Triple synergistically enhanced biosensor for biological toxin and preparation method and application thereof

Abstract

This invention discloses a triple-synergistic enhancement biotoxin SERS biosensor, its preparation method, and its application, belonging to the field of nanobiosensing and environmental pollutant detection technology. Addressing the shortcomings of existing okadaic acid detection methods—relying on large instruments, cumbersome operation, low detection sensitivity, poor anti-interference capabilities in complex shellfish matrices, and weak electromagnetic field enhancement, poor stability, and insufficient reproducibility of single SERS substrates—this invention constructs a novel Fe3O4@AuNFs-AuNPs dimer SERS substrate. Combining magnetic separation enrichment technology, noble metal plasma-coupled electromagnetic field enhancement technology, and enzyme-free catalytic hairpin self-assembly nucleic acid signal amplification technology, a triple-synergistic enhancement detection system is formed. This significantly improves the local electromagnetic field enhancement effect. Simultaneously, based on the CHA cascade hybridization amplification reaction, enzyme-free cyclic amplification of the target signal is achieved, solving the problems of weak SERS signals and inaccurate quantification caused by the low polarity and weak adsorption of okadaic acid.

Description

Technical Field

[0001] This invention belongs to the field of nanobiosensing and environmental pollutant detection, and more specifically, it relates to a triple-synergistic enhanced biotoxin SERS biosensor; at the same time, this invention also relates to the preparation method of the triple-synergistic enhanced biotoxin SERS biosensor, and the application of the triple-synergistic enhanced biotoxin SERS biosensor. Background Technology

[0002] Okadaic acid (OA) is a typical lipid-soluble polycyclic polyether diarrhetic shellfish toxin (DSP) that can cause gastrointestinal poisoning by inhibiting protein phosphatase activity and carries a carcinogenic risk. Traditional OA detection methods, such as enzyme-linked immunosorbent assay (ELISA) and high-performance liquid chromatography (HPLC), are cumbersome, time-consuming, or dependent on large instruments, making them unsuitable for rapid on-site screening. Therefore, the development of highly sensitive, highly specific OA detection technologies applicable to complex matrices is urgently needed.

[0003] SERS technology has become a cutting-edge field in trace substance analysis due to its advantages of single-molecule-level detection sensitivity, fingerprint spectral characteristics, and non-invasiveness. However, for small molecules such as okadaic acid with low polarity and weak adsorption, conventional SERS substrates are limited by insufficient local electromagnetic field enhancement efficiency and weak molecule-substrate interaction, making it difficult to achieve reliable quantitative detection. Therefore, we propose a biosensor for biotoxins with triple synergistic enhancement, as well as its preparation method and application. Summary of the Invention

[0004] The purpose of this invention is to address the shortcomings of existing technologies by proposing a triple-synergistic enhancement biotoxin SERS biosensor, its preparation method, and its application. This invention utilizes Fe3O4 magnetic nanoparticles to achieve efficient separation and enrichment of target substances in complex shellfish samples. It constructs a high-density SERS hotspot through the plasmon coupling effect of AuNFs and AuNPs dimer structures, significantly improving the local electromagnetic field enhancement effect. Simultaneously, it achieves enzyme-free cyclic amplification of the target signal based on the CHA cascade hybridization amplification reaction, solving the problem of weak SERS signals and inaccurate quantification caused by the low polarity and weak adsorption of Okada acid.

[0005] To achieve the above objectives, the present invention provides the following technical solution: A triple-synergistic enhancement of biotoxin SERS biosensor, consisting of a Fe3O4@AuNFs-OAAptamer-cDNA capture probe, a SERS tag, and a Fe3O4@AuNFs-H1 substrate unit.

[0006] Preferably, the Fe3O4@AuNFs-OA Aptamer-cDNA capture probe is composed of gold nanoflowers modified on the surface of Fe3O4 magnetic nanoparticles, and sequentially linked with okada acid aptamers and complementary strand cDNA. The SERS tag consists of a gold nanoparticle dimer modified with Au-S bonds to a hairpin probe H3 and a Raman reporter molecule 4-mercaptobenzonitrile; The Fe3O4@AuNFs-H1 substrate unit is composed of Fe3O4@AuNFs connected to hairpin probe H1; In this process, the Fe3O4@AuNFs-OA Aptamer-cDNA capture probe binds to the target OA and releases cDNA. The cDNA triggers a catalytic hairpin self-assembly reaction to form an H1-H2 double strand, which then captures the SERS tag through complementary pairing of H2 and H3, forming a Fe3O4@AuNFs-AuNPs dimer complex structure.

[0007] Preferably, the gold nanoparticle dimer is formed by AuNPs with a particle size of 30 nm through silver ion-mediated dimer structure, and its plasmonic coupling effect produces an absorption peak at 600 nm.

[0008] A method for preparing a triple-synergistic enhanced biotoxin SERS biosensor, the method comprising the following steps: S1. Preparation of the capture probe: After activating the SH-OA aptamer with TCEP, it was incubated with Fe3O4@AuNFs, and MCH was added to block non-specific sites. Then it was hybridized with cDNA to form Fe3O4@AuNFs-OA Aptamer-cDNA. S2 and SERS tag preparation: S21. Synthesize 30 nm AuNPs, and obtain AuNPs dimers by silver ion-mediated and agarose gel electrophoresis separation and purification. S22. Hairpin probe H3 was modified onto the surface of AuNPs dimer using a freeze-thaw method, and then 4-MBN was added for incubation and fixation. S3, Substrate Preparation: After annealing the hairpin probe H1, it was activated with TCEP, incubated with Fe3O4@AuNFs, and blocked with MCH to obtain the Fe3O4@AuNFs-H1 substrate. S4, Sensor Construction: S41. After incubation of the capture probe with OA, magnetic separation is performed, and the supernatant containing cDNA is collected. S42. Mix the cDNA, hairpin probe H2, and SERS tag in the supernatant with the Fe3O4@AuNFs-H1 substrate, react at 37℃ for 80 min, and then perform magnetic separation and cleaning to obtain the sensor.

[0009] Preferably, in step S3, the fixed concentration of the hairpin probe H1 is 300 nM, and the MCH blocking time is 30 min.

[0010] Preferably, the concentration of AuNPs dimers in the SERS tag of step S2 is 8 nM.

[0011] Application of the triple synergistic enhancement biotoxin SERS biosensor: The above-mentioned triple synergistic enhancement biotoxin SERS biosensor is used in the detection of shellfish toxin okadaic acid.

[0012] Preferably, 2234cm -1 Using the intensity of the 4-MBN characteristic peak as a quantitative signal, the detection linear range is 1 pg / mL to 1 μg / mL, and the detection limit is sub-pg / mL, making it suitable for complex matrix samples containing biological tissues.

