Preparation method and application of horseradish peroxidase-gold platinum nano-cluster

Abstract

The invention relates to the technical field of synthesis of gold-platinum nano-clusters, in particular to a preparation method and application of horseradish peroxidase-gold-platinum nano-clusters (HRP-AuPtNCs), and the preparation method comprises the following specific steps: preparing a horseradish peroxidase-gold-platinum nano-cluster stock solution under a specific pH condition by taking horseradish peroxidase as a protective agent and a reducing agent; under the excitation wavelength of 370 nm, the gold-platinum nano-cluster presents red fluorescence, and two maximum emission peaks exist at the positions of 480 nm and 655 nm; the gold-platinum nano-cluster can be used as an environment-friendly ratiometric fluorescent probe for quantitatively detecting folic acid in a solution. The prepared horseradish peroxidase-gold platinum nano-cluster is stable in optical property, simple and green in preparation process, good in biocompatibility, capable of rapidly and quantitatively detecting folic acid (FA) in a solution, high in detection sensitivity and small in environmental influence, and is an ideal fluorescent nano-material applied to the fields of biology and medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of gold-platinum nanocluster synthesis, and particularly relates to a preparation method and application of horseradish peroxidase-gold-platinum nanoclusters. Background Art

[0002] Horseradish peroxidase (HRP), as a labeling enzyme, is widely used in enzyme-linked immunosorbent assay. It can catalyze the substrate to be converted into a colored product, and its signal change is measured by absorption spectroscopy. With the rapid development of fluorescent materials, using fluorescence instead of absorbance to improve sensitivity, cost-effectiveness and flexibility has attracted much attention. HRP is widely distributed in the plant kingdom and is a glycoprotein composed of a colorless enzyme protein and a brown iron porphyrin. It has high activity, stability and is easy to prepare, and is an ideal ligand material for preparing nanomaterials.

[0003] Metal nanoclusters (MNCs) are a kind of (sub)nanomaterials composed of dozens of noble metal atoms, with excellent electrical, optical, catalytic and chemical properties. Due to the small size and good electron conductivity of MNCs, they can be inserted into enzyme or protein molecules as relay units for promoting electron transfer. Their remarkable feature is the strong photoluminescence property, easy synthesis, good quantum yield, adjustable fluorescence emission, large Stokes shift, high photostability, etc. Traditionally, MNCs can be passivated by a monolayer of organic ligands to produce high fluorescence intensity. The latest research results show that water-soluble MNCs with adjustable fluorescence emission are easily synthesized in various biocompatible scaffolds, making them a new biocompatible fluorescent group with broad application prospects in biological labeling and chemical / biological sensors.

[0004] Folic acid (FA) is a water-soluble vitamin that widely exists in most animal and plant products. FA plays an important role in the human body, participating in the transport and enzymatic processing of nucleic acids, amino acids, hemoglobin and methyl compounds. In addition, FA is crucial for cell growth and reproduction, as well as the formation and maturation of red blood cells. Some diseases are caused by FA deficiency, such as megaloblastic anemia, fetal neural tube defects, cardiovascular diseases and cancer. In addition, regular use of FA before and during pregnancy can reduce spinal, cranial and brain malformations caused by neural tube defects. FA is an important part of human health. Therefore, it is crucial to develop a sensitive, rapid, simple and reliable method for quantitative analysis of FA. Summary of the Invention

[0005] The object of the present invention is to solve the disadvantages existing in the prior art, and a preparation method of horseradish peroxidase-gold-platinum nanoclusters and their application in folic acid detection are proposed. Using horseradish peroxidase as a ligand molecule, horseradish peroxidase-gold-platinum nanoclusters are prepared in an aqueous solution by a one-step synthesis method. The product has unique photophysical properties, low toxicity and excellent biocompatibility, can rapidly and quantitatively detect folic acid, and has good detection sensitivity.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation method of horseradish peroxidase-gold-platinum nanoclusters, the specific steps are as follows:

[0008] Step S1: Weigh a certain amount of horseradish peroxidase powder, add 100 μL of water to dissolve it, and then add an appropriate proportion of chloroauric acid solution and chloroplatinic acid solution, and stir vigorously at room temperature for 10 min to obtain solution A;

[0009] Step S2: Add a 1 M NaOH solution to the solution A obtained in step S1, and heat the mixed solution in a water bath for 2 - 14 hours to obtain solution B;

[0010] Step S3: Filter the solution B obtained in step S2 with a 0.45 μm filter membrane, transfer the filtered solution to a dialysis bag and dialyze it in a phosphate buffer solution for 24 hours, and store the product at 4 °C to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0011] Preferably, in step S1, the concentration of horseradish peroxidase is 20 mg mL -1 , and the ratio of the chloroauric acid solution to the chloroplatinic acid solution is 3:1.

