Extracellular vesicle protein assays for non-invasive diagnostics

Abstract

Embodiments of the present invention relate to devices, methods and kits for determining a disease in a subject by selectively capturing extracellular vesicles (EVs) by click chemistry and functionalized antibodies, selectively labeling the EVs with antibodies coupled with a plurality of DNA barcodes, optionally releasing the plurality of DNA barcodes, and determining the disease in the subject. And determining the plurality of DNA barcodes to determine whether the subject has the disease or predict a stage of the disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 327,657, filed on April 5, 2022; the entire contents of which are incorporated herein by reference. Government support

[0002] This invention was made with government support under Grant Nos. CA240887, EB026421, CA255727, and CA253651 awarded by the National Institutes of Health. The government has certain rights in this invention. Sequence Listing

[0003] This application contains a sequence listing XML, which has been submitted electronically and is incorporated herein by reference in its entirety. The XML copy was created on April 5, 2023, is named 58086-575390, and is 11174 bytes in size. Background Art 1. Technical Field

[0004] The presently claimed embodiments of the invention relate to devices, methods and kits for determining a disease in a subject, and more specifically, to devices, methods and kits for determining a disease in a subject by selectively labeling, capturing and quantifying extracellular vesicles (EVs). 2. Related technical discussions

[0005] In 2020, primary liver cancer was the third leading cause of cancer-related deaths worldwide. 1 In the United States, although morbidity and mortality trends have remained stable over the past decade, 2,3 It is estimated that the number of deaths from primary liver cancer will exceed 30,000 in 2021. 4 Hepatocellular carcinoma (HCC) accounts for 80-85% of primary liver cancers. 5,6 And it mainly occurs in patients with cirrhosis or chronic hepatitis B virus (HBV) infection. 6 The poor prognosis of HCC is mainly due to the advanced stage of the disease at the time of diagnosis, and studies have shown that early detection of HCC is associated with increased acceptance of curative treatment and improved patient outcomes. 3,7 Current clinical practice guidelines recommend twice-yearly liver ultrasound HCC surveillance with or without serum alpha-fetoprotein (AFP) for high-risk patients to achieve the goal of detecting HCC in the curative phase. 8,9 However, their accuracy remains relatively low, with a sensitivity between 60%-70% at a specificity of 90%. 10-13 Therefore, novel biomarkers for early detection of HCC are still urgently needed. Extracellular vesicles (EVs) refer to nanoparticles released by both normal and tumor cells. 14,15EVs are encapsulated by a lipid bilayer membrane and contain biomolecules including DNA, RNA, proteins, metabolites, and lipids. By transferring these cargoes, tumor-derived EVs participate in intercellular communication and cancer progression. Given their presence in the circulation early during tumorigenesis, analysis of tumor-derived EVs is considered a promising liquid biopsy approach for diagnosing early cancers. 14,15 Several studies have investigated the role of EVs in the detection of HCC in high-risk patients, and the results are very promising. 14,16,17 For example, von Felden et al. and Tseng et al. demonstrated that noncoding RNAs in EVs 16 and mRNA 17 The markers could be used to distinguish Barcelona Clinic Liver Cancer (BCLC) stage 0 to A HCC from cirrhosis, respectively, with areas under the receiver operating characteristic curves (AUROCs) of 0.93. Therefore, EVs have great potential to improve current surveillance strategies and improve outcomes for HCC patients.

[0006] Given that current surveillance methods for detecting early hepatocellular carcinoma (HCC) have suboptimal sensitivity, extracellular vesicles (EVs) are promising circulating biomarkers for early detection of cancer. In this study, we aimed to develop an HCC EV-based surface protein assay for early detection of HCC. Join by reference

[0007] All publications and patent applications identified herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1A to Figure 1D A schematic workflow diagram of an implementation scheme for the use of EV click beads for the isolation and surface protein characterization of hepatocellular carcinoma cell extracellular vesicles (HCC EVs) is provided. (A) Schematic diagram of the working mechanism of EV click beads, which are covalently functionalized with tetrazine (Tz); click chemistry-mediated EV capture is combined with the use of antibodies targeting HCC-related surface markers (i.e., EpCAM, ASGPR, GPC3, and CD147) ( FIG1B ). A pair of highly reactive click chemistry motifs, namely Tz and trans-cyclooctene (TCO), are grafted onto click beads and EVs, respectively. When the plasma sample passes through the workflow, the click chemistry reaction between the Tz-grafted click beads and the TCO-grafted HCC EVs results in the immobilization of HCC EVs on the click beads ( FIG1C ). Subsequently, the captured HCC EVs ( Figure 1D). The characteristics of the obtained HCC EV surface protein markers were analyzed to distinguish HCC from cirrhosis.

[0009] Figure 2A to Figure 2B is a schematic diagram of the workflow of HCC EV SPA; (A) an embodiment of antibody-DNA barcoding for HCC EV SPA and (B) an embodiment of DNA barcode library preparation and NGS analysis.

[0010] Figure 3A to Figure 3B Schematic diagram of the workflow of HCC EV SPA; (A) an embodiment of antibody-DNA barcoding for HCC EV SPA and (B) an embodiment of DNA barcoding analysis based on the NanoString system.

[0011] Figure 4 Embodiments for preparing click beads are described.

[0012] Figure 5 An embodiment for preparing four TCO-conjugated HCC-specific antibodies is described.

[0013] Figure 6A to Fig. 6E The steps for conjugating DNA barcodes to antibodies and validating the conjugated DNA-antibody are described.

[0014] FIG. 7 provides a table of DNA barcode sequences SEQ ID NO: 1 to SEQ ID NO: 2 and probes / primers SEQ ID NO: 3 to SEQ ID NO: 8 for duplex real-time PCR.

[0015] Figure 8A to Figure 8C Characterization of HCC cell line-derived EVs is depicted. (A) Concentration and size distribution of HepG2-derived EVs (B) Expression of EV markers (CD63 and Annexin V) on HepG2-derived EVs by flow cytometry. (C) Representative TEM images of HepG2-derived EVs in bulk solution before immunogold capture.

[0016] Figure 9A to Fig.9F Provide data on optimization of HCC EV isolation by click beads.

[0017] Fig.10 Characterization of HepG2-derived EVs captured on click beads by EM is described.

[0018] Fig.11 An embodiment of the workflow of the HCC EV surface protein assay is shown for quantification of eight subpopulations of HCC EVs.

[0019] Figure 12A to Fig.12H Optimization of HCC EV surface protein assay using HCC cell line-derived EVs is provided.

[0020] Fig.13 We show the reproducibility of the HCC EV surface protein assay validated using clinical plasma samples.

[0021] Fig.14 Shown are the ROC curves of eight HCC EV subsets for discovering early HCC from cirrhosis in the UCLA training cohort.

[0022] FIG15 shows Table 2, which displays the relative signals used to assess the reproducibility of the HCC EV surface protein assay.

[0023] FIG. 16 shows Table 3, which shows univariate logistic regression analysis of eight HCC subgroups for finding early HCC from liver cirrhosis.

[0024] Fig.17 A flow chart describing an embodiment is shown.

[0025] Fig.18 A clinical study design flow chart describing study recruitment and exclusions is shown.

[0026] Figure 19A to Fig.19D Validation of the selected four HCC-related surface markers using tissue microarray (TMA) and HCC cell-derived EVs is shown.

[0027] Figure 20A to Fig.20E Shown is the HCCEV surface protein assay (SPA) used to measure HCC EV subsets and discover early-stage HCC in the UCLA training cohort.

[0028] Figure 21A to Fig. 21C HCC EV ECG score for early-stage HCC detection in the CSMC independent validation cohort is shown.

[0029] Figure 22A to Fig.22D Comparison between HCC EV ECG score and serum AFP for finding early HCC is shown. Summary of the invention

[0030] A method for determining a disease in a subject, comprising: selectively labeling an extracellular vesicle (EV) from a sample of the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group, so that the first capture agent can still be connected to the EV, and the first molecule can be connected to a second molecule from a second bioorthogonal functional group, the second molecule is complementary to the first molecule, functionalizing the DNA barcode with the second molecule, connecting the functionalized first capture agent with the functionalized DNA barcode to form a barcoded capture agent, mixing the barcoded capture agent with the sample to form a labeled sample; selectively capturing the EV from the sample of the subject, comprising: labeling the EV with a first molecule from a third bioorthogonal functional group; The second capture agent of the EV is functionalized with three molecules so that the second capture agent can still be connected to the EV, and the third molecule can also bind to a fourth molecule from a fourth bioorthogonal functional group, and the fourth molecule is complementary to the third molecule, the functionalized second capture agent is mixed with the labeled sample so that the functionalized second capture agent binds to the EV to form an activated sample, the capture surface is functionalized with the fourth molecule, and at least a portion of the activated sample is deposited on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through the binding of the fourth molecule to the third molecule; the first DNA barcode is determined by a nucleic acid test; and the presence or absence of the disease in the subject is determined by determining the first DNA barcode from the EV.

[0031] A method for determining a disease in a subject, comprising: selectively labeling an extracellular vesicle (EV) from a sample of the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group, such that the first capture agent can still be connected to the EV, and the first molecule can be connected to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule, functionalizing the DNA barcode with a second molecule, connecting the functionalized first capture agent to the functionalized DNA barcode to form a barcoded capture agent; functionalizing a second capture agent of the EV with a third molecule from a third bioorthogonal functional group, such that the second capture agent can still be connected to the EV , and the third molecule is capable of connecting to a fourth molecule from a fourth bioorthogonal functional group, the fourth molecule is complementary to the third molecule, the barcoded capture agent and the functionalized second capture agent are mixed with the sample to form an activated sample; the EV is selectively captured from the sample from the subject, including: functionalizing a capture surface with the fourth molecule, and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through the binding of the fourth molecule to the third molecule; determining the first DNA barcode by a nucleic acid test; and determining whether the subject has the disease by determining the first DNA barcode from the EV.

