Design and application of graphene oxide nano-enzyme rapid pesticide residue detection platform combined with colorimetric-catalytic signal enhancement

Abstract

The invention discloses design and application of a colorimetric-catalytic signal enhancement combined graphene oxide nano-enzyme rapid pesticide residue detection platform, and belongs to the technical field of immunochromatography detection. Graphene (GO) is used as a substrate, 30 nm Pt nanoparticles are adsorbed on the surface of the substrate to provide a colorimetric signal, and gaps of Pt NPs are filled with 5 nm AuIr nanoparticles to enhance the catalytic performance of the substrate, so that the colorimetric / catalytic dual-signal enhanced nano-enzyme is prepared. The GO (at) Pt30-AuIr nano-enzyme is synergistically assembled with Pt NPs and AuIr NPs by utilizing the large specific surface area, excellent stability and dispersibility of GO, so that the catalytic performance of the system is remarkably improved. The method has the beneficial effects that a GO-Pt30-AuIr-LFA-based detection system can realize high-sensitivity dual detection on heavy metal cadmium ions (Cd < 2 + >) and pesticide residue imidacloprid (IMI) within 20 minutes, and the detection limit reaches the level of pg / mL. The detection method provided by the research shows significant application potential in the aspect of rapid screening of small molecules in a complex matrix, and a new technical approach is provided for food safety monitoring.

Description

Technical Field

[0001] The present invention relates to the technical field of immunochromatographic detection, and in particular to the design and application of a graphene oxide nanozyme rapid pesticide residue detection platform combined with colorimetric-catalytic signal enhancement. Background Art

[0002] As a sensing technology based on paper-based materials, lateral flow immunochromatography (LFA) has been widely used in food safety, clinical diagnosis 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 label in LFA due to their good cost-effectiveness and simple preparation method. However, traditional AuNPs-LFA often exhibits weak colorimetric signals and unsatisfactory detection sensitivity due to its limitation to a single signal output mode and limited light absorption performance. In order to improve the analytical performance of LFA, researchers have developed a variety of improvement strategies. Among them, the development of new nano-labels is an important direction. By regulating the assembly structure of precious metals, such as optimizing the ratio of Au and Ag, the colorimetric performance of nano-labels can be improved. However, the enhancement effect of colorimetric signals is still limited by the simple assembly of one or two precious metals. Another strategy is to expand the signal output mode, including the introduction of multiple signal types such as fluorescence, Raman, electrochemistry and photothermal. These new signal modes do improve the detection sensitivity to a certain extent, but they generally require the use of professional instruments for signal reading, which not only increases the detection cost, but also affects the portability and practicality of the method. Therefore, it is of great research significance to develop a new LFA method that can significantly improve detection sensitivity while maintaining detection convenience.

[0003] To meet the challenge of LFA detection sensitivity, researchers have turned their attention to enzyme catalytic systems with signal amplification potential. Among them, nanozymes, as a class of engineered nanomaterials with enzyme-like catalytic activity, not only inherit the unique structural characteristics of nanomaterials, but also have the catalytic function of biological enzymes. Compared with natural enzymes, nanozymes show excellent stability and versatility, and have the advantages of easy large-scale production and cost-effectiveness. At present, a variety of nanomaterials with enzyme-like activity have been developed for biosensing, including precious metal nanoparticles, single-atom nanozymes, carbon nanomaterials and metal-organic frameworks. These nanozymes, as dual-signal nanolabels, have shown significant advantages in improving the sensitivity of traditional LFA detection. By catalyzing the color development reaction of the substrate, nanozymes can not only enhance the colorimetric signal and expand the detection range, but also achieve visible detection without additional instruments. Recent studies have shown that multi-metal nanozyme systems exhibit better catalytic performance. However, in the study of multi-metal nanozymes, how to accurately regulate the combination and ratio of different metals is still a key scientific problem that needs to be solved. The breakthrough of this challenge will provide important support for the development of high-performance nanozyme detection systems.

