Three-dimensional multilayer sheet nanozymes, their preparation methods, and their application in immunochromatographic detection of small molecule environmental pollutants.

Abstract

This invention discloses a three-dimensional multilayer sheet-like nanozyme, its preparation method, and its application in the immunochromatographic detection of small molecule environmental pollutants, belonging to the field of nanozyme and immunochromatographic detection technology. The three-dimensional sheet-like nanozyme is meticulously designed by continuously constructing Au nanoparticles (NPs) and AuPt bimetallic NPs on flexible graphene oxide (GO) nanosheets. Beneficial effects: In the nanozyme, two-dimensional GO provides a large surface area and high stability, hundreds of large AuNPs provide enhanced colorimetric ability and a large spherical surface area, and tens of thousands of AuPt satellites act as spatial catalytic sites and produce excellent peroxidase-like activity. The proposed bimodal ICA can simultaneously detect three important drugs, namely gentamicin, clenbuterol, and ractopamine, in real complex samples, greatly improving the detection performance of existing colorimetric ICA techniques based on simple catalysis, thus showing great application potential in real-time monitoring of small molecule targets under different conditions.

Description

Technical Field

[0001] This invention belongs to the field of nanozyme and immunochromatographic detection technology, specifically relating to an ultrasensitive dual-mode immunochromatographic analysis test strip based on three-dimensional sheet-like nanozyme-mediated colorimetric catalytic dual-signal amplification, its preparation method, and its application. Background Technology

[0002] Small molecule drugs, such as antibiotics and veterinary drugs, are widely used in global livestock farming to prevent animal diseases and promote animal growth and reproduction. However, some of these drugs are strictly limited or prohibited from overuse (such as gentamicin (GM), clenbuterol (CLE), and ractopamine (RAC)) because they are difficult to metabolize in animals and can cause serious health problems in humans (such as hepatotoxicity, nephrotoxicity, and bone marrow toxicity) through accumulation. Furthermore, drug residues are easily released into the environment through animal feces (including feces and urine), adversely affecting the ecological environment. Immunochromatography has been considered the most popular point-of-care testing (POCT) technology over the past 40 years due to its unique advantages of simplicity, speed, real-time analysis, visual recognition, good specificity, and low cost.

[0003] In recent years, researchers have focused on a class of nanomaterials—nanozymes—possessing strong and stable peroxidase-like activity—to develop sensitive, instrument-free ICA methods. Nanozymes offer two unique advantages over other nanotags on immunochromatographic platforms: First, they can significantly enhance the colorimetric signal on the T-line of immunochromatographic bands by catalyzing the chromogenic substrate. This effect effectively improves detection sensitivity. Second, the catalytic process in nanozyme immunochromatography is rapid (requiring only 2-5 minutes) and direct (involving a single step). Therefore, nanozyme immunochromatography retains the advantages of visual identification and ease of operation. Various nanozymes with different material compositions and structures have been successfully introduced into immunochromatography, demonstrating a significant ability to improve sensitivity (up to 5-64 times) through catalytic signal amplification.

[0004] The limited and uncontrollable number of active catalytic sites on current nanozymes restricts the maximum peroxidase-like activity of individual nanostructures and limits the detection range of current nanozyme-based competitive immunochromatography (typically within 3-4 orders of magnitude). Furthermore, most nanozymes are currently colloidal materials, unstable in complex environments (e.g., salt ions and matrix interference). These characteristics limit their practical applications.

[0005] Several specific areas of nanozymes need further development to improve the performance of current immunochromatographic methods: creating additional stable catalytic sites on nanozymes to enhance catalytic activity; increasing the relative surface area of ​​nanozymes to improve detection sensitivity; and optimizing nanostructures to achieve good stability in the analysis of complex samples.

[0006] Chinese patent application CN116621227A discloses a method for preparing and applying two-dimensional sheet-like manganese chalcopyrite nanozymes. The nanozymes are prepared by a hydrothermal reaction of divalent copper salts and divalent manganese salts. These nanozymes exhibit good catalase-like and peroxidase-like activities, catalyzing the production of O2 or ·OH from H2O2, and possess rapid and specific glutathione consumption capabilities. They also demonstrate good killing ability against tumor cells. However, the catalytic activity and stability of these two-dimensional sheet-like manganese chalcopyrite nanozymes are relatively poor and require further improvement. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to solve the problems of low catalytic activity and poor stability of current nanozymes.