[0032] According to the assay method of any one of the aforementioned embodiments, the second capture agent selected for capturing the EV is a tumor-specific capture agent and binds to surface markers specifically expressed by tumor-derived EVs.

[0033] The assay method of any of the preceding embodiments, wherein selectively capturing extracellular vesicles (EVs) from a sample from a subject is performed prior to selectively labeling the EVs from the sample from the subject with a first DNA barcode.

[0034] The determination method according to any one of the aforementioned embodiments further includes: repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with a second DNA barcode, so that the EVs are labeled with different first capture agents bound to different regions of the EVs, and the first DNA barcode and the second DNA barcode are distinguishable from each other; determining the second DNA barcode using a nucleic acid test; and determining whether the subject has the disease by determining the first DNA barcode and the second DNA barcode from the EV.

[0035] The determination method according to the aforementioned embodiment further includes: repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with more DNA barcodes, so that the EVs are labeled with multiple different capture agents that each binds to different regions of the EV, and the first DNA barcode and the more DNA barcodes are each distinguishable from each other; using multiple nucleic acid tests to determine the more DNA barcodes; and determining the surface protein expression characteristics by determining the multiple DNA barcodes.

[0036] The assay method according to the aforementioned embodiment further comprises: repeating selective labeling, selective capture, DNA barcode determination, and surface protein expression signature determination multiple times to predict disease stage and / or distinguish different disease stages. Each selective capture uses a capture agent that binds to a portion of the EV that is different from the portion that the other capture agents are used to selectively capture.

[0037] The assay method according to any one of the preceding embodiments further comprises: releasing the DNA barcode from the captured EV prior to assay.

[0038] The assay method according to any of the aforementioned embodiments is implemented by selectively capturing extracellular vesicles (EVs) from a sample from a subject prior to selectively labeling the EVs from the sample from the subject with a plurality of DNA barcodes.

[0039] The assay method according to the aforementioned embodiment, wherein the surface protein expression signature differentiates between different disease stages.

[0040] According to the assay method of the aforementioned embodiment, the expression of EpCAM+CD63+ on HCC EV, the expression of CD147+CD63+ on HCC EV and the expression of GPC3+CD63+ Expression on HCC EVs was higher in patients with hepatocellular carcinoma (HCC) compared with patients with cirrhosis.

[0041] An assay method according to any of the preceding embodiments, wherein the capture agent enabling capture of the EV is functionalized with a third bioorthogonal functional group selected from trans-cyclooctene (TCO), alkynes and cyclooctyne, and wherein the capture surface is functionalized with a fourth bioorthogonal functional group selected from tetrazine (Tz) and azide.

[0042] The assay method according to any one of the preceding embodiments, wherein the capture surface is the surface of beads in suspension.

[0043] The assay method according to any one of the preceding embodiments, wherein the capture surface is a surface of a microfluidic device.

[0044] The assay method according to any one of the preceding embodiments, wherein the assay comprises using multiplex PCR.

[0045] The assay method according to any of the preceding embodiments, wherein the assay comprises using a next generation sequencing process.

[0046] A kit for determining a disease in a subject according to any one of the preceding embodiments, The invention comprises: a plurality of capture agents, each of which binds to a different region of an EV; a plurality of molecules having complementary bioorthogonal functional groups; a plurality of DNA barcodes; and instructions for labeling the EV with the plurality of DNA barcodes, capturing the EV, measuring the plurality of DNA barcodes, and determining whether the subject has the disease or predicting the stage of the disease through the measurement.

[0047] The kit according to the aforementioned embodiment further comprises instructions for releasing the plurality of DNA barcodes prior to assaying. DETAILED DESCRIPTION

[0048] Some embodiments of the present invention are discussed in detail below. When describing the embodiments, specific terms are used for clarity. However, the present invention is not intended to be limited to the selected specific terms. Those skilled in the relevant art will recognize that other equivalent components can be used and other methods can be developed without departing from the broad concepts of the present invention. All references cited anywhere in this specification are incorporated by reference as if each were individually incorporated.

[0049] The term "bioorthogonal chemistry" refers to any chemical reaction between azide and alkyne functional groups that are substantially inert or bioorthogonal to biomolecules and aqueous environments and that do not themselves interfere with natural biochemical processes. In some embodiments, a molecule containing a highly reactive azide group reacts with a molecule containing a strained alkyne. The azide and alkyne "click" together and the two molecules are connected by the newly formed triazole group. The reaction may or may not require a catalyst, such as copper, light, or electricity.

[0050] A non-limiting example of a bioorthogonal reaction for use according to embodiments of the present invention is a 1,3-dipolar cycloaddition between an azide-containing molecule and a cyclooctyne, as depicted in the following diagram:

[0051] The term DNA barcode as used herein is intended to refer to a known DNA sequence that is orthogonal to the subject's organism so as to be clearly distinguishable from the subject's genome. The term "barcodes" refers to a unique sequence that can be used to distinguish nucleic acids even after the nucleic acids are mixed together. In some embodiments, the DNA barcode can be functionalized and coupled to a capture agent.

[0052] Current HCC surveillance is based on ultrasound with or without AFP, although their diagnostic performance is suboptimal. A statistical model named GALAD score derived from sex, age, and three serum biomarkers (i.e., AFP-L3, AFP, and decarboxyprothrombin) has shown high sensitivity in a phase II biomarker study for HCC discovery. 26，27 However, recent phase III studies have shown overall low accuracy, emphasizing the need for better biomarkers for HCC monitoring. 28 Liquid biopsy has emerged as another promising strategy for HCC surveillance. 14，29，30 For example, several circulating tumor DNA-based assays that measure molecular signatures have been proposed for the discovery of HCC and have received breakthrough device designation from the Food and Drug Administration. 31-33 In addition, as previous studies have shown, EVs such as miRNA, 14 Non-coding RNA, 16 mRNA, 17 Biomolecular cargoes such as mitochondria and proteins are also considered as potential biomarkers for early detection of HCC.

[0053] Functionalization of capture surfaces

[0054] Some embodiments of the present invention include capture surfaces for capturing extracellular vesicles. Further non-limiting examples of capture surfaces are the surface of beads in suspension or the surface of beads that may contain a variety of silicon nanostructures. Such capture surfaces may be functionalized with molecules having bioorthogonal functional groups that are complementary to the bioorthogonal functional groups of the functionalized capture agent. Figure 4 ) In one aspect of the embodiment, beads suitable for covalent capture of terminal azide / alkyne-coupled molecules are used to capture extracellular vesicles (EVs) from a sample.

[0055] The shape of the capture surface is not critical. For example, a sphere or bead can be grafted using silane chemistry with at least one molecule having a bioorthogonal functional group that can be linked to a complementary bioorthogonal functional group of another molecule that can functionalize the capture agent. In other embodiments, the capture surface can comprise a variety of nanostructures, such as nanowires, nanofibers, nanotubes, nanopillars, nanospheres, or nanoparticles.

[0056] When the sample is placed on the functionalized capture surface, the extracellular vesicles grafted by at least one capture agent are selectively captured due to the click chemistry reaction. The one or more capture agents used will depend on the type of extracellular vesicles targeted. Conventional capture agents are suitable for use in some embodiments of the present invention. Non-limiting examples of capture agents include antibodies, nucleic acids, oligopeptides or polypeptides, cell receptors, ligands, nucleic acid aptamers, biotin, avidin, coordination complexes, synthetic polymers, carbohydrates, lipid derivatives, and combinations thereof.

[0057] In other embodiments, the capture surface is coated with streptavidin and the capture agent is biotinylated to facilitate immobilization of EVs.

[0058] The capture surface of the embodiments can be covered by a microfluidic chaotic mixer, allowing for configuration of throughput flow, for example, to enhance physical contact between the sample and at least a portion of the functionalized capture surface. Non-limiting examples of microfluidic chaotic mixers include polydimethylsiloxane (PDMS) chaotic mixers.

[0059] Some embodiments of the present invention relate to a method for determining a disease in a subject. For example, the subject can be a human or an animal. The method includes selectively labeling an extracellular vesicle (EV) from a sample of the subject with a first DNA barcode, selectively capturing EV from the sample, optionally releasing the first DNA barcode from the captured EV, determining a nucleic acid sequence from the first DNA barcode, and determining whether the subject has a disease by determining the first DNA barcode from the EV.

[0060] Choice of capture agent

[0061] The choice of capture agent for EV labeling or EV capture depends solely on the disease being diagnosed or assessed. In some embodiments, the capture agent is selected to achieve the desired sensitivity and specificity for identifying and labeling and / or capturing tumor-derived EVs. In one aspect of the embodiment, the capture agent is an antibody that specifically binds to a tumor-associated surface marker present on tumor-derived EVs. Non-limiting examples of surface markers include antigens, polypeptides, receptors, or surface proteins. In one aspect of the embodiment, the antibody may be selected for its specificity for tumor-associated surface markers, which are themselves differentially expressed between tumor-derived EVs and normal cell-derived EVs or between tumor-derived EVs at different stages of the disease. Differential expression refers to high expression (upregulated), low expression (downregulated), limited expression, or no expression of a surface marker in tumor-associated EVs. In another aspect of the embodiment, a marker may be preferentially selected for its lack of expression in EVs derived from non-disease cells. For example, in a method for capturing EVs derived from tumors such as HCC, multiple antibodies may be selected for their specificity for HCC-EVs, HCC- The EVs are selected by binding to surface markers that are highly expressed on CTCs, HCC cell lines, and primary tumor tissues, but not expressed on leukocytes. For example, in a method of labeling EVs derived from a tumor such as HCC, a variety of antibodies (not necessarily from the tumor) can be selected because they bind to surface markers expressed on EVs. Non-limiting examples of HCC markers include EpCAM, ASGPR, CD147, and GPC-3. Non-limiting examples of HCC EV capture agents include anti-EpCAM, anti-ASGPR, anti-CD147, and anti-GPC-3 antibodies. Non-limiting examples of EV capture agents include anti-CD63 and anti-CD9 antibodies.

