A nano-enzyme for catalyzing a color developing reaction and a preparation method thereof
By preparing Cu-PQQ metal-organic hybrid materials, the stability and catalytic activity issues of nanozyme materials during preparation and modification were solved, enabling simple and efficient detection of colorimetric reactions, which is suitable for immunoassay.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ANYANG NORMAL UNIV
- Filing Date
- 2024-05-08
- Publication Date
- 2026-06-30
Smart Images
Figure CN118477691B_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of nanozyme materials, and in particular to nanozymes based on copper ions and pyrroloquinoline quinone (PQQ) metal-organic hybrid materials and their preparation methods, belonging to the field of chemistry. Background Technology
[0002] Immunoassays have demonstrated wide applications in disease diagnosis, food safety, environmental monitoring, and public safety. Due to their high catalytic activity and substrate specificity, natural enzymes are currently the most commonly used signal markers in immunoassays. However, natural enzymes suffer from high preparation and purification costs, difficult storage, poor stability, and low recyclability. Nanozymes (including metal nanoparticles, metal oxides, carbon-based materials, and metal-organic hybrids) possess catalytic activity similar to peroxidases or superoxide dismutases. Compared to natural enzymes, nanozymes offer advantages such as high stability, tunable structure, low cost, ease of preparation, and recyclability. Nanozymes modified with specific biorecognition elements can be used for photoelectric and electroimmunoassays. However, the surface modification steps for nanozymes are often complex, and the modified molecules may reduce the number of catalytically active sites and decrease catalytic activity, affecting the sensitivity of analytical detection. Metal-organic hybrids are a class of crystalline materials formed by the coordination of metal ions with organic ligands. Nanozymes based on this type of material possess advantages such as large specific surface area, abundant structural units, tunable pore size, and good biocompatibility, and have broad application prospects in catalytic reactions, analytical detection, and biomedicine. However, the catalytic centers of most enzyme-like metal-organic hybrid materials originate from secondary metal structural units contained in metal ions or organic ligands, such as metal-organic framework materials composed of iron ions, cobalt ions, copper ions, cerium ions, or metalloporphyrins. Meanwhile, reports and applications of nanozymes with metal ions and organic ligands as active centers are relatively few. Summary of the Invention
[0003] The purpose of this invention is to provide a nanozyme for catalyzing colorimetric reactions and its preparation method, wherein the nanozyme is a Cu-PQQ metal-organic hybrid material.
[0004] To achieve the objective of this invention, the following technical solution is adopted: a nanozyme for catalyzing colorimetric reactions, wherein the nanozyme is a Cu-PQQ metal-organic hybrid material.
[0005] Furthermore, the nanozyme has a particle size of 100-1000 nm.
[0006] A method for preparing a nanozyme for catalytic colorimetric reactions, wherein the nanozyme is a Cu-PQQ metal-organic hybrid material, is disclosed by the following preparation method: polyvinylpyrrolidone is dissolved in a mixed solution of N,N-dimethylformamide and ethanol to obtain solution A; copper nitrate and PQQ are dissolved in N,N-dimethylformamide to obtain solution B; solution B is added to solution A and then sonicated; the mixed solution is then transferred to a polytetrafluoroethylene reaction vessel and heated at 90 to 120 degrees Celsius for 6-10 hours; after cooling to room temperature, the product is centrifuged, washed, and vacuum dried to obtain the Cu-PQQ metal-organic hybrid material.
[0007] The positive and beneficial technical effects of the present invention are as follows: the materials of the present invention are simple to prepare and the conditions are mild. When used for colorimetric reactions, they have the advantages of being simple, sensitive, intuitive and rapid, and requiring no special instruments. The specific implementation methods are described in detail below. Attached Figure Description
[0008] Figure 1 This is a scanning electron microscope image of Cu-PQQ metal-organic hybrid material.
[0009] Figure 2 This is a particle size distribution diagram of Cu-PQQ metal-organic hybrid materials.
[0010] Figure 3 These are the UV-Vis absorption spectra of TMB oxidation catalyzed by different substances.
[0011] Figure 4 This demonstrates the feasibility of the application example analysis method. Ab2-biotin is a biotinylated PSA detection antibody.
[0012] Figure 5 This is an example analysis method for detecting ultraviolet absorption spectra of PSA at different concentrations.
[0013] Figure 6 This is a curve showing the relationship between absorption intensity and PSA concentration in an application example.
