SERS method for high-sensitivity detection of aquatic product pathogenic bacteria

Abstract

According to the scheme, target bacteria (such as vibrio parahaemolyticus) are subjected to high-sensitivity SERS detection by utilizing a sandwich structure principle, Au (at) Ag-MPBA / PEG-Ab1 is prepared as an SERS probe for quantitative detection of the target bacteria, and enrichment of the target bacteria is effectively realized by combining a magnetic composite nano material Fe3O4 (at) TA / Ag NPs-Ab2, so that the SERS detection of the pathogenic bacteria in the aquatic products is realized. A sandwich structure detection system with high sensitivity and specificity to vibrio parahaemolyticus is formed, finally, signal amplification is achieved by preparing a two-dimensional flexible functional regenerated SERS substrate of a high-density SERS'hot spot 'with a bamboo cellulose membrane as a raw material, so that enrichment and sensitivity detection of vibrio parahaemolyticus is rapidly completed, the detection range is 10-107 CFU / mL, and the detection sensitivity is high. The method has great potential in the aspects of food safety monitoring and infectious disease diagnosis.

Description

Technical Field

[0001] This invention relates to the fields of aquatic product testing technology and pathogenic bacteria analysis and detection technology, and in particular to a highly sensitive SERS method for detecting pathogenic bacteria in aquatic products. Background Technology

[0002] Vibrio parahaemolyticus is a Gram-negative, halophilic marine vibrio widely distributed in seawater and sediments of temperate and tropical waters. It is a common foodborne pathogen in nearshore aquatic products (such as shrimp, crab, shellfish, and fish) worldwide. This bacterium is a facultative anaerobe, with an optimal growth temperature of 25-37℃ and a requirement of 3%-6% sodium chloride. It struggles to survive in freshwater, but its spores can tolerate low temperatures and dryness, and may remain dormant in refrigerated or frozen aquatic products. This bacterium is widely present in nearshore aquatic products such as shrimp, crab, shellfish, and fish. It multiplies rapidly in the high temperatures of summer and autumn (June-October), easily causing mass contamination during aquaculture, processing, and transportation, leading to unsold or recalled aquatic products and economic losses to the industry. Furthermore, it can cause infection by producing heat-resistant direct hemolysin (TDH) and heat-related hemolysin (TRH). Humans can become infected by ingesting bacteria with a concentration exceeding 10... 5 Consuming CFU / g of aquatic products can easily lead to acute gastroenteritis symptoms, including abdominal pain, diarrhea, vomiting, and fever. In severe cases, it can cause dehydration and shock, and in rare severe cases, sepsis may occur. Therefore, the detection and control of Vibrio parahaemolyticus is of great significance to the development of aquaculture and the health of consumers.

[0003] Currently, the traditional culture and isolation method is the primary gold standard for detecting Vibrio parahaemolyticus. This method, which uses selective culture media for enrichment and identification, offers high accuracy but has a long detection cycle (3-5 days) and relies on skilled technicians, making it unsuitable for rapid screening. In addition, some studies have used colloidal gold immunochromatography and real-time fluorescence PCR to detect Vibrio parahaemolyticus; however, these methods mostly require expensive laboratory equipment, skilled personnel, and time-consuming operations. These limitations hinder their application in rapid on-site detection.

[0004] Compared to traditional methods, surface-enhanced Raman spectroscopy (SERS), as a molecular-level detection and analysis method based on Raman scattering, avoids complex sample pretreatment processes and provides highly sensitive, rapid, simple, and non-destructive qualitative and quantitative analysis. However, due to the extremely complex environment and low concentration of Vibrio parahaemolyticus in seafood samples, quantitative detection presents significant challenges. Therefore, specific identification is needed to eliminate interference from other substances in the environment and enhance the intensity of the SERS signal.

[0005] Currently, there are few published or authorized patents that utilize SERS technology for bacterial detection. Patent CN115901719A discloses a bacterial detection device, preparation method, detection, and application based on a multilayer core-shell silver nanowire filtration network. However, the ZIF-8 material used in this method is prone to clumping in aqueous solution, leading to a decrease in its adsorption capacity; it also slowly deliquesces in air, exhibiting some hygroscopicity. These characteristics make it difficult for ZIF-8 to maintain its high adsorption efficiency and long-term stability in practical applications. Patent CN108872194B discloses a sandwich-structured SERS method for detecting pathogens, used to detect and identify three pathogenic bacteria. However, the antimicrobial peptides used have broad-spectrum antibacterial activity but are ineffective against certain specific bacteria, making accurate quantitative analysis difficult. Patent CN116148239A discloses a nano-"sandwich" bacterial detection system with multiple SERS signal enhancement and its preparation method, but it uses concanavalin A, a lectin protein. Due to its broad-spectrum antibacterial properties, it shares the same problem as the aforementioned antimicrobial peptides: its specificity makes it difficult to perform accurate quantitative analysis of single, specific bacteria. Patent CN117074385A discloses a highly stable SERS substrate and its application for rapid detection of pathogens. This method is simple to operate, has a short testing time, and requires low operator skill, but it cannot effectively isolate and enrich pathogens in samples. Patent CN112033949B discloses a rapid detection method for spoilage bacteria in aquatic products using a SERS biosensor, but the MOFs material used in this method also... There are stability defects; Patent CN118777280A discloses a novel SERS-Apt sensor for detecting Tau protein and its usage method. The aptamer used in this method has certain advantages in stability, cost, and production. It is specific only for a specific target, but may be affected by interference from similar structures, leading to a higher false positive rate. Compared with antibodies, which have more mature and reliable performance advantages at the current stage of technology, it cannot effectively meet the high sensitivity and high reliability quantitative detection requirements of complex organisms (such as bacteria); Patent CN115015217A discloses a dual-plasma-based... The superstructured sandwich SERS biosensor, its preparation and application, targets small molecule drugs. Its detection strategy differs from the design logic of this invention. It adopts an indirect sandwich mode of "antibody-antigen-secondary antibody (IgM)," relying on the simultaneous binding of two large molecule antibodies to a small molecule antigen. This is inherently challenging in terms of steric hindrance and binding efficiency, which may lead to unstable sensitivity. At the same time, IgM, as a secondary amplification signal molecule, binds to bacteria in a non-specific manner (IgM can bind to various pathogen-related molecular patterns), which can easily introduce background noise and lead to the loss of specific recognition ability for specific bacterial species.

[0006] Therefore, developing a rapid, simple, low-cost, highly sensitive, and easy-to-use detection method for pathogenic bacteria in aquatic products (such as Vibrio parahaemolyticus) is a research topic with significant practical implications. Summary of the Invention

[0007] In view of this, the purpose of this invention is to propose a highly sensitive SERS method for the detection of pathogenic bacteria in aquatic products. This method utilizes Au@Ag-MPBA / PEG-Ab1 as a SERS probe for the quantitative detection of target bacteria, combined with the magnetic composite nanomaterial Fe3O4@TA / Ag NPs-Ab2 to effectively enrich the target bacteria, thus forming a sandwich-structured detection system with high sensitivity and specificity for Vibrio parahaemolyticus. Finally, signal amplification is achieved by preparing a two-dimensional flexible functionalized regenerated SERS substrate with high-density SERS "hot spots" using bamboo cellulose membrane as raw material. This allows for rapid enrichment and sensitive detection of Vibrio parahaemolyticus, demonstrating great potential in food safety monitoring and infectious disease diagnosis.

