Methods and devices for characterizing nanovesicles and their bound or associated targets

Abstract

The present invention relates to methods for detecting and / or characterizing nanovesicles in a sample, or methods for detecting targets bound to or associated with the nanovesicles, wherein the sample is contacted with nanoparticles that are capable of binding to the surface of the nanovesicles and forming a nanoshell surrounding the nanovesicles in situ. In a preferred embodiment, the nanovesicles are exosomes labeled with a fluorescent probe, and the nanoparticles are gold nanoparticles (AuNPs). The present invention also relates to kits or microfluidic chips for performing these methods, as well as methods for determining the prognosis of cancer in a subject by performing the methods.

Description

Technical Field

[0001] The inventions described herein generally relate to the field of biotechnology. Specifically, the invention relates to methods for detecting and / or characterizing nanovesicles or methods for detecting targets bound to or associated with the nanovesicles. The invention also relates to kits or microfluidic chips for performing these methods. Background Art

[0002] Recently, exosomes have become promising circulating biomarkers. Exosomes are known for their biophysical and biomolecular composition. They are nanoscale membrane vesicles (30 to 150 nm in diameter) that are actively released by a variety of mammalian cells, and most especially dividing cancer cells. Exosomes contain rich molecular content (including proteins, nucleic acids, lipids, and various modifications), either as genetic components from parent cells or as membrane-associated molecules. As powerful messengers of intercellular communication, exosomes play an important role in mediating disease progression. For example, cancer cells actively produce and utilize exosomes to promote tumor growth. Exosomes are released the most by rapidly dividing cancer cells. Exosomal contents not only mediate intercellular communication, but also regulate the microenvironment to promote cancer metastasis. This orchestrated release and functional activity highlight the clinical potential of exosomes as more reflective circulating biomarkers.

[0003] Despite this clinical potential, direct and specific analysis of exosomes in natural biofluids remains technically challenging, especially for clinical translation. Specifically, clinical biofluids are compositionally diverse and contain nanoscale vesicles as well as abundant non-vesicular, free molecules. Current detection of exosome populations in such complex mixtures relies primarily on biophysical or biochemical characterization, performed in a separate or sequential manner. In biophysical preparations, vesicles of a characteristic size can be isolated by traditional ultracentrifugation or advanced sorting strategies; however, these methods require extensive processing, face contamination with other similarly sized protein aggregates, and lack biomolecular confirmation of vesicle identity. On the other hand, biochemical assays typically use affinity enrichment based on common exosomal markers to capture and measure vesicles. This approach often misses vesicle subpopulations and / or is susceptible to interference from biochemically identical but histologically distinct molecular targets (e.g., non-vesicular, free protein antigens).

[0004] Therefore, it is generally desirable to overcome or ameliorate one or more of the above-mentioned difficulties. Summary of the Invention

[0005] Disclosed herein is a method for detecting and / or characterizing nanovesicles in a sample, the method comprising the following steps:

[0006] a) contacting the sample with nanoparticles or precursors of nanoparticles, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicles and forming a nanoshell around the vesicles in situ; and

[0007] b) illuminating the sample and measuring an optical signal of the sample to detect and / or characterize nanovesicles in the sample.

[0008] Furthermore, disclosed herein is a method for detecting one or more targets bound to or associated with nanovesicles in a sample, the method comprising the steps of:

[0009] a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the nanovesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each of the targets; and

[0010] b) illuminating the sample and measuring the emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

[0011] Furthermore, disclosed herein is a microfluidic chip for performing the method defined herein.

[0012] Furthermore, disclosed herein are kits for performing the methods defined herein.

[0013] Furthermore, disclosed herein is a method of determining the prognosis of cancer in a subject by simultaneously detecting or characterizing one or more targets that are bound to or associated with nanovesicles in a sample from the subject and are indicative of the nature of the cancer, the method comprising:

[0014] a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicles and forming a nanoshell surrounding the vesicles in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicles and providing a unique emission fluorescence wavelength for each of the targets; and

[0015] b) illuminating the sample and measuring absorbance and / or emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Embodiments of the invention are described below, by way of non-limiting example only, with reference to the accompanying drawings, in which:

[0017] Figure 1 Templated nanoplasmons for multiparametric profiling of exosomes. (a) Schematic diagram of the TPEX (Templated Plasmonic Exosome) platform. This technology is designed to measure exosomal markers and comprises three functional steps. First, exosomes are labeled with fluorescent molecular probes and AuNPs. Although the AuNPs remain well dispersed when associated with non-vesicular, free proteins, they assemble to the exosome periphery through electrostatic interactions. Excess unbound probes and AuNPs are not removed. In the presence of gold salts, the AuNPs serve as seeds for in situ gold growth. The dispersed AuNPs undergo minimal growth and produce slight shifts in their absorption spectra, leading to small changes in the probe's fluorescence signal. Exosome-bound AuNPs, on the other hand, develop into nanoshells; these nanostructures are templated to the vesicle size and exhibit a large red shift in their plasmon resonance, effectively quenching the fluorescence signal of probes bound to the same vesicles. Therefore, the TPEX fluorescence signal is multiparametric and is used for exosome biophysical characterization and biomarker composition. (b) Transmission electron micrographs of TPEX products. In the presence of free protein, AuNPs remain well dispersed (before) and exhibit small particle growth after treatment with gold salts (after). When incubated with exosomes, AuNPs bind to the periphery of the vesicles (before) and develop into large spherical particles after gold growth (after). Scale bar: 20 nm. (cd) Photos of the microfluidic device and smartphone-based optical detector. Absorbance and fluorescence measurements can be performed on the integrated platform using different LED light sources and filter configurations. Scale bar: 1 cm.

[0018] Figure 2 : TPEX absorbance analysis. (a) Optical simulation of templates of different sizes. Based on the microscopic characterization of the formed TPEX nanostructures, the plasmon resonance peaks of gold nanoshells developed on templates of different sizes are simulated (left). For exosome-sized templates (30 to 150 nm, red shading), the resulting plasmon peak is mainly located at >600 nm. The red dashed line represents the average peak wavelength formed by this template diameter range, and it is located at 750 nm. The electric field distribution at 750 nm of a single AuNP (naked or free protein-associated particle) and a gold nanoshell formed after gold growth (exosome-templated) is plotted (right). represents the particle size after gold growth. This simulation confirmed that nanoshells templated with exosome size can generate strong plasmon resonance at 750 nm. (b) Adjustment of the TPEX response range relative to the template diameter. Templates of different sizes were incubated with AuNPs of different diameters to form gold nanoshells. The TPEX absorbance measurement (A) is defined as the ratio of the absorbance at 750 nm and 540 nm, and the difference (ΔA) is defined as the difference before and after gold growth. Using 9-nm AuNPs, the TPEX response range can be optimized to match the exosome size, thereby maximizing the signal caused by exosomes. (c) Experimental evaluation of biological samples. Exosomes from human colorectal adenocarcinoma (DLD-1) were added to vesicle-depleted FBS (dFBS) and TPEX analysis was performed using 9-nm AuNPs. In all reactions, the resulting absorbance (left) and diameter change (right) were measured. The diameter change was analyzed by dynamic light scattering. Only samples containing exosomes showed a large signal increase, while reactions in PBS (i.e., bare AuNPs) and dFBS (i.e., free protein) showed negligible changes. (d) Correlation between TPEX absorbance analysis and exosome concentration. Exosomes from four cell lines (DLD-1, HTC116, MKH45, and SNU484) were counted by nanoparticle tracking analysis and evaluated by TPEX absorbance analysis. All measurements were performed in triplicate, and in (bc), data are shown as mean ± sd. *P < 0.05, ***P < 0.0005, NS, indicates not significant; Student's t-test; au indicates arbitrary units.

[0019] Figure 3: Multiplex fluorescence analysis of exosomal molecular markers. (a) TPEX fluorescence analysis. To evaluate whether TPEX nanoshells can be used to quench co-localized fluorescent probes, PDA nanoparticles were prepared as templates of defined size and conjugated to a fluorescent dye (A647). The templates were treated with the TPEX reaction, and the resulting changes in fluorescence (ΔF, top) and absorbance (ΔA, bottom) were measured. Both analyses showed similar trends and confirmed the optimized template size response range for exosome diameter. (b) Assay specificity of exosomal markers. Whole exosomes containing CD63 (derived from DLD-1) (top) and free CD63 (bottom) were incubated with fluorescent aptamers (anti-CD63 and scrambled control) for TPEX measurement. Only whole exosomes showed significant signal, while free CD63 samples showed negligible signal. Among the different fluorescent dyes tested (FITC, RB, and A647), the aptamer modified with A647 (emission 665 nm, closest to the TPEX absorbance of 750 nm) showed the greatest signal difference. (c) Multiplex profiling of exosome markers. Exosomes were incubated with different fluorescent aptamers and used for TPEX analysis alone (singleplex) or as a mixture (multiplex). The multiplex fluorescence spectra were consistent with the singleplex spectra (top) and showed accurate marker expression profiles across cell lines (bottom). (d) Molecular detection sensitivity. The detection limit was determined by titrating known amounts of exosomes and measuring their TPEX signals associated with CD63. The detection limit of the ELISA was independently evaluated based on chemiluminescence. All measurements were performed in triplicate, and the fluorescence analysis was normalized to the respective sample-matched scrambled controls. In a, b, and d, data are shown as mean ± sd. *P<0.05, **P<0.005, ***P<0.0005, NS, not significant; Student's t-test; au, arbitrary unit.

