and prostatic fluid.
  • a sample can be a viral or bacterial sample, a sample obtained from an environmental source, such as
  • a “biological sample” is derived or obtained from a living organism.
  • the organism can be a whole organism or can be cells or organs grown in culture.
  • a “biological sample” also refers to a cell or population of cells or a quantity of tissue or fluid from a subject. Most often, a sample has been removed from a subject, but the term “biological sample” can also refer to cells or tissue analyzed in vivo, i.e., without removal from the subject. Often, a “biological sample” will contain cells from a subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine.
  • a biological sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary, secondary, or metastatic tumor, e.g., breast, ovarian, pancreatic, biliary tract, colorectal, glioblastoma, lung tumors or a cell block from pleural fluid.
  • a biological sample includes primary ascites cells.
  • Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues.
  • a biological sample can be provided by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells or cellular extracts (e.g., isolated by another person, at another time, and/or for another purpose).
  • Archival tissues such as those having treatment or outcome history may also be used.
  • Biological samples include, but are not limited to, tissue biopsies, scrapes (e.g., buccal scrapes), whole blood or other bodily fluids, such as plasma, serum, urine, saliva, cell culture, urine, ascites, pleural fluid, Broncho Alveolar Lavage Fluid (BALF), or cerebrospinal fluid.
  • tissue biopsies scrapes (e.g., buccal scrapes)
  • whole blood or other bodily fluids such as plasma, serum, urine, saliva, cell culture, urine, ascites, pleural fluid, Broncho Alveolar Lavage Fluid (BALF
  • samples analyzed by the systems and methods described herein may have been processed, e.g., by purification or enrichment of EVs contained therein, prior to analysis.
  • an “extracellular vesicle” refers to a naturally occurring or synthetic vesicle that includes a cavity inside.
  • the EVs comprise a lipid bilayer membrane enclosing contents of the internal cavity.
  • An EV can include, but is not limited to, an ectosome, a microvesicle, a microparticle, an exosome, an oncosome, an apoptotic body, a liposome, a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, a matrix vesicle, or a multivesicular body.
  • An EV has a dimension of up to about 10 microns, but are typically about 1000 nm or less.
  • Exosomes and microvesicles are types of EVs, and can be shed by eukaryotic cells, or budded off of the plasma membrane, to the exterior of the cell. These membrane-bound vesicles are heterogeneous in size with diameters ranging from about 10 nm to about 5000 nm. The methods and compositions described herein are equally applicable for microvesicles of all sizes.
  • exosome also refers to protein complexes containing exoribonucleases that are involved in mRNA degradation and the processing of small nucleolar RNAs (snoRNAs), small nuclear RNAs (snRNAs) and ribosomal RNAs (rRNA).
  • small nucleolar RNAs snoRNAs
  • snRNAs small nuclear RNAs
  • rRNA ribosomal RNAs
  • Such protein complexes do not have membranes and are not “microvesicles” or “exosomes,” and thus are not EVs, as those terms are used here in.
  • the term “patient” and “subject” are used interchangeably to refer to a human or animal, such as a vertebrate, e.g., a mammal.
  • mammals include, without limitation, primates, rodents, domestic animals, or game animals.
  • Primates include chimpanzees, monkeys, and macaques, e.g., Rhesus.
  • Rodents include mice, rats, rabbits, and hamsters.
  • Domestic and game animals include cows, horses, pigs, deer, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, avian species, e.g., chicken, duck, and ostrich, and fish, e.g., trout, bass, and salmon.
  • Patient or subject includes any subset of the foregoing, e.g., all of the above, but excluding one or more groups or species such as humans, primates, or rodents.
  • the subject is a mammal, e.g., a primate, e.g., a human.
  • a subject can be male or female.
  • a subject can be any stage of development, e.g., embryo, fetus, infant, child, pre-adolescent, adolescent, young adult, mature adult, and elderly adult.
  • the female subject can be pregnant or not.
  • the subject can be a patient or a subject in a clinical setting.
  • the subject can be suspected of, or at risk for, having or developing a disease or disorder, or may have already been diagnosed as having a disease or disorder.
  • the subject may be a patient undergoing treatment.
  • a “capture agent” refers to any agent having specific binding for EVs generally (e.g., an exosome) or target EVs. Binding may be to a marker, e.g., a biomarker, which is present on all EVs, or to a subset of target EVs. Typically, the capture agent specifically binds to a biomarker fully or partially present on the external surface of the EVs (referred herein as an extravesicular marker), although in some embodiments, the capture agent specifically binds to a marker that is present on the interior of the EV (referred herein as an intravesicular marker).
  • the capture agent is immobilized on the surface of a plasmonic nanostructure that is contacted to the sample (e.g., the sensing area).
  • capture agents include, without limitation, nucleic acids, oligonucleotides, peptides, polypeptides, aptamers, antigens, polyclonal antibodies, monoclonal antibodies, single chain antibodies (scFv), antibody portions, F(ab) fragments, F(ab')2 fragments, Fv fragments, small organic molecules, polymers, compounds from a combinatorial chemical library, inorganic molecule, or any combination thereof.
  • a “nucleic acid,” as described herein, can be RNA or DNA, and can be single or double stranded, and can be, for example, a nucleic acid encoding a protein of interest, a polynucleotide, an oligonucleotide, a nucleic acid analogue, for example, peptide-nucleic acid (PNA), pseudo-complementary PNA (pc-PNA), locked nucleic acid (LNA) etc.
  • PNA peptide-nucleic acid
  • pc-PNA pseudo-complementary PNA
  • LNA locked nucleic acid
  • Nucleic acid sequences include, for example, but are not limited to, nucleic acid sequences that act as transcriptional repressors, antisense molecules, ribozymes, small inhibitory nucleic acid sequences, for example, but not limited to, RNAi, shRNAi, siRNA, micro RNAi (mRNAi), antisense oligonucleotides etc.
  • DNA is defined as deoxyribonucleic acid.
  • polynucleotide is used herein interchangeably with “nucleic acid” to indicate a polymer of nucleosides.
  • a polynucleotide is composed of nucleosides that are naturally found in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine) joined by phosphodiester bonds.
  • the term encompasses molecules including nucleosides or nucleoside analogs containing chemically or biologically modified bases, modified backbones, etc., whether or not found in naturally occurring nucleic acids, and such molecules can be used for certain applications.
  • polypeptide refers to a polymer of amino acids.
  • protein and “polypeptide” are used interchangeably herein.
  • a peptide is a relatively short polypeptide, typically between about 2 and 60 amino acids in length.
  • Polypeptides used herein typically contain amino acids such as the 20 L-amino acids that are most commonly found in proteins. However, other amino acids and/or amino acid analogs known in the art can be used.
  • One or more of the amino acids in a polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a phosphate group, a fatty acid group, a linker for conjugation, functionalization, etc.
  • polypeptide that has a non-polypeptide moiety covalently or noncovalently associated therewith is still considered a “polypeptide.”
  • modifications include glycosylation and palmitoylation.
  • Polypeptides can be purified from natural sources, produced using recombinant DNA technology, synthesized through chemical means such as conventional solid phase peptide synthesis, etc.
  • An “antigen” is defined herein as a substance inducing an immune response.
  • the antigenic determinant group is termed an epitope, and the epitope in the context of a carrier molecule (that can optionally be part of the same molecule, for example, botulism neurotoxin A, a single molecule, has three different epitopes.
  • antigens are foreign to the animal in which they produce immune reactions.
  • antibodies can include polyclonal and monoclonal antibodies and antigen-binding derivatives, or portions or fragments thereof.
  • Well-known antigen binding fragments include, for example, single domain antibodies (dAbs; which consist essentially of single VL or VH antibody domains), Fv fragment, including single chain Fv fragment (scFv), Fab fragment, and F(ab')2 fragment. Methods for the construction of such antibody molecules are well known in the art.
  • dAbs single domain antibodies
  • Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding fragments include, inter aha, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), single domain antibodies, chimeric antibodies, diabodies and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.
  • Fab, Fc, pFc', F(ab') 2 and Fv are employed with standard immunological meanings (see, e.g., Klein, Immunology (John Wiley, New York, N.Y, 1982); Clark, W. R. (1986) The Experimental Foundations of Modern Immunology (Wiley & Sons, Inc., New York); and Roitt, I. (1991) Essential Immunology, 7th Ed., (Blackwell Scientific Publications, Oxford).
  • reporter group refers to a composition capable of producing or enhancing a detectable optical signal indicative of the presence of the target in a sample.
  • 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 metal, e.g., gold, semiconductor, plastic, polymer, and glass.
  • reporter groups is a QUAD marker (Mucin 1, cell surface associated (MUC1), Epidermal Growth Factor Receptor (EGFR), epithelial cellular adhesion molecule (EpCAM), and human epidermal growth factor receptor 2 (HER2)),
  • MUC1 cell surface associated
  • EGFR Epidermal Growth Factor Receptor
  • EpCAM epithelial cellular adhesion molecule
  • HER2 human epidermal growth factor receptor 2
  • label refers to a composition capable of producing or enhancing a detectable signal indicative of the presence of the target in a sample.
  • the term “marker” or “biomarker” refers to a molecule that is associated with an EV and can bind to a capture agent for detecting the EV.
  • a marker can be any components of an EV that can be recognized by a capture agent. Examples of markers include, without limitation, proteins, or nucleic acids or a component of the lipid bilayer that makes up the membrane of the EV Useful markers include receptors (e.g., extracellular) and channel components.
  • a marker can be either an extravesicular or an intravesicular marker, as defined herein.
  • a marker can be present on all EVs in a sample, or on a subset of EVs in a sample. A marker that is common to all EVs in a sample is referred to herein as a pan-EV marker.
  • QUAD biomarkers refers to MUC1, EpCAM, HER2, and EGFR.
  • bonds e.g., ionic, covalent, polar, or hydrogen bonds.
  • bonds e.g., ionic, covalent, polar, or hydrogen bonds.
  • linker groups e.g., PEG, or affinity ligands as described herein.
  • an “affinity ligand” is defined herein as a molecule that is directly attached or fixed to a molecular spacer or to a substrate or nanostructure, and also can be directly attached to a capture agent.
  • an affinity ligand can be a capture agent.
  • an affinity ligand physically links a molecular spacer (or substrate or nanostructure) and a capture agent (or molecular spacer) together.
  • the affinity ligand is a first member of a specific binding pair.
  • the capture agents may be the second member of the specific binding pair.
  • an affinity ligand can include, but is not limited to, a nucleic acid, oligonucleotide, peptide, polypeptide, antigen, polyclonal antibody, monoclonal antibody, single chain antibody (scFv), an antibody portion, F(ab) fragment, F(ab')2 fragment, Fv fragment, small organic molecule, polymer, compounds from a combinatorial chemical library, inorganic molecule, or any combination thereof.
