Preparation method and application of surface-enhanced raman scattering immunodetection system for prostate cancer exosome

Abstract

The present invention discloses a preparation method and application of a surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes. The method is characterized by comprising the steps of preparing a copper oxide nanowire-silver immunosubstrate; preparing a gold nanoimmunoprobe; dropwise adding a test solution containing prostate cancer exosomes onto the copper oxide nanowire-silver immunosubstrate, then dropwise adding a gold nanoimmunoprobe solution onto the immunosubstrate to which the exosomes are attached, and combining the immunoprobe with the prostate cancer exosomes through antigen-antibody specific binding to form a sandwich immunostructure, thereby obtaining the surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes. The method has the advantages of high specificity and low sensitivity, which facilitates the highly sensitive detection of trace amounts of prostate cancer exosomes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of prostate cancer exosome detection, and in particular to a preparation method and application of a surface-enhanced Raman scattering immunodetection system for prostate cancer exosomes. BACKGROUND

[0002] As one of the most serious malignancies in men, the mortality rate of prostate cancer ranks fifth among all cancers. In order to achieve early screening of cancer, prostate specific antigen (PSA) detection has been recognized as a reliable standard method. However, PSA detection lacks sufficient specificity. This is because the concentration of PSA in serum can also be significantly increased due to other prostate diseases such as prostate hypertrophy and inflammation. Therefore, it is urgent for the clinic to develop biomarkers with good specificity to improve the diagnostic accuracy of prostate cancer. As vesicles secreted by cells, exosomes with a size of 30 to 150 nm play a crucial role in intercellular communication and material exchange. Compared with normal cells, tumor cells usually produce more exosomes, and the membrane surface of the exosomes has a large number of specifically expressed proteins. If exosomes with specifically expressed proteins are used as cancer markers, it will provide a new technical solution for early cancer screening and monitoring. However, existing exosome determination methods, such as chemiluminescence and enzyme-linked immunoassay, have problems such as complex determination process, need for professional analysis, and low sensitivity, which do not meet the clinical needs. Therefore, it is urgent to develop a system that can detect trace amounts of exosomes secreted by early cancer cells. SUMMARY

[0003] The technical problem to be solved by the present application is to provide a preparation method and application of a surface-enhanced Raman scattering immunodetection system for prostate cancer exosomes with high specificity and sensitivity.

[0004] The technical solution adopted by the present application to solve the above technical problems is: a preparation method of a surface-enhanced Raman scattering immunodetection system for prostate cancer exosomes, comprising the following steps:

[0005] (1) Preparation of copper oxide nanowire-silver immunosubstrate

[0006] After the copper foil is heated to 500℃ in air for 1 hour to obtain copper oxide nanowires, the copper oxide nanowires are sputtered with silver at 0.4 Pa and 40 W for 1 minute using a small ion sputtering machine to obtain copper oxide nanowire-silver. The copper oxide nanowire-silver is placed in a 10-100 millimolar 10 millimolar dithiobis-succinimidyl propionate solution for 2 hours, washed with ethanol, and then placed in a mixed solution of equal volumes of a 10 milligram per milliliter N-hydroxysuccinimide solution and a 10 milligram per milliliter 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution to activate the carboxyl group. The copper oxide nanowire-silver is then placed in 2-20 microliters of a 10 microgram per milliliter CD63 protein solution and left to react for 1 hour. After washing with 20-200 microliters of a 1 milligram per milliliter BSA solution, a copper oxide nanowire-silver immunization substrate is obtained.

[0007] (2) Preparation of gold nanometer immunization probes

[0008] After 40-400 microliters of a 10 milligram per milliliter sodium citrate solution and 1-10 milliliters of a 10 milligram per milliliter chloroauric acid solution are reacted at 120℃ for 1 hour to obtain gold nanometer particles, 10-100 microliters of a 10 millimolar dithiobis-succinimidyl propionate solution and 20-200 microliters of a 1 millimolar rhodamine B solution are added. The precipitate is collected by centrifugation, washed with ethanol, and then redissolved in a PBS solution. The gold nanometer particles are then placed in a mixed solution of equal volumes of a 10 milligram per milliliter N-hydroxysuccinimide solution and a 10 milligram per milliliter 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution to activate the carboxyl group. A 2-20 microgram EpCAM antibody solution is then added, left to react for 1 hour, and then 20-200 microliters of a 1 milligram per milliliter BSA solution is added. After washing by centrifugation, a gold nanometer immunization probe is obtained.

