Simultaneous characterization of rnas and proteins in extracellular vesicles and lipoproteins

Abstract

The present disclosure relates to methods and systems for simultaneously detecting RNAs and proteins in situ, in Extracellular Vesicles (EVs) and / or Lipoproteins (LPs), isolated from a sample obtained from a subject via one or more capture antibodies.

Description

[0001] SIMULTANEOUS CHARACTERIZATION OF RNAS AND PROTEINS

[0002] IN EXTRACELLULAR VESICLES AND LIPOPROTEINS

[0003] CROSS REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 578,377, filed August 24, 2023, which is incorporated by reference herein in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under Grant No. GR128402 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] The sequence listing submitted on August 23, 2024, as an .XML file entitled “103361- 585W01_ST26.xml” created on August 19, 2024, and having a file size of 65,589 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0009] FIELD

[0010] The present disclosure relates to methods and systems for simultaneously detecting RNAs and proteins in situ, in Extracellular Vesicles (EVs) and / or Lipoproteins (LPs), via one or more capture antibodies.

[0011] BACKGROUND

[0012] Extracellular vesicles (EVs) are small membranous vesicles secreted by cells that are trafficked intercellularly and present in various biofluids. EVs are involved in various biological processes from immunomodulation to embryonic development. In cancer, EVs promote drug resistance, immunosuppression, the epithelial-to-mesenchymal transition, disruption of the blood-brain barrier, and organotropism. However, the biomolecular composition of EVs is highly heterogeneous, with proteins, RNAs, DNAs, lipids, and metabolites reflecting their tissue of origin. Despite the potential use of EVs in the clinic for diagnostics, current methods for isolating and characterizing EVs are technically challenging, partly due to the co-isolation of physically similar particles present in complex biofluids, such as lipoproteins (LPs). As such, isolation methods are cumbersome and irreproducible, yielding isolation-dependent vesicular profiles. On the other hand, conventional characterization methods, such as western blot (WB), enzyme-linked immunosorbent assay (ELISA), quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR), next-generation sequencing (NGS), and mass spectroscopy (MS) require vesicular lysis to obtain intraluminal contents. Emerging evidence on the complexity of single EV and particle (siEVP) co-isolates in complex biofluids and cell culture media, suggests that the lysis of EVPs convolutes the interparticle complexity of biofluids and mutes their inherent heterogeneity. Therefore, there is an unmet need to develop technologies that provide an accurate and efficient analysis of the biomolecular content in siEVPs without compromising the structural integrity of the particles.

[0013] Several analytical methods are frequently employed to quantify the physical and biomolecular characteristics of intact single EVs (siEVs), including optical and non-optical techniques. Nanoparticle tracking analysis (NTA), tunable resistive pulse sensing (TRPS), and microfluidic resistive pulse sensing (MRPS) are routinely used to measure the size and concentration of siEVs, with the minimum detectable size in the 50 - 100 nm range. However, NTA, TRPS, and MRPS integrate siEVP signals non-specifically due to limitations in phenotyping. Atomic force microscopy (AFM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM) are often utilized to provide morphological and mechanical properties of siEVs while surpassing the optical limit of diffraction. Although some distinguishing characteristics amongst the siEVPs are present, physically similar siEVPs are often undiscemible, and results are user-dependent. Incorporating immunogold labeling with TEM can provide additional phenotyping of siEVP surface proteins, but the technique is low throughput, labor intensive, and rarely quantitative. Accordingly, nanoflow cytometry (nF CM), which can detect siEVPs as small as 40 nm, based on the intensity of side-scattered photons, can identify subpopulations of siEVs via surface protein composition by incorporating fluorescently labeled antibodies. However, reduced multiplexed capability, inability to detect low-expressing biomarkers, particle swarming due to required concentrations, and extensive calibration requirements have limited their use. Optical techniques can also be applied to tunable signal-enhancing surfaces, such as plasmonic and interferometric surfaces, to examine siEV surface protein composition via immunoselective immobilization. Furthermore, antibody-DNA conjugates incorporating random-tag sequences in a proximity barcoding assay with NGS have been used to improve the simultaneous profiling of surface proteins in siEVs. Although these promising technologies have demonstrated their ability to resolve subpopulations of siEVs from different tissues, the complex intraluminal cargo of siEVs, such as nucleic acids, still requires the same rigor and optimization. On the other hand, evidence on the bioactivity of LP -transported miRNA and LP -bound proteins has inspired novel engineering approaches for their quantification. However, in situ biomarker quantification has not been performed at a single LP (siLP) resolution. The molecular heterogeneity of extracellular vesicles (EVs) and the co-isolation of physically similar particles, such as lipoproteins (LPs), confounds and limits the sensitivity of EV bulk biomarker characterization.

[0014] What is needed are new systems and methods for detecting RNAs and proteins in situ, in Extracellular Vesicles (EVs) and / or Lipoproteins (LPs).

[0015] SUMMARY

[0016] Disclosed herein are methods and systems for simultaneously detecting an RNA and a protein in situ, comprising tethering a plurality of an Extracellular Vesicle (EV) and / or a Lipoprotein (LP), isolated from a sample obtained from a subject, to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises of anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof for EV and / or LP capture.

[0017] Accordingly, in one aspect disclosed herein, is a method of simultaneously detecting an RNA and a protein in situ. In some embodiments, the method comprises obtaining a sample from a subject; isolating an EV and / or an LP from the sample; tethering a plurality of the EV and / or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti- ApoB, or a combination thereof; binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0018] Accordingly, in one aspect disclosed herein, is a method of simultaneously detecting an RNA and a protein in situ. In some embodiments, the method comprises obtaining a sample from a subject; isolating an EV and / or an LP from the sample; tethering a plurality of the EV and / or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl, or anti-ApoB; binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattem array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0019] In some embodiments, the method further comprises fluorescently imaging the micropattern array to capture an image data; and detecting an occurrence of a single EV and / or an LP expressing the first target type of the molecular cargo based on a fluorescent spot of a first color associated with the detection antibody in the image data captured; detecting an occurrence of a single EV and / or an LP expressing the second target type of the molecular cargo based on a fluorescent spot of a second color associated with the molecular beacon in the image data captured; and detecting an occurrence of the single EV and / or the LP expressing both the first target type of the molecular cargo, and the second target type of the molecular cargo based on a fluorescent spot of a third color in the image data captured.

[0020] In some embodiments, the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB. In some embodiments, the one or more capture antibodies comprises anti-CD63. In some embodiments, the one or more capture antibodies comprises anti-CD9.

[0021] In some embodiments, the glass substrate is coated with poly-L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG). In some embodiments, the PEG is covalently bound to the PLL through N-hydroxysuccinimide (NHS) chemistry. In some embodiments, the PEG is methoxy-poly(ethylene glycol)- succinimidyl valerate (mPEG-SVA).

[0022] In some embodiments, the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).

[0023] In some embodiments, the micropattern array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm.

[0024] In some embodiments, the one or more capture antibodies are biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies bind to surface proteins expressed on the EV and / or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof. In some embodiments, the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof. In some embodiments, RNA encodes AXL , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p.

[0025] In some embodiments, the detection antibody is conjugated with one or more fluorophores for fluorescent imaging.

[0026] In some embodiments, the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NO: 1-14.

[0027] In some embodiments, fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM). In some embodiments, TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near a surface of the glass substrate to visualize signals from immobilized EV and / or an LP.

[0028] In some embodiments, the sample is saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid.

[0029] In some embodiments, the subject is human.

[0030] In one aspect disclosed herein, is a system for simultaneously detecting an RNA and a protein in situ. The system comprises one or more capture antibodies attached to a micropattem array on a glass substrate, a of detection antibody and a molecular beacon and a fluorescent imaging device to capture image data. In some embodiments, the one or more capture antibodies comprises either anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof. In some embodiments, the detection antibody is configured to bind to a first target type of molecular cargo. In some embodiments, the molecular beacon is configured to bind to a second target type of molecular cargo. In some embodiments, the first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA.

[0031] In some embodiments, the one or more capture antibodies further comprises anti- CD63. In some embodiments, the one or more capture antibodies further comprises anti-CD9. In some embodiments, the one or more capture antibodies comprises anti-ApoAl . In some embodiments, the one or more capture antibodies comprises anti-ApoB. In some embodiments, the glass substrate is coated with poly-L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG). In some embodiments, the PEG is covalently bound to the PLL through N-hydroxysuccinimide (NHS) chemistry. In some embodiments, the PEG is methoxy-poly(ethylene glycol)- succinimidyl valerate (mPEG-SVA).

[0032] In some embodiments, the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).

[0033] In some embodiments, the micropattem array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm.

[0034] In some embodiments, the one or more capture antibodies are biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies binds to surface proteins expressed on the EV and / or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

[0035] In some embodiments, the system further comprises a first color associated with the detection antibody in a captured image data in a first channel. In some embodiments, the system further comprises a second color associated with the molecular beacon in the captured image data in a second channel. In some embodiments, the system comprises a third color in the captured image data in a third channel.

[0036] In some embodiments, the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof. In some embodiments, RNA encodes AXL , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p.

[0037] In some embodiments, the detection antibody is conjugated with one or more fluorophores for fluorescent imaging.

[0038] In some embodiments, the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NO: 1-14.

[0039] In some embodiments, fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM). BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0041] FIGS. 1 A-1F show detection of siEVPs with theS1EVPPRA. FIG. 1 A shows a schematic representation of the assay is condensed into three steps: (i)S1EVPPRA fabrication in which coverslips functionalized with poly-L-lysine (PLL) and subsequently methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA) are photoetched into micropattem arrays via a digital-micromirror (DMD)-based UV projection system, (ii) single extracellular vesicle and particle (siEVP) capture in which NeutraAvidin (NA) is physisorbed onto the micropattem arrays to immobilize biotinylated antibodies against epitopes on the surfaces of siEVP to sort and capture siEVPs, and (iii) detection of multiple biomolecular species on siEVPs in which fluorescently labeled antibodies and molecular beacons (MBs) are utilized to detect proteins and RNAs, respectively, via total internal reflection fluorescence microscopy (TIRFM). FIG. IB shows CD63 and hsa-miR-21-5p on Gli36-derived single EVs (siEVs) are detected and colocalized with theS1EVPPRA. The control sample (phosphate-buff ered saline; PBS) demonstrates a negligible fluorescence signal. FIG. 1C shows TIRFM images quantified as distributions of fluorescence intensity to depict the expression of CD63 and hsa-miR-21-5p at a single-particle level for the different samples. FIG. ID shows TIRFM images quantified as bar graphs of relative fluorescence intensity (RFI) for CD63 and hsa-miR-21-5p in siEVs captured in the device and the negative controls, including PBS, IgG, and scramble (N = 4, error bars indicate the standard deviation, ****p < 0.0001). FIG. IE shows scanning electron microscopy (SEM) of Gli36-derived added to theS1EVPPRA confirms the presence of siEVs immobilized on the micropattemed surface. FIG. IF shows the size distribution and concentration of Gli36-derived siEVs measured by tunable resistive sense pulsing (TRPS). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0042] FIGS. 2A-2E show specificity and sensitivity of RNA and protein detection in siEVs. FIG. 2A shows that siEVs enriched with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p are detected by the corresponding MBs targeting cel-miR-39-3p, cel-miR-54-3p, and cel-miR- 238-3p, whereas control samples demonstrate negligible fluorescence signal. FIG. 2B shows bar graphs of the RFIs of siEVs with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p with their corresponding MBs are higher than the different control conditions tested, including siEVs detected with unmatched MBs (a / b / c2, a / b / c3), siEVPs from human serum (a / b / c4), and PBS (a / b / c5). The denotations are provided in FIG. 10 (N = 3, error bars indicate the standard deviation, ****p < 0.0001). FIG. 2C shows that RFI for the detection of cel-miR-39-3p in the engineered siEVs increases with increasing concentrations of the cel-miR-39-3p plasmid transfected into the cells (N = 3, error bars indicate the standard deviation). FIG. 2D shows that theS1EVPPRA is compared against standard qRT-PCR for cel-miR-39-3p from the engineered EVs secreted from transfected Gli36 cells with a plasmid concentration of 400 ng / pL (N = 3, error bars indicate the standard deviation). The dotted line illustrates the linear fitting of the linear range (R2= 0.92; ANOVA, p = 0.0026). Representative images and distributions of fluorescence intensity are provided in FIG. 11. FIG. 2E shows theS1EVPPRA compared against a standard ELISA for detecting EGFR from EVs isolated from Gli36 cells (N = 3, error bars indicate the standard deviation). The dotted line illustrates the linear fitting of the linear range (R2= 0.98; ANOVA, p = 0.0001). Representative images and distributions of fluorescence intensity are provided in FIG. 12. All micropatterns were functionalized with an anti- CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0043] FIGS. 3A-3G show multiplexed detection of siEVs across various biomolecules. FIG. 3 A shows the colocalization of tetraspanins on Gli36-derived siEVs via multi-protein detection with theS1EVPPRA show siEVs expressing CD63, CD9, CD81, CD63 and CD9, CD9 and CD81, CD81 and CD63, and all three tetraspanins. FIG. 3B shows bar graphs of the quantification of the colocalization rates of the tetraspanins are depicted with respect to their additive colors (N = 3, n = 25, error bars indicate the standard deviation). FIG. 3C shows the colocalization of multiple RNA species within Gli36-derived siEVs via multi -RNA detection with theS1EVPPRA show siEVs expressing AAZ-1, hsa-miR-9-5p, hsa-miR-21-5p, AXL-1 and hsa-miR-9-5p, hsa- miR-9-5p and hsa-miR-21-5p, hsa-miR-21-5p and AXL-1, and all three RNA species. FIG. 3D shows bar graphs of the quantification of the colocalization rates of the multiple RNA species are depicted with respect to their additive colors (N = 3, n = 25, error bars indicate the standard deviation). FIG. 3E shows the colocalization of multiple biomolecular species within Gli36- derived siEVs via protein-RNA co-detection with theS1EVPPRA show siEVs expressing CD63, hsa-miR-9-5p, hsa-miR-21-5p, CD63 and hsa-miR-9-5p, hsa-miR-9-5p and hsa-miR-21-5p, hsa-miR-21-5p and CD63, and all three biomolecular species. FIG. 3F shows bar plots of the quantification of the colocalization rates of the multiple biomolecular species depicted with respect to their additive colors (N = 3, n = 25, error bars indicate the standard deviation). FIG. 3G shows insets of the colocalization of protein, mRNA, and miRNA within Gli36-derived siEVs with theS1EVPPRA show siEVs expressing CD63, AXL-2, hsa-miR-21-5p, CD63 and AXL-2, AXL-2 and hsa-miR-21-5p, hsa-miR-21-5p and CD63, and all three biomarkers. The representative images from which the insets were derived, and the quantifications thereof are presented in FIG. 17. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0044] FIGS. 4A-4G show single intervesicular and interparticle heterogeneity analysis. FIG. 4A shows western blot (WB) analyses on CD63, CD9, CD81, EGFR, annexin Al, and ARF6 on cell and EV lysates. FIG. 4B shows radar plots of the multidimensional data analysis of RFIs across multiple micropatterns functionalized solely with antibodies targeting CD9, CD63, annexin Al, ARF6, and EGFR with IgG as an isotype control, demonstrate variable detection levels for CD63, CD9, CD81, EGFR, hsa-miR-9-5p, hsa-miR-21-5p, GAPDH, AXL-2, p53. FIG. 4C shows a heatmap of the multidimensional data analysis after linear discriminant (FIG. 17) and literature-based analysis is condensed to demonstrate expression across clustered subpopulations, including “classical” exosomes, ectosomes, tumor-derived, and an isotype control. FIG. 4D shows single lipoproteins (siLPs) and siEVs are sorted on micropatterns functionalized with an anti-ApoB / ApoAl antibody cocktail and detected for ApoAl, ApoB, and CD63. FIG. 4E shows TIRFM images are quantified as bar graphs of the RFIs of the siEVP for the ApoB+ / ApoAl+subpopulation (N = 3, error bars indicate the standard deviation, ****p < 0.0001). FIG. 4F shows siLPs and siEVs are sorted on micropatterns functionalized with an anti-CD63 / CD9 antibody cocktail and detected for ApoAl, ApoB, and CD63. FIG 4G shows TIRFM images are quantified as bar graphs of the RFIs of the siEVPs for the CD63+ / CD9+subpopulation (N = 3, error bars indicate the standard deviation, ****p < 0.0001, ***p < 0.001, *p < 0.05). All scale bars are 10 pm unless stated otherwise.

