Nano-enzyme-mediated SERS (Surface Enhanced Raman Scattering) immunodetection method without Raman nano-label

Abstract

The invention discloses a nano-enzyme mediated SERS (Surface Enhanced Raman Scattering) immunodetection method without a Raman nano-label, which can be used for detecting low-concentration biomarkers and improving the sensitivity of SERS detection. The method comprises the following steps: step 1, combining a metal nano-enzyme with a recognition antibody to obtain a nano-enzyme modified by the recognition antibody; combining the capture antibody with an SERS immune substrate to obtain a capture antibody modified SERS immune substrate; step 2, dropwise adding an antigen to be detected on the SERS immune substrate modified by the capture antibody, and incubating; after immune recognition, washing the SERS immune substrate with a PBS buffer solution; nano-enzyme modified by the recognition antibody is dropwise added to the SERS immune substrate and incubated, and the nano-enzyme modified by the recognition antibody and the SERS immune substrate modified by the capture antibody are combined through an antigen; 3, a color developing solution is dropwise added into the SERS immune substrate, and after incubation, quantitative detection is conducted on antigens through a Raman spectrometer.

Description

Technical Field

[0001] The present invention relates to a method for detecting biomarkers by combining nanozyme with surface-enhanced Raman scattering, and in particular to a nanozyme-mediated Raman-free nano-label SERS immunoassay method. Background Art

[0002] In the prior art, since target biomolecules usually do not have Raman activity, SERS immunoassays based on metal nanoparticle monolayer superlattice films usually use the method of detecting signal molecules (Raman molecules) with high Raman scattering cross sections as internal standards. Figure 1 As shown, the method mainly includes three steps: Step 1: Metal nanoparticles are bound to Raman molecules to obtain Raman molecule-modified Raman nanotags. These Raman nanotags are then bound to recognition antibodies to obtain recognition antibody-modified Raman nanotags. Step 2: Capture antibodies are bound to a metal nanoparticle monolayer superlattice film to obtain an immunogenic substrate. Step 3: The antigen to be detected is added, and the recognition antibody-modified Raman nanotags and the capture antibody-modified immunogenic substrate bind to the antigen through the antigen, forming a double-antibody sandwich structure. Finally, the Raman molecule signal is detected using a Raman spectrometer to detect the antigen. Step 1 requires ensuring that the Raman molecule is both stably bound to the nanoparticle surface and has a strong Raman signal. Dye molecules (such as Nile Blue) have a large scattering cross-section and a strong Raman signal. However, because they bind to the nanoparticle surface through electrostatic adsorption, the Raman molecules are easily dropped onto the SERS substrate during the formation of the double-antibody sandwich structure in Step 3, resulting in false positive signals in SERS detection. Raman molecules bound by chemical bonds (such as 4-mercaptobenzoic acid) have weaker Raman signals than dye molecules. Therefore, the requirements for SERS substrates and Raman spectrometers are high, which is not conducive to the real-time, rapid and ultra-sensitive detection of biomarkers at low or low concentrations. Specifically, the Raman molecules carried in the Raman nanotags have difficulty entering the hotspot area of ​​the SERS substrate (i.e., the gaps between closely arranged metal nanoparticles, usually <5 nm) due to steric hindrance, resulting in the inability to achieve optimal enhancement of their Raman signals. This spatial limitation makes the sensitivity of the system heavily dependent on the surface electromagnetic field effect of the nanotag itself, which in turn places higher requirements on the performance of the detection instrument. At present, high-sensitivity detection still relies on precision equipment such as laser confocal Raman spectrometers. Therefore, the development of a new SERS immunoassay method that combines high sensitivity with ease of operation remains an important issue that needs to be broken through in the current field of biosensors. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a nanozyme-mediated Raman nanotag-free SERS immunoassay method, which can use a portable Raman spectrometer to detect biomarkers, especially low-concentration biomarkers, and improve the sensitivity of SERS detection.

