RNA analysis from extracellular particles for treatment of brain cancer

Abstract

The present disclosure relates to biochip assays and methods of use thereof to detect, diagnosis, grade, characterize, and treat brain cancers, including but not limited to glioblastoma multiforme (GBM).

Description

[0001] Docket No. 103362-036WO1

[0002] RNA ANALYSIS FROM EXTRACELLULAR PARTICLES FOR TREATMENT OF BRAIN CANCER

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63 / 690,506, filed September 4, 2024, 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. U18TR003807 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] REFERENCE TO SEQUENCE LISTING

[0008] The sequence listing submitted on September 4, 2025, as an .XML file entitled “103362- 036WOl_ST26.xml” created on August 31, 2025, and having a file size of 46,583 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

[0009] FIELD

[0010] The present disclosure relates to biochip assays and methods of use thereof to detect, diagnosis, grade, characterize, and treat brain cancers, including but not limited to glioblastoma multiforme (GBM).

[0011] BACKGROUND

[0012] Glioblastoma multiforme (GBM) is the most malignant form of gliomas and the most lethal primary brain tumors in adults. According to the World Health Organization (WHO), gliomas can be divided into grades I to IV depending on the degree of malignancy, of which grade IV defined as GBM. Current standard treatment for patients with GBM consists of a combination of surgery, radiotherapy, and chemotherapy with temozolomide (TMZ). However, the median survival for GBM patients remains 14 to 16 months after diagnosis, with 70% of patients showing disease progression one year after diagnosis, and less than 5% of patients survive over five years. The initial diagnosis of GBM is achieved by neuroimaging, and followed by tissue biopsies to definitively diagnose, grade and characterize the tumor. Immunohistochemistry and molecular analyses are used to further detect the combined loss of chromosome, the mutation and / or expression P53, the presence of isocitrate dehydrogenase 1 (IDH1) mutation and the methylation status of O-6-methylguanine-DNA methyltransferase (MGMT) promotor. However, these Docket No. 103362-036WO1 biomarkers or clinical features from tissue biopsy samples cannot distinguish glioma true progression from pseudoprogression and are not suited to monitoring the tumor activity during the treatment. Thus, what is needed in the art is an assay and / or method of detecting GBM biomarkers for definitive diagnosis, grading, and characterization of glioblastomas.

[0013] SUMMARY

[0014] The present disclosure providesS1EVRNA biochips and assays comprisingS1EVRNA biochips for detecting, diagnosing, and treating brain cancers including but not limited to glioblastoma multiforme (GBM). The present disclosure also provides methods of detecting, diagnosing, and / or treating a subj ect with GBM using theS1EVRNA biochips and assays comprisingS1EVRNA biochips.

[0015] In some aspects, disclosed herein is a biochip assay comprising a titanium (Ti) and gold (Au) coated biochip, wherein the Ti and Au coated biochip comprises a linker, a neutravidin coating, a biotin conjugated capture antibody, and a molecular beacon (MB) probe, wherein the biochip assay comprises a) contacting a liquid sample with the Ti and Au coated biochip, wherein the liquid sample comprises a subpopulation of extracellular vesicles (EVs), b) binding the MB probe to at least one mRNA, at least one miRNA, or a combination thereof located on an extracellular vesicle (EV), c) imaging the EV using an imaging modality to detect the at least one mRNA, at least one miRNA, or a combination thereof, wherein the at least one mRNA comprises Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); or at least one microRNA (miRNA) comprises miR-9-5p, miR- 124-3p, miR122-5p or miR1246-5p.

[0016] In some embodiments, the biochip assay comprising the linker comprises 20- tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH. In some embodiments, the biochip assay comprising the biotin conjugated capture antibody binds to one or more surface proteins, including but not limited to CD63 and CD9, located on the EV.

[0017] In some embodiments, the biochip assay comprising the MB probe comprises one or more fluorescent dyes. In some embodiments, the biochip assay comprising the MB probe comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the biochip assay comprising the fluorescent imaging modality comprises total reflection fluorescence microscopy (TIRFM). In some embodiments, the biochip assay comprising the liquid sample comprises blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF). Docket No. 103362-036WO1

[0018] In some aspects, disclosed herein is a method of detecting a signature of glioblastoma (GBM) in a subject, the method comprising a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; and c) detecting the signature of GBM, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122- 5p or miR1246-5p.

[0019] In some aspects, disclosed herein is a method of treating a glioblastoma (GBM) in a subject in need thereof, the method comprising: a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC 14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; c) detecting a signature of GBM, d) diagnosing the subject with a GBM when an area under the curve (AUC) from the signature is at least 0.8 compared to a control subject, and e) administering to the subject an anti -cancer agent to decrease the GBM relative to an untreated control, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N- ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122-5p or miR1246-5p.

[0020] In some embodiments, the method of any preceding aspect comprises the biotin conjugated capture antibody binding to an EV subpopulation from the liquid sample. In some embodiments, the method of any preceding aspect comprises the biotin conjugated capture antibody binding to one or more surface proteins, including but not limited to CD63 and CD9 located on the EV.

[0021] In some embodiments, the method of any preceding aspect comprises the MB probe binding the least one mRNA or the at least one miRNA. In some embodiments, the method of any preceding aspect comprises the MB probe comprising one or more fluorescent dyes for a fluorescent imaging modality. In some embodiments, the method of any preceding aspect comprises the MB probe comprising one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

[0022] In some embodiments, the method of any preceding aspect comprises the fluorescent imaging modality comprising total reflection fluorescence microscopy (TIRFM). In some Docket No. 103362-036WO1 embodiments, the method of any preceding aspect comprises the liquid sample comprising blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

[0023] BRIEF DESCRIPTION OF FIGURES

[0024] The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.

[0025] FIGS. 1A, IB, 1C, ID, IE, and IF show the design and characterization of single extracellular vesicular RNA (S1EVRNA) biochip assay. Figure 1 A shows the schematic ofS1EVRNA biochip assay. Figure IB shows the scanning electron microscope (SEM) images of EVs purified by TFF. Figure 1C shows the EV size and concentration measured by qNano. Figure ID shows the tetraspanin exosome biomarker (CD63, CD9 and CD81) expressions in EVs from HD and GBM serum samples. Figure IE shows the NCAN expression in EVs from SNB75 cells and patient serum sorted by CD63, CD81 and CD9. Figure IF shows the NCAN expression in EVs in human microglial cells and GBM cells sorted by mixed capture antibodies (CD63 / CD9). All data were presented as means (n = 2 wells). P values were determined by the two-way ANOVA test. **P < 0.01.

[0026] FIGS. 2A, 2B, 2C, and 2D show the EV mRNA and miRNA profile in 20 GBM patient and 20 healthy control serum samples. Figure 2A shows the volcano plot of mRNA and miRNA profile. Figure 2B shows the heat map of EV mRNA (NRGN, TMEM30C, NCAN and NSF) and EV miRNA (miR-122-5p, miR-9-5p, miR1246-5p and miR-124-3p) expression. Figure 2C shows the box plot of NRGN, TMEM30C, NCAN and NSF mRNA expression levels. Figure 2D shows the box plots of miR-122-5p, miR-9-5p, miR1246-5p and miR-124-3p expression. Data were presented as means (n = 2 wells, each well with 100 images). / ? values were determined by Mann- Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.

[0027] FIGS. 3 A, 3B, 3C, 3D, 3E, and 3F show the comparison of RNA expression byS1EVRNA biochip assay and qRT-PCR in GBM cells. NRGN mRNA (Figure 3A), TMEM30C mRNA (Figure 3B), miR-9-5p (Figure 3C), miR122-5p (Figure 3D) expression in GBM cells (Gli36, SNB75 and U251) and GBM cell derived EVs. Figures 3E and 3F show the calibration curve of NRGN mRNA and miR-9-5p expressions in SNB75 cell derived EVs spiked healthy donor serum in comparison with qRT-PCR. Dotted lines in E and F indicate detection limit forS1EVRNA biochip assay and qRT-PCR.

