Protein molecule mediated monatomic catalyst as well as preparation method and application thereof

Abstract

The invention discloses a protein molecule mediated monatomic catalyst as well as a preparation method and application thereof, and relates to the technical field of chemical biosensing and biological detection. A protein molecule mediated monatomic catalyst is constructed by coordinating platinum atoms with amino and carboxyl in a protein framework, and specifically, a protein solution and a potassium chloroplatinite solution are subjected to a mixed reaction. The obtained monatomic catalyst has high catalytic activity, high selectivity and good stability, the preparation method is simple, the cost is low, the sensitivity is high, rapid and accurate detection can be achieved, and a protein guiding strategy has expandability, biocompatibility and adaptability to various proteins; a multifunctional platform is provided for nano-catalysis in biosensing, diagnosis and precise oncology, meanwhile, a new thought is provided for wider application of the SACs in the field of biological medicine, and a foundation is laid for innovation of protein engineering catalysis in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical biosensing and biological detection, and in particular to a single-atom catalyst mediated by protein molecules and a preparation method and application thereof. Background Art

[0002] Peroxidase is a class of enzymes that can catalyze the decomposition of hydrogen peroxide (H2O2). They play a key role in many redox reactions in organisms. However, although natural enzymes have many advantages as a biocatalyst, they also have some defects, such as high preparation and purification costs, poor operational stability, high environmental sensitivity, and difficulty in recycling and reuse. These defects limit the further development and application of natural enzymes as a biocatalyst. Based on this, researchers have been looking for alternatives, such as the development of recombinant enzymes, engineered enzymes, nanozymes, and chemical enzymes, to overcome the limitations of natural enzymes and improve their performance and stability in various applications. At present, nanomaterials, metal oxides, precious metals and metal-organic frameworks, porphyrins, biomolecules, metal complexes and polymers, as well as many small molecules and ions, have been found to have peroxidase-mimicking activity, which provides new ideas for the related research of peroxidase-mimicking enzymes.

[0003] Single-atom catalysts (SACs) are a new type of catalyst in which metal atoms are dispersed on a substrate in a single form, with extremely high atomic utilization and unique catalytic properties. In recent years, the research and application of single-atom catalysts have developed rapidly. The metal atom utilization of single-atom catalysts is close to 100%, which is especially important for precious metal catalysts because they can significantly reduce costs. At the same time, stability and durability are also hot topics in current research in practical applications. The stability of the catalyst can be improved by adjusting the interaction between the metal and the substrate. Although the research on single-atom catalysts has made significant progress, there are still some challenges, such as large-scale synthesis, long-term stability, and performance in complex industrial environments. Future research will continue to address these problems and promote the application of single-atom catalysts in various fields.

[0004] Peroxidase mimics can be used as key components of biosensors to detect H2O2 or other chemicals (such as glucose). First, in the body, the role of peroxidase mimics is essential for maintaining redox balance and protecting cells from oxidative damage. Second, in the fields of medicine and biotechnology, the research and development of efficient peroxidase mimics is of great significance for the treatment of diseases associated with oxidative stress. Peroxidase mimics usually work in conjunction with other antioxidant systems (such as the glutathione system, superoxide dismutase, etc.) to maintain redox balance in cells. Since H2O2 is a reactive oxygen species (ROS), excess H2O2 is harmful to cells. Peroxidase mimics can protect cells from oxidative stress damage by decomposing H2O2.