[0009] (3) Preparation of a sandwich structure-based immunization detection system

[0010] After 20-200 microliters of a solution to be tested containing prostate cancer exosomes is added dropwise to the copper oxide nanowire-silver immunization substrate and left to react at 37℃ for 2 hours, the immunization substrate captures the prostate cancer exosomes through antigen-antibody specific binding. After 10-100 microliters of a 10 milligram per milliliter gold nanometer immunization probe solution is added dropwise to the immunization substrate with the exosomes attached and left to react at 37℃ for 2 hours, the immunization probe is combined with the prostate cancer exosomes through antigen-antibody specific binding to form a sandwich immunization structure, and a surface-enhanced Raman scattering immunization detection system for prostate cancer exosomes is obtained.

[0011] The application of the surface-enhanced Raman scattering immunodetection system of the prostate cancer exosome prepared by the preparation method is characterized in that the surface-enhanced Raman scattering immunodetection system of the prostate cancer exosome is subjected to spectral measurement by using a Raman spectrometer, and the concentration of the prostate cancer exosome in a to-be-measured solution can be calculated according to the quantitative relationship between the spectral intensity and the concentration of the prostate cancer exosome.

[0012] Compared with the prior art, the application has the advantages that the application first discloses a preparation method of a surface-enhanced Raman scattering immunodetection system of a prostate cancer exosome and application thereof, the copper oxide nanowire-silver immunosubstrate is prepared by a thermal oxidation method and an ion sputtering method, the method is simple, efficient and easy to mass-produce. The synthesized copper oxide nanowire is staggered and interconnected to form a reticular structure surface, which is conducive to efficient linkage of the prostate cancer exosome. In particular, the silver nanoparticles formed by ion sputtering are closely arranged on the surface of the copper oxide nanowire to form a high-density hot spot area, and a significant electromagnetic enhancement effect is obtained. The formation of the semiconductor heterojunction structure inside the copper oxide nanowire is conducive to efficient charge transfer, thereby triggering a significant chemical enhancement effect. The synergistic effect of the electromagnetic and chemical enhancement effects can further improve the SERS performance of the copper oxide nanowire-silver immunosubstrate, and the electromagnetic enhancement effect of the gold nanometer immunoprobe is conducive to high-sensitivity detection of trace prostate cancer exosomes. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 It is a scanning electron microscope photo of the copper oxide nanowire-silver immunosubstrate prepared in the embodiment 1 of the application;

[0014] Figure 2 It is a scanning electron microscope photo of the gold nanometer immunoprobe prepared in the embodiment 1 of the application;

[0015] Figure 3 It is a scanning electron microscope photo of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and the gold nanometer immunoprobe prepared in the embodiment 1 of the application after immunoreaction with a to-be-measured antigen;

[0016] Figure 4 It is a Raman detection result of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and the gold nanometer immunoprobe prepared in the embodiment 1 of the application after immunoreaction with a to-be-measured antigen;

[0017] Figure 5 It is a Raman detection result of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and the gold nanometer immunoprobe prepared in the embodiment 1 of the application after immunoreaction with different concentrations of to-be-measured antigens (the concentration is 2.79×10 10 particles per milliliter to 2.79×10 2Raman spectrum obtained by Raman detection of the substrate after immunoreaction of the particles (per milliliter) ;

[0018] Figure 6 Figure 4 is a graph showing the change of the characteristic peak intensity at 1646 cm -1 in the Raman spectrum of the prostate cancer exosome marker detection system prepared in Example 1 with the concentration of the antigen to be detected;

[0019] Figure 7 Figure 5 is a scanning electron microscope image of the copper oxide nanowire-silver immunosubstrate prepared in Example 2;

[0020] Figure 8 Figure 6 is a scanning electron microscope image of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and gold nanometer immunization probe prepared in Example 2 after immunoreaction with the antigen to be detected;

[0021] Figure 9 Figure 7 is the Raman detection result of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and gold nanometer immunization probe prepared in Example 2 after immunoreaction with the antigen to be detected;

[0022] Figure 10 Figure 8 is a scanning electron microscope image of the copper oxide nanowire-silver immunosubstrate prepared in Example 3;

[0023] Figure 11 Figure 9 is a scanning electron microscope image of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and gold nanometer immunization probe prepared in Example 3 after immunoreaction with the antigen to be detected;

[0024] Figure 12 Figure 10 is the Raman detection result of the sandwich immunization structure of the copper oxide nanowire-silver immunosubstrate and gold nanometer immunization probe prepared in Example 3 after immunoreaction with the antigen to be detected. DETAILED DESCRIPTION

[0025] The application will be further described in detail below with reference to the accompanying drawings.