[0028] FIGS. 4 A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, 41, and 4 J show the mRNA expression in GBM patient serum byS1EVRNA biochip assay. NRGN (Figure 4A), TMEM30C (Figure 4B), NCAN (Figure 4C), NSF (Figure 4D) mRNA expression and representative images in HD and GBM Docket No. 103362-036WO1 patient serum. TIRF microscopy images were enlarged from 80 pm x 80 pm to 20 pm x 20 pm to show bright spots. TFI: Relative Fluorescence Intensity Ratio of TMEM30C to NRGN (Figure 4E), Ratio of NSF and NRGN (Figure 4F), Ratio of NCAN and NRGN (Figure 4G) were calculated using their RFI. Figure 4H shows the ROC curves of four mRNA expressions as a single biomarker. Figure 41 shows the ROC curves of three mRNA and NRGN ratio as a biomarker. Figure 4J shows the ROC curve of best combination among four mRNAs. Pairwise comparison P values were determined by the Mann-Whitney U test. ***P < 0.001.

[0029] FIGS. 5A, 5B, 5C, 5D, and 5E show the miRNA expression in GBM patient serum byS1EVRNA biochip assay. miR124-3p (Figure 5A), miR-9-5p (Figure 5B), miR-122-5p (Figure 5C), miR-1246-5p (Figure 5D) expression and representative images in HD and GBM patient serum. TIRF microscopy images were enlarged from 80 pm x 80 pm to 20 pm x 20 pm to show bright spots. TFI: Relative Fluorescence Intensity (Figure 5E) ROC curves of four miRNA expressions as a single biomarker. Pairwise comparison P values were determined by the Mann-Whitney U test. *** < 0.001.

[0030] FIGS. 6 A, 6B, 6C, and 6D show the combination of miR-9-5p and mRNA / NRGN ratio as GBM biomarkers. Figure 6A shows the ROC curves for EV numbers and combination of miR-9- 5p and mRNA to NRGN ratio. Figure 6B shows the scatter plot of TMEM30C / NRGN ratio vs. miR-9-5p. Figure 6C shows the scatter plot of NCAN / NRGN ratio vs. miR-9-5p. Figure 6D shows the scatter plot of NSF / NRGN ratio vs. miR-9-5p.

[0031] FIG. 7 shows the TMEM30C and miR-122-5p expression in single EVs sorted by CD63 or CD9 or mixed antibodies. Single EVs from GBM patients (P27 and P31) and healthy donors (HD 14 and HD 16) were used to measure TMEM30C expression. All data were presented as means (n = 2~3 wells). P values were determined by the two-way ANOVA test. *P < 0.05.

[0032] FIGS. 8A and 8B show the heatmap of highly differentiated mRNAs and miRNA. Figure 8A shows the 37 highly differentiated and down-regulated mRNAs. Figure 8B shows the highly differentiated and up-regulated miRNAs.

[0033] FIGS. 9A and 9B show the calibration curve of TMEM30C mRNA (Figure 9A) and miR- 122-5p (Figure 9B) expressions in SNB75 cell derived EVs spiked healthy donor serum in comparison with qRT-PCR. Dotted line in Figures 9A and 9B indicates detection limit for limit forS1EVRNA biochip assay and qRT-PCR.

[0034] FIGS. 10A, 10B. 10C, and 10D show the combination of miR-124-3p with the ratios of mRNA / NRGN as GBM biomarkers. Figure 10A shows the ROC curves for combination of miR- 124-3p and mRNA to NRGN ratio. Figure 10B shows the scatter plot of TMEM30C / NRGN ratio Docket No. 103362-036WO1 vs. miR-124-3p. Figure IOC shows the scatter plot of NCAN / NRGN ratio vs. miR-124-3p. Figure 10D shows the scatter plot of NSF / NRGN ratio vs. miR-124-3p.

[0035] FIGS. 11 A, 11B, 11C, and 11D show the combination of miR-122-5p with the ratios of mRNA / NRGN as GBM biomarkers. Figure 11 A shows the ROC curves for combination of miR- 122-5p and mRNA to NRGN ratio. Figure 1 IB shows the scatter plot of TMEM30C / NRGN ratio vs. miR-122-5p. Figure 11C shows the scatter plot of NCAN / NRGN ratio vs. miR-122-5p. Figure 1 ID shows the scatter plot of NSF / NRGN ratio vs. miR-122-5p.

[0036] FIGS. 12 A, 12B, 12C, and 12D shows the combination of miR-1246-5p with the ratios of mRNA / NRGN as GBM biomarkers. Figure 12A shows the ROC curves for combination of miR- 1246-5p and mRNA to NRGN ratio. Figure 12B shows the Scatter plot of TMEM30C / NRGN ratio vs. miR-1246-5p. Figure 12C shows the scatter plot of NCAN / NRGN ratio vs. miR-1246- 5p. Figure 12D shows the scatter plot of NSF / NRGN ratio vs. miR-1246-5p.

[0037] DETAILED DESCRIPTION

[0038] The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.

[0039] 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.

[0040] Terminology

[0041] 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 Docket No. 103362-036WO1 “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.

[0042] The following definitions are provided for the full understanding of terms used in this specification.

[0043] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0044] 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 are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0045] 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. Docket No. 103362-036WO1 An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0046] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

[0047] "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.

[0048] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0049] A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5 -carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule. Docket No. 103362-036WO1

[0050] A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5 -carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.

[0051] A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.

[0052] A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.

[0053] The term “mRNA” refers to messenger ribonucleic acid, or single stranded molecule of RNA that corresponds to the genetic sequence of a gene, and is translated by a ribosome in the process of synthesizing a protein. mRNA is created during the process of transcription, where a gene is converted into a primary transcript mRNA (or pre-mRNA). The primary transcript is further processed through RNA splicing to only contain regions that will encode protein. mRNA can also be targeted for epigenetic modifications, such as methylation, to impact mRNA translation, nuclear retention, nuclear export, processing, and splicing.

[0054] The term “miRNA” refers to micro ribonucleic acid, also known as miRNA, microRNA, or pRNA, are small single stranded, non-coding RNA molecules, usually containing 20-25 nucleotides. miRNAs are commonly involved in RNA silencing, which refers to the complementarity of miRNA molecules to mRNA molecules, and further their ability to regulate gene expression. Docket No. 103362-036WO1siEVRNA biochips and biochip assays

[0055] The present disclosure providesS1EVRNA biochips and assays comprisingS1EVRNA biochips for detecting, diagnosing, and treating brain cancers including but not limited to glioblastoma multiforme (GBM).

[0056] An RNA biochip assay is a high-throughput test using a miniaturized device, or biochip, to detect and analyze RNA molecules, such as for example mRNA and miRNA, simultaneously. These assays employ specific molecular probes to hybridize with target RNA molecules, enabling the detection of various mRNA, miRNAs, and other types of RNA molecules, often with the goal of understanding gene expression, gene regulation, and diseases diagnostics. Key advantages to RNA biochip assays include, but are not limited to speed, sensitivity, and point-of-care diagnostics by reducing sample volume and assay time compared to traditional methods like quantitative realtime polymerase chain reactions (qRT-PCR). The present disclosure further optimizes these assays by targeting extracellular vesicle (EV) subpopulations comprising specific oligonucleotides and polypeptides which can be indicative of specific disease states, including but not limited to cancer.

[0057] Extracellular vesicles (EVs) are membrane-derived vesicles enclosed by phospholipid bilayers. EVs include vesicles of varied sizes from the smallest exosomes to the largest apoptotic bodies. EVs express various receptor and ligand molecules from source cells and are capable of interacting with target cells through these molecules. As EVs harbor various cargos such as proteins, mRNAs, and miRNAs derived from source cells, EVs are considered to be important mediators of intercellular communications. EVs bear functional and structural resemblance to synthetic drug carriers similar to liposomes, exosomes can be further used for drug delivery.

[0058] In some aspects, disclosed herein is a biochip assay comprising a titanium (Ti) and gold (Au) coated biochip, wherein the Ti and Au coated biochip comprises a linker, a neutravidin coating, a biotin conjugated capture antibody, and a molecular beacon (MB) probe, wherein the biochip assay comprises a) contacting a liquid sample with the Ti and Au coated biochip, wherein the liquid sample comprises a subpopulation of extracellular vesicles (EVs), b) binding the MB probe to at least one mRNA, at least one miRNA, or a combination thereof located on an extracellular vesicle (EV), c) imaging the EV using an imaging modality to detect the at least one mRNA, at least one miRNA, or a combination thereof, wherein the at least one mRNA comprises Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); and / or at least one microRNA (miRNA) comprising miR-9- 5p, miR-124-3p, miR122-5p or miR1246-5p.

