Preparation of photo-activated quinone-mimicking activity gold nanoclusters and application in biological thiols analysis

Abstract

The application belongs to the field of nanomaterials and biotechnology, and particularly relates to preparation of photo-activated oxidase-like gold nanoclusters and application of the same in biological thiol analysis. An alkaline solution of 6-methyl-2-thiouracil is added to a chloroauric acid solution, the product is purified by ultrafiltration centrifugation after stirring reaction at room temperature to obtain photo-activated oxidase-like gold nanoclusters. The photo-activated oxidase-like gold nanoclusters have an enzyme activity "switch" caused by photo-activation, so that the reaction is controllable, the enzyme-like activity is high, excellent enzyme-like activity can be obtained for biological detection at a sample concentration of only 100 μg / mL, and the photo-activated oxidase-like gold nanoclusters have good stability, so that simple, rapid, efficient and sensitive biochemical analysis biosensing, biochemical analysis and drug treatment can be established.

Description

Technical Field

[0001] This invention belongs to the fields of nanomaterials and biotechnology, specifically relating to the preparation of photoactivated oxidase-like active gold nanoclusters and their application in biothiol analysis. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Nanozymes are a class of enzyme-mimicking materials that possess both the unique properties of nanomaterials and catalytic functions. They offer advantages such as low cost, good stability, relatively mild reaction conditions, high catalytic efficiency, and the ability to be mass-produced, making them a viable alternative to natural enzymes. Furthermore, nanozymes exhibit relatively stable structures and maintain catalytic activity even under extreme conditions. Utilizing their physicochemical properties, nanozymes are widely used in environmental monitoring, in vivo disease diagnosis and treatment, and antibacterial applications, demonstrating broad application prospects.

[0004] Among the many discovered nanozymes, gold nanozymes (gold nanozymes) have attracted widespread attention from researchers due to their diverse enzyme-like properties. Compared to other nanozymes, gold nanozymes possess advantages such as high stability, good biocompatibility, and ease of surface modification, thus being considered one of the most important nanozymes. The catalytic mechanism of gold nanozymes is highly complex, and their enzyme-like activity is generally influenced by factors such as size and morphology, dispersion and aggregation, environmental pH, environmental temperature, surface modification, composition, ionic, molecular, and optical interactions.

[0005] Gold nanoclusters, as a type of gold nanozyme, are a novel fluorescent nanomaterial developed in recent years. Due to significant differences in their optical, electrical, and chemical properties compared to ordinary gold nanoparticles, they possess unique photoelectric and chemical properties. Gold nanoclusters can exhibit oxidase-like activity, catalyzing electron transfer processes between molecular oxygen and substrate molecules, showing broad application prospects in molecular detection, disease diagnosis, and other fields. However, the oxidase-like activity of gold nanoclusters is uncontrollable, and its accuracy is easily affected by changes in activity during use. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a method for preparing photoactivated oxidase-like active gold nanoclusters and their application in biothiol analysis. The photoactivated oxidase-like active gold nanoclusters of the present invention achieve photo-modulated enzyme activity in materials for biosensing, biochemical analysis, and drug therapy without the need for hydrogen peroxide. This avoids the drawbacks of introducing hydrogen peroxide while solving problems in existing technologies such as uncontrollable enzyme catalytic activity of nanomaterials, high synthesis costs, complex procedures, and difficult operation.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0008] In a first aspect, the present invention provides a method for preparing photoactivated oxidase-like active gold nanoclusters, comprising the following steps:

[0009] An alkaline solution of 6-methyl-2-thiouracil was added to a chloroauric acid solution, and the reaction was carried out by stirring at room temperature. The product was then purified by ultrafiltration and centrifugation to obtain photoactivated oxidase-like gold nanoclusters.

[0010] Preferably, the molar ratio of chloroauric acid to 6-methyl-2-thiouracil is 1:2.5-2.6.

[0011] More preferably, the concentration of 6-methyl-2-thiouracil in the alkaline solution is 75-85 mmol / L, and the concentration of chloroauric acid solution is 20-30 mmol / L.

[0012] More preferably, the volume ratio of the alkaline solution of 6-methyl-2-thiouracil to the chloroauric acid solution is 0.75-0.85:1.

[0013] Preferably, the stirring reaction time is 0.9-1.1 h.

[0014] In a second aspect, the present invention provides a photoactivated oxidase-like active gold nanocluster, which is obtained by the preparation method described in the first aspect.

[0015] Thirdly, the present invention provides the application of photoactivated oxidase-like active gold nanoclusters as described in the second aspect in the analysis of biothiols.

