A platinum glutamate complex and its preparation method and application

Abstract

The present invention discloses a glutamate platinum complex, a preparation method thereof, and an application thereof. The structural formula of the glutamate platinum complex is shown in formula (1). A glutamic acid solution and a potassium chloroplatinite solution are mixed and reacted for 1 to 2 hours to obtain a glutamate platinum complex having peroxidase mimicking activity. The glutamate platinum complex is used as a mimic enzyme in detecting the content of alkaline phosphatase, in detecting the content of cholesterol, and in cascading with a natural enzyme. The method of the present invention is simple, low in cost, and highly sensitive, capable of achieving rapid and accurate detection, and has very important reference value for the detection of other biological macromolecules and diseases, providing new opportunities for the development of future functionally enhanced biosensors and biorecognition systems.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical biosensing and biological detection, and particularly relates to a glutamate platinum complex and a preparation method and application thereof. Technical Field

[0003] Peroxidase, as one of the most effective biocatalysts, has high catalytic activity and substrate specificity, and plays a vital role in industry, agriculture, medicine and biology. However, the inherent defects of natural enzymes, such as high preparation and purification costs, low operational stability, high sensitivity to the environment, and difficulties in recycling and reuse, limit their further application. Based on this, the rapid exploration and development of various artificial enzyme mimics have been promoted, which have lower costs, higher durability and better physicochemical stability against harsh environments than natural enzymes. Including nanomaterials, metal oxides, precious metals and metal-organic frameworks, porphyrins, biomolecules, metal complexes and polymers, many small molecules and ions have also been found to have peroxidase mimic activity, which provides new and promising materials for the research related to peroxidase mimics.

[0004] Alkaline phosphatase (ALP) is a homodimeric protease that mediates numerous biological processes, including molecular transport, metabolism, and gene expression. ALP has been recognized as a potential target for various diseases, with its levels associated with disorders of the liver, prostate, bone tissue, and endocrine system. Abnormal ALP levels are intertwined with certain conditions, such as breast and prostate cancer, bone disease, hepatobiliary disease, bone damage, dental inflammation, and liver dysfunction. Medically, it is considered a key biomarker for numerous conditions. Therefore, monitoring ALP levels is crucial for better diagnosis and treatment of these conditions.

[0005] Cholesterol is an essential lipid for humans. It is a structural unit of the hormone system, the sole precursor of steroid hormones, and is involved in the synthesis of many vitamins. High cholesterol levels in serum are associated with numerous diseases, including coronary heart disease, cardiovascular disease, hypertension, myocardial infarction, and cancer. However, low cholesterol levels can lead to hypocholesterolemia, which may lead to hemorrhagic stroke. Therefore, it is extremely important to develop a simple, inexpensive, and reliable method for measuring cholesterol in serum samples.

[0006] Reducing small molecules, including antioxidants (ascorbic acid, tartaric acid, and tannic acid), biothiols (GSH, Cys, and Hcy), and dopamine, can inhibit the oxidation reaction catalyzed by peroxidase mimetic enzymes, resulting in signal attenuation and thus achieving quantitative detection.

[0007] Hydrogen peroxide is a product of numerous enzymatic reactions. Cholesterol, glucose, uric acid, and lactic acid, among other substances, undergo oxidation reactions under the action of cholesterol oxidase, glucose oxidase, urate oxidase, and lactate oxidase, respectively, to produce hydrogen peroxide. When oxidases are combined with peroxidase mimics, the hydrogen peroxide content can be used to indirectly detect the relevant oxidase substrates.

