A kind of MC-LR type microcystin detection test strip based on gold nanorod SERS immunoprobe

Abstract

This invention discloses a Raman immunoassay probe based on gold nanorods, comprising gold nanorods, a signal labeling molecule modified on the gold nanorods, and a microcystin-LR (MC-LR) specific monoclonal antibody molecule. The inventors also established a method for preparing the corresponding Raman immunoassay probe. Based on this, the inventors developed a test strip for MC-LR microcystin detection based on a gold nanorod SERS immunoassay probe. It uses gold nanorods as the SERS labeling substrate, enhancing the Raman signal to produce a stronger and more stable signal, significantly improving detection sensitivity and signal reproducibility. Simultaneously, the combination of highly specific immunorecognition elements ensures the method's anti-interference ability and accuracy in complex matrices, thus providing a practical solution for achieving highly sensitive, stable, and quantitative rapid detection of MC-LR, suitable for rapid on-site screening and quantitative detection of trace MC-LR in water and aquatic products.

Description

Technical Field

[0001] This invention belongs to the field of microcystin detection technology, and particularly relates to an MC-LR type microcystin detection test strip based on a gold nanorod SERS immunoprobe. Background Technology

[0002] Microcystins (MCs) are a class of hepatotoxic cyclic heptacapeptide toxins produced by cyanobacterial blooms, with MC-LR being the most common and abundant subtype in water bodies. MC-LR toxins are highly stable and degrade slowly, accumulating in drinking water and aquatic products and seriously endangering human health, becoming a major global environmental and food safety issue. To address MC pollution, detection technologies have evolved from biotoxicity testing and instrumental analysis to immunological and spectroscopic analysis. Early biotoxicity and cytotoxicity testing methods (such as mouse tests) could reflect total toxicity, but lacked specificity and were time-consuming, failing to meet the need for rapid and accurate detection. Subsequent instrumental analytical methods (such as HPLC and HPLC-MS) became the mainstream for laboratory confirmation due to their high sensitivity and specificity, capable of separating and quantifying different subtypes; however, their reliance on large equipment, complex sample preparation, and high cost made them unsuitable for rapid on-site screening. Immunological methods (such as ELISA), based on antigen-antibody reactions, are relatively fast, but struggle to achieve accurate risk assessment. Raman spectroscopy offers advantages such as speed and portability, but its inherently weak signal makes it insufficient for detecting trace amounts of metallic minerals (MCs). To address this, surface-enhanced Raman scattering (SERS) technology has emerged. Through the plasmon resonance effect of noble metal nanostructures, the Raman signal can be amplified by millions to billions of times. Currently, two main strategies have been developed: label-free SERS (directly detecting the characteristic spectra of MCs) and labeled SERS immunoassay (using SERS labels as signal reporter units), providing a new direction for highly sensitive and rapid detection of MCs by light-sensitive scattering (LSR).

[0003] Despite the existence of various detection methods, current technologies still have significant limitations for rapid on-site detection of MC-LR in water and aquatic products: colloidal gold immunochromatographic test strips lack sufficient sensitivity and cannot accurately quantify; traditional ELISA methods struggle to accurately quantify target subtypes due to antibody cross-reactivity; conventional Raman spectroscopy has too low sensitivity; and unlabeled SERS detection is severely affected by coexisting substances in complex real-world samples, resulting in poor selectivity and reproducibility. While labeled SERS immunoassays (such as SERS immunochromatography) theoretically possess both high sensitivity and specificity, they still face a key bottleneck: the signal intensity and stability of conventional SERS markers (such as single metal nanoparticles) are limited, restricting the accuracy and reliability of quantitative detection. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a rapid, sensitive and accurate MC-LR type microcystin toxin detection strip based on gold nanorod SERS immunoprobes.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: Raman immunoprobes based on gold nanorods include gold nanorods, as well as signal marker molecules and microcystin-LR (MC-LR) specific monoclonal antibody molecules modified on the gold nanorods.

[0006] The signal marker molecule is 4-mercaptobenzoic acid (4-MBA).