[0045] FIGS. 5A-5C show sequencing of cellular and vesicular RNA in glioma cell lines and validation with theS1EVPPRA. FIG. 5A shows cellular and vesicular mRNA (left) and miRNA (right) are sequenced across six glioma cell lines, including SF268, SF295, SF539, SNB19, SNB75, and U251, revealing the upregulation of NSF, hsa-miR-9-5p, NCAN, hsa-miR-1246- 5p in cells and EVs. FIG. 5B shows that NSF, hsa-miR-9-5p, NCAN, and hsa-miR-1246-5p are profiled in EVs (solid line) and cells (dash line) from the six different glioma cell lines by bulk RNA characterization. FIG.5C shows bar graphs of the RFIs of NSF, hsa-miR-9-5p, NCAN, and hsa-miR-1246-5p in siEVs from the six different glioma cell lines with theS1EVPPRA, showing comparable RNA expression trends (N = 3, error bars indicate the standard deviation). Representative images and distributions of fluorescence intensity are provided in FIGS. 19-22. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail. FIGS. 6A-6C show differential expression of GBM-associated vesicular RNA species in siEVPs from GBM patient serum. FIG. 6A shows representative TIRFM images of siEVPs expressing NSF, hsa-miR-9-5p, NCAN, and hsa-miR-1246-5p in TFF-purified serum from GBM patients and healthy donors characterized with theS1EVPPRA. FIG. 6B shows box plots of the RFIs for MS' / ’', hsa-miR-9-5p, NCAN, and hsa-miR-1246-5p in GBM patients and healthy donors (N = 10, *** > < 0.001, **p < 0.01). FIG. 6C shows representative distributions of fluorescence intensity for the siEVP detection of NSF, hsa-miR-9-5p, NCAN, and hsa-miR- 1246-5p in TFF-purified serum from patient and healthy donors, indicate variable expression and homogenous profiles amongst mRNAs and heterogeneous profiles amongst miRNAs. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0046] FIG. 7 shows homogenous physisorption of NA. UV photoetching of the mPEG monolayer under the optimized conditions (a dose of 20 mJ / mm2and grayscale value of 50%) allows for the homogenous adsorption of NA in distinct micropattems.

[0047] FIGS. 8A-8B show EV recovery rates after partial permeabilization. FIG. 8 A shows the concentration of EVs before and after incubation with the TE buffer and PBS at 4 °C and 37 °C as measured by TRPS remains constant (N= 3, error bars indicate the standard deviation). FIG. 8B shows cryo-TEM images of small and large EVs under treatment without (left) and with (right) the TE buffer reveals intact vesicular membrane structures. The inset demonstrates the intact lipid bilayer post-treatment. The scale bar is 100 nm.

[0048] FIGS. 9A-9C show specificity of RNA detection via MBs and effect of partial permeabilization. FIG. 9A shows hsa-miR-21-5p and cel-miR-39-3p are detected with theS1EVPPRA in Gli36-derived siEVs with and without partial permeabilization of the lipid membrane with the TE buffer, along with negative controls. FIG. 9B shows TIRFM images are quantified as bar graphs of the RFIs and indicate the necessity for partial permeabilization (N= 2, error bars indicate the standard deviation, ***p < 0.001). FIG. 9C shows TIRFM images are quantified as bar graphs of the RFIs for internal and external epitope detection with fluorescently labeled antibodies with and without partial permeabilization (N = 3, error bars indicate the standard deviation, ***p < 0.001). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0049] FIGS. 10A-10B show cross-reactivity of engineered siEVs. FIG. 10A shows a schematic of transfection via electroporation, where Gli36 cells are transfected with cel-miR-39-3p, cel- miR-54-3p, and cel-miR-238-3p plasmids. FIG. 10B shows representative TIRFM images for the controls in FIG. 2B with the respective denotations are shown for unmatched MBs (a / b / c2, a / b / c3), siEVPs from human serum (a / b / c4), and PBS (a / b / c5). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0050] FIGS. 11 A-l IB show the sensitivity of theS1EVPPRA for RNA detection. FIG. 11 A shows representative TIRFM images of FIG. 2D in which a serial dilution of engineered EVs enriched with cel-miR-39-3p is detected with theS1EVPPRA. FIG. 11B shows TIRFM images are quantified as distributions of fluorescence intensity. All micropattems were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0051] FIGS. 12A-12B show the sensitivity of theS1EVPPRA for protein detection. FIG. 12A shows representative TIRFM images of FIG. 2E in which a serial dilution of Gli36-derived EVs is detected for EGFR with theS1EVPPRA. FIG. 12B shows TIRFM images are quantified as distributions of fluorescence intensity. All micropatterns were functionalized with an anti- CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0052] FIG. 13 shows the crosstalk specificity of fluorescent channels. Fluorescently labeled antibodies targeting CD63, CD81, and CD9 are illuminated by different wavelengths (488, 561, and 640 nm) and are excited only by their corresponding channel (N= 2, error bars indicate the standard deviation, ****p < 0.0001). All micropatterns were functionalized with an anti- CD63 / CD9 antibody cocktail.

[0053] FIGS. 14A-14C show multi-protein detection via single-particle interferometric reflectance imaging sensing (SP-IRIS). FIG. 14A shows representative SP-IRIS images for a micropattern functionalized with CD9 and an enlarged inset of the micropattern show siEVs expressing CD63, CD9, CD81, CD63 and CD9, CD9 and CD81, CD81 and CD63, and all three groups. FIG. 14B shows bar graphs for the number of siEVs captured on each micropattern are presented against an isotype control (N= 3, error bars indicate the standard deviation; Dunnett’s test, ****p < 0.0001, ***p < 0.001, **p < 0.01). FIG. 14C shows bar graphs of the quantification of the colocalization rates of the tetraspanins are depicted with respect to their additive colors (N = 3, error bars indicate the standard deviation). All scale bars are 10 pm unless stated otherwise.

[0054] FIGS. 15 A-l 5D show multiple target detection on the AXL mRNA strand. FIG. 15 A shows how three regions of the AXL mRNA strand are detected and multiplexed with theS1EVPPRA on Gli36-derived siEVs with the first region (AA7.- I ), the second region (AA7.-2), and the third region (AAZ-3), the colocalization of.4A7.- l andAXL-2, AXL-2 and^AZ-3, AXL-3 and AAZ-1, and all three regions. FIG. 15B shows that TIRFM images are quantified as distributions of fluorescence intensity for the single region targets on Gli36-derived EVs and the respective controls. FIG. 15C shows that TIRFM images are quantified as bar graphs of the RFIs for the single region targets on Gli36-derived EVs and the respective controls (N = 2, error bars indicate the standard deviation). Fig. 15D shows bar graphs of the quantification of the colocalization rates of the multiple AXL regions on the RNA strand are depicted with respect to their additive colors (N = 3, n = 25, error bars indicate the standard deviation). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0055] FIGS. 16A-16B show multiplexed miRNA detection in engineered siEVs. FIG. 16A shows the colocalization of multiple miRNAs in the engineered siEVs with theS1EVPPRA show siEVs expressing cel-miR-39-3p, cel-miR-54-3p, cel-miR-238-3p, cel-miR-39-3p and cel-miR-54-3p, cel-miR-54-3p and cel-miR-238-3p, cel-miR-238-3p and cel-miR-39-3p, and all three miRNAs. FIG. 16B shows bar graphs of the quantification of the colocalization rates of the miRNA are depicted with respect to their additive colors (N= 3, n = 25, error bars indicate the standard deviation). All micropattems were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0056] FIGS. 17A-17B show co-detection of protein, mRNA, and miRNA in siEVs. FIG. 17A shows the colocalization of protein, mRNA, and miRNA in Gli36-derived siEVs with theS1EVPPRA show siEVs expressing CD63, AXL-2, hsa-miR-21-5p, CD63 and AXL-2, AXL-2 and hsa-miR-21-5p, hsa-miR-21-5p and CD63, and all three biomarkers. FIG. 17B shows bar graphs of the quantification of the colocalization rates of the three biomolecular species depicted with respect to their additive colors (N = 3, n = 25, error bars indicate the standard deviation). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0057] FIGS. 18A-18B shows linear discriminant analysis on subpopulation heterogeneity. FIG. 18A shows heterogeneity amongst the subpopulations is demonstrated with linear discriminant analysis on the nine biomarkers. FIG. 18B shows the clustering of subpopulations is observed with linear discriminant analysis after grouping the nine biomarkers into miRNA, mRNA, and protein biomolecular species.

[0058] FIGS. 19A-19E show composition of serum-derived siEVPs post-TFF filtration. FIG. 19A shows images of unprocessed serum and the retentate and permeate after TFF illustrate the removal of soluble protein from the retentate. FIG. 19B shows a bar graph of the soluble protein content and a scatter plot for the recovery rate of siEVPs on healthy donor serum illustrates a loss of soluble protein and retention of siEVPs. FIG. 19C shows WB analyses on unprocessed serum, isolated very low-density LP / low-density LP (VLDL / LDL), isolated high-density LP (HDL), and the TFF retentate for EV biomarkers (annexin Al, ARF6, CD63, and CD9) and LP biomarkers (ApoB and ApoAl), indicate a co-isolation of the particles after TFF. FIG. 19D shows WB analyses in cell and EV lysates demonstrate a lack of signal for LP biomarkers. FIG. 19E shows TEM images of Gli36-derived EVs and serum-isolated LPs show different morphologies.

[0059] FIGS. 20A-20C show the detection of apolipoprotein corona on siEVs. FIG. 20A shows a schematic of the preparation of siEVs with apolipoprotein corona, where TFF-purified EVs are incubated with EV-depleted plasma (EVDP) for 30 minutes at room temperature (RT) and subsequently purified with size-exclusion chromatography (SEC). FIG. 20B shows representative TIRFM images for siEVs with apolipoprotein corona and the respective control demonstrate the detection of ApoAl and ApoB on siEVs. FIG. 20C shows TIRFM images are quantified as bar graphs of the RFIs for Gli36-derived siEVs with apolipoprotein corona and the respective controls (N = 3, error bars indicate the standard deviation, *p < 0.05). All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0060] FIGS. 21A-21B show MS' / ’' detection with theS1EVPPRA. FIG. 21A shows representative TIRFM images for FIG. 5C demonstrating the detection of NSF in siEVs. FIG. 21B shows distributions of fluorescence intensity for the expression of NSF in siEVs across the six glioma cell lines as detected by theS1EVPPRA show homogeneous profiles. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0061] FIGS. 22A-22B show NCAN detection with theS1EVPPRA. FIG. 22 A shows representative TIRFM images for FIG. 5C demonstrating the detection of NCAN in siEVs. FIG. 22B shows distributions of fluorescence intensity for the expression of NCAN in siEVs across the six glioma cell lines as detected by theS1EVPPRA show homogeneous profiles. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0062] FIGS. 23A-23B show hsa-miR-9-5p detection with theS1EVPPRA. FIG. 23A shows representative TIRFM images for FIG. 5C demonstrating the detection of hsa-miR-9-5p in siEVs. FIG. 23B shows distributions of fluorescence intensity for the expression of hsa-miR- 9-5p in siEVs across the six glioma cell lines as detected by theS1EVPPRA show heterogeneous profiles. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0063] FIGS. 24A-24B show hsa-miR-1246-5p detection with theS1EVPPRA. FIG. 24 A shows representative TIRFM images for FIG. 5C demonstrating the detection of hsa-miR-1246-5p in siEVs. FIG. 24B shows distributions of fluorescence intensity for the expression of hsa-miR- 1246-5p in siEVs across the six glioma cell lines as detected by theS1EVPPRA show heterogeneous profiles. All micropatterns were functionalized with an anti-CD63 / CD9 antibody cocktail, and all scale bars are 10 pm unless stated otherwise.

[0064] DETAILED DESCRIPTION

[0065] Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising”, and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

[0067] The following definitions are provided for the full understanding of terms used in this specification.

[0068] Terminology

[0069] The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

[0070] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a pharmaceutical carrier” includes mixtures of two or more such carriers, and the like. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10”as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0071] The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2-Aminoadipic acid, N-Ethylasparagine, 3-Aminoadipic acid, Hydroxylysine, P-alanine, P-Amino-propionic acid, allo-Hydroxylysine acid, 2-Aminobutyric acid, 3-Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6- Aminocaproic acid, Isodesmosine, 2-Aminoheptanoic acid, allo-Isoleucine, 2- Aminoisobutyric acid, N-Methylglycine, sarcosine, 3 -Aminoisobutyric acid, N- Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4-Diaminobutyric acid, N- Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3- Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.

[0072] The term "antibody" is used in the broadest sense, and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies). Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins having the same structural characteristics. While antibodies exhibit binding specificity to a specific target, immunoglobulins include both antibodies and other antibody-like molecules which lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end.

[0073] The term "antibody fragment" refers to a portion of a full-length antibody, generally the target binding or variable region. Examples of antibody fragments include Fab, Fab1, F(ab')2 and Fv fragments. The phrase "functional fragment or analog" of an antibody is a compound having qualitative biological activity in common with a full-length antibody. For example, a functional fragment or analog of an anti-IgE antibody is one which can bind to an IgE immunoglobulin in such a manner so as to prevent or substantially reduce the ability of such molecule from having the ability to bind to the high affinity receptor, FcsRI. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')2 fragments. An "Fv" fragment is the minimum antibody fragment which contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (VH-VL dimer). It is in this configuration that the three CDRs of each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer target binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for a target) has the ability to recognize and bind target, although at a lower affinity than the entire binding site. "Single-chain Fv" or "sFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for target binding. "Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.

[0074] As used herein, “codon” refers to the genetic code used by living cells to translate information encoded by genetic material (DNA or mRNA sequences of nucleotide triplets) into protein. This term also refers to the genetic code that specifies which amino acids will be added next during protein synthesis.

[0075] A “control” is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

[0076] The term “detect” or “detecting” refers to an output signal released for the purpose of sensing of physical phenomenon. An event or change in environment is sensed and signal output released in the form of light.

[0077] As used herein, “enhance”, “enhanced”, “enhancement”, “enhancing”, and any grammatical variations thereof as used herein, refers to an act of intensifying, increasing, or further improving the quality, value, or extent of a biological function, composition, compound, cell, or tissue.

