An exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification and its application

Abstract

The present invention discloses an exosome fluorescence detection kit and application based on in-situ initiated ARGET ATRP signal amplification. The kit includes: carboxyl magnetic beads, BSA, EDC, NHS, BMP, EGFR antibody, Apt, FA, AA, CuBr2, ME6TREN, DMSO, and PBS buffer. The kit significantly improves the detection sensitivity based on the fluorescence performance of FA and the in-situ initiated ARGET ATRP signal amplification strategy. Through calculation, it is found that there is a good linear relationship between the logarithm of exosome concentration in the range of 5×10 4 ~5×10 9 exosomes / mL and the fluorescence signal intensity. The linear equation is: F(au) = 35956lgC exosomes -118474, and the correlation coefficient (R 2 ) is 0.997. The detection limit (LOD) of this method for detecting exosomes is 1.161×10 4 exosomes / mL (S / N = 3). Compared with the linear range and detection limit for detecting exosomes in other literatures, the fluorescence signal detection kit designed by the present invention has a wider detection range and a lower detection limit.

Description

Technical Field

[0001] The present invention relates to an exosome fluorescence detection kit and application based on in-situ initiated ARGET ATRP signal amplification, belonging to the field of bioanalysis technology. Background Art

[0002] Exosomes are microvesicles released by various cells, carrying proteins, nucleic acids, lipids, etc. closely related to their sources, and play important roles in cell communication, tumor development, immune response, etc. It has been found that epidermal growth factor receptor (EGFR), CD91, CD317, etc. are highly expressed in exosomes derived from non-small cell lung cancer. Due to the presence of these exosome proteins, exosomes can be used as biomarkers for the early diagnosis of non-small cell lung cancer. In addition, exosomes have advantages in terms of abundance, stability, and accessibility compared with other biomarkers. Therefore, exosomes are used for early lung cancer screening and real-time monitoring of the dynamic progress and metastasis of lung cancer, which is of great significance in improving the lung cancer screening rate and achieving early diagnosis and treatment of lung cancer.

[0003] Common methods for qualitative analysis of exosomes include: electron microscopy analysis, enzyme-linked immunosorbent assay, and nanoparticle tracking analysis. Electron microscopy analysis can provide information about the size and morphology of exosomes, but due to the loss of exosomes during dehydration and embedding, this method cannot effectively provide quantitative information, and the equipment is expensive and requires professional technical personnel to operate, limiting its application. In recent years, methods such as enzyme-linked immunosorbent assay, nanoparticle tracking analysis, Western blotting, colorimetry, fluorescence method, surface plasmon resonance method, etc. have been developed to quantitatively analyze exosomes. Enzyme-linked immunosorbent assay (ELISA), although it can detect exosomes by recognizing exosome characteristic proteins, requires labeling and has low sensitivity. Nanoparticle tracking analysis (NTA) can visualize individual exosomes, but the main drawback is that lipoprotein particles and protein aggregates may interfere with the determination of exosomes in the light scattering mode, and the measured exosome concentration range is limited (only 10 6 to 10 9 exosomes per milliliter). Biosensing methods have been applied to the quantitative detection of exosomes due to their advantages such as simple operation, rapid response, high sensitivity, and strong specificity. Exosomes can be specifically recognized by antibodies with strong affinity, and the fluorescence biosensor formed by combining with fluorescence labeling can directly detect exosomes. However, the binding ratio of traditional sensor signal molecules to targets is not high, and the use of signal amplification strategies can effectively improve the sensitivity.

[0004] As a signal amplification strategy, polymerization can not only improve the sensitivity of biosensing methods in biosensors, but also make the reaction conditions of the sensors milder, the operation more convenient, and the cost lower. Atom transfer radical polymerization (ATRP) technology, which has attracted much attention in recent years, has been widely used due to its advantages of simplicity, low cost, and controllability. Among them, activators regenerated by electron transfer atom transfer radical polymerization (ARGET ATRP) has oxygen tolerance due to the presence of strong reducing reagents, and greatly reduces the dosage of transition metal catalysts in the transition state. It has the advantages of simple operation, green economy, etc., and is favored by many researchers. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an exosome fluorescence detection kit and application based on in-situ initiated ARGET ATRP signal amplification.