[0004] To solve the above problems, the embodiments of the present invention provide the following technical solutions: A nanozyme-mediated Raman nanotag-free SERS immunoassay method, characterized in that the method comprises: Step 1: combining the metal nanozyme with the recognition antibody to obtain the recognition antibody-modified nanozyme; Using a single-layer superlattice film as a SERS immunosubstrate, combining the capture antibody with the SERS immunosubstrate to obtain a capture antibody-modified SERS immunosubstrate; Step 2: Add the antigen to be detected onto the capture antibody-modified SERS immune substrate and incubate; after immune recognition, rinse the SERS immune substrate with PBS buffer; then, add the recognition antibody-modified nanozyme onto the SERS immune substrate and incubate. The recognition antibody-modified nanozyme binds to the capture antibody-modified SERS immune substrate through the antigen to form a double-antibody sandwich structure; rinse the SERS immune substrate with PBS buffer; Step 3: Add tetramethylbenzidine colorimetric solution to the SERS immunoassay substrate, incubate, and then quantitatively detect the antigen using a Raman spectrometer.

[0005] As a preferred example, in step 1, the method for obtaining the antibody-modified nanozyme comprises: Adjust the pH value of the nanozyme solution to the same as the isoelectric point of the recognition antibody to be added; The recognition antibody solution is added to the nanozyme solution. After incubation, bovine serum albumin solution is added to the solution. After incubation, centrifugation is performed, the supernatant is removed, and the solution is washed with PBS. After centrifugation, the supernatant is removed and the solution is resuspended with PBS buffer to obtain the recognition antibody-modified nanozyme.

[0006] As a preferred example, in step 1, the final concentration of the recognition antibody in the nanozyme solution is 10 μg / mL; the mass fraction of the BSA solution is 10%, the volume ratio of the BSA solution to the nanozyme solution is 1:10; and the volume ratio of the PBS buffer solution to the nanozyme solution is 0.25:1.

[0007] As a preferred example, in step 1, the method for obtaining a SERS immune substrate modified with a capture antibody is as follows: placing a silver nanoparticle monolayer superlattice film in a plasma cleaning machine for irradiation to remove the PS ligand on the surface of the silver nanoparticle monolayer superlattice film, then dripping the capture antibody, incubating to form a SERS immune substrate; rinsing the SERS immune substrate with PBS buffer; dripping BSA solution onto the immune substrate, incubating, and rinsing it with PBS buffer several times to obtain a SERS immune substrate modified with a capture antibody.

[0008] As a preferred example, the capture antibody concentration is 25 μg / mL, the BSA solution mass fraction is 10%, and the volume ratio of the BSA solution to the capture antibody is 5:1.

[0009] As a preferred example, in step 2, the volume ratio of the antigen and the recognition antibody-modified nanozyme is 5:2.

[0010] As a preferred example, in the dual-antibody sandwich structure, different determinants on the surface of the antigen are respectively combined with the recognition antibody on the surface of the recognition antibody-modified nanozyme and the recognition sites of the capture antibody in the SERS immune substrate, forming a structure in which the antigen is located in the middle layer, the recognition antibody-modified nanozyme is located in the top layer, and the capture antibody-modified SERS immune substrate is located in the bottom layer.

[0011] As a preferred example, in step 2, the concentration of the antigen to be detected is 100 fg / mL ~ 1 ng / mL.

[0012] As a preferred example, in step 3, in the TMB color developing solution, the final concentration of hydrogen peroxide is 0.3%, and the final concentration of TMB is 1.4 mM.

[0013] As a preferred example, in step 3, the Raman spectrometer is a portable Raman spectrometer, the laser power of the Raman spectrometer is 200 mW, the exposure time is 5 s, and the test is performed at room temperature.