[0026] The following examples are used to illustrate the application, but are not intended to limit the scope of the application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art, and the raw materials used are commercially available. The Raman spectrum detector BWS415 used in the examples is purchased from B&W Tek Inc., USA.

[0027] Example 1

[0028] A preparation method of a surface-enhanced Raman scattering immunodetection system for prostate cancer exosomes comprises the following steps:

[0029] 1. Preparation of copper oxide nanowire-silver immunosubstrate

[0030] After copper foil was heated to 500°C in air for 1 hour to obtain copper oxide nanowire, silver was sputtered on the copper oxide nanowire using a small ion sputtering machine at 0.4 Pa and 40 W for 1 minute to obtain copper oxide nanowire-silver. The copper oxide nanowire-silver was placed in a 10-ml solution of dithiobis-succinimidyl propionate with a concentration of 10 mmol for 2 hours, then washed with ethanol, and then placed in a mixed solution of equal volume of N-hydroxysuccinimide solution with a concentration of 10 mg / ml and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution with a concentration of 10 mg / ml to activate the carboxyl group. Then, the copper oxide nanowire-silver was placed in a 2-μl solution of CD63 protein with a concentration of 10 μg / ml and allowed to stand for 1 hour. After washing with 20 μl of BSA solution with a concentration of 1 mg / ml, the copper oxide nanowire-silver immunosubstrate was obtained.

[0031] Figure 1 A scanning electron microscope photograph of the copper oxide nanowire-silver immunosubstrate prepared in this example is shown in FIG. 1. As can be seen from FIG. 1, the prepared copper oxide is in a linear structure, and the surface is covered with a large number of silver nanoparticles. Figure 1

[0032] 2. Preparation of gold nanometer immunoprobesAfter 40 μl of sodium citrate with a concentration of 10 mg / ml and 1 ml of chloroauric acid with a concentration of 10 mg / ml were reacted at 120°C for 1 hour to obtain gold nanoparticles, 10 μl of dithiobis-succinimidyl propionate with a concentration of 10 mmol / ml and 20 μl of rhodamine B with a concentration of 1 mmol / ml were added, and the precipitate was collected by centrifugation, washed with ethanol, and then redissolved in a PBS solution. Then, the gold nanoparticles were placed in a mixed solution of equal volume of N-hydroxysuccinimide solution with a concentration of 10 mg / ml and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution with a concentration of 10 mg / ml to activate the carboxyl group. Then, 2 μg of EpCAM antibody solution was added, and the gold nanoparticles were allowed to stand for 1 hour. After 20 μl of BSA solution with a concentration of 1 mg / ml was added, the gold nanometer immunoprobes were obtained after washing by centrifugation.

[0033] Figure 2 A scanning electron microscope photograph of the gold nanometer immunoprobes prepared in this example is shown in FIG. 2. As can be seen from FIG. 2, the prepared gold nanometer immunoprobes are in a spherical structure, and the diameter is about 20 nm. Figure 2

[0034] 3. Preparation of an immunodetection system based on a sandwich structure

[0035] ​The 20 microliters of the solution containing the prostate cancer exosome to be detected is dropped onto the copper oxide nanowire-silver immune substrate, and reacted at 37°C for 2 hours. After the prostate cancer exosome is captured by the immune substrate through specific antigen-antibody binding, 10 microliters of gold nanometer immune probe solution with a concentration of 10 milligrams per milliliter is dropped onto the immune substrate with the exosome attached, and reacted at 37°C for 2 hours. The immune probe is combined with the prostate cancer exosome through specific antigen-antibody binding to form a sandwich immune structure, that is, a surface-enhanced Raman scattering immune detection system of the prostate cancer exosome is obtained.

[0036] Figure 3 The scanning electron microscope photos of the sandwich immune structure after the copper oxide nanowire-silver immune substrate and the gold nanometer immune probe prepared in this embodiment are combined with the antigen to be detected through the immune reaction are shown in FIG. 6. Figure 3 It can be seen that the gold nanometer immune probe is successfully adsorbed on the surface of the copper oxide nanowire-silver immune substrate through the specific immune reaction between the antigen and the antibody.

