Method for detecting extracellular vesicle protein and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a method for detecting extracellular vesicle protein and application thereof. According to the invention, the design is optimized on the basis of the first-generation SENSORS technology, and a second-generation BarFllare high-sensitivity detection technology is developed. According to the technology, a gold nanoparticle-mediated three-function fluorescent probe controllable release system is integrated, a superparamagnetic nanoparticle-mediated fluorescent probe captures enrichment and enhances a fluorescent signal through a spherical hot spot aggregation effect, the detection sensitivity is improved by 31 times compared with that of a traditional ELISA method, and high-sensitivity detection (1.37-7.30 parts / mu L) of EVs protein is achieved. Compared with the first-generation SENSORS technology, the BarFllare detection time is shortened by 43.75%, and the cost is reduced by about 63%.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for detecting extracellular vesicle proteins and its application. Background Technology

[0002] The inventors' previous work integrated novel nanomaterials, primer exchange reaction (PER)-mediated tandem signal amplification, and the CRISPR / Cas12a system to construct a novel, highly sensitive protein detection technology—SENSORS (YuWang). # Shan Xing # Yi-Wei Xu # Qing-Xia Xu # Ming-Fang Ji # Yu-Hui Peng # ,Ya-Xian Wu,Meng Wu,Ning Xue,Biao Zhang,Shang-Hang Xie,Rui-Dan Zhu,Xin-Yuan Ou,Qi Huang,Bo-Yu Tian,Hui-Lan Li,Yu Jiang,Xiao-Bin Yao,Jian-Pei Li,Li Ling,Su-Mei Cao*,Qian Zhong*,Wan-Li Liu*,Mu-Sheng Zeng*.Highly sensitive detection platform-based diagnosis of oesophageal squamous cell carcinoma in China:amulticentre,case-control,diagnostic study.Lancet Digital Health.2024Oct;6(10):e705-e717.).

[0003] While SENSORS technology has made significant progress in improving detection sensitivity, it still has shortcomings such as cumbersome operation, long processing time, and limited automation, which urgently need to be optimized and make it difficult to meet the high-throughput detection requirements of large-scale population screening.

[0004] Therefore, in order to overcome these limitations, this invention has developed a new generation of detection technology based on SENSORS technology. Summary of the Invention

[0005] The first aspect of the present invention is to provide a method for detecting extracellular vesicle proteins.

[0006] A second aspect of the present invention is to provide a detection kit.

[0007] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0008] A first aspect of the present invention provides a method for detecting extracellular vesicle proteins (BarFlare, a combination of Barcode and Fluorescence), comprising the following steps:

[0009] 1) Capture extracellular vesicles and the target protein they carry;

[0010] 2) Add biotinylated specific binding substances to bind to the target protein;

[0011] 3) Add avidin and gold nanoparticle probes sequentially;

[0012] 4) Add dithiothreitol;

[0013] 5) Magnetic nanoparticles modified with avidin;

[0014] 6) Detect fluorescence signals.

[0015] In some embodiments of the present invention, the biotinylated specific binding substance specifically binds to the target protein.

[0016] In some embodiments of the present invention, the specific binding substance includes an antibody or a nucleic acid aptamer.

[0017] In some embodiments of the present invention, the method is used for purposes other than disease detection or treatment.

[0018] In some embodiments of the present invention, esophageal squamous cell carcinoma cells are used as the target for detection of extracellular vesicle proteins. The purpose is solely to detect extracellular vesicle proteins, not to directly obtain the health status of living human or animal bodies. Those skilled in the art can detect any extracellular vesicle protein in any sample according to the detection purpose.

[0019] In some embodiments of the present invention, the target protein includes, but is not limited to: MMP13, SCCA1, SCCA2, CEA, EpCAM, CD71, CD109, VEGF, EGFR, CA15-3, CA125, etc.

[0020] In some embodiments of the present invention, the method of step 1) is as follows:

[0021] Extracellular vesicles and their loaded target proteins are captured using microplates coated with specific antibodies containing markers on the surface of extracellular vesicles.

[0022] In some embodiments of the present invention, the extracellular vesicle surface marker is CD63.

[0023] In some embodiments of the present invention, the microporous plate includes a U-shaped bottom microporous plate or a V-shaped bottom microporous plate.

[0024] In some embodiments of the present invention, the microporous plate includes a V-bottom microporous plate (Beyotime#FPT018).

[0025] In some embodiments of the present invention, in step 3), the gold nanoparticle probe includes a probe sequence and gold nanoparticles.

[0026] In some embodiments of the present invention, the 5' end of the probe sequence is modified with biotin and the 3' end is modified with a thiol group.

[0027] In some embodiments of the present invention, the probe sequence is bound to the surface of gold nanoparticles.

[0028] In some embodiments of the present invention, the probe sequence is modified with a fluorescent group.

[0029] In some embodiments of the present invention, the nucleotide sequence length of the probe sequence is 30–70 bp.

[0030] In some embodiments of the present invention, the probe sequence includes a random sequence, a PolyA sequence, a PolyT sequence, a PolyC sequence, or a combination of a random sequence and a PolyA sequence, a PolyT sequence, or a PolyC sequence.

[0031] In some embodiments of the present invention, the probe sequence includes:

[0032] Barcode1: Biotin-ACACTTTGACCCCCG / iCy5 / GATACGACGCACTTT-SH (SEQ ID NO: 1);

[0033] Barcode2: Biotin-TGTACACTCTTATTG / iCy5 / ATCGGGATACTTTAG-SH (SEQ ID NO: 2);

[0034] Barcode3: Biotin-AGCCACAAGATGTCA / iCy5 / TTAAGCACCAAGACT-SH (SEQ ID NO: 3);

[0035] Bio-T30-Cy5-SH: Biotin-TTTTTTTTTTTTTTTT / iCy5 / TTTTTTTTTTTTTTTT-SH (SEQ IDNO: 4);

[0036] Bio-A30-Cy5-SH: Biotin-AAAAAAAAAAAAAAA / iCy5 / TAAAAAAAAAAAAAAAA-SH (SEQ IDNO: 5);

[0037] Bio-C30-Cy5-SH: Biotin-CCCCCCCCCCCCCCC / iCy5 / TCCCCCCCCCCCCCCCC-SH (SEQ IDNO: 6);

[0038] Barcode1-A10: ACACTTTGACCCCCGGATACGACGCACTTTAAAAAAAAAA-SH (SEQ ID NO: 7);

[0039] Barcode 1-A20:

[0040] ACACTTTGACCCCCGGATACGACGCACTTTAAAAAAAAAAAAAAAAAAAAA-SH (SEQ ID NO: 8);

[0041] Barcode 1-A30:

[0042] ACACTTTGACCCCCGGATACGACGCACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-SH (SEQID NO: 9);

[0043] Barcode 1-A40:

[0044] ACACTTTGACCCCCGGATACGACGCACTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-SH (SEQ ID NO: 10);

[0045] In some embodiments of the present invention, the fluorescent group includes, but is not limited to: FAM, HEX, ROX, Cy3, Cy5, etc.

