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CN-119869517-B - Three-dimensional multilayer flaky nano-enzyme, preparation method thereof and application thereof in immunochromatography detection of small molecular environmental pollutants

CN119869517BCN 119869517 BCN119869517 BCN 119869517BCN-119869517-B

Abstract

The invention discloses a three-dimensional multilayer flaky nano-enzyme, a preparation method thereof and application thereof in immunochromatography detection of small molecular environmental pollutants, and belongs to the technical field of nano-enzyme and immunochromatography detection. Three-dimensional sheet nanoenzymes are carefully designed by continuously building Au Nanoparticles (NPs) and AuPt bimetallic NPs on flexible Graphene Oxide (GO) nanoplatelets. Beneficial effects in nanoenzymes, two-dimensional GO provides tremendous surface area and high stability, hundreds of large aunps provide enhanced colorimetry and large area spherical surfaces, and tens of thousands of AuPt satellites act as spatial catalytic sites and produce excellent peroxidase-like activity. The proposed dual-mode ICA can detect three important drugs, namely gentamicin, clenbuterol and ractopamine, in a real complex sample simultaneously, and greatly improves the detection performance of the existing colorimetric ICA technology based on simple catalysis, so that huge application potential is shown in the aspect of monitoring small molecular targets in real time under different conditions.

Inventors

  • ZHENG SHUAI
  • WANG CHONGWEN
  • WANG SHU
  • GU BING
  • BAI WENLONG
  • LIU YONG

Assignees

  • 中国科学院合肥物质科学研究院

Dates

Publication Date
20260505
Application Date
20241108

Claims (10)

  1. 1. The application of the three-dimensional flaky GO-Au-AuPt nano-enzyme in immunochromatographic detection of small molecular environmental pollutants is characterized in that the small molecular environmental pollutants are one or more of clenbuterol and ractopamine, and the preparation method of the three-dimensional flaky GO-Au-AuPt nano-enzyme comprises the following steps: (1) Fully mixing the monolayer graphene aqueous solution and the polyetherimide aqueous solution under an ultrasonic condition to obtain GO-PEI nano-sheets; (2) Carrying out ultrasonic reaction on the GO-PEI nano-sheets obtained in the step (1) and a negatively charged Au NPs aqueous solution again to obtain two-dimensional GO-Au nano-sheets, wherein the particle size of the Au NPs is 20-100 nm; (3) Dispersing the GO-Au nano-sheets obtained in the step (2) into water by ultrasonic, fully mixing the GO-Au nano-sheets with PEI aqueous solution under ultrasonic conditions to obtain GO-Au-PEI, adding the AuPt NPs solution into the GO-Au-PEI solution, and carrying out ultrasonic treatment again to obtain the three-dimensional flaky GO-Au-AuPt nano-enzyme, wherein the particle size of the AuPt NPs is 5-20 nm.
  2. 2. The use according to claim 1, wherein in step (1), the monolayer graphene has a particle size of 300-700 nm.
  3. 3. The use according to claim 2, wherein in step (1) the monolayer graphene has a particle size of 500 nm.
  4. 4. The use according to claim 2, wherein in step (1) the monolayer graphene has a particle size of 700 nm.
  5. 5. The use according to claim 1, wherein in step (1) the concentration of the aqueous polyetherimide solution is between 0.5 and 4 mg/mL.
  6. 6. The use according to claim 5, wherein in step (1) the concentration of the aqueous polyetherimide solution is 0.5 mg/mL.
  7. 7. The use according to claim 5, wherein in step (1) the concentration of the aqueous polyetherimide solution is 4 mg/mL.
  8. 8. The use according to claim 1, wherein in step (2) the Au NPs have a particle size of 20 nm and in step (3) the AuPt NPs have a particle size of 5 nm.
  9. 9. The use according to claim 1, wherein 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. 10. The use according to claim 9, wherein in steps (1), (2) and (3), the ultrasonic frequency of the ultrasonic treatment is 50 KHZ and the ultrasonic treatment time is 30 min.

