Au@Pt NCs nanoscale enzyme probe for detecting new type of nicotine pesticide, and preparation method and application thereof

Abstract

The application discloses an Au@PtNCs nanometer enzyme probe for detecting novel nicotine pesticides, and a preparation method and application thereof, and belongs to the field of rapid detection of biosensors. The application provides an Au@PtNCs nanometer enzyme probe, which is generated by combining AuNCs and PtNCs and coupling with secondary antibodies, and can effectively realize rapid and specific detection of novel nicotine pesticides such as imidacloprid. The linear range of the biosensor probe provided by the application for detecting imidacloprid pesticides is 0.1-100 mu g / L, and the detection limits LOD of colorimetric method and fluorescence method can respectively reach 0.127 and 0.119 mu g / L.

Description

Technical Field

[0001] This invention relates to an Au@PtNCs nanozyme probe for detecting novel neonicotinoid pesticides, its preparation method, and its application, belonging to the field of rapid detection by biosensors. Background Technology

[0002] Novel neonicotinoid pesticides are difficult to decompose in the environment, and their accumulation in ecosystems poses a serious threat to biodiversity. Pesticides can be ingested or absorbed through the food chain, causing serious harm to human health, leading to dizziness, nausea, difficulty breathing, cancer, and irreversible damage to the liver and kidneys. Imidacloprid (IMI), a common novel neonicotinoid pesticide, is a broad-spectrum and highly effective pesticide that can be effectively applied to corn, potatoes, garlic, tomatoes, and various crops. Due to excessive use of imidacloprid, residues remain in fruits, vegetables, water, and soil. It eventually enters the food chain or poses a serious risk to human health. This can lead to many chronic diseases, such as weight loss, persistent weakness, tachycardia or bradycardia, memory loss, and difficulty concentrating. Therefore, there is an urgent need to develop accurate, sensitive, rapid, and effective methods to detect novel neonicotinoid pesticide residues in fruit, vegetable, and environmental samples.

[0003] Traditional biological probes for detecting novel neonicotinoid pesticide residues suffer from structural instability, limited detection metrics, low sensitivity, and slow signal response. In recent years, the use of nanozymes to recognize biomolecules, immobilized on the surface of biosensors, and combined with nanomaterials through nanozyme catalysis, has garnered significant attention. Furthermore, gold nanoclusters (AuNCs), as fluorescent nanomaterials, exhibit strong surface-enhanced fluorescence at their ends. This fluorescence is not only tunable in intensity but also highly stable and has a relatively high quantum yield. Platinum nanoclusters (PtNCs), as nanomaterials with high peroxidase-like activity, possess excellent catalytic performance and stability. Gold-platinum nanoclusters (Au@PtNCs), as a nanocomposite material, exhibit good biocompatibility and can be used in ELISA reaction systems. Utilizing their peroxidase-like catalysis, they emit a colorimetric-fluorescence dual-mode reaction signal, further improving the sensitivity and accuracy of imidacloprid pesticide (IMI) detection and achieving rapid detection.

[0004] However, at present, there are few nanozyme probes made of gold-platinum bimetallic composite nanomaterials, and how to improve the detection sensitivity and response speed is an urgent problem to be solved.

[0005] Based on this, the present invention aims to construct an “OFF-enhanced ON” colorimetric-fluorescence visualization quenching nanozyme probe based on the Au@PtNCs-TMB-H2O2 reaction system and FRET and SEF effects for the detection of imidacloprid (IMI). Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art by designing a novel core-shell structure Au@PtNCs nanozyme probe with uniform size and morphology, good dispersibility, and controllable composition for the detection of novel neonicotinoid pesticides.

[0007] Another objective of this invention is to provide a method for preparing and applying the above-mentioned core-shell structured Au@PtNCs nanozyme.

[0008] This invention provides a core-shell structured Au@PtNCs nanoprobe, in which metallic Au forms the core and metallic Pt forms the shell.

[0009] In order to obtain nanoclusters with superior catalytic performance, the average particle size of the above-mentioned core-shell structure Au@PtNCs nanoclusters is 10-40 nm, and the molar ratio of Au to Pt is 1:0.5 to 1:4. This invention provides an Au@PtNCs nanozyme, which is prepared according to the following method: (1) After mixing and stirring glutathione and chloroauric acid, sodium hydroxide solution was added and stirring was continued. The mixture was then reacted at 60~75℃ and 500~900 rpm for 24~36 h. The final solution color was yellow, and AuNCs nanocluster gold seeds were obtained. (2) Disperse the AuNCs nanocluster gold seeds from step (1) into water to obtain an AuNCs nanocluster gold seed solution. After stirring and mixing with polyvinylpyrrolidone, add hexachloroplatinic acid and L-ascorbic acid. React at 60~75 ℃ and 500~900 rpm for 12~14 h. After cooling, Au@PtNCs nanoclusters are obtained, which are Au@PtNCs nanozymes.

[0010] In one embodiment of the present invention, in step (1), the molar ratio of chloroauric acid to glutathione is (0.5~2):(0.5~2); the concentration of chloroauric acid is 10~20 mM, the concentration of glutathione is 50~100 mM; the volume of chloroauric acid added is 0.4~0.8 mL, and the volume of glutathione added is 0.16~0.32 mL.

[0011] In one embodiment of the present invention, after the glutathione and chloroauric acid are mixed, the mixture is stirred for 5 to 10 minutes at 20 to 25°C and 500 to 600 rpm, and a sodium hydroxide solution with a concentration of 0.5 to 1 mM is added. The mixture is then stirred for 24 to 26 minutes at 800 to 900 rpm.

[0012] In one embodiment of the present invention, in step (2), the molar ratio of AuNCs nanocluster gold seed solution to hexachloroplatinic acid is (1~4):(1~4); the molar ratio of PVP to L-ascorbic acid is (1:200):(1:200). In one embodiment of the present invention, the concentration of the AuNCs nanocluster gold seed solution is 5~10 mM, the concentration of hexachloroplatinic acid is 5~10 mM, the concentration of polyvinylpyrrolidone is 0.5~1 mM, and the concentration of L-ascorbic acid is 50~100 mM. The volume of the AuNCs nanocluster gold seed solution is 150~300µL, the volume of hexachloroplatinic acid is 300~600µL, the volume of polyvinylpyrrolidone is 100~200µL, and the volume of L-ascorbic acid is 200~400µL. In one embodiment of the present invention, the stirring conditions with polyvinylpyrrolidone are: 20~25°C, 800~900 rpm for 5~14 min.

[0013] In one embodiment of the present invention, the Au@PtNCs nanoclusters obtained in step (2) further include a purification step, wherein the prepared nanocluster solution is ultrasonically treated, filtered, centrifuged and then reconstituted to obtain purified Au@PtNCs nanoclusters.

[0014] In one embodiment of the present invention, the Au@PtNCs nanoprobe has peroxidase-like (POD) activity; In one embodiment of the present invention, the molar ratio of the metal Au to the metal Pt is 1:(1~4).

[0015] The present invention provides a detection sensor containing the Au@PtNCs nanozyme described above.

[0016] This invention also provides an Au@PtNCs nanozyme probe, which is prepared according to the following steps: (1) The above Au@PtNCs nanozyme was added to ethanol to prepare Au@PtNCs ethanol solution; (2) Add the Au@PtNCs ethanol solution obtained in step (1) to the APTES solution and stir at 25~30℃ and 500~700rpm for 12~14 h; (3) Centrifuge the Au@PtNCs solution obtained after the reaction in step (2), and resuspend the precipitate in PBS buffer to prepare Au@PtNCs dispersion; (4) Centrifuge the Au@PtNCs dispersion prepared in step (3), discard the supernatant, and resuspend it with MES buffer to obtain Au@PtNCs solution; add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution to Au@PtNCs solution in sequence, and react at 4~8℃ and 300~500 rpm for 12~14 h to obtain the reaction system; (5) Add 1~2 μg / μL of goat anti-mouse to the reaction system obtained in step (4), react at 4~8℃ and 300~500 rpm for 12~14 h, centrifuge, discard the uncoupled supernatant, and resuspend the precipitate with PBS buffer to obtain Au@PtNCs nanozyme probe.

