High-stability nano enzyme and virus detection application thereof
By combining highly stable gold-core platinum-shell nanozymes with nucleic acid molecular regulation, the problems of long detection time and high equipment requirements in existing virus detection methods have been solved, realizing rapid, simple and low-cost virus detection, which is suitable for rapid diagnosis and prevention and control of infectious disease pathogens.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-06
- Publication Date
- 2026-04-03
AI Technical Summary
Existing virus detection methods, such as virus isolation and culture, antibody detection, and nucleic acid detection, suffer from problems such as long processing time, high equipment requirements, high false positive rates, and high costs, making it difficult to meet the needs for rapid, convenient, and high-throughput virus detection during epidemics.
A highly stable gold-core platinum-shell nanozyme is used, and its activity is regulated by nucleic acid molecules. The nanozyme coated with polystyrene sulfonic acid and polydimethyldiallyl ammonium chloride is used to detect viruses, simplifying the operation and reducing equipment requirements.
It enables rapid, simple, and low-cost virus detection with a wide response range and high detection sensitivity, eliminating the need for complex operations and expensive equipment, and achieving a detection limit at the ng/mL level.
Smart Images
Figure CN121775908A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a highly stable nanozyme and its application in virus detection, belonging to the field of biological detection reagents. Background Technology
[0002] Infectious diseases are a class of diseases caused by various pathogens that can be transmitted between humans, animals, or between humans and animals. The most common infectious diseases, such as influenza, COVID-19, and hepatitis B, are caused by viruses. Because viruses reside inside cells, treatment is relatively difficult and mainly relies on antiviral drugs and the body's immune system to fight them off.
[0003] Rapid diagnosis and isolation of infected viruses can effectively block their transmission routes, thereby preventing large-scale infection and spread.
[0004] Laboratory diagnosis of infectious diseases can employ methods such as virus isolation and culture, antibody testing, and nucleic acid detection. Virus isolation is the gold standard for laboratory testing of hand-foot-mouth disease (HFMD). However, this method is time-consuming, susceptible to many influencing factors, and requires a biosafety level 2 laboratory, which cannot meet the need for simultaneous processing of large numbers of samples during viral outbreaks. Current antibody detection methods include neutralization tests and enzyme-linked immunosorbent assays (ELISA). Neutralization tests are a precise diagnostic tool with type specificity. However, antigen drift can lead to negative results in neutralization tests, and other negative HFMD infections can cause abnormally high antibody levels, resulting in false positives. Furthermore, the production of neutralizing antibodies in patient serum takes time, which is not conducive to early diagnosis of infectious diseases. ELISA, similar to neutralization tests, diagnoses related diseases through the specific reaction of antigens and antibodies. ELISA can detect IgG and IgM antibodies present in patient serum. This method is rapid, sensitive, and easy to operate, making it suitable for widespread clinical use. After viral infection, IgM-specific antibodies can be detected in the serum of patients one day after the onset of illness. However, cross-reactivity often exists between IgM-specific antibodies, resulting in a high false-positive rate. Furthermore, because IgM-specific antibodies persist in the body for a long time, a positive ELISA result cannot determine whether the infection is a past or recent infection. With the rapid development of molecular biology techniques, viral nucleic acid detection technology has gradually become the main method for pathogen detection. Compared with antibody serological testing, nucleic acid testing can detect patients in the window period and identify infected individuals early. However, it has high requirements for testing equipment or platforms, requiring a fluorescence quantitative PCR instrument with fluorescence signal acquisition. High-sensitivity fluorescence PCR instruments are expensive, and the requirements for laboratory cleanliness and operators are also high, limiting the application of molecular diagnostic technology in clinical diagnosis.
[0005] Currently, nucleic acid molecular detection technology is gradually becoming one of the main methods for detecting infectious disease viruses. Establishing a simple, low-cost, and high-throughput method is of great significance for the early diagnosis, treatment, and prevention and control of infectious diseases. Nanozymes are simple to synthesize and have easily controllable catalytic activity, possessing both the high catalytic activity of natural enzymes and the stability and economy of nanoparticles. Utilizing the highly stable gold-core platinum-shell nanozymes to regulate catalytic activity, establishing a detection method for infectious disease pathogens based on gold-core platinum-shell nanozymes can provide data support and reference for the rapid diagnosis of infectious disease pathogens, and also has significant practical implications for the prevention and control of infectious diseases and the protection of public health. Summary of the Invention
[0006] To address the aforementioned problems, this invention provides a highly stable nanozyme and its application in virus detection, utilizing the regulation of the activity of the highly stable noble metal nanozyme by nucleic acid molecules to achieve its virus detection.
