Construction method and application of peroxidase simulant with adjustable catalytic activity

Abstract

The invention belongs to the field of catalytic sensing of bionic nano-enzymes, and discloses a construction method and application of a peroxidase simulant with adjustable catalytic activity. According to the invention, chloroauric acid, glutathione and a dopa-containing peptidomimetic ligand are used as raw materials, and the gold nanocluster AuNCs with Fe < 2 + > chelating ability is prepared by a one-step hydrothermal method. And simply mixing the AuNCs and Fe < 2 + > with different concentrations in 1xPBS to obtain the peroxidase simulant AuNCs-Fe < 2 + > with different catalytic activities. The catalytic activity of peroxidase displayed by the gold nanocluster AuNCs is greater than that of linear superposition when the single gold nanocluster AuNCs and Fe < 2 + > play roles. On the basis of the controllable catalytic ability, the sensitive detection of H2O2 can be realized, the detection limit is extremely low, and the catalyst can also be used for determining cell samples to identify tumor cells, and has excellent prospects in the fields of biosensing, disease detection and the like.

Description

[Technical Field]

[0001] This invention belongs to the field of biomimetic nanoenzymes for catalytic sensing, specifically relating to a peroxidase mimic enzyme AuNCs@Fe with tunable catalytic activity. 2+ The construction of the compound and its application in the detection of hydrogen peroxide (H2O2) and the identification of tumor cells. [Background Technology]

[0002] While natural enzymes possess high specificity due to the precise spatial conformational matching between their active sites and substrates, their protein-like nature leads to poor stability and demanding reaction conditions, severely limiting their large-scale applications. In contrast, nanozymes, as nanomaterials with enzyme catalytic activity, not only exhibit high stability and ease of large-scale synthesis, but also allow for precise control of catalytic activity and selectivity through the manipulation of nanomaterial size and composition, making them an ideal alternative to biological enzymes (CN115739182B, 2024.01.16). Among these, peroxide mimics have attracted significant attention due to their ability to catalyze the decomposition of H2O2 to produce reactive oxygen species. Currently, various nano-peroxide mimics have been developed, including transition metal oxides (CN118719120B, 2024.12.03) and noble metal clusters (CN120169352A, 2025.06.20). Gold nanoclusters, with their molecular-like properties, high specific surface area, excellent optical properties, and stable catalytic ability, show great potential in peroxide mimic research, becoming a foundational material and research hotspot in analytical detection, biomedicine, and other fields.

[0003] While peroxide mimicry enzymes based on nanomaterials have been widely studied and applied in current H2O2 detection methods, they still face technical bottlenecks in practical scenarios, including insufficient immobilization and regulation of catalytic activity. Currently developed peroxide mimicry enzyme systems, such as transition metal oxides and noble metal clusters, primarily depend on the inherent structure and composition of the materials themselves for catalytic activity. Once the synthesis process is complete, their catalytic activity is difficult to dynamically adjust based on differences in the detection scenario and sample characteristics. In the detection of complex biological samples, due to the presence of interfering substances in the sample matrix and significant fluctuations in H2O2 concentration, mimicry enzyme systems with fixed catalytic activity often fail to simultaneously meet the dual requirements of detection sensitivity and specificity. [Summary of the Invention]

[0004] The main objective of this invention is to construct a peroxidase-mimicking nanoassembly with tunable catalytic activity by utilizing dopa-containing peptide ligands, thereby solving the problem that traditional peroxidase-mimicking enzymes have fixed activity and cannot be regulated according to demand.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention relates to the components and sequences of peptide-like ligands, including the following:

[0007] (1) The total number of linking units of the formed peptide-like compounds is 3-6.

[0008] (2) L-DOPA and natural amino acids form different sequences in a specific order and are linked by amide bonds. The sequence can be DpCDp (DOPA-cysteine-DOPA) or CDpE (cysteine-DOPA-glutamic acid), preferably DpCDp.