[0046] In some embodiments of the present invention, the fluorescent group is located at position 25 from the 5' end of the biotinylated end.

[0047] In some embodiments of the present invention, the probe sequence is subjected to TCEP reduction of the 3' end thiol group.

[0048] In some embodiments of the present invention, the concentration of TCEP is 1–100 μM, preferably 10–50 μM.

[0049] In some embodiments of the present invention, the probe sequence is labeled on gold nanoparticles by a freeze-thaw method.

[0050] In some embodiments of the present invention, the freezing temperature of the freeze-thaw method is -200 to 0°C.

[0051] In some embodiments of the present invention, the freezing time of the freeze-thaw method is 1 to 200 minutes.

[0052] In some embodiments of the present invention, the biotinylated specific binding substance includes a biotinylated specific antibody and a biotinylated specific nucleic acid aptamer, wherein the concentration of the biotinylated specific antibody is 10-500 ng / mL and the concentration of the biotinylated specific nucleic acid aptamer is 20-500 nmol / L.

[0053] In some embodiments of the present invention, the avidin includes, but is not limited to, avidin, streptavidin, and ovalbumin.

[0054] In some embodiments of the present invention, the concentration of the avidin used is 1-100 nmol / L.

[0055] In some embodiments of the present invention, the concentration of the gold nanoparticle probe is 25-500 pmol / L.

[0056] In some embodiments of the present invention, the concentration of the avidin-modified magnetic nanoparticles used is 0.1-10 mg / mL.

[0057] A second aspect of the present invention provides a kit for detecting extracellular vesicle proteins, the kit comprising a microplate modified with antibodies against extracellular vesicle surface markers, a biotinylated specific binding substance, a gold nanoparticle probe, and avidin-modified magnetic nanoparticles.

[0058] In some embodiments of the present invention, the extracellular vesicle surface marker is CD63.

[0059] In some embodiments of the present invention, the biotinylated specific binding substance specifically binds to the target protein.

[0060] In some embodiments of the present invention, the specific binding substance includes an antibody or a nucleic acid aptamer.

[0061] In some embodiments of the present invention, the gold nanoparticle probe includes a probe sequence and gold nanoparticles.

[0062] In some embodiments of the present invention, the 5' end of the probe sequence is modified with biotin and the 3' end is modified with a thiol group.

[0063] In some embodiments of the present invention, the probe sequence is bound to the surface of gold nanoparticles.

[0064] In some embodiments of the present invention, the probe sequence is modified with a fluorescent group.

[0065] The beneficial effects of this invention are:

[0066] This invention optimizes and develops a second-generation BarFlare high-sensitivity detection technology based on the first-generation SENSORS technology. This technology integrates a gold nanoparticle-mediated controlled release system of a trifunctional fluorescent probe, superparamagnetic nanoparticle-mediated fluorescent probe capture and enrichment, and a spherical hotspot aggregation effect to enhance the fluorescence signal. Its detection sensitivity is 31 times higher than that of traditional ELISA methods, achieving high-sensitivity detection of EVs proteins (1.37-7.30 particles / μL). Compared to the first-generation SENSORS technology, BarFlare detection time is reduced by 43.75%, and cost is reduced by approximately 63%. Attached Figure Description

[0067] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0068] Figure 1 This is a schematic diagram illustrating the technical principle of the BarFlare detection method of the present invention.

[0069] Figure 2 To validate the Enricher-mediated capture-enrichment strategy and hotspot-enhanced fluorescence signal, the following are included: (A) Schematic diagram of the Enricher-mediated capture-enrichment strategy; (BC) Fluorescence intensity analysis (B) and corresponding LOD (limit of detection) of different concentrations of fluorescent barcode probes before and after magnetic enrichment on streptavidin-modified Fe3O4 nanoparticles (Enricher); (C) Inverted fluorescence microscopy imaging results of spherical hotspot signals formed by magnetic field enrichment under the condition of fluorescent barcode probes at indicated concentrations; (E) Based on the fluorescence microscopy platform, a Python algorithm using Gaussian filtering and Otsu threshold segmentation was established for image quantitative analysis of spherical hotspot signals; (F) Based on the fluorescence microplate reader platform, direct fluorescence quantitative analysis of spherical hotspot signals was performed; (G) Correlation analysis of hotspot fluorescence intensity obtained from the fluorescence microplate reader platform and the fluorescence microscopy platform; LOD, limit of detection. Figure 2 The probe sequence used is SEQ ID NO: 25.

[0070] Figure 3 Results of optimizing the spatial position of the fluorophore on the barcode DNA fluorescent probe, wherein: (AB) fluorescence intensity of Cy5 relative to the biotinylated end on the trifunctional probe was analyzed using a fluorescence microplate reader platform (A) and a fluorescence microscope platform (B).

[0071] Figure 4 The results show the characterization and validation of the Transformer probe, including: (A) Characterization of AuNPs before (left) and after (right) DNA functionalization using transmission electron microscopy; (BC) Measurement of the zeta potential (B) and particle size (C) of DNA-functionalized AuNPs (Transformer) using dynamic light scattering; (D) Measurement of the maximum absorption wavelength of the Transformer using UV-Vis absorption spectroscopy; (E) Validation of the design strategy for controlled release of fluorescent probes induced by DTT; (F) Linear correlation between SNR and Transformer probe concentration; (G) Feasibility validation of BarFlare detection of serially diluted SA target proteins; AuNPs, gold nanoparticles; SNR, signal-to-noise ratio; DTT, dithiothreitol; SA, streptavidin; LOD, limit of detection.

[0072] Figure 5 The results of the optimization of Transformer probe preparation conditions include: (A) diameter of gold nanoparticles (AuNPs), (B) length of barcode probes, (C) design of barcode probe sequences, (D) concentration of barcode probe feed, (E) thiol reduction conditions, and (F) particle freeze-thaw labeling procedure.

[0073] Figure 6 The results of the BarFlare detection condition optimization include: (A) optimization of different solid-phase microplate configurations for BarFlare detection; (B) detailed configuration of the Beyotime#FPT018 96-well V-shaped microplate; and (C) optimization of the excitation focal length of the fluorescence microplate reader.

[0074] Figure 7 The results show the sensitivity comparison of different V-shaped and U-shaped microplate configurations in fluorescent barcode probe detection.

[0075] Figure 8 For the comparison of analytical performance of the BarFlare and ELISA methods, where: (A) linear range and LOD assessment of BarFlare and (B) ELISA methods for detecting MMP13 protein; ELISA, enzyme-linked immunosorbent assay; MMP13, matrix metalloproteinase-13; LOD, limit of detection; OD, optical density.

[0076] Figure 9 The feasibility of recognizing Eca-109-derived extracellular vesicles (EVs) based on antibodies and nucleic acid aptamers was verified. Specifically: (A, B) feasibility verification based on antibodies Anti-SCC (A) and Anti-MMP13 (B); (C, D) feasibility verification based on the nucleic acid aptamer CD63 apt (C) and a random sequence control (D). EVs, extracellular vesicles; aptamer; SCC, squamous cell carcinoma antigen; MMP13, matrix metalloproteinase-13.