[0008] To achieve the above-mentioned technical objectives, the technical solution adopted by this invention is as follows: A highly sensitive SERS method for detecting pathogenic bacteria in aquatic products includes: using Au@Ag-MPBA / PEG-Ab1 as a SERS signal probe for quantitative detection of target bacteria, combining it with magnetic composite nanomaterial Fe3O4@TA / Ag NPs-Ab2 to enrich the target bacteria, forming a sandwich-structured detection system for detecting the target bacteria, and then placing the detection system on a two-dimensional flexible SERS substrate prepared from bamboo cellulose membrane. The SERS signal is amplified through this two-dimensional flexible SERS substrate, thereby rapidly completing the enrichment and sensitive detection of target bacteria using the SERS method.

[0009] As one possible implementation method, this solution further includes the following steps: S1. Dissolve bamboo fiber in a pre-cooled solvent system and stir vigorously at low temperature to obtain a cellulose suspension; S2. Centrifuge the cellulose suspension, transfer the supernatant to a polytetrafluoroethylene mold, and then remove the solvent at room temperature to obtain a preformed membrane (i.e., regenerated bamboo cellulose membrane). Then immerse the preformed membrane in a regeneration solution formed by mixing ethanol and water, repeatedly soak, wash and replace the regeneration solution to remove residual solvent, and then dry to obtain a neutral regenerated cellulose membrane, denoted as RCM. S3. Prepare tannic acid (TA) solution as a reducing agent and chloroauric acid solution as an oxidizing agent. Place RCM in the reducing agent solution and oxidizing agent solution in sequence, and stir in a water bath at room temperature in the dark. After taking them out, wash them several times with ultrapure water and then dry them to finally obtain RCM@Au NPs, which is a two-dimensional flexible SERS substrate prepared by bamboo cellulose membrane. S4. Fe3O4 magnetic nanoparticles were synthesized by a solvothermal / coprecipitation method, and then modified with tannic acid. After modification, they were mixed with silver nitrate solution to load silver nanoparticles, namely Fe3O4@TA / Ag NPs. Subsequently, they were mixed with anti-target bacterial antibody Ab2 to obtain functionalized magnetic nanoparticles modified with antibody Ab2, namely Fe3O4@TA / Ag NPs-Ab2. S5. 4-MPBA is mixed with silver-coated gold nanoparticles to obtain Au@Ag-MPBA, and then mixed with thiol-polyethylene glycol-carboxyl aqueous solution and anti-target bacterial monoclonal antibody Ab1 to obtain Au@Ag-MPBA / PEG-Ab1, i.e., SERS signal probe. S6. The Fe3O4@TA / Ag NPs-Ab2 obtained in S4 was used as a capture probe and mixed with the target bacteria. Then, Au@Ag-MPBA / PEG-Ab1 obtained in S5 was added as a SERS probe and incubated to form a "magnetic nanoparticle-bacteria-SERS probe" sandwich structure detection system. The sandwich structure detection system was separated under the action of an external magnetic field. S7. The sandwich structure detection system obtained in S6 is dropped onto the RCM@Au NPs two-dimensional flexible SERS substrate obtained in S3, and then the target bacteria are quantitatively detected by SERS.

[0010] As a preferred implementation option, preferably, in this scheme S1, the method for preparing bamboo fiber includes: crushing bamboo raw materials, passing them through a 100-mesh sieve to obtain fine fiber powder, then treating the fine fiber powder with 1.5%-2% NaOH solution and 6%-10% NaOH solution in sequence to remove lignin and hemicellulose impurities, and then washing the product until neutral to obtain bamboo fiber; In S1, the solvent system is pre-cooled to -2℃ to 4℃, and its composition includes: 8-12 wt% urea, 3-5 wt% caprolactam, 7-9 wt% sodium hydroxide, and the balance being water; In S1, bamboo fiber is dissolved in a pre-cooled solvent system and then vigorously stirred at a speed of 450-550 rpm for 30-50 minutes at a low temperature of -2 ℃ to 4 ℃ to obtain a cellulose suspension.

[0011] As a preferred implementation option, in S2 of this scheme, the regenerated solution is formed by mixing ethanol and water at a volume ratio of 2:1; when immersing and washing the preformed membrane, the regenerated solution is replaced every 30-60 minutes, and a total of 2-4 replacements are made.

[0012] As a preferred implementation option, in S3 of this scheme, the solvent for preparing the tannic acid solution is phosphate buffer or Tris-HCl buffer with a pH of 7.5-8.0, and the mass concentration of the tannic acid solution is 0.4-1 wt%; the concentration of the chloroauric acid solution is 0.8-1.2 mmol·L⁻¹. -1 .

[0013] As a preferred implementation option, in S3 of this scheme, RCM is first immersed in a tannic acid solution and stirred at 450-550 rpm in the dark for 8-11 hours. After removal, it is washed 3-5 times with ultrapure water and dried at 35-45℃ to obtain RCM-TA. Then, RCM-TA is immersed in a chloroauric acid solution and stirred at room temperature and 450-550 rpm in the dark for 0.5-1 hours. After removal, the membrane is washed 3-5 times with ultrapure water and dried under vacuum at 35-45℃ to obtain RCM@AuNPs loaded with high-density gold nanoparticles, which is a two-dimensional flexible SERS substrate prepared from bamboo cellulose membrane.

[0014] As a preferred implementation option, the solvothermal synthesis of Fe3O4 magnetic nanoparticles mentioned in S4 of this scheme can be specifically as follows: 1.35 g of FeCl3·6H2O is dissolved in 40 mL of ethylene glycol and stirred for 30 min. During stirring, 3.6 g of anhydrous sodium acetate and 1.1 g of PEG are added to the mixture, and the mixture is heated to 60 °C until completely dissolved. When the reactants are completely dissolved, a distinct yellow solution is obtained. The yellow mixed solution is transferred to a hydrothermal reactor and placed at a constant temperature of 200 °C for 6 h. A strong magnet is used to adhere to the outer wall of the flask to adsorb the black precipitate. After the precipitate has completely aggregated (about 5 min), the supernatant is slowly poured off. 20 mL of deionized water is added to the flask, the magnet is removed, and the precipitate is dispersed by sonication for 5 min. The precipitate is adsorbed again with a magnet, and the supernatant is discarded. The precipitate is washed three times with deionized water and ethanol, respectively, and then dried in a drying oven at 60 °C for 12 h to obtain Fe3O4 magnetic bead powder.

[0015] As a preferred implementation option, S4 of this scheme includes: preparing Fe3O4 magnetic nanoparticles into a 1-1.5 wt% Fe3O4 aqueous dispersion, then adding 1-1.5 wt% tannic acid solution to it at a volume ratio of 1:1-2, stirring and reacting at 300 rpm for 40-120 min, and then magnetically separating, washing, and redispersing in an equal volume of water to complete the modification treatment; In S4, the concentration of the silver nitrate solution is 5-8 mmol·L⁻¹. -1 The volume ratio of Fe3O4 aqueous dispersion, tannic acid solution, and silver nitrate solution is 1:1:2.

[0016] As a preferred implementation option, in S4 of this scheme, Fe3O4@TA / Ag NPs are dispersed in PBS buffer, then anti-target bacterial antibody Ab2 and sulfonyl-NHS ester crosslinking agent are added, the reaction is stirred at room temperature, bovine serum albumin (BSA) is added to a final concentration of 1.2% (w / v), and the reaction is continued at room temperature to block non-specific sites. The product is washed with PBST magnetic separation, and finally resuspended in PBS buffer containing BSA to obtain Fe3O4@TA / Ag NPs-Ab2 as a capture probe.