[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The highly stable nanozyme of this invention is prepared by the following steps. 1) Prepare a gold core platinum shell nanoenzyme solution coated with sodium polystyrene sulfonate. Take the gold core platinum shell nanoenzyme solution and add polystyrene sulfonic acid solution (PSS solution). After the solution is thoroughly mixed, take it in a water bath. The PSS fully coats the core platinum shell nanoenzyme. Then remove the supernatant. The precipitate is the nanorod coated with polystyrene sulfonic acid layer. 2) Preparation of gold core platinum shell nanoenzyme solution coated with polydiallyldimethylammonium chloride: The solution of nanorods coated with polystyrene sulfonic acid layer was sonicated again in deionized water. Then, polydiallyldimethylammonium chloride solution (PDDAC solution) was added to the nanorod solution. After the solution was thoroughly mixed, it was placed in a water bath to ensure that polydiallyldimethylammonium chloride (PDDAC) was fully coated. After the coating was complete, the nanorod solution was centrifuged to remove the supernatant and the precipitate was polystyrene sulfonic acid solution (PSS solution) polydiallyldimethylammonium chloride (PDDAC) coated gold core platinum shell nanoenzyme.
[0008] According to the aforementioned highly stable nanozyme, the preparation process of the gold-core platinum-shell nanozyme solution in step 1) is as follows: 1.1) First, prepare a gold seed solution. Add hexadecyltrimethylammonium bromide aqueous solution to tetrachloroauric acid aqueous solution, mix well, and then add sodium borohydride aqueous solution under magnetic stirring. After stirring, stop and let stand for 2 hours to obtain a gold seed solution containing gold seeds. 1.2) To prepare the gold nanorod solution, take an aqueous solution of hexadecyltrimethylammonium bromide, add an aqueous solution of tetrachloroauric acid, an aqueous solution of silver nitrate, and an aqueous solution of sulfuric acid, mix well, and then add an aqueous solution of ascorbic acid. The resulting mixed solution changes from orange-red to colorless. Then add the gold seed solution prepared in step 1.1); mix well and place in a 30°C constant temperature water bath; after 16 hours, a gold nanorod solution is formed. 1.3) Preparation of gold core platinum shell nanoenzyme solution: Take the gold nanorod solution into a test tube, and add deionized water, potassium tetrachloroplatinate aqueous solution and ascorbic acid aqueous solution to it in turn and mix well; then put it into a constant temperature water bath at 30°C and react for 3 hours. Then centrifuge twice at 12,000 rpm to remove unreacted ions and excess hexadecyltrimethylammonium bromide to obtain purified gold core platinum shell nanoenzyme solution.
[0009] According to the highly stable nanozyme, in step 1.2), the gold nanorod solution is purified by placing the prepared gold nanorod solution in a constant temperature water bath at 30°C, and then centrifuging it twice at 12,000 rpm for 10 minutes each time to remove unreacted ions and excess hexadecyltrimethylammonium bromide, thereby obtaining a purified gold nanorod solution. Deionized water is then added to control the concentration of gold nanorods in the purified gold nanorod solution to be 0.5 mM.
[0010] According to the described high-stability nanozyme, in step 1), 1 mL of purified gold-core platinum-shell nanozyme solution was taken and 50 μL of 20 mg / mL PSS solution was added. After thoroughly mixing the solution, it was placed in a 30°C water bath for at least 3 hours to ensure complete PSS coating. Then, it was centrifuged twice at 12,000 rpm for 10 minutes each time, and the supernatant was removed. The precipitate was a polystyrene sulfonate-coated nanorod.
