A method for colorimetric-sers dual-mode detection and photothermal sterilization for specific detection of vibrio parahaemolyticus and application thereof

Abstract

The application discloses a colorimetric-SERS dual-mode detection and photothermal sterilization method for specifically detecting Vibrio parahaemolyticus and application thereof, and belongs to the technical field of food safety biology. The application takes Vibrio parahaemolyticus as a target, and prepares a multifunctional magnetic composite nanomaterial through layer-by-layer assembly. Firstly, the superparamagnetic separation performance of the magnetic composite nanomaterial is combined with magnetic chromatography to achieve the effect of rapidly separating bacteria from a complex food matrix. Secondly, the colorimetric and SERS sensing signals of the magnetic composite nanomaterial are used to quantitatively detect bacteria in a short time, high precision and high stability in a dual-mode. Finally, the photothermal performance of the magnetic composite nanomaterial is used to kill the detected bacteria by photothermal sterilization, so that the hazard control at the back end of detection is completed.

Description

Technical Field

[0001] This invention relates to a method for specific detection of Vibrio parahaemolyticus using colorimetric-SERS dual-mode detection and photothermal sterilization, and its application, belonging to the field of food safety biotechnology. Background Technology

[0002] Vibrio parahaemolyticus is a halophilic, Gram-negative bacterium that can grow naturally in marine or estuarine environments. It is a foodborne pathogen, typically caused by consuming raw or undercooked seafood, resulting in symptoms such as acute gastroenteritis and primary septicemia. With global warming, rising ocean temperatures have directly led to further proliferation and spread of Vibrio parahaemolyticus, making it a major cause of seafood poisoning worldwide.

[0003] The detection of Vibrio parahaemolyticus has primarily focused on seafood. However, traditional methods for detecting Vibrio parahaemolyticus in food have numerous shortcomings and fail to meet the growing demand for food safety. Plate counting methods are cumbersome, labor-intensive, and time-consuming; molecular biological methods are not ideal in terms of detection speed, and their sensitivity is limited by the initial bacterial count in the sample, requiring enrichment culture to achieve optimal sensitivity; immunological methods are expensive, antibody preparation is complex, and they are susceptible to interference from the environment and contaminants. Therefore, it is necessary to establish a rapid and simple method for detecting Vibrio parahaemolyticus to control food safety. Summary of the Invention

[0004] [Technical Issues]

[0005] First, most existing materials for detecting Vibrio parahaemolyticus consist of two parts: a recognition element and a signal probe. Few studies have developed materials that integrate both recognition and dual-signal processing. Second, traditional detection methods suffer from long detection times, expensive equipment, and cumbersome operation, while immunoassays have relative problems such as long processing times, low specificity, and unstable antibodies. Third, existing research on Vibrio parahaemolyticus detection technologies has not considered the control of pathogenic bacteria at the post-detection stage after quantitative detection.

[0006] [Technical Solution]

[0007] This invention provides a method for preparing multifunctional magnetic composite nanomaterials for the specific identification, detection, and killing of Vibrio parahaemolyticus, comprising the following steps:

[0008] (1) Fe3O4 nanomaterials with superparamagnetism were prepared by solvothermal method;

[0009] (2) MOF-919 (Fe-Cu) was grown in situ on the surface of Fe3O4 by hydrothermal reaction to obtain Fe3O4@MOF (Fe-Cu) composite material;

[0010] (3) By modifying the surface of Fe3O4@MOF(Fe-Cu) with mercapto groups, and then loading gold nanostars onto the surface of the composite material, Fe3O4@MOF(Fe-Cu)-GNS composite material was prepared.

[0011] (4) Fe3O4@MOF(Fe-Cu)-GNS-MBA composite material was prepared by mixing and incubating Fe3O4@MOF(Fe-Cu)-GNS with 4-mercaptophenylboronic acid (4-MBA);

[0012] (5) By co-incubating Fe3O4@MOF(Fe-Cu)-GNS-MBA with Vibrio parahaemolyticus aptamer, Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt composite material, namely multifunctional magnetic composite nanomaterial, was obtained.

[0013] In one embodiment of the present invention, in step (1), sodium citrate dihydrate and ferric chloride hexahydrate are dispersed in ethylene glycol, followed by the addition of anhydrous sodium acetate, mixed well, and then placed in a high-pressure reactor for heating and reaction. After the reaction is completed, the mixture is cooled, the solid is separated and collected, washed, and dried to obtain Fe3O4 nanomaterials.

[0014] In one embodiment of the present invention, in step (1), the solvothermal condition is a reaction at 200°C for 10 hours.

[0015] In one embodiment of the present invention, in step (1), the mass ratio of ferric chloride hexahydrate, sodium citrate dihydrate, and anhydrous sodium acetate is 1:(0.1-0.5):(1-2).

[0016] In one embodiment of the present invention, in step (1), the concentration of ferric chloride hexahydrate relative to ethylene glycol is 0.01-0.05 g / mL; specifically, 0.03 g / mL may be selected.

[0017] In one embodiment of the present invention, in step (2), FeCl3·6H2O and Cu(NO3)2·3H2O are dispersed in an organic solvent, H2PyC is added, and after dissolution and mixing, the Fe3O4 nanomaterials obtained in step (1) are added, mixed, and transferred to a high-temperature reactor for heating and reaction. After the reaction is completed, the mixture is cooled, the solid is separated and collected, washed, and dried to obtain the Fe3O4@MOF(Fe-Cu)-GNS composite material.

[0018] In one embodiment of the present invention, in step (2), the heating reaction is carried out at 100°C for 12 hours.

[0019] In one embodiment of the present invention, in step (2), the mass ratio of FeCl3·6H2O, Cu(NO3)2·3H2O, and H2PyC is (0.5-1):(2-3):1.

[0020] In one embodiment of the present invention, in step (2), the concentration of H2PyC relative to the organic solvent is 5-8 mg / mL.

[0021] In one embodiment of the present invention, in step (2), the organic solvent may be N,N-dimethylformamide (DMF).

[0022] In one embodiment of the present invention, in step (3), gold nanostars are prepared by the following method:

[0023] Sodium citrate solution and chloroauric acid solution were mixed and heated, then cooled to obtain a gold seed solution. HCl solution and HAuCl solution were mixed, and the gold seed solution was added while stirring. At the same time, AgNO3 solution and ascorbic acid solution were quickly added and stirred until homogeneous to obtain a gold nanostar solution.

