Platinum-modified mesoporous gold nanoszyme and preparation method and application thereof

Abstract

The present application belongs to the technical field of nanomaterial sensing and detection, and specifically relates to a platinum-modified mesoporous gold nanosensor and a preparation method and application thereof. The present application utilizes liposomes as a surfactant template to synthesize nanoparticle mesoporous gold with a porous structure under the action of a reducing agent ascorbic acid, and then grows platinum nanoparticles in situ on the surface of the mesoporous gold to obtain platinum-modified mesoporous gold nanosensor MGNSs@Pt. The mesoporous gold nanosensor has super-high peroxidase-like activity and can catalyze the reaction of hydrogen peroxide and a chromogenic agent to produce a change in the color of the solution. The linear relationship between the absorption light intensity of the solution and hydrogen peroxide or glucose can be used to realize rapid detection of the two detection objects. The present application has the advantages of high efficiency, high sensitivity, low cost, simple operation and the like.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial sensing and detection technology, specifically relating to a platinum-modified mesoporous gold nanozyme, its preparation method, and its application. Background Technology

[0002] Peroxidase-like nanozymes are a class of inorganic nanomaterials with peroxidase-mimicking capabilities, including metals (e.g., Pd and Pt), metal oxides (e.g., Fe₂O₃ and V₂O₅), carbon materials, metal-organic frameworks, and their composites. Peroxidase-like nanozymes offer many advantages over natural enzymes, such as low cost, high stability, and ease of preparation and storage, thus possessing significant application value in biosensing and biomedicine. However, the catalytic efficiency of some peroxidase-like nanozymes does not meet expectations, and may even be lower than that of natural enzymes, posing a significant challenge to their further application.

[0003] To date, platinum-based nanoparticles have attracted widespread attention and research due to their excellent properties in catalytic activity, biocompatibility, and structural controllability. It has been reported that small-sized platinum nanoparticles exhibit higher peroxidase-like activity compared to larger-sized platinum nanoparticles due to their greater number of active sites and higher affinity for H₂O₂. However, small-sized platinum nanoparticles are difficult to recover and tend to aggregate in solution, leading to a significant reduction in active sites on the nanocluster surface, further causing a sharp decline in stability and catalytic efficiency. To avoid this problem, one solution is to immobilize platinum particles on supports, such as MOFs, carbon, and silicon-based nanomaterials. These nanosupports not only improve the dispersibility and stability of platinum particles but also enhance their utilization rate. However, most support materials have low specific surface areas due to their large size or lack of porosity, meaning platinum particles can only be modified on the outer surface of the support, resulting in poor loading capacity. In contrast, porous materials such as mesoporous silica nanospheres (MSNSs) show great potential for loading platinum particles due to their large surface area and tunable pore size. However, due to limitations in pore size and affinity for platinum particles, the loading capacity of MSNSs remains limited, typically below 10 wt%. More importantly, MSNSs can only serve as supports for platinum particles and do not enhance their catalytic activity. Therefore, synthesizing nanocarriers with both high loading capacity and platinum particle enhancement capabilities is crucial for peroxidase-like nanozymes.

[0004] Mesoporous metallic materials are a novel type of nanomaterial possessing porous metallic frameworks with pore sizes ranging from 2 to 50 nanometers. Due to their superior physicochemical properties, they hold great promise in numerous applications, such as surface-enhanced Raman scattering (SERS) detection, photoacoustic imaging, photothermal therapy, and electrocatalysis. First, they exhibit exceptional optical properties, such as localized surface plasmon resonance (LSPR), which is absent in silicon-based and carbon-based mesoporous materials. Second, their internal and external pore structures provide a higher specific surface area compared to ordinary non-porous gold nanoparticles, offering abundant loading sites for other catalysts. Third, the unique porous structure also accelerates electron transfer and modulates catalyst stability, thereby enhancing catalytic activity. Currently, no studies have explored the modification of mesoporous gold nanospheres with metal catalysts. Loading metal catalysts onto mesoporous gold also faces several challenges, such as the difficulty for metal catalysts to penetrate the pore channels, leading to reduced loading capacity and decreased catalytic activity. Therefore, there is an urgent need to design mesoporous gold nanospheres with appropriate pore sizes and high loading capacities. Summary of the Invention

[0005] The purpose of this invention is to propose a novel and highly efficient platinum-modified mesoporous gold nanozyme MGNSs@Pt and its preparation method, and to use this nanozyme for rapid, efficient, and low-cost detection of hydrogen peroxide and glucose.