[0059] In some embodiments, the biochip assay comprising the linker comprises 20- tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH. In Docket No. 103362-036WO1 some embodiments, the linker of any preceding aspect further comprises PEG-SH. In some embodiments, the biochip assay comprising the biotin conjugated capture antibody binds to one or more surface proteins, including but not limited to CD63 and CD9, located on the EV.

[0060] In some embodiments, the biochip assay comprising the MB probe comprises one or more fluorescent dyes. Non-limiting examples of fluorescent dyes used in any disclosed aspect herein includes luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFPl), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, yellow fluorescent protein for energy transfer (YPet), super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T-Sapphire, Fucci, mK02, m0range2, m Apple, Sirius, Azurite, EBFP, EBFP2, Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 561, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, BODIPY FL, Courmarin, Cy3, Cy5, Fluorescein (FITC), Oregon Green, Pacific Blue, Pacific Green, Pacific Orange, PE-Cyanine7, PerCP-Cyanine5.5, Tetramethylrhodamine (TRITC), Texas Red, DAPI, Propidium Iodide, SYTO 9, SYTOX Green, TO-PRO-3, Allophycocyanin (APC), R-Phycoerythrin (R-PE), Cyan Fluorescent Protein (CFP), Green Fluorescent Protein (GFP), Red Fluorescent Protein (RFP), Blue Fluorescent Protein (BFP), 1,5 IAEDANS; 1,8-ANS; 4- Methylumbelliferone; 5-carboxy-2,7- dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein; 5- Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy- X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2- methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITS A; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; Docket No. 103362-036WO1 APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP / GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bisBTC; Blancophor FFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492 / 515; Bodipy493 / 503; Bodipy500 / 510; Bodipy; 505 / 515; Bodipy 530 / 550; Bodipy 542 / 563; Bodipy 558 / 568; Bodipy 564 / 570; Bodipy 576 / 589; Bodipy 581 / 591; Bodipy 630 / 650-X; Bodipy 650 / 665-X; Bodipy 665 / 676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl -Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP / YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Di chlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Diehl orodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC 18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635- NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™ / Fluo-3; Fura-2; Fura-2 / BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation Docket No. 103362-036WO1 (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein / Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow / Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE- TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO- 3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF 1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; Docket No. 103362-036WO1 SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; Y0-PR0-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.

[0061] In some embodiments, the biochip assay comprising the MB probe comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance. In some embodiments, the MB probe comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more LNAs. As used herein, the term “locked nucleic acid (LNA)”, also referred to as “bridged nucleic acid”, refers to a modified nucleic analog where a methylene bridge between the 2’-0 and 4’carbon atoms of the ribose sugar “locks” the sugar rings into a confirmation ideal for complementary binding. The structural rigidity of the LNA increases the LNA’s binding affinity to RNA, resulting in higher thermal stability (i.e., melting temperatures). It should be noted that LNAs can be incorporated into oligonucleotides (such as primers and probes) to enhance their stability, increase nuclease resistance, and increased specificity to target sequences.

[0062] In some embodiments, the biochip assay comprising the fluorescent imaging modality includes, but is not limited to total reflection fluorescence microscopy (TIRFM).

[0063] In some embodiments, the biochip assay comprising the liquid sample comprises blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

[0064] Methods of using asiEVRNA biochip

[0065] The present disclosure also provides methods detecting, diagnosing, and / or treating a subject with GBM using aS1EVRNA biochip or an assay thereof.

[0066] In some aspects, disclosed herein is a method of detecting a signature of glioblastoma (GBM) in a subject, the method comprising a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; and c) detecting the signature of GBM, Docket No. 103362-036WO1 wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); and / or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122-5p or miR1246-5p.

[0067] In some aspects, disclosed herein is a method of diagnosing a glioblastoma (GBM) in a subject, the method comprising: a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i)a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; c) detecting a signature of GBM, and d) diagnosing the subject with a GBM when an area under the curve (AUC) from the signature is at least 0.8 compared to a control subject, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N-ethylmaleimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); and / or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122-5p or miR1246-5p.

[0068] In some embodiments, the method of any preceding aspect detects and / or diagnosis a grade I, grade II, grade III, and / or grade IV of glioblastoma. Thus, the method of any preceding aspect can detect and / or diagnose slow growing glioblastoma tumors, intermediate or moderately growing glioblastoma tumors, and aggressive or fast-growing glioblastoma tumors.

[0069] In some aspects, disclosed herein is a method of treating a glioblastoma (GBM) in a subject in need thereof, the method comprising: a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC 14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; c) detecting a signature of GBM, d) diagnosing the subject with a GBM when an area under the curve (AUC) from the signature is at least 0.8 compared to a control subject, and e) administering to the subject an anti -cancer agent to decrease the GBM relative to an untreated control, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N- ethylmaleimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); and / or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122-5p or miR1246-5p.

[0070] In some embodiments, the method of any preceding aspect treats and / or prevents a grade I, grade II, grade III, and / or grade IV of glioblastoma. Thus, the method of any preceding aspect Docket No. 103362-036WO1 can treats and / or prevents a slow growing glioblastoma tumors, intermediate or moderately growing glioblastoma tumors, and aggressive or fast-growing glioblastoma tumors.

[0071] In some embodiments, the method of any preceding aspect comprises the biotin conjugated capture antibody binding to an EV subpopulation from the liquid sample. In some embodiments, the method of any preceding aspect comprises the biotin conjugated capture antibody binding to one or more surface proteins, including but not limited to CD63 and CD9 located on the EV.

[0072] In some embodiments, the method of any preceding aspect comprises the MB probe binding the least one mRNA or the at least one miRNA. In some embodiments, the method of any preceding aspect comprises the MB probe comprising one or more fluorescent dyes for a fluorescent imaging modality. In some embodiments, the method of any preceding aspect comprises the MB probe comprising one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

[0073] In some embodiments, the method of any preceding aspect comprises the fluorescent imaging modality comprising total reflection fluorescence microscopy (TIRFM). In some embodiments, the method of any preceding aspect comprises the liquid sample comprising blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

[0074] The method of any preceding aspect applies to glioblastoma and any grade or stage thereof. In some embodiments, the method of any preceding aspect further comprises administering an anti-cancer agent includes, but is not limited to a chemotherapeutic agent, a small molecule inhibitor, an immunotherapeutic agent, a natural anti-cancer toxin, or a designed anti -cancer toxin.

[0075] Exemplary biotherapeutic anti-cancer agents include, but are not limited to, interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and / or immunomodulatory agents (e.g., IL- 1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)).

[0076] Exemplary chemotherapeutic agents include, but are not limited to, anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), antiandrogens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfm (BPD- MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, Docket No. 103362-036WO1 vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA- paclitaxel, Taxoprexin), polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel -EC- 1 (paclitaxel bound to the erbB2-recognizing peptide EC- 1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9- aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5- fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. l-methyl-4- phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AGO 13736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK / ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS- 690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and / or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, Docket No. 103362-036WO1 temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, caminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, caminomycin, aminopterin, and hexamethyl melamine.

