Surface-enhanced Raman sensor based on golden spiked club structure, preparation method of surface-enhanced Raman sensor and application of surface-enhanced Raman sensor in AD marker detection

Abstract

The invention discloses a surface-enhanced Raman sensor based on a golden spiked club structure, a preparation method of the surface-enhanced Raman sensor and application of the surface-enhanced Raman sensor in AD marker detection. The sensor comprises a golden spiked club substrate surface modified with a capture antibody and a golden ball DTNB-Au-Ab2 with a Raman label, the preparation process of the gold mace substrate surface modified with the capture antibody comprises the following steps: carrying out magnetron sputtering on a gold nano-film with the thickness of 10-100 nm on the substrate surface, and then depositing a gold mace structure on the surface of the gold nano-film; dropwise adding P-tau181 to the surface of the golden spiked club structure to capture an antibody to obtain the golden spiked club. The preparation process of the gold ball DTNB-Au-Ab2 with the Raman label comprises the following steps: preparing a gold ball solution with the particle size of 13-60 nm by using a sodium citrate reduction method; the method comprises the following steps: adding a DTNB solution into a gold ball solution to obtain a gold ball-DTNB solution, and then modifying the surface of DTNB with a P-tau181 detection antibody through an amide reaction to obtain the gold ball DTNB-Au-Ab2 with a Raman label. The kit is used for ultra-sensitive detection of the AD marker P-tau181 in blood. The detection limit of the P-tau181 can reach 1.3 fM, and the linear range of the P-tau181 is 1 fM to 1 nM.

Description

Technical Field

[0001] The invention belongs to the field of SERS detection, and specifically relates to a surface enhanced Raman sensor based on a gold mace structure, a preparation method thereof, and an application thereof in AD marker detection. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease with a very long duration. According to statistics from the National Health Commission, by 2022, there will be 10 million AD patients among the elderly over 60 years old in my country, making China the country with the largest number of AD patients in the world, which has brought huge economic and mental burdens to the country, society and patients' families. However, there is no good drug that can reverse or cure the disease, so early diagnosis, early detection and early intervention are very important.

[0003] The present application constructs a SERS sensor based on a gold mace structure for ultrasensitive detection of the AD marker P-tau181 in the blood. The quantitative analysis of the marker can be used for early diagnosis of AD. Summary of the invention

[0004] The object of the present invention is to provide a surface enhanced Raman sensor based on a gold mace structure, a preparation method thereof and an application thereof in the detection of AD markers.

[0005] Based on the above purpose, the present invention adopts the following technical solutions: A method for preparing a surface enhanced Raman sensor based on a gold mace structure, comprising a gold mace substrate surface modified with a capture antibody and a gold ball DTNB-Au-Ab2 with a Raman label, wherein the preparation process of the gold mace substrate surface modified with a capture antibody is as follows: (1) On a clean substrate surface, magnetron sputter a gold nanofilm with a thickness of 10 nm to 100 nm, and then deposit a gold mace structure on the surface of the gold nanofilm; (2) P-tau181 capture antibody is added dropwise to the surface of the golden mace structure to obtain a golden mace substrate surface modified with the capture antibody; The preparation process of gold sphere DTNB-Au-Ab2 with Raman label is as follows: A. Prepare a gold ball solution with a particle size of 13nm~60nm using sodium citrate reduction method; B. Add DTNB solution to the gold ball solution to obtain a gold ball-DTNB solution. Then, modify the P-tau181 detection antibody to the DTNB surface through an amide reaction to obtain a gold ball DTNB-Au-Ab2 with a Raman label.