[0020] Figure 4: Exosome analysis in a complex context. (a) TPEX analysis of mock clinical samples. Samples were prepared by spiking exosomes from six human cell lines into vesicle-depleted human serum. In these spiked samples, the exosomal marker CD63 and putative cancer markers (including CD24, EpCAM, and MUC1) were measured. All protein measurements of the spiked samples were performed by traditional single-plex sandwich ELISA and by multiplex TPEX analysis on a microfluidic platform. This analysis was compared with the marker signature of pure exosomes (obtained from exosomes before spiking). For each marker analyzed, TPEX analysis showed better consistency reflecting expression trends across cell lines. (b) Correlation of TPEX measurements with pure exosome signatures. TPEX detection showed good correlation with pure exosome analysis (left), while conventional ELISA measurements performed on the same spiked samples showed significantly poorer correlation (right). All measurements were performed in triplicate for their respective sample-matched scrambled controls. Data were assay-normalized and shown as mean in a and mean ± SD in b.

[0021] Figure 5 : TPEX analysis of patient prognosis. (a) Analysis of protein markers in clinical cancer ascites (n = 20; 12 colorectal cancer and 8 gastric cancer) using multiplex TPEX to measure vesicle-associated target markers (top) and conventional single-plex ELISA to measure total target markers (bottom). TPEX analysis showed different protein expression profiles compared with ELISA analysis. (b, c) Receiver operating characteristic (ROC) curves of TPEX (b) and ELISA (c) regression models for ascites samples of colorectal cancer (left), gastric cancer (center), and two cancer types (right). ROC curves were constructed using single markers or combinations (mixes) of target markers. TPEX analysis showed higher prognostic classification accuracy for both cancers compared with ELISA assays. All measurements were performed in triplicate against respective sample-matched scrambled controls. Data were assay-normalized and shown as means in (a).

[0022] Figure 6 Size and molecular characterization of extracellular vesicles. (a) Dynamic light scattering analysis of the size distribution of free protein in depleted fetal bovine serum (dFBS) and (b) dynamic light scattering analysis of the size distribution of extracellular vesicles derived from a human colorectal adenocarcinoma cell line (DLD-1). (c) Transmission electron micrograph of DLD-1 vesicles. Scale bar: 20 nm. (d) Western blot analysis of vesicle lysates. Lysates were immunoblotted for exosome markers (CD63, ALIX, HSP70, TSG101, Flotillin 1).

[0023] Figure 7 Microscopic and spectroscopic characterization of TPEX products. Size distribution of gold nanomaterials before (top) and after (bottom) TPEX gold growth when incubated with (a) free protein and (b) exosomes. All measurements were determined by transmission electron microscopy (TEM) analysis. (c,d) Absorbance analysis of the corresponding gold nanomaterials. AU represents arbitrary units.

[0024] Figure 8 Schematic diagram of the microfluidic platform. Exploded view of the device. The platform is assembled from two polydimethylsiloxane (PDMS) layers and consists of a valve layer and a microchannel layer, which construct torque-activated valves for sequential flow control and a serpentine mixer for efficient labeling, respectively.

[0025] Figure 9 : Operation of TPEX device.

[0026] Figure 10 Optical simulations of TPEX gold nanostructures. (a) Electric field simulations of gold nanostructures generated from bare AuNPs (9-nm AuNPs only) and templated by exosomes of varying sizes. Simulated electric field distribution at wavelengths of 540 nm (top) and 750 nm (bottom). (b) Simulated absorbance of the resulting gold nanostructures as a function of template diameter. Nanoparticles templated with bare AuNPs and nanoshells templated with exosomes exhibit strong resonances at 540 nm and 750 nm, respectively. au denotes arbitrary units.

[0027] Figure 11 : Experimental validation under different template diameters. (a) Size distribution of various polydopamine templates determined by dynamic light scattering analysis. (b) Variation of template diameter with sodium hydroxide volume. (c) Experimental absorption spectrum of templated nanomaterials after growth. The experimental validation is consistent with the simulated absorbance. Specifically, in the absence of target template (i.e., bare AuNP), a single resonance peak was observed around 540 nm; when reacting with templates of increasing diameter, an additional resonance peak appeared at 750 nm. All measurements were performed in triplicate, and in ab, the data are shown as mean ± sd. Au represents arbitrary units.

[0028] Figure 12 Characterization of AuNPs of different sizes. (a) Absorbance measurements of AuNPs of different sizes before and after functionalization with polyethyleneimine (PEI). (b) Transmission electron micrographs of AuNPs of different sizes after PEI functionalization. Scale bar: 50 nm. (c) Size distribution of the prepared AuNPs determined by TEM analysis, confirming the monodispersity of the preparation. AU represents arbitrary units.

[0029] Figure 13TPEX assay of exosomes and free proteins. (a) Zeta potential and (b) hydrodynamic diameter of exosomes and free proteins (dFBS) measured under different experimental conditions (+, presence; -, absence). All measurements were performed in triplicate, and data are shown as mean ± SD.

[0030] Figure 14 Extracellular vesicles isolated from different cell sources. Extracellular vesicles isolated from colorectal cancer cells (a) DLD-1, (b) HCT116, and gastric cancer cells (c) MKN45, (d) SNU484. All vesicles were characterized using nanoparticle tracking assays.

[0031] Figure 15 TPEX absorbance analysis of exosomes. (a) Absorbance spectra of different exosome counts after TPEX reaction. Exosomes derived from the DLD-1 cell line were quantified using nanoparticle tracer analysis and subjected to TPEX reaction. (b) TPEX absorbance sensitivity. The limit of detection was determined by titrating known amounts of exosomes and measuring their associated TPEX absorbance changes. All measurements were performed in triplicate, and in (b), data are shown as mean ± SD. AU represents arbitrary units.

[0032] Figure 16 : Synthesis of aptamers with branched fluorescence. Step 1: Acrylation of the aptamer with a diacrylate molecule via aza-Michael addition. Step 2: Addition of 4-arm-PEG to the modified aptamer via reaction of primary amines with acryloyl groups. Step 3: Tagging of the PEGylated aptamer with a fluorophore via ester conjugation. After each step, the modified aptamer was purified from excess reagent by size-selective filtration (molecular cutoff, 3000).

[0033] Figure 17 : Performance evaluation of fluorescent aptamers. (a) Anti-CD63 aptamers were prepared using branched fluorescent (3 dyes) or single fluorescent molecules (1 dye). The aptamers for TPEX reaction were used with exosomes, and the absorbance (top) and fluorescence (bottom) changes were measured. Although the two aptamer preparations showed comparable absorbance changes, the 3-dye preparation showed better fluorescence signals. All measurements were performed on their respective scrambled control aptamers. (b) Detection limit of 1-dye aptamers. Exosomes were diluted and measured with 1 dye anti-CD63 aptamer. All measurements were performed in triplicate, and the data are shown as mean ± sd. *P < 0.05, ***P < 0.0005, NS indicates not significant; Student's t-test; au indicates arbitrary units.

[0034] Figure 18: TPEX analysis using antibodies and miRNA probes. (a) Compared with ELISA analysis (bottom), TPEX analysis using different fluorescent antibodies (top) shows accurate protein marker expression profiles across cell lines. (b) Compared with PCR analysis (bottom), TPEX analysis using different fluorescent DNA probes for miRNA targets (top) shows accurate miRNA marker expression profiles across cell lines. All measurements were performed in triplicate. The analysis was normalized to the corresponding sample-matched IgG isotype control antibody or scrambled control in a and b, respectively.

[0035] Figure 19 : Smartphone-based detector. (a) Spectra of different LED light sources. (b) Configurations of the smartphone-based detector for absorbance and fluorescence detection, respectively. (c) Correlation of TPEX measurements between the smartphone-based detector and a commercial plate reader. The smartphone-based detector showed good performance correlation with the commercial plate reader (R 2 =0.9945). All measurements were performed in triplicate, and in (c), data are shown as mean ± SD. AU represents arbitrary units.

[0036] Figure 20 Comparison of CD63 expression levels in clinical samples. (a) Exosome counts in a clinical ascites sample determined by nanoparticle tracing assay. (b) TPEX analysis of CD63 in a clinical sample. (c) ELISA analysis of total CD63 protein in a clinical sample. TPEX analysis of CD63 better reflects exosome counts, as determined by the gold standard nanoparticle tracing assay, whereas ELISA analysis of total CD63 protein shows poor agreement with counts. DETAILED DESCRIPTION

[0037] Disclosed herein is a method for detecting and / or characterizing nanovesicles in a sample, the method comprising the following steps:

[0038] a) contacting the sample with nanoparticles or precursors of nanoparticles, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicles and forming a nanoshell around the vesicles in situ; and

[0039] b) illuminating the sample and measuring an optical signal of the sample to detect and / or characterize nanovesicles in the sample.

[0040] In one embodiment, the formation of nanoshells induces localized plasmon resonance and / or increased optical absorbance in the infrared region.

[0041] In one embodiment, the spectral properties (absorbance) of the nanoshells are tuned to differentiate the size of extracellular nanovesicles and the corresponding vesicle counts.

