Method for preparing dual-mode immune structure based on surface-enhanced raman substrate and up-conversion luminescent probe, and application thereof

Abstract

A method for preparing a dual-mode immune structure based on a surface-enhanced Raman substrate and an up-conversion luminescent probe, and an application thereof. The dual-mode immune structure based on a surface-enhanced Raman substrate and an up-conversion luminescent probe comprises a copper / black phosphorus / silver nanoflower composite immunosubstrate and a NaGdF4:Yb3+ / Er3+ up-conversion nanoparticle immunoprobe, wherein the copper / black phosphorus / silver nanoflower composite immunosubstrate is prepared using an electrochemical reduction method, and the NaGdF4:Yb3+ / Er3+ up-conversion nanoparticle immunoprobe is prepared using a hydrothermal method. During the application to cancer detection, assembling an immunosubstrate and an immunoprobe realizes the combination of up-conversion luminescence and surface-enhanced Raman spectroscopy technology for realizing dual-mode detection of cancer biomarkers. Thus, simple operation and high detection sensitivity are achieved, thereby facilitating efficient and precise clinical screening and identification of cancer biomarkers.

Description

Preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and upconversion luminescence probes and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of material engineering and nanotechnology, and particularly relates to a preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and upconversion luminescence probes and application thereof. BACKGROUND

[0002] In recent years, cancer has become an important factor of premature death and poses a serious threat to people's health. Prostate cancer is the most common malignant tumor and has been recognized as the third leading cause of male cancer-related deaths. Realizing early detection and dynamic monitoring is crucial for effective treatment of most cancer patients, and prostate-specific antigen (PSA) has been widely recognized as a key biomarker. However, the current mainstream detection methods of PSA are relatively single, with long cycle, complicated operation steps, low detection accuracy, etc. Compared with traditional fluorescent materials, upconversion materials, as a kind of fluorescent material that can emit short-wave light under red or infrared light excitation, can convert low-energy long-wave light into high-energy short-wave light and have a relatively narrow absorption spectrum and emission spectrum, and are particularly suitable for specific marker testing in the medical field. At the same time, surface-enhanced Raman scattering (SERS) is another highly efficient spectral detection technology with extremely high detection sensitivity and molecular fingerprint recognition capability, and also has good application prospects in clinical detection. However, there is currently no report on the combination of upconversion luminescence and SERS spectral technology to realize dual-mode detection of cancer markers. In fact, the dual-mode detection technology based on upconversion luminescence and SERS can realize the complementary advantages of the two detection methods and further improve the accuracy and sensitivity of cancer detection.

[0003] SUMMARY

[0004] An advantage of the present application is to provide a preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and upconversion luminescence probes and application thereof. The copper / black phosphorus / silver nanoflower composite immune substrate and NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immune probes are assembled and applied to the detection of cancer, realizing dual-mode detection of cancer markers by combining upconversion luminescence and SERS spectral technology, which is more accurate, efficient and has high detection sensitivity.

[0005] Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, which comprehensively utilizes the advantages of high upconversion luminescence efficiency, good stability and strong anti-interference ability, and the characteristics of strong SERS spectrum fingerprint recognition ability and high detection sensitivity, and is conducive to realizing efficient and accurate screening and identification of cancer markers in a clinical environment.

[0006] Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, wherein compared with traditional fluorescent materials, upconversion materials can emit short-wave light under red or infrared light excitation as a fluorescent material, can convert low-energy long-wave light into high-energy short-wave light, and have a relatively narrow absorption spectrum and emission spectrum, and are particularly suitable for specific marker testing in the medical field.

[0007] Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, wherein surface-enhanced Raman scattering (SERS) is another efficient spectral detection technology, has extremely high detection sensitivity and molecular fingerprint recognition ability, has good application prospects in clinical detection, and has important significance for improving the accuracy and sensitivity of cancer detection.

[0008] Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, wherein a Raman spectrometer is used to measure the fluorescence spectrum or Raman spectrum of a complex of the immune probe and the copper / black phosphorus / silver nanoflower composite immune substrate, the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the cancer marker antigen concentration and the fluorescence characteristic peak or Raman characteristic peak intensity, and the result is relatively accurate. 3+ / Er 3+ Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, wherein a Raman spectrometer is used to measure the fluorescence spectrum or Raman spectrum of a complex of the immune probe and the copper / black phosphorus / silver nanoflower composite immune substrate, the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the cancer marker antigen concentration and the fluorescence characteristic peak or Raman characteristic peak intensity, and the result is relatively accurate.

[0009] Another advantage of the present application is to provide a preparation method and application of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, which has a simple preparation method, low cost and convenient use, and is suitable for clinical popularization and application.

[0010] According to an aspect of the present application, a preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe is provided, which comprises the following steps:

[0011] (S10) preparing a copper / black phosphorus / silver nanoflower composite immune substrate; and

[0012] (S20) preparing a NaGdF4:Yb 3+ / Er 3+Upconversion nanoparticle immunoprobes.

[0013] Wherein in the step (S10), a copper / black phosphorus / silver nanoflower composite immunization substrate is prepared by electrochemical reduction, and in the step (S20), NaGdF4:Yb 3+ / Er 3+ Upconversion nanoparticle immunoprobes.

[0014] Wherein the step (S10) comprises the following steps: (S101) adding black phosphorus into an NMP solution to prepare a black phosphorus solution, ultrasonic treatment, centrifugation, collecting light brown liquid, washing and then adding into deionized water to obtain a black phosphorus nanosheet suspension; (S102) ultrasonic treatment of the black phosphorus nanosheet solution obtained in step (S101), collecting the precipitate after centrifugal washing, dissolving the precipitate into deionized water to form a suspension, and electrodeposition, thereby obtaining a copper / black phosphorus nanosheet substrate; (S103) taking the copper / black phosphorus nanosheet substrate prepared in step (S102) as a working electrode, an Ag / AgCl electrode as a reference electrode, a platinum sheet as a counter electrode, and an aqueous silver nitrate solution as an electrolyte, and performing electrochemical reaction, thereby obtaining a copper / black phosphorus / silver nanoflower composite substrate; (S104) immersing the copper / black phosphorus / silver nanoflower composite substrate prepared in step (S103) in a DMF solution, then washing with a PBS solution, and then transferring the above substrate to an EDC / NHS PBS solution, immersing, then adding a PBS solution containing an antibody PSMA dropwise, immersing, and deionized water washing, thereby obtaining a copper / black phosphorus / silver nanoflower composite immunization substrate.

