Design and application of a rapid pesticide residue detection platform based on graphene oxide nanoszyme combined with colorimetric-catalytic signal enhancement

Abstract

The application discloses a design and application of a graphene oxide nanoscale enzyme rapid pesticide residue detection platform combining colorimetric-catalytic signal enhancement, and belongs to the technical field of immunochromatographic detection. The graphene (GO) is used as a substrate, 30nm Pt nanoparticles are adsorbed on the surface of the graphene to provide a colorimetric signal, and 5nm AuIr nanoparticles are filled into the gap of the Pt NPs to enhance the catalytic performance, so that the nanoscale enzyme with colorimetric / catalytic dual signal enhancement is prepared. 30 The GO@Pt 30 -AuIr nanoscale enzyme utilizes the large specific surface area, excellent stability and dispersibility of GO to synergistically assemble with Pt NPs and AuIr NPs, so that the catalytic performance of the system is significantly improved. Beneficial effects: the detection system of the GO-Pt 2+ -AuIr-LFA can realize high-sensitivity dual detection of heavy metal cadmium ions (Cd 2+ ) and pesticide residues imidacloprid (IMI) within 20 minutes, and the detection limit reaches the pg / mL level. The detection method provided in the research exhibits significant application potential in rapid screening of small molecules in a complex matrix, and provides a new technical approach for food safety monitoring.

Description

Technical Field

[0001] This invention relates to the field of immunochromatographic detection technology, specifically to the design and application of a rapid pesticide residue detection platform combining colorimetric-catalytic signal enhancement with graphene oxide nanoenzymes. Background Technology

[0002] Lateral-flow immunochromatography (LFA), a paper-based sensing technology, has been widely applied in food safety, clinical diagnostics, and environmental monitoring due to its advantages of simple operation, rapid detection, and cost-effectiveness. Gold nanoparticles (AuNPs) have become the most commonly used signal tags in LFA due to their good cost-effectiveness and simple preparation methods. However, traditional AuNPs-LFA often exhibits weak colorimetric signals and unsatisfactory detection sensitivity due to limitations in a single signal output mode and limited light absorption performance. To improve the analytical performance of LFA, researchers have developed various improvement strategies. Among them, developing novel nanotags is an important direction. By controlling the assembly structure of noble metals, such as optimizing the ratio of Au and Ag, the colorimetric energy of nanotags can be improved. However, relying solely on the simple assembly of one or two noble metals still has limited effect on enhancing the colorimetric signal. Another strategy is to expand the signal output modes, including introducing multiple signal types such as fluorescence, Raman, electrochemical, and photothermal. These novel signal methods do improve detection sensitivity to some extent, but they generally require specialized instruments for signal reading, which not only increases detection costs but also affects the portability and practicality of the method. Therefore, developing novel LFA methods that can significantly improve detection sensitivity while maintaining detection convenience is of great research significance.

[0003] To address the challenge of low-sensitivity LFA detection, researchers have turned their attention to enzyme catalytic systems with signal amplification potential. Among these, nanozymes, as a class of engineered nanomaterials with enzyme-like catalytic activity, not only inherit the unique structural characteristics of nanomaterials but also possess the catalytic functions of biological enzymes. Compared to natural enzymes, nanozymes exhibit superior stability and multifunctionality, while also offering advantages such as ease of large-scale production and cost-effectiveness. Currently, various nanomaterials with enzyme-like activity have been developed for biosensing, including noble metal nanoparticles, single-atom nanozymes, carbon nanomaterials, and metal-organic frameworks. These nanozymes, acting as dual-signal nanotags, demonstrate significant advantages in improving the detection sensitivity of traditional LFA. By catalyzing substrate colorimetric reactions, nanozymes can not only enhance colorimetric signals and expand the detection range but also achieve visible detection without additional instruments. Recent studies have shown that multi-metal nanozyme systems exhibit even superior catalytic performance. However, in the research of multi-metal nanozymes, how to precisely control the combination and ratio of different metals remains a key scientific problem that urgently needs to be solved. A breakthrough in this challenge will provide important support for the development of high-performance nanozyme detection systems.