[0013] Preferably, the detection is achieved through the following steps: The shellfish sample to be tested was homogenized and centrifuged, and the supernatant was incubated with the capture probe. After magnetic separation, the supernatant containing cDNA was collected to construct a sensor; SERS detection was performed by drop-coating onto a silicon wafer at a diameter of 2234 cm⁻¹. -1 Peak intensity difference is used to quantify OA concentration.

[0014] Technical effects and advantages of the present invention: The triple synergistic enhancement of the biotoxin SERS biosensor, its preparation method and application provided by the present invention, compared with the traditional technology, the present invention constructs a nanostructure Fe3O4@AuNFs-AuNPs dimer, that is, the detection sensitivity is improved by utilizing the plasmon coupling of AuNFs and AuNPs dimers and the CHA cascade signal amplification, and at the same time, the separation and enrichment effect of aptamer-functionalized magnetic beads is utilized to achieve rapid, accurate and highly sensitive on-site detection of shellfish toxin OA through triple synergistic enhancement. Attached Figure Description

[0015] Figure 1 This is a schematic diagram illustrating the construction of a SERS sensor based on Fe3O4@AuNFs-AuNPs dimer and its triple synergistic enhancement in detecting shellfish toxin OA. Figure 2 Characterization of Fe3O4@AuNFs; Figure a shows the UV absorption spectra of AuNPs, Fe3O4, Fe3O4-PEI, Fe3O4@AuNPs, and Fe3O4@AuNFs; Figure b shows the Zeta potential analysis; Figure c shows the particle size analysis. Figure 3 Morphology and elemental characterization of Fe3O4@AuNFs; In the figures: Figures a-c are TEM images of Fe3O4, Fe3O4@seed crystals, and Fe3O4@AuNFs; Figure d is a SEM image; Figure e is an EDX elemental mapping image. Figure 4 Zeta potential analysis of Fe3O4@Au NFs-OA Aptamer-cDNA synthesis process; Figure 5 Characterization diagram of AuNPs dimer-H3 SERS tag; Figure a shows the UV spectra of AuNPs and AuNPs dimers; Figure b shows the particle size analysis of AuNPs dimers. Figure 6 TEM characterization image; In the figures: Figure a is a TEM characterization image of AuNPs; Figure b is a TEM characterization image of AuNPs dimers; Figure 7 SERS tag characterization diagram; In the figures: Figure a shows the Zeta plot of AuNPs and AuNPs dimer; Figure b shows the gel electrophoresis plot of AuNPs and AuNPs dimer after H3 modification. Figure 8 Characterization diagram of the SERS sensor: Figure a shows the zeta potential analysis of the Fe3O4@AuNFs-AuNPs dimer preparation. Figure b shows the particle size analysis of Fe3O4@AuNFs-AuNPs dimer; Figure 9 EDS elemental mapping of Fe3O4@AuNFs-AuNPs dimer; Figure 10 Agarose gel electrophoresis analysis for cascaded signal amplification; Figure 11 Figure showing the feasibility analysis of using SERS sensors for OA toxin detection; Figure a shows the SERS spectra of OA toxin detected by the sensor with and without CHA cyclic amplification; Figure b shows the SERS spectra of OA toxin detected by the SERS sensor and the blank control group. Figure 12 Raman spectroscopy for detecting OA toxins with different SERS tags; In the figures: Figure a shows the individual SERS tag signal; Figure b shows the SERS spectrum for detecting OA toxin. Figure 13 To detect OA toxins by immobilizing different concentrations of H1 on Fe3O4@AuNFs-H1 substrate; In the figure: Figure a is the SERS spectrum; Figure b is the spectrum at 2234 cm⁻¹. -1 A bar chart showing the difference between the intensity of the characteristic peak and the blank background signal of the SERS signal; Figure 14 To optimize the spectra and bar charts of MCH at different blocking times; In the figures: Figure a shows the SERS spectrum of the blank control; Figure b shows the SERS spectrum for detecting OA toxin; Figure c shows the SERS spectrum at 2234 cm⁻¹. -1 A bar chart plotting the intensity of the characteristic peak signal, the blank background signal, and their difference; Figure 15 Spectra and bar charts of OA toxins detected at different AuNPs dimer-H3 SERS tag concentrations; In the figure: Figure a is the SERS spectrum; Figure b is the spectrum at 2234 cm⁻¹. -1 A bar chart plotting the difference between the intensity of the characteristic peak signal and the blank background signal; Figure 16 Spectra and bar charts for detecting OA toxins at different incubation times using the one-pot method; In the figure: Figure a is the SERS spectrum; Figure b is the spectrum at 2234 cm⁻¹. -1 A bar chart plotting the difference between the intensity of the characteristic peak signal and the blank background signal; Figure 17 The stability and repeatability spectra and bar charts of the Fe3O4@AuNFs-AuNPs dimer substrate; In the figures: Figures ab show the spectra and bar charts of 15 randomly selected points; Figures cd show the spectra and bar charts of 1 μg / mL OA SERS signals measured on 5 self-assembled substrates prepared in different batches. Figure 18 Spectrograms and bar charts for detecting other marine toxins; In the figure: Figure a is the SERS spectrum; Figure b is the spectrum at 2234 cm⁻¹. -1 A bar chart showing the difference between the peak value and the blank background; Figure 19 Spectra and linear curves of OA toxin at different concentrations detected by SERS sensor; In the figure: Figure a is the SERS spectrum; Figure b is the spectrum at 2234 cm⁻¹. -1 A linear curve plotting the difference between the input and the blank background; Figure 20SERS spectrum for detecting OA in spiked sample. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0017] This invention provides a triple synergistic enhancement biotoxin SERS biosensor, its preparation method, and its application. It avoids the problems of poor antibody stability and large non-specific adsorption interference in traditional immunoassay detection, effectively improving the sensitivity, specificity, and matrix adaptability of the detection system. It can realize rapid, accurate, and on-site detection of trace amounts of okadaic acid in shellfish samples, breaking through the technical bottleneck of traditional single-enhancement sensing mechanisms. It has broad application prospects in the fields of food safety testing and aquatic ecological toxin screening. The biosensor consists of a Fe3O4@AuNFs-OA Aptamer-cDNA capture probe, a SERS tag, and a Fe3O4@AuNFs-H1 substrate unit; the preparation method is as follows: Step 1: Preparation of the probe (1) Synthesis of Au seed crystals (8nm) Mix 1 mL of HAuCl4 (1 wt%) with 99 mL of ultrapure water under magnetic stirring for 15 minutes. Then, add 1 mL of Na3Cit (1 wt%) to the solution under a 30°C water bath and mix well. After that, add dropwise a mixture of 1 mL of NaBH4 (0.075 wt%) and 1 mL of Na3Cit (1 wt%) to the solution, stir for 30 minutes, and store at room temperature for later use.