[0014] Compared with the prior art, the nanozyme-mediated Raman nanotag-free SERS immunoassay method of the embodiment of the present invention can use a portable Raman spectrometer to detect biomarkers, especially low-concentration biomarkers, and improve the sensitivity of SERS detection. The method includes: step 1, combining a metal nanozyme with a recognition antibody to obtain a nanozyme modified with a recognition antibody; using a single-layer superlattice film as a SERS immunosubstrate, combining a capture antibody with the SERS immunosubstrate, and obtaining a SERS immunosubstrate modified with a capture antibody; step 2, dropping the antigen to be detected on the SERS immunosubstrate modified with the capture antibody and incubating; after immune recognition, rinsing the SERS immunosubstrate with PBS buffer; then, dropping the nanozyme modified with the recognition antibody on the SERS immunosubstrate and incubating, the nanozyme modified with the recognition antibody and the SERS immunosubstrate modified with the capture antibody through the antigen to form a double antibody sandwich structure; rinsing the SERS immunosubstrate with PBS buffer; step 3, dropping TMB color development solution on the SERS immunosubstrate, and after incubation, quantitatively detecting the antigen using a Raman spectrometer. This method achieves highly sensitive and label-free detection of biomarkers by combining a single-layer superlattice membrane immunosubstrate with high SERS activity with a metal nanoenzyme immunoprobe with high catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the schematic diagram of the existing SERS immunoassay with Raman nanotags; Figure 2 This is a schematic diagram of the SERS immunoassay principle of an embodiment of the present invention; Figure 3 is a spectrum data diagram of Example 1 of the present invention; Figure 4 Spectral data diagrams of Examples 2 and 3 of the present invention. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described in detail below with reference to the accompanying drawings.

[0017] Surface-enhanced Raman scattering (SERS), a rapid characterization technique leveraging the enhanced electromagnetic field on the surface of metal nanoparticles, can enable the detection of chemical and biological molecules. When metal nanoparticles are brought into close proximity to form a highly ordered two-dimensional assembly, known as a monolayer superlattice, plasmon coupling between adjacent particles generates significantly enhanced electromagnetic fields in the interparticle spaces, known as "hotspots." The SERS signal of molecules entering these hotspots is significantly enhanced, enabling detection down to the single-molecule level. Portable Raman spectrometers are low-cost, easy-to-use, and rapid tools for on-site Raman spectroscopy. Due to their compact size and simplicity, the laser spot size used to excite the Raman signal is typically in the 100 μm range (compared to 1 μm for large confocal Raman instruments). This results in low resolution and sensitivity in the detection results. The present invention combines a portable Raman spectrometer with a SERS substrate based on a metal nanoparticle monolayer superlattice film to achieve highly sensitive, convenient and reliable immunoassay.

[0018] like Figure 2 As shown, a nanozyme-mediated Raman nanotag-free SERS immunoassay method according to an embodiment of the present invention comprises: Step 1: Combine the metal nanozyme with the recognition antibody to obtain the recognition antibody-modified nanozyme; use the single-layer superlattice film as the SERS immune substrate, combine the capture antibody with the SERS immune substrate, and obtain the capture antibody-modified SERS immune substrate.

[0019] Preferably, in step 1, the method for obtaining the antibody-modified nanozyme comprises: Step 101: Adjust the pH of the nanozyme solution to the same value as the isoelectric point of the antibody to be added. The optimal pH for nanoparticle-antibody binding is near the antibody's isoelectric point. Adjusting the pH of the nanozyme solution to the same value as the isoelectric point of the antibody to be added allows for efficient antibody binding. Different antibodies have different isoelectric points. The pH of the nanozyme solution is determined based on the antibody's isoelectric point.

[0020] Step 102: Add the recognition antibody solution to the nanozyme solution. After incubation, add a BSA (bovine serum albumin) solution to the solution. After incubation, centrifuge, remove the supernatant, wash with PBS (phosphate buffer saline), and reconstitute with PBS buffer to obtain a recognition antibody-modified nanozyme. The final concentration of the recognition antibody in the nanozyme solution is 10 μg / mL; the mass fraction of the BSA solution is 10%, the volume ratio of the BSA solution to the nanozyme solution is 1:10, and the volume ratio of the PBS buffer to the nanozyme solution is 0.25:1.