[0037] 4. The immune sandwich structure (including the immune probe and the immune substrate) obtained after the above steps is subjected to spectral measurement by using the Raman spectrometer, and the prostate cancer exosome to be detected can be detected.

[0038] Figure 4 The Raman spectrum obtained by using the copper oxide nanowire-silver immune substrate and the gold nanometer immune probe prepared in this embodiment to detect the sandwich immune structure after the immune reaction with the antigen to be detected is shown in FIG. 7. Figure 4 It can be seen that the strong Raman characteristic spectrum of the marker molecule rhodamine B can be detected, and the Raman signal intensity at 1646 cm -1 reaches 12243.

[0039] Figure 5 The Raman spectrum obtained by using the copper oxide nanowire-silver immune substrate and the gold nanometer immune probe prepared in this embodiment to detect the substrate after the immune reaction with different concentrations of the exosome to be detected (the concentration is 2.79×10 10 particles per milliliter to 2.79×10 2 particles per milliliter) is shown in FIG. 8. Figure 5 It can be seen that as the concentration of the exosome to be detected decreases, the Raman characteristic spectrum intensity of the marker molecule rhodamine B gradually decreases, and when the concentration of the exosome to be detected decreases to 2.79×10 2 particles per milliliter, the Raman characteristic peak of the marker molecule is still obvious relative to the background signal.

[0040] Figure 6 The Raman spectrum obtained by using the copper oxide nanowire-silver immune substrate and the gold nanometer immune probe prepared in this embodiment to detect the substrate after the immune reaction with different concentrations of the exosome to be detected (the concentration is 2.79×10 -1The characteristic peak intensity of the exosomes changes with the concentration of the exosomes to be tested. It can be seen from the fitting that when the concentration of the exosomes to be tested increases from 2.79×10 10 The number of particles per milliliter changed to 2.79×10 2 At 100 particles per milliliter, the intensity of the Raman peak changes linearly with concentration. Fitting results show that this trend conforms to the linear equation Y = 1486.58 * LogX - 2911.67, with a standard deviation of 0.995, where X is the exosome concentration and Y is the Raman peak intensity. Using this linear equation, the detection limit for exosomes was calculated to be 89 particles per milliliter.

[0041] Example 2

[0042] A method for preparing a surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes comprises the following steps:

[0043] 1. Preparation of copper oxide nanowire-silver immunoassay substrate

[0044] The copper foil was heated to 500° C. in air and maintained for 1 hour to obtain copper oxide nanowires. Silver was then sputtered on the copper oxide nanowires at 0.4 Pa and 40 watts for 1 minute using a small ion sputtering machine to obtain copper oxide nanowire-silver. The copper oxide nanowire-silver was placed in 50 ml of a 10 mmol dithiobis succinimidyl propionate solution to react for 2 hours and then washed with ethanol. It was then placed in a mixed solution of equal volumes of 10 mg / ml N-hydroxysuccinimide solution and 10 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to activate the carboxyl group. It was then placed in 10 μl of a 10 μg / ml CD63 protein solution and allowed to react for 1 hour. After washing with 100 μl of a 1 mg / ml BSA solution, the copper oxide nanowire-silver immunosubstrate was obtained.

[0045] Figure 7 The scanning electron microscope photo of the copper oxide nanowire-silver immunoassay substrate prepared in this example is shown. Figure 7 It can be seen that the prepared copper oxide has a linear structure and its surface is covered by a large number of aggregated silver nanoparticles.

[0046] 2. Preparation of gold nano-immune probe 200 microliters of sodium citrate with a concentration of 10 milligrams per milliliter and 5 milliliters of chloroauric acid with a concentration of 10 milligrams per milliliter were reacted at 120°C for 1 hour to obtain gold nanoparticles, then 50 microliters of dithiobis succinimidyl propionate with a concentration of 10 millimoles per milliliter and 100 microliters of rhodamine B with a concentration of 1 millimole per milliliter were added, the precipitate was collected by centrifugation, washed with ethanol, and then redissolved in a PBS solution, then placed in a mixed solution of equal volumes of N-hydroxysuccinimide solution with a concentration of 10 milligrams per milliliter and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution with a concentration of 10 milligrams per milliliter to activate the carboxyl group, then 10 micrograms of EpCAM antibody solution was added, and after standing for 1 hour of reaction, 100 microliters of BSA solution with a concentration of 1 milligram per milliliter was added, and the gold nano-immune probe was obtained after centrifugal washing.

