Multi-metal nano-enzyme as well as preparation method and application thereof

Abstract

The invention provides a multi-metal nano-enzyme as well as a preparation method and application thereof, and belongs to the field of nano-enzyme materials. The preparation method of the multi-metal nano-enzyme comprises the steps that a chloroauric acid solution and a sodium citrate solution are mixed for a first reduction reaction, and gold nanoparticles are obtained; dispersing the gold nanoparticles, mixing the dispersed gold nanoparticles with a chloroplatinic acid solution and an L-ascorbic acid solution, and carrying out a second reduction reaction to obtain Au / Pt nanoparticles; and mixing the Au / Pt nano particles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol for pretreatment, and then adding an ethylene glycol solution of a sodium hexachloroiridate (III) hydrate for a third reduction reaction to obtain the multi-metal nano enzyme. The multi-metal nano-enzyme prepared by the preparation method provided by the invention has high substrate affinity, catalytic performance and stability, and the multi-metal nano-enzyme has high detection sensitivity and stability on tumor markers when being used for lateral flow chromatography test strips.

Description

Technical Field

[0001] This invention relates to the field of nanoenzyme materials, and in particular to a multimetal nanoenzyme, its preparation method, and its application. Background Technology

[0002] Tumor markers, such as alpha-fetoprotein (AFP), are among the most common biomarkers in the diagnosis and prognostic assessment of liver cancer. However, the commonly used detection methods generally rely on specific instruments and equipment, require professional operating skills, and have complex, time-consuming, and costly detection procedures. These factors, to some extent, limit the widespread application of these technologies in resource-limited environments or in real-time detection scenarios.

[0003] Lateral flow chromatography test strips offer advantages such as rapid detection, ease of operation, and low cost, and have been widely used for point-of-care testing of biomarkers in food, the environment, and clinical settings. However, currently commonly used lateral flow chromatography test strips for detecting alpha-fetoprotein (AFP) biomarkers use gold nanoparticles as labeled probes, and their detection sensitivity needs further improvement. Summary of the Invention

[0004] The purpose of this invention is to provide a multimetal nanozyme, its preparation method, and its applications. The multimetal nanozyme prepared by the method provided by this invention exhibits high catalytic performance and stability, and the multimetal nanozyme used in lateral flow chromatography test strips demonstrates high detection sensitivity and stability for tumor markers.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing multimetal nanozymes, comprising: (1) Chloroauric acid solution and sodium citrate solution were mixed to carry out the first reduction reaction to obtain gold nanoparticles; (2) The gold nanoparticles obtained in step (1) are dispersed and mixed with chloroplatinic acid solution and L-ascorbic acid solution to carry out a second reduction reaction to obtain Au / Pt nanoparticles; the concentration of L-ascorbic acid solution is 25~35 mmol / L; the addition rate of L-ascorbic acid solution is 15~25 mL / h; (3) The Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol obtained in step (2) are mixed and pretreated, and then a ethylene glycol solution of sodium hexachloroiridate (III) hydrate is added to carry out a third reduction reaction to obtain a multi-metal nanozyme; the temperature of the pretreatment is 110~120 ℃; the concentration of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 0.05~0.15 mg / mL; the addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 15~25 mL / h; the temperature of the third reduction reaction is 170~180 ℃.

[0006] Preferably, the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution in step (1) is (24~26):(96~98).

[0007] Preferably, in step (3), the mass ratio of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide and polyvinylpyrrolidone is (2~5):(70~90):(40~60):(5~20).

[0008] Preferably, in step (3), the concentration of Au / Pt nanoparticles in the mixture formed by mixing Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol is 1~1.5 mg / mL.

[0009] Preferably, the mixing of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol in step (3) specifically involves: 1) Au / Pt nanoparticles are mixed with a portion of ethylene glycol to form mixture A; 2) Mix L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and the remaining ethylene glycol to form mixture B; 3) Mix the mixture A and the mixture B for the third time.

[0010] Preferably, the pretreatment time in step (3) is 0.5~1.5 h.

[0011] Preferably, in step (3), the mass ratio of Au / Pt nanoparticles to sodium hexachloroiridate (III) hydrate in the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is (2~5):(1~2).

[0012] Preferably, the time for the third reduction reaction in step (3) is 1~2 h.

[0013] The present invention also provides a multi-metal nanozyme prepared by the preparation method described in the above technical solution.

[0014] The present invention also provides the application of the multimetal nanozyme described in the above technical solution in a side-flow chromatography test strip.

[0015] This invention provides a method for preparing a multimetal nanozyme, comprising: (1) mixing chloroauric acid solution and sodium citrate solution to carry out a first reduction reaction to obtain gold nanoparticles; (2) dispersing the gold nanoparticles obtained in step (1) and mixing them with chloroplatinic acid solution and L-ascorbic acid solution to carry out a second reduction reaction to obtain Au / Pt nanoparticles; the concentration of the L-ascorbic acid solution is 25~35 mmol / L; the addition rate of the L-ascorbic acid solution is 15~25 mL / h; (3) mixing the Au / Pt nanoparticles obtained in step (2), L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol for pretreatment, and then adding an ethylene glycol solution of sodium hexachloroiridate (III) hydrate to carry out a third reduction reaction to obtain a multimetal nanozyme; the pretreatment temperature is 110~120 ℃; the concentration of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 0.05~0.15 mg / mL; the addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 15~25 mL / h; the temperature of the third reduction reaction is 170~180 ℃.

[0016] This invention first prepares gold nanoparticles by reducing chloroauric acid with sodium citrate, then reduces and deposits platinum on the surface of the gold nanoparticles to obtain Au / Pt nanoparticles. Finally, using the Au / Pt nanoparticles as the core, precise epitaxial growth of iridium is achieved through key surface pretreatment to obtain Au / PtIr nanoparticles. During the pretreatment at 110–120 °C, the adsorption kinetics of polyvinylpyrrolidone (PVP) are enhanced, forming a dense and stable protective layer on the Au / Pt surface, providing colloidal stability for subsequent high-temperature reactions. Simultaneously, the Br in potassium bromide… - Preferential adsorption occurs on highly active edges and crystal faces, saturating and stabilizing surface active sites through coordination, thus homogenizing the overall reaction activity. L-Ascorbic acid provides a mild reducing environment, cleaning surface oxides and impurities. The three work synergistically to regulate the metal surface to a stable and uniform chemical state, laying the foundation for subsequent epitaxial growth. In the subsequent high-temperature reaction, by precisely controlling the concentration and addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate and the temperature of the third reduction reaction, the strong reducing properties of ethylene glycol at high temperatures are utilized to reduce Ir... 3+ The Ir is rapidly reduced to its atomic state, enabling ultra-slow, atomically precise epitaxial growth, ultimately forming a continuous, dense, smooth, and highly crystalline Ir shell. PVP effectively prevents particle aggregation during this process, ensuring good product dispersibility, and ultimately yielding Au / PtIr multimetal nanozymes with stable structure, uniform dispersion, and excellent catalytic performance.