[0008] Our experiments demonstrate that platinum glutamate complexes possess a certain degree of catalytic activity. In the presence of platinum glutamate complexes, H₂O₂ is rapidly decomposed, and hydrogen peroxide is catalytically decomposed to produce hydroxyl radicals. These hydroxyl radicals oxidize TMB (3,3',5,5'-tetramethylbenzidine), with different concentrations of oxidized TMB exhibiting different colors. Based on this, we developed a dual-mode detection method that allows for both visual semi-quantitative detection of the analyte using color changes and quantitative determination of the analyte's exact content through absorbance. Our experiments compared the effects of small-molecule platinum glutamate complexes with those of large-molecule nanozymes on the activity of natural enzymes, demonstrating that small-molecule platinum glutamate complexes have minimal interference with natural enzyme activity and surpass nanozymes in cascade reactions. Summary of the Invention

[0009] The first object of the present invention is to provide a glutamate platinum complex, the structural formula of the glutamate platinum complex is shown in formula (1):

[0010]

[0011] A second object of the present invention is to provide a method for preparing the above-mentioned glutamate platinum complex, which comprises the following steps: mixing a glutamic acid solution and a potassium chloroplatinite solution and reacting them for 2 hours to obtain a glutamate platinum complex having peroxidase mimicking activity.

[0012] Furthermore, the pH value of the glutamic acid solution is first adjusted to neutral, and then the glutamic acid solution and the potassium chloroplatinite solution are sequentially added to the round-bottom flask in a volume ratio of 1:1; wherein the concentration of the glutamic acid solution is 0.02 mol / L, and the concentration of the potassium chloroplatinite solution is 0.01 mol / L, and the reaction is stirred at 70°C for 2 hours.

[0013] The third object of the present invention is to provide an application of the glutamate platinum complex obtained by the preparation method of the glutamate platinum complex, that is, the application of the glutamate platinum complex as a mimetic enzyme in detecting the content of alkaline phosphatase:

[0014] First, different concentrations of alkaline phosphatase, L-ascorbic acid-2-phosphate, and Tris-HCl buffer solution are mixed for one hour. Then, glutamate platinum complex, hydrogen peroxide, 3,3',5,5'-tetramethylbenzidine (TMB) solution, and acetic acid buffer solution are added to the system. Different concentrations of alkaline phosphatase decompose L-ascorbic acid-2-phosphate to produce different concentrations of ascorbic acid, which inhibits color development to different degrees, that is, the generated Ox-TMB exhibits different colors. Then, based on the relationship between the absorbance value of Ox-TMB at a wavelength of 652 nm and the alkaline phosphatase concentration, the alkaline phosphatase content in the test sample is calculated.

[0015] Furthermore, the alkaline phosphatase, L-ascorbic acid-2-phosphate, and Tris-HCl buffer solution were added in a volume ratio of 1:1:1, wherein the concentration of the added L-ascorbic acid-2-phosphate was 0.002 mol / L, the concentration of the added Tris-HCl buffer solution was 0.02 mol / L, and the pH value was 8.0. The reaction was carried out in a 37°C water bath for 1 hour;

[0016] In the TMB color development step, the concentration of the added glutamate platinum complex was 0.0005 mol / L, the concentration of the acetate buffer solution was 0.2 mol / L, its pH value was 4.0, the concentration of hydrogen peroxide was 0.1 mol / L, the concentration of TMB was 0.004 mol / L, and the volume ratio of the four solutions was 1:4.8:0.2:1.

[0017] The fourth object of the present invention is to provide an application of the glutamate platinum complex obtained by the preparation method of the glutamate platinum complex, that is, an application of the glutamate platinum complex as a mimetic enzyme in detecting the content of cholesterol:

[0018] First, different concentrations of cholesterol, cholesterol oxidase and PBS buffer solution are mixed for 10 minutes, and then glutamate platinum complex, 3,3',5,5'-tetramethylbenzidine (TMB) solution and acetate buffer solution are added to the system. The different contents of hydrogen peroxide produced by cholesterol oxidase decomposing different concentrations of cholesterol result in different colors of Ox-TMB. Then, based on the relationship between the absorbance value of Ox-TMB at a wavelength of 652nm and the cholesterol concentration, the cholesterol content in the sample to be tested is calculated.

[0019] Furthermore, the cholesterol oxidase, cholesterol, and PBS buffer solution were added in a volume ratio of 1:1:1, wherein the concentration of the added cholesterol oxidase was 2 U / mL, the concentration of the PBS buffer solution was 0.01 mol / L, and the pH value was 7.4. The reaction was carried out in a 37°C water bath for 10 minutes;

[0020] In the TMB color development step, the concentration of the added glutamate platinum complex was 0.0005 mol / L, the concentration of the acetate buffer solution was 0.2 mol / L, its pH value was 4.0, the concentration of the TMB solution was 0.02 mol / L, and the volume ratio of the three solutions was 0.5:5.9:0.6.

