Preparation method and application of silver fiber substrate for detecting alpha-synuclein molecules of Parkinson's disease

Abstract

The invention discloses a preparation method of a silver fiber substrate for detecting alpha-synuclein molecules of Parkinson's disease. The preparation method specifically comprises the following steps: step 1, cleaning silver fibers; 2, physically scraping the cleaned silver fibers to form a substrate with strip-shaped scratches or net-shaped scratches on the surfaces of the silver fibers, cleaning and drying the silver fibers, and then putting the dried silver fibers into absolute ethyl alcohol to be stored; and step 3, putting the silver fiber stored in the absolute ethyl alcohol in the step 2 into concentrated sulfuric acid, reacting for a certain time under a water bath condition, and washing with deionized water to obtain the silver fiber SERS substrate. The problem that in the prior art, the preparation process of the silver nanoparticle aggregate SERS substrate is complex is solved. The invention further discloses application of the silver fiber substrate for detecting the alpha-synuclein molecules of the Parkinson's disease in SERS detection of biomolecules, and a detection means is provided for detecting and distinguishing PD disease patients, PSP disease patients and MSA disease patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, health and surface enhanced Raman scattering detection methods, and relates to a preparation method of a silver fiber substrate for detecting Parkinson's disease α-synuclein molecules. The present invention also relates to the application of the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules in SERS detection of biological molecules. Background Art

[0002] Surface enhanced Raman scattering (SERS) is a nanotechnology-based spectral analysis method that can significantly enhance the Raman scattering signal of molecules, thereby achieving high-sensitivity detection of extremely low concentration substances. SERS is a commonly used detection technique that can achieve single-molecule SERS nanoparticle detection in some cases when molecules are adsorbed on corrugated metal surfaces.

[0003] SERS technology plays an important role in the detection of α-synuclein molecules. SERS provides a very sensitive, non-destructive target detection platform technology that can achieve high-sensitivity, high-precision, and water-free single-molecule detection, and can reveal the structural information of the analyte. The application field of SERS technology is very wide, covering biomedicine, environmental monitoring, food safety, cultural heritage protection and other fields. In biomedicine, SERS can be used to detect biological molecules, pathogens, drugs and their metabolites, and help study the chemical processes and structural changes inside cells.

[0004] The existing SERS technology basically uses silver nanoparticle aggregate SERS substrate for detection, but the preparation process of the silver nanoparticle aggregate SERS substrate is complicated, and the use of the silver nanoparticle aggregate SERS substrate for experiments has high requirements on experimental equipment and experimental conditions.

[0005] In addition, Parkinson's disease (PD) is a progressive movement disorder whose diagnosis is difficult, especially in the early stages, because its symptoms overlap with other diseases, such as essential tremor, progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) (Nature 2020: 578, 273–277). In addition, apart from limited clinical and neuroimaging evaluations (which are often expensive), there is currently no established objective and effective method to detect and distinguish between PD patients, PSP patients, and MSA patients (Brain 2010: 133, 713–726) (Nature Medicine 2022: 28, 2207–2215). Summary of the invention

[0006] The purpose of the present invention is to provide a method for preparing a silver fiber substrate for detecting α-synuclein molecules of Parkinson's disease, thereby solving the problem of complex preparation process of a silver nanoparticle aggregate SERS substrate in the prior art.

[0007] Another object of the present invention is to provide a silver fiber substrate for detecting Parkinson's disease α-synuclein molecules in the SERS detection of biological molecules, so as to provide a detection means for detecting and distinguishing PD disease patients, PSP disease patients and MSA disease patients.

[0008] The technical solution adopted by the present invention is a method for preparing a silver fiber substrate for detecting Parkinson's disease α-synuclein molecules, which is specifically implemented according to the following steps:

[0009] Step 1, cleaning the silver fiber or silver wire;

[0010] Step 2, physically scratching the silver fiber cleaned in step 1 to form a substrate with strip scratches or mesh scratches on its surface, then cleaning and drying the physically scratched silver fiber, and then storing the dried silver fiber in anhydrous ethanol;

[0011] Step 3, placing the silver fiber stored in anhydrous ethanol in step 2 into concentrated sulfuric acid, then reacting for a certain period of time in a water bath, and then rinsing with deionized water to obtain a silver fiber SERS substrate.