  • binding pairs include antigen-antibody, hapten-antibody, or antibody-antibody pairs, complementary oligonucleotides or polynucleotides, avidinbiotin, streptavidin-biotin, hormone-receptor, ligand-receptors, lectin-carbohydrate, IgG- protein A, nucleic acid-nucleic acid binding protein, and nucleic acid-anti-nucleic acid antibody.
  • specific binding refers to a chemical interaction between two molecules, compounds, cells, and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target.
  • specific binding can refer to an affinity of the first entity for the second target entity that is at least 10 times greater than the affinity for the non-target entity.
  • a reagent specific for a given target is one that exhibits specific binding for that target under the conditions of the assay being utilized.
  • specific binding is indicated by a dissociation constant on the order of ⁇ 1 O’ 8 M, ⁇ 10" 9 M, ⁇ 10" 10 M or below.
  • Polyethylene glycol (PEG) is referred to herein as a possible component of the nano-plasmonic array and is used as a molecular spacer. A variety of forms and combinations of PEG are envisioned for use as such spacers.
  • Polyethylene glycol (PEG) is a polyether compound with many applications from industrial manufacturing to medicine. The structure of PEG is (note the repeated element in parentheses): H — (O — CH 2 -CH 2 )n-OH.
  • PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular weight.
  • PEG, PEO, or POE refers to an oligomer or polymer of ethylene oxide.
  • PEG refers to oligomers and polymers with a molecular mass below 20,000 g/mol
  • PEO refers to polymers with a molecular mass above 20,000 g/mol
  • POE refers to a polymer of any molecular mass.
  • PEG and PEO are liquids or low-melting solids, depending on their molecular weights.
  • Different forms of PEG are also available, depending on the initiator used for the polymerization process — the most common initiator is a monofunctional methyl ether PEG, or methoxypoly(ethylene glycol), abbreviated mPEG.
  • Lower-molecular- weight PEGs are also available as purer oligomers, referred to as monodisperse, uniform, or discrete.
  • Branched PEGs have three to ten PEG chains emanating from a central core group.
  • Star PEGs have 10 to 100 PEG chains emanating from a central core group.
  • Comb PEGs have multiple PEG chains normally grafted onto a polymer backbone.
  • a “long-chain polyethylene glycol (PEG)” or “long PEG” is defined herein as a PEG polymer having a molecular weight equal to or higher than 750 Da.
  • a “short-chain PEG” or “short PEG” is defined herein as a PEG polymer having a molecular weight equal to or less than 500 Da.
  • expression level refers to the number of mRNA molecules and/or polypeptide molecules encoded by a gene of interest that are present in a cell or sample.
  • FIGs. lAto 1G illustrate an example of a nano-plasmonic array for multiplexed single EV analysis.
  • FIG. 1 A is a diagram of the procedural steps of multiplexed single EV analysis.
  • FIG. IB is an image of periodic nanoholes in the nano-plasmonic array.
  • FIG. 1 C is an image showing the enhanced electromagnetic fields on the nanohole surface of the nano-plasmonic array.
  • FIG. ID are images of fluorescent nanospheres on glass and the nano-plasmonic substrates.
  • FIG. IE is a histogram of pixel intensities of a glass substrate and the nano-plasmonic substrate.
  • FIG. IF is a bar graph showing fluorescence intensity of fluorescent nanostructures on glass and the nano-plasmonic substrate.
  • FIG. 1 G is an image showing a magnified view of the nano-plasmonic array.
  • FIGs. 2A and 2B illustrate an example of an optical system for the nano- plasmonic array for multiplexed single EV analysis.
  • FIG. 2A is a schematic diagram of an optical system for multiplexed single EV analysis.
  • FIG. 2B is a microscope image of the nano-plasmonic array.
  • FIGs. 3 A to 3 J are images and graphs of fluorescence used to characterize a system for detecting target EVs.
  • FIG. 3A is a series of images of substrates coated different fluor ophore-conjugated biotin-binding proteins.
  • FIG. 3B is a bar graph showing intensity profiles of different fluor ophore-conjugated biotin-binding proteins.
  • FIG. 3C is a bar graph showing fluorescence intensity of different fluorophore-conjugated biotinbinding proteins.
  • FIG. 3D is a graph showing the absorption/emission spectra of different fluorophore-conjugated biotin-binding proteins.
  • FIG. 3E is a pair of images of EVs captured on glass and substrates of the nano-plasmonic arrays.
  • FIG. 3F is a graph showing intensities of captured EVs on glass and substrates of the nano-plasmonic arrays.
  • FIG. 3G is a bar graph showing the number of EVs on glass and substrates of the nano-plasmonic arrays.
  • FIG. 3H is a pair of images of EVs captured on glass and substrates of the nano-plasmonic arrays with an adhesive layer.
  • FIG. 31 is a graph showing intensities of captured EVs on glass and substrates of the nano-plasmonic arrays with an adhesive layer.
  • FIG. 3 J is a bar graph showing the number of EVs on glass and substrates of the nano-plasmonic arrays with an adhesive layer.
  • FIGs. 4A to 4C illustrate single EV measurements.
  • FIG. 4A is a series of microscope images of captured EVs with different fluorescent antibodies.
  • FIG. 4B is a series of graphs showing intensities of EVs with different fluorescent antibodies in different positions on the substrate.
  • FIG. 4C is a series of bar graphs showing the number/fraction of EVs with different fluorescent antibodies.
  • FIGs. 5Ato 5E illustrate measurements of tumor markers of captured EVs.
  • FIG. 5 A is a series of microscope images of EVs labeled against tumor marker EGFR.
  • FIG. 5B is a series of microscope images of EVs labeled against tumor marker EGFRvIII.
  • FIG. 5C is a representation of gels showing EGFR expression in different EVs.
  • FIG. 5D is a bar graph showing EGFR fractions in different EVs.
  • FIG. 5E is a bar graph showing EGFRvIII fractions in different EVs.
  • FIGs. 6Ato 6G illustrate measurements of tumor markers in EV-spiked plasma samples.
  • FIG. 6A is a flow chart showing a decision tree to classify EV populations.
  • FIG. 6B and FIG. 6C are series of graphs showing EV populations positive for different markers.
  • FIG. 6D to FIG. 6G are bar graphs showing EV detection and marker profiling.
  • FIGs. 7A and 7B illustrate an example of a characterization of EVs isolated from Gli36-WT and Gli36-EGFRvIII.
  • FIG. 7A is a graph showing a size distribution of EVs isolated from Gli36-WT and Gli36-EGFRvIII.
  • FIG. 7B is a series of images of gels showing expression levels of EVs isolated from Gli36-WT and Gli36-EGFRvIII.
  • FIGs. 8A and 8B illustrate an example of a characterization of EVs varied in concentrations.
  • FIG. 8A is a series of microscope images of EVs labeled against different markers.
  • FIG. 8B is a series of bar graphs showing the fractions of EVs labeled against different markers under different EV concentrations.
  • FIGs. 9A and 9B illustrate negative controls to demonstrate test sensitivity and specificity of the captured EVs.
  • FIG. 9A is a pair of microscope images showing sensitivity and specificity of EVs labeled against EGFR.
  • FIG. 9B is a pair of microscope images showing sensitivity and specificity of EVs labeled against EGFRvIII.
  • FIGs. lOAto 10D is a series of schematic diagrams that illustrate an example of a method for fabricating a nano-plasmonic array including gold nanorods.
  • FIG. 10A is a schematic diagram of a step of imprinting nanorods on a resist layer.
  • FIG. 1 OB is a schematic showing a step of patterning an imprinting mold.
  • FIG. IOC is a schematic diagram of a step of depositing gold nanorods and lifting off of the mask.
  • FIG. 1 OD is a schematic diagram that shows the use of the nano-plasmonic array to detect EVs that bind to the gold nanorods.
  • FIGs. 11 A to 1 ID illustrate an example of a method for detecting target EVs.
  • FIG. 11A is a schematic diagram that illustrates an example of a nano-plasmonic array including nanorods for plasmon-enhanced single EV sensing technology (NEXT), as described herein.
  • FIG. 1 IB is a representation of a microscope image of an EV bound to a nanorod.
  • FIG. 11 C is a representation of a microscope image of a nanorod array.
  • FIG. 1 ID is a representation of a microscope image of EVs bound to different locations on nanorod.
  • FIG. 1 lE-a is a schematic of an example of a fabrication procedure for 3D Au nanoparticles (AuNPs) on Au nanopillars (NPOP) structure, as described herein.
  • FIG. 1 lE-b is a photograph of wafer-scale (90 cm 2 area) chip.
  • FIGs. 1 lE-c and 1 lE-d are scanning electron images of nanopillars formed on a PET substrate, and 3D NPOP structures formed on coated nanopillars (scale bar, 200 nm).
  • FIG. 1 lE-e is a transmission electron image of 3D NPOP structures.
  • the inset shows the size distribution of AuNPs on the nanopillars.
  • FIG. 1 lE-f is a high-resolution transmission electron image showing a zoomed-in image of the white dashed box in (e).
  • FIG. HE-g shows a finite element method (FEM) simulation of the 3D NPOP structure shown in the large dashed box in 1 lE-e.
  • FEM finite element method
  • FIG. 12A is a graph showing scattering intensities of nanorods of different sizes.
  • FIG. 12B is a graph showing scattering intensities changes in dark field imaging.
  • FIGs. 13 A to 13H illustrate finite-difference time-domain (FDTD) simulations showing optical resonances of nanorod and nanodisk arrays in different sizes.
  • FIG. 13A is a representation of side view of EV captured on a nanorod.
  • FIG. 13B is a representation of a top view of a nanodisk.
  • FIG. 13C is an image of top view of a nanodisk.
  • FIG. 13D is a graph showing scattering intensities of nanodisks having different diameters.
  • FIG. 13E is a graph showing peak shifts of nanodisks having different diameters.
  • FIG. 13F is a representation of a top view of a nanorod.
  • FIG. 13G is a graph showing scattering intensities of nanorods having different diameters.
  • FIG. 13H is a graph showing
  • FIGs. 14Ato 14F illustrate three FDTD simulation scenarios showing spectral shifts of dark-field scattering upon EV binding to nanodisks in different locations and distances to the substrate.
  • FIG. 14A is a representation of Scenario 1 of a first EV binding location and its detected peak wavelength, along with a corresponding graph.
  • FIG. 14B is a representation of Scenario 2 of a second EV binding location and its detected peak wavelength, along with a microscope image showing electromagnetic waves.
  • FIG. 14C is a representation of Scenario 3 of a third EV binding location and its detected peak wavelength, along with a microscope image showing electromagnetic waves.
  • FIG. 14A is a representation of Scenario 1 of a first EV binding location and its detected peak wavelength, along with a corresponding graph.
  • FIG. 14B is a representation of Scenario 2 of a second EV binding location and its detected peak wavelength, along with a microscope image showing electromagnetic waves.
  • FIG. 14C is a representation of Scenario 3 of
  • FIG. 14D is a representation of dark-field scattering spectra of nanodisks upon EV binding in varying distances (z) to the surface, showing the different level of spectral shifts depending on the distance between the EV and nanodisk.