[0004] Chinese patent application CN107583627A discloses an Au nanoparticle / graphene oxide composite material, its preparation method, and its applications. This composite material consists of graphene oxide and Au nanoparticles uniformly loaded on its sheet surface. The Au nanoparticles are connected to the graphene oxide sheets via poly(4-vinylpyridine). The preparation method involves dropwise addition of a solution of poly(4-vinylpyridine)-modified graphene oxide into an Au nanosol, followed by centrifugation, washing, and drying to obtain the product. This composite material can be used for the selective adsorption and enrichment of negatively charged fluorescent dyes in solution, and their detection can be achieved using surface-enhanced Raman spectroscopy. However, the performance of this composite material is poor and it cannot be used for immunochromatographic detection; therefore, further improvement is needed. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to design and apply a rapid pesticide residue detection platform that combines colorimetric-catalytic signal enhancement with graphene oxide nanoenzymes.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] The first objective of this invention is to provide a film-like nanomaterial with enhanced colorimetric / catalytic dual signals based on a combination of graphene oxide (GO) nanofilms and noble metals, wherein Pt NPs and AuIr NPs are coated on the surface of graphene oxide to provide colorimetric signals and catalytic properties, respectively, thereby generating a colorimetric / catalytic dual signal mode in immunochromatography.

[0008] The second objective of this invention is to improve a method for preparing the aforementioned dual-signal film-like composite nanostructure. This strategy is based on electrostatic self-assembly, in which Pt NPs and AuIr NPs are adsorbed layer by layer onto the surface of graphene oxide nanofilms. The preparation process is simple, efficient, and reproducible, and can be used to prepare film-like nanotags of different noble metals or alloys.

[0009] A third objective of this invention is to provide an immunochromatographic detection system based on membrane-like composite nanomaterials for the simultaneous detection of the heavy metal Cd. 2+ And pesticide residue IMI.

[0010] To achieve the above objectives, the technical solution of the present invention is as follows:

[0011] The first aspect of the present invention proposes a nanofilm label based on graphene oxide, wherein the nanofilm label is a GO-Pt-AuIr composite nanofilm, with a single layer of graphene oxide (GO) as a carrier, and Pt particles and AuIr alloy particles uniformly loaded on the GO surface to form a GO nanofilm coated with Pt particles and AuIr alloy particles; a polyethyleneimine (PEI) layer is disposed between the Pt particles, AuIr alloy particles and GO.

[0012] Preferably, the mass ratio of GO, PEI, Pt, and AuIr is 1:1:(300-400):(350-450); more preferably, it is 1:1:321:366.

[0013] Preferably, the particle size range of the monolayer graphene oxide is 200–400 nm.

[0014] Preferably, the molecular weight of the PEI is 5,000 to 80,000.

[0015] Preferably, the Pt particles are larger than the AuIr alloy particles, and the AuIr alloy particles fill the gaps between the Pt particles.

[0016] Preferably, the average particle size of the Pt particles is 30 nm.

[0017] Preferably, the average particle size of the AuIr alloy particles is 5 nm.

[0018] A second aspect of the present invention provides a method for preparing the above-mentioned nanofilm label, comprising the following steps:

[0019] (1) Preparation of GO-PEI nanofilm: Mix monolayer graphene oxide with deionized water, then add polyethyleneimine (PEI) solution, and sonicate to obtain PEI-coated graphene oxide nanofilm (i.e. GO-PEI nanofilm).

[0020] (2) Preparation of GO-Pt composite nanofilm: Pt NPs solution was added to GO-PEI nanofilm and ultrasonic treatment was performed to obtain PtNPs coated graphene oxide nanofilm (i.e. GO-Pt composite nanofilm).

[0021] (3) Preparation of GO-Pt-AuIr composite nanofilm: Add AuIr NPs solution to GO-Pt composite nanofilm and sonicate to obtain graphene oxide nanofilm coated with Pt NPs and AuIr NPs (i.e. GO-Pt-AuIr composite nanofilm).

[0022] Preferably, in step (1), the concentration of the PEI solution is 0.1 to 10 mg / mL, and more preferably 0.2 mg / mL.

[0023] Preferably, in steps (1), (2), and (3), the ultrasonic treatment time is 10 to 60 minutes, preferably 30 minutes.

[0024] Preferably, the method further includes: adding a 2-nitrobenzoic acid (DTNB) solution to the GO-Pt-AuIr composite nanofilm and performing ultrasonic treatment to form a carboxylated GO-Pt-AuIr composite nanofilm.

[0025] A third aspect of this invention provides a method for preparing a nanozyme probe, comprising the following steps: dispersing the above-mentioned nanofilm tag in PBST buffer, then adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), sonicating, and adding the heavy metal Cd. 2+ The specific antibody against pesticide residue IMI was shaken at room temperature until fully mixed, then bovine serum albumin (BSA) was added for blocking. After the reaction was completed, the product was washed with PBST buffer to obtain the final product.

