Preparation method, product and application of AgNS (at) (Mn-ZIF-8)-AuNPs assembly

Abstract

The invention relates to the field of SERS (Surface Enhanced Raman Scattering) detection, in particular to a preparation method, a product and application of an AgNS (at) (Mn-ZIF-8)-AuNPs assembly. The assembly is composed of an AgNS core, a ZIF-8 shell layer wrapped with metal elements and an AuNPs satellite growing on the surface of the shell layer, and is of a core-shell-satellite structure. The assembly is used for detecting OTA through SERS-LFIA, has good specificity and stability, can simply and rapidly detect OTA in grains in a trace amount, and has important reference significance for accurate analysis of mycotoxin in the field of food safety.

Description

Technical Field

[0001] This invention relates to the field of SERS detection, and in particular to a method for preparing AgNS@(Mn-ZIF-8)-AuNPs assemblies, products, and applications. Background Technology

[0002] Ochratoxin A (OTA), one of the most common mycotoxins contaminating feed and food, is widely distributed and highly toxic, posing a serious threat to human and animal health and lives. To achieve sensitive and rapid detection of OTA, it is necessary to further improve the sensitivity and accuracy of the SERS-LFIA method. The design and preparation of SERS probes are key to improving the detection performance of the SERS-LFIA method. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing AgNS@(Mn-ZIF-8)-AuNPs assemblies, as well as the products and applications thereof. The assemblies exhibit good specificity and stability when used for the detection of OTA using the SERS-LFIA method.

[0004] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is a method for preparing AgNS@(Mn-ZIF-8)-AuNPs assemblies, comprising the following steps: Step 1: Add an alkaline solution to the hydroxylamine solution and mix well. Then add a silver source solution, let it stand, and then add sodium citrate solution to react and obtain AgNS solution. Step 2: The precipitate obtained after centrifugation of the AgNS solution is dispersed in polyvinylpyrrolidone solution and stirred, then mixed with 4-ATP solution, allowed to stand, and the precipitate obtained after centrifugation is dispersed in anhydrous methanol to obtain AgNS@4-ATP methanol solution; Step 3: Add a mixed solution of Zn source and Mn source to the AgNS@4-ATP methanol solution, then add ligand solution and mix well to react and obtain AgNS@(Mn-ZIF-8) core-shell structure; disperse the AgNS@(Mn-ZIF-8) core-shell structure in water to obtain AgNS@(Mn-ZIF-8) solution; Step 4: Mix the AgNS@(Mn-ZIF-8) solution with the gold source solution, add sodium borohydride solution to react, and obtain the AgNS@(Mn-ZIF-8)-AuNPs assembly.

[0005] The second technical solution of the present invention is an AgNS@(Mn-ZIF-8)-AuNPs assembly prepared according to the above preparation method.

[0006] The third technical solution of the present invention is a method for preparing a SERS immune probe, which involves mixing an anti-OTA monoclonal antibody with an AgNS@(Mn-ZIF-8)-AuNPs assembly and a solvent, incubating the mixture, centrifuging it, adding BSA solution, and allowing it to stand to obtain the SERS immune probe.

[0007] The fourth technical solution of the present invention is a SERS immune probe prepared according to the above preparation method.

[0008] The fifth technical solution of the present invention is the application of the above-mentioned AgNS@(Mn-ZIF-8)-AuNPs assembly or the above-mentioned SERS immune probe in the detection of OTA by SERS-LFIA method.

[0009] Compared with the prior art, the present invention has the following beneficial effects: This invention prepares an AgNS@(Mn-ZIF-8)-AuNPs core-shell-satellite assembly with improved synergistic enhancement properties. This assembly was then used for the detection of OTA via SERS-LFIA. The application of this assembly with electromagnetic field-chemical synergistic enhancement properties to the SERS-LFIA detection of OTA demonstrates good specificity and stability, exhibiting excellent linearity within a detection range of 0.05 fg / mL to 1 μg / mL, with a detection limit as low as 0.021 fg / mL. This simple, rapid, and trace-level SERS-LFIA method for the detection of OTA in grains has significant reference value for the accurate analysis of mycotoxins in the field of food safety. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0011] Figure 1 This is a schematic diagram of the preparation process of AgNS@(X-ZIF-8)-AuNPs assemblies.

[0012] Figure 2 High-resolution TEM images and EDS elemental distribution maps of the core-shell structure are shown; (a) is AgNS@(Cu-ZIF-8); (b) is AgNS@(Cd-ZIF-8); (c) is AgNS@(Mn-ZIF-8). The distributions of Ag, Zn, Cu, Cd and Mn in the figures are marked in red, green, dark blue, light blue and yellow, respectively.

[0013] Figure 3The UV-Vis absorption spectra are for different element doping types and amounts; where (a) is AgNS@(Cu-ZIF-8); (b) is AgNS@(Cd-ZIF-8); and (c) is AgNS@(Mn-ZIF-8).

[0014] Figure 4 The bar chart shows the SERS spectral signal intensity of AgNS@(X-ZIF-8) under different elemental doping types and amounts. Based on the fact that the 4-ATP characteristic signal molecule is located at 1135 cm⁻¹, the signal intensity is determined. -1 Plot the signal intensity of the non-perfectly symmetric peak at the location (n=3).

[0015] Figure 5 High-resolution transmission electron microscope (TEM) images and EDS elemental distribution diagrams of the core-shell-satellite assembly are shown; where (a) represents AgNS@(Cu-ZIF-8)-AuNPs; (b) represents AgNS@(Cd-ZIF-8)-AuNPs; and (c) represents AgNS@(Mn-ZIF-8)-AuNPs.

[0016] Figure 6 The images show the UV-Vis absorption spectra before and after the growth of AuNPs; where (a) represents AgNS@(Cu-ZIF-8)-AuNPs; (b) represents AgNS@(Cd-ZIF-8); and (c) represents AgNS@(Mn-ZIF-8).