[0017] In one embodiment of the present invention, in step (1), the concentration of the Au@PtNCs ethanol solution is 0.5~10 mg / mL; the volume of the Au@PtNCs ethanol solution is 1~2 mL. In one embodiment of the present invention, in step (2), the volume ratio of Au@PtNCs ethanol solution to APTES solution is 1:10 to 1:20; the concentration of APTES solution is 10 to 20 mM. In one embodiment of the present invention, in step (4), the concentration of the Au@PtNCs solution obtained after resuspending with MES buffer is 0.5~1 mg / ml, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 5~10 mM, the concentration of the N-hydroxysuccinimide solution is 10~20 mM, and the concentration ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to the N-hydroxysuccinimide solution is (1~2):(1~2). The volume of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 50-100 μL; the volume of the N-hydroxysuccinimide solution is 50-100 μL. The present invention also provides a kit or device for detecting imidacloprid pesticides, wherein the kit or device contains the above-mentioned Au@PtNCs nanozyme probe.

[0018] This invention also provides the application of Au@PtNCs nanozyme probes in the detection of imidacloprid pesticides or in the preparation of products for detecting imidacloprid pesticides.

[0019] This invention also provides a method for detecting imidacloprid pesticides. The method involves coating and sealing the surface of the wells of an ELISA plate with imidacloprid-BSA conjugate antigen; sequentially adding the test sample and imidacloprid monoclonal antibody to the wells of the ELISA plate and incubating; adding the aforementioned Au@PtNCs nanozyme probe to the system after incubation and continuing incubation; discarding the liquid after incubation and adding TMB chromogenic solution to the wells; and detecting the concentration of imidacloprid pesticides in the test sample using a colorimetric or fluorescence method.

[0020] In one embodiment of the present invention, the concentration of the imidacloprid-BSA conjugate antigen is 0.1~8 μg / mL; In one embodiment of the present invention, the blocking solution is a 5% (w / w) skim milk solution obtained by dissolving skim milk in PBS solution; In one embodiment of the present invention, the coating method is to incubate at 4°C for 12-18 h, and the blocking method is to add blocking solution to the wells of the coated ELISA plate and incubate at 37°C for 1 h. In one embodiment of the present invention, the concentration of the added imidacloprid monoclonal antibody is 0.5 μg / mL; the sample and imidacloprid monoclonal antibody are mixed at a volume ratio of 1:1 and incubated at 37°C for 1 h; In one embodiment of the present invention, Au@PtNCs nanozyme probe at a concentration of 0.5~1 mg / mL is added to the system after incubation and then incubated at 37°C for 1 h.

[0021] In one embodiment of the present invention, the colorimetric method is as follows: 5 mM TMB colorimetric solution is added to the well plate, and the reaction is carried out in the dark for 15 min until the positive wells turn blue. The ultraviolet absorbance value at 652 nm is then measured. The concentration of imidacloprid pesticide in the sample to be tested is calculated using the following formula. Colorimetric inhibition rate (%) = ; The linear equation is Y = 29.91203X + 30.941, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.993; In one embodiment of the present invention, the fluorescence method is as follows: 5 mM TMB colorimetric solution is added to the well plate, and the reaction is carried out in the dark for 15 min. During the colorimetric process, 120 μL of Rhodamine 6G solution is added to quench the fluorescence, and the fluorescence intensity at 550 nm is measured under 525 nm wavelength excitation. The concentration of imidacloprid pesticide in the sample to be tested was calculated using the following formula; The fluorescence inhibition rate (%) is: ; The linear equation is Y = 27.8396X + 28.43217, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.991 In some embodiments, the novel neonicotinoid pesticide is imidacloprid (IMI).

[0022] This invention also provides a preparation method and application for extracting and detecting imidacloprid pesticides from fruits and vegetables, the method comprising the following steps: (1) Imidacloprid pesticide is extracted from bananas mainly by liquid-solid extraction, which requires first grinding and homogenizing to collect a uniform slurry; (2) Add 20 mL of methanol (extraction solvent) to 10 g of slurry, then add 4 g of anhydrous magnesium sulfate (dehydrating agent) and 1 g of sodium chloride (salting-out agent), vortex for 2 minutes, and finally extract by ultrasound and centrifugation; (3) Add the sample extracted in the previous step to a 15 mL centrifuge tube. The centrifuge tube contains a small amount of solid-phase extraction adsorbent (150 mg PSA + 15 mg GCB) and 900 mg MgSO4. Vortex for 2 minutes, and finally perform ultrasonic and centrifugal extraction. (4) Dilute the sample 10 times with methanol to obtain the supernatant, purify the sample through a 0.22 μm organic filter membrane, and finally add PBS buffer solution to ensure that the methanol ratio is 10%; (5) Construct a standard curve for the detection of imidacloprid pesticide. Prepare 7 to 9 standard concentration solutions of imidacloprid pesticide with different concentrations in the range of 0 to 100 μg / mL, including a blank imidacloprid pesticide solution. Add the Au@PtNCs nanozyme probe described in the first technical solution. In colorimetric mode, as the concentration of imidacloprid pesticide increases, the specific binding sites of the Au@PtNCs nanozyme probe surface and the BSA-hapten on the microtiter plate decrease, the catalytic effect decreases, and finally the catalytic signal at 652 nm steadily decreases. In fluorescence mode, as the concentration of imidacloprid pesticide increases, the catalytic signal at 652 nm steadily weakens, the fluorescence quenching weakens, resulting in an enhanced fluorescence signal. Finally, obtain the mapping relationship between the imidacloprid pesticide concentration and the colorimetric-fluorescence signal intensity.

[0023] (6) Disperse the spiked sample to be tested in methanol. The spiked sample can be processed in sequence according to steps (1) to (3). Add the Au@PtNCs nanozyme probe described in the first technical solution, and record the linear relationship between the ultraviolet absorption light intensity value of the Au@PtNCs nanozyme probe in colorimetric mode and the fluorescence intensity value in fluorescence mode and the concentration of imidacloprid pesticide and the recovery rate. The recovery rate of the spiked sample to be tested can be determined by the mapping relationship obtained in step (5).

[0024] The above detection method has a low detection limit, high sensitivity, and a good mapping function relationship.

[0025] In some implementations, the sample in step (1) needs to be frozen with liquid nitrogen after being chopped.

[0026] In some embodiments, the sample in step (2) is preferably extracted by ultrasonic power of 300 W, temperature of 25°C, centrifugation speed of 4000 rpm, and time of 5 minutes.

[0027] In some embodiments, the concentration of the PBS buffer solution in step (4) is preferably 10 mM and the pH value is preferably 7.4.

[0028] In some implementations, the pH of the MES buffer in step (5) is preferably 5.5.

[0029] In some embodiments, the centrifugation speed in step (5) is preferably 12000 r / min, the time is preferably 10 min and 15 min respectively, and the low temperature environment needs to be maintained at 4°C.

[0030] In some embodiments, the ratio of the diluted supernatant to the PBS buffer in step (5) is preferably 1:(5-9), and the supernatant is preferably 55 μL to 100 μL.

[0031] In some embodiments, the concentrations of TMB and H2O2 in step (5) are preferably 5 mM and 50 mM, respectively.

[0032] In some embodiments, the centrifugation speed in step (6) is preferably 7500 r / min, the time is preferably 5 min, and it is preferably carried out at a low temperature environment of 4°C.

[0033] In some embodiments, the concentration of imidacloprid pesticide in step (6) is preferably in the range of 0.1 to 100 μg / L in colorimetric mode and in the range of 0.1 to 100 μg / L in fluorescence mode.

[0034] Beneficial effects This invention provides an Au@PtNCs nanozyme probe technology and its preparation method for detecting novel neonicotinoid pesticides. The invention optimizes the reagent ratio, temperature, pH, rotation speed, and time in the reaction system of a core-shell structured Au@PtNCs nanoclusters, ultimately synthesizing Au@PtNCs nanoclusters with small particle size, uniform morphology distribution, strong fluorescence intensity, and strong plasmon resonance peaks. Further optimization of the reaction conditions in the TMB + H2O2 reaction system, such as temperature, pH, time, and substrate ratio, yields an Au@PtNCs nanozyme with the highest peroxidase-like activity. This invention is used for the specific binding of imidacloprid to the Au@PtNCs nanozyme probe. In systems using novel neonicotinoid pesticides in vegetables and fruits with detection probes, as the concentration of imidacloprid pesticide increases, the UV absorption intensity at 652 nm gradually decreases, while the fluorescence intensity at 404 nm increases under 320 nm excitation, forming a certain mapping function relationship. In the presence of imidacloprid, the Au@PtNCs nanozyme probe, acting as a biorecognition element, preferentially binds to the imidacloprid antibody. Meanwhile, the imidacloprid pesticide and OVA-hapten compete for the antibody on the Au@PtNCs in the 96-well plate. Increasing the imidacloprid concentration reduces the binding of the Au@PtNCs nanozyme probe to the OVA hapten and also decreases the concentration of Au@PtNCs on the 96-well plate, thus generating a corresponding signal. Colorimetric signals show an inverse linear relationship with imidacloprid concentration, while fluorescence signals show a positive linear relationship, thereby determining the imidacloprid pesticide content in the sample. Attached Figure Description

[0035] Figure 1 This is a synthetic route diagram of the Au@PtNCs nanozyme in this invention.