[0077] Figure 10 The analytical performance results of antibody-based and nucleic acid aptamer-based BarFlare assays are presented, including: the linear range and limit of detection (LOD) for antibody-based BarFlare assays of EV-derived SCC (A) and EV-derived MMP13 (B), and the linear range and LOD for nucleic acid aptamer-based BarFlare assays of EV-derived CEA (C) and EV-derived EpCAM (D); EVs, extracellular vesicles; LOD, limit of detection; SCC, squamous cell carcinoma antigen; MMP13, matrix metalloproteinase-13.

[0078] Figure 11 This is to validate the specificity of BarFlare technology in protein detection in EVs, where: the levels of Calnexin (A) and TSG101 (B) proteins in serially diluted Eca-109 cell-derived EVs were detected using BarFlare technology; EVs are extracellular vesicles. Detailed Implementation

[0079] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0080] The technical principle of this invention is as follows: BarFlare assay technology integrates a multi-step immune capture and signal amplification approach: First, an Anti-CD63 antibody is pre-coated in a V-shaped microplate to specifically capture CD63-positive EVs from serum samples; then, a biotinylated detection antibody is added to selectively bind to the target protein on the EVs; next, streptavidin protein and functionalized AuNPs gold nanoparticle probes (referred to as Transformers) are added sequentially. These gold nanoparticles are coupled with a trifunctional fluorescent DNA probe, which is modified with a biotin group at the 5' end, a Cy5 fluorescent group in the middle region, and a thiol group at the 3' end. Through the biotin-streptavidin interaction, a pentamer complex is formed, consisting of the capture antibody-EVs-detection antibody-streptavidin-Transformer, thereby transducing the protein signal into a fluorescent probe signal. Subsequently, dithiothreitol (DTT) was added to trigger a ligand substitution reaction that released fluorescent probes. These probes were then captured and enriched by streptavidin-modified superparamagnetic Fe3O4 nanoparticles (referred to as Enrichers). A magnetic field was applied using a magnetic rack to cause these nanoparticles to accumulate at the bottom of the pores of a V-shaped microplate, forming spherical aggregates. Under excitation light, these high-density hotspot aggregates exhibited significantly enhanced fluorescence signals. Finally, fluorescence signal quantification was performed using a dual-modal platform of fluorescence microscopy and a fluorescence microplate reader, and the fluorescence intensity was positively correlated with the level of extracellular vesicle target proteins.

[0082] The relevant experimental materials are as follows:

[0083] Reagents and Consumables: Human MMP13 protein ELISA kit (R&D, DY511), SCCA1 / SCCA2 protein ELISA kit (cloud-clone, KSB814Hu01); Anti-MMP13 antibody (Abcam, ab51072, ab39012); Anti-SerpinB3 / SCCA+SerpinB4 / SCCA-2 antibody (Abcam, ab254255); Recombinant Anti-ALIX antibody [EPR15314-33]-N-terminal (Abcam, ab186728); Exosome antibody combination (Calnexin, CD9, CD63, CD81, Hsp70, TSG101) (Abcam, ab275018); Anti-CD63 polyclonal antibody (Proteintech, 25682-1-AP); Goat anti Rabbit-HRP antibody (Invitrogen, 31460); Exosome isolation kit EXOSOME HUMANCD63ISOL / DET (ThermoFisher, 10606D); RPMI 1640 medium (Life Technologies, Carlsbad, CA); fetal bovine serum (Invitrogen, Carlsbad, CA); KSFM medium (Invitrogen, Carlsbad, CA); gold nanoparticles (Ruixi Biotechnology, R-G030005); streptavidin (solarbio, S9170); streptavidin magnetic beads (Roche); DTT (ThermoFisher, A39255); PBS buffer (pH 7.4, ThermoFisher); magnetic rack (Invitrogen); microplate (Greiner) bio-one#655001, Cloud-clone#KSB814Hu01, Jet#TCP011096, Corning#42592, Corning#3897, Beyondtime#FPT018, Jet#TCP002096).

[0084] Primers: All primers were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. (Due to technical limitations of the company, some Cy5 group modifications could not be performed on A, C, and G, so the modified bases were replaced with T). The primers were purified by HPLC or PAGE. The sequences of all synthesized primers are shown in Table 1.

[0085] Table 1 Primer sequences

[0086]

[0087]

[0088]

[0089] Example 1: Feasibility Verification of BarFlare Technology

[0090] This embodiment first verifies the feasibility of the design strategy of using streptavidin-modified superparamagnetic Fe3O4 nanoparticles (named Enricher) to capture and enrich trifunctional fluorescent barcode probes.

[0091] 1. Feasibility verification of the signal enrichment and amplification strategy based on Enricher

[0092] 1) Set up different concentration gradients of the barcode DNA fluorescent probe (SEQ ID NO: 25): 50 nM, 10 nM, 2 nM, 400 pM, 80 pM, 16 pM, 3.2 pM and 0 pM (PBS control).

[0093] 2) Set up enrichment and non-enrichment groups. Enrichment group: Transfer 50 μL of barcode DNA probes of different concentrations to V-type microplates, then add 6 μL / well of Enricher (streptavidin-modified superparamagnetic Fe3O4 nanoparticles, concentration 0.72 mg / mL), and incubate at 37℃ and 800 rpm for 20 min. Non-enrichment group: Transfer 50 μL of barcode DNA probes of different concentrations to V-type microplates, then add 6 μL / well of PBS, and incubate at 37℃ and 800 rpm for 20 min.

[0094] 3) Place the V-shaped microplate on a 96-well magnetic rack in the dark for magnetic separation. After 5 minutes, measure the fluorescence intensity using a Tecan fluorescence microplate reader with the following parameters: excitation wavelength 635 nm, emission wavelength 680 nm, gain 227 (Manual), and Z-axis position 28500 μm. Calculate the semi-quantitative result using the following formula: F / F0 = average(Fs / Fc), where Fs represents the fluorescence intensity value of samples with different concentrations of barcode DNA probes, and Fc represents the fluorescence intensity value of the PBS control sample.

[0095] 2. Optimization of the fluorescent group position of the barcode DNA fluorescent probe

[0096] 1) Bio-T30-10Cy5, Bio-T30-15Cy5, Bio-T30-20Cy5, Bio-T30-25Cy5, and Bio-T30-30Cy5 were selected for fluorescence intensity testing. The method is as follows: 1) Poly(T30) oligonucleotide probes with different positions (modified with Cy5 fluorescent groups at the 10th T base [SEQ ID NO: 21], 15th T base [SEQ ID NO: 22], 20th T base [SEQ ID NO: 23], 25th T base [SEQ ID NO: 24], and 30th T base [SEQ ID NO: 25] from the 5' end were synthesized, and biotin groups were labeled at their 5' ends.

[0097] 2) Transfer 100 μL of 10 pM probe to V-type microplates, then add 6 μL of streptavidin-modified magnetic beads (Enricher) per well, and incubate at 37°C on a shaker for 20 min.