[0021] In step 102, the recognition antibody solution is added to the nanozyme solution. After incubation, an antibody-modified nanozyme solution is formed. BSA solution is then added to the solution to block sites on the nanozyme surface that are not modified by the antibody, thereby preventing nonspecific binding and false positive results in subsequent steps. After incubation, centrifugation is performed, the supernatant is removed, and the solution is washed with PBS to remove excess antibody and free BSA. After centrifugation, the supernatant is removed and the solution is resuspended in PBS buffer to obtain the recognition antibody-modified nanozyme.

[0022] Preferably, in step 1, the method for obtaining a capture antibody-modified SERS immunosubstrate is as follows: placing a silver nanoparticle (Ag NPs) monolayer superlattice film in a plasma cleaner for irradiation to remove PS (Polystyrene) ligands on the surface of the silver nanoparticle monolayer superlattice film, then dropwise adding a capture antibody and incubating to form a SERS immunosubstrate; dropwise adding a BSA solution to the immunosubstrate, incubating, and then rinsing multiple times with PBS buffer to remove excess antibody and free BSA, thereby obtaining a capture antibody-modified SERS immunosubstrate. The capture antibody concentration is 25 μg / mL, the BSA solution mass fraction is 10%, and the volume ratio of BSA solution to capture antibody is 5:1.

[0023] Step 2: Add the antigen to be detected onto the SERS immune substrate modified with the capture antibody and incubate; after immune recognition, rinse the SERS immune substrate with PBS buffer; then, add the nanozyme modified with the recognition antibody onto the SERS immune substrate and incubate. The nanozyme modified with the recognition antibody and the SERS immune substrate modified with the capture antibody are combined through the antigen to form a double antibody sandwich structure; rinse the SERS immune substrate with PBS buffer.

[0024] In step 2, in the dual-antibody sandwich structure, different determinants on the surface of the antigen are respectively combined with the recognition antibody on the surface of the recognition antibody-modified nanozyme and the recognition sites of the capture antibody in the SERS immune substrate, forming a structure in which the antigen is located in the middle layer, the recognition antibody-modified nanozyme is located in the top layer, and the capture antibody-modified SERS immune substrate is located in the bottom layer.

[0025] Preferably, in step 2, the volume ratio of the antigen to the recognition antibody-modified nanozyme is 5:2. Under this ratio, different concentrations of antigen can completely cover the surface of the single-layer superlattice film, reducing errors.

[0026] Preferably, in step 2, the concentration of the antigen to be detected is 100 fg / mL to 1 ng / mL. Even for antigens with extremely low concentrations, the method of the present application can detect biomarkers with high sensitivity.

[0027] Step 3: Add tetramethylbenzidine (TMB) colorimetric solution to the SERS immunoassay substrate and incubate. Nanozyme-catalyzed TMB generates a blue oxidation product, which is then quantitatively detected using a Raman spectrometer. The TMB colorimetric solution is a mixture of TMB and hydrogen peroxide. The final concentration of hydrogen peroxide in the TMB colorimetric solution is 0.3%, for a total of 1.4 mM. For example, to prepare 10 mL of TMB colorimetric solution, add 9.9 mL of H₂O, 100 μL of 30% hydrogen peroxide, and 3.365 mg of TMB (molecular weight 240.34) to create a TMB colorimetric solution with a final hydrogen peroxide concentration of 0.3% and a TMB concentration of 1.4 mM.

[0028] In step 3, the Raman spectrometer is a portable Raman spectrometer, the laser power is 200 mW, the exposure time is 5 seconds, and the test is performed at room temperature. The spot diameter of the laser confocal Raman spectrometer is 1 μm, while the spot diameter of the portable Raman spectrometer used in the present invention is 150 μm. The room temperature is 18-22°C.