[0026] A fourth aspect of the present invention provides a nanozyme probe prepared by the above-described preparation method.

[0027] The fifth aspect of this invention proposes the application of the aforementioned nanomembrane tags or nanozyme probes in the immunochromatographic detection of heavy metal Cd. 2+ Application in and / or pesticide residues such as imidacloprid (IMI).

[0028] The beneficial effects of this invention are as follows:

[0029] (1) This invention proposes a film-like composite nanomaterial based on graphene oxide (GO), which has the effect of enhancing both colorimetric and catalytic signals. Compared with traditional spherical materials, GO, as a typical two-dimensional carbon nanomaterial, has the characteristics of large specific surface area, strong conductivity, good flexibility, and excellent solution stability and dispersibility. It provides an ideal material basis for constructing a high-performance detection platform.

[0030] (2) The method for preparing the film-like composite nanomaterials proposed in this invention adopts a PEI-mediated layer-by-layer self-assembly method. Through the self-assembly loading of PEI, Pt NPs and AuIr NPs were successfully loaded onto the surface of graphene oxide.

[0031] (3) The nanozyme tag based on graphene oxide proposed in this invention supports colorimetric / catalytic dual signal mode on the immunochromatographic platform, which greatly broadens the detection range of immunochromatographic analysis and improves detection sensitivity.

[0032] (4) Pt NPs and AuIr NPs were loaded onto the surface of graphene oxide. The resulting GO@Pt film... 30-AuIr consists of a highly stable flexible graphene oxide and noble metal Pt NPs and AuIr NPs. The graphene oxide provides a highly stable substrate and a broad reaction interface, the Pt NPs provide a strong colorimetric signal, and the AuIr NPs greatly enhance the catalytic performance.

[0033] (5) This study proposes a novel nanozyme design strategy. By precisely controlling the assembly of single-metal and multi-metal nanozymes on a GO membrane substrate, the catalytic activity of the system was significantly enhanced. Specifically, we innovatively adsorbed large-size Pt nanoparticles (30 nm) onto the GO surface and filled the gaps between small-size AuIr nanoparticles (5 nm) with small-size AuIr nanoparticles (5 nm), constructing a composite nanozyme system with a dual-signal enhancement effect. This unique layered design not only fully utilizes the large specific surface area of ​​GO to provide more catalytic sites, but also achieves colorimetric-catalytic dual-signal enhancement through the synergistic effect of Pt and AuIr nanoparticles. More importantly, the organic combination of the three components, GO, Pt, and AuIr, produces a significant synergistic effect, greatly improving the sensitivity and stability of the detection system while expanding the detection range. This multi-component nanozyme design based on membrane materials provides a new approach for the development of high-performance biosensing platforms.

[0034] (6) The invented film-like colorimetric / catalytic dual-signal enhancement nanotag based on the combination of graphene oxide and noble metals consists of a highly stable flexible graphene oxide and noble metals Pt NPs and AuIr NPs. Graphene oxide provides a highly stable substrate and a broad reaction interface, Pt NPs provide a strong colorimetric signal, and AuIr NPs greatly enhance the catalytic performance. This invention innovatively adopts a PEI-mediated layer-by-layer self-assembly strategy to orderly assemble large-size Pt NPs and small-size AuIr NPs, successfully constructing a GO-Pt nanotag with dual-signal enhancement characteristics. 30 -AuIr nanozymes. This unique hierarchical structure design allows small-sized AuIr NPs to effectively fill the gaps between Pt NPs, significantly improving the performance of the detection system. The GO-Pt proposed in this invention... 30 -AuIr nanozymes have also been used for dual-mode analysis of heavy metals and pesticide residues in Dendrobium, a medicinal and edible herb. They have demonstrated good stability and accuracy in complex samples, fully demonstrating their practical application value. Furthermore, they have shown great potential on immunochromatographic platforms, serving as a simple, low-cost, and accurate immunoassay tool for on-site monitoring of agricultural drugs. Attached Figure Description

[0035] Figure 1This is a schematic diagram of the preparation method of graphene oxide-based nanofilm tags in Examples 1 and 2 of the present invention.

[0036] Figure 2 This is a structural characterization diagram of the graphene oxide-based nanofilm tag of Example 2 of the present invention, wherein... Figure 2 ad is GO-Pt 30 and GO-Pt 30 - The overall morphology of AuIr and its local magnified images, Figure e shows the potential change results during the material preparation process, Figure fg shows the lattice spacing of Pt NPs, Au NPs and Ir NPs, Figure h shows the high-angle annular dark-field scanning transmission electron microscopy imaging, Figure i shows the elemental mapping analysis, and Figure jo shows the X-ray photoelectron spectrum and the high-resolution XPS spectrum of each element.