[0017] Figure 7 The SERS spectra of AgNS@(X-ZIF-8)-AuNPs with different metal element doping types are shown.

[0018] Figure 8 The diagram shows the calculation of the overall bandgap value of the assembly; where (a) represents AgNS@(Cu-ZIF-8)-AuNPs; (b) represents AgNS@(Cd-ZIF-8)-AuNPs; and (c) represents AgNS@(Mn-ZIF-8)-AuNPs.

[0019] Figure 9 To record the electrochemical impedance spectroscopy of an electrode modified with AgNS@(X-ZIF-8)-AuNPs assembly in a mixed electrolyte containing potassium ferricyanide.

[0020] Figure 10 The SERS spectra are obtained by SERS detection of OTA standard solutions of different concentrations using the SERS-LFIA method.

[0021] Figure 11 It is 1135 cm -1 The linear fit between the Raman signal intensity at a given location and the logarithm of the OTA concentration.

[0022] Figure 12 This presents the specificity experimental results of SERS-LFIA based on AgNS@(Mn-ZIF-8)-AuNPs for OTA detection.

[0023] Figure 13 The results show the stability of the SERS-LFIA method based on AgNS@(Mn-ZIF-8)-AuNPs for OTA detection. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Unless otherwise specified, "room temperature" in this invention refers to 15-30°C.

[0030] Building upon the successful amplification of SERS signals via electromagnetic field-chemical synergistic enhancement achieved by core-shell-satellite assemblies with metal-doped ZIF-8 shells, this study further designs and improves the morphology and structure of the assemblies from two aspects: electromagnetic field enhancement and chemical enhancement. This includes achieving SERS electromagnetic field enhancement by altering the morphology of the nanonucleus and achieving SERS chemical enhancement by changing the elemental doping species in the ZIF-8 shell.

[0031] First, regarding electromagnetic field enhancement, the selection and preparation of the core morphology play a decisive role in the SERS activity of composite noble metal nanoparticles. Due to the "lightning rod" effect, more high-density charges with strong electromagnetic energy, i.e., "hot spots," can be generated at the edges, tips, or internal gaps of anisotropic plasmonic materials. Therefore, the enhancement effect of anisotropic noble metal nanoparticles with sharp tips on the local electromagnetic field is much greater than that of nanospheres. This invention selects AgNS as the core for the preparation of the assembled SERS substrate. AgNS often has more "hot spots" at the branch tips, which can significantly improve the SERS signal from the perspective of electromagnetic field enhancement. Second, regarding chemical enhancement, since zinc-based metal-organic framework (ZIF-8) materials have strong scalability, in addition to doping Cu in the ZIF-8 shell, other types of metal elements can be doped for flexible design and improvement, such as doping Mn and Cd. Furthermore, previous studies have reported that doping these metal elements into ZIF-8 can have a certain effect on reducing the ZIF-8 band gap. Therefore, using X-ZIF-8 shells with metal element doping in the synthesis of assemblies can greatly improve the chemical enhancement potential of the assemblies.

[0032] This invention develops a novel SERS nanoprobe capable of detecting OTA using the SERS-LFIA method. The probe consists of an AgNS core, an outer ZIF-8 shell doped with metal elements, and a core-shell-satellite assembly (AgNS@(X-ZIF-8)-AuNPs) with AuNPs satellites grown on the shell surface. The enhancement of the overall SERS activity of the assembly by doping the ZIF-8 shell with three different metal elements, Cu, Cd, and Mn, was compared. In this structure, the AgNS branch surface has a denser distribution of electromagnetic "hot spots," which, combined with the metal-doped X-ZIF-8 shell, gives the assembly excellent electromagnetic field-chemical synergistic enhancement performance. The study found that using the Mn-ZIF-8 shell maximizes the enhancement of the assembly's SERS signal intensity. The AgNS@(Mn-ZIF-8)-AuNPs assembly was used to detect OTA using the SERS-LFIA method, demonstrating significant research and application value in the detection of mycotoxins in the field of food safety.

[0033] The first aspect of this invention provides a method for preparing AgNS@(Mn-ZIF-8)-AuNPs assemblies, comprising the following steps: Step 1: Add an alkaline solution to the hydroxylamine solution and mix well. Then add a silver source solution, let it stand, and then add sodium citrate solution to react and obtain AgNS solution. Step 2: The precipitate obtained after centrifugation of the AgNS solution is dispersed in polyvinylpyrrolidone solution and stirred, then mixed with 4-ATP solution, allowed to stand, and the precipitate obtained after centrifugation is dispersed in anhydrous methanol to obtain AgNS@4-ATP methanol solution; Step 3: Add a mixed solution of Zn source and Mn source to the AgNS@4-ATP methanol solution, then add ligand solution and mix well to react and obtain AgNS@(Mn-ZIF-8) core-shell structure; disperse the AgNS@(Mn-ZIF-8) core-shell structure in water to obtain AgNS@(Mn-ZIF-8) solution; Step 4: Mix the AgNS@(Mn-ZIF-8) solution with the gold source solution, add sodium borohydride solution to react, and obtain the AgNS@(Mn-ZIF-8)-AuNPs assembly.

[0034] In a preferred embodiment of the present invention, in step 1, the concentration of the hydroxylamine solution is 60 mM; the alkaline solution is a 50 mM sodium hydroxide solution; the silver source solution is a 1 mM silver nitrate solution; the concentration of the sodium citrate solution is 40-43 mM; the volume ratio of the hydroxylamine solution to the alkaline solution, the silver source solution, and the sodium citrate solution is 1:1:18:0.2; and the reaction conditions are set as follows: shaking at 100 rpm and 25°C for 15 min.