[0005] Glucose oxidase is an enzyme that catalyzes the decomposition of glucose. It is very important in glucose metabolism in organisms and in industrial applications. Glucose oxidase can decompose glucose into gluconic acid and H2O2. The H2O2 produced by the reaction can be further decomposed into water and oxygen by peroxidase or other catalysts to prevent H2O2 accumulation from damaging cells. Monitoring blood glucose levels is essential for personal health and disease management, especially in the diabetic population. For diabetic patients, regular monitoring of blood glucose levels is a core part of managing diabetes and helps adjust diet, exercise and drug treatment plans. Maintaining blood glucose within the normal range for a long time can reduce the risk of diabetic complications such as cardiovascular disease, kidney disease, retinopathy and neuropathy. Timely monitoring of blood glucose can help prevent and identify hypoglycemia. Understanding changes in blood glucose levels can help patients adjust their diet and exercise habits to better control blood glucose. The two-step method for detecting glucose is a common method for screening and diagnosing diabetes. This method is divided into two stages: the first stage: 50 grams of oral glucose load test (OGTT); in this stage, the subject drinks a solution containing 50 grams of glucose in a non-fasting state. Then the glucose concentration in plasma or serum is measured after 1 hour. If the result exceeds the set threshold (usually 130 to 140 mg / dL), the screening result is considered positive and further diagnostic testing is required. Stage 2: 100-gram oral glucose tolerance test (OGTT); for individuals with positive screening results, a solution containing 100 grams of glucose is drunk in the fasting state, and blood glucose levels are measured 1 hour, 2 hours, and 3 hours later. If the blood glucose value at at least two time points reaches or exceeds a specific threshold (for example, 180 mg / dL at 1 hour, 155 mg / dL at 2 hours, and 140 mg / dL at 3 hours), diabetes can be diagnosed. This blood glucose testing method is effective, but the testing process is cumbersome and time-consuming.

[0006] Blood sugar, uric acid, etc. are very important biochemical indicators, which are critical for evaluating the metabolic health of the human body. Therefore, it is of great significance to develop simple, cheap and reliable detection methods for important metabolites of the human body such as blood sugar and uric acid. Summary of the invention

[0007] In view of the technical problems that the detection methods of important human metabolites such as blood sugar in the above-mentioned prior art have the advantages of complicated detection process and long time consumption, the present invention provides a single-atom catalyst mediated by a protein molecule and a preparation method thereof, and the obtained single-atom catalyst is used for the detection of human metabolites such as blood sugar and uric acid that are closely related to personal health and disease management. The method is not only simple and low in cost, but also highly sensitive and can achieve rapid and accurate detection.

[0008] The single-atom catalysts (SACs) mediated by protein molecules provided by the present invention are constructed by coordinating platinum atoms with amino groups and carboxyl groups within a protein framework.

[0009] The method for preparing the above-mentioned single-atom catalyst mediated by protein molecules is to mix a protein solution with a potassium chloroplatinite solution to react, thereby obtaining a single-atom catalyst having peroxide mimicking enzyme activity and maintaining the original protein activity, that is, a single-atom catalyst mediated by protein molecules.

[0010] Furthermore, proteins include natural enzyme proteins and non-enzyme proteins.

[0011] Furthermore, the natural enzyme protein includes at least one of glucose oxidase (GOx), alkaline phosphatase (ALP), D-amino acid oxidase (DAO), lactate oxidase (LOX), cholesterol oxidase (COD) or urate oxidase (UAO), and the non-enzyme protein includes at least one of bovine serum albumin (BSA), human serum albumin (HSA), hemoglobin (Hb) and myoglobin antibody (MYO), and the corresponding single-atom catalysts mediated by protein molecules are GOx-Pt, ALP-Pt, DAO-Pt, LOX-Pt, COD-Pt, UAO-Pt, BSA-Pt, HSA-Pt, Hb-Pt, MYO-Pt, respectively.

[0012] Furthermore, the usage ratio of the protein solution to the potassium chloroplatinite solution is 3-8 mg / ml: 0.001-0.005 mol / L, preferably 4-6 mg / ml: 0.001-0.003 mol / L, and more preferably 5-6 mg / ml: 0.002-0.003 mol / L.

[0013] Furthermore, the reaction is carried out at a pH of 7.0 to 7.8, preferably 7.4 to 7.6; the reaction temperature is 35 to 39° C., more preferably 36 to 38° C., most preferably 37° C.; and the reaction time is 20 to 24 hours.

[0014] Furthermore, it also includes post-dialysis treatment after the reaction is completed, such as dialyzing the reaction product with a dialysis bag with a cutoff of 8000Da at 4°C for 12 hours, replacing the dialysate every 4 hours, and storing the dialyzed solution in a refrigerator at 4°C for later use.

