Copper growth enhanced plasma assay for separation-free exosome analysis

Abstract

An assay for detecting target extracellular vesicles in a sample effectively utilizes a copper shell grown on a gold nanostructure labeling the target extracellular vesicles to enhance a scattered signal, thereby improving assay sensitivity.

Description

Background Art

[0001] Extracellular vesicles are secreted by different types of cells and can ultimately accumulate and circulate in various body fluids. Due to the unique role of extracellular vesicles in biogenesis, extracellular vesicles secreted by diseased cells can preserve important biomolecules including proteins and nucleic acids. These biomolecules are closely related to the progression and prognosis of various diseases, such as various cancers, liver diseases, infectious diseases, etc. Therefore, extracellular vesicles may be able to serve as new markers for disease diagnosis and prognosis. Although extracellular vesicles are usually secreted and present in large amounts in circulation, extracellular vesicles from diseased cells only constitute a small fraction of the extracellular vesicles present in body fluids, especially in the early stages of the disease. Traditional methods require time-consuming and labor-intensive steps, involving separation steps and subsequent immuno / molecular methods to detect the molecular contents of interest carried by extracellular vesicles.

[0002] Traditional techniques utilize plasmonic nanosensors. For example, the enhanced Rayleigh scattering of gold nanospheres / gold nanorods enables the separation-free nanoparticle plasmon-enhanced scattering (nPES) method to be used for extracellular vesicle detection. Antibody-conjugated gold nanoparticles have also been used to label target extracellular vesicles to induce strong scattering signals for imaging and quantification. However, there is still a trade-off between sensitivity, throughput, and instrument accessibility. For example, high-magnification analysis can achieve ultrasensitive detection but requires skilled users and expensive optical configurations that are not easily accessible. In contrast, low-magnification analysis can provide a cost-effective and even portable method, but the sensitivity is insufficient to detect low concentrations of target extracellular vesicles.

[0003] Several strategies have also been developed to modulate the plasmonic signal of gold nanoparticles, including the assembly and etching of multiple gold nanoparticles. However, the aggregation / depolymerization of nanoparticles requires specially designed cross-linkers, additional washing steps, and may have low efficiency on solid substrates. Dissolving Au 0 into Au 1 by oxidation to etch the surface of gold nanoparticles requires a strong oxidant, which may damage extracellular vesicles and have an adverse effect on the results. In addition, smaller gold nanoparticles are generally considered to exhibit lower Rayleigh scattering, which may weaken the signal. Summary of the Invention

[0004] There is currently a need for an improved assay for sensing trace target extracellular vesicles. The assays and methods of the present disclosure enhance the plasmonic signal to enhance the scattering signal for extracellular vesicle detection. The assays and methods of the present disclosure can have increased sensitivity and provide a one-step method. The processes of the present disclosure can be carried out in a shorter time frame, such as approximately 10 minutes, making them more clinically applicable.

[0005] According to the present disclosure, an assay for detecting target extracellular vesicles in a sample containing or suspected of containing target extracellular vesicles and non-target extracellular vesicles may include: providing a substrate coated with a capture antibody that can bind to both the target extracellular vesicles and the non-target extracellular vesicles; contacting the substrate with the sample under conditions sufficient to allow the target extracellular vesicles and the non-target extracellular vesicles to bind to the capture antibody, thereby immobilizing the target extracellular vesicles and the non-target extracellular vesicles on the substrate; contacting the substrate with the immobilized target extracellular vesicles and non-target extracellular vesicles with a labeling reagent that includes a gold nanostructure associated with a detection antibody that can bind only to the target extracellular vesicles, wherein when the detection antibody binds to the target extracellular vesicles, the target extracellular vesicles are labeled with the gold nanostructure; incubating the substrate containing the labeled target extracellular vesicles with a copper growth reagent that includes copper ions, a reducing agent, and a structure-directing agent under conditions sufficient to induce reduction of the copper ions and formation of a copper shell around the gold nanostructure; and detecting a scattering signal from the gold nanostructure on which the copper shell has formed.

[0006] According to the present disclosure, there is provided a kit for performing an assay for detecting target extracellular vesicles in a sample containing or suspected of containing target extracellular vesicles and non-target extracellular vesicles, the kit may include: a substrate coated with a capture antibody that can bind to both the target extracellular vesicles and the non-target extracellular vesicles; a labeling reagent that includes a gold nanostructure associated with a detection antibody; a copper growth reagent that includes copper ions, a reducing agent, and a structure-directing agent; and instructions for performing the assay, the assay including: contacting the substrate coated with the capture antibody with the sample to immobilize the target extracellular vesicles and the non-target extracellular vesicles on the substrate; contacting the immobilized target extracellular vesicles and non-target extracellular vesicles with the labeling reagent that includes the gold nanostructure associated with the detection antibody, whereby the detection antibody selectively binds to the target extracellular vesicles, thereby labeling the target extracellular vesicles; incubating the substrate containing the labeled target extracellular vesicles in a copper growth solution to form a copper shell around the gold nanostructure; and detecting a scattering signal from the gold nanostructure on which the copper shell has formed. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 is a schematic diagram of an assay according to the present disclosure.

[0008] Figure 2A is a dark-field image before copper growth enhancement.

[0009] Figure 2B is a dark-field image after copper growth enhancement.

[0010] Figure 3 A includes (i) SEM images and (ii) TEM images of bare gold nanorods.

[0011] Figure 3 B is a graph showing the surface plasmon response of bare gold nanorods (50 nm).

[0012] Figure 3 C includes (i) SEM images and (ii) TEM images of gold nanorods grown with a copper shell thereon according to the present disclosure.

[0013] Figure 3 D is a graph showing the surface plasmon response of gold nanorods (200 nm) grown with a copper shell thereon.

[0014] Figure 4A is a representative dark-field image of copper-enhanced scattering signals on gold nanorods serially diluted 2-fold from 625 μg / mL to 0 μm / mL.

[0015] Figure 4B is a representative dark-field image of copper-enhanced scattering signals on gold nanospheres serially diluted 2-fold from 625 μg / mL to 0 μm / mL.

[0016] Figure 5A and 5B is a graph showing the correlation of scattered monomers relative to the initial NP concentration for (A) AuS samples and (B) AuR samples with and without exposure to copper nanoshell growth conditions (dashed lines represent log-log correlations, horizontal lines represent signals generated by the blank matrix; gray bars represent signals outside the linear range).

[0017] Figure 6A is the nanoparticle (NP) scattering intensity detected after 5 minutes of growth of copper nanoshells on AuR cores without a reducing agent, with sodium citrate, glutathione (GSH), sodium borohydride (NaBH4), or NaVc added, or on NaVc without AuR.