[0035] In a preferred embodiment of the present invention, in step 2, the concentration of the polyvinylpyrrolidone solution is 12.5 g / L; the concentration of the 4-ATP solution is 20 mM; the volume ratio of AgNS solution to polyvinylpyrrolidone solution, 4-ATP solution and anhydrous methanol is 10:10:1:10; and the standing time is 1 h.

[0036] The solid obtained after centrifuging the AgNS solution in step 2 is dispersed in a polyvinylpyrrolidone solution and stirred overnight; the purpose of stirring overnight is to replace the sodium citrate on the surface of AgNS with polyvinylpyrrolidone.

[0037] In a preferred embodiment of the present invention, in step 3, the Zn source is Zn(NO3)2; the Mn source is Mn(NO3)2; the concentration of the Zn source in the mixed solution is 25 mM; the amount of Mn source added in the mixed solution is 0%~15% of the amount of Zn source, and not 0; the ligand solution is a 50 mM 2-methylimidazole solution.

[0038] In a preferred embodiment of the present invention, in step 3, the volume ratio of AgNS@4-ATP methanol solution to mixed solution, ligand solution and water is 17:0.5:0.75:10; the reaction conditions are set as follows: first stir for 5 min, then let stand at room temperature for 1 h.

[0039] In this invention, as the amount of Mn doping increases, the SERS activity of the assembly first increases and then decreases. Therefore, the preferred method of this invention is to limit the amount of Mn source added to the mixed solution to the range of parameters mentioned above.

[0040] In a preferred embodiment of the present invention, in step 4, the gold source solution is an aqueous solution of chloroauric acid tetrahydrate with a concentration of 0.025 mM; the concentration of sodium borohydride solution is 18.5 mM; the volume ratio of AgNS@(Mn-ZIF-8) solution to gold source solution and sodium borohydride solution is 10:10:0.24; the reaction conditions are set as follows: stirring at 600 rpm for 10 min.

[0041] A second aspect of the present invention provides an AgNS@(Mn-ZIF-8)-AuNPs assembly prepared according to the above-described preparation method.

[0042] The third aspect of the present invention provides a method for preparing a SERS immune probe, wherein an anti-OTA monoclonal antibody is mixed with an AgNS@(Mn-ZIF-8)-AuNPs assembly and a solvent and incubated, then centrifuged, and BSA solution is added and allowed to stand to obtain the SERS immune probe.

[0043] This invention does not have any special requirements for the sequence of anti-OTA monoclonal antibodies; commercially available anti-OTA monoclonal antibodies are all suitable for this invention.

[0044] A fourth aspect of the present invention provides a SERS immune probe prepared according to the above-described preparation method.

[0045] The fifth aspect of the present invention provides the application of the above-described AgNS@(Mn-ZIF-8)-AuNPs assembly or the above-described SERS immune probe in the detection of OTA by SERS-LFIA method.

[0046] This invention also utilizes NC membranes to prepare test strips for OTA detection. The preparation method of the test strips is a conventional technique in the field and is not the focus of this invention's patent protection; therefore, it will not be described in detail here.

[0047] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0048] The chemical reagents used in this invention are shown in Table 1: Table 1

[0049]

[0050] The instruments and equipment used in this invention are shown in Table 2: Table 2

[0051] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0052] Unless otherwise specified, the solvent for each solution in the embodiments of the present invention is "water".

[0053] Example 1 1. Experimental Section 1.1 Preparation of AgNS First, take 1 mL (60 mM) of hydroxylamine solution (the first reducing agent) in a sample vial, then add 1 mL (50 mM) of sodium hydroxide solution to adjust the pH of the solution. Gently shake the sample vial for 1 min to mix the solution thoroughly. Then, slowly add 18 mL (1 mM) of silver nitrate solution dropwise. As the silver nitrate solution is added, the color of the mixed solution changes from colorless to dark gray. After standing at room temperature for 5 min, add 200 μL (41.3 mM) of sodium citrate solution (the second reducing agent). Shake in a shaker at 100 rpm and 25°C for 15 min to obtain the AgNS solution.

[0054] 1.2 Preparation of AgNS@(X-ZIF-8) core-shell structure The AgNS solution (20 mL) was centrifuged at 6000 rpm for 10 min and then dispersed in 20 mL (12.5 g / L) polyvinylpyrrolidone solution, stirred overnight at 200 rpm. Then, it was mixed with 2 mL (20 mM) 4-ATP solution and allowed to stand at room temperature for 1 h. After centrifugation at 5500 rpm for 10 min, it was dispersed in 20 mL anhydrous methanol to obtain the AgNS@4-ATP methanol solution. Next, 17 mL of the AgNS@4-ATP methanol solution was placed in an Erlenmeyer flask and slowly stirred in an ice-water bath. Then, 0.5 mL of a mixed solution of Zn(NO3)2 and X(NO3)2 (Zn(NO3)2 concentration of 25 mM) was added and stirred at 500 rpm for 10 min. In this study, X(NO3)2 was Cu(NO3)2, Cd(NO3)2, or Mn(NO3)2, and mixed solutions with different X(NO3)2 doping amounts (0%, 5%, 10%, and 15% of the molar concentration of Zn(NO3)2 compared to 25 mM Zn(NO3)2) were prepared and used. Then, 0.75 mL (50 mM) of 2-methylimidazole solution was added, stirred for 5 min, and allowed to stand at room temperature for 1 h. Finally, the resulting reaction mixture was centrifuged at 5000 rpm for 10 min to obtain the AgNS@(X-ZIF-8) core-shell structure. After washing three times with ultrapure water, it was dispersed in 10 mL of ultrapure water to obtain the AgNS@(X-ZIF-8) solution for further use.