[0062] Tumor heterogeneity includes spatial heterogeneity within each tumor and inter-patient heterogeneity in terms of genetic and molecular diversity between tumors from different patients. Similar to parental tumor cells, tumor-derived EVs are highly heterogeneous, emphasizing the necessity of integrating different surface protein markers to develop liquid biopsy-based diagnostic assays to detect early-stage cancers including HCC. HCC tissue microarrays showed that four surface protein markers (i.e., EpCAM, CD147, GPC3, and ASGPR1) were complementarily expressed on the surface of tumor cells. Complementary expression patterns of surface protein markers integrate different subpopulations of tumor-derived EVs for the discovery of highly heterogeneous tumors. For example, surface protein markers other than ASGPR1 can be included in the score; although ASGPR1 shows strong positivity in HCCTMA. It is well known that ASGPR1 is a liver-specific marker that is also expressed on hepatocytes in patients with cirrhosis.

[0063] In some embodiments, EVs are labeled with more than one capture agent to optimize the specificity and sensitivity of the method. Given that EVs can be secreted by highly heterogeneous cell populations, it is conceivable that EVs derived from the same tumor may express different surface markers. In one aspect of the embodiment, multiple capture agents may be necessary to label EVs, allowing for sensitive and specific discovery of tumor-derived EVs at all disease stages. For a given clinical sample, the expression of multiple labeled capture agents can be assessed, providing an expression signature that may be characteristic of the disease stage. Integrating click chemistry-mediated EV capture and multiple capture agents such as antibodies provides a more sensitive and specific method for labeling, capturing, and measuring tumor-derived EVs with minimal background levels.

[0064] Affinity capture: click chemistry

[0065] In some embodiments, the capture surface is functionalized with a molecule comprising a bioorthogonal functional group. Non-limiting examples of bioorthogonal functional groups include azide motifs, preferably Tz motifs. The capture agent that immobilizes EVs on the surface is functionalized with a molecule having a bioorthogonal functional group selected from trans-cyclooctene (TCO), alkyne, and cyclooctyne derivatives. Figure 5 ) In one aspect of the embodiment, the cyclooctyne derivative comprises dibenzylcyclooctyne (DBCO) or biarylazacyclooctyne ketone (BARAC). In one aspect of the embodiment, a pair of highly reactive click chemistry motifs including but not limited to Tz and TCO are grafted onto the capture substrate and the capture agent, respectively. In one aspect of the embodiment, in view of their balanced chemical properties regarding stability and reactivity, the reverse electron demand Diels-Alder cycloaddition (rate constant of 10 4 M -1 ·s -1 ) can be selected. The connection between the Tz-grafted capture surface and the TCO-grafted capture agent is insensitive to biomolecules, water, and oxygen, resulting in specific, rapid, and irreversible immobilization of EVs, improving capture efficiency and reducing nonspecific capture of particles in the background. This approach allows for more efficient coupling of TCO-grafted antibodies to most EVs in a small volume of solution, facilitating click chemistry-mediated capture of EVs onto click beads.

[0066] In some embodiments, the capture agent capable of capturing EVs is functionalized with a third bioorthogonal functional group selected from trans-cyclooctene (TCO), alkynes, and cyclooctyne, and wherein the capture surface is functionalized with a fourth bioorthogonal functional group selected from tetrazine (Tz) and azide. In one aspect of the embodiment, the third molecule having the third bioorthogonal functional group and the fourth molecule having the fourth bioorthogonal functional group are present in a molar ratio between 2:1 and 10:1. ( Figure 4 and Figure 5 ) In one aspect of the embodiment, the capture agent capable of capturing EVs involves a surface marker specifically expressed by tumor-derived EVs.

[0067] In some embodiments, the capture agent is functionalized with a molecule having a bioorthogonal functional group that can be linked to a complementary bioorthogonal functional group of a functionalized DNA barcode, while enabling it to bind to its surface markers on the EV bilayer membrane. (Figure 6). In one aspect of the embodiment, the coupling of the capture agent and the DNA barcode is implemented in a molar ratio between 1:2 and 1:10.

[0068] In some embodiments, DNA barcodes are coupled to capture agents for targeting EV markers. For example, DNA barcode 1 (SEQ ID NO: 1) and DNA barcode 2 (SEQ ID NO: 2) are coupled to antibodies targeting CD63 and CD9 (FIG. 6A and FIG. 7). In some aspects of the embodiments, the DNA barcodes are coupled to capture agents having a molar ratio of 1: In another aspect of the embodiment, the capture agent is functionalized with a molecule comprising a biorthogonal functional group complementary to the azide motif. A non-limiting example of a complementary biorthogonal functional group is DBCO. (Diarylcyclooctyne) The functionalization of the capture agent is carried out in a molar ratio of 1:2 to 1:20, preferably 1:10.

[0069] In some embodiments, a cleavage motif is incorporated between each pair of DNA barcode and capture agent, allowing cleavage and recovery of the DNA barcode for subsequent quantification by multiplex PCR or NGS or any other nucleic acid test. Non-limiting examples of cleavage motifs are disulfide motifs cleavable with DTT or UV irradiation-triggered linkers cleavable with UV irradiation.

[0070] In some embodiments, other affinity capture methods may be used, such as, but not limited to, biotin-streptavidin.

[0071] Selective labeling of EVs

[0072] An embodiment relates to a method for determining a disease in a subject, comprising: selectively labeling extracellular vesicles (EVs) from a sample from the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group so that the first capture agent can still be attached to the EV and the first molecule is capable of attaching to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule, functionalizing the DNA barcode with the second molecule, attaching the functionalized first capture agent to the functionalized DNA barcode to form a barcoded capture agent, and mixing the barcoded capture agent with the sample to form a labeled sample.

[0073] Embodiments relate to a method for determining a disease in a subject, comprising: selectively labeling an extracellular vesicle (EV) from a sample of the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group, such that the first capture agent is still attachable to the EV, and the first molecule is capable of attaching to a second molecule from a second bioorthogonal functional group, the second molecule being complementary to the first molecule, functionalizing the DNA barcode with a second molecule, attaching the functionalized first capture agent to the functionalized DNA barcode to form a barcoded capture agent, functionalizing the second capture agent of the EV with a third molecule from a third bioorthogonal functional group, such that the second capture agent is still attachable to the EV, and the third molecule is capable of attaching to a fourth molecule from a fourth bioorthogonal functional group, the fourth molecule being complementary to the third molecule, mixing the barcoded capture agent and the functionalized tumor-specific capture agent with the sample to form an activated sample,

[0074] In some embodiments, the first capture agent is an antibody that binds to a surface marker specifically expressed on EVs, but not necessarily to a surface marker specifically expressed by tumor-derived EVs. When assaying HCC EVs, non-limiting examples include anti-CD63 and anti-CD9.

[0075] In some embodiments, the method comprises simultaneously labeling EVs with a functionalized second / tumor-specific capture agent and at least one DNA-capture agent conjugate. In other embodiments, the method comprises sequentially labeling EVs with a functionalized second / tumor-specific capture agent followed by labeling of at least one DNA-capture agent conjugate. In other embodiments, the method comprises sequentially labeling EVs with at least one DNA-capture agent conjugate followed by a functionalized second / tumor-specific capture agent.

[0076] In some embodiments, the second capture agent is an antibody that binds to a surface marker specifically expressed on tumor-derived EVs.

[0077] Selective capture of EVs

[0078] In some embodiments, selectively capturing extracellular vesicles (EVs) from a sample from a subject is performed prior to selectively labeling the EVs from the sample from the subject with one of a plurality of DNA barcodes.

[0079] An embodiment of the method for selectively capturing extracellular vesicles (EVs) from a sample from a subject further includes: functionalizing a second capture agent of the EV with a third molecule from a third bioorthogonal functional group so that the second capture agent can still be attached to the EV and the third molecule is capable of attaching to a fourth molecule from a fourth bioorthogonal functional group, the fourth molecule being complementary to the third molecule, mixing the functionalized second capture agent with the labeled sample so that the functionalized second capture agent binds to the EV to form an activated sample, functionalizing a capture surface with the fourth molecule, and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through the binding of the fourth molecule to the third molecule; determining the first DNA barcode by a nucleic acid test; and determining whether the sample has the disease by determining the first DNA barcode from the EV.

[0080] An embodiment of the method for selectively capturing extracellular vesicles (EVs) from a sample from a subject further includes: functionalizing a capture surface with the fourth molecule, and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through binding of the fourth molecule to the third molecule; determining the first DNA barcode by a nucleic acid test; and determining whether the subject has the disease by determining the first DNA barcode from the EV.

[0081] In some aspects of the embodiments, releasing the first DNA barcode from the captured EV is performed prior to determining the first DNA barcode.

[0082] In one aspect of the embodiment, the second capture agent used to capture the selected EV is a tumor-specific capture agent that binds to a surface marker specifically expressed by tumor-derived EVs.