[0018] Figure 6B is the NP scattering signal detected after 10 minutes of growth of copper nanoshells with AuR and NaVc in the presence of the specified Cu 2+ ion and PEI ratio.

[0019] Figure 7 is a graph showing the extracellular vesicle detection performance of ELISA, an assay using bare gold nanorods, and an assay using copper-enhanced gold nanorods according to the present disclosure; the assay according to the present disclosure demonstrates the widest dynamic range and an LOD of 1800 EVs / μl.

[0020] Figure 8ALAM-EV signal maps detected in the sera of pediatric tuberculosis (confirmed and unconfirmed) cases and non-tuberculosis (unlikely) cases measured by the AuR assay.

[0021] Figure 8B LAM-EV signal maps detected in the sera of pediatric tuberculosis (confirmed and unconfirmed) cases and non-tuberculosis (unlikely) cases measured by the Cu-NEI.

[0022] Figure 8C is a graph showing the ROC curves and AUC values (in parentheses) of serum LAM EV AuR and Cu-NEI signals, as well as the binary (positive / negative) sputum Xpert MTB / RIF and urine LAM assay results.

[0023] Figure 9 A is a graph showing the exoPD-L1 levels detected by Cu-NEI, which enables PD-L1+ analysis of the NSCLC population.

[0024] Figure 9 B is a graph showing the exoPD-L1 levels detected by ELISA.

[0025] Figure 9 C is a graph showing the comparison of PD-L1 biopsy scores between the good response group and the poor response group.

[0026] Figure 9 D is Figure 9 The ROC curves of three different methods for A-9C.

[0027] Figure 9 E is a graph showing the combined response of ExoPD-L1 Cu-NEI and biopsy scores.

[0028] Figure 9 F is a graph showing the combined response of ExoPD-L1 ELISA and biopsy scores.

[0029] Figure 9 G is a graph showing the Pearson correlation between ExoPD-L1 and PFS.

[0030] Figure 9 H is a graph showing the Pearson correlation between biopsy and PFS.

[0031] Figure 10A and 10B are selected area electron diffraction (SAED) patterns of (A) AuR nuclei and (B) AuS nuclei.

[0032] Figure 10C and 10Dare SEM images of (C) AuR@Cu and (D) AuS@Cu NPs (scale bar: 500 nm).

[0033] Figure 10E and 10F are simulated light scattering and electric fields generated by the interaction of plane waves with (E) tetrahedral or (F) cubic nanoshell models and the neighboring atmosphere.

[0034] Figure 10F and 10G are plots showing the total heat loss of (G) Cu NP cubes and tetrahedra with 200 nm side lengths and (H) Cu NP cubes with a specified 1 / 2 side length.

[0035] Figure 11A Include TEM images of copper nanoshells formed on AuR NPs and AuS NPs (scale bar: 100 nm).

[0036] Figure 11B is an image showing the geometric shape distribution of AuR@Cu and AuS@Cu nanoshells, which is determined by calculating the number and percentage of each shape in three SEM images of each sample (239 AuR@Cu and 188 AuS@Cu particles).

[0037] Figure 11C is an image showing the calculated scattering profiles of COMSOL-simulated cubic and spherical Cu nanoshells with 200 nm side lengths and diameters. The complex refractive indices of copper spheres and cubes are obtained from the optical material database, where the heat map bar represents W / m 3 .

[0038] Figure 11D and 11E Include DFM images of the overall and single NP (insert) scattering signals generated by (D) AuR and AuR@Cu NPs and (E) AuS and AuS@Cu NPs.

[0039] Figure 12A Shows time-lapse DFM images of the in-situ growth reaction of AuR@Cu nanoshells (scale = 200 μm).

[0040] Figure 12B Shows Figure 12A a heat map contour plot corresponding to the intensity of its scattering signal, representing pixel intensity on a scale of 0 to 255.

[0041] Figure 12C Shows the scattering signals of AuR and AuS samples incubated in the presence (AuR@Cu and AuS@Cu) or absence (AuR and AuS) of Cu nanoshell growth reagents during a 20-minute recording period.

[0042] Figure 12D was detected at the specified time after subtracting the baseline AuR or AuS signal of the AuR@Cu and AuS@Cu growth reactions ( Figure 6A and 6B the mean ± SE of the three reactions shown).

[0043] Figure 13 Includes DFM images of the precipitate of the Cu nanoshell growth reaction after co-incubating the input AuR NPs with Cu 2+ solution and PEI supplemented with the specified reducing agent.

[0044] Figure 14A Are graphs showing the signal changes of NPs of AuR@Cu vs. AuR and AuS@Cu vs. AuS.

[0045] Figure 14B Is a graph showing the optimization of the input AuR concentration on the Cu-NEI platform.

[0046] Figure 14C and 14D Are graphs showing the normalized intensities of serial dilutions of (E) IgG standards measured by AuR immunoassay and Cu-NEI and (F) EV standards analyzed by ELISA and Cu-NEI, where the data were normalized to the blank controls of their respective assays and plotted to show their correlation with a four-parameter logistic curve. All graphs show the mean ± SE values of three technical replicates of each sample.

[0047] Figure 15A Is a TEM image (scale bar: 200 nm) of an EV standard sample isolated from the HCT116 cell line.

[0048] Figure 15B Is a size distribution plot of an EV standard sample isolated from the HCT116 cell line by NTA assay.

[0049] Figure 15C Is an image of protein immunoblot data showing the expression of two EV markers (CD81 and CD63) in the protein lysates of these EV samples.

[0050] Figure 15D Is a graph showing the correlation between the EV concentration quantified by NTA and the EV protein concentration measured by BCA (R 2 = 0.98).

[0051] Figure 16A Is a TEM image (scale bar: 100 nm) of HCT116 cell-derived EVs immunolabeled with an AuR probe labeled with a CD63 antibody.

[0052] Figure 16B It is a graph showing the EV size distribution measured by NTA in EV samples incubated with Cu nanoshell growth reagent and (A) co-incubated with BSA-blocked AuRs; or (B) not co-incubated with BSA-blocked AuRs; or (C) PBS was added and then centrifuged to remove AuR@Cu or self-nucleated CuNPs. One-way ANOVA with Bonferroni correction was used to analyze the differences.

[0053] Figure 16C It is a graph showing the EV concentration and recovery detected before (gray bars) and after centrifugation to remove these NPs when EV standard samples were co-incubated with (A) unblocked AuR@Cu NPs or (B) BSA-blocked AuR@Cu NPs.

[0054] Figure 17A It is an image showing the LAM expression detected using EVs isolated from Mtb-infected or uninfected THP-1 or Mtb culture filtrate protein (CFP) (used as a positive control).