[0012] Preferably, in step S2, NaOH adjusts the solution pH to 13, the water bath temperature is 37 °C, and the reaction time is 12 hours.

[0013] The present invention also provides an application of horseradish peroxidase-gold-platinum nanoclusters obtained by the above preparation method in folic acid detection. Dilute the horseradish peroxidase-gold-platinum nanoclusters with deionized water, add different concentrations of folic acid and mix evenly, and incubate at room temperature. Under the excitation wavelength condition, as the folic acid concentration gradually increases, the fluorescence intensity of the horseradish peroxidase-gold-platinum nanoclusters at 480 nm gradually increases, while the fluorescence intensity at 655 nm remains unchanged, realizing ratio detection.

[0014] Preferably, incubate at room temperature for 1 minute, and under the excitation wavelength condition of 370 nm, ratio detection is realized.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. The present invention uses horseradish peroxidase as a ligand molecule and prepares horseradish peroxidase-gold-platinum nanoclusters in an aqueous solution by a one-step synthesis method. The product has unique photophysical properties, low toxicity, good stability and excellent biocompatibility.

[0017] 2. The horseradish peroxidase-gold-platinum nanoclusters prepared by the present invention have a fast and simple method for detecting folic acid and good detection sensitivity, and are ideal fluorescent nanomaterials applied to the biological and medical fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the excitation spectrum and emission spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters of the present invention;

[0019] Figure 2 It is the fluorescence spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters synthesized at different reaction times of the present invention;

[0020] Figure 3 It is the fluorescence spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters synthesized at different gold-platinum ratios of the present invention;

[0021] Figure 4 It is the fluorescence spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters synthesized at different temperatures of the present invention;

[0022] Figure 5 It is the fluorescence spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters synthesized at different horseradish peroxidase concentrations of the present invention;

[0023] Figure 6 It is the fluorescence spectrum diagram of the horseradish peroxidase-gold-platinum nanoclusters synthesized at different pH values of the present invention

[0024] Figure 7 It is the fluorescence emission spectrum diagram of the horseradish peroxidase-gold-platinum nanocluster solution with different concentrations of folic acid added in the present invention;

[0025] Figure 8 It is the linear relationship diagram between the folic acid concentration and the fluorescence intensity of the horseradish peroxidase-gold-platinum nanoclusters in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings, so that those skilled in the art can better understand the advantages and features of the present invention, and thus make a clearer definition of the protection scope of the present invention. The embodiments described in the present invention are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work belong to the protection scope of the present invention.

[0027] Refer toFigures 1 - 8 , to improve the luminescence performance of the product, the following steps are included:

[0028] Weigh a certain amount of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid and 50 μL of chloroplatinic acid, and stir vigorously for 10 min; mix evenly with 1 M sodium hydroxide solution, adjust the pH of the solution, and heat it in a water bath for 1 hour; use a fluorescence spectrometer to detect the emission spectrum of the product. Prolong the reaction time and observe the fluorescence spectra of the product at different reaction times. As the reaction time increases, the fluorescence intensity gradually increases and reaches the strongest when the reaction time is 12 hours; therefore, 12 hours is selected as the optimal reaction time for preparing horseradish peroxidase-gold-platinum nanoclusters.

[0029] Weigh a certain amount of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid and 50 μL of chloroplatinic acid, and stir vigorously for 10 min; mix evenly with 1 M sodium hydroxide solution, adjust the pH of the solution, and heat it in a water bath for 12 hours. Use a fluorescence spectrometer to detect the emission spectrum of the product. Change the volume of chloroplatinic acid to adjust the concentration ratio of chloroauric acid to chloroplatinic acid, and observe the fluorescence spectrum. When the concentration ratio of chloroauric acid to chloroplatinic acid is 3:1, the fluorescence intensity is the strongest; therefore, the concentration ratio of chloroauric acid to chloroplatinic acid of 3:1 is selected as the optimal ratio for preparing horseradish peroxidase-gold-platinum nanoclusters.

[0030] Weigh a certain amount of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid and 33.3 μL of chloroplatinic acid, and stir vigorously for 10 min; mix evenly with 1 M sodium hydroxide solution, adjust the pH of the solution, and heat it in a 25 °C water bath for 12 hours. Use a fluorescence spectrometer to detect the emission spectrum of the product. Increase the water bath temperature and observe the fluorescence spectrum. As the water bath temperature increases, the fluorescence intensity gradually increases. When the reaction temperature is 37 °C, the fluorescence intensity is the best; therefore, 37 °C is selected as the optimal temperature for preparing horseradish peroxidase-gold-platinum nanoclusters.