[0024] In one embodiment of the present invention, step (3) specifically includes: dissolving Fe3O4@MOF(Fe-Cu) nanomaterials in toluene, then mixing them with a methanol solution of ethylene dithiol, stirring and reacting to obtain thiol-modified Fe3O4@MOF(Fe-Cu) material; then dispersing the thiol-modified Fe3O4@MOF(Fe-Cu) material in pure water, adding gold nanostar solution, stirring and reacting to obtain Fe3O4@MOF(Fe-Cu)-GNS.

[0025] In one embodiment of the present invention, in step (4), Fe3O4@MOF(Fe-Cu)-GNS is dispersed in PBS solution to prepare a 1 mg / mL dispersion, and then 1 mg / mL 4-MBA ethanol solution is added. After mixing and incubation, the solid is separated, collected, washed, and dried. The volume ratio of the dispersion to the 4-MBA ethanol solution is 10:1.

[0026] In one embodiment of the present invention, in step (5), Fe3O4@MOF(Fe-Cu)-GNS-MBA is dispersed in PBS solution to prepare a 1 mg / mL material dispersion; the Vibrio parahaemolyticus aptamer is dispersed in PBS solution to prepare a 200 nM aptamer dispersion; then the ligand dispersion and TCEP solution are mixed and activated at room temperature, and then the material dispersion is added and incubated for a period of time. After incubation, the solid is collected, washed, and dried. The concentration of the TCEP solution is 10 mM; the volume ratio of the ligand dispersion, TCEP solution, and material dispersion is 1:0.1:1.

[0027] Based on the above method, this invention provides a multifunctional composite magnetic nanomaterial for detecting and killing Vibrio parahaemolyticus.

[0028] The multifunctional magnetic composite nanomaterial Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt exhibits the following five characteristics: ① superparamagnetic properties; ② nanozyme activity; ③ SERS performance; ④ photothermal conversion performance; ⑤ specific recognition performance.

[0029] In the multifunctional magnetic composite nanomaterial of the present invention,

[0030] (1) Bimetallic MOF nanomaterials doped with transition metals have excellent nanozyme activity because they combine the highly ordered porosity of MOF materials with atomically dispersed bimetallic nodes.

[0031] (2) Gold nanostars are highly anisotropic gold nanoparticles composed of a central core and many tips. Due to their excellent local surface plasmon resonance (LSPR), gold nanostars have broad prospects in the construction of SERS or fluorescent biosensors. In addition, due to their excellent photothermal properties, gold nanostars have strong absorption in the near-infrared region, which can convert light energy into heat energy, thereby achieving the effect of heat absorption and sterilization. Combining sensitive detection with efficient sterilization is the most valuable way to control the source of foodborne pathogens and avoid secondary pollution.

[0032] (3) Magnetic nanomaterials are easy to control under an external magnetic field, have a large surface area, and are easy to modify, which gives them a significant advantage in enriching trace bacteria in food matrices.

[0033] (4) In addition, nucleic acid aptamers, as biorecognition elements, have been combined with signals such as surface-enhanced Raman scattering (SERS) and colorimetry to construct specific, sensitive and rapid aptamer sensing methods, which are widely used in food contaminant detection and other fields.

[0034] Based on the above, this invention combines nanozymes, SERS, photothermal sterilization, magnetic separation, and aptamers to prepare a multifunctional composite magnetic nanomaterial that can be used for dual-mode detection and photothermal killing of Vibrio parahaemolyticus, thereby developing a highly specific, accurate, rapid, and safe detection and killing method for pathogenic bacteria.

[0035] This invention also provides a method for specific detection of Vibrio parahaemolyticus using colorimetric-SERS dual-mode detection and photothermal sterilization, and its application, mainly comprising the following steps:

[0036] 1) The prepared multifunctional magnetic composite nanomaterial was co-incubated with Vibrio parahaemolyticus to specifically capture bacteria from food matrix;

[0037] 2) Magnetophoresis chromatography was used to separate the bacteria-material complex and then used it for subsequent detection.

[0038] 3) For the isolated bacteria-material complex, the nanoenzyme colorimetric signal and SERS signal of the composite magnetic nanomaterial were used to quantitatively detect Vibrio parahaemolyticus and prepare a standard curve;

[0039] 4) For Vibrio parahaemolyticus in the test solution, the photothermal properties of magnetic composite nanomaterials are used to kill the bacteria by photothermal action, thus completing the hazard control at the back end of the test.

[0040] 5) The detection and sterilization methods studied above were applied to seafood samples to conduct a practical performance study on Vibrio parahaemolyticus.

[0041] This invention also provides a method for dual-mode detection and photothermal sterilization of Vibrio parahaemolyticus using colorimetric-SERS technology, based on multifunctional magnetic composite nanomaterials combined with magnetophoretic chromatography detection technology, including the following steps:

[0042] (a) The above-mentioned multifunctional magnetic composite nanomaterials were added to a series of target Vibrio parahaemolyticus solutions of known concentrations, incubated, separated by magnetic chromatographic chromatography, and detected by colorimetric and SERS dual-mode to obtain the corresponding UV absorbance and SERS intensity; the concentration of target Vibrio parahaemolyticus was linearly correlated with UV absorbance and SERS intensity to obtain the corresponding standard curves.

[0043] (b) Add multifunctional magnetic composite nanomaterials to the sample solution to be tested, incubate, separate by magnetic chromatographic chromatography, use colorimetric and SERS dual-mode detection, measure and calculate the ultraviolet absorbance and SERS intensity, substitute into the standard curve, and obtain the concentration of the target Vibrio parahaemolyticus.

[0044] (c) After the colorimetric and SERS dual-mode detection is completed, near-infrared light is used to photothermally kill Vibrio parahaemolyticus in the test solution in order to complete the hazard control at the back end of the detection.

[0045] In one embodiment of the present invention, in step (a) or (b), the amount of multifunctional magnetic composite nanomaterial added relative to the target Vibrio parahaemolyticus solution or the sample solution to be tested is 150 μg / mL.

[0046] In one embodiment of the present invention, in step (a) or (b), after incubation, the material is separated from the mixture by magnetic separation and then dispersed in PBS for magnetic chromatographic separation.