[0006] This invention first provides a method for preparing platinum-modified mesoporous gold nanozymes (MGNSs@Pt), the specific steps of which are as follows:

[0007] (1) Preparation of liposomes: Dispalmitoylphosphatidylcholine and cholesterol in a molar ratio of (50-60):(40-50) were completely dissolved in 10-20 mL of chloroform. After stirring for 20-60 minutes, the organic solvent was removed by rotary evaporation. Subsequently, ascorbic acid (AA) solution with a concentration of 0.05-0.5 M was added to the liposome residue until the solute concentration was 0.5-2 mg / mL. After sonication for 5-60 minutes, the liposome solution was centrifuged at 10000 rpm for 5-60 minutes, and the supernatant was collected to obtain a liposome solution containing ascorbic acid.

[0008] (2) Preparation of mesoporous gold: 200-800 μL of 4-10 mM chloroauric acid trihydrate solution was added dropwise to the liposome solution prepared in step (1) at a stable and constant rate (20-1000 μL / min) using a micro-injection pump; the reaction was carried out for 30-90 minutes, the product was collected by centrifugation, and washed three times with deionized water to remove residual reactants and liposomes, to obtain three-dimensional nanospheres of mesoporous gold with a uniform mesoporous structure, denoted as MGNSs;

[0009] (3) Platinum-modified mesoporous gold: Dilute the mesoporous gold nanospheres prepared in step (2) with deionized water to 500-2000 μL, add 10-40 μL of polyvinylpyrrolidone (5-30 wt%) to the solution; after sonication for 15-20 minutes, add 40-200 μL of ascorbic acid solution (concentration 100-400 mg / mL) to the mixture. -1 The mixture was incubated with 20-100 μL of chloroplatinic acid solution (concentration 0.01-0.08 M) at 25-100 °C with gentle stirring for 0.5-2 h. Finally, the platinum-modified mesoporous gold was collected by centrifugation and denoted as MGNSs@Pt. The gold was then washed and purified three times by centrifugation and finally redispersed in deionized water.

[0010] The platinum-modified mesoporous gold nanozyme prepared in this invention, denoted as MGNSs@Pt, exhibits extremely high peroxidase-like activity. It can be used for colorimetric detection of hydrogen peroxide and glucose. The specific steps are as follows.

[0011] (a) Detection of hydrogen peroxide:

[0012] (1) Add 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and H2O2 standard solutions of different concentrations to NaAc / HAc buffer solution with pH=3-5; add 0.2-1.5 μg of MGNSs@Pt and incubate at 45-60℃ for 5-40 minutes; then use a UV-Vis spectrophotometer or microplate reader to read the absorbance of the solution at 652 nm to obtain the concentration-absorbance curve;

[0013] (2) Add the sample to be tested into the buffer solution and test the absorbance of the solution at 652 nm according to the method in step (1). Obtain the concentration of hydrogen peroxide in the sample by means of the concentration-absorbance curve.

[0014] (II) Glucose detection:

[0015] (1) Mix glucose with 0.1-1 mg / mL -1 Glucose oxidase was added to NaAc / HAc buffer at pH 3-5 and incubated at 20-50℃ for 10-30 minutes. Then, 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and 0.2-1.5 μg of MGNSs@Pt were added. The mixture was incubated at 45-60℃ for 10-30 minutes. The absorbance of the solution at 652 nm was then read using a UV-Vis spectrophotometer or microplate reader to obtain a concentration-absorbance curve.