[0006] Further, the preparation process of the gold mace structure is as follows: using a gold nanofilm as a working electrode, Ag / AgCl as a reference electrode, a platinum sheet electrode as a counter electrode, and a mixed solution of chloroauric acid and silver nitrate as an electrolyte, electro-depositing on the surface of the gold nanofilm by a constant voltage method, the deposition time is 30s~600s, the potential applied during the electro-deposition is -0.1~-0.6V, the concentration of chloroauric acid in the mixed solution is 0.5mmol / L~25mmol / L, and the concentration of silver nitrate is 0.5μmol / L~50μmol / L. Preferably, in the mixed solution, the molar ratio of silver nitrate to chloroauric acid is 1:50.

[0007] Furthermore, the concentration of the capture antibody is 5-15 μmol / L, preferably, the concentration of the capture antibody is 10 μmol / L, and the addition amount of the capture antibody is 1.5*10 -5 ~2.0*10 -4 μmol / mm 2 After adding the capture antibody, incubation for 0.5~2 h is required.

[0008] Furthermore, the preparation process of the gold ball is as follows: prepare a chloroauric acid solution with a concentration of 1~5 mmol / L and a sodium citrate solution with a concentration of 15~25 mmol / L; heat the chloroauric acid solution to boiling, quickly add the sodium citrate solution, and keep boiling, continue stirring for 10~20 minutes, and the molar ratio of chloroauric acid to sodium citrate is 1:(1~10).

[0009] Furthermore, the process of the gold ball-DTNB solution is as follows: add (0.5~1) mL, 5~15mM dinitrobenzoic acid solution to every 1mL of the gold ball solution, and react in a nitrogen atmosphere at room temperature for 20~30 hours to obtain the solution.

[0010] Furthermore, the specific process of modifying the P-tau181 detection antibody onto the DTNB surface through amide reaction is as follows: add EDC solution and NHS solution to the gold ball-DTNB solution to activate the carboxyl groups on the DTNB surface, then add the P-tau181 detection antibody, react at 35-40°C for 1-3 hours, and centrifuge to remove unbound antibodies.

[0011] Further, the concentration of the EDC solution is 0.1-0.5 mol / L, the concentration of the NHS solution is 0.01 mol / L-0.1 mol / L, the concentration of the detection antibody is 5-15 μmol / L, and 50-150 μL of EDC solution and NHS, and 2-15 μL of detection antibody solution need to be added to each 1 mL of the gold ball-DTNB solution. Preferably, during the amide reaction, the molar ratio of EDC to NHS is 4:1.

[0012] The surface enhanced Raman sensor based on the gold mace structure is prepared by the above method.

[0013] The above-mentioned surface enhanced Raman sensor based on the gold mace structure is used in the detection of AD marker P-tau181. The process is as follows: (1) First, 2-10 μL of a series of concentrations of AD marker standard samples were added to the surface of a gold mace substrate modified with a capture antibody, and the mixture was reacted at 35-40°C for 0.1-1 hour. After rinsing with PBS, DTNB-Au-Ab2 was added and incubated at 35-40°C for 0.1-1 hour. The Raman signal was detected by a Raman spectrometer, with the logarithm of the concentration as the horizontal axis and 1332 cm -1 The Raman signal intensity at is taken as the ordinate, and a standard curve is drawn to obtain the standard curve equation; (2) Add 2-10 μL of the sample to be tested to the surface of another gold mace substrate modified with the capture antibody, react at 35-40°C for 0.1-1 hour, rinse with PBS, then add DTNB-Au-Ab2, incubate at 35-40°C for 0.1-1 hour, and detect the Raman signal with a Raman spectrometer, recording at 1332 cm -1 The Raman signal intensity at the location is substituted into the standard curve equation to calculate the concentration of the target AD marker in the sample to be tested.

[0014] In the present application, the AD marker is P-tau181 protein, the AD marker standard is the P-tau181 protein standard, and the concentrations of P-tau181 protein are 10fM, 100fM, 1pM, 10 pM, 100pM, and 1nM, respectively.