[0004] Publication number is the Chinese patent application document of CN107583627A, discloses a kind of Au nanoparticle / graphene oxide composite material and its preparation method and application, this composite material is made up of graphene oxide and Au nanoparticles uniformly loaded on its lamella surface, and described Au nanoparticles are connected on the lamella of graphene oxide by poly-4-vinyl pyridine.This preparation method drips the solution of graphene oxide modified by poly-4-vinyl pyridine into Au nano sol and reacts, and obtains product through centrifugal, washing and drying steps.This composite material can be used for selective adsorption, enrichment solution with negatively charged fluorescent dye, and detects it by surface enhanced Raman scattering spectrum.But the performance of this composite material is poor and can not be used for immunochromatographic detection, therefore, still need to be further improved. Summary of the invention

[0005] The technical problem to be solved by the present invention is how to provide a design and application of a graphene oxide nanozyme rapid pesticide residue detection platform combined with colorimetric-catalytic signal enhancement.

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

[0007] The first object of the present invention is to provide a film-like colorimetric / catalytic dual signal enhanced nanomaterial based on a combination of graphene oxide (GO) nanomembrane and precious metals, wherein Pt NPs and AuIr NPs are coated on the surface of graphene oxide to provide colorimetric signals and catalytic properties, respectively, to generate a colorimetric / catalytic dual signal mode in an immunochromatographic method.

[0008] The second purpose of the present invention is to improve a method for preparing the dual-signal film-like composite nanostructure. The strategy is based on electrostatic self-assembly, and Pt NPs and AuIr NPs are adsorbed layer by layer on the surface of graphene oxide nanofilm. The preparation process is simple, efficient, and repeatable, and can be used to prepare film-like nanotags of different precious metals or alloys.

[0009] The third object of the present invention is to provide an immunochromatographic detection system based on a membrane-like composite nanomaterial for simultaneous detection of heavy metal Cd 2+ and pesticide residue IMI.

[0010] To achieve the above object, 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, Pt particles and AuIr alloy particles are uniformly loaded on the GO surface to form a GO nanofilm coated with Pt particles and AuIr alloy particles; a polyethyleneimine (PEI) layer is arranged 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 of the single-layer graphene oxide is in the range of 200 to 400 nm.

[0014] Preferably, the molecular weight of the PEI is 5000-80000.

[0015] Preferably, the Pt particles are larger than AuIr alloy particles, and the AuIr alloy particles are filled in 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] The second aspect of the present invention provides a method for preparing the above-mentioned nanofilm label, comprising the following steps:

[0019] (1) preparing GO-PEI nanomembrane: mixing a monolayer of graphene oxide with deionized water, adding a polyethyleneimine (PEI) solution, and ultrasonically treating the mixture to obtain a PEI-coated graphene oxide nanomembrane (i.e., a GO-PEI nanomembrane);

[0020] (2) Preparation of GO-Pt composite nanomembrane: adding Pt NPs solution to GO-PEI nanomembrane and ultrasonic treatment to obtain PtNPs-coated graphene oxide nanomembrane (i.e., GO-Pt composite nanomembrane);

[0021] (3) Preparation of GO-Pt-AuIr composite nanomembrane: Adding AuIr NPs solution to the GO-Pt composite nanomembrane and ultrasonically treating it to obtain a graphene oxide nanomembrane coated with Pt NPs and AuIr NPs (i.e., GO-Pt-AuIr composite nanomembrane).

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

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

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

[0025] The third aspect of the present invention provides a method for preparing a nanozyme probe, comprising the following steps: dispersing the above-mentioned nanomembrane tag in PBST buffer, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), ultrasonically treating, adding heavy metal Cd 2+ and the specific antibody of pesticide residue IMI, oscillate at room temperature until fully mixed, then add bovine serum albumin (BSA) for blocking; after the reaction is completed, wash the product with PBST buffer to obtain the product.

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

[0027] The fifth aspect of the present invention proposes that the above-mentioned nanomembrane tag or nanoenzyme probe is used in immunochromatographic detection of heavy metal Cd 2+ and / or application of pesticide residue imidacloprid (IMI).

[0028] The beneficial effects of the present invention are:

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

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

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

[0032] (4) Pt NPs and AuIr NPs were loaded onto the surface of graphene oxide. 30-AuIr is composed of a highly stable flexible graphene oxide and precious metal 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.