[0015] The above-mentioned single-atom catalyst mediated by protein molecules or the single-atom catalyst mediated by protein molecules obtained by the above-mentioned preparation method is used to detect the content of human metabolites, and the human metabolites include one of glucose, uric acid, D-amino acids, cholesterol, lactic acid or phosphate in serum. The detection time is short and the accuracy is high.

[0016] Furthermore, when the single-atom catalyst mediated by the protein molecule is GOx-Pt, the above application includes the following specific steps: S1. Add GOx-Pt, 3,3',5,5'-tetramethylbenzidine solution (TMB), and acetate buffer solution into a centrifuge tube, then add different concentrations of glucose into each centrifuge tube and react for 20-40 min, measure the UV-visible absorption spectrum and the absorbance at 652 nm (maximum absorption wavelength), and perform linear fitting with glucose concentration as the horizontal axis and absorbance as the vertical axis to obtain the glucose standard curve; S2. Dilute the serum sample to be tested 10-30 times with phosphate buffer solution (PBS), take 50 μL of serum, add 50 μL of glucose standard of different concentrations respectively, then add GOx-Pt, acetate buffer solution, H2O2 and TMB, measure the absorbance at 652 nm after 20-40 min, and then calculate the glucose content according to the standard curve.

[0017] Furthermore, in S2, the concentration of GOx-Pt added is 0.0004~0.0006mol / L (preferably 0.0005mol / L), the concentration of acetate buffer solution is 0.1~0.3mol / L (preferably 0.2mol / L), the pH is controlled at 3.0~5.0 (preferably 4.0), the concentration of hydrogen peroxide is 0.05~0.15mol / L (preferably 0.1mol / L), the concentration of TMB is 0.003~0.005mol / L (preferably 0.004mol / L), and the added volume ratio of GOx-Pt, acetate buffer solution, H2O2 and TMB is 0.9~1.1:4.8~5.2:0.15~0.25:0.9~1.1.

[0018] GOx-Pt has a certain catalytic ability. In the presence of GOx-Pt, H2O2 is rapidly catalytically decomposed to produce hydroxyl radicals, which oxidize TMB (3,3',5,5'-tetramethylbenzidine). Different concentrations of oxidized TMB show different colors. Therefore, the color change can be used to visualize the semi-quantitative detection of the analyte, and the absorbance can be used to quantitatively detect the accurate content of the analyte. Glucose oxidase can catalyze the decomposition of glucose to produce H2O2, and then H2O2 will be catalytically decomposed by GOx-Pt with peroxidase activity, so that TMB can be oxidized for colorimetric determination. The experimental results show that GOx-Pt retains glucose oxidase activity and is endowed with peroxidase activity. Using this dual enzyme activity, the cumbersome steps of two-step glucose detection can be omitted, and glucose detection can be directly performed in one step. By adjusting different proteins to obtain the corresponding enzyme properties, the level of human-related diseases can be monitored. For example, GOx is replaced by UAO, so that UAO-Pt acquires the dual enzyme properties of peroxidase mimetic enzyme and uricase, and this property is used to detect uric acid. The uric acid level can be preliminarily judged based on the different concentrations of H2O2 produced by urate oxidase decomposing uric acid, that is, the different colors of the generated Ox-TMB.

[0019] The above-mentioned single-atom catalyst mediated by protein molecules or the single-atom catalyst mediated by protein molecules obtained by the above-mentioned preparation method is used to prepare anti-cancer drugs. By encapsulating the single-atom catalyst mediated by protein molecules in the pH-responsive ZIF-8 framework, it can show selective activation in the acidic tumor microenvironment and achieve effective anti-cancer treatment.

[0020] Compared with the prior art, the present invention has the following beneficial effects: The single-atom catalyst prepared by the present invention exhibits high catalytic activity, high selectivity and good stability. The preparation method of the present invention can synthesize SAC with specific enzyme activity: non-enzyme protein obtains peroxidase-like activity, while enzyme protein (such as glucose oxidase) realizes dual enzyme function.