[0015] Wherein the step (S20) comprises the following steps: (S201) mixing a rare earth nitrate RE(NO3)3·6H2O (RE=60-80% Gd, 15-35% Yb, 5% Er) with an aqueous citric acid solution, stirring, adding an aqueous sodium hydroxide solution to the above solution, stirring, adding an aqueous sodium fluoride solution, continuing to stir, obtaining a precursor solution, and transferring to a stainless steel reaction kettle with a polytetrafluoroethylene liner for closed reaction. After the reaction is completed, cooling, centrifugation, taking out the reaction product and washing with ethanol and deionized water, drying, thereby obtaining NaGdF4:Yb 3+ / Er 3+ nanoparticles; (S202) dissolving the NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (201) in an ethanol solution to obtain a NaGdF4:Yb 3+ / Er 3+nanoparticle solution, then add R6G ethanol solution, mix, stir, centrifuge and wash with ethanol, and store in deionized water; (S203) add NHS / EDC PBS solution to the above solution, shake in a shaker, centrifuge, disperse the mixture in PBS again, then add antibody EpCAM containing solution to the above solution, incubate, centrifuge, remove residual antibody, wash with PBS buffer, add BSA solution to the colloidal solution, stir, store at room temperature, and finally obtain EpCAM-NaGdF4:Yb 3+ / Er 3+ The upconversion nanoparticle immunoprobes are dispersed in PBS buffer and stored at 4°C for standby use.

[0016] In the step (S101), the concentration of the black phosphorus solution is 1-10 mg / mL, ultrasonic treatment is carried out at 3°C for 6-8 h, and after grading centrifugation at 5000 r / min for 15 min, the light brown liquid is collected, the dark brown precipitate is removed, and the collected light brown liquid is washed with deionized water and ethanol.

[0017] In the step (S102), electrodeposition is carried out in an electrolytic cell, and a two-electrode system is used in the electrodeposition process, in which a platinum sheet is an anode and a copper foil is a cathode, wherein the copper foil is cleaned with acetone, ethanol and deionized water for multiple times, and the cathode electrodeposition is carried out at a voltage of 10-20 V for 5-10 min to obtain a copper / black phosphorus nanoplate substrate.

[0018] In the step (S103), the concentration of the silver nitrate electrolyte is 2-6 mmol / mL, the deposition voltage is -0.2 V to -0.6 V, and the deposition time is 20-100 s, and in the step (S104), the copper / black phosphorus / silver nanoflower composite substrate prepared in the step (S103) is soaked in a DMF solution for 2 h, then washed with a PBS solution for multiple times, and then transferred to an EDC / NHS PBS solution (1:1, 10 mg / mL) for soaking at room temperature for 1 h to activate the carboxyl group, then dropwise add a PBS solution containing prostate cancer exosome specific antibody PSMA with a concentration of 2-6 mg / mL, soak at room temperature for 2 h, wash with deionized water for multiple times to obtain a copper / black phosphorus / silver nanoflower composite immunosubstrate.

[0019] In the step (S201), the concentration of the aqueous citric acid solution is 0.4-0.8 mol / L, the concentration of the aqueous sodium hydroxide solution is 5-10 mmol / mL, and the concentration of the aqueous sodium fluoride solution is 1-3 mmol / mL, and the reaction is carried out at 180°C for 12 h in a stainless steel reaction kettle with a polytetrafluoroethylene liner, and the reaction product is taken out by centrifugation and repeatedly washed with ethanol and deionized water, and the product is dried in air at 80°C for 12 h to obtain NaGdF4:Yb 3+ / Er3+ nanoparticles; in the step (S202), the NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in the step (201) are dissolved in 5-10 mL of ethanol solution to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticle solution, then, 10-20 mL of R6G (10 -3 mol / L) ethanol solution is added for mixing, after stirring at room temperature for 12 h, the excess unreacted R6G is removed by centrifugation at 7000 r / min for 10 min, and the nanoparticles are washed with ethanol for several times and then stored in 5 mL of deionized water; in the step (S203), 1-3 mL of NHS / EDC PBS solution (1 / 4, 10 mg / mL) is added to the above solution, and the carboxyl group is activated by reaction in a shaking incubator at 37 °C for 1 h to improve the protein connection efficiency, the excess PBS solution is removed by centrifugation at 6000 r / min for 10 min, the mixture is dispersed in 1 mL of PBS (PBS / H2O = 1 / 4) again, 20-40 μg of prostate cancer exosome specific antibody EpCAM is added to the above solution, and incubated for 1 h, and then centrifuged at 8000 r / min for 10 min to remove the residual antibody, and then washed with PBS buffer for 3 times, 1% BSA solution is added to the colloidal solution for stirring, and then stored at room temperature for 1 h, and finally the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes are dispersed in 2 mL of PBS buffer and stored at 4 °C for standby.

[0020] According to another aspect of the present application, the present application also provides a dual-mode immunological structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, comprising: a copper / black phosphorus / silver nanoflower composite immunological substrate and NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes.

[0021] In the application detection process, the immunological substrate and the immunoprobes are assembled, the PBS solution containing the antigen to be detected is added dropwise to the copper / black phosphorus / silver nanoflower composite immunological substrate, and the antigen and the antibody are allowed to react at 37 °C for 2 h, the antigen to be detected is removed by washing with PBS solution for several times, 10-20 μL of NaGdF4:Yb 3+ / Er 3+ dual-mode immunoprobes are added dropwise to the copper / black phosphorus / silver nanoflower composite immunological substrate adsorbed with the antigen to be detected, and reacted at 37 °C for 2 h, and then the excess unreacted NaGdF4:Yb3+ / Er 3+ Dual-mode immunoprobes, i.e. NaGdF4:Yb 3+ / Er 3+ Dual-mode immunoprobes and copper / black phosphorus / silver nanoflower composite immunosubstrate prostate cancer exosome detection structure, using a Raman spectrometer to measure the spectrum of the above immunoreaction obtained NaGdF4:Yb 3+ / Er 3+ Immunoprobes and copper / black phosphorus / silver nanoflower composite immunosubstrate composite, according to the linear relationship between the concentration of cancer marker antigen and the intensity of Raman characteristic peak, the concentration of the cancer marker antigen to be measured is calculated.

[0022] Wherein the antigen is prostate specific antigen PSA, alpha-fetoprotein AFP, ferritin antigen or carbohydrate antigen CA199.

[0023] According to another aspect of the present application, the present application also provides an application of a dual-mode immunological structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, wherein the dual-mode immunological structure based on the surface-enhanced Raman substrate and the upconversion luminescence probe is suitable for application in tumor detection, especially in prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer detection.