[0047] In one embodiment of the present invention, in step (a) or (b), the conditions for magnetic chromatographic separation are: using a 25 wt% PEG aqueous solution as the separation channel.

[0048] In one embodiment of the present invention, in step (a) or (b), colorimetric detection acquires the ultraviolet absorbance value at 652 nm; SERS detection acquires the absorbance value at 1068 cm⁻¹. -1 SERS intensity at the location.

[0049] [Beneficial Effects]

[0050] This invention targets Vibrio parahaemolyticus and prepares a multifunctional magnetic composite nanomaterial through layer-by-layer assembly. First, the superparamagnetic separation properties of this magnetic composite nanomaterial, combined with magnetophoretic chromatography, enable rapid separation of bacteria from complex food matrices. Second, the colorimetric and SERS sensing signals of this magnetic composite nanomaterial allow for short-time, high-precision, and high-stability dual-mode quantitative detection of bacteria. Finally, the photothermal properties of this magnetic composite nanomaterial enable photothermal killing of detected bacteria, thereby achieving hazard control at the back end of the detection process.

[0051] Existing detection technologies for Vibrio parahaemolyticus are mostly single-mode detection, and there are few developments for dual-mode quantitative detection methods. This invention selects to utilize colorimetric and SERS dual-mode signals and uses the prepared multifunctional magnetic composite nanomaterials to quantitatively detect bacteria.

[0052] Most existing studies on pathogen detection technologies do not consider the control of pathogen hazards at the detection end. This invention chooses to use near-infrared light to irradiate the detection solution and utilizes the prepared multifunctional magnetic composite nanomaterials to perform photothermal killing of bacteria, thereby completing the hazard control at the detection end. Attached Figure Description

[0053] Figure 1The following are characterization data for various materials in Example 2: TEM images of Fe3O4 (A), Fe3O4@MOF(Fe-Cu) (B), Fe3O4@MOF(Fe-Cu)-GNS (C), and gold nanostars (D); Fourier transform infrared spectrum (E) of Fe3O4@MOF(Fe-Cu); powder X-ray diffraction pattern (F) of Fe3O4@MOF(Fe-Cu)-GNS; Zeta potential diagrams (G) of Fe3O4 nanoparticles, Fe3O4@MOF, and Fe3O4@MOF-GNS; XPS spectrum (H) of Fe3O4@MOF-GNS; Zeta potential diagrams (I) of FMG, FMG-MBA, and FMG-MBA-Apt; and UV-Vis absorption spectra before and after aptamer modification (J).

[0054] Figure 2 The results of magnetic property tests on various materials in Example 3 include: superparamagnetic test images (A) of Fe3O4, Fe3O4@MOF(Fe-Cu) and Fe3O4@MOF(Fe-Cu)-GNS; and photographs (B) showing the rapid response of Fe3O4, Fe3O4@MOF and Fe3O4@MOF-GNS within 30 seconds under an external magnetic field.

[0055] Figure 3 The image shows a comparison of optimized colorimetric conditions for the composite material and physical mixing in Example 3, including: different pH conditions (A), different temperature conditions (B), and different compounding methods (C).

[0056] Figure 4 The optimized SERS signals of the composite materials obtained at different 4-MBA concentrations in Example 3 are shown below; including: a comparison chart of SERS signals at different 4-MBA concentrations (A); 1 mg·mL -1 SERS signals (B) of Fe3O4@MOF(Fe-Cu)-GNS-MBA and pure MBA obtained by 4-MBA; 1 mg·mL -1 Comparison of SERS intensities of Fe3O4@MOF(Fe-Cu)-GNS-MBA obtained by 4-MBA over five usage cycles (C).

[0057] Figure 5 The results of the photothermal performance of the composite material in Example 3 are shown in the figure; including: temperature change of Fe3O4@MOF(Fe-Cu)-GNS during 5 minutes of exposure time (A); temperature change of material suspension and pure water (control) over time (B); and photothermal conversion capacity change of Fe3O4@MOF(Fe-Cu)-GNS over five use cycles (C).

[0058] Figure 6To optimize the capture efficiency of the composite material in Example 3, the morphological images of Fe3O4@MOF-GNS-MBA-Apt and Vibrio parahaemolyticus binding are shown in (A); and the capture efficiency of Vibrio parahaemolyticus is compared with different amounts of material added (B).

[0059] Figure 7 This is a comparison chart showing the optimized PEG concentration separation by magnetophoresis in Example 4.

[0060] Figure 8 The results of the isolation and detection of Vibrio parahaemolyticus in Example 4 include: optical images (A) of the pipette tip after magnetic chromatographic separation of Vibrio parahaemolyticus containing different concentrations of the bacteria; changes in ultraviolet absorbance intensity (B) for different concentrations of Vibrio parahaemolyticus; changes in SERS intensity (D) for different concentrations of Vibrio parahaemolyticus; and standard curves of Vibrio parahaemolyticus concentration versus ultraviolet absorbance intensity (C) and SERS intensity (E).

[0061] Figure 9 This is a comparison chart showing the control of Vibrio parahaemolyticus hazard in the test solution using the photothermal properties of the material in Example 5.

[0062] Figure 10 The image shows the colony formation of the spiked sample solution in Example 5 after photothermal treatment for 4 minutes.

[0063] Figure 11 This is a schematic diagram illustrating the preparation and detection application of the multifunctional magnetic composite nanomaterial of this invention. (a) shows the preparation process of the multifunctional magnetic composite nanomaterial; (b) is a schematic diagram of the dual-mode detection and photothermal sterilization process for Vibrio parahaemolyticus. Detailed Implementation

[0064] Example 1: Preparation process of multifunctional magnetic composite nanomaterials

[0065] 1. Preparation of Fe3O4 nanomaterials

[0066] The Fe3O4 nanomaterials with carboxyl modification were prepared by a one-step hot solvent method: 0.664 g of sodium citrate dihydrate and 2.4 g of ferric chloride hexahydrate were weighed and dissolved in 80 mL of ethylene glycol by sonication. Then, 3.84 g of anhydrous sodium acetate was added and the mixture was magnetically stirred for 1 h. The resulting dark-colored solution was placed in a high-pressure reactor and reacted at 200 °C for 10 h. After the reaction was completed, the mixture was cooled to room temperature. The product was washed several times with pure water and anhydrous ethanol, and then dried at 60 °C to obtain carboxyl-modified Fe3O4 nanomaterials for later use.