[0078] "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom, Thus, a gene encodes a protein if transcription and translation of mRNA occurs.

[0079] An “epitope” or “antigenic determinant” refers to the part of an antigen, a molecular structure, or foreign particulate that can bind to a specific antibody or T-cell receptor. The presence of antigens or epitopes of antigens within a host can illicit an immune response. A “fluorophore” is a fluorescent chemical compound that can re-emit light upon light excitation. The chemicals are sometimes used alone as a tracer in fluids, as a due for staining certain structures, as an enzyme substrate, or as a probe / indicator. More commonly they are covalently bonded to a macromolecule to serve as a marker for bioactive reagents (i.e.: antibodies, peptides, nucleic acids, etc.) Fluorophores are notably used to stain tissues, cells, or materials in a variety of analytical methods such as fluorescent imaging and spectroscopy.

[0080] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0081] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0082] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0083] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0084] The term “increased” or “increase” as used herein generally means an increase by a statistically significant amount, for example “increased” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10- fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level so long as the increase is statistically significant.

[0085] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

[0086] As used herein, the terms “may,” “optionally,” and “may optionally” are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation “may include an excipient” is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.

[0087] A “protein”, "polypeptide", or “peptide” each refer to a polymer of amino acids and does not imply a specific length of a polymer of amino acids. Thus, for example, the terms peptide, oligopeptide, protein, antibody, and enzyme are included within the definition of polypeptide. This term also includes polypeptides with post-expression modification, such as glycosylation (e.g., the addition of a saccharide), acetylation, phosphorylation, and the like.

[0088] The term “nucleic acid” as used herein means a polymer composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides. The term "nucleobase" refers to the part of a nucleotide that bears the Watson / Crick base-pairing functionality. The most common naturally-occurring nucleobases, adenine (A), guanine (G), uracil (U), cytosine (C), and thymine (T) bear the hydrogen-bonding functionality that binds one nucleic acid strand to another in a sequence specific manner.

[0089] The terms “ribonucleic acid” and “RNA” as used herein mean a polymer composed of ribonucleotides.

[0090] The terms “deoxyribonucleic acid” and “DNA” as used herein mean a polymer composed of deoxyribonucleotides.

[0091] As used throughout, by a "subject" (or a “host”) is meant an individual. Thus, the "subject" can include, for example, domesticated animals, such as cats, dogs, etc., livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.) mammals, non-human mammals, primates, non-human primates, rodents, birds, reptiles, amphibians, fish, and any other animal. The subject can be a mammal such as a primate or a human. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject.

[0092] Disclosed herein are the components to be used to prepare the disclosed compositions as to be used in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. If a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C- F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods. Methods of simultaneously detecting an RNA and a protein in situ

[0093] Extracellular vesicles (EVs) are particles that are released by cells into the extracellular space. They are lipid bilayer-delimited clusters of different sizes, cargo, and surface markers. EVs are carriers that can transport a variety of cargo, including proteins, lipids, nucleic acids, and metabolites.

[0094] Lipoproteins are spherical particles made of fat and protein that transport lipids (such as, for example, cholesterol) throughout a human body's bloodstream to the cells. They play a vital role in transporting lipids between organs, forming structural components in nervous tissue, and helping transmit electrical impulses.

[0095] The two main groups of lipoproteins are called HDL (high-density lipoprotein) or "good" cholesterol and LDL (low-density lipoprotein) or "bad" cholesterol.

[0096] Extracellular vesicles and lipoproteins can interact to form functional complexes such as single EV and particle (siEVP) co-isolates. Such complexes have been observed in biofluids (such as, for example, saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid) from healthy human donors and in various in vitro disease models (such as, for example, breast cancer and hepatitis C infection).

[0097] Apolipoprotein Al (ApoAl) is a protein that is a major component of high-density lipoprotein (HDL) and plays a role in lipid metabolism and transport. ApoAl helps move cholesterol and phospholipids from inside cells to the cell's outer surface, converts cholesterol into a form that can be integrated into HDL and helps transform free cholesterol into cholesterol ester, which can then be transported to the liver for degradation.

[0098] Apolipoprotein B (ApoB) is a protein that carries lipids in the bloodstream, including cholesterol and fats. It's a building block of low-density lipoproteins (LDLs), intermediatedensity lipoproteins (IDLs), and very low-density lipoproteins (VLDLs), which are also known as "bad" cholesterol. ApoB is encoded by the APOB gene and is produced in the liver and small intestine. There are two forms of ApoB that circulate in the body: ApoB48, which comes from the small intestine, and ApoB 100, which comes from the liver. ApoB48 serves in the absorption of dietary fats from the intestine. ApoB 100 is necessary for the assembly of VLDL in the liver and is the primary ligand for LDL receptor-mediated clearance of LDL particles from the blood.

[0099] In some aspects, disclosed herein is a method of simultaneously detecting an RNA and a protein in situ, comprising: obtaining a sample from a subject; isolating an Extracellular Vesicle (EV) and / or a Lipoprotein (LP) from the sample; tethering a plurality of the EV and / or the LP to a micropattem array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl anti-ApoB, or a combination thereof; and binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0100] In some aspects, disclosed herein is a method of simultaneously detecting an RNA and a protein in situ. In some embodiments, the method comprises obtaining a sample from a subject; isolating an EV and / or an LP from the sample; tethering a plurality of the EV and / or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof; binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattem array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0101] In some embodiments, the second target type of the molecular cargo is a DNA fragment.

[0102] In some embodiments, the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB. In some embodiments, the one or more capture antibodies comprises anti-CD63. In some embodiments, the one or more capture antibodies comprises anti-CD9.

[0103] In some aspects, disclosed herein is a method of simultaneously detecting an RNA and a protein in situ, comprising: obtaining a sample from a subject; isolating an Extracellular Vesicle (EV) and / or a Lipoprotein (LP) from the sample; tethering a plurality of the EV and / or the LP to a micropattem array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl or anti-ApoB; and binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0104] In some aspects, disclosed herein is a method of simultaneously detecting an RNA and a protein in situ. In some embodiments, the method comprises obtaining a sample from a subject; isolating an EV and / or an LP from the sample; tethering a plurality of the EV and / or the LP to a micropattern array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-ApoAl or anti-ApoB; binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

[0105] In some embodiments, the second target type of the molecular cargo is a DNA fragment.

[0106] In some embodiments, the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB.

[0107] In some embodiments, the glass substrate was photoetched using the PRIMO optical module (for example, Alveole) mounted on an automated inverted microscope (for example, Nikon Eclipse Ti Inverted Microscope System).

[0108] In one aspect disclosed herein, the method disclosed in any of the preceding aspects further comprises: fluorescently imaging the micropattern array to capture an image data; detecting an occurrence of a single EV and / or an LP expressing the first target type of the molecular cargo based on a fluorescent spot of a first color associated with the detection antibody in the image data captured; detecting an occurrence of the single EV and / or the LP expressing the second target type of the molecular cargo based on a fluorescent spot of a second color associated with the molecular beacon in the image data captured; and detecting an occurrence of the single EV and / or the LP expressing both the first target type of the molecular cargo, and the second target type of the molecular cargo based on a fluorescent spot of a third color in the image data captured.

[0109] In some embodiments, the method further comprises fluorescently imaging the micropattern array to capture an image data; and detecting an occurrence of a single EV and / or an LP expressing the first target type of the molecular cargo based on a fluorescent spot of a first color associated with the detection antibody in the image data captured; detecting an occurrence of a single EV and / or an LP expressing the second target type of the molecular cargo based on a fluorescent spot of a second color associated with the molecular beacon in the image data captured; and detecting an occurrence of the single EV and / or an LP expressing both the first target type of the molecular cargo, and the second target type of the molecular cargo based on a fluorescent spot of a third color in the image data captured.

[0110] In some embodiments, the glass substrate is coated with poly-L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG). Some other coatings that can be used on glass surfaces include but are not limited to epoxy silane, 3- D Hydrogel, aminosilane, streptavidin, poly-L-Lysine, aldehydesilane, or 3-D Polymer.

[0111] In some embodiments, the PEG is covalently bound to the PLL through N- hydroxysuccinimide (NHS) chemistry. Polyethylene glycol (PEG) is a synthetic polymer that can be categorized into different types based on molecular weight, synthesis geometry, specific functional groups, and applications. Some types of PEG include: Branched PEG, Star PEG, Comb PEG, Alkyne-PEG, PEG 200, PEG 300, PEG 400, PEG 600, PEG 4000, PEG 6000, and PEG 8000.

[0112] In some embodiments, the PEG is methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA). Physical adsorption or physisorption is the process wherein adsorbate molecules are held to the surface of an adsorbent by weak Van der Waals forces.

[0113] In some embodiments, the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).

[0114] In some embodiments, the micropattern array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm. In some embodiments, each individual circle has a diameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0115] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,

[0116] 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

[0117] 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

[0118] 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

[0119] 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,

[0120] 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 m. In some embodiments, each individual circle has the diameter of about 20 pm. In some embodiments, each individual circle has a center-to-center spacing of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

[0121] 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

[0122] 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87,

[0123] 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,

[0124] 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,

[0125] 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,

[0126] 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,

[0127] 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,

[0128] 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 pm in relation to an adjacent circle. In some embodiments, each individual circle has a center-to-center spacing of about 80 pm in relation to an adjacent circle.

[0129] In some embodiments, the one or more capture antibodies are biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies bind to surface proteins expressed on the EV and / or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

[0130] In some embodiments, the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof. In some embodiments, RNA encodes AXL , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p.

[0131] In some embodiments, DNA fragments such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and mitochondrial DNA (mtDNA) In some embodiments, the detection antibody is conjugated with one or more fluorophores, (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.

[0132] In some embodiments, the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NO: 1-14. In some embodiments, the molecular beacon comprises SEQ ID NO: 1. In some embodiments, the molecular beacon comprises SEQ ID NO: 2. In some embodiments, the molecular beacon comprises SEQ ID NO: 3. In some embodiments, the molecular beacon comprises SEQ ID NO: 4. In some embodiments, the molecular beacon comprises SEQ ID NO: 5. In some embodiments, the molecular beacon comprises SEQ ID NO: 6. In some embodiments, the molecular beacon comprises SEQ ID NO: 7. In some embodiments, the molecular beacon comprises SEQ ID NO: 8. In some embodiments, the molecular beacon comprises SEQ ID NO: 9. In some embodiments, the molecular beacon comprises SEQ ID NO: 10. In some embodiments, the molecular beacon comprises SEQ ID NO: 11. In some embodiments, the molecular beacon comprises SEQ ID NO: 12. In some embodiments, the molecular beacon comprises SEQ ID NO: 13. In some embodiments, the molecular beacon comprises SEQ ID NO: 14.

[0133] In some embodiments, fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM). In some embodiments, TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near a surface of the glass substrate to visualize signals from immobilized EV and / or an LP.

[0134] In some embodiments, detection of the EV and / or LP comprises flowcytometry.

[0135] TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section (usually less than 200 nanometers) that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid glass at its interface with a liquid medium, an electromagnetic field (also known as an evanescent wave) is generated at the solid-liquid interface with the same frequency as the excitation light. The intensity of the evanescent wave exponentially decays with distance from the surface of the solid so that only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. Two-dimensional images of the fluorescence can then be obtained, although there are also mechanisms in which three- dimensional information on the location of vesicles or structures in cells can be obtained.

[0136] In some embodiments, the sample is a biofluid. In some embodiments the biofluid is saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid.

[0137] In some embodiments, the subject is a human.

[0138] Systems for simultaneously detecting an RNA and a protein in situ

[0139] In one aspect disclosed herein, is a system for simultaneously detecting an RNA and a protein in situ, comprising: one or more of capture antibodies attached to a micropattem array on a glass substrate, wherein the one or more of capture antibodies comprises anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof; a detection antibody and a molecular beacon, wherein the detection antibody is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.

[0140] In some embodiments, disclosed herein is a method for simultaneously detecting an RNA and a protein in situ. The system comprises a one or more of capture antibodies attached to a micropattern array on a glass substrate, a detection antibody and a molecular beacon and a fluorescent imaging device to capture image data. In some embodiments, the one or more of capture antibodies comprises either anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB or a combination thereof. In some embodiments, the detection antibody is configured to bind to a first target type of molecular cargo. In some embodiments, the molecular beacon is configured to bind to a second target type of molecular cargo. In some embodiments, the first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA.

[0141] In some embodiments, the second type of molecular cargo is a DNA fragment.

[0142] In one aspect disclosed herein, is a system for simultaneously detecting an RNA and a protein in situ, comprising: a plurality of capture antibodies attached to a micropattern array on a glass substrate, wherein the plurality of capture antibodies comprises anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof; a plurality of detection antibodies and a plurality of molecular beacons, wherein the plurality of detection antibodies is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.

[0143] In some embodiments, disclosed herein is a method for simultaneously detecting an RNA and a protein in situ. The system comprises a plurality of capture antibodies attached to a micropattern array on a glass substrate, a plurality of detection antibodies and a plurality of molecular beacons and a fluorescent imaging device to capture image data. In some embodiments, the plurality of capture antibodies comprises either anti-CD63, anti-CD9, anti- ApoAl, anti-ApoB or a combination thereof. In some embodiments, the plurality of detection antibodies is configured to bind to a first target type of molecular cargo. In some embodiments, the plurality of molecular beacons is configured to bind to a second target type of molecular cargo. In some embodiments, the first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA.

[0144] In some embodiments, the second type of molecular cargo is a DNA fragment.

[0145] In some embodiments, the one or more capture antibodies comprises anti-CD63. In some embodiments, the one or more capture antibodies comprises anti-CD9. In some embodiments, the one or more capture antibodies comprises anti-ApoAl. In some embodiments, the one or more capture antibodies comprises anti-ApoB.

[0146] In one aspect disclosed herein, is a system for simultaneously detecting an RNA and a protein in situ, comprising: one or more of capture antibodies attached to a micropattem array on a glass substrate, wherein the one or more of capture antibodies comprises anti-ApoAl or anti-ApoB; a detection antibody and a molecular beacon, wherein the detection antibody is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.

[0147] In some embodiments, disclosed herein is a method for simultaneously detecting an RNA and a protein in situ. The system comprises a one or more of capture antibodies attached to a micropattern array on a glass substrate, a detection antibody and a molecular beacon and a fluorescent imaging device to capture image data. In some embodiments, the one or more of capture antibodies comprises either anti-ApoAl or anti-ApoB. In some embodiments, the detection antibody is configured to bind to a first target type of molecular cargo. In some embodiments, the molecular beacon is configured to bind to a second target type of molecular cargo. In some embodiments, the first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA.

[0148] In some embodiments, the second type of molecular cargo is a DNA fragment.

[0149] In some embodiments, the one or more capture antibodies comprises anti-ApoAl . In some embodiments, the one or more capture antibodies comprises anti-ApoB.

[0150] In one aspect disclosed herein, is a system for simultaneously detecting an RNA and a protein in situ, comprising: a plurality of capture antibodies attached to a micropattern array on a glass substrate, wherein the plurality of capture antibodies comprises anti-ApoAl or anti-ApoB; a plurality of detection antibodies and a plurality of molecular beacons, wherein the plurality of detection antibodies is configured to bind to a first target type of molecular cargo, and wherein the molecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.