[0077] Some examples of small molecule inhibitors of cancer include but are not limited to Imatinib (Gleevec) (BCR-ABL, PDGFR, SCF, KIT Inhibitor), Dasatinib (Sprycel) (BCR-ABL, SRC family (SRC, LCK, YES, FYN), and KIT, EPHA2, PDGFRp Inhibitor), Nilotinib (Tasigna) (BCR-ABL, PDGFRB, KIT Inhibitor), Bosutinib (Bosulif) (BCR-ABL, SRC-family (SRC, LYN, and HCK) Inhibitor), Ponatinib (Iclusig) (BCR-ABL, BCR-ABL (T315I), VEGFR, PDGFR, FGFR, EPH receptors, SRC families of kinases, KIT, RET, TIE2, FLT3 Inhibitor), Asciminib (Scemblix) (BCR-ABL, BCR-ABL (T315I) Inhibitor), Ripretinib (Quinlock) (KIT, PDGFRA, PDGFRA mutations, PDGFRB, TIE2, VEGFR2, BRAF Inhibitor), Avapritinib (Ayvakit) ( KIT, KIT D816V, KIT exon 11, 11 / 17, and 17 mutants, PDGFRA and PDGFRA D842 mutants, PDGFRB, and CSFR1 Inhibitor), Gefitinib (Iressa) (EGFR and HER family Inhibitor), Erlotinib (Tarceva) (EGFR and HER family Inhibitor), Afatinib (Gilotrif) (EGFR and HER family Inhibitor), Osimertinib (Tagrisso) (EGFR and HER family Inhibitor), Dacomitinib (Vizimpro) (EGFR and HER family Inhibitor), Mobocertinib (Exkivity) (EGFR and HER family Inhibitor), Lapatinib (Tykerb) (EGFR and HER family Inhibitor), Neratinib (Nerlynx) (EGFR and HER family Inhibitor), Tucatinib (Tukysa) (EGFR and HER family Inhibitor), Osimertinib (Tagrisso) (EGFR and HER family Inhibitor)., Crizotinib (Xalkori) (ALK, HGFR, c-Met, ROS1, RON), Ceritinib (Zykadia) (ALK, IGF-1R, InsR, ROS1), Alectinib (Alecensa) (ALK, RET), Brigatinib (Alunbrig) (ALK, ROS1, IGF-1R, FLT-3, EGFR deletion and point mutations inhibitors), Lorlatinib (Lorviqua) (ALK, ROS1, TYK1, FER, FPS, TRKA, TRKB, TRKC, FAK, FAK2, ACK inhibitor), Capmatinib (Tabrecta) (MET, MET exon 14 skipping inhibitor), Tepotinib (Tepmetko) (MET, MET exon 14 skipping inhibitor), Pralsetinib (Gavreto) (wild-type RET, oncogenic RET fusions (CCDC6-RET), RET mutations (RET V804L, RET V804M and RET M918T) inhibitor), Selpercatinib (Retevmo) (wild-type RET, multiple mutated RET isoforms inhibitor), Erdafitinib (Balversa) (FGFR1, FGFR2, FGFR3, FGFR4, RET, CSF1R, PDGFRA, PDGFRB, FLT4, KIT, VEGFR2 inhibitor), Pemigatinib (Pemazyre) (FGFR1, FGFR2, FGFR3 inhibitor), Infigratinib (Truseltiq) (FGFR1, FGFR2, FGFR3, FGFR4 inhibitor), Larotrectinib (Vitrakvi) (NTRK1, NTRK2, NTRK3 inhibitor), Entrectinib (Rozlytrek) (NTRK1, NTRK2, NTRK3, ROS1, ALK, Docket No. 103362-036WO1 JAK2, TNK2 inhibitor), Midostaurin (Rydapt) (FLT3 inhibitor), Gilteritinib (Xospata) (FLT3 inhibitor), Pexidartinib (Turalio) (CSF1R, KIT, FLT3 with ITD mutation Inhibitor), Vemurafenib (Zelboraf) (mutated forms of BRAF, wild-type BRAFCRAF, ARAF, SRMS, ACK1, MAP4K5, FGR inhibitor), Dabrafenib (Tafinlar) (BRAF V600E, BRAF V600K, and BRAF V600D, wildtype BRAF, CRAF, SIK1, NEK11, LIMK1 inhibitor), Encorafenib (Braftovi) (BRAF V600E, wild-type BRAF, CRAF, JNK1, JNK2, JNK3, LIMK1, LIMK2, MEK4, STK36 inhibitor), Trametinib (Mekinist) (MEK1, MEK2 Inhibitor), Cobimetinib (Cotellic) (MEK1, MEK2 Inhibitor), Binimetinib (Mektovi) (MEK1, MEK2 Inhibitor), Selumetinib (Koselugo) (MEK1, MEK2 Inhibitor), Idelalisib (Zydelig) (PI3K5 inhibitor), Copanlisib (Aliqopa) (PI3Ka, PI3K5 Inhibitor), Duvelisib (Copiktra) ((PI3Ka, PI3K5 Inhibitor), Alpelisib (Piqray) (PI3Ka inhibitor), Umbralisib (Ukoniq) (PI3K5, CKls inhibitor), Ruxolitinib (Jakafi) (JAK1, JAK2 inhibitor), Fedratinib (Impact) (JAK2 inhibitor), Palbociclib (Ibrance) (CDK4, CDK6 Inhibitor), Ribociclib (Kisqali) (CDK4, CDK6 Inhibitor), Abemaciclib (Verzenio) (CDK4, CDK6 Inhibitor), Trilaciclib (Cosela) (CDK4, CDK6 Inhibitor), Ibrutinib (Imbruvica) (BTK Inhibitor), Acalabrutinib (Calquence) (BTK Inhibitor), Zanubrutinib (Brukinsa) (BTK Inhibitor), Enasidenib (Idhifa) (IDH1 and IDH2), Ivosidenib (Tibsovo) (IDH1 and IDH2 Inhibitor), Tirbanibulin (Klisyri) (SRC Inhibitor).

[0078] Some examples of immunomodulatory agents include but are not limited to Amivantamab, Cetuximab, Nimotuzumab, Panitumumab, Bevacizumab, Ramucirumab, Nivolumab, Pembrolizumab, Cemiplimab, Atezolizumab, Avelumab, Durvalumab, Ertumaxomab, Margetuximab, Pertuzumab, Trastuzumab, Trastuzumab duocarmazine, Trastuzumab emtansine, Rituximab, Human or humanized anti-CD20 antibodies, Ibritumomab, Brentuximab (+mono methyl auristatin E), Alemtuzumab, and Ipilimumab (Yervoy).

[0079] Exemplary natural or designed anti-cancer agents include but are not limited to Pseudomonas exotoxin A (PE, ETA), Diphtheria toxin (DT), Ricin, Shiga toxin (Stx), Abrin, Barnase, Binase, Anthrax toxin, KillerRed, miniSOG, Granzyme B, Botulinum neurotoxin, Listeriolysin O or Streptolysin-O.

[0080] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

[0081] By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. Docket No. 103362-036WO1

[0082] EXAMPLES

[0083] The following examples are set forth below to illustrate the compositions, devices, 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 disclosure which are apparent to one skilled in the art.

[0084] Example 1: Single extracellular vesicular RNA signature as diagnostic biomarkers for glioblastoma detection.

[0085] Glioblastoma multiform (GBM) is a highly lethal primary brain cancer that is resistant to conventional immunotherapies. However, currently, there is a lack of reliable biomarkers for clinical practice. Extracellular vesicles (EVs) present in biofluids are reliable biomarkers for cancer diagnosis. Herein, a non-invasive biochip assay was developed to detect single EV (siEV) mRNA and miRNA expressions and evaluated them as GBM biomarkers. EVs from cell culture medium, healthy control serum, and GBM patient serum were purified using tangential flow filtration (TFF). Morphology, size, and numbers of EVs were characterized by TEM and qNano, respectively. EVs from healthy control and GBM patient serum were compared using bulk mRNA and miRNA sequencing analysis. mRNA and miRNA were selected as GBM biomarkers and the expression in siEVs were quantified by a fluorescent nucleic acid detection method using a single EV RNA biochip assay (S1EVRNA). TheS1EVRNA biochip assay has been benchmarked against standard PCR methods and shown to be 100- to 10,000- fold higher in sensitivity across a variety of RNAs. The AUC of ROC for siEV NRGN, TMEM30C, NCAN and NSF expression was 0.8 ~ 0.9, when using siEV RNA as single biomarker. Notably, calculating the ratio using up-regulated siEV mRNA with down-regulated siEV NRGN resulted in a significant increase in AUC (> 0.9). In contrast, the AUC of ROC for miR-9-5p, miR-124-3p, miR-122-5p and miR-1246-5p was 0.7-0.8, when using siEV RNA as single biomarker. Interestingly, combining miR-9-5p with ratio of mRNA / NRGN, the AUC / ROC reached 1.000 for NCAN / NRGN, NSF / NRGN and TMEM30C / NRGN, showing an outstanding level for GBM diagnosis. The non-invasiveS1EVRNA biochip assay outperformed qRT-PCR for EV RNA detection. The miRNA and mRNA signatures established herein can be used for blood-based GBM diagnosis.