[0083] Surface protein expression signatures predicting disease stage

[0084] In some embodiments, EVs are selectively labeled with 2 antibodies, each coupled to a distinguishable DNA barcode. In another aspect of the embodiment, the assay method further comprises: repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with a second DNA barcode, such that the EVs are labeled with different capture agents that bind to different regions of the EVs, and the first and second DNA barcodes are distinguishable from each other; optionally releasing the second DNA barcode from the captured EVs; determining the second DNA barcode using a nucleic acid test; and determining whether the subject has the disease by determining the first and second DNA barcodes from the EVs.

[0085] In another aspect of the embodiment, the assay method further comprises: repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with further multiple DNA barcodes, so that the EVs are labeled with multiple different capture agents that each binds to different regions of the EV, and the first and further multiple DNA barcodes are each distinguishable from each other; optionally releasing the further DNA barcodes from the captured EVs; determining the further DNA barcodes using a multiple nucleic acid test; and determining the surface protein expression characteristics by determining the multiple DNA barcodes.

[0086] In some embodiments, the assay method further comprises: repeating multiple times selective labeling, selective capture, determination of DNA barcodes, and determination of surface protein expression characteristics to predict disease stage and / or distinguish different disease stages. Each selective capture uses a capture agent that binds to a portion of the EV that is different from the portion that other capture agents use to selectively capture. In one aspect of the embodiment, the assay method comprises releasing the DNA barcode from the captured EV prior to the assay.

[0087] Through a series of bioinformatics and validation processes, the method of the embodiment can identify surface protein expression features that distinguish disease stages. In addition, identifying surface protein expression features not only has clinical applications, but also provides new understanding of the progression of the disease.

[0088] In some embodiments, a simple HCC EV surface protein assay (SPA) was designed and developed to distinguish early HCC from cirrhosis (Figure 1). Using a method based on click chemistry-mediated EV purification and dual real-time immuno-PCR, eight HCC EV subsets (i.e., EpCAM + CD63 + HCC EV, CD147 + CD63 + HCC EV, GPC3 + CD63 + HCC EV, ASGPR1 + CD63 + HCC EV、EpCAM + CD9 + HCC EV, CD147 + CD9 + HCC EV, GPC3 + CD9 + HCC EVs and ASGPR1 + CD9 + HCC EV). According to the obtained HCC EV surface protein characteristics (i.e. EpCAM+ CD63 + HCC EV, CD147 + CD63 + HCC EV, GPC3 + CD63 + HCC EV) A logistic regression model named HCC EV ECG score was established to distinguish early HCC from cirrhosis. According to the International Cancer Association (ILCA) biomarker development guidelines, a phase 2 biomarker study was conducted to evaluate the effect of HCC EV ECG score for the detection of early HCC. (Figure 19 to Figure 22)

[0089] Determination

[0090] In some embodiments, the determination may include the use of a PCR process. In some embodiments, the determination may include the use of multiple PCR when multiple DNA barcodes need to be quantified. (Figure 2). In one aspect of the embodiment, the determination is performed by immuno-PCR. In another aspect of the embodiment, the DNA barcode is quantified by next-generation sequencing (NGS) as a downstream readout of our HCC EV surface protein assay (SPA), allowing a significant increase in multiplexing capacity to discover and quantify up to 50 to 100 surface protein targets on HCC EV in a single assay. Figure 2 summarizes the two-step workflow of this new HCC EV SPA. This is intended to be a non-limiting example of an embodiment involving HCC. In some embodiments, the determination may include the use of a PCR process. In some embodiments, the determination may include the use of a NanoString process. These amplification methods can be implemented according to protocols well known in the art.

[0091] To quantify tumor-derived EV subpopulations, the present embodiment of EV SPA integrates two powerful technologies: click beads for purification of tumor-derived EVs and dual real-time immuno-PCR for quantification of purified tumor-derived EV subpopulations. Compared with traditional immunoaffinity-based EV capture methods, click beads achieve more rapid and irreversible purification of HCC EVs by utilizing click chemistry reactions between beads and targeted EVs. Notably, the connection between the mTz-grafted capture surface and the TCO-grafted EVs not only improves the capture efficiency but also reduces nonspecific EV capture from the background.

[0092] Embodiments utilizing multiplex real-time immuno-PCR combine the advantages of i) the flexibility and robustness of immunoassays and ii) the sensitivity of PCR. Immuno-PCR uses specific antibodies grafted with DNA barcodes, enabling multiplex detection of several antigens. Compared to similar enzyme amplification immunoassays, real-time immuno-PCR typically exhibits a 10- to 1000-fold increase in sensitivity. Therefore, the integration of click beads and dual / multiplex real-time immuno-PCR enables tumor-derived EV SPA to sensitively and specifically quantify tumor-derived EV subpopulations for each individual. Perhaps most importantly, the exemplary embodiment assay requires only a very small amount of plasma (approximately 400 μL) to obtain the patient's EV surface protein expression signature.

[0093] In some embodiments, determination of multiple barcodes provides surface protein expression signatures that distinguish samples at different disease stages. In one aspect of the embodiment, the presence of EpCAM+CD63+HCC EVs, CD147+CD63+HCC EVs, and GPC3+CD63+HCC EVs were observed in HCC patients compared to those in cirrhosis patients. Example

[0094] Figure 1 is provided here to help describe some concepts according to some embodiments of the present invention. However, the general concepts of the present invention are not limited to labeling EVs with only two DNA barcodes. They can be labeled with a single DNA barcode in some embodiments, or with more than two barcodes in other embodiments. Embodiments that have been developed include even up to 50 or 100 DNA barcodes attached to the EV. However, the general concepts of the present invention are not limited to a specific number of DNA barcodes. Although HCC is described as a specific example, the general concepts of the present invention are not limited to just HCC.

[0095] Fabrication of functionalized click beads.

[0096] Click beads were prepared by designing a three-step chemical modification procedure ( Figure 4): (i) Silica microbeads with natural hydroxyl groups (silica MBs, Bangs Laboratories, Inc) were first treated with nitric acid (2 mol / L, Sigma-Aldrich) for 30 min and then washed with DI water and ethanol in sequence. (ii) The resulting silica MBs were then resuspended in (3-aminopropyl)triethoxysilane / ethanol solution (4%, v / v, Sigma-Aldrich) for 45 min to introduce terminal amine groups. The amine-terminated silica microbeads (NH2-modified MBs) were then washed three times with ethanol. (iii) In order to graft the mTz motif, the NH2-modified MBs were incubated with methyltetrazine-sulfo-NHS ester (3.8 mM, click chemistry tool) in PBS (200 μL, PH = 8.5) for 1 hour. The resulting mTz-grafted MBs were washed three times with DI water. After drying under a stream of nitrogen, the mTz-grafted MBs were stored at 4°C and retrieved before use. (APTES, aminopropyltriethoxysilane; DI, deionized; DMSO, dimethyl sulfoxide; MBs, microbeads; mTz, methyltetrazine; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; PEG, polyethylene glycol; RT, room temperature).

[0097] Preparation of four TCO-conjugated HCC-specific antibodies

[0098] Goat anti-human EpCAM (R&D Systems, Inc., reconstituted at 0.2 mg / mL, diluted 200-fold to 2000-fold in the sample), rabbit anti-human ASGPR1 (LifeSpan BioSciences, Inc., 1 mg / mL, diluted 1000-fold to 10000-fold in the sample), sheep anti-human GPC3 (R&D Systems, Inc., reconstituted at 0.2 mg / mL, diluted 200-fold to 2000-fold in the sample), and goat anti-human CD147 (R&D Systems, Inc., reconstituted at 0.5 mg / mL, diluted 1000-fold to 2000-fold in the sample) were incubated with TCO-PEG4-NHS ester (0.5 mM, click chemistry tool Bioconjugate Technology Company) in PBS at a molar ratio of 1:4 for 30 minutes at room temperature. Prepare separate TCO-conjugated antibodies before use. (Fig. 21) (ASGPR1, asialoglycoprotein receptor 1; DMSO, dimethyl sulfoxide; EpCAM, epithelial cell adhesion molecule; GPC3, glypican 3 protein; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; PEG, polyethylene glycol; RT, room temperature; TCO, trans-cyclooctene). HCC EV SPA relies on the use of different HCC-associated surface protein markers to target and purify HCC EV subsets in plasma samples. Recent studies have demonstrated that the use of surface markers such as EpCAM 23 and ASGPR1 24 The feasibility of selectively characterizing HCC EVs in plasma samples by using HCC-associated surface markers. In particular, a study 24 showed that EVs carrying surface protein markers AnnexinV, EpCAM, CD147, and ASGPR1 were significantly increased in the plasma of HCC patients compared with cirrhotic patients and healthy individuals. EpCAM, GPC3, and ASGPR1 are HCC-associated surface markers. 25Therefore, four candidate HCC EV surface protein markers (i.e., EpCAM, ASGPR1, GPC3, and CD147) were selected for validation. Since the surface proteins of tumor-derived EVs can reflect the surface proteins of parental tumor cells, we first used 708 HCC TMAs generated from resected HCC specimens archived by the UCLA Department of Pathology to validate the expression of the four candidate markers. Representative TMA H&E staining and IHC staining of the four selected HCC-related surface protein markers are shown in Figures 19A to 19B, summarizing the IHC quantitative results of the TMAs. Among the four selected markers, CD147 exhibited the highest positive rate - among 708 HCC tumors (excluding cases where tumor tissue was not captured on the TMA slides), 576 (83.5%), 88 (12.8%), and 21 (3.0%) were stained strongly (3 + ), moderately (2 + ), and weakly (1 + ), respectively. For the commonly used epithelial marker EpCAM, only 36 (5.2%), 59 (8.5%), and 165 (23.8%) were stained strongly (3 + ), moderately (2 + ), and weakly (1 + ), respectively. As for the HCC-specific marker GPC3, 64 (9.2%), 118 (17.0%), and 270 (38.8%) were stained strongly (3 + ), moderately (2 + ), and weakly (1 + ), respectively. As expected, most HCC tissues were positive for the liver-specific marker ASGPR1, with 549 (80.5%), 92 (13.5%), and 28 (4.1%) stained strongly (3 + ), moderately (2 + ), and weakly (1 +), demonstrating the primary liver origin of HCC tissue. Overall, 99.7% (701 out of 703) stained positive for at least one marker (24.6% of all four markers were positive, 47.7% of any three markers were positive, 24.6% of any two markers were positive, and 2.8% of any one marker was positive, Figure 19C), which highlights the complementary expression of the four surface protein markers in heterogeneous HCC tissues and lays a solid foundation for the use of these HCC-related antibodies in HCC EV SPA. After identifying, selecting and implementing TMA validation of the four HCC-related surface proteins, their presence in EVs derived from two HCC cell lines was validated by Western Blotting. As shown in Figure 19C, all four HCC-related surface protein markers were detected in HepG2-derived EVs. In Hep3B-derived EVs, EpCAM and GPC3 were detected.