[0029] The method of this embodiment combines a single-layer superlattice film with high SERS activity with a nanozyme with high catalytic activity and easy surface modification of antibodies. The SERS immunosubstrate uses a single-layer superlattice film, and adjacent particles will generate a significantly enhanced electric field between the particles due to the plasmon coupling effect. This method does not require the modification of Raman signal molecules on the nanozyme. This method uses metal nanozymes to rapidly catalyze hydrogen peroxide to oxidize TMB to form positively charged TMB. + , can easily combine with the negatively charged single-layer superlattice film through electrostatic interaction, making TMB + It is effectively deposited in the hotspot area of ​​the monolayer superlattice film, thus generating a strong SERS signal. +As the SERS spectra of in situ generated Raman molecules with high scattering cross sections, such as Figure 2 As shown, the dual signal amplification effect of metal nanozyme catalysis and SERS immunosubstrate enhancement enables quantitative analysis of extremely low-concentration antigens using a portable Raman spectrometer. The method of this example utilizes a hydrogen peroxide-nanozyme-TMB system to detect biomarkers, eliminating the step of nanoparticle-modified Raman tags. This effectively reduces the problem of false positives caused by Raman tag detachment while improving the sensitivity of SERS detection.

[0030] The method of this embodiment has broad applicability. It is not only compatible with different SERS-enhanced immunosorbent substrates and nanozymes, but also utilizes metallo-nanozymes as nanoprobes instead of HRP-labeled secondary antibodies linked to recognition antibodies, enabling low-concentration detection of multiple biomarkers. This method has the advantages of high sensitivity, simple operation, and high universality.

[0031] The following examples and comparative examples illustrate the superior performance of the method of this embodiment. In the following examples and comparative examples, the portable Raman spectrometer is a Polaris-R80 manufactured by Polaris Scientific Instruments Co., Ltd. in China. All materials used are commercially available. The Alzheimer's disease biomarker pTau-181 (plasma phosphorylated Tau protein 181), anti-Tau-181 (Tau protein 181 antibody, used as a capture antibody), and anti-pTau-181 (phosphorylated Tau protein 181 antibody, used as a recognition antibody) were all purchased from Suzhou Renduan Biopharmaceutical Technology Co., Ltd.

[0032] Example 1 Detection of Alzheimer's Disease Biomarker pTau-181 A nanozyme-mediated Raman-free nanolabel SERS immunoassay method comprising: Step 1, preparing a nanozyme that recognizes antibody modification, specifically comprising: Step 101: preparing platinum nanozymes, i.e., Au@Ag-Pt NPs, specifically comprising: Step 1011. Prepare 15 nm gold nanoparticles (Au NPs) by adding 3 mL of a 1% Na3Ct aqueous solution to a boiling water solution containing 1 mL of 25 mM HAuCl4. Add 1 mL of 25 mM HAuCl4 to a mixture of 92.8 mL of deionized water and 0.45 mL of 1% Na3Ct, and add 4.2 mL of 15 nm Au NPs and 1 mL of 25 mM HQ (hydroquinone) with stirring. After reacting at room temperature for 2 hours, 50 nm Au NPs are obtained for later use. Step 1012: Mix 100 mL of the 50 nm Au NPs obtained above with 0.5 mL of 0.1 M L-AA (L-Ascorbic acid). Add 2.5 mL of 10 mM AgNO3 while stirring. React at room temperature for 1 hour to obtain Au@AgNPs for later use. Step 1013: Place 100 mL of the Au@Ag NPs obtained above in a three-necked flask and heat to boiling under vigorous stirring. Add 4 mL of a 1 mM H2PtCl6 aqueous solution at a rate of 5 mL / h through a syringe pump. Cool the obtained Au@Ag-PtNPs to room temperature and add 500 μL of 1% Na3Ct as a stabilizer for later use.

[0033] Step 102, preparing a nanozyme modified with the recognition antibody anti-pTau-181, specifically comprising: Step 1021: Take 1 mL of Au@Ag-Pt NPs nanozyme and adjust the nanozyme to an optimal pH of 5 (the isoelectric point of the recognition antibody is 5).