[0042] Without wishing to be bound by theory, the inventors have developed a platform / method for multi-parameter molecular profiling of these same vesicles directly in native clinical biofluids by simultaneously assessing their biophysical and biomolecular composition. This technology, termed "Template Plasmonics for Exosomes" (TPEX), utilizes the formation of gold nanoshells assembled and grown in situ on vesicles to enable specific analysis of exosomal biomarkers. For biophysical selectivity, nanoshell formation is templated on the vesicle membrane and tuned to discriminate exosome size. For biomolecular selectivity, the unique plasmon signature of the nanoshells can quench fluorescent probes through matching and localized energy transfer, which occurs only when they are targeted and bound to the same vesicle. The resulting optical signals (i.e., absorbance and fluorescence) enable multiplexed selective analysis of multiple exosomal biomarkers (e.g., proteins and miRNAs), while remaining unresponsive to non-vesicular, free molecular targets. When implemented on a smartphone-based microfluidic sensor, TPEX technology enables rapid and multiplexed analysis of exosomal targets with exceptional performance (1 μl sample, within 15 minutes). The inventors further applied the developed platform to examine natural clinical ascites samples. This technology not only revealed exosomal biomolecular signatures within a complex biological context but also revealed exosomal subpopulations of biomarkers compared to total biomarkers, enabling more accurate prognosis differentiation in cancer patients.

[0043] Methods as referred to herein may comprise contacting a sample with a nanoparticle or a precursor thereof, wherein the nanoparticle or precursor is capable of binding to the surface of a nanovesicle and forming a nanoshell surrounding the vesicle in situ. For example, in embodiments, the in situ formation of a nanoshell can be catalyzed by adding both a nanoparticle and a precursor of the nanoparticle (e.g., a metal salt, e.g., a gold salt or a silver salt) to the nanoparticle.

[0044] As used herein, the term "nanoparticle" refers to a particle having a nanometer-scale particle size, less than 1 micron in diameter. For example, the nanoparticle may have a particle size of at most about 50 nm. In another example, the nanoparticle may have a particle size of at most about 40 nm. In another example, the nanoparticle may have a particle size of at most about 30 nm. In another example, the nanoparticle may have a particle size of at most about 20 nm. In another example, the nanoparticle may have a particle size of at most about 10 nm. In another example, the nanoparticle may have a particle size of at most about 6 nm. In one embodiment, the gold nanoparticles have a diameter ranging from 1 to 4 nm, 2 to 6 nm, 3 to 7 nm, 4 to 8 nm, 5 to 9 nm, 6 to 10 nm, 7 to 11 nm, 8 to 12 nm, 9 to 13 nm, 10 to 14 nm, 11 to 15 nm, 12 to 16 nm, 13 to 17 nm, 14 to 18 nm, 15 to 19 nm, or 16 to 20 nm.

[0045] The nanoparticles can be plasma materials. Alternatively, the nanoparticles can be coated with plasma materials. In one embodiment, the nanoparticles are metal nanoparticles. The metal nanoparticles can be made of metals (such as gold, silver or titanium) or can be made of alloys of different metals. In one embodiment, the metal nanoparticles are gold nanoparticles. In one embodiment, the gold nanoparticles have a diameter range between 7 and 11 nm. In an alternative embodiment, the nanoparticles comprise an organic polymer.

[0046] In one embodiment, the precursor is a metal salt that, together with the nanoparticles, is capable of forming a nanoshell surrounding the nanovesicle. In one embodiment, the metal salt is a gold salt. The metal salt may be in solution before contacting with the sample.

[0047] As used herein, "nanovesicle" may refer to a naturally occurring or synthetic vesicle that includes a cavity inside. A nanovesicle may include a lipid bilayer membrane that encloses the contents of the lumen. A nanovesicle may include a liposome, an exosome, an extracellular vesicle, a microvesicle, an apoptotic vesicle (or apoptotic body), a vacuole, a lysosome, a transport vesicle, a secretory vesicle, a gas vesicle, a matrix vesicle, or a multivesicular body. A nanovesicle may have a size of about 1000 nm or less, about 900 nm or less, about 800 nm or less, about 700 nm or less, about 600 nm or less, about 500 nm or less, about 450 nm or less, about 400 nm or less, about 350 nm or less, about 300 nm or less, about 250 nm or less, about 240 nm or less, about 230 nm or less, about 220 nm or less, about 210 nm or less, about 200 nm or less, about 190 nm or less. or less, about 180 nm or less, about 170 nm or less, about 160 nm or less, about 150 nm or less, about 140 nm or less, about 130 nm or less, about 120 nm or less, about 110 nm or less, about 100 nm or less, about 90 nm or less, about 80 nm or less, about 70 nm or less, about 60 nm or less, about 50 nm or less, about 40 nm or less, about 30 nm or less, about 20 nm or less, or about 10 nm or less.

[0048] In one embodiment, the nanovesicles are exosomes. The term "exosome" refers to vesicles that are shed by eukaryotic cells or bud from the plasma membrane to the outside of the cell. Exosomes can vary in size, ranging from about 10 nm to about 5000 nm in diameter.

[0049] Disclosed herein is a method for detecting one or more targets bound to or associated with nanovesicles in a sample, the method comprising the steps of:

[0050] a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the nanovesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each of the targets; and

[0051] b) illuminating the sample and measuring the emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

[0052] In one embodiment, the optical properties of the fluorescent molecular probe are matched to the spectral compatibility of the nanoshell to enhance the detection signal.

[0053] In one embodiment, the optical properties of the fluorescent molecular probe are matched to the spectral compatibility of the nanoshell to distinguish between biomarkers present in or associated with extracellular vesicles of different sizes.

[0054] The target to be detected can be bound to or associated with the nanovesicles in the sample. The target can be referred to as a "biomarker". For example, the target can be a nucleic acid, lipid, protein, peptide, metabolite, or glycopeptide that is bound to or associated with the nanovesicles. In one embodiment, the target is a nucleic acid (such as RNA). In one embodiment, the target is a protein, such as a membrane protein or a membrane-associated protein. In one embodiment, the target is a lipid. In one embodiment, the target is a metabolite. Targets as referred to herein can also include modified proteins, nucleic acids, lipids, and metabolites. In one embodiment, the methods defined herein can distinguish different types of targets that can be bound to or associated with nanovesicles.

[0055] In one embodiment, the target is a cancer biomarker. The cancer biomarker may be, for example, CD24, EpCAM, or MUC1.

[0056] In one embodiment, the target is an exosomal biomarker. The exosomal biomarker may be CD63.

[0057] As described herein, the term "nucleic acid" can be RNA or DNA, and can be single-stranded or double-stranded, and can be, for example, a nucleic acid, a polynucleotide, an oligonucleotide, a nucleic acid analog encoding a protein of interest. Such nucleic acid sequences include, for example, but not limited to, nucleic acid sequences encoding proteins (e.g., acting as transcriptional repressors), antisense molecules, ribozymes, small inhibitory nucleic acid sequences, such as, but not limited to, RNAi, shRNAi, siRNA, microRNAi (mRNAi), antisense oligonucleotides, etc.

[0058] The terms "protein" and "polypeptide" are used interchangeably and refer to any polymer of amino acids (dipeptides or higher) linked by peptide bonds or modified peptide bonds. Polypeptides of less than about 10 to 20 amino acid residues are generally referred to as "peptides". The polypeptides of the present invention may contain non-peptide components, such as carbohydrate groups. Carbohydrate and other non-peptide substituents can be added to the polypeptide by the cell that produces the polypeptide and will vary with the cell type. Polypeptides are defined herein according to their amino acid backbone structure; substituents, such as carbohydrate groups, are generally not specified but may still be present.

[0059] In one embodiment, the method involves contacting the sample with an excess of nanoparticles required to form the nanoshell.

[0060] One or more fluorescent molecular probes can be capable of specifically binding to one or more targets bound to or associated with the nanovesicles. This can provide a unique (or specific) emission fluorescence wavelength for each of the targets, thereby enabling each of the targets to be distinguished from each other.

[0061] Fluorescent molecular probes can be nucleic acids, aptamers, antibodies or small molecules. Fluorescent molecular probes can be molecular probes connected to fluorescent dyes, such as nucleic acids, aptamers, antibodies or small molecules. Examples of fluorescent dyes for fluorescence labeling include fluorescent dyes with fluorescein, rhodamine, coumarin, Cy, EvoBlue, oxazine, carbopyronin, naphthalene, biphenyl, anthracene, phenanthrene, pyrene, carbazole or the like as backbones or derivatives of these fluorescent dyes. Examples of fluorescent dyes include but are not limited to fluorescein, rhodamine B and Alexa Fluor 647.

[0062] The term "aptamer" refers to an oligonucleotide that can conform in three dimensions to bind to another molecule with high affinity and specificity. Aptamers are typically identified from large random sequence libraries, but natural aptamers are also present in riboswitches. Aptamers can be broadly divided into: nucleic acid (DNA or RNA) aptamers, which consist of oligonucleotide chains (usually shorter); or peptide aptamers, which consist of short variable peptide domains attached to a protein scaffold at both ends. Aptamers, such as peptides produced by phage display or monoclonal antibodies (MAbs), are able to specifically bind to a selected target and, by binding, block or otherwise alter the function of the target molecule to which they bind. Aptamers are typically identified from a random sequence oligonucleotide library by an in vitro selection process (such as, for example, SELEX). Aptamers have been generated for more than 100 proteins, including growth factors, transcription factors, enzymes, immunoglobulins, and receptors. Aptamers are typically 10 to 15 kDa in size (30 to 45 nucleotides), bind their targets with subnanomolar affinity, and discriminate against closely related targets (eg, they typically do not bind other proteins from the same gene family).