[0008] The present invention solves the above-mentioned technical problems through the following technical means:

[0009] The first aspect of this invention provides a method for preparing a three-dimensional sheet-like GO-Au-AuPt nanozyme, comprising the following steps:

[0010] (1) A single-layer graphene aqueous solution and a polyetherimide (PEI) aqueous solution were thoroughly mixed under ultrasonic conditions to obtain GO-PEI nanosheets;

[0011] (2) The GO-PEI nanosheets obtained in step (1) are reacted again with negatively charged Au NPs aqueous solution under ultrasound to obtain two-dimensional GO-Au nanosheets.

[0012] (3) Disperse the GO-Au nanosheets obtained in step (2) into water by ultrasonication, and then mix them thoroughly with the PEI aqueous solution under ultrasonic conditions to obtain GO-Au-PEI; add the AuPt NPs solution into the GO-Au-PEI solution and ultrasonically treat it again to obtain the three-dimensional sheet-like GO-Au-AuPt nanozyme.

[0013] Preferably, in step (1), the particle size of the single-layer graphene is 300-700 nm, and more preferably 500 nm;

[0014] Preferably, in step (1), the concentration of the polyetherimide (PEI) aqueous solution is 0.5-4 mg / mL.

[0015] Preferably, in step (2), the particle size of Au NPs is 20-100 nm, preferably 20 nm.

[0016] Preferably, in step (3), the particle size of AuPt NPs is 5-20 nm, preferably 5 nm.

[0017] Preferably, in steps (1), (2), and (3), the ultrasonic frequency of the ultrasonic treatment is 40-60 kHz, and the ultrasonic treatment time is 10-60 min, preferably 30 min.

[0018] A second aspect of the present invention provides a three-dimensional sheet-like GO-Au-AuPt nanozyme prepared by the above preparation method.

[0019] A third aspect of the present invention proposes the application of the above-mentioned three-dimensional sheet-like GO-Au-AuPt nanozyme in the immunochromatographic detection of small molecule environmental pollutants.

[0020] Preferably, the small molecule environmental pollutant is one or more of gentamicin (GM), clenbuterol (CLE), and ractopamine (RAC).

[0021] A fourth aspect of the present invention provides an immunochromatographic detection method based on the above-mentioned three-dimensional sheet-like GO-Au-AuPt nanozyme material, comprising the following steps:

[0022] The three-dimensional sheet-like GO-Au-AuPt nanozyme is mixed with the sample solution to be tested and then sprayed onto the sample pad of an immunochromatographic (ICA) test strip. The changes in color intensity on the three T lines can be directly observed and evaluated using a handheld instrument with ImageJ software to achieve qualitative determination of the target drug residue. The T-line area of ​​the nitrocellulose (NC) membrane of the immunochromatographic (ICA) test strip is sprayed with three haptens: GM-BSA, CLE-BSA, and RAC-BSA.

[0023] Preferably, the concentration of GM-BSA is 0.4 mg / mL, the concentration of CLE-BSA is 0.6 mg / mL, and the concentration of RAC-BSA is 0.08 mg / mL.

[0024] The beneficial effects of this invention are as follows:

[0025] 1. This invention utilizes 2D GO nanosheets as a carrier to develop a three-dimensional (3D) sheet-like nanozyme (GO-Au-AuPt) with a flexible nanostructure, large surface interface, and numerous spatial catalytic sites. The designed GO-Au-AuPt nanozyme consists of four functional domains: a 2D GO nanosheet as a flexible substrate, providing a scalable surface interface and excellent stability; an AuNP layer to generate an enhanced colorimetric signal for direct detection and provide a periodic three-dimensional structure for constructing catalytic sites; a dense AuPt bimetallic nanozyme layer, providing excellent peroxidase-like activity and constructing numerous spatial catalytic sites for effectively catalytically amplifying the colorimetric signal; and a surface-coupled antibody for target molecule specificity and effective binding, thus improving the catalytic activity and stability of the nanozyme.

[0026] 2. This invention proposes an ultrasensitive, multi-mode, and flexible dual-mode ICA platform based on 3D sheet-like nanozymes for real-time and field monitoring of three important small molecule drugs, namely GM, CLE, and RAC.

[0027] 3. The GO-Au-AuPt immunochromatographic system proposed in this invention has an at least 88-fold increased sensing range for target small molecules after catalysis, and its excellent detection performance (accuracy, stability and specificity) in real environment and food samples has been verified.

[0028] 4. The 3D GO-Au-AuPt nanozyme material prepared in this invention is based on a polyethyleneimine (PEI)-guided electrostatic self-assembly method. A layer of large AuNPs (20 nm) and a layer of dense small AuPt NPs (5 nm) are continuously coated on the surface of flexible GO nanosheets, ingeniously preparing a 3D GO / Au-AuPt nanozyme with excellent colorimetric / catalytic activity. The former provides a large contact area and a strong colorimetric signal, while the latter greatly enhances the catalytic activity.