[0151] In some embodiments, disclosed herein is a method for simultaneously detecting an RNA and a protein in situ. The system comprises a plurality of capture antibodies attached to a micropattern array on a glass substrate, a plurality of detection antibodies and a plurality of molecular beacons and a fluorescent imaging device to capture image data. In some embodiments, the plurality of capture antibodies comprises anti-ApoAl or anti-ApoB. In some embodiments, the plurality of detection antibodies is configured to bind to a first target type of molecular cargo. In some embodiments, the plurality of molecular beacons is configured to bind to a second target type of molecular cargo. In some embodiments, the first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA.

[0152] In some embodiments, the second type of molecular cargo is a DNA fragment.

[0153] In some embodiments, the one or more capture antibodies comprises anti-ApoAl . In some embodiments, the one or more capture antibodies comprises anti-ApoB. In some embodiments, the glass substrate is coated with poly-L-lysine (PLL) through physisorption prior to coating the glass substrate with a polyethylene glycol (PEG). Some other coatings that can be used on glass surfaces include but are not limited to epoxy silane, 3-D Hydrogel, aminosilane, streptavidin, poly-L-Lysine, aldehydesilane, or 3-D Polymer.

[0154] In some embodiments, the PEG is covalently bound to the PLL through N- hydroxysuccinimide (NHS) chemistry. Polyethylene glycol (PEG) is a synthetic polymer that can be categorized into different types based on molecular weight, synthesis geometry, specific functional groups, and applications. Some types of PEG molecules include branched PEG, star PEG, comb PEG, alkyne-PEG, PEG 200, PEG 300, PEG 400, PEG 600, PEG 4000, PEG 6000 and PEG 8000.

[0155] In some embodiments, the PEG is methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA). Physical adsorption or physisorption is the process wherein adsorbate molecules are held to the surface of an adsorbent by weak Van der Waals forces.

[0156] In some embodiments, the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4-benzoylbenzyl- trimethylammonium chloride (PLPP).

[0157] In some embodiments, the micropattem array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm. In some embodiments, each individual circle has a diameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

[0158] 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

[0159] 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,

[0160] 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,

[0161] 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,

[0162] 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127,

[0163] 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146,

[0164] 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165,

[0165] 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,

[0166] 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 pm. In some embodiments, each individual circle has the diameter of about 20 pm. In some embodiments, each individual circle has a center-to-center spacing of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0167] 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,

[0168] 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

[0169] 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,

[0170] 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,

[0171] 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166,

[0172] 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,

[0173] 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 m in relation to an adjacent circle. In some embodiments, each individual circle has a center-to-center spacing of about 80 pm in relation to an adjacent circle.

[0174] In some embodiments, the one or more capture antibodies is biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the one or more capture antibodies binds to surface proteins expressed on the EV and / or the LP. In some embodiments, the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

[0175] In some embodiments, the plurality of capture antibodies is biotinylated and attached to the micropattern array by binding to a physically adsorbed Neutravidin layer on the micropattern array. In some embodiments, the plurality of capture antibodies binds to surface proteins expressed on the EV and / or the LP. In some embodiments, the plurality of capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

[0176] In some embodiments, the system further comprises a first color associated with the detection antibody in a captured image data in a first channel. In some embodiments, the system further comprises a second color associated with the molecular beacon in the captured image data in a second channel. In some embodiments, the system comprises a third color in the captured image data in a third channel.

[0177] In some embodiments, the system further comprises a first color associated with the plurality of detection antibodies in a captured image data in a first channel. In some embodiments, the system further comprises a second color associated with the plurality of molecular beacons in the captured image data in a second channel. In some embodiments, the system comprises a third color in the captured image data in a third channel.

[0178] In some embodiments, the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof. In some embodiments, RNA encodes AXL- , AXL-2, AXL-3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel-miR- 39-3p, cel-miR-54-3p or cel-miR-238-3p. In some embodiments, DNA fragments such as single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and mitochondrial DNA (mtDNA)

[0179] In some embodiments, the detection antibody is conjugated with one or more fluorophores (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.

[0180] In some embodiments, the plurality of detection antibodies is conjugated with one or more fluorophores (such as, for example, Alexa Fluor 488, Alexa Fluor 546, Alexa Flour 647, Alexa Fluor 55, FITC or CoraLite 594) for fluorescent imaging.

[0181] In some embodiments, the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the molecular beacon is selected from any one of SEQ ID NOs: 1-14. In some embodiments, the molecular beacon comprises SEQ ID NO: 1. In some embodiments, the molecular beacon comprises SEQ ID NO: 2. In some embodiments, the molecular beacon comprises SEQ ID NO: 3. In some embodiments, the molecular beacon comprises SEQ ID NO: 4. In some embodiments, the molecular beacon comprises SEQ ID NO: 5. In some embodiments, the molecular beacon comprises SEQ ID NO: 6. In some embodiments, the molecular beacon comprises SEQ ID NO: 7. In some embodiments, the molecular beacon comprises SEQ ID NO: 8. In some embodiments, the molecular beacon comprises SEQ ID NO: 9. In some embodiments, the molecular beacon comprises SEQ ID NO: 10. In some embodiments, the molecular beacon comprises SEQ ID NO: 11. In some embodiments, the molecular beacon comprises SEQ ID NO: 12. In some embodiments, the molecular beacon comprises SEQ ID NO: 13. In some embodiments, the molecular beacon comprises SEQ ID NO: 14.

[0182] In some embodiments, the plurality of molecular beacons comprises one or more fluorescent dye sequences for fluorescent imaging. In some embodiments, the plurality of molecular beacons comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the plurality of molecular beacons is selected from any one of SEQ ID NOs: 1-14. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 1. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 2. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 3. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 4. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 5. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 6. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 7. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 8. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 9. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 10. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 11. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 12. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 13. In some embodiments, the plurality of molecular beacons comprises SEQ ID NO: 14.

[0183] In some embodiments, fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM). In some embodiments, detection of the EV and / or LP comprises flowcytometry.

[0184] EXAMPLES

[0185] The following examples are set forth below to illustrate the compositions, cells, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

[0186] Example 1: Engineering a Tunable Micropattern- Array Assay to Sort Single Extracellular Vesicles and Particles to Detect RNA and Protein In Situ

[0187] Herein disclosed is a single-EV and particle (siEVP) protein and RNA assay (S1EVPPRA) to simultaneously detect mRNAs, miRNAs, and proteins in subpopulations of EVs and LPs. TheS1EVPPRA immobilizes and sorts, particles via positive immunoselection onto micropatterns and focuses biomolecular signals in situ. By detecting extracellular vesicles and particles (EVPs) at a single-particle resolution, theS1EVPPRA outperformed the sensitivities of bulkanalysis benchmark assays for RNA and protein. To assess the specificity of RNA detection in complex biofluids, EVs from various glioma cell lines were processed with small RNA sequencing, whereby two mRNAs and two miRNAs associated with glioblastoma multiforme (GBM) were chosen for cross-validation. Despite the presence of single-EV-LP co-isolates in serum, theS1EVPPRA detected GBM-associated vesicular RNA profiles in GBM patient siEVPs. TheS1EVPPRA effectively examines intravesicular, intervesicular, and interparticle heterogeneity with diagnostic promise.

[0188] Recently, super-resolution microscopy has been applied to detect and quantify fluorescent signals at the sub-vesicular level affording unprecedented detection limits to aid in interpreting siEV heterogeneity. Direct stochastic optical reconstruction microscopy (dSTORM) has been utilized to spatially locate the presence of proteins on siEVs and reconstruct siEVs in three dimensions. Furthermore, super-resolution microscopy has advanced towards visualizing siEVs in complex biofluids, such as quantitative single-molecule localization microscopy (qSMLM), which detected the protein content of siEVs from plasma, as well as total internal reflection fluorescence microscopy (TIRFM), which detected protein, miRNA, and mRNA in siEVs in plasma and serum utilizing liposomal fusion. However, using liposomes alters the native structure of siEVs and can lead to higher background signals due to electrostatic interactions with the liposomes. Therefore, a facile assay to multiplex protein and RNA in siEVPs from complex biofluids without altering their native structure while also considering interactions with siLPs is needed.

[0189] Herein described is the siEVP protein and RNA assay (S1EVPPRA), capable of multiplexing protein and RNA biomarker detection at a single-particle resolution. The assay consists of an array of micropatterns surrounded by a non-biofouling polymer film that can be functionalized with various antibodies to sort and immobilize siEVPs. In this investigation, ADP-ribosylation factor 6 (ARF6), annexin Al, CD63, and CD9 were targeted as EV-specific epitopes; epidermal growth factor receptor (EGFR) as a tumor-specific epitope; and apolipoprotein Al (ApoAl) and apolipoprotein B (ApoB) as LP-specific epitopes to immobilize and quantify siEVP subpopulations, revealing intervesicular and interparticle heterogeneity. RNA-targeting molecular beacons (MBs) and fluorescently labeled antibodies generated signals for mRNA, miRNA, and protein on siEVPs, which were then visualized by TIRFM and quantified via automatic image acquisition. By focusing signals via in situ detection at a single-particle resolution, theS1EVPPRA exceeded the detection limit for both qRT-PCR and ELISA by three orders of magnitude without tedious lysis and amplification steps. With the enhanced sensitivity of siEVP analyses, single-LP-EV (siLP-EV) co-isolates were discovered expressing CD63 by subjecting serum-isolated siLPs to CD63 / CD9-mediated capture on theS1EVPPRA, which were obscured by bulk-analysis methods. Furthermore, the combinatorial multiplexing of various biomarkers across biomolecular species in siEVs allowed the investigation of siEV intravesicular heterogeneity. The in-situ RNA detection of intact siEVPs in complex biofluids was validated by performing small RNA sequencing (sRNA-seq) on EVs harvested from six glioma cell lines to identify glioblastoma multiforme (GBM)-associated RNA and extending theS1EVPPRA to profile siEVPs isolated from the serum of GBM patients. This is the first assay that enables the simultaneous and low-dose profiling of protein, miRNA, and mRNA on siEVPs without altering their native structure, lending unique applications for liquid biopsies and biomolecular discovery.

[0190] Materials and Methods

[0191] Materials: 0.01 % (w / v) poly-L-lysine (PLL; MilliporeSigma, Burlington, MA), 5 kDa mPEG-SVA (ThermoFisher Scientific, Waltham, MA), 0.1 M 4-(2 -hydroxy ethyl)- 1- piperazineethanesulfonic acid (HEPES) buffer (pH = 8.50; ThermoFisher Scientific), 4- benzoylbenzyl-trimethylammonium chloride (PLPP; Alveole, France), NeutrAvidin (NA; ThermoFisher Scientific), bovine serum albumin (BSA; Millipore Sigma), trisethylenediaminetetraacetic acid (TE) buffer (pH = 8.05; ThermoFisher Scientific), E. coli (VB200815-101 Izys), E. coli (VB200815-1012qpx), E. coli (VB200815-1013ugb) (VectorBuilder Inc., Chicago, IL). Capture and detection antibodies used in the study are provided in Table 3. Capture antibodies (except the select few pre-biotinylated) were biotinylated using an EZ-Link™ micro Sulfo-NHS-biotinylation kit (ThermoFisher Scientific). All MBs used in the study are provided in Table 4.

[0192] Substrate fabrication: Coverslips were cleaned with ethanol and then deionized (DI) water via sonication for 3 min. The surface of the coverslip was treated with oxygen plasma for 1 minute to activate the surface. A small drop of 0.01 % (w / v) PLL was placed onto parafilm on which the treated coverslip was then placed for an even distribution of the PLL. After incubating the coverslip for 30 minutes at room temperature, the PLL-coated coverslip was rinsed with DI water and dried with nitrogen flow. Following the same method, 100 mg / mL of mPEG-SVA diluted in 0.1 M HEPES was evenly distributed on the PLL-coated coverslip. The coverslip was incubated at room temperature for 1 hour before rinsing with DI water and drying with a nitrogen airflow.

[0193] Device fabrication and surface modification: The passivated coverslip was photoetched using the PRIMO optical module (Alveole) mounted on an automated inverted microscope (Nikon Eclipse Ti Inverted Microscope System, Melville, NY). Briefly, grayscale images were translated into UV light via a digital-micromirror device (DMD) that allows for a maskless illumination of different UV intensities correlating to the corresponding grayscale values. Following the passivation of the coverslip, PLPP gel was diluted in 96 % ethanol to distribute the gel evenly throughout the surface of the coverslip. After the ethanol evaporated, a silicone spacer (W x L 3.5 mm x 3.5 mm, 64 wells; Grace Bio Labs, Bend, OR) was placed on the PEG-coated coverslip. A five-by-five array of 20-pm diameter circles spaced 80 pm center-to-center was exposed onto the coverslip with the PRIMO optical module. To optimize the RFI between samples and their controls micropatterns at different grayscale values, including 0, 25, 50, 75, 95, and 100 % with UV doses, including 10, 20, and 30 mJ / mm2 were examined (Table 1). After the UV illumination, the photoetched coverslip was washed under a stream of DI water and dried by nitrogen flow. A microscopy slide (ThermoFisher Scientific) was placed under the coverslip, and the 64-well ProPlate microarray system (Grace Bio Labs) was placed gently on the photoetched coverslip. The assembled array was secured by self-cut Delrin snap clips (Grace Bio Labs) to avoid leakage or potential contamination. The photoetched coverslip was rehydrated in phosphate-buffered saline (PBS) for 15 minutes before further functionalizing the micropatterns.

[0194] Cell culture: U251 and Gli36 glioma cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM). SF268, SF295, SF539, SNB19, and SNB-75 glioma cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. All cell culture media was prepared with 10 % (v / v) fetal bovine serum (FBS) and 1 % (v / v) penicillin-streptomycin. Cell lines were cultured to 70 % confluence at 37 °C in a 5 % CO2 incubator. Before EV collection, cells were washed with PBS three times, after which the cells were incubated in serum-free media. After two days of cell culture, the EV-enriched cell culture media was collected and centrifuged at 2,000 x g for 10 minutes at room temperature to separate cell debris before further analysis.

[0195] Human tumor specimen collection: GBM patient serum was obtained under Institutional Review Board (IRB)-approved protocols at MD Anderson Cancer Center (PA 19- 0661) following national guidelines. All patients signed informed consent forms during clinical visits before surgery and sample collection. Patients did not receive compensation in return for their participation in this study.

[0196] Healthy donor serum and plasma collection: 10 mL of whole blood from healthy donors was collected into BD Serum Separation Tubes (SST; Thermo Fisher Scientific) and BD Plasma Preparation Tubes (PPT; Thermo Fisher Scientific) for serum and plasma collection, respectively. SSTs were gently placed upright to coagulate for 60 minutes after being rocked 10 times. PPTs were rocked 10 times. Both SSTs and PPTs were centrifuged at room temperature at 1,100 * g for 10 min. After The serum and plasma were stored in 1 mL aliquots at -80 °C. All blood samples were collected under an approved IRB at The Ohio State University (IRB #2018H0268).