[0063] In one embodiment, the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. The aptamer may be conjugated to a fluorescent dye.

[0064] "Antibody" refers to a molecule with binding affinity to a target antigen. It should be understood that the term extends to immunoglobulins, immunoglobulin fragments, and non-immunoglobulin derived protein frameworks that exhibit antigen binding activity. Representative antigen-binding molecules that can be used for implementing the present invention include polyclonal and monoclonal antibodies and their fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) and domain antibodies (including, for example, shark and camelid antibodies), fusion proteins comprising antibodies, and immunoglobulin molecules comprising any other modified configuration of antigen binding / recognition sites. Antibodies include antibodies of any kind, such as IgG, IgA, or IgM (or its subclass), and the antibody does not need to be of any specific kind.

[0065] In one embodiment, the antibody is selected from the group consisting of an anti-CD63 antibody, an anti-CD24 antibody, an anti-EpCAM antibody, and an anti-MUCl antibody.

[0066] The method can include illuminating the sample and measuring the emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle. The detection can involve enhanced fluorescence quenching that identifies the unique emitted fluorescence of each of the targets.

[0067] The term "enhanced fluorescence quenching" can refer to an increase in the level of fluorescence quenching compared to a reference. For example, the reference can be the fluorescence emitted in the absence of one or more targets bound to or associated with the nanovesicle.

[0068] In one embodiment, the sample is a sample that has been obtained from a subject. In one embodiment, the subject is a subject suffering from cancer.

[0069] The term "sample" may refer to any sample derived from or containing cells, organisms (bacteria, viruses), lysed cells or organisms, cell extracts, nuclear extracts, components of cells or organisms, extracellular fluid, culture medium for in vitro culture of cells or organisms, blood, plasma, serum, gastrointestinal secretions, urine, ascites, tissue or tumor homogenates, synovial fluid, feces, saliva, sputum, cystic fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen, lymph fluid, tears, pleural fluid, nipple aspirates, breast milk, external parts of the skin, the respiratory, intestinal and genitourinary tracts, and prostatic fluid.

[0070] The sample can be a biological sample, which refers to the fact that it originates from or is derived from a living organism. The organism can be in vivo (e.g., a whole organism) or can be in vitro (e.g., a cell or organ grown in a culture medium). A "biological sample" also refers to a cell or cell mass or a certain amount of tissue or fluid from a subject. In most cases, the sample has been taken out from the subject, but the term "biological sample" can also refer to a cell or tissue analyzed in vivo, i.e., not taken out from the subject. Typically, a "biological sample" will comprise a cell from the subject, but the term can also refer to a non-cellular biological material, such as the non-cellular part of blood, saliva or urine. A biological sample can be from the resection of a primary, secondary or metastatic tumor, a bronchoscopic biopsy or a core needle biopsy, or a cell block from pleural fluid. In addition, a biological sample aspirated by fine needle is also useful. In one embodiment, the biological sample is ascites. A biological sample also includes an explant derived from a patient's tissue and a primary cell culture and / or a transformed cell culture. Biological samples can be provided by removing a cell sample from a subject, but can also be accomplished by using previously isolated cells or cell extracts (e.g., isolated by another person at another time and / or for another purpose). Archival tissues, such as those with a history of treatment or outcome, can also be used. Biological samples include, but are not limited to, tissue biopsies, scrapings (e.g., oral scrapings), whole blood, plasma, serum, urine, saliva, cell culture, or cerebrospinal fluid. The samples referred to herein may have been used to perform purification or enrichment of nanovesicles, such as exosomes.

[0071] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized, in part, by unregulated cell growth. As used herein, the term "cancer" refers to both non-metastatic and metastatic cancers, including early-stage cancers and late-stage cancers. The term "precancerous lesion" refers to a symptom or growth that typically precedes or develops into cancer. "Non-metastatic" means that the cancer is benign or remains at the primary site and has not yet infiltrated the lymphatic or vascular system or tissues other than the primary site. Typically, a non-metastatic cancer is any cancer that is a stage 0, I or II cancer, and occasionally a stage III cancer. "Early-stage cancer" means a cancer that is non-invasive or metastatic or is classified as a stage 0, I or II cancer. The term "late-stage cancer" typically refers to a stage III or IV cancer, but may also refer to a stage II cancer or a substage II cancer. One skilled in the art will understand that the classification of a stage II cancer as an early-stage cancer or a late-stage cancer depends on the specific type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, stomach cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer, carcinoma, melanoma, brain cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, multiple myeloma, rectal cancer, and esophageal cancer. In one example, the cancer is colorectal cancer or stomach cancer.

[0072] As used herein, the term "subject" includes any human or non-human animal. In one embodiment, the subject is a human. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, etc.

[0073] In one embodiment, characterization of the one or more targets comprises measuring the level of the one or more targets bound to or associated with the nanovesicles.

[0074] Disclosed herein are microfluidic chips for performing the methods defined herein. The microfluidic chip may include one or more microfluidic channels (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more microfluidic channels). Using microfluidics in the methods described herein significantly reduces the sample amount required for detection.

[0075] Microfluidic channels can have multiple functions. Each channel can be fluid independent (e.g., have its own fluid inlet and outlet). For example, microfluidic channels can be used to facilitate mixing of a sample with a fluorescent molecular probe, or to facilitate mixing of a sample with nanoparticles. These steps can be performed simultaneously or sequentially. Finally, microfluidic channels can also be used to facilitate in situ growth of nanoshells around nanovesicles. Microfluidic channels can also be used to transfer a reaction mixture to a collection chamber for on-chip or smartphone-based fluorescence measurements. In one embodiment, the microfluidic chip is as follows Figure 9 The chip shown.

[0076] Disclosed herein are kits for performing the methods defined herein. The kits may include reagents such as fluorescent molecular probes for binding to one or more targets bound to or associated with nanovesicles, nanoparticles and precursors capable of binding to the surface of nanovesicles and forming nanovesicles in situ. The kits may further include buffers, instructions for use, and the like. The kits may also provide a microfluidic chip as defined herein for performing the methods disclosed herein.

[0077] Disclosed herein are methods of determining the prognosis of cancer in a subject by simultaneously detecting or characterizing one or more targets that are bound to or associated with nanovesicles in a sample from the subject and are indicative of a nature of the cancer, the method comprising:

[0078] a) contacting the sample with nanoparticles, precursors, and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicles and forming a nanoshell surrounding the vesicles in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicles and providing a unique emission fluorescence wavelength for each of the targets; and

[0079] b) illuminating the sample and measuring absorbance and / or emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

[0080] As used herein, the term "prognosis" refers to a prediction of the likely course and outcome of a clinical condition or disease. A patient's prognosis is typically determined by evaluating disease factors or symptoms that indicate a favorable or unfavorable course or outcome of the disease. As used herein, the phrase "determining a prognosis" refers to a process by which a person skilled in the art can predict the course or outcome of a condition in a patient. The term "prognosis" does not refer to the ability to predict the course or outcome of a condition with 100% accuracy. Rather, those skilled in the art will understand that the term "prognosis" refers to an increased probability that a certain course or outcome will occur in the future; that is, patients exhibiting a given condition are more likely to experience that course or outcome than those who do not exhibit that condition. Prognosis can be expressed as the amount of time a patient is expected to survive. Alternatively, prognosis can refer to the likelihood that a disease will enter remission or the amount of time a disease can be expected to remain in remission. Prognosis can be expressed in a variety of ways; for example, a prognosis can be expressed as the percentage chance that a patient will be alive after one year, five years, ten years, etc. Alternatively, a prognosis can be expressed as the average number of months a patient is expected to survive a given condition or disease. A patient's prognosis can be considered a relative expression, with many factors influencing the final outcome. For example, for patients with certain diseases, prognosis might appropriately be expressed as the likelihood that the condition can be treated or cured, or the likelihood that the disease will enter remission, whereas for patients with more severe conditions, prognosis might more appropriately be expressed as the likelihood of survival within a specific time period.

[0081] In one embodiment, the cancer is colorectal cancer or gastric cancer. The method as defined herein may refer to determining the prognosis of a cancer (such as colorectal cancer or gastric cancer) in a subject. The method may comprise obtaining a sample from the subject. For example, the sample may be clinical cancer ascites from the subject.

[0082] In one embodiment, a subject with cancer is determined to have a good prognosis when having an expected (or predicted) overall survival of more than ten months. In another embodiment, a subject with cancer is considered to have a poor prognosis when having an expected (or predicted) overall survival of less than five months.

[0083] In one embodiment, the one or more targets include a target selected from the group consisting of CD63, CD24, EpCAM, and MUCl.

[0084] In one embodiment, the method comprises treating the subject. As used herein, the term "treating" can refer to (1) preventing or delaying the onset of one or more symptoms of a disease; (2) inhibiting the development of a disease or one or more symptoms of a disease; (3) alleviating the condition, i.e., causing amelioration of the disease or at least one or more symptoms of a disease; and / or (4) causing a reduction in the severity of one or more symptoms of a disease.