[0034] Step 1022: Add the recognition antibody anti-pTau-181 solution to the above-mentioned nanozyme solution (the final concentration of the recognition antibody is 10 μg / mL), incubate at 37°C for 30 minutes, then add 100 μL of 10% BSA solution to the above-mentioned solution, incubate for 20 minutes, centrifuge, remove the supernatant, wash twice with PBS, and re-dissolve with 0.25 mL of PBS buffer to obtain the recognition antibody anti-pTau-181 modified nanozyme (i.e., SERS nanotag).

[0035] Step 103, preparing a SERS immunosubstrate modified with capture antibody anti-Tau-181, specifically comprising: placing a silver nanoparticle monolayer superlattice film in a plasma cleaner for 1 minute to obtain a SERS immunosubstrate; dropping 10 μL of 25 μg / mL capture antibody anti-Tau-181 on the SERS immunosubstrate, incubating at 37°C for 30 minutes, and then rinsing the surface of the monolayer superlattice film multiple times with PBS buffer; dropping 50 μL of 10% BSA solution on the above-mentioned SERS immunosubstrate, and incubating at 37°C for 20 minutes, and rinsing multiple times with PBS buffer to obtain a SERS immunosubstrate modified with capture antibody anti-Tau-181, which was stored at 4°C for future use.

[0036] Step 2: Biomarker Detection: Using an immunosandwich assay, perform the assay according to a standard ELISA protocol. 50 μL of antigen sample (i.e., Alzheimer's disease biomarker pTau-181) at varying concentrations (ranging from 100 fg / mL to 1 ng / mL) was dripped onto a SERS immunoassay substrate modified with the capture antibody anti-Tau-181 and incubated at 37°C for 30 minutes. Following immunorecognition, the SERS immunoassay substrate was rinsed multiple times with PBS buffer. Subsequently, 20 μL of the nanozyme modified with the recognition antibody anti-pTau-181 was dripped onto the recognition site and incubated at 37°C for 30 minutes. The SERS immunoassay substrate was then rinsed with PBS buffer.

[0037] Step 3: Add 60 μL of TMB colorimetric solution to the SERS immunoassay substrate, incubate at 37°C for 30 minutes, and then detect using a portable Raman spectrometer. During the test, the laser power was 200 mW and the exposure time was 5 seconds. All Raman experiments were performed at room temperature.

[0038] The detection results of the portable Raman spectrometer are as follows Figure 3 As shown. Among them, Figure 3 (A) TMB at different concentrations of pTau-181 + SERS spectrum of Figure 3 (A) It can be seen that: at 1183 cm -1 、1332 cm -1 and 1602 cm -1 Typical TMB was observed + The main Raman peak of 1602 cm was selected based on the above results. -1 The Raman intensity at TMB + The characteristic peaks of pTau-181 were detected to quantify the concentration of pTau-181. Figure 3 (B) indicates TMB + At 1602 cm -1 The fitting curve of the Raman characteristic peak intensity at φ varies with the concentration of pTau-181, and the linear equation is: y =2073.72 log 10 [ x ]+17591.33, R 2 =0.96. From the figure we can see that: TMB + The SERS intensity of TMB decreased significantly with the decrease of pTau-181 antigen concentration. In the range of 100 fg / mL to 1 ng / mL, + There is a linear relationship between the Raman intensity and the logarithm of the pTau-181 concentration, and the fitted linear equation is: y =2073.72 log10 [ x ]+17591.33, R 2 =0.96. The minimum detection concentration of this SERS-based enzyme immunoassay is 100 fg / mL. Figure 3 (C) TMB at 1 pg / mL pTau-181 + SERS spectrum waterfall diagram. As can be seen from the figure: Taking 1 pg / mL pTau-181 standard as an example, 6 points were randomly selected to collect TMB at 1 pg / mL antigen. + SERS spectra. Figure 3 (C) is a waterfall plot of the SERS spectra obtained in the presence of 1 pg / mL antigen. It can be seen that the Raman spectra are highly consistent. Figure 3 (D) TMB of 1 pg / mL pTau-181 + The SERS spectrum at 1602 cm -1 The Raman characteristic peak intensity histogram at 1 pg / mL is shown in Figure 1, with RSD = 2.82%. Figure 3 (C) 1602 cm -1 The characteristic peak intensity at the plotted peak intensity and detection times histogram and calculated the RSD, RSD = 2.82% ( Figure 3 D). This demonstrates that, when using a portable Raman spectrometer, the single-layer superlattice film as a SERS immunoassay substrate for pTau-181 detection still exhibits high reproducibility. Therefore, the proposed biosensor, based on the integration of SERS and enzyme catalysis, has great potential for large-scale AD screening.