[0017] This invention applies the prepared multi-metal nanozyme to a lateral flow chromatography test strip. By utilizing the Pt contained in the multi-metal nanozyme, it provides high redox activity, efficiently adsorbing and dissociating H₂O₂ to generate highly reactive oxygen species such as hydroxyl radicals (·OH), rapidly oxidizing chromogenic substrates (such as TMB), producing color changes, achieving signal amplification, and improving detection sensitivity. Ir enhances stability and oxygen-related reaction performance, ensuring signal reproducibility. Simultaneously, Ir has unique adsorption characteristics for oxygen species, which can regulate the oxygen coverage on the Pt surface, preventing the active sites of Pt from being poisoned by excessively strong oxygen-containing intermediates, thus maintaining the high activity of Pt. Furthermore, Ir itself also possesses certain peroxidase activity, producing a synergistic or complementary effect with Pt, improving the catalytic chromogenic substrate effect of the multi-metal nanozyme, thereby enhancing the accuracy and sensitivity of the lateral flow chromatography test strip for detecting tumor markers. Results show that the multi-metal nanozyme prepared by the method provided in this invention exhibits high chromogenic activity for the substrate TMB. K m With a concentration of only 0.194 mM and a specific activity of 198.58 U / mg, the catalytic activity did not significantly decrease during a 90-day testing period, indicating that the obtained multi-metal nanozyme has high substrate affinity, catalytic performance, and stability. The multi-metal nanozyme showed good linearity in the detection of tumor markers (alpha-fetoprotein) using lateral flow chromatography test strips in the range of 0.01 to 1000 ng / mL, with a visual limit of detection (vLOD) as low as 0.2 ng / mL, which is nearly 250 times higher than that of traditional Au-LFA (50 ng / mL). After 30 days of storage, the RSD of the detection signal remained at a low level of 1.24%, demonstrating high detection sensitivity and stability. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the preparation of the multimetal nanozyme and the multimetal nanozyme-antibody probe in Application Example 1 of the present invention. Figure 2 This is a TEM image of the gold nanoparticles in Example 1 of the present invention; Figure 3 This is a TEM image of the Au / Pt nanoparticles in Example 1 of the present invention; Figure 4 This is a TEM image of the multimetal nanozyme in Example 1 of the present invention; Figure 5 This is a statistical diagram showing the particle size distribution of gold nanoparticles, Au / Pt nanoparticles, and multimetal nanozymes in Example 1 of the present invention. Figure 6 The UV-Vis absorption spectra of the gold nanoparticles, Au / Pt nanoparticles, and multimetal nanozymes in Example 1 of this invention are shown. Figure 7This is an EDS image of the multimetal nanozyme in Example 1 of the present invention; Figure 8 This is the XPS full spectrum of the multimetal nanozyme in Example 1 of the present invention; Figure 9 This is a high-resolution Au 4f XPS spectrum of the multimetal nanozyme in Example 1 of the present invention; Figure 10 This is a high-resolution Pt 4f XPS spectrum of the multimetal nanozyme in Example 1 of the present invention; Figure 11 This is a high-resolution Ir 4f XPS spectrum of the multimetal nanozyme in Example 1 of the present invention; Figure 12 This is a Zeta potential diagram of the multimetal nanozyme and multimetal nanozyme-antibody probe in Example 1 of the present invention; Figure 13 The UV-Vis absorption spectra of the multimetal nanozyme, multimetal nanozyme-antibody probe, and antibody in Application Example 1 of this invention are shown below. Figure 14 The image shows the UV-Vis absorption spectrum of the oxidation of the TMB chromogenic substrate by the multimetal nanozyme in Example 1 of this invention in the presence or absence of H2O2. Figure 15 The image shows the UV-Vis absorption spectrum of the multimetal nanozyme in Example 1 of this invention on the oxidation of OPD chromogenic substrate in the presence or absence of H2O2. Figure 16 The image shows the UV-Vis absorption spectrum of the multimetal nanozyme in Example 1 of this invention on the oxidation of ABTS chromogenic substrate in the presence or absence of H2O2. Figure 17 The UV-Vis absorption spectra of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes (Au / PtIr) catalyzing TMB color development in Example 1 of this invention are shown. Figure 18 The fluorescence spectra and reaction process diagrams of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes (Au / PtIr) in Example 1 of this invention, respectively, are used as probes to detect hydroxyl radicals (·OH). Figure 19 The Michaelis-Menten curve and corresponding double reciprocal plot of the multimetal nanozyme in Example 1 of the present invention with TMB as a variable substrate at a fixed H2O2 concentration; Figure 20 The image shows the Michaelis-Menten curve and the corresponding double reciprocal plot of the multimetal nanozyme in Example 1 of the present invention with H2O2 as a variable substrate at a fixed TMB concentration. Figure 21 This is a comparison chart of the specific activity data of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes in Example 1 of the present invention; Figure 22 This is a graph showing the relative activity of multimetal nanozymes in catalyzing TMB oxidation after different storage periods in Example 1 of the present invention. Figure 23 This is a diagram illustrating the detection mechanism of the side-flow chromatography test strip in an application example of the present invention; Figure 24 The calibration curves for the test results obtained by the conventional gold nanoparticle side-flow chromatography method in the test examples of this invention are shown in the left figure, which shows the gray values ​​of the T line at different concentrations, the inset in the left figure shows the strip photographs taken at different concentrations, and the right figure shows the calibration curves. Figure 25 The calibration curves for the test results obtained by the multi-metal nanozyme side-flow chromatography method in the test examples of this invention are shown in the left figure, which shows the gray values ​​of the T line at different concentrations, the inset in the left figure shows the strip photographs taken at different concentrations, and the right figure shows the calibration curves. Figure 26 The calibration curves of the test results obtained by the side-flow chromatography method of the multi-metal nanozyme after catalytic amplification by TMB / H2O2 substrate in the test examples of the present invention are shown in the left figure, which shows the gray values ​​of the T line at different concentrations, the inset in the left figure shows the strip photographs taken at different concentrations, and the right figure shows the calibration curves. Figure 27 This is a graph showing the specificity evaluation of Au / PtIr-LFA for AFP in the test examples of this invention; Figure 28 This is a repeatability test diagram of Au / PtIr-LFA at AFP concentrations of 10, 50, and 250 ng / mL in the test examples of this invention; Figure 29 This is a stability test graph of Au / PtIr-LFA after 1, 15, and 30 days of storage in the test examples of this invention; Figure 30 This is a flowchart of the actual clinical sample testing process for Au / PtIr-LFA in the test examples of this invention; Figure 31 This is a heatmap of the detection results of the actual clinical sample of Au / PtIr-LFA in the test examples of this invention; Figure 32 Box plot showing the distribution of positive and negative samples in the actual clinical samples of Au / PtIr-LFA in the test examples of this invention; Figure 33 This is a linear correlation graph showing the correlation between the Au / PtIr-LFA detection method and the clinical standard chemiluminescence immunoassay (CLIA) in the test examples of this invention. Detailed Implementation

[0019] This invention provides a method for preparing multimetal nanozymes, comprising: (1) Chloroauric acid solution and sodium citrate solution were mixed to carry out the first reduction reaction to obtain gold nanoparticle dispersion; (2) The gold nanoparticle dispersion obtained in step (1) is mixed with chloroplatinic acid solution and L-ascorbic acid solution to carry out a second reduction reaction to obtain Au / Pt nanoparticles; (3) After pretreatment by mixing Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol obtained in step (2), a third reduction reaction is carried out by adding an ethylene glycol solution of sodium hexachloroiridate (III) hydrate to obtain a multi-metal nanozyme.

[0020] In this invention, a chloroauric acid solution and a sodium citrate solution are mixed to carry out a first reduction reaction to obtain a gold nanoparticle dispersion.

[0021] In one embodiment of the present invention, the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution can be (24~26):(96~98), or it can be 25:97. The present invention limits the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution to the above range to obtain gold nanoparticles of suitable size (11~15 nm).