[0085] Disclosed herein is a method for detecting cancer in a subject by simultaneously detecting or characterizing one or more targets that bind to or associate with nanovesicles in a sample from the subject and indicate the presence of cancer, the method comprising:

[0086] a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the nanovesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each of the targets; and

[0087] b) illuminating the sample and measuring absorbance and / or emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

[0088] In one embodiment, the method comprises determining the likelihood of the presence (or absence) of cancer in a subject.

[0089] In one embodiment, the method further comprises treating the subject found to have cancer.

[0090] Disclosed herein is a method of treating cancer in a subject by simultaneously detecting or characterizing one or more targets that bind to or associate with nanovesicles in a sample from the subject and indicate the presence of cancer, the method comprising:

[0091] a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the nanovesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each of the targets;

[0092] b) illuminating the sample and measuring absorbance and / or emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets; and

[0093] c) treating a subject found to have cancer.

[0094] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted as an alternative (or).

[0095] As used in this application, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "agent" includes a plurality of agents, including mixtures thereof.

[0096] "About" means an amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length varies by as much as 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% from the referenced amount, level, value, number, frequency, percentage, dimension, size, quantity, weight or length.

[0097] Throughout the specification and the statements that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

[0098] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not, and should not be taken as, an acknowledgement or approval or any form of suggestion that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

[0099] It will be understood by those skilled in the art that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications that fall within the spirit and scope. The invention also includes all steps, features, compositions and compounds referred to or indicated in this specification, whether singly or collectively, and any and all combinations of any two or more of said steps or features.

[0100] Example

[0101] method

[0102] Cell culture

[0103] All human cancer cell lines were obtained from the American Type Culture Collection. DLD-1, HCT116, and GLI36vIII were grown in Dulbecco's Modified Essential Medium (Hyclone) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin (Gibco). MKN45, SNU484, and PC9 were cultured in RPMI-1640 medium (Hyclone) supplemented with 10% FBS and 1% penicillin-streptomycin. All cell lines were tested for mycoplasma contamination (MycoAlert Mycoplasma Detection Kit, Lonza, LT07-418).

[0104] Exosome isolation and quantification

[0105] Before collecting vesicles, cells at passages 1-15 were cultured in vesicle-depleted medium (containing 5% vesicle-depleted dFBS) for 48 hours. All culture media containing extracellular vesicles were filtered through 0.2-μm membrane filters (Millipore) and separated by differential centrifugation (first at 10,000 g, then at 100,000 g). Independent quantification of vesicle concentration was performed using a nanoparticle tracer analysis (NTA) system (NS300, Nanosight). The vesicle concentration was adjusted to obtain approximately 50 vesicles in the field of view for optimal counting. All NTA measurements were performed using the same system settings to maintain consistency.

[0106] Synthesis and characterization of AuNPs

[0107] Unless otherwise stated, all chemicals used for synthesis and modification were purchased from Sigma-Aldrich. AuNPs were prepared using the sodium citrate method. Briefly, AuNPs of varying sizes were synthesized by varying the amount of sodium citrate in the reaction. In a typical synthesis, to prepare AuNPs with a diameter of 9 nm, 50 ml of anhydrous trisodium citrate (0.6 mg / ml) was heated to boiling. Subsequently, 250 μl of gold(III) chloride trihydrate (HAuCl4·3H2O, 20 mg / ml) was rapidly injected into the boiling solution and allowed to react for 30 minutes to generate AuNPs. After cooling to room temperature, 9 ml of the prepared solution was mixed with 1 ml of polyethyleneimine (PEI, 10% in water) to replace the surface ligands on the AuNPs. The PEI-coated AuNPs were then centrifuged at 20,000 g for 1 hour to remove excess reactants and resuspended and kept at 4°C for future use. For AuNP characterization, the particle core diameter was measured using a transmission electron microscope (JEOL 2010F). The hydrodynamic diameter and zeta potential of AuNPs were determined using a Zetasizer Nano ZS instrument (Malvern). Three times 14 measurement runs were performed. The Z-average diameter and polydispersity were analyzed. For each measurement, the autocorrelation function and polydispersity index were monitored to ensure sample quality for sizing. The optical absorbance of AuNPs was measured using spectrometry (Tecan).

[0108] Synthesis and characterization of PDA particles

[0109] To prepare PDA nanoparticles of different sizes as target templates, 1 ml of dopamine hydrochloride (0.5 mg / ml in water) was mixed with different volumes of sodium hydroxide solution (4 mg / ml, volume varied between 1 and 50 μl). The mixture was incubated at 25°C for 12 hours under stirring to generate PDA particles with a clear diameter. All particles were stored at 4°C for later use. The particle size distribution was determined by dynamic light scattering analysis, as described above. In order to label PDA particles with corresponding fluorophores (e.g., fluorescein, rhodamine B, and Alexa Fluor 647), fluorescent dyes dissolved in dimethyl sulfoxide were added to PDA solutions (0.5 mg / ml). The mixture was incubated at 25°C for 12 hours and then sample purification was performed. The fluorescence intensity was measured by a microplate reader (Tecan).

[0110] Preparation of fluorescent aptamers

[0111] All aptamer sequences used in this study can be found in Table 1. DNA sequences modified with primary amine groups at the 3' end were purchased from Integrated DNA Technologies and dissolved in water at a final concentration of 10 μM. To enhance the fluorescence properties of the aptamers, individual aptamer sequences were tagged with three fluorescent molecules. Specifically, 100 μl of aptamer solution was reacted with 10 μl of N,N-methylenebisacrylamide (1 mM) at 37°C for 12 hours to generate acrylated aptamers. This purified reaction was added to an excess of 4-arm poly(ethylene glycol) with free amines (4-arm-PEG2K-NH2, molecular weight = 2000, 100 μM, 40 μl) at 37°C for 12 hours. Finally, a fluorescent dye (e.g., Alexa Fluor 647) was conjugated to the free amines on the polyethylene glycol aptamer. After each reaction step, the modified aptamer was purified by centrifugal filters (Amicon, molecular cutoff = 3000) to remove excess reactants. The purified fluorescent aptamers were stored at -20 °C for future use.

[0112] Table 1. List of aptamers, antibodies and sequences used.

[0113]

[0114] Optical simulation

[0115] All 3D finite-difference time-domain (FDTD) simulations were performed using a commercial software package (FDTD Solutions, Lumerical). Based on transmission electron microscopy analysis of the formed nanostructures, the exosome-templated gold nanoshells were modeled as core-shell structures with a dielectric core with a refractive index (RI) of 1.4 (35) surrounded by a 9 nm thick gold shell. The complex dielectric constant of gold was obtained from reference (36). When simulating the field distribution of AuNPs bound to free protein, the AuNPs with a final diameter of 14 nm after growth were modeled as attached to 3 nm of protein, as characterized by dFBS experiments. A uniform mesh of 2 nm was applied in all directions. In all simulations, the formed gold nanostructures were illuminated from the top with a plane wave, and the transmission (absorbance) spectra were recorded from the bottom. The simulated electric field distribution and absorption spectra were used to identify the corresponding resonance peaks of the nanostructures templated with exosomes and free protein, respectively.

[0116] TPEX absorbance measurement

[0117] To experimentally evaluate and validate the optical simulations, TPEX assays were first performed on PDA nanoparticles of varying diameters. These PDA nanoparticles served as target templates with a well-defined size distribution. Briefly, 5 μl of PDA solution was incubated with 5 μl of AuNP solution at room temperature for 15 minutes to allow AuNPs to self-assemble on the PDA surface. Without any purification, a mixture containing 10 μl of 3% hydrogen peroxide, 35 μl of PBS buffer, and 40 μl of gold salt (HAuCl4·3H2O, 1 mg / ml) was added to the reaction. The reaction was incubated for 15 minutes to allow templated in situ gold growth. Absorption spectra were recorded before and after gold growth to compare experimental results with those from simulations. To investigate the influence of AuNP diameter in modulating the TPEX absorbance response, PDA nanoparticles were incubated with AuNPs of varying sizes prior to the gold growth reaction. 9-nm AuNPs were selected for all subsequent TPEX measurements to maximize and match the TPEX response range with published exosome diameters. The optimized TPEX assay was further applied to biological samples. Extracellular vesicles and vesicle-depleted FBS (dFBS) were prepared by differential centrifugation as described above. All samples were characterized by NTA and dynamic light scattering analysis. Biological samples were treated with AuNPs and gold-grown as described above for the PDA reaction. The corresponding absorption spectra before and after gold growth were measured by spectroscopy.

[0118] TPEX fluorescence assay

[0119] To detect molecular markers, a TPEX fluorescence assay was developed. The assay was optimized with a fluorescent anti-CD63 aptamer. Using exosomes isolated from cell lines and free CD63 protein (Proteintech), the samples were incubated with 0.5 μl of fluorescent aptamer (10 μM) for 30 minutes. Subsequently, 5 μl of AuNPs (9 nm) were added to the reaction and incubated for 15 minutes. As described above, 10 μl of hydrogen peroxide (3%), 35 μl of PBS buffer, and 40 μl of gold salt (HAuCl4·3H2O, 1 mg / ml) were added to the reaction without any purification. For multiple fluorescence detection, different fluorescent aptamers were added to the sample and incubated simultaneously before AuNP incubation. For all TPEX fluorescence measurements, a sample matching control incubated with the scrambled aptamer was included. The fluorescence intensity before and after the TPEX reaction was measured.