[0197] LP isolation: Healthy donor serum was subjected to the low-density LP / very-low- density LP (LDL / VLDL) and high-density LP (HDL) purification kit (Cell Biolabs, San Diego, CA). 1 mL of serum on ice, a dextran solution, and precipitation solution A was added and incubated on ice for 5 min. The sample was centrifuged at 6,000 * g for 10 minutes at 4°C. The supernatant was removed for further HDL processing, while the remaining pellet was subj ected to further LDL purification. For LDL purification, the pellet was resuspended in 40 pL of a bicarbonate solution and centrifuged at 6,000 x g for 10 minutes at 4°C, whereby the supernatant was transferred to ImL of lx precipitation solution B and centrifuged at 6,000 x g for 10 minutes at 4°C. The pellet was resuspended with 20 pL NaCl solution, added to ImL of lx precipitation solution C, and centrifuged at 6,000 x g for 10 minutes at 4 °C. The last process was repeated and after centrifugation, the pellet was resuspended in 20 pL of a NaCl solution. For HDL isolation, the supernatant was added to 60 pL of a dextran solution and 150 pL of precipitation solution A and was then incubated for 2 hours at room temperature and centrifuged at 16,000 x g for 30 minutes at 4°C. The pellet was resuspended in 500 pL of an HDL resuspension buffer and centrifuged at 6,000 x g for 10 minutes at 4°C. The pellet was resuspended in 600 pL of a lx HDL wash solution, incubated on a rocker for 30 minutes at 4°C, and centrifuged at 6,000 x g for 10 minutes at 4°C. The HDL supernatant was transferred to 90 pL of a dextran removal solution, while the LDL resuspension was added to 80 pL of the dextran removal solution. The mixtures were incubated for 1 hour at 4°C and centrifuged at 6,000 x g for 10 minutes at 4°C by which the supernatants were recovered into a 20-kDa Slide- A-Lyzer® MINI Dialysis devices (Thermo Fisher Scientific) and incubated in PBS for 1 day.

[0198] Engineered-EV RNA model system: Cell transfection was conducted via a cellular nanoporation (CNP) biochip. Briefly, a single layer of Gli36 cells (~8 x 106) was spread overnight on a 1 cm x 1 cm 3D CNP silicon chip surface. Individual CNP chips were transfected separately with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p plasmids at 400 ng / pL concentration in PBS. For multi-plasmid transfection, a weight ratio of 1 : 1 : 1 was pre-mixed at a 400 ng / pL concentration each in PBS. The plasmid solutions were injected into the cells via nanochannels using a 150 V electric field for 10 pulses, at 10 ms durations and 0.1 s intervals. EVs were collected from the cell supernatant 24 hours after cell transfection. Tangential flow filtration (TFF) EVP purification: The EV-enriched cell culture media and serum samples were introduced into a TFF system as described by previous techniques to purify EVPs. In brief, cell culture media or serum was circulated through a 500 kDa TFF hollow fiber filter cartridge, where EVPs were retained and enriched in the system (~ 5 mL), while free proteins and nucleic acids permeated through the filter. Constant-volume diacycles of PBS were performed until pure EVPs were obtained (350 mL of PBS). The EVPs were further enriched by centrifuging the sample within a 10 kDa centrifugal unit at 3,000 x g at 4 °C until a final volume of 100 pL was achieved. Protein concentrations were measured using a Micro BCA™ Protein Assay Kit (ThermoFisher Scientific), according to the manufacturer’s protocol.

[0199] Apolipoprotein corona: EVs with apolipoprotein corona were prepared according to an established protocol. Briefly, plasma was diluted into PBS 1 : 1 and passed through a 2-pm filter then a 0.8-pm filter. The filtered plasma was ultracentrifuged at 20,000 x g at 16 °C for 40 min. The supernatant was collected and was ultracentrifuged at 100,000 x g at 4 °C for 16 hr. The supernatant was collected and referred to hereafter as EV-depleted plasma (EVDP). 60 pL of TFF-purified EVs harvested from Gli36 cells grown in serum-free conditions were incubated in 500 pL of EVDP for 30 minutes at room temperature. After the incubation, the solution was purified via size-exclusion chromatography (SEC) with the qEV (Izon Sciences, Boston, MA), according to the manufacturer’s protocol. The samples were concentrated to 109particles / mL with a 3 kDa centrifugal unit at 3,000 x g at 4°C. The purified EVs with apolipoprotein corona were immediately added to theS1EVPPRA.

[0200] TRPS: The qNano Gold (Izon Sciences) was employed to quantify the size and concentration of EVPs via NP100 (50 - 330 nm) and NP600 (275 - 1570 nm) nanopore membranes. A pressure of 10 mbar and a voltage of 0.48 and 0.26 V was applied for the NP 100 and the NP600, respectively. Polystyrene nanoparticles (CPC 100 and CPC400) were used to calibrate the samples.

[0201] Designing MB: MBs (listed 5'-3') targeting RNAs detected in this study are provided in Table 4. The designed MBs were custom synthesized and purified using high-performance liquid chromatography (HPLC; Integrated DNA Technologies, Coralville, IA). Locked nucleic acid nucleotides (depicted as +) were incorporated into the oligonucleotide strands to improve the thermal stability and nuclease resistance of the MBs for incubation at 37 °C. siEVP capture using thesiEVPPRA: 0.1 mg / mL of NA was added to the chip and allowed to adsorb onto the photoetched micropatterns for 30 min. The chip was washed with PBS thoroughly to remove excess NA. A blocking solution of 3 % BSA and 100 mg / mL of mPEG-SVA was added to avoid unwanted non-specific binding. Subsequently, biotinylated anti-CD63 and anti-CD9 were added at 20 pg / mL each and allowed to sit overnight at 4 °C. For subpopulation-based sorting, anti-CD63, anti-CD9, anti-EGFR, anti-ARF6, anti-annexin Al, anti-ApoAl, anti-ApoB, and IgG were added separately at 20 pg / mL each and allowed to sit overnight at 4 °C. 3 % BSA was added for 1 hour to further block after washing away the capture antibodies. A concentration of 109parti cles / mL (apart from dilution experiments, which employed 106- 1011particles / mL) was then added and allowed to tether to the antibodies for 2 hours at room temperature. Unbounded EVPs were washed away with PBS and further blocked with 3 % BSA for 1 hour. siEVP protein and RNA staining: 10 pg / pL of MBs diluted in a lx TE buffer was added to the immobilized siEVPs for 1 hour at 37 °C. As for protein detection, 0.4 pg / mL of the fluorescently labeled antibodies were diluted into a solution of 1 % BSA was added to the EVP sample for 1 hour at room temperature. Residual detection probes were washed away with PBS before imaging. For single biomarker analysis, sole detection probes were added. To analyze multiple proteins or RNAs, the probes were added sequentially, fluorescently labeled antibodies were added first, followed by MBs.

[0202] Image analysis: Images of fluorescently labeled siEVPs were obtained by TIRFM (Nikon Eclipse Ti Inverted Microscope System, Melville, NY) with a lOOx oil immersion lens. An automatic algorithm was used to quantify the TIRFM images by detecting all bright signals determined via the defined outline of each bright signal by localizing the fluctuating fluorescence intensities throughout the image. The background noise was removed using a Wavelet de-noising method, and the net signal for all bright signals was obtained. The sum of all the bright signals within each micropattern was employed to calculate the TFI of the sample alongside distributions of fluorescence intensity of the siEVPs. The TFI of samples was normalized to the average TFI of the negative controls as the RFI.

[0203] ELISA: EGFR protein expression levels in Gli36-derived EVs were quantified using an EGFR Human ELISA kit (ThermoFisher Scientific). EVs were spiked in healthy donor serum at concentrations ranging from 0 to 1011particles / mL while maintaining the serum- derived EVP concentration at 109particles / mL. EGFR concentrations were quantified according to the manufacturer’s instructions. qRT-PCR: cel-miR-39-3p levels within the engineered EVs were quantified using qRT-PCR. EVs were spiked in healthy donor serum at concentrations ranging from 0 to 1011 parti cles / mL while maintaining the serum-derived EVP concentration at 109parti cles / mL. Total RNA from the EVPs was isolated and purified using an RNeasy Mini Kit and an miRNeasy Serum / Plasma kit (Qiagen, Hilden, Germany), respectively, according to the manufacturer’ s instructions. cDNA was synthesized from the total RNA using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA) on a thermal cycler (Veriti 96-Well Thermal Cycler; Applied Biosystems). cel-miR-39-3p expression was quantified using a TaqMan Gene Expression assay (Assay Id: HsOl 125301 ml; ThermoFisher Scientific) on a Real-Time PCR System (Applied Biosystems).

[0204] Immunoblotting: Gli36 cells, Gli36-derived EV, serum-isolated VLDL / LDL, serum- isolated HDL, unprocessed serum, and TFF-purified serum samples were lysed in radioimmunoprecipitation assay (RIP A) buffer (ThermoFisher Scientific, Waltham, MA) with the addition of Pierce protease and phosphatase inhibitor (ThermoFisher Scientific) for 15 minutes on ice. Protein concentrations were quantified using a Micro BCA™ Protein Assay Kit (ThermoFisher Scientific), according to the manufacturer’s protocol. Equivalent amounts of sample proteins in a Laemmli buffer with 2-Mercaptoethanol (Millipore Sigma) were electrophoresed on 4 - 20 % Mini-PROTEAN® TGX Stain-Free gels (Bio-Rad, Hercules, CA) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad). The membranes were blocked and then probed with primary antibody diluted in tris-buffered saline with 0.1% Tween® 20 (TBS-T) overnight at 4 °C and then with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 hour at room temperature (Table 3). Immunoreactivity was determined using enhanced chemiluminescence solutions (Bio-Rad) and visualized using a Bio-Rad ChemiDoc™ MP imaging system.

[0205] Single-particle interferometric reflectance imaging sensing (SP-IRIS): Silicon chips coated with tetraspanins (Unchained Labs, Boston, MA) were incubated for 1 hour at room temperature with 5 * 108Gli36-derived EVs diluted in a final volume of 60 pL of incubation buffer A. After the incubation, the silicon chips were washed 3 times for 3 minutes on an orbital plate shaker with wash solution B. The chips were scanned with the Exo View™ R200 reader (Unchained Labs) with the ExoScanner software (Unchained Labs). The particle size was allowed to scatter from 50 nm to 200 nm. The data was analyzed using Exo Viewer software (Unchained Labs).

[0206] Scanning electron microscopy (SEM): Gli36-derived EVs were immobilized to the micropatterned coverslip overnight at 4°C. The immobilized siEVs were fixed in a 2 % glutaraldehyde (Millipore Sigma) and 0.1 M sodium cacodylate solution (Electron Microscopy Sciences, Hatfield, PA) for 3 hours. EVs were incubated in 1 % osmium tetraoxide (Electron Microscopy Sciences) and 0.1 M sodium cacodylate for 2 hours after washing with a 0.1 M sodium cacodylate solution. Subsequently, the sample was dehydrated with increasing ethanol concentrations (50, 70, 85, 95, and 100 %) for 30 minutes each. Later, the CO2 critical point dryer (Tousimis, Rockville, MD) was applied to dry the sample. Lastly, a ~ 2 nm layer of gold coating was deposited on the surface using a sputtering machine (Leica EM ACE 600, Buffalo Grove, IL) and was imaged using an SEM (Apreo 2, FEI, ThermoFisher Scientific).

[0207] TEM: Two 20-pL DI water droplets and two 20-pL droplets of UranyLess EM contrast stain (Electron Microscopy Science) droplets were placed on parafilm. TEM grids were plasma treated for 1 minute before 10 pL of Gli36-derived EVs and serum-isolated LPs were drop-cast onto the treated surface. The samples were incubated on the TEM surface for 1 minute and then blotted away with filter paper. The TEM grids were washed immediately by dipping into the DI water droplet, blotted with filter paper, and repeated with the other droplet. The same technique was repeated for the contrast stain with 22 s incubations. The TEM grid was kept in the grid box overnight to completely dry before imaging. TEM imaging was carried out with a Tecnai TF-20 operating at 200kV.

[0208] Cryogenic TEM (Cryo-TEM): 3-pL aliquots of EV samples with and without a lx TE buffer incubated at 37 °C for 2 hours were added to lacey 300-mesh copper specimen grids (Product #01883; Ted Pella Inc., Redding, CA). Excess liquid was blotted away for 4 s with Whatman™ grade 1 filter papers (ThermoFisher Scientific), after which the grid was immediately plunged into liquid ethane with the Vitrobot Mark IV system (ThermoFisher Scientific) to rapidly form a thin layer of amorphous ice. The grid was then transferred under liquid nitrogen to a Glacios™ Cryo-TEM (ThermoFisher Scientific). Lastly, images were collected with a Felcon™ direct electron detector (ThermoFisher Scientific).

[0209] RNA sequencing: RNA, including miRNA, was isolated from cells and the cell- derived EVs using the miRNeasy kit (Qiagen). The RNA was eluted with 50 pl of nuclease- free water and the quality was assessed using an RNA (Pico) chip on an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). A sRNA-seq library construction method that utilizes adapters with four degenerated bases to reduce adapter-RNA ligation bias was used to characterize the miRNA. Size selection was performed using a Pippin HT automated size-selection instrument (Sage Science, Beverly, MA), and library concentrations were measured with the NEBNext Library Quant Kit (New England Biolabs, Ipswich, MA). The libraries were pooled to a final concentration of 2 nM and run on a NextSeq sequencer (Illumina, San Diego, CA). The sRNA-seq data was analyzed with sRNAnalyzer. The quantity of miRNA was determined based on the number of mapped reads that were normalized with Count Per Mapped Million (CPM). RNA from cells and EVs were analyzed using Agilent Human Whole Genome 8 x 60 microarrays with fluorescent probes prepared from isolated RNA samples using Agilent QuickAmp Labeling Kit according to the manufacturer’s instructions (Agilent). Gene expression information was obtained with Agilent’s Feature Extractor and processed with the in-house SLIM pipeline.

[0210] Colocalization efficiency: An open-source plugin for Imaged called EzColocalization was employed to visualize and measure the colocalization of EV biomarkers from acquired TIRFM images.

[0211] Statistical analysis: Statistics were performed using the JMP Pro 14 software (JMP, Cary, NC), whereby statistical significances were inferred with the satisfaction of p < 0.05. Data are expressed as the mean ± SD.

[0212] Results siEVP analysis with theS1EVPPRA TheS1EVPPRA was fabricated with the PRIMO optical module (FIG. 1A). Glass coverslips were coated with poly-L-lysine (PLL) through physisorption. Methoxy-poly(ethylene glycol)-succinimidyl valerate (mPEG-SVA) was covalently bound to the surface through N-hydroxysuccinimide (NHS) chemistry, creating a non-biofouling surface. A five-by-five array of 20-pm diameter circles was photoetched from the mPEG monolayer via UV projections translated by a digital -micromirror device (DMD) in the presence of 4-benzoylbenzyl-trimethylammonium chloride (PLPP) as a photoactivator (FIG. 1 A-i). The photoetching of the mPEG monolayer promotes the adsorption of proteins61, such as NeutrAvidin (NA), that can further be functionalized via biotin motifs. Therefore, biotinylated antibodies against surface proteins expressed on EVPs were immobilized strictly within the micropatterns to selectively sort siEVPs (FIG. lA-ii). siEVP proteins were tagged with fluorescently labeled antibodies, while RNA species, including mRNAs and miRNAs, were tagged with MBs (FIG. 1 A-iii). Lastly, TIRFM was utilized to visualize the signals from immobilized siEVPs, as TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near the glass surface. The micropattem-based design thus allows for a facile multiplexed analysis of siEVPs by immediately identifying and colocalizing signals in different regions of the glass surface. The photoetching level correlates to the grayscale value of a digital template and the

[0213] UV dose. Therefore, various configurations of grayscale and dose were tested to generate micropatterns to maximize siEVP signals and minimize noise. EVs were harvested from Gli36 cells, a human glioma cell line, which were grown in serum-free media to minimize LP-EV interactions during EV collection. The collected EVs, purified by tangential flow filtration (TFF), alongside a negative control, phosphate-buffered saline (PBS), were tested on the various micropattern configurations, which were functionalized with antibodies targeting CD63 and CD9, common membrane proteins constitutively expressed in various subpopulations of EVs. The captured EVs were then detected with a fluorescently labeled antibody against CD63. A 50 % grayscale value and a 20 mJ / mm2 dose rendered the highest fluorescence intensity on siEVs relative to the control and minimized the non-specific binding of the fluorescently labeled antibody to the micropatterns (Table 1). Furthermore, the optimized grayscale value and dose demonstrated the homogenous adsorption of NA with specificity to the photoetched micropatterns (FIG. 7).