[0039] Example 2 Detection of Alzheimer's Disease Biomarker pTau-181-Spiked Artificial Serum

[0040] The method and steps were the same as those in Example 1, except that an artificial serum sample spiked with the Alzheimer's disease biomarker pTau-181 was used instead of the antigen sample in step 2 of Example 1. The final concentration of pTau-181 in the artificial serum sample spiked with the Alzheimer's disease biomarker pTau-181 was 1 pg / mL.

[0041] Example 3 Detection of artificial serum The method and steps are the same as those in Example 1, except that artificial serum is used instead of the antigen sample in step 2 of Example 1.

[0042] like Figure 4 (A) shows the TMB content in artificial serum and pTau-181 spiked artificial serum samples in Examples 2 and 3.+ From the figure, we can see that in the SERS spectrum detected by the SERS immunosubstrate in artificial serum, 1330cm -1 and 1602 cm -1 A weak Raman peak appeared at 1182 cm. This may be because artificial serum contains a variety of proteins (such as albumin, globulin, etc.), which may have some sites that can interact with anti-pTau-181, thereby causing non-specific binding. In contrast, under this detection system, the SERS spectrum of the pTau-181 spiked artificial serum sample showed a strong Raman peak at 1182 cm. -1 、1330 cm -1 and 1602 cm -1 Significant Raman peaks were observed at 1602 cm -1 The peak intensity at 100 μM was twice that of the nonspecific signal in artificial serum. This result demonstrates that the detection system can effectively distinguish extremely low concentrations of pTau-181 even in complex biological environments, showing good specificity and sensitivity. This further demonstrates the potential of this detection system in biological assays, especially its ability to detect target molecules in complex samples.

[0043] like Figure 4 (B) shows that in Example 2, when 1 pg / mL pTau-181 was spiked with artificial serum, TMB + The SERS spectrum waterfall plot shows that the six groups of SERS spectra show high consistency in signal intensity and characteristic peak positions, indicating that the detection system has good uniformity and repeatability in the detection results of different positions on the single-layer superlattice film.

[0044] like Figure 4 As shown in (C), in Example 2, when 1 pg / mL pTau-181 was spiked with artificial serum, the peak at 1602 cm -1 The Raman characteristic peak intensity histogram at 370 nm has RSD=7.39%. Figure 4 (C) Further shows that the single-layer superlattice film has a high -1 The RSD of the characteristic peak intensity at was 7.39%. This result demonstrates that the SERS immunosubstrate based on the single-layer superlattice membrane exhibits reliable repeatability and stability in detecting antigens in artificial serum. This excellent performance provides important evidence for the application of this single-layer superlattice membrane in real biological samples, particularly for the quantitative detection of pTau-181 in human serum samples.

[0045] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art will appreciate that the present invention is not limited to the specific embodiments described above. The specific embodiments and descriptions in the specification are intended only to further illustrate the principles of the present invention. The basic principles, main features, and advantages of the present invention are shown and described above without departing from the spirit and scope of the present invention. Those skilled in the art will appreciate that various changes and modifications may be made, and such changes and modifications are intended to fall within the scope of the invention as claimed.

