A preparation method for ultrasensitive detection of metalloenzyme for hydrogen peroxide

Abstract

The invention discloses a preparation method of a metal enzyme for ultrasensitive detection of hydrogen peroxide. The preparation method is to first use terephthalic acid as an organic ligand and FeCl3·6H2O as a metal source, obtain an iron-based metal-organic framework by hydrothermal reaction and high-temperature calcination, and then take the iron-based metal-organic framework and nitrogen-doped graphene and add them to an ethanol solution to prepare an iron-based metal-organic framework / nitrogen-doped graphene metal enzyme. The preparation method of the present invention is simple, low in energy consumption and low in environmental harm; the prepared metal enzyme has a catalase-like property and can realize rapid, sensitive and highly selective electrochemical detection of hydrogen peroxide, and has potential application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of electrochemical analysis, and particularly relates to a method for preparing a metal organic framework-nitrogen-doped graphene metalloenzyme for ultrasensitive detection of hydrogen peroxide. Background Art

[0002] Hydrogen peroxide is a highly oxidizing agent that decomposes into water and oxygen when exposed to strong light or short-wave radiation. Due to its oxidizing, bleaching and bactericidal properties, it is widely used in industrial production, urban sewage treatment, aseptic packaging, medicine and beauty industries. In industry, it is mainly used to produce peroxyacids and peroxyacid salts; in the pharmaceutical industry, hydrogen peroxide mainly plays a role in sterilization and disinfection; and in the beauty industry, hydrogen peroxide is often added to cosmetics because it can remove stains, achieving the effect of whitening the skin and cleaning metabolites. However, the abuse of hydrogen peroxide can cause human gene mutations, animal cancer, accelerate human cell aging, and cause a variety of skin diseases. In severe cases, it may even cause breathing difficulties and visual impairment. Therefore, the development of a fast and highly sensitive hydrogen peroxide detection method plays an important role in environmental protection, food safety and human health.

[0003] At present, the main methods used for hydrogen peroxide detection include chemiluminescence, high performance liquid chromatography, colorimetry and fluorescence, but most of these detection methods have problems such as high cost, complex operation and long detection time, and cannot meet the requirements of rapid on-site detection. In addition, the electrochemical sensors currently used for hydrogen peroxide detection are mostly constructed with catalase or peroxidase modified electrodes, and enzymes are easily affected by factors such as temperature, humidity and chemical environment and lose their activity, thereby affecting their stability and reproducibility in use, making enzyme-based electrochemical sensors generally restricted. In this context, an iron-based metal-organic framework / nitrogen-doped graphene metalloenzyme for ultrasensitive electrochemical detection of hydrogen peroxide has great practical significance and use value. Summary of the invention

[0004] In view of the technical problems of low detection sensitivity and high development cost in the current traditional hydrogen peroxide detection, the present invention provides a method for preparing a metal organic framework-nitrogen-doped graphene metalloenzyme for ultra-sensitive detection of hydrogen peroxide, which has the advantages of high sensitivity, simple operation, low cost, short time consumption, low detection limit, small dosage, etc., and thus stands out from many methods for detecting hydrogen peroxide.

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

[0006] A method for preparing a metal organic framework-nitrogen doped graphene metalloenzyme comprises the following steps:

[0007] Step 1: First, 9.9 mmol FeCl3 6H 2 O and 9.9 mmol terephthalic acid were added into 100 mL DMF and stirred to dissolve, then heated under reflux in a 100 °C oil bath for a period of time and then cooled naturally;

[0008] Step 2, the solution obtained in step 1 is rapidly centrifuged in a high-speed centrifuge, washed twice with DMF and once with anhydrous ethanol, and then dried in a vacuum drying oven at 70° C. for 12 hours to obtain an iron-based metal-organic framework material;

[0009] Step 3, grinding the iron-based metal-organic framework material synthesized in step 2 into fine powder and calcining it in nitrogen at 600° C. for 3 h;

[0010] Step 4: Weigh nitrogen-doped graphene (Xianfeng Nano, CAS No.: 7440-44-0) in a certain proportion and ultrasonically disperse it in deionized water to prepare 0.5 mg mL -1 Suspension;

[0011] Step 5, take 5 mL of the suspension prepared in step 4 and mix it with 5 mL of anhydrous ethanol, weigh 5 mg of the product ground into powder after calcination in step 3 and add it to the mixed solution, and perform ultrasonic dispersion;

[0012] Step 6, using a high-speed centrifuge to quickly centrifuge at 9000 rpm / min for 5 minutes, washing with anhydrous ethanol for 3 times, and drying in a vacuum drying oven at 70° C. for 12 hours to obtain a metal organic framework-nitrogen-doped graphene metalloenzyme.

[0013] Furthermore, in step 1, the molar ratio of the single metal Fe to the ligand terephthalic acid is 1:1.