[0024] Wherein the dual-mode immunological structure based on the surface-enhanced Raman substrate and the upconversion luminescence probe includes a copper / black phosphorus / silver nanoflower composite immunosubstrate and NaGdF4:Yb 3+ / Er 3+ Upconversion nanoparticle immunoprobes, wherein the copper / black phosphorus / silver nanoflower composite immunosubstrate is prepared by electrochemical reduction, and NaGdF4:Yb 3+ / Er 3+ The upconversion nanoparticle immunoprobes are prepared by a hydrothermal method. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a scanning electron microscope photograph of NaGdF4:Yb 3+ / Er 3+ Nanoparticles prepared in Example 1;

[0026] Figure 2 is a scanning electron microscope photograph of NaGdF4:Yb 3+ / Er 3+ Nanoparticles prepared in Example 2;

[0027] Figure 3 is a scanning electron microscope photograph of NaGdF4:Yb 3+ / Er 3+ Nanoparticles prepared in Example 3;

[0028] Figure 4 is a scanning electron microscope photograph of the copper / black phosphorus / silver flower SERS substrate prepared in Example 1;

[0029] Figure 5 is a scanning electron microscope photograph of the copper / black phosphorus / silver flower SERS substrate prepared in Example 2;

[0030] Figure 6 is a scanning electron microscope photograph of the copper / black phosphorus / silver flower SERS substrate prepared in Example 3;

[0031] Figure 7 is a SERS spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes;

[0032] Figure 8 is a SERS spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes;

[0033] Figure 9 is a SERS spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes;

[0034] Figure 10 is an upconversion spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes;

[0035] Figure 11 is an upconversion spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes;

[0036] Figure 12 is an upconversion spectrum of the prostate cancer exosome detection structure prepared in Example 1 detecting exosomes. DETAILED DESCRIPTION

[0037] The following description is presented to enable any person skilled in the art to practice the application as claimed. The preferred embodiments disclosed herein are only examples of the application and alternative embodiments, modifications, improvements, equivalents, and the like can be made thereto without departing from the spirit and scope of the application as set forth in the appended claims. The present application is defined by the appended claims.

[0038] The technical solution adopted by the present application is a preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, comprising the following steps:

[0039] (S10) preparing a copper / black phosphorus / silver nanoflower composite immune substrate by electrochemical reduction;

[0040] (S20) preparing NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immune probes; and

[0041] (S30) assembling a prostate cancer exosome detection structure.

[0042] Wherein (S10) comprises the following steps:

[0043] (S101) A black phosphorus solution is prepared by adding black phosphorus into an NMP solution, ultrasonic treatment, centrifugation, collection of light brown liquid after centrifugation, washing with deionized water and ethanol, and adding into deionized water to obtain a black phosphorus nanosheet suspension;

[0044] (S102) After ultrasonic treatment of the black phosphorus nanosheet solution obtained in step (S101), centrifugal washing is performed, the precipitate is collected, the precipitate is dissolved into deionized water to form a suspension, and electrodeposition is performed using a two-electrode system, so as to obtain a copper / black phosphorus nanosheet substrate;

[0045] (S103) The copper / black phosphorus nanosheet substrate prepared in step (S102) is used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, a platinum sheet is used as a counter electrode, and a silver nitrate aqueous solution is used as an electrolyte, and electrochemical reaction is performed, so as to obtain a copper / black phosphorus / silver nanoflower composite substrate;

[0046] (S104) The copper / black phosphorus / silver nanoflower composite substrate prepared in step (S103) is soaked in a DMF solution, then washed with a PBS solution for multiple times, then the above substrate is transferred to an EDC / NHS PBS solution, soaked to activate the carboxyl group, then a PBS solution containing a prostate cancer exosome specific antibody PSMA is added dropwise, soaked again, and then washed with deionized water for multiple times, so as to obtain a copper / black phosphorus / silver nanoflower composite immunosubstrate.

[0047] Wherein the step (S20) comprises the following steps:

[0048] (S201) A rare earth nitrate RE(NO3)3·6H2O (RE = 60-80% Gd, 15-35% Yb, 5% Er) is mixed with an aqueous citric acid solution, stirred vigorously, an aqueous sodium hydroxide solution is slowly added, stirred vigorously, an aqueous sodium fluoride solution is slowly added, and stirring is continued, the obtained precursor solution is transferred into a stainless steel reaction kettle with a polytetrafluoroethylene liner, sealed, and reacted at 180°C for 12h, after the reaction is completed, the reaction product is taken out by centrifugation, and washed repeatedly with ethanol and deionized water, dried, and NaGdF4:Yb 3+ / Er 3+ nanoparticles are obtained;

[0049] (S202) The NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (201) are dissolved in an ethanol solution to obtain NaGdF4:Yb 3+ / Er 3+The nanoparticle solution was mixed with the R6G ethanol solution, stirred, centrifuged to remove excess unreacted R6G, washed with ethanol several times, and stored in deionized water.

[0050] (S203) A PBS solution of NHS / EDC was added to the above solution, and the mixture was reacted in a shaker to activate the carboxyl group. After centrifugation, the excess PBS solution was removed. The mixture was dispersed in PBS again after centrifugation. Then, the prostate cancer exosome-specific antibody EpCAM was added to the above solution, incubated, centrifuged, and the residual antibody was removed. The PBS buffer was washed three times. The BSA solution was added to the colloidal solution and stirred at room temperature. Finally, the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ The upconversion nanoparticle immunoprobes were dispersed in PBS buffer and stored at 4°C for standby use.

[0051] In the step (S30), the PBS solution containing the antigen to be detected was added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate, and was placed at 37°C for 2h to allow the immunoreaction between the antigen and the antibody to proceed fully. The antigen to be detected was removed by washing with PBS solution several times. Then, the NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes were added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate adsorbed with the antigen to be detected, and were reacted at 37°C. The excess unreacted NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes, i.e., the NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes and the copper / black phosphorus / silver nanoflower composite immunosubstrate were used for prostate cancer exosome detection. The NaGdF4:Yb 3+ / Er 3+ The complex of the immunoprobes and the copper / black phosphorus / silver nanoflower composite immunosubstrate was subjected to spectral measurement. According to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak, the concentration of the cancer marker antigen to be detected was calculated.

[0052] The raw materials used were commercially available. The Raman spectrum detector BWS415 used in the examples was purchased from B&W Tek Inc. in the United States. The antigen used in the following examples was prostate specific antigen PSA, but was not limited to prostate specific antigen PSA, and could also be alpha-fetoprotein antigen AFP, ferritin antigen, and carbohydrate antigen CA199, etc.

