Exosome surface protein SERS (Surface Enhanced Raman Scattering) signal amplification method based on in-situ chemical growth

Abstract

The invention discloses an exosome surface protein SERS (Surface Enhanced Raman Scattering) signal amplification method based on in-situ chemical growth, and relates to the field of Raman spectrum detection. The invention aims to solve the problems of weak signal and incapability of realizing single exosome detection due to limited number, discrete distribution and large spacing of adsorbed noble metal nanoparticles in the existing SERS (Surface Enhanced Raman Scattering) immunoassay method. The method comprises the following steps: specifically adsorbing a gold nano-seed with a small size (10nm) of a modified antibody to the surface of an exosome through antigen-antibody interaction, growing the gold seed into gold nano-particles with a particle size (greater than 70nm) suitable for SERS (Surface Enhanced Raman Scattering) detection by using in-situ chemical growth, enabling the gold nano-particles to be in close contact with one another, and labeling Au NPs by using DTNB (Dimethylthio-N-Phosphate). And finally, obtaining a measurement result in a mode of SRES surface scanning measurement of an average value. By introducing an in-situ growth method, the detection sensitivity is effectively improved.

Description

Technical Field

[0001] The present invention relates to the field of Raman spectroscopy detection, and particularly to a method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth. Background Art

[0002] With the gradually increasing incidence and severity of prostate cancer, it has become an important problem threatening male health. Prostate cancer is characterized by hidden early symptoms and a high risk of metastasis. Approximately 70% of patients are in the early local stage without obvious symptoms. However, as the disease progresses to the invasive stage, the mortality rate of patients increases significantly. Currently, the existing invasive detection methods for patients mainly rely on PSA detection and pathological detection. Among them, PSA detection has a high false positive rate, and in pathological detection, needle biopsy is usually affected by tumor heterogeneity, causing great harm and easily leading to the progression of the disease. Therefore, studying new liquid biopsy methods, actively monitoring the health of patients, and obtaining the patient's condition information as early as possible and carrying out corresponding treatments have become the key to improving the survival rate and quality of patients.

[0003] Small extracellular vesicles (sEVs), also known as exosomes, are phospholipid bilayer structures secreted by cells with a size within 200 nm. The exosome surface is rich in various proteins, which have been studied to be related to the occurrence and progression of prostate cancer. For example, epithelial cell adhesion molecule (EpCAM) is highly expressed on the surface of prostate cancer exosomes and is related to cell adhesion; prostate-specific membrane antigen (PSMA) is a highly specific transmembrane glycoprotein of prostate cancer and is highly related to the malignancy of the tumor; integrin α3 (ITGα3) is involved in the adhesion, migration and invasion of tumor cells and is positively correlated with the Gleason score of prostate cancer; CD63 is one of the exosome transmembrane proteins and is highly expressed in the serum of cancer patients, serving as a general detection marker for exosomes. Precise detection of the above exosome proteins is expected to achieve precise stratified management of prostate cancer patients. Currently, existing detection methods, such as flow cytometry and mass spectrometry methods, rely on large equipment and professional personnel, with high costs and unable to achieve portable detection. Other methods, such as enzyme-linked immunosorbent assay (ELISA), immunofluorescence and colorimetric detection methods, have low sensitivity. Due to the small number of cancer-derived exosomes carrying specific target proteins (<10 6 per milliliter), it is difficult to meet the detection requirements.

[0004] Surface-enhanced Raman spectroscopy immunoassay (SERS immunoassay), which features high sensitivity, fast detection speed, and multiplex detection capabilities, has attracted wide attention in recent years. In existing SERS immunoassay methods, antibody-modified noble metal nanoparticles are generally targeted and adsorbed on the surface of exosomes through the "antigen-antibody" interaction to form a "sandwich" structure, and the quantitative detection of exosome surface proteins is achieved by detecting the SRES signals of specific probe molecules. Due to the use of specific antibodies, it has higher specificity compared to non-labeled SERS analysis methods and has become the preferred solution for precision medicine detection. However, the SERS immunoassay work reported so far mainly has the problems of weak signals and the inability to achieve single-exosome detection. The main reasons are as follows: on the one hand, since exosomes are structures with a diameter of less than 200 nm and the number of exosomes carrying target proteins is limited, only a "limited number" and "discrete distribution" of noble metal nanoparticles can be adsorbed on the exosome surface; on the other hand, the spacing between the noble metal nanoparticles adsorbed on the exosome surface is relatively large, and the electromagnetic field coupling is weak, unable to form effective SERS hot spots. In recent years, scientists have tried various means to solve the above problems, such as synthesizing gold nanoparticles with higher SERS activity, improving the antigen-antibody binding efficiency through an electrophoresis mixing device, and increasing the number of probe molecules. These methods can improve the detection sensitivity to a certain extent, but they still do not solve the problems of "limited number", "discrete distribution", and "large spacing" of the adsorbed noble metal nanoparticles, and the signal enhancement brought is limited.

[0005] Therefore, through a new SRES signal amplification strategy, improving the adsorption quantity of noble metal nanoparticles and reducing the particle spacing in the SRES detection of exosome proteins to produce a better electromagnetic field enhancement effect and enhancing the detection sensitivity to the single-exosome level has important research significance. Summary of the Invention

[0006] The purpose of the present invention is to solve the problems of weak signals and the inability to achieve single-exosome detection existing in the existing SERS immunoassay methods due to the "limited quantity", "discrete distribution", and "large spacing" of the adsorbed noble metal nanoparticles, and to provide a method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth.

[0007] The present invention provides a method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth. In the present invention, gold nanoseeds (Au seeds@Antibody) with a smaller size (10 nm) modified with antibodies are specifically adsorbed onto the surface of exosomes through antigen-antibody interaction. In-situ chemical growth is used to grow Au seeds@Antibody into Au NPs with a particle size suitable for SERS detection (>70 nm) and in close contact with each other. 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) is used to label the Au NPs. Finally, the measurement results are obtained by measuring the average value through SRES surface scanning. By introducing the in-situ growth method, the number of Au NPs adsorbed on the exosome surface is significantly increased, the gap between Au NPs is reduced, the detection sensitivity (detection limit: 57.4 particles / mL) is effectively improved, and the detection range (10 2 -10 8 particles / mL) is broadened.

[0008] A method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth is specifically completed according to the following steps:

[0009] I. Preparation of Anti-CD63 modified PDMS concave trap array:

[0010] ①. Use a plasma cleaner to hydroxylate the PDMS concave trap structure array, then place it in an anhydrous ethanol solution of APTES and heat for a period of time. After taking it out, wash it with anhydrous ethanol and dry it with nitrogen to obtain an amino-modified concave trap array;

[0011] ②. Immerse the amino-modified concave trap array in a PBS solution of glutaraldehyde and soak for a period of time. After taking it out, rinse it several times with the PBS solution to obtain an aldehyde-modified concave trap array;

[0012] ③. Immerse the aldehyde-modified concave trap array in a PBS solution of Anti-CD63 and soak for a period of time. After taking it out, immerse it in a PBS solution of bovine serum albumin with a certain concentration and block for a period of time. Finally, rinse it several times with PBS to obtain an Anti-CD63 modified PDMS concave trap array;

[0013] II. Preparation of antibody-modified Au seeds:

[0014] ①. Take a certain volume of gold seeds, and successively add a 5,5'-dithiobis(2-nitrobenzoic acid) solution and a dithiobis(propionic acid succinimidyl ester) solution to the gold seeds, and let it stand for a certain time to obtain an Au@DTNB solution;

[0015] ②. Take the Au@DTNB solution, centrifuge it at a certain speed, resuspend it in a certain volume of PBS solution, add an antibody, let it stand for a certain time, add an aqueous solution of bovine serum albumin at a certain concentration, block for a certain time, centrifuge and disperse it in a PBS solution of bovine serum albumin at a certain concentration to obtain Au seeds@Antibody;

[0016] III. SERS detection of exosome surface proteins based on in-situ chemical growth:

[0017] ①. Drop the exosome solution onto the Anti-CD63 modified PDMS concave well array, let it stand for a period of time, and wash it several times with a PBS solution of bovine serum albumin at a certain concentration to obtain an Anti-CD63 modified PDMS concave well array containing exosomes;

[0018] ②. Take a certain volume of Au seeds@Antibody and add it to the Anti-CD63 modified PDMS concave well array containing exosomes, let it stand for a certain time, and then wash it several times with a PBS solution of bovine serum albumin at a certain concentration to obtain Nanowell-Au seeds;

[0019] ③. Take a certain volume of the in-situ growth solution and add it to the Nanowell-Au seeds obtained in step III②, let it stand for a certain time, then wash it several times with a PBS solution of bovine serum albumin at a certain concentration, add a certain volume of 5,5'-dithiobis(2-nitrobenzoic acid) solution, let it stand for a certain time, and wash it several times with PBS to obtain Nanowell-Au NPs, thus completing a SERS signal amplification method for exosome surface proteins based on in-situ chemical growth.