[0098] 3) Place the V-shaped microplate on a 96-well magnetic rack in the dark for magnetic separation. After 5 minutes, measure the fluorescence intensity using a Tecan fluorescence microplate reader. The parameters are set as follows: excitation wavelength 635 nm, emission wavelength 680 nm, gain 227 (Manual), and Z-axis position 28500 μm. Calculate the semi-quantitative result using the following formula: F / F0 = average(Fs / Fc), where Fs represents the fluorescence intensity value of the sample and Fc represents the fluorescence intensity value of the control group.

[0099] 4) Place the V-shaped microplate under a fluorescence microscope for observation, focus in bright field, and then switch to the Cy5 fluorescence channel to capture images.

[0100] 3. Preparation and characterization of trifunctional fluorescent probe-functionalized gold nanoparticles (AuNPs) probes

[0101] 100 μL of 10 nM 15 nm AuNPs was mixed with 3 μL of 100 μM trifunctional DNA fluorescent probe (5' biotin-labeled, 25th TiCy5, 3' thiolated, SEQ ID NO: 4). After thorough mixing, the solution was immediately placed in a -80°C freezer, frozen in dry ice for 5 minutes, or in a -20°C freezer for 2 hours to allow rapid freezing. Then, the solution was removed and slowly thawed at room temperature in the dark, allowing DNA molecules to effectively approach the AuNPs surface through the compression of the ice crystals to form Au-S bonds. Subsequently, the solution was centrifuged at 13000 rpm at 4°C for 20 minutes to precipitate the AuNPs particles. The supernatant was carefully discarded to remove unbound DNA molecules. Then, 1 mL of 0.05% PBST solution was added, inverted to mix, and centrifuged at 13000 rpm at 4°C for 20 minutes. The supernatant was discarded, and this washing step was repeated twice. Finally, AuNPs were resuspended in 100 μL of 0.05% PBST solution and mixed thoroughly to prepare trifunctional fluorescent DNA-coupled AuNPs probes, which were then stored at 4°C in the dark for later use.

[0102] To characterize the successful coupling of AuNPs particles with trifunctional fluorescent DNA, AuNPs probes before and after functionalization were dispersed in deionized water, dropped onto a copper grid, and air-dried. Morphological analysis was performed using high-resolution transmission electron microscopy. Surface properties of AuNPs, including particle size distribution, average diameter, and zeta potential, were evaluated using dynamic light scattering (DLS) analysis with a Zetasizer Ultra instrument (Malvern). The maximum absorption wavelength of AuNPs particles in the 300–700 nm spectral range was determined using a UV-Vis absorption spectrometer (Tecan Spark™ 10M).

[0103] 4. Performance Analysis

[0104] This embodiment first tests and verifies the performance of BarFlare technology in detecting traditional single protein targets and compares it with the traditional detection method ELISA. EV protein detection is not yet involved: taking the detection of MMP13 protein as an example, MMP13 protein was serially diluted twofold as a detection standard, and both BarFlare and ELISA methods were used for detection. The linear range and detection limit (LOD = 3δ / S, where δ represents the standard deviation of the blank control [n = 3], and S represents the slope of the calibration curve) of the two methods for detecting the MMP13 target protein were compared.

[0105] The procedure for detecting traditional protein targets using enzyme-linked immunosorbent assay (ELISA) is as follows: First, resuspend the MMP13 capture antibody (R&D, Catalog #DY511) powder in PBS to a working concentration of 4 μg / mL. Add 100 μL / well to each well of an ELISA plate (Catalog #42592), seal, and incubate overnight at 4°C to ensure complete antibody coating. The next day, wash the plate three times with PBST solution containing 0.05% Tween-20 (400 μL / well), allowing it to stand for 1 minute each time to remove unbound antibody. After the final wash, invert the plate and blot dry. Then, add 300 μL / well of 1% BSA blocking buffer (prepared in PBS) and block at room temperature for 2 hours to reduce non-specific binding. After blocking, wash the plate three times and blot dry. Add 100 μL of protein standard (concentration 62.5-4000 pg / mL) to each well and incubate at room temperature for 2 hours to allow the MMP13 protein in the serum to fully bind to the coating antibody. Wash the plate three times and blot dry after incubation. Add 100 μL / well of MMP13 detection antibody (R&D, Catalog#DY511) at a working concentration of 100 ng / mL (prepared with 1% BSA) and incubate at room temperature for 2 hours to allow the detection antibody to bind to the MMP13 protein bound to the coating antibody. Wash the plate three times and blot dry after incubation. Add 100 μL / well of streptavidin-conjugated horseradish peroxidase (SA-HRP) (R&D, Catalog#DY511) diluted 1:200 (prepared with 1% BSA) and incubate at room temperature in the dark for 20 minutes to allow SA-HRP to bind to the biotin group on the detection antibody. Wash the plate three times and blot dry after incubation. Finally, substrate chromogenic solution A (H2O2) and substrate chromogenic solution B (TMB) were mixed at a 1:1 ratio, and 100 μL / well of chromogenic solution was added. The mixture was incubated at room temperature in the dark for 20 minutes to allow horseradish peroxidase to catalyze the substrate color development. The reaction was terminated by adding 50 μL / well of stop solution (2N H2SO4). After mixing, the absorbance values ​​at 450 nm (detection wavelength) and 620 nm (calibration wavelength) were measured using an automated multi-wavelength microplate reader. A standard curve was established using the results of MMP13 protein standards tested under the same conditions and from the same batch to quantitatively analyze the expression level of MMP13 protein in the samples.

[0106] The BarFlare procedure for detecting traditional protein targets is as follows: First, resuspend the MMP13 capture antibody (R&D, Catalog#DY511) powder in PBS to a working concentration of 4 μg / mL. Coat 100 μL / well of a 96-well V-type microplate (Beyotime#FPT018) and incubate overnight at 4°C to allow the antibody to fully adsorb to the bottom of the microplate. The following day, the plates were blocked with 5% skim milk and incubated at 37°C for 1 hour to block unbound sites and reduce nonspecific binding. The plates were washed three times with PBST buffer containing 0.05% Tween-20, and then different concentrations of protein standards (0.39-24.69 pg / mL) were added to each well. The plates were incubated at 37°C for 1 hour, with PBS as a blank control. The plates were washed three more times, and biotin-labeled Anti-SCC antibody (1:1000, incubated at 37°C for 1 hour; KSB814Hu01, cloud-clone) or Anti-MMP13 antibody (100 ng / mL, incubated at 37°C for 1 hour; DY511, R&D system) or biotin-labeled nucleic acid aptamer (100 nM, incubated with 5 mM Tween-20 buffer) were added. Prepare MgCl2 in PBS solution and incubate at room temperature for 1 hour. Then, add streptavidin protein (10 nM, incubate at room temperature for 20 minutes; S9170, Solarbio) and Transformer (125 pM, incubate at room temperature in the dark for 20 minutes, SEQ ID NO: 4) sequentially. Wash the plate three times, add 100 μL of 100 mM dithiothreitol (A39255, ThermoFisher) to each well, and incubate at 37°C on a shaker for 1 hour. Then, add 6 μL / well of streptavidin-modified magnetic beads (Enricher) and incubate at 37°C on a shaker for 20 minutes. Finally, place the plate on a 96-well magnetic rack in the dark for 5 minutes for magnetic separation. Measure the fluorescence intensity using a Tecan fluorescence microplate reader with the following parameters: excitation wavelength 635 nm, emission wavelength 680 nm, gain 227 (Manual), and Z-axis position 28500 μm. The semi-quantitative results are calculated using the following formula: F / F0 = average(Fs / Fc), where Fs represents the fluorescence intensity value of the sample to be tested and Fc represents the fluorescence intensity value of the control sample.