[0011] According to the high-stability nanozyme, in step 2), 100 μl of PSS-coated nanorod solution is re-ultrasonically dispersed in 1.0 mL of deionized water. Then, 50 μL of 20 mg / mL PDDAC solution is added to the nanorod solution. After the solution is thoroughly mixed, it is placed in a 30°C water bath for at least 3 hours to ensure that the PDDAC is fully coated. After the coating is complete, the nanorod solution is centrifuged at 12,000 rpm for 8 minutes once, and the supernatant is removed. The precipitate is a gold core platinum shell nanozyme coated with polystyrene sulfonic acid and polydimethyldiallyl ammonium chloride.
[0012] According to the high-stability nanozyme, the activity of the gold-core platinum-shell nanozyme coated with polystyrene sulfonic acid and polydiallyl ammonium chloride prepared in step 2) was tested. Phosphate buffer solution, TMB solution, and H2O2 solution were added to a cuvette and mixed evenly. Then, the gold-core platinum-shell nanozyme solution coated with PSS or PDDAC was added. The mixture was placed in a fixed tank at a temperature of 37°C. The TMB oxidation product had a characteristic absorption peak at 650 nm. The catalytic activity of the nanozyme was detected by observing the color change of the solution. After 10 minutes, the solution changed from colorless to blue, indicating that the gold-core platinum-shell nanozyme coated with polystyrene sulfonic acid and polydiallyl ammonium chloride can react with the TMB solution, that is, it has good peroxidase activity.
[0013] This invention relates to the application of a highly stable nanozyme for virus detection, specifically for the detection of varicella-zoster virus. The method includes the following steps: utilizing the regulation of the activity of the highly stable noble metal nanozyme by nucleic acid molecules to achieve virus detection, phosphate buffer solution, TMB solution, H2O2 solution, and labeled DNA solution are added to a test tube and mixed thoroughly. Then, a gold-core platinum-shell nanozyme solution coated with polystyrene sulfonate (PSS) and polydimethyldiallylammonium chloride (PDDAC) is added. The mixture is reacted in a water bath, and the UV absorption spectrum of the above mixed solution is measured using a UV-Vis spectrophotometer. A standard working curve is plotted based on the relationship between absorbance values and changes in varicella-zoster virus DNA concentration.
[0014] This invention utilizes the regulation of the activity of highly stable noble metal nanozymes by nucleic acid molecules to achieve virus detection. It features a wide response range, high detection sensitivity, and strong specificity. It eliminates the need for additional fluorescent molecule modification of the DNA to be tested, avoiding complex operations and the need for expensive fluorescence detection equipment. It is visually identifiable, and the final detection reaction takes only 5 minutes, overcoming the drawbacks of quantitative real-time PCR detection, which has high requirements for equipment, operation, and operating environment. In nucleic acid detection, it achieves a detection limit down to the ng / mL level.
[0015] This invention enables the establishment of a stable, rapid, and simple nucleic acid detection method, which has potential for wide application in the detection of clinical samples. Attached Figure Description
[0016] Figure 1 This is a schematic diagram of the structure of the highly stable gold-core platinum-shell nanozyme prepared in this invention. Figure 1 A), and a schematic diagram of the virus diagnosis process in which it participates ( Figure 1 B); Figure 2 The gold nanorods prepared in this invention ( Figure 2 A) Gold-core platinum-shell nanorods ( Figure 2 B), PSS-coated gold-core platinum-shell nanorods ( Figure 2 C) and PDDAC-coated gold-core platinum-shell nanozymes ( Figure 2 D) Transmission electron microscope images of the solution; Figure 3 These are the equivalent diameter and zeta potential values of the highly stable gold-core platinum-shell nanozyme prepared in this invention during the process of being placed at room temperature for four months. Figure 4 These are the peroxidase activity monitoring results of the PSS-coated gold core platinum shell nanorods and PDDAC-coated gold core platinum shell nanoenzyme solutions prepared in this invention. Figure 5 The relative activity of peroxidase in the highly stable gold-core platinum-shell nanozyme solution prepared in this invention during the process of being placed at room temperature for four months. Figure 6 This is a standard working curve for the colorimetric detection of varicella-zoster virus using a highly stable gold-core platinum-shell nanozyme solution prepared in this invention. Detailed Implementation
[0017] The contents of this invention will be described in detail below: The preparation and application of highly stable nanozymes of the present invention include the following steps: (1) Preparation of gold core platinum shell nanoenzyme solution: Gold nanorods were synthesized by seed growth method, and then potassium chloroplatinate was reduced by ascorbic acid to obtain gold core platinum shell nanoenzyme solution. The gold core platinum shell nanoenzyme is a core-shell nanostructure with gold nanorods as the core and island-shaped platinum nanoparticles as the shell. Due to the presence of the surfactant hexadecyltrimethylammonium bromide (CTAB), the surface of the nanoenzyme is positively charged.