Description

Three-dimensional multilayer flaky nano-enzyme, preparation method thereof and application thereof in immunochromatography detection of small molecular environmental pollutants Technical Field The invention belongs to the technical field of nano-enzyme and immunochromatography detection, and particularly relates to an ultrasensitive dual-mode immunochromatography analysis test strip based on three-dimensional sheet-shaped nano-enzyme-mediated colorimetric catalysis dual-signal amplification, and a preparation method and application thereof. Background Small molecule drugs, such as antibiotics and veterinary drugs, are widely used in the global livestock industry to prevent animal diseases and promote animal growth and reproduction. However, some of the drugs are severely limited or prohibited from being excessively used (such as Gentamicin (GM), clenbuterol (CLE) and Ractopamine (RAC) because they are difficult to metabolize in animals and cause serious health problems in humans through enrichment (such as hepatotoxicity, nephrotoxicity and myelotoxicity). Furthermore, drug residues are easily discharged into the surrounding environment through animal feces (including feces and urine) to adversely affect the ecological environment, immunochromatography analysis is considered to be the most popular point-of-care (POCT) technique in the past 40 years because of their unique advantages of simplicity, rapid and real-time analysis, visual recognition, good specificity and low cost. In recent years, researchers have focused on a class of nanomaterials, i.e., nanoezymes with strong and stable peroxidase-like activity, to develop instrument-free sensitive ICA methods. Nanoezymes have two distinct advantages over other nanotags on immunochromatographic platforms, firstly, they can greatly enhance the colorimetric signal on the T-line of the immunochromatographic strip by catalyzing the chromogenic substrate. This effect can effectively improve the detection sensitivity. Second, the catalytic process of nanoenzyme immunochromatography is rapid (only 2-5 minutes are required) and straightforward (involving one step). Therefore, the nano enzyme immunochromatography maintains the characteristics of visual recognition and simple operation. Various nanoezymes with different material compositions and structures have been successfully introduced into immunochromatography and demonstrated by catalytic amplification of colorimetric signals for the ability to significantly increase sensitivity (up to 5-64 fold). The few and uncontrollable active catalytic sites on current nanoezymes limit the maximum peroxidase-like activity of individual nanostructures and limit the detection range (typically in the order of 3-4 orders of magnitude) of current competitive immunochromatography based on nanoezymes. In addition, most nanoenzymes are currently colloidal materials that are unstable in complex environments (e.g., salt ions and matrix interference). These characteristics limit their practical use. Several particular areas of nanoenzymes need to be further enhanced to improve the performance of current immunochromatographic methods by creating additional stable catalytic sites on the nanoenzymes to improve catalytic activity, increasing the relative surface area of the nanoenzymes to improve detection sensitivity, and optimizing the nanostructures to achieve good stability in complex sample analysis. The Chinese patent application document with publication number of CN116621227A discloses a preparation method and application of two-dimensional flaky manganese copper ore nano-enzyme, and the flaky manganese copper ore nano-enzyme is prepared by adopting a hydrothermal reaction of cupric salt and divalent manganese salt, and has good hydrogen peroxidase-like activity and peroxidase-like activity, can catalyze H 2O2 to generate O 2 or OH, has rapid specific glutathione consumption capability, and has good killing capacity on tumor cells. However, the catalytic activity and stability of the two-dimensional lamellar manganese copper ore nano enzyme are poor, and the two-dimensional lamellar manganese copper ore nano enzyme needs to be further improved. Disclosure of Invention The invention aims to solve the technical problems of low catalytic activity and poor stability of the existing nano enzyme. The invention solves the technical problems by the following technical means: The first aspect of the invention provides a preparation method of three-dimensional flaky GO-Au-AuPt nano-enzyme, which comprises the following steps: (1) Fully mixing a single-layer graphene aqueous solution and a Polyetherimide (PEI) aqueous solution under an ultrasonic condition to obtain a GO-PEI nano-sheet; (2) Carrying out ultrasonic reaction on the GO-PEI nano-sheet obtained in the step (1) and the negatively charged Au NPs aqueous solution again to obtain a two-dimensional GO-Au nano-sheet; (3) And (3) ultrasonically dispersing the GO-Au nano-sheet obtained i