  • FIG. 14E is a representation of electromagnetic fields in a cross-section of a nanodisk on a glass substrate, showing the concentrated electromagnetic fields on top of the nanodisk surface.
  • FIG. 14F is a presentation of electromagnetic fields on a nanodisk on a glass substrate in a top view, showing the concentrated electromagnetic fields along the sides of the nanodisk.
  • FIGs. 15Ato 15D illustrate an example of EV binding detection by measuring dark-field scattering intensity changes.
  • FIG. 15A is a representative dark-field scattering image of nanodisks before EV binding.
  • FIG 15B is a representative dark-field scattering image of nanodisks after EV binding.
  • FIG 15C is a representative graph showing the changes in the dark-field scattering intensity over time for EVs on the nanodisk shown in FIG. 15A and FIG. 15B.
  • FIG 15D is a representative graph showing the changes in the dark-field scattering intensity over time for controls on the nanodisk shown in FIG. 15 A and FIG. 15B.
  • FIGs. 16Ato 16C illustrate an example of EV binding detection by measuring spectral shifts of dark-field scattering of nanodisks and corresponding graphs of the single nanodisk spectrum of the wavelength vs normalized scaling.
  • FIG. 17 illustrates plasmon enhancements of dark- field imaging and three different fluorescent signals (TRITC, Cy5, and Cy5.5) for different diameters of nanodisks from 80 to 200 nm.
  • FIGs. 18Ato 18D illustrate an example of plasmon enhancements of dark-field and fluorescence signals in different sizes of nanodisks.
  • FIG. 18A is a plasmon intensity of dark-field corresponding to different diameters of nanodisks.
  • FIG. 18B is a plasmon intensity of TRITC corresponding to different diameters of nanodisks.
  • FIG. 18C is a plasmon intensity of Cy5 corresponding to different diameters of nanodisks.
  • FIG. 18D is a plasmon intensity of Cy5.5 corresponding to different diameters of nanodisks.
  • FIG. 19A is a series of fluorescence images of AF555- (left) or AF647-labeled (right) EVs on glass or NPOP substrate in different concentrations.
  • FIG. 19B is a pair of graphs for comparison of EV counting numbers in different dilution factors on glass or NPOP substrates (left graph, AF555-labeled EVs; right graph, AF647-labeled EVs).
  • FIG. 19C is a pair of histogram plots for comparison of EV intensities (1: 1600 dilution) in glass and NPOP substrate (left graph, AF555-labeled EVs; right graph, AF647-labeled EVs).
  • FIG. 19D is a bar graph that shows quantitative analysis of signal enhancement.
  • the NPOP substrate enhanced the signal intensities by 2-fold (AF555) and 9-fold (AF647) compared with the glass substrate.
  • FIG. 20 is a schematic showing an example of a workflow of a plasmon-enhanced EV assay as described herein.
  • FIG. 21 A is a schematic of an example of a multi-channel detection strategy for single EV analysis with a gold nanopillar array (NPOP substrate) as described herein.
  • An NPOP substrate was functionalized by SH-PEG-COOH (1.0 kDA) and EVs were captured by EDC/NHS activation. Next, the captured EVs were fluorescently stained by marker-specific antibodies. The EVs were then labeled with different fluorescence- conjugated wheat germ agglutinin (WGA). Finally, the multi-color labeled EVs were imaged and analyzed.
  • FIG. 21 B is a series of representative images for EV capture by functionalization with mercaptoundecanoic acid (MU A) (1.7 nm length), SH-PEG-COOH (0.3 kDA, 2.8 nm length), and SH-PEG-COOH (1.0 kDa, 7 nm length) with or without EDC/NHS activation (scale bars, 50 pm).
  • MU A mercaptoundecanoic acid
  • SH-PEG-COOH 0.3 kDA, 2.8 nm length
  • SH-PEG-COOH 1.0 kDa, 7 nm length
  • FIG. 21 C is a bar graph of EV counting showed SH-PEG-COOH showed the least non-specific EV binding (ns, not significant; ****P ⁇ 0.0001 compared with the mock treatment, as assessed by two-way ANOVA with Bonferroni's multiple comparisons test). Error bars are shown as mean ⁇ SD from the ten different images.
  • FIG. 21 D is a series of representative images for different color-labeled EVs (green) and markers (IgG or HER2)(red). The percentages of co-localization (Marker/EV merged images) were analyzed for evaluating the differential marker expression. The colocalized signals (B/A) were indicated by white arrowheads. Scale bars, 10 pm.
  • 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 bars, 10 pm).
  • QUAD marker MUC1, HER2, EGFR, and EpCAM
  • FIG. 22B is a bar graph for the percentages of co-localization for the EVs and QUAD markers. Error bars are shown as mean ⁇ SD from the four different images.
  • FIGs. 22C to 22D are heatmaps that show the differential abundances of QUAD markers in four different tumor cell line-derived EVs, as assessed by single EV analysis (22C) and originated cell lines, as assessed by flow cytometry (22D).
  • FIGs. 23 A to 23C are a series of graphs that show the plasmon enhancement on the NPOP substrates for Cy7 fluor ophores. Imaging EVs in the Cy7 channel has been challenging due to weak fluorescence signals generated from the dye and channel. However, by using the plasmon enhancement, we could significantly amplify EV fluorescence signals when EVs are captured on the NPOP substrate compared to other plain substrates (glass or gold). This led to higher EV detection sensitivity.
  • FIG. 24A is a series of representative images of CD63 (vs. IgG) detection on EVs.
  • FIG. 24B is a pair of graphs showing EV count and marker positive EV count results for CF63 and IgG.
  • EVs can be, but are not limited to, ectosomes, microvesicles, microparticles, exosomes, oncosomes, apoptotic bodies, liposomes, vacuoles, lysosomes, transport vesicles, secretory vesicles, gas vesicles, matrix vesicles, or multivesicular bodies.
  • EVs carry multiple surface biomarkers, which can be used as indicators to monitor or diagnose certain diseases, e.g., cancers, cardiovascular, neurodegenerative, and infectious diseases, among others.
  • the new systems and methods can be used for detecting and diagnosing Alzheimer’s and other neurodegenerative diseases as well as detecting viruses, bacteria, and/or parasites, e.g., by analyzing immune cells that contain materials from the infective agents.
  • EVs have a dimension of up to about 10 microns, but are typically 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 1000 nm or less, about 900 nm or less, about 800 nm or less, about 700
  • EVs often provide weak detection signals, especially when the EV sample does not include a sufficient number of EVs or when there is a low abundance of protein and/or intravesicular markers, which can make it challenging to perform sensitive, robust, and standardized assays that can determine the composition and molecular profiles of EVs in clinical samples.
  • the present disclosure provides a solution to these problems and enables targeting single EVs by amplifying their individual optical signals to achieve an accurate and precise multiplexed analysis of the target EV.
  • Analyzing single EVs can reveal unique molecular profiles of cell-specific EVs, which will further promote clinical use of EVs, e.g., to construct a comprehensive EV “atlas” per different biological parameters (e.g., cellular origin, cell state).
  • the nano-plasmonic systems of the present disclosure enable multiplexed single EV analyses of target membrane and intravesicular markers with improved sensitivities.
  • the optical signal e.g., fluorescence
  • the enhancement can be achieved, for example, by using a substrate with a periodic array of nanostructures, such as nanoholes, nanorods, nanodisks, nanowells, nanosquares, nanopillars, nanogrooves, or any suitable periodic or non-periodic metallic nanostructures.
  • a copper or aluminum film or substrate can be used for UV illumination, and silver and gold can be used for visible wavelength illumination.
  • the substrate if used under a metal film, is a non-metal, non-conducting substrate such as glass or plastic, but metal, metal oxides, and semiconductors can also be used as substrates.
  • a periodic array of Au nanoholes support surface plasmon resonances extended in a long range (about 100 nm) which is suitable for EVs.
  • the resonance wavelength can be tuned by adjusting the nanohole periodicity and size. The same can be done with nanostructures in the form of nanorods or nanodisks.
  • the nano-plasmonic extracellular vesicle analysis with enhanced fluorescence detection (nPLEX-FL) described herein, along with similar methods using other optical signals, provide a simple, robust signal amplification strategy that improves the detection sensitivity and achieves multiplexed EV analysis.
  • the nano-plasmonic arrays used herein include a substrate, a plurality of nanostructures on or in the substrate, and a plurality of affinity ligands fixed on or adjacent to the nanostructures.
  • Different surface chemistries conjuggates to affinity ligands
  • the metals used to make the nanostructures and the substrates e.g., glass
  • 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.
  • the plurality of affinity ligands is fixed 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.
  • affinity ligands can be used in the nano-plasmonic arrays based on a corresponding EV preparation.
  • the substrate of the nano-plasmonic arrays can include a biotin-binding protein (e.g., avidin) as the affinity ligands attached on the substrate, then the EVs or target EVs are required to comprise a corresponding biotin as the capture agent to be captured by the nano-plasmonic arrays.
  • the substrate comprises semiconductors, non-conductors, plastics, or any suitable transparent substrates. Methods for attaching a corresponding capture agent to EVs are described below.
  • the system includes arrays of metal, e.g., gold, silver, copper, or aluminum, nanostructures, e.g., nanoholes, nanorods, nanodisks, or nanopillars, e.g., in sub-200 nm dimensions, which can be occupied by single EVs.
  • metal e.g., gold, silver, copper, or aluminum
  • nanostructures e.g., nanoholes, nanorods, nanodisks, or nanopillars, e.g., in sub-200 nm dimensions, which can be occupied by single EVs.
  • gold nanorods have a high sensitivity down to single molecule detection and precise tunability of resonant wavelengths by adjusting nanorod dimensions.
  • the arrays of nanostructures can be made using standard nanoimprint lithography techniques with good reproducibility through advanced imprinting and deposition processes.
  • Dense arrays (e.g., 10 5 arrays per cm 2 ) of metal, e.g., gold, nanostructures, e.g., nanorods, can be made using a new nanoimprint lithography method that can pattern gold nanorod arrays in a wafer-scale through simple imprinting and gold deposition processes (FIGs. 10A-10D).
  • the technique utilizes a reusable silicon mold with nano-patterns that are transferred to a target substrate coated with a thin resist layer (FIGs. 10A-10B). After imprinting, gold is deposited onto the patterned area; subsequent removal of the resist will leave nanorod arrays on a glass substrate (FIG. IOC).
  • Periodic nanoholes are made by patterning a thin (50 to 200 nm thick) gold film on a substrate. Nanoholes can be directly patterned by focused ion-beam milling or through lithography and metal etching. Deep ultraviolet (DUV) lithography is used to make 200 nm periodic circular patterns on a resist spun-coated on the gold film. Furthermore, the underlying gold film is etched by reactive ion etching or ion milling using the resist as an etch mask. Resist removal reveals gold nanohole patterns made in the gold film.