[0033] (5) This study proposed a novel nanozyme design strategy. By precisely regulating the assembly of single-metal and multi-metal nanozymes on a GO film substrate, the catalytic activity of the system was significantly improved. Specifically, we took advantage of the complementary characteristics of the strong colorimetric signal of large-sized nanoparticles and the excellent catalytic performance of small-sized nanoparticles, innovatively adsorbed large-sized Pt nanoparticles (30nm) on the GO surface, and filled small-sized AuIr nanoparticles (5nm) in the gaps between Pt particles to construct a composite nanozyme system with dual signal enhancement effect. This unique hierarchical 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 GO, Pt and AuIr produced 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 film materials provides a new idea for the development of high-performance biosensing platforms.

[0034] (6) The invented film-like colorimetric / catalytic dual signal enhanced nanotag based on the combination of graphene oxide and precious metals is composed of a highly stable flexible graphene oxide and precious 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. The present invention innovatively adopts a PEI-mediated layer-by-layer self-assembly strategy to orderly assemble large-sized Pt NPs and small-sized AuIrNPs, and successfully constructs a GO-Pt nanotag with dual signal enhancement characteristics. 30 -AuIr nanozymes. This unique hierarchical structure design enables small-sized AuIr NPs to effectively fill the gaps between Pt NPs, significantly improving the performance of the detection system. 30 -AuIr nanozyme was also used for dual-mode analysis of heavy metals and pesticide residues in the edible and medicinal Chinese herb Dendrobium officinale. It showed good stability and accuracy in complex samples, fully confirming its practical application value. It also showed great potential on the immunochromatographic platform and can be used as an easy-to-operate, low-cost, and accurate immunoassay tool for on-site monitoring of agricultural drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1Schematic diagram of the preparation method of graphene oxide-based nanofilm labels of Examples 1 and 2 of the present invention.

[0036] Figure 2 This is a structural characterization diagram of a nanofilm tag based on graphene oxide according to 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 image, Figure e is the result of potential change during the material preparation process, Figure fg shows the lattice spacing of Pt NPs, Au NPs and Ir NPs, Figure h is high-angle annular dark field scanning transmission electron microscopy imaging, Figure i is element mapping analysis, and Figure jo is X-ray photoelectron spectroscopy and high-resolution XPS spectra of each element.

[0037] Figure 3 This is a graph showing the optimized results of the immunochromatography of the nanozyme probe of Example 3 of the present invention, wherein Figure 3 a is the optimization result of IMI film concentration, Figure 3 b is Cd 2+ The optimization results of the film stripping concentration.

[0038] Figure 4 This is a graph showing the results of chromatography time optimization for the immunochromatography of the nanozyme probe of Example 3 of the present invention.

[0039] Figure 5 This is a graph showing the optimization results of the catalytic time of the immunochromatography of the nanozyme probe of Example 3 of the present invention.

[0040] Figure 6 The different Pt 30 Detection performance evaluation diagram of graphene oxide nanofilm labels with different ratios of Au / AuIr. Figure 6 a shows three groups with different ratios of GO-Pt 30 -TEM image of AuIr( Figure 6 a-Ⅰ~Ⅲ) and three groups with different ratios of GO-Pt 30 -AuIr nanozyme colorimetric signal and in the same TMB and H 2 O 2 Comparison of catalytic ability under reaction conditions ( Figure 6 a-IV), Figure 6 b is Pt 30 When the ratio of / AuIr is 11%, 33%, and 67%, the GO-Pt 30 -AuIr-LFA detection of Cd 2+ The colorimetric image ( Figure 6 b-Ⅰ) and catalytic images ( Figure 6 b-Ⅱ), Figure 6c is the T-line colorimetric image corresponding to the above detection image ( Figure 6 c-Ⅰ) and catalytic images ( Figure 6 c-Ⅱ) Signal heat map, Figure 6 de is the test containing 0 / 0, 0 / 200, 200 / 0 and 200 / 200 ng / mL Cd 2+ / IMI colorimetric image of the sample ( Figure 6 d-Ⅰ)、catalytic image ( Figure 6 e-Ⅰ), the signal intensity change of T line before and after catalysis ( Figure 6 d, e-Ⅱ). Figure 6 f is the SEM image of the internal structure of the T-line area of ​​the LFA strip, and the negative control test results ( Figure 6 f-Ⅰ), positive test ( Figure 6 f-Ⅱ).