[0018] (2) Preparation of highly stable gold core platinum shell nanoenzyme solution. The highly stable gold core platinum shell nanoenzyme solution completes the coating of two polymer layers through electrostatic interaction. First, the surface of the gold core platinum shell nanoenzyme is coated with negatively charged sodium polystyrene sulfonate (PSS) polymer, so that the surface of the nanoenzyme is negatively charged. Then, the surface of the gold core platinum shell nanoenzyme is further coated with positively charged polydimethyl diallyl ammonium chloride (polydimethyl diallyl ammonium chloride (PDDAC)) polymer, so that the surface of the nanoenzyme is positively charged, thus completing the double coating of the highly stable gold core platinum shell nanoenzyme.
[0019] (3) Monitoring the activity changes of highly stable gold-core platinum-shell nanozymes: The oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) catalyzed by highly stable gold-core platinum-shell nanozymes was used as a model system to study and test the changes in enzyme activity.
[0020] (4) The detection of infectious disease viruses is achieved by using the regulation of the activity of highly stable noble metal nanozymes by nucleic acid molecules. When there is no target virus, the PCR amplification reaction of DNA molecules does not contain nucleic acid products, so the highly stable gold core platinum shell nanozyme will normally oxidize the substrate and produce a color reaction. When there is a virus, the nucleic acid molecules in the PCR product will inhibit the enzyme activity of the gold core platinum shell nanozyme, which will significantly reduce the oxidase or peroxidase activity of the highly stable gold core platinum shell nanozyme and correspondingly reduce the color signal generated by the oxidation of the chromogenic substrate. Figure 1 This diagram illustrates how nucleic acid molecules can regulate the activity of highly stable noble metal nanozymes to detect viruses.
[0021] The following embodiments are merely preferred embodiments of the present invention, provided to facilitate a better understanding of the invention, and should not be construed as limiting the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; and unless otherwise specified, the experimental materials used were purchased from conventional biochemical reagent manufacturers.
[0022] Example 1: Preparation of the gold-core platinum-shell nanoenzyme solution according to the present invention. 1) Preparation of gold seed solution, Take 7.5 mL of 0.1 M hexadecyltrimethylammonium bromide aqueous solution, add 100.4 mL of 24.7 mM tetrachloroauric acid aqueous solution, mix well, and dilute the volume to 9.4 mL. Under magnetic stirring, add 0.6 mL of 0.01 M sodium borohydride aqueous solution (prepared before use and placed in ice water), stir for three minutes, stop stirring, and let stand for 2 hours to obtain a gold seed solution containing gold seeds. The concentration of gold in the gold seed solution is 0.25 mM. 2) Preparation of gold nanorod solution, Take 100 mL of a 0.1 M hexadecyltrimethylammonium bromide aqueous solution, add 2.05 mL of a 24.7 mM tetrachloroauric acid aqueous solution, 1 mL of a 10 mM silver nitrate aqueous solution, and 2 mL of a 0.5 M sulfuric acid aqueous solution, mix well, and then add 800 mL of a 0.1 M ascorbic acid aqueous solution. The resulting mixed solution changes from orange-red to colorless. Then add 240 mL of the gold seed solution prepared in step 1; mix well, and place in a 30°C constant temperature water bath; the solution begins to show color after 20 minutes, and after 16 hours, it finally turns dark red, indicating the formation of a gold nanorod solution; characterize the prepared gold nanorods by transmission electron microscopy, as shown in the image. Figure 2 As shown in A. From Figure 2 As can be seen from A, the prepared gold nanorods are approximately 80 nanometers long and 20 nanometers in diameter.