[0029] 5. The analytical performance of current nanozyme ICAs has been comprehensively improved. These improvements include increased sensitivity and detection throughput, and expanded quantitative range. A 3D GO / Au-AuPt nanozyme was prepared by electrostatically guided layer-by-layer self-assembly of a colorimetric element (AuNP) and a catalytic element (5 nm AuPt), integrating the advantages of GO, AuNP, and AuPt, resulting in enhanced colorimetric and catalytic activity compared to previously reported spherical nanozymes. Results show that our GO / Au-AuPt-based nanozyme ICA allows for colorimetric-catalytic dual-mode detection of three important small molecule drugs (GM, CLE, and RAC) under different conditions. Through simple in-situ catalysis with peroxidase substrates, the detection sensitivity and range of our proposed ICA in direct mode can be instantaneously improved by 15-fold and 83-fold, respectively.

[0030] 6. Furthermore, GO / Au-AuPt-based ICA has been successfully applied to the detection of real environmental samples (i.e., lakes and rivers) and food samples (i.e., pork and chicken), demonstrating the great potential of our multilayer sheet nanozymes in practical applications. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the preparation method of the three-dimensional sheet-like nanoenzyme material in Example 1 of the present invention, wherein... Figure 1 a is a schematic diagram of the synthesis of three-dimensional sheet-like GO / Au-AuPt nanozymes. Figure 1 b is a diagram showing the antibody modification preparation of GO / Au-AuPt nanozymes;

[0032] Figure 2This is a schematic diagram of a GO / Au-AuPt competing ICA circuit, where... Figure 2 a(I) is a schematic diagram of the pre-catalytic detection of positive GM, CLE, and RAC test strips. Figure 2 a(II) is a schematic diagram of the pre-catalytic testing strip for negative GM, CLE, and RAC. Figure 2 a(III) is a schematic diagram of the test strip detection after catalysis of positive GM, CLE and RAC. Figure 2 a(IV) is a schematic diagram of the test strip detection after catalysis of negative GM, CLE, and RAC samples. Figure 2 b is the detection mode for detecting GO / Au-AuPt competing ICA. First, semi-quantitative visual detection is performed, and then quantitative detection of grayscale signals is performed using ImageJ.

[0033] Figure 3 This is a structural characterization diagram of the three-dimensional sheet-like nanoenzyme material of Example 1 of the present invention. Figure 3 a is a morphology diagram of 2D graphene oxide nanofilms. Figure 3 b is a morphology diagram of 20nm gold nanoparticles. Figure 3 c is a morphology diagram of 5nm gold-platinum core-shell nanoparticles. Figure 3 de shows the morphology of GO / Au nanosheets at different magnifications (scale bars are 200 nm and 20 nm, respectively). Figure 3 f is a magnified TEM image of gold nanoparticles adsorbed on the GO / Au surface. Figure 3 gh represents the morphology of 3DGO / Au-AuPt nanosheets at different magnifications (scale bars are 200 nm and 20 nm, respectively). Figure 3 i is a magnified TEM image of gold-platinum satellite particles adsorbed on the surface of the sheet-like nanoenzyme. Figure 3 j represents the Zeta potential of the 3D sheet-like nanozyme and its intermediate products. Figure 3 k represents the EDS line scan result. Figure 3 l represents the element distribution map. Figure 3 mp is the elemental distribution diagram for GO / Au-AuPt: carbon (red), oxygen (green), gold (blue), and platinum (yellow);

[0034] Figure 4 This is a graph showing the enzyme catalytic activity results of the three-dimensional sheet-like nanoenzyme material in Example 1 of the present invention, wherein... Figure 4 a is a schematic diagram of the 3D multiple catalytic sites of GO / Au-AuPt nanozymes. Figure 4 b is the XPS full spectrum of the GO / Au-AuPt nanozyme. Figure 4 c represents the corresponding high-resolution XPS spectrum. Figure 4d shows the UV-Vis spectra of the prepared GO, GO / Au, GO / Au-AuPt nanosheets, gold nanoparticles (20 nm), and gold-platinum nanoparticles (5 nm). Inset: The left side shows color photographs of solutions of GO (1), GO / Au (2), GO / Au-AuPt (3), gold nanoparticles (4), and gold-platinum nanoparticles (5). Figure 4 e is a graph evaluating the peroxidase activity of sheet-like GO / Au-AuPt nanozymes. Figure 4 f is a graph showing the change in UV-Vis absorption peak intensity of TMB catalytic oxidation by GO / Au-AuPt as a function of storage time. Figure 4 g is a comparison of the catalytic activities of different nanosheets when using TMB / H2O2 as a substrate. Figure 4 h represents the steady-state kinetics analysis of the GO / Au-AuPt nanozyme. Figure 4 i represents the steady-state kinetic analysis diagram of the GO / Au-AuPt nanozyme. Figure 4 j is the calculated near-field electromagnetic field distribution of GO / Au nanosheets. Figure 4 k is the calculated near-field electromagnetic field distribution of GO / Au-AuPt nanozymes;