[0016] Fourthly, the present invention provides a method for the analysis of biothiols, comprising the following steps:

[0017] The photoactivated oxidase-active gold nanoclusters, substrate, and biothiols described in the second aspect were dissolved in a sodium acetate-acetic acid buffer solution. After irradiation with ultraviolet light, the absorbance of the solution at 652 nm was measured. The difference in absorbance between the addition of different biothiol concentrations and the absence of biothiol was used as the ordinate, and the biothiol concentration was used as the abscissa to establish a linear relationship between biothiol concentration and absorbance. The concentration of biothiols was calculated from the absorbance.

[0018] Preferably, the substrate comprises 3,3',5,5'-tetramethylbenzidine, and the biothiol comprises glutathione or cysteine.

[0019] Preferably, the concentration of the sodium acetate-acetic acid buffer solution is 0.09-0.11 mol / L, and the pH is 3; the concentration of the photoactivated oxidase-like active gold nanoclusters is 95-105 μg / mL; the power of the ultraviolet lamp is 4-6 W, and the irradiation time is 4-6 min.

[0020] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0021] The photoactivated oxidase-like active gold nanoclusters of the present invention have an enzyme activity "switch" brought about by photoactivation, which makes the reaction controllable and has high enzyme-like activity. Excellent enzyme-like activity can be obtained with a sample concentration of only 100 μg / mL for biological detection. They have good stability and can establish simple, rapid, efficient and sensitive biochemical analysis, biosensing, biochemical analysis and drug therapy.

[0022] The synthesis method of photoactivated oxidase-like active gold nanoclusters is simple and efficient. Mass production can be achieved by stirring at room temperature for 1 hour. Separation and purification are relatively simple, without any complicated subsequent processes, and the raw materials are inexpensive and readily available. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0024] Figure 1 This is a low-magnification electron microscope image of the Au-MTU material in Example 1;

[0025] Figure 2 Infrared spectra of MTU and Au-MTU of Example 1;

[0026] Figure 3 The graph shows the activity of Au-MTU photoactivator enzyme, where the absorbance at 652nm is arranged from largest to smallest as Au-MTU+TMB Light, Au-MTU+TMB Dark, TMB Light, and TMB Dark.

[0027] Figure 4 EPR singlet oxygen capture by Au-MTU under 0 min and 5 min illumination;

[0028] Figure 5 Linear response plot of GSH concentration;

[0029] Figure 6 Linear response plot of Cys concentration;

[0030] Figure 7 Diagram illustrating the AND logic gate;

[0031] Figure 8 Diagram for exploring the INH logic gate;

[0032] Figure 9 This is a diagram for exploring NOR logic gates. Detailed Implementation

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0034] Example 1

[0035] 0.8 mL of 80 mmol / L 6-methyl-2-thiouracil (MTU) solution dissolved in NaOH was added to 1 mL of 25 mmol / L HAuCl4·4H2O solution. After stirring at room temperature for 1 h, the product was purified by ultrafiltration centrifugation (Millipore, 50 kDa) to obtain pure photoactivated oxidase-like gold nanoclusters (Au-MTU), which were stored at 4 °C.

[0036] like Figure 1 As shown, Au-MTU exhibits a nanoscale nanocluster morphology. For example... Figure 2 As shown, compared to MTU, the SH bond peak disappears in the infrared spectrum of Au-MTU, indicating that Au-S bonds have formed between gold and MTU. Electron microscopy and infrared spectroscopy confirm the successful synthesis of Au-MTU.

[0037] The photoactivating enzyme activity of Au-MTU was investigated by measuring the UV-Vis absorption spectra of solutions containing dispersed Au-MTU under conditions of presence and absence of 3,3',5,5'-tetramethylbenzidine (TMB) and presence and absence of UV light irradiation. Figure 3 As shown, when TMB is added and irradiated with ultraviolet light, the reaction system exhibits a blue-green color and absorption at 652 nm, indicating that Au-MTU can catalyze the colorimetric reaction of TMB under light irradiation. Figure 4As shown, the singlet oxygen production of Au-MTU under 0 min and 5 min of light irradiation was detected by EPR singlet oxygen capture. Au-MTU showed oxidase-like activity under light excitation, but did not show oxidase-like activity under no light irradiation, indicating that the oxidase-like activity of Au-MTU is controllable.