[0055] Figure 17B It is an image showing the LAM-EV Cu-NEI specificity, which was determined using EVs from THP-1 macrophage cultures exposed to microbial pathogens causing human infections, including Streptococcus pneumoniae (S.pn), Klebsiella pneumoniae (K.pn), Escherichia coli (E.coli), Staphylococcus aureus (S.a), and Pseudomonas aeruginosa (Ps.a). Data show the mean ± SE values of three technical replicates and differences of *p<0.05, **p<0.01, and ***p<0.001, which were determined by one-way parametric ANOVA combined with Dunnett's post hoc test.

[0056] Figure 18 It is a schematic diagram of the Cu-NEI method, where the growth of single-crystalline (compared to polycrystalline) nanoshells on NP probes for target biomarkers significantly improves the detection sensitivity.

[0057] Figure 19A It is a graph showing the AuR and Cu-NEI EV-LAM signal intensities of tuberculosis and non-tuberculosis samples, where each bar represents the mean ± SE of three results for these samples, and the dashed line represents the threshold of positive EV-LAM signal determined by ROC curve analysis.

[0058] Figure 19B It is a graph showing the normalized intensity (N.I.) of LAM-EV AuR or Cu-NEI signals obtained from 5-fold serial dilutions of tuberculosis-positive serum samples. Data show the mean ± SE results of three replicates.

[0059] Figure 20A is a chart showing the clinical outcomes of the tuberculosis and non-tuberculosis groups, where the tuberculosis group was divided into subgroups diagnosed according to microbiological findings (culture or Xpert results; group A), or subgroups diagnosed according to at least two of the following criteria: symptoms suggestive of tuberculosis, positive response to anti-tuberculosis treatment (ATT), chest X-ray consistent with tuberculosis, or tuberculosis contacts, or tuberculin skin test (TST) consistent with Mycobacterium tuberculosis infection.

[0060] Figure 20B is a Venn diagram for classifying tuberculosis cases according to Cu-NEI, Mtb culture, and Xpert results.

[0061] Figure 20C is a table comparing the sensitivity, specificity, and accuracy estimates, as well as the 95% confidence interval (95% CI) values determined using the clinical and research test results and the reported Mtb EV AuR and Cu-NEI results of 31 pediatric patients shown in Figure 20A Establishment of the 95% confidence interval (95% CI) values using the clinical and research test results and the reported Mtb EV AuR and Cu-NEI results of 31 pediatric patients shown. DETAILED DESCRIPTION OF THE INVENTION

[0063] The methods and assays of the present disclosure utilize a Cu growth strategy that can increase the size of traditional gold nanostructures and alter their geometry, such as gold nanorod (AuR) nanoparticles that exhibit excellent binding kinetics prior to crystalline Cu growth, to maximize the resulting local surface plasmon resonance (LSPR). The methods and assays of the present disclosure can increase the signal intensity induced by nanoparticles by an average of 71-fold over a wide linear concentration range. The methods and assays of the present disclosure can also beneficially enhance the signal of biomarker assays using nanoparticle probes. For example, when Cu growth is induced on antibody-conjugated Au nanoparticles that bind to a target biomarker on extracellular vesicles (EVs), a detection limit of 39 EVs / μL was estimated.

[0064] The assays of the present disclosure utilize a Cu growth method that is capable of enhancing individual signals of nanoparticle-enhanced immunoassays (Cu-NEI) to enhance the scattered signal for extracellular vesicle detection. The assays of the present disclosure are capable of advantageously providing improved detection sensitivity and are easy to operate, making them a clinically applicable procedure. The use of Cu growth can also provide colorimetric-based detection, which is useful in techniques such as lateral flow strip assays.

[0065] The reduction of copper can be controlled to avoid overgrowth and non-specific reduction caused by interfering substances present in complex biological systems. In the seed-mediated growth method, the growth of nanoparticles depends to a large extent on the crystal structure of the initial nanoparticle matrix. Two widely used gold matrices include gold nanorods (AuR) and gold nanospheres (AuS). AuR nanoparticles tend to exhibit a single-crystal structure because their geometry allows Au atoms to align along the length of their longest symmetry axis, and zero-valent Cu atoms tend to align with this crystal structure to serve as seeds for the growth of single-crystal Cu nanoshells. This crystal structure is essentially absent in AuS nanoparticles because their full rotational symmetry is not conducive to the regular alignment of their Au atoms. The selected area electron diffraction (SAED) images of AuR and AuS particles used for Cu nanoshell growth mainly detected single crystals and polycrystals.

[0066] Referring Figure 1 , according to the present disclosure, an assay can include contacting a substrate having a first capture antibody with a sample containing extracellular vesicles. The sample can include target extracellular vesicles and non-target extracellular vesicles. The capture antibody is selected to bind to all extracellular vesicles in the sample, thereby immobilizing the extracellular vesicles on the substrate. Then, the substrate having the immobilized extracellular vesicles is contacted with a gold nanostructure associated with a detection antibody. The detection antibody is a target EV-specific antibody that binds only to the target extracellular vesicles, thereby selectively labeling the target extracellular vesicles with the gold nanostructure. Subsequently, the assay further includes incubating the substrate in a copper growth reagent solution to perform site-specific copper shell (Cu shell) growth on the gold nanostructure. During incubation, copper ions present in the reagent are reduced to metallic copper and selectively form a copper shell around the gold nanoparticles. Finally, the assay can include detecting the scattering signal of the sample after Cu shell growth. The Cu shell enhances the scattering signal compared to the signal from the gold nanostructure alone. This can enhance the detection sensitivity of the assay, especially when the target extracellular vesicles are present in the sample at a low concentration.

[0067] The assay can include a washing step. For example, after immobilizing the extracellular vesicles on the substrate, the unbound portion of the sample can be washed off the substrate. Additional washing can be performed, for example, after labeling the target extracellular vesicles with the detection antibody and the gold nanostructure to remove, for example, unbound nanostructures. Finally, after incubation with the copper growth reagent, a washing step can be performed before imaging and detecting the scattering signal to remove residual reagent. For example, the substrate can be washed after blocking. Washing can be performed multiple times, for example, 3 times. PBS can be used for the washing step to prevent rupture of the vesicle structure. Before performing DFM photography, the substrate should be washed with distilled water to remove unbound particles and salts.

[0068] The sample can be any sample containing extracellular vesicles. For example, body fluids can be used as samples, including but not limited to urine, blood, plasma, serum, saliva, milk, amniotic fluid, cerebrospinal fluid, synovial fluid, BALF, CSF, tears, sweat, etc.

