Preparation method and application of gold-copper nanoparticles

Abstract

The application discloses a preparation method and application of gold copper nanoparticles, characterized by the following steps: 150 muL of copper nitrate trihydrate (0.1 M) solution, 200 muL of ascorbic acid (0.1 M) solution, 150 muL of chloroauric acid tetrahydrate (20 mM) solution and 200 muL of cysteine (0.1 M) solution are sequentially added into a centrifugal tube with a specification of 5 mL, and then the solutions are fully mixed and uniformly distributed; under the condition of room temperature, the solutions are statically placed for 24 hours; after reaction, the solutions are centrifugally washed in distilled water for three times, the centrifugal speed is 8000 rpm / min, then the solutions are dispersed in twice distilled water, and stored at 4 DEG C for standby use. The prepared gold copper nanoparticles have strong affinity with an enzyme substrate TMB under the condition of physiological pH value, the AuCuNPs have strong catalytic performance, and can catalyze the oxidation of TMB under the condition of physiological pH value.

Description

Technical Field

[0001] This invention belongs to the field of polymer nanomaterials, specifically relating to a method for preparing gold-copper nanoparticles (AuCuNPs) with peroxidase activity and their applications. Background Technology

[0002] Nanozymes, as next-generation artificial enzymes, can effectively replace natural enzymes in a wide range of applications, including biosensing, environmental monitoring, disease diagnosis, and biomedicine. Especially in analytical sensing, colorimetric sensing methods based on the combination of nanozymes and the chromogenic reagent TMB are favored by researchers due to their simplicity and applicability to a variety of analytes. However, these colorimetric detection methods typically suffer from low resolution, serving only as semi-quantitative analytical methods. Accurate quantitative analysis usually requires large instruments such as ultraviolet spectrophotometers, which generally limits their application in practical sample detection. In recent years, photothermal detection methods have become an attractive strategy in analytical sensing because they do not require large instruments and can achieve precise analyte quantification using only a common thermometer. Importantly, the oxidation products of some chromogenic reagents (TMB) have been shown to have good photothermal effects, providing a new opportunity to develop simple photothermal detection methods based on nanozymes.

[0003] Hydrogen peroxide (H2O2) is an important intermediate product in many biological reactions. As one of the most common and important molecules in biological tissues, it can efficiently catalyze electron donation. Since abnormal levels of H2O2 can trigger diseases such as myocardial infarction, Alzheimer's disease, Parkinson's disease, and cancer, exploring effective and accurate sensors for H2O2 detection is crucial. Currently reported methods for H2O2 detection include fluorescence, chemiluminescence, spectrophotometry, chromatography, electrochemistry, and colorimetry. Among these, colorimetry is considered the most promising method due to its advantages such as low detection limits, high sensitivity, high selectivity, simple operation, and low cost. Horseradish peroxidase (HRP)-based sensors exhibit exceptional selectivity in traditional electrochemical H2O2 detection due to the enzyme's high sensitivity and specificity. However, enzyme sensors generally suffer from low stability due to the inherent weaknesses of natural enzymes, including catalytic activity variability under environmental conditions, low storage and operational stability, and high preparation and purification costs. These drawbacks limit the practical application of enzyme-catalyzed hydrogen peroxide sensors. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing gold-copper nanoparticles and their applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing gold-copper nanoparticles, characterized in that: a one-pot method is used for preparation at room temperature, and the specific steps are as follows: Add 150 μL of copper nitrate trihydrate (0.1 M) solution, 200 μL of ascorbic acid (0.1 M) solution, 150 μL of chloroauric acid tetrahydrate (20 mM) solution, and 200 μL of cysteine ​​(0.1 M) solution to a 5 mL centrifuge tube in sequence. Mix thoroughly and let stand at room temperature for 24 h. Then, wash the reaction solution three times by centrifugation in distilled water at 8000 rpm / min. Disperse the solution in double-distilled water and store at 4 °C for later use.

[0006] The application of the gold-copper nanoparticles is characterized by the ability to catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) under physiological pH conditions and in the presence of hydrogen peroxide, generating a blue oxidation product (TMBox); and to quantitatively analyze and detect H2O2 using TMB as a colorimetric reagent.