[0055] 1.3 Preparation of AgNS@(X-ZIF-8)-AuNPs core-shell-satellite structure The above AgNS@(X-ZIF-8) solution (10 mL) was added to 10 mL of 0.025 mM chloroauric acid tetrahydrate aqueous solution. Then, 240 μL of 18.5 mM sodium borohydride solution was added, and the mixture was stirred at 600 rpm for 10 min. After centrifugation at 3500 rpm for 10 min, the AgNS@(X-ZIF-8)-AuNPs core-shell-satellite assembly was obtained.

[0056] 1.4 Preparation of antibody-conjugated SERS immunoprobes 20 μL (0.1 mg / mL) of anti-OTA monoclonal antibody was added to the prepared optimal AgNS@(X-ZIF-8)-AuNPs assembly solution and incubated at 37 °C for 1 h. After centrifugation at 3000 rpm for 10 min, 1 mL (0.1%) BSA solution was added and allowed to stand for 30 min to block unsaturated sites. Finally, the OTA monoclonal antibody-modified AgNS@(X-ZIF-8)-AuNPs (SERS immunoprobe) was centrifuged at 2500 rpm for 10 min to remove excess antibody from the solution. The obtained SERS immunoprobe was dispersed in 0.5 mL (1% wt) PBS buffer (pH 7.4) and stored at 4 °C.

[0057] 1.5 Preparation of test strips First, the NC membrane was cut using a cutter and then activated by soaking in PBS buffer (1% wt, pH 7.4) for 30 min. Next, the NC membrane was dried in an oven at 37°C. Then, appropriate amounts of OTA-BSA conjugate and goat anti-mouse IgG were dispensed onto the detection line (T line) and control line (C line), respectively, and dried in an oven at 37°C. Finally, the SERS test strips were assembled, divided, and stored at 4°C.

[0058] 1.6 OTA Detection Methods The OTA detection method consists of two parts: sample pretreatment and OTA detection. The first step is the pretreatment of the grain sample. In this experiment, corn sold locally in Baoding City, Hebei Province, was selected as the research sample. 20 g of corn sample was weighed and thoroughly ground in a mortar. After passing through a 1 mm sieve, it was added to 100 mL of a mixed solution prepared with methanol and water in a 3:1 volume ratio and mixed thoroughly. After standing at room temperature for 10 min, the mixed solution was filtered through 0.22 μm polytetrafluoroethylene filter paper to obtain the sample extract.

[0059] The following is the detection procedure for OTA using the SERS-LFIA method. First, 8 μL of the prepared SERS immunoprobe is mixed with 20 μL of sample extract and incubated at room temperature for 10 min. Then, the mixture is introduced onto the sample pad of the test strip and incubated for 15 min. Finally, SERS detection is performed on the T line. For the specificity test of SERS-LFIA, 100 ng / mL of FB1, T-2, DON, and ZEN solutions, and 1 ng / mL of OTA solution are mixed with the SERS immunoprobe and incubated at room temperature for 10 min. The mixture is then distributed onto the sample pad of the test strip, incubated for 15 min, and SERS detection is performed on each T line. For the stability test of SERS-LFIA, the SERS immunoprobe and test strip are stored at 4°C and tested once a week. The detection procedure involves mixing the SERS immunoprobe with 1 ng / mL of OTA solution and incubating at room temperature for 10 min. The mixed solution was then introduced onto the sample pad of the test strip, and after incubation for 15 min, the T line was analyzed for SERS.

[0060] 1.7 SERS and Liquid Chromatography Detection Conditions The Raman spectroscopy detection conditions in this experiment are as follows: the excitation wavelength is 633 nm, the test range is 800-1800 nm, the laser power is 17 mw, and the integration time is 1 s.

[0061] The liquid chromatography detection conditions for the grain sample extract in this experiment were as follows: XDB-C18 column (4.6 mm × 250 mm, 5 μm) was used; the mobile phase was methanol:water = 7:3; the flow rate of the mobile phase was 0.5 mL / min; the excitation wavelength of the fluorescence detector was 333 nm and the emission wavelength was 460 nm; the column temperature was 40℃.

[0062] 2. Results 2.1 The synthesis process of the AgNS@(X-ZIF-8)-AuNPs core-shell-satellite assembly is as follows: Figure 1 As shown, AgNS was first synthesized using a two-step reduction method, successively using hydroxylamine and sodium citrate as reducing agents. AgNS exhibited sharp branches and uniform arm lengths. Then, the SERS signaling molecule 4-ATP was modified onto the AgNS surface, followed by in-situ coating with ZIF-8 doped with metal element X, synthesizing the AgNS@(X-ZIF-8) core-shell structure. Finally, AuNPs satellites were grown on the shell surface, preparing three assemblies: AgNS@(Cu-ZIF-8)-AuNPs, AgNS@(Cd-ZIF-8)-AuNPs, and AgNS@(Mn-ZIF-8)-AuNPs.

[0063] 2.2 Characterization of AgNS@(X-ZIF-8) Core-Shell Structure: First, the core-shell structure prepared by coating X-doped ZIF-8 nanoparticles on the surface of AgNS nanoparticles was analyzed. Here, the X-doped shell refers to the layer in which Cu is doped during the synthesis of ZIF-8. 2+ Cd 2+ and Mn 2+ Replacing a small amount of Zn 2+ As part of the metal ion source, it participates in the coordination bonding with the organic ligand zeolite imidazole molecule, forming three doped and modified X-ZIF-8 materials. Coating them onto the surface of AgNS yields different AgNS@(X-ZIF-8) core-shell structures. For example... Figure 2 As shown, the morphology and structure of the synthesized AgNS@(X-ZIF-8) were characterized using high-resolution transmission electron microscopy. Figure 2 (a), (b), and (c) are high-resolution TEM images and EDS elemental distribution maps of AgNS@(Cu-ZIF-8), AgNS@(Cd-ZIF-8), and AgNS@(Mn-ZIF-8), respectively. The TEM images show that the AgNS at the core has well-developed branches with uniform arm length, and is uniformly coated with an X-ZIF-8 shell. EDS analysis was performed to further demonstrate the synthesis of the structure and the elemental doping in the ZIF-8 shell. The presence and distribution of Ag in the EDS elemental distribution maps confirm the synthesis of the AgNS nanonucleus, while the presence and distribution of Zn confirm the coating of the ZIF-8 shell. In particular, the presence of small amounts of Cu, Cd, and Mn at the location of the ZIF-8 shell demonstrates that a small amount of Zn in the shell has been successfully substituted into Cu, Cd, and Mn, respectively.