[0031] Weigh 0.5 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid and 33.3 μL of chloroplatinic acid, and stir vigorously for 10 min; mix evenly with 1 M sodium hydroxide solution, adjust the pH of the solution, and heat it in a 37 °C water bath for 12 h. Use a fluorescence spectrometer to detect the emission spectrum of the product. Increase the concentration of horseradish peroxidase and observe the fluorescence spectrum. As the concentration of horseradish peroxidase increases, the fluorescence intensity gradually increases. When using 20 mg mL -1 of horseradish peroxidase, the fluorescence intensity is the strongest; therefore, 20 mg mL -1 of horseradish peroxidase is selected as the optimal concentration for preparing horseradish peroxidase-gold-platinum nanoclusters.

[0032] Weigh 2 mg of horseradish peroxidase and dissolve it in 100 μL of water (final concentration is 20 mg / mL -1 ), then add 100 μL of chloroauric acid and 33.3 μL of chloroplatinic acid, and stir vigorously for 10 min; mix evenly with 1 M sodium hydroxide solution, adjust the pH of the solution to 9, heat it in a water bath at 37 °C for 12 h, and detect the emission spectrum of the product using a fluorescence spectrometer. Adjust the volume of sodium hydroxide to change the pH of the solution, observe the fluorescence spectrum, and as the pH increases, the fluorescence intensity gradually increases. When the pH of the solution is 13, the fluorescence intensity reaches the strongest; therefore, select pH = 13 as the optimal pH for the preparation of horseradish peroxidase-gold-platinum nanoclusters.

[0033] Using horseradish peroxidase-gold-platinum nanoclusters as a fluorescence probe to detect folic acid:

[0034] Dilute horseradish peroxidase-gold-platinum nanoclusters with deionized water, add a certain amount of folic acid, incubate the mixture at room temperature for 1 minute. Under the excitation wavelength of 370 nm, as the concentration of folic acid gradually increases, the fluorescence intensity of horseradish peroxidase-gold-platinum nanoclusters gradually increases.

[0035] (I) Preparation and optimization of horseradish peroxidase-gold-platinum nanoclusters

[0036] Example 1: Weigh 2 mg of horseradish peroxidase powder, dissolve it in 100 μL of water, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0037] Example 2: Weigh 2 mg of horseradish peroxidase powder, dissolve it in 100 μL of water, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 2 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0038] Example 3: Weigh 2 mg of horseradish peroxidase powder, dissolve it in 100 μL of water, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 4 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0039] Example 4: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM. Stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 6 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0040] Example 5: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM. Stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 8 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0041] Example 6: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM. Stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 10 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0042] Example 7: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM. Stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0043] Example 8: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM. Stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat it in a water bath at 37 °C for 14 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0044] Example 9: For the horseradish peroxidase-gold-platinum nanoclusters obtained in Example 1, Example 2, Example 3, Example 4, Example 5, Example 6, Example 7, and Example 8, as Figure 2 shown, at an excitation wavelength of 370 nm, by comparing their respective fluorescence emission intensities, it was determined that when the reaction time was 12 hours, the fluorescence emission intensity was the strongest.

[0045] Example 10: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 20 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with a 1 M sodium hydroxide solution, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0046] Example 11: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, add 100 μL of chloroauric acid with a concentration of 1.5 mM and 50 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, then add a 1 M sodium hydroxide solution and mix evenly, adjust the pH of the solution to 13, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0047] Example 12: For the horseradish peroxidase-gold-platinum nanoclusters obtained in Example 1, Example 10, and Example 11, as Figure 3 shown, at an excitation wavelength of 370 nm, by comparing their respective fluorescence emission intensities, it is determined that when the concentration ratio of chloroauric acid to chloroplatinic acid is 3:1, the fluorescence emission intensity is the strongest.

[0048] Example 13: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with a 1 M sodium hydroxide solution, and heat in a water bath at 50 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0049] Example 14: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with a 1 M sodium hydroxide solution, and heat in a water bath at 25 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0050] Example 15: For the ovalbumin-gold-platinum nanoclusters obtained in Example 1, Example 13, and Example 14, as Figure 4 shown, at an excitation wavelength of 370 nm, by comparing their respective fluorescence emission intensities, it is determined that when the reaction temperature is 37 °C, the fluorescence emission intensity is the strongest.

[0051] Example 16: Weigh 1 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0052] Example 17: Weigh 0.5 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 13 with 1 M sodium hydroxide solution, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0053] Example 18: For the horseradish peroxidase-gold-platinum nanoclusters obtained in Example 1, Example 16, and Example 17, as Figure 5 shown, at an excitation wavelength of 370 nm, by comparing their respective fluorescence emission intensities, it was determined that when the concentration of horseradish peroxidase was 20 mg mL -1 the fluorescence emission intensity was the strongest.

