Preparation method and application of pepsin-platinum nanoclusters with peroxidase-like activity

Abstract

The present invention relates to the technical field of glucose detection materials, and more particularly to a method for preparing a pepsin-platinum nanocluster with peroxidase-like activity and an application thereof, including: using pepsin as a template, preparing a pepsin-platinum nanocluster under specific pH conditions, and having an optimal emission at 410nm under an excitation wavelength of 320nm. The platinum nanocluster prepared by the present invention has both peroxidase-like activity and fluorescence characteristics, and its peroxidase-like activity is used for colorimetric detection of glucose. At the same time, the platinum nanocluster can be used for quantitative detection of glucose in a concentration range of 1-10μM, and the detection limit is as low as 1.57nM.

Description

Technical Field

[0001] The invention relates to the technical field of glucose detection materials, and in particular to a preparation method and application of a pepsin-platinum nanocluster with peroxidase-like activity. Background Art

[0002] Glucose is the main component of plant and animal carbohydrates in biological systems and plays an important role in life processes. It is not only a source of energy for living cells, but also a metabolic intermediate for the synthesis of other complex molecules. In addition, the level of glucose in the blood is also one of the indicators for assessing human health. Abnormal changes in the glucose content in human urine or blood are diagnostic indicators for diabetes or hypoglycemia. Blood glucose concentration remains relatively stable due to the regulation of the nervous system and hormones. When these regulations lose their original balance, hyperglycemia or hypoglycemia occurs. Therefore, accurate and rapid determination of blood glucose content is of great significance in clinical diagnosis. In addition, accurate determination of glucose is also very important in food analysis.

[0003] The colorimetric determination of glucose has attracted widespread attention. Natural enzymes, especially peroxidases, are widely used in colorimetric methods and have efficient catalytic ability and excellent selectivity. However, natural enzymes have many defects, such as high extraction cost, easy deterioration, poor stability under harsh conditions, etc., which limit their application. In order to overcome these shortcomings, a variety of artificial synthetic materials with enzyme-mimicking activity have been developed, such as metal oxides, graphene, nanomaterials and metal-organic frameworks. Compared with natural enzymes, artificial enzyme mimics have the advantages of simple preparation, easy modification, low price, strong specificity and high sensitivity, which can overcome the shortcomings of natural enzymes such as high price, limited source, and susceptibility to inactivation due to environmental influences. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a preparation method and application of pepsin-platinum nanoclusters with peroxidase-like activity. The prepared platinum nanoclusters can be used for colorimetric sensing of glucose based on the peroxidase-like activity.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing pepsin-platinum nanoclusters with peroxidase-like activity, the specific steps are as follows:

[0007] Step 1: Weigh the concentration to 20-40 mg mL -1 280 μL of pepsin solid was added to dissolve it in water, 80 μL of 10 mM chloroplatinic acid solution was added to the pepsin solution, and the mixture was rapidly stirred for 5 minutes at room temperature to obtain solution A;

[0008] Step 2, adding a 1M sodium hydroxide solution to the solution A obtained in step 1, adjusting the pH to 9-13, mixing evenly to obtain a mixed solution, and heating in a water bath at 25-55° C. for 0-10 hours to obtain a solution B;

[0009] Step 3: The solution B obtained in step 2 was dialyzed in a phosphate buffer for 24 hours to remove unreacted raw materials, and the platinum nanoclusters were purified and stored at 4°C.

[0010] Preferably, in step 1, the concentration of pepsin is 35 mg mL -1 .

[0011] Preferably, in step 2, the pH is adjusted to 13, the reaction temperature is 50° C., and the reaction time is 8 h.

[0012] The present invention also provides an application of pepsin-platinum nanoclusters with peroxidase-like activity obtained by the above-mentioned preparation method in glucose detection.

[0013] Preferably, the detection is performed by using a simulated enzyme colorimetric method, and the detection method comprises the following steps:

[0014] Step 1, incubate different concentrations of glucose and glucose oxidase at 37°C for 20 minutes;

[0015] Step 2: Then add 50 μL of pep-PtNCs and an appropriate amount of 3,3',5,5'-tetramethylbenzidine TMB, place in NaAc-Hac buffer, adjust the pH to 4, dilute to 500 μL, mix thoroughly and place at room temperature for reaction for 30 min;

[0016] Step 3: Scan the spectrum with a UV-visible spectrophotometer and measure the absorbance at 652 nm (A 652 ) for quantification and visual colorimetric detection of glucose.

[0017] Preferably, the linear range of glucose detection is 1-10 μM, and the detection limit is 1.57 nM.