[0069] The substrate can be any suitable substrate. For example, the substrate can be a glass slide. For example, the substrate can be a multi-well plate. For example, the substrate can have a coating or film to define a porous surface on the substrate. For example, a porous PDMS membrane can be coated on a substrate such as glass to provide the glass substrate with a PDMS membrane that defines a diaphragm on the glass slide to divide the substrate surface into multiple pores. The substrate can be surface-modified using any known surface modification and modification techniques. For example, the substrate can be coated with an antibody-binding coating such as. For example, the substrate can be coated with a protein A / G coating. For example, the substrate can be a glass surface with silane, and the silane can immobilize the capture antibody. For example, the substrate can be an SLB surface.

[0070] Coat the substrate with a first capture antibody. Any suitable first capture antibody for capturing extracellular vesicles can be used. For example, CD81, CD63, or CD9 antibodies are recognized common markers of extracellular vesicles and can immobilize extracellular vesicles on the surface.

[0071] For example, the capture antibody can be coated on the substrate and washed to remove unbound antibodies. For example, it has been found that washing 3 times with PBS can effectively remove unbound antibodies. The concentration of the capture antibody can be from about 1 ug / mL to 20 ug / mL. Substrates with aminoalkylsilane surfaces and / or protein A / G surfaces can bind antibodies. However, it has been observed that protein A / G binds antibodies in a favorable orientation.

[0072] Contact the substrate with the sample under conditions sufficient to allow the extracellular vesicles to bind to the first capture antibody. For example, the sample can be incubated overnight with the substrate containing the capture antibody at about 4°C, or incubated for about 0.5 hour to about 4 hours at about 20°C to about 40°C.

[0073] Optionally, the sample can be centrifuged to remove contaminants before incubation with the substrate. For example, contaminants can be removed with particles after centrifugation, and the supernatant can be incubated with the substrate. The sample can be centrifuged at, for example, 10000 rpm for 30 minutes to about 1.5 hours.

[0074] Those skilled in the art can select the detection antibody according to the target extracellular vesicles to be captured. The detection antibody is capable of interacting with biomarkers expressed on the surface of extracellular vesicles. For example, the detection antibody can be biotinylated for an adhesive with an avidin-modified Au structure. The collected biomarkers can facilitate disease diagnosis or progression detection. For example, the disease can be cancer or an infectious disease.

[0075] The detection antibody may be present in an amount of about 1 μg / ml to about 5 μg / ml.

[0076] A substrate having extracellular vesicles immobilized thereon and target extracellular vesicles labeled with gold nanostructures is incubated in a copper growth reagent for a period of time sufficient to reduce copper ions present in the copper growth reagent and form a copper shell around the gold nanostructures. For example, the incubation time can be about 5 minutes to about 30 minutes, about 8 minutes to about 10 minutes, about 10 minutes to about 20 minutes. Other suitable incubation times include about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 minutes, and any value therebetween or any range defined between any such values.

[0077] The copper growth reagent comprises copper ions (Cu 2+ ), a structure-directing agent, and a reducing agent. The concentration of the reducing agent is present in an amount exceeding that of the copper ions and the structure-directing agent.

[0078] The structure-directing agent mediates the controlled nanostructured crystallization of copper to form a core-shell structure. In the presence of the reducing agent, Cu 0 grows together with the structure-directing agent, thereby forming a nanostructured shell around the gold core. The structure-directing agent may be a branched polymer. The structure-directing agent may be, for example, polyethyleneimine (PEI), polyvinyl alcohol; polyacrylic acid (PAA), polyvinylpyrrolidone (PVP); poly-L-lysine (PL).

[0079] The ratio of copper ions to the structure-directing agent may be about 120:1 to about 60:1, about 100:1 to about 80:1, about 95:1 to about 65:1, about 110:1 to about 70:1, about 105:1 to 75:1. Referring Figure 6B to, it was observed that at ratios below 60:1, the structure-directing agent failed to chelate copper ions in a reducing environment to form nanostructures, while ratios above 120:1 resulted in an undesirably high background on the surface protein A / G pre-coated glass slides.

[0080] The reducing agent may be or include ascorbic acid and / or citrate. For example, the reducing agent may be sodium (+)-L-ascorbate (NaVc). Generally, the reducing agent needs to be selected such that it can reduce Cu ions to metallic Cu available for in-situ Cu growth. Referring Figure 6A, various reducing agents were tested. Sodium citrate and glutathione had too weak reducing ability to reduce enough copper shells within an appropriate time, resulting in non-specific background on the surface pre-coated with protein A / G. NaBH4 led to the self-nucleation of the structure-directing agent-Cu complex, which consumed most of the copper ions without depositing on the gold nuclei, resulting in limited formation of copper shells after washing. However, the harsh reducing environment of NaBH4 might be harmful to the integrity of the lipid bilayer structure of extracellular vesicles. NaVc had moderate reducing ability, which could reduce copper ions to metallic copper on the surface of gold nuclei while limiting the self-nucleation of the structure-directing agent-Cu complex within an appropriate incubation time. It was found that sodium ascorbate had appropriate reducing ability at room temperature and incubation times for clinical practice.

[0081] A kit for performing an assay according to the present disclosure may include: a matrix coated with a capture antibody, a gold nanostructure associated with a detection antibody, a copper growth reagent, and instructions for contacting the matrix with a sample to immobilize extracellular vesicles contained therein by binding to the capture antibody, labeling the sample with the gold nanostructure by binding the gold nanostructure to the target extracellular vesicles by binding to the detection antibody, and incubating the sample with the copper growth reagent to form a copper shell around the gold nanostructure.

[0082] Reference Figure 2A and 2B , the comparison of dark-field images before ( Figure 2A ) and after ( Figure 2B ) Cu growth illustrates the signal enhancement achieved by the assay of the present disclosure. As the Cu shell grew, a 100- to 3000-fold increase in signal intensity was observed.

[0083] The shape and size of the gold nanorods were confirmed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) ( Figure 3 A). Gold nanorods with dimensions of 25 nm x 71 nm exhibited an inherent surface plasmon resonance (SPR) effect. COMSOL modeling was used to simulate the SPR on the surface of cold nanorods under the action of an electric field ( Figure 3 B). Referring to Figure 3 C, SEM and TEM imaging showed that the reduced copper formed large particles, and the shape of the formed copper particles was observed to be a uniform cubic or tetrahedral copper shell. TEM imaging confirmed the formation of a cubic copper nanoshell around the gold nanorod core. The simulation of the SPR of the copper shell / gold nanorod using the COMSOL model showed strong refraction at the edges and corners of the cube for the copper shell ( Figure 3 D).