[0107] This embodiment further evaluates the performance of BarFlare technology based on antibody and nucleic acid aptamer recognition patterns in detecting EV target proteins. EV samples purified from Eca-109 cell supernatant were serially diluted and used as detection standards. Specific antibodies were used for recognition when detecting SCC and MMP13 proteins, while corresponding specific nucleic acid aptamers (SEQ ID NO: 11-19) were used for recognition when detecting targets such as EpCAM, EGFR, VEGF, CD71, CD109, CD63, CA15-3, CA125, and CEA. This allows for the evaluation of the linear range and limit of detection (LOD) of BarFlare technology in detecting EV target proteins (LOD calculation method as before).

[0108] The BarFlare method for detecting EV protein targets is as follows: First, dilute the Anti-CD63 polyclonal antibody (#25682-1-AP, proteintech) at a ratio of 1:1000, and coat a 96-well V-type microplate (Beyotime#FPT018) with 100 μL / well. Incubate overnight at 4°C to allow the antibody to fully adsorb to the bottom of the microplate. The following day, the plates were blocked with 5% skim milk and incubated at 37°C for 1 hour to block unbound sites and reduce nonspecific binding. The plates were washed three times with PBST buffer containing 0.05% Tween-20, and then diluted serum samples (25 μL serum + 75 μL PBS solution) were added to each well. The plates were incubated at 37°C for 1 hour, with PBS as a blank control. The plates were washed three more times, and biotin-labeled Anti-SCC antibody (1:1000, incubated at 37°C for 1 hour; KSB814Hu01, cloud-clone) or Anti-MMP13 antibody (100 ng / mL, incubated at 37°C for 1 hour; DY511, R&D system) or biotin-labeled nucleic acid aptamer (100 nM, incubated with 5 mM PBS) was added. Prepare MgCl2 in PBS solution and incubate at room temperature for 1 hour. Then, add streptavidin protein (10 nM, incubate at room temperature for 20 minutes; S9170, Solarbio) and Transformer (125 pM, incubate at room temperature in the dark for 20 minutes, SEQ ID NO: 4) sequentially. Wash the plate three times, add 100 μL of 100 mM dithiothreitol (A39255, ThermoFisher) to each well, and incubate at 37°C on a shaker for 1 hour. Then, add 6 μL / well of streptavidin-modified magnetic beads (Enricher) and incubate at 37°C on a shaker for 20 minutes. Finally, place the plate on a 96-well magnetic rack in the dark for 5 minutes for magnetic separation. Measure the fluorescence intensity using a Tecan fluorescence microplate reader with the following parameters: excitation wavelength 635 nm, emission wavelength 680 nm, gain 227 (Manual), and Z-axis position 28500 μm. The semi-quantitative results are calculated using the following formula: F / F0 = average(Fs / Fc), where Fs represents the fluorescence intensity value of the sample to be tested and Fc represents the fluorescence intensity value of the control sample.

[0109] 5. Experimental Results

[0110] 1) Feasibility verification of the signal enrichment and amplification strategy based on Enricher

[0111] When detecting different concentrations of fluorescent barcode probes (SEQ ID NO: 25), the enricher, under the influence of a magnetic field, captured and enriched the fluorescent probes, forming high-density spherical hotspot aggregates. This significantly enhanced the fluorescence signal in the excited state. The signal intensity after enrichment was significantly higher than before enrichment. The limit of detection (LOD) increased from 69.43 pM (Y = 16.99X + 3.411, R² = 0.9997, where Y is the F / F0 value and X is the fluorescent probe concentration) to 0.65 pM (Y = 273.3X + 8.984, R² = 0.9961, where Y is the F / F0 value and X is the fluorescent probe concentration), representing a 107-fold increase in sensitivity compared to before enrichment. Figure 2 (AC).

[0112] This experiment does not detect specific proteins. By verifying the changes in fluorescence signal intensity, it demonstrates that this method can amplify the signal and improve sensitivity.

[0113] 2) Optimization of the fluorescent group position of the barcode DNA fluorescent probe

[0114] This embodiment further optimized the spatial arrangement between the fluorophore and magnetic nanoparticles on the trifunctional fluorescent probe, and found that the highest fluorescence quantum yield could be detected when the Cy5 fluorophore was located at the 25th thymine base from the 5' end of the biotinylated end. Figure 3 ). Figure 3 The probe sequences used for positions 10, 15, 20, 25, and 30 correspond to SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25, respectively.

[0115] To further and more intuitively observe and detect the spherical hotspot aggregates formed after the Enricher captures and enriches the fluorescent probes, fluorescence images of the magnetically enriched spherical hotspots were captured using an inverted fluorescence microscope in the Cy5 channel. The results showed that the brightness of the spherical aggregates gradually increased with increasing fluorescent barcode probe concentration, and the brightness exhibited a significant positive correlation with the probe concentration. Figure 2 (D).

[0116] This embodiment develops a general Python algorithm for the quantitative analysis of these hotspot image signals. The algorithm uses Gaussian filtering and Otsu thresholding to create a mask, thereby binarizing the image and ultimately outputting the average gray value within the spherical hotspot region. Figure 2(D). This embodiment found that the average gray value calculated by the algorithm shows a good linear correlation with the barcode probe concentration. Figure 2 Moreover, these results were highly consistent with the fluorescence intensity measurements output by the microplate fluorescence microplate reader platform, and a significant positive correlation was shown between the two detection modalities (r = 0.9872, P = 0.0002). Figure 2 (F, G). The above results suggest that the design strategy of forming high-density fluorescent hotspot aggregates based on the enricher capture and enrichment of trifunctional fluorescent probes in BarFlare technology has been verified as feasible.

[0117] 3) Representation of Transformer

[0118] Next, the present invention characterized the gold nanoparticle (AuNPs) probe (named Transformer, with the barcode probe sequence SEQ ID NO: 4) functionalized with trifunctional fluorescent probe.