[0015] Preferably, during the detection, the instrument conditions of the Raman spectrometer are: using a helium-neon laser, the excitation light source wavelength is 785nm, and the spectral range is 800-2000cm -1 , the detection frequency is 50ms, the scanning time is 10s~60s, and the warm-up time is 10min.

[0016] Compared with the prior art, the present invention has the following advantages: (1) During electrodeposition, the ratio of gold ions to silver ions is adjusted to prepare a gold-silver nanoalloy structure in the shape of a mace, which has excellent SERS performance; (2) Based on the SERS substrate, a SERS sensor for detecting AD blood marker P-tau181 was constructed; (3) The detection limit of P-tau181 can reach 1.3fM, and the linear range is 1fM-1nM; (4) The practical value of the SERS sensor was verified by testing 8 clinical blood samples, and it was confirmed that the content of P-tau181 in blood samples was significantly different between AD patients and normal people. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the SEM image of the gold mace; Figure 2 This is a standard curve diagram of the surface enhanced Raman sensor based on the gold mace structure prepared in Example 1 for detecting AD marker P-tau181; Figure 3 This is a comparison of the results of detecting 8 blood samples using the surface enhanced Raman sensor based on the gold mace structure prepared in Application Example 1. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0019] Example 1 A preparation method of a surface enhanced Raman sensor based on a gold mace structure, the process is as follows: (1) Preparation of surface enhanced Raman (SERS) substrate: On the surface of a clean glass sheet (2 mm in diameter), a layer of gold nanofilm with a thickness of 30 nm was sputtered by magnetron sputtering, and then the gold mace structure was deposited by surface electrodeposition. The specific process is as follows: the gold nanofilm was used as the working electrode, Ag / AgCl was used as the reference electrode, the platinum electrode was used as the counter electrode, and a mixed aqueous solution of chloroauric acid and silver nitrate was used as the electrolyte. The concentration of chloroauric acid in the mixed aqueous solution was 0.5 mmol / L, and the concentration of silver nitrate was 10 μmol / L. The deposition voltage was -0.2 V, and the deposition time was 300 s. A gold mace structure was grown on the surface of the sputtered gold nanofilm, and its SEM image is shown in the figure. Figure 1 As shown, Figure 1 As shown, the gold mace structure is about 20 μm in length and 0.5 μm in diameter. Many thorn-like structures with lengths ranging from about 1 to 5 μm grow in the vertical direction, thus forming a gold nanostructure shaped like a mace.

[0020] (2) Preparation of SERS detection part (DTNB-Au-Ab2): Gold spheres with a diameter of 13 nm to 60 nm were prepared by sodium citrate reduction method. The specific steps are as follows: Prepare a 2 mM chloroauric acid aqueous solution and a 20 mM sodium citrate aqueous solution. Heat the chloroauric acid aqueous solution to boiling, quickly add the sodium citrate aqueous solution, and keep it boiling. Continue stirring for 15 minutes and cool to room temperature. When the volume ratio of the sodium citrate aqueous solution to the chloroauric acid aqueous solution is 1:1, gold spheres with a diameter of 13 nm are obtained. When the volume ratio of the sodium citrate aqueous solution to the chloroauric acid aqueous solution is 1:10, gold spheres with a diameter of about 50 to 60 nm are obtained.

[0021] The size of the gold spheres can also be controlled by the reaction time. The longer the reaction time, the larger the size of the gold spheres.

[0022] To 1 mL of gold ball (diameter 13 nm) solution, add 0.5 mL of 10 mM DTNB (dinitrobenzoic acid) aqueous solution and react in nitrogen atmosphere at room temperature for 24 hours to obtain a gold ball-DTNB solution. Then, take 1 mL of the gold ball-DTNB solution, add 100 μL of 0.2 mol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution and 100 μL of 0.05 mol / L N-hydroxysuccinimide (NHS) aqueous solution to activate the surface carboxyl groups, and then add 5 μL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution. L, 10 μmol / L P-tau181 detection antibody (the detection antibody is the P-tau181 detection antibody of RUDA Company, model number is RDM003, prepared into 10 μmol / L using PBS with pH=7.4), react at 37°C for 2 hours, 10,000 rpm, centrifuge for 20 minutes, then remove the supernatant, and then re-add 1 mL PBS (pH=7.4) buffer to redissolve the gold sphere with antibody and SERS label to obtain the gold sphere DTNB-Au-Ab2 with Raman label.