[0016] (2) Add the pretreated sample to the buffer solution and test the absorbance of the solution at 652 nm according to the method in step (1). Obtain the concentration of hydrogen peroxide in the sample by the concentration-absorbance curve.

[0017] (III) Detection of glucose content in human serum:

[0018] Dilute human serum samples 2-100 times with HAc-NaAc buffer (pH 3-5) and mix with 0.5 mg / mL -1 Mix glucose oxidase; after incubating at 20-50℃ for 10-30 minutes, add 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and 0.2-1.5 μg of MGNSs@Pt respectively; incubate at 45-60℃ for 10-30 minutes; then use a UV-Vis spectrophotometer or ELISA reader to read the absorbance of the solution at 652 nm, and substitute it into the standard curve equation obtained from the concentration-absorbance curve to obtain the glucose content in serum.

[0019] The present invention has the following main features and advantages

[0020] (1) The method of synthesizing nanozymes is simple and has high catalytic efficiency: This invention utilizes the high catalytic efficiency of platinum particles and loads them onto a gold particle support with a porous structure, which can effectively avoid the aggregation of platinum particles and ensure sufficient catalytic sites; at the same time, the properties of gold nanomaterials are utilized, and their surface can enhance the catalytic efficiency of platinum when in contact with platinum particles.

[0021] (2) The detection method is simple, fast and low cost: The present invention utilizes the nanozyme MGNSs@Pt to catalyze the decomposition of hydrogen peroxide under acidic conditions. The hydroxyl radicals generated therefrom can oxidize the chromogenic substrate, thereby changing 3,3',5,5'-tetramethylbenzidine from colorless to blue. Therefore, the content of hydrogen peroxide or glucose in the sample can be confirmed by visual observation.

[0022] (3) High sensitivity, good linear relationship between absorbance and concentration, among which:

[0023] The linear equation obtained for the detection of hydrogen peroxide by MGNSs@Pt nanozymes is ΔA = 0.0019C. H2O2 +0.0183, linear relationship (R 2 The value was 0.999 between 0.12 and 250 μM; the linear equation for glucose detection by MGNSs@Pt nanozymes was ΔA = 0.0013C. glu +0.0076, linear relationship (R 2 The value was 0.992 at concentrations between 0.5 and 750 μM; where C H2O2 and C gluThese represent the concentrations of hydrogen peroxide (H2O2) and glucose, respectively.

[0024] (4) Fast and efficient: The detection of glucose in human serum using this MGNSs@Pt nanozyme can be completed within 45 minutes;

[0025] (5) Portable and easy to test on site: The hydrogen peroxide and glucose in the nanoenzyme can solve the problem of testing site and can be tested on site.

[0026] Compared with existing technologies, the nanozyme MGNSs@Pt of this invention is simple to manufacture and inexpensive. This nanozyme is used to detect hydrogen peroxide and glucose molecules in standards, exhibiting a good linear relationship between absorbance and concentration, and high sensitivity. This nanozyme is also used to detect glucose levels in serum, providing efficient, rapid, portable, and easy-to-use on-site detection. Attached Figure Description

[0027] Figure 1 Transmission electron microscopy (TEM) images of the prepared MGNSs and MGNSs@Pt.

[0028] Figure 2 The X-ray diffraction pattern of MGNSs@Pt is shown.

[0029] Figure 3 The peaks represent the UV absorption peaks generated by MGNSs@Pt reacting with the substrate.

[0030] Figure 4 Optimization of conditions for the MGNSs@Pt catalytic reaction.

[0031] Figure 5 A schematic diagram illustrating the principle of MGNSs@Pt detection of hydrogen peroxide and glucose.

[0032] Figure 6 Linear relationship between the concentration of hydrogen peroxide and the absorption peak detected by MGNSs@Pt.