  • DUV Deep ultraviolet
  • Array chips are designed through comprehensive three-dimensional computational calculations, and we found in one example that the nanorod dimension of 80 nm (length) x 30 nm (width) x 20 nm (height) achieved maximum sensitivity for 100- nm EV (mean diameter) detection, and array sensor dimensions and sensitivities are experimentally tested.
  • the sub- 100 nm dimension of gold nanorods also allows single EV capture on each nanorod.
  • Gold nanorod arrays with 3 pm separation between nanorods allow even distribution of EVs on the nanorod arrays; signals from individual EVs are clearly resolved using a 1 Ox or higher objective.
  • the total number of nanorods in a chip is readily scalable with a nano imprint mold size.
  • microarray spotter MicroSys, Digilab Inc.
  • 0.1 pL solutions are transferred from a 96- well plate and spotted on designed areas with good reproducibility ( ⁇ 5% variation).
  • Temperature and humidity are controlled inside the spotter chamber for consistent sample spotting and incubation conditions.
  • a metal, e.g., gold, nanostructure, e.g., nanorod exhibits a unique dark-field light scattering peak at a resonant wavelength.
  • EV binding to the nanorod surface increases a local refractive index, red-shifting the peak wavelength.
  • the spectral shift i.e., EV binding
  • the intensity measurement method can be used for high throughput parallel signal reading from entire arrays in a field-of-view. This approach is much faster than sequential spectral measurements used in past systems and methods.
  • the dark-field imaging is also compatible with epifluorescence measurements for molecular EV profiling in the same setup.
  • EVs e.g., tumor-derived EVs
  • FDTD finite difference time-domain
  • the signal also correlates with the size of captured EVs, facilitating EV size measurements.
  • NTA nanoparticle tracking analysis
  • readout signals from entire arrays can be measured simultaneously.
  • the intensity measurements provide much higher throughput in readouts of vast arrays than spectral measurements.
  • temperature controllers to stabilize the light source temperature and/or increase the number of signal averages to reduce background noises.
  • FIGs. 11 A to FIG. 1 ID illustrate an example of a nano-plasmonic array comprising nanorods for plasmon-enhanced single EV sensing technology (NEXT).
  • FIG 11 A is a schematic drawing of NEXT chip sensor consisting of gold nanorod arrays made in grid. The small surface area of nanorods allows for single EV binding on each nanorod.
  • FIG. 1 IB is a scanning electron micrograph (SEM) of a nanosphere captured on a gold nanorod (scale bar: 50 nm).
  • FIG. 11C is an SEM of a nanorod array made by electron-beam lithography, but such nanoarrays can also be made with nanoimprint lithography (scale bar: 500 nm).
  • 1 ID is a SEM showing binding of single nanospheres, mimicking EVs, on nanorods (scale bar: 200 nm). Captured EVs are labeled by immunofluorescence probes for high throughput multichannel analyses using plasmon enhanced fluorescence detection. The captured EVs are evenly distributed by a distance between nanorods; this will improve the accuracy of analysis.
  • FIG. 1 lEa-g shows new 3D plasmonic nanostructures composed of spherical Au nanoparticles (AuNPs) on 3D Au nanopillars (NPOP) with a 1 nm-thick uniform spacer layer between the AuNPs and the nanopillars.
  • the method is based on enhanced surface diffusion of adsorbed Au atoms (i.e., adatoms) on a low-energy surface; the Au adatoms diffuse into defective sites of the underlying film and form clustered atoms (i.e., AuNPs).
  • This process enables the direct and selective formation of spherical Au nanoparticles through a simple Au deposition process without high-temperature annealing.
  • the AuNPs density can be highly increased by introducing more nucleation sites on the 3D rough Au nanopillar surface.
  • the 3D NPOP structures provide high-density hotspots and large effective volumes of molecular binding sites for highly sensitive surface-enhanced Raman spectroscopy (SERS) and plasmon-enhanced fluorescence (PEF).
  • SERS surface-enhanced Raman spectroscopy
  • PEF plasmon-enhanced fluorescence
  • FIG. 1 lE-a shows a schematic of the fabrication procedure for the 3D NPOP substrates.
  • a polyethylene terephthalate (PET) substrate was etched with Ar plasma to form polymer nanopillars.
  • a 100 nm- thick Au film was deposited onto the nanopillars by either conventional sputtering or thermal evaporation to form high-density Au-coated nanopillars.
  • a self-assembled monolayer (SAM) of 1H,1H,2H,2H- perfluorodecanethiol (PFDT) was vapor-deposited onto the Au nanopillars.
  • SAM self-assembled monolayer
  • FIG. 1 lE-b shows a photograph of wafer-scale (90 cm 2 area) chip fabrication.
  • FIGs. 1 lE-c and 1 lE-d are scanning electron images of nanopillars formed on a PET substrate, and 3D NPOP structures formed by deposition of 20 nm of Au onto the PFDT- coated nanopillars (scale bar, 200 nm).
  • FIG. 1 lE-e shows a transmission electron image of 3D NPOP structures.
  • the inset shows the size distribution of AuNPs on the nanopillars.
  • FIG. 1 lE-f is a high-resolution transmission electron image showing a zoomed-in image of the white dashed box in (e).
  • the thickness of the PFDT layer between the AuNP and nanopillar is about 1 nm.
  • FIG. 1 lE-g shows a finite element method (FEM) simulation of the 3D NPOP structure shown in a dashed box in 1 lE-e.
  • FEM finite element method
  • Biological samples are obtained, e.g., from a human or other subject, and cells can be cultured in culture media, such as Dulbecco’s modified Eagle’s medium (DMEM, Cellgro). Media can be supplemented with serum, e.g., 10% Fetal Bovine Serum, antibiotics, e.g., penicillin and/or streptomycin, and kept under 5% CO2 (see, e.g., Min et al., Plasmon-Enhanced Biosensing for Multiplexed Profiling of Extracellular Vesicles, Advanced Biosystems, 2020, 4, 200003. DOI: 10.1002/adbi.202000003, which is incorporated herein by reference in its entirety, including all figures and reference citations).
  • serum e.g., 10% Fetal Bovine Serum
  • antibiotics e.g., penicillin and/or streptomycin
  • CO2 see, e.g., Min et al., Plasmon-Enhanced Biosensing for Multiplexe
  • EVs can be isolated using both standard ultracentrifugation (UC) and sizeexclusion chromatography (SEC) methods. Furthermore, EVs are isolated from the medium for the next process. For UC, the filtrates are concentrated, e.g., by 100,000 x g for 1 hour. After the supernatant is removed, the EV pellet is washed, e.g., with PBS and centrifuged again, e.g., at 100,000 x g for 1 hour. The EV pellet is resuspended in buffer or serum, e.g., in PBS.
  • UC ultracentrifugation
  • SEC sizeexclusion chromatography
  • EVs can be selected using different biomarkers and respective affinity binding pairs and their manufactures directions.
  • the EV analysis is performed based on the nPLEX-FL protocols described herein, which includes using multiple fluorescent labels, Raman signals, and dark-field scattering signals to detect target EVs for EV analysis.
  • fluorescence detection EVs are labeled by fluorescence probes conjugated with affinity ligands.
  • Raman detection molecules on the surface membrane or inside of EVs can be directly detected or EVs are labeled by Raman probes conjugated with affinity ligands.
  • Raman detection scattering signals from EVs can be directly detected without any labeling.
  • the nanostructures of the nano-plasmonic arrays are labeled with affinity ligands that bind, e.g., specifically bind, to EVs or are bound to capture agents that specifically bind to EVs, and then the substrate is exposed to a biological sample for a sufficient time to ensure that the substrate is bound to a sufficient number of EVs.
  • biotinylated EVs are captured on neutravidin-coated nanostructures, followed by EV fixation and permeabilization in a fix/perm solution.
  • Surface passivation can be achieved by placing the surface (with or without EVs) in a blocking solution (Superblock PBS, Thermo Fisher) for 20 minutes. This step is important to minimize undesired nonspecific binding.
  • the captured EVs are stained via two-step indirect labeling: first with primary antibodies then with compatible secondary antibodies. Thorough washing is done between steps.
  • the EVs are labeled with capture agents, such as streptavidin. Finally, the labeled EVs are attached to the nanostructures via the capture agents with a mounting solution and covered with a glass coverslip.
  • Antibodies that can be used in the present disclosure are listed in Table 1 below. Primary antibodies are used to specifically bind to a specific biomarker on the surface of the EVs, and secondary antibodies are used to specifically bind to the primary antibodies. Furthermore, the secondary antibodies are conjugated with a reporter group, e.g., a fluorescent probe, to be used in image processing, or a capture agent such as streptavidin.
  • the assay buffer can be, for example, a BD perm/wash buffer solution (BD Biosciences).
  • Antibodies and Dilution Factors Any other antibodies can also be used in the present disclosure based on the specific use. Considering different biomarkers of EVs, a corresponding antibody can be selected. EV biomarkers associated with different diseases and purposes are listed in Table 2 below.
  • the image processing of the captured EVs is performed using image analysis software, such as ImageJ® and CellProfiler®.
  • image analysis software such as ImageJ® and CellProfiler®.
  • the streptavidin imaging channel is used to identify location of captured EVs and define regions of interests as masks.
  • the corresponding fluorescent images from target molecules are aligned using ImageJ® plugins (Align slices in the stack). At each mask position, average pixel intensities are obtained. The signal is corrected by subtracting background signal surrounding the mask.
  • QUAD biomarkers can be used for multiplexed analysis of surface markers on EVs, e.g., for methods for diagnosing cancer, e.g., cancer of epithelial origin, by detecting the presence of tumor cells in a sample, based (at least in some embodiments) on the quantification of levels of four biomarkers, e.g., MUC1, EGFR, EpCAM, and HER2.
  • MUC1, EGFR, EpCAM e.g., MUC1, EGFR, EpCAM, and HER2.
  • MUC-1, HER2, EGFR, and EpCAM provide the highest diagnostic accuracy, but combining these four markers established a very high level of accuracy that is superior to conventional clinical analysis. See, e.g., US Patent No. 10,712,343, which is incorporated herein by reference in its entirety.
  • additional or alternative biomarkers may also be used in the multiplexed analysis, and two or three or four or five or six or more different biomarkers can be investigated.
  • the tumor is a pancreas, lung, breast, prostate, kidney, stomach, esophagus, bladder, endometrial, cervix, biliary, thyroid, ovary, brain, head and neck, oral, melanoma, liver, or colon tumor.
  • the methods include obtaining a sample from a subject, isolating EVs from the sample, and evaluating the presence and/or level of MUC-1, HER2, EGFR, and EpCAM in the EVs, and comparing the presence and/or level with one or more references, e.g., a control reference that represents a normal level of MUC-1, HER2, EGFR, and EpCAM, e.g., a level in an unaffected subject, and/or a disease reference that represents a level of the QUAD biomarkers in a subject having cancer.