[0120] TPEX analysis

[0121] Based on optical simulations and experimental verification, TPEX absorbance and fluorescence measurements are defined as follows:

[0122] ΔA=after A–before A

[0123] Where Aafter = TPEX absorbance signal (A) after AuNP incubation and gold growth, Abefore = TPEX absorbance signal (A) after AuNP incubation but before gold growth, and A = A750 / A540

[0124] A750 and A540 are the absorbance intensities at wavelengths of 750 nm and 540 nm, respectively.

[0125] ΔF = 1 – F sample / F control

[0126] Where Fsample = fluorescence intensity of sample incubated with target probe with different emission spectrum after gold growth, Fcontrol = fluorescence intensity of matched control sample incubated with scrambled fluorescent probe after gold growth.

[0127] TPEX antibody and miRNA detection

[0128] For TPEX measurements using antibodies, exosomes were isolated from various cell lines, and the samples were incubated with fluorescent antibodies (anti-CD63, BD Biosciences and anti-CD24, eBioscience, 1 μg / ml). AuNPs and a mixture of gold salts were added to the reaction without any purification, as described above, and the resulting fluorescence changes were measured. All measurements were compared to a gold standard ELISA assay using the same antibodies (see below for details).

[0129] For TPEX miRNA detection, whole exosomes were additionally fixed and permeabilized (BD Biosciences) and then labeled with fluorescent DNA probes against miRNA targets (Integrated DNA Technologies, 10 μM). As described above, AuNPs and a mixture of gold salts were added to the reaction without any purification, and the resulting fluorescence changes were measured. All measurements were compared to the gold standard Taqman assay (Thermo Scientific) by polymerase chain reaction (PCR, Applied Biosystems).

[0130] Microfluidic device fabrication

[0131] A prototype microfluidic device consisting of three regions ( Figure 6). Briefly, a 50 μm thick casting mold was patterned with SU-8 photoresist and a silicon wafer using a cleanroom mask aligner (SUSS MicroTec) and developed after UV exposure. Polydimethylsiloxane (PDMS, Dow Corning) and a crosslinker were mixed in a 10:1 ratio and cast on the SU-8 mold. First, the polymer was cured at 75°C for 30 minutes. Then, multiple nylon screws and hexagonal nuts (RS components) were placed on their respective channels on the PDMS film and embedded in the PDMS, followed by a final curing step.

[0132] Microfluidic TPEX assay

[0133] The microfluidic assay steps are as follows Figure 9 As shown. In a typical procedure, 1 μl of biological sample and 0.3 μl of fluorescent aptamer solution (10 μM) are loaded into the microchannel through inlet 1 and inlet 2, respectively. The solution is thoroughly mixed in the serpentine channel to promote aptamer labeling of exosome membrane biomarkers. A mixture containing 1 μl of AuNPs, 2 μl of hydrogen peroxide (3%), and 8 μl of PBS buffer pre-loaded at inlet 3 is introduced into the reaction and mixed in the microchannel at a flow rate of 2 μl / min for 5 minutes. Finally, 7 μl of gold salt (HAuCl4·3H2O, 1 mg / ml) pre-loaded at inlet 4 is added to the reaction and mixed in the microchannel for 3 minutes. The resulting fluorescence intensity is recorded by a smartphone-based optical sensor.

[0134] Smartphone-based sensors

[0135] To enable smartphone analysis of microfluidic TPEX assays, a system consisting of four components ( Figure 1 Sensor in (c): 3D-printed optical cage, three-color LED light source, three filters, and a magnifying glass. The optical cage was fabricated using a desktop 3D printer (EnvisionTEC, Aureus) from UV-curable resin (HTM 140). The custom-made LED light source (Chaoziran S&T) features three LED diodes with central wavelengths of 365 nm, 540 nm, and 750 nm. Figure 19(a). Three bandpass filters with center wavelengths of 520 nm, 590 nm, and 665 nm were used to measure fluorescein, rhodamine B, and Alexa Fluor 647, respectively. A magnifying lens (Thorlabs LA4280) was placed in front of the smartphone camera to improve image quality. The assembled system measured 45 mm (width) × 45 mm (length) × 50 mm (height) and was equipped with two sliding slots for quick connection to a smartphone (Apple). Sensor performance was evaluated against a commercial microplate reader (Tecan) for different fluorescent dyes and intensities. Figure 19 (c)

[0136] Western blot

[0137] Exosomes isolated by ultracentrifugation were lysed in radioimmunoprecipitation assay (RIPA) buffer containing protease inhibitors (Thermo Scientific) and quantified using the bicinchoninic acid assay (BCA assay, Thermo Scientific). Protein lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (PVDF, Invitrogen), and immunoblotted with antibodies against protein markers: CD63 (Invitrogen), Alix (Cell Signaling), HSP70 (BioLegend), LAMP-1 (BD Biosciences), Flotillin 1 (BD Biosciences), and TSG101 (BD Biosciences). After incubation with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling), immunodetection was performed using enhanced chemiluminescence (ThermoScientific).

[0138] ELISA

[0139] Capture antibody (5 μg / ml) was adsorbed onto an ELISA plate (Thermo Scientific) and blocked in PBS containing 1% BSA before incubation with the sample. After washing with PBST (PBS containing 0.05% Tween 20), detection antibody (1 μg / ml) was added and incubated for 2 hours at room temperature. After incubation with a horseradish peroxidase-conjugated secondary antibody (Thermo Scientific) and a chemiluminescent substrate (Thermo Scientific), chemiluminescence intensity was measured (Tecan).

[0140] Transmission electron microscopy

[0141] The sample solution was directly deposited on the surface of a formvar-carbon film-coated copper grid (Latech).The dried sample was imaged using a transmission electron microscope (JEOL 2010F).

[0142] Clinical measurements

[0143] This study was approved by the Institutional Review Boards of the National University Hospital (2016 / 01088) and SingHealth (2015 / 2479). All subjects were recruited according to an IRB-approved protocol after obtaining informed consent. Ascites samples were collected from patients with colorectal and gastric cancer, centrifuged at 500 g for 10 minutes, and filtered through a 0.2-μm membrane filter (Millipore). All samples were deidentified and stored at −80°C before TPEX measurement.

[0144] For clinical TPEX analysis, ascites samples were used directly. Ascites samples were incubated with fluorescent aptamers for different biomarkers and subjected to TPEX reaction (i.e., AuNP incubation and in situ gold growth). For all TPEX measurements, a scrambled control matched to the patient sample was included. TPEX analysis was performed relative to this control to demonstrate the nonspecific binding of the aptamer. Clinical assessments of patient characteristics were determined independently. Specifically, patient prognosis was determined by overall survival from the time the ascites was collected. When the overall survival exceeded ten months, the patient was considered to have a good prognosis. Conversely, if the overall survival was less than five months, the patient was determined to have a poor prognosis. All TPEX measurements were performed blindly and were not affected by these clinical assessments.

[0145] Statistical analysis

[0146] All measurements were performed in triplicate and data are presented as mean ± standard deviation. Significance testing was performed by two-tailed Student's t-test. For comparisons between samples, multiple pairs of samples were tested and the obtained P values ​​were adjusted using Bonferroni correction for multiple hypothesis testing. Adjusted P < 0.05 was determined to be significant. Correlation analysis was performed using linear regression to determine goodness of fit (R 2 ). For clinical analysis, TPEX and ELISA measurements were used to develop a multiple linear regression scoring model for disease prognosis classification. In order to avoid overfitting and evaluate performance, a leave-one-out cross-validation was performed. For a single marker, a receiver operating characteristic (ROC) curve was determined from marker expression. For multi-marker analysis, an ROC curve was drawn based on the regression score. Statistical analysis was performed using R (v.3.5.0) and GraphPad Prism (v.7.0c).

[0147] Example 1

[0148] TPEX Platform

[0149] The TPEX platform is designed to distinguish and measure exosome markers (i.e., composition and binding markers) from non-vesicular, free molecules. It consists of three functional steps: dual labeling, development of templated nanoplasmons, and signal detection ( Figure 1 (a) In the first step, a complex biological mixture (e.g., exosomes and free proteins) is incubated with fluorescent molecular probes (e.g., aptamers) and gold nanoparticles (AuNPs). Although AuNPs remain monodisperse when associated with free proteins, they assemble to the periphery of the exosomes through electrostatic interactions with the exosome membrane due to the entropy-driven formation of the protein corona. Excess unbound probes and AuNPs are not removed. In the next step, AuNPs serve as seeds for in situ nanomaterial growth. AuNPs associated with free proteins (or unbound AuNPs) undergo slower growth and show minimal red shift in their absorption spectra. In contrast, AuNPs bound to the exosome surface develop into nanoshells templated by the vesicle size, giving rise to strong localized plasmon resonances in the infrared region (23). The TPEX platform exploits the resulting distinct differences in nanomaterial morphology and plasmon properties to achieve simultaneous and multiselective measurements of exosome markers. Specifically, the spectral compatibility of the nanoshells, using the exosome membrane as a template, was tuned to discriminate between exosome sizes (i.e., selective for the biophysical properties of exosomes); enhanced fluorescence quenching of the probe was observed only when the probe targeted and colocalized on the same vesicle as the formed nanoshell (i.e., selective for the molecular marker). Because signal changes caused by free protein are minimal, the TPEX platform enables direct quantification of exosome markers in native biofluids, avoiding any purification.