[0214] To detect the presence of RNA within siEVs tethered to the micropatterns, a fluorescently labeled antibody against CD63 and a MB targeting hsa-miR-21-5p, an abundant vesicular miRNA in GBM, were used as detection probes simultaneously, and visualized via TIRFM. Each signal represented a siEV expressing CD63, while each signal representing a siEV carrying hsa-miR-21-5p in the acquired TIRFM images. Each colocalized signal thus demonstrated the colocalization of both biomarkers. Conversely, fluorescence signals in the control were significantly lower, indicating the ability ofS1EVPPRA to selectively multiplex different biomolecular species in siEVs (FIG. IB). Furthermore, the TIRFM images could be quantified as distributions of fluorescence intensity of the siEVs to analyze the heterogenous expression of biomarkers on siEVs (FIG. 1C) or to quantify and statistically compare various samples utilizing relative fluorescence intensities (RFI; the total fluorescence intensity of signals detected in the sample divided by the average total fluorescence intensity of signals detected in PBS within the five-by-five array): where TFI is the total fluorescence intensity, s is the fluorescence intensity from the jth signal, and n is the number of signals within the ith micropattern. The RFIs of siEVs for CD63 and hsa-miR-21-5p were 12.28 ± 0.37 and 11.21 ± 1.45, respectively (Dunnett’ s test, p < 0.0001 for CD63 and hsa-miR-21-5p), whereas IgG capture and detection for scramble miRNA produced negligible signals (FIG. ID; Dunnett’s test, p = 1.00 for IgG and p = 0.60 for scramble). Therefore, CD63 / CD9-mediated capture was used hereafter to test the sensitivity, specificity, and colocalization of signals. After EV immobilization, scanning electron microscopy (SEM) was performed on the device to further validate the fluorescence signals observed on the micropatterns as originating from siEVs. The SEM images revealed single, round particles, confirming the presence of siEVs tethered on the micropatterns (FIG. IE). TRPS measurements on the Gli36-derived EV sample used for theS1EVPPRA demonstrated a mean-siEV diameter of -150 nm (FIG. IF), consistent with the size of the vesicles observed by SEM. Thus, theS1EVPPRA selectively captures siEVs within the micropatterns and multiplexed protein and RNA signals via immunoaffinity and MB hybridization.

[0215] Specificity and sensitivity of RNA detection in siEVs: Although various methods are available to detect proteins on siEVPs, detecting RNA at a single-particle resolution without altering or damaging the integrity of the vesicles remains challenging. Therefore, it was aimed to optimize the specificity and sensitivity of RNA detection in siEVs from Gli36 cells with theS1EVPPRA. TO detect vesicular RNA, MBs were diluted in a tris-ethylenediaminetetraacetic acid (TE) buffer that is frequently used to solubilize and protect nucleic acids against degradation. On the other hand, TE contains ethylenediaminetetraacetic acid (EDTA), which electrostatically intercalates into the lipid bilayer causing its fluidization, and a tris buffer, which synergizes with EDTA. Therefore, it was hypothesized that TE buffer be used to stabilize the MBs and partially permeabilize the lipid bilayer of siEVs, allowing the MBs to reach the lumen of intact siEVs and hybridize with the desired RNA sequences. To test this, both the integrity of siEVs and the specificity of probes to intraluminal targets post-treatment with the TE buffer were quantified. The changes in EV concentration when incubating in the TE buffer and PBS were negligible when incubated at 4 °C (Student’s two-tailed t-test, p = 0.88) and 37 °C (Student’s two-tailed t-test, p = 0.65), implying the extent of permeabilization by the TE buffer neither induced aggregation nor dissolved the vesicular structures (FIG. 8A). Furthermore, cryogenic TEM (cryo-TEM) revealed that the TE buffer did not compromise the lipid bilayer present on large and small EVs (FIG. 8B). To ensure the specificity of the MBs to the desired intraluminal RNA targets with theS1EVPPRA, hsa-miR-21-5p, a miRNA abundant in Homo sapiens, and cel-miR-39-3p, a non-human miRNA abundant in Caenorhabditis elegans, were tested in siEVs derived from Gli36 cells. Gli36-derived siEVs detected with MBs targeting hsa-miR-21-5p exhibited single fluorescent signals within the micropattem when diluted in the TE buffer (FIG. 9A). The MB formulation diluted in the TE buffer produced a fluorescence signal that was 6.83 ± 0.57 times higher than the formulation without the TE buffer (Tukey's HSD, p = 0.0002), indicating the necessity for partial permeabilization. Furthermore, the siEV signals obtained from partial permeabilization with the TE buffer were 9.57 ± 0.95 times higher than the negative control (Tukey's HSD, p = 0.0002), ensuring the specificity of the MB to intraluminal hsa-miR-21-5p. In contrast, Gli36-derived siEVs detected with MBs targeting cel-miR-39-3p within the TE buffer and PBS demonstrated a negligible difference when compared to their respective controls (ANOVA, p = 0.82), thus demonstrating the ability ofS1EVPPRA to target specific RNA sequences within partially permeabilized siEVs (FIG. 9B). Furthermore, internal protein epitopes were enhanced with partial permeabilization (Student’s two-tailed t-test, p < 0.0001), while external membrane protein detection after partial permeabilization was significantly similar (Student’s two-tailed t-test, p = 0.12), indicating that partial permeabilization preserved protein signals necessary for colocalization analyses (FIG. 9C). Therefore, the TE buffer partially permeabilizes the lipid bilayer of siEVs via membrane fluidization ensuring the integrity of siEVs and the delivery of probes into the lumen of intact siEVs.

[0216] To evaluate the robustness of RNA specificity usingS1EVPPRA, Gli36 cells were transfected via electroporation to express the non-human miRNAs: cel-miR-39-3p, cel-miR- 54-3p, and cel-miR-238-3p (FIG. 10A). siEVs harvested from the transfected cells were then detected with MBs targeting cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238-3p. The engineered siEVs enriched with non-human miRNAs were successfully detected as single fluorescent signals within the micropatterns with MBs targeting the corresponding miRNA, while control samples showed a negligible number of signals (FIG. 2A). To ascertain a lack of cross-reactivity between the MBs and the other non-human miRNA, the three different engineered siEVs were tested against all the MBs targeting the non-human miRNA. Only the MBs targeting the corresponding non-human miRNA enriched within the engineered siEVs could be detected, whereas all disparate MBs presented a background level of signals (FIG. 10B). Similarly, siEVPs purified from healthy donor serum presented few signals utilizing the MBs targeting non-human miRNA (FIG. 10B). Specifically, cel-miR-39-3p-enriched siEVs detected by MBs targeting cel-miR-39-3p exhibited a RFI of 9.10 ± 2.07, while serum-derived siEVPs and the disparate MBs produced an average RFI of 1.08 ± 0.11 (FIG. 2B; Dunnett’s test, p < 0.0001 for the corresponding MB, p = 1.00 for serum-derived siEVPs and all disparate MBs); cel-miR-54-3p-enriched siEVs detected by MBs targeting cel-miR-54-3p exhibited a RFI of 9.43 ± 1.68, while serum-derived siEVPs and the disparate MBs produced an average RFI of 1.14 ± 0.15 (FIG. 2B; Dunnett’s test, p < 0.0001 for the corresponding MB, p > 0.96 for serum-derived siEVPs and all disparate MBs); and cel-miR-238-3p-enriched siEVs detected by MBs targeting cel-miR-238-3p exhibited a RFI of 8.73 ± 2.52, while serum-derived siEVPs and the disparate MBs produced an average RFI of 1.03 ± 0.10 (FIG. 2B; Dunnett’s test, p < 0.0001 for the corresponding MB, p = 1.00 for serum-derived siEVPs and all disparate MBs). Furthermore, theS1EVPPRA could discriminate between the siEVs secreted from cells transfected with varying plasmid concentrations with the EV concentration held at a constant 109 parti cles / mL insofar as the RFI of the siEVs correlated positively with increasing plasmid concentrations (Pearson's correlation coefficient, p < 0.0001 for r = 0.90), demonstrating the sensitivity of the assay to quantify nucleic acid concentrations within siEVs (FIG. 2C).

[0217] Provided the high sensitivity for detecting vesicular RNA, theS1EVPPRA was compared against the benchmark method for bulk RNA detection, qRT-PCR. EVs harvested from Gli36 cells enriched with 400 ng / pL of the cel-miR-39-3p plasmid were diluted serially into EVPs isolated from healthy donor serum and were detected with theS1EVPPRA and qRT-PCR for cel- miR-39-3p. TheS1EVPPRA exhibited a linear range at 106— 1011vesicles / mL (R2 = 0.92; ANOVA, p = 0.0026), outperforming qRT-PCR, which became undetectable below a concentration of 109vesicles / mL (FIG. 2D). Events of non-specificity of the siEVs were observed outside the micropatterns only at the higher end of the dilutions, likely due to saturation of the micropattern (FIG. 11 A). Furthermore, the maxima in the fluorescence intensity distributions of the siEVs remained consistent across the dilution, indicating the decrease in RFI as a direct measurement of the dilution of siEVs (FIG. 11B) Similarly, the sensitivity of theS1EVPPRA for protein detection in siEVs was compared to ELISA, the benchmark bulk-analysis method. Gli36-derived EVs were diluted serially into EVPs isolated from healthy donor serum and were detected with both methods for a cytoplasmic epitope of EGFR, a transmembrane protein upregulated in GBM-associated EVs with external and intraluminal epitopes. Again, theS1EVPPRA exhibited a linear range at 106- 1011vesicles / mL (R2 = 0.98; ANOVA, p = 0.0001), whereas ELISA could not detect EGFR below a concentration of 109vesicles / mL (FIG. 2E) with similar observations to RNA detection (FIGS. 12A-12B). Thus, the ability of theS1EVPPRA to focus intact siEVs and highlight biomarkers of interest at minimal concentrations affords a unique ability to colocalize biomolecular species on siEVs and explore intravesicular heterogeneity, which cannot be realized by bulk-analysis methods. Simultaneous detection of various biomolecular species in siEVs: To first determine the ability of theS1EVPPRA to multiplex various probes at a single-particle resolution, a tetraspanin analysis was performed on the siEVs, a commonplace procedure for in situ screening of siEVs. Therefore, Gli36-derived siEVs were screened for CD63, CD9, and CD81 with fluorescently labeled antibodies targeting the respective tetraspanins, whereby each antibody was chosen to excite at distinct wavelengths. The fluorescence signals were pseudocolored as the primary colors of light such that the colocalization of two detection probes could be visualized, while white signals illustrated the colocalization of all detection probes (FIG. 3 A). Furthermore, the fluorophores only emitted light when matched by their corresponding excitation wavelengths (Tukey’s HSD, p < 0.0001 for the matched channels with siEVs only), ensuring the validity of the colocalization as originating from the co-expression of the tetraspanins (FIG. 12). FIG. 3B shows the colocalization efficiencies for CD63 and CD9 (20.08 ± 2.09 %), CD81 and CD9 (19.31 ± 1.59 %), CD63 and CD81 (20.84 ± 2.52 %), and all three proteins (2.16 ± 0.58 %). As mentioned earlier, various methods exist to simultaneously detect proteins on siEVs, such as single-particle interferometric reflectance imaging sensing (SP- IRIS). Therefore, theS1EVPPRA was compared with a commercial SP-IRIS, the Exo View. Both theS1EVPPRA and the Exo View produced similar signals whereby the colocalization of the tetraspanins were illustrated (FIG. 14A). Similar to theS1EVPPRA, the Exo View differentiated positive signals from their isotype control at lower signal-to-noise ratios due to high levels of non-specificity (FIG. 14B). Although the colocalization profiles are higher on the Exo View, interestingly, the highest frequency of colocalization tends to be the complementary color to the antibody used to capture the siEVs; specifically, CD9+ / CD81+ siEVs for CD63-mediated capture, CD63+ / CD81+ siEVs for CD9-mediated capture, and CD9+ / CD63+ siEVs for CD81- mediated capture (FIG. 14C). Using an antibody cocktail as performed with theS1EVPPRA appears to normalize the bias to the capture antibody (FIG. 3B; Levene’s test, p = 0.0049). While the colocalization frequencies are higher on the Exo View, theS1EVPPRA provides higher signal-to-noise ratios for protein detection (~12 for theS1EVPPRA versus ~3 for the Exo View), an ability to colocalize with nucleic acid cargo, working concentrations one order of magnitude lower, and multiple technical replicates for a reliable colocalization analysis.

[0218] Although there are various methods to co-detect proteins on siEVs in situ, the colocalization of RNA in siEVs has not yet been achieved. Therefore, different regions of an mRNA strand were detected simultaneously within siEVs. Given the length of mRNA strands, three MBs were designed to emit distinct wavelengths when hybridized to different regions of an mRNA that translates AXL, which is abundant in GBM. All three regions of the AXL mRNA were detected in siEVs as single fluorescent signals, which, similar to multi-protein detection, were pseudo-colored to reveal colocalization events (FIG. 15 A). The distributions of fluorescence intensity for the single AXL regions detected by the MBs were similar (FIG. 15B). Moreover, the RFIs of the three different regions on the AXL mRNA were negligibly different (ANOVA, p = 0.51), demonstrating a uniform and noncompetitive affinity of the MBs to the different regions of the mRNA strand (FIG. 15C). FIG. 15D shows the colocalization efficiencies for HAL- 1 and AXL-2 (26.89 ± 2.61 %), AXL-2 andHAL-3 (28.57 ± 3.24 %), AXL- 1 and AXL-3 (23.05 ± 6.21 %), and all three regions (2.87 ± 1.03 %). Having shown the ability to colocalize signals on the same RNA biomarker in siEVs with theS1EVPPRA, it was aimed to detect multiple distinct miRNAs and thus utilized the engineered EVs. Therefore, siEVs harvested from Gli36 cells transfected with cel-miR-39-3p, cel-miR-54-3p, and cel-miR-238- 3p plasmids were detected by their respective MBs as was previously performed, but in conjunction, revealing the co-expression of multiple miRNAs within the same siEV (FIG. 16A). FIG. 16B shows the colocalization efficiencies for cel-miR-39-3p and cel-miR-54-3p (32.94 ± 1.47 %), cel-miR-54-3p and cel-miR-238-3p (31.10 ± 1.03 %), cel-miR-238-3p and cel-miR- 39-3p (31.26 ± 2.90 %), and all three miRNAs (5.51 ± 0.51 %).