[0067] 2. Preparation of Fe3O4@MOF (Fe-Cu) nanomaterials

[0068] The mixture was prepared by a solvothermal reaction: FeCl3·6H2O (35.5 mg) and Cu(NO3)2·3H2O (114.5 mg) were added to 10 mL of DMF, followed by the addition of H2PyC (54.0 mg). After dissolving the mixture by ultrasonication, 100 mg of carboxyl-modified Fe3O4 was added. The mixture was stirred until homogeneous and then transferred to a high-temperature reactor and kept at 100 °C for 12 h. After cooling to room temperature, the solid was collected by centrifugation, washed multiple times with water, and then vacuum dried at 60 °C for 12 h to obtain Fe3O4@MOF (Fe-Cu) nanomaterials for later use.

[0069] 3. Preparation of gold nanostar solution

[0070] Under vigorous stirring, sodium citrate aqueous solution (Na3C6H5O7·2H2O) (1wt%, 15mL) was added to chloroauric acid aqueous solution (HAuCl4) (1mM, 100mL) at 85℃ and heated for 15 minutes. The solution was then cooled to obtain a gold seed solution, which was stored at 4℃ protected from light. At room temperature, in a 150mL glass bottle, HCl solution (1M, 100mL) was first added to HAuCl4 aqueous solution (0.25mM, 100mL). With moderate stirring, 1mL of the sodium citrate-stabilized gold seed solution was added, and simultaneously, AgNO3 aqueous solution (1mM, 1mL) and ascorbic acid aqueous solution (100mM, 0.5mL) were rapidly added. After stirring for 1 minute, the gold nanostar solution (Au NS solution) was obtained. This gold nanostar solution was stored at 4℃ protected from light for a long period.

[0071] 4. Preparation of Fe3O4@MOF(Fe-Cu)-GNS nanomaterials

[0072] Weigh 0.1 g of Fe3O4@MOF(Fe-Cu) nanomaterials, dry at 120℃ for 12 h, add 10 mL of anhydrous toluene, sonicate to dissolve, then add 1 mL of ethylene dithiol in methanol (0.24 mol / L), stir at room temperature for 24 h, wash with pure water and anhydrous ethanol, and dry at 60℃ to obtain thiol-modified Fe3O4@MOF(Fe-Cu) material for later use. Weigh 50 mg of thiol-modified Fe3O4@MOF material, sonicate to dissolve in 10 mL of pure water, and stir vigorously with 50 mL of the above-prepared Au NS solution at room temperature for 3 h. After the reaction, wash several times with ethanol to remove unlinked Au NS. Finally, vacuum dry at 60℃ to obtain Fe3O4@MOF(Fe-Cu)-GNS nanomaterials for later use.

[0073] 5. Preparation of Fe3O4@MOF(Fe-Cu)-GNS-MBA nanomaterials

[0074] Take 1 mL of Fe3O4@MOF(Fe-Cu)-GNS PBS solution (1 mg / mL) and add 100 μL of 4-MBA ethanol solution (1 mg / mL), and mix at room temperature for 1 h. After the mixture is finished, wash away the unreacted 4-MBA with pure water and ethanol, and dry to obtain Fe3O4@MOF(Fe-Cu)-GNS-MBA nanomaterials.

[0075] 6. Preparation of multifunctional magnetic composite nanomaterials of Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt

[0076] 1 mL of PBS solution (200 nM) containing Vibrio parahaemolyticus aptamers was mixed with 0.1 mL of freshly prepared TCEP (10 mM) and activated at room temperature for 1 h. This mixture was then added to 1 mL of PBS solution (1 mg / mL) containing Fe3O4@MOF(Fe-Cu)-GNS-MBA and incubated overnight at 37 °C. Finally, the mixture was washed three times with PBS buffer (10 mM, pH 7.4) to obtain the multifunctional magnetic composite nanomaterial Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt. The obtained multifunctional magnetic composite nanomaterial can be reconstituted in PBS buffer and stored in a refrigerator for later use.

[0077] The Vibrio parahaemolyticus aptamer sequence 5'-SH-TTTTTTTTTCAACGAAACAGTGACTCGTTG-3' was synthesized by Shanghai Sangon Biotech Co., Ltd.

[0078] Example 2 Characterization of Multifunctional Magnetic Composite Nanomaterials

[0079] 1. Characterization of Fe3O4, Fe3O4@MOF(Fe-Cu) and Fe3O4@MOF(Fe-Cu)-GNS

[0080] according to Figure 1 The synthesized Fe3O4 nanoparticles exhibited good uniformity and monodispersity, with an average particle size of approximately 281.08 nm. Subsequently, MOF (Fe-Cu) was grown in situ on the Fe3O4 nanoparticles, resulting in Fe3O4@MOF(Fe-Cu) with a relatively clear core-shell structure and an average particle size increase to approximately 301.84 nm, indicating that MOF(Fe-Cu) was successfully coated on the surface of the Fe3O4 nanoparticles. Then, gold nanostars prepared via a two-step method were attached to the Fe3O4@MOF(Fe-Cu) surface, preparing Fe3O4@MOF-GNS through gold-sulfur chemical bonding. TEM images showed that high-density gold nanostars were uniformly distributed on the Fe3O4@MOF surface, indicating successful gold nanostar loading.

[0081] The core-shell structure of Fe3O4@MOF(Fe-Cu) was further verified by measuring Fourier transform infrared spectroscopy. Figure 1 As shown, 625~582cm- 1 The peak at 997 cm⁻¹ is the stretching vibration peak of Fe-O. -1 The area at 1630, 1440, 1330 and 800 cm⁻¹ is a characteristic band of N-Cu-N vibration. -1 The tensile vibrations corresponding to C=O, CC, CN, and CH are shown at the corresponding positions. Characterization results indicate that the Fe3O4 surface synthesized via a high-temperature hydrothermal method contains a large number of carboxyl groups; comparison of Fe3O4 and MOF (Fe-Cu) in the figure proves that the bimetallic Fe-Cu MOF was successfully grown on the Fe3O4 surface. After functionalizing the core-shell material with ethylenedithiol, although at 2581 cm⁻¹... -1 No obvious SH vibration peak was observed, but at 682 cm⁻¹... -1 A small, sharp new peak appeared, corresponding to the stretching vibration peak of CS. This result proves that thiol groups have been successfully modified on the surface of the core-shell structure.