[0022] In an embodiment of the present invention, the solvent of the chloroauric acid solution is water; the concentration of the chloroauric acid solution is 100 mmol / L; the solvent of the sodium citrate solution is water; and the concentration of the sodium citrate solution is 38.8 mmol / L.

[0023] In one embodiment of the present invention, the chloroauric acid solution can be heated to 95-100°C before mixing with the sodium citrate solution. The present invention does not impose any special limitations on the specific mixing operation; mixing operations commonly used by those skilled in the art can be employed.

[0024] In one embodiment of the present invention, the temperature of the first reduction reaction can be 95~100 °C; the time of the first reduction reaction can be 15~25 min, or 20 min. By limiting the temperature and time of the first reduction reaction to the above ranges, the present invention can ensure that gold nanoparticles of suitable size (11~15 nm) are obtained.

[0025] In one embodiment of the present invention, the concentration of gold nanoparticles in the gold nanoparticle dispersion can be 0.5~1.5 mmol / L or 0.8~1.2 mmol / L.

[0026] After obtaining the gold nanoparticle dispersion, the present invention mixes the gold nanoparticle dispersion with chloroplatinic acid solution and L-ascorbic acid solution to carry out a second reduction reaction to obtain Au / Pt nanoparticles.

[0027] In one embodiment of the present invention, the molar ratio of gold nanoparticles in the gold nanoparticle dispersion, chloroplatinic acid in the chloroplatinic acid solution, and L-ascorbic acid in the L-ascorbic acid solution can be (0.8~1.2):(1.1~1.3):(5.5~6.5), or it can be 0.9:1.2:6. By limiting the molar ratio of gold nanoparticles in the gold nanoparticle dispersion, chloroplatinic acid in the chloroplatinic acid solution, and L-ascorbic acid in the L-ascorbic acid solution to the above range, platinum can be deposited on the surface of the gold nanoparticles to obtain Au / Pt nanoparticles.

[0028] In one embodiment of the present invention, the concentration of the L-ascorbic acid solution can be 25-35 mmol / L or 30 mmol / L; the addition rate of the L-ascorbic acid solution can be 15-25 mL / h or 20 mL / h. The present invention limits the concentration and addition rate of the L-ascorbic acid solution to the above ranges to form high-quality Au / Pt nanoparticles.

[0029] In an embodiment of the present invention, the solvent of the chloroplatinic acid solution is water; the concentration of the chloroplatinic acid solution is 3 mmol / L; the solvent of the L-ascorbic acid solution is water; and the concentration of the L-ascorbic acid solution is 30 mmol / L.

[0030] In one embodiment of the present invention, the gold nanoparticle dispersion can be heated to 80-85°C before mixing with the chloroplatinic acid solution and L-ascorbic acid solution. The present invention does not impose any special limitations on the specific mixing operation; mixing operations commonly used by those skilled in the art can be employed.

[0031] In one embodiment of the present invention, the temperature of the second reduction reaction can be 80-85 °C; the time of the second reduction reaction can be 25-35 min, or 30 min. Limiting the temperature and time of the second reduction reaction to the above ranges allows for better deposition of platinum on the surface of gold nanoparticles.

[0032] As one embodiment of the present invention, after the second reduction reaction is completed, the reaction product obtained from the second reduction reaction can be centrifuged to obtain Au / Pt nanoparticles.

[0033] The present invention does not have any special limitations on the centrifugation operation. The Au / Pt nanoparticles can be separated by centrifugation operations commonly used by those skilled in the art.

[0034] After obtaining Au / Pt nanoparticles, the present invention pretreats the Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol, and then adds an ethylene glycol solution of sodium hexachloroiridate (III) hydrate to carry out a third reduction reaction to obtain a multi-metal nanozyme.

[0035] In one embodiment of the present invention, the mass ratio of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, and polyvinylpyrrolidone can be (2~5):(70~90):(40~60):(5~20), (2.5~4.5):(72~88):(42~58):(7~18), (3~4):(74~86):(44~56):(9~16), or (3~3.5):(78~85):(48~55):(9~15). Limiting the mass ratio of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, and polyvinylpyrrolidone to the above range allows for better pretreatment of the Au / Pt nanoparticle surface.

[0036] In one embodiment of the present invention, the concentration of Au / Pt nanoparticles in the mixture formed by Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol can be 1~1.5 mg / mL, 1~1.3 mg / mL, or 1.1 mg / mL. Limiting the concentration of Au / Pt nanoparticles in the mixture formed by Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol to the above range promotes pretreatment.

[0037] In one embodiment of the present invention, the mixture of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol can specifically be: 1) Au / Pt nanoparticles are mixed with a portion of ethylene glycol to form mixture A; 2) Mix L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and the remaining ethylene glycol to form mixture B; 3) Mix the mixture A and the mixture B for the third time.

[0038] In one embodiment of the present invention, the ethylene glycol portion may be 2 / 5 to 3 / 5 of the total volume of ethylene glycol.

[0039] The present invention does not have any special limitations on the operation of the first mixing, the second mixing and the third mixing, as long as the raw materials are evenly dispersed.

[0040] In one embodiment of the present invention, the pretreatment can be carried out under stirring conditions; the pretreatment temperature is 110~120 °C, preferably 115~120 °C; the pretreatment time can be 0.5~1.5 h, or 1~1.5 h. In the present invention, the pretreatment can be carried out by heating to the pretreatment temperature in an oil bath. The present invention limits the pretreatment temperature to 110~120 °C, which enhances the adsorption kinetics of polyvinylpyrrolidone (PVP), forming a dense and stable protective layer on the Au / Pt surface, providing colloidal stability for subsequent high-temperature reactions; simultaneously, the Br in potassium bromide... - Preferential adsorption occurs on highly active edges and crystal faces, and the surface active sites are stabilized through coordination saturation, thus homogenizing the overall reaction activity. L-Ascorbic acid provides a mild reducing environment, cleaning surface oxides and impurities. The three work together to regulate the metal surface to a stable and uniform chemical state, laying the foundation for subsequent epitaxial growth.

[0041] In one embodiment of the present invention, the pretreated mixture can be heated after the pretreatment is completed; the heating temperature can be the same as the temperature of the third reduction reaction.

[0042] In this invention, the concentration of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 0.05~0.15 mg / mL, preferably 0.08~0.12 mg / mL; the addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 15~25 mL / h, preferably 20 mL / h. As one embodiment of this invention, the ethylene glycol solution of sodium hexachloroiridate (III) hydrate can be added using a syringe pump. By limiting the concentration and addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate to the above ranges, this invention enables ultra-slow, atomically precise epitaxial growth of Ir to form a continuous, dense, smooth, and highly crystalline Ir shell, thereby improving the stability and catalytic performance of multi-metal nanozymes.

[0043] In one embodiment of the present invention, the mass ratio of the Au / Pt nanoparticles to the sodium hexachloroiridate (III) hydrate in the ethylene glycol solution of sodium hexachloroiridate (III) hydrate can be (2~5):(1~2), (2.5~4):(1~1.5), or (3~3.5):(1~1.2). Limiting the mass ratio of Au / Pt nanoparticles to the sodium hexachloroiridate (III) hydrate in the ethylene glycol solution of sodium hexachloroiridate (III) hydrate to the above ranges can better improve the stability and catalytic performance of the multi-metal nanozyme.