[0035] Figure 5 This is a flowchart of the experimental procedure for simultaneous detection of ractopamine, clenbuterol, and gentamicin using three-dimensional sheet-like nanozyme immunochromatography in Example 1 of the present invention. Figure 5 Figure a shows the feasibility test diagram of the GO / Au-AuPt-based immunochromatographic detection method, where (I) is an image of the T region with and without the target small molecule in direct detection mode; (II,III) are scanning electron microscope images of the T region with and without the target small molecule in direct detection mode; (IV) is an image of the T region with and without the target small molecule in catalytic detection mode; and (V,VI) are scanning electron microscope images of the T region with and without the target small molecule in catalytic detection mode. Figure 5 bc represents the GO / Au-AuPt-based immunochromatographic assay for assessing cross-reactivity, where (I) is an image of the three T lines; and (II) is the corresponding colorimetric signal. Figure 5 b represents the direct detection mode; Figure 5 c represents the catalytic detection mode; Figure 5 d is a diagram showing the optimized buffer for GO / Au-AuPt immunochromatographic assays for detecting GM, CLE, and RAC. Figure 5 e is a graph showing the optimized reaction time for GO / Au-AuPt immunochromatographic assay when detecting GM, CLE, and RAC;

[0036] Figure 6 This is a graph showing the detection results of the three-dimensional sheet-like nanozyme immunochromatographic assay for simultaneous detection of ractopamine, clenbuterol, and gentamicin in Example 1 of the present invention, compared with other immunoassay methods. Figure 6Figure a is a schematic diagram of a dual-mode competitive immunochromatographic assay based on GO / Au-AuPt for the simultaneous detection of three drug residues. Figure 6 bc shows the results of immunochromatographic detection of different concentrations of RAC / CLE / GM, where (I) is a photograph of the detection strip; and (II) is a detailed colorimetric signal. Figure 6 b represents the result before the catalytic reaction; Figure 6 c represents the result after the catalytic reaction; Figure 6 d is a thermogram analysis result of the T region of the GO / Au-AuPt immunochromatographic assay strip. Figure 6 e.g., a calibration curve based on colorimetric and catalytic signals, where... Figure 6 e represents the calibration curve of GM; Figure 6 f is the calibration curve for CLE; Figure 6 g represents the calibration curve of RAC. Figure 6 hj represents the ELISA detection result, where (I) is the detection image and (II) is the corresponding calibration curve. Figure 6 h represents the detection result of GM; Figure 6 i represents the detection result of CLE; Figure 6 j represents the RAC detection result; Figure 6 k represents the results of immunochromatographic assays based on gold nanoparticles: (I) shows the detection results of GM; (II) shows the detection results of CLE; (III) shows the detection results of RAC.

[0037] Figure 7 The results of the three-dimensional sheet-like nanozyme immunochromatography of Example 1 of the present invention were analyzed in actual environments and food samples. Figure 7 'a' refers to the testing of pork samples; Figure 7 b represents the testing of chicken samples; Figure 7 c represents the testing of lake water samples; Figure 7 d represents the detection of river water samples, where: (I) shows the photographs of the detection strips before and after the catalytic reaction and the colorimetric signals corresponding to the T line; (II) represents the change in signal intensity of the T region of the detection strips before and after the catalytic reaction. Detailed Implementation

[0038] 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.

[0039] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0040] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0041] Example 1:

[0042] A method for preparing a three-dimensional sheet-like GO-Au-AuPt nanozyme includes the following steps: (a schematic diagram of the preparation process is shown below) Figure 1 (As shown)

[0043] (1) Preparation of GO-PEI nanosheets: 10 mL of 500 nm monolayer graphene (GO) solution was mixed with PEI (0.5 mg / mL) aqueous solution at a volume ratio of 1:4 and subjected to strong ultrasonic treatment for 30 min (ultrasonic frequency 50 kHz). PEI was successfully coated onto the surface of the graphene nanosheets. After 30 min, the nanosheets were separated by centrifugation (10000 rpm, 10 min) to obtain GO-PEI nanosheets. The nanosheets were washed twice with deionized water to remove excess PEI and then resuspended in 10 mL of deionized water.