[0021] (2) The preparation method of the present invention is simple, low-cost, and highly sensitive, and can achieve rapid and accurate detection. Compared with the traditional SAC synthesis method, the protein-guided strategy of the present invention has scalability, biocompatibility, and adaptability to a variety of proteins, providing a multifunctional platform for nanocatalysis in biosensing, diagnosis, and precision oncology. It also provides new ideas for the wider application of SAC in the biomedical field and lays the foundation for future innovations in protein engineering catalysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the GOx-Pt dual enzyme catalytic activity.

[0023] Figure 2 The absorbance bar graph of Ox-TMB at 652 nm shows that different proteins of the present invention obtain peroxidase mimicking properties after reacting with potassium chloroplatinite.

[0024] Figure 3 The present invention shows the peroxidase mimicking properties obtained after different amino acids react with potassium chloroplatinite, and is a bar graph of the absorbance of Ox-TMB at 652 nm. In the graph, Thr represents threonine, Ala represents alanine, Gln represents glutamine, Phe represents phenylalanine, Val represents valine, Lys represents lysine, Glu represents glutamic acid, Ser represents serine, Gly represents glycine, Arg represents arginine, His represents histidine, Pro represents proline, Cys represents cysteine, Leu represents leucine, Tyr represents tyrosine, and Trp represents tryptophan.

[0025] Figure 4 The color development picture is to verify that the GOx-Pt obtained in Example 1 has both the properties of peroxidase mimetic enzyme and glucose oxidase.

[0026] Figure 5 To verify that the GOx-Pt obtained in Example 1 has the properties of peroxidase mimicking, the UV-visible absorption spectrum and color development picture are provided.

[0027] Figure 6 To verify that the GOx-Pt obtained in Example 1 has the properties of glucose oxidase, a UV-visible absorption spectrum and a color development picture are provided.

[0028] Figure 7 The GOx-Pt obtained in Example 1 catalyzes hydrogen peroxide to produce hydroxyl radicals under different pH conditions, causing TMB to be oxidized and turn blue. The absorbance bar graph of Ox-TMB at 652 nm.

[0029] Figure 8 The UV-visible absorption spectrum and color change diagram of the GOx-Pt obtained in Example 1 when detecting a glucose standard solution.

[0030] Fig. 9 is the linear equation of glucose concentration and the absorbance of oxidized TMB at 652 nm.

[0031] Fig.10 Cytotoxicity plots for GOx-Pt complexes encapsulated within the pH-responsive ZIF-8 framework.

[0032] Fig.11 This is the color diagram for the preliminary exploration of UAO-Pt detection of uric acid. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto. Example 1

[0034] Preparation of GOx-Pt single atom catalyst: The pH of 5 mg / ml GOx was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, and a magnetic bar was added. The reaction was stopped when the solution changed from light yellow to dark brown. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000 Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The obtained product was a GOx-Pt single-atom catalyst, and the schematic diagram of its dual enzyme catalytic activity is shown in FIG. Figure 1 shown.

[0035] The color image of GOx-Pt is as follows Figure 4 As shown, Figure 4 It shows that the GOx-Pt single atom catalyst prepared in Example 1 can catalyze the oxidation of TMB by hydrogen peroxide to generate blue Ox-TMB. After adding GOx-Pt, hydrogen peroxide and TMB to the left centrifuge tube, the solution turns blue. After adding GOx-Pt, glucose and TMB to the middle centrifuge tube, the solution also turns blue. After adding GOx-Pt, water and TMB to the right centrifuge tube, there is almost no color. Figure 5 and Figure 6 , indicating that the GOx-Pt prepared in Example 1 catalyzes the oxidation of TMB by hydrogen peroxide with a characteristic absorption peak at 652nm, while glucose oxidase or potassium chloroplatinite alone cannot catalyze the oxidation of TMB by hydrogen peroxide to turn it blue, and there is no characteristic absorption peak at 652nm, which proves that GOx-Pt has the properties of peroxidase mimics and has the strongest catalytic activity under pH=4 conditions, as shown in Figure 7 shown. Example 2

[0036] GOx-Pt for glucose detection: First, 10 centrifuge tubes were taken and numbered 1-10, and 100 μL GOx-Pt, 200 μL 5mmol / L TMB, and 500 μL pH=4.0, 0.2mol / L acetate buffer solution were added to each centrifuge tube. Then, 200 μL glucose standard solution (initial concentrations were: 2, 1, 0.8, 0.5, 0.25, 0.1, 0.08, 0.05, 0.025, 0 mmol / L) was added to each centrifuge tube. The UV-visible absorption spectrum and color change diagram of GOx-Pt on glucose standard solution are shown in the figure below. Figure 8 shown.