[0082] Powder X-ray diffraction analysis further verified the successful loading of gold nanostars onto the surface of the Fe3O4@MOF core-shell structure. Figure 1 As shown, the diffraction peaks of Fe3O4 correspond to 2θ values ​​of 30.06°, 35.54°, 43.22°, 52.68°, 57.28°, and 62.58°; the diffraction peak of gold nanostars has a 2θ value of 38.08°. Furthermore, the XPS spectra of Fe3O4@MOF-GNS confirmed the presence of Fe, Cu, Au, C, and O elements in the prepared composite material, demonstrating the successful loading of the bimetallic MOF (Fe-Cu) and gold nanostars onto the Fe3O4 surface.

[0083] Furthermore, the zeta potential changes of Fe3O4 nanoparticles, Fe3O4@MOF, and Fe3O4@MOF-GNS verified the sequential modification of the MOF shell and gold nanostars on the surface of Fe3O4 nanoparticles. The synthesized carboxylated Fe3O4 endowed the magnetic particles with a negative surface charge (zeta potential: -4.62 mV). In-situ growth of a bimetallic Fe-Cu MOF onto the carboxylated Fe3O4 surface endowed the core-shell structure with a positive surface charge (zeta potential: 2.86 mV). Subsequently, the interaction between the core-shell structure and the negatively charged gold nanostars resulted in a negative surface charge on the composite structure (zeta potential: -14.03 mV). These results collectively demonstrate the successful preparation of the designed Fe3O4@MOF(Fe-Cu)-GNS.

[0084] 2. Characterization of Fe3O4@MOF(Fe-Cu)-GNS-MBA and Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt nanomaterials

[0085] To further determine the changes in the charge properties of the nanoparticles after each modification step, the connection between Fe3O4@MOF(Fe-Cu)-GNS (hereinafter referred to as FMG) and the aptamer was characterized using UV-Vis absorption spectroscopy and Zeta potential detection instruments. The results are shown in the figure. The average potential of FMG (Fe3O4@MOF(Fe-Cu)-GNS) was -14.03 mV. Subsequently, the average potential of FMG-MBA (Fe3O4@MOF(Fe-Cu)-GNS-MBA) was -21.3 mV, showing a certain degree of decrease. Finally, after modification with the Vibrio parahaemolyticus aptamer, the resulting composite material FMG-MBA-Apt (Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt) had an average potential of -30.1 mV, exhibiting significant electronegativity. This is because the surface of nucleic acid aptamers contains abundant negatively charged phosphate groups. After modification, a large number of aptamers bind to the surface of FMG, resulting in a more pronounced electronegativity on the FMG-MBA surface. This further proves that the Vibrio parahaemolyticus aptamer was successfully modified onto FMG. Furthermore, since the aptamer has a characteristic UV absorption peak at 260 nm, the connection between the aptamer and the FMG-MBA material can be characterized by monitoring changes in the absorbance at 260 nm in the reaction supernatant. Figure 1 As shown, compared to the aptamer solution of the same concentration, the absorbance at 260 nm in the reaction supernatant decreased significantly. These results all indicate that the signal molecules and aptamers were successfully loaded onto the surface of the composite material.

[0086] Example 3: Performance exploration and preparation condition optimization of multifunctional magnetic composite nanomaterials.

[0087] 3.1 Investigation of Magnetic Properties

[0088] The superparamagnetism of Fe3O4, Fe3O4@MOF(Fe-Cu), and Fe3O4@MOF(Fe-Cu)-GNS was analyzed by vibrating sample magnetometry under an applied magnetic field. Figure 2 As shown, the saturation magnetizations of the prepared Fe3O4 nanoparticles, Fe3O4@MOF(Fe-Cu), and Fe3O4@MOF(Fe-Cu)-GNS are 64.40, 55.95, and 47.55 emu·g, respectively. -1Compared to Fe3O4 nanoparticles, Fe3O4@MOF and Fe3O4@MOF-GNS exhibit reduced saturation magnetization, likely due to the magnetic shielding effect of the MOF shell and gold nanostars coated on the surface of the Fe3O4 nanoparticles. Nevertheless, similar to Fe3O4 nanoparticles, Fe3O4@MOF and Fe3O4@MOF-GNS demonstrate a rapid response within 30 seconds under an external magnetic field, ensuring rapid and efficient magnetic separation and enrichment of target analytes from complex samples.

[0089] 3.2 Activity exploration and optimization of preparation conditions of Fe3O4@MOF(Fe-Cu)-GNS nanozymes

[0090] In this embodiment, the peroxidase-like activity of Fe3O4@MOF(Fe-Cu)-GNS particles was investigated by catalyzing the oxidation of TMB with H2O2. The enzyme-like activity of oxTMB could be characterized by detecting its UV absorption peak at 652 nm.

[0091] The colorimetric reaction system (1 mL) consisted of: HAc-NaAc solution (0.1 M, pH 4.5, 850 μL), H2O2 (1 M, 50 μL), and TMB (10 mM, 50 μL), with the composite material added at a concentration of 50 mg (1 mg / mL, 50 μL).

[0092] (1) The effects of pH and temperature on the enzyme activity of Fe3O4@MOF(Fe-Cu)-GNS were compared.

[0093] The optimal efficiency of Fe3O4@MOF(Fe-Cu)-GNS in HAc-NaAc buffer solutions with pH values ​​ranging from 4 to 6 was specifically investigated. The material exhibited the strongest enzyme activity at pH 4.5; however, its peroxidase-like activity decreased sharply above pH 4.5. These results indicate that pH 4.5 is the optimal pH value. To determine the optimal temperature for peroxidase-like activity, the enzyme activity was evaluated within a range of 20°C to 50°C. The results showed that enzyme activity decreased above 40°C, thus 40°C was selected as the optimal temperature for further analysis.

[0094] (2) Comparison of composite methods

[0095] In the same colorimetric reaction system (TMB+H2O2), a certain mass of the composite material (Fe3O4@MOF(Fe-Cu)-GNS) (100mg) and a physical mixture of materials with equal amounts of each substance in the composite material (83.2mg Fe3O4+6.3mg MOF(Fe-Cu)+10.5mg gold nanostars) were added as comparisons. The enzyme-like activity was compared by using the signal intensity of the hydroxyl radicals generated by catalyzing H2O2 to catalyze the conversion of TMB (colorless) to oxTMB (blue).