[0021] The beneficial effects of the present invention are as follows:

[0022] The present invention utilizes the property of a glutamate platinum complex to catalyze the decomposition of hydrogen peroxide, and based on the fact that ascorbic acid can inhibit visual detection and alkaline phosphatase can decompose L-ascorbic acid-2-phosphate to produce ascorbic acid, thereby achieving the purpose of detecting alkaline phosphatase. The present invention also utilizes the property of a glutamate platinum complex to catalyze the decomposition of hydrogen peroxide, and based on the fact that cholesterol oxidase decomposes cholesterol to produce hydrogen peroxide, thereby achieving the purpose of detecting cholesterol. The present invention uses the glutamate platinum complex to detect alkaline phosphatase and cholesterol, etc., has high sensitivity and is simple to operate, and can accurately measure the concentrations of alkaline phosphatase and cholesterol.

[0023] The present invention is a highly sensitive technique for detecting alkaline phosphatase and cholesterol, enabling rapid and accurate detection of these enzymes. This allows platinum glutamate complexes, known as enzyme mimics, to successfully replace traditional enzymes in enzyme-linked immunosorbent assays (ELISAs). Platinum glutamate complexes offer unparalleled advantages over macromolecular enzymes in terms of price and stability. More importantly, they avoid the problem of proteases losing their biological activity. Furthermore, by introducing TMB, which displays different colors under different states, a visual assay is established, resulting in high sensitivity and stability.

[0024] The fifth object of the present invention is to provide the application of the glutamate platinum complex obtained by the preparation method of the glutamate platinum complex, that is, the application of the glutamate platinum complex as a mimetic enzyme in a cascade with a natural enzyme:

[0025] First, a glutamate platinum complex is activated with EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide). Then, a corresponding protease, such as glucose oxidase, is added to the solution and allowed to react at room temperature for 2 hours. The solution is then dialyzed using a dialysis bag to obtain a conjugate with dual enzyme activity. A certain amount of the conjugate is placed in a centrifuge tube, and a glucose solution and a TMB solution are added thereto. Glucose oxidase first catalyzes the decomposition of glucose to produce hydrogen peroxide, which is then decomposed by the glutamate platinum complex to produce hydroxyl radicals, which oxidize the colorless TMB to blue Ox-TMB, indicating that the conjugate has dual enzyme activity.

[0026] Furthermore, the EDC, NHS, glutamate platinum complex and glucose oxidase were added in a volume ratio of 0.4:0.1:1:1, wherein the added EDC concentration was 5 mg / mL, the NHS concentration was 5 mg / mL, the glutamate platinum complex concentration was 0.001 mol / L, and the glucose oxidase concentration was 2 mg / mL; the EDC and NHS activated glutamate platinum complex was incubated in a 37°C water bath for 30 minutes, the activated glutamate platinum complex was reacted with glucose oxidase at room temperature for another 2 hours, and then dialyzed using a dialysis bag with a cutoff of 8000 Da;

[0027] In the TMB color development step, the concentration of the added TMB solution is 0.005 mol / L, the concentration of the glucose solution is 0.005 mol / L, and the conjugate is the original solution after dialysis. The volume ratio of the three is 1:1:1.

[0028] By treating with the above-mentioned method of the present invention, TMB solutions of different colors can be obtained. This color change can be regulated according to the concentration of hydrogen peroxide, and the detection of alkaline phosphatase and cholesterol is achieved through changes in color or absorbance. By treating with the method of the present invention, the effects of glutamate platinum complexes as small molecules and nanozymes as large molecules on the activity of natural enzymes are compared, showing that glutamate platinum complexes as small molecules have little interference with the activity of natural enzymes and surpass nanozymes in cascade reactions. Compared with the existing technology, the method of the present invention is simple, low-cost, highly sensitive, and can achieve rapid and accurate detection. It also has very important reference value for the detection of other biological macromolecules and diseases, providing new opportunities for the development of future enhanced biosensors and biorecognition systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the mass spectrum of the glutamate platinum complex prepared in the embodiment of the present invention.