[0012] Preferably, step 1 is specifically:

[0013] The silver fiber was placed in an ultrasonic cleaner for cleaning for a certain period of time, and then rinsed with anhydrous ethanol.

[0014] In step 1, the silver fiber is cleaned using an ultrasonic cleaner for 10-30 minutes and then rinsed once using anhydrous ethanol.

[0015] In step 2, the silver fibers cleaned in step 1 are cut into pieces with a length of 0.2-1 cm.

[0016] Preferably, in step 2, sandpaper is used to scratch the surface of the silver fiber so that the surface has strip scratches and mesh scratches as a base.

[0017] Preferably, step 2 uses 800-grit sandpaper for physical scraping as follows:

[0018] Preparation of a silver substrate with strip scratches: Use sandpaper to scratch along the length of the silver fibers to prepare the first type of silver substrate with strip scratches;

[0019] Preparation of a silver substrate with mesh scratches: On the basis of preparing a silver substrate with strip scratches, the scratching direction is changed to prepare strip scratches that intersect with the first strip scratches, thereby obtaining a silver substrate with mesh scratches.

[0020] Preferably, in step 2, the physically scraped silver fibers are cleaned with an ultrasonic cleaner, and after cleaning, they are dried and stored in anhydrous ethanol.

[0021] Preferably, in step 3, the water bath temperature is 60-90° C., and the water bath reaction time of the silver fiber in concentrated sulfuric acid is 1-10 min.

[0022] The second technical solution adopted by the present invention is: the silver fiber substrate for detecting Parkinson's disease (PD) α-synuclein molecules prepared by the preparation method is used in the SERS detection of biological molecules.

[0023] Preferably, the prepared silver fiber substrate for detecting Parkinson's disease α-synuclein (α-Syn) molecules is immersed in saliva samples of PD patients, Progressive Supranuclear Palsy (PSP) patients and Multiple System Atrophy (MSA) patients to adsorb α-Syn molecules, and a 785nm laser is used to perform SERS detection on the silver fiber substrate, and the SERS spectra of α-Syn in the saliva samples of PD patients, PSP patients and MSA patients are compared to obtain the detection results.

[0024] The beneficial effects of the present invention are:

[0025] The present invention only needs to use an ultrasonic cleaner, ordinary sandpaper and concentrated sulfuric acid to prepare a silver fiber SERS substrate. The method is simple. When the method is used for experimental application, the experimental equipment and conditions are low. In the embodiment of the present invention, the smooth silver fiber or silver wire is polished by sandpaper of different meshes (roughness), malachite green (MG) is used as a probe molecule to detect the performance of the silver fiber substrate, and the silver fiber substrate is irradiated with light of a wavelength of 785nm. By comparing the difference between the SERS spectra of the MG concentration of the silver fiber substrate prepared by different polishing methods and the SERS spectra of the standard MG concentration, the SERS performance difference of each substrate is analyzed, and the silver fiber substrate is chemically corroded by heating with concentrated sulfuric acid. The SERS performance of the silver fiber substrate prepared by the present invention is tested, which proves that the method of the present invention can prepare a silver fiber substrate with better SERS performance. At the same time, the silver fiber substrate prepared by the present invention can detect and distinguish PD disease, PSP disease and MSA disease, thereby providing a reliable diagnostic basis for symptomatic treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a photograph of the silver fiber (length 1 cm) corroded by concentrated sulfuric acid prepared in Example 3 of the present invention;

[0027] Figure 2 is an X-ray diffraction pattern of the silver fiber of the silver fiber SERS substrate prepared in Example 3 of the present invention;

[0028] Figure 3 is the electromagnetic field distribution of the silver fiber SERS substrate prepared in Example 3 of the present invention;

[0029] Figure 4 This is a comparison of the strip scratches and the mesh scratches obtained by sanding with 80-grit sandpaper in Example 2 using a scanning electron microscope;

[0030] Figure 5 is a scanning electron microscope image of the silver fiber SERS substrate of the present invention after being polished twice with sandpaper of different mesh sizes;

[0031] Figure 6 The present invention is a silver fiber SERS substrate polished 2 times and 4 times with 800-grit sandpaper, and the electron microscope image is 200 times magnified and the scanning electron microscope image is 500 times magnified;

[0032] Figure 7 The MG10 used in Comparative Example 1 and Example 3 of the present invention -5 Raman spectra of silver fiber substrates polished 4 times with 320-mesh, 800-mesh, and 1200-mesh sandpapers of different concentrations;