[0006] In order to achieve the above purpose, one of the technical solutions of the present invention is:

[0007] An exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification, comprising the following raw materials: carboxyl magnetic beads, BSA, EDC, NHS, BMP, EGFR antibody, Apt, FA, AA, CuBr2, ME6TREN, DMSO, PBS buffer.

[0008] Further, some raw materials need to be prepared into solutions when used. Among them, the concentration of carboxyl magnetic beads is 10 mg / mL, the concentration of Apt solution is 1 μM, the concentration of EGFR antibody solution is 1 μg / mL, the concentration of EDC solution is 0.1 M, the concentration of NHS solution is 0.025 M, the concentration of EDC in the EDC / NHS mixed solution is 0.2 M, the concentration of NHS is 0.05 M, the concentration of BSA solution is 0.01 g / mL, the concentration of BMP solution is 30 mM, the concentration of AA solution is 2 mM, the concentration of FA solution is 15 mM, the concentrations of CuBr2 and ME6TREN in the CuBr2 / ME6TREN solution are both 10 mM, and the PBS buffer is 0.1 M, pH 7.4.

[0009] Further, the sequence of Apt is shown in SEQ ID NO.1.

[0010] One of the technical solutions of the present invention is: A method for using an exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification, comprising the following steps:

[0011] (1) Pretreatment of magnetic beads: Take carboxyl magnetic beads in a centrifuge tube, wash and magnetically separate.

[0012] (2) Apt-modified magnetic beads: Add PBS buffer solution, Apt solution, EDC solution, and NHS solution to the magnetic beads pretreated in step (1), and react;

[0013] (3) BSA-modified magnetic beads: Wash and magnetically separate the magnetic beads modified in step (2), add BSA solution and PBS buffer solution, and react;

[0014] (4) Exosome-modified magnetic beads: Wash and magnetically separate the magnetic beads modified in step (3), add the solution to be detected and PBS buffer solution, and react;

[0015] (5) anti-EGFR-BMP-modified magnetic beads: Wash and magnetically separate the magnetic beads modified in step (4), add anti-EGFR-BMP solution and PBS buffer solution, and react;

[0016] (6) FA-modified magnetic beads: Wash and magnetically separate the magnetic beads modified in step (5), add CuBr2 / Me6TREN solution, FA solution, AA solution, and PBS buffer solution, and react;

[0017] (7) Fluorescence detection:

[0018] ① After completing the above reactions, wash and magnetically separate the magnetic beads modified in step (6);

[0019] ② Add PBS buffer solution to the magnetic beads that have been magnetically separated after washing in step ① as the sample, use PBS buffer solution as the blank control, perform fluorescence detection, and calculate the exosome concentration based on the fluorescence signal intensity.

[0020] Further, the reaction conditions in step (2) are: react overnight at 37 °C; the reaction temperature in steps (3)-(6) is 36-38 °C, and the time is 1-1.5 h.

[0021] Further, the anti-EGFR-BMP connection step is: Mix the EGFR antibody solution and the EDC / NHS mixed solution in equal volumes. After activation, add the BMP solution in an equal volume to the EGFR antibody solution, react, and connect through an amide bond.

[0022] Further, the activation temperature is 36-38 °C, and the time is 4 h; the reaction temperature is 36-38 °C, and the time is 1 h.

[0023] Further, the fluorescence detection conditions are: excitation wavelength 489 nm, slit width 2 nm.

[0024] One of the technical solutions of the present invention is: an application of the said kit in detecting exosomes.

[0025] The construction process of the exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification is as follows Figure 1 shown in Figure A. First, the carboxyl groups on the MBs were activated with a mixed solution of EDC and NHS. Then, Apt modified with an amino group at the end was immobilized on the MBs through an amide reaction. At the same time, the initiator BMP of ATRP was linked to the EGFR antibody through an amide reaction. The excess active sites on the surface of the MBs were blocked with BSA. After adding exosomes, the Apt linked to the MBs specifically captured the exosomes. Next, the EGFR antibody was linked to the exosomes through specific antigen-antibody binding to form a sandwich structure. Finally, BMP linked to the EGFR antibody served as an initiator, ascorbic acid (AA) served as a reducing agent, FA served as a fluorescent monomer, and CuBr2 / Me6TREN served as a catalyst to initiate the ARGET ATRP reaction, forming a long-chain fluorescent polymer.