[0037] Figure 3 This is a graph showing the optimized membrane-spreading concentration results of the nanozyme probe in Immunochromatography according to Example 3 of the present invention. Figure 3 a represents the optimized concentration of IMI membrane. Figure 3 b is Cd 2+ The optimized results of the membrane concentration.

[0038] Figure 4 This is a graph showing the optimized chromatography time of the nanozyme probe in Example 3 of the present invention.

[0039] Figure 5 The figure shows the optimization results of the catalytic time of the nanozyme probe in immunochromatography of Example 3 of the present invention.

[0040] Figure 6 Different Pt values ​​in the immunochromatographic system of Example 3 of this invention 30 A graph evaluating the detection performance of graphene oxide nanofilm tags with an AuIr ratio. Figure 6 a represents three groups with different proportions of GO-Pt 30 -AuIr TEM image ( Figure 6 a-Ⅰ~Ⅲ) and three groups of GO-Pt with different proportions 30 Comparison of colorimetric signals and catalytic activity of AuIr nanozymes under the same TMB and H2O2 reaction conditions ( Figure 6 a-IV), Figure 6 b is Pt 30 When the proportion of / AuIr is 11%, 33%, and 67%, based on GO-Pt 30 -AuIr-LFA detection of Cd 2+ Colorimetric images ( Figure 6 b-Ⅰ) and catalytic images ( Figure 6 b-Ⅱ), Figure 6 c is the T-line colorimetric image corresponding to the above-mentioned detection image. Figure 6c-Ⅰ) and catalytic images ( Figure 6 signal heatmap of c-II), Figure 6 de is the test result for Cd containing 0 / 0, 0 / 200, 200 / 0, and 200 / 200 ng / mL. 2+ Colorimetric images of the samples from / IMI ( Figure 6 d-Ⅰ), catalytic images ( Figure 6 e-Ⅰ), the signal intensity change of the T line before and after catalysis (e-Ⅰ), Figure 6 d, e-Ⅱ). Figure 6 f is a SEM image of the internal structure of the T-line region of the LFA band, with results from the negative control experiment. Figure 6 f-Ⅰ), positive test ( Figure 6 f-Ⅱ).

[0041] Figure 7 This is an experimental flowchart of the immunochromatographic detection of insulators and imidacloprid using the nanozyme probe of Example 4 of the present invention. The left side of the figure shows the detection mode of the colorimetric signal, and the right side shows the detection mode of the catalytic signal.

[0042] Figure 8 The figures show the experimental results of the simultaneous immunochromatographic detection of insulators and imidacloprid using the nanozyme probe of Example 4 of this invention, and a comparison with other immunochromatographic detection methods. The results are shown in colorimetric mode. Figure 8 a) Detection results under catalytic mode ( Figure 8 b), heatmap results ( Figure 8 c), the standard curve of colorimetric signal intensity change ( Figure 8 d, f-Ⅰ), standard curve of catalytic signal intensity change ( Figure 8 d、f-Ⅱ), Figure 8 e is based on GO-Pt 30 -AuIr-LFA detection of IMI and Cd 2+ Changes in the detection range Figure 8 g is the ELISA method for detecting Cd. 2+ As a result, Figure 8 h is based on AuNP-LFA detection of Cd. 2+ The result.

[0043] Figure 9 This is a graph showing the immunochromatographic analysis results of the nanozyme probe of Example 4 of the present invention in an actual sample of Dendrobium. Figure 9 a is based on GO-Pt 30 -AuIr-LFA detection of IMI and Cd 2+ Pre-catalytic detection results image. Figure 9 b is based on GO-Pt 30 -AuIr-LFA detection of IMI and Cd 2+ The detection results after catalysis are shown in the figure.

[0044] Figure 10 This is a repeatability verification diagram of the nanozyme probe of Example 4 of the present invention using immunochromatography, wherein... Figure 10 a represents IMI and Cd 2+ The detection results before catalysis were obtained when the detection concentration was 10 / 10 ng / mL (Group 1) and 0.1 / 0.1 ng / mL (Group 2). Figure 10 a-Ⅰ is the detected strip image. Figure 10 a-Ⅱ represents the signal strength change of the corresponding T line; Figure 10 b represents the detection results after catalysis at two different concentrations, where... Figure 10 b-Ⅰ is the detected strip image. Figure 10 b-Ⅱ represents the signal strength change of the corresponding T line.