[0119] Transmission electron microscopy (TEM) results showed that, compared to bare gold nanoparticles that lack surface protection and are prone to aggregation, the Transformer coupled with the trifunctional fluorescent probe exhibited a stable and uniform particle distribution due to the formation of a protective layer by surface functionalization of negatively charged nucleic acid molecules, resulting in electrostatic repulsion. Figure 4 (A). Dynamic light scattering (DLS) results showed an increased zeta potential (from -40.34±0.8472mV to -28.79±0.9750mV) and increased particle size (from 23.34±1.671nm to 51.39±3.173nm) in the Transformer probe functionalized with the trifunctional fluorescent probe. Full-spectrum analysis also showed a redshift in the maximum absorption wavelength of the Transformer probe compared to the bare gold particle control (from 521nm to 525nm). All these results confirm that the Transformer probe was successfully coupled with the trifunctional fluorescent barcode probe. Figure 4 (BD). The addition of dithiothreitol (DTT) significantly increased the fluorescence intensity of the Transformer probe solution (from 115.3 ± 3.215 au to 1119 ± 96.88 au), demonstrating the feasibility of the AuNP-based strategy for controlling the release of fluorescent barcode probes. Figure 4(E). In this embodiment, the concentration of free fluorescent probes released into the solution was calculated using a standard curve, and the loading rate of ssDNA fluorescent probes on the Transformer surface was determined based on the concentration of AuNPs particles. Each Transformer particle surface could be labeled with 163±5 ssDNA fluorescent probes, which is consistent with the results reported in the literature (Liu B, Liu J. Freezing Directed Construction of Bio / Nano Interfaces: Reagentless Conjugation, Denser Spherical Nucleic Acids, and Better Nanoflares. J Am Chem Soc. 2017; 139(28): 9471-4.).

[0120] Next, in this embodiment, the Transformer probe was subjected to a concentration gradient dilution, followed by DTT-mediated fluorescent barcode release and Enricher-mediated capture and enrichment detection. The results showed a clear signal-response relationship between the fluorescence signal-to-noise ratio (SNRs) and the Transformer probe concentration (Y = 0.01104X + 0.7888, R0). 2 =0.9997, where Y is the F / F0 value and X is the Transformer probe concentration), and the calculated LOD is 23.17 fM ( Figure 4 The result (F) indicates that the integrated combination of the design strategy proposed in this embodiment is feasible. To verify the feasibility of applying BarFlare technology to protein target detection, this embodiment uses streptavidin (SA) as a simulated protein target. SA protein diluted at a series of concentration gradients (100 fM - 1 nM) was coated into the wells of a microplate before detection. The results showed a strong linear correlation between fluorescence SNRs and SA concentration (Y = 3.701X - 5.155, R0). 2 =0.9760, where Y is the F / F0 value and X is lg[SA protein concentration]), and the calculated LOD is 26.44 fM ( Figure 4 (G). The above results fully demonstrate the feasibility of BarFlare technology for protein target detection, and its high sensitivity.

[0121] Example 2: Optimization of BarFlare Technical Conditions

[0122] This embodiment systematically optimizes various parameters and conditions for Transformer particle preparation, including AuNP particle size, fluorescent barcode probe length, sequence design, feed concentration, thiol reduction conditions, and freeze-thaw labeling procedure. While keeping other experimental conditions constant, different AuNP particle sizes (5, 15, 30 nm), different lengths of fluorescent barcode probes (30, 40, 50, 60, 70 nt, corresponding to SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, respectively), different sequences of barcode probes (T30, A30, C30, SEQ ID NO: 4, 5, 6, and 30 nt length random nucleic acid sequences Barcode 1, Barcode 2, Barcode 3, SEQ ID NO: 1, 2, 3), different feed concentrations of barcode probes (1, 2, 3, 4 μM), and whether or not TCEP reduction of the thiol groups was used (with or without 3 μL) were all considered. By comparing the normalized absorbance values ​​corresponding to the maximum absorption wavelength of each group of particle samples using 1 mol / L TTCEP incubation at room temperature for 30 min and different freeze-thaw labeling procedures (dry ice freezing for 1 min, -80℃ freezing for 5 min, -20℃ freezing for 2 h), it was found that using 15 nm diameter gold nanoparticles, a 3 μM concentration of a 30 nucleotide poly(T) sequence, a trifunctional fluorescent barcode probe reduced by TCEP, and then freezing at -20℃ for 2 h, the highest concentration of functional Transformer particle probes could be prepared under these conditions. Figure 5 These conditions were used as standard experimental procedures in subsequent experiments.

[0123] In addition, this embodiment also optimized the geometric design of the solid-phase carrier and tested seven different configurations of ELISA plate solid-phase carriers from different companies, including four flat-bottomed ELISA plates (Greiner bio-one#655001, Cloud-clone#KSB814Hu01, Jet#TCP011096, Corning#42592), two V-shaped ELISA plates (Corning#3897, Beyotime#FPT018) and one U-shaped ELISA plate (Jet#TCP002096).

[0124] Taking the detection of MMP13 as an example, referring to the BarFlare process in Example 1 and the optimized conditions in Example 2, different bottom plates were used for detection (the Barcode probe sequence used is SEQ ID NO: 4).

[0125] The results showed that both the V-shaped bottom plate (Beyotime#FPT018 and Corning#3897) and the U-shaped bottom plate (Jet#TCP002096) configurations could effectively promote the aggregation of magnetic nanoparticles at the bottom of the plate, forming spherical high-density hotspot aggregates. Figure 6 (A). Further comparison of the performance of these three solid-phase supports in the detection of low-concentration fluorescent barcode probes with gradient dilution was conducted. This embodiment found that Beyotime#FPT018 can achieve the lowest detection limit (detecting barcode probes as low as 5 pM). Figure 7 Therefore, in this embodiment, the Beyotime#FPT018 96-well V-type microplate was selected as the preferred solid-phase carrier for subsequent experiments. Detailed specifications of this microplate, including its configuration, plate height, well depth, and pore diameter, are as follows: Figure 6 As shown in Figure B. Furthermore, this embodiment also optimized the excitation focal length of the fluorescence microplate reader, finding that the signal-to-noise ratio (F / F0 value) reached its maximum when the Z-axis focal position was 28,500 μm. Figure 6 (C). Ultimately, this embodiment selected the above-mentioned optimal experimental conditions and detection parameters for subsequent research.

[0126] Example 3: Performance Comparison of Protein Analysis Using the BarFlare Method

[0127] After completing the comprehensive validation and condition optimization of the BarFlare technology, this embodiment compares the protein analysis performance of BarFlare with that of traditional ELISA methods.

[0128] Using serially diluted MMP13 protein as a standard target, two methods were used for simultaneous detection. The results showed that as the concentration of MMP13 protein gradually increased, the fluorescence intensity (FI) value of the BarFlare technique also gradually increased, exhibiting a clear signal-concentration response relationship (Y = 0.09234X + 1.074, RFlare). 2 =0.9940, where Y is the F / F0 value and X is the MMP13 protein concentration), and the calculated LOD is 2.99 pg / mL. Figure 8 (A); The optical density (OD) value detected by traditional ELISA also shows a strong linear correlation with the target protein concentration (Y = 0.001501X - 0.09003, R). 2 =0.9918, where Y is the optical density OD value and X is the MMP13 protein concentration), the calculated LOD is 93.00 pg / mL ( Figure 8(See Table B). The above results indicate that the BarFlare technology exhibits a lower limit of detection and a 31-fold improvement in detection sensitivity compared to the traditional ELISA method. Furthermore, the intra-assay repeatability test (n=6) of the BarFlare technology showed that when detecting high (24.69 pg / mL), medium (6.17 pg / mL), and low (1.54 pg / mL) concentrations of the MMP13 protein target, the output F / F0 values ​​were 3.60±0.33, 2.53±0.16, and 1.81±0.21, respectively, with relative standard deviations (RSDs) of 9.27%, 6.34%, and 11.53% (Table 2), respectively. This suggests that under optimal detection conditions and parameters, the BarFlare technology demonstrates robust analytical performance and high reproducibility.