[0064] 2.3 Effect of Different Doping Elements and Doping Amounts on SERS Performance: To investigate the effect of different doping elements on the SERS performance of the core-shell structure, AgNS@(X-ZIF-8) synthesized using mixed solutions of Zn(NO3)2 and Cu(NO3)2, Cd(NO3)2, or Mn(NO3)2 in different proportions during the shell synthesis process was subjected to spectroscopic analysis. First, the optical properties of the samples were analyzed by UV-Vis absorption spectroscopy. Figure 3 In the UV-Vis absorption spectrum shown, Figure 3(a), (b), and (c) are the UV-Vis absorption spectra of AgNS@(Cu-ZIF-8), AgNS@(Cd-ZIF-8), and AgNS@(Mn-ZIF-8), respectively. In all these spectra, a distinct absorption peak (LSPR peak) of AgNS is present at approximately 400 nm. The spectra named Cu 0%, Cd 0%, and Mn 0% are the absorption spectra of AgNS@(ZIF-8). It can be seen that compared to AgNS, the LSPR peak after the shell coating undergoes a redshift, which is due to the shell coating altering the dielectric constant of the structure. Furthermore, in each spectra, as the doping molar ratio increases (5%, 10%, and 15%, where the percentage refers to the amount of X(NO3)2 added during preparation being 5%, 10%, and 15% of the amount of Zn(NO3)2), the redshift of the absorption spectra of AgNS@(X-ZIF-8) with Cu, Cd, and Mn doped shells increases continuously. This phenomenon may be due to the doping of various metal elements further altering the dielectric constant of the AgNS@ZIF-8 structure, thereby affecting the LSPR behavior and causing a change in the position of the absorption peak.

[0065] Then, SERS spectroscopy analysis was performed on AgNS@(Cu-ZIF-8) (Cu doping amounts of 5%, 10%, and 15%), AgNS@(Cd-ZIF-8) (Cd doping amounts of 5%, 10%, and 15%), and AgNS@(Mn-ZIF-8) (Mn doping amounts of 5%, 10%, 15%, and 20%) with different metal element doping types and amounts at an excitation wavelength of 633 nm. 4-ATP was used as the SERS signal molecule. The signal was obtained by observing that 4-ATP is located at 1135 cm⁻¹. -1 The SERS signal intensity at the non-perfectly symmetrical peak at the location was plotted as follows: Figure 4 The bar chart shown is for n=3. First, the signal amplification capabilities of each AgNS@(X-ZIF-8) synthesized with different X(NO3)2 doping concentrations are compared. It can be seen that as the doping concentration increases, the signal amplification capability at 1135 cm⁻¹... -1 The SERS signal intensity at each location initially increases and then decreases. AgNS@(Cu-ZIF-8), AgNS@(Cd-ZIF-8), and AgNS@(Mn-ZIF-8) exhibit the best SERS enhancement effects when the doping concentrations of Cu, Cd, and Mn are 10%, 10%, and 15%, respectively. Then, the different AgNS@(X-ZIF-8) components are compared at the optimal elemental doping concentrations. The figure shows that at 1135 cm⁻¹... -1The SERS signal enhancement at the point of origin is as follows: Mn 15% > Cu 10% > Cd 10%. Therefore, it is concluded that when a mixed solution doped with Mn(NO3)2 at a molar ratio of 15% is used for the preparation of the shell, AgNS@(Mn-ZIF-8) with the best SERS chemical enhancement effect can be obtained.

[0066] Because the Zn(NO3)2 and Mn(NO3)2 dissolved in the mixed solution are not completely used for coordination bonding with zeolite imidazole molecules during the synthesis of Mn-ZIF-8, residual Zn will remain in the solution. 2+ and Mn 2+ The presence of Zn and Mn was determined using ICP-MS to quantify the Zn and Mn content in AgNS@(Mn-ZIF-8), which exhibited the best SERS enhancement performance. As shown in Table 3, the Zn content in 0.2 mL of sample solution was 27.07 μg / mL, and the Mn content was 6.78 μg / mL. This led to the calculation that when the Mn(NO3)2 doping molar ratio was 15%, the corresponding Mn:Zn ratio in the shell was 2:7. Combined with the previous SERS spectral results, it was concluded that the structure exhibited the best SERS enhancement performance when the Mn:Zn ratio in the shell of AgNS@(Mn-ZIF-8) was 2:7.