[0020] Advantages of the present invention:

[0021] I. In the present invention, by using the Anti-CD63 modified PDMS concave well array (Nanowell-AntiCD63) as a substrate to adsorb exosomes, taking advantage of the high specific surface area morphology structure of the concave well array and the high specificity of the immunoaffinity separation method, it can not only achieve the rapid and efficient adsorption of exosomes, but also improve the SERS enhancement effect of the subsequent in-situ growth of Au NPs;

[0022] II. In the present invention, gold nanoseeds (Au seeds@Antibody) with a smaller size (10nm) and surface-modified antibodies are used as probes to target specific proteins on the exosome surface. Compared with the larger-sized (>70nm) Au NPs probes reported in the literature, it can effectively increase the number of particle adsorption;

[0023] III. In the present invention, in-situ chemical growth is used to grow Au seeds@Antibody to Au NPs with a particle size suitable for SERS detection (>70 nm) and in close contact with each other; the electromagnetic field enhancement formed by the coupling between Au NPs effectively improves the detection sensitivity (the lower detection limit is 57.4 particles / mL) and broadens the detection range (10 2 -10 8 particles / mL). Compared with the SERS detection sensitivity (the lower detection limit is 3.68×10 4 particles / mL) and the detection range (10 5 -10 8 ) of the direct adsorption method, it is improved by about three orders of magnitude;

[0024] IV. The method provided by the present invention can flexibly adjust the antibody type to achieve multi-target detection of exosome surface proteins, and can realize the invasive analysis of prostate cancer cells and patients according to the protein expression level; the present invention provides a new idea and method for the detection of surface proteins of small particle substances such as EVs and viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of a SERS signal amplification method for exosome surface proteins based on in-situ chemical growth and its application in the detection of prostate cancer exosomes;

[0026] Figure 2 (a) is a schematic diagram of the preparation process of Nanowell-AntiCD63, and (b)-(i) are scanning electron microscope images of different concentrations of LNCaP cell EVs (10 2 -10 8 particles / mL) adsorbed on the Nanowell-AntiCD63 prepared in Experiment 1;

[0027] Figure 3 (a) is a schematic diagram of the preparation process of Au seeds@Anti-EpCAM, and (b)-(g) are optical images, transmission electron microscope images, particle size analysis diagrams, ultraviolet-visible absorption spectra, hydrated kinetic particle size analysis diagrams, and Zeta potential diagrams of the Au seeds, Au@DTNB, and Au seeds@Anti-EpCAM prepared in Experiment 1;

[0028] Figure 4 (a)-(d) are respectively optical images, ultraviolet-visible absorption spectra, hydrated kinetic particle size analysis diagrams, and Zeta potential diagrams of the Au seeds@Anti-PSMA, Au seeds@Anti-CD63, and Au seeds@Anti-ITGα3 prepared in Experiment 3;

[0029] Figure 5 TEM images of EVs from different prostate cancer cells prepared for Experiment 2;

[0030] Figure 6 Nanoparticle tracking analysis images of EVs from different prostate cancer cells prepared for Experiment 2;

[0031] Figure 7 TEM images of Au@anti-EpCAM with different particle sizes adsorbing LNCaP cell EVs prepared for Experiment 1 and Experiment 4, where (a)-(b) are 10 nm and were prepared for Experiment 1; (c)-(d) are 45 nm and were prepared for Experiment 4;

[0032] Figure 8 Comparison diagram of the adsorption effects of the direct adsorption and in-situ growth methods: (a) is a schematic diagram of the operation steps of the in-situ growth method; (b) is an SEM image of the direct adsorption method in Experiment 4; (c) is an SEM image of the in-situ growth method in Experiment 1; (d) is an SEM image of the in-situ growth method in Experiment 5; (e) is an SEM image of the in-situ growth method in Experiment 6; (e)-(h) are FDTD electromagnetic field intensity simulation images constructed from the SEM images in (b)-(e), where the exosomes are produced by LNCaP cells and the concentration is 10 8 per milliliter, and the antibody is Anti-EpCAM;

[0033] Figure 9 (a) is a comparison diagram of the average SERS spectra obtained by detecting LNCaP cell exosomes with a concentration of 10 8 per milliliter in Experiment 4, Experiment 5, Experiment 1, and Experiment 6; (b) is a bar chart of the intensity comparison at a Raman shift of 1330 cm -1 where the antibody is Anti-EpCAM;

[0034] Figure 10 (a) is a comparison diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 per milliliter in Experiment 7; (b) is a bar chart of the intensity comparison at a Raman shift of 1330 cm -1 in (a); (c) is a comparison diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 per milliliter in Experiment 8; (d) is a bar chart of the intensity comparison at a Raman shift of 1330 cm -1 in (c); (e) is a comparison diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8SERS average spectral comparison diagrams obtained from LNCaP cell EVs at a concentration of 10 cells / mL; (f) is a bar chart of the intensity comparison at a Raman shift of 1330 cm in Figure (e), where the antibody is Anti-EpCAM; -1 at this position, where the antibody is Anti-EpCAM;

[0035] Figure 11 (a) is the SERS average spectral comparison diagram obtained by different test methods for detecting LNCaP cell EVs at a concentration of 10 cells / mL in Experiment Ten; (b) is the bar chart of the intensity comparison at a Raman shift of 1330 cm in Figure (a); (c) is the bar chart of the intensity comparison at a Raman shift of 1330 cm obtained by repeating the method described in Experiment One 10 times for detecting LNCaP cell EVs at a concentration of 10 cells / mL in Experiment Eleven; (d) is the SRES average spectral comparison diagram obtained by the method of Experiment One for detecting EVs at different concentrations (10 - 10 cells / mL); (e) is the SRES average spectral comparison diagram obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); 8 SERS average spectral comparison diagrams obtained from LNCaP cell EVs at a concentration of 10 cells / mL; (b) is the bar chart of the intensity comparison at a Raman shift of 1330 cm in Figure (a); (c) is the bar chart of the intensity comparison at a Raman shift of 1330 cm obtained by repeating the method described in Experiment One 10 times for detecting LNCaP cell EVs at a concentration of 10 cells / mL in Experiment Eleven; (d) is the bar chart of the intensity comparison at a Raman shift of 1330 cm obtained by the method of Experiment One for detecting EVs at different concentrations (10 - 10 cells / mL); (e) is the bar chart of the intensity comparison at a Raman shift of 1330 cm obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); -1 at this position; (c) is the bar chart of the intensity comparison at a Raman shift of 1330 cm obtained by repeating the method described in Experiment One 10 times for detecting LNCaP cell EVs at a concentration of 10 cells / mL in Experiment Eleven; (d) is the SRES average spectral comparison diagram obtained by the method of Experiment One for detecting EVs at different concentrations (10 - 10 cells / mL); (e) is the SRES average spectral comparison diagram obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); 8 at this position obtained by detecting LNCaP cell EVs at a concentration of 10 cells / mL; (d) is the SRES average spectral comparison diagram obtained by the method of Experiment One for detecting EVs at different concentrations (10 - 10 cells / mL); (e) is the SRES average spectral comparison diagram obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); -1 at this position; (d) is the SRES average spectral comparison diagram obtained by the method of Experiment One for detecting EVs at different concentrations (10 - 10 cells / mL); (e) is the SRES average spectral comparison diagram obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); 2 -10 8 at this position; (e) is the SRES average spectral comparison diagram obtained by the method of Experiment Four for detecting EVs at different concentrations (10 - 10 cells / mL); (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); 2 -10 8 at this position; (f) is the linear fitting picture of the intensity at a Raman shift of 1330 cm in Figures (d)-(e); -1 at this position;

[0036] Figure 12 (a) is a schematic diagram of the method for detecting four proteins (EpCAM, PSMA, CD63, and ITGα3) in EVs of different prostate cancer cells; (b) is the SERS average spectral comparison diagram obtained by detecting the EpCAM content in EVs of five different cells by the method described in Experiment One; (c) is the SERS average spectral comparison diagram obtained by detecting the PSMA content in EVs of five different cells by the method described in Experiment One; (d) is the SERS average spectral comparison diagram obtained by detecting the CD63 content in EVs of five different cells by the method described in Experiment One; (e) is the SERS average spectral comparison diagram obtained by detecting the ITGα3 content in EVs of five different cells by the method described in Experiment One; (f)-(i) are bar charts of the intensity comparison at a Raman shift of 1330 cm in Figures (b)-(e); -1 at this position. Specific Embodiments

[0037] Specific Embodiment One: A method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth is specifically completed according to the following steps:

[0038] I. Preparation of Anti-CD63 modified PDMS concave well array:

[0039] ①. Use a plasma cleaner to hydroxylate the PDMS concave well structure array, then place it in an anhydrous ethanol solution of APTES and heat for a period of time. After taking it out, wash it with anhydrous ethanol and dry it with nitrogen to obtain an amino-modified concave well array;