[0044] In one embodiment of the present invention, the temperature of the third reduction reaction is 170~180 °C, preferably 170~175 °C; the time of the third reduction reaction can be 1~2 h, or 1.5~2 h. By limiting the temperature of the third reduction reaction to the above range, the present invention enables ethylene glycol to have a strong reducing ability at high temperatures, thereby reducing Ir... 3+ The Ir is rapidly reduced to atomic state by ethylene glycol, enabling precise atomic-level epitaxial growth of Ir to form a continuous, dense, smooth, and highly crystalline Ir shell, thereby improving the stability and catalytic performance of the multi-metal nanozyme. By limiting the time of the third reduction reaction to the above range, the full progress of the reduction reaction can be guaranteed.

[0045] As one embodiment of the present invention, after the third reduction reaction is completed, the product of the third reduction reaction can be centrifuged and washed sequentially to obtain a multi-metal nanozyme.

[0046] The present invention does not have any special limitations on the centrifugation operation. Any centrifugation operation commonly used by those skilled in the art can be used to separate the multi-metal nanozymes.

[0047] In one embodiment of the present invention, the washing may involve washing once with acetone and then washing twice with water.

[0048] This invention prepares gold nanoparticles by reducing chloroauric acid with sodium citrate, then deposits platinum on the surface of the gold nanoparticles to obtain Au / Pt nanoparticles. Finally, using the Au / Pt nanoparticles as the core, precise epitaxial growth of iridium is achieved through key surface pretreatment to obtain Au / PtIr nanoparticles. During the pretreatment at 110–120 °C, the adsorption kinetics of polyvinylpyrrolidone (PVP) are enhanced, forming a dense and stable protective layer on the Au / Pt surface, providing colloidal stability for subsequent high-temperature reactions. Simultaneously, the Br in potassium bromide… - Preferential adsorption occurs on highly active edges and crystal faces, saturating and stabilizing surface active sites through coordination, thus homogenizing the overall reaction activity. L-Ascorbic acid provides a mild reducing environment, cleaning surface oxides and impurities. The three work synergistically to regulate the metal surface to a stable and uniform chemical state, laying the foundation for subsequent epitaxial growth. In the subsequent high-temperature reaction, by precisely controlling the concentration and addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate and the temperature of the third reduction reaction, the strong reducing properties of ethylene glycol at high temperatures are utilized to reduce Ir... 3+ The Ir is rapidly reduced to its atomic state, enabling ultra-slow, atomically precise epitaxial growth, ultimately forming a continuous, dense, smooth, and highly crystalline Ir shell. PVP effectively prevents particle aggregation during this process, ensuring good product dispersibility, and ultimately yielding Au / PtIr multimetal nanozymes with stable structure, uniform dispersion, and excellent catalytic performance.

[0049] The present invention also provides a multi-metal nanozyme prepared by the preparation method described in the above technical solution.

[0050] The present invention also provides the application of the multimetal nanozyme described in the above technical solution in a side-flow chromatography test strip.

[0051] In one embodiment of the present invention, the multimetal nanozyme is first prepared as a multimetal nanozyme-antibody probe when used in a side-flow chromatography test strip.

[0052] In an application example of the present invention, the preparation method of the multi-metal nanozyme-antibody probe is as follows: 200 μL of a multimetal nanozyme dispersion with a concentration of 0.9–1.2 mg / mL was added to a 100 mmol / L borate buffer solution with pH=9 to adjust the pH of the solution to 7–8. After mixing thoroughly, the solution was allowed to stand for 15 min. Then, 8 μL of an anti-alpha-fetoprotein monoclonal antibody with a concentration of 1 mg / mL was dispersed in the above solution, and 1 μL of a 5 wt% polyvinylpyrrolidone solution was added. The mixture was mixed thoroughly and vortexed at room temperature for 1 h. Then, 20 μL of a 10 mmol / L boric acid solution (pH=10) containing 5 wt% bovine serum albumin was added, and the solution was incubated at room temperature (20–25 °C) for 30 min. Finally, the solution was washed with a 10 mmol / L borate buffer solution and centrifuged (8000 rpm, 6 min) to obtain the multimetal nanozyme-antibody probe.

[0053] In an application example of the present invention, the multimetal nanozyme-antibody probe is used in the form of a multimetal nanozyme-antibody probe dispersion; the preparation of the multimetal nanozyme-antibody probe dispersion is as follows: the multimetal nanozyme-antibody probe is dispersed in 50 μL of a 5 mmol / L boric acid buffer solution (the boric acid solution also contains 1 wt% bovine serum albumin, 3 wt% sucrose and 0.2 wt% Tween 20) to obtain the multimetal nanozyme-antibody probe dispersion.

[0054] In this invention, the side-flow chromatography test strip includes a polyvinyl chloride card and a sample pad, a conjugate pad, an NC membrane, and an absorbent pad assembled on the polyvinyl chloride card from left to right; the conjugate pad is adsorbed with the multi-metal nanozyme-antibody probe described in the above technical solution.

[0055] In an application example of the present invention, the preparation method of the lateral flow chromatography test strip is as follows: 1 mg / mL of anti-alpha-fetoprotein antibody and 0.3 mg / mL of goat anti-mouse IgG antibody are sprayed onto the NC membrane at a rate of 0.5 μL / cm to form T lines and C lines. The sample pad and conjugate pad are treated with blocking buffer (0.01 M phosphate buffer solution containing 0.5 wt% bovine serum albumin, 0.1 wt% Tween 20 and 2 wt% sucrose) and dried overnight at room temperature (20~25 ℃). Then, the conjugate pad is soaked in a multi-metal nanozyme-antibody probe dispersion until the conjugate pad is thoroughly wetted and then dried. During the assembly process, the sample pad, conjugate pad, NC membrane and absorbent pad are assembled onto a polyvinyl chloride card from left to right. Each part of the lateral flow chromatography test strip overlaps with the adjacent part by 2 mm, and the strip is cut into 3.8 mm widths to obtain the lateral flow chromatography test strip.

[0056] As one embodiment of the present invention, when the lateral flow chromatography test strip is used for tumor marker detection, a substrate solution can be used to amplify the detection signal.

[0057] In one embodiment of the present invention, the substrate solution may contain H2O2; the content of H2O2 in the substrate solution may be 5~10 mmol / L.

[0058] This invention applies the prepared multi-metal nanozymes to lateral flow chromatography test strips. By utilizing the Pt contained in the multi-metal nanozymes, high redox activity can be provided, which can efficiently adsorb and dissociate H2O2 to generate highly active hydroxyl radicals (·OH) and other reactive oxygen species, rapidly oxidizing chromogenic substrates (such as TMB), producing color changes, achieving signal amplification, and improving detection sensitivity. Ir can enhance stability and oxygen-related reaction performance, ensuring signal reproducibility. At the same time, Ir has unique adsorption characteristics for oxygen species, which can adjust the oxygen coverage on the Pt surface, preventing the active sites of Pt from being poisoned by excessively strong oxygen-containing intermediates, thereby maintaining the high activity state of Pt. In addition, Ir itself also has a certain peroxidase activity, producing a synergistic or complementary effect with Pt, improving the catalytic chromogenic substrate effect of the multi-metal nanozymes. The synthesized highly active and stable multi-metal nanozymes can be used in lateral flow chromatography test strips, which can improve their accuracy and sensitivity in detecting tumor markers.