[0099] Western Blotting to detect four HCC-related surface protein markers in EVs derived from HCC cell lines

[0100] Western blot analysis was performed to quantify four HCC-related surface protein markers from EVs. The collected HepG2-derived and Hep3B-derived EVs were lysed in an appropriate volume of radioimmunoprecipitation assay buffer on ice for 30 min and then centrifuged at 14,000 g for 20 min. The protein concentration was assessed using the Qubit assay (Thermo Fisher Scientific). Loading buffer (G-Biosciences) was added and the protein samples were then heated to 95°C for 5 min. The protein samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes and blocked for 1 h in TBS-T (containing 0.1% Tween-20) containing 5% skim milk. The membranes were first incubated with antibodies against β-actin (Sigma-AldrichChemie, Steinheim, Germany), EpCAM, CD147, GPC3, and ASGPR1 at 4°C overnight, and then the above primary antibodies were dissolved in the corresponding blocking solution and then incubated with appropriate secondary antibodies for 1 h. The results were visualized using an enhanced chemiluminescence system.

[0101] Conjugation of DNA barcodes to antibodies and validation of conjugated DNA-antibodies

[0102] DNA barcodes with sequences SEQ ID NO: 1 and SEQ ID NO: 2 were coupled to antibodies for targeting EV markers CD63 and CD9, respectively ( FIG. 6A ). (i) Generation of N3-DNA: DNA barcodes were purchased from IDT, as well as sequences ( FIG. 7 , Table 1 ). N3-DNA was generated by incubating the DNA barcodes with azido-PEG4-NHS (Click Chemistry Tools) at a molar ratio of 1:20 for 4 hours at room temperature. Excess azido-PEG4-NHS was removed using an Amicon Ultra-0.5 centrifugal filter unit (10K, Millipore). (ii) Preparation of DBCO antibodies: For anti-CD63 and anti-CD9, a 40K ZebaTM spin desalting column (Thermo Scientific) was used for buffer exchange with 50 mM borate buffered saline (BBS, pH 8.4). Antibodies were incubated with NHS-PEG5-DBCO (Click Chemistry Tools) at a molar ratio of 1:10 for 45 minutes at room temperature. Excess NHS-PEG5-DBCO was removed using an Amicon Ultra-0.5 centrifugal filter unit (50K, Millipore). (iii) DNA-antibody coupling: SPAAC was used to functionalize DBCO-antibody using N3-DNA overnight at 4°C. Coupling of antibody and DNA was performed in PBS at a molar ratio of 1:2, 1:4 and 1:10 to determine the functionalization efficiency. Excess N3-DNA was removed twice using an Amicon Ultra-0.5 centrifugal filter unit (50 kDa, Millipore). The DNA-antibody was stored in a glycerol / PBS (1:1) solution at -20°C and retrieved before use. (Figure 6B) The coupling between the DNA barcode and anti-CD63 was detected by gel electrophoresis. Briefly, DNA, DNA-N3 and DNA antibody-conjugates were run on a 2% agarose gel (0.5×TBE) using DNA SafeStain (Lamda Biotech) and scanned on a gel recording system. (Figure 6C) After purification by Amicon centrifugal filters, DNA-conjugated EV-specific antibodies were characterized by dual real-time immuno-PCR. The linearity of the dual real-time immuno-PCR used for detection was (Figure 6D) DNA1-anti-CD63, R 2 =0.993, and ( Fig. 6E )DNA2-anti-CD9, R 2 = 0.997. (DBCO, diarylcyclooctyne; EV, extracellular vesicle; IDT, integrated DNA technologies; NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; PEG, polyethylene glycol; SPAAC, strain-promoted azide-alkyne cycloaddition.

[0103] Optimization of click-bead isolation of hepatocellular carcinoma EVs

[0104] The yield of HCC EVs isolated by clicking beads was evaluated by quantitative methods (Figure 9A). Artificial plasma samples were prepared by spiking 10 μL aliquots of HCC cell line-derived EV pellets into 90 μL of plasma from healthy donors. Since AFP transcripts were not present in the plasma of the healthy donors we tested, the yield of EVs isolated by clicking beads from HCC cell lines can be calculated using RT-ddPCR assays according to the following equation:

[0105] In short, 700 μL QIAzol lysis reagent (Qiagen) was used to lyse the original 10 μL aliquots of HCC EV and HCC EV separated by clicking beads. RNA was extracted using miRNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized using Maxima H Minus reverse transcriptase kit (Thermo Scientific) according to the manufacturer's instructions. Dual droplet digital PCR was used on the QX200 system (Bio-Rad Laboratories, Inc.) to test the cDNA of AFP transcripts, and the resulting data were analyzed using QuantaSoftTM software (Bio-Rad Laboratories., Inc.). Primers and probes for AFP were purchased from Thermo Fisher Scientific (AFP, Hs01040598_m1448489). (Fig. 9B to Fig. 9E) Using artificial plasma samples spiked with HepG2-derived EVs, the separation yield of click beads was observed at different amounts of TCO-anti-EpCAM, TCO-anti-CD147, TCO-anti-GGPC3, and TCO-anti-ASGPR1. Data are presented as mean ± SD of three independent determinations. Asterisks indicate the optimal amount of antibody conjugated to each TCO. ( Fig.9F ) The optimal separation yield of TCO-coupled antibody click beads was observed using HepG2-derived EV-spiked and Hep3B-derived spiked artificial plasma samples, respectively.

[0106] Characterization of HepG2-derived EVs captured on click beads

[0107] The identity of HepG2-derived EVs captured on click beads was verified using anti-CD63 immunogold staining in TEM imaging. EVs were identified and highlighted using 12 nm gold nanoparticles using anti-CD63. Fig.10) Scale bar, 100 nm. Yellow arrows: gold nanoparticles.

[0108] Optimizing HCC EV surface protein assays using HCC cell line-derived EVs

[0109] Four subpopulations of HCC EVs (EpCAM + The workflow of HCC EV surface protein determination was determined by using artificial plasma samples, in which 10 7 HepG2-derived EVs were spiked with 100 μL fetal bovine serum (FBS) to prepare. (Figure 12A) Duplex real-time immuno-PCR was used to quantify the copy number of DNA1-anti-CD63 labeled on the purified subpopulations of EV samples. (Figure 12B) Comparison of relative signals from HCC EV surface protein assay under different blocking conditions. 5% BSA combined with PFBS achieved the best blocking results. (Figure 12C) DNA1-anti-CD63 had a higher signal from the HCC EV surface protein assay compared to the DNA-IgG control. (Figure 12D) The relative signal of the HCC EV surface protein assay was used to detect four subpopulations of HCC EVs. The highest signal was detected in the CD147+ subpopulation. Good linearity was achieved in the HCC EV surface protein assay for detecting HepG2 cell-derived EVs using DNA1-anti-CD63, (Figure 12E) EpCAM + Subgroup, R 2 =0.992; (Fig. 12F) CD147 + Subgroup, R 2 =0.989; (G) GPC3 + Subgroup, R 2 =0.999; and ( Fig.12H )ASGPR1 + Subgroup, R 2 =0.980.

[0110] Validation of the reproducibility of HCC EV surface protein assays using clinical plasma samples

[0111] Four HCC EV subsets (EpCAM + CD63 + HCC EV, CD147 + CD63 + HCC EV, GPC3 + CD63 + HCC EV, ASGPR1 + CD63 +Reproducibility of HCC EV quantification. Fig.13 ) The coefficient of variation (CV%) for each subpopulation of each sample is listed in Figure 15 (Table 2).

[0112] ROC curves of eight HCC EV subsets for early HCC detection from cirrhosis in the UCLA training cohort

[0113] EpCAM + CD63 + HCC EV, CD147 + CD63 + HCC EV, GPC3 + CD63 + HCC EV, ASGPR1 + CD63 + HCC EV、EpCAM + CD9 + HCC EV, CD147 + CD9 + HCC EV, GPC3 + CD9 + HCC EVs and ASGPR1 + CD9 + ROC curves of HCC EV for differentiating early HCC from cirrhosis in the UCLA training cohort. Fig.14 )

[0114] Antibody-DNA barcoding for HCC EV SPA

[0115] A total of 100 μL of plasma from each patient was added to an Eppendorf tube, and TCO-conjugated HCC-related antibodies: TCO-anti-EpCAM (25 ng), TCO-anti-CD147 (25 ng), TCO-anti-GPC3 (25 ng), and TCO-anti-ASGPR1 (25 ng) were also added. TCO-antibody-labeled plasma samples were incubated with click beads (0.1 mg) for 30 minutes to separate each subpopulation of EVs, relying on the capture of TCO-antibodies on click beads mediated by covalent chemistry. Click beads with immobilized HCC EVs were collected by centrifugation at 10,000 g for 3 minutes. All plasma samples received click beads with only one freeze-thaw cycle.