[0038] Au-MTU, TMB, and biothiols (glutathione (GSH) or cysteine ​​(Cys)) were added to a 0.1 mol / L NaAc-HAc buffer solution at pH 3. The final concentration of Au-MTU was 100 μg / mL, and the final concentration of TMB was 0.4 mM. The solution was irradiated with a 6W UV lamp at room temperature for 5 min, and the absorbance at 652 nm was measured. The difference in absorbance between the solution with different biothiol concentrations and the solution without biothiol was plotted on the ordinate, and the biothiol concentration was plotted on the abscissa to establish the relationship between biothiol concentration and absorbance. Figure 5 As shown, there is a linear relationship between the concentration of glutathione and the difference in absorbance. Figure 6 As shown, the absorbance difference also changes linearly with the concentration of cysteine. Therefore, Au-MTU can achieve the quantitative detection of biothiols.

[0039] Au-MTU generates singlet oxygen under photoactivated conditions, which oxidizes the substrate TMB to produce a colorimetric reaction. This colorimetric reaction enables the visual determination of biothiols such as glutathione and cysteine, and a logic gate is then constructed to output the signal.

[0040] like Figure 7 As shown, an absorbance of 0.3 at 652 nm was used as the threshold of the AND logic gate. TMB was used as the gate unit, and illumination and the addition of Au-MTU were used as the two input signals. The absence and presence of these two conditions were defined as "0" and "1," respectively. Only when both illumination and Au-MTU were present was TMB oxidized to TMBox, generating a strong signal at 652 nm greater than 0.3, resulting in a distinctly blue solution color and an output of "1" (output signal (1 1)). When either illumination or Au-MTU was present or absent, the output was "0" (output signal (00, 0 1, 1 0)), the UV-Vis absorption intensity was less than 0.3, and the solution was almost colorless. The results provide a truth table, strongly demonstrating the successful construction of the AND logic gate.

[0041] like Figure 8As shown, similar to the AND logic gate, the INH logic gate uses the simultaneous presence of TMB and illumination as a logic unit, with GSH and Au-MTU as the two input signals. The introduction of GSH greatly suppresses the reaction between TMB and Au-MTU under illumination. Only when Au-MTU is input into the logic alone will the output result be "1" (output signal is (0 1)). In other cases, the output result is "0" (output signal is (0 0, 1 0, 1 1)). The design of the INH logic gate can be used for the detection of GSH. When the system contains GSH and reaches a certain concentration, the output result will change from "1" (without GSH) to "0" (with GSH), and the solution color will change from blue to a nearly colorless, faint pale blue, thus achieving a visual logic gate detection with both signal output and intuitive results.

[0042] like Figure 9 As shown, the NOR logic gate is established with Au-MTU, TMB, and illumination conditions all present and used as logic units, with GSH and Cys as input signals. The presence of Cys, GSH, and their mixtures can significantly suppress the photoactivated color rendering system of the Au-MTU-TMB platform. The output signal "1" corresponds to the fact that blue can only be obtained and presented when both GSH and Cys inputs are absent (0 0). In other cases, the absorption intensity is less than 0.3, and the output signal is "0".

[0043] By combining colorimetric detection with three logic gates, and establishing visual logic gate detection through the output signals of the three logic gates, a promising paradigm for ultra-sensitive and highly reliable biosensing is provided.

[0044] Example 2

[0045] 0.8 mL of 80 mmol / L MTU solution dissolved in NaOH solution was added to 1 mL of 25.6 mmol / L HAuCl4·4H2O solution. After stirring at room temperature for 1 h, the product was purified by ultrafiltration centrifugation (Millipore, 50 kDa) to obtain pure photoactivated oxidase-like active gold nanoclusters (Au-MTU), which were stored at 4 °C.

[0046] Au-MTU, TMB, and biothiols (GSH or Cys) were added to a 0.1 mol / L NaAc-HAc buffer solution with pH=3. The final concentration of Au-MTU was 100 μg / mL. The solution was irradiated with a 6W UV lamp for 5 min at room temperature, and the absorbance at 652 nm was measured. The difference in absorbance between the solution with different biothiol concentrations and the solution without biothiols was plotted on the ordinate, and the biothiol concentration was plotted on the abscissa to establish the relationship between biothiol concentration and absorbance.

[0047] Example 3

[0048] 0.8 mL of 80 mmol / L MTU solution dissolved in NaOH solution was added to 1 mL of 24.6 mmol / L HAuCl4·4H2O solution. After stirring at room temperature for 1 h, the product was purified by ultrafiltration centrifugation (Millipore, 50 kDa) to obtain pure photoactivated oxidase-like active gold nanoclusters (Au-MTU), which were stored at 4 °C.