[0026] The reaction mechanism of ATRP is as follows Figure 1 shown in Figure B. First, AA reduced Br-Cu II / Me6TREN + to Cu I / Me6TREN + . Cu I / Me6TREN + captured the Br of the initiator (Pn-Br). Br- underwent an addition reaction with the monomer (FA). At the same time, the excess reducing agent (AA) caused Br-Cu II / Me6TREN + to be continuously reduced to Cu I / Me6TREN + , thus initiating a new round of polymerization.

[0027] Advantages of the present invention:

[0028] 1. The kit significantly improves the detection sensitivity based on the fluorescence performance of FA and the in-situ initiated ARGET ATRP signal amplification strategy.

[0029] 2. The kit has the advantages of high efficiency, convenient operation, environmental friendliness, and good detection performance in normal human serum, and has reliable practical application value.

[0030] 3. Under the optimal experimental conditions, the analytical performance of the kit within a certain range of exosome concentrations was studied. Magnetic beads were modified with different concentrations of exosomes and the fluorescence signal intensity was detected. The strongest fluorescence signal was at 514 nm. It was found through calculation that the logarithm of the exosome concentration was in the range of 5×10 4 ~5×10 9There is a good linear relationship between the range of exosomes / mL and the fluorescence signal intensity, and the linear equation is: F (au) = 35956lgC exosomes - 118474, and the correlation coefficient (R 2 ) is 0.997. The limit of detection (LOD) of this method for detecting exosomes is 1.161 × 10 4 exosomes / mL (S / N = 3). Compared with the linear range and limit of detection for detecting exosomes in other literatures, the fluorescence signal detection kit designed by the present invention has a wider detection range and a lower limit of detection. The above experimental results show that the exosome fluorescence detection kit based on the in-situ initiated ARGET ATRP signal amplification strategy has a wide effective detection range, high sensitivity and good analytical performance. Brief Description of the Drawings

[0031] Figure 1 It is the construction process (A) of the detection kit of the present invention and the reaction principle (B) of ARGET ATRP. Pn-Br = alkyl bromide, K a = activation rate constant, K da = inactivation rate constant, K p = propagation rate constant.

[0032] Figure 2 It is the feasibility analysis diagram of fluorescence detection.

[0033] Figure 3 It is the SEM images of magnetic beads without exosome modification (A) and magnetic beads with exosome modification (B). The images of magnetic beads without exosome modification (C) and magnetic beads with exosome modification (D) under fluorescence confocal microscope.

[0034] Figure 4 It is the optimization of the reaction time between exosomes and Apt (A), the optimization of the reaction time between EGFR antibody and exosomes (B), the optimization of ATRP reaction time (C), and the optimization of FA monomer concentration (D).

[0035] Figure 5 It is the fluorescence intensity of magnetic beads after adding exosomes with different concentrations (A) and the linear relationship between fluorescence intensity and the logarithm of exosome concentration (B).

[0036] Figure 6 It is the fluorescence response of the kit to exosomes from different cell sources (A) and the anti-interference (5% normal human serum) situation of the kit (B). Detailed Embodiments

[0037] The following further elaborates on the specific embodiments of the present invention in conjunction with the examples.

[0038] The carboxyl magnetic beads are purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.

[0039] The CD63 protein aptamer (Apt) used was synthesized by Shanghai Sangon Biotech Co., Ltd. and purified by HPLC-CE. The aptamer sequence is as follows:

[0040] 5’-CACCCCACCTCGCTCCCGTGACACTAATGCTA-C6-NH2-3’ (SEQ ID NO.1).

[0041] The EGFR antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd., Cat No. 66455-1-lg.

[0042] Example 1: An exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification

[0043] This kit includes the following raw materials: carboxyl magnetic beads, bovine serum albumin (BSA), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), 2-bromo-2-methylpropionic acid (BMP), EGFR antibody, CD63 protein aptamer (Apt), fluorescein O-acrylate (FA), ascorbic acid (AA), CuBr2, ligand tris-(N,N-dimethylaminoethyl)amine (ME6TREN), dimethyl sulfoxide (DMSO), PBS buffer.