[0014] Figure 7 It is the selectivity of the application example analysis method. Detailed Implementation
[0015] To more fully explain the implementation of the present invention, implementation examples are provided. These implementation examples are merely illustrative of the process and do not limit the scope of the present invention. The present invention is illustrated by the following embodiments, but is not limited to the following embodiments. Any variations are included within the technical scope of the present invention.
[0016] The following examples use PSA as an example for detailed illustration, and the antibody is an antibody against PSA. Further explanation of the accompanying drawings follows. Figure 1 This is a scanning electron microscope (SEM) image of a Cu-PQQ metal-organic hybrid material. From... Figure 1 As can be seen, the synthesized hybrid material is granular. Figure 2 This is a particle size distribution diagram of Cu-PQQ metal-organic hybrid materials. From... Figure 2 As can be seen, the average size of the hybrid material is about 530 nm, and the dispersion is good. Figure 3 This shows the catalytic effect of different substances on the oxidation of TMB. Curves 1-3 are the UV absorption curves of H2O2 oxidation of TMB under PQQ (curve 1), copper ions (curve 2), and Cu-PQQ hybrid material (curve 3), respectively. Figure 3 As can be seen from the data, Cu-PQQ metal-organic hybrid materials exhibit the best catalytic effect. Figure 4 The feasibility of this method is demonstrated, indicating that only after the enzyme-labeled plate captures PSA and biotinylated PSA detection antibodies can the rSA-Cu-PQQ hybrid material be effectively captured, thereby catalyzing the oxidation of TMB.
[0017] Figure 5 The UV-Vis absorption spectra of the solutions at different concentrations of PSA, namely 0, 0.1, 1, 10, 100, 250, 500 and 1000 pg / mL. Figure 6 The relationship between the absorption intensity of the solution at 650 nm and the PSA concentration is given by PSA concentrations of 0.1, 1, 10, 100, 250, 500 and 1000 pg / mL. Figure 6 The PSA concentrations in the illustrations are 0.1, 1, 10, 100 and 250 pg / mL, respectively, and the absorption intensity is linearly related to the PSA concentration. Figure 7 This refers to the selectivity of the immunoassay method. Numbers 1 to 6 correspond to: 10 pg / mL bovine serum albumin, 10 ng / mL immunoglobulin G, 10 ng / mL alpha-fetoprotein, 10 ng / mL thrombin, 250 pg / mL PSA, and a mixture containing 250 pg / mL PSA and 10% fetal bovine serum. Figure 7 It can be seen that this method only responds to PSA, and other components in fetal bovine serum do not interfere with the detection of PSA. Table 1 shows the detection results of PSA in actual samples using this immunoassay method.
[0018] Example:
[0019] I. Examples of Cu-PQQ metal-organic hybrid materials:
[0020] Specifically, the following steps were performed: 200 mg of polyvinylpyrrolidone was dissolved in a mixture of 4 mL of N,N-dimethylformamide and 4 mL of ethanol to obtain solution A; 23.2 mg of copper nitrate and 5.4 mg of PQQ were dissolved in 4 mL of N,N-dimethylformamide to obtain solution B; solution B was added to solution A and sonicated for approximately 20 minutes; then the mixture was transferred to a polytetrafluoroethylene reactor and heated at 100°C for approximately 8 hours; after cooling to room temperature, the product was washed by centrifugation and vacuum dried to obtain Cu-PQQ organometallic hybrid material, as shown in the morphology... Figure 1 Particle size distribution Figure 2 As shown.
[0021] II. Application Examples of Cu-PQQ Metal-Organic Hybrid Materials:
[0022] (1) Preparation of Cu-PQQ hybrid materials functionalized with recombinant streptavidin protein:
[0023] 0.1 mg of Cu-PQQ organometallic hybrid material was dispersed in 1 mL of phosphate buffer solution, and 1 mL of phosphate buffer solution containing 1 mg of recombinant streptavidin protein rSA was added. The mixture was shaken for about 1 hour, centrifuged at 5000 rpm for 10 minutes, and the lower precipitate was centrifuged and washed three times before being dispersed in 10 mL of phosphate buffer solution (10 mM, pH 7.4). The prepared rSA-Cu-PQQ was stored at 4 °C for later use.