[0018] (2) Synthesis of Fe3O4@AuNFs 10 mg of Fe3O4 nanoparticles were mixed with PEI solution (5 mg·mL⁻¹). -1After PEI successfully self-assembled on Fe3O4, it was mixed and bathed in a metal bath for 2 hours. The mixture was then washed three times with ultrapure water using magnetic separation. Subsequently, Fe3O4@PEI nanoparticles were mixed with Au seed crystals (5 mL) and sonicated in a water bath for 30 min, followed by standing for 1 h to form Fe3O4@Au seed crystals. The product was washed three times with ultrapure water under manual shaking to remove excess Au seed crystals, and the Fe3O4@Au seed crystals were collected using a magnet. Finally, based on the above ultrasonic treatment conditions, the prepared Fe3O4@Au seed crystals were dispersed in 50 mL of HAuCl4 solution, and then 0.5 mL of NH3·H2O (100 mg·mL⁻¹) was rapidly added. -1 After 5 minutes, add 0.15 g PVP-K30, sonicate for 1 hour, wash the final product with ultrapure water under manual shaking to remove excess PVP-K30, and concentrate to 5 mL.

[0019] (3) Preparation of Fe3O4@AuNFs-OA Aptamer-cDNA Mix 30 μL of 100 μM SH-OA Aptamer with 30 μL of 10 mM TCEP solution and activate for 1 hour. Then add 300 μL of Fe3O4@AuNFs solution and incubate for 12 hours with gentle shaking. After washing with reaction buffer, concentrate to 100 μL and add 100 μL of 100 mM MCH. Let stand for 10 minutes. Finally, wash the obtained Fe3O4@AuNFs-cDNA with PB buffer with manual shaking, collect it with a magnet, and redisperse it in 0.5×TBE solution.

[0020] Step 2: Preparation of SERS Tags (1) Synthesis of 30 nm AuNPs Gold spheres with a particle size of 30 nm were prepared by seed growth method. 150 mL of ultrapure water was added to a clean three-necked flask and heated in an oil bath for one hour. After boiling, 348 μL of 127 mM HAuCl4 was quickly added. Two minutes later, 0.0157 g of Na3Cit dissolved in 1 mL of ultrapure water was immediately added. During the heating process for one hour, the color of the solution changed from light yellow to purple, then to blue, and finally to red. After boiling for one hour, the three-necked flask was placed in a 90°C oil bath for equilibration for 30 minutes. 660 μL of 180 mM Na3Cit was added. Two minutes later, 660 μL of 75 mM HAuCl4 was quickly added. After 40 minutes, the addition of Na3Cit and HAuCl4 was repeated three times. After reacting for 40 minutes, heating was stopped. After the colloidal gold solution was cooled to room temperature, 750 μL of 100 mg / mL BSPP solution was added, and the solution was stored overnight at room temperature in the dark. The UV absorption spectrum of the gold spheres was scanned using a UV spectrophotometer. A small amount of NaCl particles were added to 150 mL of colloidal gold solution. After the gold spheres precipitated and turned blackish-gray, they were centrifuged at 3000 rpm for 5 min. The supernatant was completely discarded, and the precipitate was resuspended with 1 mg / mL BSPP, concentrated to 1 mL, and stored at 4 °C. The particle size and distribution of the prepared colloidal gold were determined by a particle size analyzer, and the particle size and uniformity were further observed using a transmission electron microscope.

[0021] (2) Preparation of AuNPs dimers Add a small amount of NaCl particles to 1 mL of colloidal gold after centrifugation and concentration, causing the gold spheres to precipitate and turn blackish-gray. Centrifuge for 30 seconds in a handheld centrifuge, discard the supernatant, and resuspend the precipitate in a 1.5 mL centrifuge tube with 1 mg / mL BSPP for later use. In six 1.5 mL low-adsorption centrifuge tubes, 2 μL of 5×TBE buffer was added to each tube, followed by 1.9, 1.8, 1.7, 1.6, 1.5, and 1.4 μL of H2O, respectively. After mixing, 8 μL of 5 mg / mL FSDNA solution was added to each tube, and the mixture was mixed again. Then, 3.1, 3.2, 3.3, 3.4, 3.5, and 3.6 μL of 100 mM AgNO3 solution were added, respectively. After rapid mixing, 5 μL of concentrated colloidal gold solution with salt was added to each tube, and the mixture was mixed. After standing for 20 min, 0.5 μL of 75% glycerol was added to each tube. A 1.2% agarose gel was prepared in advance, and electrophoresis was performed using the solidified agarose gel. The voltage was set to 90 V, the current to 60 mA, and the time to 30 min. The optimal reaction system was selected based on the electrophoresis results, and a scale-up experiment was conducted. The selected optimal reaction system was scaled up 50 times, and electrophoretic separation was performed using solidified agarose gel. The voltage was set to 120V, the current to 90mA, and the time to 60min. Cut off the gel block containing AuNPs dimer bands and mince it, collect it in a 50 mL centrifuge tube, soak it overnight in 0.5×TBE buffer to disperse the AuNPs dimers in the gel into the 0.5×TBE buffer, aspirate the AuNPs dimer solution and aliquot it into centrifuge tubes for concentration, centrifuge at 3500 rpm for 15 min; discard the supernatant, collect the precipitate and make up to 100 μL, scan the UV absorption spectrum with a UV spectrophotometer, and observe its uniformity and particle size with a transmission electron microscope.

[0022] Preparation of SERS tags H3, i.e., the complementary chain of thiolated H2, can be used directly without further processing, and no DTT or TCEP treatment is required; the thiolated probe H3 and the Raman molecule 4-MBN are fixed on the surface of AuNPs dimer through gold-sulfur (Au–S) bonds; Thiolized Apt sample H3 (100 μM, 3 μL) was mixed with AuNPs dimer (10 nM, 100 μL), and the mixture was then frozen at -20 °C for 2 hours. After thawing at room temperature, centrifugation and washing were performed, and 4-MBN (final concentration 10) was added. -3 M) Let stand for 1 hour, wash with ultrapure water by centrifugation, and disperse in deionized water for later use.