[0041] Figure 7 This is an experimental flow chart of the immunochromatography of the nanozyme probe of Example 4 of the present invention for simultaneously detecting insulators and imidacloprid. The left side of the figure shows the detection mode of the colorimetric signal, and the right side of the figure shows the detection mode of the catalytic signal.

[0042] Figure 8 The experimental results of the immunochromatographic detection of the nanozyme probe of Example 4 of the present invention for simultaneously detecting the insulator and imidacloprid are shown in Figure 1 and compared with other immunochromatographic detection methods, wherein the detection results in the colorimetric mode ( Figure 8 a), Detection results in catalytic mode ( Figure 8 b), heat map results ( Figure 8 c), 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+ The detection range changes, Figure 8 g is ELISA method to detect Cd 2+ As a result, Figure 8 h Detection of Cd based on AuNP-LFA 2+ result.

[0043] Fig. 9 This is a graph showing the analysis results of the immunochromatography of the nanozyme probe of Example 4 of the present invention in the actual sample Dendrobium officinale, wherein Fig. 9 a is based on GO-Pt 30 -AuIr-LFA detection of IMI and Cd 2+ The test results before catalysis. Fig. 9 b is based on GO-Pt 30 -AuIr-LFA detection of IMI and Cd2+ Figure 2. Detection results after catalysis.

[0044] Fig.10 This is a repeatability verification diagram of the immunochromatography of the nanozyme probe of Example 4 of the present invention, wherein Fig.10 a is IMI and Cd 2+ The test results before catalysis when the test concentration was 10 / 10 ng / mL (first group) and 0.1 / 0.1 ng / mL (second group), where Fig.10 a-Ⅰ is the detection band image, Fig.10 a-Ⅱ is the corresponding T line signal intensity change; Fig.10 b is the detection result after catalysis under two groups of concentrations, where Fig.10 b-Ⅰ is the detection band image, Fig.10 b-Ⅱ is the corresponding change in signal intensity of T line.

[0045] Fig.11 This is a specificity verification diagram of the immunochromatography of the nanozyme probe of Example 4 of the present invention, wherein Fig.11 a is IMI and Cd before catalysis 2+ The specific test results are Fig.11 a-Ⅰ is the detection band image, Fig.11 a-Ⅱ is the corresponding T line signal intensity change; Fig.11 b shows IMI and Cd after catalysis 2+ The test results, among which Fig.11 b-Ⅰ is the detection band image, Fig.11 b-Ⅱ is the corresponding change in signal intensity of T line. DETAILED DESCRIPTION

[0046] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0047] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0048] If no specific technology or conditions are specified in the examples, they can be carried out according to the technology or conditions described in the literature in the field or according to the product instructions. Unless otherwise specified, the quantitative tests in the following examples are repeated more than three times, and the results are averaged.

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

[0050] H 2 PtCl6·6H 2 O solution (5mM, 30mL) was added to 240mL boiling deionized water and reacted for 1min, followed by the addition of 5.5mL of a mixture of 1% sodium citrate and 0.05% citric acid and the reaction for 30s. Then 2.75mL of a newly prepared solution (a mixture of 0.08% NaBH4, 1% sodium citrate and 0.05% citric acid) was quickly added, and the heating was stopped after reacting for 10min, and the mixture was cooled to room temperature to obtain a 5nm Pt NP solution. 3mL of Pt NPs with a size of 5nm was added to 90mL deionized water and mixed with 0.135mL of 0.4M H2PtCl6·6H2O solution. After heating to boiling, 1.5mL of 1.25% L-ascorbic acid reduced with 1% sodium citrate was added to the mixture, and the heating was stopped after reacting for 30min, and the mixture was cooled to room temperature to obtain Pt NPs with a diameter of 30nm.