[0116] After HCC EVs were fixed to click beads, 10 EV-specific antibody-DNA barcode conjugates, including DNA1-anti-CD63, DNA2-anti-CD9, DNA3-anti-CD81, DNA4-anti-Annexin V, and other DNA barcode-conjugated EV-specific antibodies, were incubated with immobilized HCC EVs. Next, click beads with antibody-DNA barcode conjugates were collected by centrifugation at 10,000 g for 3 minutes, and then complementary DNA was incubated, which consisted of a DNA barcode binding moiety and a PCR handle required for subsequent NGS library preparation. A photocleavable linker was added between each pair of DNA barcodes and HCC EV-specific antibodies, allowing UV irradiation-triggered DNA barcode photocleavage and recovery for NGS quantification.

[0117] DNA barcoding library preparation and NGS analysis

[0118] The collected DNA barcodes were then subjected to a library preparation process that began with the extension of three deoxycytidine triphosphates (dCTPs) from the 3' end of the complementary DNA by the unique activity of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). Then, a template switching (TS) primer with three guanosine triphosphates (rGTP) at its 3' end hybridized with the protruding dCTP portion of the complementary DNA, and a template switching reaction then occurred, generating a complementary sequence to the TS primer. Subsequent polymerase chain reaction (PCR) with a forward and reverse primer set that binds to the TS primer and the complementary sequence of the PCR handle, respectively, amplified the DNA barcodes, which can be referred to as regions of interest (ROIs). A second round of PCR was also performed to further add the P5 / P7 adapter and a unique sample index, and the final library product was analyzed by using the Illumina NGS system. (Figure 2) In addition, the feasibility of the NanoString system as a downstream readout for HCC EV SPA was also evaluated, as shown in Figure 3.

[0119] Detection of four HCC-related surface protein markers in HCC tissue microarray (TMA)

[0120] In a HCC tissue microarray (TMA) generated from 708 resected HCC specimens archived at the University of California, Los Angeles (UCLA), hematoxylin and eosin (H&E) and immunohistochemistry (IHC) staining for four HCC-related surface protein markers were performed. HCC tissues were fixed in 10% neutral formalin for 24 hours and embedded in paraffin according to the tissue standard operating procedures of the UCLA Pathology Department. Five-micrometer-thick serial tissue microarray sections were cut from formalin-fixed paraffin blocks and mounted on poly-L-lysine-coated glass slides. Standard IHC staining was performed according to a protocol optimized for each antibody, which included anti-EpCAM (Cell Signaling, Danvers, MA), anti-CD147 (R&D Systems, Minneapolis, MN), anti-glypican-3 (GPC3; Santa Cruz Biotechnology, Santa Cruz, CA), and anti-asialoglycoprotein receptor (ASGPR; Abcam, Cambridge, UK). All sections were scanned using an Aperio ScanScope AT high-throughput scanning system and converted to high-resolution digital images magnified 40-fold. A pathologist (Y.Z.) used a four-point scale for staining intensity (none, 0; weak, 1 + ; moderate, 2 + ; strong, 3 + ) to evaluate the IHC staining results. An IHC staining was considered positive if the HCC tissue showed weak (1 + ), moderate (2 + ), or strong (3 + ) staining for the tested antibody. (Figure 19)

[0121] Study population

[0122] In this phase 2 biomarker (case-control) study, a total of 106 participants (45 untreated early HCC patients and 61 cirrhotic patients) were enrolled as a training cohort at the Ronald Reagan UCLA Medical Center from October 2016 to August 2021 to develop and optimize a HCC EV SPA for differentiating early HCC from cirrhosis. A total of 72 participants (35 untreated early HCC patients and 37 cirrhotic patients) were enrolled as an independent validation cohort at the Cedars-Sinai Medical Center (CSMC) from October 2019 to October 2021. According to the UCLA Institutional Review Board (IRB) protocol number 14-000197 and the CSMC Institutional Review Board protocol number 00000066, all participants provided written informed consent for this study.

[0123] Diagnosis of HCC according to the American Association for the Study of Liver Diseases clinical practice guidelines: 8 (i) histology or (ii) imaging classified as Liver Imaging Reporting and Data System 5. Early HCC was defined as BCLC stage 0 or A. Cirrhosis was defined based on histological and imaging features of cirrhosis (nodular surface) or portal hypertension (splenomegaly or portosystemic collaterals). Patients with cirrhosis were ensured to be free of HCC by (i) follow-up of at least six months after blood collection or (ii) negative contrast-enhanced multiphase computed tomography or magnetic resonance imaging within two weeks of blood collection. All HCC cases were untreated at the time of blood collection. Patients were excluded if they had concomitant tumors.

[0124] The etiologies of underlying liver disease were defined as follows. HBV infection was confirmed by HBsAg positivity. Hepatitis C virus (HCV) infection was confirmed by (i) HCV RNA or (ii) chronic liver disease with anti-HCV positivity. Alcoholic liver disease (ALD) was confirmed by (i) a documented history of ALD or (ii) a history of significant alcohol abuse or addiction. Nonalcoholic fatty liver disease or nonalcoholic steatohepatitis was confirmed by (i) histological or radiological evidence of fatty infiltration or inflammation without any history of significant alcohol intake (less than 20 g per day), or (ii) clinical evidence of risk factors for fatty liver disease, such as metabolic syndrome, without any history of significant alcohol intake.

[0125] Clinical study design flow chart describing the recruitment and exclusion of the study cohort. ( Fig.17 ) Blood samples were collected from 106 and 73 eligible participants from UCLA (training cohort) and CSMC (independent validation cohort), respectively. In brief, logistic regression was used in the training cohort (UCLA cohort, n = 106) to determine the HCCEV subpopulation significantly associated with early HCC relative to cirrhosis, and a logistic regression model (i.e., HCC EV ECG score) was established to detect early HCC from cirrhosis. Leave-one-out cross-validation was used to evaluate the performance of the HCC EV ECG score established in the training cohort. After one participant was excluded due to coexistence with other tumors, the HCC EV ECG score was externally validated in the independent validation cohort (CSMC cohort, n = 72).

[0126] HCC EV Surface Protein Assay (SPA) for measuring HCC EV subpopulations and detecting early-stage HCC in a UCLA training cohort

[0127] Heatmap summarizing the relative duplex real-time immuno-PCR readouts of plasma samples from patients with early-stage HCC (BCLC stage 0 to A, n=45) and patients with cirrhosis (n=61). (Figure 20A) CD63 expression was observed in HCC patients (Figure 20B) compared to those in cirrhosis patients. + and (Fig. 20C) CD9+ The immunoPCR signal of HCC EV subpopulation was significantly higher. (Fig. 20D) ROC curve of HCC EV ECG score, which is composed of EpCAM + CD63 + HCC EV, CD147 + CD63 + HCC EVs and GPC3 + CD63 + The signal of HCC EV was calculated using HCC EVSPA and used to detect early HCC from cirrhosis in the UCLA training cohort. Fig.20E ) ROC curve of the HCC EV ECG score after leave-one-out cross-validation for early HCC detection from cirrhosis in the UCLA training cohort.

[0128] Blood sample processing

[0129] Peripheral venous blood samples were collected from participants with written informed consent according to the IRB protocols of UCLA and CSMC. Blood samples were collected using 10.0 mL ethylenediaminetetraacetic acid vacuum tubes (BD Medical, Fisher Cat. # BD 366643-1). Whole blood was centrifuged at 530 g for 10 minutes, and then the supernatant was recovered and centrifuged at 4600 g for 10 minutes. The final supernatant was collected as a plasma sample and stored in a -80 ° C refrigerator. All plasma samples were retrieved before receiving HCC EV SPA.

[0130] General information about HCC EV SPA

[0131] To perform HCC EV SPA, (i) click beads (i.e., methyltetrazine [mTz]-modified microbeads) were first prepared according to a chemical modification procedure ( Figure 4 ), (ii) preparation of four trans-cyclooctene (TCO)-conjugated HCC-related antibodies using a copper-free click chemistry coupling method ( Figure 5 ), (iii) DNA barcodes were coupled to anti-CD63 and anti-CD9 to target EV markers and subsequently validated ( Figure 6 ). To evaluate the performance of click beads in capturing HCC EVs, we prepared HCC cell line-derived EVs according to our previous protocol. 17 Characterization of HepG2-derived EVs (Figure 8) was performed according to the Minimum Information for the Study of Extracellular Vesicles (MISEV) 2018 published by the International Society for Extracellular Vesicles 21 and MISEV 2014 22 Figures 8 and 9 summarize the evaluation of click beads for capturing HCC EVs.

[0132] Characterization of HCC cell line-derived EVs

[0133] EVs derived from HCC cell lines were prepared according to our previous protocol. 3 and MISEV 2014 4 Guidelines for characterization of HepG2-derived EVs. (Figure 8A) The concentration and size distribution of HepG2-derived EVs (diluted 1:100 in PBS) were measured by nanoparticle tracking analysis using NanoSight NS300 (Malvern Instruments Ltd, Malvern, UK). The size of HepG2-derived EVs ranged from 80nm to 550nm. (Figure 8B) The expression of EV markers (CD63 and Annexin V) on HepG2-derived EVs was analyzed by flow cytometry. ( Figure 8C ) Representative TEM images of HepG2-derived EVs in bulk solution before capture with immunogold labeling. EVs were identified and highlighted with gold nanoparticles using anti-CD63. Scale bar, 100 nm. Briefly, EVs fixed in PBS were incubated with anti-CD63 (Abcam, mouse, 1:100 dilution) for 30 min. The samples were then incubated with nanogold-conjugated anti-mouse IgG (12 nm, 1:50 dilution) for 1 h. The gold-labeled samples were dropped onto carbon-coated copper grids and incubated for 10 min before being wiped off the grids. After rinsing with water 5 times, the grids were dried for TEM imaging.