[0033] Figure 7 Linear relationship between glucose concentration and absorption peak for MGNSs@Pt detection. Detailed Implementation

[0034] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0035] The embodiments described above are some embodiments of the present invention. All other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0036] Example 1

[0037] Preparation of mesoporous gold: Dipalmitoylphosphatidylcholine and cholesterol in a molar ratio of 50:50 were completely dissolved in 10 mL of chloroform. After stirring for 30 minutes, the organic solvent was removed by rotary evaporation. Subsequently, 0.3 M ascorbic acid (AA) solution was added to the liposome residue until the solute concentration was 1 mg / mL. After sonication for 30 minutes, the liposome solution was centrifuged at 10,000 rpm for 20 minutes, and the supernatant was collected to obtain a liposome solution containing ascorbic acid. 400 μL of 8 mM chloroauric acid trihydrate solution was added dropwise to the liposome solution at a stable and constant rate (500 μL / min) using a microinjection pump. The reaction was carried out for 45 minutes, and the product was collected by centrifugation and washed three times with deionized water to remove residual reactants and liposomes, yielding three-dimensional mesoporous gold nanospheres with a uniform mesoporous structure, denoted as MGNSs. Figure 1 Left middle image.

[0038] Example 2

[0039] Platinum-modified mesoporous gold: Dilute a certain amount of mesoporous gold with deionized water to 1000 μL, add 20 μL of 20 wt% polyvinylpyrrolidone to the solution; after sonication for 30 minutes, add 80 μL of a solution with a concentration of 100-400 mg / mL to the mixture. -1 The ascorbic acid solution and 40 μL of 0.01-0.08 M chloroplatinic acid solution were mixed and incubated at 65 °C with gentle stirring for 1 h. Finally, the platinum-modified mesoporous gold nanozyme, denoted as MGNSs@Pt, was collected by centrifugation. Figure 1 The image is shown in the middle right corner. Its related crystal structure is shown below. Figure 2 The color development of the TMB substrate is shown in the figure. Figure 3 The reactivity at different temperatures, with varying TMB and H2O2 concentrations is shown in the figure. Figure 4 .

[0040] Example 3

[0041] The test target is hydrogen peroxide

[0042] 140 μL of TMB (1.1 mM) and 140 μL of hydrogen peroxide solutions of different concentrations were prepared using HAc-NaAc buffer (pH = 4, 0.02 M). After thorough mixing, 20 μL of MGNSs@Pt nanozyme solution (1.18 μg) was added, and the mixture was reacted at 55 °C for 15 minutes. The absorption spectrum was then measured using a UV-Vis spectrophotometer. The concentration-absorbance curve for hydrogen peroxide detection is shown in [Figure number missing]. Figure 6 ,from Figure 6 As can be seen, the linear equation of the standard curve is ΔA = 0.0019C. H2O2 +0.0183(R 2=0.999), with a linear range of 0.12-250 μM. The color response of solutions with different concentrations of hydrogen peroxide is shown in [reference needed]. Figure 6 In the upper part, the detection limit for hydrogen peroxide by the UV-Vis spectrophotometer is 0.1 μM.

[0043] Example 4

[0044] The test subject is glucose.

[0045] Different concentrations of glucose solutions and 0.5 mg / mL glucose oxidase (GOx) were mixed in HAc-NaAc buffer solution (pH = 4, 0.02 M). After incubation at 37 °C for 30 minutes, 300 μL of the glucose reaction solution was taken, and 1.1 mM TMB and 1.18 μg MGNSs@Pt were added. The mixture was incubated at 55 °C for 20 minutes, and the absorption spectra were measured using a UV-Vis spectrophotometer. The concentration-absorbance curve for hydrogen peroxide detection is shown in [Figure number missing]. Figure 7 ,from Figure 7 As can be seen from this, the linear equation of the standard curve is ΔA = 0.0013C. glu +0.0076(R 2 =0.992), with a linear range of 0.5-750 μM. The color response of solutions with different concentrations of hydrogen peroxide is shown in [reference needed]. Figure 7 In the upper part, the detection limit for hydrogen peroxide by the UV-Vis spectrophotometer is 0.4 μM.