[0023] Step 3: Preparation of Fe3O4@AuNFs-H1 substrate The hairpin probe DNA (H1 and H2) were annealed separately, that is, heated at 95℃ for 5 min, then slowly cooled to 25℃ and placed at 4℃ for 15 min to stabilize the hairpin structure. Subsequently, 6 μL of 300 μM H1 and 6 μL of 10 mM TCEP were mixed thoroughly and incubated at 25 °C for 1 h to dissociate disulfide bonds. Then, 300 μL of Fe3O4@AuNFs was added and incubated at room temperature for 12 h. After washing with reaction buffer, the mixture was concentrated to 100 μL. Then, 100 μL of 10 mM MCH was added and allowed to stand for 10 min to seal the exposed surface of Fe3O4@AuNFs and avoid non-specific adsorption. After washing with water by magnetic separation, Fe3O4@AuNFs-H1 substrate was obtained.

[0024] Step 4: Construction of the Fe3O4@AuNFs-AuNPs dimer SERS sensor First, mix 100 μL of the capture probe Fe3O4@AuNFs-OA Aptamer-cDNA with 100 μL of the target toxin OA and incubate at 25°C for 1 hour. Collect the supernatant, which is the mixed solution containing cDNA, by magnetic separation and store it at 4°C for later use. Take 5 μL of cDNA, 10 μL of H2 (1 μM) and 20 μL of SERS tag and mix them evenly. Then add them to 20 μL of Fe3O4@AuNFs-H1 substrate and mix evenly. React in a PCR instrument at 37℃ for 80 min. After the reaction is completed, remove unbound SERS tags by magnetic separation and washing 3 times. Concentrate to 10 μL to obtain Fe3O4@AuNFs-AuNPs dimer SERS sensor.

[0025] It should be noted that the DNA sequence listing of the materials used in the above steps is as follows: Table 1 DNA Sequence Listing

[0026] Based on the above, biosensors can also be optimized in the following ways: For the selection of SERS tag materials, 20 μL of SERS tags prepared from AuNPs or AuNPs dimers were mixed with 5 μL of cDNA and 10 μL of H2 (1 μM), and then added to 20 μL of Fe3O4@AuNFs-H1 substrate. The mixture was reacted in a PCR instrument at 37℃ for 80 min. After magnetic separation and washing three times, the mixture was concentrated to 10 μL, dropped onto a silicon wafer, dried, and then used for SERS detection. The concentration of H1 fixed on the Fe3O4@AuNFs-H1 substrate was optimized by incubating 300 μL of Fe3O4@AuNFs overnight with 6 μL of hairpin H1 solutions of 10 nM, 50 nM, 100 nM, 200 nM, 300 nM, 400 nM, and 500 nM, respectively. After magnetic separation and washing, the solution was concentrated to 100 μL for the preparation of Fe3O4@AuNFs-AuNPs dimer sensor. The solution was then concentrated to 10 μL, drop-coated onto a silicon wafer, dried, and subjected to SERS detection.

[0027] To optimize the MCH blocking time, 200 μL of 10 mM MCH was mixed with an equal volume of Fe3O4@AuNFs-H1 substrate and allowed to stand in the dark for different times (0 min, 5 min, 10 min, 20 min, 30 min, 40 min, 50 min, 60 min). After magnetic separation and cleaning, the mixture was used to prepare Fe3O4@AuNFs-AuNPs dimer sensors, which were then drop-coated onto silicon wafers, dried, and subjected to SERS detection.

[0028] The one-pot incubation time was optimized. When preparing Fe3O4@AuNFs-AuNPs dimer sensors, the one-pot incubation time was 5 min, 10 min, 20 min, 40 min, 60 min, 80 min, 100 min and 120 min respectively. After magnetic separation and cleaning, the mixture was concentrated into 10 μL and dropped onto a silicon wafer, dried and then subjected to SERS detection.

[0029] The biosensors prepared above have the following properties: To ensure homogeneity, 100 μL of the capture probe Fe3O4@AuNFs-OA Aptamer-cDNA was mixed with 100 μL of the target toxin OA at a concentration of 1 μg / mL. The mixture was incubated at 25°C for 1 h. The supernatant, which is the mixed solution containing cDNA, was collected by magnetic separation and stored at 4°C for later use. Subsequently, the Fe3O4@AuNFs-AuNPs dimer SERS sensor was prepared by the one-pot incubation method described above. 10 μL of sample was dropped onto the silicon wafer substrate, and after drying, 15 test sites were randomly selected for SERS detection.

[0030] To assess reproducibility, 100 μL of the capture probe Fe3O4@AuNFs-OA Aptamer-cDNA was mixed with 100 μL of the target toxin OA at a concentration of 1 μg / mL and incubated at 25°C for 1 h. The supernatant, i.e., the mixed solution containing cDNA, was collected by magnetic separation and stored at 4°C for later use. Subsequently, the Fe3O4@AuNFs-AuNPs dimer SERS sensor was prepared by one-pot incubation as described above. 10 μL of sample was dropped onto a silicon substrate, and five different batches of substrates were prepared. After drying, SERS detection was performed.

[0031] Specificity was assessed by detecting OA, TTX, MC-LR, STX, and DA separately, with the concentration of other toxins at 10 μg / mL and OA toxin at 1 μg / mL. Parallel tests and signal acquisition were performed under the above optimal reaction conditions. The specificity recognition ability and anti-interference performance of this sensing method were systematically evaluated by comparing the Raman response intensity of different toxins detected.

[0032] This proposal also suggests the application of a triple-synergistic SERS biosensor for detecting the shellfish toxin okadaic acid; the specific method is as follows: 1. Detection of OA standard solutions using SERS sensors: OA toxin standard solutions of different concentrations were prepared, with toxin concentrations of 10... -6 -1μg / mL; Then, 100 μL of the capture probe Fe3O4@AuNFs-OA Aptamer-cDNA was mixed with 100 μL of the target toxin OA and incubated at 25°C for 1 hour. The supernatant, i.e., the mixed solution containing cDNA, was collected by magnetic separation and stored at 4°C for later use. A Fe3O4@AuNFs-AuNPs dimer SERS sensor was prepared using a one-pot method. 10 μL of sample was added to a silicon wafer substrate, dried, and then subjected to SERS detection. The characteristic peak was observed at 2234 cm⁻¹. -1 The difference between the SERS signal intensity at the treatment site and the blank control group was used as the basis for quantification, and a standard curve for OA toxin was finally established.