[0086] Introduction

[0087] Liquid biopsy is a non-invasive technique that provides the opportunity of detecting, analyzing and monitoring circulating biomarkers in various body fluids such as blood or CSF Docket No. 103362-036WO1 instead of cancer tissue. GBM biomarkers were evaluated and measured in liquid biopsies, including CTCs, ctDNA and extracellular vesicles. The CTC detection rate in GBM patients ranged from 20% to 77% depending on the CTC isolation method. Therefore, standardized protocols for CTC isolation and characterization are urgently needed. It has been found that ctDNA was able to detect many of the same mutations as the primary tumor in GBM patients. However, the release of ctDNA into the bloodstream is relatively limited due to the blood brain barrier (BBB). As a result, the detection rates of ctDNA are often low, making it more challenging to detect tumor-specific mutations. In contrast, extracellular vesicles (EVs) have shown promise as tumor biomarker. EVs are membrane-bound vesicles that are secreted by cells under physiological conditions and mediate intercellular communication. EVs carry a variety of cargoes, including mRNA, miRNA, DNA, and proteins, and these cargoes are protected within EVs. Studies have revealed EVs as biomarkers for GBM diagnosis. EGFRvIII protein, the most common oncogenic variant of the GBM receptor, has been demonstrated in EVs from glioma cells and cell lines transfected with EGFRvIII. EV miR-21 was one of the first miRNAs shown in GBM patients for diagnostic purposes. Consequently, more serum exosomal miRNAs, such as miR- 301a, miR-29b, miR-222, miR-124-3p, and miR-9-5p, are evaluated as biomarkers in GBM patients. Exosomal microRNAs have shown potential as GBM biomarker, but microRNAs as a single biomarker are not sufficient to distinguish between GBM patients and healthy donors. Furthermore, multiple EV microRNAs combined into classifiers or signatures were evaluated in GBM diagnosis and better distinguished GBM patients from healthy donors compared to single miRNAs. However, there are currently no biomarkers or clinical features that can be used to detect GBM.

[0088] In order to screen the EV RNA biomarkers in GBM diagnosis, transcriptomic analyses were performed using twenty GBM patients and twenty healthy donors by sRNA-seq and microarrays. Four mRNAs, including Neurogranin (NRGN), Neurocan (NCAN), N- ethylmal eimide sensitive factor (NSF) and transmembrane 30C (TMEM30C) and four miRNAs, including miR-9-5p, miR-124-3p, miR122-5p and miR1246-5p were selected from the sequencing results and literatures. Using a single EV RNA biochip assay (S1EVRNA), mRNA and miRNA expression was measured in CD63 and CD9 positive EV subpopulations and further evaluated as GBM biomarkers. TheS1EVRNA biochip assay demonstrated 100 to 10,000-fold higher sensitivity compared to standard PCR methods. When using mRNA or miRNA as a single biomarker, the AUC of ROC was between 0.8 and 0.9, whereas when combining mRNA / NRGN ratio and miR- 9-5p as a signature, a perfect AUC of ROC was achieved. Therefore, the miRNA and mRNA signatures established byS1EVRNA biochip assay can be used for blood-based GBM diagnosis. Docket No. 103362-036WO1

[0089] Results

[0090] Single extracellular vesicular RNA (siEVRNA) biochip assay

[0091] The scheme of a thin titanium / gold (Ti / Au) coated single extracellular vesicle RNA (S1EVRNA) biochip assay showed in Figure 1 A. A glass coverslip, coated with a nanometer layer of Ti / Au, was treated with a linker solution of 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa- tricontane-1 -thiol (WC14) and biotin-PEG-SH in / ?-mercaptoethanol. The coverslip was then assembled to a plastic spacer featuring an array of 4 mm wells. To construct theS1EVRNA biochip assay, neutravidin solution was introduced and anchored onto the biochip surface through biotin- neutravidin interactions. Then, biotin conjugated capture antibodies were applied to adhere to the biochip through biotin-neutravidin interactions, facilitating the capture of a selected EV subpopulation from a liquid sample. After washing, detection of a target RNA within the captured EV subpopulation using molecular beacon (MB) probe. Lastly, high-resolution total internal reflection fluorescence (TIRF) microscopy was employed to record one hundred fluorescence images in a single well. Then, fluorescence image analysis was performed through a computer algorithm that generates a fluorescence intensity histogram of the bright spots detected in the TIRF images. A suitable histogram cutoff was established to minimize the influence of background noises and to emphasize the EVs containing the bright sports in the images.

[0092] A biochip coated with a thin gold layer and PEG treatment could sensitively quantify target RNAs in EVs for an early cancer diagnosis. Nguyen et al also showed that Ti-Au coated biochip using capture antibodies could detect single-EV mRNA biomarkers in predicting patient responses to immunotherapy (Nguyen, L. T. H., Zhang, J., Rima, X. Y., Wang, X., Kwak, K. J., Okimoto, T., Amann, J., Yoon, M. J., Shukuya, T., Chiang, C. L., Walters, N., Ma, Y., Belcher, D., Li, H., Palmer, A. F., Carbone, D. P., Lee, L. J., & Reategui, E. (2022). An immunogold single extracellular vesicular RNA and protein (Au SERP) biochip to predict responses to immunotherapy in non-small cell lung cancer patients. Journal of Extracellular Vesicles, 11(9), el2258). In both designs, EVs were captured on the biochip using cationic lipoplex nanoparticles (CLNs) via electrostatic fusion, and EV RNA cargos were detected with MBs encapsulated within the CLNs. The present disclosure provides free MBs were used instead of MB encapsulated in CLNs decreased non-specific binding caused by CLNs. To enhance MB to EV internalization, MBs were diluted with TE buffer. Without using streptavidin-conjugated gold NP, the total cost was dramatically reduced.

[0093] EVs from GBM patient serum was isolated using tangential flow filtration (TFF) and then characterized by SEM (Figure IB) and qNANO (Figure 1C). Compared to other EV isolation Docket No. 103362-036WO1 methods (UC, SEC and TEI), TFF can remove 98% human serum albumin in the patient serum. EVs had a mean particle size of -185 nm. To maximize EV capture, CD63 and CD9 expressions were determined in EVs from HD and GBM patient serum. Both HD and GBM patients showed similar CD63 and CD9 expression (Figure ID). The GBM biomarker, NCAN expression was compared after EVs captured with individual CD63, CD9 and CD81. Since CD81 captured EVs had a lower NCAN mRNA expression than CD63 or CD9 captured EVs in GBM cells and GBM patient serum, CD63 and CD9 were chosen as capture antibodies (Figure IE) and an antibody mixture of CD63 / CD9 with an equal weight ratio was used in subsequent studies because an antibody mixture is more effective than an individual antibody in patient serum derived EVs (Figure 7). Using a CD63 / CD9 mixture as a capture antibody, EV NCAN mRNA expression was significantly higher in GBM cell (Gli36 and SNB75) EVs than in human normal microglial cell (HMC3) EVs (Figure IF), showing thatS1EVRNA biochip assay can be used for mRNA biomarker evaluation.

[0094] EV RNA profile in GBM patients compared to healthy control

[0095] Several studies of exosomal miRNA in GBM patients have been reported, however, these studies were focused on predefined miRNAs and relatively small groups of patient samples. A comprehensive analysis of mRNA and miRNA profiles in serum EVs from GBM patients has not been performed. Herein, circulating EV mRNA and miRNA profiles from patients with histopathologically confirmed IDHWTGBM (n = 20) were compared to age- and gender-matched healthy controls (n = 16 - 20) via microarrays and sRNA-seq. The differentially expressed mRNA and miRNA were identified if their differential expression met a fold change (FC) > 1.5 in either direction or unadjusted p values < 0.05. It was found that 165 and 1508 mRNAs were significantly up- and down- regulated in GBM patients compared to healthy control, respectively. After analyzing 37 highly differentiated and down-regulated mRNAs (Figure 2A), NRGN (FC = - 4.25), which is only associated with brain function, was discovered. Among the highly differentiated and up-regulated mRNAs (FC>2), TMEM30C was selected because it is located in an integral component of the membrane and showed a high AUC of ROC (Figure 2A). In addition, NCAN and NSF mRNAs were also selected even though they are not highly differentiated mRNAs. EV mRNA profile from GBM patients and healthy control showed the differences in NCAN expression between GBM patients and healthy control (PO.01), but notNSF expression (P>0.05). NCAN and NSF mRNAs was previously discovered by GBM cells and GBM cell EVs and demonstrated as GBM biomarkers. The heatmap and AUC of ROC for selected mRNAs including NRGN, TMEM30C, NCAN and NSF are shown in Figure 2B and C and consequently evaluated as a biomarker byS1EVRNA biochip assay. Docket No. 103362-036WO1 Comparing miRNA expression profiles, 200 and 6 miRNAs were identified as significantly up- and down- regulated in GBM patients compared to healthy control, respectively (Figure 2A). After analyzing 10 highly differentiated and upregulated miRNAs (Figure 8), miR- 122-5p was selected. miR-122-5p has been studied extensively as a biomarker for other cancers, it has not been evaluated as a GBM biomarker. The other differentially expressed miRNAs identified herein had been previously evaluated as GBM biomarkers. miR-9-5p and miR-1246-5p were also selected because both miRNAs were highly expressed in GBM cell EVs compared to GBM cells. Unfortunately, miR-9-5p or miR-1246-5p expressions were not detected in some patients and healthy control samples using sRNA-sequencing studies. This discrepancy in the expression of miR-9-5p and miR-1246-5p between GBM cell EVs and GBM serum EVs, may be due to low serum EV miR-9-5p or miR-1246-5p concentrations. Some studies have reported that miR-124-3p in serum exosomes of high-grade glioma (HGG) patients is significantly higher than that of low-grade glioma (LGG) and healthy individuals. Nevertheless, EV miR-124-5p expression was not detected by sRNA-sequencing. Furthermore, miR-9-5p, and miR-124-3p have been reported to be associated with brain function. Considering the siEV RNA biochip assay with 100 times higher sensitivity than standard qRT-PCR, 4 miRNAs include miR-122-5p, miR-9-5p, miR-124-3p and miR1246-5p were selected. The heatmap and the AUC of ROC for selected miRNAs including miR-122-5p, miR-9-5p, miR-124-3p and miR1246-5p are shown in Figure 2B and D.siEVRNA biochip assay enables single EV mRNA and miRNA detection with high sensitivity