[0047] 3. Preparation of immune detection system based on sandwich structure

[0048] 100 microliters of the solution to be tested containing prostate cancer exosomes were added dropwise to the copper oxide nanowire-silver immune substrate, and after 2 hours of reaction at 37°C, the prostate cancer exosomes were captured by the immune substrate through specific antigen-antibody binding, then 50 microliters of gold nano-immune probe solution with a concentration of 10 milligrams per milliliter was added dropwise to the immune substrate with exosomes attached, and after 2 hours of reaction at 37°C, the prostate cancer exosomes were combined with the gold nano-immune probe to form a sandwich immune structure through specific antigen-antibody binding, and thus the surface-enhanced Raman scattering immune detection system for prostate cancer exosomes was obtained.

[0049] Figure 8 The scanning electron microscope photos of the sandwich immune structure after the copper oxide nanowire-silver immune substrate and the gold nano-immune probe prepared in this embodiment were immunoreacted with the antigen to be tested are shown in FIG. 2. Figure 8 As can be seen, the gold nano-immune probe was successfully adsorbed on the surface of the copper oxide nanowire-silver immune substrate through specific immune reaction between the antigen and antibody.

[0050] 4. The immune sandwich structure (including the immune probe and the immune substrate) obtained after the above steps was subjected to spectral measurement by a Raman spectrometer, and the prostate cancer exosomes to be tested could be detected.

[0051] Figure 9 The Raman spectrum obtained by Raman detection of the sandwich immune structure after the copper oxide nanowire-silver immune substrate and the gold nano-immune probe prepared in this embodiment were immunoreacted with the antigen to be tested is shown in FIG. 3. Figure 9 As can be seen, the strong Raman characteristic spectrum of the marker molecule rhodamine B could be detected, and the Raman signal intensity at 1646 cm -1 reached 11056.

[0052] Example 3

[0053] A method for preparing a surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes, characterized by comprising the following steps:

[0054] 1. Preparation of copper oxide nanowire-silver immunoassay substrate

[0055] The copper foil was heated to 500° C. in air and maintained for 1 hour to obtain copper oxide nanowires. Silver was then sputtered on the copper oxide nanowires at 0.4 Pa and 40 watts for 1 minute using a small ion sputtering machine to obtain copper oxide nanowire-silver. The copper oxide nanowire-silver was placed in 10-100 ml of a 10 mmol dithiobis succinimidyl propionate solution and reacted for 2 hours. After washing with ethanol, it was placed in a mixed solution of equal volumes of 10 mg / ml N-hydroxysuccinimide solution and 10 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to activate the carboxyl group. The solution was then placed in 20 μl of a 10 μg / ml CD63 protein solution and allowed to react for 1 hour. After washing with 200 μl of a 1 mg / ml BSA solution, the copper oxide nanowire-silver immunosubstrate was obtained.

[0056] Figure 10 The scanning electron microscope photo of the copper oxide nanowire-silver immunoassay substrate prepared in this example is shown. Figure 10 It can be seen that the prepared copper oxide has a linear structure and its surface is covered by a large number of aggregated silver nanoparticles.

[0057] 2. Preparation of gold nanoimmunoprobes

[0058] 400 μL of 10 mg / mL sodium citrate and 10 mL of 10 mg / mL chloroauric acid were reacted at 120°C for 1 hour to obtain gold nanoparticles. Then, 100 μL of 10 mmol / mL dithiodisuccinimidyl propionate and 200 μL of 1 mmol / mL rhodamine B were added. The precipitate was collected by centrifugation, washed with ethanol, and redissolved in PBS solution. It was then placed in a mixed solution of equal volumes of 10 mg / mL N-hydroxysuccinimide solution and 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to activate the carboxyl group. 20 μg of EpCAM antibody solution was added. After standing for 1 hour, 200 μL of 1 mg / mL BSA solution was added. After centrifugation and washing, the gold nanoimmunoprobe was obtained.