[0129] Table 2. In-batch performance evaluation of BarFlare technology for protein target detection

[0130]

[0131] MMP13, matrix metalloproteinase-13; SD, standard deviation; RSD, relative standard deviation.

[0132] Example 4: Sensitivity test of the BarFlare method for detecting EV proteins

[0133] 1. Experimental Methods

[0134] 1) Design of antibodies and nucleic acid aptamers

[0135] When detecting EVs-related MMP13 and SCC protein biomarkers, the biotinylated detection antibodies in the Human MMP13 Protein ELISA Kit (R&D, DY511) and the SCCA1 / SCCA2 Protein ELISA Kit (cloud-clone, KSB814Hu01) were used; when detecting EVs-related EpCAM, EGFR, VEGF, CD71, CD109, CD63, CA15-3, CA125 and CEA protein biomarkers, the references (Jia W, Ren C, Wang L, et al. CD109 is identified as a potential nasopharyngeal carcinoma biomarker using aptamer selected by cell-SELEX. Oncotarget 2016; 7(34):55328-42; Tian F, Zhang S, Liu C, et al. Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer. Nat Commun ... Nat Commun 2016; 7(34):55328-42; Nat Commun 2016; 7(34):55328-42; Nat Commun 2016; 7(34):55 2021;12(1):2536;Liu C,Zhao J,Tian F,et al.Low-cost thermophoretic profiling of extracellular-vesiclesurface proteins for the early detection and classification of cancers.NatBiomed Eng 2019;3(3):183-93;Wu X,Liu H,Han D,et al.Elucidation and Structural Modeling of CD71 as a Molecular Target for Cell-Specific Aptamer Binding.J AmChem Soc 2019;141(27):10760-9) reported the nucleic acid aptamer sequence, added 15 poly(T) at the 3' end of the sequence, and modified the 3' end with biotin (SEQ ID NO: 11-19).

[0136] 2) Differential centrifugation method for separation and purification of EVs

[0137] First, after passage and expansion of Eca-109 cells, they were cultured in 1640 medium containing 10% FBS until the cell density reached approximately 70%-80%. The original medium supernatant was discarded, and the cells were washed twice with PBS. Then, the medium was replaced with FBS-free 1640 medium and cultured for another 48 hours. The cell culture supernatant was collected and centrifuged at 500g for 10 minutes at 4°C to remove dead cells and larger cell debris. The supernatant was then transferred to a new 50mL centrifuge tube and centrifuged at 3000g for 20 minutes at 4°C to further remove cell debris. The supernatant was then transferred to a 35mL ultracentrifuge tube and centrifuged at 12000g for 20 minutes at 4°C to remove large vesicles and apoptotic bodies. Finally, the supernatant was transferred to a new 35mL ultracentrifuge tube and centrifuged at 100000g for 70 minutes at 4°C to precipitate EVs to the bottom of the tube. The supernatant was discarded, and the precipitate was washed with PBS solution filtered through a 0.22 μm membrane to remove residual culture medium components and impurities. Finally, the washed EV precipitate was centrifuged again at 100,000 g for 70 minutes at 4°C, the supernatant was discarded, and the EVs were resuspended in 100 μL of PBS solution to obtain a purified Eca-109 cell-derived EV suspension. The particle morphology of EVs isolated from Eca-109 and NE-1 cells was observed using a transmission electron microscope (TEM) JEM-1200EX (JEOL, Japan) with negative staining. The particle size and concentration of Eca-109 and NE-1 cell-derived EVs were determined using a NanoSight NS300 nanoparticle tracking analysis (NTA) instrument (Malvern Instruments Ltd, UK) to evaluate the EV isolation and purification efficiency.

[0138] 2. Experimental Results

[0139] To establish a methodology for the application of BarFlare technology in EV protein detection, this embodiment used EVs SCC, EVs MMP13, and EVs CD63 proteins as proof-of-concept models for methodological validation. Antibody-based and nucleic acid aptamer-based signal recognition and signal transduction elements were designed, and the feasibility of these two designs in EV protein recognition was preliminarily verified. After capturing EVs isolated and purified from the supernatant of the ESCC cell line Eca-109 using Anti-CD63 magnetic beads, the cells were sequentially incubated with either Anti-SCC or Anti-MMP13 primary antibody and a fluorescent secondary antibody. In this embodiment, a ring fluorescence signal was observed around the magnetic beads in the Cy5 channel, while no fluorescence signal was detected in the control magnetic beads without Eca-109 EVs under the same conditions. Figure 9 (AB). Binding verification experiments using CD63-specific nucleic acid aptamers showed a clear fluorescent signal around the magnetic beads in the Cy5 channel, while no fluorescent signal was detected in the control of random nucleic acid sequences of the same length. Figure 9(CD). These results suggest the applicability of SCC antibodies, MMP13 antibodies, and nucleic acid aptamers in EV protein recognition, laying the foundation for establishing an EV protein detection platform based on BarFlare technology.

[0140] This embodiment then uses Eca-109 cell-derived EVs purified by differential centrifugation as a reference standard to establish and evaluate the linear range and LOD of BarFlare for detecting SCC, MMP13, CEA, and EpCAM proteins derived from EVs. The results showed that as the concentration of Eca-109 EVs gradually increased, the fluorescence intensity values ​​of BarFlare detection of different EV protein targets also gradually increased, exhibiting a clear signal-concentration response relationship. The antibody-based BarFlare detection technology demonstrated high sensitivity, capable of detecting SCC as low as 2.97 particles / μL. + EVs(Y=0.2067X+0.4949,R 2 =0.9989, where Y is the F / F0 value, X is log2 [Eca-109 EVs particle concentration], and MMP13 is 1.37 particles / μL. + EVs(Y=0.0816X+0.8675,R 2 =0.9685, where Y is the F / F0 value and X is log2[Eca-109 EVs particle concentration])( Figure 10 (AB); the BarFlare technique based on nucleic acid aptamers also showed considerable sensitivity for CEA. + EVs and EpCAM + The LOD of EVs was 7.30 particles / μL (Y = 0.1154X + 0.6223, R). 2 =0.9528, where Y is the F / F0 value, X is log2 [Eca-109 EVs particle concentration]) and 2.33 particles / μL (Y = 0.4222X - 1.021, R 2 =0.9799, where Y is the F / F0 value and X is log2[Eca-109 EVs particle concentration])( Figure 10 (CD). The above results indicate that this embodiment successfully constructed a BarFlare technology based on antibodies and nucleic acid aptamers for the detection of EV protein biomarkers, and its sensitivity is high.