[0096] Reverse transcription-quantitative PCR (RT-qPCR) has been employed as a standard method for detecting and quantifying RNA in both cell lines and clinical samples. Here, the expression of NRGN, TMEM30C, miR-122-5p and miR-9-5p expression were compared byS1EVRNA biochip assay and RT-qPCR method using three GBM cells and GBM cell derived EVs. RT-qPCR results showed that all three GBM cells had high Neurogranin (NRGN) and miR-9-5p expression while all three GBM cells had low transmembrane 30C (TMEM30C) and miR-122-5p expression (Figures 3 A, 3B, 3C, and 3D). In contrast, EVs derived from three GBM cells exhibited low NRGN, miR-122-5p and miR-9-5p expression with Ct of > 33 when using IxlO10EVs. TMEM30C expression was not detected by RT-qPCR when using IxlO10EVs (Figures 3A, 3B, 3C, and 3D). UsingS1EVRNA biochip assay, all four RNA expressions were detected at single EV levels with high mRNA expressions for NRGN, TMEM30C and miR-122-5p (Figures 3A, 3B, and 3D) and low miRNA expression for miR-9-5p (Figure 3C).

[0097] Next, various amounts of EVs derived from BNB75 cells were spiked in 1 mL of healthy donor (HD) serum to evaluate the linearity and limit of detection (LOD) by RT-qPCR andS1EVRNA Docket No. 103362-036WO1 biochip assay.S1EVRNA biochip showed a good linearity in the EV concentration range of 1E7 to 1E11 EVs / mL for NRGN (Figure 3E), TMEM30C (Figure 9A), miR-9-5p (Figure 3F) and miR- 122-5p (Figure 9B) expression with an LOD of 1E7 / mL for 4 RNAs. The qRT-PCR measurements for averaged EV RNA expressions are also given in Figures 3E, 3F and Figure 9A and 9B. Detection limit for NRGN and TMEM30C mRNAby RT-qPCR was 1E11 EVs / mL while miR-9-5p and miR-122-5p by RT-PCR was 1E8 and 1E10 EVs, respectively. These results showed that theS1EVRNA biochip assay is at lease more than 100-times more sensitive than those standard methods. mRNA expression in GBM patient serum bysiEVRNA biochip assay

[0098] Next, the four mRNA (NRGN, TMEM30C, NCAN and NSF) were evaluated as GBM biomarkers usingS1EVRNA biochip assay. A cohort of GBM patient serum with stage IV (n = 37 from MD Anderson medical center) and Healthy control (n = 20 from Zenbio, Inc) were chosen for the investigation. Clinical characteristics of those patients are given in Table 1. EVs from serum samples were purified using TFF and 20 pL of the purified EVs was processed with theS1EVRNA biochip assay. Representative TIRF images for NRGN, TMEME30C, NCAN and NSF are shown in Figures 4A, 4B, 4C, and 4D. Among the four mRNAs, RFI of siEV NRGN expression was significantly decreased in GBM patient serum compared to HDs (Figure 4A, p < 0.001). In contrast, RFI of TMEM30C (Figure 4B), NCAN (Figure 4C) and NSF (Figure 4D) expression was significantly increased in patient with GBM compared to HDs (p < 0.001 for all three mRNA). As a single biomarker, the AUC of ROC reached to 0.849, 0.900, 0.886 and 0.837 for NRGN, NCAN, NSF and TMEM30C, respectively (Figure 4E). No difference was observed in NSF mRNA expression between GBM patients and healthy control by RNA microarray sequencing.

[0099] Since RFI of NRGN mRNA expression decreased in siEVs from GBM patient serum compared to HDs, ratio was calculated by dividing RFI of NCAN, NSF and TMEM30C with RFI of NRGN to maximize differences between GBM patients and HDs. The ratio of TMEM30C / NRGN (Figure 4E), NCAN / NRGN (Figure 4F) and NSF / NRGN (Figure 4G) discriminated GBM patient from HDs well with AUC / ROC of 0.950, 0.925 and 0.920, respectively (Figure 41). miRNA expression in GBM patient serum bysiEVRNA biochip assay miRNA as a GBM biomarker was also evaluated in EVs from GBM patients and healthy donors usingS1EVRNA biochip assay. Representative TIRF images for miR-124-3p, miR-9-5p, miR-1246-5p and miR-122-5p are shown in Figures 5A, 5B, 5C, and 5D. miRNA expression was successfully detected including miR-9-5p and miR-124-3p. RFI of miR124-3p (Figure 5A), miR- Docket No. 103362-036WO1 9-5p (Figure 5B) and miR-1246-5p (Figure 5C) in EVs were significantly increased in GBM patient serum compared to HDs (p < 0.001). RFI of miR-122-5p expression (Figure 5D) was also increased in EVs from GBM compared to HDs (p < 0.05). As a single biomarker, AUC of ROC reached to 0.811, 0.770, 0.799 and 0.732 for miR-124-3p, miR-9-5p, miR-1246-5p and miR-122- 5p, respectively (Figure 5E). When 2, 3, and 4 miRNAs were combined as signature, the highest AUC of ROC was 0.8730, 0.8865, 0.9041 for miR124-3p / miR-1246-5p, miR124-3p / miR-1246- 5pmiR-9-5p and miR124-3p / miR-1246-5p / miR-9-5p / miR-122-5p, respectively. mRNA and miRNA combination for GBM detection

[0100] Then it was further contemplated whether combination of mRNA and miRNA could be good predictors for GBM diagnosis withS1EVRNA assay. There is not any report that mRNA and miRNA combination as cancer or GBM biomarker. Since mRNA / NRGN ratio revealed a higher AUC than single mRNA (Figures 4E, 4F, and 4G), a ROC curve analysis in combining of mRNA / NRGN ratio with miRNA were performed. Among the four miRNA tested, miR-9-5p itself is considered acceptable biomarker with AUC / ROC of 0.770 (Figure 5B). Interestingly, combining miR-9-5p with ratio of mRNA / NRGN, the AUC / ROC reached to 1.000 for NCAN / NRGN, NSF / NRGN and TMEM30C / NRGN (Figure 6A), showing an outstanding level for cancer diagnosis. The scatter plot in Figures 6B, 6C, and 6D show that all HD samples had both low miR-9-5p expression and low mRNA / NRGN ratio (i.e. L / L) in serum, but GBM patients revealed either high miR-9-5p and high ratio of mRNA / NRGN (i.e. H / H), high miR-9-5p and low mRNA / NRGN ratio (i.e. H / L), or low miR-9-5p and high mRNA / NRGN ratio (i.e. L / H) in serum. To distinguish patients with GBM from HD in clinical setting, the distinct cut-off values of RFI were determined. The cut-off values for TMEM30C / NRGN (Figure 6B), NCAN / NRGN and NSF / NRGN were 2.34, 2.00, and 1.57, respectively. miR-124-3p showed considerably higher accuracy in differentiation between GBM patients and healthy donors (AUC = 0.811), showing the siEV miR-124-3p as a better biomarker for GBM diagnosis. Combining miR-124-3p with the mRNA / NRGN ratio, the AUC of ROC was 0.968, 0.950, and 0.967 for NCAN / NRGN, NSF / NRGN and TMEM30C / NRGN, respectively (Figure 10A). The scatter plot in Figures 10B, 10C, and 10D show that all HD samples had both low miR-124-3p expression and low mRNA / NRGN ratio (i.e. L / L) in serum siEVs. However, some GBM patient samples also had low miR-124-3p expression and low mRNA / NRGN ratio (i.e. L / L) in serum siEVs.