[0033] Figure 8 The silver nano-substrate MG10 in Example 3 of the present invention -5 Raman spectra of concentration;

[0034] Fig. 9 is an electron microscope comparison of the silver fiber SERS substrate before and after being corroded by concentrated sulfuric acid in Example 2 and Example 3 of the present invention;

[0035] Fig.10 The silver fiber SERS substrate MG10 after 1 min of grinding and corrosion in Example 3 and non-grinding and corrosion in Comparative Example 4 -5 Raman spectra;

[0036] Fig.11 MG10 of different corrosion times of the silver fiber substrate polished 4 times with 800-grit sandpaper in Example 5 -5 Raman spectroscopy;

[0037] Fig.12 The silver-based MG10 was polished with 800-grit sandpaper for different times in Example 4. -5Raman spectroscopy;

[0038] Fig.13 The MG10 is the silver fiber substrate polished 4 times with 800-grit sandpaper and corroded for 5 minutes in Example 7, and then cleaned with ultrasonic and deionized water. -5 Raman spectroscopy;

[0039] Fig.14 The silver substrate polished 4 times with different mesh sizes in Example 1 was polished on MG10 -5 Raman spectra under

[0040] Fig.15 is the Raman spectra of MG solutions of different concentrations of the silver fiber substrate polished 4 times and etched for 5 min using 800-grit sandpaper in Example 8;

[0041] Fig.16 The MG10 of Example 8 is a silver fiber substrate that was polished 4 times with 800 mesh and etched for 5 minutes. -5 Waterfall chart of

[0042] Fig.17 This is the SERS spectrum of α-synuclein molecules in saliva samples of Parkinson's patients using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 50mW;

[0043] Fig.18 This is the SERS spectrum of α-synuclein molecules in saliva samples of patients with progressive supranuclear palsy (PSP) using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 50mW;

[0044] Fig.19 This is the SERS spectrum of α-synuclein molecules in saliva samples of patients with multiple system atrophy (MSA) using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 50mW;

[0045] Fig. 20 This is a bar graph of the ratio of the β-folded structure to the α-amorphous structure of α-synuclein molecules in saliva samples of patients with Parkinson's disease (PD), progressive supranuclear palsy (PSP), and multiple system atrophy (MSA) using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 50mW.

[0046] Fig.21 This is a SERS spectrum of α-synuclein molecules in saliva samples of Parkinson's patients using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 100mW;

[0047] Fig. 22This is the SERS spectrum of α-synuclein molecules in saliva samples of patients with progressive supranuclear palsy using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 100mW;

[0048] Fig.23 This is the SERS spectrum of α-synuclein molecules in saliva samples of patients with multiple system atrophy using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 100mW;

[0049] Fig.24 This is a handheld Raman spectrometer that uses an excitation light wavelength of 785nm and a power of 100 milliwatts. It is a bar graph of the ratio of the β-folded structure to the α-amorphous structure of α-synuclein molecules in saliva samples of Parkinson's patients, progressive supranuclear palsy patients, and multiple system atrophy patients. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0051] The preparation method of the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules of the present invention is specifically implemented according to the following steps:

[0052] Step 1, cleaning the silver fiber, specifically: placing the silver fiber in an ultrasonic cleaner for cleaning for 10-30 minutes to remove surface pollutants and moisture, and then rinsing once with anhydrous ethanol for further cleaning;

[0053] Step 2, cutting 0.2-1 cm of the silver fiber cleaned in step 1, using 800-mesh sandpaper to scratch the surface of the silver fiber so that the surface has strip scratches or mesh scratches as a base, and then cleaning the physically scratched silver fiber with an ultrasonic cleaner, and storing it in anhydrous ethanol after cleaning and drying to prevent oxidation;

[0054] Step 3, placing the silver fiber stored in anhydrous ethanol in step 2 into concentrated sulfuric acid, reacting for 1-10 minutes at a water bath temperature of 60-90° C., and then rinsing with deionized water to obtain a silver fiber SERS substrate.

[0055] Among them, step 2 uses sandpaper to perform physical scraping specifically as follows:

[0056] Preparation of a silver substrate with strip scratches: Use sandpaper to scratch along the length of the silver fibers to prepare the first type of silver substrate with strip scratches;

[0057] Preparation of a silver substrate with mesh scratches: On the basis of preparing a silver substrate with strip scratches, the scratching direction is changed to prepare strip scratches that intersect with the first strip scratches, thereby obtaining a silver substrate with mesh scratches.