[0014] Furthermore, the reaction time of step 1 is 10-12h.

[0015] Furthermore, the dosage of step 4 is 2.5 mg of nitrogen-doped graphene (N content 3.0 wt%-5.0 wt%) in 5 mL of deionized water.

[0016] Furthermore, the mass ratio of the nitrogen-doped graphene suspension and the iron-based metal-organic framework material weighed in a certain proportion in step 5 is 1:2.

[0017] The metal organic framework-nitrogen doped graphene metalloenzyme prepared by the above method is used in the preparation of hydrogen peroxide detection products.

[0018] The above application comprises the following steps:

[0019] Step 1, accurately weigh 2.5 mg of the metal organic framework-nitrogen doped graphene metalloenzyme ground into fine powder, add it to a mixed solution of 400 μL of anhydrous ethanol, 75 μL of deionized water and 25 μL of Nafion, and ultrasonicate for 2 h to evenly disperse the material to prepare a 5 mg / mL suspension;

[0020] Step 2, 10 μL of the suspension was modified onto a polished glassy carbon electrode, and then irradiated under an infrared lamp for 5 minutes to prepare an ultrasensitive electrochemical sensor for detecting hydrogen peroxide;

[0021] Step 3, using cyclic voltammetry, with the electrochemical sensor prepared in step 2 as the working electrode, the platinum wire as the counter electrode, and the saturated Ag / AgCl electrode as the reference electrode, the electrochemical CV detection parameters are set as follows: starting potential -0.5V, ending potential +0.5V, standing time 2s, scan rate 50mV / s, and scan segment number 8, to detect the hydrogen peroxide sample to be tested.

[0022] The metal enzyme of the invention enables the modified electrode to have a larger electrochemically active specific surface area, has a faster mass transfer rate in the electrochemical process, can improve the reaction activity, and thus can catalyze the progress of the hydrogen peroxide reduction reaction.

[0023] Beneficial effects:

[0024] The present invention provides a method for preparing a metal organic framework-nitrogen-doped graphene metalloenzyme for ultrasensitive detection of hydrogen peroxide, which has the following beneficial effects compared with the prior art:

[0025] 1. -0.28 V in the cyclic voltammetry diagram of the present invention is a characteristic reduction peak of hydrogen peroxide.

[0026] 2. The present invention uses non-toxic and easily available raw materials, low cost, and low energy consumption to prepare carbon materials with good conductivity and large specific surface area. The prepared materials are successfully applied to electrochemical sensing of hydrogen peroxide.

[0027] 3. The present invention has a relatively wide linear range of 0.001-200 μM when detecting hydrogen peroxide.

[0028] 4. The present invention has a lower detection limit of 0.0005 μM when detecting hydrogen peroxide.

[0029] 5. The present invention has a smaller relative standard deviation value when detecting hydrogen peroxide in human serum using the standard addition method. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 is the SEM image of the metal organic framework-nitrogen doped graphene metalloenzyme of the present invention ( Figure 1 A) and TEM images ( Figure 1 B).

[0031] Figure 2 This is a CV diagram of the metal organic framework-nitrogen doped graphene metalloenzyme of the present invention used as a modified electrode to detect hydrogen peroxide. DETAILED DESCRIPTION

[0032] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified. The reaction starting materials used in the following examples of the present invention are commercially available analytically pure terephthalic acid, FeCl 3 6H 2 O, DMF, nitrogen-doped graphene (N content 3.0wt%-5.0wt%), anhydrous ethanol, 30% hydrogen peroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, etc. The electrolyte in the performance test was 10mM H 2 O 2 The experiments were all carried out at room temperature.

[0033] In the following examples, the morphology of the prepared iron-based metal-organic framework metalloenzyme was observed using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The performance test was performed using a Shanghai Chenhua CHI660E electrochemical workstation in 10 mM H 2 O 2 CV tests were performed in solution.

[0034] Example 1

[0035] A method for preparing a metal organic framework-nitrogen-doped graphene metalloenzyme for ultrasensitive detection of hydrogen peroxide comprises the following steps:

[0036] Step 1, preparation of materials: First, 9.9 mmol FeCl 3 6H 2 O and 9.9 mmol terephthalic acid were added into 100 mL DMF and stirred to dissolve, then heated under reflux in a 100 °C oil bath for 10 h and cooled naturally;

[0037] Step 2, the solution obtained in step 1 is rapidly centrifuged in a high-speed centrifuge, then washed with DMF twice and anhydrous ethanol once, and dried in a vacuum drying oven at 70° C. for 12 hours to obtain an iron-based metal-organic framework material;

[0038] Step 3: Grind the iron-based metal-organic framework material synthesized in step 2 into fine powder and heat it at 600 °C in a nitrogen atmosphere at a heating rate of 2 °C min -1 , calcined for 3h;