[0023] 3) Preparation of gold-core platinum-shell nanoenzyme solution The prepared gold nanorod solution was allowed to stand in a constant temperature water bath at 30°C, and then centrifuged twice at 12,000 rpm for 10 minutes each time to remove unreacted ions and excess hexadecyltrimethylammonium bromide, resulting in a purified gold nanorod solution. Deionized water was then added to control the concentration of gold nanorods in the purified solution to 0.5 mM. 1 mL of the centrifuged purified gold nanorod solution was placed in a test tube, and 1 mL of deionized water, 40 mL of 2 mM potassium tetrachloroplatinate aqueous solution, and 8 mL of 0.1 M ascorbic acid aqueous solution were added sequentially and mixed thoroughly. The solution was then placed in a constant temperature water bath at 30°C. After reacting for 3 hours, the solution changed from a maroon color to a dark gray color (indicating the formation of the gold core platinum shell nanostructure). It was then centrifuged twice at 12,000 rpm for 10 minutes each time to remove unreacted ions and excess hexadecyltrimethylammonium bromide, resulting in a purified gold core platinum shell nanoenzyme solution. The structure of the prepared gold-core platinum-shell nanoenzyme solution was characterized by transmission electron microscopy (TEM), such as... Figure 2 As shown in B.
[0024] Example 2: Preparation of a highly stable gold-core platinum-shell nanoenzyme solution. 1) Preparation of sodium polystyrene sulfonate-coated gold core platinum shell nanoenzyme solution, Take 1 mL of the purified gold-core platinum-shell nanozyme solution after centrifugation, add 50 μL of 20 mg / mL PSS solution (containing 60 mM NaCl), mix thoroughly, and place in a 30°C water bath for at least 3 hours to ensure complete PSS coating. Then centrifuge twice at 12000 rpm for 10 minutes each time, remove the supernatant, and re-disperse the precipitate sonically in 100 μL of deionized water for later use. Characterize the structure of the prepared sodium polystyrene sulfonate polymer-coated gold-core platinum-shell nanozyme solution by transmission electron microscopy, as shown in the figure. Figure 2 As shown in C.
[0025] 2) Preparation of polydiallyldiammonium chloride-coated gold-core platinum-shell nanoenzyme solution. 100 μl of PSS-coated nanorod solution was re-ultrasonicated and dispersed in 1.0 mL of deionized water. Then, 50 μL of 20 mg / mL PDDAC solution (containing 60 mM NaCl) was added to the nanorod solution, and the solution was thoroughly mixed and placed in a 30°C water bath for at least 3 hours to ensure complete PDDAC coating. After complete coating, the nanorod solution was centrifuged at 12000 rpm for 8 minutes at a time, the supernatant was removed, and the precipitate was re-ultrasonicated and dispersed in 100 μL of deionized water for later use. The structure of the prepared polydimethyldiallyl ammonium chloride-coated gold-core platinum-shell nanozyme solution was characterized by transmission electron microscopy (TEM). Figure 2 As shown in D.
[0026] The particle size and surface potential changes of polydimethyldiallylammonium chloride-coated gold-core platinum-shell nanozymes were measured using a dynamic light scattering laser particle size analyzer after being placed at room temperature for four months. (Here, since the particle size measured by dynamic light scattering assumes that the particles are spherical, while our gold-core platinum-shell nanozymes are not spherical, the particle size results given are effective diameters.) Figure 3 The results showed that the measured particle size did not change significantly during the four-month period, and the surface potential remained basically constant. This indicates that the gold core and platinum shell nanozyme, which is double-coated with polymer, has good dispersibility in solution and also shows that it has high stability.
[0027] Example 3: Monitoring the activity changes of the highly stable gold-core platinum-shell nanozyme prepared according to the present invention. Add 0.93 mL of 0.1 M pH 4.5 phosphate buffer solution, 10 μL of 100 mM TMB solution, and 50 μL of 1 M H₂O₂ solution to a cuvette, mix thoroughly, and then add 10 μL of PSS or PDDAC-coated gold-core platinum-shell nanozyme solution. Place the mixture in a temperature-controlled bath at 37 °C. The TMB oxidation product exhibits a characteristic absorption peak at 650 nm, and the catalytic activity of the nanozyme can be detected by observing the color change of the solution. Figure 4 It can be seen that the solution changed from colorless to blue after about 10 minutes, which shows that the PSS or PDDAC-coated gold core platinum shell nanozyme solution can develop color in TMB solution, that is, it has good peroxidase activity, and the PDDAC-coated gold core platinum shell nanozyme solution has a better color development effect. Figure 5 The prepared high-stability gold-core platinum-shell nanozyme solution showed almost no change in the relative activity of its peroxidase during four months of storage at room temperature, indicating that the gold-core platinum-shell nanozyme with its surface double-coated by a polymer has high catalytic stability.
