A surface-enhanced Raman sensor based on a golden mace structure, its preparation method and application in the detection of AD markers

Abstract

The present invention discloses a surface-enhanced Raman sensor based on a golden mace structure, its preparation method and application in the detection of AD markers. The sensor includes a surface of a golden mace substrate modified with a capture antibody and a gold sphere DTNB-Au-Ab2 with a Raman tag. The preparation process of the surface of the golden mace substrate modified with the capture antibody is as follows: magnetron sputter a gold nanometer thin film with a thickness of 10 nm to 100 nm on the substrate surface, and then deposit a golden mace structure on the surface of the gold nanometer thin film; dropwise add a P-tau181 capture antibody on the surface of the golden mace structure to obtain it. The preparation process of the gold sphere DTNB-Au-Ab2 with a Raman tag is as follows: prepare a gold sphere solution with a particle size of 13 nm to 60 nm by the sodium citrate reduction method; add a DTNB solution to the gold sphere solution to obtain a gold sphere-DTNB solution, and then modify the P-tau181 detection antibody on the surface of DTNB through an amide reaction to obtain the gold sphere DTNB-Au-Ab2 with a Raman tag. It is used for the ultrasensitive detection of the AD marker P-tau181 in blood. The detection limit of P-tau181 can reach 1.3 fM, and the linear range is 1 fM - 1 nM.

Description

Technical Field

[0001] The present invention belongs to the field of SERS detection, and particularly relates to 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. Background Art

[0002] Alzheimer's disease is a neurodegenerative disease with a very long duration. However, there is currently no good drug that can reverse or cure this disease. Therefore, 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 blood. The quantitative analysis of this marker can be used for early diagnosis of AD. Summary of the Invention

[0004] The purpose 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:

[0006] A preparation method of a surface-enhanced Raman sensor based on a gold mace structure, including 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 the capture antibody is as follows:

[0007] (1) On a clean substrate surface, a gold nanometer thin film with a thickness of 10 nm to 100 nm is magnetron sputtered, and then a gold mace structure is deposited on the surface of the gold nanometer thin film;

[0008] (2) A P-tau181 capture antibody is dropped on the surface of the gold mace structure to obtain a gold mace substrate surface modified with the capture antibody;

[0009] The preparation process of the gold ball DTNB-Au-Ab2 with a Raman label is as follows:

[0010] A. A gold ball solution with a particle size of 13 nm to 60 nm is prepared by the sodium citrate reduction method;

[0011] B. A DTNB solution is added to the gold ball solution to obtain a gold ball-DTNB solution, and then a P-tau181 detection antibody is modified on the surface of DTNB through an amide reaction to obtain a gold ball DTNB-Au-Ab2 with a Raman label.

[0012] Further, the preparation process of the golden mace structure is as follows: using a gold nanofilm as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet electrode as the counter electrode, with a mixed solution of chloroauric acid and silver nitrate as the electrolyte, electro-depositing on the surface of the gold nanofilm by the constant voltage method, the deposition time is 30 s to 600 s, the applied potential during electro-deposition is -0.1 to -0.6 V, the concentration of chloroauric acid in the mixed solution is 0.5 mmol / L to 25 mmol / L, and the concentration of silver nitrate is 0.5 μmol / L to 50 μmol / L. Preferably, in the mixed solution, the molar ratio of silver nitrate to chloroauric acid is 1:50.

[0013] Further, the concentration of the capture antibody is 5 to 15 μmol / L. Preferably, the concentration of the capture antibody is 10 μmol / L. Based on the substrate area, the addition amount of the capture antibody is 1.5×10 -5 ~2.0×10 -4 μmol / mm 2 , and after adding the capture antibody, it needs to be incubated for 0.5 to 2 h.

[0014] Further, the preparation process of the gold nanoparticles is as follows: preparing a chloroauric acid solution with a concentration of 1 to 5 mmol / L and a sodium citrate solution with a concentration of 15 to 25 mmol / L; heating the chloroauric acid solution to boiling, quickly adding the sodium citrate solution, and maintaining the boiling state, and continuing to stir for 10 to 20 minutes, and the molar ratio of chloroauric acid to sodium citrate is 1:(1 to 10).

[0015] Further, the process of the gold nanoparticles - DTNB solution is as follows: adding (0.5 to 1) mL of a 5 to 15 mM dinitrobenzoic acid solution to every 1 mL of the gold nanoparticle solution, and reacting in a nitrogen atmosphere at room temperature for 20 to 30 hours to obtain it.