[0053] Example 1

[0054] A preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, comprising the following steps:

[0055] (1) A copper / black phosphorus / silver nanoflower composite immune substrate is prepared by an electrochemical reduction method

[0056] A. Black phosphorus is added to an NMP solution to prepare a black phosphorus solution with a concentration of 1 mg / mL, then the solution is ultrasonically treated at 3°C for 6 h, and after grading centrifugation at 5000 r / min for 15 min, the light brown liquid is collected, the dark brown precipitate is removed, and the collected light brown liquid is washed with deionized water and ethanol for 3 times, and then added to 10 mL of deionized water to obtain a black phosphorus nanosheet suspension;

[0057] B. The black phosphorus nanosheet solution obtained in step (1) A is ultrasonically treated, and the precipitate is collected after centrifugal washing, and then the precipitate is dissolved in deionized water to form a suspension with a concentration of 0.5 mg / mL, and 10 mL of the suspension is transferred to an electrolytic cell for electrodeposition, and a double electrode system is used in the electrodeposition process: a platinum sheet as an anode and a copper foil as a cathode (the copper foil is cleaned with acetone, ethanol and deionized water for multiple times), and the cathode is electrodeposited at a voltage of 10 V for 5 min to obtain a copper / black phosphorus nanosheet substrate;

[0058] C. The copper / black phosphorus nanosheet substrate prepared in step (1) B is used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, and a platinum sheet is used as a counter electrode, 10 mL of an aqueous solution containing 2 mmol / mL of silver nitrate is used as an electrolyte, the deposition voltage is controlled at-0.2 V, and the deposition time is controlled at 20 s, and an electrochemical reaction is carried out to obtain a copper / black phosphorus / silver nanoflower composite substrate;

[0059] D. The copper / black phosphorus / silver nanoflower composite substrate prepared in step (1) C is soaked in 2 mL of a DMF solution for 2 h, and then washed with a PBS solution for multiple times, and then the substrate is transferred to 1 mL of an EDTA / NHS PBS solution (1:1, 10 mg / mL) for soaking at room temperature for 1 h to activate the carboxyl group. Then 10 μL of a PBS solution containing a prostate cancer exosome specific antibody PSMA (with a concentration of 2 mg / mL) is added dropwise, and the substrate is soaked at room temperature for 2 h, and then washed with deionized water for multiple times to obtain a copper / black phosphorus / silver nanoflower composite immune substrate;

[0060] (2) NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immune probe

[0061] A. 1 mmol of rare earth nitrate RE(N03)3-6H20 (RE = 60% Gd, 35% Yb, 5% Er) was mixed with 10 mL of 0.4 mol / L aqueous solution of citric acid and stirred vigorously for 30 min. Then, 0.2 mL of 5 mmol / mL aqueous solution of sodium hydroxide was slowly added to the above solution and stirred vigorously for 15 min. Subsequently, 8 mL of 1 mmol / mL aqueous solution of sodium fluoride was slowly added and stirred for another 30 min. The obtained precursor solution was then transferred into a 40 mL stainless steel reactor with a polytetrafluoroethylene liner and sealed. The reactor was placed in an oven at 180 °C and reacted for 12 h. After the reaction, the reactor was naturally cooled to room temperature. The reaction product was removed by centrifugation and repeatedly washed with ethanol and deionized water. Finally, the product was dried in air at 80 °C for 12 h to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticles.

[0062] B. 0.1 mmol of NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (2) A was dissolved in 5 mL of ethanol solution to obtain a NaGdF4:Yb 3+ / Er 3+ nanoparticle solution. Subsequently, 10 mL of R6G (10 -3 mol / L) ethanol solution was added and stirred at room temperature for 12 h. The excess unreacted R6G was removed by centrifugation at 7000 r / min for 10 min, and the nanoparticles were washed with ethanol several times and stored in 5 mL of deionized water.

[0063] C. 1 mL of NHS / EDC PBS solution (1 / 4, 10 mg / mL) was added to the above solution and reacted at 37 °C for 1 h to activate the carboxyl group and improve the protein connection efficiency. The solution was centrifuged at 6000 r / min for 10 min to remove the excess PBS solution. After centrifugation, the mixture was again dispersed in 1 mL of PBS (PBS / H20 = 1 / 4). Subsequently, 20-40 μg of prostate cancer exosome-specific antibody EpCAM was added to the above solution and incubated for 1 h. The residual antibody was removed by centrifugation at 8000 r / min for 10 min, and the PBS buffer was washed for 3 times. 1% BSA solution was added to the colloidal solution and stirred at room temperature for 1 h. Finally, the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes were dispersed in 2 mL of PBS buffer and stored at 4 °C for standby use.

[0064] (3) Prostate cancer exosome detection structure assembly

[0065] Add PBS solution containing the antigen to be tested dropwise onto a copper / black phosphorus / silver nanoflower composite immunomodulator and incubate at 37°C for 2 hours to allow the immune reaction between antigen and antibody to proceed fully. Wash repeatedly with PBS solution to remove excess unreacted antigen. Then add 10 μL of NaGdF4:Yb 3+ / Er 3+ Dual-mode immunoprobes were dropped onto a copper / black phosphorus / silver nanoflower composite immunoassay substrate adsorbed with the antigen to be tested, and reacted at 37°C for 2 hours. Excess unreacted NaGdF4:Yb was then washed away. 3+ / Er 3+ Dual-mode immune probes, i.e., NaGdF4:Yb 3+ / Er 3+ Detection of prostate cancer exosomes using a dual-mode immunoassay probe and a copper / black phosphorus / silver nanoflower composite immunoassay substrate. The structure of the NaGdF4:Yb obtained after the above immunoassay was analyzed using Raman spectroscopy. 3+ / Er 3+ The complex of the immune probe and the copper / black phosphorus / silver nanoflower composite immune substrate was subjected to spectral measurement. Based on the linear relationship between the concentration of the cancer biomarker antigen and the intensity of the Raman characteristic peak, the concentration of the cancer biomarker antigen to be tested was calculated.

[0066] Figure 1 shows the NaGdF4:Yb prepared in this embodiment. 3+ / Er 3+ Scanning electron microscope image of upconversion luminescent nanoparticles. As can be seen from Figure 1, the prepared nanomaterials have a spherical shape.

[0067] Figure 4 shows the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman substrate prepared in this embodiment. As can be seen from Figure 4, silver nanoparticles are uniformly coated on the surface of black phosphorus nanosheets.

[0068] Figure 7 shows the NaGdF4:Yb prepared in this embodiment. 3+ / Er 3+ Raman spectra of upconversion luminescent nanoparticle immunoprobes and copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunoassay substrates were obtained by Raman detection of the substrates after immunoreaction with different concentrations of analyte antigens (10 mg / mL to 100 mg / mL). The figures show that the Raman characteristic spectral intensity of the labeled molecule gradually decreases with decreasing analyte antigen concentration. Even at a concentration of 100 mg / mL, the Raman characteristic peak of the labeled molecule remains significantly distinct relative to the background signal; this concentration represents the detection limit of this method for the analyte antigen.