[0036] Figure 2 The mapping morphology characterization diagrams for AuNC, PtNCs, and Au@PtNCs are shown.

[0037] Figure 3 The particle size distribution diagrams are for AuNC, PtNCs, and Au@PtNCs.

[0038] Figure 4 STEM and lattice fringes and X-ray diffraction (XRD) of AuNC, PtNCs, and Au@PtNCs composite nanoclusters.

[0039] Figure 5 X-ray photoelectron spectroscopy (XPS) of AuNC, PtNCs, and Au@PtNCs composite nanoclusters.

[0040] Figure 6Comparison of UV absorption spectra (left) and fluorescence spectra (right) of AuNC, PtNCs, and Au@PtNCs composite nanoclusters.

[0041] Figure 7 The results of the comparative test on the optimization of Au@PtNCs nanozymes under different reaction pH (left) and temperature (right) conditions in the TMB + H2O2 reaction system are shown.

[0042] Figure 8 The results of the optimization test of the catalytic activity of Au@PtNCs nanozymes in the TMB + H2O2 reaction system (left) and H2O2 (right) are shown, along with the kinetic curve of the Au@PtNCs nanozymes.

[0043] Figure 9 Dynamic light scattering (DLS) characterization of Au@PtNCs nanozymes before and after conjugation with secondary antibody (goat anti-mouse).

[0044] Figure 10 The curves show the changes in UV absorption intensity (left) and fluorescence intensity (right) before and after conjugation of Au@PtNCs nanozyme with secondary antibody (goat anti-mouse).

[0045] Figure 11 Test results optimized for Au@PtNCs nanoprobes under different coating antigen and imidacloprid concentrations.

[0046] Figure 12 The graphs and standard curves show the changes in UV absorption intensity (left) and fluorescence intensity (right) after the Au@PtNCs nanozyme probe recognizes imidacloprid (IMI). Detailed Implementation

[0047] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0048] This invention provides a core-shell structured Au@PtNCs nanozyme probe, the preparation and principle of which are described below.

[0049] By adjusting the solvent ratios of gold (Au), platinum (Pt), ligands glutathione (GSH), polyvinylpyrrolidone (PVP), and reducing agent L-ascorbic acid (VC), and optimizing reaction conditions such as temperature, time, rotation speed, and pH, Au@PtNCs nanoclusters with smaller particle size, stable and uniform morphology, strong fluorescence intensity, and strong plasmon resonance peaks were screened and characterized.

[0050] The selected Au@PtNCs nanoclusters were subjected to optimized reaction conditions in a TMB + H2O2 reaction system, such as temperature, pH, time, and the ratio of the reaction substrates TMB and H2O2, to obtain an Au@PtNCs nanozyme with the highest peroxidase-like activity.

[0051] Based on the high peroxidase-like activity of this Au@PtNCs nanozyme In colorimetric mode As the concentration of imidacloprid pesticide increases, the specific binding sites of the Au@PtNCs nanozyme probe surface to the OVA-hapten on the microtiter plate decrease, resulting in reduced catalytic activity and a significant change in the amplified catalytic signal at 652 nm. This is also based on fluorescence resonance energy transfer (FRET) and surface-enhanced fluorescence (SEF) techniques. In fluorescence mode An "OFF-enhanced ON" switch was constructed. As the concentration of imidacloprid pesticide increased, the catalytic signal at 652 nm steadily weakened. The fluorescence intensity was quenched from "OFF" to "enhanced ON", resulting in a significant fluorescence amplification signal. The weakening of fluorescence quenching led to the enhancement of the fluorescence signal. Finally, the mapping relationship between the concentration of imidacloprid pesticide and the colorimetric-fluorescence signal intensity was obtained.

[0052] As an embodiment of the present invention, an Au@PtNCs nanozyme probe for detecting novel neonicotinoid pesticides is described, and the synthetic route of the probe is as follows: Figure 1 First, gold nanoclusters (AuNCs) need to be synthesized, and then linked with platinum through metal bonds to synthesize gold-platinum nanoclusters (Au@PtNCs). Finally, they are coupled with secondary antibody (goat anti-mouse) to form Au@PtNCs nanozyme probes.

[0053] Raw materials used in the examples: The chloroauric acid (HAuCl4, 99%), chloroplatinic acid (H2PtCl6, 99%), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), 3-aminopropyltriethoxysilane (APTES, 99.9%), and methanol (99.9%) used in this invention were purchased from Sinopharm Chemical Reagent Co., Ltd.; L-ascorbic acid (VC, 99%), polyvinylpyrrolidone (PVP, 99%), sodium borohydride (NaBH4, 99%), dimethyl sulfoxide (DMSO, 99.9%), and glutathione (GSH, 99%) were also used. 3,3',5,5'-Tetramethylbenzidine (TMB), citric acid dihydrate (Na3C6H5O7·2H2O, 99%), hydrogen peroxide (30%), and hexadecyltrimethylammonium bromide (CTAB, 99%) were all purchased from Suzhou Great Medical Technology Co., Ltd.; imidacloprid standard solution (IMI), dinotefuran standard solution (DIN), thiamethoxam standard solution (TMX), dimethoate standard solution (DIM), chlorpyrifos standard solution (CPF), and cypermethrin standard solution were all purchased from Shanghai Mairui Biochemical Technology Co., Ltd.; ethanol (70%), MES buffer (pH 6.0), PBS buffer (pH 7.4), carbonate buffer (CBS) (pH 9.6), and PBST (PBS + 0.05% Tween-20) were all purchased from Beyotime Biotechnology Co., Ltd.; imidacloprid monoclonal antibody and imidacloprid-BSA conjugate antigen were both purchased from Fisher-Price (Hangzhou) Medical Research Co., Ltd.

[0054] All reagents used in the following examples were not further treated before use. The glassware and magnetic stirring rotors used were soaked in freshly prepared aqua regia (volume of concentrated hydrochloric acid:volume of concentrated nitric acid = 3:1) for 1 hour, rinsed with plenty of water for 5 minutes, rinsed several times with ultrapure water, and finally dried for use to ensure that there were no metal ions remaining in the glass containers.

[0055] The detection methods involved in the following embodiments: Method for calculating the colorimetric inhibition rate: Colorimetric inhibition rate (%) = ; Note: The blank control only contains antibody and buffer solution, without standard.

[0056] Calculation method for colorimetric recovery rate: Colorimetric recovery rate (%): Detection concentration / Spiked concentration × 100%; Note: The detection concentration was obtained from the standard curve of inhibition rate versus IMI concentration; Method for calculating colorimetric RSD: .

[0057] Methods for calculating the inhibition rate using fluorescence assay: The fluorescence inhibition rate (%) is: ; Note: Blank control: only antibody and buffer solution are added, no standard is added; Negative control: only buffer solution is added, no antibody and standard are added. Calculation method for fluorescence recovery rate: Fluorescence recovery rate (%): detection concentration / spiked concentration × 100%; Note: The detection concentration was obtained from the standard curve of inhibition rate versus IMI concentration; Methods for calculating RSD using fluorescence method: Fluorescence RSD (%): .

[0058] Example 1: Synthesis of gold-platinum nanoclusters (Au@PtNCs) The specific steps are as follows: 1. Synthesis of Au@PtNCs nanoclusters (1) Glutathione-modified gold seed solution: 0.8 mL of 20 mM chloroauric acid (HAuCl4) was added to 0.32 mL of 100 mM glutathione (GSH) (i.e., the molar ratio of HAuCl4 to GSH was 1:2). The mixture was stirred vigorously at 25 °C at a speed of 500 rpm for 5 min. Then, 0.1 mL of 1 M NaOH and 8.88 mL of ultrapure water were added to bring the total volume to 10.0 mL. The mixture was stirred continuously at 500 rpm until the turbid solution became clear and transparent. The clear and transparent solution was then placed in a 70 °C water bath and stirred vigorously at 800 rpm for 24 h. The final solution color was yellow, thus obtaining the AuNCs nanocluster gold seed solution.