[0141] Example 5: Specificity test for detecting EVs protein using the BarFlare method

[0142] 1. Experimental Methods and Materials

[0143] Anti-Calnexin (ab92573) and Anti-TSG101 (ab133586) were used for antibody detection. Biotin conjugation was performed first (using the EZ-Link Sulfo-NHS-LC-LC-Biotin Biotin Conjugation Kit [Thermo Scientific, A35358]). Following the BarFlare procedure in Example 1, Eca-109 cell-derived EVs purified by differential centrifugation were used as reference standards. Different concentrations of gradient dilutions (1000, 500, 250, 125, 62.5 particles / μL) were set to detect the signal response of Calnexin and TSG101 proteins in the samples.

[0144] 2. Experimental Results

[0145] This embodiment first tested the response of BarFlare to non-EV proteins and EV-related membrane inclusion proteins to verify the specificity of BarFlare technology in EV protein detection. Calnexin is an endoplasmic reticulum chaperone protein, mainly located in the endoplasmic reticulum, assisting in the correct folding of newly synthesized glycoproteins; it is currently believed not to be present in EV components. TSG101 is a core component of ESCRT-I (endosome sorting and transport complex), participating in the formation of multivesicular endosomes; as one of the marker proteins of exosomes, it is currently believed to be mainly located within the exosome lumen. In this embodiment, when using BarFlare technology to detect Calnexin and TSG101 proteins in gradient-diluted Eca-109 EVs, the output fluorescence signals were found to be at low levels and not significantly correlated with EV particle concentration. Figure 11 This low responsiveness to Calnexin and TSG101 indicates that the BarFlare technology has the ability to specifically detect EV membrane proteins and can effectively distinguish between EV membrane proteins and non-EV proteins, as well as EV intraluminal proteins.

[0146] Next, this embodiment tested the detection precision of the BarFlare technology. BarFlare was used to detect high (1000.00 particles / μL), medium (125.00 particles / μL), and low (15.63 particles / μL) concentrations of Eca-109. For EVs particles, the F / F0 values ​​of the intra-batch repeatability test (n=6) were 3.36±0.31, 2.76±0.23, and 2.23±0.26, with relative standard deviations (RSDs) of 9.18%, 8.44%, and 11.68%, respectively; the F / F0 values ​​of the inter-batch repeatability test (n=6) were 3.46±0.42, 2.64±0.24, and 1.93±0.20, with relative standard deviations (RSDs) of 12.13%, 9.16%, and 10.13%, respectively (Table 3), highlighting the robust analytical performance and high detection repeatability of BarFlare.

[0147] Table 3. Intra- and inter-batch performance evaluation of BarFlare technology for EV protein target detection.

[0148]

[0149] EV, extracellular vesicles; MMP13, matrix metalloproteinase-13; SD, standard deviation; RSD, relative standard deviation.

[0150] To evaluate the anti-serum matrix interference (SCC) ability of BarFlare, this example uses SCC at concentrations of 500 particles / μL, 125 particles / μL, and 31.25 particles / μL. + EVs were added to 20% and 100% fetal bovine serum for recovery tests. Results showed that high, medium, and low concentrations of SCC were detected in 20% serum. + The concentrations detected in EVs were 508.68±134.86 particles / μL, 135.16±35.75 particles / μL, and 29.89±17.95 particles / μL, with corresponding recoveries of 101.74%, 108.13%, and 95.64%. High, medium, and low concentrations of SCC were detected in 100% serum. +The detected concentrations of EVs were 594.58±186.75 particles / μL, 134.13±7.39 particles / μL, and 38.04±23.57 particles / μL, with corresponding recoveries of 118.92%, 107.31%, and 121.72% (Table 4). These results confirm that the BarFlare technique has strong anti-interference ability in detecting EV proteins in complex biological samples.

[0151] Table 4. Recovery of SCC protein from Eca-109 EVs using BarFlare technology.

[0152]

[0153] EVs, extracellular vesicles; SCC, squamous cell carcinoma antigen.

[0154] In summary, this invention optimizes and develops a second-generation BarFlare high-sensitivity detection technology based on the first-generation SENSORS technology. This technology integrates a gold nanoparticle-mediated trifunctional fluorescent probe controllable release system, superparamagnetic nanoparticle-mediated fluorescent probe capture and enrichment, and spherical hotspot aggregation effect to enhance the fluorescence signal. Its detection sensitivity is 31 times higher than the traditional ELISA method, achieving high-sensitivity detection of EVs proteins (1.37-7.30 particles / μL). Compared to the first-generation SENSORS technology, BarFlare detection time is reduced by 43.75%, and cost is reduced by approximately 63%.

[0155] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A method for detecting extracellular vesicle proteins, comprising the following steps: 1) Capture extracellular vesicles and the target protein they carry; 2) Add biotinylated specific binding substances to bind to the target protein; 3) Add avidin and gold nanoparticle probes sequentially; 4) Add dithiothreitol; 5) Magnetic nanoparticles modified with avidin; 6) Detect fluorescence signals; The biotinylated specific binding substance specifically binds to the target protein; The specific binding substance includes antibodies or nucleic acid aptamers; The method is used for purposes other than disease detection and treatment.

2. The method according to claim 1, characterized in that: The method for step 1) is as follows: Extracellular vesicles and their loaded target proteins are captured using microplates coated with specific antibodies containing markers on the surface of extracellular vesicles.

3. The method according to claim 2, characterized in that: The extracellular vesicle surface marker is CD63.

4. The method according to claim 2, characterized in that: The microporous plate includes a U-shaped bottom microporous plate or a V-shaped bottom microporous plate.

5. The method according to claim 1, characterized in that: In step 3), the gold nanoparticle probe includes a probe sequence and gold nanoparticles; The probe sequence is modified with biotin at the 5' end and with a thiol group at the 3' end. The probe sequence binds to the surface of gold nanoparticles; The probe sequence is modified with a fluorescent group.

6. The method according to claim 5, characterized in that: The nucleotide sequence of the probe is 30–70 bp in length.

7. The method according to claim 5, characterized in that: The probe sequence was restored using TCEP.

8. The method according to claim 5, characterized in that: The probe sequence was labeled onto gold nanoparticles using a freeze-thaw method; The freezing temperature of the freeze-thaw method is -200 to 0°C; The freezing time for the freeze-thaw method is 1 to 200 minutes.

9. The method according to claim 5, characterized in that: The fluorescent group is located at position 25, starting from the 5' end of the biotinylated end.

10. A kit for detecting extracellular vesicle proteins, characterized in that: The kit includes microplates modified with antibodies against extracellular vesicle surface markers, biotinylated specific binding substances, gold nanoparticle probes, and avidin-modified magnetic nanoparticles. Preferably, the extracellular vesicle surface marker is CD63; Preferably, the biotinylated specific binding substance specifically binds to the target protein; Preferably, the specific binding substance includes an antibody or a nucleic acid aptamer; Preferably, the gold nanoparticle probe comprises a probe sequence and gold nanoparticles; Preferably, the 5' end of the probe sequence is modified with biotin and the 3' end is modified with a thiol group; Preferably, the probe sequence is bound to the surface of gold nanoparticles; Preferably, the probe sequence is modified with a fluorescent group.

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