[0058] Use sandpaper to polish several times to obtain a multi-polished silver base.

[0059] The silver fiber substrate for detecting Parkinson's disease α-synuclein molecules prepared by the preparation method of the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules is used in SERS detection of biological molecules.

[0060] During application, the prepared silver fiber substrate for detecting Parkinson's disease α-synuclein (α-Syn) molecules is immersed in saliva samples of PD patients, Progressive Supranuclear Palsy (PSP) patients and Multiple System Atrophy (MSA) patients to adsorb α-Syn molecules. The silver fiber substrate is subjected to SERS detection using a 785nm laser, and the SERS spectral differences of α-Syn in the saliva samples of PD patients, PSP patients and MSA patients are compared to obtain the detection results.

[0061] Example 1

[0062] The preparation process of polishing the silver fiber SERS substrate using 800-grit sandpaper specifically includes the following steps:

[0063] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0064] Step 2: Physical polishing: use 800-grit sandpaper to polish 4 times along the length of the silver fiber to form uniform strip scratches;

[0065] Step 3: After polishing, the polished silver fiber substrate is cleaned again with an ultrasonic cleaner to remove residues, and then dried and stored in anhydrous ethanol to prevent oxidation.

[0066] Example 2

[0067] In this example, MG (malachite green) solutions of different concentrations were used to test the performance of the prepared silver fiber SERS substrate, specifically:

[0068] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0069] Step 2: Physical polishing: Use 800-grit sandpaper to polish 4 times along the length of the silver fiber to form uniform strip scratches.

[0070] Step 3: The polished silver fiber substrate was soaked in MG solutions of different concentrations (10^-5mol / L to 10^-10mol / L) for 10 minutes at each concentration.

[0071] Step 4: Use a 785 nm laser to perform SERS detection on the immersed silver fiber substrate, and record and analyze the obtained SERS spectrum data.

[0072] Example 3

[0073] This embodiment describes the chemical corrosion treatment process of the silver fiber SERS substrate, which specifically includes the following steps:

[0074] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0075] Step 2: Physical polishing: Use 800-grit sandpaper to polish 4 times along the length of the silver fiber to form uniform strip scratches.

[0076] Step 3: Chemical etching: Place the polished silver fiber substrate into concentrated sulfuric acid preheated to 90°C, react for 1 minute, and then rinse with deionized water to form a hydrophilic surface.

[0077] Step 4: SERS performance verification: Use 10^-5mol / L MG solution to perform SERS test on the corroded silver fiber substrate to verify the effect of chemical corrosion on SERS signal enhancement.

[0078] The photo of the silver fiber prepared in this example after being corroded by concentrated sulfuric acid is shown in FIG. Figure 1 As shown, the X-ray diffraction pattern of the silver fiber of the prepared silver fiber SERS substrate is as follows Figure 2 As shown in Figure 2, the electromagnetic field distribution of the silver fiber SERS substrate is Figure 3 shown.

[0079] Example 4

[0080] This example discusses the effect of different polishing times on the performance of the silver fiber SERS substrate, specifically including the following steps:

[0081] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0082] Step 2: Use 800-grit sandpaper to polish the silver fiber 2 times and 4 times respectively, and compare the effects of different polishing times on the surface morphology and SERS performance.

[0083] Step 3: After polishing, the polished silver fiber substrate is cleaned again with an ultrasonic cleaner to remove residues, and then dried and stored in anhydrous ethanol to prevent oxidation.

[0084] Step 4: Perform SERS performance tests on silver fiber substrates with different polishing times, using 10^-5mol / L MG solution, and analyze the effect of polishing times on SERS signal enhancement.

[0085] Example 5

[0086] This example describes the application of the silver fiber SERS substrate in an actual saliva sample, which specifically includes the following steps:

[0087] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0088] Step 2: Physical polishing: Use 800-grit sandpaper to polish 4 times along the length of the silver fiber to form uniform strip scratches.

[0089] Step 3: The polished and chemically etched silver fiber substrate is immersed in pre-treated saliva samples of PD (Parkinson's) patients and healthy subjects to adsorb α-Syn molecules.