[0150] To confirm the morphological changes of the nanomaterials caused by TPEX, transmission electron microscopy (TEM) analysis was performed before and after gold growth ( Figure 1 In the presence of free protein ( Figure 6 In (a), AuNPs (average diameter = 9.2 nm) remained well dispersed and exhibited minimal particle growth after TPEX reaction. When incubated with exosomes derived from a human colorectal adenocarcinoma cell line (DLD-1), the AuNPs (average diameter = 9.2 nm) remained well dispersed and exhibited minimal particle growth after TPEX reaction. Figure 6 bd), AuNPs were bound to the periphery of the vesicles. TEM analysis further confirmed the presence of large spherical particles after nanomaterial growth, consistent with the formation of gold nanoshells using exosomes as templates ( Figure 7 The absorption spectrum of the formed nanomaterial is in good agreement with the TEM characterization ( Figure 7To facilitate TPEX measurements of complex clinical biofluids, this technique was implemented in a miniaturized microfluidic system ( Figure 1 The device incorporates a serpentine mixer for efficient labeling and a torque-activated valve for fluid control ( Figure 8 ) and is designed to simplify TPEX assay operation ( Figure 9 ). In addition, the microfluidic system can be loaded onto a custom-designed smartphone-based optical detector ( Figure 1 In (d), absorbance and fluorescence measurements are achieved using different configurations of LED light sources and filter settings. Image-based data acquisition and analysis can be automated through a smartphone interface.

[0151] Exosome-templated nanoplasmons

[0152] To evaluate the effect of biomarker template size on TPEX plasma performance and thus optimize the technique for exosome size, numerical simulations were first performed for a range of template diameters ( Figure 2 (a) Based on the TEM characterization of the formed nanostructures ( Figure 1 Zhongb and Figure 7 ), simulating the growth of a 9nm gold nanolayer on the surface of an exosome-sized template. The simulation results show that for exosome diameters (30 to 150nm), the resulting plasmon resonance peak is mainly located at >600nm (with an average peak position at 750nm), which is different from that of smaller templates (e.g., bare AuNPs or AuNPs associated with free proteins) ( Figure 2 The electric field distribution and normalized absorption spectra further confirmed that the exosome-templated nanoshells and bare AuNP-templated nanoparticles exhibited strong resonances at 750 nm and 540 nm, respectively ( Figure 10 ).

[0153] To experimentally verify the simulation results, polydopamine (PDA) nanoparticles were prepared as templates of different sizes with a well-defined diameter distribution ( Figure 11 The templates were then incubated with AuNPs (average diameter = 9.2 nm). The absorption spectra obtained after the templated nanomaterials grew confirmed the simulation results. In the absence of the target template (i.e., bare AuNPs), a single resonance peak formed around 540 nm. When reacting with templates of increasing size, an additional resonance peak appeared at 750 nm ( Figure 11 Therefore, the TPEX absorbance measurement (A) is defined as the ratio of the absorbance at 750 nm and 540 nm, and the difference (ΔA) before and after gold growth is used to assess the formation of large templated nanoshells. Interestingly, it was found that by using different sizes of AuNPs ( Figure 12), the response range of TPEX absorbance for templates of different diameters can be finely adjusted ( Figure 2 (b). Therefore, 9-nm AuNPs were selected for all subsequent TPEX measurements to match the response range of exosome diameter (30 to 150 nm), thereby maximizing the signal due to exosomes and minimizing background interference from other smaller biological entities. The optimized TPEX absorbance analysis (ΔA) was further validated with biological samples. Exosomes from human colorectal adenocarcinoma (DLD-1) were spiked into vesicle-depleted FBS (dFBS) and subjected to TPEX reactions ( Figure 6 The corresponding absorbance analysis reflects the good selectivity for exosomes. Specifically, ΔA shows a large increase only in the presence of exosomes, while the changes in the reactions in PBS (i.e., naked AuNPs) and dFBS (i.e., free proteins) are negligible ( Figure 2 Similar selectivity was observed in the resulting particle size changes before and after gold growth, as determined by dynamic light scattering analysis ( Figure 2 This good specificity of TPEX is attributed to the assay design that exploits multiple biophysical properties of vesicles in forming distinct plasmonic properties; the negatively charged vesicle membrane facilitates electrostatic binding of AuNPs, and the vesicles themselves serve as scaffolds for developing size-compatible gold nanoshells whose plasmonic properties are templated by the vesicle diameter ( Figure 13 The system for determining exosome concentration was evaluated by means of the specificity of TPEX absorbance analysis. Exosomes from different cell sources (DLD-1, HCT116, MKN45 and SNU484, Figure 14 ) were diluted to different concentrations and quantified by gold standard nanoparticle tracer analysis before addition of dFBS. In all spiked samples tested, TPEX absorbance analysis allowed direct determination of exosome concentrations ( Figure 15 ) and demonstrated good correlation with gold standard measurements (R 2 =0.931)( Figure 2 (d)

[0154] Multiplex fluorescence detection of exosome markers

[0155] This technique was then extended to multiplex detection of exosomal molecular markers. The plasmonic properties of TPEX nanoshells were exploited to quench colocalized fluorescent probes. To evaluate this technique, PDA nanoparticles of various sizes were prepared and a fluorescent dye (A647) was attached to the PDA surface. The nanoparticles were subjected to TPEX reaction (i.e., AuNP incubation and gold growth) and the changes in their fluorescence intensity (ΔF) and absorbance signal (ΔA) were monitored ( Figure 3(a). Both assays showed similar trends and demonstrated an optimized template size response range for exosome diameter. TPEX fluorescence analysis was then applied for exosome marker assessment. Using CD63 (a tetraspanin found and characterized in most exosomes) as a positive control target, two samples were prepared to assess the specificity of the technique: intact exosomes containing CD63 (derived from the DLD-1 cell line) and free CD63 protein ( Figure 3 (b) Samples were incubated with fluorescent aptamers (anti-CD63 and scrambled control) for TPEX measurement. Each aptamer was modified with three identical fluorescent molecules ( Figure 16 ) to enhance its signal performance ( Figure 17 Importantly, three different types of fluorescent dyes (i.e., fluorescein / FITC, rhodamine B / RhB, and Alexa Fluor 647 / A647) were evaluated, chosen for their different excitation and emission spectra, to examine the effect of resonance spectral matching on TPEX analysis. Of all the fluorescent dyes tested, TPEX showed a significant signal only in the presence of exosomes, while the signal displayed for free CD63 protein was negligible. The A647-modified aptamer, whose emission peak (665 nm) was closest to the TPEX absorbance (750 nm), showed the greatest signal difference ( Figure 3 Consistent with published reports, these observations suggest that TPEX fluorescence quenching is influenced by electron transfer from the gold nanoshell surface (i.e., distance effect) as well as spectral matching (i.e., plasmon and fluorescence).

[0156] A multiplex TPEX assay was developed for the simultaneous detection of multiple exosomal markers in a single test using different fluorescent aptamers. Exosomes from human cancer cells (i.e., DLD-1 and MKN45) were incubated with different fluorescent aptamers and used for TPEX measurements individually (singleplex) or as a mixture (multiplex). Figure 3 (c). The multiplex fluorescence spectra are consistent with the single spectra and can accurately reveal the marker expression profile. In addition, this multiplex TPEX assay can be adapted for protein measurement using fluorescent antibodies and extended to in situ analysis of all miRNAs in exosomes ( Figure 18 The molecular detection sensitivity of this technique was further confirmed by titration analysis ( Figure 3 (d) Exosome counts were measured by nanoparticle tracer analysis. The measured TPEX response, determined by CD63 aptamer analysis, correlated with exosome counts, and a detection limit of approximately 1500 exosomes was determined. This observed sensitivity is >10 times higher than that of the ELISA assay. 3 times.

[0157] In situ analysis in complex backgrounds

[0158] Next, the TPEX platform was evaluated to measure exosome marker signatures against the complex biological background of natural biofluids (i.e., human serum). Simulated clinical samples were prepared by adding exosomes from various human cell lines (i.e., DLD-1, HCT116, MKN45, GLI36vIII, and PC9) to vesicle-depleted human serum. Based on published literature, the following protein markers were measured: the exosomal marker CD63 and the expression of putative cancer markers (including CD24, EpCAM, and MUC1). The TPEX platform was used to measure exosome markers against the complex biological background of natural biofluids (i.e., human serum ... Figure 1 cd) TPEX analysis of spiked samples showed good performance correlation with commercial microplate readers ( Figure 19 ).

[0159] For all serum-spiked samples, a conventional sandwich ELISA assay was also performed for comparative analysis (see Table 1 for a list of aptamers and antibodies). For each marker analyzed, TPEX analysis showed better consistency in reflecting expression trends across cell lines compared to pure exosome signatures (obtained from the same exosomes before spiking). Figure 4 Specifically, TPEX analysis of spiked samples showed good correlation with pure exosome characteristics (R 2 =0.9299, Figure 4 b, left), whereas ELISA measurements of the same spiked samples showed significantly poorer correlation (R 2 =0.03211, Figure 4 (middle b, right). This performance difference is attributed to the multi-selectivity of TPEX in measuring exosome markers directly against a complex background (i.e., exosome biophysical properties and biomarker composition). However, ELISA analysis is only sensitive to the marker and can be affected by free-floating forms of the target protein (e.g., unbound proteins in human plasma).