[0040] ②. Immerse the amino-modified concave well array in a PBS solution of glutaraldehyde for a period of time. After taking it out, rinse it several times with the PBS solution to obtain an aldehyde-modified concave well array;

[0041] ③. Immerse the aldehyde-modified concave well array in a PBS solution of Anti-CD63 for a period of time. After taking it out, immerse it in a PBS solution containing a certain concentration of bovine serum albumin to block for a period of time, and finally rinse it several times with PBS to obtain an Anti-CD63 modified PDMS concave well array;

[0042] II. Preparation of antibody-modified Au seeds:

[0043] ①. Take a certain volume of gold seeds, and successively add 5,5′-dithiobis(2-nitrobenzoic acid) solution and dithiobis(succinimidyl propionate) solution to the gold seeds, and let it stand for a certain time to obtain Au@DTNB solution;

[0044] ②. Take the Au@DTNB solution, centrifuge it at a certain speed, resuspend it in a certain volume of PBS solution, add the antibody, let it stand for a certain time, add an aqueous solution of a certain concentration of bovine serum albumin, block for a certain time, and after centrifugation, disperse it in a PBS solution containing a certain concentration of bovine serum albumin to obtain Au seeds@Antibody;

[0045] III. SERS detection of exosome surface proteins based on in-situ chemical growth:

[0046] ①. Drop the exosome solution onto the Anti-CD63 modified PDMS concave well array, let it stand for a period of time, and wash it several times with a PBS solution containing a certain concentration of bovine serum albumin to obtain an Anti-CD63 modified PDMS concave well array containing exosomes;

[0047] ②. Take a certain volume of Au seeds@Antibody and add it to the Anti-CD63 modified PDMS concave well array containing exosomes, let it stand for a certain time, and then wash it several times with a PBS solution containing a certain concentration of bovine serum albumin to obtain Nanowell-Au seeds;

[0048] ③. Take a certain volume of the in situ growth solution and add it to the Nanowell-Au seeds obtained in step 3②, let it stand for a certain time, then wash it several times with a PBS solution of a certain concentration of bovine serum albumin, then add a certain volume of 5,5'-dithiobis(2-nitrobenzoic acid) solution, let it stand for a certain time, and wash it several times with PBS to obtain Nanowell-Au NPs, thus completing a method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth.

[0049] Specific embodiment 2: This embodiment differs from the specific embodiment 1 in that the preparation method of the PDMS concave well structure array described in step 1① is completed according to the following steps:

[0050] Ⅰ. Clean a silicon wafer with a size of 0.5 cm×0.5 cm in O2 plasma for 60s to 120s at a power of 100W to 200W to obtain a hydrophilized silicon wafer;

[0051] Ⅱ. Add polystyrene microspheres to a mixture of water and ethanol, mix them evenly by ultrasonication, and obtain a polystyrene microsphere dispersion with a mass fraction of 1.25% to 2.5%; transfer the polystyrene microsphere dispersion with a mass fraction of 1.25% to 2.5% to a hydrophilized silicon wafer by using a syringe, construct a single-layer polystyrene microsphere array on the surface of the silicon wafer, and then heat and cure at 90° C. to 110° C. for 60 min to 90 min;

[0052] The diameter of the polystyrene microspheres in step II is 650nm-700nm; the volume ratio of water to ethanol in the water-ethanol mixture is 1:1;

[0053] III. Cast 5 g to 10 g of PDMS glue on the monolayer polystyrene microsphere array, heat at 60°C to 90°C for 3 h to 5 h, peel off the film, wash with dichloromethane 2 to 4 times, and blow dry with nitrogen to obtain a PDMS concave well structure array;

[0054] The mass ratio of the PDMS main agent to the curing agent in the PDMS glue described in step III is (8.5-9.5):1, and the model of the PDMS glue is Dow Corning SYLGARD184;

[0055] The size of the PDMS concave well structure array described in step III is 0.5 cm×0.5 cm, and the average opening diameter of the concave well is 485 nm. The other steps are the same as those in the first embodiment.

[0056] Specific Embodiment 3: The differences between this embodiment and one of Specific Embodiments 1 or 2 are as follows: The power of the plasma cleaner described in step ① of step one is 100W - 200W; the time for hydroxylation treatment in step ① of step one is 60s - 120s; the mass fraction of the APTES absolute ethanol solution in step ① of step one is 2% - 5%; the heating temperature in step ① of step one is 60°C - 65°C, and the heating time is 3h - 6h. Other steps are the same as those in Specific Embodiment 1 or 2.

[0057] Specific Embodiment 4: The differences between this embodiment and one of Specific Embodiments 1 to 3 are as follows: The mass fraction of glutaraldehyde in the glutaraldehyde PBS solution in step ② of step one is 2.5% - 5.0%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the soaking time in step ② of step one is 50 min - 60 min; the concentration of the PBS solution in step ② of step one is 10 mmol / L, and the pH value is 7.4; the number of times of rinsing with the PBS solution in step ② of step one is 2 - 3 times. Other steps are the same as those in Specific Embodiments 1 to 3.

[0058] Specific Embodiment 5: The differences between this embodiment and one of Specific Embodiments 1 to 4 are as follows: The concentration of the Anti - CD63 antibody in the Anti - CD63 PBS solution in step ③ of step one is 2 μg / mL - 10 μg / mL, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the concentration of bovine serum albumin (BSA) in the BSA PBS solution in step ③ of step one is 5% - 10%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the number of times of rinsing with PBS in step ③ of step one is 2 - 3 times; the soaking time in step ③ of step one is 50 min - 60 min; the blocking time is 50 min - 60 min. Other steps are the same as those in Specific Embodiments 1 to 4.

[0059] Specific Embodiment Six: The difference between this embodiment and any one of Specific Embodiments One to Five is as follows: The preparation method of the gold seeds described in Step 2① is specifically as follows: Add 5 mL of a sodium citrate solution with a mass fraction of 0.9% - 1% to 100 mL of a HAuCl4 solution with a mass fraction of 0.01% heated to boiling, stir for 15 min - 20 min to obtain gold seeds; the concentration of the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution described in Step 2① is 9.5 mmol / L - 10.5 mmol / L; the concentration of the dithiobis(succinimidyl propionate) (DSP) solution described in Step 2① is 9.5 mmol / L - 10.5 mmol / L; the volume ratio of the gold seeds, 5,5'-dithiobis(2-nitrobenzoic acid) solution, and dithiobis(succinimidyl propionate) solution described in Step 2① is (0.9 mL - 1.1 mL):1 μL:0.2 μL; the standing time described in Step 2① is 8 h - 12 h. Other steps are the same as those in Specific Embodiments One to Five.

[0060] Specific Embodiment Seven: The difference between this embodiment and any one of Specific Embodiments One to Six is as follows: The antibody described in Step 2② is Anti-EpCAM, Anti-PSMA, Anti-CD63, or Anti-ITGα3; the rotation speed of centrifugation described in Step 2② is 15000 rpm - 18000 rpm, and the centrifugation time is 10 min; in Step 2②, take 0.9 - 1.1 mL of the Au@DTNB solution, centrifuge at a certain rotation speed, resuspend it in 190 μL - 210 μL of a PBS solution with a concentration of 0.1 mmol / L and a pH value of 7.4, add 0.25 μg - 1.25 μg of the antibody, let it stand for 30 min - 60 min, add 10 μL - 20 μL of an aqueous solution of bovine serum albumin (BSA) with a mass fraction of 1% - 2%, block for 30 min - 60 min, and after centrifugation, disperse the solid matter in 100 μL - 200 μL of a PBS solution of bovine serum albumin (BSA) with a mass fraction of 1% - 2% to obtain Au seeds@Antibody. Other steps are the same as those in Specific Embodiments One to Six.

[0061] Specific Embodiment Eight: The difference between this embodiment and any one of Specific Embodiments One to Seven is as follows: The solvent of the exosome solution described in Step 3① is PBS, serum, or cell culture medium; the concentration of the exosome solution described in Step 3① is 10 2 particles / mL - 10 8 particles / mL; the ratio of the volume of the exosome solution to the surface area of the Anti-CD63 modified PDMS well array is (30 μL - 50 μL):0.25 cm 2; The standing time described in Step 3① is 30 min to 60 min; the mass fraction of the PBS solution of bovine serum albumin (BSA) described in Step 3① is 1% to 2%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the number of times of washing with the PBS solution containing a certain concentration of bovine serum albumin in Step 3① is 2 to 3 times; the volume ratio of the Au seeds@Antibody described in Step 3② to the surface area of the Anti-CD63 modified PDMS well array containing exosomes is (30 μL to 50 μL):0.25 cm 2 ; The standing time described in Step 3② is 15 min to 90 min; the mass fraction of the PBS solution of bovine serum albumin (BSA) described in Step 3② is 1% to 2%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the number of times of washing with the PBS solution containing a certain concentration of bovine serum albumin in Step 3② is 2 to 3 times. Other steps are the same as those in the first to seventh specific embodiments.