Claims

1. A nanozyme-mediated Raman nanotag-free SERS immunoassay method, characterized in that: The method comprises: Step 1: combining the metal nanozyme with the recognition antibody to obtain the recognition antibody-modified nanozyme; Using a single-layer superlattice film as a SERS immunosubstrate, combining the capture antibody with the SERS immunosubstrate to obtain a capture antibody-modified SERS immunosubstrate; Step 2: Add the antigen to be detected onto the capture antibody-modified SERS immune substrate and incubate; after immune recognition, rinse the SERS immune substrate with PBS buffer; then, add the recognition antibody-modified nanozyme onto the SERS immune substrate and incubate. The recognition antibody-modified nanozyme binds to the capture antibody-modified SERS immune substrate through the antigen to form a double-antibody sandwich structure; rinse the SERS immune substrate with PBS buffer; Step 3: Add tetramethylbenzidine colorimetric solution to the SERS immunoassay substrate, incubate, and then quantitatively detect the antigen using a Raman spectrometer.

2. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 1, characterized in that: In step 1, the method for obtaining the antibody-modified nanozyme comprises: Adjust the pH value of the nanozyme solution to the same as the isoelectric point of the recognition antibody to be added; The recognition antibody solution is added to the nanozyme solution. After incubation, bovine serum albumin solution is added to the solution. After incubation, centrifugation is performed, the supernatant is removed, and the solution is washed with PBS. After centrifugation, the supernatant is removed and the solution is resuspended with PBS buffer to obtain the recognition antibody-modified nanozyme.

3. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 2, characterized in that: In step 1, the final concentration of the recognition antibody in the nanozyme solution is 10 μg / mL; the mass fraction of the BSA solution is 10%, the volume ratio of the BSA solution to the nanozyme solution is 1:10; and the volume ratio of the PBS buffer solution to the nanozyme solution is 0.25:

1.

4. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 1, characterized in that: In step 1, the method for obtaining the capture antibody-modified SERS immunosubstrate is: The silver nanoparticle monolayer superlattice film was placed in a plasma cleaning machine for irradiation to remove the PS ligands on the surface of the silver nanoparticle monolayer superlattice film, and then the capture antibody was added dropwise and incubated to form a SERS immunosubstrate; The SERS immunosubstrate was rinsed with PBS buffer; BSA solution was added dropwise to the immunosubstrate, incubated, and then rinsed multiple times with PBS buffer to obtain a SERS immunosubstrate modified with capture antibodies.

5. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 4, characterized in that: The capture antibody mass concentration is 25 μg / mL, the BSA solution mass fraction is 10%, and the volume ratio of the BSA solution to the capture antibody is 5:

1.

6. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 1, characterized in that: In step 2, the volume ratio of the antigen and the recognition antibody-modified nanozyme is 5:

2.

7. The nanozyme-mediated Raman tag-free SERS immunoassay method according to claim 1, characterized in that: In the dual-antibody sandwich structure, different determinants on the surface of the antigen are respectively combined with the recognition antibody on the surface of the recognition antibody-modified nanozyme and the recognition site of the capture antibody in the SERS immune substrate, forming a structure in which the antigen is located in the middle layer, the recognition antibody-modified nanozyme is located in the top layer, and the capture antibody-modified SERS immune substrate is located in the bottom layer.

8. The nanozyme-mediated Raman tag-free SERS immunoassay method according to claim 1, characterized in that: In step 2, the concentration of the antigen to be detected is 100 fg / mL to 1 ng / mL.

9. The nanozyme-mediated Raman nanotag-free SERS immunoassay method according to claim 1, characterized in that: In step 3, in the tetramethylbenzidine colorimetric solution, the final concentration of hydrogen peroxide is 0.3%, and the final concentration of tetramethylbenzidine is 1.4 mM.

10. The nanozyme-mediated Raman-free nanotag SERS immunoassay method according to claim 1, characterized in that: In step 3, the Raman spectrometer is a portable Raman spectrometer, the laser power of the Raman spectrometer is 200 mW, the exposure time is 5 s, and the test is performed at room temperature.

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