[0054] Example 19: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 11 with 1 M sodium hydroxide solution, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0055] Example 20: Weigh 2 mg of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add 100 μL of chloroauric acid with a concentration of 1.5 mM and 33.3 μL of chloroplatinic acid with a concentration of 1.5 mM, stir vigorously for 10 min, adjust the pH of the solution to 9 with 1 M sodium hydroxide solution, and heat in a water bath at 37 °C for 12 hours to obtain horseradish peroxidase-gold-platinum nanoclusters.

[0056] Example 21: For the horseradish peroxidase-gold-platinum nanoclusters obtained in Example 1, Example 19, and Example 20, as Figure 6 shown, at an excitation wavelength of 370 nm, by comparing their respective fluorescence emission intensities, it was determined that when the pH was 13, the fluorescence emission intensity was the strongest.

[0057] (2) Detection of folic acid using horseradish peroxidase-gold-platinum nanoclusters as a fluorescence probe

[0058] Example 22: Dilute the horseradish peroxidase-gold-platinum nanocluster stock solution with deionized water, then add a folic acid solution with a final concentration of 10 - 90 μM. After incubating at room temperature for 1 minute, detect the fluorescence spectrum at an excitation wavelength of 370 nm using a fluorescence spectrometer. As Figure 7 , 8 shown, at an excitation wavelength of 370 nm, the fluorescence intensity at 480 nm gradually increases, while the fluorescence intensity at 655 nm remains unchanged. The change in fluorescence intensity at 480 nm shows a linear relationship with the folic acid concentration.

[0059] As can be seen from the above, at an excitation wavelength of 370 nm (Ex1), the horseradish peroxidase-gold-platinum nanoclusters exhibit red fluorescence with fluorescence emission peaks at 480 nm (Em1) and 655 nm (Em2); it can be used as an effective environmentally friendly ratiometric fluorescence probe for the quantitative detection of folic acid in solution.

[0060] In summary, the present invention uses horseradish peroxidase as a ligand molecule and prepares horseradish peroxidase-gold-platinum nanoclusters in aqueous solution by a one-step synthesis method. The product has unique photophysical properties, low toxicity, and excellent biocompatibility, can rapidly and quantitatively detect folic acid with good detection sensitivity.

[0061] The descriptions and practices disclosed in the present invention are easy to think about and understand for those of ordinary skill in the art. Without departing from the principles of the present invention, several improvements and refinements can also be made. Therefore, the modifications or improvements made without deviating from the spirit of the present invention should also be regarded as within the protection scope of the present invention.

Claims

1. A method for preparing horseradish peroxidase-gold platinum nanoclusters, characterized in that: The specific steps are as follows: Step S1: Weigh a certain mass of horseradish peroxidase powder, add 100 μL of water to dissolve it, then add chloroauric acid and chloroplatinic acid solutions with appropriate concentrations, and stir vigorously for 10 min to obtain solution A; Step S2: Add 1M NaOH solution to solution A obtained in step S1, heat the mixed solution in a water bath for 2 - 14 hours to obtain solution B; Step S3: Filter the solution B obtained in step S2 through a 0.45 μm filter membrane, transfer the filtered solution to a dialysis bag and dialyze it in a phosphate buffer for 24 hours, and store the product at 4 °C to obtain horseradish peroxidase-gold-platinum nanoclusters.

2. The preparation method of horseradish peroxidase-gold-platinum nanoclusters according to claim 1, characterized in that, In the step S1, the concentration of horseradish peroxidase is 20 mg / mL -1 , and the concentration ratio of chloroauric acid to chloroplatinic acid is 3:

1.

3. The preparation method of horseradish peroxidase-gold-platinum nanoclusters according to claim 1, characterized in that, In step S2, sodium hydroxide adjusts the solution pH to 13, the heating temperature is 37 °C, and the reaction time is 12 hours.

4. Use of the horseradish peroxidase-gold-platinum nanoclusters obtained by the preparation method according to any one of claims 1-3 in the quantitative detection of folic acid, characterized in that, Dilute the horseradish peroxidase-gold-platinum nanoclusters with deionized water, add folic acid with different concentrations and mix evenly, and incubate at room temperature. Under the excitation wavelength condition, as the folic acid concentration gradually increases, the fluorescence intensity of the horseradish peroxidase-gold-platinum nanoclusters at 480 nm gradually increases, while the fluorescence intensity at 655 nm remains unchanged, realizing ratio detection.

5. The application according to claim 4, wherein Incubate for 1 minute at room temperature, and ratio detection can be achieved under the excitation wavelength condition of 370 nm.

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