[0062] Specific Embodiment Nine: The difference between this embodiment and one of the first to eighth specific embodiments is that the in-situ growth solution described in Step 3③ is composed of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution and hydrogen peroxide. Among them, the concentration of the sodium oleate solution is 9.8 mmol / L to 10.2 mmol / L, the concentration of the chloroauric acid solution is 9.8 mmol / L to 10.2 mmol / L, the concentration of the polyvinylpyrrolidone solution is 45 mg / mL to 55 mg / mL, and the mass fraction of hydrogen peroxide is 28% to 30%; the volume ratio of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution and hydrogen peroxide in the in-situ growth solution described in Step 3③ is (200 μL to 500 μL):(30 μL to 70 μL):(150 μL to 350 μL):(10 μL to 50 μL):(30 μL to 50 μL); the mass fraction of the PBS solution of bovine serum albumin (BSA) described in Step 3③ is 1% to 2%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4; the concentration of the 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution described in Step 3③ is 0.1 mmol / L; the volume ratio of the Nanowell-Auseeds, in-situ growth solution, and 5,5'-dithiobis(2-nitrobenzoic acid) solution described in Step 3③ is (30 μL to 50 μL):(420 μL to 1020 μL):(30 μL to 50 μL); the standing time described in Step 3③ is 15 min to 20 min; the number of washing times is 2 to 3 times. Other steps are the same as those in the first to eighth specific embodiments.

[0063] Embodiment 10: The difference between this embodiment and any one of Embodiments 1 to 9 is that: the Nanowell-Au NPs obtained in Step ③ are subjected to SERS spectral surface scanning by a confocal Raman spectrometer to obtain the spectral average value; if the SERS characteristic peaks collected are 1330 cm -1 , 1550 cm -1 , 1050 cm -1 , the SERS signal of the exosome surface protein is obtained; the objective lens magnification of the confocal Raman spectrometer is 100X, the laser wavelength is 785 nm, the power is 20 mW, and the integration time is 0.5 s; the surface scan area is 10×10 μm 2 , the step size is 1 μm, a total of 100 points, each exosome sample is measured three times repeatedly, and three different surface scan areas are randomly tested each time; the preprocessing process of the SERS spectrum is divided into three steps: smoothing, baseline correction and normalization. Among them, smoothing uses Savitzky-Golay filtering, the window number is 20, and baseline correction uses the asymmetric reweighted penalty least squares smoothing method. Other steps are the same as those in Embodiments 1 to 9.

[0064] The following examples are used to verify the beneficial effects of the present invention:

[0065] Experiment 1: A method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth is specifically completed according to the following steps:

[0066] I. Preparation of Anti-CD63 modified PDMS concave well array (Nanowell-AntiCD63):

[0067] ①. Use a plasma cleaner to hydroxylate the PDMS concave well structure array, then place it in an anhydrous ethanol solution of APTES and heat for a period of time, take it out, wash it with anhydrous ethanol, and dry it with nitrogen to obtain an amino-modified concave well array;

[0068] The power of the plasma cleaner described in Step ① is 150 W;

[0069] The time of the hydroxylation treatment described in Step ① is 120 s;

[0070] The mass fraction of the anhydrous ethanol solution of APTES described in Step ① is 5%;

[0071] The heating temperature described in Step ① is 65 °C, and the heating time is 6 h;

[0072] The preparation method of the PDMS concave well structure array described in Step ① is completed according to the following steps:

[0073] Ⅰ. A silicon wafer with a size of 0.5 cm × 0.5 cm was cleaned in O2 plasma for 75 seconds at a power of 150 W to obtain a hydrophilized silicon wafer;

[0074] Ⅱ. Add polystyrene microspheres to a mixture of water and ethanol, mix them evenly by ultrasonication, and obtain a polystyrene microsphere dispersion with a mass fraction of 1.25%; transfer the polystyrene microsphere dispersion with a mass fraction of 1.25% to a hydrophilized silicon wafer using a syringe, construct a single-layer polystyrene microsphere array on the surface of the silicon wafer, and then heat and cure it at 100°C for 70 minutes;

[0075] The diameter of the polystyrene microspheres described in step II is 670 nm;

[0076] III. Pour 7.5 g of PDMS glue on the monolayer polystyrene microsphere array, heat at 65°C for 4.5 h, peel off the film, wash twice with dichloromethane and blow dry with nitrogen to obtain a PDMS concave well structure array;

[0077] The mass ratio of the PDMS main agent to the curing agent in the PDMS glue described in step III is 9:1, and the model of the PDMS glue is Dow Corning SYLGARD184;

[0078] The size of the PDMS well structure array described in step III is 0.5 cm×0.5 cm, and the average opening diameter of the well is 485 nm;

[0079] ②, immersing the amino-modified concave well array in a PBS solution of glutaraldehyde for a period of time, taking it out and rinsing it several times with a PBS solution to obtain an aldehyde-modified concave well array;

[0080] The mass fraction of glutaraldehyde in the PBS solution of glutaraldehyde described in step 1② is 2.5%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0081] The soaking time described in step 1② is 60min;

[0082] The concentration of the PBS solution described in step 1② is 10mmol / L, and the pH value is 7.4;

[0083] The number of times of washing with PBS solution in step 1② is 3 times;

[0084] ③, immerse the aldehyde-modified concave well array in the PBS solution of Anti-CD63 for 60 minutes, take it out and immerse it in a PBS solution of a certain concentration of bovine serum albumin for blocking for 60 minutes, and finally rinse it with PBS several times to obtain the Anti-CD63 modified PDMS concave well array (Nanowell-AntiCD63);

[0085] In the Anti-CD63 PBS solution described in Step 1③, the concentration of the Anti-CD63 antibody is 5 μg / mL, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0086] In the PBS solution of bovine serum albumin (BSA) described in Step 1③, the concentration of bovine serum albumin (BSA) is 10%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0087] The number of times of rinsing with PBS in Step 1③ is 3 times;

[0088] II. Preparation of antibody-modified Au seeds (Au seeds@Antibody):

[0089] ①. Take a certain volume of Au seeds, and sequentially add 5,5'-dithiobis(2-nitrobenzoic acid) solution (DTNB) and dithiobis(propionate succinimidyl ester) (DSP) solution to the Au seeds, and let it stand for a certain time to obtain an Au@DTNB solution;

[0090] The specific preparation method of the Au seeds described in Step 2① is as follows: Add 5 mL of a 1% sodium citrate solution by mass to 100 mL of a 0.01% HAuCl4 solution heated to boiling, and stir for 15 min to obtain Au seeds;

[0091] The concentration of the DTNB solution described in Step 2① is 10 mmol / L;

[0092] The concentration of the DSP solution described in Step 2① is 10 mmol / L;

[0093] The volume ratio of the Au seeds, DTNB solution, and DSP solution described in Step 2① is 1 mL:1 μL:0.2 μL;

[0094] The standing time described in Step 2① is 8 h;

[0095] ②. Take 1.0 mL of the Au@DTNB solution, centrifuge at a certain speed, resuspend it in 200 μL of a PBS solution with a concentration of 0.1 mmol / L and a pH value of 7.4, add 1 μg of antibody, let it stand for 60 min, add 20 μL of an aqueous solution of 1% bovine serum albumin (BSA) by mass to block for 60 min, and after centrifugation, disperse the solid matter in 200 μL of a 1% bovine serum albumin (BSA) PBS solution to obtain Au seeds@Anti-EpCAM;

[0096] The antibody described in Step 2② is Anti-EpCAM;

[0097] The rotation speed of the centrifugation described in Step 2② is 18000 rpm, and the centrifugation time is 10 min;

[0098] III. SERS detection of exosome surface proteins based on in-situ chemical growth:

[0099] ① Drop the exosome solution onto the Anti-CD63 modified PDMS nanowell array (Nanowell-AntiCD63), let it stand for a period of time, and wash it several times with a PBS solution containing a certain concentration of bovine serum albumin to obtain an Anti-CD63 modified PDMS nanowell array containing exosomes;

[0100] The solvent of the exosome solution described in Step 3① is a PBS solution with a concentration of 10 mmol / L and a pH value of 7.4;

[0101] The concentration of the exosome solution described in Step 3① is 1.0×10 8 per milliliter;

[0102] The ratio of the volume of the exosome solution to the surface area of the Anti-CD63 modified PDMS nanowell array described in Step 3① is 30 μL:0.25 cm 2 ;

[0103] The standing time described in Step 3① is 30 min;

[0104] The mass fraction of the PBS solution of bovine serum albumin (BSA) described in Step 3① is 1%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0105] The number of times of washing with a PBS solution containing a certain concentration of bovine serum albumin in Step 3① is 3 times;

[0106] ② Take a certain volume of Au seeds@Anti-EpCAM and add it to the Anti-CD63 modified PDMS nanowell array containing exosomes, let it stand for a certain time, and then wash it several times with a PBS solution containing a certain concentration of bovine serum albumin to obtain Nanowell-Au seeds;

[0107] The ratio of the volume of Au seeds@Antibody to the surface area of the Anti-CD63 modified PDMS nanowell array containing exosomes described in Step 3② is 30 μL:0.25 cm 2 ;

[0108] The standing time described in Step 3② is 30 min;

[0109] In step ③②, the mass fraction of the PBS solution of bovine serum albumin (BSA) is 1%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0110] In step ③②, the number of times of washing with the PBS solution of bovine serum albumin at a certain concentration is 2 times;

[0111] ③ Add a certain volume of the in-situ growth solution to the Nanowell-Au seeds obtained in step ③②, let it stand for 20 min, then wash it several times with the PBS solution of bovine serum albumin at a certain concentration, and then add a certain volume of 5,5'-dithiobis(2-nitrobenzoic acid) solution, let it stand for 20 min, and wash it several times with PBS to obtain Nanowell-Au NPs, thus completing a method for amplifying the SERS signal of exosome surface proteins based on in-situ chemical growth.