[0059] In this invention, the preparation flowchart of the multimetal nanozyme-antibody probe is as follows: Figure 1 As shown, from Figure 1It can be seen that gold nanoparticles (Au) react with chloroplatinic acid (H2PtCl6) and L-ascorbic acid solution (AA) to obtain Au / Pt nanoparticles (Au / Pt). Au / Pt nanoparticles react with L-ascorbic acid (AA), potassium bromide, polyvinylpyrrolidone, ethylene glycol (EG), and sodium hexachloroiridate (III) hydrate (Na3IrCl6) to obtain a multimetal nanozyme (Au / PtIr). The multimetal nanozyme is then combined with an antibody to obtain a multimetal nanozyme-antibody probe (Au / PtIr-mAb).

[0060] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0061] In the embodiments of this invention, chloroplatinic acid (H2PtCl6), polyvinylpyrrolidone (PVP, MW=55000), sodium hexachloroiridium(III) hydrate (Na3IrCl6), and chloroauric acid (HAuCl4) were purchased from Shanghai Aladdin Co., Ltd.; L-ascorbic acid (AA), 3,3',5,5'-tetramethylbenzidine (TMB), Tween 20, phosphate-buffered saline (PBS), and goat anti-mouse IgG were purchased from Shanghai Sangon Biotech Co., Ltd.; alpha-fetoprotein antigen (AFPAntigen), anti-alpha-fetoprotein antibody (labeling), and anti-alpha-fetoprotein antibody (coating) were purchased from Shanghai Lingchao Biotechnology Co., Ltd.; nitrocellulose membranes (NC membranes), sample pads, conjugate pads, and absorbent pads were all provided by Sartorius (Göttingen, Germany). Unless otherwise stated, all other chemicals were of analytical grade.

[0062] Example 1 A method for preparing a multi-metal nanozyme is as follows: (1) 100 mL of 1 mmol / L chloroauric acid aqueous solution was heated to 100 °C and mixed with 10 mL of 38.8 mmol / L sodium citrate aqueous solution. The first reduction reaction was carried out at 100 °C for 20 min to obtain a 0.9 mmol / L gold nanoparticle dispersion (denoted as Au); the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution was 25:97. (2) Take 10 mL of the gold nanoparticle dispersion obtained in step (1) and heat (preheat) it to 80 °C. Then add 4 mL of 3 mmol / L chloroplatinic acid solution and add 2 mL of 30 mmol / L L-ascorbic acid solution at a rate of 20 mL / h using a syringe pump. After the addition is complete, carry out the second reduction reaction at 80 °C for 30 min. Then cool to 25 °C and centrifuge to obtain Au / Pt nanoparticles (denoted as Au / Pt). The molar ratio of gold nanoparticles in the gold nanoparticle dispersion, chloroplatinic acid in the chloroplatinic acid solution and L-ascorbic acid in the L-ascorbic acid solution is 0.9:1.2:6. (3) The Au / Pt nanoparticles obtained in step (2) are first mixed with 3 / 5 volume of ethylene glycol to form mixture A. L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and the remaining ethylene glycol are second mixed to form mixture B. Mixture A and mixture B are third mixed. The mixture is then pretreated for 1 h by heating in an oil bath to 120 °C with stirring. Then it is heated to 170 °C and a 0.1 mg / mL ethylene glycol solution of sodium hexachloroiridate (III) hydrate is added at a rate of 20 mL / h using a syringe pump. The third reduction reaction is carried out at 170 °C for 1.5 h (the third reduction reaction begins when the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is added). The product of the third reduction reaction is then centrifuged at 12000 rpm for 15 minutes. The sample was washed once with acetone and then twice with water to obtain a multi-metal nanozyme (denoted as Au / PtIr). The mass ratio of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, and polyvinylpyrrolidone was 3.3:80:50:10. The concentration of Au / Pt nanoparticles in the mixture formed by Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol was 1.1 mg / mL. The mass ratio of Au / Pt nanoparticles to sodium hexachloroiridate (III) hydrate in the ethylene glycol solution was 3.3:1.

[0063] Application Example 1 The application of the multimetal nanozyme described in Example 1 in a lateral flow chromatography test strip, wherein the multimetal nanozyme is first prepared as a multimetal nanozyme-antibody probe when used in the lateral flow chromatography test strip; The preparation method of the multimetal nanozyme-antibody probe is as follows: The multimetal nanozyme prepared in Example 1 is prepared into a multimetal nanozyme dispersion with a concentration of 1 mg / mL. 200 μL of the multimetal nanozyme dispersion is added to a 100 mmol / L borate buffer solution with pH=9 to adjust the pH of the solution to 7-8. After mixing evenly, the solution is allowed to stand for 15 min. Then, 8 μL of anti-alpha-fetoprotein monoclonal antibody with a concentration of 1 mg / mL is dispersed in the above solution, and 1 μL of polyvinylpyrrolidone with a concentration of 5 wt% is added. The solution is mixed evenly and vortexed at room temperature for 1 h. Then, 20 μL of 10 mmol / L boric acid solution (pH=10) containing 5 wt% bovine serum albumin is added, and the solution is incubated at room temperature (25 ℃) for 30 min. Finally, the solution is washed with 10 mmol / L borate buffer solution and centrifuged (8000 rpm, 6 min) to obtain the multimetal nanozyme-antibody probe (denoted as Au / PtIr-mAb). The multimetal nanozyme-antibody probe was dispersed in 50 μL of a 5 mmol / L boric acid solution (the boric acid solution also contained 1 wt% bovine serum albumin, 3 wt% sucrose and 0.2 wt% Tween 20) to obtain a multimetal nanozyme-antibody probe dispersion. A lateral flow chromatography test strip comprises a polyvinyl chloride card and a sample pad, a conjugate pad, an NC membrane, and an absorbent pad, which are assembled sequentially from left to right on the polyvinyl chloride card; the conjugate pad is adsorbed with the aforementioned multimetal nanozyme-antibody probe. The preparation method of the lateral flow chromatography test strip is as follows: 1 mg / mL of anti-alpha-fetoprotein antibody and 0.3 mg / mL of goat anti-mouse IgG antibody are sprayed onto the NC membrane at a rate of 0.5 μL / cm to form T and C lines, respectively. The sample pad and conjugate pad are treated with blocking buffer (0.01 M phosphate buffer solution containing 0.5 wt% bovine serum albumin, 0.1 wt% Tween 20 and 2 wt% sucrose) and dried overnight at room temperature (20~25 ℃). Then, the conjugate pad is soaked in multi-metal nanozyme-antibody probe dispersion until it is thoroughly wetted and then dried. During assembly, the sample pad, conjugate pad, NC membrane and absorbent pad are assembled onto a polyvinyl chloride card from left to right. Each part of the lateral flow chromatography test strip overlaps with the adjacent part by 2 mm, and the strip is cut into 3.8 mm widths to obtain the lateral flow chromatography test strip (denoted as Au / PtIr-LFA).

[0064] Test case The morphology of the gold nanoparticles, Au / Pt nanoparticles, and multimetal nanozymes in Example 1 was observed using transmission electron microscopy (TEM). The TEM image of the gold nanoparticles in Example 1 is shown below. Figure 2 As shown, the TEM image of Au / Pt nanoparticles is as follows. Figure 3As shown, the TEM image of the multimetal nanozyme is as follows. Figure 4 As shown, from Figures 2-4 As can be seen, the three types of synthesized nanoparticles are uniform in size and well dispersed. The gold nanoparticles are spherical, while the Au / Pt nanoparticles and the multi-metal nanozymes exhibit irregular morphological structures on their surfaces. Furthermore, the multi-metal nanozymes have a more compact surface structure due to the introduction of Ir.