[0096] The results are as follows Figure 3 As shown, both colorimetric reaction systems in the two experimental groups exhibited distinct absorption peaks, but the highest absorption peak appeared in the composite material Fe3O4@MOF(Fe-Cu)-GNS system. The results indicate that the composite material (Fe3O4@MOF(Fe-Cu)-GNS) prepared in Example 1 possesses excellent peroxidase-like catalytic activity, significantly superior to simply physically mixing Fe3O4, MOF-919 (Fe-Cu), and gold nanostars. This suggests a synergistic effect between the composite structures, further enhancing the overall enzyme-like activity of the composite material.

[0097] 3.3 SERS performance investigation and preparation condition optimization

[0098] To better functionalize the material with signal molecules and aptamer probes for highly sensitive detection of Vibrio parahaemolyticus, this invention optimized the concentration of 4-MBA linked to the material. Therefore, different concentrations of 4-MBA (0.001, 0.01, 0.1, 1, 10 mg·mL⁻¹) were linked via Au-S bonds. -1 The 4-MBA concentration was modified onto the surface of the material, and its Raman signal intensity was measured to evaluate the Raman enhancement effect on the material. Results are as follows: Figure 4 As shown, the Raman signal intensity of Fe3O4@MOF(Fe-Cu)-GNS-MBA gradually increased with increasing 4-MBA concentration, and reached a maximum at a 4-MBA concentration of 1 mg·mL⁻¹. -1 It reaches its peak value at a certain time, and then gradually decreases. This indicates that 1 mg·mL -1 The optimal incubation concentration for 4-MBA is when the concentration exceeds 1 mg / mL. -1 The subsequent decrease in Raman signal intensity is likely due to the aggregation of the material caused by high concentrations of 4-MBA, which reduces the Raman signal intensity and affects the detection effect. Therefore, the concentration of 1 mg·mL⁻¹ was determined to be lower than that of 4-MBA. -1The optimal concentration of 4-MBA on Fe3O4@MOF(Fe-Cu)-GNS was determined. Furthermore, by investigating the SERS stability of the material, this embodiment found that the material maintained excellent Raman signal intensity over five usage cycles.

[0099] 3.4 Investigation of photothermal properties and optimization of preparation conditions

[0100] The photothermal inactivation ability of Fe3O4@MOF(Fe-Cu)-GNS to inactivate bacteria was investigated. Since the Au NS prepared in Example 1 has a broad LSPR absorption peak around 675 nm, a 0.25 cm⁻¹ [glucose concentration] was used. 2 Red light spot (660nm, 2.0W / cm²) 2 Irradiation was performed, and the temperature changes of the material suspension and pure water (control) over time were recorded.

[0101] from Figure 5 As can be seen, Fe3O4@MOF-GNS significantly increased the temperature from 20.8℃ to 65.1℃ within a 5-minute exposure period, while pure water, as a control, only increased the temperature by 0.4℃. These results indicate that Fe3O4@MOF(Fe-Cu)-GNS possesses excellent photothermal conversion capabilities. Furthermore, by investigating the photothermal stability of the material, it was found that its photothermal performance maintained excellent photothermal conversion capabilities even after five usage cycles. In summary, the Fe3O4@MOF(Fe-Cu)-GNS material prepared in this invention exhibits good photothermal properties and can be used for convenient sterilization of bacteria in the test solution after quantitative detection, thereby improving the safety of single-use material analysis, and is particularly suitable for the detection of pathogenic bacteria.

[0102] 3.5 Investigation of capture performance and optimization of preparation conditions

[0103] like Figure 6 The morphological images of Fe3O4@MOF-GNS-MBA-Apt and Vibrio parahaemolyticus binding were confirmed by scanning electron microscopy. The prepared composite material can accurately target and tightly adhere to the surface of Vibrio parahaemolyticus through the specific action of the aptamer. To obtain a good bacterial capture effect, the capture conditions were optimized by adjusting the dosage range of Fe3O4@MOF-GNS-MBA-Apt. To determine the appropriate amount of material to be added, Vibrio parahaemolyticus (10 5 cfu·mL -1 ) was used as the upper limit for the capture model. In PBS (10 mM, pH 7.4), the concentration was determined by adding 1 mL of Vibrio parahaemolyticus solution (10 mM). 5 cfu·mL -1Different amounts of Fe3O4@MOF-GNS-MBA-Apt were added to achieve specific capture of bacteria, followed by incubation at 37°C for 60 minutes, and then separation of the material-bacteria complex using a magnetic rack. Figure 6 It was found that when the dosage of Fe3O4@MOF-GNS-MBA-Apt was continuously increased to 150 μg, the capture efficiency significantly improved, reaching 97.8%. However, when the dosage exceeded 150 μg, the capture efficiency did not improve significantly. These results indicate that 150 μg of material can be considered a suitable dosage for further analysis. These results fully demonstrate that the aptamer-modified material exhibits excellent specific capture performance against Vibrio parahaemolyticus and can be used for further detection and analysis.

[0104] Example 4: A method for detecting Vibrio parahaemolyticus content in shrimp samples using magnetophoresis chromatography combined with multifunctional magnetic composite nanomaterials.

[0105] 4.1 Optimization of PEG concentration for separating free material and bacteria-material complexes in magnetic phoresis chromatography

[0106] The multifunctional material (Fe3O4@MOF-GNS-MBA-Apt, 150 μg) was added to a solution containing Vibrio parahaemolyticus (10 μg). 5 cfu·mL -1 After incubation in 1 mL of PBS for 60 minutes, the material was separated from the mixture by magnetic separation and then dispersed in 50 μL of PBS for magnetic phoresis separation. Before magnetic phoresis separation, the liquid at the tip of the pipette contained both free material and bacterial-material complexes.