[0039] Step 4, weigh 2.5 mg of nitrogen-doped graphene (Xianfeng Nano CAS No.: 7440-44-0)) and ultrasonically disperse it in deionized water for 2 h to prepare a 0.5 mg / mL suspension;

[0040] Step 5, take 5 mL of the suspension prepared in step 4 and mix it with 5 mL of anhydrous ethanol, weigh 5 mg of the product ground into fine powder after calcination in step 3 and add it to the mixed solution, and perform ultrasonic dispersion for 2 hours;

[0041] Step 6, use a high-speed centrifuge to quickly centrifuge at 9000 rpm / min for 5 minutes, wash with anhydrous ethanol three times, and dry in a vacuum drying oven at 70° C. for 12 hours to obtain an iron-based metal-organic framework / nitrogen-doped graphene heterojunction metal enzyme.

[0042] The Fe-based metal-organic framework / nitrogen-doped graphene heterojunction metalloenzymes were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Figure 1 As shown, the material morphology is a polygonal structure and its distribution is relatively uniform.

[0043] Example 2

[0044] Electrochemical sensing of hydrogen peroxide: The prepared iron-based metal-organic framework / nitrogen-doped graphene heterojunction metalloenzyme was used for electrochemical detection of hydrogen peroxide. The specific experimental steps are as follows:

[0045] Step 1, accurately weighing 2.5 mg of the iron-based metal-organic framework / nitrogen-doped graphene heterojunction metalloenzyme prepared in Example 1 and ground into fine powder using an analytical balance, adding the mixture to a mixed solution of 400 μL of anhydrous ethanol, 75 μL of deionized water and 25 μL of Nafion, ultrasonicating for 2 h to uniformly disperse the material, and preparing a 5 mg / mL suspension;

[0046] Step 2: Use a 10 μL pipette to take 10 μL of the suspension twice, 5 μL at a time, and modify it onto a polished glassy carbon electrode. Irradiate it under an infrared lamp for 5 minutes to prepare an ultrasensitive electrochemical sensor for detecting hydrogen peroxide.

[0047] Hydrogen peroxide detection was performed by cyclic voltammetry (CV), a three-electrode system consisting of a working electrode (material-modified glassy carbon electrode), a counter electrode (platinum wire) and a reference electrode (saturated Ag / AgCl electrode). 9.99 mL of PBS with a pH of 7 was added with 10.21 μL of hydrogen peroxide to prepare a 10 mM hydrogen peroxide solution. The electrochemical CV detection parameters were set as follows: starting potential -0.5 V, end potential +0.5 V, standing time 2 s, scan rate 50 mV / s, scan segment number 8, and the working curve was obtained, as shown in FIG. Figure 2 shown.

Claims

1. Application of metal organic framework-nitrogen doped graphene metalloenzyme in the preparation of hydrogen peroxide detection products, characterized in that: The metal organic framework-nitrogen doped graphene metalloenzyme is prepared by the following steps: Step 1, adding FeCl3·6H2O and terephthalic acid into DMF and stirring to dissolve, wherein the molar ratio of FeCl3·6H2O to terephthalic acid is 1:1, heating under reflux in an oil bath at 100°C and then cooling naturally; In step 1, the amount of FeCl3·6H2O is 9.9 mmol, the amount of terephthalic acid is 9.9 mmol, the amount of DMF is 100 mL, and the reaction time is 10-12 h; Step 2, the solution obtained in step 1 is quickly centrifuged in a high-speed centrifuge, washed with DMF and anhydrous ethanol, and then dried in a vacuum drying oven at 70° C. for 12 hours to obtain an iron-based metal-organic framework material; Step 3, grinding the iron-based metal-organic framework material synthesized in step 2 into fine powder and calcining it in nitrogen at 600° C. for 3 h; Step 4, weighing nitrogen-doped graphene and ultrasonically dispersing it in deionized water to prepare a 0.5 mg / L suspension, wherein the amount of nitrogen-doped graphene used is 2.5 mg, and the amount of deionized water used is 5 mL; Step 5, the suspension prepared in step 4 and anhydrous ethanol are mixed in a volume ratio of 1:1, the product ground into powder after calcination in step 3 is added to the mixed solution, the mass ratio of nitrogen-doped graphene to the product ground into powder after calcination in step 3 is 1:2, and ultrasonic dispersion is performed, the amount of the suspension is 5 mL, the amount of anhydrous ethanol is 5 mL, and the amount of the product ground into powder after calcination in step 3 is 5 mg; Step 6, using a high-speed centrifuge to quickly centrifuge at 9000 rpm / min for 5 minutes, washing with anhydrous ethanol for 3 times, and drying in a vacuum drying oven at 70° C. for 12 hours to obtain a metal organic framework-nitrogen-doped graphene metalloenzyme.

Images