[0112] The in-situ growth solution described in step ③③ is composed of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution and hydrogen peroxide. Among them, the concentration of the sodium oleate solution is 10 mmol / L, the concentration of the chloroauric acid solution is 10 mmol / L, the concentration of the polyvinylpyrrolidone solution is 50 mg / mL, and the mass fraction of hydrogen peroxide is 30%;

[0113] In the in-situ growth solution described in step ③③, the volume ratio of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution and hydrogen peroxide is 500 μL:70 μL:350 μL:50 μL:50 μL;

[0114] In step ③③, the mass fraction of the PBS solution of bovine serum albumin (BSA) is 1%, the concentration of the PBS solution is 10 mmol / L, and the pH value is 7.4;

[0115] In step ③③, the concentration of the 5,5'-dithiobis(2-nitrobenzoic acid) solution is 0.1 mmol / L;

[0116] In step ③③, the volume ratio of Nanowell-Au seeds, in-situ growth solution, and 5,5'-dithiobis(2-nitrobenzoic acid) solution is 30 μL:1020 μL:50 μL;

[0117] Perform SERS spectral surface scanning on the Nanowell-Au NPs obtained in step ③③ by a confocal Raman spectrometer to obtain the spectral average value; if the SERS characteristic peaks collected are 1330 cm -1 、1550 cm -1 、1050 cm -1, the SERS signal of the exosome surface protein is obtained;

[0118] The objective lens magnification of the confocal Raman spectrometer described is 100X, the laser wavelength is 785 nm, the power is 20 mW, and the integration time is 0.5 s; the area of surface scanning is 10×10 μm 2 , the step size is 1 μm, a total of 100 points, each exosome sample is measured three times, and three different surface scanning areas are randomly tested each time;

[0119] The preprocessing process of the SERS spectrum in step three ③ is divided into three steps: smoothing, baseline correction and normalization. Among them, smoothing uses Savitzky-Golay filtering, the window number is 20, and baseline correction uses the asymmetric reweighted penalized least squares smoothing method.

[0120] Experiment 2: Extraction of prostate cancer cell EVs. First, five prostate cells with different degrees of invasiveness are cultured. Among them, RWPE-1 cells are normal prostate epithelial cells; RWPE2 are regional prostate cancer cells; LNCaP cells are androgen-dependent prostate lymphatic metastasis cell lines with medium invasiveness; PC3 is an androgen-independent, bone metastasis cell line with strong invasiveness; DU145 is a prostate brain metastasis cell line with strong invasiveness. When the cells grow to 80% density, discard the existing culture medium, rinse twice with PBS solution with a pH value of 7.4, replace with serum-free medium, continue to culture for 24-48 hours, collect the supernatant, and apply a rotation speed of 300 g in turn. After 10 minutes, precipitate and remove cells and other substances; rotate at 2000 g for 10 minutes to precipitate and remove dead cells and other substances; rotate at 10000 g for 30 minutes to remove cell debris and microvesicles and other substances. Use an ultracentrifuge to apply a rotation speed of 120000 g to the supernatant for 70 minutes, and centrifuge twice in total. Finally, disperse the obtained EVs in PBS (pH = 7.4) to obtain an EVs sample (prostate cancer exosome solution) with a concentration of 1.0×10 8 per milliliter.

[0121] Figure 2 (a) is a schematic diagram of the preparation process of Nanowell-AntiCD63, and (b)-(i) are scanning electron microscope images of different concentrations of LNCaP cell EVs (10 2 -10 8 per milliliter) adsorbed on the Nanowell-AntiCD63 prepared in Experiment 1;

[0122] From Figure 2(b)-(i) It can be seen that LNCaP cell EVs were successfully adsorbed on the surface of Nanowell-AntiCD63 through immune affinity. As the concentration of LNCaP cell EVs decreased, the number of EVs on Nanowell-AntiCD63 continuously decreased. At a concentration of 10 2 per milliliter, discrete individual EVs could still be seen adsorbed in the wells.

[0123] Figure 3 (a) Schematic diagram of the preparation process of Au seeds@Anti-EpCAM. (b)-(g) Optical images, transmission electron microscope images, particle size analysis diagrams, ultraviolet-visible absorption spectra, hydrodynamic particle size analysis diagrams, and Zeta potential diagrams of Auseeds, Au@DTNB, and Au seeds@Anti-EpCAM prepared in Experiment 1;

[0124] From Figure 3 (c)-(d) It can be seen that the Au seeds had a small particle size and a uniform distribution. The average particle size measured by statistical analysis was 10 nm. From Figure 3 (e) The ultraviolet-visible absorption spectrum showed that the absorption peaks of the particles red-shifted after coating with DTNB and antibodies. The absorption peaks of the three kinds of particles were 519 nm, 523 nm, and 530 nm respectively. This was due to the change in the refractive index around the Au NPs caused by the coating material. The hydrodynamic particle sizes of the three kinds of particles were measured by dynamic light scattering and were 16 nm, 17 nm, and 27 nm respectively. The particles maintained good dispersibility after coating with antibodies and did not agglomerate. The Zeta potentials of the particles were -17.1 mV, -61.9 mV, and -35.4 mV respectively. The change in the Zeta potential of the particles indicated that the substances were successfully coated and maintained good water solubility.

[0125] Experiment 3: Preparation of gold nanoseeds modified with different antibodies (Au seeds@Anti-PSMA, Au seeds@Anti-CD63, Au seeds@Anti-ITGα3): The difference between this experiment and Experiment 1 was that the antibody types described in step 2② were changed to Anti-PSMA, Anti-CD63, and Anti-ITGα3 respectively. Three kinds of gold nanoseeds were obtained in step 2②: Au seeds@Anti-PSMA, Au seeds@Anti-CD63, and Au seeds@Anti-ITGα3. Other steps and parameters were the same as those in Experiment 1.

[0126] Figure 4(a)-(d) are the optical pictures, ultraviolet-visible absorption spectra, hydrodynamic particle size analysis diagrams, and Zeta potential diagrams of Au seeds@Anti-PSMA, Au seeds@Anti-CD63, and Au seeds@Anti-ITGα3 prepared in Experiment 3, respectively;

[0127] As Figure 4 can be seen, by changing the antibody type, three different antibody-modified probes were successfully prepared, all of which have good monodispersity and water solubility and no aggregation occurred.

[0128] Figure 5 are the TEM pictures of different prostate cancer cell EVs prepared in Experiment 2;

[0129] As Figure 5 shown, the sizes of all 4 EVs are below 200 nm, presenting a "saucer-shaped" structure. The phospholipid bilayer wrapping can be clearly seen, indicating that EVs were successfully obtained by the ultracentrifugation method.

[0130] Figure 6 are the nanoparticle tracking analysis images of different prostate cancer cell EVs prepared in Experiment 2;

[0131] The test results show that the average particle size of EVs is between 100 - 200 nm, which is consistent with the reported range of EVs diameters in the literature, indicating that the obtained EVs have a high purity.