[0007] The above-mentioned method for preparing Raman immunoassay probes involves electrostatic adsorption and covalent coupling of 4-mercaptobenzoic acid-modified gold nanorods with microcystin-LR (MC-LR) specific monoclonal antibodies via the carboxyl group at the end of the 4-mercaptobenzoic acid molecule and the amino group (-NH2) in the microcystin-LR (MC-LR) specific monoclonal antibody molecule, thereby achieving directional immobilization of the antibody on the surface of the nanoparticles. After coupling, the probes are blocked using bovine serum albumin (BSA) solution to block non-specific binding sites. Finally, the purified immunoassay probes are obtained by centrifugation and washing, and stored at 4°C for later use.

[0008] 4-Mercaptobenzoic acid-modified gold nanorods were prepared as follows: 10 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) solution was mixed with 100 μL of 1% chloroauric acid solution, and after thorough stirring, 600 μL of freshly prepared chloroauric acid solution was quickly added. The solution was prepared by adding ice water, which immediately turned brown. After standing at room temperature for 2 hours, a gold seed solution was obtained. Separately, 9.5 mL of 0.1 M CTAB solution was taken, and 500 μL of 0.01 M chloroauric acid solution was added sequentially. The solution and 500 μL of 0.1 M hydroquinone solution were stirred thoroughly until the solution became colorless and transparent. Then, 200 μL of the above gold seed solution was added, and the mixture was stirred gently for 10 s. The solution was then placed in the dark and allowed to stand for 6–12 h to obtain a gold nanorod (AuNR) solution. 1 mL of the gold nanorod (AuNR) solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in ultrapure water to the original volume. This purification process was repeated twice to remove residual CTAB. 10 mL of the purified gold nanorod (AuNR) solution was taken, and 2 μL of 10 mM 4-mercaptobenzoic acid (4-MBA) ethanol solution was added. The mixture was stirred at room temperature for 2 h to obtain gold nanorods (AuNR@4-MBA) material with 4-MBA surface modification. The material was stored at 4 °C in the dark.

[0009] The above preparation method involves taking 1 mL of the prepared gold nanorods (AuNR@4-MBA) material and using... The pH of the solution was adjusted to the range of 8.0–8.5 and allowed to stand for equilibration for 5 min. Then, 10 µL of MC-LR monoclonal antibody was added and incubated with continuous shaking at room temperature for 1 h to allow the antibody to stably bind to the carboxyl groups on the particle surface through electrostatic interaction. Next, 100 µL of 10% bovine serum albumin (BSA) blocking solution was added and the mixture was further treated with shaking at room temperature for 1 h to saturate unbound sites and reduce nonspecific adsorption. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 8 min and the supernatant was carefully removed. Finally, the precipitate was resuspended in 200 µL of phosphate buffer containing 0.1% BSA to obtain a stable SERS immunoprobe, which was stored at 4°C in the dark for later use.

[0010] The application of the above-mentioned Raman immunoprobes or the Raman immunoprobes prepared by the above-mentioned method in the detection and recognition of MC-LR type microcystin.

[0011] The MC-LR type microcystin test strip includes a nitrocellulose membrane (NC membrane), a sample pad and absorbent paper, and a PVC base plate. The nitrocellulose membrane is coated with MC-LR-BSA complete antigen and goat anti-mouse IgG polyclonal antibody, which serve as the test line (T line) and the control line (C line), respectively.

[0012] The above test strips are used in the detection and identification of MC-LR type microcystin.

[0013] To address the issue of insufficient sensitivity in on-site screening of microcystin products, the inventors plan to construct a dual-signal mode SERS-LFIA detection system by combining highly sensitive surface-enhanced Raman scattering (SERS) technology with immunochromatography. To this end, the inventors developed a Raman immunoprobe based on gold nanorods, comprising gold nanorods, a signal labeling molecule modified on the gold nanorods, and a microcystin-LR (MC-LR) specific monoclonal antibody molecule. Simultaneously, the inventors established a corresponding method for preparing the Raman immunoprobe. Based on this, the inventors developed an MC-LR type microcystin test strip based on a gold nanorod SERS immunoprobe. It uses gold nanorods as the SERS labeling substrate, and the enhancement effect produces a stronger and more stable Raman signal, significantly improving detection sensitivity and signal reproducibility. Simultaneously, the combination of a highly specific immunorecognition element ensures the method's anti-interference ability and accuracy in complex matrices, thus providing a practical solution for achieving highly sensitive, stable, quantitative, and rapid detection of MC-LR microcystins. In summary, the test strip of this invention has the characteristics of high sensitivity, high specificity, and good repeatability, ensuring reliability and practicality in real sample testing.