[0023] Surface enhanced Raman (SERS) sensor: First, add 5 μL of 10 μM P-tau181 capture antibody on the surface of the golden mace structure (the capture antibody used is RUDA's P-tau181 capture antibody, model RDM004, prepared into 10 μmol / L using PBS at pH = 7.4), incubate at 37°C for 1 hour, and rinse with PBS at pH = 7.4 to obtain the golden mace substrate surface modified with the capture antibody. 5 μL of P-tau181 protein of different concentrations (10 fmol / L, 100 fmol / L, 1 pmol / L, 10 pmol / L, 100 pmol / L, 1 nmol / L) was added to the surface of the gold mace substrate modified with the capture antibody, incubated at 37°C for 1 hour, rinsed three times with PBS (pH=7.4), and then 5 μL of DTNB-Au-Ab2 was added. After incubation at 37°C for 1 hour, the Raman signal was detected by Raman spectrometer, with the logarithm of the concentration as the horizontal axis and 1332 cm -1 The Raman signal intensity at is taken as the vertical axis, and a standard curve is drawn, such as Figure 2 As shown, the standard curve equation is Y=1542.6x+22044, R 2 =0.9917, and the detection limit was 1.3fM according to the standard curve equation.

[0024] The instrument conditions of the Raman spectrometer are as follows: using a helium-neon laser, an excitation light source wavelength of 785 nm, and a spectral range of 800-2000 cm -1 , the detection frequency is 50ms, the scanning time is 10s~60s, and the warm-up time is 10min.

[0025] Application Example 1 Take 5 μL of human serum sample, add 5 μL of PBS with pH = 7.4 to dilute, mix well and add 5 μL to the surface of the gold mace substrate modified with capture antibody, react at 37°C for 1 hour, then rinse three times with PBS (pH = 7.4) to remove unbound sample solution, then add 5 μL of DTNB-Au-Ab2, react at 37°C for 1 hour, rinse off unbound DTNB-Au-Ab2 molecules with PBS (pH = 7.4), dry, and measure its Raman signal with a Raman spectrometer, recording at 1332 cm -1 The Raman signal intensity at .

[0026] Eight blood samples (four healthy control samples and four AD patient samples) were tested according to the above method. The Raman signal intensities of the eight blood samples are shown in Figure 2. Figure 3 As shown by Figure 3 It can be seen that there is a significant difference in the content of P-tau181 between AD patients and healthy controls (*** indicatesp <0.001). The Raman signal intensity of 8 blood samples can also be put into the standard curve equation to calculate the concentration of the target (P-tau181 protein) in the sample.

Claims

1. A method for preparing a surface enhanced Raman sensor based on a gold mace structure, characterized in that: The invention comprises a gold mace substrate surface modified with a capture antibody and a gold ball DTNB-Au-Ab2 with a Raman label, wherein the preparation process of the gold mace substrate surface modified with a capture antibody is as follows: (1) On a clean substrate surface, magnetron sputter a gold nanofilm with a thickness of 10 nm to 100 nm, and then deposit a gold mace structure on the surface of the gold nanofilm; (2) P-tau181 capture antibody is added dropwise to the surface of the golden mace structure to obtain a golden mace substrate surface modified with the capture antibody; The preparation process of gold sphere DTNB-Au-Ab2 with Raman label is as follows: A. Prepare a gold ball solution with a particle size of 13nm~60nm using sodium citrate reduction method; B. Add DTNB solution to the gold ball solution to obtain a gold ball-DTNB solution. Then, modify the P-tau181 detection antibody to the DTNB surface through an amide reaction to obtain a gold ball DTNB-Au-Ab2 with a Raman label.

2. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 1, characterized in that: The preparation process of the gold mace structure is as follows: using gold nanofilm as the working electrode, Ag / AgCl as the reference electrode, platinum electrode as the counter electrode, and a mixed solution of chloroauric acid and silver nitrate as the electrolyte, electrodeposition is performed on the surface of the gold nanofilm by a constant voltage method, and the deposition time is 30s~600s. The potential applied during the electrodeposition is -0.1~-0.6V, the concentration of chloroauric acid in the mixed solution is 0.5mmol / L~25mmol / L, and the concentration of silver nitrate is 0.5μmol / L~50μmol / L.

3. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 1, characterized in that: The concentration of capture antibody is 5~15μmol / L. Based on the substrate area, the amount of capture antibody added is 1.5*10 -5 ~2.0*10 -4 μmol / mm 2 After adding the capture antibody, incubation for 0.5~2 h is required.

4. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 1, characterized in that: The preparation process of the gold ball is as follows: prepare a chloroauric acid solution with a concentration of 1~5 mmol / L and a sodium citrate solution with a concentration of 15~25 mmol / L; heat the chloroauric acid solution to boiling, quickly add the sodium citrate solution, and keep boiling, continue stirring for 10~20 minutes, the molar ratio of chloroauric acid to sodium citrate is 1:(1~10).

5. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 1, characterized in that: The process of preparing the gold ball-DTNB solution is as follows: add (0.5-1) mL of 5-15 mM dinitrobenzoic acid solution to every 1 mL of the gold ball solution, and react in a nitrogen atmosphere at room temperature for 20-30 hours to obtain the solution.

6. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 1, characterized in that: The specific process of modifying the P-tau181 detection antibody onto the DTNB surface through amide reaction is as follows: add EDC solution and NHS solution to the gold ball-DTNB solution, then add P-tau181 detection antibody, react at 35-40°C for 1-3 hours, and centrifuge to remove unbound antibodies.

7. The method for preparing a surface enhanced Raman sensor based on a gold mace structure according to claim 6, characterized in that: The concentration of EDC solution is 0.1~0.5mol / L, the concentration of NHS solution is 0.01mol / L~0.1 mol / L, and the concentration of detection antibody is 5~15μmol / L. For every 1mL of gold ball-DTNB solution, 50~150μL of EDC solution and NHS and 2~15μL of detection antibody solution need to be added.

8. A surface enhanced Raman sensor based on a gold mace structure prepared by the method according to any one of claims 1 to 7.

9. Use of the surface enhanced Raman sensor based on the gold mace structure according to claim 8 in detecting AD marker P-tau181.

10. The use according to claim 9, characterized in that: The process is as follows: (1) First, 2-10 μL of a series of concentrations of AD marker standard samples were added to the surface of a gold mace substrate modified with a capture antibody, and the mixture was reacted at 35-40°C for 0.1-1 hour. After rinsing with PBS, DTNB-Au-Ab2 was added and incubated at 35-40°C for 0.1-1 hour. The Raman signal was detected by a Raman spectrometer, with the logarithm of the concentration as the horizontal axis and 1332 cm -1 The Raman signal intensity at is taken as the ordinate, and a standard curve is drawn to obtain the standard curve equation; (2) Add 2-10 μL of the sample to be tested to the surface of another gold mace substrate modified with the capture antibody, react at 35-40°C for 0.1-1 hour, rinse with PBS, then add DTNB-Au-Ab2, incubate at 35-40°C for 0.1-1 hour, and detect the Raman signal with a Raman spectrometer, recording at 1332 cm -1 The Raman signal intensity at the location is substituted into the standard curve equation to calculate the concentration of the target AD marker in the sample to be tested.

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