[0037] The UV-visible absorption spectrum and absorbance at 652 nm were measured after 30 min of reaction, and a linear fit was performed with glucose concentration as the abscissa and absorbance as the ordinate to obtain the glucose standard curve.

[0038] Sample processing and recovery determination: First, different human serum samples were diluted 20 times with phosphate buffer solution (PBS), and 50 μL of serum was taken and 50 μL of glucose standards of different concentrations were added respectively. The pH value of PBS buffer solution was 7.2. Then, 500 μL of acetic acid buffer solution with pH = 4.0 and a concentration of 0.2 mol / L, 100 μL of 0.5 mmol / L GOx-Pt, 100 μL of 0.004 mol / L TMB, and 20 μL of 0.1 mol / L H2O2 were added. After 30 minutes of action, the absorbance at 652 nm was measured. The concentration of glucose can be obtained according to the standard curve. The absorbance at 652 nm is linearly related to the glucose concentration in the range of 0-0.2 mmol / L (such as Fig. 9 As shown), R 2 =0.992, and the detection limit calculated based on 3δ / K is 9 μmol / L. Actual samples were tested, and different concentrations of glucose were added to serum samples for recovery experiments. The recovery range was 98.64%~102.86%, as shown in Table 1. The results show that it meets the requirements of quantitative analysis of actual biological samples and has high accuracy.

[0039] Table 1

[0040]

[0041] The above results show that the present invention utilizes the property of glucose oxidase to decompose glucose into hydrogen peroxide, and then performs visual detection based on the property of GOx-Pt to rapidly catalyze and decompose H2O2, thereby achieving the purpose of one-step glucose detection. GOx-Pt is used to detect glucose in serum, which has high sensitivity, simple operation, and can quickly and accurately determine the glucose concentration.

[0042] The advantages of GOx-Pt in measuring blood sugar are mainly reflected in two aspects: first, GOx-Pt has incomparable advantages over biological enzyme macromolecules in terms of price and stability. More importantly, GOx-Pt does not have the problem of biological proteases easily losing their biological activity. On the other hand, the introduction of TMB shows different colors in different states, establishes visual detection, and has high detection sensitivity and stability.

[0043] Example 3 Encapsulation of GOx-Pt Complexes within pH-responsive ZIF-8 Framework Cytotoxicity Assay: See also Fig.10, cultured human hepatoma cells (Hcclm3) and mouse fibroblasts (L929), washed the cells twice with 2 mL PBS buffer, added 1 mL pancreatin to the cell culture flask, and added 4 mL DMEM (Dulbecco's Modified Eagle's Medium) medium to stop digestion after the cells became round, centrifuged at 1000 r / min for 5 min, removed the supernatant, then added 4 mL DMEM medium to the centrifuge tube and gently blow the cells, then added 100 μL of cells with a density of 50,000 / mL to the 96-well microplate, disinfected the microplate and cultured it in a cell culture incubator for one day until the cells adhered to the wall. 100 μL of sterilized material was added to each well of the experimental group, and 3 groups were paralleled; 100 μL of sterilized ultrapure water was added to each well of the control group, and cell viability was evaluated after one day.

[0044] Cell viability assessment: Add 20μL 5mg / mL thiazolyl blue (MTT) to each well, sterilize the microplate and place it in a cell culture incubator for 4h, then remove the supernatant, add 150μL dimethyl sulfoxide (DMSO) to each well to dissolve the formazan crystals generated in the living cells, then place the microplate on a shaker for 5-7min, and then measure its absorbance at 490nm. The effect of the material on cell viability is expressed by cell survival rate: Cell survival rate (%) = (number of surviving cells / total number of cells) × 100% = (OD490 of the experimental group / OD490 of the control group) × 100%.