[0033] 2. SERS sensor for spiked detection of OA in actual clam meat samples: 1 g of clam meat tissue was crushed, mixed with ultrapure water at room temperature, shaken for 30 min, and then centrifuged at 4000 rpm for 10 min. Collect the supernatant and dilute it 40 times to achieve the target tissue content. Prepare three samples of clam meat tissue supernatant and add different volumes of OA standard solution to prepare different tissue sample concentrations (0.1, 0.01 and 0.001 μg / mL). Take 100 μL of each sample and mix it with the prepared SERS capture probe. After incubation at 25 °C for 20 min, collect the supernatant, which is a mixed solution containing cDNA, by magnetic separation. Store it at 4 °C for later use. Use the above one-pot incubation method to obtain the SERS sensor, drop it onto a silicon wafer for SERS detection, and analyze the detection results.

[0034] The detection method achieves multiple signal amplification and triple synergistic enhancement through target-induced chain substitution reaction and hybridization chain amplification strategy. Its working principle is as follows: Figure 1 : In the detection system, the Fe3O4@AuNFs composite material is first incubated with the OA ligand and its complementary strand cDNA to form the Fe3O4@AuNFs-OA Aptamer-cDNA composite structure. When the target analyte OA is present, the cDNA is competitively released into the solution because the binding affinity of the aptamer to the target molecule is higher than that to the cDNA.

[0035] The supernatant containing cDNA was collected by magnetic separation and mixed with Fe3O4@AuNFs substrate pre-modified with hairpin probe H1. The cDNA can specifically recognize and open the hairpin structure of H1. At this time, the hairpin probe H2 added to the system has more complementary base pairs with H1, and can replace cDNA to hybridize with H1, forming a more stable H1-H2 double strand, while displacing the cDNA. The replaced cDNA can enter the next round of the cycle reaction, triggering a new round of H1 opening and hybridization with H2, thereby achieving signal cycle amplification. During the formation of the H1-H2 doublet, the 3' extension sequence of the H2 chain can complementarily pair with the hairpin probe H3 modified on the SERS tag, thereby capturing the SERS tag onto the surface of Fe3O4@AuNFs and forming a Fe3O4@AuNFs-AuNPs dimer structure. The AuNPs dimer forms a nanoscale gap between the tip of the gold nanoflower and the gold nanoparticle, generating a high-density electromagnetic hot spot that significantly enhances the SERS signal. At the same time, the local surface plasmon resonance effect of the gold nanoflower itself further enhances the electric field strength, achieving triple synergistic amplification of the signal. When the target OA is not present in the system, cDNA cannot be competitively released, the hairpin probe H1 remains closed, and the subsequent hybridization chain reaction cannot be triggered. Therefore, the SERS sensor cannot be constructed, and the detected background signal is extremely low. The characteristic molecular fingerprint spectrum of the target can be obtained through SERS detection, achieving highly sensitive and specific detection of OA.

[0036] Example 1 Preparation and characterization of capture probes and SERS tags: The synthesized Fe3O4@AuNFs were characterized by UV-Vis, Zeta potential analysis, and dynamic light scattering (DLS) particle size analysis; Figure 2 As shown, in the ultraviolet-visible absorption spectroscopy analysis, the experimental results show that different nanomaterials exhibit significantly different optical absorption characteristics.

[0037] AuNPs exhibit a sharp surface plasmon resonance (SPR) absorption peak at a wavelength of 520 nm. The peak position and shape further confirm that AuNPs have a uniform particle size distribution. Meanwhile, Fe3O4 nanoparticles exhibit a relatively flat and broad absorption curve in the entire visible to near-infrared spectral region (400-1000 nm), without obvious characteristic absorption peaks, which is consistent with the optical properties of Fe3O4 as a magnetic semiconductor. The absorption curve of the PEI-modified Fe3O4-PEI sample is similar to that of pure Fe3O4, but the absorption intensity at 800 nm is slightly increased. The Fe3O4@AuNFs complex shows a significantly enhanced broad absorption peak in the near-infrared region near 800 nm. The appearance of the characteristic absorption band clearly confirms the successful formation of the gold nanoflower structure. Zeta potential characterization revealed that the original Fe3O4 nanoparticles exhibited a strong positive charge state (+51.71 mV). After modification with PEI, the Zeta potential of Fe3O4-PEI decreased to +42.32 mV. Coating its surface with a layer of gold nanoflowers changed the charge from positive to negative (-13.05 mV). DLS measurement showed that the diameter of the composite material was approximately 663 nm, an increase from the original magnetic beads' 280 nm, demonstrating the successful synthesis of the Fe3O4@AuNFs composite material.

[0038] Further characterization using TEM and energy-dispersive X-ray spectroscopy elemental mapping revealed that seed crystals were successfully assembled on the surface of the magnetic beads, and gold nanoflower structures were formed through in-situ growth. Figure 3 As shown in Figure a, as Figure 3 EDX elemental analysis shown in Figure e indicates the presence of four elements—Fe, Au, O, and N—on the Fe3O4@AuNFs substrate, confirming that the seed crystals were successfully assembled onto the surface of the magnetic beads using polyethyleneimine, thus synthesizing Fe3O4@AuNFs nanomaterials.

[0039] The Fe3O4@AuNFs composite material was modified by co-incubating it overnight with the dissociated SH-OA aptamer and its complementary cDNA. The changes in surface charge before and after modification were characterized using zeta potential. Figure 4As shown, compared to the unmodified Fe3O4@AuNFs (-13.05mV), the potential decreased to -22.22mV after modification with the aptamer; and after binding to the complementary strand cDNA, the potential further decreased to -26.05mV, due to the large amount of negative charge carried by the phosphate groups in the nucleic acid molecular backbone. The above results together indicate that the Fe3O4@AuNFs-OA Aptamer-cDNA complex was successfully prepared.

[0040] The synthesized AuNPs dimers were characterized by UV-Vis and DLS particle size analysis, such as... Figure 5 As shown in Figure a, AuNPs monomers exhibit a surface plasmon resonance absorption peak at 525 nm, and the AuNPs dimer spectrum shows a new absorption peak near 600 nm, with the absorbance of this peak being lower than that of the gold sphere monomers. The aforementioned spectral changes confirm the occurrence of plasmon coupling effects during AuNPs dimer formation. The DLS particle size distribution is shown in the figure below. Figure 5 As shown in Figure b, the AuNPs dimer has a particle size of 128.26 nm, which is significantly larger than that of a single AuNP. In summary, UV-Vis spectroscopy confirms the spectral redshift and the generation of new peaks caused by plasma coupling, while DLS results directly confirm the formation of the AuNPs dimer size. The two characterizations corroborate each other and jointly confirm the successful synthesis of AuNPs dimers.