[0045] Figure 11 This is an immunochromatographic verification diagram of the nanozyme probe of Example 4 of the present invention, wherein... Figure 11 a represents IMI and Cd before catalysis. 2+ The specific detection results, among which Figure 11 a-Ⅰ is the detected strip image. Figure 11 a-Ⅱ represents the signal strength change of the corresponding T line; Figure 11 b represents IMI and Cd after catalysis. 2+ The test results, among which Figure 11 b-Ⅰ is the detected strip image. Figure 11 b-Ⅱ represents the signal strength change of the corresponding T line. Detailed Implementation

[0046] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0048] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0049] Pt NPs: The preparation method is as follows:

[0050] Add 30 mL of 5 mM H₂PtCl₆·6H₂O solution to 240 mL of boiling deionized water and react for 1 min. Then add 5.5 mL of a mixture of 1% sodium citrate and 0.05% citric acid and react for 30 s. Next, quickly add 2.75 mL of freshly prepared solution (a mixture of 0.08% NaBH₄, 1% sodium citrate, and 0.05% citric acid) and react for 10 min. Stop heating and cool to room temperature to obtain a 5 nm Pt NP solution. Add 3 mL of 5 nm Pt NPs to 90 mL of deionized water and mix with 0.135 mL of 0.4 M H₂PtCl₆·6H₂O solution. After heating to boiling, add 1.5 mL of 1.25% L-ascorbic acid reduced with 1% sodium citrate to the mixture. React for 30 min, stop heating, and cool to room temperature to obtain 30 nm diameter Pt NPs.

[0051] AuIr NPs: Preparation method is as follows:

[0052] First, prepare a 500mL Erlenmeyer flask and rinse it twice with deionized water. Then, add 4mL of 1% chloroauric acid and 3.5mL of 1% sodium citrate to 400mL of deionized water and stir gently at 600rpm until homogeneous. The solution will turn pale yellow. Next, add 12mL of sodium borohydride (101.6mM). The solution will change from pale yellow to bright red. Stir at 600rpm for 4-6 hours to obtain 3nm gold nanoparticles.

[0053] Sodium citrate (0.034M, 12mL) and sodium hexachloroiridate (Na3IrCl6·xH2O, 2mM, 40mL) were stirred until homogeneous. The pH was adjusted to 7-9 with NaOH, and the mixture was stirred at 80℃ for 30 minutes. Then, sodium borohydride (NaBH4, 0.3M, 2mL) was added, and the mixture was stirred at 80℃ for another 30 minutes. The solution color gradually changed from pale yellow to grayish-brown. Finally, the solution was cooled to room temperature and stored away from light to obtain IrNPs.

[0054] Au-Ir nanoparticles were synthesized using a seed-mediated method. First, 80 mL of sodium hexachloroiridate (2M) and 24 mL of 1% sodium citrate were added to 400 mL of Au NPs, respectively. The pH was adjusted to 7-9 with NaOH, and the mixture was stirred at 80°C for 30 minutes. Then, freshly prepared sodium borohydride (NaBH4, 0.1M, 2 mL) was quickly added, and stirring continued for another 30 minutes. Heating was then turned off, and the mixture was stirred at room temperature for 12 hours before being stored in the dark.

[0055] Example 1:

[0056] A graphene oxide-based nanofilm label is a GO-Pt-AuIr composite nanofilm. The label uses monolayer graphene oxide (GO) (average particle size 300 nm) as a carrier, with Pt particles (average particle size 30 nm) and AuIr alloy particles (average particle size 5 nm) uniformly loaded on the GO surface. The AuIr alloy particles fill the gaps between the Pt particles, forming a GO nanofilm coated with Pt and AuIr alloy particles. A polyethyleneimine (PEI) layer (molecular weight 12000) is disposed between the Pt particles, AuIr alloy particles, and GO. The mass ratio of GO, PEI, Pt, and AuIr is 1:1:321:366.

[0057] The above-mentioned method for preparing nanofilm tags (illustrated as follows) Figure 1 (As shown), including the following steps:

[0058] (1) Preparation of GO-PEI nanofilms:

[0059] Mix 5 mL of 300 nm monolayer graphene oxide (GO) (2 mg / mL) with 20 mL of deionized water, sonicate for 5 min, centrifuge to separate the graphene oxide nanosheets, and treat with 2 mL of polyethyleneimine (PEI) aqueous solution (0.2 mg / mL) under sonication for 30 min to obtain GO-PEI nanofilm. Rinse twice with deionized water to remove excess PEI, and resuspend in 10 mL of deionized water to obtain GO-PEI solution.