[0107] First, this embodiment examines the magnetophoretic separation effect of free materials and bacterial-material complexes under conditions where no magnetic field is applied and separation is achieved solely through the gravitational force of the bacteria. Figure 7 The first column shows that, without a magnetic field, the free material and the bacteria-material complex showed slight sedimentation in the 15wt% PEG aqueous solution channel within 10 minutes, but gravity alone could not break through the 20wt% PEG aqueous solution channel within 10 minutes. Therefore, gravity could not separate the free material and the bacteria-material complex in the 20wt% PEG aqueous solution channel. Next, the magnetophoretic separation effect under a magnetic field was investigated for materials containing only free material. Figure 7As shown in the second column, under the influence of a magnetic field, free material can overcome the viscous resistance in the 15wt% and 20wt% PEG channels and undergo particle sedimentation within 10 minutes through magnetic force, but it cannot overcome the 25wt% PEG channel within 10 minutes. Therefore, free material cannot be separated by magnetic force in the 25wt% PEG channel. Based on the above investigation of individual forces (bacterial gravity and magnetic force) in magnetophoretic chromatography, this embodiment further investigates the magnetophoretic separation effect of free material and bacterial-material complex under the influence of a magnetic field to achieve the best separation effect. Figure 7 As shown in the third column, under the combined forces of the magnetic field and bacterial gravity, the bacterial-material complex can overcome the viscous resistance in the 15-25 wt% PEG channel and undergo particle sedimentation within 10 minutes, and can also weakly pass through the 30 wt% PEG channel. Therefore, in the 25 wt% PEG channel, the free material and the bacterial-material complex can be separated by the combined forces of magnetophoresis and gravity. After separation by magnetophoresis, the bacterial-material complex is collected at the bottom of the PEG channel, while the free material remains in the PEG channel and cannot reach the bottom. Considering the accuracy of signal collection, the optimal PEG concentration in magnetophoresis was determined to be 25 wt%.

[0108] 4.2 Constructing a colorimetric-SERS dual-mode analytical method for the detection of Vibrio parahaemolyticus based on the colorimetric and SERS properties of multifunctional magnetic composite nanomaterials

[0109] Composite materials were used to incubate and capture Vibrio parahaemolyticus bacterial suspensions containing different concentrations. After magnetophoresis, the precipitate particles at the bottom of the pipette were collected and dispersed in PBS (100 μL). The suspension (50 μL) was transferred to a pre-prepared colorimetric reaction system: HAc-NaAc solution (0.1 M, pH 4.5, 850 μL); H2O2 (1 M, 50 μL); TMB (10 mM, 50 μL). The reaction mixture was incubated in the dark at 40 °C for 15 minutes, and its absorbance at 652 nm was measured. The remaining suspension (50 μL) was dropped onto a clean glass slide, and the SERS signal was scanned using a confocal Raman spectrometer under excitation at 785 nm.

[0110] Colorimetric method for detecting Vibrio parahaemolyticus. For example... Figure 8 As shown, the solution color gradually deepens to a dark blue as the concentration of Vibrio parahaemolyticus increases. The concentration of Vibrio parahaemolyticus in each solution was quantitatively determined by measuring the ultraviolet absorbance at 652 nm. The absorption peak intensity of the solution increased with increasing bacterial concentration. A good linear relationship was found between the absorbance of the colorimetric detection system and the logarithm of the Vibrio parahaemolyticus concentration, with a linear regression equation of y = 0.303x - 0.101(R²). 2 =0.997). In 10 1-10 5 cfu·mL -1 Within the specified concentration range, the detection limit for Vibrio parahaemolyticus is 9 cfu·mL⁻¹. -1 .

[0111] SERS detection of Vibrio parahaemolyticus. For example... Figure 8 As shown, the SERS signal intensity in the system also increased with increasing Vibrio parahaemolyticus concentration. 4-MBA was selected at 1068 cm⁻¹. -1 The SERS intensity at the site was quantitatively detected. A good linear relationship was found between the relative SERS intensity of the SERS detection system and the logarithm of the Vibrio parahaemolyticus concentration, with a linear regression equation of y = 118.6x - 96.27 (R²). 2 =0.9893). In 10 1 -10 5 cfu·mL -1 Within the specified concentration range, the detection limit for Vibrio parahaemolyticus is 7 cfu·mL⁻¹. -1 .

[0112] To test the analytical performance of the sensor, the content of Vibrio parahaemolyticus in artificially contaminated fresh shrimp samples was simultaneously detected using both the traditional plate counting method and the proposed dual-mode detection method.

[0113] The results are summarized in Table 1. There was no significant difference between the results obtained by the two methods. The recoveries of Vibrio parahaemolyticus in fresh shrimp samples by colorimetric and SERS methods were 94.6%–99.1% and 89.3%–96.7%, respectively. The results showed good consistency with the spiked concentration, proving that the colorimetric-SERS aptamer sensor developed in this paper can be used for the determination of Vibrio parahaemolyticus in complex food samples.

[0114] Table 1 shows the dual-mode detection of spiked shrimp samples developed using this invention.

[0115]

[0116] Example 5: A method for photothermal killing of Vibrio parahaemolyticus in shrimp sample test solution using multifunctional magnetic composite nanomaterials.

[0117] Under NIR light source illumination, the detection solution inside the well was recorded using a photothermal imager (to ensure its sterilization effect, 10 μL was selected in this embodiment). 5 cfu mL -1 The detection solution was used as the upper limit of detection to explore the optimal sterilization time (temperature changes over time). Figure 9It can be seen that with increasing illumination time, the material targeting *Vibrio parahaemolyticus* in the test solution exhibits excellent photothermal effect, causing the overall temperature of the test solution to rise sharply from 20.5℃ to 64.7℃ within 5 minutes. The photothermal sterilization effect was directly observed by applying the light-treated test solution directly to a plate. Figure 9 As can be seen, the control group plates without light treatment had a large number of dense colonies; while the colony counts on plates treated with photothermal therapy for different durations were all lower than those in the control group, showing a rapid decline. It is noteworthy that most microorganisms can be inactivated by high temperatures above 50°C. Because the material surface was modified with an aptamer specifically targeting Vibrio parahaemolyticus, the bacteria-material complex was tightly bound, resulting in effective localized high-temperature sterilization after near-infrared light irradiation. Figure 9 It was observed that after 3 minutes of light treatment, only a very small number of colonies (18 CFU) grew on the agar plate, and the survival rate of Vibrio parahaemolyticus in the test solution was only 4.36%. After 4 minutes of light treatment, all the Vibrio parahaemolyticus captured in the test solution was killed. Therefore, it was determined that near-infrared light irradiation for 4 minutes should be used for sterilization in the back-end of the detection process.