[0069] Figure 10 shows the NaGdF4:Yb prepared in this embodiment. 3+ / Er 3+The upconversion luminescence spectrum of the upconversion luminescent nanoparticle immunoprobes and the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunosubstrate after the immunoreaction with different concentrations of the antigen to be detected (the concentration is 10 mg / mL to 100 mg / mL) is detected by luminescence. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the upconversion luminescence spectrum intensity gradually decreases, and when the concentration of the antigen to be detected decreases to 100 mg / mL, the upconversion luminescence spectrum peak is still obvious relative to the background signal. This concentration is the detection limit of the antigen to be detected in the present scheme.

[0070] Example 2

[0071] A preparation method of a dual-mode immunological structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, comprising the following steps:

[0072] (1) Adopting an electrochemical reduction method to prepare a copper / black phosphorus / silver nanoflower composite immunosubstrate

[0073] A. Black phosphorus is added to an NMP solution to prepare a black phosphorus solution with a concentration of 5 mg / mL, then the solution is ultrasonically treated at 3°C for 7 h, and after grading centrifugation at 5000 r / min for 15 min, the light brown liquid is collected, the dark brown precipitate is removed, and the collected light brown liquid is washed with deionized water and ethanol for 3 times, and then added to 15 mL of deionized water to obtain a black phosphorus nanosheet suspension;

[0074] B. After ultrasonic treatment of the black phosphorus nanosheet solution obtained in step (1) A, the precipitate is collected after centrifugal washing, and the precipitate is dissolved in deionized water to form a suspension with a concentration of 0.8 mg / mL. 15 mL is transferred to an electrolytic cell for electrodeposition. A double electrode system is used in the electrodeposition process: a platinum sheet as an anode and a copper foil as a cathode (the copper foil is cleaned with acetone, ethanol and deionized water several times), and the cathode is electrodeposited at 15 V for 8 min to obtain a copper / black phosphorus nanosheet substrate;

[0075] C. The copper / black phosphorus nanosheet substrate prepared in step (1) B is used as a working electrode, an Ag / AgCl electrode is used as a reference electrode, and a platinum sheet is used as a counter electrode. 15 mL of an aqueous solution containing 4 mmol / mL of silver nitrate is used as an electrolyte, the deposition voltage is controlled at -0.4 V, and the deposition time is controlled at 60 s for electrochemical reaction to obtain a copper / black phosphorus / silver nanoflower composite substrate;

[0076] D. The copper / black phosphorus / silver nanoflower composite substrate prepared in step (1) C was immersed in 4 mL of DMF solution for 2 h, followed by multiple washing with PBS solution, and then the substrate was transferred to 2 mL of EDC / NHS PBS solution (1:1, 10 mg / mL) and immersed at room temperature for 1 h to activate the carboxyl group. Then 20 μL of PBS solution containing prostate cancer exosome specific antibody PSMA (concentration of 4 mg / mL) was added dropwise and immersed at room temperature for 2 h, followed by multiple washing with deionized water to obtain a copper / black phosphorus / silver nanoflower composite immunosubstrate;

[0077] (2) NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes were prepared by the hydrothermal method

[0078] A. 1-3 mmol of rare earth nitrate RE(NO3)3·6H2O (RE = 70% Gd, 25% Yb, 5% Er) was mixed with 15 mL of 0.6 mol / L aqueous citric acid solution and stirred vigorously for 30 min. Then 0.3 mL of 8 mmol / mL aqueous sodium hydroxide solution was slowly added to the solution and stirred vigorously for 15 min. Then 9 mL of 2 mmol / mL aqueous sodium fluoride solution was slowly added, and the stirring was continued for 30 min. The obtained precursor solution was then transferred to a 50 mL stainless steel reactor with a polytetrafluoroethylene liner, sealed, and reacted at 180°C for 12 h. After the reaction was completed, the solution was naturally cooled to room temperature. The reaction product was centrifuged and washed repeatedly with ethanol and deionized water. Finally, the product was dried in air at 80°C for 12 h to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticles.

[0079] B. 0.2 mmol of the NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (2) A were dissolved in 8 mL of ethanol solution to obtain a NaGdF4:Yb 3+ / Er 3+ nanoparticle solution. Then 15 mL of R6G (10 -3 mol / L) ethanol solution was added and stirred at room temperature for 12 h. The excess unreacted R6G was removed by centrifugation at 7000 r / min for 10 min, and the product was washed repeatedly with ethanol and stored in 5 mL of deionized water;

[0080] C.2 mL of NHS / EDC PBS solution (1 / 4, 10 mg / mL) was added to the above solution, and the carboxyl group was activated to improve the protein connection efficiency by reacting for 1 h in a 37°C shaking table. After centrifugation at 6000 r / min for 10 min, the excess PBS solution was removed. After centrifugation, the mixture was again dispersed in 1 mL of PBS (PBS / H2O = 1 / 4). Subsequently, 30 μg of prostate cancer exosome specific antibody EpCAM was added to the above solution, and incubated for 1 h. After centrifugation at 8000 r / min for 10 min, the residual antibody was removed, and PBS buffer was washed for 3 times. 1% BSA solution was added to the colloidal solution and stirred for 1 h at room temperature. Finally, the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ The upconversion nanoparticle immunoprobes were dispersed in 2 mL of PBS buffer and stored at 4°C for standby use.

[0081] (3) Prostate cancer exosome detection structure assembly

[0082] The PBS solution containing the antigen to be tested was added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate, and was placed at 37°C for 2 h to allow the immunoreaction between the antigen and the antibody to proceed fully. The excess unreacted antigen to be tested was removed by washing with PBS solution multiple times. Then, 15 μL of NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes were added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate adsorbed with the antigen to be tested, and were reacted at 37°C for 2 h. The excess unreacted NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes, i.e., NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes and the copper / black phosphorus / silver nanoflower composite immunosubstrate were used for prostate cancer exosome detection structure. The NaGdF4:Yb 3+ / Er 3+ The complex of the immunoprobes and the copper / black phosphorus / silver nanoflower composite immunosubstrate was subjected to spectral measurement. According to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak, the concentration of the cancer marker antigen to be tested was calculated.

[0083] Figure 2 shows the scanning electron microscope photos of the NaGdF4:Yb 3+ / Er 3+ The scanning electron microscope photos of the upconversion luminescent nanoparticles. As can be seen from Figure 1, the prepared nanomaterials have a spherical shape.

[0084] Figure 5 shows the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman substrate prepared in this embodiment. As can be seen from Figure 4, silver nanoparticles are uniformly coated on the surface of black phosphorus nanosheets.