[0084] Reference Figure 4A and 4B, dark-field imaging was further used to evaluate the enhancement of the scattering signal. Antibody-coated gold nanorods and gold nanospheres were attached to protein A / G-functionalized slides through the interaction between the antibody and protein A / G. 2-fold serial dilutions of 625 - 0 μg / ml were used, and gold nanostructures were covalently bound in gradients to evaluate the sensitivity enhancement achieved in the presence of the copper shell. According to the present disclosure, a copper shell was grown on the gold nanostructures by incubating the slide with the gold nanostructures fixed thereon in a copper growth reagent under conditions sufficient to reduce copper ions to metallic copper and form a copper shell structure. The presence of the copper shell was observed to significantly enhance the signal. Refer to Figure 5A and 5B , the scattering signal intensity of the gold nanorods was enhanced by 10 2 to 10 3 times in the presence of the copper shell, while the signal intensity of the gold nanospheres was enhanced by 10 - 10 2 times. The signal enhancement of the copper shell - gold nanorods at higher gold nanorod concentrations caused overexposure of the CCD on DMF, so Figure 5A was not reported. Example

[0085] Example 1

[0086] To evaluate the ability of extracellular vesicles to tolerate the AuR@Cu growth conditions, standard extracellular vesicles isolated from the HCT-116 line (NovusBiologicals) were used. These EV fractions were observed to have the expected size distribution, CD63 and CD81 biomarker expression, and the expected morphology of small EVs derived from this cell, and revealed a strong correlation between the sample protein concentration and the EV abundance determined by nanoparticle tracking analysis (NTA) (Figure 15). It was also found that these EV samples bound to AuR NPs conjugated with anti-CD63 antibodies ( Figure 16A ). To evaluate the potential of the copper shell growth conditions to disrupt the antigen-antibody probe interaction or destabilize the lipid bilayer to attenuate biomarker detection, we incubated these EVs with PBS or AuR blocked with ±BSA in the copper nanoshell growth reagent, then centrifuged to remove AuR@Cu or self-nucleating Cu NPs, and analyzed the EV supernatant by NTA to evaluate potential changes in EV diameter or abundance. The EVs in all these samples showed similar size distributions ( Figure 16B ), although fewer EVs were detected in the Cu growth reaction, which we assume is due to the non-specific interaction and precipitation of EVs caused by the AuR@Cu NPs or self-nucleating Cu NPs generated in these samples. This conclusion is supported by the following observation: after centrifugation of the EV samples incubated with BSA-blocked AuR@Cu samples and unblocked AuR@Cu samples, 85% and 38% of the input EVs were recovered, respectively.Figure 16C ) indicating that the copper growth buffer condition did not disrupt the integrity of EVs.

[0087] In this experiment, to test the ability of Cu-NEI to detect EV surface biomarkers, EV standards of known concentration were incorporated into an EV-depleted fetal bovine serum matrix and then captured on a detection slide pre-coated with an antibody against the EV surface protein CD81, incubated with a biotinylated antibody specific for the EV surface protein CD63, and then washed and hybridized with an avidin-conjugated AuR or horseradish peroxidase probe. The Cu-NEI LOD and ELISA LOD measured in this analysis are as Figure 14D shown to be 39 and 5187 EVs / μL respectively -1 , while the LOD obtained from the signal detected by the AuR probe before Cu nanoshell growth was 800 EVs / μL -1 ( Figure 16A ). Thus, the sensitivity of the Cu-NEI EV assay is approximately 20-fold and 130-fold higher than that of the AuR method and ELISA method respectively, and it shows that its linear range is also approximately two orders of magnitude wider than that of EV ELISA.

[0088] These results indicate that the growth of AuR@Cu on the captured EVs did not disrupt these EVs, although the interaction between the growing AuR@Cu crystals and the assay plate during the Cu growth reaction may prevent their loss during EV lysis. To address this possibility, the captured EVs were incubated with or without a non-denaturing lysis solution before or after the AuR@Cu growth reaction. It was found that when the samples were blocked with BSA before the Cu growth reaction, the pre-addition of lysis buffer completely eliminated the Cu-NEI assay signal, and the lysis of EVs after the Cu growth reaction had a similar but smaller effect ( Figure 16B , meaning that EV integrity is required to generate and retain the Cu-NEI signal on the captured EVs).

[0089] Example 2

[0090] To test the exosome detection of the assay of the present disclosure, standard exosomes derived from the HCT-116 line (NovusBiologicals) were used, and CD81 and CD63 were used as the capture antibody and detection antibody respectively. Standard extracellular vesicles were incorporated into exosome-depleted human serum. The concentration range of the extracellular vesicles was 30 x 10 7 particles / μL to 6 x 10 7 particles / μL. Samples were processed using the assay of the present disclosure and standard ELISA procedures, and compared using the same capture and detection antibodies.

[0091] The assay of the present disclosure is performed using a protein A / G-coated matrix coated with a CD81 capture antibody thereon. The matrix is a porous slide, and the surface of each well is coated with protein A / G. Then the sample is dropped onto the matrix, and the extracellular vesicles are immobilized by the interaction between the extracellular vesicles and the capture antibody. Gold nanorods modified with a CD63 detection antibody are added, and the target extracellular vesicles are labeled with the gold nanorods by detecting the binding between the detection antibody and the extracellular vesicles. All extracellular vesicles expressing the CD63 biomarker are labeled with gold nanorods modified with the CD63 antibody. Thereafter, the matrix is incubated in a copper growth reagent containing Cu 2+ ions and PEI (at a ratio of 60:1), while sodium ascorbate is used as a reducing agent. The matrix is incubated in the copper growth reagent for 10 minutes to allow the growth of the copper shell.

[0092] For comparison, ELISA is performed using the same capture and detection antibodies according to standard procedures known in the art.

[0093] As Figure 7 shown, the assay of the present disclosure is capable of detecting extracellular vesicles at a concentration as low as about 1.8 x 10 3 particles / μL, which is 100 times lower than that of ELISA using the same capture and detection antibodies. In addition, the dynamic range of extracellular vesicle detection of the assay of the present disclosure is much wider than that of traditional ELISA.