[0132] Experiment 4: Detection of exosome surface proteins was achieved by the direct adsorption method, which was specifically completed according to the following steps:

[0133] I. Preparation of Anti-CD63 modified PDMS concave well array (Nanowell-AntiCD63):

[0134] ①. Use a plasma cleaner to hydroxylate the PDMS concave well structure array, then place it in an anhydrous ethanol solution of APTES for heating. After taking it out, wash it with anhydrous ethanol and dry it with nitrogen to obtain an amino-modified concave well array;

[0135] The size of the PDMS concave well structure described in step I ① is 0.5 cm × 0.5 cm, which is cast by the self-assembly structure of polystyrene microspheres with a diameter of 670 nm, and the average opening diameter of the concave well is 485 nm;

[0136] The plasma cleaning conditions described in step I ① are a power of 150 W and a time of 120 s;

[0137] The mass fraction of the anhydrous ethanol solution of APTES in step I ① is 5%, the heating temperature is 65 °C, and the heating time is 6 h;

[0138] ② Immerse the amino-modified well array in a PBS solution of glutaraldehyde and let it stand for a period of time. After taking it out, rinse it with PBS solution to obtain an aldehyde-modified well array;

[0139] In step ② of step one, the concentration of the PBS solution of glutaraldehyde is 2.5%;

[0140] In step ② of step one, the soaking time is 60 min and the number of washing times is 3 times;

[0141] ③ Immerse the aldehyde-modified well array in a PBS solution of a certain concentration of Anti-CD63 for a period of time. After taking it out, soak it in a PBS solution of a certain concentration of BSA for a period of time to block, and finally rinse it with PBS to obtain an Anti-CD63-modified PDMS well array (Nanowell-AntiCD63);

[0142] In step ③ of step one, the concentration of the Anti-CD63 solution is 5 μg / mL and the soaking time is 60 min;

[0143] In step ③ of step one, the concentration of the BSA solution is 10% and the number of washing times is 3 times;

[0144] II. Preparation of antibody-modified Au nanoparticles (Au NPs@Antibody):

[0145] ① Take a certain volume of nanoparticles, and successively add DTNB and DSP solutions, and let it stand for a certain time to obtain Au NPs@DTNB;

[0146] The preparation method of the gold nanoparticles in step ① of step two is specifically as follows: quickly add 1 mL of a 1% sodium citrate solution to 100 mL of a 0.01% HAuCl4 solution heated to boiling, and stir for 15 minutes to form;

[0147] In step ① of step two, the volume of the gold nanoparticles is 1 mL;

[0148] In step ① of step two, the concentrations of the DTNB and DSP solutions are both 10 mM, the volumes are 1 μL and 0.2 μL respectively, and the standing time is 8 h;

[0149] ② Take the Au NPs@DTNB solution, centrifuge it at a certain speed, resuspend it in a certain volume of PBS solution, add an antibody, let it stand for a certain time, add an aqueous solution of a certain concentration of BSA, block for a certain time, centrifuge and disperse it in a PBS solution of a certain concentration of BSA to finally obtain Au NPs@Antibody;

[0150] In step ② of step two, the centrifugation speed is 6000 rpm, the time is 10 min, and the volume of the PBS solution is 200 μL;

[0151] In step ② of step two, the added amount of the antibody is 1 μg, the standing time is 60 min, and the antibody type is Anti-EpCAM;

[0152] In step ② of step two, the concentration of the BSA aqueous solution is 1%, the volume is 20 μL, and the blocking time is 60 min;

[0153] In step ② of step two, the concentration of the BSA PBS solution is 1%, and the volume is 200 μL;

[0154] III. SERS detection of exosome surface proteins based on the direct adsorption method:

[0155] ① Drop the exosome solution onto Nanowell-AntiCD63, let it stand for a certain time, and wash it with a PBS solution containing a certain concentration of BSA;

[0156] In step ① of step three, the solvent of the exosome solution is the PBS solution, the volume is 30 μL, and the time is 30 min;

[0157] In step ① of step three, the concentration of the BSA PBS solution is 1%, and the number of washing times is 3 times;

[0158] ② Take a certain volume of Au NPs@Antibody and add it to the Nanowell-AntiCD63 processed in step ①, let it stand for a certain time, and then wash it with a PBS solution containing a certain concentration of BSA to obtain Nanowell-Au NPs;

[0159] In step ② of step three, the volume of the Au NPs@Antibody is 30 μL, and the time is 30 min;

[0160] In step ② of step three, the concentration of the BSA PBS solution is 1%, and the number of washing times is 2 times;

[0161] ③ Perform SERS spectral surface scanning on the nanowell obtained in ② with a confocal Raman spectrometer, and calculate the spectral average value;

[0162] If the collected SERS characteristic peaks are 1330 cm -1 、1550 cm -1 、1050 cm -1 , the SERS signal of the exosome surface protein is obtained;

[0163] In Step ③, the objective lens magnification of the confocal Raman spectrometer was 100X, the laser wavelength was 785 nm, the power was 20 mW, and the integration time was 0.5 s; the area of surface scanning was 10×10 μm 2 , the step size was 1 μm, with a total of 100 points. Each exosome sample was measured three times, and three different surface scanning areas were randomly tested each time;

[0164] The preprocessing process of the SERS spectrum in Step ③ was divided into three steps: smoothing, baseline correction, and normalization. Among them, Savitzky-Golay filtering was used for smoothing, with a window number of 20; and the asymmetric reweighted penalized least squares smoothing method was used for baseline correction.

[0165] Figure 7 TEM images of Au@anti-EpCAM with different particle sizes adsorbed on LNCaP cell EVs prepared for Experiment 1 and Experiment 4. Among them, (a)-(b) are 10 nm, prepared in Experiment 1; (c)-(d) are 45 nm, prepared in Experiment 4;

[0166] It can be seen from Figure 7 that when the particle size of Au@Anti-EpCAM was 45 nm, only a very limited number of Au NPs were adsorbed on the surface of EVs. This was because its particle size was comparable to that of EVs, and the steric hindrance between Au NPs led to limited adsorption quantity. When the particle size of Au@Anti-EpCAM was 10 nm, the Au NPs adsorbed on the surface of EVs increased significantly. The size of 10 nm Au seeds was significantly smaller than that of EVs. As shown in Figure 7 (d), the adsorption of multiple Au seeds can be achieved on the surface of a single EV, providing abundant nucleation sites for subsequent in-situ growth and contributing to the dense coating of Au NPs on the surface of a single EV.

[0167] Experiment 5: The difference between this experiment and Experiment 1 was that in the in-situ growth solution described in Step ③, the volume ratio of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution, and hydrogen peroxide was 500 μL: 30 μL: 150 μL: 50 μL: 30 μL. Other steps and parameters were the same as those in Experiment 1.

[0168] Experiment 6: The difference between this experiment and Experiment 1 was that in the in-situ growth solution described in Step ③, the volume ratio of water, sodium oleate solution, chloroauric acid solution, polyvinylpyrrolidone solution, and hydrogen peroxide was 500 μL: 110 μL: 550 μL: 50 μL: 70 μL. Other steps and parameters were the same as those in Experiment 1.

[0169] Figure 8Comparison chart of adsorption effects for the direct adsorption and in-situ growth methods: Among them, (a) is a schematic diagram of the operation steps of the in-situ growth method; (b) is an SEM image of the direct adsorption method in Experiment 4; (c) is an SEM image of the in-situ growth method in Experiment 1; (d) is an SEM image of the in-situ growth method in Experiment 5; (e) is an SEM image of the in-situ growth method in Experiment 6; (e)-(h) are FDTD electromagnetic field intensity simulation images constructed based on the SEM images in Figures (b)-(e), respectively, where the exosomes are produced by LNCaP cells and the concentration is 10 8 particles / mL, and the antibody is Anti-EpCAM;

[0170] From Figure 8 (b), it can be seen that when the direct adsorption method is used, only discrete and limited amounts of Au NPs are adsorbed on the surface of the concave trap, and the spacing between the Au NPs is large, which is not conducive to generating a strong electromagnetic field enhancement. As Figure 8 (c)-(e) show, when the in-situ growth method is used, the number of Au NPs adsorbed on the surface of the concave trap is significantly increased. By changing the volume of each component in the in-situ growth solution, the size of the Au NPs can be regulated. As the amount of chloroauric acid added increases, the average diameters of the Au NPs are 53 nm, 68 nm, and 88 nm, respectively. In addition, as the chloroauric acid increases, the spacing between the Au NPs continuously shortens, and when the volume is 350 μL, close contact of the Au NPs is achieved. The electromagnetic field distribution around the particles was simulated by the finite-difference time-domain approximation (FDTD), and it was found that the particle spacing plays a key role in the increase of the electromagnetic field. When the Au NPs are in close contact, the electromagnetic field enhancement multiple is significantly increased, which is mainly caused by the hot spots generated by the electromagnetic field coupling between the Au NPs. Therefore, through the in-situ growth method, we achieved close contact of the Au NPs on the surface of the exosomes, thereby greatly increasing the electromagnetic field enhancement multiple, which helps to improve the SERS detection effect.

[0171] Figure 9 (a) is a comparison chart of the average SERS spectra obtained by detecting LNCaP cell exosomes with a concentration of 10 8 particles / mL in Experiments 4, 5, 1, and 6; (b) is a column chart of the intensity comparison at a Raman shift of 1330 cm -1 , where the antibody is Anti-EpCAM;

[0172] Figure 9 It can be seen that the in-situ growth method described in Experiment 1 achieved the strongest SERS enhancement effect. Compared with the direct adsorption method, the SERS signal of the EpCAM protein in LNCaP cell EVs with a concentration of 10 8 particles / mL detected by the in-situ growth method in Experiment 1 was increased by 23.5 times.