[0017] Preferably, as an example, the specific procedure for modifying antibody Ab2 in S4 of this scheme is as follows: 20 mg Fe3O4@AgNPs are dispersed in 5 mL MES buffer (0.1 mol·L⁻¹). -1 In a solution of 10 mg EDC and 20 mg NHS (pH 6.0), the carboxyl groups on the surface of magnetic nanoparticles were activated by gentle stirring at 200 rpm for 30 min at room temperature. The activated particles were then magnetically separated and rapidly transferred to 2-5 mL of PBS buffer (0.01 M, pH 7.4). 200 µg of antibody Ab2 was added, and the mixture was slowly rotated at 200 rpm for 2 h at 4 °C. Finally, BSA was added to bring the final concentration to 1% (w / v), and the reaction was continued at 4 °C for 1 h to complete the blocking, yielding Fe3O4@TA / Ag NPs-Ab2.

[0018] As a preferred implementation option, S5 of this solution preferably includes: Silver-coated gold nanoparticles were formulated into an Au@Ag NPs dispersion. A 4-mercaptophenylboronic acid (4-MPBA) ethanol solution was added to the dispersion. After stirring, centrifugation, and washing in the dark, the particles were resuspended in the solution to obtain Au@Ag-MPBA. A mercapto-polyethylene glycol-carboxyl aqueous solution was then added to Au@Ag-MPBA. The mixture was stirred at room temperature in the dark, centrifuged, washed, and resuspended in MES buffer to obtain Au@Ag-MPBA / PEG-COOH. EDC and NHS solutions were then added, and the mixture was activated by stirring at room temperature. The particles were collected by centrifugation and resuspended in PBS. An anti-target bacterial monoclonal antibody Ab1 was immediately added, and the reaction was carried out at 4°C for 2 h. BSA was then added to a final concentration of 1%, and the reaction was continued at 4°C for 30 min. The particles were washed by centrifugation with PBST and finally resuspended in PBS containing 1% BSA to obtain Au@Ag-MPBA / PEG-Ab1, which serves as a SERS signal probe.

[0019] As an example of actual data input, S5 of this solution includes the following sub-steps: 1) Add 100 µL of 1 mM 4-MPBA ethanol solution to 10 mL of Au@Ag NPs dispersion, stir at room temperature in the dark for 2 h at 200 rpm; centrifuge and wash 3 times (8000 rpm, 10 min) to completely remove unbound MPBA, and then resuspend in 5 mL of ultrapure water to obtain Au@Ag-MPBA; 2) Add 50 µL of 1 mM HS-PEG-COOH solution, react gently with stirring overnight at room temperature in the dark (12 h, 200 rpm), then centrifuge and wash twice (8000 rpm, 10 min), and resuspend in 2 mL MES buffer (0.1 M, pH 6.0) to obtain Au@Ag-MPBA / PEG-COOH; 3) Add 20 µL of freshly prepared EDC solution (50 mg / mL) and 40 µL of NHS solution (50 mg / mL), and gently stir at room temperature (200 rpm, 30 min); centrifuge at high speed (12000 rpm, 15 min) to collect activated particles, carefully remove the supernatant and immediately resuspend the particles in 1 mL of PBS buffer (0.01 M, pH 7.4); add 200 µL of Ab1 at a concentration of 1 mg / mL, and react the mixture at 4 °C for 2 h; add BSA to a final concentration of 1%, and continue reacting at 4 °C for 30 min to block unreacted activation sites; centrifuge and wash (8000 rpm, 10 min, with PBST containing 0.05% Tween-20) 3 times to thoroughly remove unbound antibodies; finally, resuspend the Au@Ag-MPBA / PEG-Ab1 probe in 1 mL of PBS (storage buffer) containing 1% BSA and store at 4 °C protected from light.

[0020] As a preferred implementation option, S6 of this scheme includes: mixing Fe3O4@TA / AgNPs-Ab2 suspension, which serves as the capture probe, with the target bacteria, then incubating at 37°C with shaking at 200 rpm for 60 minutes, followed by magnetic separation, discarding the supernatant, resuspending and washing with PBST, repeating this process multiple times, adding Au@Ag-MPBA / PEG-Ab1, which serves as the SERS signal probe, to the washed complex, incubating, then magnetically separating again, discarding the supernatant, resuspending and washing with PBST, and repeating this process multiple times until the supernatant is clear, obtaining the final purified "magnetic nanoparticle-bacteria-SERS probe" sandwich structure detection system, and resuspending it in PBS.

[0021] As an example of actual data, this protocol S6 uses PBS (pH 7.4) containing 1% BSA as the incubation and dilution buffer, and the specific operation procedure is as follows; 1) Mix 50 µL of Fe3O4@TA / Ag NPs-Ab2 suspension with 1 mL of target bacterial sample and incubate gently at 37°C with 200 rpm for 60 minutes. After incubation, place the centrifuge tube on a strong magnetic separator for 2 minutes to allow the magnetic complex to aggregate to the tube wall. Carefully aspirate the supernatant and add 500 µL of washing buffer (PBST: PBS containing 0.05% Tween-20) to resuspend the particles. Repeat the washing 2-3 times to remove unbound bacteria, sample matrix and other impurities to obtain the "magnetic nanoparticle-bacteria" complex. 2) Add 30 µL of SERS probe (Au@Ag-MPBA / PEG-Ab1) suspension to the washed "magnetic nanoparticle-bacteria" complex, and make up the total volume to 100 µL with PBS containing 1% BSA. Incubate at 37°C with gentle shaking at 200 rpm in the dark for 1 h. After placing the reaction tube on a magnetic separator for 2 min, carefully aspirate the supernatant, add 1 mL of PBST (0.1% Tween-20) to gently but thoroughly resuspend the particles, and repeat the washing at least 3-4 times until the supernatant is completely clear. Resuspend the finally purified "magnetic nanoparticle-bacteria-SERS probe" sandwich complex in 50 µL of PBS buffer.

[0022] As a preferred implementation option, preferably, in S7 of this scheme, the SERS detection conditions are: 785nm laser, integration time 5000ms, laser power 30mW, and objective lens magnification 20x.

[0023] As a preferred implementation option, the target bacteria described in this scheme is Vibrio parahaemolyticus.

[0024] By adopting the above technical solution, the present invention has the following beneficial effects compared with the prior art: 1. The method provided in this solution uses regenerated bamboo cellulose membrane as the substrate material for loading gold nanoparticles, which has the advantages of low cost and environmental protection. At the same time, the prepared two-dimensional flexible SERS substrate has the characteristics of being more stable, uniform and reproducible than traditional gold nanoparticle or silver sol substrates.

[0025] 2. The regenerated bamboo cellulose membrane obtained in this scheme has strong hydrophilicity, water absorption, high porosity and high specific surface area. After loading gold nanoparticles, the volume of the regenerated cellulose membrane decreases suddenly during the drying process, and the gold nanoparticles loaded on it become more compacted, which is more conducive to the formation of high-density SERS "hot spots" and greatly improves the sensitivity of SERS detection.

[0026] 3. The Au@Ag-MPBA / PEG-Ab1 probe design involved in this scheme utilizes the PEG chain to form a highly hydrated, dynamic, flexible brush on the probe surface, effectively preventing biomacromolecules such as proteins and cell debris from non-specifically adsorbing onto the nanoparticle surface through hydrophobic or electrostatic interactions, thus effectively reducing detection background. It can also prevent the probe from salting out or non-specifically aggregating in physiological saline and protein environments, maintaining long-term dispersion stability. At the same time, the PEG spacer arm achieves spatial separation and functional independence between Ab1 and 4-MPBA (4-mercaptophenylboronic acid) through flexible connection, allowing the antibody's specificity to complement the high affinity of 4-MPBA for bacteria, ultimately improving the capture efficiency, specificity, and stability of the Au@Ag-MPBA / PEG-Ab1 system for pathogenic bacteria.