[0028] Add 0.93 mL of 0.1 M pH 4.5 phosphate buffer, 10 μL of 100 mM TMB solution, 50 μL of 1 M H₂O₂ solution, and 20 μL of 1 μg / mL target DNA solution to a test tube, mix well, and then add 10 μL of PSS or PDDAC-coated gold-core platinum-shell nanozyme solution. Place the mixture in a temperature-controlled bath at 37°C, and observe the color change of the solution to detect the catalytic activity of the nanozyme. Figure 4 It can be seen that the solution changed from colored to colorless after about 10 minutes, which indicates that the peroxidase activity of the PSS or PDDAC-coated gold core platinum shell nanozyme solution was inhibited by the target DNA, and therefore could not develop color in the TMB solution.
[0029] Example 4 utilizes the regulation of the activity of highly stable noble metal nanozymes by nucleic acid molecules to achieve virus detection. Take 0.93 mL of 0.1 M pH 4.5 phosphate buffer solution, 10 μL of 100 mM TMB solution, 50 μL of 1 M H2O2 solution, and 20 μL of target DNA solutions of different concentrations and add them to a test tube. Mix well and then add 10 μL of PSS or PDDAC-coated gold-core platinum-shell nanozyme solution. React in a 37 °C water bath for 5 minutes and measure the UV absorption spectrum of the above mixed solution using a UV-Vis absorption spectrophotometer.
[0030] A standard working curve was plotted based on the relationship between absorbance values and changes in varicella-zoster virus DNA concentration. The results are as follows: Figure 6 As shown, the response range of the varicella-zoster virus DNA to be tested is 1 ng / mL to 1000 ng / mL, the linear range is 1 ng / mL to 10 ng / mL, and the detection limit is 1 ng / mL.
[0031] The applicant declares that the present invention is illustrated through the above embodiments to demonstrate its detailed features and methods, but the present invention is not limited to the above detailed features and methods, that is, it does not mean that the present invention must rely on the above detailed features and methods to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the components used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A highly stable nanozyme, characterized in that, The preparation steps are as follows: 1) Prepare a gold core platinum shell nanoenzyme solution coated with sodium polystyrene sulfonate. Take the gold core platinum shell nanoenzyme solution and add polystyrene sulfonic acid solution (PSS solution). After the solution is thoroughly mixed, take it in a water bath. The PSS fully coats the core platinum shell nanoenzyme. Then remove the supernatant. The precipitate is the nanorod coated with polystyrene sulfonic acid layer. 2) Preparation of gold core platinum shell nanoenzyme solution coated with polydiallyldimethylammonium chloride: The solution of nanorods coated with polystyrene sulfonic acid layer was sonicated again in deionized water. Then, polydiallyldimethylammonium chloride solution (PDDAC solution) was added to the nanorod solution. After the solution was thoroughly mixed, it was placed in a water bath to ensure that polydiallyldimethylammonium chloride (PDDAC) was fully coated. After the coating was complete, the nanorod solution was centrifuged to remove the supernatant and the precipitate was polystyrene sulfonic acid solution (PSS solution) polydiallyldimethylammonium chloride (PDDAC) coated gold core platinum shell nanoenzyme.