[0016] Further, the specific process of modifying the P-tau181 detection antibody onto the DTNB surface through an amide reaction is as follows: adding an EDC solution and an NHS solution to the gold nanoparticles - DTNB solution, after activating the carboxyl groups on the DTNB surface, then adding the P-tau181 detection antibody, and reacting at 35 to 40 °C for 1 to 3 hours, and centrifuging to remove the unbound antibody to obtain it.

[0017] Further, the concentration of the EDC solution is 0.1 to 0.5 mol / L, the concentration of the NHS solution is 0.01 mol / L to 0.1 mol / L, the concentration of the detection antibody is 5 to 15 μmol / L, and 50 to 150 μL of the EDC solution and NHS, as well as 2 to 15 μL of the detection antibody solution need to be added to every 1 mL of the gold nanoparticles - DTNB solution. Preferably, during the amide reaction, the molar ratio of EDC to NHS is 4:1.

[0018] The surface-enhanced Raman sensor based on the golden mace structure prepared by the above method.

[0019] Application of the above surface-enhanced Raman sensor based on the golden mace structure in detecting AD biomarker P-tau181. The process is as follows:

[0020] (1) First, add 2-10 μL of a series of concentrations of AD biomarker standard samples to the surface of a golden mace substrate modified with a 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. Using the logarithm of the concentration as the abscissa and the Raman signal intensity at 1332 cm -1 as the ordinate, plot the standard curve to obtain the standard curve equation;

[0021] (2) Add 2-10 μL of the sample to be tested to the surface of another golden mace substrate modified with a 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. Record the Raman signal intensity at 1332 cm -1 and substitute it into the standard curve equation to calculate the concentration of the target AD biomarker in the sample to be tested.

[0022] In this application, the AD biomarker is P-tau181 protein, and the AD biomarker standard samples are P-tau181 protein standard samples with the concentrations of P-tau181 protein being 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, and 1 nM in sequence.

[0023] Preferably, during detection, the instrument conditions of the Raman spectrometer are: using a helium-neon laser, the excitation light source wavelength is 785 nm, the spectral range is 800-2000 cm -1 , the detection frequency is 50 ms, the scanning time is 10 s - 60 s, and the preheating time is 10 min.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] (1) During electrodeposition, by regulating the ratio of gold ions and silver ions, a gold-silver nanoalloy structure with a mace shape is prepared, and this structure has good SERS performance;

[0026] (2) Based on this SERS substrate, a SERS sensor for detecting AD blood biomarker P-tau181 is constructed;

[0027] (3) The detection limit of P-tau181 can reach 1.3 fM, and the linear range is 1 fM - 1 nM;

[0028] (4) By detecting 8 clinical blood samples, the practical value of this SERS sensor was verified, and it was confirmed that there were significant differences in the content of P-tau181 in blood samples between AD patients and normal people. Description of the Drawings

[0029] Figure 1 is the SEM image of the golden mace;

[0030] Figure 2 is the standard curve graph for detecting the AD biomarker P-tau181 by the surface-enhanced Raman sensor based on the golden mace structure prepared in Example 1;

[0031] Figure 3 is the result comparison of detecting 8 blood samples by the surface-enhanced Raman sensor based on the golden mace structure prepared in Application Example 1. Detailed Description of the Invention

[0032] The technical solutions of the present invention will be further described in detail below in conjunction with the drawings and embodiments.

[0033] Example 1

[0034] A preparation method of a surface-enhanced Raman sensor based on a golden mace structure is as follows:

[0035] (1) Preparation of the surface-enhanced Raman (SERS) substrate: On the surface of a clean glass slide (diameter 2 mm), a 30-nm-thick gold nanometer film is sputtered by magnetron sputtering, and then the golden mace structure is deposited by surface electrodeposition. The specific process is as follows: Using the gold nanometer film as the working electrode, Ag / AgCl as the reference electrode, and a platinum sheet electrode as the counter electrode, a mixed aqueous solution of chloroauric acid and silver nitrate is used as the electrolyte. The concentration of chloroauric acid in the mixed aqueous solution is 0.5 mmol / L, the concentration of silver nitrate is 10 μmol / L, the deposition voltage is -0.2 V, and the deposition time is 300 s. The golden mace structure grows on the surface of the sputtered gold nanometer film, and its SEM image is as Figure 1 shown, as Figure 1 shown, the length of the golden mace structure is about 20 μm, the diameter is about 0.5 μm, and many thorn-like structures with lengths ranging from about 1 to 5 μm grow in the vertical direction, thus forming a gold nanostructure in the shape of a mace.