[0134] HCC EV SPA for quantification of eight subpopulations of HCC EVs

[0135] HCC EV SPA is a three-step workflow (Figures 1 and 16 and Fig.10 and Fig.11 Internal control fetal bovine serum (FBS) was used instead of plasma samples to measure the background binding of DNA1-anti-CD63 and DNA2-anti-CD9 to click beads.

[0136] Step 1: Sequential labeling of each subpopulation of HCC EVs in plasma

[0137] A total of 400 μL of plasma from each patient was evenly aliquoted into four Eppendorf tubes. In the first incubation, the four 100 μL plasma aliquots were incubated with the respective TCO-conjugated HCC-related antibodies: TCO-anti-EpCAM (50 ng), TCO-anti-CD147 (25 ng), TCO-anti-GPC3 (50 ng), and TCO-anti-ASGPR1 (50 ng). In the second incubation, these aliquots were corktailed with DNA-conjugated EV-specific antibodies: DNA1-anti-CD63 and DNA2-anti-CD9 at room temperature for 30 minutes. TCO-conjugated HCC-related antibodies were not added to the internal controls incubated only with DNA1-anti-CD63 and DNA2-anti-CD9.

[0138] Step 2: Covalent chemistry-mediated capture of HCC EV subpopulations on click beads

[0139] Click Bead 19 Blocked with protein-free buffer and then with 5% bovine serum albumin (BSA) solution before use. Plasma samples labeled with TCO-antibodies and DNA barcodes were then incubated with click beads (0.1 mg) for 30 min to separate each subpopulation of EVs. Click beads with separated EVs were collected by centrifugation at 10000 g for 2 min. All plasma samples received click beads with only one freeze-thaw cycle. Internal controls were treated according to the same protocol.

[0140] Step 3: On-bead duplex real-time immuno-PCR for quantification of each subpopulation of HCC EVs

[0141] The click beads with separated EVs were resuspended in 500 μL PBS containing 0.2% BSA and then collected by centrifugation at 10,000 g for 2 minutes. After 3 washing steps with PBS containing 0.2% BSA, the click beads were then washed 2 times with PBS to wash off unbound DNA1-anti-CD63 and DNA2-anti-CD9. After 5 washing steps, 90 μL of double distilled water was added to the sediment of each well and mixed thoroughly. 9 μL of the mixed suspension was loaded into the real-time PCR system (CFX Connect TM Real-time PCR detection system, Bio-Rad). Primers and probes for DNA barcodes were purchased from Integrated DNA Technologies (IDT), and the sequences are listed in Figure 7 (Table 1). The raw data of the real-time PCR run was evaluated using the corresponding software system (Bio-Rad CFX Manager 3.1, Bio-Rad). The relative signal was calculated using the following equation:

[0142] Relative signal = 2 -(Cq9样本)-Cq(内部对照))

[0143] Optimization and validation of HCC EV SPA using HepG2-derived EVs

[0144] Optimization and validation of HCC EV SPA using HepG2-derived EVs Fig.10 We first used 5% BSA, Pierce TM The click beads were blocked with protein-free (PBS) blocking buffer (PFBS, Thermofisher, #37572). Then, EpCAM of HepG2-derived EVs was quantified using DNA-IgG control (anti-PSA, monoclonal mouse IgG1, Abcam) conjugate and DNA-anti-CD63, respectively. + To verify the efficacy of DNA-anti-CD63, a 10 4 , 10 5 and 10 6 EV subpopulations were isolated by mixing with DNA-anti-CD63 for 30 min at room temperature. FBS samples without HepG2-derived EVs were implemented as internal controls. Then, samples labeled with TCO-antibodies and DNA-anti-CD63 were incubated with EV click beads (0.1 mg) for 30 min for EV subpopulation separation. Click beads with isolated EVs were collected by centrifugation at 10000 g for 2 min. After 5 washing steps, click beads were measured as described above.

[0145] Evaluation of the reproducibility of HCC EV SPA

[0146] To test the general applicability of the HCC EV SPA, the reproducibility of the HCC EV SPA was evaluated by calculating the coefficient of variation percentage (%CV) relative to the signal. Two plasma samples from patients with cirrhosis and two plasma samples from patients with HCC were processed with the HCC EV SPA in triplicate by the operator at different time points. The reproducibility evaluation of the HCC EV SPA is summarized in Fig.11 and Figure 15 (Table 2).

[0147] Statistical analysis

[0148] For descriptive statistics, continuous variables were reported as medians and interquartile ranges, and categorical variables were reported as numbers and percentages. Comparisons of continuous and categorical variables between groups were completed using the Mann-Whitney U test and Fisher's exact test or square test, respectively.

[0149] In this retrospective phase 2 biomarker (case-control) study, the sample size was calculated using the paired DeLong test to compare the HCC EV SPA or serum AFP 10 The AUC of the model was calculated using a sample size of 48 (24 HCC and 24 controls), which was expected to have 90% power to detect a difference between the AUROCs of HCC EV SPA and serum AFP for early HCC detection, assuming that our assay had an AUROC of 0.93 and serum AFP 17 The AUROC of 50.3% was 0.69 when the correlation between the assays was assumed to be 0.5. The power was obtained from a two-sided test with a significance level of 0.05.

[0150] To determine which HCC EV subpopulations were significantly associated with early HCC relative to cirrhosis (P < 0.05), univariate logistic regression analysis was applied in the training cohort (UCLA cohort; 45 HCC and 61 cirrhosis). The HCC EV subpopulations that were highly associated with HCC at a significance level of 0.005 were included as the final logistic regression model (i.e., HCC EV ECG score) for the detection of early HCC from cirrhosis. The Youden Index was used to determine the optimal cutoff value for the HCC EV ECG score. Leave-one-out cross-validation was applied to evaluate the performance of the HCC EV ECG score in the training cohort. The HCC EV ECG score was externally validated in an independent validation cohort (CSMC cohort; 35 HCC and 37 cirrhosis). Figure 19 summarizes the overall study design. According to the ILCA guidelines for the development of HCC biomarkers, 20 The sensitivity, specificity, and AUROC of the HCC EV ECG score were evaluated in the training and validation cohorts to distinguish early-stage HCC from high-risk cirrhosis. The AUROC between the HCC EV ECG score and AFP was compared using the paired DeLong test.

[0151] All statistical analyses were performed using MedCalc software (version 20.015; MedCalc software Ltd, Ostend, Belgium), GraphPad Prism (version 9.2.0; GraphPad software, Inc., CA, USA), and R statistical software (version 4.0.2; R Foundation, Vienna, Austria) with two-sided tests and a significance level of 0.05.

[0152] Quantification of eight HCC EV subsets in the UCLA cohort to differentiate early-stage HCC from high-risk cirrhosis

[0153] After optimizing HCC EV SPA using artificial samples (Figures 9 to 12) and confirming reproducibility ( Fig.13and Fig.17 (Table 2)), 45 early-stage HCC patients and 61 high-risk patients with cirrhosis were enrolled at UCLA as a training cohort for testing the HCC EV SPA. The demographic and clinical characteristics of the UCLA cohort are shown in Table 1. The age, gender, and race / ethnicity of HCC and cirrhotic control patients were similar. Approximately three-quarters of HCC patients (73%) had well-compensated liver disease (Child-Pugh A), compared with nearly half (44%) of cirrhotic controls. Among HCC patients, 82% had cirrhosis. 80% of HCC patients had BCLC cancer stage A, and the rest were BCLC stage 0. The optimized HCC EV SPA was used to obtain the HCC EV surface protein signature of the UCLA cohort ( Fig.17 Briefly, four HCC-associated antibodies (i.e., anti-EpCAM, anti-CD147, anti-GPC3, and anti-ASGPR1) were identified to purify EVs, and two conjugated DNA-antibodies targeting EV markers (i.e., DNA1-anti-CD63 and DNA2-anti-CD9) were used to report the presence of EVs. + CD63 + HCC EV, CD147 + CD63 + HCC EV, GPC3 + CD63 + HCCEV、ASGPR1 + CD63 + HCC EV、EpCAM + CD9 + HCC EV, CD147 + CD9 + HCC EV, GPC3 + CD9 + HCC EVs and ASGPR1 + CD9 + The readouts obtained for the HCC EVs are summarized in Figure 8A. Overall, significantly higher signals were observed in the HCC group compared to those found in the cirrhosis group (P < 0.005 in all subgroups; Figures 8B to Figure 8C ).

[0154] Development of the HCC EV ECG score for differentiating early-stage HCC from high-risk cirrhosis

[0155] To evaluate the ability of HCC EV surface protein signatures (based on quantification of eight subpopulations of HCC EVs) to distinguish early-stage HCC from high-risk cirrhosis, the UCLA cohort was used as a training cohort, as well as a supplemental Fig.10The diagnostic performance of each HCC EV subset was summarized. Among the eight HCC EV subsets, CD147 + CD63 + HCC EVs and GPC3 + CD63 + HCC EVs had the highest AUROC, 0.91 (95% confidence interval [CI] = 0.86-0.96) and 0.86 (95% CI = 0.79-0.94), respectively. In addition, univariate logistic regression analysis was performed to determine which HCC EV subpopulations were significantly associated with early HCC relative to cirrhosis (Figure 16, Table 3). Three HCC EV subpopulations with a significance level of 0.005 that were highly associated with HCC were selected in the final logistic regression model: EpCAM + CD63 + HCCEV、CD147 + CD63 + HCC EVs and GPC3 + CD63 + HCC EV, used to detect early HCC from cirrhosis, is called the HCC EVECG score.