[0030] Figure 2 The color development picture is used to verify that the glutamate platinum complex of the present invention has the property of peroxidase mimicking.

[0031] Figure 3 The UV-visible absorption spectrum is used to verify that the glutamate platinum complex of the present invention has the property of peroxidase mimicking.

[0032] Figure 4 The absorbance histogram of Ox-TMB at 652 nm is shown in Figure 2, which shows that the platinum glutamate complex of the present invention catalyzes hydrogen peroxide to produce hydroxyl radicals, causing TMB to be oxidized and turn blue under different pH conditions.

[0033] Figure 5 The diagram shows the UV-visible absorption spectrum and color change diagram when detecting alkaline phosphatase standard solution according to the embodiment of the present invention.

[0034] Figure 6 is the linear equation of the alkaline phosphatase concentration and the absorbance ΔA of oxidized TMB at 652 nm (ΔA=A0-A, A0 is the absorbance corresponding to the absence of alkaline phosphatase, and A is the absorbance corresponding to the addition of different concentrations of alkaline phosphatase).

[0035] Figure 7 The figures are the UV-visible absorption spectrum and color change diagram when detecting cholesterol standard solution according to the embodiment of the present invention.

[0036] Figure 8 is the linear equation of cholesterol concentration and the absorbance of oxidized TMB at 652 nm.

[0037] Figure 9 This is a color development picture showing that the glutamate platinum complex and glucose oxidase conjugate of the present invention has dual enzyme activity.

[0038] Figure 10 The UV-visible absorption spectra show the effects of different substances on the activity of glucose oxidase after coupling with glucose oxidase. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below with reference to experimental examples and accompanying drawings.

[0040] Example 1

[0041] Preparation of glutamate platinum complex mimic enzyme:

[0042] First, the pH value of the glutamic acid solution was adjusted to neutral, and then the glutamic acid solution and the potassium chloroplatinite solution were added to the round-bottom flask in sequence. The volume ratio of the glutamic acid solution and the potassium chloroplatinite solution was 1:1, wherein the concentration of the glutamic acid solution was 0.02 mol / L and the concentration of the potassium chloroplatinite solution was 0.01 mol / L. The two were vigorously stirred in a 70°C oil bath for about 2 hours, and the reaction was stopped. The reaction solution was subjected to multiple vacuum distillations to obtain a relatively pure product. The product was subjected to mass spectrometry detection, and its mass spectrum is shown as follows: Figure 1 Its structural formula is shown in formula (1):

[0043]

[0044] See also Figure 2 , indicating that the glutamate platinum complex prepared in this example can catalyze the oxidation of TMB with hydrogen peroxide to produce blue Ox-TMB. When the glutamate platinum complex, hydrogen peroxide, and TMB were added to the left centrifuge tube, the solution turned blue. When glutamate, hydrogen peroxide, and TMB were added to the middle centrifuge tube, almost no color was developed. When potassium chloroplatinite, hydrogen peroxide, and TMB were added to the right centrifuge tube, almost no color was developed. Figure 3, indicating that the glutamate platinum complex prepared in this example has a characteristic absorption peak at 652nm, while glutamate 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 the glutamate platinum complex has peroxidase mimicking properties and has the strongest catalytic activity under pH = 4 conditions, as shown in FIG. Figure 4 shown.

[0045] Example 2

[0046] Glutamate platinum complexes mimic enzyme properties for the detection of alkaline phosphatase:

[0047] First, add 100 μL alkaline phosphatase, 100 μL 0.002 mol / L L-ascorbic acid-2-phosphate, and then add 100 μL 0.02 mol / L Tris-HCl buffer solution (pH value of Tris-HCl buffer solution is 8.0) to the centrifuge tube, and react at 37°C for 60 minutes; then add 480 μL 0.2 mol / L acetate buffer solution (pH value of acetate buffer solution is 4.0), 100 μL 0.5 mmol / L glutamate platinum complex, 100 μL 0.004 mol / L TMB, and 20 μL 0.1 mol / L H2O2. After 20 minutes, the color of the solution will be as follows: Figure 5 shown. Figure 5 From left to right, 0, 0.5, 1, 2.5, 3, 4, 5, 6, 7.5, 8, 10, 12, 15, 18, 20, 30, and 50 U / L of alkaline phosphatase were added. Figure 5 It can be seen that the higher the alkaline phosphatase concentration, the lighter the TMB color. Based on the absorbance of the oxidized TMB at 652nm, the relationship between the calculated ΔA (ΔA=A0-A, A0 is the absorbance corresponding to the absence of alkaline phosphatase, A is the absorbance corresponding to the addition of different concentrations of alkaline phosphatase) and the alkaline phosphatase concentration in the range of 0-12U / L can obtain a standard curve with a good linear relationship, such as Figure 6 As shown, the content of alkaline phosphatase in the unknown sample can be calculated according to the linear equation.

[0048] Sample processing and recovery determination:

[0049] First, different human serum samples were diluted 50-fold. 50 μL of serum was taken and 50 μL of alkaline phosphatase standard of different concentrations was added, 100 μL of 0.002 mol / L L-ascorbic acid-2-phosphate solution, and 100 μL of 0.02 mol / L Tris-HCl buffer solution were added. The pH value of the Tris-HCl buffer solution was 8.0, and the reaction was carried out at 37°C for 60 minutes. Subsequently, 480 μL of acetate buffer solution (0.2 M, pH = 4.0), 100 μL of 0.5 mmol / L glutamate platinum complex, 100 μL of 0.004 mol / L TMB, and 20 μL of 0.1 mol / L H2O2 were added. After 20 minutes, the absorbance at 652 nm was measured. The concentration of alkaline phosphatase was determined based on the standard curve. Calculation showed that the recovery rate of alkaline phosphatase detection was between 97.8% and 104.7%, as shown in Table 1. This meets the requirements for quantitative analysis of actual biological samples and has high accuracy.

[0050] Table 1

[0051]

[0052] Example 3

[0053] Glutamate platinum complexes mimic enzyme properties for cholesterol detection:

[0054] First, add 100 μL of 2U / mL cholesterol oxidase and 100 μL of different concentrations of cholesterol into a centrifuge tube, then add 100 μL of 0.01mol / L PBS buffer solution (pH value of PBS buffer solution is 7.4), and react at 37°C for 10 minutes; then add 590 μL of 0.2mol / L acetate buffer solution (pH value of acetate buffer solution is 4.0), 50 μL of 0.0005mol / L glutamate platinum complex, and 60 μL of 0.02mol / L TMB, and after incubating in a 45°C water bath for 30 minutes, the color of the solution will be as follows: Figure 7 As shown, Figure 7 From left to right, 80, 70, 60, 50, 40, 30, 20, 10, 7.5, 5, 2.5, 1, 0.75, 0.5, 0.1, and 0 μmol / L of cholesterol were added. Figure 7 It can be seen that the higher the cholesterol concentration, the darker the color of oxidized TMB. According to the relationship between the absorbance of oxidized TMB at 652nm and the cholesterol concentration, a standard curve with a good linear relationship can be obtained, such as Figure 8 As shown, the cholesterol content in the unknown sample can be calculated based on the linear equation.

[0055] Sample processing and recovery determination:

[0056] First, different human serum samples were diluted 50-fold. 100 μL of serum was taken and 100 μL of cholesterol standards of varying concentrations was added to each sample. 100 μL of 2 U / mL cholesterol oxidase was also added, and the mixture was reacted at 37°C for 10 minutes. Subsequently, 590 μL of acetate buffer (0.2 M, pH 4.0), 50 μL of 0.5 mmol / L glutamate platinum complex, and 60 μL of 20 mmol / L TMB were added. After incubation at 45°C for 30 minutes, the absorbance at 652 nm was measured. The cholesterol concentration was calculated based on the standard curve. Calculations showed that the cholesterol recovery rate was between 100.1% and 104.0%, as shown in Table 2. This method meets the requirements for quantitative analysis of actual biological samples and demonstrates high accuracy.