[0067] Table 3 Results of quantitative determination of Zn and Mn content by inductively coupled plasma mass spectrometry (ICP-MS)

[0068] 2.4 Characterization of AgNS@(X-ZIF-8)-AuNPs core-shell-satellite assemblies: To further enhance the SERS activity of the structure, AuNPs were grown on the surface of AgNS@(X-ZIF-8) structures with optimal Cu, Cd, and Mn doping levels, preparing three AgNS@(X-ZIF-8)-AuNPs core-shell-satellite assemblies capable of generating more "hot spots." First, the structure and morphology of the synthesized assemblies were characterized, such as... Figure 5 As shown. Figure 5Images (a), (b), and (c) show high-resolution TEM images and EDS elemental distribution maps of AgNS@(Cu-ZIF-8)-AuNPs, AgNS@(Cd-ZIF-8)-AuNPs, and AgNS@(Mn-ZIF-8)-AuNPs assemblies, respectively. The TEM images reveal that in all assemblies, the central structure is a nanonucleus composed of multi-branched AgNS, with an intermediate X-ZIF-8 shell doped with different metal elements. The AuNPs, acting as satellites, are uniformly and densely distributed on the shell surface. The EDS elemental distribution map of the AgNS@(X-ZIF-8)-AuNPs assembly further confirms the elemental composition of each component. The presence and distribution of Ag confirm the synthesis of AgNS, while the presence and distribution of Zn confirm the encapsulation of ZIF-8. Furthermore, the elemental distribution diagrams of each assembly show that small amounts of Cu, Cd, and Mn elements are present at the locations of Zn, respectively, demonstrating the successful encapsulation of Cu-ZIF-8, Cd-ZIF-8, and Mn-ZIF-8 shells. The distribution of Au elements in the outermost layer proves the successful growth of AuNPs.

[0069] Subsequently, the synthesized assembly underwent spectral testing. First, UV-Vis absorption spectroscopy was performed, such as... Figure 6 As shown. Figure 6 (a), (b), and (c) are comparisons of the UV-Vis absorption spectra of AgNS@(Cu-ZIF-8), AgNS@(Cd-ZIF-8), and AgNS@(Mn-ZIF-8) before and after satellite growth, respectively. The figures show that growing AuNPs outside different core-shell structures causes a slight redshift and broadening of the absorption peak (LSPR peak). This is because the presence of the shell layer after satellite growth leads to strong interactions and localized surface plasmon coupling between the AgNS nanonucleus and the AuNP satellites, as well as between the satellites themselves, increasing the extinction coefficient and thus altering the absorption peak position. Furthermore, the figures show that the redshift is similar for core-shell structures doped with different metal elements after AuNP growth, indicating a similar degree of influence of AuNP growth on the localized surface electric field. This is because, although AuNP growth enhances the SERS activity of the assembly through electromagnetic field enhancement, the overall effect is still relatively stable. However, the differences in SERS enhancement among the three AgNS@(X-ZIF-8)-AuNPs assemblies mainly stem from the different chemical enhancement effects resulting from doping with different metal elements. Therefore, the differences in localized surface plasmon coupling among the three assemblies are not significant.

[0070] To obtain assemblies with optimal electromagnetic field-chemical synergistic enhancement performance, SERS spectroscopy was performed on assemblies doped with different metal elements, namely AgNS@(Cu-ZIF-8)-AuNPs, AgNS@(Cd-ZIF-8)-AuNPs, and AgNS@(Mn-ZIF-8)-AuNPs. The detection results are as follows: Figure 7 As shown, by comparing the SERS spectra of different assemblies, it was observed that the three assemblies exhibited a similar pattern at 1135 cm⁻¹. -1 The SERS signal intensity at the point of origin was: AgNS@(Mn-ZIF-8)-AuNPs > AgNS@(Cu-ZIF-8)-AuNPs > AgNS@(Cd-ZIF-8)-AuNPs. This result indicates that the assembly with the Mn-doped shell exhibits the best SERS enhancement effect, corresponding to the previously observed best SERS enhancement performance of AgNS@(Mn-ZIF-8). Therefore, in subsequent experiments detecting OTA using the SERS-LFIA method, the AgNS@(Mn-ZIF-8)-AuNPs assembly will be selected for the preparation of the SERS immunoassay probe.

[0071] 2.5 Chemical enhancement analysis of AgNS@(X-ZIF-8)-AuNPs assemblies The SERS chemical enhancement of this invention is still reflected in the preparation of core-shell-satellite assemblies with metal-doped shells. Based on the inherent CT performance of the ZIF-8 shell, metal doping introduces additional electronic states, promoting the CT efficiency of the structure and resulting in better chemical enhancement performance of the assembly. Furthermore, AuNPs grown on the shell surface can act as electron acceptors to further promote CT. In this experiment, different elements Cu, Cd, and Mn were used for doping to further investigate the influence of elemental doping on the chemical enhancement effect, preparing core-shell-satellite assemblies with optimal SERS enhancement performance. The following chemical enhancement analysis was performed accordingly.

[0072] First, the Tauc Plot method is used based on theoretical formulas. Using UV-Vis absorption spectral data, the band gap values ​​of the three assemblies were calculated respectively. Figure 8 As shown, Figure 8Images (a), (b), and (c) show the calculated overall bandgap values ​​of AgNS@(Cu-ZIF-8)-AuNPs, AgNS@(Cd-ZIF-8)-AuNPs, and AgNS@(Mn-ZIF-8)-AuNPs, respectively. The bandgap values ​​of these assemblies are 1.63 eV, 1.68 eV, and 1.57 eV, respectively. These results indicate that among the three synthesized core-shell-satellite assemblies, AgNS@(Mn-ZIF-8)-AuNPs has the smallest bandgap value. This demonstrates that among doped metal elements, Mn doping can provide the highest charge concentration, maximally reducing the conduction band energy of ZIF-8 and accelerating electron transfer.

[0073] Next, electrochemical impedance spectroscopy (EIS) was used to test the impedance and CT capabilities of the three assemblies, AgNS@(Cu-ZIF-8)-AuNPs, AgNS@(Cd-ZIF-8)-AuNPs, and AgNS@(Mn-ZIF-8)-AuNPs. Figure 3-9 The figure shows the Nyquist plots of the three assemblies. After modifying the assemblies onto the surface of an indium tin oxide (ITO) electrode and testing them, impedance spectra with different semicircular diameters in the high-frequency region were obtained. It can be seen from the figure that the semicircular diameter of AgNS@(Cd-ZIF-8)-AuNPs > AgNS@(Cu-ZIF-8)-AuNPs > AgNS@(Mn-ZIF-8)-AuNPs. Since the semicircular diameter in the high-frequency region is inversely proportional to the CT velocity of the material, it can be demonstrated that the core-shell-satellite assembly with the Mn-ZIF-8 shell has the lowest impedance and the fastest CT velocity. This is consistent with the previous results showing that the assembly with the Mn-doped shell has the best SERS signal intensity and the lowest bandgap. In summary, the AgNS@(Mn-ZIF-8)-AuNPs core-shell-satellite assembly can maximize CT and achieve effective amplification of the SERS signal through a chemical enhancement mechanism, thus giving this assembly the best electromagnetic field-chemical synergistic enhancement performance.