[0065] The particle size distribution of the gold nanoparticles, Au / Pt nanoparticles, and multimetal nanozymes in Example 1 was statistically analyzed, and the resulting particle size distribution statistics are shown in the figure below. Figure 5 As shown, from Figure 5 As can be seen, the average size of the nanoparticles gradually increases as the reaction proceeds, which strongly confirms the successful stepwise deposition of metal elements.

[0066] The UV-Vis absorption peaks of the gold nanoparticles, Au / Pt nanoparticles, and multimetal nanozymes in Example 1 were measured using a UV-2600 UV spectrophotometer, further confirming their structural evolution. The obtained UV-Vis absorption spectra are shown below. Figure 6 As shown, from Figure 6 As can be seen, only spherical gold nanoparticles exhibit a significant surface plasmon resonance (SPR) absorption peak at 519 nm. After introducing Pt and Ir and forming a sea urchin-like structure, the SPR peaks of Au / Pt nanoparticles and multi-metal nanozymes completely disappear. The main reason for this is that the shell metal (Pt / Ir) quenches the SPR effect through electron scattering and changes in the dielectric environment.

[0067] Energy-dispersive X-ray spectroscopy (EDS) elemental mapping analysis was performed on the multi-metal nanozyme in Example 1 using field emission transmission electron microscopy. The EDS image of the multi-metal nanozyme in Example 1 is shown below. Figure 7 As shown, from Figure 7 As can be seen, the signals of Au, Pt and Ir in the multimetal nanozyme are clearly visible and uniformly distributed throughout the structure, which preliminarily proves the successful construction of the ternary nanostructure.

[0068] The elemental chemical state of the surface of the multimetal nanozyme in Example 1 was analyzed using X-ray photoelectron spectroscopy. The XPS full spectrum of the multimetal nanozyme is shown below. Figure 8 As shown, the high-resolution Au 4f XPS spectrum of the multimetal nanozyme is as follows: Figure 9 The high-resolution Pt 4f XPS spectrum is shown below. Figure 10 As shown, the high-resolution Ir 4f XPS spectrum is as follows: Figure 11 As shown, from Figures 8-11 It can be seen that these binding energy data are in good agreement with the standard values, confirming that Au, Pt, and Ir all exist in the zero-valent metallic state.

[0069] In summary, through the above series of characterizations, we have fully confirmed the successful synthesis of urchin-shaped Au / PtIr trimetallic nanoparticles.

[0070] The zeta potential was measured using a Zetasizer Nano ZS90 potentiometer corresponding to the multimetal nanozyme-antibody probe in Example 1. The zeta potential graph of the multimetal nanozyme-antibody probe in Example 1 is shown below. Figure 12 As shown, from Figure 12 As can be seen, the Zeta potential of the modified antibody-modified multimetal nanozyme changed significantly from -15.3 mV to -33.5 mV. This negative shift directly proves that the antibody has been loaded onto the surface of the multimetal nanozyme. In addition, it indicates that the modified antibody-modified multimetal nanoparticles (Au / PtIr-mAb) have better colloidal stability and monodispersity due to the enhanced electrostatic repulsion, which is beneficial for its application in immunochromatography.

[0071] The UV-Vis absorption peaks of the multi-metal nanozyme-antibody probe in Example 1 were measured using a UV-2600 UV spectrophotometer. The UV-Vis absorption spectrum of the multi-metal nanozyme-antibody probe in Example 1 is shown below. Figure 13 As shown, from Figure 13 Further evidence can be found in the results showing that the effect of antibody modification on the surface properties of nanoparticles is consistent with the results of Zeta potential.

[0072] The catalytic performance of the multimetal nanozyme in Example 1 was tested: the multimetal nanozyme was added to solutions of three commonly used substrates (TMB, OPD and ABTS) to test the difference in catalytic performance of the multimetal nanozyme with and without H2O2 in the substrate solution.

[0073] The UV-Vis absorption spectrum of the multimetal nanozyme in Example 1 on the oxidation of the chromogenic substrate TMB in the presence or absence of H2O2 is shown below. Figure 14 As shown, the UV-Vis absorption spectrum of the oxidation of the chromogenic substrate OPD is as follows: Figure 15 As shown, the UV-Vis absorption spectrum of the oxidation of the chromogenic substrate ABTS is as follows: Figure 16 As shown. From Figures 14-16It can be seen that Au / PtIr effectively catalyzes the conversion of colorless TMB, OPD, and ABTS into blue, yellow, and green products, respectively, producing characteristic absorption peaks at 652 nm, 450 nm, and 416 nm. In contrast, without the addition of H2O2, the color of the reaction solution and the absorption signal show no significant changes. This indicates that Au / PtIr mainly exhibits peroxidase (POD)-like activity, while its intrinsic oxidase-like activity is negligible. This characteristic helps to reduce the interference of dissolved oxygen and improve the selectivity for H2O2 detection.

[0074] Using TMB as a substrate, the POD-like activities of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes (Au / PtIr) in Example 1 were compared. The UV-Vis absorption spectra of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes (Au / PtIr) catalyzing TMB color development are shown below. Figure 17 As shown. From Figure 17 It can be seen that all three can catalyze the production of blue products from TMB, but the Au / PtIr reaction system has the deepest solution color and the absorption peak intensity at 652 nm is significantly higher than the other two.

[0075] The mechanism of enhanced catalytic activity was verified by using terephthalic acid (TA) as a fluorescent probe. The fluorescence spectra and reaction process diagrams of gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes (Au / PtIr) in Example 1, using terephthalic acid (TA) as probes, for detecting hydroxyl radicals (·OH) are shown below. Figure 18 As shown, from Figure 18 It can be seen that TA can easily react with ·OH to form highly fluorescent 2-hydroxyterephthalic acid (TAOH); the fluorescence spectrum shows that the fluorescence intensity of the Au / PtIr system is significantly higher than that of the other two, indicating that it has the strongest ability to generate ·OH, which preliminarily proves that Au / PtIr has the best peroxidase-like activity.

[0076] Kinetic parameter analysis was performed on the multimetal nanozyme in Example 1. The Michaelis-Menten curve and corresponding double reciprocal plot were obtained with TMB as a variable substrate at a fixed H2O2 concentration, as shown below. Figure 19 As shown, the Michaelis-Menten curves and corresponding double reciprocal plots obtained with H2O2 as a variable substrate at a fixed TMB concentration are as follows: Figure 20 As shown, from Figure 19 and 20 It can be seen that multimetal nanozymes have a strong affinity for the substrate TMB. K mThe value was only 0.194 mM. Specific activity analysis was performed on the gold nanoparticles (Au), Au / Pt nanoparticles (Au / Pt), and multimetal nanozymes in Example 1. The comparison of specific activity data is shown in the figure below. Figure 21 As shown, from Figure 21 As can be seen, the multimetal nanozyme exhibits a high specific activity of 198.58 U / mg.

[0077] The multimetal nanozyme of the present invention targets TMB and H2O2. K m Value and V max The values ​​are compared with existing technologies, and the comparison data is shown in Table 1.

[0078] Table 1. The effects of the multimetal nanozyme of the present invention on TMB and H2O2. K m Value and V max Summary of comparisons between values ​​and existing technologies

[0079] As can be seen from Table 1, the catalytic rate of the multimetal nanozyme of the present invention is also improved compared with the previously reported nanozymes.