[0094] Example 3

[0095] The diagnostic performance of the assay according to the present disclosure was tested using tuberculosis (TB)-specific extracellular vesicles from a tuberculosis population, which included 21 positive cases (including 5 confirmed tuberculosis cases and 16 unconfirmed tuberculosis cases) and 10 negative cases (or unlikely to be tuberculosis). The patient population is a diagnostically challenging group, in which children infected with HIV are at high risk of tuberculosis-related morbidity. The glycolipid lipoarabinomannan (LAM) is located on Mtb and is related to its virulence. It can be located on the macrophage cell membrane and circulate in the peripheral blood. These Mtb-specific extracellular vesicles can diagnose tuberculosis or non-tuberculosis by targeting LAM on the extracellular vesicle membrane. In some positive cases, the signal may be weak due to the low number of TB-specific extracellular vesicles in circulation. The diagnostic ability of the Cu-NEI assay according to the present disclosure is through its detection of the Mtb glycolipid biomarker lipoarabinomannan (LAM) on the surface of serum EVs from tuberculosis individuals and non-tuberculosis individuals ( Figure 17A) The ability to evaluate. It is reported that LAM is highly enriched on EV membranes, and detecting LAM on EVs isolated from different body fluids can be used for the diagnosis of tuberculosis. Notably, the anti-LAM antibody used in this study targets the Mtb-specific LAM motif, so it should be able to provide sufficient diagnostic specificity when used as a detection antibody in our serum-based Cu-NEI EV tuberculosis diagnostic assay.

[0096] The assay according to the present disclosure was performed as described in Example 2, except that TB serum samples were used instead of exosome-depleted human serum spiked with extracellular vesicles. Refer to Figure 18 A, this assay directly captures EVs from serum and incubates them directly with the LAM-specific AuR probe, which serves as a matrix for the growth of AuR@Cu nanoshells to enhance the detection of low-abundance LAM-positive EV signals.

[0097] This Cu-NEI method differentiates EVs released from Mtb-infected macrophages from those produced by equivalent cultures infected with five other common human pathogens, including several pathogens causing respiratory infections ( Figure 17B ). Given the need for new diagnostic methods to improve tuberculosis diagnosis in children, especially those affected by HIV, and because traditional tuberculosis diagnostic methods perform poorly in these populations, this potential tuberculosis diagnostic method was used to analyze serum samples obtained from a cohort of HIV-positive young children aged 0.2 to 9.7 years. These children were thus classified as tuberculosis cases confirmed by microbiological findings (confirmed TB), or classified into an unconfirmed TB group or a non-TB group according to other clinical data by an algorithm (Table 1).

[0098]

[0099]

[0100] No bacteriological confirmation was obtained and did not meet the criteria for unconfirmed TB

[0101] It was observed that when analyzed using the AuR immunoassay or Cu-NEI, the expression of serum EV LAM was significantly different, although the Cu-NEI signal was stronger and there was less overlap between TB individuals and non-TB individuals (confirmed TB and unconfirmed TB compared to non-TB), which helped to improve the discrimination of their samples when using the threshold determined by receiver operating characteristic (ROC) curve analysis (Figure 8D) ( Figure 8A 、 8B and 19A). Figure 8A and 8BShows mean ± SE values, where individual data points represent the mean of three technical replicates, and p-values were calculated using a two-tailed Mann-Whitney test. The dashed line represents the cut-off value for positive LAM-EV signals determined by receiver operating characteristic (ROC) curve analysis of the indicated AuR and Cu-NEI data ( Figure 14B ). The area under the curve values generated from the ROC curve data were 0.78 and 0.92, respectively, indicating that the AuR and Cu-NEI results were much more capable of differentiating between these groups than the values determined using the corresponding Xpert and urine LAM data (0.60 and 0.51).

[0102] When comparing the AuR and Cu-NEI results with those of the NIH criteria for pediatric tuberculosis diagnosis, no significant association was found between these two sets of data. All microbiologically confirmed tuberculosis cases showed positive serum EV LAM results when analyzed by AuR and Cu-NEI assays, while respiratory culture and Xpert results were positive in three and four of these five cases, respectively ( Figure 20A , Group A). However, in the clinically diagnosed tuberculosis group, fewer positive serum EV LAM results were detected, with fewer positive results generated by the AuR assay than by Cu-NEI, and the proportions detected by both assays in clinically diagnosed cases were lower than those in microbiologically confirmed tuberculosis cases (38% compared to 69% sensitivity; 6 / 16 and 11 / 16; Figure 20A , Group B), which may reflect lower EV LAM abundance in the clinically diagnosed group. However, for individuals with available results, the positive EV LAM results detected by the AuR assay and Cu-NEI were much more numerous in both groups than those detected by the urine LAM assay (1 / 4 and 1 / 13). Notably, the positive urine LAM rates observed in these two tuberculosis groups were similar to those in the non-tuberculosis group (1 / 7), highlighting the poor diagnostic utility of this test. There was no obvious strong consistency pattern between Cu-NEI positive results and individuals with tuberculosis-related symptoms (10 out of 15 matches) or CXR results (9 out of 12 matches).

[0103] Cu-NEI positive results for serum EV LAM expression detected 15 out of 21 tuberculosis cases in this cohort, including all microbiologically diagnosed tuberculosis cases and most (69%; 11 / 16) clinically diagnosed tuberculosis cases ( Figure 20B ), and thus could directly detect disease groups that were not detected by other diagnostic tests, thereby showing higher overall diagnostic performance than other direct tests ( Figure 20C)。The Cu-NEI assay can rapidly detect low-abundance serum EV biomarkers for disease diagnosis, which is of great significance for tuberculosis control and may also have potential implications for other diseases where similar EV biomarkers may circulate at low levels. In the current example, the diagnosis of pediatric tuberculosis cases may take weeks to months if not diagnosed by PCR-based tests such as Xpert, as Mtb culture may take up to 8 weeks to provide a final result, and the standard aggregation for clinical diagnosis also takes weeks (symptom duration) or months (positive response to tuberculosis treatment), and may be difficult to assess in combination with other infections or complications. The superior performance of the Cu-NEI assay compared to the AuR assay can also be verified by examining the LAM-EV intensity after serial dilution ( Figure 19B ). After serial dilution of a serum sample with strong LAM-EV expression, the intensity of the AuR assay decreased to 15% of the initial intensity after 5-fold dilution, while the intensity of the Cu-NEI assay remained at 70% for the sample after 125-fold dilution, indicating that Cu-NEI can amplify the signal of low-level EVs, thus achieving better diagnostic performance in EV-based assays.

[0104] Example 4

[0105] The assay of the present disclosure also showed better performance than PD_L1 biopsy scores and ELISA results in predicting treatment response after immunotherapy. In a non-small cell lung cancer study, extracellular vesicle quantification related to PD-L1 was performed to evaluate its association with immunotherapy patients. The assay of the present disclosure was performed as described in Example 1, using clinical serum samples from non-small cell lung cancer patients as samples.