[0044] Some raw materials need to be prepared into solutions when used. Among them, the concentration of carboxyl magnetic beads is 10 mg / mL, the concentration of Apt solution is 1 μM, the concentration of EGFR antibody solution is 1 μg / mL, the concentration of EDC solution is 0.1 M (the solvent is PBS buffer), the concentration of NHS solution is 0.025 M (the solvent is PBS buffer), the concentration of EDC in the EDC / NHS mixed solution is 0.2 M, the concentration of NHS is 0.05 M (the solvent is PBS buffer), the concentration of BSA solution is 0.01 g / mL, the concentration of BMP solution is 30 mM, the concentration of AA solution is 2 mM, the concentration of FA solution is 15 mM, the concentrations of CuBr2 and ME6TREN in the CuBr2 / ME6TREN solution are both 10 mM (the solvent is DMSO), and the PBS buffer is 0.1 M, pH 7.4.

[0045] Example 2: Method for using the kit

[0046] (1) Pretreatment of magnetic beads: Take 20 μL of carboxyl magnetic beads in a centrifuge tube, add 200 μL of PBS buffer and wash 2 times, and magnetically separate to remove the supernatant;

[0047] (2) Apt-modified magnetic beads: Add 150 μL of PBS buffer, 10 μL of aptamer (Apt) solution, 20 μL of EDC solution, and 20 μL of NHS solution to the pretreated magnetic beads in step (1), and react overnight in a constant temperature oscillator at 37 °C;

[0048] (3) Bovine serum albumin (BSA)-modified magnetic beads: Wash the magnetic beads modified in step (2) 2 times with 200 μL of PBS buffer and magnetically separate to remove the supernatant. Add 20 μL of BSA solution and 180 μL of PBS buffer, and react in a constant temperature oscillator at 37 °C for 1 h;

[0049] (4) Exosome-modified magnetic beads: Wash the magnetic beads modified in step (3) 2 times with 200 μL of PBS buffer and magnetically separate to remove the supernatant. Add 10 μL of the solution to be detected (containing 5×10 8 exosomes / mL) and 90 μL of PBS buffer, and react in a constant temperature oscillator at 37 °C for 80 min;

[0050] (5) Magnetic beads modified with anti-EGFR-BMP conjugated with BMP: Wash the magnetic beads modified in step (4) 2 times with 200 μL of PBS buffer and magnetically separate to remove the supernatant. Add 20 μL of the anti-EGFR-BMP solution conjugated with BMP in advance and 180 μL of PBS buffer, and react in a constant temperature oscillator at 37 °C for 80 min; Among them,

[0051] The conjugation step of anti-EGFR-BMP is: Mix the EGFR antibody solution and the EDC / NHS mixed solution in equal volumes. After activating at 37 °C for 4 h, add the BMP solution in an equal volume to the EGFR antibody solution and react at 37 °C for 1 h, and connect through an amide bond.

[0052] (6) Fluorescein O-acrylate (FA)-modified magnetic beads (ATRP reaction): Wash the magnetic beads modified in step (5) 2 times with 200 μL of PBS buffer and magnetically separate to remove the supernatant. Add 20 μL of CuBr2 / Me6TREN solution, 20 μL of FA solution, 20 μL of AA solution, and 140 μL of PBS buffer, and react in a constant temperature oscillator at 37 °C for 80 min;

[0053] (7) Fluorescence detection:

[0054] ① After completing the above reaction, wash the magnetic beads modified in step (6) with 200 μL of DMSO and magnetically separate, repeat 2 times, and then wash with 200 μL of PBS buffer and magnetically separate, repeat 2 times;

[0055] ② Add 600 μL of PBS buffer to the magnetic beads separated by magnetic separation after washing in step ① as the sample, and use 600 μL of PBS buffer as the blank control. Under the conditions of an excitation wavelength of 489 nm and a slit width of 2 nm, detect the fluorescence signal intensity with a fluorescence spectrophotometer, and calculate the exosome concentration based on the fluorescence signal intensity.

[0056] Example 3: Feasibility analysis

[0057] To verify the feasibility of this fluorescence detection kit, the present invention compared the fluorescence intensities of magnetic beads modified with different components. As Figure 2 shown, in the absence of Apt (curve b), exosomes (curve c), anti-EGFR-BMP (curve d), and FA (curve e) modification of the magnetic beads, the fluorescence signal was very weak, and the maximum value was only 1.11×10 4 a.u. However, when all modification steps were completed (curve a), the fluorescence intensity at 514 nm reached 1.455×10 5 a.u. These results indicate that each modification step is essential in the experiment, clearly demonstrating the feasibility of this method for detecting exosomes.