[0014] In summary, this invention retains the advantages of traditional immunochromatography, such as ease of operation and rapid response, while utilizing SERS technology to significantly enhance the Raman signal, thereby improving the detection sensitivity for MC-LR. Simultaneously, by integrating colorimetric and Raman dual-signal output modes, cross-validation can be achieved between qualitative screening and quantitative analysis, effectively improving the accuracy and reliability of the detection results. This invention is suitable for rapid on-site screening and quantitative detection of trace MC-LR in water bodies and aquatic products. Attached Figure Description

[0015] Figure 1 This is the UV-Vis spectrum of AuNR.

[0016] Figure 2 This is a macroscopic TEM image of the particle size and distribution of AuNR.

[0017] Figure 3 These are microscopic TEM images of the particle size and distribution of AuNR.

[0018] Figure 4 Images showing the detection results of a negative sample (left) and a positive sample (right). Figure 5 This is a graph showing the results of pH optimization. In the graph: A represents the addition of 1, 2, 3, ... The colorimetric effects of negative and positive samples in the solution are shown, where "+" and "-" represent positive samples (30 µg / L) and negative samples, respectively; B is the SERS signal corresponding to A, with the top 4 lines being negative and the bottom 4 lines being positive.

[0019] Figure 6 The graph shows the results of optimizing the amount of labeled antibody. In the graph: A shows the colorimetric effect of adding 4, 6, 8, and 10 μL of MC-LR antibody, where "+" and "-" represent positive samples (30 µg / L) and negative samples, respectively; B shows the SERS signal corresponding to A, with the top 4 lines being negative and the bottom 4 lines being positive.

[0020] Figure 7 The figure shows the results of optimizing the T-line coating dosage. In the figure: A shows the color development effect of MC-LR-BSA with T-line coating dosages of 5, 10, and 15 μL, where "+" and "-" represent positive samples (30 µg / L) and negative samples, respectively; B shows the SERS signal corresponding to A, with the top 3 lines being negative and the bottom 3 lines being positive.

[0021] Figure 8 This is a graph showing the LOD measurement results. In the graph: A represents the color development effect of MC-LR standard solutions with concentrations of 0 (negative), 5, 10, 15, 20, and 25 μg / L, respectively; B represents the SERS signal corresponding to A.

[0022] Figure 9This is a graph showing the specificity assessment results. In the graph: A is a negative sample (PB solution), with 30 µg / L MC-LR and 200 µg / L aflatoxin. ), DON (vomiting toxin), T-2 toxin, fumonisin The color development effects of ochratoxin A (OTA) and zearalenone (ZEN) are shown in Figure 1; B represents the SERS signal corresponding to A. Detailed Implementation

[0023] To further illustrate how the present invention is implemented, the following detailed examples are provided. Unless otherwise specified, all raw materials and reagents used are commercially available products; all quantitative experiments were performed in triplicate, and the average value of the results was taken.

[0024] 1. Preparation of gold nano-seed solution Take a clean glass container, add 10 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) solution, and then add 100 μL of 1% chloroauric acid. The solution was thoroughly mixed. While continuously stirring, 600 μL of freshly prepared and ice-bath-cooled sodium borohydride (NaBH4) solution (0.01 mol / L) was rapidly added to the mixture. Upon completion of the addition, the mixture immediately turned brown, indicating that the gold nanoseeds had initially formed. The resulting solution was allowed to stand at room temperature for 2 hours to obtain a stable gold nanoseed solution.

[0025] 2. Preparation of 4-mercaptobenzoic acid modified gold nanorods (AuNR@4-MBA) (1) Take another clean glass container and add 9.5 mL of 0.1 M CTAB solution. Under continuous stirring, add 500 μL of 0.01 mol / L chloroauric acid solution and 150 μL of 0.01 M silver nitrate solution to the container in sequence. The solution was thoroughly mixed. Then, 500 μL of a 0.1 M hydroquinone solution was added, at which point the solution gradually changed from pale yellow to colorless and transparent. Finally, 200 μL of the gold nanoparticle seed solution prepared in the previous step was added to the mixture, and the mixture was slowly stirred for 10 seconds before stopping. The reaction system was then placed in a dark environment and allowed to stand for 6–12 h to obtain a gold nanorod colloidal solution. The resulting product was stored at room temperature in the dark.