  • a control reference that represents a normal level of MUC-1, HER2, EGFR, and EpCAM
  • the methods include determining a value, e.g., a normalized expression value, for each of the biomarkers (MUC-1, HER2, EGFR, and EpCAM), and calculating a test score from the sum of each of the levels. This score can then be compared to a reference score, wherein the presence of a test score above (e.g., equal to or above, or simply above) the reference score indicates the presence of cancer in the subject. See, e.g., US Patent No. 10,712,343.
  • a test score e.g., a normalized expression value
  • the methods can also be used to monitor a patient, e.g., to determine whether a treatment has been effective, or whether a subject is experiencing a recurrence, or whether treatment resistance is emerging.
  • the methods include detecting the presence and/or level of biomarkers in EVs obtained from a subject over time, e.g., in a first or baseline sample, and then in one or more subsequent EV samples, e.g., over a period of one or more weeks or months.
  • a decrease over time in the presence or levels of the biomarkers indicates an improvement in the disease, e.g., that a treatment administered in the intervening time is effective to cause a decrease in a number of cancerous cells or tumor burden.
  • a treatment administered in the intervening time is effective to cause a decrease in a number of cancerous cells or tumor burden.
  • the QUAD biomarkers may be used in this analysis, additional or alternative biomarkers may also be used in the multiplexed analysis of surface markers on EVs.
  • No change in the presence or levels of the biomarkers indicates no change in disease, e.g., that any intervening treatment was not effective to cause a decrease in a number of cancerous cells or tumor burden (though in particularly aggressive disease the treatment may have been effective to halt progression, which may be a treatment goal).
  • An increase over time in the presence or levels of the biomarkers indicates that the disease is progressing, e.g., there has been an increase in a number of cancerous cells and/or tumor burden. Such an increase may also be indicative of a poor prognosis, e.g., an increased likelihood of mortality.
  • the methods include the use of antibodies or antigen fragments thereof, or oligonucleotides, that specifically bind (i.e., do not bind substantially to other molecules) to the biomarkers.
  • 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, which retain the ability to bind antigen.
  • the antibody can be polyclonal, monoclonal, recombinant, chimeric, de-immunized or humanized, fully human, nonhuman, (e.g., murine), or single chain antibody.
  • the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind to an Fe receptor.
  • the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fe receptor, e.g., it has a mutagenized or deleted Fe receptor binding region.
  • the new methods and nano-plasmonic arrays can be used to analyze single EVs in multiple scenarios.
  • tumor-derived EVs contain protein and RNA markers reflective of primary tumor cells, and the new nano-plasmonic array sensors can rapidly and sensitively detect tumor EVs directly from clinical samples.
  • EV analyses offer compelling clinical potential for diagnosing cancers and monitoring longitudinal tumor response to therapy.
  • the single EV detection platforms as described herein enable to identify individual EVs derived from tumors or specific organs and detect specific target molecules on the membrane or inside of EVs from the target subpopulation, otherwise diluted or undetected by EVs from non-target origins.
  • the molecular profiling of EVs from targetspecific tumors or organs can indicate the molecular status of originating cells.
  • FIG. 20 depicts the overall workflow of the plasmon-enhanced EV assay.
  • EVs are isolated from human specimens (e.g., blood or other biofluids) or cell culture supernatant.
  • the isolated EVs are captured on a plasmonic substrate with nanostructures, e.g., gold nanostructures (e.g., nanopillars, nanodisks, nanorods, nanoholes, nanoslits, nanoparticles, random structures), either by covalent bonding/binding or by affinity ligands.
  • nanostructures e.g., gold nanostructures (e.g., nanopillars, nanodisks, nanorods, nanoholes, nanoslits, nanoparticles, random structures), either by covalent bonding/binding or by affinity ligands.
  • the surface of plasmonic substrates (made of gold, silver, copper, aluminum, platinum, or their combination) is functionalized with a linker (e.g., PEG or carbon chains with functional groups) or biomolecule adhesion layer to capture all EVs on the surface via non-specific interactions.
  • linker e.g., PEG or carbon chains with functional groups
  • biomolecule adhesion layer e.g., biomolecule adhesion layer
  • marker-positive, target EV subpopulations are captured on the surface via affinity ligands/capture agents (e.g., antibodies, aptamers, peptides, nucleic acids).
  • affinity ligands/capture agents e.g., antibodies, aptamers, peptides, nucleic acids.
  • the first case is the capture of all EVs.
  • the second case is the capture of target EVs.
  • the captured EVs are then immune-labeled with fluorophores for marker analysis.
  • the markers include, but are not limited to, surface proteins, intravesicular proteins, lipids, and nucleic acids.
  • Ligands used to capture EVs on the substrate can be also used for marker labeling. For example, QUAD markers (MUC1, HER2, EGFR, and EpCAM) can be used. In the first case, only target EVs among all EVs are labeled using target EV-specific capture agents bound to reporter groups. The capture agents must specifically bind to markers found only on target EVs, and not on all EVs.
  • all EVs that are bound to the nanostructures and/or the substrate are target EVs, and so all can be labeled with reporter groups, and thus the reporter groups can be bound to capture agents that bind to any EVs (since only target EVs are present on the substrate).
  • target EV-specific reporter groups can be used in both cases. The end goal is to label with reporter groups only the target EVs.
  • the labeled target EVs are imaged, e.g., by a fluorescence microscope. As shown in FIG. 20, images from multiple fluorescence channels can be obtained for multiplexed analysis of EV markers at the single EV level. The images are then analyzed to detect the total number of EVs and their concentrations, 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.
  • 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.
  • FIG. 21 A is a schematic that depicts EV capture on gold nanopillar arrays.
  • the gold nanopillars can be decorated with gold nanoparticles to increase the plasmon enhancements.
  • the gold nanopillar diameter can be 20 - 500 nm and the height can be 20 - 5000 nm.
  • the diameter of decorating nanoparticles can be 10 - 100 nm.
  • the schematic shows the case of non-specific EV capture via PEG-COOH after the activation of carboxylated groups by EDC/NHS.
  • antibodies can be immobilized on the nanopillar surface to capture marker-specific EVs.
  • the captured EVs are labeled by fluorophore-conjugated antibodies.
  • Electrodynamic computation can be performed using the finite-difference time-domain (FDTD) method.
  • FDTD finite-difference time-domain
  • FIGs. 1A to 1G illustrate an example of a nano-plasmonic array for multiplexed single EV analysis.
  • FIG. 1 A illustrates the procedural steps of the multiplexed single EV analysis starting from capturing EVs, e.g., target EVs, labeling the captured target EVs, taking images of the labelled target EVs, and analyzing the images of target EVs. EVs are captured on the nanohole surface and immune-stained by fluorescent detection probes, and then labeled target EVs are imaged in different fluorescence channels, and their intensities are analyzed.
  • capturing EVs e.g., target EVs
  • labeling the captured target EVs taking images of the labelled target EVs
  • analyzing the images of target EVs e.g., analyzing the images of target EVs.
  • EVs are captured on the nanohole surface and immune-stained by fluorescent detection probes, and then labeled target EVs are imaged in different fluorescence channels
  • EVs are captured on the Au nanohole surface via affinity ligands (e.g., capturing biotinylated EVs on avidin-coated Au nanohole surface).
  • the target EVs which can be some or all of the captured EVs are then immune-stained by fluorescently labeled antibodies in different color channels (typically 3 to 4 colors).
  • the fluorescence signals are amplified by surface plasmon resonances (SPR) excited by the underlying Au nanohole structures.
  • SPR surface plasmon resonances
  • FIG. IB illustrates a scanning electron micrograph of periodic nanoholes in the nano-plasmonic array.
  • the diameter of a nanohole is about 200 nm and the periodicity is 500 nm.
  • the scale bar in FIG. IB is 1 pm.
  • the nanostructure in FIG. IB is optimized as a SPR substrate, and the substrate is a 100-nm thick Au film.
  • FIG. 1 C illustrates a finite-difference time-domain simulation showing the enhanced electromagnetic fields confined on the nanohole surface of the nano-plasmonic array.
  • the strong fields are responsible for plasmon-enhanced fluorescence signals.
  • the periodic nanohole granting on the chip surface concentrates electromagnetic fields with the maximum field intensity up to 300-fold.
  • the resonance fields extend to 110 nm in the z-direction, which mostly covers small EVs (e.g., exosomes with an average diameter of 100 nm).
  • the fluorescence radiation can be further enhanced by the interaction of the Au nanostructure with proximal fluorophores in the resonance range.
  • FIG. ID illustrates images of fluorescent nanospheres (using Cy5, 200 nm) on glass and the present nano-plasmonic substrates.
  • the scale bar in FIG. ID is 10 pm.
  • the plasmon-enhanced fluorescence by Au nanohole structures of the nPLEX chip using fluorescent nanospheres (Cy5, 200 nm) is tested in comparison with a glass substrate.
  • FIG. IE illustrates an example of histograms of pixel intensities of a glass substrate and the present nano-plasmonic substrate. It shows that the fluorescence intensities of individual nanospheres are significantly higher on the nPLEX-FL substrate (two-tailed t-test, p ⁇ 0.0001).
  • IF illustrates a mean fluorescence intensity of fluorescent nanospheres on glass and the nano-plasmonic substrates.
  • the mean fluorescence intensity is increased by a factor of 18, and the signal -to-noise ratio (given by signal divided by 3 -times standard deviation of blank) is increased by a factor of 20, from 17.7 (glass) to 358 (nPLEX-FL).
  • the coefficient of variation given by the ratio of the standard deviation to the mean, for fluorescence intensities between the glass (36.2%) and nPLEX-FL substrates (33.6%), indicating the signal amplification does not increase the intensity variation.
  • FIG. 1G illustrates a finite-difference time-domain simulation that shows the enhanced electromagnetic fields around a nanohole.
  • the scanning electron microscopy shows EVs captured by functionalized Au nanohole chip.
  • the dotted circles represent the enhanced electromagnetic field distribution around the nanoholes.
  • FIG. 2A illustrates an example of an optical system for the nano-plasmonic array for multiplexed single EV analysis (NEXT readout system), that integrates dark-field imaging with multi-channel fluorescence imaging.
  • FIG. 2A illustrates an example of an upright microscope setup 10 for dark-field (transmission) and epifluorescence dual-mode imaging.
  • the charge-coupled device (CCD) 20 of the optical setup can be used to capture electromagnetic radiation emitted, scattered, or reflected by reporter groups (e.g., fluorescent antibodies) on the labeled target EVs captured on the nano-plasmonic array.
  • reporter groups e.g., fluorescent antibodies
  • the upright microscope setup 10 comprises a filter set 22 to process/filter electromagnetic radiation, a microscope stage 26 on which a substrate with a nano-plasmonic array is placed under an objective 24, a camera, e.g., CCD, 20 to capture the radiation processed by the filter set 22.
  • the system also includes a dark field condenser 28, arranged below the stage 26, a primary light source 30 (e.g., an LED light source) arranged to illuminate the microscope stage 26 from above, and a secondary light source 32, arranged to illuminate the microscope stage 26 from below through a darkfield condenser 28.