[0074] 2.6 OTA Detection Principle This experiment detected OTA using the SERS-LFIA method. The detection process combines SERS technology with a competitive immunoassay, utilizing the principle that toxins and antigens compete for binding with a limited number of antibodies. Specifically, an AgNS@(Mn-ZIF-8)-AuNPs assembly with optimal electromagnetic field-chemical synergistic enhancement properties was selected and combined with an OTA monoclonal antibody to prepare a SERS immunoprobe. An OTA-coated antigen-modified NC membrane strip was used as the test line. The synthesized SERS immunoprobe was mixed with the sample and dropped onto a sample pad. Higher OTA concentrations in the sample result in more OTA binding to the OTA-modified SERS immunoprobe, leading to fewer immunoprobes on the antigen detection line that capture the SERS immunoprobes through antigen-antibody specific binding. Therefore, fewer AgNS@(Mn-ZIF-8)-AuNPs immunoprobes on the detection line result in a weaker SERS signal after SERS detection, thus enabling quantitative analysis of OTA. In short, higher OTA concentrations lead to lower SERS detection signals, showing a negative correlation.

[0075] 2.7 Establishment of the Standard Curve Based on the aforementioned OTA detection principle, OTA standard solutions of different concentrations were analyzed to evaluate the analytical performance of the AgNS@(Mn-ZIF-8)-AuNPs core-shell-satellite assembly in the SERS-LFIA method for OTA detection. Eleven OTA standard samples ranging from 0.05 fg / mL to 1 μg / mL were prepared, and these standard solutions of different concentrations were analyzed by SERS under optimal detection conditions. The detection results are as follows: Figure 10 As shown, SERS spectra of OTA standard solutions at different concentrations were obtained. The figures show that as the concentration of the OTA standard solution gradually increased from 0.05 fg / mL to 1 μg / mL, the characteristic molecule of 4-ATP was located at 1135 cm⁻¹. -1 The SERS signal intensity gradually decreased at that location. OTA concentration showed a negative correlation with SERS signal intensity.

[0076] To quantify the relationship between OTA concentration and SERS signal intensity, multiple measurements were performed on OTA standard samples at each concentration. The obtained SERS spectral data were then linearly fitted to establish a model. Figure 11 The standard curve for OTA detection by the SERS-LFIA method is shown (n=3). (From...) Figure 11 It can be seen that within the concentration range of 0.05 fg / mL to 1 μg / mL, 1135 cm -1 The linear relationship between the SERS signal intensity and the logarithm of OTA concentration at a given location is Y = 15007 - 2178X, R2 =0.996. Using the formula LOD=3σ / S, the detection limit of this method is calculated to be 0.021 fg / mL.

[0077] 2.8 Specificity and stability testing This study investigated the specificity and stability of AuNS@(Mn-ZIF-8)-AuNPs assemblies for OTA detection using the SERS-LFIA method. Firstly, regarding the specificity of this SERS-LFIA method for OTA detection, four other fungal toxins—FB1, T-2, DON, and ZEN—were selected as interfering toxins. The concentration of OTA was 1 ng / mL, and the concentrations of all interfering toxins were 100 ng / mL. After applying the five toxins to the SERS-LFIA method, the test results obtained by SERS spectral analysis are as follows: Figure 12 As shown, it can be seen that only when OTA is used for detection does the obtained 1135 cm⁻¹ -1 The SERS signal at the site was significantly reduced. However, when using other interfering toxins for detection, even with higher toxin concentrations, the obtained SERS signal intensity remained similar to that of the blank sample. This result demonstrates that this SERS-LFIA method based on AgNS@(Mn-ZIF-8)-AuNPs assemblies has good selectivity for OTA.

[0078] In the stability experiment of the SERS-LFIA method for OTA detection, the SERS immunoassay probe and test strip were stored at 4°C for four weeks (28 days). OTA was tested once every week at 0.1 ng / mL. A total of five tests were performed within the four-week timeframe (1 day, 7 days, 14 days, 21 days, and 28 days). The test results are as follows: Figure 13 As shown in the figure, it can be observed that during storage in a 4°C refrigerator, the measured value was 1135 cm⁻¹ every week. -1 The SERS signal intensity at each location decreased slightly compared to the previous week. The fifth detection result showed a decrease of only 9.825% ± 0.525% in SERS signal intensity compared to the first detection result, demonstrating that this SERS-LFIA method based on AgNS@(Mn-ZIF-8)-AuNPs probe can maintain good stability for OTA detection within 4 weeks.

[0079] 2.9 Actual Sample Testing Finally, to verify the effectiveness of the SERS-LFIA method based on the AgNS@(Mn-ZIF-8)-AuNPs assembly in detecting OTA in actual samples, corn was selected as the spiked sample for detection. The spiked grain sample solution was mixed with the SERS immunoprobe containing AgNS@(Mn-ZIF-8)-AuNPs and then used for SERS-LFIA detection, yielding recoveries for samples with different concentrations of spikes. The detection results are shown in Table 4. The recovery rate of OTA detected by this SERS-LFIA method was 96.55%-105.43%, with an RSD of 1.36%-4.12%. In contrast, the recovery rate of OTA detected by liquid chromatography was 98.38%-104.39%, with an RSD of 1.69%-4.17%. It can be seen that the detection results of the two detection methods are consistent, thus indicating that the SERS-LFIA detection method based on AgNS@(Mn-ZIF-8)-AuNPs assembly has good reliability in the quantitative evaluation of OTA.