[0007] The beneficial effects of this invention are: the preparation process is simple, the reaction conditions are mild and can be completed at room temperature, and the prepared gold-copper nanoparticles (AuCuNPs) have strong oxidase activity under physiological pH conditions, which solves the problem of low nanozyme activity under physiological pH conditions and is conducive to expanding the analytical and detection applications of nanozymes in the biological and medical fields. Attached Figure Description

[0008] Figure 1 Figure (ai) shows the feasibility verification of AuCuNPs synthesis. Figure 2 To determine the absorbance values ​​(A) of the reaction solution under different conditions 652 nm )picture; Figure 3 Feasibility diagram for H2O2 colorimetric sensing; Figure 4 The graph shows the colorimetric performance of H2O2. Detailed Implementation

[0009] Example 1 Add 150 μL of 0.1 M copper nitrate trihydrate solution, 200 μL of 0.1 M ascorbic acid solution, 150 μL of 20 mM chloroauric acid tetrahydrate solution, and 200 μL of 0.1 M cysteine ​​solution to a 5 mL centrifuge tube in sequence. Mix thoroughly and let stand at room temperature for 24 h. Then, wash the reaction solution three times by centrifugation in distilled water at 8000 rpm / min. Disperse the solution in double-distilled water and store at 4 °C for later use.

[0010] (I) Feasibility verification of AuCuNPs synthesis To investigate the catalytic activity of the one-pot preparation of AuCuNPs, different materials were prepared by combining various reagents. The feasibility of the AuCuNPs nanozyme preparation method was further demonstrated through catalytic activity comparisons. ai represent: Cu(NO3)2+AA, Cu(NO3)2+Cys, HAuCl4+AA, HAuCl4+Cys, Cu(NO3)2+AA+HAuCl4, Cu(NO3)2+AA+Cys, Cu(NO3)2.H2O+HAuCl4+Cys, AA+HAuCl4+Cys, and Cu(NO3)2+AA+HAuCl4+Cys. The synthesis process of these materials is similar to that of the AuCuNPs prepared above. Finally, the enzyme-like activity of the nine prepared materials was studied. Figure 1 (A - Tris-HCl buffer solutions of different concentrations under physiological pH 7.4; B - different buffer solutions under physiological pH 7.4) Under the same reaction conditions, when equal amounts of material were added, it was found that only the AuCuNPs nanozyme synthesized in process i had catalase-like activity, which could catalyze the oxidation of TMB to produce blue TMBox, and the solution color turned blue, thus proving the reliability of the synthesis method.

[0011] (ii) The activity of nanozymes is closely related to the pH of the solution and the concentration of the buffer solution. AuCuNPs (100 μL), TMB (0.5 mM), and H2O2 (0.25 mM) were added to different buffer systems, respectively. After reacting the mixed solutions at room temperature for 35 min, the absorbance of the solutions at 652 nm was recorded using a UV-Vis spectrophotometer. Figure 2 As shown, AuCuNPs exhibit strong catalytic activity for TMB at physiological pH and Tris-HCl (2 mM).

[0012] (III) Experiment to verify the feasibility of colorimetric sensing strategy for H2O2 detection from Figure 3As shown in (A – photograph and UV-Vis spectrum; B – UV-Vis bar chart), the colorless TMB solution does not exhibit a significant UV-Vis characteristic absorption peak in the 400-1000 nm range, and AuCuNPs cannot directly catalyze the oxidation of TMB to form TMBox, resulting in no color change. However, upon adding H₂O₂ to the AuCuNPs-TMB system, the absorbance at 652 nm significantly increases, and the solution color changes from colorless to deep blue. Therefore, we have successfully established a colorimetric sensor for detecting H₂O₂.

[0013] (iv) Study on the colorimetric sensing performance of H2O2 First, 100 μL of AuCuNPs and 100 μL of TMB (10 mM) were added to 1700 μL of Tris-HCl (pH 7.4, 2 mM) buffer solution, respectively. Then, different volumes of AuCuNPs were added to the above mixed solution. After reacting at room temperature for 35 min, the solution color was observed, and the change in absorbance was measured by visible-near-infrared absorption spectroscopy. Figure 4 As shown, a strong linear relationship is observed, and the linear regression equation is A = 4.416 + 3.857 Log C. H2O2 R 2 =0.997, linear range: 140 μM~300 μM, and the detection limit is calculated to be 75.63 μM based on (S / N=3).

Claims

1. A method for preparing gold-copper nanoparticles, characterized in that: The preparation was carried out using a one-pot method at room temperature, and the specific steps are as follows: Add 150 μL of copper nitrate trihydrate (0.1 M) solution, 200 μL of ascorbic acid (0.1 M) solution, 150 μL of chloroauric acid tetrahydrate (20 mM) solution, and 200 μL of cysteine ​​(0.1 M) solution to a 5 mL centrifuge tube in sequence. Mix thoroughly and let stand at room temperature for 24 h. Then, wash the reaction solution three times by centrifugation in distilled water at 8000 rpm / min. Disperse the solution in double-distilled water and store at 4 °C for later use.

2. The application of gold-copper nanoparticles according to claim 1, characterized in that: Under physiological pH conditions and in the presence of hydrogen peroxide, it can catalytically oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue oxidation product (TMBox); using TMB as a colorimetric reagent, H2O can be quantitatively analyzed and detected. 2。

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