[0057] Table 2

[0058]

[0059] Example 4

[0060] Glutamate platinum complex mimics the enzyme cascade with glucose oxidase:

[0061] First, 2 mL of 0.001 mol / L glutamate platinum complex was activated with 0.8 mL of 5 mg / mL EDC and 0.2 mL of 5 mg / mL NHS. The mixture was incubated in a 37°C water bath for 30 minutes. Then, 2 mL of 2 mg / mL glucose oxidase was added to the solution and the reaction was allowed to proceed at room temperature for 2 hours. The solution was then dialyzed using an 8000 Da cutoff dialysis bag to obtain a conjugate with dual enzyme activity. The conjugation procedures for gold nanoclusters (Au NCs) and gold nanoparticles (Au NPs) to glucose oxidase were similar to those described above.

[0062] Take 0.2mL of the conjugate into centrifuge tube a and centrifuge tube b respectively. Add 0.2mL of 0.005mol / L glucose solution and 0.2mL of 0.005mol / L TMB to centrifuge tube a. Add 0.2mL of double distilled water and 0.2mL of 0.005mol / L TMB to centrifuge tube b as a control. Glucose oxidase first catalyzes the decomposition of glucose to produce hydrogen peroxide. Then, the glutamate platinum complex can decompose hydrogen peroxide to produce hydroxyl radicals, which oxidize the colorless TMB to blue ox-TMB, indicating that the conjugate has dual enzyme activity. Figure 9 The test tube on the left is centrifuge tube a, and the test tube on the right is centrifuge tube b. In the presence of glucose, the solution in centrifuge tube a turns from colorless to blue, while in the absence of glucose, the solution in centrifuge tube b shows almost no color.

[0063] In addition, four centrifuge tubes a, b, c, and d were taken respectively, and 0.2 mL of glucose oxidase, 0.2 mL of 0.005 mol / L glucose, 0.2 mL of 0.005 mol / L TMB, and 0.1 mL of horseradish peroxidase were added to centrifuge tube a, and centrifuge tube a was used as the control group; 0.2 mL of the conjugate of glutamate platinum complex and glucose oxidase, 0.2 mL of 0.005 mol / L glucose, 0.2 mL of 0.005 mol / L TMB, and 0.1 mL of horseradish peroxidase were added to centrifuge tube b; 0.2 mL of the conjugate of gold nanoclusters and glucose oxidase, 0.2 mL of 0.005 mol / L glucose, 0.2 mL of 0.005 mol / L TMB, and 0.1 mL of horseradish peroxidase were added to centrifuge tube c; 0.2 mL of the conjugate of gold nanoparticles and glucose oxidase, 0.2 mL of 0.005 mol / L glucose, 0.2 mL of 0.005 mol / L TMB and 0.1 mL horseradish peroxidase were reacted at room temperature for half an hour, and the UV-visible absorption spectrum was measured. Figure 10 As shown, after the platinum glutamate complex is coupled to glucose oxidase, its characteristic absorption peak in the presence of natural peroxidase is higher than that of control group a, indicating that it has less effect on glucose oxidase activity. In contrast, the characteristic absorption peaks of gold nanoclusters and gold nanoparticles, as macromolecules, are lower than that of control group a after coupling with glucose oxidase, indicating that they have a greater impact on glucose oxidase activity. Compared with nanozymes, the small molecule platinum glutamate complex has less interference with the activity of the natural enzyme, reflecting its unique advantages.

Claims

1. A platinum glutamate complex, the structural formula of which is shown in formula (1):

2. A method for preparing the glutamate platinum complex as claimed in claim 1, characterized in that The method comprises the following steps: mixing a glutamic acid solution and a potassium chloroplatinite solution for reaction for 2 hours to obtain a glutamic acid platinum complex having peroxidase mimicking activity.

3. The method for preparing the platinum glutamate complex according to claim 2, wherein: First, the pH value of the glutamic acid solution is adjusted to neutral, and then the glutamic acid solution and the potassium chloroplatinite solution are added to the round-bottom flask in sequence with a volume ratio of 1:1; wherein the concentration of the glutamic acid solution is 0.02 mol / L, and the concentration of the potassium chloroplatinite solution is 0.01 mol / L, and the reaction is stirred at 70°C for 2 hours.

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