[0026] Characterization results of the obtained AuNR particle size and distribution by ultraviolet-visible spectroscopy and transmission electron microscopy are as follows: Figures 1 to 3 As shown. By Figure 1As shown, UV-Vis spectral analysis reveals a significant LSPR double peak at approximately 520 nm and 850 nm. This characteristic not only indicates excellent nanorod size uniformity but also reflects the high consistency of its structure. Electron microscopy shows that the synthesized AuNR particles are well dispersed, exhibiting no obvious agglomeration, and displaying a regular rod-like structure with good uniformity. The major axis length is approximately 100 nm, the minor axis length is 30 nm, and the aspect ratio is 3.33. The solution exhibits a bright brownish-red color, displaying a unique optical appearance.

[0027] (2) Take 1 mL of the prepared gold nanorod solution and place it in a 1.5 mL centrifuge tube. Centrifuge at 8000 rpm for 10 min and carefully remove the supernatant. Redisperse the precipitate to the original volume with ultrapure water. Repeat the above centrifugation and washing operation twice to completely remove residual CTAB from the system. Take 10 μL of the washed and resuspended gold nanorod dispersion and add 2 μL of 10 mM 4-mercaptobenzoic acid (4-MBA) ethanol solution. Stir to mix evenly and react at room temperature for 2 h to obtain the 4-MBA modified gold nanorod complex (AuNR@4-MBA). Store the product at 4℃ in the dark for subsequent analysis.

[0028] 3. Preparation of immune probes Take 1 mL of the gold nanorod composite (AuNR@4-MBA) prepared in the above steps, and use... The pH of the solution was adjusted to 8.0–8.5 and allowed to stand for equilibration for 5 min. Then, 10 µL of MC-LR monoclonal antibody (Source: Dai Rui. Preparation and Preliminary Application of Monoclonal Antibodies for Three Algal Toxins [D]. China Agricultural University, 2014.) was added, and the mixture was continuously shaken and incubated at room temperature for 1 h to allow the antibody to bind stably to the carboxyl groups on the particle surface through electrostatic interaction. Next, 100 µL of 10% bovine serum albumin (BSA) blocking solution was added, and the mixture was shaken at room temperature for another 1 h to saturate unbound sites and reduce nonspecific adsorption. After the reaction, the mixture was centrifuged at 10,000 rpm for 8 min, and the supernatant was carefully removed. Finally, the precipitate was resuspended in 200 µL of phosphate buffer containing 0.1% BSA to obtain a stable SERS immunoprobe, which was stored at 4℃ in the dark for later use.

[0029] 4. Sample testing Prepare the MC-LR test sample solution using 10 mM pH 7.4 phosphate buffer containing 0.05% Tween-20 as the sample diluent. Take 80 μL of the diluted sample solution and mix it thoroughly with 20 μL of the prepared immunoprobe. Vertically insert the sample end of the assembled test strip into the well of a 96-well plate containing the above mixture, and perform the chromatography reaction at room temperature. After the chromatography is complete (approximately 15–20 min), observe the color development results of the test line (T line) and control line (C line) on the test strip.

[0030] The result interpretation method is as follows: if both the C-line and T-line show visible bands, the result is considered negative; if only the C-line shows color while the T-line does not, the result is considered positive. Typical color development patterns are shown below. Figure 4 As shown, the left image is a negative sample, where both the C and T lines show visible bands; the right image is a positive sample, where only the C line shows color while the T line does not.

[0031] 5. pH optimization for labeling reaction Take 0.5 mL of AuNR@4-MBA solution and place it into eight 1.5 mL centrifuge tubes. Add 1, 2, 3, and 4 μL of the solution to each tube respectively. Solution, pH adjusted, and a series of immunoprobes prepared; corresponding test strips were used to detect Raman signals in negative and 50 μg / L MC-LR positive samples, respectively. Optimal Raman signal intensity ratio (T-line signal / C-line signal) and signal-to-noise ratio were used to determine the optimal... Dosage.

[0032] Optimization results are as follows Figure 5 As shown in Figure A, when 3 μL of 0.1 M K₂CO₃ solution is added, the C and T lines of the test strip show the deepest color; as... Figure 5 As shown in Figure B, the Raman signal of line T is strongest when 3 μL of K2CO3 is added.