[0060] (2) Preparation of GO-Pt 30 Composite nanofilms:

[0061] Add 10 mL of Pt NPs (5 mg / mL) to the prepared GO-PEI solution and sonicate for 30 min to form a graphene oxide nanofilm (GO-Pt) coated with Pt NPs. 30 After centrifugation and washing once with deionized water, GO-Pt was dispersed in 10 mL of deionized water. 30 Solution.

[0062] (3) Preparation of GO-Pt 30 -AuIr composite nanofilm:

[0063] In the GO-Pt prepared above 30 20 mL of AuIr NPs (1 mg / mL) was added to the solution, and the mixture was sonicated for 30 min to obtain a graphene oxide nanofilm (GO-Pt) coated with Pt NPs and AuIr NPs. 30 -AuIr). After centrifugation and washing with deionized water, excess AuIr NPs were removed. Stored in 10 mL of ethanol for later use.

[0064] In this embodiment, GO nanosheets and GO-PEI nanofilms are used in step (1), and GO-Pt is prepared in step (2). 30 Composite nanofilm, GO-Pt prepared in step (3) 30 Transmission electron microscopy (TEM) image of the AuIr composite nanofilm as follows: Figure 2 As shown in the image, Pt NPs and AuIr NPs are orderly assembled on the surface of GO nanosheets.

[0065] Example 2:

[0066] The difference between this embodiment and Example 1 is that the prepared GO-Pt-AuIr composite nanofilm is subjected to carboxylation treatment, that is, step (4) is also included:

[0067] Add 10 mL of GO-Pt 30 -AuIr composite nanofilms were ultrasonically treated with 0.1 mL of 2-nitrobenzoic acid (DTNB) solution (10 mM) to form carboxylated GO-Pt. 30 -AuIr composite nanofilm (denoted as GO-Pt) 30 -AuIr-D). (A schematic diagram of the preparation is shown below) Figure 1 (As shown)

[0068] Example 3:

[0069] A method for preparing a nanozyme probe includes the following steps: carboxylated GO-Pt obtained in Example 2... 30 AuIr composite nanofilm (1 mL) was dispersed in 0.05% PBST buffer (0.5 mL), then EDC (5 μL) and NHS (10 μL) were added, and the mixture was sonicated for 10 min. Heavy metal Cd was then added. 2+ The specific antibody against pesticide residue IMI was mixed thoroughly by shaking at room temperature for 2 hours, followed by the addition of 10% BSA and blocking for 1 hour. After the reaction was complete, the product was washed with 0.05% PBST buffer to obtain GO-Pt. 30 -AuIr nanozyme probe.

[0070] Applying nanozyme probes to a dual-channel immunochromatographic system for detection:

[0071] Includes a sample pad for loading the sample solution and an absorbent pad for capillary action, with two independent detection lines (Cd). 2+Nitrocellulose membranes containing BSA (IMI-BSA) and a control line (goat anti-mouse IgG) were prepared. The prepared NC membranes were affixed to a PVC substrate, assembled with the sample pad and absorbent pad, and dried in a constant temperature oven at 37°C. Finally, the assembled cards were cut into individual strips using a cutter and stored in a vacuum desiccator for later use; the antibody modification amount on the label was 5–8 mg / mL; Cd22-C ... 2+ The concentration of -BSA is 0.1–1 mg / mL, the concentration of IMI-BSA is 0.05–0.1 mg / mL; the concentration of goat anti-mouse IgG sprayed on the quality control line is 0.5–2 mg / mL; the width of the immunochromatographic test strip is 3.5 mm.

[0072] During immunochromatographic assays, the nanozyme probe and the buffer solution are directly loaded onto the sample pad of the immunochromatographic strip. After the chromatographic reaction is complete, the colorimetric and catalytic signals on the test strip are read. The run buffer is formulated with 1% PBST.

[0073] This embodiment optimizes the operating conditions of the competitive immunochromatography to achieve the best performance of the dual-channel detection platform. The optimization results of the antigen concentration captured on the detection line are as follows: Figure 3 As shown, when Cd is detected on the line 2+ When the concentrations of -BSA and IMI-BSA (coated antigen) were 0.1 mg / mL and 0.05 mg / mL, respectively, the competitive immunochromatography based on graphene oxide nanofilm tags showed the highest competitive inhibition rate against the target. The optimization results for chromatography time are as follows: Figure 4 As shown, a chromatography time of 15 min is sufficient to achieve a strong colorimetric signal and the highest competitive inhibition rate in competitive immunochromatography using graphene oxide-based nanofilm tags. The optimization results for the catalytic time are as follows: Figure 5 As shown, the best catalytic performance and the highest competitive inhibition rate can be achieved within a catalytic time of 3 minutes.