  • a primary light source 30 e.g., an LED light source
  • the dark-field scattering imaging is used to illuminate the nano-plasmonic array from below to capture emitted, scattered, or reflected electromagnetic radiation from the nano-plasmonic array through the objective 24.
  • the second light source 32 and the dark-field condenser 28 of the dark-field scattering imaging system can be disposed above the objective 24 to illuminate the microscope stage 26 from above.
  • 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.
  • dark- field scattering signals can be also amplified by an underlying plasmonic substrate.
  • FIG. 2B illustrates an example of a dark-field scatter image of the nanostructure arrays.
  • An inset shows a zoomed image of the nanostructure arrays.
  • the nanostructure arrays may be nanorod arrays, and each nanorod is separated by 2 pm.
  • nPLEX-FL chips were prepared using the lithography methods described above. The chip was incubated overnight at room temperature with thiolated biotin polyethylene glycol (PEG) (10 x 10 -3 m in PBS, PG2-BNTH-lk, Nanocs). After washing with PBS, an equimolar mixture of streptavidin molecules conjugated with either Alexa Fluor 488, Cy3, Cy5, or Cy5.5 (Biolegend) was incubated for 10 min.
  • PEG biotin polyethylene glycol
  • the concentration of each fluorescence dye was diluted to be 2.5 pg mL-1, except Alexa Fluor 488-conjugated streptavidin (25 pg mL-1 in PBS) due to the weak fluorescence signal compared to other channels.
  • FIG. 3 A is a series of fluorescence images of the nano-plasmonic arrays/chips coated with four colors of fluorophore-conjugated streptavidin (AF488, Cy3, Cy5, and Cy5.5).
  • the scale bar in FIG. 3 A is 20 pm.
  • Nanohole arrays were made in a 100 x 100 pm 2 sized square area highlighted by white dashed boxes, e.g., the white dashed box shown in the fluorescence image using AF488.
  • FIG. 3B illustrates cross-sectional intensity profiles along the grey horizontal dashed lines in FIG. 3 A.
  • the plasmon enhancement in different fluorescence channels was tested using a molecular monolayer.
  • the Au nanohole surface was functionalized using thiolated biotin polyethylene glycol derivatives (thiol-PEG-biotin), and fluorophore-conjugated streptavidin molecules are immobilized on the biotinylated Au surface.
  • the Au surface was functionalized with thiol-PEG-biotin (IkDa , 6-8 nm) and neutravidin (60kDa, 4-5 nm), which results in an adhesion layer of 10-13 nm in thickness.
  • FIG. 3 A and FIG. 3B show the strong signal enhancements in the 100 x 100 pm 2 sized square area of nanohole gratings (highlighted by a white dashed box) compared to the flat Au area (outside of the square, FIG. 3B).
  • FIG. 3C illustrates examples of an enhancement factor (EF) of fluorescence intensity in different fluorescence channels.
  • the signal enhancement is most dominated in the Cy5 channel, and the EF of fluorescence intensity in the nanohole area in comparison to flat Au areas is 23 -fold.
  • FIG. 3D illustrates an example of a plasmon-supported light transmission spectrum through nanohole arrays overlaid with the absorption/emission spectra of fluorophores.
  • the Cy5.5 and Cy3 intensities are also increased by 17 and 9 folds, respectively, when the AF488 signal is only increased by 3-fold.
  • These EFs in the different channels can be explained by spectral overlaps between plasmon-supported light transmission through nanoholes and absorption/emission spectral of fluorophores.
  • the light transmission peak is measured at 667 nm, which is most overlapped with Cy5 absorption (649 nm) and emission (666 nm) peaks followed by Cy5.5 and Cy3.
  • FIGs. 3E to 31 illustrate examples of plasmonic enhancements on EVs.
  • FIG. 3E indicates that biotinylated EVs are captured on glass and substrates of the nano- plasmonic arrays, and subsequently labeled the captured EVs with streptavidin- conjugated dyes. The captured EVs are labeled with Cy5-conjugated streptavidin, and then imaged.
  • the scale bar in FIG. 3E is 10 pm.
  • FIG.3H indicates biotinylated EVs captured on the device surface coated with an L-3,4-dihydroxyphenylalanine (L-DOPA)- based bioadhesive layer, and the captured EVs were labeled with AF488-conjugated streptavidin, and then imaged.
  • FIG. 31 illustrates a comparison of the mean fluorescence intensities and
  • FIG. 3 J illustrates the number of captured EVs in FIG. 3H in the region of interest (ROI) between the nanohole chip and glass substrate.
  • An L-DOPA-based bioadhesive layer is used to capture EVs in the same densities on different substrates (glass and Au) and investigated fluorescence intensities and detectable EV counts.
  • FIG. 3F illustrates examples of histograms of pixel intensities of captured EVs of FIG. 3E.
  • the averaged signal enhancement factors in terms of fluorescence intensity after background correction were measured to be 1.54 for AF488 and 8.60 for Cy5.
  • the overall enhancement is less significant than the streptavidin monolayer coating in FIG. 3C likely due to localized electromagnetic fields, which are strongest near the surface shown in FIG. 3C.
  • FIG. 3G illustrates the number of detected EVs of FIG. 3E between the nanohole chip and glass substrate.
  • the fluorescence intensity was normalized by background signals defined by the sum of the mean fluorescence intensity in the absence of EV and three times the standard deviation.
  • glioblastoma cell lines for testing Gli36-WT and Gli36-EGFRvIII (overexpressing human EGFRvIII).
  • Gli36-WT and Gli36-EGFRvIII are biomarkers of interest for glioblastoma as amplification of EGFR and its variant (EGFRvIII) occur frequently in glioblastoma.
  • CD-pan ubiquitous EV tetraspanin combination 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 FIGs. 6A and 6B).
  • EVs were isolated from conditioned cell culture media. Nanoparticle tracking analysis showed that the isolated EVs used in this study have a size distribution ranging 50-200 nm with an average diameter of 100 nm, also confirmed by transmission electron micrographs.
  • the isolated EVs were biotinylated, diluted in pure buffer (1-10 10 8 EVs mL-1 phosphate-buffered saline (PBS)), and captured on the neutravidin-coated gold nanohole surface.
  • PBS phosphate-buffered saline
  • the captured EVs were immune-labeled against membrane (i.e., CD63, EGFR) and/or intravesicular markers (i.e., GAPDH) and imaged under a fluorescence microscope.
  • the average blob size of the detected vesicles in fluorescence images was about 500 nm (8 pixels with a pixel size of 63 nm).
  • FIG. 4A shows representative nPLEX-FL images of biotinylated EVs labeled against CD-pan (AF488), streptavidin (Cy3), and GAPDH (Cy5).
  • GAPDH GAPDH
  • FIG. 4 A illustrates that EVs from the Gli36-WT cell line are biotinylated and captured on the nanohole surface. Individual EV are detected through staining with fluorescent Cy3 -streptavidin (top left). For molecular profiling, EVs are labeled with fluorescent antibodies against transmembrane EV markers (CD63) and intravesicular markers (GAPDH). Multiple EV markers are chosen to detect and classify single EVs based on marker expression levels. AF488 dye to high abundance/easy-to- detect markers and Cy5 to low abundance/hard-to-detect markers are assigned.
  • FIG. 488 dye to high abundance/easy-to- detect markers and Cy5 to low abundance/hard-to-detect markers are assigned.
  • FIG. 4A shows representative nPLEX-FL images of Gli36-WT derived EVs labeled against CD63 (AF488) and GAPDH (Cy5).
  • GAPDH is chosen as a representative intravesicular marker, which is commonly used as a control for many other quantitative methods (e.g., Western blotting, qPCR).
  • FIG. 4B illustrates line scans showing high signal-to-noise for the chosen markers in this example. Gray shading highlights EV positions. Line scan shows high signal-to- noise and heterogeneity for the chosen markers on individual vesicles.
  • FIG. 4C illustrates EV subtyping.
  • the raw intensity data shown in FIG. 4B was then analyzed for the marker expression and EV subtyping.
  • marker-positive and marker-negative which can be separated by the intensity cutoff of 100.
  • Roughly half the captured vesicles had CD63 (46%), and of the CD63+ EVs, a fraction expressed GAPDH (58%). It is confirmed that strong overlapping (>95%) of GAPDH+ EVs with CD63+ EVs.
  • FIGs. 5A to FIG. 5E illustrate an example of a measurement of tumor markers of captured EVs to demonstrate tumor diagnostic potential of the new systems and methods.
  • EVs from three different cell lines (GH36-WT, Gli36-EGFRvIII, MCF7) were biotinylated and captured on a nanohole array surface, and EVs were labeled with fluorescent antibodies against the CD-pan marker panel (CD9, CD63, and CD81) as well as tumor markers which comprise EGFR in FIG. 5A and EGFRvIII in FIG. 5B.
  • Spots with dotted circles indicate tumor marker-positive EVs in FIGs. 5A and 5B.
  • FIG. 5C illustrates a 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.
  • the bar graphs in FIGs. 5D and 5E illustrate EV subtyping.
  • Fraction (%) EVCD- pan+Target+ / EVcD-pan+.
  • Fraction (%) EVCD- pan+Target+ / EVcD-pan+.
  • FIG. 5D a significant fraction of Gli36-WT EV were positive for EGFR (54%) whereas a small fraction of Gli36-EGFRvIII were positive for EGFR (7%).
  • FIG. 5E somewhat over 10% of Gli36-EGFRvIII vesicles were positive for EGFRvIII, while Gli36-WT and MCF7 showed the EGFRvIII-positive fractions ( ⁇ 1%) below the threshold for statistical significance.
  • a negative control was prepared with the same procedure with no EV incubation.
  • particles labeled with Cy3-conjugated streptavidin were first detected and prescreened by size exclusion ( ⁇ 1 pm) to exclude large aggregates from the analysis.
  • size exclusion ⁇ 1 pm
  • EVs positive for CD-pan markers CD9, CD63, and CD81
  • the prescreened EVs were sub-gated with target glioblastoma markers of EGFR or EGFRvIII.
  • FIGs. 6B and 6C show biomarker distribution analyses on a single-EV level.
  • We observed 10- 15% positivity of streptavidin-positive particles for CD-pan markers FIG. 6E.
  • PBS pure-buffer
  • the detected EVs positive for CD-pan were screened for target markers of EGFR and EGFRvIII.
  • glioblastoma EVs can be used to detect EGFRvIII mutation proteins.
  • Glioblastoma (GBM) cell lines were used for testing: Gli36-WT and Gli36-EGFRvIII, a clone of Gli36 EV that is positive for EGFRvIII mutation.
  • EVs were collected from conditioned cell culture media and membrane filtered, biotinylated, immobilized on the nanohole array chip surface, and immune- labeled against membrane (i.e., CD63, EGFR) and/or intravesicular markers (i.e., GAPDH).