[0009] The method for preparing gold nanoclusters (AuNCs) according to the present invention includes the following steps:

[0010] (1) Mix chloroauric acid solution and glutathione solution evenly and let stand at room temperature for a period of time. Then add dopa-containing peptide ligand solution and react in a high-temperature water bath for an appropriate time. After the reaction is complete, ultrafiltration purification is performed to obtain gold nanoclusters AuNCs.

[0011] (2) In step (1), the ratio of the three raw materials is chloroauric acid, glutathione and dopa-like peptide ligand = 2:3.2-2.6:0.6-1.2, preferably, the ratio is 2:3.0:0.8.

[0012] In step (1), the settling time is 5-15 minutes, preferably 10 minutes.

[0013] In step (1), the reaction temperature is 65-80℃, preferably 70℃.

[0014] In step (1), the reaction time is 26-32 hours, preferably 30 hours.

[0015] This invention relates to AuNCs@Fe peroxide mimic enzymes with tunable catalytic activity. 2+ The construction method includes the following steps:

[0016] Using DpCDp-functionalized AuNCs with different concentrations of Fe 2+ Mix in 1xPBS buffer solution.

[0017] Among them, Fe 2+ The concentration ranges from 0 to 160 μM.

[0018] This invention relates to AuNCs@Fe peroxide mimic enzymes with tunable catalytic activity. 2+ The method for detecting H2O2 consists of the following steps:

[0019] (1) AuNCs and Fe of different concentrations 2+ Different Fe2+ Chelated concentrations of AuNCs@Fe 2+ It was incubated separately with 3,3',5,5'-tetramethylbenzidine (TMB) / H2O2 in an acetate-sodium acetate buffer solution.

[0020] The concentration of TMB is 1.0-5.0 mM, preferably 3.0 mM.

[0021] The pH of the acetate-sodium acetate buffer solution is 2.0-5.0, preferably 3.0.

[0022] The incubation time is 10-30 minutes, preferably 15 minutes.

[0023] (2) Utilizing AuNCs and Fe with different 2+ Chelated concentrations of peroxidase mimic AuNCs@Fe 2+ Incubate in a mixture with the TMB / H2O2 system.

[0024] (3) The absorbance values ​​of the multiple solutions obtained in steps (1) and (2) are measured using a UV-Vis spectrophotometer.

[0025] (4) Take the absorption value at 652 nm of the absorption spectrum obtained in step (3), and plot the AuNCs and Fe... 2+ and AuNCs@Fe 2+ A comparison chart of catalytic activities.

[0026] (5) Prepare H2O2 solutions of different concentrations and react them with TMB / AuNCs and TMB / AuNCs@Fe, respectively. 2+ Mixed incubation.

[0027] (6) Measure the absorbance of the solutions obtained in step (5) using a UV-Vis spectrophotometer.

[0028] (7) Take the absorption values ​​of a series of absorption spectra obtained in step (6) at 652 nm, and obtain the linear relationship between the absorption value and the H2O2 concentration.

[0029] This invention relates to AuNCs@Fe peroxide mimic enzymes with tunable catalytic activity. 2+ The method for identifying tumor cells comprises the following steps:

[0030] Mouse fibroblasts (NIH3T3) with low H2O2 content and Chinese hamster ovary cells (CHO) were selected as the negative group, while human lung cancer cells (A549) and human liver cancer cells (HepG2) with high H2O2 content were selected as the positive group. TMB / AuNCs@Fe 2+The mixed solution was incubated with cell lysates of the four cell types described above. The UV-Vis absorption spectra of each sample were then measured.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] (1) AuNCs@Fe peroxidase with tunable catalytic activity 2+ The preparation is simple, requiring only the addition of gold nanoclusters (AuNCs) and Fe obtained via a one-step hydrothermal method. 2+ It can be obtained by simply mixing under neutral conditions.

[0033] (2) By controlling the Fe on the surface of gold nanoclusters AuNCs 2+ The amount of chelation required can easily achieve the desired effect on AuNCs@Fe. 2+ Precise regulation of catalytic activity to meet the requirements under certain conditions.