[0074] The performance of the dual-mode competitive immunochromatography proposed in this embodiment is highly dependent on the ratio of the two metals. We introduced three Pt metals into the same immunochromatographic system. 30 The volumetric ratios of / AuIr (11%, 33%, and 67%) were used to determine Pt. 30 The impact of the / AuIr ratio on our method. Detection results are as follows: Figure 6 As shown.

[0075] We can conclude that, considering the dual-signal enhancement effect of both colorimetric and catalytic signals, Pt 30 The proportion of GO@Pt to AuIrNPs was 33%. 30 -AuIr can produce more stable colorimetric and catalytic signals.

[0076] SEM images of the T-line region in immunochromatographic assays showed a large amount of membrane-like GO-Pt in the negative control experiment. 30 -AuIr tag( Figure 6 f-Ⅰ), while no nanofilm was found in the positive test (f-Ⅰ). Figure 6 f-Ⅱ), indicating that GO-Pt 30 -AuIr tags function correctly on immunochromatographic systems. In evaluating the detection performance of the immunochromatographic assay, we used four different concentrations of Cd... 2+ / IMI samples (1:0 / 0, 2:0 / 200, 3:200 / 0, 4:200 / 200 ng / mL) were used to evaluate GO@Pt. 30 The selectivity of the T-line on the AuIr-LFA. For example... Figure 6 Medium colorimetric image ( Figure 6 d-Ⅰ) and catalytic images ( Figure 6 e-Ⅰ) shows that only samples containing the target pollutant can suppress the colorimetric / catalytic signal in the corresponding T region. For example... Figure 6 The signal intensities of the T lines before and after catalysis, as shown by d and e-II, clearly demonstrate the good selectivity of our method, Cd 2+ There was no cross-reactivity with IMI detection.

[0077] Example 4:

[0078] This embodiment uses different concentrations of Cd 2+ IMI (200–0.0122 ng / mL) was added simultaneously to PBS solution (10 mM, pH 7.4) to simulate samples co-contaminated with multiple small molecule pollutants. This example was used for Cd. 2+ The flowchart of the immunochromatographic assay with dual-channel IMI detection is as follows: Figure 7 As shown. A competitive immunochromatographic system based on graphene oxide nanofilm tags for the detection of Cd. 2+ The analysis results of IMI.

[0079] Based on GO-Pt in colorimetric mode 30 The detection results of the AuIr immunochromatographic test strip are as follows: Figure 8 As shown in a, based on GO@Pt 30 In the -AuIr immunochromatographic colorimetric mode, Cd 2+ The vLOD values ​​for IMI were 0.195 and 0.0976 ng / mL, respectively. Meanwhile, the cutoff values ​​for the two target molecules were 1.562 and 0.781 ng / mL, respectively.

[0080] GO@Pt in catalytic mode 30 The results of the immunochromatographic assay for AuIr are as follows: Figure 8 As shown in b, Cd2+ The COV values ​​of IMI were increased to 100 and 50 ng / mL, respectively. Figure 8 c is based on GO-Pt 30 The heatmap results of AuIr are as follows: Figure 8 As shown in c; based on GO@Pt 30 The colorimetric signal intensity changes in the detection region of the -AuIr immunochromatographic bands are as follows: Figure 8 As shown in d and f, Cd is plotted through calculation. 2+ The fitted curves of IMI showed that the limits of detection (LODs) before catalysis were 24.66 and 71.25 pg / mL, respectively, and further decreased to 7.86 and 7.02 pg / mL after catalysis.

[0081] This embodiment further evaluated the reliability of dual-mode immunochromatography by testing actual samples (Dendrobium nobile, a traditional Chinese medicine). Figure 9 As shown, the colorimetric and catalytic signal results of the Dendrobium sample test strips are consistent with those of the PBS sample.