[0085] Figure 8 is the upconversion luminescence spectrum of the NaGdF4:Yb 3+ / Er 3+ Figure 10 shows the Raman spectrum of the upconversion luminescent nanoparticle immunoprobes and copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunosubstrate after immunoreaction with different concentrations of the antigen to be detected (concentrations of 10 mg / mL to 100 mg / mL) and Raman detection of the substrate. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and until the concentration of the antigen to be detected decreases to 100 mg / mL, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal. This concentration is the detection limit of the antigen to be detected in this scheme.

[0086] Figure 11 is the upconversion luminescence spectrum of the NaGdF4:Yb 3+ / Er 3+ Figure 10 shows the Raman spectrum of the upconversion luminescent nanoparticle immunoprobes and copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunosubstrate after immunoreaction with different concentrations of the antigen to be detected (concentrations of 10 mg / mL to 100 mg / mL) and Raman detection of the substrate. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and until the concentration of the antigen to be detected decreases to 100 mg / mL, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal. This concentration is the detection limit of the antigen to be detected in this scheme.

[0087] Example 3

[0088] A method for preparing a dual-mode immunological structure based on a surface-enhanced Raman substrate and an upconversion luminescent probe, comprising the following steps:

[0089] (1) Using an electrochemical reduction method to prepare a copper / black phosphorus / silver nanoflower composite immunosubstrate

[0090] A. Black phosphorus was added to an NMP solution to prepare a 10 mg / mL black phosphorus solution, which was then ultrasonically treated at 3°C for 8 h using a cell ultrasonic crusher. After fractionation centrifugation at 5000 r per min for 15 min, the light brown liquid was collected, and the dark brown precipitate was removed. The collected light brown liquid was washed with deionized water and ethanol three times, and then added to 20 mL of deionized water to obtain a black phosphorus nanosheet suspension;

[0091] B. The black phosphorus nanosheet solution obtained in step (1) A was ultrasonically treated, and the precipitate was collected after centrifugal washing. The precipitate was dissolved in deionized water to form a suspension with a concentration of 1.0 mg / mL. 10-20 mL of the suspension was transferred to an electrolytic cell for electrodeposition. A two-electrode system was used in the electrodeposition process: a platinum sheet as the anode and a copper foil as the cathode (the copper foil was cleaned with acetone, ethanol and deionized water several times). The cathode was electrodeposited at a voltage of 20 V for 10 min to obtain a copper / black phosphorus nanosheet substrate;

[0092] C. The copper / black phosphorus nanosheet substrate prepared in step (1) B was used as the working electrode, an Ag / AgCl electrode was used as the reference electrode, and a platinum sheet was used as the counter electrode. 20 mL of an aqueous solution containing 6 mmol / mL of silver nitrate was used as the electrolyte. The deposition voltage was controlled at -0.6 V, and the deposition time was controlled at 100 s to perform the electrochemical reaction. A copper / black phosphorus / silver nanoflower composite substrate was obtained.

[0093] D. The copper / black phosphorus / silver nanoflower composite substrate prepared in step (1) C was soaked in 6 mL of a DMF solution for 2 h, and then washed with a PBS solution several times. Subsequently, the substrate was transferred to 3 mL of a PBS solution containing EDC / NHS (1:1, 10 mg / mL) and soaked at room temperature for 1 h to activate the carboxyl group. Then, 30 μL of a PBS solution containing a prostate cancer exosome-specific antibody PSMA (concentration: 2-6 mg / mL) was added dropwise, and the substrate was soaked at room temperature for 2 h. Subsequently, the substrate was washed with deionized water several times to obtain a copper / black phosphorus / silver nanoflower composite immunosubstrate.

[0094] (2) NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes

[0095] A. 1-3 mmol of rare earth nitrate RE(NO3)3·6H2O (RE = 80% Gd, 15% Yb, 5% Er) was mixed with 20 mL of a 0.8 mol / L citric acid aqueous solution and stirred vigorously for 30 min. Then, 0.4 mL of a 10 mmol / mL sodium hydroxide aqueous solution was slowly added to the above solution and stirred vigorously for 15 min. Subsequently, 10 mL of a 3 mmol / mL sodium fluoride aqueous solution was slowly added, and the stirring was continued for 30 min. The obtained precursor solution was then transferred to a 60 mL stainless steel reactor with a polytetrafluoroethylene liner and sealed. The reactor was placed in an oven at 180°C and reacted for 12 h. After the reaction was completed, the reactor was naturally cooled to room temperature. The reaction product was centrifuged and washed repeatedly with ethanol and deionized water. Finally, the product was dried in air at 80°C for 12 h to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticles.

[0096] B. The NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (2) A were taken 0.3 mmol and dissolved in 10 mL ethanol solution to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticle solution, then, 20 mL R6G (10 -3 mol / L) ethanol solution was added and mixed, after stirring at room temperature for 12 h, the excess unreacted R6G was removed by centrifugation at 7000 r / min for 10 min and washed with ethanol for several times, and then stored in 5 mL deionized water;

[0097] C. 3 mL NHS / EDC PBS solution (1 / 4, 10 mg / mL) was added to the above solution, and the carboxyl group was activated to improve the protein connection efficiency by reacting for 1 h in a 37 °C shaking table. After centrifugation at 6000 r / min for 10 min, the excess PBS solution was removed. After centrifugation, the mixture was again dispersed in 1 mL PBS (PBS / H2O = 1 / 4). Then, 20-40 μg of prostate cancer exosome specific antibody EpCAM was added to the above solution, incubated for 1 h, centrifuged at 8000 r / min for 10 min, and the residual antibody was removed. PBS buffer was washed for 3 times. 1% BSA solution was added to the colloidal solution and stirred for 1 h at room temperature. Finally, the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes were dispersed in 2 mL PBS buffer and stored at 4 °C for standby use.

[0098] (3) Prostate cancer exosome detection structure assembly

[0099] The PBS solution containing the antigen to be detected was added dropwise to the copper / black phosphorus / silver nanoflower composite immunobase, which was placed at 37 °C for 2 h to allow the immune reaction between the antigen and the antibody to proceed fully. The excess unreacted antigen to be detected was removed by washing with PBS solution for several times. Then, 20 μL NaGdF4:Yb 3+ / Er 3+ bimodal immunoprobes were added dropwise to the copper / black phosphorus / silver nanoflower composite immunobase adsorbed with the antigen to be detected, and reacted at 37 °C for 2 h. The excess unreacted NaGdF4:Yb 3+ / Er 3+ bimodal immunoprobes were obtained, which were based on NaGdF4:Yb 3+ / Er 3+ bimodal immunoprobes and the copper / black phosphorus / silver nanoflower composite immunobase prostate cancer exosome detection structure. The NaGdF4:Yb3+ / Er 3+ The complex of the immunological probe and the copper / black phosphorus / silver nanoflower composite immunological substrate is subjected to spectral measurement, and the concentration of the cancer marker antigen to be detected is calculated according to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak.