[0041] The morphology and structure of the material were characterized and analyzed using TEM, such as Figure 6 The images shown are TEM images of AuNPs monomers and AuNPs dimers, respectively. Figure 6 As observed in Figure a, AuNPs exhibit a good spherical morphology, with uniform particle distribution and good dispersion. Figure 6 Figure b shows a TEM image of the AuNPs dimer. It is clearly observed that two AuNPs are connected together by van der Waals forces or surface ligand interactions to form an AuNPs dimer structure. The two nanoparticles in the AuNPs dimer are closely adjacent but still maintain a complete spherical outline, without obvious fusion or deformation. The TEM characterization results above directly confirm the successful synthesis of AuNPs monomers and AuNPs dimers, and the prepared nanoparticles have good morphological uniformity and dispersibility.

[0042] The surface charge properties and migration behavior of AuNPs monomers, AuNPs dimers, and H3-modified samples were characterized using Zeta potential analysis and agarose gel electrophoresis. Figure 7Figure a shows a comparison of the Zeta potentials of AuNPs dimer before and after H3 modification. The AuNPs dimer exhibits a negative potential characteristic with a Zeta potential of -38.22 mV, indicating that the surface of the AuNPs dimer carries a negative charge. When AuNPs dimers are modified with H3, their Zeta potential is further reduced to approximately -44.69 mV, and the amount of negative charge is significantly increased. Since the phosphate backbone of DNA single strands itself has negatively charged groups, it indicates that H3 has been successfully modified on the surface of AuNPs dimers. Furthermore, the increase in the absolute value of the Zeta potential makes the modified nanoparticles more electrostatically stable in the dispersion system, which is beneficial for preventing the aggregation and sedimentation of the material.

[0043] Further analysis of AuNPs, AuNP dimers, and their H3 modifications was performed using agarose gel electrophoresis. Figure 7 As shown in Figure b, the gel electrophoresis pattern shows the migration of samples in the four lanes: The first lane contains AuNPs monomers, which appear as red stripes and are located at the bottom of the lane, where they migrate the longest distance. The second lane is AuNPs-H3, and the position of the red band has shifted significantly upward, indicating that the particle migration rate is reduced after modification with H3. The third lane contains AuNPs dimers, which appear as blue-purple bands (the color change reflects the shift of the plasma resonance peak caused by the change in the aggregation state of AuNPs), and the position of the bands is higher than that of the first lane; Lane 4 is for AuNPs dimer-H3, with the blue-purple band at the highest position and the shortest migration distance; The above gel electrophoresis results and Zeta potential analysis results corroborate each other: for AuNPs monomers or AuNPs dimers, the migration distance of the H3-modified samples in the gel was significantly shortened. The modification of H3 changed the charge density and distribution on the particle surface, affecting its electrophoretic mobility in an electric field. The combined results of Zeta potential and gel electrophoresis confirm that H3 molecules have been successfully modified on the surface of AuNPs and AuNPs dimers.

[0044] Example 2 The fabrication and characterization of SERS sensors were carried out by using Zeta potential analysis and dynamic light scattering technology to characterize the Fe3O4@AuNFs-AuNPs dimer composite material. like Figure 8 As shown in Figure a, the trend of Zeta potential changes shows: The initial Zeta potential of Fe3O4@AuNFs was approximately -13 mV, which decreased to approximately -25 mV after H1 modification, further decreased to approximately -27 mV after H2 modification, and finally decreased to approximately -32 mV after connecting AuNPs dimers. The gradual decrease in the Zeta potential confirms the successful modification of the material surface by H1, H2 and AuNPs dimers; like Figure 8 As shown in Figure b, the average kinetic particle size of the Fe3O4@AuNFs-AuNPs dimer is approximately 925.14 nm, which is significantly larger than that of the single Fe3O4@AuNFs. The AuNPs dimer was successfully bridged on the Fe3O4@AuNFs surface.

[0045] The morphology and elemental composition of the Fe3O4@AuNFs-AuNPs dimer composite material were characterized by TEM and energy-dispersive X-ray spectroscopy. like Figure 8 As shown, the SEM image and EDS elemental mapping of the Fe3O4@AuNFs-AuNPs dimer reveal spherical particle aggregation with AuNPs dimer nanoparticles attached to the surface. The EDS elemental mapping results show dense distribution of Fe and O elements, confirming the presence of Fe3O4. Au elements cover the entire particle surface, indicating that AuNFs and AuNPs dimers are successfully attached to the material surface. The EDS energy dispersive spectroscopy confirms that the material contains the main elements Fe, O, and Au, with Au having the highest content. These results collectively confirm the successful synthesis of the Fe3O4@AuNFs-AuNPs dimer composite material.

[0046] Example 3 Feasibility analysis of CHA cascade amplification reaction, agarose gel electrophoresis results as follows: Figure 10 As shown, the CHA cascade signal amplification sensing mechanism was characterized by 2% agarose gel electrophoresis; Lanes 1, 2, 3, and 4 correspond to H1, H2, H3, and cDNA, respectively. They capture short DNA strands that are contested when the probe binds to the target toxin. They can specifically recognize H1, open its hairpin structure, and hybridize with it. When hairpin H1 was incubated with cDNA, a new band appeared in lane 5, indicating that cDNA could effectively open hairpin H1 and bind to it. Lane 6 is a mixture of H1, cDNA and H2. Compared with lane 5 (H1+cDNA), a new high molecular weight band appears in lane 6, indicating that the CHA amplification reaction successfully produced the H1-H2 double-stranded product under cDNA triggering. Lane 7 is a mixture of H1+cDNA+H2+H3. Compared with lane 6, its band migration rate is reduced, indicating that the H1-H2 double strand further binds with H3 to form a complex with a higher molecular weight, thus confirming that the CHA cascade signal amplification was successfully achieved. The above electrophoresis results show that the sensing system can effectively respond to the target toxin and achieve efficient signal amplification through the CHA cascade reaction; To verify the feasibility of the prepared SERS sensor for detecting OA toxins, two concentration gradients of OA toxins were detected: from Figure 11 As shown in Figure a, the sensor's response signal to OA toxin is weak when CHA cyclic amplification is not introduced; After introducing CHA cyclic amplification, the characteristic peak intensity of the signal molecule at the same concentration was significantly enhanced, reaching 4.5 to 6 times that without CHA, indicating that the CHA cascade signal amplification strategy can effectively improve detection sensitivity. like Figure 11 As shown in Figure b, the blank control group showed almost no obvious Raman signal, indicating that the SERS substrate background was clean and interference was minimal. When the OA concentration was 0.1 μg / mL, the characteristic peak of the signal molecule was 2234 cm⁻¹. -1 The peak values ​​were significantly higher than those in the blank control. When the OA concentration increased to 1 μg / mL, the signal intensity of each characteristic peak was significantly enhanced, indicating that the SERS signal was positively correlated with the OA concentration and had the potential for quantitative analysis. In addition, the characteristic peaks in the Raman silent region were not interfered with by the complex matrix. The above results show that the SERS sensor based on CHA cascade signal amplification can effectively respond to OA toxin and has good detection feasibility.