[0036] (2) Preparation of the SERS detection part (DTNB-Au-Ab2): Gold nanoparticles with diameters ranging from 13 nm to 60 nm were prepared by the sodium citrate reduction method. The specific steps are as follows: Prepare an aqueous solution of chloroauric acid with a concentration of 2 mM and an aqueous solution of sodium citrate with a concentration of 20 mM. Heat the chloroauric acid aqueous solution to boiling, quickly add the sodium citrate aqueous solution, and maintain the boiling state while continuing to stir for 15 minutes. Then cool to room temperature. When the volume ratio of the sodium citrate aqueous solution to the chloroauric acid aqueous solution is 1:1, gold nanoparticles with a diameter of 13 nm are obtained. When the volume ratio is 1:10, gold nanoparticles with a diameter of approximately 50 - 60 nm are obtained.

[0037] The size of the gold nanoparticles can also be controlled by adjusting the reaction time. The longer the reaction time, the larger the size of the gold nanoparticles.

[0038] To 1 mL of the gold nanoparticle (13 nm in diameter) solution, add 0.5 mL of a 10 mM DTNB (dinitrobenzoic acid) aqueous solution, and react for 24 hours in a nitrogen atmosphere at room temperature to obtain a gold nanoparticle-DTNB solution. Then, take 1 mL of the gold nanoparticle-DTNB solution, add 100 μL of a 0.2 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) aqueous solution and 100 μL of a 0.05 mol / L N-hydroxysuccinimide (NHS) aqueous solution. After activating the surface carboxyl groups, add 5 μL of a 10 μmol / L P-tau181 detection antibody (the detection antibody is the P-tau181 detection antibody from RUDA Company, model RDM003, prepared as 10 μmol / L using PBS with pH = 7.4), react at 37 °C for 2 hours, centrifuge at 10,000 revolutions per minute for 20 minutes, then remove the supernatant, and then re-add 1 mL of PBS (pH = 7.4) buffer to redissolve the gold nanoparticles with antibodies and SERS tags, thus obtaining the gold nanoparticles DTNB-Au-Ab2 with Raman tags.

[0039] Surface-Enhanced Raman (SERS) Sensor: First, 5 μL of 10 μM P-tau181 capture antibody (the capture antibody used is the P-tau181 capture antibody from RUDA Company, model RDM004, prepared as 10 μmol / L with PBS at pH = 7.4) was dropped onto the surface of the gold mace structure and incubated at 37 °C for 1 hour. After rinsing with PBS at pH = 7.4, the surface of the gold mace substrate modified with the capture antibody was obtained. 5 μL of P-tau181 protein with different concentrations (prepared with PBS at pH = 7.4 with concentrations of 10 fmol / L, 100 fmol / L, 1 pmol / L, 10 pmol / L, 100 pmol / L, and 1 nmol / L in sequence) was dropped onto 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, its Raman signal was detected with a Raman spectrometer. With the logarithm of the concentration as the abscissa and the Raman signal intensity at 1332 cm -1 as the ordinate, a standard curve was plotted, as shown in Figure 2 . The standard curve equation was Y = 1542.6x + 22044, and R 2 = 0.9917. The detection limit of 1.3 fM was obtained from the standard curve equation.

[0040] Among them, during detection, the instrument conditions of the Raman spectrometer were as follows: Helium-neon laser was used, the excitation light source wavelength was 785 nm, the spectral range was 800 - 2000 cm -1 , the detection frequency was 50 ms, the scanning time was 10 s to 60 s, and the preheating time was 10 min.

[0041] Application Example 1

[0042] 5 μL of human serum sample was taken, diluted with 5 μL of PBS at pH = 7.4, and after mixing evenly, 5 μL was taken and dropped onto the surface of the gold mace substrate modified with the capture antibody, reacted at 37 °C for 1 hour. Then, it was rinsed three times with PBS (pH = 7.4) to remove the unbound sample solution, and then 5 μL of DTNB-Au-Ab2 was added. After reacting at 37 °C for 1 hour, the unbound DTNB-Au-Ab2 molecules were rinsed off with PBS (pH = 7.4), dried, and its Raman signal was measured with a Raman spectrometer, and the Raman signal intensity at 1332 cm -1 was recorded.