[0033] 6. Optimization of labeled antibody dosage Take 6 equal volumes (0.5 mL) of AuNR@4-MBA solution, adjust to the optimized pH value, and add 4, 6, 8, and 10 μL of MC-LR monoclonal antibody respectively to prepare immune probes with different antibody loadings. After assembling the test strips, positive and negative samples were detected respectively. The ratio of T-line Raman signal inhibition rate to background signal was used as the evaluation index to determine the optimal antibody labeling amount.

[0034] Optimization results are as follows Figure 6 As shown in Figure A, when 8 μL of MC-LR monoclonal antibody was added, the C and T lines on the test strip showed the deepest color development, as indicated by Figure A. Figure 6 As shown in B, the Raman signal of line T is strongest at this time.

[0035] 7. Test strip assembly process MC-LR-BSA and goat anti-mouse IgG polyclonal antibody were diluted to 2 mg / mL using 10 mM phosphate buffer (pH 7.4). Using a three-dimensional spraying apparatus, MC-LR-BSA was coated onto a nitrocellulose membrane (NC membrane) at a rate of 1 μL / cm to serve as the detection line (T line), with a spacing of 5 mm. Goat anti-mouse IgG was used as the control line (C line). The coated NC membrane was dried at 37°C for 3 h. Subsequently, the sample pad, NC membrane, and absorbent paper were sequentially attached to a PVC substrate, with each layer overlapping by 1-2 mm. The composite membrane was cut into 3 mm wide test strips using an automatic strip cutter and placed in a light-proof bag containing desiccant, stored at 4°C for later use.

[0036] During the test, the sample end of the test strip is inserted into the test solution containing the immunoassay probe for chromatography. After 20 minutes, the colorimetric results are observed, and the SERS signals of the T and C lines are collected using a Raman spectrometer. The quantitative analysis of MC-LR is achieved based on the established standard curve.

[0037] 8. Optimize the amount of T-line wrapping used. MC-LR-BSA solutions (1.15 μg / mL) with concentration gradients of 5, 10, and 15 μL were prepared and coated onto NC membranes using a three-dimensional spraying apparatus to form the T-line. After assembling the test strips, negative and 30 μg / L MC-LR positive samples were tested, and the T-line was acquired at 1078 cm⁻¹ using Raman spectroscopy. The optimal coating concentration is the concentration with the highest Raman signal and a clear distinction between positive and negative signals.

[0038] Optimization results are as follows Figure 7 As shown in Figure A, when 10 μL of MC-LR-BSA solution was added, the C and T lines on the test strip showed the deepest color and a clear distinction between positive and negative results, as indicated by Figure A. Figure 7 As shown in B, the Raman signal of line T is strongest at this time, and the distinction between positive and negative signals is also obvious.

[0039] 9. Sensitivity Measurement MC-LR standard solutions were prepared using a negative matrix at concentrations of 0 (negative), 5, 10, 15, 20, and 25 μg / L. 80 μL of each standard solution was mixed with 20 μL of the immunoprobe, and the results were detected using test strips. The results were recorded visually and at the T line at 1078 cm⁻¹. The Raman signal intensity at the location was measured. The limit of detection (LOD) was defined as the concentration corresponding to the mean negative signal minus three standard deviations.

[0040] Sensitivity test results are as follows Figure 8 As shown in Figure A, when 15 μL of MC-LR positive sample was added, the T line on the test strip clearly disappeared, as shown in Figure A. Figure 8As shown in Figure B, the Raman signal of line T is significantly reduced at this point. A standard curve was plotted with the concentration of the MC-LR standard solution on the x-axis and the SERS signal intensity on the y-axis. The linear equation obtained in the range of 0–20 μg / L is y = Based on this, the LOD of the SERS test strip for MC-LR based on the Raman signal was calculated by subtracting three times the standard deviation from the mean of the negative signal.

[0041] 10. Specificity evaluation Select a negative sample (PB solution) with 200 µg / L aflatoxin. ), DON (vomiting toxin), T-2 toxin, fumonisin Ochratoxin A (OTA) and zearalenone (ZEN) were used as interfering agents, and their specificity was evaluated against MC-LR (30 µg / L). After adding the immunoprobe to each group of samples, Raman spectra were collected using test strips, and the specificity was evaluated by the signal intensity at 1078 cm⁻¹.