[0047] Example 4 To construct a high-performance SERS sensor, the preparation conditions were optimized, including the selection of SERS tag material, the concentration of H1 fixed on the Fe3O4@AuNFs-H1 substrate, the MCH blocking time, the concentration of AuNPs dimers, and the one-pot incubation detection time. Two SERS tags were prepared by modifying AuNPs and AuNPs dimers with H3 using a freezing method. The material with better performance was screened by detecting OA toxins. like Figure 12 As shown, the performance of the prepared SERS tag was evaluated by comparing the characteristic peaks of the SERS tag itself with those obtained from detecting 1 μg / mL OA toxin. The results showed that when the SERS tag uses AuNPs dimer as the substrate material, both its own signal peak at 1586 cm⁻¹... -1 The characteristic peak value after detecting OA toxin was 2234 cm⁻¹ in the Raman silent region. -1It outperforms SERS tags based on AuNPs, given that AuNPs dimer-H3 has superior SERS performance; The detection of 1 μg / mL OA toxin was performed by incubating different concentrations of H1 on Fe3O4@AuNFs-H1 substrate. Figure 13 As shown, the SERS signal intensity gradually increases with the gradual increase of H1 concentration, and then tends to stabilize. When the H1 concentration is low, the substrate surface sites are not fully occupied. With the increase of H1 concentration, a plateau is reached at 300 nM. Since the number of H1 binding sites on the Fe3O4@AuNFs substrate surface is limited, when the H1 concentration reaches 300 nM, the substrate surface binding sites are basically completely occupied. Further increasing the H1 concentration cannot bind more SERS tags and it is difficult to improve the SERS signal intensity. Therefore, the optimal H1 concentration for Fe3O4@AuNFs-H1 substrate is 300 nM. To avoid nonspecific adsorption, the MCH blocking time was optimized to effectively eliminate nonspecific binding while ensuring optimal SERS signal intensity for toxin detection. Figure 14 As shown in Figure a, the peak value of the characteristic peak of the blank control group gradually decreased with the increase of the blocking time, indicating that the blocking time can reduce non-specific adsorption. like Figure 14 As shown in Figure b, the OA toxin detection signal was strongest when the blocking time was 30 min; when the blocking time was extended to 60 min and 90 min, the signal intensity decreased. Extending the blocking time caused the H1 active site to be partially covered, which affected the subsequent specific binding. like Figure 14 As shown in Figure c, with 2234cm -1 The difference between the intensity of the characteristic peak signal and the blank background signal is used as the evaluation index, and the signal difference is the largest when the closure time is 30 min. The concentration of SERS tags was optimized by detecting the target toxin OA, such as... Figure 15 As shown in Figure a, as the SERS tag concentration increased from 2 nM to 8 nM, the 2234 cm⁻¹ -1 The signal intensity at the characteristic peak increases, and when it reaches 10 nM, the signal intensity tends to stabilize and decreases slightly. like Figure 15 As shown in Figure b, when the SERS tag concentration is 8 nM, the difference between the signal intensity and the blank background is the largest, and the detection sensitivity is the best. As the SERS tag concentration increases, the number of tags bound to the substrate increases, and the signal is enhanced. When the concentration reaches 8 nM, the binding sites on the substrate surface are saturated, and excessive accumulation causes the 4-MBN signal molecule on the tag to be masked, and the signal is no longer enhanced. One-pot incubation time is a key factor affecting detection performance; its essence lies in providing sufficient reaction time for the CHA-catalyzed hairpin assembly reaction, such as... Figure 16 As shown in Figure a, as the incubation time increased from 5 min to 80 min, 2234 cm -1 The signal intensity of the characteristic peak showed a significant upward trend. When the incubation time continued to increase to 100 min and 120 min, the signal intensity tended to stabilize and decrease. like Figure 16 As shown in Figure b, when the incubation time is 80 min, 2234 cm -1 When the difference between the characteristic peak signal intensity and the blank background signal reaches its maximum value, as the incubation time increases, the cDNA fully binds to the recognition element H1 on the SERS substrate, triggering the CHA cascade reaction. A large number of H1-H2 double strands are generated, which then bind to the SERS tag, and the cDNA enters the next cycle of amplification. When the incubation time reaches 80 minutes, the CHA reaction is basically completed, and no product is generated if the incubation time is extended further.

[0048] Example 5

[0049] The homogeneity and reproducibility of the SERS substrate are key indicators for evaluating its practical application performance, directly determining the reliability of the detection results and the accuracy of quantitative analysis. Figure 17 As shown, 15 test sites were randomly selected on the surface of the same batch of substrates to collect and analyze SERS signals of DA toxin. The RSD of the characteristic peak intensity was only 2.5%, indicating that the substrate has signal uniformity.

[0050] Five independent batches of SERS substrates were further selected and DA toxin was detected under the same test conditions. The RSD of the detection signal between different batches was 2.2%, which proved that the preparation process was stable and controllable and the substrate performance had excellent batch-to-batch reproducibility. The above results show that the SERS substrate prepared in this work has high uniformity and good reproducibility. OA and TTX, MC-LR, STX, and DA were detected separately, with OA concentration of 1 μg / mL and TTX, MC-LR, STX, and DA concentration of 10 μg / mL. Figure 18 As shown, the specificity detection results indicate that OA toxin was detected at 2234 cm⁻¹. -1 The characteristic peak signal intensity is higher than that of TTX, MC-LR, STX and DA, indicating that the prepared SERS sensor has excellent specific recognition ability for OA. The SERS sensor based on CHA cascade signal amplification can still achieve high specificity detection of OA in complex matrices and has practical application potential.

[0051] Example 6 The prepared SERS sensor exhibited excellent quantitative analytical performance in the detection of OA toxins, such as... Figure 19 As shown, with 2234cm -1 A standard curve was plotted using the characteristic peak intensity versus the logarithm of OA concentration. The linear equation is as follows: Y = 6394.94 + 1043.39 X Correlation coefficient R 2 = 0.9948, indicating excellent linearity across six orders of magnitude from 1 μg / mL to 1 pg / mL; like Figure 19 As shown in Figure a, as the OA concentration increases from 10... -6 Gradually increase μg / mL to 1 μg / mL, 2234cm -1 The characteristic peak signal intensity increased accordingly, and the characteristic peak positions were consistent at each concentration, indicating that the signal originated from the signal molecule 4-MBN and had good specificity. The small error bar indicates that the detection method has good repeatability and stability. The above results show that the SERS sensor based on CHA cascade signal amplification can be used for the quantitative detection of OA toxin.