[0156] HCC EVE ECG score is defined as: HCC EV ECG score = -9.54338 + 0.13544 * [EpCAM + CD63 + HCCEV]+0.35729*[CD147 + CD63 + HCC EV]+0.37513*[GPC3 + CD63 + HCC EV]

[0157] The HCC EV ECG score showed excellent accuracy for distinguishing early HCC patients from cirrhotic controls in the training cohort with an AUROC of 0.95 (95% CI = 0.90-0.99; Figure 20D). At the optimal cutoff value of -0.40, the sensitivity and specificity of the HCC EVECG score for detecting early HCC were 91% (95% CI = 79%-96%) and 90% (95% CI = 80%-95%), respectively. In addition, the leave-one-out cross-validation of the training cohort confirmed an AUROC of 0.95 (95% CI = 0.88-0.98; Fig.20E ), the accuracy of the model with a sensitivity of 89% (95% CI = 76%-96%) and a specificity of 89% (95% CI = 78%-95%).

[0158] HCC EV ECG score reveals performance of early-stage HCC in an independent validation cohort

[0159] To further validate the HCC EV ECG score for early HCC detection, 35 early HCC patients and 37 high-risk patients with cirrhosis recruited from CSMC were analyzed as independent validation cohorts. The clinical characteristics of the CSMC cohort are shown in Figure 7, Table 1, and the HCC EV surface protein characteristics of the CMSC cohort are summarized in Figure 21A. There were no significant differences between the CSMC and UCLA cohorts in terms of patient characteristics. As expected, EpCAM + CD63 + HCC EV, CD147 + CD63 + HCC EVs and GPC3 + CD63 + The readings of HCCEV were significantly higher in HCC patients than in cirrhosis patients (P<0.001; FIG. 21B ).

[0160] In the CSMC validation cohort, the AUROC of the HCC EV ECG score for detecting early HCC remained excellent at 0.93 (95% CI = 0.87-0.99; Fig. 21C ). When the cutoff value was -0.65, the sensitivity was 94% (95% CI = 81%-99%), and the specificity was 81% (95% CI = 65%-92%). When the cutoff value was set to -0.40, the same as the optimal critical value determined in the training cohort, the HCC EV ECG score still had a high accuracy in detecting early HCC, with a sensitivity of 91% (95% CI = 77%-98%) and a specificity of 81% (95% CI = 65%-92%).

[0161] Comparison and subgroup analysis of HCC EV ECG score and serum AFP for early detection of HCC

[0162] After validating the HCC EV ECG score in an independent cohort, we compared the performance of the HCC EV ECG score with serum AFP in detecting early HCC in all participants in this study (UCLA+CSMC cohort, n=172). Individuals without serum AFP records (n=4) were excluded from the analysis. As shown in Figure 22A, the HCC EV ECG score was superior to serum AFP in distinguishing early HCC from cirrhosis (AUROC=0.94 [95%CI=0.90-0.97] vs. 0.79 [95%CI=0.72-0.86], P<0.001). At the determined cutoff value of -0.40, the sensitivity of the HCC EV ECG score was 91% (95%CI=82%-96%) and the specificity was 86% (95%CI=78%-92%). For serum AFP, when the standard cutoff value of 20 ng / mL was used, the sensitivity was 45% (95% CI = 34%-57%) and the specificity was 98% (95% CI = 94%-100%).

[0163] The HCC EV ECG score performed well in patient subgroups stratified by etiology (viral vs. non-viral) and Milan criteria. In patients with chronic viral hepatitis (n = 64), the AUROC was 0.95 (95% CI = 0.90-1.00; Figure 22B); on the other hand, the AUROC for patients with non-viral etiology (n = 114) was 0.94 (95% CI = 0.88-0.99; Figure 22C). When HCC patients were restricted to patients with tumors according to the Milan criteria (n = 166), the HCC EV ECG score still had excellent performance for the detection of HCC, with an AUROC of 0.93 (95% CI = 0.89-0.97; Fig.22D ), with a sensitivity of 90% (95% CI = 80%-96%) and a specificity of 87% (95% CI = 78%-93%). 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Claims

1. A method for determining a disease in a subject, comprising: Selectively labeling extracellular vesicles (EVs) from a sample from the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group such that the first capture agent is still attachable to the EV and the first molecule is capable of attaching a second molecule from a second bioorthogonal functional group that is complementary to the first molecule, functionalizing the DNA barcode with a second molecule, linking the functionalized first capture agent to the functionalized DNA barcode to form a barcoded capture agent, mixing the barcoded capture agent with the sample to form a labeled sample, selectively capturing the EVs from the sample from the subject, comprising: functionalizing the second capture agent of the EV with a third molecule from a third bioorthogonal functional group such that the second capture agent can still be attached to the EV and the third molecule is capable of attaching a fourth molecule from a fourth bioorthogonal functional group that is complementary to the third molecule, The functionalized second capture agent is mixed with the labeled sample so that the functionalized second capture agent binds to the EV to form an activated sample, functionalizing the capture surface with said fourth molecule, and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through binding of the fourth molecule to the third molecule; determining the first DNA barcode by a nucleic acid test; and The presence or absence of the disease in the subject is determined by measuring the first DNA barcode from the EV.

2. A method for determining a disease in a subject, comprising: Selectively labeling extracellular vesicles (EVs) from a sample from the subject with a first DNA barcode, comprising: functionalizing a first capture agent of the EV with a first molecule from a first bioorthogonal functional group such that the first capture agent can still be attached to the EV and the first molecule is capable of attaching a second molecule from a second bioorthogonal functional group that is complementary to the first molecule, functionalizing the DNA barcode with a second molecule, linking the functionalized first capture agent to the functionalized DNA barcode to form a barcoded capture agent, functionalizing the second capture agent of the EV with a third molecule from a third bioorthogonal functional group such that the second capture agent can still be attached to the EV and the third molecule is also able to attach a fourth molecule from a fourth bioorthogonal functional group that is complementary to the third molecule, mixing the barcoded capture agent and the functionalized second capture agent with the sample to form an activated sample, selectively capturing the EVs from the sample from the subject, comprising: functionalizing the capture surface with said fourth molecule, and depositing at least a portion of the activated sample on at least a portion of the functionalized capture surface, thereby selectively capturing the EV through binding of the fourth molecule to the third molecule; determining the first DNA barcode by a nucleic acid test; and The presence or absence of the disease in the subject is determined by measuring the first DNA barcode from the EV.

3. The assay method according to any one of claims 1-2, wherein the second capture agent selected for capturing the EV is a tumor-specific capture agent that can bind to a surface marker specifically expressed by tumor-derived EVs.

4. The assay method according to any one of claims 1-3, wherein selectively capturing extracellular vesicles (EVs) from a sample from a subject is performed before selectively labeling the EVs from the sample from the subject with a first DNA barcode.

5. The assay method according to any one of claims 1 to 4, further comprising: Repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with a second DNA barcode, such that the EVs are labeled with different capture agents that bind to different regions of the EVs, and the first DNA barcode and the second DNA barcode are distinguishable from each other; determining the second DNA barcode using a nucleic acid test; as well as The presence or absence of the disease in the subject is determined by measuring the first DNA barcode and the second DNA barcode from the EV.

6. The assay method according to any one of claims 1 to 5, further comprising: Repeatedly selectively labeling the extracellular vesicles (EVs) from the sample with more DNA barcodes, such that the EVs are labeled with a plurality of different capture agents that each bind to a different region of the EV, and the first DNA barcode and the more DNA barcodes are distinguishable from each other; Determining the greater number of DNA barcodes using a multiplex nucleic acid test; as well as The surface protein expression signature is determined by measuring the multiple DNA barcodes.

7. The assay method according to any one of claims 1 to 6, further comprising: Selective labeling, selective capture, determination of DNA barcoding, and determination of surface protein expression signatures are repeated multiple times to predict disease stage and / or distinguish between different disease stages.

8. The assay method according to any one of claims 1 to 7, further comprising: The DNA barcode or the multiple DNA barcodes are released from the captured EVs prior to measurement.

9. The assay method of claim 8, wherein the surface protein expression signature distinguishes clinical samples at different disease stages.

10. The assay method of claim 9, wherein the expression of EpCAM+CD63+ on HCC EVs, the expression of CD147+CD63+ on HCC EVs, and the expression of GPC3+CD63+ on HCC EVs are higher in HCC patients compared to cirrhosis patients.

11. The assay method according to any one of claims 1 to 10, wherein the capture surface is the surface of beads in suspension.

12. The assay method according to any one of claims 1 to 11, wherein the capture surface is a surface of a microfluidic device.

13. An assay method according to any one of claims 1-12, wherein the capture agent capable of capturing the EV is functionalized with a third bioorthogonal functional group selected from the group consisting of trans-cyclooctene (TCO), alkynes and cyclooctyne, and wherein the capture surface is functionalized with a fourth bioorthogonal functional group selected from the group consisting of tetrazine (Tz) and azide.

14. The assay method according to any one of claims 1 to 13, wherein the assay comprises using multiplex PCR.

15. The assay method according to any one of claims 1-14, wherein the assay comprises using a next generation sequencing process.

16. A kit for use in a method for determining a disease in a subject according to any one of claims 1 to 15, the kit comprising: a plurality of capture agents, each of the capture agents binding to a different region of an EV of the subject; A variety of molecules with complementary bioorthogonal functional groups; Multiple DNA barcodes; as well as Instructions for labeling the EVs with the multiple DNA barcodes, capturing the EVs, determining the multiple DNA barcodes, and determining by determination whether the subject has the disease or the disease stage of the subject.

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