[0044] (2) Preparation of GO-Au nanosheets: Add 150 mL of negatively charged Au NP (20 nm) solution to the prepared GO-PEI solution and mix. Sonicate the mixture for 30 minutes (ultrasonic frequency 50 kHz), separate the obtained GO-Au nanosheets by centrifugation (7000 rpm, 6 minutes), and wash once with deionized water to remove excess Au NP.

[0045] (3) Preparation of GO-Au-AuPt composite nanozyme: The prepared GO-Au composite nanosheets were ultrasonically dispersed in 10 mL of deionized water, then mixed with 30 mL of PEI (0.5 mg / mL) aqueous solution, and subjected to strong ultrasonic treatment for 30 min (ultrasonic frequency 50 kHz). GO-Au-PEI was collected by centrifugation, washed twice to remove excess PEI, and redispersed in deionized water (10 mL). Subsequently, AuPt NP (5 nm) solution was added to the GO-Au-PEI solution and ultrasonically treated to obtain GO-Au nanosheets modified with AuPt NP. The GO-Au-AuPt composite nanozyme was collected by centrifugation (6000 rpm, 6 min).

[0046] An immunochromatographic detection method for three-dimensional sheet-like GO-Au-AuPt nanozyme material includes the following steps: The three-dimensional sheet-like GO-Au-AuPt nanozyme prepared in this embodiment is mixed with the sample solution to be tested and then sprayed onto the sample pad of an immunochromatographic (ICA) test strip. The changes in color intensity on the three T lines are directly observed and evaluated using a handheld instrument equipped with ImageJ software, enabling qualitative determination of the target drug residue. The T-line region of the nitrocellulose (NC) membrane of the immunochromatographic (ICA) test strip is sprayed with three haptens: GM-BSA, CLE-BSA, and RAC-BSA. The concentration of GM-BSA is 0.4 mg / mL, the concentration of CLE-BSA is 0.6 mg / mL, and the concentration of RAC-BSA is 0.08 mg / mL. Goat anti-mouse antibody is sprayed on the control line (C line) region to ensure normal operation of the detection. The concentration of goat anti-mouse IgG sprayed on the control line is 1 mg / mL.

[0047] Figure 2 The schematic diagram shows the principle of an immunochromatographic detection method for three-dimensional sheet-like GO-Au-AuPt nanozyme materials, including the following steps:

[0048] The detection principle of competitive ICA based on GO / Au-AuPt is as follows: Figure 2 The process is explained in section a. First, a multiplex ICA test strip is constructed, consisting of a sample pad, an absorbent pad with three parallel test lines (T lines), a control line (C line), and a capillary force-providing pad. Second, the prepared immunoGO / Au-AuPt nanozyme is directly mixed with the sample solution and then loaded onto the sample pad of the ICA test strip. When the sample solution contains drug residues, these target molecules are preferentially captured by the corresponding immunonanozyme, thereby hindering the immunoGO / Au-AuPt from binding to the T-line hapten and eliminating their colorimetric signal. Figure 2 a(I)-(II) represents schematic diagrams of the positive and negative test strips before competitive ICA catalysis using GO / Au-AuPt. Under the catalytic conditions of AEC, H2O2, and nanomaterials, the visual detection signal intensity of positive and negative samples was enhanced. Figure 2 a(III-IV)). Figure 2 b demonstrates the detection mode of the GO / Au-AuPt competitive ICA, where the changes in sensitivity before and after catalysis are mainly reflected in the cut-off value and vLOD. First, we performed semi-quantitative detection of the sensitivity visually to determine the initial sensitivity value; second, we used the grayscale reading software ImageJ to quantitatively detect the sensitivity changes before and after catalysis. The structural characterization of the three-dimensional sheet-like nanozyme material prepared in this embodiment is as follows: Figure 3 As shown.

[0049] The enzyme activity of the 3D GO-Au-AuPt composite nanozyme material prepared in this embodiment was verified.