[0101] Similar to miR-124-3p, improved diagnostic efficacy was observed when combining miR- 122-5p or miR1246-5p with the mRNA / NRGN ratio. The AUC of ROC was 0.984, 0.981 and 0.978 for NCAN / NRGN, NSF / NRGN and TMEM30C / NRGN combined with miR-122-5p, Docket No. 103362-036WO1 respectively (Figures 11 A, 1 IB, 11C, and 1 ID). Combining miR1246-5p with the mRNA / NRGN, the AUC of ROC reached 0.968, 0.967 and 0.950 for NCAN / NRGN, NSF / NRGN and TMEM30C / NRGN, respectively (Figures 12A, 12B, 12C, and 12D).

[0102] Discussion

[0103] Herein, it was demonstrated that a non-invasiveS1EVRNA biochp assay for siEV mRNA and siEV miRNA detection has higher sensitivity than qRT-PCR. Using mRNA and miRNA profile data, four mRNA (NRGN, TMEM30C, NCAN and NSF) and four miRNA (miR-122-5p, miR-9-5p, miR124-3p and miR-1246-5p) were selected as GBM biomarker were further evaluated usingsiEVRNA biochip assay. AUC of ROC for NRGN, TMEM30C, NCAN and NSF mRNA was 0.8 ~ 0.9, when using siEV mRNA as single biomarker. The combination of three up-regulated siEV mRNA as signature (TMEM30C / NCAN / NSF) improved the diagnostic accuracy, yielding an AUC of 0.974 (95% CI 0.9420-1.007, p < 0.0001). Surprisingly, calculating the ratio using up- regulated siEV mRNA with down-regulated siEV NRGN resulted in a significant increase in AUC (> 0.9). In contrast, miRNA (miR-122-5p, miR-9-5p, miR-124-3p and miR-1246-5p) as a single biomarker or signature discriminated between GBM patients and healthy controls, but had a lower AUC than mRNA. Consistent with these results, the combination of exosome miR-21, miR-222 and 124-3p improved the diagnostic accuracy, yielding an AUC of 0.87. Surprisingly, combining miRNA and mRNA / NRGN ratio significantly improved the AUC of ROC. Moreover, a perfect AUC of ROC was obtained when combining miR-9-5p with ratio of NCAN / NRGN, TEME30 / NRGN and NSF / NRGN.

[0104] Extensive studies have reported that most miRNAs are involved in GBM. miR-9-5p is overexpressed in glioblastoma stem cell line (NCH421k) and corresponding sEVs compared to healthy astrocytes. Additionally, in patients with GBM and low-grade glioma, higher miR-9-5p expression was associated with improved survival (p = 0.012). Therefore, EV miR-9-5p has been identified as a promising candidate for GBM diagnosis and prognosis. The results herein demonstrated that miR-9-5p itself is considered acceptable biomarker with AUC / ROC of 0.770. When combined with the mRNA / NRGN ratio, miR-9-5p completely distinguished GBM patients from healthy controls. miR-124-3p is one of the most abundantly expressed miRNAs in neural tissue and overexpressed in the sEVs of GBM stem cell lines (NCH644) compared to the sEVs of all other GBM cell lines and astrocytes. In addition, several studies have found that miR-124-3p is downregulated in GBM, and this alters tumor cell growth, survival, migration, and chemoresistance. Although miR-124-3p by itself or combined with mRNA / NRGN ratio showed a Docket No. 103362-036WO1 relatively high AUC in the present disclosure, miR-124-3p is a more suitable candidate for GBM prognosis and chemotherapy monitoring.

[0105] Meanwhile, miR-1246-5p and miR-122-5p were found to play an oncogenic role in breast, colon, lung, pancreas, and ovarian cancers, as well as melanoma and glioma. miR-1246-5p overexpression was previously demonstrated in GBM patient EVs. miR-122-5p overexpression was first evaluated in siEVs from GBM patient serum compared to healthy control. Since miR- 122-5p and miR-1246-5p were highly expressed in other cancers.

[0106] In conclusion, combining siEV miR-9-5p expression and mRNA / NRGN ratio can provide for GBM diagnosis.

[0107] Materials and Methods

[0108] RNA sequencing.

[0109] The mRNA profile was analyzed by microarray. Total RNA was isolated from EVs purified from about 600ul of serum 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). RNA from EVs derived from HD and GBM were analyzed using Agilent Human Whole Genome 8x60 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.

[0110] The sequencing with array based methods, the high number of reads mapped to various ribosomal and hemoglobin RNAs makes the sequencing based methods less efficient for EV mRNA characterization.

[0111] The miRNA profile was analyzed by small RNA sequencing. Total RNA was isolated from EVs purified from about 200ul of serum using the miRNeasy kit. 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 sizeselection 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 RNA was determined based on the number of mapped reads that were normalized with Count Per Mapped Million (CPM).

[0112] Cell culture Docket No. 103362-036WO1

[0113] U251 and Gli36 glioma cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM). SNB-75 glioma cell lines were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium. Cells were maintained in culture medium supplemented with 10% fetal bovine serum, 100 U / mL of penicillin and 100 pg / mL of streptomycin at 37°C in a 5% CO2 / 95% air humidified atmosphere. When EV collection, cells were washed with PBS three times and then incubated in serum-free media for 48 h. The EV-enriched cell culture media was collected and centrifuged at 2000 x g for 10 min at room temperature to separate cell debris before further analysis.

[0114] GBM patient serum collection

[0115] 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.

[0116] Healthy donor serum collection

[0117] 10 mL of whole blood from healthy donors was collected into BD Serum Separation Tubes (SST; Thermo Fisher Scientific). SSTs were gently placed upright to coagulate for 60 min after being rocked 10 times. The SSTs were centrifuged at room temperature at 1,100 x g for 10 min. The serum was stored in 1 mL aliquots at -80 °C. All blood samples were collected under an approved IRB at The Ohio State University (IRB #2018H0268). Another 20 healthy donor serum were purchased from Zenbio Inc.

[0118] EV purification by TFF

[0119] The EV-enriched cell culture media and serum samples were introduced into a TFF system as described by our previous technique to purify EVs. In brief, cell culture media or serum were centrifuged at 2500xg for lOmin and then the supernatant was circulated through a 500 kDa TFF hollow fiber filter cartridge, where EVs were retained and enriched in the system (~ 2 mL), while free proteins and nucleic acids permeated through the filter. Constant-volume diacycles of PBS were performed until pure EVs were obtained (350 mL of PBS). The EVPs were further enriched by centrifuging the sample within a 10 kDa centrifugal unit at 3000 x g at 4 °C until a final volume of 100 pL was achieved.

[0120] EV concentration and size detection

[0121] The qNano Gold (Izon Sciences, Boston, MA) was employed to quantify the size and concentration of EVs via NP200 (50 - 330 nm) nanopore membranes. A pressure of 10 mbar and a voltage of 0.48 were applied. Polystyrene nanoparticles (CPC 100) were used to calibrate the samples. Docket No. 103362-036WO1S1EVRNA biochip fabrication

[0122] A cleaned high precision glass coverslip (D263M Glass, 24x75 nm rectangle, 0.15 mm thickness, Schott AG, Germany) was first activated using a UV-ozone cleaner for 15 min. Thin layers of 2 nm thick Ti and 10 nm thick Au were sequentially deposited using a Denton-e-beam evaporator (DV-502A, Moorestown, NJ). The Au-coated glass was immersed into a linker solution (1%) in ethanol (200 Proof, Fisher Scientific) for overnight at RT. The linker solution composed with l-thiahexa(ethylene oxide) lipidic anchor molecule WC14 [20-tetradecyloxy- 3,6,7,12,15,18, 22-heptaoxahexa-tricontane-l -thiol], PEG-SH, biotin-PEG-SH and a lateral spacer P-mercaptoethanol (P-ME) (molar ratio = 5: 1 : 1 :93). The coverslip was then washed three times with ethanol and air-dried.