[0080] The specific activity of the multimetal nanozyme of the present invention is compared with that of the prior art, and the comparison data are shown in Table 2.

[0081] Table 2 Summary of the specific activity of the multi-metal nanozymes of the present invention compared with that of the prior art

[0082] As can be seen from Table 2, the specific activity of the multimetal nanozyme of the present invention is superior to most reported peroxidase-like nanozymes.

[0083] The peroxidase-like activity of the multimetal nanozyme in Example 1 was evaluated after different storage periods. The absorbance at 652 nm of the catalyzed TMB oxidation products was monitored, and the activities of freshly prepared multimetal nanozymes and those stored for 15 to 90 days were compared. The relative activities of the multimetal nanozymes catalyzing TMB oxidation after different storage periods are shown in the graph below. Figure 22 As shown (day 0 is defined as the day with the highest absorbance value of 100% relative activity, and the absorbance values ​​of oxTMB at 652 nm after different storage periods are plotted), from Figure 22 As can be seen, its catalytic activity did not significantly decrease during the 90-day testing period. This result demonstrates that multimetal nanozymes possess excellent long-term stability, laying the foundation for their reliable application in diagnostic tools requiring long-term storage or transportation.

[0084] When lateral flow chromatography test strips are used for tumor marker detection, the detection mechanism diagram is as follows: Figure 23 As shown, from Figure 23 As can be seen, the AFP antigen in the sample binds to the multimetal nanozyme-antibody probe on the binding pad during chromatography. The resulting complex continues to migrate to the detection line (T line) and is captured by the immobilized capture antibody, forming a sandwich structure of "capture antibody-antigen-nanozyme probe". Unbound probes are captured by the secondary antibody on the control line (C line). Therefore, positive samples will produce bands on both the T line and the C line, while negative samples will only show color on the C line. When the lateral flow chromatography test strip is used for tumor marker detection, the detection signal can be amplified using a substrate solution. Specifically, after the immune reaction is completed, a TMB / H2O2 mixed substrate solution is added to the test strip. The multi-metal nanozymes enriched on the T line exert their peroxidase-like activity, catalyzing the oxidation of colorless TMB to generate the blue product oxTMB, thereby converting the specific immune recognition signal into a significant colorimetric signal, achieving cascade amplification of the signal.

[0085] To evaluate the quantitative detection performance of the lateral flow chromatography test strips, a series of AFP standards at different concentrations (0–1000 ng / mL) were used to conduct parallel comparisons of three detection modes: conventional gold nanoparticle lateral flow chromatography (Au-LFA); multimetal nanozyme lateral flow chromatography (Au / PtIr-LFA before catalysis, i.e., without substrate solution); and multimetal nanozyme lateral flow chromatography amplified by TMB / H2O2 substrate catalysis (Au / PtIr-LFA after catalysis).

[0086] The calibration curve of the test results obtained by the traditional gold nanoparticle side-flow chromatography method is as follows: Figure 24 As shown, the left image displays the T-line grayscale values ​​for different concentrations, the inset in the left image shows the band photographs taken at different concentrations, and the right image is the calibration curve. Insets 1-12 in the left image correspond to AFP concentrations of 0, 0.01, 0.1, 0.2, 0.5, 1, 10, 50, 100, 200, 500, and 1000 ng / mL, respectively. Figure 24 As can be seen, the T line gradually darkens with increasing AFP concentration, and the calibration curves all show typical concentration dependence.

[0087] The calibration curve of the test results obtained by the multi-metal nanozyme side-flow chromatography method is as follows: Figure 25 As shown, the left image displays the T-line grayscale values ​​for different concentrations, the inset in the left image shows the band photographs taken at different concentrations, and the right image is the calibration curve. Insets 1-12 in the left image correspond to AFP concentrations of 0, 0.01, 0.1, 0.2, 0.5, 1, 10, 50, 100, 200, 500, and 1000 ng / mL, respectively. Figure 25It can be seen that even before catalytic amplification, the basic color intensity and signal response of its T-line are slightly higher than those of the traditional Au-LFA, indicating that the nanozyme itself has superior optical properties.

[0088] The calibration curve of the test results obtained by the side-flow chromatography method of multi-metal nanozymes catalyzed by TMB / H2O2 substrate is shown below. Figure 26 As shown, the left image represents the T-line grayscale values ​​for different concentrations, the inset in the left image represents the band photographs taken at different concentrations, and the right image represents the calibration curve. Insets 1-12 in the left image correspond to AFP concentrations of 0, 0.01, 0.1, 0.2, 0.5, 1, 10, 50, 100, 200, 500, and 1000 ng / mL, respectively. Figure 26 As can be seen, the T line of the catalyzed test strip changes from gray to blue, becoming clearly distinguishable even at low concentrations, and the color intensity is significantly enhanced. Calibration curve analysis shows that the catalyzed method exhibits good linearity in the range of 0.01 to 1000 ng / mL, with a visual limit of detection (vLOD) as low as 0.2 ng / mL, which is nearly 50 times higher than that before catalysis (10 ng / mL) and traditional Au-LFA (50 ng / mL), respectively.

[0089] The detection limit, detection range, and detection time of the Au / PtIr-LFA of the present invention are compared with those of the prior art, and the comparison data are shown in Table 3.

[0090] Table 3 summarizes the comparison data of the detection limit, detection range, and detection time of the Au / PtIr-LFA of the present invention with those of AFP detection in the prior art.

[0091] As shown in Table 3, the Au / PtIr-LFA of this invention not only retains the core advantages of traditional LFA, such as visual readability and ease of operation, but also successfully overcomes its low sensitivity bottleneck by integrating nanozyme catalytic amplification. Specifically, this Au / PtIr-LFA achieves excellent detection limits (0.2 ng / mL) and a wide linear range (0.01~1000 ng / mL) while maintaining a detection speed of within 12 min. Compared with detection methods that rely on large instruments, it has broader application prospects.

[0092] To verify the specificity of Au / PtIr-LFA for AFP, a specificity experiment was conducted: 100 μL of other biomarkers (100 ng / mL) were tested, including C-reactive protein (CRP), bovine serum albumin (BSA), carcinoembryonic antigen (CEA), carbohydrate antigen 199 (CA199), and carbohydrate antigen 125 (CA125). The specific steps were as follows: the biomarkers were serially diluted to 100 ng / mL in PBS, and then the solution was added dropwise to the sample pad. As the reaction proceeded, the color intensity of the T-line was read by visual observation and software assistance, achieving semi-quantitative and quantitative detection. The specificity experiment results are as follows: Figure 27 As shown, from Figure 27 As can be seen, only the AFP group produced a significant signal response, while the signals of other interfering groups were no different from the negative control, confirming that this method has high selectivity for AFP and can effectively eliminate the influence of potential interfering substances in complex samples.

[0093] Repeatability was evaluated by testing AFP samples at low, medium, and high concentrations (10, 50, and 250 ng / mL), and stability was evaluated after storage at 50 ng / mL for different times (1, 15, and 30 days). The relative standard deviation (RSD) was calculated by quantitatively analyzing the T-line gray values. The repeatability experimental graphs of Au / PtIr-LFA at AFP concentrations of 10, 50, and 250 ng / mL are shown below. Figure 28 As shown, from Figure 28 As can be seen, the relative standard deviation ranges from 1.05% to 1.58%, indicating that the method has excellent batch-to-batch repeatability. Stability test results after 1, 15, and 30 days of storage are shown in the figures below. Figure 29 As shown, from Figure 29 As can be seen, even after 30 days of storage, the RSD of the detection signal remains at a low level of 1.24%, indicating that Au / PtIr-LFA can maintain stable detection performance after long-term storage.