[0024] (2) Detection of PSA
[0025] Add 100 µL of phosphate buffer containing PSA to the ELISA plate covered with capture antibody, react for about 30 minutes, and wash three times with 200 µL of washing buffer. Then add 100 µL of phosphate buffer containing biotinylated PSA detection antibody (50 ng / mL), incubate for about 30 minutes, and wash once with 200 µL of phosphate buffer. Then add 100 µL of the above phosphate buffer containing rSA-Cu-PQQ hybrid material, incubate at room temperature for about 30 minutes, and wash three times with 200 µL of phosphate buffer. Finally, add 100 µL of acetate buffer (10 mM, pH 4.0) containing 1 mM TMB and 100 mM H2O2, incubate at room temperature for about 30 minutes, and observe the color change of the solution with the naked eye or measure the absorbance of the solution at 650 nm using a UV-Vis spectrophotometer. Figure 5 These are the UV-Vis absorption spectra of solutions at different concentrations of PSA, from... Figure 5As can be seen, the higher the concentration of PSA, the higher the absorption intensity of the solution, and the solution color gradually changes from colorless to blue. This phenomenon is caused by the oxidized TMB generated in the solution, indicating that colorimetric analysis and detection of PSA can be achieved based on the change in absorption intensity of the generated oxidized TMB at 650 nm. A spectrophotometer can detect PSA at a concentration of 0.1 pg / mL, with a linear range of 0.1 ~ 250 pg / mL (see...). Figure 6 ).
[0026] (3) Selective experiment
[0027] The procedure is the same as the application example, except that PSA is replaced with the substance to be tested, and the other conditions remain unchanged. The experimental results are as follows. Figure 7 As shown in the figure, only PSA can cause a significant increase in absorption intensity and a blue color in the solution. The changes in UV absorbance caused by other substances are negligible. Furthermore, substances in fetal bovine serum have almost no effect on the detection of PSA, indicating that this method has good selectivity for the detection of PSA.
[0028] Actual sample testing experiment
[0029] The procedure was the same as in the application example, except that the PSA was replaced with a diluted actual sample, and all other conditions remained unchanged. The experimental results are shown in Table 1. As can be seen from Table 1, the detection results obtained by this method are consistent with those obtained by commercially available enzyme-linked immunosorbent assay (ELISA) kits.
[0030] sample This method (ng / mL) Enzyme-linked immunosorbent assay (ng / mL) 1 1.2 ± 0.1 1.1 ± 0.1 2 1.3 ± 0.1 1.4± 0.1 3 2.7 ± 0.2 2.9 ± 0.3
[0031] After PSA is captured by the antibody on the ELISA plate, the detection antibody and rSA-Cu-PQQ hybrid material are immobilized on the surface of the ELISA plate. The copper ions and PQQ in the captured hybrid material catalyze the oxidation of TMB, turning the solution blue. This change can be monitored visually or with a UV-Vis spectrophotometer. The specific technical solution is as follows: Add 100 µL of phosphate buffer solution containing PSA to the ELISA plate covered with capture antibody, react for about 30 minutes, and wash three times with 200 µL buffer; then add phosphate buffer solution containing 100 µL of biotinylated detection antibody, incubate for about 30 minutes, and wash once with 200 µL buffer; then add phosphate buffer solution containing rSA-Cu-PQQ hybrid material, incubate for about 30 minutes, and wash three times with 200 µL buffer; then add 100 µL of acetate buffer (10 mM, pH 4.0) containing 1 mM TMB and 100 mM H2O2, incubate at room temperature for about 30 minutes, and observe the color change of the solution with the naked eye or measure the absorbance of the solution at 650 nm with a UV-Vis spectrophotometer.
[0032] After a detailed description of the embodiments of the present invention, those skilled in the art will clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent applications. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention shall fall within the scope of the technical solution of the present invention, and the present invention is not limited to the embodiments of the examples given in the specification.
Claims
1. A nanoscale enzyme catalyzing a chromogenic reaction, characterized in that: The nanozyme is a Cu-PQQ metal-organic hybrid material. The following preparation method was used: polyvinylpyrrolidone was dissolved in a mixed solution of N,N-dimethylformamide and ethanol to obtain solution A; copper nitrate and PQQ were dissolved in N,N-dimethylformamide to obtain solution B; solution B was added to solution A and then sonicated; then the mixed solution was transferred to a polytetrafluoroethylene reactor and heated at 90 to 120 degrees Celsius for 6 to 10 hours; after cooling to room temperature, the product was washed by centrifugation and vacuum dried to obtain Cu-PQQ organometallic hybrid material.
2. The nanozyme for catalyzing colorimetric reactions according to claim 1, characterized in that: The nanozyme has a particle size of 100-1000 nm.