[0034] (3) Benefiting from AuNCs@Fe 2+ Its high catalytic activity enables sensitive and accurate quantitative detection of H2O2 with an extremely low detection limit, and it is also applicable to the determination of cell samples. [Attached Image Description]

[0035] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used to explain this description but do not constitute a limitation thereof. The drawings are as follows:

[0036] Figure 1 Transmission electron microscope images of gold nanoclusters (AuNCs) provided for this invention.

[0037] Figure 2 The UV-Vis absorption spectrum of gold nanoclusters AuNCs provided by this invention.

[0038] Figure 3 The gold nanoclusters AuNCs provided by this invention and Fe nanoclusters of different concentrations 2+ The resulting peroxidase AuNCs@Fe with tunable catalytic activity was obtained after mixing. 2+ The ultraviolet-visible absorption spectrum.

[0039] Figure 4 The gold nanoclusters AuNCs and Fe provided by this invention were determined using the TMB colorimetric method. 2+ Different Fe 2+ Chelated concentrations of AuNCs@Fe 2+ Comparison of peroxidase catalytic activities.

[0040] Figure 5 Different concentrations of H2O2 were reacted with TMB / AuNCs and TMB / AuNCs@Fe, respectively.2+ Linear calibration curve of absorbance at 652 nm versus H2O2 concentration after system mixing and incubation.

[0041] Figure 6 To utilize AuNCs@Fe 2+ The H2O2 content in cell samples was determined.

Detailed Implementation Methods

[0042] The present invention will be described in more detail below through specific embodiments, which are intended to explain the invention and not to limit it.

[0043] Example 1

[0044] The preparation method of gold nanoclusters (AuNCs) is as follows:

[0045] (1) Mix 0.50 mL of 20 mM chloroauric acid solution and 0.75 mL of 20 mM GSH solution evenly, let stand at room temperature for 10 min, then add 0.40 mL of 10 mM DpCDp solution, use ultrapure water to make up to 5.0 mL, mix thoroughly, and place in a 70℃ water bath for 30 h.

[0046] (2) After taking out the sample, cool it to room temperature and purify it by ultrafiltration to obtain an aqueous solution of AuNCs. Store it in a refrigerator at 4°C in the dark.

[0047] Example 2

[0048] AuNCs@Fe peroxidase with tunable catalytic activity 2+ The construction method is as follows:

[0049] (1) Prepare 50 μL FeCl2 aqueous solutions with concentrations of 0 μM, 60 μM, 120 μM, 300 μM, 600 μM, 900 μM, 1200 μM, 1560 μM, and 1920 μM using ultrapure water. Measure 200 μL of the gold nanoclusters (AuNCs) solution obtained in Example 1 and mix them with FeCl2 aqueous solutions of different concentrations, and add 350 μL of 1xPBS (pH = 7.4) buffer solution.

[0050] (2) After reacting at room temperature for 120 s, the solution was transferred to a cuvette and the UV-Vis absorption spectra were measured.

[0051] Example 3

[0052] AuNCs@Fe, a peroxidase with tunable catalytic activity 2+ Determination of H2O2 content in solution:

[0053] (1) Mix 200 μL of AuNCs solution from Example 1 with 50 μL of FeCl2 aqueous solutions of different concentrations (60 μM, 120 μM, 300 μM), and add 350 μL of 1xPBS buffer solution to prepare different Fe... 2+ AuNCs@Fe chelation concentrations (5 μM, 10 μM, 25 μM) 2+ .

[0054] (2) Take 33 μL of the AuNCs solution obtained in Example 1, 100 μL of FeCl2 aqueous solutions of different concentrations (25 μM, 50 μM, 125 μM), and 100 μL of the three groups of AuNCs@Fe obtained in step (1) of Example 3. 2+ The solutions were mixed with 60 μL TMB (25 mM) + 20 μL H2O2 (50 mM) and then brought to a final volume of 500 μL using an acetate-sodium acetate buffer solution. After incubation for 15 min, the absorbance of all samples was measured.