[0059] (2) Synthesis of gold-platinum nanoclusters The AuNCs nanocluster gold seed solution obtained in step (1) was diluted to 10 mM. 300 µL of the 10 mM AuNCs nanocluster gold seed solution was placed in a 25°C water bath and stirred vigorously (800 rpm). Then, 200 µL of 1 mM polyvinylpyrrolidone (PVP) was added and stirred (800 rpm) for 5 minutes. Then, 600 µL of 10 mM hexachloroplatinic acid (H2PtCl6) (i.e., the molar ratio of AuNCs to H2PtCl6 is 1:2) and 400 µL of 100 mM L-ascorbic acid (VC) (i.e., the molar ratio of VC to PVP is 200:1) were added. The mixture was stirred (800 rpm) and heated to 70°C. After the solution color changed from yellow to black, the reaction was continued for 12 h. Heating was then stopped and the mixture was cooled to room temperature to obtain the Au@PtNCs nanocluster solution.

[0060] (3) Purify Au@PtNCs nanoclusters.

[0061] The prepared Au@PtNCs nanocluster solution was treated with ultrasound (power: 200W) for 5 minutes, and then the solution was ultrafiltered and centrifuged using a 3 kDa ultrafiltration tube. The solution in the filter tube was then taken and centrifuged 3 more times according to the above steps. The final filtrate was redissolved in 1 mL of ultrapure water. The final solution was stored at 4 °C for further use.

[0062] The ultrafiltration centrifugation speed needs to be kept stable at 6500~7000 r / min, the temperature at 4℃, and the time at 15~25min.

[0063] The resulting Au@PtNCs nanoclusters are denoted as Au@PtNCs nanoclusters-1.

[0064] 2. Synthesis of Au@PtNCs nanoclusters under different reaction conditions (1) Specifically the same as step 1, except that the molar ratio of HAuCl4 and GSH in step (1) of step 1 is adjusted to 1:1 or 2:1; Au@PtNCs nanoclusters-2 (1:1) and Au@PtNCs nanoclusters-3 (2:1) were prepared according to the method in step 1. (2) Specifically the same as step 1, except that the ratio of AuNCs and H2PtCl6 in step (2) of step 1 is adjusted to 1:1 or 2:1; Au@PtNCs nanoclusters-4 (1:1) and Au@PtNCs nanoclusters-5 (2:1) were prepared according to the method in step 1. (3) Specifically the same as step 1, except that the ratio of PVP to VC in step (2) of step 1 is adjusted to 1:1 and 200:1; Au@PtNCs nanoclusters-6 (1:1) and Au@PtNCs nanoclusters-7 (200:1) were prepared according to the method in step 1. 3. The Au@PtNCs nanoclusters prepared in steps 1 and 2 were characterized respectively. The results show: (1) Determination of the molar ratio of HAuCl4 and GSH 1) Au@PtNCs nanoclusters-1: Au@PtNCs nanoclusters-1 were synthesized under the following conditions: a molar ratio of HAuCl4 to GSH of 1:2, a molar ratio of AuNCs to H2PtCl6 of 1:2, and a molar ratio of PVP to VC of 1:200. Based on dynamic light scattering (DLS) characterization, the particle size was found to be in the range of 20-30 nm. Based on mapping morphology characterization, a stable and uniform morphology was obtained. 2) Au@PtNCs nanoclusters-2: Au@PtNCs nanoclusters-2 were synthesized under the following conditions: a molar ratio of HAuCl4 to GSH of 1:1, a molar ratio of AuNCs to H2PtCl6 of 1:2, and a molar ratio of PVP to VC of 1:200. According to the dynamic light scattering (DLS) characterization, the particle size ranged from 60 to 80 nm, and the particle size distribution was uneven and the particles were relatively large. 3) Au@PtNCs nanoclusters-3: Under the conditions of a molar ratio of HAuCl4 to GSH of 2:1, a molar ratio of AuNCs to H2PtCl6 of 1:2, and a molar ratio of PVP to VC of 1:200, the synthesized Au@PtNCs nanoclusters were characterized by dynamic light scattering (DLS) and showed a particle size range of 40-70 nm, with uneven particle size distribution and relatively large particles.

[0065] (2) Determination of the ratio of AuNCs and H2PtCl6 1) Au@PtNCs nanoclusters-4: Under the conditions of a molar ratio of HAuCl4 to GSH of 1:2, a molar ratio of AuNCs to H2PtCl6 of 1:1, and a molar ratio of PVP to VC of 1:200, the synthesized Au@PtNCs nanoclusters were characterized by dynamic light scattering (DLS) and showed a particle size range of 20-50 nm, with uneven particle size distribution and relatively large particles. 2) Au@PtNCs nanoclusters-5: Under the conditions of a molar ratio of HAuCl4 to GSH of 1:2, a molar ratio of AuNCs to H2PtCl6 of 2:1, and a molar ratio of PVP to VC of 1:200, the synthesized Au@PtNCs nanoclusters were characterized by dynamic light scattering (DLS) and showed a particle size range of 90-100 nm, with uneven particle size distribution and relatively large particles.

[0066] (3) Determination of the molar ratio of PVP and VC 1) Au@PtNCs nanoclusters-6: Under the conditions of a molar ratio of HAuCl4 to GSH of 1:2, a molar ratio of AuNCs to H2PtCl6 of 1:2, and a molar ratio of PVP to VC of 1:1, the synthesized Au@PtNCs nanoclusters were characterized by dynamic light scattering (DLS) and showed a particle size range of 50-80 nm, with uneven particle size distribution and relatively large particles. 2) Au@PtNCs nanoclusters-7: Under the conditions of a molar ratio of HAuCl4 to GSH of 1:2, a molar ratio of AuNCs to H2PtCl6 of 1:2, and a molar ratio of PVP to VC of 200:1, the synthesized Au@PtNCs nanoclusters were characterized by dynamic light scattering (DLS) and showed a particle size range of 40-90 nm, with uneven particle size distribution and relatively large particles.

[0067] 4. The Au@PtNCs nanoclusters-1 prepared in step 1, which showed the best results, were characterized. (1) Synthesis of AuNCs nanoclusters and PtNCs nanoclusters 1) Synthesis of AuNCs nanoclusters: AuNCs nanocluster gold seed solution was prepared according to step (1) of step 1 above; after purifying the above AuNCs nanocluster gold seed solution according to step (3) of step 1, glutathione-modified AuNCs nanocluster solution was prepared. 2) Synthesis of PtNCs nanoclusters: Preparation method of polyvinylpyrrolidone-modified PtNCs nanoclusters solution: 1.5 mL of 20 mM chloroplatinic acid (H2PtCl6) was added to 1 mL of 10 mM polyvinylpyrrolidone (PVP). The mixture was stirred vigorously at 45 °C at a speed of 800 rpm for 5 min. Then, 1 mL of 10 mM L-ascorbic acid (VC) solution was slowly added to the mixture. The reaction was continued for 12 h. The final color of the mixture turned dark brown, indicating that PtNCs nanoclusters were successfully synthesized.

[0068] Au@PtNCs nanoclusters-1 were prepared according to the method in Example 1.

[0069] (2) The AuNCs nanoclusters, PtNCs nanoclusters and Au@PtNCs nanoclusters-1 were characterized respectively.

[0070] The results show: 1) By Figure 2 It can be seen that mapping morphology characterization of AuNC, PtNCs, and Au@PtNCs reveals them to be clustered nanomaterials with a gold-platinum core-shell structure. 2) By Figure 3 It can be seen that comparing the particle size distribution diagrams of single metals (AuNC, PtNCs) and bimetals (Au@PtNCs) ( Figure 3 The size of bimetallic particles varies compared to that of monometallic particles. 3) By Figure 4 As can be seen, X-ray diffraction (XRD) shows that the center positions of the single metals Au and Pt are approximately 2θ = 38.2. ° and 39.8 ° The crystal structure is well-preserved, and the Au@PtNCs nanoclusters-1 have a 2θ of 39.0 between the two monometals. ° Good crystallization was formed at that location, and based on STEM and comparison with lattice fringes, d was obtained. Au =0.235 nm, d Pt =0.226 nm ( Figure 4 ); 4) By Figure 5 As can be seen, X-ray photoelectron spectroscopy (XPS) shows that the characteristic peaks of Au4f and Pt4f are located at 84.0 and 72.0 eV, respectively. Therefore, Au@PtNCs nanoclusters-1 are mainly composed of Au(0) as the core and Pt(I) distributed on the surface. Figure 5 ); 5. Detection of enzyme activity in Au@PtNCs nanoclusters-1, compared with that of natural horseradish peroxidase (HRP): (1) The specific reaction is: The total volume of the enzymatic reaction system was 2 mL: 100 μL of H2O2 solution (50 mM) and 100 μL of TMB (5 mM) were added to HAc-NaAc buffer (0.1 M) at pH=4. The reaction was carried out at 37 ℃ for 1 min. Then, 50 µL of Au@PtNCs nanocluster-1 solution (0.5 mg / mL) prepared in Example 1 was added (with 50 µL of HRP at a concentration of 0.05 mg / mL as a control). The reaction was carried out at 37 ℃ for 10 min. The change in absorbance at 652 nm was detected using an ELISA reader.