[0050] First, XPS spectra showed that the sheet-like nanozymes contained no other impurity elements but did contain essential elements. UV-Vis spectroscopy verified that Au NP in the GO-Au-AuPt composite nanozyme was the source of the colorimetric signal. Figure 3 d). The figure shows that the UV-Vis spectrum of bare GO has no absorption peak; however, after the AuNP layer is assembled, the UV-Vis spectra of GO-Au and GO-Au-AuPt show a clear and broad absorption peak at 530 nm. We then used TMB as a chromogenic substrate to study the peroxidase activity of the 3D nanozyme. Figure 3 e). This demonstrates that 3D GO-Au-AuPt nanosheets rapidly catalyze TMB solutions in the presence of H2O2 and NaAc, exhibiting strong catalytic activity. Furthermore, the GO-Au-AuPt nanozyme we prepared still exhibited high and stable peroxidase-mimicking activity after long-term storage (at least 60 days). Figure 3 f). Furthermore, we quantitatively evaluated the peroxidase-like activity of GO / Au-AuPt using enzyme kinetic equations. Using TMB as a variable, we found that GO-Au-AuPt exhibited superior enzyme activity in both Vmax and Km (f). Figure 3 h).

[0051] To optimize the feasibility and operating conditions of multichannel detection in competitive immunochromatography based on graphene-supported nanozymes, in order to achieve the best performance of the multichannel composite platform.

[0052] Scanning electron microscopy (SEM) and test strip experiments showed the detection results of GO-Au-AuPt-ICA for CLE before and after AEC catalytic amplification. AEC catalysis (from slightly black to deep red) effectively enhanced the colorimetric signal intensity on the T line of negative samples, while no observable signal appeared in the same area of ​​positive samples. This indicates that the catalytic reaction improved the sensitivity of GO-Au-AuPt-ICA without affecting the detection specificity. Figure 4 a). Subsequently Figure 4 Results from bc indicate that the multiplex ICA bands based on the GO-Au-AuPt nanozyme exhibit good selectivity for the simultaneous detection of three drug residues before and after catalysis, with no cross-reactivity. Considering the large size of GO-Au-AuPt, this invention optimizes some key parameters of the multiplex ICA method to achieve optimal detection performance. Figure 4The results showed that, among these solutions, 1% PBST buffer (10 mM, pH 7.4) containing 0.5% milk produced the highest competitive inhibition rate against RAC / CLE / GM as the electrophoresis buffer. Furthermore, this invention optimized the chromatographic time of the nanozyme run and the colorimetric enhancement catalytic time, demonstrating that the proposed ICA method provided optimal detection performance at a run time of 15 minutes and a catalytic time of 3 minutes. Figure 4 e).

[0053] The detection procedure and experimental results of RAC / CLE / GM based on graphene-supported immunochromatographic assay are described. First, a multiplex ICA test strip was constructed, consisting of a sample pad, an NC membrane with three parallel T lines and one C line, and an absorbent pad providing capillary force. Second, the prepared immuno-GO-Au-AuPt nanozyme was directly mixed with the sample solution and then loaded onto the sample pad of the ICA test strip. Figure 5 a) When the sample solution contains drug residues, the target molecule will be preferentially captured by the corresponding immunonanozyme, thereby hindering the immunobinding of GO-Au-AuPt to the T-line hapten and eliminating its colorimetric signal. If there are no target drug residues in the sample, the sheet-like nanozyme can specifically recognize the pre-fixed hapten on the T region, generating an observable colorimetric band on the corresponding T line for visual observation. Therefore, direct observation and evaluation of the color intensity changes on the three T lines using a handheld instrument with ImageJ software can rapidly and qualitatively determine the target drug residues. Figure 5 b).

[0054] The analytical performance of sheet-like nanozymes-based ICA was first evaluated using samples with different concentrations of RAC (10-0.00005 ng / mL), CLE (100-0.0005 ng / mL), and GM (100-0.0005 ng / mL). Figure 6 At different concentrations, Figure 6 b(I) indicates that the weakest visually perceptible colorimetric signal in the T region was caused by RAC, CLE, and GM concentrations of 0.04, 0.36, and 0.36 ng / mL, respectively. Figure 6 b(II) represents the grayscale value of the colorimetric signal. Therefore, the COVs of GO-Au-AuPt-ICA in competitive mode for RAC, CLE, and GM are 0.12, 1.1, and 1.1 ng / mL, respectively, and the vLODs for RAC, CLE, and GM are 0.013, 0.12, and 0.12, representing the minimum target concentrations that produce a darker color intensity on the T line compared to the blank group concentration. Figure 6When RAC / CLE / GM were measured after c-catalysis, the COV and vLOD of GO-Au-AuPt-ICA increased to 10 / 100 / 100 and 0.001 / 0.013 / 0.013 ng / mL, respectively. Figure 6 c(II) is the gray value of the colorimetric signal. Figure 6 d describes the changes in colorimetric signal intensity measured on three T lines using ImageJ before and after catalysis.