[0106] The combination of ExoPD-L1 and biopsy scores was able to distinguish between good responder groups and poor responder groups ( Figure 9 C), and ultimately outperformed the combination of ELISA and biopsy scores (Figure 5F). ExoPD-L1 detected by the assay of the present disclosure was negatively correlated with the progression-free survival (PFS) time ( Figure 9 G), while the biopsy score had little correlation with PFS ( Figure 9 H). This further indicates that the assay of the present disclosure provides stable and powerful performance in extracellular vesicle analysis and can be used to predict treatment response after PD1 / PD-L1 immunotherapy.

[0107] Example 5

[0108] To induce the growth of Cu nanoshells, AuR and AuS NPs were incubated with a CuCl2 solution containing polyethylenimine (PEI) (as a structure-directing agent to facilitate controlled Cu deposition) and sodium ascorbate (NaVc) (as a reducing agent). Subsequently, the resulting NPs were analyzed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) to evaluate the relative percentages of the nanoshell structures produced in these reactions ( Figure 11A and 11B as well as Figure 10C and 10D ). The reaction using the AuR substrate for Cu nanoshells mainly produced cubic and tetrahedral NPs (about 79%), which is consistent with the common geometries of single-crystalline Cu NPs; while the reaction using the AuS substrate mainly produced asymmetric NPs (about 84%) lacking well-defined faces and edges ( Figure 11A and 11B ), and similar results were also detected in the SEM images of these NPs ( Figure 10C and 10D ). According to the prediction of Mie theory, the edges of cubic and tetrahedral NPs should exhibit stronger plasmonic scattering activity. Therefore, Cu-coated AuR nanoshell NPs (AuR@Cu) are expected to produce a stronger plasmonic signal than the corresponding Cu-coated AuS nanoshell NPs (AuS@Cu). This is consistent with our observations based on Mie theory when simulating the scattering of an incident plane wave from AuR@Cu and AuS@Cu using nanocubes and nanospheres of the same size (200 nm side length or diameter) ( Figure 11C ), where the choice of these shapes reflects the regular and amorphous shapes of the NPs produced in these reactions. The maximum dissipation loss of this nanocube is 5 times that of the observed nanosphere (40000 W / m 3 compared to 8000 W / m 3 ). Similar strong plasmonic scattering was also observed at the edges and corners of the corresponding tetrahedra with side lengths matching the cubes ( Figure 10E and 10F ). By simulating the heat loss of these three NP shapes in the visible spectrum (380 nm - 700 nm) as an indirect means to model their overall scattering activity ( Figure 10G ), it was found that the heat loss gradually decreased from cubes to tetrahedra to spherical NPs (6.2 compared to 3.0 compared to 2.1 x 10 -17 W / NP). Similar analysis also found that the heat loss increased with the increase in NP size ( Figure 10H ). This scattering difference was manifested in the analysis of AuR NPs before and after in-situ AuR@Cu growth ( Figure 11D)。Accordingly, AuS@Cu NPs produced a relatively weak scattering signal under the same imaging conditions (50 ms exposure time). Figure 11E )。

[0109] Example 6

[0110] By varying the reducing agent used in the reaction, the effects of different reducing agents on the growth of single-crystalline Cu nanoshells were analyzed. In these reactions, the amounts of AuR and Cu growth solutions were kept constant, and the structure-directing agent was polyethyleneimine (PEI). At room temperature, glutathione (GSH), sodium citrate, and sodium borohydride (NaBH4) had little effect on Cu nanoshell deposition over a certain period of time, while NaVc produced a signal enhancement that was approximately 10 times higher than the detection of any other reducing agent. Figure 6A )。Without being bound by theory, it is believed that NaBH4 moderately increased the scattering, which may be due to the formation of a large number of insoluble Cu seeds that consumed the CuCl2 solution, reduced the formation of AuR@Cu, and may have created a strong reducing environment that could disrupt protein-protein interactions or lipid bilayer stability. In contrast, the moderate reducing activity of NaVc reduced Cu 2+ ions to metallic Cu, which attached to the AuR core without significantly inducing the self-nucleation of the PEI-Cu complex in the absence of AuR. Figure 6A and 13 )。

[0111] Reference Figure 6B was used to study the effect of adding different amounts of PEI on the growth of AuR@Cu under constant concentrations of CuCl2 and NaVc. The amounts of Cu 2+ :PEI from 600:1 to 6:1 were tested. It was observed that the plasmon signal intensity reached a peak at a CuCl2:PEI molar ratio of 60:1, while there was no significant difference in signal intensity in reactions lacking PEI or CuCl2 or with a CuCl2:PEI ratio ≤ 30:1. However, the background signal increased at a CuCl2:PEI ratio ≥ 300:1 because an increased signal was detected in these samples in the absence of the AuR matrix. The formation of AuR@Cu was detected to occur rapidly at room temperature in reactions using these optimized conditions by scattering. Figure 12A and 12B )。The changes in plasmon scattering intensity over time of AuS and AuR probes with and without treatment with Cu nanoshell growth reagents were compared. Figure 12C ) and it was found that the AuS and AuR signals remained constant over time, while the AuR@Cu signal increased more rapidly (4.8x10 6 compared to 4.3x10 5a.u. / min), and eventually higher than the AuS@Cu signal (4.8x10 7 Compared to 4.3x10 6 a.u.). Notably, after 10 minutes of growth, the AuR@Cu signal was 2.8x10 3 times higher than the AuR signal and only increased by 1.7 times after a total incubation period of 20 minutes; while the AuS@Cu signal reached a plateau after a 10-minute incubation period and was 200 times higher than the AuS signal ( Figure 12D ).

[0112] Example 7

[0113] Over a wide concentration range, a strong correlation was detected between the NP concentration and scattering of AuS and AuR NPs, with or without Cu growth. Although the growth of the nanoshells shifted these correlations to lower concentrations and improved their linearity ( Figure 5A and 5B ). However, when using AuR NPs as the Cu growth matrix, the increase in scattering was always greater and more reproducible, as reflected in the estimated limits of detection (LOD) of AuR@Cu NPs and AuS@Cu NPs (1.9 compared to 5.2 μg mL -1 ). This difference in signal increase was even more evident when normalizing the Cu nanoshell signal to the signal generated by its NP matrix at each concentration ( Figure 14A ).

[0114] To determine the AuR probe concentration required to produce the maximum Cu-NEI signal-to-noise ratio, biotinylated IgG was incubated on a multi-well plate pre-coated with protein A / G to allow binding of a series of diluted streptavidin-conjugated AuR probes, followed by signal amplification with Cu shell growth. The AuR probe concentration (6.25 μg mL -1 ) that produced the strongest and most reproducible signal was used for all subsequent analyses. Then, a series of diluted biotinylated IgG was incubated with the optimized AuR concentration to evaluate the signal difference with and without the Cu shell amplification step ( Figure 14C ), and the estimated LOD values of the Cu-NEI and AuR assays were 0.064 ng mL -1 and 1.6 ng mL -1 .