Claims

1. A method for preparing platinum-modified mesoporous gold nanozymes, characterized in that, The specific steps are as follows: (1) Preparation of liposomes: Dispalmitoylphosphatidylcholine and cholesterol in a molar ratio of (50-60):(40-50) were completely dissolved in 10-20 mL of chloroform. After stirring for 20-60 minutes, the organic solvent was removed by rotary evaporation. Subsequently, ascorbic acid solution with a concentration of 0.05-0.5M was added to the liposome residue until the concentration of solute was 0.5-2 mg / mL. After sonication for 5-60 minutes, the liposome solution was centrifuged at 10000 rpm for 5-60 minutes, and the supernatant was collected to obtain a liposome solution containing ascorbic acid. (2) Preparation of mesoporous gold: 200-800 μL of chloroauric acid trihydrate solution with a concentration of 4-10 mM was added dropwise into the liposome solution prepared in step (1) at a stable and constant rate of 20-1000 μL / min using a micro-injection pump; the reaction was carried out for 30-90 minutes, the product was collected by centrifugation, and washed three times with deionized water to remove residual reactants and liposomes, to obtain three-dimensional nanosphere mesoporous gold with a uniform mesoporous structure, denoted as MGNSs; (3) Platinum-modified mesoporous gold: Dilute the mesoporous gold prepared in step (2) with deionized water to 500-2000 μL, add 10-40 μL of 5-30 wt% polyvinylpyrrolidone to the solution; after sonication for 15-20 minutes, add 40-200 μL of a solution with a concentration of 100-400 mg / mL to the mixture. -1 The ascorbic acid solution and 20-100 μL of 0.01-0.08 M chloroplatinic acid solution were mixed and incubated at 25-100℃ with gentle stirring for 0.5-2 h. Finally, the platinum-modified mesoporous gold nanozyme, denoted as MGNSs@Pt, was collected by centrifugation.

2. A platinum-modified mesoporous gold nanozyme obtained by the preparation method of claim 1.

3. The application of a platinum-modified mesoporous gold nanozyme as described in claim 2 in the detection of hydrogen peroxide and glucose.

4. The application according to claim 3, characterized in that, The specific steps are as follows: (a) Detection of hydrogen peroxide: (1) Add 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and H2O2 standard solutions of different concentrations to NaAc / HAc buffer solution with pH=3-5; add 0.2-1.5 μg of MGNSs@Pt and incubate at 45-60 °C for 5-40 minutes; then use a UV-Vis spectrophotometer or microplate reader to read the absorbance of the solution at 652 nm to obtain the concentration-absorbance curve; (2) Add the sample to be tested into the buffer solution and test the absorbance of the solution at 652 nm according to the method in step (1). Obtain the concentration of hydrogen peroxide in the sample by means of the concentration-absorbance curve. (II) Glucose detection: (1) Mix glucose with 0.1-1 mg / mL -1 Glucose oxidase was added to NaAc / HAc buffer at pH 3-5 and incubated at 20-50℃ for 10-30 minutes. Then, 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and 0.2-1.5 μg of MGNSs@Pt were added. The mixture was incubated at 45-60℃ for 10-30 minutes. The absorbance of the solution at 652 nm was then read using a UV-Vis spectrophotometer or microplate reader to obtain a concentration-absorbance curve. (2) Add the pretreated sample to the buffer solution and test the absorbance of the solution at 652 nm according to the method in step (1). Obtain the concentration of hydrogen peroxide in the sample by means of the concentration-absorbance curve. (III) Detection of glucose content in human serum: Dilute human serum samples 2-100 times with HAc-NaAc buffer (pH 3-5) and mix with 0.5 mg mL -1 The glucose oxidase was mixed; after incubation at 20-50℃ for 10-30 minutes, 0.5-1.5 mM of 3,3',5,5'-tetramethylbenzidine and 0.2-1.5 μg of MGNSs@Pt were added respectively; after incubation at 45-60℃ for 10-30 minutes; then the absorbance of the solution at 652 nm was read using a UV-Vis spectrophotometer or ELISA reader, and the result was substituted into the standard curve equation obtained from the concentration-absorbance curve to obtain the glucose content in the serum.

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