[0082] These results clearly demonstrate that immunochromatography based on graphene oxide nanofilm tags exhibits good stability and accuracy in the quantitative analysis of heavy metals and pesticide residues in complex real-world samples. We analyzed mixed samples (containing Cd) at two different concentrations (1:10 / 102:0.1 / 0.1 ng / mL). 2+ Five tests were conducted on both GO-Pt and IMI to further confirm our proposed GO-Pt-based approach. 30 The detection stability and accuracy of the AuIr-LFA method are shown in the results. Figure 10 As shown. When the concentration of the target contaminant in the detection solution is the same, the immunochromatographic test strips in the same group all show stable and consistent colorimetric signals. Containing 10 / 10 ng / mL and 0.1 / 0.1 ng / mL Cd... 2+ The relative standard deviations of the samples from both GO-Pt and IMI were less than 9.6%. These results demonstrate that our established GO-Pt model... 30 The AuIr-LFA method exhibits good stability. Heavy metal ion interferences include chromium ions (Cr). 3+ ), lead ions (Pb) 2+ For pesticide residue interferences, carbendazim (CBZ) and acetamiprid (ACE) are selected. Figure 11 The display shows that when only Cd is present 2+ When IMI exists, GO-Pt 30 The colorimetric and catalytic signals on the T-line of -AuIr-LFA are completely suppressed. Therefore, when a high concentration of interfering substances (200 ng / mL) is present in the detection solution, GO-Pt... 30The colorimetric and catalytic signals on the T-line of -AuIr-LFA remained stable without significant changes, indicating that our designed GO-Pt... 30 The AuIr-LFA method exhibits excellent specificity in detecting target contaminants.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanofilm tag based on graphene oxide, characterized in that, The nanofilm label is a GO-Pt-AuIr composite nanofilm, with monolayer graphene oxide (GO) as the carrier, and Pt particles and AuIr alloy particles uniformly loaded on the GO surface to form a GO nanofilm coated with Pt particles and AuIr alloy particles; a polyethyleneimine (PEI) layer is disposed between the Pt particles, AuIr alloy particles and GO.

2. The graphene oxide-based nanofilm label according to claim 1, characterized in that, The mass ratio of GO, PEI, Pt, and AuIr is 1:1:(300~400):(350~450).

3. The graphene oxide-based nanofilm label according to claim 2, characterized in that, The mass ratio of GO, PEI, Pt, and AuIr is 1:1:321:

366.

4. The graphene oxide-based nanofilm label according to claim 1, characterized in that, The particle size range of the monolayer graphene oxide is 200~400nm; the molecular weight of the PEI is 5000~80000; the Pt particles are larger than the AuIr alloy particles, and the AuIr alloy particles fill the gaps between the Pt particles.

5. The graphene oxide-based nanofilm label according to claim 1, characterized in that, The average particle size of the Pt particles is 30 nm; the average particle size of the AuIr alloy particles is 5 nm.

6. The method for preparing the graphene oxide-based nanofilm tag according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of GO-PEI nanofilm: Mix monolayer graphene oxide with deionized water, add polyethyleneimine (PEI) solution, and sonicate to obtain GO-PEI nanofilm; (2) Preparation of GO-Pt composite nanofilm: Pt NPs solution was added to GO-PEI nanofilm and ultrasonic treatment was performed to obtain GO-Pt composite nanofilm; (3) Preparation of GO-Pt-AuIr composite nanofilm: Add AuIr NPs solution to GO-Pt composite nanofilm and sonicate to obtain GO-Pt-AuIr composite nanofilm.

7. The preparation method according to claim 6, characterized in that, In step (1), the concentration of PEI solution is 0.1-10 mg / mL; in steps (1), (2), and (3), the ultrasonic treatment time is 10-60 min.

8. The preparation method according to claim 7, characterized in that, In step (1), the concentration of the PEI solution is 0.2 mg / mL.

9. The preparation method according to claim 7, characterized in that, In steps (1), (2), and (3), the ultrasonic treatment time is 30 min.

10. The preparation method according to claim 6, characterized in that, Also includes: A 2-nitrobenzoic acid solution was added to the GO-Pt-AuIr composite nanofilm, and the mixture was subjected to ultrasonic treatment to form a carboxylated GO-Pt-AuIr composite nanofilm.

11. A method for preparing a nanozyme probe, characterized in that, Includes the following steps: The nanofilm label according to any one of claims 1 to 5 is dispersed in PBST buffer, then EDC and NHS are added, followed by sonication, and then the heavy metal Cd is added. 2+ The specific antibody against pesticide residue IMI was shaken at room temperature until fully mixed, then bovine serum albumin was added and the mixture was blocked. After the reaction was completed, the product was washed with PBST buffer to obtain the final product.

12. The nanozyme probe prepared by the method of claim 11.

13. The nanofilm tag according to any one of claims 1 to 5 or the nanofilm tag prepared by the preparation method according to any one of claims 6 to 10, and the nanozyme probe according to claim 12, for the immunochromatographic detection of heavy metal Cd. 2+ And / or its application in pesticide residues such as imidacloprid.

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