[0055] (3) Prepare aqueous solutions of H2O2 with concentrations of 0 mM, 0.125 mM, 0.250 mM, 0.625 mM, 1.25 mM, 2.50 mM, 5.00 mM, 12.5 mM, 25.0 mM, 37.5 mM, and 50.0 mM. Mix 20 μL of each H2O2 aqueous solution with 60 μL of TMB (25.0 mM) solution, and then add 100 μL of AuNCs solution (490 μg / mL) and 100 μL of Fe... 2+ AuNCs@Fe with a chelation concentration of 25 μM 2+ (500 μg / mL) was added to the system, and finally, the volume was adjusted to 500 μL using acetate-sodium acetate buffer solution. After reacting for 15 min, the UV-Vis absorption spectra of all samples were measured.

[0056] Figure 5 Plotting H₂O₂ concentration (0-200 μM) on the x-axis and the absorbance of the solution at 652 nm on the y-axis, we obtained the results using AuNCs and Fe. 2+ AuNCs@Fe with a chelation concentration of 25 μM 2+ Linear curves of H2O2 concentration and absorbance when used as a peroxidase. The figure shows a good linear relationship between H2O2 concentration and absorbance within the concentration range of 0-200 μM. When AuNCs are used, the obtained linear equation is y = 0.0009x + 0.0018, R0 2 =0.988, detection limit is 1.17 μM; using Fe 2+ AuNCs@Fe with a chelation concentration of 25 μM 2+When the linear relationship is obtained, the equation is y = 0.0075x + 0.0614, R0 2 =0.996, detection limit is 0.27μM.

[0057] Example 4

[0058] Use Fe 2+ AuNCs@Fe with a chelation concentration of 25 μM 2+ Used for the identification of tumor cells:

[0059] Four cell lines—NIH3T3, CHO, A549, and HepG2—were cultured in vitro. When they reached 80-90% confluence, they were detached from the culture flask using a cell scraper and dissolved in buffer solution. 100 μL of the Fe2+ prepared in Example 3 was then sequentially measured. 2+ AuNCs@Fe with a chelation concentration of 25 μM 2+ The solution and 100 μL of cell lysate from different cells were placed in centrifuge tubes and reacted at room temperature for 15 min. The solutions were then transferred to cuvettes and the UV-Vis absorption spectra were measured.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it; moreover, the above embodiments are only preferred embodiments of the present invention, and not all of them.

Claims

1. A method for constructing and applying a peroxidase mimic with tunable catalytic activity, characterized in that, By using organic molecules containing thiol and dopa groups as a bridge, catalytically active gold nanoclusters and Fe(II) can be combined in a certain ratio. By adjusting the ratio of gold nanoclusters to Fe(II) in the complex, the catalytic activity of the prepared peroxide mimic enzyme can be precisely controlled.

2. The simulated peroxidase according to claim 1, characterized in that, The overall catalytic activity is greater than the linear superposition of the individual catalytic activities of gold clusters and Fe(II), and it has a highly efficient peroxidase catalytic ability.

3. The organic molecular ligand according to claim 1, characterized in that, It is a peptide-like compound in which L-DOPA, cysteine, and other natural amino acids are linked together in a certain sequence using amide bonds (total number of linking units 3≤n≤6).

4. The gold nanocluster assembly unit according to claim 1, characterized in that, It has the ability to catalyze the generation of hydroxyl radicals from hydrogen peroxide.

5. The peroxidase mimicry according to claim 1, characterized in that, When mixed with H2O2, it can generate hydroxyl radicals and cause the 3,3',5,5'-tetramethylbenzidine (TMB) indicator to change color. Given a fixed concentration of peroxidase mimic and TMB (3.0 mM), the TMB absorbance showed a linear relationship with the H2O2 concentration in the range of 0-200 μM. The detection limit for H2O2 was 1.17 μM when the chelation concentration was 0 μM, and the detection limit for H2O2 reached 0.27 μM when the chelation concentration was 25 μM.

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