[0045] Example 4 Preparation of UAO-Pt single atom catalyst: The pH of 5 mg / ml urate oxidase (UAO) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to milky white turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a UAO-Pt single-atom catalyst.

[0046] Example 5 UAO-Pt was used to monitor uric acid levels. First, 8 centrifuge tubes were taken and numbered 1-8. 200 μL of UAO-Pt obtained in Example 4 was added to each tube. Then, 200 μL of uric acid (initial concentrations were: 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05) was added to each centrifuge tube. The tubes were heated in a 37°C water bath for 15 min to produce hydrogen peroxide. Then, 200 μL of 5 mM TMB and 500 μL of pH=4.0 0.2 M acetate buffer solution were added. The color change after 30 min of reaction was observed. Fig.11 It can be observed that the colors become lighter in sequence, and the color of high concentration uric acid is darker. The experimental results are in line with expectations.

[0047] Example 6 Preparation of ALP-Pt single atom catalyst: The pH of 5 mg / ml alkaline phosphatase (ALP) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to milky white turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was an ALP-Pt single-atom catalyst.

[0048] Example 7 Preparation of DAO-Pt single atom catalyst: The pH of 5 mg / ml D-amino acid oxidase (DAO) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to milky white turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a DAO-Pt single-atom catalyst. Example 8

[0049] Preparation of LOX-Pt single atom catalyst: The pH of 5 mg / ml lactate oxidase (LOX) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution was observed to change from a transparent state to a light black liquid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with an 8000Da cutoff, and the dialysate was replaced every 4 hours. The dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a LOX-Pt single-atom catalyst. Example 9

[0050] Preparation of COD-Pt single atom catalyst: The pH of 5 mg / ml cholesterol oxidase (COD) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to milky white turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a COD-Pt single-atom catalyst. Example 10

[0051] Preparation of BSA-Pt single atom catalyst: The pH of 5 mg / ml bovine serum albumin (BSA) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to milky white turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a BSA-Pt single-atom catalyst. Embodiment 11

[0052] Preparation of HSA-Pt single atom catalyst: The pH of 5 mg / ml human serum albumin (HSA) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution was observed to change from a transparent state to a milky white liquid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da, and the dialysate was replaced every 4 hours. The dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a HSA-Pt single-atom catalyst.

[0053] Example 12 Preparation of MYO-Pt single atom catalyst: The pH of 5 mg / ml myoglobin antibody (MYO) was adjusted to 7.4, mixed with an equal volume of 0.002 mol / L K2PtCl4, placed in a round-bottom flask, added with a magnetic bar, and reacted at 37°C. The reaction was stopped when the solution changed from transparent to transparent turbid. The reaction product was dialyzed at 4°C for 12 hours using a dialysis bag with a cutoff of 8000Da. The dialysate was replaced every 4 hours, and the dialyzed solution was stored in a refrigerator at 4°C for use. The resulting product was a MYO-Pt single-atom catalyst.

[0054] Different proteins acquire peroxidase mimicking properties after reacting with potassium chloroplatinite. The absorbance bar graph of Ox-TMB at 652 nm is shown in the figure below. Figure 2 shown. Figure 2 It shows that bovine serum albumin (BSA), human serum albumin (HSA), alkaline phosphatase (ALP), D-amino acid oxidase (DAO), cholesterol oxidase (COD), and urate oxidase (UAO) react with platinum ions to form complexes BSA-Pt, HSA-Pt, ALP-Pt, DAO-Pt, COD-Pt, and UAO-Pt, respectively, and then H2O2 and TMB are added for color development. The results show that the above complexes can all be colored and have absorbance at 652nm. Therefore, it can be inferred that most complexes of proteins and platinum ions have peroxidase mimicking activity. Some antibodies, such as myoglobin antibodies, can also react with potassium chloroplatinite to obtain peroxidase mimicking properties.

[0055] To elucidate the mechanism behind this catalytic effect, the present invention analyzed the peroxidase-like activity of platinum with single amino acids commonly found in proteins (e.g. Figure 3 The absorption spectra of these platinum-amino acid complexes showed significant activity at 652 nm, among which the Arg-Pt complex showed the strongest response.