[0071] (2) Optimization of reaction conditions: 1) The specific conditions are the same as in step (1) above, except that the pH is adjusted to 2, 4, 6, 8, 10, and 12 respectively; The results show: Au@PtNCs nanozyme reaction: pH adjusted to 2. 4 At 6, 8, 10, and 12 o'clock, the enzyme activities of Au@PtNCs nanozymes were 0.975, ... 1.449 1.359, 1.175, 1.153, 0.738; HRP enzyme reaction: The absorbance at pH values ​​of 2, 4, 6, 8, 10, and 12 were 0.735, 1.336, 1.163, 1.012, 0.673, and 0.327, respectively. 2) The specific conditions are the same as in step (1) above, except that the reaction temperature is adjusted to 4℃, 25℃, 40℃, 60℃, 80℃ and 100℃ respectively. The results show: Au@PtNCs nanozyme reaction: The temperature was adjusted to 4℃ and 25℃ respectively. 40℃ At 60℃, 80℃, and 100℃, the enzyme activities of Au@PtNCs nanozymes were 0.492, 0.984, and 0.984, respectively. 1.477 1.342, 1.120, 1.084; HRP enzyme reaction: The absorbance at 4℃, 25℃, 40℃, 60℃, 80℃, and 100℃ were 1.665, 2.519, 1.805, 1.161, 0.528, and 0.201, respectively. 3) The specific conditions are the same as in step (1) above, except that: after adding Au@PtNCs nanozyme solution, different reaction times are set to 10, 20, 30, 40, 50 and 60 min respectively; the reaction temperature is adjusted to 40℃; The results show: Au@PtNCs nanozyme reaction: When the reaction time was adjusted to 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, the enzyme activities of Au@PtNCs nanozymes were 0.804, 1.279, 1.424, 1.646, and 1.863, respectively. 1.948 ; HRP enzyme reaction: The absorbance at times of 10, 20, 30, 40, 50, and 60 min were 0.883, 1.473, 1.863, 2.265, 1.542, and 1.049, respectively. The experimental results are as follows: 1) By Figure 6 As shown in the UV absorption spectrum on the left, AuNCs, PtNCs, and Au@PtNCs nanoclusters all exhibit a significant absorption peak at 652 nm. Among the three, Au@PtNCs shows the highest absorbance, indicating the highest POD-like enzyme activity. This demonstrates that Au@PtNCs nanozymes possess high peroxidase-like activity in the TMB-H2O2 system. Under acidic conditions (HAc-NaAc buffer, pH=4.0), H2O2 decomposes at the catalytic center on the nanozyme surface, forming intermediates ·OH and H2O. The ·OH intermediate then captures an H ion from TMB. + The colorless TMB is oxidized and converted into the blue product oxTMB.

[0072] 2) By Figure 6 As shown in the fluorescence spectra on the right, AuNC, PtNCs, and Au@PtNCs nanoclusters all exhibit significant fluorescence at 760 nm. Based on the intensity comparison, AuNCs shows the highest fluorescence intensity, while the fluorescence of Au@PtNCs nanoclusters, which combine two single metals, falls in the middle. The results demonstrate that Au@PtNCs nanoclusters possess optical properties (UV absorption intensity and fluorescence intensity).

[0073] Within 60 minutes, the UV absorption values ​​of Au@PtNCs nanozyme and HRP gradually increased with time, and after reaching a certain level, the UV absorption value of HRP gradually decreased.

[0074] 3) By Figure 7As shown on the left side, with the increase of the pH value of the HAc-NaAc buffer, the UV absorption values ​​of both Au@PtNCs nanocluster enzyme and HRP gradually increase. After reaching a certain level, the UV absorption values ​​gradually decrease with further increases in pH. The UV absorption intensity reaches its maximum at pH 4 for both Au@PtNCs nanocluster enzyme and HRP. Therefore, pH 4 is usually chosen for the HAc-NaAc buffer, and Au@PtNCs nanocluster enzyme exhibits higher catalytic activity than HRP.

[0075] 4) By Figure 7 As shown on the right side, with increasing temperature, the UV absorption value of Au@PtNCs nanozymes gradually increases, reaching a certain level before gradually decreasing with further temperature increases. The catalytic activity of Au@PtNCs nanozymes reaches its maximum at approximately 40℃; therefore, a reaction temperature of around 40℃ is typically chosen, as Au@PtNCs nanozymes exhibit high stability in catalytic activity. In contrast, the absorbance value of HRP decreases rapidly with increasing temperature after reaching a certain level.

[0076] Therefore, in summary, the stability of the catalytic activity of Au@PtNCs nanozymes is higher than that of HRP.

[0077] 6. Steady-state dynamics measurement (1) The specific method is as follows: HAc-NaAc buffer (0.1 M, pH=4.0) and 50 µL of Au@PtNCs solution (control group: deionized water) with a concentration of 0.5 mg / mL are added to a total volume of 2 mL of enzyme reaction system. Then, 100 µL of TMB with a concentration of 5 mM and 100 µL of H2O2 solution with a concentration of 50 mM are added. Under the condition of keeping warm at 37℃, the change of UV absorbance at 652 nm of the system within 60 min is detected.

[0078] (2) Optimization of reaction conditions The specific conditions are the same as above, except that the H2O2 concentrations are set to be 10 mM, 20 mM, 40 mM, 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, and 160 mM respectively. The results showed that the absorbance changes ΔA were: 0.050, 0.097, 0.121, 0.146, 0.158, 0.182, 0.186, 0.187, and 0.188. Rate V (absorbance change rate ΔA / min): 2.12, 4.13, 5.16, 6.22, 6.74, 7.79, 7.93, 8.00, 8.05; The specific conditions are the same as above, except that different concentrations of TMB are set as follows: 1, 5, 10, 15, 20, 25, 30, 35, and 40 mM. The results showed that the absorbance changes ΔA were: 0.020, 0.071, 0.094, 0.119, 0.131, 0.153, 0.159, 0.160, and 0.161. Rate V (absorbance change rate ΔA / min): 0.87, 3.04, 4.01, 5.07, 5.59, 6.54, 6.78, 6.85, 6.90; The experimental results are as follows: By measuring the reaction rate at different substrate concentrations and fitting the Michaelis-Menten equation, K was obtained. m (Michaelis constant, reflecting the enzyme's affinity for its substrate: K) m The smaller the size, the stronger the affinity) and V max (Maximum reaction rate, reflecting the upper limit of the enzyme's catalytic capacity), determined by... Figure 8 Steady-state kinetics curves show that the Au@PtNCs nanozyme in the TMB + H2O2 reaction system was determined to contain TMB (… Figure 8 (on the left) and H2O2 ( Figure 8 The steady-state kinetic parameters Km and Vmax (on the right side of the diagram) are shown. The results indicate that the POD-like activity of the Au@PtNCs nanozyme conforms to Michaelis-Menten kinetics.

[0079] Based on the calculation results: The Km and Vmax values ​​of Au@PtNCs nanozymes against TMB solution were 0.03077 mM and 16.39 × 10⁻⁶ mM, respectively. -8 .

[0080] The Km and Vmax values ​​of Au@PtNCs nanozymes against H2O2 solution were 0.03404 mM and 9.99 × 10⁻⁶ mM, respectively. -8 .

[0081] Compared with HRP and other nanozymes, Au@PtNCs nanozymes have a lower Km value and a larger Vmax. Comparative Example 1: The specific steps are the same as in Example 1, except that the different morphologies and particle sizes of the materials are adjusted. Gold nanospheres (AuNPs) and gold nanorods (AuNRs) were prepared respectively. Morphological characterization of single-metal gold showed that AuNPs were spherical with a particle size of 5-20 nm, while AuNRs were rod-shaped with a particle size of 10-50 nm. The reaction was carried out under the optimal reaction conditions according to step 5 of Example 1. The results showed that the enzyme activity of AuNPs nanozyme was 0.461~0.714; the enzyme activity of AuNRs nanozyme was 0.239~0.603.

[0082] Example 2: Construction of Au@PtNCs nanozyme probe The specific steps are as follows: 1. Functionalization of Au@PtNCs surfaces.

[0083] (1) The Au@PtNCs nanocluster-1 solution synthesized in step 1 of Example 1 was freeze-dried, 10 mg of powder was added to 1 ml of anhydrous ethanol, and ultrasonically dispersed for 5-10 min with an ultrasonic cleaner so that Au@PtNCs were uniformly dispersed in ethanol again.