[0173] Experiment VII: The difference between this experiment and Experiment I is that the amounts of antibody added in Step ② are 0.25 μg, 0.5 μg, 0.75 μg, and 1.25 μg respectively, to obtain Au seeds@Anti-EpCAM modified with different amounts of antibody. Other steps and parameters are the same as those in Experiment I.

[0174] Experiment VIII: The difference between this experiment and Experiment I is that the standing times in Step ③ are 10 min, 20 min, 40 min, 60 min, and 90 min respectively, to obtain the detection results under different adsorption times of Au seeds@Anti-EpCAM; other steps and parameters are the same as those in Experiment I.

[0175] Experiment IX: The difference between this experiment and Experiment I is that the concentrations of DTNB solution in Step ③ are 10 mmol / L, 1 mmol / L, and 0.01 mmol / L respectively, to obtain the detection results modified with different DTNB concentrations; other steps and parameters are the same as those in Experiment I.

[0176] Figure 10 (a) is the comparative diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 cells / mL in Experiment VII; (b) is the columnar diagram of the intensity comparison at a Raman shift of 1330 cm -1 in Figure (a); (c) is the comparative diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 cells / mL in Experiment VIII; (d) is the columnar diagram of the intensity comparison at a Raman shift of 1330 cm -1 in Figure (c); (e) is the comparative diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 cells / mL in Experiment IX; (f) is the columnar diagram of the intensity comparison at a Raman shift of 1330 cm -1 in Figure (e), where the antibody is Anti-EpCAM;

[0177] It can be seen from Figure 10 (a)-(b) that as the amount of antibody added in the probe increases, the SERS signal gradually increases. Considering the material cost, the optimal value is obtained when the amount of antibody added is 1 μg. It can be seen from Figure 10 (c)-(d) that the optimal value is obtained when the incubation time after adding the probe is 30 minutes. It can be seen from Figure 10 (e)-(f) that the detection effect is the best when the concentration of the labeling molecule DTNB is 0.1 mM.

[0178] Experiment X: The specific experimental method for the specific detection of this system is as follows:

[0179] Remove the step of adding Au NPs@Antibody in step 3② of Experiment 1 to obtain a control group (-Au seeds) without the addition of Au seeds; remove the step of adding antibodies in step 2② of Experiment 1 to obtain a control group without antibodies (-Anti-EpCAM); remove the step of adding exosomes in step 3① of Experiment 1 to obtain a control group without exosomes (-EVs); remove the step of adding the in-situ growth solution in step 3③ of Experiment 1 to obtain a control group without in-situ growth (-In situ growth); other steps and parameters are the same as those in Experiment 1.

[0180] Experiment XI: The experimental method for detecting the repeatability of this system is as follows:

[0181] Perform ten repeated experiments using the parameters described in Experiment 1, and calculate the standard deviation of the final average value results to obtain the repeatability measurement results.

[0182] Figure 11 (a) is a comparative diagram of the average SERS spectra obtained by detecting LNCaP cell EVs with a concentration of 10 8 cells / ml using different experimental methods in Experiment X; (b) is a column chart of the intensity comparison at a Raman shift of 1330 cm -1 in Figure (a); (c) is a column chart of the intensity comparison at a Raman shift of 1330 cm 8 obtained by detecting LNCaP cell EVs with a concentration of 10 -1 cells / ml by repeating the method described in Experiment 1 ten times in Experiment XI; (d) is a comparative diagram of the average SRES spectra obtained by detecting EVs with different concentrations (10 2 -10 8 cells / ml) using the method of Experiment 1; (e) is a comparative diagram of the average SRES spectra obtained by detecting EVs with different concentrations (10 2 -10 8 cells / ml) using the method of Experiment 4; (f) is a linear fitting picture of the intensity at a Raman shift of 1330 cm -1 in Figures (d)-(e);

[0183] It can be seen from Figure 11 (a)-(b) that the system has excellent specificity for the EpCAM protein in LNCaP cell EVs. As Figure 11 (c) shows, the RSD value of the average of ten repeated measurements is 4.19%, indicating that the surface system has good repeat measurement stability. By using the in-situ growth method described in Experiment 1 for different concentrations (10 2 -10 8The EVs (number / mL) were detected, and the logarithmic average value of the detected spectral intensity was linearly fitted with the logarithmic average value of the concentration. The fitting formula was logI 1330 = 0.3468logC Evs + 0.06959, and the goodness of fit R 2 was 0.997. The lower limit of detection calculated by 3σ / k was 57.464 particles / mL. The same sample was detected using the direct adsorption method described in Experiment 4, and the obtained linear fitting formula was logI 1330 = 0.3273logC EVs - 1.0049, and the goodness of fit R 2 was 0.996. The lower limit of detection calculated by 3σ / k was 3.68×10 4 particles / mL. By introducing the in-situ growth method, the number of Au NPs adsorbed on the surface of exosomes was significantly increased, the gap between Au NPs was reduced, the detection sensitivity was effectively improved, and the detection range was broadened.

[0184] Figure 12 (a) Schematic diagram of the method for detecting four proteins (EpCAM, PSMA, CD63, and ITGα3) in EVs of different prostate cancer cells; (b) Comparative diagram of SERS average spectra obtained by detecting the EpCAM content in EVs of five different cells using the method described in Experiment 1; (c) Comparative diagram of SERS average spectra obtained by detecting the PSMA content in EVs of five different cells using the method described in Experiment 1; (d) Comparative diagram of SERS average spectra obtained by detecting the CD63 content in EVs of five different cells using the method described in Experiment 1; (e) Comparative diagram of SERS average spectra obtained by detecting the ITGα3 content in EVs of five different cells using the method described in Experiment 1; (f)-(i) Column chart of intensity comparison at a Raman shift of 1330 cm -1 in the figures (b)-(e).

[0185] Figure 12Among the four proteins detected, EpCAM is highly expressed on the surface of prostate cancer EVs and is related to cell adhesion; PSMA is a transmembrane glycoprotein highly specific to prostate cancer and is highly correlated with tumor malignancy; ITGα3 is involved in the adhesion, migration, and invasion of tumor cells and is positively correlated with the Gleason score of prostate cancer; CD63 is one of the transmembrane proteins of EVs and is highly expressed in the serum of cancer patients, serving as a general detection marker for EVs. In the detection results of EpCAM and PSMA proteins, the intensity of LNCaP cells is higher, and the intensity of PC3 and DU145 cells decreases in turn; in the detection results of CD63 protein, the intensity of PC3 cells is higher; in the detection results of ITGα3, the intensity of DU145 cells is higher, and the intensity of PC3 and LNCaP cells decreases in turn; in addition, the expression of EpCAM and PSMA in RWPE2 cells is higher than that in RWPE1 cells. In summary, the detection of four EV surface proteins related to prostate cancer patients was achieved through in-situ chemical growth.

Claims

1. A method for amplifying SERS signals of exosome surface proteins based on in situ chemical growth, characterized in that The method is specifically completed according to the following steps:

1. Preparation of Anti-CD63 modified PDMS well array: ①, using a plasma cleaning machine to perform hydroxylation treatment on the PDMS well structure array, then placing it in an APTES anhydrous ethanol solution and heating it for a period of time, taking it out and cleaning it with anhydrous ethanol, and drying it with nitrogen to obtain an amino-modified well array; ②, immersing the amino-modified concave well array in a PBS solution of glutaraldehyde for a period of time, taking it out and rinsing it several times with a PBS solution to obtain an aldehyde-modified concave well array; ③, immerse the aldehyde-modified concave well array in the PBS solution of Anti-CD63 for a period of time, take it out and immerse it in a PBS solution of a certain concentration of bovine serum albumin for a period of time, and finally rinse it with PBS several times to obtain the Anti-CD63 modified PDMS concave well array; 2. Preparation of antibody-modified Au seeds: ① Take a certain volume of gold seed, add 5,5′-dithiobis(2-nitrobenzoic acid) solution and dithiobis(succinimidyl propionate) solution to the gold seed in sequence, and let it stand for a certain period of time to obtain Au@DTNB solution; ② Take the Au@DTNB solution, centrifuge it at a certain speed, resuspend it in a certain volume of PBS solution, add the antibody, let it stand for a certain time, add an aqueous solution of a certain concentration of bovine serum albumin, block it for a certain time, centrifuge it and disperse it in a PBS solution of a certain concentration of bovine serum albumin to obtain Au seeds@Antibody; 3. SERS detection of exosome surface proteins based on in situ chemical growth: ①, drip the exosome solution onto the Anti-CD63 modified PDMS well array, let it stand for a while, and wash it several times with a PBS solution of a certain concentration of bovine serum albumin to obtain the Anti-CD63 modified PDMS well array containing exosomes; ② Take a certain volume of Au seeds@Antibody and add it to the PDMS well array modified with Anti-CD63 containing exosomes, let it stand for a certain period of time, and then wash it several times with a PBS solution containing a certain concentration of bovine serum albumin to obtain Nanowell-Au seeds; ③. Take a certain volume of the in situ growth solution and add it to the Nanowell-Au seeds obtained in step 3②, let it stand for a certain time, then wash it several times with a PBS solution of a certain concentration of bovine serum albumin, then add a certain volume of 5,5'-dithiobis(2-nitrobenzoic acid) solution, let it stand for a certain time, and wash it several times with PBS to obtain Nanowell-Au NPs, thus completing a method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth.

2. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The preparation method of the PDMS concave well structure array described in step 1① is completed according to the following steps: Ⅰ. Clean a silicon wafer with a size of 0.5 cm×0.5 cm in O2 plasma for 60s to 120s at a power of 100W to 200W to obtain a hydrophilized silicon wafer; Ⅱ. Add polystyrene microspheres to a mixture of water and ethanol, mix them evenly by ultrasonication, and obtain a polystyrene microsphere dispersion with a mass fraction of 1.25% to 2.5%; transfer the polystyrene microsphere dispersion with a mass fraction of 1.25% to 2.5% to a hydrophilized silicon wafer by using a syringe, construct a single-layer polystyrene microsphere array on the surface of the silicon wafer, and then heat and cure at 90° C. to 110° C. for 60 min to 90 min; The diameter of the polystyrene microspheres in step II is 650nm-700nm; the volume ratio of water to ethanol in the water-ethanol mixture is 1:1; III. Cast 5 g to 10 g of PDMS glue on the monolayer polystyrene microsphere array, heat at 60°C to 90°C for 3 h to 5 h, peel off the film, wash with dichloromethane 2 to 4 times, and blow dry with nitrogen to obtain a PDMS concave well structure array; The mass ratio of the PDMS main agent to the curing agent in the PDMS glue described in step III is (8.5-9.5):1, and the model of the PDMS glue is Dow Corning SYLGARD184; The size of the PDMS concave well structure array described in step III is 0.5 cm×0.5 cm, and the average opening diameter of the concave well is 485 nm.

3. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The power of the plasma cleaning machine described in step 1① is 100W~200W; the time of the hydroxylation treatment described in step 1① is 60s~120s; the mass fraction of the anhydrous ethanol solution of APTES described in step 1① is 2%~5%; the heating temperature described in step 1① is 60℃~65℃, and the heating time is 3h~6h.

4. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The mass fraction of glutaraldehyde in the PBS solution of glutaraldehyde described in step 1② is 2.5% to 5.0%, the concentration of the PBS solution is 10mmol / L, and the pH value is 7.4; the immersion time described in step 1② is 50min to 60min; the concentration of the PBS solution described in step 1② is 10mmol / L, and the pH value is 7.4; the number of times the PBS solution is used for rinsing in step 1② is 2 to 3 times.

5. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The concentration of Anti-CD63 antibody in the Anti-CD63 PBS solution described in step 1③ is 2μg / mL~10μg / mL, the concentration of PBS solution is 10mmol / L, and the pH value is 7.4; the concentration of bovine serum albumin (BSA) in the bovine serum albumin PBS solution described in step 1③ is 5%~10%, the concentration of PBS solution is 10mmol / L, and the pH value is 7.4; the number of times PBS is used for washing in step 1③ is 2 times~3 times; the immersion time described in step 1③ is 50min~60min; the blocking time is 50min~60min.

6. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The preparation method of the gold seed described in step 2① is as follows: add 5mL of sodium citrate solution with a mass fraction of 0.9% to 1% to 100mL of HAuCl4 solution with a mass fraction of 0.01% heated to boiling, and stir for 15min to 20min to obtain the gold seed; the concentration of the 5'-dithiobis(2-nitrobenzoic acid) solution described in step 2① is 9.5mmol / L to 10.5mmol / L; the concentration of the dithiobis(succinimidyl propionate) solution described in step 2① is 9.5mmol / L to 10.5mmol / L; the volume ratio of the gold seed, 5,5'-dithiobis(2-nitrobenzoic acid) solution and dithiobis(succinimidyl propionate) solution described in step 2① is (0.9mL to 1.1mL):1μL:0.2μL; the standing time described in step 2① is 8h to 12h.

7. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The antibody described in step 2② is Anti-EpCAM, Anti-PSMA, Anti-CD63 or Anti-ITGα3; the centrifugal speed described in step 2② is 15000rpm~18000rpm, and the centrifugal time is 10min; in step 2②, 0.9~1.1mL Au@DTNB solution is taken, centrifuged at a certain speed, resuspended in 190μL~210μL PBS solution with a concentration of 0.1mmol / L and a pH value of 7.4, 0.25μg~1.25μg antibody is added, let stand for 30min~60min, 10μL~20μL of an aqueous solution of bovine serum albumin with a mass fraction of 1%~2% is added, and the solution is blocked for 30min~60min. After centrifugation, the solid matter is dispersed in 100μL~200μL PBS solution with a mass fraction of 1%~2% bovine serum albumin to obtain Au seeds@Antibody.

8. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The solvent of the exosome solution in step 3① is PBS, serum or cell culture medium; the concentration of the exosome solution in step 3① is 10 2 pcs / ml~10 8 The ratio of the volume of the exosome solution described in step 3① to the surface area of ​​the PDMS well array modified with Anti-CD63 is (30 μL to 50 μL): 0.25 cm 2 ; The standing time described in step 3① is 30min~60min; The mass fraction of the bovine serum albumin (BSA) PBS solution described in step 3① is 1%~2%, the concentration of the PBS solution is 10mmol / L, and the pH value is 7.4; The number of times of washing with a PBS solution of a certain concentration of bovine serum albumin in step 3① is 2~3 times; The volume ratio of Au seeds@Antibody described in step 3② to the surface area ratio of the PDMS concave well array modified with Anti-CD63 containing exosomes is (30μL~50μL):0.25cm 2 ; The standing time described in step 3② is 15min~90min; The mass fraction of bovine serum albumin (BSA) PBS solution described in step 3② is 1%~2%, the concentration of PBS solution is 10mmol / L, and the pH value is 7.4; The number of times of washing with a certain concentration of bovine serum albumin PBS solution in step 3② is 2 times~3 times.

9. The method for amplifying the SERS signal of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The in-situ growth solution described in step 3 ③ is a mixture of water, sodium oleate solution, tetrachloroauric acid solution, polyvinyl pyrrolidone solution and hydrogen peroxide, wherein the concentration of the sodium oleate solution is 9.8mmol / L to 10.2mmol / L, the concentration of the tetrachloroauric acid solution is 9.8mmol / L to 10.2mmol / L, the concentration of the polyvinyl pyrrolidone solution is 45mg / mL to 55mg / mL, and the mass fraction of hydrogen peroxide is 28% to 30%; the in-situ growth solution described in step 3 ③ is water, sodium oleate solution, tetrachloroauric acid solution, polyvinyl pyrrolidone solution and hydrogen peroxide. The volume ratio of ketone solution and hydrogen peroxide is (200μL~500μL):(30μL~70μL):(150μL~350μL):(10μL~50μL):(30μL~50μL); the mass fraction of bovine serum albumin (PBS) solution in step 3③ is 1%~2%, the concentration of PBS solution is 10mmol / L, and the pH value is 7.4; the concentration of 5,5'-dithiobis(2-nitrobenzoic acid) solution in step 3③ is 0.1mmol / L; the Nanowell-Au The volume ratio of seeds, in situ growth solution, and 5,5'-dithiobis(2-nitrobenzoic acid) solution is (30μL~50μL):(420μL~1020μL):(30μL~50μL); the standing time described in step 3③ is 15min~20min; the number of cleanings is 2 to 3 times.

10. The method for amplifying SERS signals of exosome surface proteins based on in situ chemical growth according to claim 1, characterized in that The Nanowell-Au NPs obtained in step 3③ were scanned by confocal Raman spectrometer to obtain the average spectrum. If the SERS characteristic peak collected was 1330 cm -1 、1550cm -1 、1050cm -1 , that is, the SERS signal of the exosome surface protein is obtained; the objective lens magnification of the confocal Raman spectrometer is 100X, the laser wavelength is 785nm, the power is 20mW, the integration time is 0.5s; the surface scanning area is 10×10μm 2 , the step size was 1 μm, a total of 100 points, each exosome sample was measured three times, and three different scan areas were randomly tested each time; the preprocessing process of SERS spectrum was divided into three steps: smoothing, baseline correction and normalization. The smoothing adopted Savitzky-Golay filtering with a window number of 20, and the baseline correction adopted asymmetric reweighted penalized least squares smoothing method.

Images