  • the isolated EVs used in this study have a size distribution ranging 50-200 nm with an average diameter of 100 nm and the high purity determined by western blotting for ubiquitous EV protein markers (CD9, CD63, and CD81, FIG. 7B).
  • the avidin-functionalized Au chip showed high specificity for biotinylated EV capture, which was confirmed by electron microscopy.
  • FIG. 7A illustrates a size distribution graph of Gli36-WT and Gli36- EGFRvIII EVs obtained by nanoparticle tracking analysis (NTA).
  • FIG. 7B illustrates Western blot measurements of Gli36-WT and Gli36-EGFRvIII EVs to determine pan-CD marker expression levels (CD9/CD63/CD81) in bulk.
  • FIG. 8 A illustrates EVs from the OVCA429 cell line biotinylated and captured on the nano-plasmonic array device. EVs were collected from conditioned cell culture media and membrane filtered, biotinylated, and immobilized on the nanohole array chip surface. Captured EVs were labeled against the pan-CD marker, which is a combination of CD9, CD63, and CD81 (AF488), and EGFR (Cy5). EVs were artificially color-coded for visual aid. The scale bar in FIG. 8 A is 10 pm.
  • the three bar graphs in FIG. 8B illustrate various EV concentrations (4-fold difference) and the number of captured EV. Regardless of the EV concentrations, roughly half the CD-pan+ EVs expressed EGFR. We identified individual vesicles by staining EV with Cy3 -streptavidin.
  • FIG. 9A to FIG. 9B illustrate negative controls to demonstrate test sensitivity and specificity of the captured EVs.
  • EVs from the MCF7 cell line were collected from conditioned cell culture media and membrane filtered, biotinylated, and immobilized on the nanohole array chip surface.
  • EVs were labeled with the CDpan marker which is a combination of CD63/CD81/CD9 (AF488) and EGFR (shown in FIG. 9A) or EGFRvIII (Cy5) (shown in FIG. 9B).
  • CDpan marker which is a combination of CD63/CD81/CD9 (AF488) and EGFR (shown in FIG. 9A) or EGFRvIII (Cy5) (shown in FIG. 9B).
  • the negative control (e.g., no EVs) was prepared with the same procedure with no EV incubation.
  • the results in FIGs. 9A to FIG. 9B indicate a statistical significance of the present nano-plasmonic array sensor system for detecting target EVs.
  • FIG. 12A to FIG. 12B illustrate examples of the optical characterization of nanorod sensor arrays as described herein.
  • the graph of FIG. 12A illustrates the results of finite-difference time-domain (FDTD) simulations showing optical resonance peaks for different sizes of nanorods having lengths of 40, 60, 80, 100, and 120 nm.
  • the optical tuning is important to maximize fluorescence signal enhancement by the nanorod’s surface plasmon resonance.
  • the nano-plasmonic array can have a specific size/dimension for each of the nanorods based on a size of the target EVs and/or other requirements (e.g., to detect a specific wavelength of SPR) in analysis steps.
  • the graph in FIG. 12B shows the experimental results of spectral shifts and intensity changes of dark-field scattering as the surface refractive index increases from 1.33 to 1.45.
  • Water and ethanol mixtures in different mix rations were prepared and applied to the nanorod arrays to vary the refractive indices from 1.33 to 1.45.
  • the spectral shifts were measured and plotted against the surface refractive index.
  • the peak wavelength with the surface refractive index of 1.33 was used as a reference to calculate spectral shifts.
  • EV binding to the nanorods increases the surface refractive index, shifting the resonance peak. The EV binding event can be detected by measuring either spectral shifts or scattering light intensity changes.
  • FIG. 13 A is a representation of a side view of an EV captured on a nanorod.
  • FIG. 13B is a representation of top view of a nanodisk in an array.
  • FIG. 13C is a scanning electron microscope image of a top view of an EV on a nanostructure.
  • FIG. 13D is a graph showing scattering intensities (a.u.) of nanodisks having different diameters of 40, 60, 80, 100, 120, 140, 160, 180, or 200 nm, calculated by FDTD simulations. As shown, the scattering intensity increases with the diameter of the nanodisk, and the wavelength of peak scattering intensity also increases with nanodisk diameter.
  • FIG. 13E is a graph showing peak shifts (nm) of nanodisks having different diameters from 40 to 200 nm. As shown, the peak shift is highest for a nanodisk having a diameter of 40 nm, drops sharply from 60 to 80 nm diameters, and then continues to decrease as diameter increases until leveling off at about 180 nm.
  • FIG. 13F is a representation of a top view of a nanorod having length L and a width of 30 nm.
  • FIG. 13G is a graph showing scattering intensities (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 wavelength and with length of the nanorods.
  • FIG. 13H is a graph showing peak shifts (nm) of nanorods having different lengths from 40 to 120 nm. As shown, the peak shift decreases with nanorod length
  • the nano-plasmonic array can be designed to have nanostructures with specific sizes/dimensions of nanorods or nanodisks based on a size of the target EVs as well as other requirements (e.g., to detect a specific wavelength of SPR) for detecting target EVs. Any shapes similar to nanorods or nanodisks can be also used as plasmonic nanostructures to amplify fluorescent and dark-field signals.
  • FIG. 14Ato FIG. 14C illustrate FDTD simulations showing spectral shifts of dark-field scattering upon EV binding to nanodisks in different locations and distances to the substrate.
  • FIG. 14A is a representation of Scenario 1 of a first EV binding location and its detected peak wavelength, along with a corresponding graph.
  • FIG. 14B is a representation of Scenario 2 of a second EV binding location and its detected peak wavelength, along with a microscope image showing electromagnetic waves.
  • FIG. 14C is a representation of Scenario 3 of a third EV binding location and its detected peak wavelength, along with a microscope image showing electromagnetic waves concentrated on the nanodisk surface. The results show single EV binding to the nanodisk surface in various binding scenarios can be detected by measuring spectral shifts of dark-field scattering peak wavelength.
  • FIG. 15Ato FIG. 15D illustrate an example of EV binding detection by measuring dark- field scattering intensity changes in real time.
  • Timeline (1) as shown in FIG. 15 A, is before EV binding to the nanodisk in the center of the array (middle circle).
  • Timeline (2) as shown in FIG. 15B, is after EV binding to the nanodisk in the center of the array (middle circle).
  • the graph of FIG. 15C shows real time measurements showing an abrupt intensity change at time point (2) upon EV binding to the center nanodisk.
  • FIG. 15C shows that the intensity is increased at 100 seconds when an EV binds to the nanodisk, as the signal difference shown in FIG. 15Aand FIG. 15B (middle circle).
  • the graph of FIG. 15D shows real time measurements showing no abrupt intensity changes of two controls that show no binding to any of the nanodisks in the array.
  • FIG 15D shows the changes in the dark- field scattering intensity over time for the control nanodisks (Control 1 and Control 2 in FIG. 15Aand FIG. 15B), which have no affinity ligands. There is no EV binding and thus no intensity change is measured.
  • the vertical dashed lines (1) and (2) in FIG. 15C and FIG. 15D indicate the time points when FIG. 15 A and FIG. 15B are taken.
  • FIGs. 16Ato 16C illustrate the results of how EV binding can be detected by measuring spectral shifts of dark- field scattering by the nanodisks.
  • EV binding was confirmed by overlays with EV fluorescence images. EVs were labeled by specific fluorescent probes to locate the nanodisks that have captured EVs.
  • FIGs. 16Ato 16C show overlaid images of fluorescence channels for EVs and dark-field scattering of nanodisks, which show EVs are captured on the nanodisks (circles in the images). Dark-field scattering spectra before and after EV binding on those nanodisks are shown on the right, demonstrating that EV binding to the nanodisk surface induces spectral shifts.
  • FIG. 16A shows a central nanodisk that shows a fluorescent signal indicating EV binding.
  • the accompanying graph of a single nanodisk spectrum shows a slight spectral shift, where no shift means a perfect overlay, in normalized scattering before or after EV binding.
  • the image of FIG. 16B shows a central nanodisk with a change in fluorescence indicating EV binding.
  • the accompanying graph of a single nanodisk spectrum shows a pronounced rightward (increase in peak wavelength) shift in normalized scattering after EV binding.
  • FIG. 16C shows a nanodisk that shows a fluorescent signal indicating EV binding.
  • the accompanying graph of a single nanodisk spectrum shows no significant shift in normalized scattering before or after EV binding, where no shift means a perfect overlay.
  • the nano-plasmonic arrays can be designed to have nanodisks having specific diameters based on a corresponding dark- field/fluorescent labels for detecting target EVs.
  • a monolayer of fluorescence molecules was formed on the top of the nanodisk arrays, and the fluorescence signals on the nanodisk and substrate were measured.
  • FIG. 17 illustrates plasmon enhancements of dark-field and three different fluorescent signals (TRITC, Cy5, and Cy5.5) for nanodisks having different diameters of 80, 100, 120, 140, 160, 180, and 200 nm.
  • FIGs. 18Ato 18D are graphs that illustrate plasmon enhancements of dark-field and fluorescence signals for different sizes of nanodisks.
  • FIG. 18A is a graph of 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 declines at 200 nm.
  • FIG. 18B is a graph of plasmon intensity of TRITC corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200 nm, and as shown the level increases sharply with diameter up to 120 nm and then declines sharply to 140 nm, declines slightly to 180 nm, and then increases again to 200 nm.
  • FIG. 18C is a graph of plasmon intensity of Cy5 corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200, and as shown the level decreases slightly from 80 to 100 nm, then increases sharply with diameter up to 120 nm and then declines to 200 nm.
  • FIG. 18D is a graph of plasmon intensity of Cy5.5 corresponding to different diameters of nanodisks of 80, 100, 120, 140, 160, 180, and 200 nm, and as shown the level remains the same from 80 to 100 nm, increases sharply with diameter from 100 up to 140 nm, then declines sharply to 180 nm, and then declines slightly to 200 nm.
  • TRICT and Cy5 show the maximum intensity when they are coated on 120 nm (diameter) nanodisks while Cy5.5 showed the maximum intensity on the 140 nm diameter nanodisk.
  • EVs were isolated from a cell culture of Gli36-WT (ATCC), Gli36-EGFRvIII (generated from Gli36-WT through lentivirus transduction), and MCF-7 cells (ATCC) grown in DMEM (Cellgro), OVCA429 cells (ATCC) cultured in RPMI-1640 medium (Cellgro). Media were supplemented with 10% fetal bovine serum (FBS, Thermo Fisher), 100 U/mL penicillin, and 100 pg/mL streptomycin (Cellgro) at 37°C in 5% CO2. Furthermore, cell lines were tested and were free of mycoplasma contamination (MycoAlertTM mycoplasma detection kit, Lonza).
  • EVs were incubated in DMEM with 1% exosome-depleted FBS (Thermo Fisher) for 48 hours before EV collection.
  • the conditioned medium was collected and centrifuged, e.g., at 300 x g for 5 minutes, and then supernatant was filtered through, e.g., through a 0.2 pm membrane filter (Millipore Sigma).