[0084] (2) Slowly add 1 ml of the pretreated Au@PtNCs ethanol solution (10 mg / ml) from step (1) to 10 mL of 10 mM APTES (3-(azidopropyl)triethoxysilane) solution (i.e., the ratio of Au@PtNCs ethanol solution to APTES solution is 1:10). Stir the reaction on a magnetic stirrer (500 rpm) at room temperature (25℃) for 12 hours, with ultrasonic assistance during the reaction. Sonicate for 5-10 minutes every 4 hours (power: 10-20 W) to promote the reaction. This allows the silanol groups generated by the hydrolysis of APTES to undergo a condensation reaction with the hydroxyl groups and other groups on the surface of Au@PtNCs, thereby introducing amino groups onto the surface of Au@PtNCs.

[0085] (3) Centrifuge the aminated Au@PtNCs solution obtained after the reaction in step (2). The centrifugation speed should be kept stable at 8000~10000 r / min, the temperature should be 4℃, and the time should be kept at 10~15 min. Finally, resuspend the precipitate in PBS buffer and store it in a refrigerator at 4℃ for later use to prepare the Au@PtNCs dispersion.

[0086] 2. Effects of different reaction conditions on the surface functionalization of Au@PtNCs The specific steps are the same as in step 1, except that the ratio of Au@PtNCs ethanol solution to APTES solution is 1:20. The results showed that the ratio of Au@PtNCs ethanol solution to APTES solution was 1:10 to 1:20. Characterization revealed that the particle size changed with different ratios. When the ratio was 1:10, the functionalized particle size was 25 nm to 30 nm, an increase of about 5 nm. When the ratio was 1:20, the particle size increased by about 20 nm, reaching 40 nm to 50 nm.

[0087] Therefore, the final ratio of Au@PtNCs ethanol solution to APTES solution was chosen to be 1:10, which enabled the surface functionalization of Au@PtNCs.

[0088] 3. Au@PtNCs conjugated with secondary antibodies.

[0089] Centrifuge 1 mL of Au@PtNCs dispersion prepared in step 1 (8000~10000 r / min, temperature 4℃, time 10~15 min), discard the supernatant, and resuspend in 1 mL of MES buffer (pH = 6.0) to obtain Au@PtNCs solution.

[0090] Add 5 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) solution and 10 mM NHS (N-hydroxysuccinimide) solution sequentially to Au@PtNCs solution (i.e., the ratio of EDC to NHS is 1:2), shake at 4 ℃ for 3 h to fully activate the carboxyl groups and obtain the reaction system.

[0091] Then, add 5 μL of secondary antibody (goat anti-mouse) with a concentration of 2 μg / μL to the reaction system, gently shake at 4℃ for 12 h, and finally centrifuge (5000~7000 r / min, temperature 4℃, time 5~10 min), discard the uncoupled supernatant, resuspend the precipitate with PBS buffer (pH 7.4), and store it in a 4℃ refrigerator for later use.

[0092] 4. Effects of different reaction conditions on the conjugation effect of Au@PtNCs to secondary antibodies. The specific steps are the same as in step 3, except that the ratio of EDC to NHS usage is adjusted to 1:1. The specific steps are the same as in step 3, except that the ratio of EDC to NHS usage is adjusted to 2:1. The results showed that when the ratio of EDC to NHS solution was 1:2 (step 3), Au@PtNCs nanozyme probes with a particle size of 50-60 nm, high ion resonance peaks, and high fluorescence intensity were obtained by characterization through dynamic light scattering (DLS), ultraviolet absorption spectroscopy, and fluorescence spectroscopy.

[0093] Depend on Figure 9 Dynamic light scattering (DLS) revealed that the particle size of the Au@PtNCs nanozyme probe continuously increased from the synthesis of monometallic to bimetallic compounds and after coupling with the secondary antibody (step 3), a phenomenon consistent with the synthesis rules of the complex.

[0094] Depend on Figure 10 As can be seen from the UV absorption spectrum in the left figure, the Au@PtNCs nanozyme has a characteristic absorption peak at 520 nm, and the secondary antibody has an absorption peak at 280 nm. After the two are coupled, the absorption wavelength of the Au@PtNCs nanozyme probe (step 3) shows a certain degree of red shift. The fluorescence spectrum in the right figure shows that the fluorescence emission peak of the Au@PtNCs nanozyme at 505 nm was measured under excitation at a wavelength of 395 nm. After the two are coupled, the fluorescence emission peak shows a certain degree of red shift. The above phenomena indicate that the Au@PtNCs nanozyme and the secondary antibody have been successfully coupled.

[0095] Example 3: Construction of Standard Curve The construction of a standard curve for detecting imidacloprid pesticide based on Au@PtNCs nanozyme probe (step 3 of Example 2) includes the following steps: 1. The optimal concentrations of the coating antigen and antibody were optimized using the checkerboard method.

[0096] The specific steps are as follows: 1) Dilute the imidacloprid-BSA conjugate antigen with carbonate buffer (CBS) to concentrations of 0.1, 0.5, 1, 2, 4, and 8 μg / mL. Add 100 μL of the diluted antigen solution to each well of a 96-well plate, one dilution per row. Seal the wells of the 96-well plate with sealing film to prevent liquid evaporation. Wrap the plate in plastic film and incubate at 4°C overnight (12-18 h) to allow the antigen to fully adsorb onto the surface of the wells. After incubation, discard the coating solution. Add 200 μL of washing buffer (PBST) to each well, allowing it to soak for 30 s each time, washing 3-5 times, and then pat dry on absorbent paper. Add 200 μL of blocking buffer (5% skim milk dissolved in PBS) to each well, incubate the plate at 37°C for 1 h, discard the blocking buffer, wash 3-5 times with washing buffer, and then pat dry on absorbent paper.

[0097] 2) Prepare a standard solution of 10 μg / mL by dissolving imidacloprid pesticide standard in methanol. Dilute 4 mg / mL imidacloprid monoclonal antibody (primary antibody) with PBS buffer at different dilution ratios of 1:2000, 1:4000, 1:8000 and 1:64000. Mix the imidacloprid standard and imidacloprid monoclonal antibody at a 1:1 volume ratio. Add 100 μL of the mixture to each well after patting dry in step 1), incubate at 37°C for 1 hour for each dilution, discard the blocking solution, wash 3-5 times with washing solution, and then pat dry on absorbent paper.

[0098] 3) Using the Au@PtNCs nanozyme probe described in Example 2 as a biorecognition molecule, dilute the Au@PtNCs nanozyme probe stock solution prepared in Example 2 to 1:2000 with PBS buffer; Add 100 μL to each well after patting dry in step 2), incubate at 37°C for 1 hour, discard the liquid in the wells, wash the microplate 3-5 times with washing buffer, and pat dry.

[0099] 4) Add 100 μL of TMB colorimetric solution to each well and react at room temperature in the dark for 15 min. Develop the color until the positive well turns blue, and quickly measure the UV absorbance at 652 nm.

[0100] The results show: 1) When the imidacloprid-BSA conjugate antigen is greater than 1 μg / mL and the primary antibody is less than or equal to 1:4000, the OD value is close to 2.0 (e.g., 4 μg / mL + 1:2000 combination OD=2.281). At this time, the antigen or antibody is in excess and the binding reaches saturation. When imidacloprid standard is added subsequently, the competitive inhibition effect will be weakened (the signal change range is narrow).

[0101] 2) When the imidacloprid-BSA conjugate antigen is less than 0.1 μg / mL or the primary antibody is ≥1:16000, the OD value is <0.5 (e.g., 0.1 μg / mL + 1:64000 combination OD=0.186), the signal is too weak and the inhibitory effect of low concentration imidacloprid cannot be distinguished.

[0102] 3) Based on the signal intensity being between 1.0 and 2.0, and provided that the signal requirements are met, a lower coating antigen concentration and a higher primary antibody dilution factor should be prioritized to further reduce costs.

[0103] 4) Considering both cost and sensitivity, the concentration of the imidacloprid-BSA conjugate antigen was selected as 0.5 μg / mL, and the dilution factor of the primary antibody was 1:8000. The results are as follows: Figure 11 As shown.

[0104] 2. Construction of the fitted curve (1) Coating and blocking antigen The imidacloprid-BSA conjugate antigen was prepared to a concentration of 0.5 μg / mL using carbonate buffer (CBS). 100 μL of the diluted antigen solution was added to each well of a 96-well plate. The wells were sealed with sealing film to prevent evaporation. The plate was then wrapped in plastic film and incubated overnight (12–18 h) at 4°C to allow the antigen to fully adsorb onto the surface of the wells. After incubation, the coating solution was discarded. 200 μL of washing buffer (PBST) was added to each well, and the plate was allowed to soak for 30 seconds each time, washing 3–5 times. The plate was then patted dry on absorbent paper. 200 μL of blocking buffer (5% skim milk dissolved in PBS) was added to each well. The plate was incubated at 37°C for 1 h. The blocking buffer was discarded, and the plate was washed 3–5 times with washing buffer, then patted dry on absorbent paper.