[0051] AuIr NPs: The preparation method is as follows:

[0052] First, prepare a 500mL conical flask, wash it twice with deionized water, then add 4mL 1% chloroauric acid and 3.5mL 1% sodium citrate to 400mL deionized water, stir at 600rpm, and the solution will turn pale yellow. Then add 12mL sodium borohydride (101.6mM). The solution turns from pale yellow to bright red, and stir at 600rmp for 4-6h to obtain 3nm gold nanoparticles.

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

[0054] Au-Ir nanoparticles were synthesized by seed-mediated method. First, 80 mL of sodium hexachloroiridate (2 M) and 24 mL of 1% sodium citrate were added to 400 mL of Au NPs. The pH was adjusted to 7-9 with NaOH, stirred at 80 °C for 30 minutes, and then the newly prepared sodium borohydride (NaBH4, 0.1 M, 2 mL) was quickly added. After stirring for 30 minutes, the heating was turned off and stirred at room temperature for 12 hours. The mixture was kept away from light.

[0055] Embodiment 1:

[0056] A graphene oxide-based nanofilm label, which is a GO-Pt-AuIr composite nanofilm, takes a single-layer graphene oxide (GO) (average particle size of 300 nm) as a carrier, Pt particles (average particle size of 30 nm) and AuIr alloy particles (average particle size of 5 nm) are uniformly loaded on the GO surface, and the AuIr alloy particles are filled in the gaps between the Pt particles to form a GO nanofilm coated with the Pt particles and the AuIr alloy particles; a polyethyleneimine (PEI) layer (the molecular weight of PEI is 12000) is arranged between the Pt particles, the AuIr alloy particles and the GO; wherein the mass ratio of GO, PEI, Pt and AuIr is 1:1:321:366.

[0057] The preparation method of the above-mentioned nanofilm label (schematic diagram as shown in Figure 1 As shown), comprising the following steps:

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

[0059] 5 mL of 300 nm single-layer graphene oxide GO (2 mg / mL) was mixed with 20 mL of deionized water, ultrasonicated for 5 min, and the graphene oxide nanosheets were separated by centrifugation. The mixture was treated with 2 mL of polyethyleneimine (PEI) aqueous solution (0.2 mg / mL) under ultrasonic conditions for 30 min to obtain a GO-PEI nanomembrane, which was rinsed twice with deionized water to remove excess PEI and resuspended in 10 mL of deionized water to obtain a GO-PEI solution.

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

[0061] 10 mL of Pt NPs (5 mg / mL) was added to the prepared GO-PEI solution and ultrasonicated for 30 min to form a graphene oxide nanofilm coated with Pt NPs (GO-Pt 30 ). After centrifugation and washing with deionized water once, GO-Pt 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 ultrasonicated for 30 min to obtain graphene oxide nanofilms coated with Pt NPs and AuIr NPs (GO-Pt 30 -AuIr). Excess AuIr NPs were removed by centrifugation and washed with deionized water. The mixture was stored in 10 mL of ethanol for later use.

[0064] In this embodiment, the GO nanosheets and GO-PEI nanofilms in step (1) and the GO-Pt 30 Composite nanofilm, GO-Pt prepared in step (3) 30 -AuIr composite nanofilm transmission electron microscopy (TEM) Figure 2 The image shows that Pt NPs and AuIr NPs are orderly assembled on the surface of GO nanosheets.

[0065] Embodiment 2:

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

[0067] 10mL GO-Pt 30 -AuIr composite nanofilm was treated with 0.1 mL 2-nitrobenzoic acid (DTNB) solution (10 mM) by ultrasonication to form carboxylated GO-Pt 30 -AuIr composite nanofilm (denoted as GO-Pt 30 -AuIr-D). (The schematic diagram of the preparation is shown in Figure 1 (shown)

[0068] Embodiment 3:

[0069] A method for preparing a nanozyme probe comprises the following steps: 30 -AuIr composite nanofilm (1 mL) was dispersed in 0.05% PBST buffer (0.5 mL), EDC (5 μL) and NHS (10 μL) were added, and ultrasonic treatment was performed for 10 min. 2+ and the specific antibody of pesticide residue IMI, shake at room temperature for 2 hours to mix thoroughly, then add BSA (10%) and block for 1 hour; after the reaction, wash the product with 0.05% PBST buffer to obtain GO-Pt 30 -AuIr nanozyme probe.