[0055] These results indicate that the GO-Au-AuPt-guided AEC / H2O2 catalytic reaction improves the visual sensitivity (vLOD) and detection range (COV) of the colorimetric ICA method by approximately 10-fold and 77-fold, respectively. Figure 6 For example, the LOD values ​​of GO-Au-AuPt-ICA for RAC, CLE, and GM were 11.24, 17.65, and 16.86 pg / mL before catalysis, respectively, and decreased to 0.74, 0.96, and 1.13 pg / mL after catalysis. This invention uses standard colorimetric ICA test strips of ELISA and AuNP to detect the same drug residues and demonstrates that GO-Au-AuPt-ICA is superior to traditional POCT methods. Figure 6 hj indicates the ELISA results for GM, CLE, and RAC, showing that the LOD values ​​for the three targeted drugs are 50.2, 440.5, and 186.3 pg / mL, respectively. Meanwhile, Figure 6 k is a photograph of the colorimetric ICA of AuNP for GM / CLE / RAC. The COV and vLOD values ​​for GM(I) of these test strips were 11 and 3.3 ng / mL, respectively; for CLE(II), 3.3 and 1.1 ng / mL, respectively; and for RAC(III), 11 and 3.3 ng / mL, respectively. The nanozyme-ICA results show that the visual sensitivity of our GO-Au-AuPt-ICA in direct mode is 9.2 times that of ordinary AuNP-ICA. Catalytic results show that the detection sensitivity of the catalytically processed ICA test strip is at least 44.4 times higher than that of ELISA. Furthermore, the vLOD and detection range of this invention are 253.8 and 9.1 times that of the standard AuNP colorimetric method, respectively. The reliability of the nanozyme immunochromatography was further evaluated by testing food samples from real-world environmental samples.

[0056] like Figure 7As shown, the results of the test strip signals in PBS were consistent in different environments. These results clearly demonstrate that graphene-supported nanozyme immunochromatography exhibits good stability, accuracy, and multiplexing capability in real-world complex samples. The GO-Au-AuPt-ICA band of this invention performs well in various complex samples and shows stable detection results in both direct and catalytic modes. As the GM / CLE / RAC spike concentrations decreased from high (10 / 10 / 1 ng / mL) to low (0.01 / 0.01 / 0.001), the colorimetric / catalytic signal intensity on the three T lines significantly increased. Furthermore, compared to direct detection, the catalytic enhancement of our GO / Au-AuPt nanozyme greatly improves the detection performance of the ICA band in real-world complex samples. The average recoveries of GO-Au-AuPt-ICA in the four matrix samples of this invention were 98.34%–103.23%, 98.25%–102.85%, and 94.55%–100.59%, respectively, with relative standard deviations (RSD) of less than 8.29%.

[0057] Example 2:

[0058] A method for preparing a three-dimensional sheet-like GO-Au-AuPt nanozyme includes the following steps: (a schematic diagram of the preparation process is shown below) Figure 1 (As shown)

[0059] (1) Preparation of GO-PEI nanosheets: 10 mL of 700 nm monolayer graphene (GO) solution and PEI (4 mg / mL) aqueous solution were mixed at a volume ratio of 1:4 and subjected to strong ultrasonic treatment for 60 min (ultrasonic frequency 40 kHz). PEI was successfully coated onto the surface of the graphene nanosheets. After 30 min, the nanosheets were separated by centrifugation (10000 rpm, 10 min) to obtain GO-PEI nanosheets. The nanosheets were washed twice with deionized water to remove excess PEI and then resuspended in 10 mL of deionized water.

[0060] (2) Preparation of GO-Au nanosheets: Add 150 mL of negatively charged Au NP (100 nm) solution to the prepared GO-PEI solution and mix. Sonicate the mixture for 60 minutes (ultrasonic frequency 40 kHz), separate the obtained GO-Au nanosheets by centrifugation (7000 rpm, 6 minutes), and wash once with deionized water to remove excess Au NP.

[0061] (3) Preparation of GO-Au-AuPt composite nanozyme: The prepared GO-Au composite nanosheets were ultrasonically dispersed in 10 mL of deionized water, then mixed with 30 mL of PEI (4 mg / mL) aqueous solution, and subjected to strong ultrasonic treatment for 60 min (ultrasonic frequency 40 kHz). GO-Au-PEI was collected by centrifugation, washed twice to remove excess PEI, and redispersed in deionized water (10 mL). Subsequently, AuPt NP (20 nm) solution was added to the GO-Au-PEI solution and ultrasonically treated to obtain GO-Au nanosheets modified with AuPt NP. The GO-Au-AuPt composite nanozyme was collected by centrifugation (6000 rpm, 6 min).