[0027] 4. The design of Fe3O4@TA / Ag NPs-Ab2 magnetic nanoparticles in this scheme ingeniously achieves the organic unity of four major functions: magnetic separation, signal transduction, interfacial chemistry, and biorecognition. The rapid enrichment of target substances by Fe3O4 meets the core requirements of modern point-of-care testing (POCT) for speed, sensitivity, specificity, and ease of operation. It is especially suitable for the efficient detection of trace target substances in complex matrices in fields such as food safety, environmental monitoring, and clinical pathogen diagnosis.

[0028] 5. The tannic acid (TA) involved in this scheme is firmly bonded to the Fe3O4 surface through strong metal-phenol coordination bonds, forming a stable and uniform coating layer to prevent the magnetic nuclei from agglomerating or dissolving in harsh environments; its o-phenol groups can reduce silver ions (Ag) in situ. + The method uses silver nanoparticles (Ag NPs) to anchor them stably on the surface, avoiding the difficulties of synthesizing Ag NPs separately and subsequent coupling. At the same time, the phenolic hydroxyl and quinone groups rich in TA can be further oxidized or derivatized to provide a large number of active groups such as carboxyl groups (-COOH), which facilitates subsequent covalent coupling with antibodies. Finally, TA itself is a natural polyphenol with good biocompatibility, which helps to reduce non-specific bioadsorption and toxicity of the material.

[0029] 6. The detection method provided in this solution combines antibodies with SERS technology. By utilizing the specific recognition and binding ability between antibodies and Vibrio parahaemolyticus, it overcomes the shortcomings of poor specificity in SERS detection. At the same time, it effectively reduces the influence of other interfering molecules in the matrix on the detection, ensuring excellent specificity.

[0030] 7. The detection method provided in this solution has strong platform versatility. By changing the specific antibody, the platform can be adapted to the detection of other bacteria, viruses or biomarkers. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 The schematic diagram of the SERS method for highly sensitive detection of pathogenic bacteria in aquatic products provided in this solution and its application. Figure 2 (AB) shows the SERS enhancement effect of RCM@Au NPs substrates prepared with TA solutions of different mass concentrations; (CD) shows the SERS enhancement effect of RCM@Au NPs substrates prepared with chloroauric acid solutions of different concentrations. Figure 3 (A) is a schematic diagram of the prepared RCM; (B) is a schematic diagram of the uniformity of the SERS enhancement effect of the prepared RCM@Au NPs substrate; (CD) is a schematic diagram of the uniformity of the SERS enhancement effect of the prepared RCM@Au NPs substrate; (EF) is a schematic diagram of the reproducibility of the SERS enhancement effect of different batches of prepared RCM@Au NPs substrates. Figure 4 XPS characterization images of RCM and RCM@Au NPs substrates prepared for this method; Figure 5 The image shows the results of contact angle testing on the RCM, RCM-TA, and RCM@Au NPs substrates prepared according to this method; Figure 6 (AC) are electron microscope images of the RCM, RCM-TA and RCM@Au NPs substrates prepared by this method; (D) are infrared characterization images of TA and the RCM, RCM-TA and RCM@Au NPs substrates prepared by this method. Figure 7 The image shows the SERS detection results after this method reacted with different concentrations of Vibrio parahaemolyticus bacterial solutions. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be particularly noted that the following embodiments are for illustrative purposes only and do not limit the scope of the invention. Similarly, the following embodiments are only some, not all, embodiments of the present invention, and all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1 Combination Figure 1 As shown in the figure, this embodiment provides a highly sensitive SERS method for detecting pathogenic bacteria in aquatic products, which specifically includes the following steps: S1. Crush the bamboo raw material and pass it through a 100-mesh sieve to obtain fine fiber powder. Treat it successively with 2% NaOH solution (80℃, 2h) and 8% NaOH solution (50℃, 1h) to remove impurities such as lignin and hemicellulose, and wash until neutral. Add the pretreated bamboo fiber (5 wt%) to a solvent system pre-cooled to 0-4℃ (composition: 8 wt% NaOH, 10 wt% urea, 4 wt% caprolactam, balance water). Stir vigorously at 4℃ and 500 rpm for 45 minutes to obtain a uniform cellulose suspension.

[0035] S2. Centrifuge the above cellulose suspension at 12000 rpm for 10 minutes. Pour the supernatant into a 1×1 cm² polytetrafluoroethylene mold and allow it to stand at room temperature for 12 hours to evaporate the solvent and form a membrane. Immerse the pre-formed membrane in a 1:1 (v / v) ethanol / water regeneration solution, changing the solution every hour for a total of 3 times to ensure complete regeneration and removal of residual solvent. Remove the membrane and dry it at room temperature to obtain a neutral regenerated cellulose membrane (RCM).

[0036] S3, prepare a 0.5 wt% tannic acid (TA) solution (solvent: pH 7.8 phosphate buffer) and 1.0 mmol·L⁻¹ - ¹ An aqueous solution of chloroauric acid (HAuCl4). RCM was first immersed in TA solution and reacted at room temperature and 500 rpm in the dark for 12 hours. After removal, it was washed three times with ultrapure water and dried at 40°C to obtain RCM-TA. RCM-TA was then immersed in HAuCl4 solution and reacted under the same conditions (room temperature, 500 rpm, in the dark) with stirring for 0.5 hours. The membrane was removed, washed three times with ultrapure water, and dried under vacuum at 40°C to obtain a flexible SERS substrate loaded with gold nanoparticles (RCM@Au NPs); Figure 6As shown in (AC), the RCM dense porous RCM prepared by the above method has gold nanoparticles densely distributed on the RCM surface, with a particle size between 100-150 nm.

[0037] S4. Dissolve 1.35 g FeCl3·6H2O in 40 mL ethylene glycol and stir for 30 minutes. Add 3.6 g anhydrous sodium acetate and 1.1 g polyethylene glycol (PEG 6000) sequentially, and heat at 60°C until completely dissolved to obtain a yellow transparent solution. Transfer the mixture to a 100 mL PTFE-lined high-pressure reactor and react at 200°C for 6 hours. After natural cooling, separate the black precipitate with a magnet and wash three times each with deionized water and ethanol. Dry at 60°C for 12 hours to obtain Fe3O4 magnetic nanoparticle powder. Prepare a 1 wt% aqueous dispersion of the Fe3O4 magnetic nanoparticle powder. Mix it with 1 wt% TA solution (Tris-HCl buffer, pH 8.5) at a volume ratio of 1:1. Stir at 300 rpm for 2 hours at room temperature. Magnetic separation, washing, and redisperse in an equal volume of water. Add 5 mmol·L⁻¹ to the above dispersion at a volume ratio of 1:2. - ¹A silver nitrate (AgNO3) solution. The reaction was carried out in a 60℃ water bath under light-protected conditions with stirring at 300 rpm for 2 hours. Magnetic separation and washing were performed to obtain the Fe3O4@TA / Ag NPs composite material. 20 mg of Fe3O4@TA / Ag NPs was dispersed in 5 mL of MES buffer (0.1 mol·L⁻¹). - ¹, pH 6.0). Add 10 mg EDC and 20 mg NHS, and stir at 200 rpm for 30 minutes at room temperature to activate the carboxyl groups on the particle surface. Magnetic separation, discard the supernatant, and quickly resuspend the activated particles in 2 mL PBS buffer (0.01 M, pH 7.4). Add 200 µg of anti-Vibrio parahaemolyticus antibody Ab2, and slowly rotate and mix at 200 rpm for 2 hours at 4°C. Add bovine serum albumin (BSA) to a final concentration of 1% (w / v), and continue the reaction at 4°C for 1 hour to block non-specific sites. Magnetic separation and washing three times with PBST (PBS containing 0.05% Tween-20), and finally resuspend in PBS containing 1% BSA, storing at 4°C to obtain the capture probe Fe3O4@TA / Ag NPs-Ab2.