[0090] Step 4: Use a handheld Raman spectrometer with an excitation light wavelength of 785 nm and a power of 50 mW to detect the silver fiber substrate of α-synuclein molecules in the saliva sample of Parkinson's disease (PD) patients, and obtain a SERS spectrum, as shown in Fig.17 As shown in the figure, by analyzing the relevant characteristic peaks of Raman spectroscopy, α-Syn in the saliva of PD (Parkinson's) patients can be identified, and then the SERS spectral differences of α-Syn in the saliva samples of Parkinson's disease (PD) patients, Progressive Supranuclear Palsy (PSP) patients and Multiple System Atrophy (MSA) patients are compared, so as to distinguish and detect PD patients.

[0091] After replacing the saliva sample of the PD (Parkinson's) patient in step 3 with the saliva sample of the progressive supranuclear palsy (PSP) patient, and then using a handheld Raman spectrometer with an excitation light of 785 nm wavelength and a power of 50 mW, the SERS spectrum of the α-synuclein molecule in the saliva sample of the progressive supranuclear palsy (PSP) patient is obtained, as shown in FIG. Fig.18As shown, by analyzing the relevant characteristic peaks of Raman spectroscopy, α-Syn in the saliva of PSP (progressive supranuclear palsy) patients can be identified, and then the SERS spectral differences of α-Syn in the saliva samples of PSP patients, PD patients and MSA patients are compared to detect PD patients.

[0092] After replacing the saliva sample of the PD (Parkinson's) patient in step 3 with the saliva sample of the multiple system atrophy (MSA) patient, and then using a handheld Raman spectrometer with an excitation light of 785 nm wavelength and a power of 50 mW, the SERS spectrum of the α-synuclein molecule in the saliva sample of the multiple system atrophy (MSA) patient is obtained, as shown in FIG. Fig.19 As shown, by analyzing the relevant characteristic peaks of Raman spectroscopy, α-Syn in the saliva of MSA (multiple system atrophy) patients can be identified, and then the SERS spectral differences of α-Syn in the saliva samples of MSA patients, PD patients and PSP patients can be compared to detect MSA disease patients.

[0093] Using a handheld Raman spectrometer with an excitation light wavelength of 785nm and a power of 50mW, the β-folded structure and α-amorphous structure of α-synuclein molecules in saliva samples of Parkinson's (PD) patients, Progressive Supranuclear Palsy (PSP) patients, and multiple system atrophy (MSA) patients were detected, and a bar graph of the ratio of the β-folded structure to the α-amorphous structure of α-synuclein molecules in saliva samples of Parkinson's (PD) patients, Progressive Supranuclear Palsy (PSP) patients, and multiple system atrophy (MSA) patients was obtained, as shown in FIG. Fig. 20 As shown, PSP patients have the largest amount of α-amorphous structure, so the ratio of α-Syn's β-folded structure to α-amorphous structure is the smallest, while MSA patients have the largest amount of β-folded structure and the smallest amount of α-amorphous structure, so the ratio of α-Syn's β-folded structure to α-amorphous structure is the largest, thereby detecting PD patients, PSP patients and MSA patients.

[0094] Based on step 4, using a handheld Raman spectrometer with an excitation light of 785 nm wavelength and a power of 100 mW, the SERS spectra of α-synuclein molecules in saliva samples of Parkinson's disease (PD) patients, the SERS spectra of α-synuclein molecules in saliva samples of Progressive Supranuclear Palsy (PSP) patients, and the SERS spectra of α-synuclein molecules in saliva samples of Multiple System Atrophy (MSA) patients were obtained, as shown in FIG. Figure 21-23 As shown, by analyzing the relevant characteristic peaks of the Raman spectrum after protein mutation, and then comparing the SERS spectrum differences of α-Syn in the saliva samples of PD patients, PSP patients and MSA patients, PD patients, PSP patients and MSA patients can be distinguished.

[0095] Then, using a handheld Raman spectrometer with an excitation light wavelength of 785 nm and a power of 100 mW, a bar graph of the ratio of the β-folded structure to the α-amorphous structure of α-synuclein molecules in saliva samples of Parkinson's (PD) patients, progressive supranuclear palsy (PSP) patients, and multiple system atrophy (MSA) patients was obtained, as shown in Figure 2. Fig.24 As shown, after using a 100 mW handheld Raman spectrometer, the temperature rise causes the α amorphous structure to mutate. The α amorphous structure of PSP patients mutates the most, resulting in the least amount of α amorphous structure, so the ratio of the β folding structure to the α amorphous structure of α-Syn is the largest, while the α amorphous structure of MSA patients mutates the least, resulting in the most α amorphous structure, so the ratio of the β folding structure to the α amorphous structure of α-Syn is the smallest, thereby detecting and distinguishing PD patients, PSP patients and MSA patients.