[0052] Example 7 The effectiveness of a triple synergistic enhancement biotoxin SERS biosensor in detecting the shellfish toxin okadaic acid was evaluated, and the recovery rate of OA toxin added to clam meat samples was detected. Actual tissue solutions containing OA at concentrations of 0.001 μg / mL, 0.01 μg / mL, and 0.1 μg / mL were selected for detection. The SERS chromatograms and recovery results are shown below. Figure 20 As shown in Table 2, the spiked recoveries of OA in clam meat matrix ranged from 91.02% to 100.77%, and the RSDs of each concentration group were less than 10%, demonstrating good detection accuracy and repeatability. The recovery rate range meets the relevant requirements for trace pollutant analysis and can meet the accurate quantitative needs of low OA content in actual samples. Under the interference of complex clam meat matrix, the method can still maintain high recovery rate and low RSD. It has excellent resistance to matrix interference and has good reliability and practicality. The results show that the SERS sensor based on Fe3O4@AuNFs-AuNPs dimer and the triple synergistic enhancement detection method of shellfish toxin okadaic acid can be used for stable and specific practical detection of OA in shellfish, providing a reliable technical solution for rapid and highly sensitive screening of OA in shellfish products. Table 2 Recovery rates of OA content in spiked samples

[0053] In summary, this invention utilizes Fe3O4 magnetic nanoparticles to achieve efficient separation and enrichment of target analytes in complex shellfish samples. It constructs high-density SERS hotspots through the plasmon coupling effect of AuNFs and AuNPs dimer structures, significantly enhancing the local electromagnetic field. Simultaneously, it achieves enzyme-free cyclic amplification of the target signal based on the CHA cascade hybridization amplification reaction, solving the problem of weak SERS signals and inaccurate quantification caused by the low polarity and weak adsorption of Okada acid. It avoids the problems of poor antibody stability and large non-specific adsorption interference in traditional immunoassay, effectively improving the sensitivity, specificity and matrix adaptability of the detection system. It can realize rapid, accurate and on-site detection of trace amounts of okadaic acid in shellfish samples, and breaks through the technical bottleneck of traditional single enhanced sensing mechanism. It has broad application prospects in the fields of food safety detection and aquatic ecological toxin screening.

[0054] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A triple-synergistically enhanced biotoxin SERS biosensor, characterized in that, This biosensor consists of a Fe3O4@AuNFs-OA Aptamer-cDNA capture probe, a SERS tag, and a Fe3O4@AuNFs-H1 substrate unit. The Fe3O4@AuNFs-OA Aptamer-cDNA capture probe consists of gold nanoflowers modified on the surface of Fe3O4 magnetic nanoparticles, which are then sequentially linked with okada acid aptamers and complementary strand cDNA. The SERS tag consists of a gold nanoparticle dimer modified with Au-S bonds to a hairpin probe H3 and a Raman reporter molecule 4-mercaptobenzonitrile; The Fe3O4@AuNFs-H1 substrate unit is composed of Fe3O4@AuNFs connected to hairpin probe H1; In this process, the Fe3O4@AuNFs-OA Aptamer-cDNA capture probe binds to the target OA and releases cDNA. The cDNA triggers a catalytic hairpin self-assembly reaction to form an H1-H2 double strand, which then captures the SERS tag through complementary pairing of H2 and H3, forming a Fe3O4@AuNFs-AuNPs dimer complex structure.

2. The triple-synergistic enhanced biotoxin SERS biosensor according to claim 1, characterized in that, The gold nanoparticle dimer is formed by AuNPs with a particle size of 30 nm through silver ion mediation to form a dimer structure, and its plasmonic coupling effect produces an absorption peak at 600 nm.

3. A method for preparing a triple-synergistically enhanced biotoxin SERS biosensor, the method being used to prepare the biosensor according to any one of claims 1-2, characterized in that, Includes the following steps: S1. Preparation of the capture probe: After activating the SH-OA aptamer with TCEP, it was incubated with Fe3O4@AuNFs, and MCH was added to block non-specific sites. Then it was hybridized with cDNA to form Fe3O4@AuNFs-OA Aptamer-cDNA. S2 and SERS tag preparation: S21. Synthesize 30 nm AuNPs, and obtain AuNPs dimers by silver ion-mediated and agarose gel electrophoresis separation and purification. S22. Hairpin probe H3 was modified onto the surface of AuNPs dimer using a freeze-thaw method, and then 4-MBN was added for incubation and fixation. S3, Substrate Preparation: After annealing the hairpin probe H1, it was activated with TCEP, incubated with Fe3O4@AuNFs, and blocked with MCH to obtain the Fe3O4@AuNFs-H1 substrate. S4, Sensor Construction: S41. After incubation of the capture probe with OA, magnetic separation is performed, and the supernatant containing cDNA is collected. S42. Mix the cDNA, hairpin probe H2, and SERS tag in the supernatant with the Fe3O4@AuNFs-H1 substrate, react at 37℃ for 80 min, and then perform magnetic separation and cleaning to obtain the sensor.

4. The method for preparing the triple synergistic enhanced biotoxin SERS biosensor according to claim 3, characterized in that, In step S3, the fixed concentration of hairpin probe H1 is 300 nM, and the MCH blocking time is 30 min.

5. The method for preparing the triple synergistic enhanced biotoxin SERS biosensor according to claim 4, characterized in that, The concentration of AuNPs dimers in the SERS tag of step S2 is 8 nM.

6. The application of a triple-synergistic enhanced biotoxin SERS biosensor, characterized in that, Application of the triple synergistic enhancement biotoxin SERS biosensor according to any one of claims 1-2 in the detection of shellfish toxin okadaic acid.

7. The application of the triple synergistic enhancement biotoxin SERS biosensor according to claim 6, characterized in that, With 2234cm -1 Using the intensity of the 4-MBN characteristic peak as a quantitative signal, the detection linear range is 1 pg / mL to 1 μg / mL, and the detection limit is sub-pg / mL, making it suitable for complex matrix samples containing biological tissues.

8. The application of the triple synergistic enhancement biotoxin SERS biosensor according to claim 7, characterized in that, The detection is achieved through the following steps: The shellfish sample to be tested was homogenized and centrifuged, and the supernatant was incubated with the capture probe. After magnetic separation, the supernatant containing cDNA was collected to construct a sensor; SERS detection was performed by drop-coating onto a silicon wafer at a diameter of 2234 cm⁻¹. -1 Peak intensity difference is used to quantify OA concentration.

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