[0100] Figure 3 shows the NaGdF4:Yb 3+ / Er 3+ The scanning electron microscope photos of the upconversion luminescent nanoparticles. As can be seen from Figure 1, the prepared nanomaterials are spherical in shape.

[0101] Figure 6 shows the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman substrate prepared in the embodiment. As can be seen from Figure 4, the silver nanoparticles are uniformly coated on the surface of the black phosphorus nanosheet.

[0102] Figure 9 is the upconversion luminescence spectrum of the NaGdF4:Yb 3+ / Er 3+ The Raman spectrum of the upconversion luminescent nanoparticle immunological probe and the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunological substrate after the immunological reaction with different concentrations of the antigen to be detected (the concentration is 10 mg / mL to 100 mg / mL) and the Raman detection of the substrate. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the Raman characteristic spectrum intensity of the labeled molecules gradually decreases, and when the concentration of the antigen to be detected decreases to 100 mg / mL, the Raman characteristic peak of the labeled molecules is still very obvious relative to the background signal. This concentration is the detection limit of the antigen to be detected in the present scheme.

[0103] Figure 12 is the upconversion luminescence spectrum of the NaGdF4:Yb 3+ / Er 3+ The upconversion luminescence spectrum of the upconversion luminescent nanoparticle immunological probe and the copper / black phosphorus / silver nanoflower composite surface-enhanced Raman immunological substrate after the immunological reaction with different concentrations of the antigen to be detected (the concentration is 10 mg / mL to 100 mg / mL) and the luminescence detection of the substrate. As can be seen from the figure, as the concentration of the antigen to be detected decreases, the upconversion luminescence spectrum intensity gradually decreases, and when the concentration of the antigen to be detected decreases to 100 mg / mL, the upconversion luminescence spectrum peak is still very obvious relative to the background signal. This concentration is the detection limit of the antigen to be detected in the present scheme.

[0104] It can be illustrated by the above examples that the preparation method of the dual-mode immune structure based on the surface-enhanced Raman substrate and the upconversion luminescence probe and the dual-mode immune structure based on the surface-enhanced Raman substrate and the upconversion luminescence probe prepared by the preparation method can be applied to the detection technology field of cancer, and comprehensively utilize the advantages of high upconversion luminescence efficiency, good stability and strong anti-interference ability, and the characteristics of strong SERS spectrum fingerprint recognition ability and high detection sensitivity, and are beneficial to realize efficient and accurate screening and identification of cancer markers in clinic. Moreover, the operation is simple, the use is convenient, the screening is fast and accurate, and the dual-mode immune structure has good application prospect in clinical detection.

[0105] It should be understood by those skilled in the art that the embodiments of the application shown in the above description and the drawings are only examples and do not limit the application. The purpose of the application has been fully and effectively achieved. The function and structural principle of the application has been shown and described in the embodiments, and the embodiments of the application can be any modification or modification without departing from the principle.

Claims

1. A method for preparing a dual-mode immunostructure based on a surface- enhanced Raman substrate and an upconversion luminescence probe, characterized in that, Comprising the following steps: (S10) preparing a copper / black phosphorus / silver nanoflower composite immunization substrate; And (S20) Preparation of NaGdF4:Yb 3+ / Er 3+ Upconversion nanoparticle immunoprobes.

2. The method for preparing a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe according to claim 1, wherein in the step (S10), a copper / black phosphorus / silver nanoflower composite immune substrate is prepared by an electrochemical reduction method, and in the step (S20), NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immune probes are prepared by a hydrothermal method.

3. A method for preparing a dual-mode immunological structure based on a surface- enhanced Raman substrate and upconversion luminescence probes according to claim 1, wherein said step (S10) comprises the following steps: (S101) adding black phosphorus to an NMP solution to prepare a black phosphorus solution, ultrasonic treatment, centrifugation, collecting light brown liquid, washing and then adding to deionized water to obtain a black phosphorus nanosheet suspension; (S102) ultrasonic treatment of the black phosphorus nanosheet solution obtained in step (S101), collecting the precipitate after washing and centrifugation, dissolving the precipitate in deionized water to form a suspension, and electrodeposition to obtain a copper / black phosphorus nanosheet substrate; (S103) taking the copper / black phosphorus nanosheet substrate prepared in step (S102) as a working electrode, an Ag / AgCl electrode as a reference electrode, a platinum sheet as a counter electrode, and an aqueous silver nitrate solution as an electrolyte to perform an electrochemical reaction to obtain a copper / black phosphorus / silver nanoflower composite substrate; (S104) soaking the copper / black phosphorus / silver nanoflower composite substrate prepared in step (S103) in a DMF solution, washing with a PBS solution, then transferring the above substrate to an EDC / NHS PBS solution, soaking, then adding a PBS solution containing the antibody PSMA, soaking, and washing with deionized water to obtain a copper / black phosphorus / silver nanoflower composite immunization substrate.

4. The method for preparing a dual-mode immunological structure based on a surface- enhanced Raman substrate and upconversion luminescence probes according to claim 3, wherein said step (S20) comprises the following steps: (S201) mixing a rare earth nitrate RE(NO3)3-6H2O (RE=60-80% Gd, 15-35% Yb, 5% Er) with an aqueous solution of citric acid, stirring, adding an aqueous solution of sodium hydroxide to the above solution, stirring, adding an aqueous solution of sodium fluoride, continuing to stir, obtaining a precursor solution, transferring to a stainless steel reactor with a polytetrafluoroethylene liner and reacting in a closed state. After the reaction is completed, cooling, centrifuging, taking out the reaction product and washing with ethanol and deionized water, drying, obtaining NaGdF4:Yb 3+ / Er 3+ nanoparticles; (S202) dissolving the NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in step (201) in an ethanol solution to obtain a NaGdF4:Yb 3+ / Er 3+ nanoparticle solution, then adding an R6G ethanol solution, mixing, stirring, centrifuging and washing with ethanol and storing in deionized water; (S203) To the above solution, a PBS solution of NHS / EDC was added, and the reaction was carried out in a shaker. The mixture was centrifuged, and then dispersed in PBS again. Subsequently, an antibody EpCAM was added to the above solution, and incubated. After centrifugation, the residual antibody was removed, and the PBS buffer was washed. A BSA solution was added to the colloid, and stirred. The colloid was stored at room temperature. Finally, the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ The upconversion nanoparticle immunoprobes were dispersed in PBS buffer, and stored at 4°C for standby use.