Claims

1. A single-atom catalyst mediated by a protein molecule, characterized in that: The protein-mediated single-atom catalyst is constructed by coordinating platinum atoms with amino and carboxyl groups within a protein framework.

2. The method for preparing a single-atom catalyst mediated by a protein molecule according to claim 1, characterized in that: The protein solution is mixed with potassium chloroplatinite solution to react, thereby obtaining a single-atom catalyst having peroxidase mimicking activity and maintaining the activity of the original protein, that is, a single-atom catalyst mediated by a protein molecule.

3. The method for preparing a single-atom catalyst mediated by a protein molecule according to claim 2, characterized in that: The proteins include natural enzyme proteins and non-enzyme proteins.

4. The method for preparing a single-atom catalyst mediated by a protein molecule according to claim 3, characterized in that: The natural enzyme protein includes at least one of glucose oxidase GOx, alkaline phosphatase ALP, D-amino acid oxidase DAO, lactate oxidase LOX, cholesterol oxidase COD or urate oxidase UAO, the non-enzyme protein includes at least one of bovine serum albumin BSA, human serum albumin HSA, hemoglobin Hb and myoglobin antibody MYO, and the corresponding single-atom catalysts mediated by protein molecules are GOx-Pt, ALP-Pt, DAO-Pt, LOX-Pt, COD-Pt, UAO-Pt, BSA-Pt, HSA-Pt, Hb-Pt, MYO-Pt.

5. The method for preparing a single-atom catalyst mediated by a protein molecule according to claim 2, characterized in that: The dosage ratio of the protein solution to the potassium chloroplatinite solution is 3-8 mg / ml: 0.001-0.005 mol / L; the reaction is carried out at a pH of 7.0-7.8; the reaction temperature is 35-39°C; and the reaction time is 20-24 hours.

6. The method for preparing a single-atom catalyst mediated by a protein molecule according to any one of claims 2 to 5, characterized in that: It also includes post-dialysis treatment after the reaction is completed.

7. Use of the single-atom catalyst mediated by a protein molecule according to claim 1 or the single-atom catalyst mediated by a protein molecule obtained by the preparation method according to any one of claims 2 to 6 in detecting the content of human metabolites.

8. The use according to claim 7, characterized in that: The human body metabolites include one of glucose, uric acid, D-amino acid, cholesterol, lactic acid or phosphate in serum.

9. The use according to claim 7, characterized in that: When the single-atom catalyst mediated by the protein molecule is GOx-Pt, the application comprises the following specific steps: S1. Add GOx-Pt, 3,3',5,5'-tetramethylbenzidine solution TMB, and acetate buffer solution into a centrifuge tube, then add different concentrations of glucose into each centrifuge tube for 20-40 min, measure the UV-visible absorption spectrum and the absorbance at 652 nm, and perform linear fitting with glucose concentration as the horizontal axis and absorbance as the vertical axis to obtain the glucose standard curve; S2. Dilute the serum sample to be tested 10-30 times with phosphate buffer solution, take 50 μL of serum, add 50 μL of glucose standard of different concentrations respectively, and then add GOx-Pt, acetate buffer solution, H2O2 and TMB. The concentration of GOx-Pt is 0.0004-0.0006 mol / L, the concentration of acetate buffer solution is 0.1-0.3 mol / L, the pH is controlled at 3.0-5.0, the concentration of hydrogen peroxide is 0.05-0.15 mol / L, the concentration of TMB is 0.003-0.005 mol / L, the volume ratio of GOx-Pt, acetate buffer solution, H2O2 and TMB is 0.9-1.1:4.8-5.2:0.15-0.25:0.9-1.

1. After 20-40 min, measure the absorbance at 652 nm, and then calculate the glucose content according to the standard curve.

10. Use of the single-atom catalyst mediated by a protein molecule according to claim 1 or the single-atom catalyst mediated by a protein molecule obtained by the preparation method according to any one of claims 2 to 6 in the preparation of anticancer drugs, characterized in that: The protein-mediated single-atom catalyst is encapsulated within the pH-responsive ZIF-8 framework.

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