[0115] Variations and other embodiments disclosed herein will come to mind and be of benefit to those skilled in the art to which the disclosed compositions and methods pertain from the teachings presented in the foregoing description. Accordingly, it is to be understood that the disclosure is not limited to the specific embodiments disclosed, and that variations and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0116] It should also be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. As used in the specification and claims, the term "comprising" can include aspects "consisting of". Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed compositions and methods pertain. In this specification and the subsequent claims, reference will be made to a number of terms defined herein.

[0117] It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has discrete components and features that can be readily separated from or combined with the features of any one of several other embodiments without departing from the scope or spirit of the disclosure. Any of the recited methods can be performed in the order of events recited or in any other order that is logically possible.

[0118] Unless otherwise indicated, the terms "a", "an", "the" and similar referents used in the context of the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural. Unless otherwise indicated herein, references to numerical ranges herein are merely intended as a shorthand method of referring individually to each separate numerical value falling within the range, and each separate numerical value is incorporated into the specification as if it were individually recited herein. Unless otherwise indicated, any and all examples or exemplary language (e.g., "such as") used herein are intended to better illustrate the disclosure and are not a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

Claims

1. An assay for detecting target extracellular vesicles in a sample containing or suspected of containing target extracellular vesicles and non-target extracellular vesicles, characterized in that, Comprising: Providing a matrix coated with a capture antibody that can bind to target extracellular vesicles and non-target extracellular vesicles; Contacting the matrix with a sample under conditions sufficient to allow the target extracellular vesicles and non-target extracellular vesicles to bind to the capture antibody, thereby immobilizing the target extracellular vesicles and non-target extracellular vesicles on the matrix; Contacting the matrix with immobilized target extracellular vesicles and non-target extracellular vesicles with a labeling reagent comprising a gold nanostructure associated with a detection antibody that can bind only to the target extracellular vesicles, wherein when the detection antibody binds to the target extracellular vesicles, the target extracellular vesicles are labeled with the gold nanostructure; Incubating the matrix containing the labeled target extracellular vesicles with a copper growth reagent comprising copper ions, a reducing agent, and a structure-directing agent under conditions sufficient to induce reduction of the copper ions and formation of a copper shell around the gold nanostructure; and Detecting the scattering signal from the gold nanostructure on which the copper shell has formed.

2. The assay method according to claim 1, wherein The gold nanostructure comprises gold nanorods and / or gold nanospheres.

3. The assay method according to claim 1 or 2, characterized in that, The matrix further comprises a protein A / G coating for binding the capture antibody to the matrix.

4. The assay according to any one of the preceding claims, characterized in that, The sample comprises one or more body fluids.

5. The assay according to claim 4, wherein, The one or more body fluids are selected from the group consisting of urine, blood, plasma, BALF, CSF, tears, sweat, or combinations thereof.

6. The assay according to any one of the preceding claims, characterized in that, The matrix is a microplate or comprises a porous PDMS membrane.

7. The assay according to any one of the preceding claims, characterized in that, The reducing agent is sodium ascorbate.

8. The assay according to any one of the preceding claims, characterized in that, The structure-directing agent is polyethyleneimine.

9. The assay according to claim 8, wherein The polyethyleneimine is branched.

10. The assay according to any one of the preceding claims, characterized in that, The ratio of copper ions to the structure-directing agent in the copper growth reagent is from 120:1 to 60:

1.

11. The assay according to claim 10, wherein The structure directing agent is polyethyleneimine (PEI), and the copper ion is Cu 2+ , and the ratio of Cu 2+ to PEI is 60:

1.

12. The assay according to any one of the preceding claims, characterized in that, The matrix is incubated in the copper growth reagent for about 5 to about 30 minutes.

13. The assay according to claim 12, wherein The matrix is incubated in the copper growth reagent for about 10 minutes.

14. The assay according to any one of the preceding claims, characterized in that, Further comprising washing the matrix after immobilizing the target extracellular vesicles and non-target extracellular vesicles on the matrix.

15. The assay according to any one of the preceding claims, characterized in that, Further comprising washing the matrix after labeling the target extracellular vesicles with the label.

16. The assay according to any one of the preceding claims, characterized in that, Further comprising washing the matrix after incubating the matrix in the copper growth reagent.

17. A kit for an assay for detecting target extracellular vesicles in a sample containing or suspected of containing target extracellular vesicles and non-target extracellular vesicles, characterized in that, The kit comprises: A matrix coated with a capture antibody that can bind to target extracellular vesicles and non-target extracellular vesicles; A labeling reagent comprising a gold nanostructure associated with a detection antibody; A copper growth reagent comprising copper ions, a reducing agent, and a structure-directing agent; and Instructions for performing the assay, the assay comprising: contacting a matrix coated with a capture antibody with a sample to immobilize the target extracellular vesicles and non-target extracellular vesicles on the matrix; contacting the immobilized target extracellular vesicles and non-target extracellular vesicles with a labeling reagent comprising a gold nanostructure associated with a detection antibody, whereby the detection antibody selectively binds to the target extracellular vesicles, thereby labeling the target extracellular vesicles; incubating the matrix containing the labeled target extracellular vesicles in a copper growth solution to form a copper shell around the gold nanostructure; and detecting the scattering signal from the gold nanostructure on which the copper shell has formed.

18. The kit according to claim 17, wherein The gold nanostructure comprises gold nanorods and / or gold nanospheres.

19. The kit according to claim 17 or 18, characterized in that, The matrix further includes a protein A / G coating for binding the capture antibody to the matrix.

20. The kit according to any one of claims 17 to 19, characterized in that The sample includes one or more body fluids.

21. The kit according to claim 20, wherein The one or more body fluids are selected from the group consisting of urine, blood, plasma, BALF, CSF, tears, sweat, or a combination thereof.

22. The kit according to any one of claims 17 to 21, characterized in that, The matrix is a porous plate or includes a porous PDMS membrane.

23. The kit according to any one of claims 17 to 22, characterized in that, The reducing agent is sodium ascorbate.

24. The kit according to any one of claims 17 to 23, characterized in that, The structure-directing agent is polyethyleneimine.

25. The kit according to any one of claims 17 to 24, characterized in that, The ratio of copper ions to the structure-directing agent in the copper growth reagent is from 120:1 to 60:

1.

26. The kit according to claim 24, characterized in that, The structure-directing agent is polyethyleneimine (PEI), and the copper ion is Cu 2+ , and the ratio of Cu 2+ to PEI is 60:1.

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