[0038] S5. First, gold nanoparticle seeds with a particle size of approximately 50 nm were prepared using the sodium citrate reduction method. 10 mL of this seed sol was taken, and the pH was adjusted to 9-10. 50 µL of 0.1 M NaOH and 300 µL of 10 mM ascorbic acid were added sequentially, followed by dropwise addition of 500 µL of 1 mM AgNO3 solution. After reacting for 15 minutes, the mixture was centrifuged and washed to obtain Au@Ag core-shell nanoparticles. 100 µL of 1 mM 4-mercaptophenylboronic acid (4-MPBA) ethanol solution was added to 10 mL of the Au@Ag NPs dispersion, and the mixture was stirred at room temperature in the dark for 2 hours (200 rpm). After centrifugation (8000 rpm, 10 min), washing three times, and resuspending in 5 mL of ultrapure water, Au@Ag-MPBA was obtained. Add 50 µL of 1 mM thiol-polyethylene glycol-carboxyl (HS-PEG-COOH, MW 5000) aqueous solution to 5 mL Au@Ag-MPBA and stir at room temperature in the dark for 12 hours (200 rpm). Centrifuge and wash twice, then resuspend in 2 mL MES buffer (0.1 M, pH 6.0) to obtain Au@Ag-MPBA / PEG-COOH. Add 20 µL of EDC solution (50 mg / mL) and 40 µL of NHS solution (50 mg / mL), and stir at room temperature for 30 minutes (200 rpm) for activation. Centrifuge (12000 rpm, 15 min) to collect the particles and resuspend in 1 mL PBS (0.01 M, pH 7.4). Immediately add 200 µL of 1 mg / mL anti-Vibrio parahaemolyticus monoclonal antibody Ab1 and react at 4°C for 2 hours. Add BSA to a final concentration of 1% and continue reacting at 4°C for 30 minutes. The sample was washed three times by centrifugation with PBST and finally resuspended in 1 mL of PBS containing 1% BSA. It was then stored at 4°C in the dark to obtain the SERS signal probe Au@Ag-MPBA / PEG-Ab1.

[0039] S6. Take 50 µL of the capture probe (Fe3O4@TA / Ag NPs-Ab2) suspension and mix it with 1 mL of sample containing different concentrations of Vibrio parahaemolyticus (in PBS containing 1% BSA). Incubate at 37°C with shaking at 200 rpm for 60 minutes. Perform magnetic separation for 2 minutes and discard the supernatant. Resuspend and wash with 500 µL of PBST (0.05% Tween-20), repeating 3 times. Add 30 µL of the SERS signal probe (Au@Ag-MPBA / PEG-Ab1) to the washed complex and bring the total volume to 100 µL with PBS containing 1% BSA. Incubate at 37°C with shaking for 60 minutes (200 rpm) in the dark. After magnetic separation, discard the supernatant. Resuspend and wash with 1 mL of PBST (0.1% Tween-20), repeating 4 times until the supernatant is clear. The final purified “magnetic nanoparticle-bacteria-SERS probe” sandwich complex was resuspended in 50 µL PBS.

[0040] S7. Add 50 µL of the sandwich composite suspension obtained in step S6 to the RCM@Au NPs flexible substrate prepared in step S1, and allow it to air dry at room temperature. Detection is performed using a confocal micro Raman spectroscopy system. The conditions are set as follows: laser wavelength 785 nm, laser power 30 mW, integration time 5000 ms, and objective magnification 20×. MPBA is acquired at approximately 1070 cm⁻¹. -1 The intensity of the characteristic Raman peak at a given location is used as the signal. A standard working curve is plotted with the logarithm of bacterial concentration on the x-axis and the intensity of the characteristic peak on the y-axis to achieve quantitative detection of Vibrio parahaemolyticus in the sample.

[0041] Based on this embodiment, in order to facilitate the optimization of parameters in step S3, this scheme takes "maximizing the intensity of SERS characteristic peak signal" as the core objective. It adopts the pre-experimental single-factor variable method and literature investigation to explore the effects of TA solution concentration and chloroauric acid solution concentration on the SERS performance of RCM@Au NPs. Through multiple sets of parallel experiments to eliminate random errors, the two concentration combinations that can produce the strongest and most stable SERS signal of the product are finally selected.

[0042] Literature review and preliminary experiments revealed that 0.5% TA and 1mM chloroauric acid were the optimal concentration combination for the formation of stable Au NPs in the preliminary experiments. Therefore, this concentration was chosen as the baseline to ensure that most parallel samples exhibited basic SERS activity, guaranteeing comparability of screening results and reducing experimental waste due to ineffective products. Tannic acid, acting as both a reducing agent and stabilizer, effectively reduced chloroauric acid at this concentration, while avoiding excessive tannic acid. Too low a concentration might result in incomplete reduction, while too high a concentration might introduce background interference or inhibit Au NP growth. At this concentration, the reduction growth rate, size, and morphology of gold nanoparticles (Au NPs) obtained from chloroauric acid were likely within an ideal range. Too high a concentration might lead to Au NP aggregation or the formation of irregular structures, reducing the SERS enhancement effect; too low a concentration might result in insufficient Au NP coverage, leading to a weak SERS signal.

[0043] In step S3, during the investigation of RCM@Au NPs preparation, preliminary experiments were conducted by varying the concentrations of TA solution and chloroauric acid solution (TA solution 0.01%, 0.05%, 0.1%, 0.5%, 1%wt; chloroauric acid solution 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mM) to derive multiple parallel examples. The optimal concentration was then determined using product characterization. Figure 2 Preliminary experimental data indicate that using a 0.5 wt% TA solution ( Figure 2 (A, B) and 1.0 mM chloroauric acid solution ( Figure 2 The RCM@Au NPs substrate prepared under the combined conditions of (C, D) exhibits the best SERS enhancement effect.

[0044] Figure 3 (A) shows the morphology of the RCM prepared in step S2 of this embodiment; to evaluate the enhancement effect, uniformity, and reproducibility of the RCM@Au NPs substrate prepared in step S3, SERS detection was performed using 4-MBA (4-mercaptobenzoic acid, C7H6O2S) as the Raman signal molecule, according to Example 1 above, and the results are as follows. Figure 3 As shown in (BF); Figure 3 (B) SERS detection of different concentrations of 4-MBA shows that the prepared RCM@Au NPs exhibit good SERS enhancement effect; Figure 3 (CD) represents 50 points randomly selected from the RCM@Au NPs substrate for 10... 5 The SERS signal detection of M 4-MBA yielded an RSD of 7.39%, proving that the RCM@Au NPs substrate has good homogeneity; Figure 3(EF) involves randomly selecting five RCM@AuNPs substrates from different batches and randomly selecting three points on each unit for SERS detection. The results show that RCM@Au NPs have good reproducibility.