[0219] To add further complexity to the multiplexed RNA detection, multiple RNA species were screened in Gli36-derived siEVs. AXL-\, hsa-miR-9-5p, and hsa-miR-21-5p were colocalized in various siEVs revealing the co-expression of mRNA and miRNA (FIG. 3C). FIG. 3D shows the colocalization efficiencies for HAL- 1 and hsa-miR-9-5p (21.15 ± 2.29 %), hsa-miR-21-5p and hsa-miR-9-5p (22.62 ± 1.08 %), HAL-1 and hsa-miR-21-5p (20.67 ± 2.58 %), and all three RNA biomarkers (2.95 ± 0.18 %). Lastly, the two methods of detection, immunoaffinity and MB hybridization, were tested together to test the ability of theS1EVPPRA to multiplex proteins and RNA simultaneously. Therefore, CD63, hsa-miR-9-5p, and hsa-miR- 21-5p were screened in Gli36-derived siEVs, revealing colocalization (FIG. 3E). FIG. 3F shows the colocalization efficiencies for CD63 and hsa-miR-9-5p (19.30 ± 1.05 %), hsa-miR- 21-5p and hsa-miR-9-5p (22.52 ± 1.90 %), hsa-miR-21-5p and CD63 (20.71 ± 2.23 %), and all three biomarkers (2.12 ± 0.48 %). Lastly, it was aimed to detect protein, mRNA, and miRNA expression in Gli36-derived siEVs. TheS1EVPPRA successfully detected the co-expression of the three biomolecular species on siEVs (FIG. 3G, FIG. 17A). FIG. 17B shows the colocalization efficiencies for CD63 and HAL-2 (21.49 ± 5.78 %), hsa-miR-21-5p and AXL-2 (14.16 ± 2.84 %), CD63 and hsa-miR-21-5p (13.68 ± 2.72 %), and all three biomarkers (0.43 ± 0.19 %). Therefore, the marriage of the two detection methods on intact siEVs with theS1EVPPRA broadens the horizon of current in situ methods, illustrating a rare display of siEV intravesicular heterogeneity with various biomolecular species.

[0220] Sorting siEVPs into subpopulations: Tailoring the surface chemistry of the micropatterns enables the examination of intervesicular heterogeneity by first sorting siEVs into subpopulations based on membrane-protein composition. Although constitutively expressed, CD63 and CD9 are expressed in higher quantities in small EVs and are considered “classical” exosomal biomarkers due to their enrichment and involvement in cargo loading despite being present in some ectosome subpopulations. On the other hand, ARF6 and annexin Al are considered ectosomal biomarkers due to their enrichment and contribution towards the budding of vesicles from the plasma membrane. Moreover, Cetuximab, a chimeric monoclonal antibody, was utilized to capture tumor-specific siEVs, which target the extracellular domain of EGFR and efficiently immobilize tumor-derived EVs from GBM patients. WB analyses confirmed that tetraspanins CD63, CD9, and CD81 were enriched in the TFF-purified EVs from Gli36 cells when compared to their cellular concentrations. Conversely, EGFR, annexin Al, and ARF6 were upregulated in Gli36 cells, but were still present in the TFF-purified EVs (FIG. 4A). Therefore, separate micropattems were decorated with antibodies targeting CD63, CD9, annexin Al, ARF6, and EGFR with IgG as a negative isotype control for siEV capture. Two miRNAs (hsa-miR-21-5p and hsa-miR-9-5p), two mRNAs (GAPDH and AXL-2 four proteins (CD63, CD9, CD81, and EGFR), and a control for RNA detection (p53), a gene downregulated in GBM, were screened individually across the several subpopulations. The expression profiles indicated variable levels across the subpopulations (FIG. 4B), with IgG unable to immobilize siEVs independent of the detection probe utilized (ANOVA, p = 0.14 for RFI, p = 0.97 for the probe) and p53 detection demonstrating negligible signals independent of the subpopulation analyzed (ANOVA, p = 0.34 for RFI, p = 0.92 for the subpopulation). Furthermore, linear discriminant analysis on the expression of the nine biomarkers demonstrated vast heterogeneity across the subpopulations with similarities amongst ARF6+ and CD9+ siEVs (FIG. 18A). However, by combining the biomarkers into their respective biomolecular species, the linear discriminant analysis revealed similarities between CD63+, CD9+, and EGFR+ siEV profiles, while ARF6+ and annexin A1+ siEVs demonstrated similarities (FIG. 18B). Therefore, the average RFI of the nine biomarkers was analyzed with respect to biogenesis pathways and the clustering from the linear discriminant analysis as “classical” exosomes (CD63+ / CD9+ siEVs), ectosomes (ARF6+ / annexin A1+ siEVs), tumor- derived siEVs (EGFR+ siEVs), and an isotype control (IgG), exhibiting an enrichment of most biomarkers in CD63+ / CD9+ siEVs (FIG. 4C). Therefore, an anti-CD63 / CD9 antibody cocktail was utilized for the remainder of the investigation to immobilize siEVPs.

[0221] Given that particles present in the blood are abundantly LPs, cholesterol-transporting particles that are often co-isolated with EVs due to their similar sizes and densities, the interaction of theS1EVPPRA with LPs was examined next. First, it was determined whether TFF, a size-exclusion purification process that was utilized on the Gli36-derived EVs, could remove LPs from healthy donor serum. While efficient at removing soluble proteins and retaining particles (FIG. 19A-B), the composition of the particles was uncertain. Accordingly, high- density LPs (HDL) and a mixture of very-low-density LPs (VLDL) and low-density LPs (LDL) were separated from the same healthy donor serum via dextran-based precipitation. The isolated LPs demonstrated an absence of annexin Al, ARF6, CD63, and CD9, and an abundance of ApoAl in the HDL fraction and ApoB in the VLDL / LDL fraction (FIG. 19C), which are absent in Gli36 cells and their EV secretions (FIG. 19D). Furthermore, the isolated LPs demonstrated dense morphologies as opposed to the classical “cup shapes” of EVs observed in TEM (FIG. 19E). TFF on the healthy donor serum demonstrated enrichment of CD63, annexin Al, and ApoB and retention with a slight loss of ARF6, CD9, and ApoAl (FIG. 19C) indicating the co-isolation of LPs and EVs after TFF purification. Therefore, Gli36- derived EVs and a mixture of the two LP isolates were deposited on theS1EVPPRA functionalized with an anti-ApoB / ApoAl antibody cocktail and screened for ApoAl, ApoB, and CD63. Positive single fluorescent signals for the ApoAl+ / ApoB+ siEVP subpopulation were obtained solely in the siLP mixture for ApoAl (Dunnett’s test, p < 0.0001 for siLPs, p = 0.61 for siEVs) and ApoB (Dunnett’s test, p < 0.0001 for siLPs, p = 0.97 for siEVs), while CD63 was absent in all siEVP formulations (ANOVA, p = 0.83), indicating the specificity of ApoB and ApoAl to siLPs (FIG. 4D-E). Interestingly, the micropattems functionalized with an anti-CD63 / CD9 antibody cocktail yielded positive signals for ApoB in siLPs (Dunnett’ s test, p = 0.0006 for siLPs, p = 0.89 for siEVs) and CD63 in siLPs and siEVs (Dunnett’s test, p = 0.016 for siLPs, p < 0.0001 for siEVs), indicating the complete specificity of Gli36-derived siEVs to the micropattems functionalized with an anti-CD63 / CD9 antibody cocktail and the rare presence of CD63 -expressing siLPs with an affinity for CD63 / CD9-mediated capture, which are obscured by the majority of LPs (FIG. 4F-G). The presence of a rare subpopulation of siLPs is confirmed by the absence of CD63 in theS1EVPPRA analysis of the ApoAl+ / ApoB+ siLP subpopulation, representing the majority of siLPs, and the absence of CD63 and CD9 in the WB analysis of HDL and VLDL / LDL samples. However, the ability to enrich these complex subpopulations of siLPs via immunopositive selection, which are possibly EV-LP hybrids or co-isolated serum-derived EVs with apolipoprotein corona, highlights the benefit of theS1EVPPRA for probing interparticle heterogeneity at minuscule quantities that are lost with bulk-analysis methods.

[0222] Due to the extensive characterization of apolipoprotein corona on EVs, their presence with theS1EVPPRA was investigated as a possibility for the observed siLP-EV co-isolates. Therefore, TFF-purified EVs harvested from Gli36 cells cultured in serum-free culture were incubated in EV-depleted plasma (EVDP) and subsequently purified to remove soluble proteins and enrich the EVs with apolipoprotein corona (FIG. 20A). The EVs incubated in EVDP were then introduced to theS1EVPPRA with CD63 / CD9-mediated capture and detected for ApoAl and ApoB. While siEVs that were not incubated in EVDP produced negligible fluorescence signal utilizing the same conditions (FIG. 4F-G) with RFIs of 0.78 ± 0.22 for ApoAl (Student’s two-tailed t-test, p = 0.19) and 0.80 ± 0.26 for ApoB (Student’s two-tailed t-test, p = 0.29), siEVs incubated in EVDP produced positive fluorescence signals for ApoAl and ApoB reminiscent of siEVP signals (FIG. 20B). Furthermore, siEVs incubated in EVDP produced RFIs of 8.95 ± 2.33 for ApoAl (Welch’s two-tailed t-test, p = 0.0274) and 41.19 ± 11.63 for ApoB (Welch’s two-tailed t-test, p = 0.0265), confirming the ability for apolipoproteins to adhere onto the EV surface (FIG. 20C). Given the complete specificity of siEVs to anti-CD63 / CD9 functionalized micropatterns and the rarity of the siLP-EV co-isolates that are simply siEVs disguised with apolipoprotein corona, the investigation was advanced to test the ability of theS1EVPPRA to detect vesicular RNA in a complex biofluid notwithstanding the observed complexity of biological samples.

[0223] Profiling siEVP RNA in glioma cell lines and GBM patient serum: To demonstrate the translational potential ofS1EVPPRA and incorporation of vesicular RNA biomarkers, transcriptomic analyses was performed on six different glioma cell lines (SF268, SF295, SF539, SNB19, SNB75, and U251) and their corresponding EVs collected from serum-free media and purified by TFF, via sRNA-seq and microarrays. To represent the pathological heterogeneity of gliomas, astrocytoma, gliosarcoma, and glioblastoma cell lines were included. Several RNAs exhibited high concentrations in both cells and EVs with miRNA showing more differential expression levels (FIG. 5A). Among the high-expressing RNAs analyzed, four transcripts, two mRNAs (NSF and NCAN) and two miRNAs (hsa-miR-9-5p and hsa-miR- 1246-5p) were selected for further analysis, due to their previous association with GBM. In general, the concentrations measured via microarrays of the four selected transcripts across the different glioma cell lines showed less variability than their corresponding EVs (Levene’s test, p = 0.0002 for NSF, p = 0.33 for hsa-miR-9-5p, p = 0.0008 for NCAN, p = 0.013 for hsa-miR- 1246-5p), indicating the differential packing of the RNA species across the cell lines (FIG. 5B). The heterogeneity of these transcripts in EVs was further explored with theS1EVPPRA at a single-particle resolution (FIG. 5C). The RFI determined by theS1EVPPRA correlated positively with the concentration of the transcripts in the EVs derived from the different glioma cell lines (Pearson's correlation coefficient, p = 0.0001 for r = 0.70); however, failed to correlate with the cellular concentrations (Pearson's correlation coefficient, r = 0.16). The discrepancy was also observed between cellular and vesicular concentrations with bulk RNA measurements (Pearson's correlation coefficient, r = 0.01), indicating the ability ofS1EVPPRA to coincide with bulk RNA detection. However, an advantage theS1EVPPRA exhibits, which bulk RNA measurements cannot achieve, is quantifying the variability of biomolecular expression in siEVPs via fluorescence profiles. The distributions of siEV fluorescence intensity demonstrated a more homogeneous expression for the mRNAs than the miRNAs across the six cell lines (FIGS. 21-24). Specifically, hsa-miR-9-5p cargo from SF268-, SF295-, SF539-, and SNB75-derived siEVs indicated more heterogeneous profiles with distribution maxima shifted to the right (FIG. 23). Similarly, hsa-miR-1246-5p cargo from SF268-, SNB75-, and SNB19- derived siEVs also demonstrated a heterogeneous expression with distribution maxima shifted to the right (FIG. 24).

[0224] Having validated the GBM-associated vesicular RNA biomarkers across various cell lines, theS1EVPPRA was used to characterize siEVPs from TFF-purified serum from GBM patients. For each individual, 20 pL of the purified serum was processed with theS1EVPPRA. A cohort of 10 GBM patients and 10 age-matched healthy individuals were chosen for the investigation (Table 2). Although the presence of siLP-EV co-isolates were demonstrated in serum, higher frequencies of positive signals for NSF, hsa-miR-9-5p, NCAN, and hsa-miR- 1246-5p were obtained from purified GBM patient serum in comparison to healthy donor serum (FIG. 6A). Furthermore, RFIs of siEVPs from GBM patients were significantly higher for NSF, NCAN, hsa-miR-9-5p, and hsa-miR-1246-5p RNAs when compared to the siEVPs from purified healthy donor serum (FIG. 6B; Mann-Whitney U test, p = 0.0002 for ASF, NCAN and hsa-miR-9-5p, p = 0.0022 for hsa-miR-1246-5p). Comparing the distributions of fluorescence intensity for the different RNA species revealed that siEVPs expressing NSF and NCAN presented more homogeneous fluorescence signals. In contrast, the distributions of fluorescence intensity for siEVPs expressing hsa-miR-9-5p and hsa-miR-1246-5p yielded broad distributions of fluorescence intensity with distribution maxima shifted to the right (FIG. 6C), which agreed with the previous observation on the siEVs across the glioma cell lines. These findings confirm the ability ofS1EVPPRA to measure the intravesicular heterogeneity of RNA in siEVPs from complex biofluids, specifically serum, and its ability to discriminate GBM patients from healthy donors while conserving vesicular profiles. The success of this work and the tunability of theS1EVPPRA opens the possibility for its potential application in liquid biopsy for cancer diagnoses in GBM and other diseases.

[0225] Discussion

[0226] The enhanced sensitivity of siEVP methods, the tunability of surface chemistry within the micropattems, and the ability to multiplex across various biomolecular species realized by the single-EV and particle (siEVP) protein and RNA assay (S1EVPPRA) lends unique opportunities to investigate intravesicular, intervesicular, and interparticle heterogeneity at a single-particle resolution. Due to the inherent heterogeneity of EVPs and the abundance of LPs in circulation, it is necessary to decode the molecular profile of EVPs and advance technologies to distinguish EVs from LPs. Highly tunable multiplexed approaches, such as theS1EVPPRA, can aid in the precise deconvolution of siEVPs. Various novel siEV technologies have adopted an in-situ approach to characterize siEVs to visualize intravesicular heterogeneity via colocalization analyses. TheS1EVPPRA is the first assay to demonstrate intravesicular heterogeneity across multiple biomolecular species with colocalization analyses, including protein and RNA, which was realized with a facile incubation with a TE buffer that stabilizes MBs, ensures the integrity of intact siEVs, preserves external epitopes, and delivers detection probes into the lumen of siEVs via partial permeabilization. While the highest colocalization rates were observed for the engineered siEVs, which is non-trivial due to the induced enrichment of synthetic miRNA species, the second-highest (highest non-synthetic) colocalization rate was observed for the co-detection of multiple regions of the AXL mRNA strand. Interestingly, only a small population of siEVs demonstrated the colocalization of all targets, further demonstrating the observed fragmentation of mRNA strands during packaging, which can compromise highly sensitive assays that require high-quality RNA, such as qRT- PCR. Lastly, interspecies colocalization yielded similar colocalization rates, indicating consistency across methods, except for the co-detection of protein, mRNA, and miRNA, which demonstrated slightly lower frequencies of mutual expression. While often utilized for colocalization analyses, it was found that colocalization was relatively low for the tetraspanins, which agrees with other reported siEV tetraspanin analyses. With theS1EVPPRA, the intravesicular heterogeneity for single biomolecular expression could be further observed as fluorescence intensity distributions. As such, higher miRNA variability than mRNA was observed in the siEVs secreted from the glioma cell lines and siEVPs in GBM patient serum. Due to the small size of intact miRNA (~22 nucleotides) and the fragmentation of mRNA in EVs, the observed variability in miRNA is a more accurate illustration of non-genetic single- cellular heterogeneity. Both methods to profile intravesicular heterogeneity were lost with bulk-analysis methods, such as WB, ELISA, qRT-PCR, sRNA-seq, and microarrays, indicating the utility of theS1EVPPRA in uncovering intravesicular heterogeneity.