  • EVs were isolated using both standard ultracentrifugation (UC) and sizeexclusion chromatography (SEC) methods: (i) for UC, the filtrates were concentrated by centrifugation at 100,000 x g for 1 hour. After the supernatant was removed, the EV pellet was washed with a buffer or saline solution, such as PBS, and centrifuged at 100,000 x g for 1 hour.
  • UC ultracentrifugation
  • SEC sizeexclusion chromatography
  • the isolated EVs were resuspended in buffer or saline solution, e.g., PBS and incubated with the capture agent, such as EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher) for a sufficient time, e.g., 30 minutes, at room temperature. For example, a 20- fold molar excess of sulfo-NHS-biotin to EV protein was used in a 0.5 mL volume. Four to six biotins were incorporated per molecule. Excess biotin was then removed utilizing the Exosome Spin Columns, MW3000 (Thermo Fisher) per the kit instructions. The prepared EVs were filtered using a 0.22 pm centrifugal filter (Ultrafree®, Millipore).
  • NPOP nanoparticles on nanopillars
  • AF555 or AF647 dyes were fluorescently labeled EVs with AF555 or AF647 dyes and the same aliquots were applied to the substrates. Because substrates are made of different materials, we coated the substrate surface with a cell-adhesion layer (Corning® CELL-TAKTM) to ensure the same surface chemistry.
  • TFP Dye was prepared as follows. First, we prepared 27.5 mM of Azido- dPEG®i2-TFP ester (Quanta Biodesign) with Anhydrous DMSO (Sigma). Then we prepared 25 mM of AFdye DBCO with Anhydrous DMSO (Sigma), and then mixed equal volume of Azido-dPEG®i2-TFP ester and AFdye DBCO at room temperature for 2 hours with a HulaMixer® (ThermoFisher Scientific). EVs were labeled as follows.
  • the substrates and EV samples were prepared as follows. We first washed the substrates, and glass was incubated in absolute ethanol for 5 minutes and washed with water. The NPOPs were washed with water, and we removed the water with nitrogen gas. We attached the PDMS (4 mm wells, 2 mm thickness) onto the substrate to make EV attachment. We prepared a cell and tissue adhesive solution (CELL-TAKTM, Corning) to attach the EVs on the substrate without surface chemistry. We then mixed 30 pg of Cell-Tak, 10 mM sodium bicarbonate, and 17 mN sodium hydroxide and added the resulting mixture into the wells and incubated for 30 minutes for surface absorption.
  • CELL-TAKTM Cell and tissue adhesive solution
  • FIGs. 19A-19D show that a higher number of EVs can be detected when EVs are captured on the NPOP substrate than on a glass substrate. This was due to signal enhancement by the plasmonic substrate that amplifies EVs’ fluorescence signals. Overall signals were amplified and higher numbers of EVs were detected for both AF555 and AF647 channels. The enhancement can occur for dyes with excitation wavelengths longer than 530 nm.
  • FIG. 19A shows fluorescence images of AF555- or AF647-labeled EVs on a glass substrate or on an NPOP substrate. The same amount of labeled EVs in different dilution factors was mounted onto the adhesive molecule-treated substrates. Scale bars, 50 pm.
  • Fig. 19B shows a pair of graphs for comparison of EV counting numbers in different dilution factors on glass (black line) or NPOP substrates (lighter line).
  • the NPOP substrate showed a higher number of EVs detected in both AF555- or AF647- labeled EVs.
  • Left panel AF555-labeled EVs; right panel, AF647-labeled EVs. Error bars represent the mean ⁇ SD.
  • FIG. 19C is a pair of histogram plots for comparison of EV intensities (1:1600 dilution) in glass (black graph) and NPOP substrate (light line). The intensities of detected EVs were higher in NPOP substrate. Left panel, AF555-labeled EVs; right panel, AF647-labeled EVs.
  • FIG. 19D is a bar graph for quantitative analysis of signal enhancement.
  • the NPOP substrate enhanced the signal intensities by 2-fold (AF555) and 9-fold (AF647) compared with the glass substrate (*P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001; ****p ⁇ 0.0001), as assessed by two-way ANOVA with Bonferroni's multiple comparisons test. Error bars are shown as mean ⁇ SD from the three different images.
  • the linker groups have two functions. First, once their carboxylated groups are activated by EDC/NHS, the linkers serve as affinity ligands and provide binding sites for EVs through covalent bonding between the activated functional groups on the linker and EVs. Second, the linker groups play a role as a passivation layer (mock) to minimize non-specific molecular binding. In this test, we showed that thiol-PEG-COOH (1.0 kDa) shows a greater difference between desired (EDC/NHS) and undesired (mock) EV capture than other tested linkers.
  • NPOP substrates and captured EVs by washing the substrate with water. We then removed the water with nitrogen gas and placed the substrates in a humid chamber. We functionalized the NPOP substrate with COOH molecules of different lengths.
  • MU A mercaptoundecanoic acid
  • SH-PEG-COOH (0.4 kDa) we prepared the 0.25 mM of SH-PEG-COOH (0.4 kDa, Nanocs) in water, added the mixture onto the NPOP substrate, incubated for 4 hours at RT, and washed with water.
  • SH-PEG- COOH (1.0 kDa) we prepared the 0.25 mM of SH-PEG-COOH (1.0 kDa, Nanocs) in water, added the mixture onto the NPOP substrate, incubated for 4 hours at RT, and washed with water.
  • NPOP substrate 0.1 M MES (pH 6.0) for Mock treatment and the mixture of 50 mM EDC (ThermoFisher Scientific) and 125 mM of sulfo-NHS (ThermoFisher Scientific) in 0.1 M MES (pH 6.0) for 7 minutes to capture the EVs by covalent bonding.
  • EDC ThermoFisher Scientific
  • sulfo-NHS ThermoFisher Scientific
  • FIG. 21B is a series of representative images for EV capture by functionalization with MUA (1.7 nm length), SH-PEG-COOH (0.3 kDA, 2.8 nm length), and SH-PEG-COOH (1.0 kDa, 7 nm length) with or without EDC/NHS activation. Scale bars, 50 pm.
  • FIG. 21 C is a bar graph of EV counting showed SH-PEG-COOH showed the least non-specific EV binding (ns, not significant; ****P ⁇ 0.0001 compare with the mock treatment, as assessed by two-way ANOVA with Bonferroni's multiple comparisons test). Error bars are shown as mean ⁇ SD from the ten different images. EDC/NHS activation seems to provide the most significant advantage with the 1.0 kD linker and no benefit with the MUA linker. Overall, the SH-PEG-COOH linkers were better than the MUA linker.
  • FIG. 21 D is a series of representative images for different color-labeled EV (green) and marker (IgG or HER2, red). The percentages of co-localization (Marker/EV) were analyzed for evaluating the differential marker expression. The co-localized signals were indicated by white arrow heads. Scale bars, 10 pm.
  • Example 16 Multi-Channel Detection for Molecular Profiling of Tumor Cell-Derived EVs using QUAD Markers by Single EV Analysis on NPOP Substrates
  • This example explores molecular profiling of tumor-derived EVs from four different breast cancer cell lines. These cell lines represent four major subtypes of breast cancer based on their HER2, ER, and PR expression levels.
  • QUAD markers MUC1, HER2, EGFR, and EpCAM
  • NPOPs and captured EVs were prepared as follows. We washed the substrate with water and removed the water with nitrogen gas and placed the NPOPs into a humid chamber. We functionalized the NPOP substrate with 0.25 mM of SH-PEG-COOH (1.0 kDa, Nanocs) and 0.75 mM of SH-mPEG (0.35 kDa, Nanocs) in water for 4 hours at RT.
  • FIGs. 22A- 22E are images that demonstrate molecular profiling of tumor cell-derived EVs for the QUAD markers by single EV analysis on NPOP substrate.
  • 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 bars, 10 pm.
  • QUAD marker MUC1, HER2, EGFR, and EpCAM
  • FIG. 22B is a bar graph for the percentages of co-localization for the EVs and QUAD markers. Error bars are shown as mean ⁇ SD from the four different images.
  • FIGs. 22C-22D are a pair of heatmaps showing the differential abundances of QUAD markers in four different tumor cell line-derived EVs, as assessed by single EV analysis (21C) and originated cell lines, as assessed by flow cytometry (21D).
  • Example 17 Plasmon Enhancements on a Cy7 Channel Imaging EVs in the Cy7 channel was challenging due to weak fluorescence signals generated from the dye and channel. However, by using the plasmon enhancement, we could significantly amplify EV fluorescence signals when EVs are captured on the NPOP substrate compared to other plain substrates (glass or gold). This led to higher EV detection sensitivity.
  • FIG. 23A is a graph that shows EV count vs. dilution factor, and the EV count was clearly highest over a variety of dilution factors (top line) compared to a gold substrate and a glass substrate (dark line dropping rapidly from the left at a dilution factor of about 90 to a dilution faction of 1000, where the EV count was zero).
  • FIG. 23B is a histogram plot of the transformation of Cy7, and again, the large curve on the right, which represents the NPOP substrate showed a significantly higher EV count than glass or gold.
  • 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.
  • the results show that the NPOP substrates are superior to glass or gold substrates in terms of total EVs captured and are far more sensitive and can thus detect EVs at far lower concentrations that glass or gold substrates.
  • EVs were labeled by TFP-AF555 dyes and captured on the NPOP substrate. Then, captured EVs were labeled by CD63 primary antibodies followed by secondary antibodies conjugated with AF647. Isotype IgG control was used as a negative control.
  • NPOP preparation and EV capture we washed the substrate with water, removed the water with nitrogen gas and placed this into a humid chamber.
  • FIGs. 24A and 24B show similar numbers of EVs captured on the two samples, decreasing with titrating EV concentrations.
  • CD63+ EV counts are significantly higher than the IgG isotype (control), and the CD63 -positive EV counts are proportional to the EV concentrations applied on the surface.

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Abstract

L'invention concerne des systèmes, des méthodes et des dispositifs destinés à être utilisés dans la détection et/ou la surveillance de vésicules extracellulaires ("VE") cibles, par exemple, pour détecter et/ou surveiller le traitement d'une maladie telle que le cancer, chez un sujet. Les systèmes peuvent comprendre des réseaux nano-plasmoniques de nanostructures agencées pour former un réseau périodique sur un substrat, et chaque nanostructure peut comprendre un nanopilier, une couche d'espacement, par exemple, revêtue sur une couche métallique, et une pluralité de nanoparticules métalliques liées à chaque nanopilier. Les réseaux nano-plasmoniques amplifient des longueurs d'onde spécifiques de rayonnement électromagnétique et peuvent être utilisés pour capturer et imager des objets cibles.
PCT/US2023/070948 2022-07-25 2023-07-25 Méthodes et systèmes d'amélioration de signaux de rayonnement électromagnétique à partir de vésicules extracellulaires WO2024026314A1 (fr)

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