2. The highly stable nanozyme according to claim 1, characterized in that, Step 1) Preparation process of gold core platinum shell nanozyme solution, 1.1) First, prepare a gold seed solution. Add hexadecyltrimethylammonium bromide aqueous solution to tetrachloroauric acid aqueous solution, mix well, and then add sodium borohydride aqueous solution under magnetic stirring. After stirring, stop and let stand for 2 hours to obtain a gold seed solution containing gold seeds. 1.2) To prepare the gold nanorod solution, take an aqueous solution of hexadecyltrimethylammonium bromide, add an aqueous solution of tetrachloroauric acid, an aqueous solution of silver nitrate, and an aqueous solution of sulfuric acid, mix well, and then add an aqueous solution of ascorbic acid. The resulting mixed solution changes from orange-red to colorless. Then add the gold seed solution prepared in step 1.1); mix well and place in a 30°C constant temperature water bath; after 16 hours, a gold nanorod solution is formed. 1.3) Preparation of gold core platinum shell nanoenzyme solution: Take the gold nanorod solution into a test tube, and add deionized water, potassium tetrachloroplatinate aqueous solution and ascorbic acid aqueous solution to it in turn and mix well; then put it into a constant temperature water bath at 30°C and react for 3 hours. Then centrifuge twice at 12,000 rpm to remove unreacted ions and excess hexadecyltrimethylammonium bromide to obtain purified gold core platinum shell nanoenzyme solution.
3. The highly stable nanozyme according to claim 2, characterized in that, In step 1.2), the gold nanorod solution is purified by placing the prepared gold nanorod solution in a constant temperature water bath at 30°C, and then centrifuging it twice at 12,000 rpm for 10 minutes each time to remove unreacted ions and excess hexadecyltrimethylammonium bromide, thereby obtaining a purified gold nanorod solution. Deionized water is then added to control the concentration of gold nanorods in the purified gold nanorod solution to be 0.5 mM.
4. The highly stable nanozyme according to claim 3, characterized in that, In step 1), take 1 mL of purified gold-core platinum-shell nanozyme solution, add 50 μL of 20 mg / mL PSS solution, mix the solution thoroughly, and place it in a 30°C water bath for at least 3 hours to ensure complete PSS coating. Then, centrifuge twice at 12,000 rpm for 10 minutes each time, remove the supernatant, and the precipitate is polystyrene sulfonate-coated nanorods.
5. The highly stable nanozyme according to claim 4, characterized in that, In step 2), 100 μl of PSS-coated nanorod solution was re-ultrasonically dispersed in 1.0 mL of deionized water. Then, 50 μL of 20 mg / mL PDDAC solution was added to the nanorod solution. After the solution was thoroughly mixed, it was placed in a 30°C water bath for at least 3 hours to ensure complete PDDAC coating. After complete coating, the nanorod solution was centrifuged at 12,000 rpm for 8 minutes at a time. The supernatant was removed, and the precipitate was a gold-core platinum-shell nanozyme coated with polystyrene sulfonic acid and polydimethyldiallyl ammonium chloride.
6. The highly stable nanozyme according to claim 1, characterized in that, For the activity test of the gold-core platinum-shell nanozyme coated with polystyrene sulfonic acid and polydiallyl ammonium chloride prepared in step 2), phosphate buffer solution, TMB solution, and H2O2 solution were added to a cuvette and mixed evenly. Then, PSS or PDDAC-coated gold-core platinum-shell nanozyme solution was added. The mixture was placed in a fixed bath at a temperature of 37°C. The TMB oxidation product showed a characteristic absorption peak at 650 nm. The catalytic activity of the nanozyme was detected by observing the color change of the solution. After 10 minutes, the solution changed from colorless to blue, indicating that the gold-core platinum-shell nanozyme coated with polystyrene sulfonic acid and polydiallyl ammonium chloride can react with the TMB solution, that is, it has good peroxidase activity.
7. A virus detection application of a highly stable nanozyme prepared according to claim 1, characterized in that, Its application in the detection of varicella-zoster virus includes the following steps: Nucleic acid molecules regulate the activity of highly stable noble metal nanozymes to achieve virus detection. Phosphate buffer solution, TMB solution, H2O2 solution, and labeled DNA solution are added to a test tube and mixed thoroughly. Then, a gold-core platinum-shell nanozyme solution coated with polystyrene sulfonate (PSS) and polydimethyldiallylammonium chloride (PDDAC) is added. The reaction is carried out in a water bath. The UV absorption spectrum of the above mixed solution is measured using a UV-Vis spectrophotometer. A standard working curve is plotted based on the relationship between absorbance values and changes in varicella-zoster virus DNA concentration.