[0096] Example 6

[0097] This embodiment adds a concentrated sulfuric acid optimized silver fiber SERS substrate preparation method to enhance the SERS signal, which specifically includes the following steps:

[0098] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0099] Step 2: Use 800-grit sandpaper to polish the silver fiber 4 times to form a strip scratch structure, and then perform post-polishing treatment.

[0100] Step 3: Chemical etching: Place the polished silver fiber substrate into concentrated sulfuric acid preheated to 90°C, react for 1 minute, and then rinse with deionized water to form a hydrophilic surface.

[0101] Step 4: Use MG solutions of different concentrations to perform SERS tests on the silver fiber substrate and optimize the preparation conditions to obtain the best SERS signal enhancement effect.

[0102] Example 7

[0103] The preparation method of the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules is specifically implemented according to the following steps:

[0104] Step 1, cleaning the silver fiber, specifically: placing the silver fiber in an ultrasonic cleaner for cleaning for 10 minutes to remove surface pollutants and moisture, and then rinsing once with anhydrous ethanol for further cleaning;

[0105] Step 2, cut 1 cm of the silver fiber cleaned in step 1, use 800-mesh sandpaper to scratch the surface of the silver fiber 4 times, so that the surface has strip scratches, and then clean the physically scratched silver fiber with an ultrasonic cleaner, and after cleaning, put it in anhydrous ethanol for storage after drying to prevent oxidation;

[0106] Step 3, placing the silver fiber stored in anhydrous ethanol in step 2 into concentrated sulfuric acid, reacting for 5 minutes at a water bath temperature of 90° C., and then rinsing with deionized water to obtain a silver fiber SERS substrate.

[0107] The silver fiber SERS substrate was subjected to SERS performance test, and 10^-5mol / L MG solution was used to analyze the influence of SERS signal enhancement effect.

[0108] Example 8

[0109] On the basis of Example 2, the polished silver fiber substrate is placed in concentrated sulfuric acid preheated to 90°C, reacted for 5 minutes, and then rinsed with deionized water to form a hydrophilic surface. The rinsed silver fiber substrate is then immersed in MG solutions of different concentrations (10^-5mol / L to 10^-10mol / L) for 10 minutes at each concentration.

[0110] Then, a 785 nm laser was used to perform SERS detection on the immersed silver fiber substrate, and the obtained SERS spectrum data was recorded and analyzed.

[0111] Comparative Example 1

[0112] The preparation process of polishing the silver fiber SERS substrate using 80-grit sandpaper specifically includes the following steps:

[0113] Step 1: Pretreatment of silver fibers: Place the silver fibers in an ultrasonic cleaner for 10 minutes to remove surface contaminants and moisture, and then rinse with anhydrous ethanol for further cleaning.

[0114] Step 2: Physical polishing: use 80 mesh, 150 mesh, 320 mesh, and 1200 mesh sandpaper to polish along the length of the silver fiber for 2 and 4 times respectively to form uniform strip scratches.

[0115] The polished silver fiber substrate was immersed in MG solution with a concentration of 10^-5 mol / L for 10 minutes, and SERS detection was performed on the immersed silver fiber substrate using a 785 nm laser, and the obtained SERS spectral data were recorded and analyzed.

[0116] Comparative Example 2

[0117] On the basis of Comparative Example 1, 80-grit sandpaper was used to polish the strip scratches and the mesh scratches. Figure 4 The following is a comparison of the strip scratches and mesh scratches obtained by sandpaper polishing using a scanning electron microscope. Figure 4 It can be seen that polishing can increase the surface area and roughness of silver fibers and improve the SERS detection performance of silver fibers.

[0118] Comparative Example 3

[0119] Compared with Example 6, the difference from Example 6 is that the time for preheating reaction corrosion in concentrated sulfuric acid in this comparative example is 5 minutes.

[0120] Comparative Example 4

[0121] Based on Example 3, the physical polishing step is removed.