5. The preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe according to claim 4, wherein in the step (S101), the concentration of the black phosphorus solution is 1-10 mg / mL, ultrasonic treatment is performed at 3°C for 6-8 h, light brown liquid is collected after fractional centrifugation at 5000 r / min for 15 min, and the deep brown precipitate is removed, and the collected light brown liquid is washed with deionized water and ethanol.

6. The preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe according to claim 5, wherein in the step (S102), electrodeposition is performed in an electrolytic cell, and a two-electrode system is used in the electrodeposition process, wherein the platinum sheet is an anode and the copper foil is a cathode, and the copper foil is cleaned with acetone, ethanol, and deionized water multiple times, and electrodeposition is performed at a voltage of 10-20 V for 5-10 min to obtain a copper / black phosphorus nanosheet substrate.

7. The preparation method of a dual-mode immune structure based on a surface-enhanced Raman substrate and an upconversion luminescence probe according to claim 6, wherein in the step (S103), the concentration of the silver nitrate electrolyte is 2-6 mmol / mL, the deposition voltage is -0.2 V to -0.6 V, and the deposition time is 20-100 s, and in the step (S104), the copper / black phosphorus / silver nanoflower composite substrate prepared in step (S103) is soaked in a DMF solution for 2 h, then washed multiple times with a PBS solution, and then transferred to an EDC / NHS PBS solution (1:1, 10 mg / mL) to soak for 1 h at room temperature to activate the carboxyl group, then a PBS solution containing the prostate cancer exosome specific antibody PSMA is added dropwise, the concentration is 2-6 mg / mL, and the soaking is performed at room temperature for 2 h, and the copper / black phosphorus / silver nanoflower composite immunization substrate is obtained after multiple washing with deionized water.

8. The method of claim 4 to 7, wherein in the step (S201), the concentration of the aqueous citric acid solution is 0.4-0.8 mol / L, the concentration of the aqueous sodium hydroxide solution is 5-10 mmol / mL, the concentration of the aqueous sodium fluoride solution is 1-3 mmol / mL, the reaction is carried out at 180°C for 12 h in a stainless steel reactor with a polytetrafluoroethylene liner under airtight condition, the reaction product is obtained by centrifugation and washed repeatedly with ethanol and deionized water, and the product is dried in air at 80°C for 12 h to obtain NaGdF4:Yb 3+ / Er 3+ nanoparticles; in the step (S202), the NaGdF4:Yb 3+ / Er 3+ nanoparticles prepared in the step (201) are dissolved in 5-10 mL of ethanol solution at a concentration of 0.1-0.3 mmol to obtain a NaGdF4:Yb 3+ / Er 3+ nanoparticle solution, then 10-20 mL of an R6G (10 -3 mol / L) ethanol solution is added and mixed, the mixture is stirred at room temperature for 12 h, the excess unreacted R6G is removed by centrifugation at 7000 r / min for 10 min, the mixture is washed repeatedly with ethanol, and then stored in 5 mL of deionized water; in the step (S203), 1-3 mL of a PBS solution of NHS / EDC (1 / 4, 10 mg / mL) is added to the above solution, the carboxyl group is activated by reaction at 37°C for 1 h to improve the protein connection efficiency, the excess PBS solution is removed by centrifugation at 6000 r / min for 10 min, the mixture is dispersed again in 1 mL of PBS (PBS / H2O = 1 / 4), 20-40 μg of prostate cancer exosome specific antibody EpCAM is added to the above solution, incubated for 1 h, centrifuged at 8000 r / min for 10 min to remove the residual antibody, washed with PBS buffer for 3 times, 1% of a BSA solution is added to the colloid and stirred, and the mixture is stored at room temperature for 1 h, and finally the obtained EpCAM-NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunoprobes are dispersed in 2 mL of PBS buffer and stored at 4°C for standby use.

9. A dual-mode immunostructure based on surface-enhanced Raman substrate and upconversion luminescence probe, characterized in that, Comprising: Copper / black phosphorus / silver nanoflower composite immunosubstrates and nagdf4:yb 3+ / er 3+ Upconversion nanoparticle immunoprobes.

10. The dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe according to claim 9, wherein the dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe is prepared by the preparation method according to any one of claims 1 to 8.

11. The dual-mode immunosructure based on surface-enhanced Raman substrate and upconversion luminescence probe according to claim 10, wherein the immunosubstrate and immunoprobe are assembled in the application of detection process, wherein, The PBS solution containing the antigen to be tested is added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate, which is placed at 37℃ for 2h, so that the immune reaction between the antigen and the antibody is fully carried out, and the excess unreacted antigen to be tested is removed by washing with PBS solution for multiple times; then 10-20μL NaGdF4:Yb 3+ / Er 3+ The dual-mode immunoprobes are added dropwise to the copper / black phosphorus / silver nanoflower composite immunosubstrate adsorbed with the antigen to be tested, and reacted at 37℃ for 2h, and the excess unreacted NaGdF4:Yb 3+ / Er 3+ Dual-mode immunoprobes, i.e. NaGdF4:Yb 3+ / Er 3+ Dual-mode immunoprobes and the prostate cancer exosome detection structure of the copper / black phosphorus / silver nanoflower composite immunosubstrate, the NaGdF4:Yb 3+ / Er 3+ immunoprobes and the copper / black phosphorus / silver nanoflower composite immunosubstrate are measured by spectroscopy, and the concentration of the cancer marker antigen to be tested is calculated according to the linear relationship between the concentration of the cancer marker antigen and the intensity of the Raman characteristic peak.

12. The dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe according to claim 11, wherein the antigen is prostate specific antigen PSA, alpha-fetoprotein AFP, ferritin antigen or carbohydrate antigen CA199.

13. Use of a dual-mode immunostructure based on a surface-enhanced Raman substrate and an upconversion luminescence probe, characterized in that, The dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe is suitable for application in tumor detection.

14. The use of the dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe according to claim 1, wherein the dual-mode immunostucture based on surface-enhanced Raman substrate and upconversion luminescence probe is suitable for application in prostate cancer, colorectal cancer, ovarian cancer or pancreatic cancer detection.

15. The use of the dual-mode immunological structure based on surface-enhanced Raman substrate and upconversion luminescence probe according to claim 1, wherein the dual-mode immunological structure based on surface-enhanced Raman substrate and upconversion luminescence probe comprises a copper / black phosphorus / silver nanoflower composite immunological substrate and NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunological probe, wherein the copper / black phosphorus / silver nanoflower composite immunological substrate is prepared by electrochemical reduction, and the NaGdF4:Yb 3+ / Er 3+ upconversion nanoparticle immunological probe is prepared by hydrothermal method.

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