[0058] Example 4: Characterization analysis

[0059] To verify the successful construction of the kit, the magnetic beads of the blank group without exosomes and the experimental group that had undergone the complete modification steps were observed under a scanning electron microscope (SEM) and a fluorescence confocal microscope. The SEM results are as Figure 3 shown in A and Figure 3 B. Compared with the magnetic beads lacking exosome modification, the surface modifiers of the magnetic beads that had undergone the complete modification steps were significantly more, relatively rough, and some magnetic beads showed visible flocculation. This is because the blank group lacking exosomes could not connect the EFGR antibody carrying the initiator, resulting in the inability to proceed with subsequent reactions. The results of the fluorescence confocal microscope test are as Figure 3 shown in C and Figure 3 D. The magnetic bead group lacking exosome modification did not show fluorescent spots under the fluorescence confocal microscope. This is because in the absence of exosomes, the EFGR antibody conjugated with the initiator (BMP) could not attach to the magnetic beads, resulting in the inability of the fluorescent monomer FA to polymerize on the surface of the magnetic beads in the absence of the initiator. In contrast, the experimental group that had undergone the complete modification steps showed a large number of fluorescent spots under the fluorescence confocal microscope. The above characterization results indicate that the construction of the kit was successful.

[0060] Example 5: Condition optimization

[0061] Several important conditions involved in the kit detection were optimized to achieve the best detection performance of the exosome fluorescence detection kit. The effects of the reaction time of exosomes with Apt and anti-EGFR antibodies, the reaction time of ATRP, and the concentration of FA monomers on the fluorescence intensity were studied.

[0062] A sufficient reaction time allows Apt to capture more exosomes, thus providing more binding sites for anti-EGFR antibodies. Similarly, the amount of antibody captured by exosomes is related to the reaction time of exosomes with anti-EGFR antibodies. Therefore, it is very important to study the reaction times of exosomes with Apt and anti-EGFR antibodies.

[0063] 1. Optimization of the reaction time between exosomes and Apt

[0064] As Figure 4 shown in A, the fluorescence intensity increased within 20 - 80 min and reached the maximum value at 80 min. After that, the fluorescence intensity tended to be stable. This indicates that as the reaction time increases, the upper limit of exosomes that can be captured by Apt attached to the magnetic beads is reached. Therefore, the optimal reaction time between exosomes and Apt is 80 min.

[0065] 2. Optimization of the reaction time between anti-EGFR antibodies and exosomes

[0066] As Figure 4 shown in B, the fluorescence intensity increased with the increase of the reaction time of anti-EGFR antibodies within 20 min - 80 min and stabilized after 80 min. This is because the anti-EGFR antibodies that exosomes can bind to have basically reached the upper limit. Therefore, the optimal reaction time between anti-EGFR antibodies and exosomes is 80 min.

[0067] The degree of polymerization of the ATRP reaction is directly related to the size of the fluorescence signal, and the concentration of the fluorescent monomer FA and the ATRP reaction time have crucial effects on the degree of the polymerization reaction. Therefore, this invention studied the effects of the ATRP reaction time and the concentration of the fluorescent monomer FA on the fluorescence signal intensity.

[0068] 3. Optimization of the ATRP reaction time

[0069] As Figure 4 shown in C, the fluorescence intensity continuously increased from 20 - 80 min and almost stopped increasing after 80 min. Therefore, the optimal reaction time of ATRP is 80 min.

[0070] 4. Optimization of the FA monomer concentration

[0071] As Figure 4As shown in D, the fluorescence signal increases with the increase of FA concentration, reaches the maximum value at 15 mM, and then stabilizes. This is because the number of initiators is limited, and it is difficult for the polymer chains to continue growing after reaching a certain length. Therefore, the optimal concentration of FA monomer is 15 mM.

[0072] Example 6: Performance Analysis

[0073] Under the optimal experimental conditions of Example 5, exosomes with different concentrations were detected to analyze the performance of the kit. As Figure 5 shown in A, in the range of 5×10 4 ~5×10 9 exosomes / mL, the fluorescence intensity increases with the increase of exosome concentration. This is because more exosomes successfully attach to the magnetic beads. The exosomes capture more EGFR antibodies carrying BMP, and the increase in initiators and binding sites leads to an increase in the number of fluorescent monomers polymerized to the magnetic beads through subsequent reactions. As Figure 5 shown in B, the fluorescence intensity and the logarithm of exosome concentration show a good linear relationship: F(au) = 35956lgC exosomes - 118474 (R 2 = 0.997), and the detection limit is 1.161×10 4 exosomes / mL (S / N = 3). Where F(au) represents the fluorescence signal intensity, and C exosomes represents the concentration of exosomes (exosomes / mL) (the error bars represent the standard deviation of three measurements). The experimental results show that the kit proposed by the present invention has a low detection limit and a wide linear range, and good analysis performance.