[0018] By adopting the above technical scheme: the prepared platinum nanoclusters have good peroxidase-like activity, and glucose is oxidized by glucose oxidase to produce H2O2. Based on the excellent peroxidase-like activity of platinum nanoclusters (pep-PtNCs), TMB is catalyzed and oxidized to turn blue in the presence of H2O2, and the TMB amino group loses an electron to become a cationic free radical and exists in the system in the form of a dimeric charge transfer complex. The dimer has a maximum absorption at 652nm. A quantitative relationship is established between the maximum absorption and the glucose concentration, thereby realizing the detection of glucose concentration.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The present invention uses pepsin as a template and adopts a one-step synthesis method to prepare pep-PtNCs, which has a simple preparation process and low price. Because its size is close to the Fermi wavelength of electrons, NCs have size-adjustable electronic transitions, show strong luminescence properties, and have excellent fluorescence and chemical stability, which can overcome the shortcomings of traditional organic dyes such as short fluorescence lifetime and easy photobleaching; and it has peroxidase-like activity and is used for catalytic colorimetric analysis.

[0021] 2. The present invention is based on the oxidation of glucose by glucose oxidase to generate H2O2. TMB reacts with H2O2 under the catalysis of the peroxidase-like enzyme of pep-PtNCs. The TMB amino group loses an electron and becomes a cationic free radical, which exists in the system as a dimeric charge transfer complex. The dimer has a maximum absorption at 652nm and is blue.

[0022] 3. The method constructed by the present invention has good determination specificity and high sensitivity, and the detection limit is as low as 1.57nM.

[0023] 4. The method constructed by the present invention can be used for the determination of glucose in serum without complicated sample pretreatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The excitation spectrum and emission spectrum of pep-PtNCs of the present invention are shown;

[0025] Figure 2 is the ultraviolet absorption spectrum of pepsin and pep-PtNCs in the present invention;

[0026] Figure 3 The UV absorption spectra and photos of H2O2 on pep-PtNCs / TMB and control groups (TMB / H2O2, TMB / pep-PtNCs) in the present invention;

[0027] Figure 4 (a) UV spectrum of the reaction with the pep-PtNCs / TMB system after incubation with 0-10 mM glucose and glucose oxidase; daylight photograph of the pep-PtNCs / TMB system; (b) the effect curve of glucose concentration on the UV absorption of the pep-PtNCs / TMB system at 652 nm, and the linear fitting curve of the absorbance of the system at 652 nm for 0-10 μM glucose. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings, so that those skilled in the art can better understand the advantages and features of the present invention, thereby making 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, not all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work are within the scope of protection of the present invention.

[0029] Embodiment 1:

[0030] A method for preparing pepsin-platinum nanoclusters with peroxidase-like activity, the specific steps are as follows:

[0031] First, the protein concentration was 35 mg mL -1 The pepsin was completely dissolved in 280 μL of water, 80 μL of 10 mM H2PtCl6 was added, the two were mixed, and stirred vigorously for 5 min. Then, 10 μL of 1 M NaOH was added, the pH was adjusted to 13, and heated in a water bath at 50 °C for 8 h to obtain platinum nanoclusters (pep-PtNCs).

[0032] The results show that the excitation wavelength of the pep-PtNCs obtained in Example 1 is 320 nm and the emission wavelength is 410 nm ( Figure 1 ), the fluorescence spectrum emission position was used to preliminarily determine the synthesis of pep-PtNCs. Secondly, the nanocluster has no ultraviolet absorption peak in the range of 400-800nm ​​( Figure 2 ), indicating that the product particle size is small, thus proving that pep-PtNCs have been successfully synthesized.

[0033] Embodiment 2:

[0034] In order to prove that pep-PtNCs have excellent peroxidase-like activity, 50 μL of pep-PtNCs, 5 mM TMB and 100 mM H2O2 were mixed with 350 μL HAc-NaAc buffer and incubated for 30 min. The absorption peak at 652 nm was detected by UV-visible absorption spectroscopy to characterize the peroxidase-like activity of pep-PtNCs.

[0035] The results show that: Figure 3As shown: in the absence of pep-PtNCs catalysis, the system with only TMB and H2O2 has almost no ultraviolet absorption peak at 652nm; in the absence of H2O2, the system with only pep-PtNCs and TMB has a very weak ultraviolet absorption peak at 652nm; in the presence of H2O2 and pep-PtNCs at the same time, they can react with TMB, making the system show an obvious absorption peak at 652nm, and the color of the solution turns dark blue, indicating that in the presence of H2O2, pep-PtNCs / H2O2 can significantly promote the oxidation process of TMB and has excellent peroxidase-like activity.

[0036] Embodiment 3:

[0037] The peroxidase-like activity of pep-PtNCs was used to detect glucose. The specific operation was as follows: different concentrations of glucose were incubated with glucose oxidase at 37°C for 20 minutes, and then 50 μL of pep-PtNCs and an appropriate amount of TMB were added to NaAc-HAc buffer (pH 4), the volume was fixed to 500 μL, and the mixture was fully mixed and allowed to react for 30 minutes at room temperature. The absorption value at 652 nm was measured in a UV-visible spectrophotometer. Substituting the absorption value into the established absorption value concentration curve, the glucose concentration and A 652 The linear relationship between the color of glucose and the concentration of glucose was recorded. Glucose was catalyzed by glucose oxidase to generate H2O2. Under the catalysis of pep-PtNCs, H2O2 produced hydroxyl radicals and then oxidized the colorless TMB to blue TMB-OX.