[0105] (2) Add standard products Prepare standard solutions of imidacloprid pesticide with methanol at concentrations of 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 μg / L. Prepare a 0.5 μg / mL concentration of imidacloprid monoclonal antibody (primary antibody) with PBS buffer (the primary antibody is diluted 1:8000). Mix the imidacloprid standard and imidacloprid monoclonal antibody at a 1:1 volume ratio. Add 100 μL of the mixture to each well after patting dry in step (1) and incubate at 37°C for 1 hour; Simultaneously set up a blank control (add only antibody and buffer, no standard) and a negative control (add only buffer, no antibody and standard). After incubation, discard the liquid in the wells, wash 3-5 times with washing buffer, and pat dry.

[0106] (3) The Au@PtNCs nanozyme probe prepared in Example 2 was used as a biorecognition molecule. The Au@PtNCs nanozyme probe was diluted with PBS buffer to 1:2000 (concentration: 0.25 μg / mL). 100 μL was added to each well after incubation in step (2), and incubated at 37°C for 1 hour. The liquid in the well was discarded, and the microplate was washed 3-5 times with washing buffer and patted dry.

[0107] (4) Detection method Method 1: Colorimetric mode.

[0108] Add 100 μL of 5 mM TMB chromogenic solution to each well after patting dry in step (3), react at room temperature in the dark for 15 min, and develop the color until the positive wells turn blue. Quickly measure the UV absorbance at 652 nm to obtain the standard curve for the colorimetric detection of Au@PtNCs nanozyme probe against imidacloprid pesticide, as shown below. Figure 12 As shown on the left.

[0109] Method 2: Fluorescence mode.

[0110] Add 100 μL of 5 mM TMB chromogenic solution to each well after patting dry in step (3), and react at room temperature in the dark for 15 min. During the color development process, add 120 μL of Rhodamine 6G solution to quench the fluorescence. Measure the fluorescence intensity at 550 nm under 525 nm excitation to obtain the standard curve for the fluorescence detection of imidacloprid pesticide by the Au@PtNCs nanozyme probe. Figure 12 As shown on the right.

[0111] The results show: (1) The correlation between imidacloprid pesticide concentration and ultraviolet absorption and fluorescence values ​​is as follows: Figure 12 .

[0112] from Figure 12 On the left side, in colorimetric mode, as the concentration of imidacloprid pesticide increases, the specific binding sites of the Au@PtNCs nanozyme probe surface and the imidacloprid-BSA conjugate antigen on the microtiter plate decrease, the catalytic activity decreases, and finally the catalytic signal at 652 nm steadily decreases.

[0113] from Figure 12 On the right side, in fluorescence mode, as the concentration of imidacloprid pesticide increases, the catalytic signal at 652 nm steadily weakens, fluorescence quenching weakens, leading to enhanced fluorescence signal.

[0114] (2) The standard curves relating imidacloprid pesticide concentration to ultraviolet absorption and fluorescence values ​​are as follows: Figure 12 Illustrations; from Figure 12 (Left side) It can be seen that when the concentration of imidacloprid pesticide is in the range of 0.1~100 μg / L, the logarithm of the imidacloprid pesticide concentration has a good linear relationship with the inhibition rate; The linear equation is Y = 29.91203X + 30.941, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.993; The formulas for calculating the inhibition rate and absorbance are as follows: Colorimetric inhibition rate (%) = Based on the fitted curve, the detection limit (LOD) in colorimetric mode was calculated to be 0.127 μg / L.

[0115] from Figure 12 (Right side) It can be seen that when the concentration of imidacloprid pesticide is in the range of 0.1~100 μg / L, the logarithm of the imidacloprid pesticide concentration has a good linear relationship with the inhibition rate; The linear equation is Y = 27.8396X + 28.43217, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.991; The formulas for calculating the inhibition rate and absorbance are as follows: Fluorescence inhibition rate (%) = ; Based on the fitted curve, the detection limit (LOD) in fluorescence mode was calculated to be 0.119 μg / L.

[0116] Example 4: Actual Sample Testing Based on the detection rate of imidacloprid in fruits and vegetables, bananas and yams were selected as actual sample analysis materials to evaluate the accuracy of Au@PtNCs nanozyme probe in detecting imidacloprid pesticide concentration in actual samples.

[0117] 1. The specific method for preparing actual samples is as follows: (1) Wash the bananas, yams and spinach separately, and blend them into a paste using a blender; (2) Take 10 g (±0.1 g) of homogenized sample and place it into a 50 mL centrifuge tube. Then add 100 μL of imidacloprid IMI standard solution (detection concentration 0.1, 10, 10, 100 ng / mL) to each sample tube. Vortex for 30 seconds, add 10 mL of methanol to each sample tube, tighten the cap, and shake for 1 minute. Add QuEChERS EN extraction salt to each centrifuge tube, tighten the cap, shake vigorously for 1 minute, centrifuge at 10000 rpm for 10 minutes, and collect the supernatant.

[0118] (3) Take 6 mL of the prepared supernatant and transfer it into a 15 mL tube containing 150 mg PSA (N-propylethylenediamine), 900 mg anhydrous magnesium sulfate and 15 mg GCB (graphitized carbon black) of a dispersive solid-phase extraction column. Seal the tube tightly and shake vigorously for 1 minute. Centrifuge the 15 mL tube at 10,000 rpm for 10 minutes.

[0119] (4) Take the supernatant, filter the supernatant through a 0.22 μm microporous membrane, and store it at 4 °C for further use.

[0120] 2. Detection of imidacloprid pesticide in samples (1) Coating and blocking antigen The imidacloprid-BSA conjugate antigen was prepared to a concentration of 0.5 μg / mL using carbonate buffer (CBS). 100 μL of the diluted antigen solution was added to each well of a 96-well plate. The wells were sealed with sealing film to prevent evaporation. The plate was then wrapped in plastic film and incubated overnight (12–18 h) at 4°C to allow the antigen to fully adsorb onto the surface of the wells. After incubation, the coating solution was discarded. 200 μL of washing buffer (PBST) was added to each well, and the plate was allowed to soak for 30 seconds each time, washing 3–5 times. The plate was then patted dry on absorbent paper. 200 μL of blocking buffer (5% skim milk dissolved in PBS) was added to each well. The plate was incubated at 37°C for 1 h. The blocking buffer was discarded, and the plate was washed 3–5 times with washing buffer, then patted dry on absorbent paper.

[0121] (2) Add samples Take the sample obtained in step 1, prepare the imidacloprid monoclonal antibody (primary antibody) with PBS buffer to a concentration of 0.5 μg / mL (the dilution factor of the primary antibody is 1:8000), and mix the sample with the imidacloprid monoclonal antibody at a volume ratio of 1:1. Add 100 μL of the mixture to each well after patting dry in step (1) and incubate at 37°C for 1 hour; Simultaneously set up a blank control (add only antibody and buffer, no standard) and a negative control (add only buffer, no antibody and standard). After incubation, discard the liquid in the wells, wash 3-5 times with washing buffer, and pat dry.

[0122] (3) The Au@PtNCs nanozyme probe prepared in Example 2 was used as a biorecognition molecule. The Au@PtNCs nanozyme probe was diluted with PBS buffer to 1:2000 (concentration: 0.25 μg / mL). 100 μL was added to each well after incubation in step (2), and incubated at 37°C for 1 hour. The liquid in the well was discarded, and the microplate was washed 3-5 times with washing buffer and patted dry.

[0123] (4) Detection method Method 1: Colorimetric mode.

[0124] Add 100 μL of 5 mM TMB colorimetric solution to each well after patting dry in step (3), react at room temperature in the dark for 15 min, and develop the color until the positive well turns blue. Quickly measure the UV absorbance at 652 nm.

[0125] Method 2: Fluorescence mode.

[0126] Add 100 μL of 5 mM TMB colorimetric solution to each well after patting dry in step (3), react at room temperature in the dark for 15 min, add 120 μL of Rhodamine 6G solution to quench fluorescence during the color development process, and measure the fluorescence intensity at 550 nm under 525 nm excitation.

[0127] The concentration of imidacloprid in the samples was detected using the standard curve of Example 3.