[0043] Eight blood samples (four samples from healthy control groups and four samples from AD patients) were detected according to the above method. The Raman signal intensities of the eight blood samples are shown in Figure 3 , and from Figure 3It can be seen that there are significant differences in the content of P-tau181 between AD patients and healthy control population samples (*** indicates p <0.001). The Raman signal intensities of 8 blood samples can also be substituted into the standard curve equation to calculate the concentration of the target substance (P-tau181 protein) in the samples.

Claims

1. A preparation method of a surface-enhanced Raman sensor based on a golden mace structure, characterized in that, The sensor includes a surface of a gold mace substrate modified with a capture antibody and a gold sphere DTNB-Au-Ab2 with a Raman tag, and the preparation process of the surface of the gold mace substrate modified with the capture antibody is as follows: (1) On the surface of a clean glass slide, a 30-nm-thick gold nano-film is magnetron sputtered, and then a gold mace structure is deposited on the surface of the gold nano-film. The preparation process of the gold mace structure is as follows: Using the gold nano-film as the working electrode, Ag / AgCl as the reference electrode, and a platinum plate electrode as the counter electrode, with a mixed solution of chloroauric acid and silver nitrate as the electrolyte, electro-deposition is carried out on the surface of the gold nano-film by the constant voltage method for 300 s. The applied potential during electro-deposition is -0.2 V, the concentration of chloroauric acid in the mixed solution is 0.5 mmol / L, and the concentration of silver nitrate is 10 μmol / L; (2) Drop the P-tau181 capture antibody on the surface of the gold mace structure to obtain the surface of the gold mace substrate modified with the capture antibody; The preparation process of the gold sphere DTNB-Au-Ab2 with a Raman tag is as follows: A. Prepare a gold sphere solution with a particle size of 13 nm to 60 nm by the sodium citrate reduction method. The preparation process of the gold spheres is as follows: Prepare a chloroauric acid solution with a concentration of 1 to 5 mmol / L and a sodium citrate solution with a concentration of 15 to 25 mmol / L; Heat the chloroauric acid solution to boiling, quickly add the sodium citrate solution, and keep it boiling. Continue to stir for 10 to 20 minutes, and then cool to room temperature to obtain the gold sphere solution. The molar ratio of chloroauric acid to sodium citrate is 1:(1 to 10); B. Add (0.5 to 1) mL of a 5 to 15 mM dinitrobenzoic acid solution to each 1 mL of the gold sphere solution, and react in a nitrogen atmosphere at room temperature for 20 to 30 hours to obtain a gold sphere-DTNB solution. Then, add an EDC solution and an NHS solution to the gold sphere-DTNB solution, and then add a P-tau181 detection antibody, and react at 35 to 40 °C for 1 to 3 hours. Centrifuge to remove the unbound antibody to obtain the gold sphere DTNB-Au-Ab2 with a Raman tag.

2. The preparation method of the surface-enhanced Raman sensor based on the golden mace structure according to claim 1, characterized in that, The concentration of the capture antibody is 5 - 15 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, it needs to be incubated for 0.5 - 2 h.

3. The preparation method of the surface-enhanced Raman sensor based on the golden mace structure according to claim 1, characterized in that, The concentration of the EDC solution is 0.1 to 0.5 mol / L, the concentration of the NHS solution is 0.01 mol / L to 0.1 mol / L, the concentration of the detection antibody is 5 to 15 μmol / L. For each 1 mL of the gold sphere-DTNB solution, 100 μL of the EDC solution and 100 μL of the NHS solution, and 2 to 15 μL of the detection antibody solution need to be added.

4. A surface-enhanced Raman sensor based on a gold mace structure prepared by the method according to any one of claims 1 to 3.

5. The non-diagnostic application of the surface-enhanced Raman sensor based on a gold mace structure according to claim 4 in detecting the AD biomarker P-tau181.

6. The application according to claim 5, wherein 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)Drop 2 - 10 μL of the sample to be tested on the surface of another gold mace substrate modified with 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 then detect the Raman signal with a Raman spectrometer. Record the Raman signal intensity at 1332 cm -1 , substitute it into the standard curve equation, and calculate the concentration of the target AD biomarker in the sample to be tested.

Images