[0080] Table 4 Comparison of the recovery rate and RSD value of OTA in spiked cereal samples by SERS-LFIA and liquid chromatography

[0081] In this invention, based on the principle that a core-shell-satellite assembly with a metal-doped ZIF-8 shell can generate SERS electromagnetic field-chemical synergistic enhancement, an AgNS@(Mn-ZIF-8)-AuNPs core-shell-satellite assembly with better synergistic enhancement performance was designed and prepared. The assembly was then tested for OTA using SERS-LFIA. The design of the assembly focused on both electromagnetic field enhancement and chemical enhancement. Firstly, regarding electromagnetic field enhancement, AgNS, with its superior local electric field enhancement capability, was used as the core. Its sharp tip generates a higher density of "hot spots," improving the SERS activity of the structure. Secondly, regarding chemical enhancement, the enhancement capabilities of three different metal elements—Cu, Cd, and Mn—doped into the ZIF-8 shell on the overall SERS activity of the assembly were compared. It was found that using the Mn-ZIF-8 shell maximized the enhancement of the assembly's SERS signal intensity. The results were validated through bandgap calculation and electrochemical impedance spectroscopy (EIS). The assembly with the Mn-doped shell exhibited the smallest bandgap and lowest impedance, resulting in a faster CT rate and improved chemical enhancement performance. Finally, this assembly with synergistic electromagnetic-chemical enhancement was applied to the SERS-LFIA detection of OTA. The detection system demonstrated good specificity and stability, exhibiting excellent linearity within a detection range of 0.05 fg / mL to 1 μg / mL, with a detection limit as low as 0.021 fg / mL. This simple, rapid, and trace-level SERS-LFIA method for the detection of OTA in grains provides important reference value for the accurate analysis of mycotoxins in the field of food safety.

[0082] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing AgNS@(Mn-ZIF-8)-AuNPs assemblies, characterized in that, Includes the following steps: Step 1: Add an alkaline solution to the hydroxylamine solution and mix well. Then add a silver source solution, let it stand, and then add sodium citrate solution to react and obtain AgNS solution. Step 2: The precipitate obtained after centrifugation of the AgNS solution is dispersed in polyvinylpyrrolidone solution and stirred, then mixed with 4-ATP solution, allowed to stand, and the precipitate obtained after centrifugation is dispersed in anhydrous methanol to obtain AgNS@4-ATP methanol solution; Step 3: Add a mixed solution of Zn source and Mn source to the AgNS@4-ATP methanol solution, then add ligand solution and mix well to react and obtain AgNS@(Mn-ZIF-8) core-shell structure; disperse the AgNS@(Mn-ZIF-8) core-shell structure in water to obtain AgNS@(Mn-ZIF-8) solution; Step 4: Mix the AgNS@(Mn-ZIF-8) solution with the gold source solution, add sodium borohydride solution to react, and obtain the AgNS@(Mn-ZIF-8)-AuNPs assembly.

2. The preparation method according to claim 1, characterized in that, In step 1, the concentration of hydroxylamine solution is 60 mM; the alkaline solution is 50 mM sodium hydroxide solution; the silver source solution is 1 mM silver nitrate solution; the concentration of sodium citrate solution is 40~43 mM; the volume ratio of hydroxylamine solution to alkaline solution, silver source solution and sodium citrate solution is 1:1:18:0.2; the reaction conditions are set as follows: shaking at 100 rpm and 25℃ for 15 min.

3. The preparation method according to claim 1, characterized in that, In step 2, the concentration of the polyvinylpyrrolidone solution was 12.5 g / L; the concentration of the 4-ATP solution was 20 mM; the volume ratio of AgNS solution to polyvinylpyrrolidone solution, 4-ATP solution and anhydrous methanol was 10:10:1:10; and the standing time was 1 h.

4. The preparation method according to claim 1, characterized in that, In step 3, the Zn source is Zn(NO3)2; the Mn source is Mn(NO3)2; the concentration of the Zn source in the mixed solution is 25 mM; the amount of Mn source added in the mixed solution is 0%~15% of the amount of Zn source, and not 0; the ligand solution is a 50 mM 2-methylimidazole solution.

5. The preparation method according to claim 1, characterized in that, In step 3, the volume ratio of AgNS@4-ATP methanol solution to mixed solution, ligand solution and water is 17:0.5:0.75:10; the reaction conditions are set as follows: stir for 5 min first, then let stand at room temperature for 1 h.

6. The preparation method according to claim 1, characterized in that, In step 4, the gold source solution is a 0.025 mM aqueous solution of chloroauric acid tetrahydrate; the sodium borohydride solution has a concentration of 18.5 mM; the volume ratio of AgNS@(Mn-ZIF-8) solution to the gold source solution and the sodium borohydride solution is 10:10:0.24; the reaction conditions are set as follows: stirring at 600 rpm for 10 min.

7. An AgNS@(Mn-ZIF-8)-AuNPs assembly prepared by the preparation method according to any one of claims 1 to 6.

8. A method for preparing a SERS immune probe, characterized in that, The anti-OTA monoclonal antibody was mixed with the AgNS@(Mn-ZIF-8)-AuNPs assembly of claim 7 and a solvent, and then incubated. After centrifugation, BSA solution was added and the mixture was allowed to stand to obtain the SERS immune probe.

9. A SERS immune probe prepared by the preparation method according to claim 8.

10. The application of the AgNS@(Mn-ZIF-8)-AuNPs assembly as described in claim 7, or the SERS immune probe as described in claim 9, in the detection of OTA by the SERS-LFIA method.

Images