[0118] In actual sample testing experiments, NIR irradiation is used immediately after quantitative detection for photothermal sterilization, further controlling pathogenic bacteria at the back end of the detection process. Figure 10 As can be seen, after NIR light irradiation for 4 minutes to sterilize the Vibrio parahaemolyticus in the three spiked samples detected by magnetophoresis, no colonies grew on the agar plates. These results fully demonstrate that 4 minutes of NIR light irradiation can completely kill Vibrio parahaemolyticus in the test solution, thus ensuring that secondary pollution to the environment will not occur due to the detection of pathogenic bacteria.

[0119] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for preparing a multifunctional magnetic composite nanomaterial for specifically recognizing, detecting, and killing Vibrio parahaemolyticus, characterized in that, Includes the following steps: (1) Fe3O4 nanomaterials with superparamagnetism were prepared by solvothermal method; (2) MOF-919 (Fe-Cu) was grown in situ on the surface of Fe3O4 by hydrothermal reaction to obtain Fe3O4@MOF(Fe-Cu) composite material; FeCl3·6H2O and Cu(NO3)2·3H2O were dispersed in an organic solvent, H2PyC was added, and after dissolution and mixing, the Fe3O4 nanomaterial obtained in step (1) was added, mixed, and transferred to a high-temperature reactor for heating and reaction. After the reaction was completed, the mixture was cooled, the solid was separated and collected, washed, and dried to obtain Fe3O4@MOF(Fe-Cu)-GNS composite material; the heating reaction conditions were 100°C for 12 h; the mass ratio of FeCl3·6H2O, Cu(NO3)2·3H2O and H2PyC was (0.5-1):(2-3):1; the concentration of H2PyC relative to the organic solvent was 5-8 mg / mL; (3) Fe3O4@MOF(Fe-Cu)-GNS composite material was prepared by modifying the surface of Fe3O4@MOF(Fe-Cu) with thiol groups and then loading gold nanostars onto the surface of the composite material. Specifically, the process included: dissolving Fe3O4@MOF(Fe-Cu) nanomaterials in toluene, then mixing them with a methanol solution of ethylene dithiol and stirring to obtain thiol-modified Fe3O4@MOF(Fe-Cu) material; then dispersing the thiol-modified Fe3O4@MOF(Fe-Cu) material in pure water, adding the gold nanostar solution, and stirring to obtain Fe3O4@MOF(Fe-Cu)-GNS; wherein the gold nanostar solution was prepared by the following method: Sodium citrate solution and chloroauric acid solution were mixed and heated, then cooled to obtain a gold seed solution; HCl solution and HAuCl solution were mixed, and the gold seed solution was added while stirring, while AgNO3 solution and ascorbic acid solution were quickly added and stirred until well mixed to obtain a gold nanostar solution. (4) Fe3O4@MOF(Fe-Cu)-GNS-MBA composite material was prepared by mixing and incubating Fe3O4@MOF(Fe-Cu)-GNS with 4-MBA; Fe3O4@MOF(Fe-Cu)-GNS was dispersed in PBS solution to prepare a 1 mg / mL dispersion, and then 1 mg / mL ethanol solution of 4-MBA was added. After mixing and incubation, the solid was separated, collected, washed, and dried; wherein the volume ratio of the dispersion to the ethanol solution of 4-MBA was 10:

1. (5) Fe3O4@MOF(Fe-Cu)-GNS-MBA-Apt composite material was prepared by mixing and incubating Fe3O4@MOF(Fe-Cu)-GNS-MBA with Vibrio parahaemolyticus aptamer.

2. The method according to claim 1, characterized in that, In step (1), sodium citrate dihydrate and ferric chloride hexahydrate are dispersed in ethylene glycol, followed by the addition of anhydrous sodium acetate, mixed thoroughly, and then placed in a high-pressure reactor for heating and reaction. After the reaction is completed, the mixture is cooled, the solid is separated and collected, washed, and dried to obtain Fe3O4 nanomaterials. The solvothermal conditions are 200℃ for 10 h. The mass ratio of ferric chloride hexahydrate, sodium citrate dihydrate, and anhydrous sodium acetate is 1:(0.1-0.5):(1-2). The concentration of ferric chloride hexahydrate relative to ethylene glycol is 0.01-0.05 g / mL.

3. A multifunctional magnetic composite nanomaterial for specifically recognizing, detecting, and killing Vibrio parahaemolyticus, prepared by the method described in claim 1 or 2.

4. A method for dual-mode detection and photothermal killing of Vibrio parahaemolyticus using colorimetric-SERS technology, characterized in that, Includes the following steps: (a) The multifunctional magnetic composite nanomaterial of claim 3 was added to a series of target Vibrio parahaemolyticus solutions of known concentration, incubated, separated by magnetic chromatographic chromatography, and detected by colorimetric and SERS dual-mode to obtain the corresponding ultraviolet absorbance and SERS intensity. The concentration of the target Vibrio parahaemolyticus was linearly correlated with the ultraviolet absorbance and SERS intensity to obtain the corresponding standard curves. (b) Add multifunctional magnetic composite nanomaterials to the sample solution to be tested, incubate, separate by magnetic chromatographic chromatography, use colorimetric and SERS dual-mode detection, measure and calculate the ultraviolet absorbance and SERS intensity, substitute into the standard curve, and obtain the concentration of the target Vibrio parahaemolyticus. (c) After the colorimetric and SERS dual-mode detection is completed, near-infrared light is used to photothermally kill Vibrio parahaemolyticus in the test solution to complete the hazard control at the back end of the detection.

5. The method according to claim 4, characterized in that, In step (a) or (b), the amount of multifunctional magnetic composite nanomaterial added relative to the target Vibrio parahaemolyticus solution or the sample solution to be tested is 150 μg / mL.

6. The method according to claim 4, characterized in that, In step (a) or (b), after incubation, the material is separated from the mixture by magnetic separation and then dispersed in PBS for magnetic chromatographic separation. The conditions for magnetic chromatographic separation are: using 25 wt% PEG solution as the separation channel.

7. The method according to any one of claims 4-6, characterized in that, In step (a) or (b), colorimetric detection acquires the ultraviolet absorbance at 652 nm; SERS detection acquires the absorbance at 1068 cm⁻¹. -1 SERS intensity at the location.

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