[0070] Application of nanozyme probes in dual-channel immunochromatographic system detection:

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

[0072] During immunochromatographic detection, the nanozyme probe and the buffer of the running buffer are directly loaded onto the sample pad of the immunochromatographic strip. After the chromatography reaction is completed, the colorimetric signal and catalytic signal on the test strip detection line are read. The running buffer formula is 1% PBST.

[0073] This example optimizes the operating conditions of the competitive immunochromatography to achieve the best performance of the dual-channel detection platform. The optimization results of the concentration of captured antigens on the detection line are as follows: Figure 3 As shown, when the detection line Cd 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 nanomembrane label had the highest competitive inhibition rate on the target. The optimization results of chromatography time are shown in Figure 2. Figure 4 As shown in Figure 1, a chromatography time of 15 min is sufficient for competitive immunochromatography based on graphene oxide nanomembrane tags to obtain a strong colorimetric signal and the highest competitive inhibition rate. The optimization results of the catalytic time are shown in Figure 1. Figure 5 As shown, the best catalytic performance can be achieved at a catalytic time of 3 minutes, and the highest competitive inhibition rate can be obtained.

[0074] The performance of the dual-mode competitive immunochromatography proposed in this example is highly dependent on the ratio of the two metals. We introduced three Pt 30 / AuIr volume ratio (11%, 33% and 67%) to determine the Pt 30 The effect of the ratio of / AuIr on our method. Figure 6 shown.

[0075] We can conclude that considering the dual signal enhancement effects of colorimetric signal and catalytic signal, Pt 30 GO@Pt with a ratio of 33% and AuIrNPs 30 -AuIr can produce more stable colorimetric and catalytic signals.

[0076] The SEM image of the T-line region of the immunochromatography showed that a large amount of film-like GO-Pt 30 -AuIr tag( Figure 6 f-Ⅰ), while no nanofilm was found in the positive test ( Figure 6 f-Ⅱ), indicating that GO-Pt 30 -AuIr tag can work properly on the immunochromatographic system. In the evaluation of the detection performance of the immunochromatographic system, we used 4 samples containing 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 the GO@Pt 30 -Selectivity of T line on AuIr-LFA. Figure 6 Medium color image ( Figure 6 d-Ⅰ) and catalytic images ( Figure 6 e-Ⅰ) shows that only samples containing target contaminants can inhibit the colorimetric / catalytic signal of the corresponding T zone. Figure 6 The signal intensity of the T line before and after catalysis shown in d and e-Ⅱ also clearly shows that our method has good selectivity. 2+ There is no cross-reaction with IMI detection.

[0077] Embodiment 4:

[0078] In this example, different concentrations of Cd 2+ and IMI (200-0.0122 ng / mL) were added to a PBS solution (10 mM, pH 7.4) to simulate a sample contaminated with multiple small molecule pollutants. 2+ The immunochromatographic experimental flow chart of IMI dual-channel detection is as follows: Figure 7 Competitive immunochromatographic system based on graphene oxide nanomembrane label for detection of Cd 2+ and IMI analysis results.

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

[0080] In catalytic mode, GO@Pt 30 -AuIr immunochromatographic test results are as follows Figure 8 As shown in b, Cd2+ The COV values ​​of SARS-CoV-2 and IMI were increased to 100 and 50 ng / mL, respectively. Figure 8 c is based on GO-Pt 30 -The heat map results of AuIr are as follows Figure 8 c; based on GO@Pt 30 -The colorimetric signal intensity changes in the detection area of ​​the AuIr immunochromatographic strip are as follows Figure 8 d and f are shown, and Cd is drawn by calculation 2+ The fitting curves of HPLC and IMI showed that the detection limits (LODs) were 24.66 and 71.25 pg / mL before catalysis, and further decreased to 7.86 and 7.02 pg / mL after catalysis.