[0122] Comparative Example 5

[0123] On the basis of Example 4, the corrosion time of concentrated sulfuric acid is set to different corrosion times, and the silver fiber substrate treated after polishing is chemically corroded.

[0124] The silver fiber SERS substrates after being polished to different mesh sizes in Example 1 and Comparative Example 1 are as follows: Figure 5-6 As shown, Figure 5 The scanning electron microscope images of the silver fiber SERS substrate polished twice with sandpaper of different mesh sizes, including: (a) scanning electron microscope image of 80 mesh polished twice; (b) scanning electron microscope image of 150 mesh polished twice; (c) scanning electron microscope image of 320 mesh polished twice; (d) scanning electron microscope image of 800 mesh polished twice; (e) scanning electron microscope image of 1200 mesh polished twice; (f) scanning electron microscope image of the original silver fiber;

[0125] Figure 6 The 200-fold magnified electron microscope images and 500-fold magnified scanning electron microscope images of the silver fiber SERS substrate of the present invention that was polished 2 times and 4 times with 800-mesh sandpaper, wherein (a) is a scanning electron microscope image magnified 200 times after being polished 2 times with 800-mesh sandpaper; (b) is a scanning electron microscope image magnified 200 times after being polished 4 times with 800-mesh sandpaper; (c) is a scanning electron microscope image magnified 500 times after being polished 2 times with 800-mesh sandpaper; (d) is a scanning electron microscope image magnified 500 times after being polished 1 time with 800-mesh sandpaper;

[0126] like Figure 7 As shown, the MG10 obtained in Comparative Example 1 and Example 3 -5The Raman spectra of the silver fiber substrate polished four times with 320-mesh, 800-mesh and 1200-mesh sandpapers of different concentrations show that the silver wire polished with 800-mesh has the most reasonable scratches and the best SERS detection performance.

[0127] Figure 8 The silver nano-substrate MG10 in Example 3 of the present invention -5 The Raman spectrum of the concentration was verified by the standard Raman probe molecule (MG molecule), which showed that the prepared silver fiber SERS substrate had good spectral detection capability.

[0128] Fig. 9 : is an electron microscope comparison of the silver fiber SERS substrate before and after concentrated sulfuric acid corrosion in Example 2 and Example 3 of the present invention. Fig. 9 The electron microscopic comparison of the silver fiber SERS substrate before and after corrosion with concentrated sulfuric acid shows that the corroded silver fiber has a higher surface nano-roughness, which is beneficial to improving the detection performance of SERS.

[0129] Fig.10 The silver fiber SERS substrate MG10 after 1 min of grinding and corrosion in Example 3 and non-grinding and corrosion in Comparative Example 4 -5 The Raman spectrum of Fig.10 This shows that the surface structure prepared by first grinding and then etching has better SERS detection performance.

[0130] Fig.11 MG10 of different corrosion times of the silver fiber substrate polished 4 times with 800-grit sandpaper in Comparative Example 5 -5 Raman spectroscopy, through Fig.11 This shows that 1-5 min is an optimal corrosion time, and the silver fiber prepared in this corrosion time process has good SERS detection performance.

[0131] Fig.12 MG10 of the silver substrate polished with 800 grit sandpaper for different times in Example 4 -5 Raman spectroscopy, through Fig.12 This indicates that the SERS performance of the silver fiber polished four times is better than that of the silver fiber polished twice, which is more conducive to SERS detection applications.

[0132] Fig.13 The silver fiber substrate in Example 7 was polished 4 times with 800-mesh sandpaper and corroded for 5 minutes, and the silver fiber substrate was polished 4 times with 800-mesh sandpaper and then post-polished (after ultrasonic and deionized water cleaning) and then corroded for 5 minutes, and MG10 was performed. -5 Raman spectroscopy shows that ultrasonic cleaning can better clean the surface of silver fibers, and SERS spectroscopy has better detection clarity.

[0133] Fig.14The MG10 with silver base polished 4 times with different mesh numbers in Comparative Example 1 -5 Raman spectroscopy, through Fig.14 It can be shown that the 800-mesh polished silver fiber has better SERS performance and is more conducive to SERS detection applications.