[0042] Specificity evaluation results as follows Figure 9 As shown in Figure A, the C and T lines of the test strips detecting negative samples and six interfering substances are clearly visible, while only the T line of MC-LR is not visible, as shown in Figure A. Figure 8 As shown in Figure B, at this time, the T line of the test strip that only detects MC-LR has almost no Raman signal. Both the colorimetric result and the SERS signal indicate that the test strip has good specificity.

[0043] 11. Repeatability of tests Intra-batch repeatability: Six test strips were randomly selected from the same batch, and three MC-LR positive samples and three negative controls were tested. The coefficient of variation (CV) of the T-line Raman signal intensity for each group was calculated. SERS results showed that the CV value of the test strips during intra-batch testing was 4.27%.

[0044] Inter-batch repeatability: Two test strips were taken from each of three different production batches, and MC-LR positive samples and negative controls were tested respectively. The CV of signal intensity for each batch and between batches was calculated. The results showed that the CV value for inter-batch testing was 8.52%.

Claims

1. A Raman immunoassay probe based on gold nanorods, characterized in that... This includes gold nanorods, as well as signaling marker molecules modified on the gold nanorods and microcystin-LR (MC-LR) specific monoclonal antibody molecules.

2. The Raman immunoassay probe according to claim 1, characterized in that: The signal marker molecule is 4-mercaptobenzoic acid (4-MBA).

3. The method for preparing the Raman immunoassay probe according to claim 2, characterized in that: 4-Mercaptobenzoic acid-modified gold nanorods were electrostatically adsorbed and covalently coupled with microcystin-LR specific monoclonal antibody to achieve directional immobilization of the antibody on the surface of nanoparticles. After conjugation, the probe was blocked using bovine serum albumin solution; finally, the purified immune probe was obtained by centrifugation and washing.

4. The preparation method according to claim 3, characterized in that: The 4-mercaptobenzoic acid-modified gold nanorods were prepared as follows: 10 mL of 0.1 M hexadecyltrimethylammonium bromide solution was mixed with 100 μL of 1% chloroauric acid solution, and after stirring until homogeneous, 600 μL of freshly prepared [solution / concentration] solution was quickly added. The ice-water solution was allowed to stand at room temperature for 2 hours to obtain the gold seed solution; separately, 9.5 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB) solution was added, followed by 500 μL of 0.01 M chloroauric acid solution. The solution and 500 μL of 0.1 M hydroquinone solution were stirred thoroughly until the solution became colorless and transparent. Then, 200 μL of the above gold seed solution was added, and the mixture was stirred gently for 10 s. The solution was then placed in a dark environment and allowed to stand for 6–12 h to obtain a gold nanorod (AuNR) solution. 1 mL of the gold nanorod solution was centrifuged at 8000 rpm for 10 min, the supernatant was discarded, and the precipitate was resuspended in ultrapure water to the original volume. This purification process was repeated twice to remove residual CTAB. 10 mL of the purified gold nanorod solution was taken, and 2 μL of 10 mM 4-mercaptobenzoic acid ethanol solution was added. The mixture was stirred at room temperature for 2 h to obtain gold nanorod materials with 4-MBA surface modification.

5. The preparation method according to claim 4, characterized in that: Take 1 mL of the prepared gold nanorod material and use... The pH of the solution was adjusted to 8.0–8.5 and allowed to stand for equilibration for 5 min. Then, 10 µL of MC-LR monoclonal antibody was added and incubated with continuous shaking at room temperature for 1 h. Next, 100 µL of 10% bovine serum albumin blocking solution was added and the mixture was further incubated with shaking at room temperature for 1 h. After the reaction was completed, the mixture was centrifuged at 10,000 rpm for 8 min and the supernatant was carefully removed. Finally, the precipitate was resuspended in 200 µL of phosphate buffer containing 0.1% BSA to obtain a stable SERS immunoprobe.

6. The application of the Raman immunoprobe according to claim 1 or the Raman immunoprobe prepared by any of the methods in claims 2-5 in the detection and recognition of MC-LR type microcystin.

7. An MC-LR type microcystin test strip, comprising a nitrocellulose membrane, a sample pad, absorbent paper, and a PVC base plate, characterized in that: The nitrocellulose membrane is coated with MC-LR-BSA complete antigen and goat anti-mouse IgG polyclonal antibody, which serve as the detection line and the quality control line, respectively.

8. The application of the test strip according to claim 7 in the detection and identification of MC-LR type microcystin.

9. The application according to claim 8, characterized in that: The objects to be detected and identified are water bodies or aquatic products.

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