[0128] 3. Results show: (1) As can be seen from Table 1, the UV absorption intensity and fluorescence intensity measured in the actual sample are basically consistent with those of the standard. The relative standard deviation range can be used to quickly and accurately detect the concentration of imidacloprid pesticide in the sample using Au@PtNCs nanozyme probe.

[0129] Table 1: Experimental results of Au@PtNCs nanozyme probe in detecting imidacloprid pesticide in real samples

[0130] 2. In addition, the detection accuracy of other methods (liquid chromatography-tandem mass spectrometry, immunochromatographic detection) was compared. The content of imidacloprid in the samples was detected by liquid chromatography-tandem mass spectrometry, immunochromatography, and Au@PtNCs nanozyme probe method, respectively. The detection limits are shown in Table 2.

[0131] The detection method using liquid chromatography-tandem mass spectrometry was referenced in "Dispersive solid phase extraction of acetamiprid and imidacloprid from vegetables using quasi-metalorganic frameworks before analysis by liquid chromatography-tandem mass spectrometry." The detection method using immunochromatography was referenced in "Novel time-resolved fluorescent-based multiplex immunochromatography test strip for simultaneous detection of pesticides in vegetables." The results are shown in Table 2.

[0132] Table 2: Comparison of Au@PtNCs nanozyme probe detection results with other detection methods

[0133] The results are shown in Table 2. As can be seen from Table 2, this method is also applicable to the detection of imidacloprid pesticides in actual samples. Compared with the existing methods for detecting novel neonicotinoid pesticides imidacloprid based on gold nanoparticles, this method has higher sensitivity and is simpler, making it convenient for rapid on-site detection.

[0134] 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. An Au@PtNCs nanozyme, characterized in that, The Au@PtNCs nanozyme was prepared according to the following method: (1) After mixing and stirring glutathione and chloroauric acid, sodium hydroxide solution was added. After stirring, the mixture was reacted at 60~75 ℃ and 500~900 rpm for 24~36 h. The final solution color was yellow, and AuNCs nanocluster gold seeds were obtained. The molar ratio of chloroauric acid to glutathione was (0.5~2):(0.5~2). The concentration of chloroauric acid was 10~20 mM, and the concentration of glutathione was 80~100 mM. After mixing glutathione and chloroauric acid, the mixture was stirred at 20~25 ℃ and 500~600 rpm for 5~10 min. A sodium hydroxide solution with a concentration of 0.5~1 mM was added, and the mixture was stirred at 800~900 rpm for 24~26 min. (2) Disperse the AuNCs nanocluster gold seeds from step (1) into water to obtain an AuNCs nanocluster gold seed solution. After stirring and mixing with polyvinylpyrrolidone, add hexachloroplatinic acid and L-ascorbic acid. React at 60~75 ℃ and 500~900 rpm for 12~24 h. After cooling, Au@PtNCs nanoclusters are obtained, which are Au@PtNCs nanozymes. The molar ratio of AuNCs nanocluster gold seed solution to hexachloroplatinic acid is (1~4):(1~4); the molar ratio of PVP to L-ascorbic acid is (1:200):(1:200); the concentration of AuNCs nanocluster gold seed solution is 5~10 mM, the concentration of hexachloroplatinic acid is 5~10 mM, the concentration of polyvinylpyrrolidone is 0.5~1 mM, and the concentration of L-ascorbic acid is 50~100 mM.

2. The Au@PtNCs nanozyme according to claim 1, characterized in that, The stirring conditions for AuNCs nanocluster gold seed solution and polyvinylpyrrolidone are: 20~25℃, 800~900 rpm for 5~10 min.

3. The Au@PtNCs nanozyme according to claim 2, characterized in that, The Au@PtNCs nanoclusters obtained in step (2) also include a purification step, wherein the prepared nanocluster solution is ultrasonically treated, filtered, centrifuged and then reconstituted to obtain purified Au@PtNCs nanoclusters.

4. An Au@PtNCs nanozyme probe, characterized in that, The probe was prepared according to the following steps: (1) Add the Au@PtNCs nanozyme according to any one of claims 1 to 3 to ethanol to prepare an Au@PtNCs ethanol solution; (2) Add the Au@PtNCs ethanol solution obtained in step (1) to the APTES solution and stir at 25~30℃ and 500~700 rpm for 12~14 h; (3) Centrifuge the Au@PtNCs solution obtained after the reaction in step (2), and resuspend the precipitate in PBS buffer to prepare Au@PtNCs dispersion; (4) Centrifuge the Au@PtNCs dispersion prepared in step (3), discard the supernatant, and resuspend it with MES buffer to obtain the Au@PtNCs solution; 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution were added sequentially to Au@PtNCs solution, and the reaction was carried out at 4~8℃ and 300~500 rpm for 3~5 h to obtain the reaction system; (5) Add 1~2 μg / μL of goat anti-mouse to the reaction system obtained in step (4), react at 4~8℃ and 300~500rpm for 12~14 h, centrifuge, discard the uncoupled supernatant, and resuspend the precipitate with PBS buffer to obtain Au@PtNCs nanozyme probe.

5. The Au@PtNCs nanozyme probe according to claim 4, characterized in that, In step (1), the concentration of the Au@PtNCs ethanol solution is 0.5~10 mg / ml; In step (2), the volume ratio of Au@PtNCs ethanol solution to APTES solution is 1:10 to 1:20; the concentration of APTES solution is 10 to 20 mM. In step (4), the concentration of the Au@PtNCs solution obtained after resuspending with MES buffer is 0.5~1 mg / ml, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 5~10 mM, the concentration of the N-hydroxysuccinimide solution is 10~20 mM, and the concentration ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to the N-hydroxysuccinimide solution is (1~2):(1~2).

6. A reagent kit or device for detecting imidacloprid pesticides, characterized in that, The kit or device contains the Au@PtNCs nanozyme probe as described in claim 4 or 5.

7. The use of the Au@PtNCs nanozyme probe according to claim 4 or 5 in the detection of imidacloprid pesticides or in the preparation of products for detecting imidacloprid pesticides.

8. A method for detecting imidacloprid pesticide, characterized in that, The method involves coating and sealing the imidacloprid-BSA conjugate antigen onto the surface of the wells of an ELISA plate; sequentially adding the test sample and imidacloprid monoclonal antibody to the wells of the ELISA plate and incubating; adding the Au@PtNCs nanozyme probe as described in claim 4 or 5 to the system after incubation, continuing incubation, discarding the liquid after incubation, adding TMB chromogenic solution to the wells, and detecting the concentration of imidacloprid pesticide in the test sample using colorimetry or fluorescence method.

9. The method according to claim 8, characterized in that, The concentration of the imidacloprid-BSA conjugate antigen is 0.1~8 μg / mL.

10. The method according to claim 9, characterized in that, The coating method involves incubating at 4°C for 12-18 hours. The blocking method involves adding blocking solution to the wells of the coated ELISA plate and incubating at 37°C for 1 hour. The blocking solution is a 5% (w / w) skim milk solution obtained by dissolving skim milk in PBS solution.

11. The method according to claim 10, characterized in that, The concentration of the added imidacloprid monoclonal antibody was 0.5 μg / mL; the sample and imidacloprid monoclonal antibody were mixed at a volume ratio of 1:1 and incubated at 37°C for 1 h.

12. The method according to claim 11, characterized in that, After incubation, Au@PtNCs nanozyme probes at a concentration of 0.25~1 μg / mL were added to the system and incubated at 37℃ for 1 h.

13. The method according to claim 12, characterized in that, The colorimetric method involves adding 5 mM TMB colorimetric solution to the well plate, reacting in the dark for 15 min, developing color until the positive wells turn blue, and measuring the ultraviolet absorbance at 652 nm. The concentration of imidacloprid pesticide in the sample to be tested was calculated using the following formula; Colorimetric inhibition rate (%) = (1 - sample OD value / blank OD value) × 100%; The linear equation is Y = 29.91203X + 30.941, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.

993.

14. The method according to claim 13, characterized in that, The fluorescence method involves adding 5 mM TMB colorimetric solution to the well plate, reacting in the dark for 15 min, adding 120 μL of Rhodamine 6G solution during the colorimetric process to quench the fluorescence, and measuring the fluorescence intensity at 550 nm under 525 nm excitation. The concentration of imidacloprid pesticide in the sample to be tested was calculated using the following formula; Fluorescence inhibition rate (%) = (1 - [(sample fluorescence value - blank fluorescence value) / (negative control fluorescence value - blank fluorescence value)] × 100%; The linear equation is Y = 27.8396X + 28.43217, where Y is the inhibition rate and X is the logarithm of the imidacloprid pesticide concentration; R 2 =0.991.

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