[0134] Fig.15 It is the Raman spectra of MG solutions of different concentrations on the silver fiber substrate polished 4 times and corroded for 5 minutes using 800-grit sandpaper in Example 8. Using the silver fiber substrate, SERS spectral peaks with good linear intensity distribution can be detected for MG molecules of different concentrations in the present invention (10^-5mol / L to 10^-10mol / L), indicating that quantitative detection of analytes can be achieved by this method.

[0135] Fig.16 The MG10 of Example 8 is a silver fiber substrate that was polished 4 times with 800 mesh and etched for 5 minutes. -5 The waterfall chart, Fig.16 This indicates that the bright silver substrate prepared by the present invention has good detection signal stability and uniformity.

[0136] Through the above embodiments and comparative examples, it can be seen that the silver fiber SERS substrate for detecting α-synuclein of the present invention is modified by physical polishing and chemical corrosion, which can effectively enhance the surface enhanced Raman scattering signal. By studying the influence of different polishing conditions and sandpaper mesh numbers on the performance of the silver fiber SERS substrate, the strip scratch structure formed by 4 polishings with 800 mesh sandpaper was finally screened out, which has the best enhancement effect on the SERS signal. The silver fiber SERS substrate can not only realize the in-situ, rapid, and non-destructive SERS detection of biomolecules, but also has potential application value in drug efficacy analysis and pathogen detection. This silver fiber SERS substrate can significantly improve the detection efficiency and reduce the analysis cycle. It is a new type of biomolecule detection tool with promotion potential.

Claims

1. A method for preparing a silver fiber substrate for detecting α-synuclein molecules in Parkinson's disease, characterized in that: The specific steps are as follows: Step 1, cleaning the silver fiber; Step 2, physically scratching the silver fiber cleaned in step 1 to form a substrate with strip scratches or mesh scratches on its surface, then cleaning and drying the physically scratched silver fiber, and then storing the dried silver fiber in anhydrous ethanol; Step 3, placing the silver fiber stored in anhydrous ethanol in step 2 into concentrated sulfuric acid, then reacting for a certain period of time in a water bath, and then rinsing with deionized water to obtain a silver fiber SERS substrate.

2. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 1, characterized in that: The step 1 is specifically as follows: The silver fiber was placed in an ultrasonic cleaner for cleaning for a certain period of time, and then rinsed with anhydrous ethanol.

3. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 2, characterized in that: In the step 1, the silver fiber is cleaned with an ultrasonic cleaner for 10-30 minutes, and then rinsed once with anhydrous ethanol.

4. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 1, characterized in that: In the step 2, the silver fibers cleaned in the step 1 are cut into pieces with a length of 0.2-1 cm.

5. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 4, characterized in that: In step 2, sandpaper is used to scratch the surface of the silver fiber so that the surface has strip scratches and mesh scratches as a substrate.

6. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 5, characterized in that: The physical scraping using 800-grit sandpaper in step 2 is specifically as follows: Preparation of a silver substrate with strip scratches: Use sandpaper to scratch along the length of the silver fibers to prepare the first type of silver substrate with strip scratches; Preparation of a silver substrate with mesh scratches: On the basis of preparing a silver substrate with strip scratches, the scratching direction is changed to prepare strip scratches that intersect with the first strip scratches, thereby obtaining a silver substrate with mesh scratches.

7. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 6, characterized in that: In the step 2, the physically scraped silver fibers are cleaned with an ultrasonic cleaner, and after cleaning, they are dried and stored in anhydrous ethanol.

8. The method for preparing the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules according to claim 5, characterized in that: In step 3, the water bath temperature is 60-90° C., and the water bath reaction time of the silver fiber in concentrated sulfuric acid is 1-10 min.

9. The silver fiber substrate for detecting α-synuclein molecules in Parkinson's disease prepared by the method for preparing the silver fiber substrate for detecting α-synuclein molecules in Parkinson's disease according to any one of claims 1 to 8 is used in SERS detection of biological molecules.

10. The use of the silver fiber substrate for detecting Parkinson's disease α-synuclein molecules in SERS detection of biological molecules according to claim 9, characterized in that: The prepared silver fiber substrate for detecting α-synuclein molecules in Parkinson's disease was immersed in saliva samples of PD patients, PSP patients and MSA patients respectively to adsorb α-Syn molecules. The silver fiber substrate was subjected to SERS detection using a 785nm laser, and the SERS spectra of α-Syn in saliva samples of PD patients, PSP patients and MSA patients were compared to obtain the detection results.

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