[0123] The treated glass coverslip was attached to a 64-well chamber (Grace Bio-Labs ProPlate® multi-well chamber, Sigma-Aldrich) and washed thoroughly with deionized (DI) water. Next, 50 pg / mL neutravidin in PBS were applied into each well of biochip for 30 min at RT on a shaker. After rinsing six times with PBS, the biochip was incubated with capture antibody mixture for overnight at 4°C. The antibody mixture includes 10 pg / mL of biotinylated anti-CD63 / anti-CD9 antibodies. After the antibodies were tethered onto the nanogold surface, the biochip was washed six times with PBS, and then blocked with 5% (w / v) BSA in PBS for 1 h at RT before EV capture. A concentration of 109particles / mL EVs (apart from dilution experiments, which employed 106- 1011parti cles / mL) were then added and allowed to tether to the antibodies for 2 h at room temperature.

[0124] MB design and RNA detection

[0125] MBs (listed 5'-3') targeting RNAs detected are provided in Table 2. 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.

[0126] For RNA detection, 10 pg / pL MBs diluted in a IX TE buffer were added to the EV captured gold biochip and incubated for 1 h at 37 °C.

[0127] 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 h at room temperature.

[0128] TIRF Image analysis

[0129] Images of fluorescently labeled siEVPs were obtained by TIRFM (Nikon Eclipse Ti Inverted Microscope System, Melville, NY) with a 100* oil immersion lens. An automatic algorithm was used to quantify the TIRFM images by detecting all bright signals determined via Docket No. 103362-036WO1 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 microdomain 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. qRT-PCR

[0130] Total RNA from cells and cell derived EVs were isolated using RNeasy mini kit (Qiagen, Inc.) according to the manufacturer’s instructions. cDNA was then synthesized from total RNA using a High-Capacity cDNA reverse transcription kit (#4368814, Applied Biosystems). Subsequently, the target mRNA expression was quantified using a TaqMan Gene Expression assay (ThermoFisher Scientific) on a real-time PCR instrument (Applied Biosystems). The primers for the target gene were listed in Table S4.

[0131] Statistical analysis

[0132] The MATLAB R2019a was used for data analysis. The data were expressed as mean ± SD. P values for pairwise comparisons were obtained using paired student’s t-test. To compare the difference in between groups, Kruskal Wallis post hoc test was used with Dunnet' s adjustment. To compare the difference between before and after operation, 1 -sample t-test was conducted to test whether the ratio equals to one. ROC curves were used to determine the sensitivity and specificity to compare AUCs of serum / plasma from different PDAC stages. The cutoff points were selected using Youden’s index, which maximizes the sum of sensitivity and specificity. All clinical samples were measured two times on a biochip.

[0133] It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. Docket No. 103362-036WO1

[0134] TABLES

[0135] Table 1. Clinical characteristics of GBM patients and healthy controls.

[0136] Table 2. Detailed information of Molecular beacon (listed 5’ -> 3’) sequence targeting RNAs. Docket No. 103362-036WO1

[0137] “+” indicates Locked Nucleic Acids (LNAs); “iCy3” refers to an internal cyanine fluorophore; “iCy5” refers to an internal cyanine fluorophore; and “3BHQ 2” refers to a Black Hole Quencher chromophore.

Claims

Docket No. 103362-036WO1CLAIMSWhat is claimed is:

1. A biochip assay comprising a titanium (Ti) and gold (Au) coated biochip, wherein the Ti and Au coated biochip comprises a linker, a neutravidin coating, a biotin conjugated capture antibody, and a molecular beacon (MB) probe, wherein the biochip assay comprises: a) contacting a liquid sample with the Ti and Au coated biochip, wherein the liquid sample comprises a subpopulation of extracellular vesicles (EVs), b) binding the MB probe to at least one mRNA, at least one miRNA, or a combination thereof located on an extracellular vesicle (EV), and c) imaging the EV using an imaging modality to detect the at least one mRNA, at least one miRNA, or a combination thereof, wherein the at least one mRNA comprises Neurogranin (NRGN), Neurocan (NCAN), N- ethylmal eimide sensitive factor (NSF), or transmembrane 30C (TMEM30C); and at least one microRNA (miRNA) comprises miR-9-5p, miR-124-3p, miR122-5p, or miR1246-5p.

2. The biochip assay of claim 1, wherein the linker comprises 20-tetradecyloxy- 3,6,7,12,15,18,22-heptaoxahexa-tricontane-l-thiol (WC14) and biotin-PEG-SH.

3. The biochip assay of claim 1 or 2, wherein the biotin conjugated capture antibody binds to one or more surface proteins located on the EV.

4. The biochip assay of claim 3, wherein the one or more surface proteins comprise CD63 or CD9.

5. The biochip assay of any one of claims 1-4, wherein the MB probe comprises one or more fluorescent dyes.

6. The biochip assay of any one of claims 1-5, wherein the MB probe comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

7. The biochip assay of any one of claims 1-6, wherein the imaging comprises total reflection fluorescence microscopy (TIRFM).Docket No. 103362-036WO18. The biochip assay of any one of claims 1-7, wherein the liquid sample comprises blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

9. A method of detecting a signature of glioblastoma (GBM) in a subject, the method comprising: a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa- tricontane-1 -thiol (WC14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; and c) detecting the signature of GBM, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122- 5p and miR1246-5p.

10. The method of claim 9, wherein the biotin conjugated capture antibody binds an EV subpopulation from the liquid sample.

11. The method of claim 9 or 10, wherein the biotin conjugated capture antibody binds to one or more surface proteins located on the EV.

12. The method of claim 11, wherein the one or more surface proteins comprise CD63 or CD9.

13. The method of any one of claims 9-12, wherein the MB probe binds the least one mRNA or the at least one miRNA.

14. The method of any one of claims 9-13, wherein the MB probe comprises one or more fluorescent dyes for a fluorescent imaging modality.Docket No. 103362-036WO115. The method of any one of claims 9-14, wherein the MB probe comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

16. The method of any one of claims 14-15, wherein the fluorescent imaging modality comprises total reflection fluorescence microscopy (TIRFM).

17. The method of any one of claims 9-16, wherein the liquid sample comprises blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

18. A method of treating a glioblastoma (GBM) in a subject in need thereof, the method comprising: a) collecting a liquid sample from the subject, b) contacting the liquid sample with aS1EVRNA biochip comprising: i) a titanium and gold coating, ii) a linker comprising 20-tetradecyloxy-3,6,7,12,15,18,22-heptaoxahexa- tricontane-1 -thiol (WC14) and biotin-PEG-SH, iii) a neutravidin coating, iv) a biotin conjugated capture antibody, and v) a molecular beacon (MB) probe; c) detecting a signature of GBM, d) diagnosing the subject with a GBM when an area under the curve (AUC) from the signature is at least 0.8 compared to a control subject, and e) administering to the subject an anti-cancer agent to decrease the GBM relative to an untreated control, wherein the signature comprises at least one messenger RNA (mRNA) comprising Neurogranin (NRGN), Neurocan (NCAN), N-ethylmal eimide sensitive factor (NSF) or transmembrane 30C (TMEM30C); or at least one microRNA (miRNA) comprising miR-9-5p, miR-124-3p, miR122- 5p or miR1246-5p.

19. The method of claim 18, wherein the biotin conjugated capture antibody binds an EV subpopulation from the liquid sample.

20. The method of claim 18 or 19, wherein the biotin conjugated capture antibody binds to one or more surface proteins located on the EV.Docket No. 103362-036WO121. The method of claim 20, wherein the one or more surface proteins comprise CD63 or CD9.

22. The method of any one of claims 18-21, wherein the MB probe binds the least one mRNA or the at least one miRNA.

23. The method of any one of claims 18-22, wherein the MB probe comprises one or more fluorescent dyes for a fluorescent imaging modality.

24. The method of any one of claims 18-23, wherein the MB probe comprises one or more locked nucleic acid (LNA) nucleotides to improve thermal stability and nuclease resistance.

25. The method of any one of claims 23-24, wherein the fluorescent imaging modality comprises total reflection fluorescence microscopy (TIRFM).

26. The method of any one of claims 18-25, wherein the liquid sample comprises blood, serum, plasma, urine, sputum, saliva, or cerebral spinal fluid (CSF).

Images