[0074] Example 7: Selectivity and Anti-Interference Ability Analysis

[0075] To verify the specificity of this method for detecting exosomes from A549 cells, exosomes from other cell sources (H460 and EC-1) were added under the same conditions and the fluorescence intensity was measured. As Figure 6 shown in A, the fluorescence intensity of the exosome groups from other cell sources (H460, EC-1) is significantly weaker than that of the exosome group from A549 cells. Their fluorescence intensities are 34.01% and 25.01% of the exosome group from A549 cells respectively, and the fluorescence intensity of the blank group (only pure PBS buffer was added in the exosome modification step) is the weakest, with no significant difference from the PBS buffer. This is because exosomes from other cell sources are not specifically recognized by Apt and EGFR antibodies, resulting in a significant reduction in the number of initiators and fluorescent monomers subsequently linked to the magnetic beads. These results indicate that the kit has good selectivity in detecting exosomes from A549 cells.

[0076] The composition of substances in human serum is complex. By comparing the fluorescence intensities of exosomes at different concentrations in PBS buffer containing 5% (v / v) normal human serum and pure PBS buffer, the anti-interference ability of the kit was verified. As Figure 6 shown in 6 B, the present invention detected the signal intensities of exosomes at three concentrations of 5×10 7 exosomes / mL, 5×10 8 exosomes / mL, and 5×10

[0077] Example 8: Reproducibility, stability, and spiking recovery

[0078] The performance of the kit in practical applications was verified by reproducibility, stability, and spiking recovery experiments. The reproducibility of the exosome detection kit was studied using intra-group and inter-group experiments (n = 5), and the results showed that the RSDs of intra-group and inter-group experiments were 3.7% and 4.6%, respectively. The fully modified magnetic beads were refrigerated at 4°C for 7 days and 14 days, and the fluorescence intensities were still 95.54% and 91.20% of the newly constructed fully modified magnetic beads. To verify the applicability of the developed sensing system for the monitoring of exosomes in real samples, three concentrations of exosomes (5×10 6 exosomes / mL, 5×10 7 exosomes / mL, 5×10 8 exosomes / mL) were selected for the spiking recovery experiment of normal human serum. As shown in Table 1, the recoveries of the spiked exosome samples were 90.60% - 108%, and the average recovery was 97.85%; the RSD range was from 2.11% - 7.03%, and the average RSD was 4.82%. The above results indicate that this method has the characteristics of good reproducibility, high stability, and good accuracy, and can be successfully applied to the detection of A549 exosomes in human serum.

[0079] Table 1. Spiking recovery experiment

[0080]

Claims

1. An exosome fluorescence detection kit based on in-situ initiated ARGET ATRP signal amplification, characterized in that, It includes the following raw materials: carboxyl magnetic beads, BSA, EDC, NHS, 2-bromo-2-methylpropionic acid (BMP), anti-EGFR antibody, Apt, fluorescein O-acrylate (FA), ascorbic acid (AA), CuBr₂ and ME6TREN; Some raw materials need to be prepared into solutions before use. Among them, the concentration of carboxyl magnetic beads is 10 mg / mL, the concentration of Apt solution is 1 μM, the concentration of anti-EGFR antibody solution is 1 μg / mL, the concentration of EDC solution is 0.1 M, the concentration of NHS solution is 0.025 M, the concentration of EDC in the EDC / NHS mixed solution is 0.2 M, the concentration of NHS is 0.05 M, the concentration of BSA solution is 0.01 g / mL, the concentration of BMP solution is 30 mM, the concentration of AA solution is 2 mM, the concentration of FA solution is 15 mM, and the concentrations of CuBr₂ and ME6TREN in the CuBr₂ / ME6TREN solution are both 10 mM; The sequence of Apt is shown as SEQ ID NO.

1.

2. The kit according to claim 1, characterized in that, It also includes DMSO and PBS buffer.

3. Use of a kit as claimed in claim 1 in the preparation of an exosome detection product.

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