[0081] This example further evaluates the reliability of the dual-mode immunochromatography by testing actual samples (Chinese medicinal material Dendrobium officinale). Fig. 9 As shown, the colorimetric and catalytic signal results of the Dendrobium sample test strips were consistent with those of the PBS sample.

[0082] These results clearly show that the immunochromatography based on graphene oxide nanomembrane label has good stability and accuracy in the quantitative analysis of heavy metals and pesticide residues in actual complex samples. We conducted a quantitative analysis of two mixed samples (containing Cd 2+ and IMI) were tested five times to further confirm our proposed 30 -AuIr-LFA method detection stability and accuracy, the results are as follows Fig.10 When the concentration of the target contaminant in the test solution is the same, the immunochromatographic test strips in the same group all show stable and consistent colorimetric signals. 2+ The relative standard deviations of the samples of GO-Pt 30 -AuIr-LFA method has good stability. Heavy metal ion interference chromium ion (Cr 3+ ), lead ion (Pb 2+ ), and the pesticide residue interference substances include carbendazim (CBZ) and acetamiprid (ACE). Fig.11 Display, when only Cd 2+ When GO-Pt 30 The colorimetric and catalytic signals on the T line of -AuIr-LFA are completely suppressed. Therefore, when there is a high concentration of interfering substances (200 ng / mL) 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 the GO-Pt 30 -AuIr-LFA method has excellent specificity in detecting target pollutants.

[0083] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanofilm label based on graphene oxide, characterized in that: The nanofilm label is a GO-Pt-AuIr composite nanofilm, which uses a single layer of graphene oxide (GO) as a carrier, and Pt particles and AuIr alloy particles are uniformly loaded on the GO surface to form a GO nanofilm coated with Pt particles and AuIr alloy particles; a polyethyleneimine (PEI) layer is arranged 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); more preferably 1:1:321:

366.

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

4. 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.

5. The method for preparing a graphene oxide-based nanofilm label according to any one of claims 1 to 4, characterized in that: The following steps are involved: (1) Preparation of GO-PEI nanofilm: a monolayer of graphene oxide is mixed with deionized water, a polyethyleneimine (PEI) solution is added, and ultrasonic treatment is performed to obtain a GO-PEI nanofilm; (2) Preparation of GO-Pt composite nanomembrane: adding Pt NPs solution to GO-PEI nanomembrane and ultrasonic treatment to obtain GO-Pt composite nanomembrane; (3) Preparation of GO-Pt-AuIr composite nanomembrane: Add AuIr NPs solution to the GO-Pt composite nanomembrane and perform ultrasonic treatment to obtain a GO-Pt-AuIr composite nanomembrane.

6. The preparation method according to claim 5, characterized in that: In step (1), the concentration of the PEI solution is 0.1 to 10 mg / mL, preferably 0.2 mg / mL; in steps (1), (2) and (3), the ultrasonic treatment time is 10 to 60 min, preferably 30 min.

7. The preparation method according to claim 5, characterized in that: Also includes: A 2-nitrobenzoic acid (DTNB) solution was added to the GO-Pt-AuIr composite nanomembrane and ultrasonic treatment was performed to form a carboxylated GO-Pt-AuIr composite nanomembrane.

8. A method for preparing a nanozyme probe, characterized in that: The following steps are involved: The nanomembrane tags were dispersed in PBST buffer, EDC and NHS were added, and then ultrasonic treatment was performed. 2+ and the specific antibody of pesticide residue IMI, oscillate at room temperature until fully mixed, then add bovine serum albumin (BSA) for blocking; after the reaction is completed, wash the product with PBST buffer to obtain the product.

9. The nanozyme probe obtained by the preparation method of the nanozyme probe according to claim 8.

10. The nanomembrane tag according to any one of claims 1 to 4 or the nanomembrane tag prepared by the preparation method according to any one of claims 5 to 7, and the nanoenzyme probe according to claim 9 are used for detecting heavy metal Cd in immunochromatography 2+ and / or application of pesticide residue imidacloprid (IMI).

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