[0059] 3. Preparation of sandwich-structured immunoassay system

[0060] The 200 microliters of the solution containing the prostate cancer exosome to be detected is dropped onto the copper oxide nanowire-silver immunosubstrate, and reacted at 37°C for 2 hours. After the prostate cancer exosome is captured by the immunosubstrate through specific antigen-antibody binding, 100 microliters of a gold nanometer immunoprober solution with a concentration of 10 milligrams per milliliter is dropped onto the immunosubstrate with the exosome attached, and reacted at 37°C for 2 hours. The prostate cancer exosome is combined with the immunoprober through specific antigen-antibody binding to form a sandwich immunization structure, that is, a surface-enhanced Raman scattering immunodetection system of the prostate cancer exosome is obtained.

[0061] Figure 11 The scanning electron microscope photos of the sandwich immunization structure after the copper oxide nanowire-silver immunosubstrate and the gold nanometer immunoprober prepared in the embodiment are combined with the antigen to be detected through immunoreaction are shown in the following figures. Figure 11 It can be seen that the gold nanometer immunoprober is successfully adsorbed on the surface of the copper oxide nanowire-silver immunosubstrate through specific immunoreaction between the antigen and antibody.

[0062] 4. The immunosandwich structure (including the immunoprober and the immunosubstrate) obtained after the above steps is subjected to spectral measurement by using a Raman spectrometer, so that the prostate cancer exosome to be detected can be detected.

[0063] Figure 12 The Raman spectrum obtained by using the copper oxide nanowire-silver immunosubstrate and the gold nanometer immunoprober prepared in the embodiment to detect the sandwich immunization structure after the immunoprober and the immunosubstrate are combined with the antigen to be detected through immunoreaction is shown in the following figure. Figure 12 It can be seen that the strong Raman characteristic spectrum of the marker molecule rhodamine B can be detected, and the Raman signal intensity at 1646 cm -1 reaches 8827.

[0064] The above description is not a limitation of the present application, and the present application is not limited to the above examples. Changes, modifications, additions or replacements made by those skilled in the art within the essential scope of the present application shall also fall within the protection scope of the present application.

Claims

1. A method for preparing a surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes, characterized in that The following steps are involved: (1) Preparation of copper oxide nanowire-silver immunoassay substrate A copper foil is heated to 500° C. in air and maintained for 1 hour to obtain copper oxide nanowires. Silver is then sputtered onto the copper oxide nanowires using a small ion sputtering machine at 0.4 Pa and 40 W for 1 minute to obtain copper oxide nanowire-silver. The copper oxide nanowire-silver is placed in 10-100 ml of a 10 mM dithiobissuccinimidyl propionate solution and reacted for 2 hours. The copper oxide nanowire-silver is then washed with ethanol and then placed in a mixture of equal volumes of a 10 mg / ml N-hydroxysuccinimide solution and a 10 mg / ml 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to activate the carboxyl groups. The copper oxide nanowire-silver immunostaining substrate is then placed in 2-20 μl of a 10 μg / ml CD63 protein solution and allowed to react for 1 hour. The substrate is then washed with 20-200 μl of a 1 mg / ml BSA solution to obtain a copper oxide nanowire-silver immunostaining substrate. (2) Preparation of gold nanoimmunoprobes 40-400 μL of 10 mg / mL sodium citrate and 1-10 mL of 10 mg / mL chloroauric acid are reacted at 120° C. for 1 hour to obtain gold nanoparticles, and then 10-100 μL of 10 mmol / mL dithiobissuccinimidyl propionate and 20-200 μL of 1 mmol / mL rhodamine B are added. The precipitate is collected by centrifugation, washed with ethanol, and redissolved in PBS solution. The precipitate is then placed in a mixed solution of equal volumes of 10 mg / mL N-hydroxysuccinimide solution and 10 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to activate the carboxyl group. 2-20 μg of EpCAM antibody solution is added, and after standing for 1 hour, 20-200 μL of 1 mg / mL BSA solution is added. The precipitate is washed by centrifugation to obtain a gold nanoimmunoprobe. (3) Preparation of sandwich-structured immunoassay system 20-200 microliters of the test solution containing prostate cancer exosomes is added to the copper oxide nanowire-silver immune substrate and reacted at 37°C for 2 hours. After the immune substrate captures the prostate cancer exosomes through antigen-antibody specific binding, 10-100 microliters of a gold nanoimmunoprobe solution with a concentration of 10 mg / ml is added to the immune substrate attached with exosomes and reacted at 37°C for 2 hours. Through antigen-antibody specific binding, the immune probe and prostate cancer exosomes are combined to form a sandwich immune structure, thus obtaining a surface-enhanced Raman scattering immunoassay system for prostate cancer exosomes.

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