[0227] Intervesicular and interparticle heterogeneity were recognized by tuning the surface chemistry on the micropattern within theS1EVPPRA to capture siEVP subpopulations. Testing various capture antibodies and detection probes on Gli36-derived siEVs further elucidated intervesicular heterogeneity between subpopulations as a function of biomarker expression. Multivariate analyses on the grouped biomolecular species revealed similar profilometric trends for CD63+, CD9+, and EGFR+ siEV subpopulations, which corresponds with the upregulation of EGFR in Gli36-derived “classical” exosomes. Furthermore, capturing siEVs by CD63 and CD9 yielded the most holistic signature across all biomolecules tested, which coincides with immunoselective immobilization strategies for siEVPs from non-small cell lung cancer patient serum. A possibility for the enhanced capture and detection utilizing an anti- CD63 / CD9 antibody cocktail to immobilize siEVs is that the cocktail does not discriminate cellular origin and rather captures siEVs from all cells. While originally considered a “classical” exosomal biomarker, CD9 is present in larger EVs albeit enriched in small EVs and is found on small ectosomes. Interestingly, the linear discriminant analysis on the single biomarker expression demonstrated similarities between CD9+ and ARF6+ siEV subpopulations. Therefore, the anti-CD63 / CD9 antibody cocktail captures exosomes and ectosomes, thus widening the breadth of capture. However, the antibody cocktail was insufficient in sorting out LPs as CD63+ / CD9+ siLP-EV co-isolates were uncovered, concurring with newer evidence on the complexity of LPs and EVs. Specifically, the co-expression of ApoAl and ApoB on the surface of EVs as protein corona with theS1EVPPRA was confirmed, which maintains bioactivity and has implications in disease progression. Various studies utilize tetraspanins to immobilize siEVs and assume negligible interactions with LPs. This erroneous assumption may result from the dilution of the subpopulation in bulk-analysis methods, further motivating the necessity for siEVP methods in uncovering interparticle heterogeneity.

[0228] Although theS1EVPPRA is highly sensitive and capable of discerning heterogeneous subpopulations amongst siEVPs, colocalization is limited to the number of compatible fluorophores, indicating the possibility for siEVP subpopulations immobilized on the micropattern that downregulate all targets. With the inherent heterogeneity of blood-derived EVPs, capturing all signals leads to a more comprehensive compositional analysis. Advances in spectral microscopy, sequential labeling, and quantum-dot synthesis, to name a few, are strategic methods for overcoming spectral overlapping in bandpass filters, the applications of which further increase the scope of targeted siEVPs. Furthermore, SP-IRIS demonstrated higher colocalization rates for the tetraspanin analyses, albeit with high levels of nonspecificity. The use of enhancing methods, such as the incorporation of an interferometric substrate or surface plasmon resonating surfaces, can further increase signals. Lastly, a complex subpopulation of siLP-EV co-isolates, which competitively interacts with the micropatterned surface was found. While their presence indicated minimal interference with the capability of theS1EVPPRA to measure GBM-associated vesicular RNA profiles in GBM patient serum, the possible contributions of siLPs as RNA carriers cannot be missed.

[0229] The siEVP method circumvented the artifact of dilution often experienced in bulkanalysis methods. As such, disease-associated proteins and RNAs were preserved in intact siEVPs thus enhancing sensitivities and allowing detection at low concentrations and volumes. The feasibility of analyzing RNAs in siEVPs from the serum of GBM patients unveiled the ability ofS1EVPPRA for liquid biopsy applications. The current study focused on GBM vesicular RNA analyses, but theS1EVPPRA is easily adapted to other diseases by customizing the surface chemistry to capture disease-specific epitopes. Furthermore, the multivariate heterogeneity analysis afforded by theS1EVPPRA aids in uncovering differences in subpopulation-dependent packaging of biomolecules and illuminate biogenesis pathways. Lastly, the ability for theS1EVPPRA to multiplex across various biomolecular species shows a qualitative perspective into siEVP heterogeneity more comprehensively than previously accomplished. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference. Those skilled in the art will appreciate that numerous changes and modifications can be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. It is, therefore, intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

[0230] TABLES

[0231] Table 1: List of antibodies. Table 2: List of MB designs.

[0232] Table 3: RFI optimization by controlling monolayer degradation.

[0233] Table 4: GBM and healthy donor information. SEQUENCES

[0234] SEQ ID NO: 1 hsa-miR-21-5p

[0235] GAATAGCTCCGTGTTATCA

[0236] SEQ ID NO: 2 hsa-miR-21-5p

[0237] T+CA A+CA / iCy5 / +TC A+GT +CT+G ATA AGC TAA CTT ATC AGA CTG AGIAbRQSp /

[0238] SEQ ID NO: 3

[0239] AXL-1

[0240] +CTC CC+C A6-FAMK / +GG A+TT T+GG CA+C TGC AGT GCC AAA TCC / 3BHQ 1 /

[0241] SEQ ID NO: 4

[0242] AXL-2

[0243] +TGG +TGT / iCy 3 / +CTA +GTT +AGT +CAC AAC TGT GTG ACT AAC TAG / 3BHQ 2 /

[0244] SEQ ID NO: 5

[0245] AXL-3

[0246] +GTG +ATT / iCy 5 / +CTG +AGC +TGG +CTG ACC AAG CCA GCT CAG / 3IAbRQSp /

[0247] SEQ ID NO: 6 hsa-miR-9-5p

[0248] +CAT +ACA / iCy3 / +GC T+AG A+TA ACC AAA +GAT TGG TTA TCT AGC / 3BHQ 2 /

[0249] SEQ ID NO: 7

[0250] NSF

[0251] +GTT +G / iFluorT / C +CCA +CTG +AGA +AGG CCT TCT CAG TGG / 3BHQ 1 /

[0252] SEQ ID NO: 8

[0253] NCAN

[0254] +GCT +CCA / iCy3 / +GG C+AT A+TC C+AC CTC ATG GAT ATG CC / 3BHQ 2 / SEQ ID NO: 9 hsa-miR-1246-5p

[0255] +CCT +GC / iCy5 / +TC+C AA+A AA+T CCA TTC GGA TTT TTG GA / 3IAbRQSp /

[0256] SEQ ID NO: 10 cel-miR-39-3p

[0257] +CAA +GC / iFluorT / +GAT +TTA +CAC +CCG GTG AGG GTG TAA ATC / 3BHQ 1 /

[0258] SEQ ID NO: 11 cel-miR-54-3p

[0259] +GTT +CTC / iCy3 / +GT C+GT C+TC A+TA TCC TGG ATA TGA GAC GAC / 3BHQ 2 /

[0260] SEQ ID NO: 12 cel-miR-238-3p

[0261] +CTT T+GA A / iCy5 / +C GT+C C+GA +GAA CAT CCA TTC TCG GAC G / 3IAbRQSp /

[0262] SEQ ID NO: 13 p53

[0263] +CTC CGT / iCy 5 / CAT GTG CTG TGA CTT CAC AGC AC A TG / BHQ3-3 /

[0264] SEQ ID NO: 14

[0265] GAPDH

[0266] +CTC +AGC +C / iFluorT / T +GAC +GGT GAA TTC ACC GTC AAG / 3BHQ 1 /

Claims

CLAIMSWhat is claimed is:

1. A method of simultaneously detecting an RNA and a protein in situ, comprising: obtaining a sample from a subject; isolating an Extracellular Vesicle (EV) and / or a Lipoprotein (LP) from the sample; tethering a plurality of the EV and / or the LP to a micropattem array on a glass substrate via one or more capture antibodies, wherein the one or more capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof; and binding a detection antibody and a molecular beacon to the EV and / or the LP tethered on the micropattern array, wherein the detection antibody is configured to bind to a first target type of a molecular cargo, and the molecular beacon is configured to bind to a second target type of the molecular cargo, wherein the first target type of the molecular cargo is a protein, and the second target type of the molecular cargo is an RNA.

2. The method of claim 1, further comprising: fluorescently imaging the micropattern array to capture an image data; detecting an occurrence of a single EV and / or an LP expressing the first target type of the molecular cargo based on a fluorescent spot of a first color associated with the detection antibody in the image data captured; detecting an occurrence of the single EV and / or the LP expressing the second target type of the molecular cargo based on a fluorescent spot of a second color associated with the molecular beacon in the image data captured; and detecting an occurrence of the single EV and / or the LP expressing both the first target type of the molecular cargo, and the second target type of the molecular cargo based on a fluorescent spot of a third color in the image data captured.

3. The method of any one of claims 1-2, wherein the one or more capture antibodies comprises anti-ApoAl.

4. The method of any one of claims 1-3, wherein the one or more capture antibodies comprises anti-ApoB.

5. The method of any one of claims 1-4, wherein the one or more capture antibodies comprises anti-CD63.

6. The method of any one of claims 1-5, wherein the one or more capture antibodies comprises anti-CD9.

7. The method of any one of claims 1-6, wherein the glass substrate is coated with poly- L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG), wherein the PEG is covalently bound to the PLL through N-hydroxysuccinimide (NHS) chemistry.

8. The method of claim 7, wherein the PEG is methoxy-poly(ethylene glycol)- succinimidyl valerate (mPEG-SVA).

9. The method of any one of claims 1-8, wherein the micropattern array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4- benzoylbenzyl-trimethylammonium chloride (PLPP).

10. The method of any one of claims 1-9, wherein the micropattern array comprises a five- by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm.

11. The method of any one of claims 1-10, wherein the one or more capture antibodies are biotinylated and attached to the micropattem array by binding to a physically adsorbed Neutravidin layer on the micropattem array.

12. The method of any one of claims 1-11, wherein the one or more capture antibodies binds to surface proteins expressed on the EV and / or the LP.

13. The method of any one of claims 1-12, wherein the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

14. The method of any one of claims 1-13, wherein the RNA is selected from a microRNA (miRNA), a messenger RNA (mRNA), or combinations thereof.

15. The method of any one of claims 1-14, wherein the RNA encodes AXL-\, AXL-2, AXL- 3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel- miR-39-3p, cel-miR-54-3p, or cel-miR-238-3p.

16. The method of any one of claims 1-15, wherein the detection antibody is conjugated with one or more fluorophores for fluorescent imaging.

17. The method of any one of claims 1-16, wherein the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging.

18. The method of any one of claims 1-17, wherein the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

19. The method of any one of claims 1-18, wherein the molecular beacon is selected from any one of SEQ ID NO: 1-14.

20. The method of any one of claims 2-19, wherein fluorescently imaging the micropattem array to capture image data comprises total internal reflection fluorescence microscopy (TIRFM).

21. The method of claim 20, wherein TIRFM produces an exponentially decaying electromagnetic wave that only excites fluorophores near a surface of the glass substrate to visualize signals from an immobilized EV and / or an LP.

22. The method of any one of claims 1-21, wherein the sample is saliva, serum, plasma, urine, sputum, nasal swab, fecal, tears, or cerebral spinal fluid.

23. The method of any one of claims 1-22, wherein the subject is a human.

24. A system for simultaneously detecting an RNA and a protein in situ, comprising: one or more of capture antibodies attached to a micropattern array on a glass substrate, wherein the one or more of capture antibodies comprises anti-CD63, anti-CD9, anti-ApoAl, anti-ApoB, or a combination thereof; a detection antibody and a molecular beacon, wherein the detection antibody is configured to bind to a first target type of molecular cargo, and wherein themolecular beacon is configured to bind to a second target type of molecular cargo wherein first target type of molecular cargo is a protein, and the second target type of molecular cargo is an RNA; and a fluorescent imaging device to capture image data.

25. The system of claim 24, wherein the one or more capture antibodies comprises anti- CD63.

26. The system of any one of claims 24-25, wherein the one or more capture antibodies comprises anti-CD9.

27. The system of any one of claims 24-26, wherein the one or more capture antibodies comprises anti-ApoAl.

28. The system of any one of claims 24-27, wherein the one or more capture antibodies comprises anti-ApoB.

29. The system of any one of claims 24-28, wherein the glass substrate is coated with poly- L-lysine (PLL) through physical adsorption prior to coating the glass substrate with a polyethylene glycol (PEG), wherein the PEG is covalently bound to the PLL through N-hydroxysuccinimide (NHS) chemistry.

30. The system of claim 29, wherein the PEG is methoxy-poly(ethylene glycol)- succinimidyl valerate (mPEG-SVA).

31. The system of any one of claims 24-30, wherein the micropattem array is photoetched on the glass substrate in presence of a photoactivator, wherein the photoactivator is 4- benzoylbenzyl-trimethylammonium chloride (PLPP).

32. The system of any one of claims 24-31, wherein the micropattern array comprises a five-by-five array of circles, wherein each individual circle has a diameter ranging from about 2 pm to about 200 pm.

33. The system of any one of claims 24-32, wherein the one or more capture antibodies is biotinylated and attached to the micropattem array by binding to a physically adsorbed Neutravidin layer on the micropattem array.

34. The system of any one of claims 24-33, wherein the one or more capture antibodies binds to surface proteins expressed on a single EV and / or an LP.

35. The system of any one of claims 24-34, wherein the one or more capture antibodies further comprises anti-EGFR, anti-ARF6, anti-annexin Al, or a combination thereof.

36. The system of any one of claims 24-35, further comprises a first color associated with the detection antibody in a captured image data in a first channel, a second color associated with the molecular beacon in the captured image data in a second channel, and a third color in the captured image data in a third channel.

37. The system of any one of claims 24-36, wherein the RNA is selected from microRNA (miRNA), messenger RNA (mRNA), or combinations thereof.

38. The system of any one of claims 24-37, wherein the RNA encodes AXL- 1, AXL-2, AXL- 3, NSF, NCAN, p53, GAPDH, hsa-miR-21-5p, hsa-miR-9-5p, hsa-miR-1246-5p, cel- miR-39-3p, cel-miR-54-3p or cel-miR-238-3p.

39. The system of any one of claims 24-38, wherein the detection antibody is conjugated with one or more fluorophores for fluorescent imaging.

40. The system of any one of claims 24-39, wherein the molecular beacon comprises one or more fluorescent dye sequences for fluorescent imaging.

41. The system of any one of claims 24-40, wherein the molecular beacon comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

42. The system of any one of claims 24-41, wherein the molecular beacon is selected from any one of SEQ ID NO: 1-14.

3. The system of any one of claims 24-42, wherein the fluorescent imaging device to capture image data comprises total internal reflection fluorescence microscopy (TIRFM).

Images