[0160] TPEX classification of clinical outcomes

[0161] To evaluate the clinical utility of TPEX, a feasibility study was finally conducted using patient ascites samples. The objectives were to address the following questions: (1) whether TPEX can be directly applied to clinical samples for multiplexed measurements, (2) the accuracy of TPEX in distinguishing exosome targets, and (3) whether TPEX signatures can distinguish other clinical features (e.g., prognosis). Cancer ascites samples (n = 20; 12 colorectal cancers and 8 gastric cancers) were obtained and assayed using a miniaturized microfluidics and detector platform ( Figure 1 cd) These samples were directly subjected to multiplex TPEX molecular analysis (1 μl per native sample) ( Figure 5 For comparison, conventional single-plex ELISA analysis was also performed to measure the total target protein in all clinical samples ( Figure 5 Middle a, bottom. Interestingly, TPEX analysis (exosome target) showed a different protein expression profile than that measured by ELISA analysis (total target), consistent with published reports. In all clinical samples tested, TPEX analysis of CD63 reflected vesicle counts as determined by the gold standard nanoparticle tracer assay, whereas ELISA analysis of total CD63 protein showed poor agreement with counts ( Figure 20 ).

[0162] Using survival data from individual patients determined by the length of survival after ascites collection, TPEX and ELISA measurements were used to develop regression scoring models for disease prognosis classification. These models were validated using leave-one-out cross-validation and analyzed by receiver operating characteristic (ROC) curve analysis ( Figure 5 The TPEX model showed higher accuracy in prognostic classification of both cancer types ( Figure 5 b, area under the curve (AUC) = 0.970), while ELISA analysis of total target protein showed lower accuracy ( Figure 5 c, AUC = 0.758). This improved TPEX performance is attributed to the following reasons. Ascites contains target protein markers in different tissue states (e.g., bound and unbound exosomes). Recent studies have shown that these proteins are released through different mechanisms and play different roles in disease progression, highlighting the potential use of exosomes as more reflective indicators of disease aggressiveness and poor prognosis. Specifically, while free-floating membrane proteins are typically released during cell death, exosomes are secreted during active tumor growth and carry a variety of cargoes to promote metastasis. Therefore, the ability of TPEX to distinguish and measure these reflective vesicle indicators can promote better disease stratification and prognosis.

[0163] discuss

[0164] Exosomes play a crucial role in mediating disease progression. Their orchestrated release from actively dividing cancer cells and their functional activities in modulating the tumor microenvironment, among other heterogeneous circulating factors found in body fluids, highlight the clinical potential of exosomes as more reflective biomarkers. Despite these recent discoveries, direct and specific analysis of exosomes in natural clinical samples remains challenging due to limitations in existing analytical methods. Specifically, exosomes possess unique biophysical and biomolecular properties. However, current detection of exosome populations relies primarily on biophysical or biochemical characterization performed independently or sequentially. Such analyses are not only prone to missing vesicle subpopulations but also fail to provide multiparametric analysis of vesicle biophysical and biomolecular composition.

[0165] To overcome these challenges, the TPEX platform was developed as a dedicated analytical platform for multi-selective molecular profiling of exosomes directly in clinical samples by simultaneously and in situ assessing the biophysical and biochemical composition of the same vesicles. This technology is well-suited for rapid and multi-parameter analysis of exosomes: (1) the assay design is multi-selective, targeting the biophysical properties of exosomes (e.g., membrane envelope and characteristic size) and the co-localized biomolecular content of the same vesicles; (2) the technology is applicable to measure a variety of exosomal biomarkers (e.g., proteins and miRNAs) but remains unresponsive to non-vesicular, free molecules; and (3) implementation with a smartphone-based sensor not only enables multimodal analysis (e.g., absorbance and fluorescence) but also simplifies the assay process to eliminate any washing steps. The entire assay can be completed in as little as 15 minutes, requiring 1 μl of native sample. Using the developed technology, it was demonstrated that the TPEX platform can distinguish biomarker tissue status (i.e., exosome-associated vs. total biomarker) and that exosome subpopulations of biomarkers can more accurately distinguish the prognosis of cancer patients.

[0166] The scientific applications of the developed technology are potentially extensive. With its strong ability to distinguish biomarker tissues in natural clinical samples, TPEX technology can be easily expanded to measure other molecules and modifications and study their binding and / or association with different vesicles. Since the growth of nanoshells is templated by vesicle biophysics, its plasma properties can be adjusted to measure extracellular vesicles of other different sizes (e.g., tumor bodies) and molecular subtypes (e.g., derived from different cell sources). By incorporating other molecular probes and advanced recognition mechanisms, further technical improvements can improve the analytical performance of the technology to measure even rare and complex molecular modifications. These studies not only contribute to comprehensive vesicle characterization, but also provide more insights into changes in the composition of secreted factors during disease progression.

[0167] The technology can also be developed and adapted to suit different clinical benefits. Specifically, the TPEX platform can be applied to discover new biomarker signatures and improve existing clinical biomarkers by incorporating multiparameter analysis of biomarker organization, vesicle biophysics and molecular composition. These developments will not only distinguish biomarker subpopulations, but also elucidate the biophysical and / or biochemical properties of related biomarkers, thereby providing new approaches for establishing accurate composite signatures. For clinical translation, the TPEX platform is rapid, sensitive and wash-free. With its robustness in local patient samples, the system can be applied to various clinical samples (e.g., serum, urine) for various diseases (e.g., cancer, neurodegenerative diseases). Further technical improvements, such as multiplexed microfluidic partitioning and array-type sensor integration, can achieve highly parallel detection and facilitate large-scale clinical validation.

Claims

1. A method for detecting one or more targets bound to or associated with nanovesicles in a sample, the method comprising the steps of: a) contacting the sample with nanoparticles or precursors of nanoparticles and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or precursors of nanoparticles are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the nanovesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each of the targets; b) illuminating the sample and measuring the emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

2. The method according to claim 1, wherein The optical properties of the fluorescent molecular probe are matched to the spectral compatibility of the nanoshell to enhance the detection signal and / or distinguish between targets present in or associated with nanovesicles of different sizes.

3. The method according to claim 1 or 2, wherein: The one or more targets are selected from the group consisting of proteins, nucleic acids and lipids.

4. The method according to claim 1 or 2, wherein: The nanovesicles are exosomes.

5. The method according to claim 1 or 2, wherein: The nanoparticles are metal nanoparticles.

6. The method according to claim 5, wherein: The metal nanoparticles are gold nanoparticles.

7. The method according to claim 6, wherein: The diameter of the metal nanoparticles ranges from 7 nm to 11 nm.

8. The method according to claim 1 or 2, wherein: The precursor is a metal salt.

9. The method according to claim 8, wherein The metal salt solution is a gold salt.

10. The method according to claim 1 or 2, wherein: The method involves contacting the sample with an excess of nanoparticles required to form the nanoshell.

11. The method according to claim 1 or 2, wherein: The fluorescent molecular probe is a nucleic acid, an aptamer, a peptide or an antibody.

12. The method according to claim 1 or 2, wherein: The fluorescent molecular probe is modified with branched fluorescence to enhance the detection signal.

13. The method according to claim 1 or 2, wherein: Characterization of the one or more targets includes measuring the level of the one or more targets bound to or associated with the nanovesicles.

14. A microfluidic chip for carrying out the method according to any one of the preceding claims.

15. A kit for performing the method according to any one of claims 1 to 13.

16. Use of a nanoparticle or a precursor of a nanoparticle and one or more fluorescent molecular probes in the preparation of a kit for determining the prognosis of cancer in a subject by simultaneously detecting or characterizing one or more targets that bind to or associate with nanovesicles in a sample from the subject and are indicative of the nature of the cancer, wherein: a) contacting the sample with nanoparticles or nanoparticle precursors and one or more fluorescent molecular probes sequentially or simultaneously, wherein the nanoparticles or nanoparticle precursors are capable of binding to the surface of the nanovesicle and forming a nanoshell surrounding the vesicle in situ, and wherein the one or more fluorescent molecular probes are capable of specifically binding to one or more targets bound to or associated with the nanovesicle and providing a unique emission fluorescence wavelength for each target; b) illuminating the sample and measuring absorbance and / or emitted fluorescence to detect one or more targets bound to or associated with the nanovesicle, wherein the detection involves enhanced fluorescence quenching that identifies a unique emitted fluorescence wavelength for each of the targets.

17. The use according to claim 16, wherein The cancer is colorectal cancer or gastric cancer.

18. The use according to claim 16 or 17, wherein The sample was clinical cancer ascites.

19. The use according to claim 16 or 17, wherein The nanoparticles are metal nanoparticles.

20. The use according to claim 19, wherein The metal nanoparticles are gold nanoparticles.

21. The use according to claim 16 or 17, wherein The precursor is a metal salt.

22. The use according to claim 21, wherein The metal salt is a gold salt.

23. The use according to claim 16 or 17, wherein The one or more targets include a target selected from the group consisting of CD63, CD24, EpCAM, and MUC1.

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