[0049] Au-MTU, TMB, and biothiols (GSH or Cys) were added to a 0.1 mol / L NaAc-HAc buffer solution with pH=3. The final concentration of Au-MTU was 100 μg / mL. The solution was irradiated with a 6W UV lamp for 5 min at room temperature, and the absorbance at 652 nm was measured. The difference in absorbance between the solution with different biothiol concentrations and the solution without biothiols was plotted on the ordinate, and the biothiol concentration was plotted on the abscissa to establish the relationship between biothiol concentration and absorbance.

[0050] Example 4

[0051] 0.8 mL of 80 mmol / L MTU solution dissolved in NaOH solution was added to 1 mL of 25 mmol / L HAuCl4·4H2O solution. After stirring at room temperature for 1 h, the product was purified by ultrafiltration centrifugation (Millipore, 50 kDa) to obtain pure photoactivated oxidase-like gold nanoclusters (Au-MTU), which were stored at 4 °C.

[0052] Au-MTU, TMB, and biothiols (GSH or Cys) were added to a 0.1 mol / L NaAc-HAc buffer solution with pH=3. The final concentration of Au-MTU was 95 μg / mL. The solution was irradiated with a 6W UV lamp for 5 min at room temperature, and the absorbance at 652 nm was measured. The difference in absorbance between the solution with different biothiol concentrations and the solution without biothiols was plotted on the ordinate, and the biothiol concentration was plotted on the abscissa to establish the relationship between biothiol concentration and absorbance.

[0053] Example 5

[0054] 0.8 mL of 80 mmol / L MTU solution dissolved in NaOH solution was added to 1 mL of 25 mmol / L HAuCl4·4H2O solution. After stirring at room temperature for 1 h, the product was purified by ultrafiltration centrifugation (Millipore, 50 kDa) to obtain pure photoactivated oxidase-like gold nanoclusters (Au-MTU), which were stored at 4 °C.

[0055] Au-MTU, TMB, and biothiols (GSH or Cys) were added to a 0.1 mol / L NaAc-HAc buffer solution with pH=3. The final concentration of Au-MTU was 105 μg / mL. The solution was irradiated with a 6W UV lamp for 5 min at room temperature, and the absorbance at 652 nm was measured. The difference in absorbance between the solution with different biothiol concentrations and the solution without biothiols was plotted on the ordinate, and the biothiol concentration was plotted on the abscissa to establish the relationship between biothiol concentration and absorbance.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of a photoactivated oxidase-like active gold nanocluster in the analysis of biothiols, characterized in that, The preparation method of the photoactivated oxidase-like active gold nanoclusters includes the following steps: An alkaline solution of 6-methyl-2-thiouracil was added to a chloroauric acid solution, and the reaction was carried out by stirring at room temperature. The product was then purified by ultrafiltration and centrifugation to obtain photoactivated oxidase-like gold nanoclusters. The molar ratio of chloroauric acid to 6-methyl-2-thiouracil is 1:2.5-2.

6.

2. The application as described in claim 1, characterized in that, The concentration of 6-methyl-2-thiouracil in an alkaline solution is 75-85 mmol / L, and the concentration of chloroauric acid solution is 20-30 mmol / L.

3. The application as described in claim 1, characterized in that, The volume ratio of alkaline solution of 6-methyl-2-thiouracil to chloroauric acid solution is 0.75-0.85:

1.

4. The application as described in claim 1, characterized in that, The stirring reaction time is 0.9-1.1 h.

5. A method for analyzing biothiols, characterized in that, Includes the following steps: The photoactivated oxidase-active gold nanoclusters, substrate, and biothiols described in any one of claims 1-4 were dissolved in a sodium acetate-acetic acid buffer solution. After irradiation with ultraviolet light, the absorbance of the solution at 652 nm was measured. The difference in absorbance between the addition of different biothiol concentrations and the absence of biothiol was used as the ordinate, and the biothiol concentration was used as the abscissa to establish a linear relationship between biothiol concentration and absorbance. The concentration of biothiol was calculated from the absorbance.

6. The method for analyzing biothiols as described in claim 5, characterized in that, The substrate includes 3,3',5,5'-tetramethylbenzidine, and the biothiol includes glutathione or cysteine.

7. The method for analyzing biothiols as described in claim 5, characterized in that, The concentration of acetic acid and sodium-acetic acid buffer solution was 0.09-0.11 mol / L, pH=3; the concentration of photoactivated oxidase-like active gold nanoclusters was 95-105 μg / mL; the UV lamp power was 4-6 W, and the irradiation time was 4-6 min.

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