[0094] To evaluate the detection performance and practicality of Au / PtIr-LFA in real clinical samples, 30 clinical serum samples were analyzed to demonstrate its feasibility for widespread use in clinical testing. All samples were diluted 10-fold with PBS before use. The detection flowchart is shown below. Figure 30 As shown, from Figure 30 It can be seen that: the blood sample is first centrifuged to obtain serum, the serum sample is diluted and then dropped onto the test strip, followed by chromatography reaction and catalytic signal amplification.

[0095] A series of AFP standard solutions of different concentrations (10, 50, and 250 ng / mL) were added to human serum diluted 10-fold and spiked. Spiking recovery experiments were conducted to evaluate the accuracy and precision of different detection modes of Au / PtIr-LFA in complex serum samples. The recovery rates and relative standard deviations of the multimetal nanozyme lateral flow chromatography method (denoted as Au / PtIr-LFA before catalysis, i.e., without substrate solution) and the multimetal nanozyme lateral flow chromatography method after TMB / H2O2 substrate catalysis (denoted as Au / PtIr-LFA after catalysis) are shown in Table 4.

[0096] Table 4. Recovery and relative standard deviation test data of multi-metal nanozyme side-flow chromatography and multi-metal nanozyme side-flow chromatography amplified by TMB / H2O2 substrate catalysis.

[0097] As shown in Table 4, the recoveries of Au / PtIr-LFA under different detection modes ranged from 95.25% to 103.04%, and the relative standard deviations (RSDs) ranged from 1.09% to 4.25%, indicating that the method has good reproducibility and accuracy in complex matrices.

[0098] 100 μL of human serum diluted 10-fold (10-fold dilution with PBS) was analyzed using Au / PtIr-LFA. The heatmap of the results for all actual clinical samples is shown below. Figure 31 As shown, from Figure 31 As can be seen, based on a cutoff value of 20 ng / mL, all samples were clearly distinguished as negative or positive, with an upper limit of the detection dynamic range reaching 1200 ng / mL; the box plot of the distribution of positive and negative samples in actual clinical testing results is shown below. Figure 32 As shown, from Figure 32 As can be seen, there is a highly statistically significant difference in the detection of AFP-positive and negative samples (p<0.0001), highlighting the excellent clinical discrimination ability of this method. Direct detection results on clinical samples show that the Au / PtIr-LFA of this invention possesses clear qualitative and quantitative capabilities.

[0099] Finally, the Au / PtIr-LFA detection method was compared with the clinical standard chemiluminescence immunoassay (CLIA), and the correlation analysis linear plot is shown below. Figure 33 As shown, from Figure 33 It can be seen that the data points measured by the two methods are closely distributed on both sides of the fitted line, showing a high degree of linear correlation. Linear regression analysis yields R0. 2 The correlation coefficient was 0.997, demonstrating the excellent quantitative correlation between the two.

[0100] This invention applies the prepared multi-metal nanozyme to a lateral flow chromatography test strip. By utilizing the Pt contained in the multi-metal nanozyme, it provides high redox activity, efficiently adsorbing and dissociating H₂O₂ to generate highly reactive oxygen species such as hydroxyl radicals (·OH), rapidly oxidizing chromogenic substrates (such as TMB), producing color changes, achieving signal amplification, and improving detection sensitivity. Ir enhances stability and oxygen-related reaction performance, ensuring signal reproducibility. Simultaneously, Ir has unique adsorption characteristics for oxygen species, adjusting the oxygen coverage on the Pt surface and preventing the active sites of Pt from being poisoned by excessively strong oxygen-containing intermediates, thus maintaining the high activity of Pt. Furthermore, Ir itself also possesses certain peroxidase activity, producing a synergistic or complementary effect with Pt, improving the catalytic chromogenic substrate effect of the multi-metal nanozyme, thereby enhancing the accuracy and sensitivity of the lateral flow chromatography test strip for detecting tumor markers. The results of the examples show that the multi-metal nanozyme prepared by the method provided in this invention exhibits high chromogenic activity for the substrate TMB. K m With a concentration of only 0.194 mM and a specific activity of 198.58 U / mg, its catalytic activity did not significantly decrease during a 90-day testing period, demonstrating high substrate affinity, catalytic performance, and stability. The multi-metal nanozyme used in the lateral flow chromatography test strip exhibited good linearity for tumor markers (alpha-fetoprotein) in the range of 0.01 to 1000 ng / mL, with a detection limit (LOD) as low as 0.2 ng / mL, nearly 250 times higher than the traditional Au-LFA (50 ng / mL). After 30 days of storage, the RSD of the detection signal remained at a low level of 1.24%, demonstrating high detection sensitivity and stability.

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a multimetal nanozyme, comprising: (1) Chloroauric acid solution and sodium citrate solution were mixed to carry out the first reduction reaction to obtain gold nanoparticle dispersion; (2) The gold nanoparticle dispersion obtained in step (1) is mixed with chloroplatinic acid solution and L-ascorbic acid solution to carry out a second reduction reaction to obtain Au / Pt nanoparticles; the concentration of L-ascorbic acid solution is 25~35 mmol / L; the addition rate of L-ascorbic acid solution is 15~25 mL / h; (3) The Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol obtained in step (2) are mixed and pretreated, and then a ethylene glycol solution of sodium hexachloroiridate (III) hydrate is added to carry out a third reduction reaction to obtain a multi-metal nanozyme; the temperature of the pretreatment is 110~120 ℃; the concentration of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 0.05~0.15 mg / mL; the addition rate of the ethylene glycol solution of sodium hexachloroiridate (III) hydrate is 15~25 mL / h; the temperature of the third reduction reaction is 170~180 ℃.

2. The production method according to claim 1, characterized by, In step (1), the molar ratio of chloroauric acid in the chloroauric acid solution to sodium citrate in the sodium citrate solution is (24~26):(96~98).

3. The production method according to claim 1, characterized by, In step (3), the mass ratio of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide and polyvinylpyrrolidone is (2~5):(70~90):(40~60):(5~20).

4. The method of claim 1, wherein, In step (3), the concentration of Au / Pt nanoparticles in the mixture formed by mixing Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone and ethylene glycol is 1~1.5 mg / mL.

5. The preparation method according to claim 1, characterized in that, The mixing of Au / Pt nanoparticles, L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and ethylene glycol in step (3) specifically involves: 1) Au / Pt nanoparticles are mixed with a portion of ethylene glycol to form mixture A; 2) Mix L-ascorbic acid, potassium bromide, polyvinylpyrrolidone, and the remaining ethylene glycol to form mixture B; 3) Mix the mixture A and the mixture B for the third time.

6. The preparation method according to claim 1, characterized in that, The preprocessing time in step (3) is 0.5~1.5 h.

7. The preparation method according to claim 1, characterized in that, In step (3), the mass ratio of Au / Pt nanoparticles to sodium hexachloroiridate (III) hydrate in the ethylene glycol solution is (2~5):(1~2).

8. The preparation method according to claim 1, characterized in that, The time for the third reduction reaction in step (3) is 1~2 h.

9. The multimetal nanozyme prepared by the preparation method according to any one of claims 1 to 8.

10. The application of the multimetal nanozyme according to claim 9 in a side-flow chromatography test strip.

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