[0045] To further confirm the successful reduction of gold nanoparticles onto the RCM, the RCM prepared in step S2 and the RCM@Au NPs prepared in step S3 of this embodiment were characterized by XPS. The results are as follows: Figure 4 (A) and Figure 4 As shown in (B), by comparing the two figures, it can be clearly seen that the characteristic band of Au appears on the XPS plot of RCM@Au NPs.

[0046] By conducting contact angle tests on the RCM, RCM-TA, and RCM@Au NPs prepared in the embodiments of the present invention, the following results were obtained sequentially. Figure 5 The characterization diagram of (AC) shows that the hydrophilicity of RCM is reduced after loading gold nanoparticles. This is because the original RCM is rich in hydroxyl groups and therefore has extremely strong hydrophilicity. The coating of gold nanoparticles reduces the hydrophilicity of RCM, but it still has excellent hydrophilicity.

[0047] To further confirm that TA was successfully grafted onto RCM, the RCM, RCM-TA, and RCM@Au NPs prepared in this embodiment were characterized by FTIR, and the results were obtained. Figure 6 (D) By comparison, it can be clearly seen that the characteristic band of TA appears on the FTIR plot of RCM@Au NPs.

[0048] The material prepared according to Example 1 was co-incubated with standard solutions of Vibrio parahaemolyticus at different concentrations, and SERS detection was performed. The detection results are as follows: Figure 7 As shown, the detection limit of this method reaches 10 CFU·mL. -1 And the curve fit R 2 =0.993, indicating that the working curve has a good linear fitting effect and can be used as the basis for subsequent calculation of Vibrio parahaemolyticus in actual samples.

[0049] Example 2 Combination Figure 1 As shown in the figure, this embodiment provides a highly sensitive SERS method for detecting pathogenic bacteria in aquatic products, which specifically includes the following steps: S1. Crush the bamboo raw material and pass it through a 150-mesh sieve to obtain finer fiber powder. Treat it sequentially with 1.5% NaOH solution (85℃, 1.5 h) and 10% NaOH solution (55℃, 45 min) to improve the purity of α-cellulose, and wash until neutral. Add the pretreated bamboo fiber (4 wt%) to a solvent system pre-cooled to 0-4℃ (composition: 9 wt% NaOH, 12 wt% urea, 5 wt% caprolactam, balance water). Stir vigorously at -2℃ and 550 rpm for 50 minutes to obtain a uniform cellulose suspension.

[0050] S2. Centrifuge the above suspension at 9500 rpm for 15 minutes. Pour the supernatant into a 2×2 cm container. 2 A pre-formed cellulose membrane (RCM) was formed by allowing the solvent to evaporate on a polytetrafluoroethylene mold at a constant temperature of 15°C for 10 hours. The pre-formed membrane was then immersed in a 2:1 volume ratio ethanol / water regeneration solution, with the solution being replaced every 45 minutes for a total of 4 times to ensure complete regeneration and removal of residual solvent. The membrane was then removed and vacuum-dried at 40°C for 6 hours to obtain a high mechanical strength regenerated cellulose membrane (RCM).

[0051] S3. Prepare a 0.5 wt% tannic acid (TA) solution (using Tris-HCl buffer at pH 8.0) and 1.0 mmol·L⁻¹ - ¹ An aqueous solution of chloroauric acid (HAuCl4). RCM was first immersed in the TA solution and reacted at room temperature and 550 rpm in the dark for 10 hours. After removal, it was washed five times with ultrapure water and dried at 45°C to obtain RCM-TA. RCM-TA was then immersed in the HAuCl4 solution and reacted under the same conditions (room temperature, 550 rpm, in the dark) for 10 hours. The membrane was then removed, washed five times with ultrapure water, and dried under vacuum at 45°C to obtain a flexible SERS substrate (RCM@Au NPs) loaded with high-density gold nanoparticles.

[0052] S4. Dissolve 1.62 g FeCl3·6H2O and 0.72 g FeCl2·4H2O in 50 mL of deionized water and stir for 15 minutes under nitrogen protection. Quickly add 15 mL of 28% ammonia solution to bring the pH to 11 and react at 80°C for 40 minutes. Without separation, directly add 50 mL of 1.5 wt% TA solution (pH 9.0) and continue the reaction for 1 hour. Add 20 mL of 8 mmol·L⁻¹ TA solution. -The Fe3O4@TA / Ag NPs composite material was obtained by magnetic separation and washing after reacting with silver nitrate (AgNO3) solution at 350 rpm for 1.5 hours in a 65°C water bath under light-protected conditions. 25 mg of Fe3O4@TA / Ag NPs was dispersed in 4 mL of PBS buffer (0.01 M, pH 7.4). 220 µg of anti-Vibrio parahaemolyticus antibody Ab2 and 12 µL of 50 mg / mL sulfo-NHS ester crosslinking agent were added, and the mixture was stirred at 250 rpm for 90 minutes at room temperature. Bovine serum albumin (BSA) was added to a final concentration of 1.2% (w / v), and the reaction was continued at room temperature for 40 minutes to block non-specific sites. The mixture was magnetically washed twice with PBST (PBS containing 0.05% Tween-20), and finally resuspended in PBS containing 1.2% BSA. It was stored at 4°C to obtain the capture probe Fe3O4@TA / Ag NPs-Ab2.

[0053] S5. First, gold nanoparticle seeds with a particle size of ~50 nm were prepared using the sodium citrate reduction method. 10 mL of this seed sol was taken, and the pH was adjusted to 10-11. 60 µL of 0.1 M NaOH and 350 µL of 15 mM ascorbic acid were added sequentially, followed by dropwise addition of 600 µL of 1.2 mM AgNO3 solution. After reacting for 20 minutes, the mixture was centrifuged and washed to obtain Au@Ag core-shell nanoparticles. 120 µL of 1 mM 4-mercaptophenylboronic acid (4-MPBA) ethanol solution and 60 µL of 0.8 mM mercapto-polyethylene glycol-carboxyl (HS-PEG-COOH, MW 5000) aqueous solution were added to 10 mL of the Au@Ag NPs dispersion, and the mixture was stirred at room temperature in the dark for 3 hours (250 rpm). The particles were washed three times by centrifugation (8000 rpm, 10 min) and resuspended in 4 mL of MES buffer (0.1 M, pH 6.0). Activation was performed by adding 25 µL of LEDC solution (50 mg / mL) and 50 µL of NHS solution (50 mg / mL) and stirring at room temperature for 25 min (250 rpm). The particles were collected by centrifugation (12000 rpm, 15 min) and resuspended in 1.2 mL of PBS (0.01 M, pH 7.4). Immediately, 220 µL of 1 mg / mL anti-Vibrio parahaemolyticus monoclonal antibody Ab1 was added, and the reaction was carried out at 4 °C for 1.5 h. BSA was added to a final concentration of 1.2%, and the reaction was continued at 4 °C for 25 min. The particles were washed three times by centrifugation with PBST and finally resuspended in 1 mL of PBS containing 1% BSA and 0.5% trehalose. The mixture was stored at 4 °C protected from light to obtain the SERS signal probe Au@Ag-MPBA / PEG-Ab1.