[0062] Example 3:

[0063] A method for preparing a three-dimensional sheet-like GO-Au-AuPt nanozyme includes the following steps:

[0064] (1) Preparation of GO-PEI nanosheets: 10 mL of 300 nm monolayer graphene (GO) solution was mixed with PEI (2 mg / mL) aqueous solution at a volume ratio of 1:4 and subjected to strong ultrasonic treatment for 60 min (ultrasonic frequency 40 kHz). PEI was successfully coated onto the surface of the graphene nanosheets. After 30 min, the nanosheets were separated by centrifugation (10000 rpm, 10 min) to obtain GO-PEI nanosheets. The nanosheets were washed twice with deionized water to remove excess PEI and then resuspended in 10 mL of deionized water.

[0065] (2) Preparation of GO-Au nanosheets: Add 150 mL of negatively charged Au NP (50 nm) solution to the prepared GO-PEI solution and mix. Sonicate the mixture for 10 minutes (ultrasonic frequency 60 kHz), separate the obtained GO-Au nanosheets by centrifugation (7000 rpm, 6 minutes), and wash once with deionized water to remove excess Au NP.

[0066] (3) Preparation of GO-Au-AuPt composite nanozyme: The prepared GO-Au composite nanosheets were ultrasonically dispersed in 10 mL of deionized water, then mixed with 30 mL of PEI (2 mg / mL) aqueous solution, and subjected to strong ultrasonic treatment for 10 min (ultrasonic frequency 60 kHz). GO-Au-PEI was collected by centrifugation, washed twice to remove excess PEI, and redispersed in deionized water (10 mL). Subsequently, AuPt NP (10 nm) solution was added to the GO-Au-PEI solution and ultrasonically treated to obtain GO-Au nanosheets modified with AuPt NP. The GO-Au-AuPt composite nanozyme was collected by centrifugation (6000 rpm, 6 min).

[0067] The structural characterization and properties of the three-dimensional sheet-like GO-Au-AuPt nanozymes prepared in Examples 2 and 3 are similar to those in Example 1.

[0068] 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. The application of a three-dimensional sheet-like GO-Au-AuPt nanozyme in the immunochromatographic detection of small molecule environmental pollutants, characterized in that, The small molecule environmental pollutant is one or more of clenbuterol and ractopamine; the preparation method of the three-dimensional sheet-like GO-Au-AuPt nanozyme includes the following steps: (1) A single-layer graphene aqueous solution and a polyetherimide aqueous solution were thoroughly mixed under ultrasonic conditions to obtain GO-PEI nanosheets; (2) The GO-PEI nanosheets obtained in step (1) are reacted again with negatively charged Au NPs aqueous solution under ultrasound to obtain two-dimensional GO-Au nanosheets; the particle size of Au NPs is 20-100 nm. (3) Disperse the GO-Au nanosheets obtained in step (2) into water by ultrasonication, and then mix them thoroughly with the PEI aqueous solution under ultrasonic conditions to obtain GO-Au-PEI; add the AuPt NPs solution into the GO-Au-PEI solution and ultrasonically treat it again to obtain three-dimensional sheet-like GO-Au-AuPt nanozyme; the particle size of AuPt NPs is 5-20 nm.

2. The application according to claim 1, characterized in that, In step (1), the particle size of the single-layer graphene is 300-700 nm.

3. The application according to claim 2, characterized in that, In step (1), the single-layer graphene particle size is 500 nm.

4. The application according to claim 2, characterized in that, In step (1), the particle size of the single-layer graphene is 700 nm.

5. The application according to claim 1, characterized in that, In step (1), the concentration of the polyetherimide aqueous solution is 0.5-4 mg / mL.

6. The application according to claim 5, characterized in that, In step (1), the concentration of the polyetherimide aqueous solution is 0.5 mg / mL.

7. The application according to claim 5, characterized in that, In step (1), the concentration of the polyetherimide aqueous solution is 4 mg / mL.

8. The application according to claim 1, characterized in that, In step (2), the particle size of Au NPs is 20 nm; in step (3), the particle size of AuPt NPs is 5 nm.

9. The application according to claim 1, characterized in that, In steps (1), (2), and (3), the ultrasonic frequency of the ultrasonic treatment is 40-60 kHz, and the ultrasonic treatment time is 10-60 min.

10. The application according to claim 9, characterized in that, In steps (1), (2), and (3), the ultrasonic frequency of the ultrasonic treatment is 50 kHz, and the ultrasonic treatment time is 30 min.

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