[0038] The results are as follows Figure 4 As shown, with the increase of glucose concentration, the absorbance detected by the UV-visible spectrophotometer at 652 nm became higher, and the two showed a good linear relationship in the range of 0-10 μM, with a linear correlation coefficient of 0.98.

[0039] Embodiment 4:

[0040] The pep-PtNCs / TMB system constructed by the present invention was used to determine glucose in serum to explore the practicality of the system in a physiological environment. Blood samples were collected from adult male Sprague-Dawley rats (200-250 g), diluted 10 times with PBS, and then a specific concentration of glucose was added to the serum as a spiked sample. First, a sufficient amount of glucose oxidase was used to incubate with glucose at 37°C for 20 minutes, and then pep-PtNCs and an appropriate amount of TMB were added and mixed thoroughly at room temperature for 30 minutes. The absorbance at 652 nm was detected by an ultraviolet spectrophotometer, and the recovery rate and relative standard deviation of the detection were calculated based on the standard curve obtained above.

[0041] The results are shown in Table 1. The relative standard deviation of the sample detection in serum is 3.10%-5.80%, and the recovery rate is 98.75%-104.33%. The relative standard deviation is small and the recovery rate is good, indicating that the system has good practical applicability and can be used for the detection of glucose in actual serum samples.

[0042] Table 1 shows the parameters for detecting glucose in serum

[0043]

[0044] In summary, the platinum nanoclusters prepared in the present invention have good peroxidase-like activity, and produce H2O2 by catalyzing the oxidation of glucose through glucose oxidase. Based on the excellent peroxidase-like activity of platinum nanoclusters (pep-PtNCs), TMB is catalyzed and oxidized to turn blue in the presence of H2O2, and the TMB amino group loses an electron to become a cationic free radical and exists in the system in the form of a dimeric charge transfer complex. The dimer has a maximum absorption at 652nm. A quantitative relationship is established between the maximum absorption and the glucose concentration, thereby realizing the detection of the glucose concentration.

[0045] The description and practice disclosed in the present invention are easy to think and understand for ordinary technicians in the technical field, and several improvements and modifications can be made without departing from the principles of the present invention. Therefore, modifications or improvements made without departing from the spirit of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing pepsin-platinum nanoclusters having peroxidase-like activity, characterized in that: The specific steps are as follows: Step 1: Weigh the concentration to 20~40 mg·mL -1 280 μL of pepsin solution was added to dissolve it in water, 80 μL of 10 mM chloroplatinic acid solution was added to the pepsin solution, and the mixture was rapidly stirred for 5 minutes at room temperature to obtain solution A; Step 2, adding a 1 M sodium hydroxide solution to the solution A obtained in step 1, adjusting the pH to 9-13, mixing evenly to obtain a mixed solution, and heating in a water bath at 25-55° C. for 0-10 hours to obtain a solution B; Step 3: The solution B obtained in step 2 was dialyzed in a phosphate buffer for 24 hours to remove unreacted raw materials, and the platinum nanoclusters were purified and stored at 4°C.

2. The method for preparing pepsin-platinum nanoclusters having peroxidase-like activity according to claim 1, characterized in that: In step 1, the concentration of pepsin was 35 mg mL -1 .

3. The method for preparing pepsin-platinum nanoclusters having peroxidase-like activity according to claim 1, characterized in that: In step 2, the pH was adjusted to 13, the reaction temperature was 50 °C, and the reaction time was 8 h.

4. An application of pepsin-platinum nanoclusters having peroxidase-like activity obtained by the preparation method according to any one of claims 1 to 3 in glucose detection, characterized in that: The application is a non-disease diagnosis application.

5. The use according to claim 4, characterized in that: The detection method is carried out by using a simulated enzyme colorimetric method, and the detection method comprises the following steps: Step 1, incubate different concentrations of glucose and glucose oxidase at 37°C for 20 min; Step 2: Then add 50 μL of pep-PtNCs and an appropriate amount of 3,3',5,5'-tetramethylbenzidine TMB in NaAc-HAc buffer, adjust the pH to 4, make the volume to 500 μL, mix thoroughly and react at room temperature for 30 min; Step 3: Use a UV-visible spectrophotometer to perform spectral scanning and quantify the absorbance at 652 nm to achieve visual colorimetric detection of glucose.

6. The use according to claim 5, characterized in that: The linear range of glucose detection was 1-10 μM, and the detection limit was 1.57 nM.

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