[0054] S6. Take 40 µL of the capture probe (Fe3O4@TA / Ag NPs-Ab2) suspension and mix it with 1 mL of sample containing different concentrations of Vibrio parahaemolyticus (in PBS containing 1% BSA). Incubate at 37°C with shaking at 250 rpm for 50 minutes. Perform magnetic separation for 2 minutes and discard the supernatant. Resuspend and wash with 600 µL of PBST (0.05% Tween-20), repeating twice. Add 35 µL of the SERS signal probe (Au@Ag-MPBA / PEG-Ab1) to the washed complex and bring the total volume to 120 µL with PBS containing 1% BSA. Incubate at 37°C with shaking in the dark for 50 minutes (250 rpm). After magnetic separation, discard the supernatant. Resuspend and wash with 1 mL of PBST (0.1% Tween-20), repeating three times until the supernatant is clear. The final purified “magnetic nanoparticle-bacteria-SERS probe” sandwich complex was resuspended in 40 µL PBS.

[0055] S7. Add 40 µL of the sandwich composite suspension obtained in step S6 to the RCM@Au NPs flexible substrate prepared in step S3, and allow it to air dry at room temperature. Detection is performed using a confocal micro Raman spectroscopy system. The conditions are set as follows: laser wavelength 785 nm, laser power 25 mW, integration time 3000 ms, and objective magnification 20×. MPBA is acquired at approximately 1070 cm⁻¹. -1 The intensity of the characteristic Raman peak at a given location is used as the signal. A standard working curve is plotted with the logarithm of bacterial concentration on the x-axis and the intensity of the characteristic peak on the y-axis to achieve quantitative detection of Vibrio parahaemolyticus in the sample.

[0056] Example 3 Combination Figure 1 As shown in the figure, this embodiment provides a highly sensitive SERS method for detecting pathogenic bacteria in aquatic products, which specifically includes the following steps: S1. Crush the bamboo raw material and pass it through an 80-mesh sieve to obtain fiber powder. Treat the powder sequentially with 2% NaOH solution (75℃, 1 h) and 6% NaOH solution (45℃, 40 min) to quickly remove impurities, and wash until neutral. Add the pretreated bamboo fiber (6 wt%) to a solvent system pre-cooled to 2℃ (composition: 7 wt% NaOH, 8 wt% urea, 3 wt% caprolactam, balance water). Stir vigorously at 2℃ and 450 rpm for 30 minutes to obtain a uniform cellulose suspension.

[0057] S2. Centrifuge the above suspension at 8500 rpm for 10 minutes. Pour the supernatant into a 0.5 × 0.5 cm² polytetrafluoroethylene mold and allow it to stand at room temperature for 8 hours to evaporate the solvent and form a membrane. Immerse the pre-formed membrane in a 1:1 (v / v) ethanol / water regeneration solution, changing the regeneration solution every 30 minutes for a total of two changes. Remove the membrane and dry it at room temperature to obtain a regenerated cellulose membrane (RCM).

[0058] S3, prepare a 0.4 wt% tannic acid (TA) solution (solvent: pH 7.5 phosphate buffer) and 0.8 mmol·L⁻¹ -1 The RCM was first immersed in an aqueous solution of chloroauric acid (HAuCl4) and reacted at room temperature and 450 rpm in the dark for 8 hours. After removal, it was washed three times with ultrapure water and dried at 35°C to obtain RCM-TA. RCM-TA was then immersed in an HAuCl4 solution and reacted at 35°C and 450 rpm in the dark for 6 hours. The membrane was then removed, washed three times with ultrapure water, and dried under vacuum at 35°C to obtain the flexible SERS substrate loaded with gold nanoparticles (RCM@Au NPs).

[0059] S4. Co-precipitation method: Dissolve 1.08 g FeCl3·6H2O and 0.40 g FeCl2·4H2O in 40 mL of deionized water. Under nitrogen protection, rapidly add 8 mL of 28% ammonia solution until the pH reaches 10. React at 70℃ for 20 minutes. Add 40 mL of 1 wt% TA solution (pH 8.5) and continue the reaction for 40 minutes. Add 15 mL of 6 mmol·L... - The Fe3O4@TA / Ag NPs composite material was obtained by stirring in silver nitrate (AgNO3) solution at 400 rpm for 1 hour in a 55°C water bath under light-protected conditions. After magnetic separation and washing, the Fe3O4@TA / Ag NPs composite material was obtained. 15 mg of Fe3O4@TA / Ag NPs was dispersed in 3 mL of PBS buffer (0.01 M, pH 7.4). 150 µg of anti-Vibrio parahaemolyticus antibody Ab2 and 8 µL of 50 mg / mL sulfo-NHS ester crosslinking agent were added, and the mixture was stirred at 300 rpm for 60 minutes at room temperature. Bovine serum albumin (BSA) was added to a final concentration of 0.8% (w / v), and the reaction was continued at room temperature for 20 minutes. After magnetic separation and washing twice, the mixture was resuspended in PBS containing 0.8% BSA and stored at 4°C to obtain the capture probe.

[0060] S5. First, gold nanoparticle seeds with a particle size of ~40 nm were prepared using the sodium citrate reduction method. 8 mL of the seed sol was taken, and the pH was adjusted to 9-10. 40 µL of 0.1 M NaOH and 250 µL of 8 mM ascorbic acid were added sequentially, followed by a rapid addition of 400 µL of 1 mM AgNO3 solution. After reacting for 10 minutes, the mixture was centrifuged and washed to obtain Au@Ag core-shell nanoparticles. 80 µL of 1 mM 4-MPBA ethanol solution was added to 8 mL of the Au@AgNPs dispersion, and the mixture was stirred at room temperature in the dark for 1 hour (300 rpm). After centrifugation and washing twice, the particles were resuspended in 3 mL of MES buffer. 15 µL of EDC solution and 30 µL of NHS solution were added, and the mixture was stirred at room temperature for 20 minutes for activation. The particles were collected by centrifugation and resuspended in 0.8 mL of PBS. 150 µL of 1 mg / mL Ab1 was added, and the mixture was reacted at room temperature for 1 hour. BSA was added to a final concentration of 0.8%, and the mixture was reacted for 15 minutes. After centrifugation and washing twice, the sample was resuspended in 0.8 mL PBS to obtain the SERS signal probe.

[0061] S6. Take 30 µL of the capture probe suspension and mix it with 0.8 mL of bacterial sample. Incubate at 37°C with shaking at 300 rpm for 30 minutes. Perform magnetic separation for 1 minute and wash once. Add 25 µL of SERS signal probe to bring the total volume to 80 µL. Incubate at 37°C in the dark with shaking for 30 minutes (300 rpm). After magnetic separation, wash twice. Resuspend the complex in 30 µL of PBS.

[0062] S7. Add 30 µL of the sandwich composite suspension to the RCM@Au NPs substrate and allow to air dry at room temperature. Detect using Raman spectroscopy: laser wavelength 785 nm, laser power 35 mW, integration time 2000 ms, objective magnification 20×. Acquire at 1070 cm⁻¹. -1 The peak intensity was quantified.

[0063] The above description is only a part of the embodiments of the present invention and does not limit the scope of protection of the present invention. Any equivalent device or equivalent process transformation made based on the content of the present invention specification and drawings, or direct or indirect application in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A highly sensitive SERS method for detecting pathogenic bacteria in aquatic products, characterized in that, It includes: Au@Ag-MPBA / PEG-Ab1 was used as a SERS signal probe for the quantitative detection of target bacteria. The target bacteria were enriched by combining the magnetic composite nanomaterial Fe3O4@TA / Ag NPs-Ab2 to form a sandwich structure detection system for the detection of target bacteria. The detection system was then placed on a two-dimensional flexible SERS substrate made of bamboo cellulose membrane. The SERS signal was amplified by the two-dimensional flexible SERS substrate, thereby rapidly completing the enrichment and sensitive detection of target bacteria by the SERS method.

2. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 1, characterized in that, It includes the following steps: S1. Dissolve bamboo fiber in a pre-cooled solvent system and stir vigorously at low temperature to obtain a cellulose suspension; S2. Centrifuge the cellulose suspension, transfer the supernatant to a polytetrafluoroethylene mold, and then remove the solvent at room temperature to obtain a preformed membrane. Then immerse the preformed membrane in a regeneration solution formed by a mixture of ethanol and water, repeatedly soak, wash and replace the regeneration solution to remove residual solvent, and then dry to obtain a neutral regenerated cellulose membrane, denoted as RCM. S3. Prepare tannic acid solution as a reducing agent and chloroauric acid solution as an oxidizing agent. Place RCM in the reducing agent solution and oxidizing agent solution in turn, and stir in a water bath at room temperature in the dark. After taking them out, wash them several times with ultrapure water and then dry them to finally obtain RCM@Au NPs, which is a two-dimensional flexible SERS substrate prepared by bamboo cellulose membrane. S4. Fe3O4 magnetic nanoparticles were synthesized by a pre-defined method, and then modified with tannic acid. They were then mixed with silver nitrate solution to load silver nanoparticles, namely Fe3O4@TA / Ag NPs. Subsequently, they were mixed with anti-target bacterial antibody Ab2 to obtain functionalized magnetic nanoparticles modified with antibody Ab2, namely Fe3O4@TA / Ag NPs-Ab2. S5. 4-MPBA is mixed with silver-coated gold nanoparticles to obtain Au@Ag-MPBA, and then mixed with thiol-polyethylene glycol-carboxyl aqueous solution and anti-target bacterial monoclonal antibody Ab1 to obtain Au@Ag-MPBA / PEG-Ab1, i.e., SERS signal probe. S6. The Fe3O4@TA / Ag NPs-Ab2 obtained in S4 was used as a capture probe and mixed with the target bacteria. Then, Au@Ag-MPBA / PEG-Ab1 obtained in S5 was added as a SERS probe and incubated to form a "magnetic nanoparticle-bacteria-SERS probe" sandwich structure detection system. The sandwich structure detection system was separated under the action of an external magnetic field. S7. The sandwich structure detection system obtained in S6 is dropped onto the RCM@Au NPs two-dimensional flexible SERS substrate obtained in S3, and then the target bacteria are quantitatively detected by SERS.

3. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, In S1, the method for preparing bamboo fiber includes: crushing bamboo raw materials, passing them through a 100-mesh sieve to obtain fine fiber powder, then treating the fine fiber powder with 1.5%-2% NaOH solution and 6%-10% NaOH solution in sequence to remove lignin and hemicellulose impurities, and then washing the product until neutral to obtain bamboo fiber. In S1, the solvent system is pre-cooled to -2℃ to 4℃, and its composition includes: 8-12 wt% urea, 3-5 wt% caprolactam, 7-9 wt% sodium hydroxide, and the balance being water; In S1, bamboo fiber is dissolved in a pre-cooled solvent system and then vigorously stirred at a speed of 450-550 rpm for 30-50 minutes at a low temperature of -2 ℃ to 4 ℃ to obtain a cellulose suspension.

4. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, In S2, ethanol and water are mixed in a volume ratio of 2:1 to form a regeneration solution. When immersing and washing the preformed membrane, the regeneration solution is replaced every 30-60 minutes, and a total of 2-4 replacements are made.

5. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, In step S3, the solvent for preparing the tannic acid solution is phosphate buffer or Tris-HCl buffer with a pH of 7.5-8.0, and the mass concentration of the tannic acid solution is 0.4-1 wt%; the concentration of the chloroauric acid solution is 0.8-1.2 mmol·L⁻¹. -1 ; In S3, RCM is first immersed in a tannic acid solution and stirred in the dark at 450-550 rpm for 8-11 hours. After removal, it is washed 3-5 times with ultrapure water and dried at 35-45℃ to obtain RCM-TA. Then, RCM-TA is immersed in a chloroauric acid solution and stirred in the dark at room temperature at 450-550 rpm for 6-11 hours. After removal, the membrane is washed 3-5 times with ultrapure water and dried under vacuum at 35-45℃ to obtain RCM@Au NPs loaded with high-density gold nanoparticles, which is a two-dimensional flexible SERS substrate prepared from bamboo cellulose membrane.

6. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, S4 include Fe3O4 magnetic nanoparticles were prepared into a 1-1.5 wt% Fe3O4 aqueous dispersion. Then, 1-1.5 wt% tannic acid solution was added to it at a volume ratio of 1:1-2. The mixture was stirred at 300 rpm for 40-120 min. After magnetic separation, washing, and redispersing in an equal volume of water, the modification treatment was completed. In S4, the concentration of the silver nitrate solution is 5-8 mmol·L⁻¹. -1 The volume ratio of Fe3O4 aqueous dispersion, tannic acid solution, and silver nitrate solution is 1:1:

2.

7. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, In S4, Fe3O4@TA / Ag NPs were dispersed in PBS buffer, and then anti-target bacterial antibody Ab2 and sulfonyl-NHS ester crosslinking agent were added. The mixture was stirred at room temperature, and then bovine serum albumin (BSA) was added to a final concentration of 1.2% (w / v). The reaction was continued at room temperature to block non-specific sites. The mixture was washed with PBST magnetic separation, and finally the product was resuspended in PBS buffer containing BSA to obtain Fe3O4@TA / Ag NPs-Ab2 as a capture probe.

8. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, S5 include: Silver-coated gold nanoparticles were formulated into an Au@Ag NPs dispersion. A 4-mercaptophenylboronic acid (4-MPBA) ethanol solution was added to the dispersion. After stirring, centrifugation, and washing in the dark, the particles were resuspended in the solution to obtain Au@Ag-MPBA. A mercapto-polyethylene glycol-carboxyl aqueous solution was then added to Au@Ag-MPBA. The mixture was stirred at room temperature in the dark, centrifuged, washed, and resuspended in MES buffer to obtain Au@Ag-MPBA / PEG-COOH. EDC and NHS solutions were then added, and the mixture was activated by stirring at room temperature. The particles were collected by centrifugation and resuspended in PBS. An anti-target bacterial monoclonal antibody Ab1 was immediately added, and the reaction was carried out at 4°C for 2 h. BSA was then added to a final concentration of 1%, and the reaction was continued at 4°C for 30 min. The particles were washed by centrifugation with PBST and finally resuspended in PBS containing 1% BSA to obtain Au@Ag-MPBA / PEG-Ab1, which serves as a SERS signal probe.

9. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in claim 2, characterized in that, S6 include: The Fe3O4@TA / Ag NPs-Ab2 suspension, used as the capture probe, was mixed with the target bacteria and then incubated at 37°C with shaking at 200 rpm for 60 minutes. After magnetic separation, the supernatant was discarded, and the mixture was resuspended and washed with PBST. This process was repeated several times. Au@Ag-MPBA / PEG-Ab1, used as the SERS signal probe, was added to the washed complex. After incubation, the mixture was magnetically separated, the supernatant was discarded, and the mixture was resuspended and washed with PBST. This process was repeated several times until the supernatant was clear, thus obtaining the final purified "magnetic nanoparticle-bacteria-SERS probe" sandwich structure detection system. The system was then resuspended in PBS.

10. The SERS method for highly sensitive detection of pathogenic bacteria in aquatic products as described in any one of claims 1 to 9, characterized in that, The target bacterium is Vibrio parahaemolyticus.

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