Preparation method of ZIF-8 / RGO / Pt / GCE sensor for monitoring release of H2O2 by zebrafish embryonic cells

Abstract

The invention relates to the technical field of sensor preparation, in particular to a preparation method of a ZIF-8 / RGO / Pt / GCE sensor for monitoring release of H2O2 by zebrafish embryonic cells, which comprises the following steps: dropwise adding a methanol solution containing 2-methylimidazole into a methanol solution containing zinc nitrate to obtain a white precipitate, and centrifuging the solution containing the white precipitate in a centrifugal machine to obtain a precipitate; the preparation method comprises the following steps: centrifuging a reduced graphene oxide suspension, drying after centrifuging to obtain a pure white solid product, dispersing the obtained pure white solid product in an absolute ethyl alcohol solution to obtain a ZIF-8 suspension, mixing the reduced graphene oxide suspension with the ZIF-8 suspension, and mixing the mixed solution with a chitosan solution to obtain the ZIF-8 / RGO composite modification material. The ZIF-8 / RGO composite modification material is dropwise added to the surface of a glassy carbon electrode, platinum nanoparticles are electrically deposited on a ZIF-8 / RGO / GCE semi-finished product, and the ZIF-8 / RGO / Pt / GCE sensor is obtained, the sensor prepared through the steps has the excellent characteristics of being rapid in response, low in detection limit, wide in linear range and the like, and H2O2 can be accurately detected in the complex environment or the cell level.

Description

Technical Field

[0001] The present invention relates to the technical field of sensor preparation, and in particular to a method for preparing a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 by zebrafish embryonic cells. Background Art

[0002] Heavy metal ions such as lead and mercury are biologically toxic and can accumulate in organisms, causing poisoning and dysfunction of important organs such as bones, nervous system and liver. Heavy metal exposure causes the body to produce excessive reactive oxygen species (ROS), which disrupts the redox balance in the body and causes oxidative damage to biological macromolecules such as proteins, nucleic acids and lipids, leading to cell apoptosis or necrosis and metabolic disorders of tissues and organs, causing body lesions and even cancer. Studies have shown that oxidative stress is the cause of almost all diseases, and the harm of heavy metal pollutants to living organisms is closely related to the oxidative stress effect. The ROS produced by oxidative stress mainly include hydrogen peroxide (H2O2), superoxide anions (O2 - ) and hydroxyl radicals (·OH), etc. Among them, H2O2 is one of the most important active oxygen species in the body. It can diffuse to the outside of the cell through the water channels of the cell membrane and is the catalytic agent of superoxide dismutase to oxidize O2 - One of the products of the reaction, and can form ·OH with the participation of transition metal ions. It can be said that the level of H2O2 in the body is related to O2 - Therefore, by detecting the content of H2O2, we can understand the overall level of ROS in cells and thus track and evaluate the oxidative stress response.

[0003] As a kind of reactive oxygen species (ROS) in organisms, the detection of hydrogen peroxide (H2O2) levels is of vital importance for studying its physiological and pathological effects in organisms. In order to accurately and quickly detect the content of hydrogen peroxide, researchers have developed a variety of methods, including chemiluminescence, fluorescence spectroscopy, chromatography, and electrochemical methods. Chemiluminescence is a highly sensitive detection method that relies on light signals generated during chemical reactions for analysis. This method is not only highly sensitive, but also can achieve continuous automatic operation, but it usually requires relatively expensive experimental equipment. Fluorescence spectroscopy uses the property of substances emitting fluorescence under ultraviolet light excitation for detection. By measuring the fluorescence intensity of these fluorescent substances, the concentration of hydrogen peroxide can be indirectly determined. Fluorescence spectroscopy has the advantages of simple operation and high sensitivity, so it has been widely used in the detection of hydrogen peroxide in biological samples. Chromatography is a technology for separating and analyzing components in a mixture. The advantages of chromatography are good sensitivity and fast detection speed, and it is suitable for the analysis of food, drugs, biochemistry, etc. Each method has its advantages and disadvantages and different applicable scenarios.

[0004] In summary, the existing methods for monitoring the release of H2O2 from zebrafish embryonic cells do not have the functions of high sensitivity, real-time monitoring, selectivity, simple operation and reversibility at the same time. Summary of the invention

[0005] The purpose of the present invention is to provide a method for preparing a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells, so as to solve the problem that the existing methods for monitoring the release of H2O2 from zebrafish embryonic cells do not have the functions of high sensitivity, real-time monitoring, selectivity, simple operation and reversibility at the same time.

[0006] To achieve the above object, the present invention provides a method for preparing a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells, comprising the following steps:

[0007] S1: adding a methanol solution containing 2-methylimidazole dropwise into a methanol solution containing zinc nitrate, and stirring at room temperature. After stirring, a white precipitate is obtained;

[0008] S2: After washing the obtained white precipitate three times with methanol solution and deionized water, the solution containing the white precipitate is placed in a centrifuge for centrifugation, and the white solid obtained by centrifugation is transferred to a vacuum drying oven for drying to obtain a pure white solid product;

[0009] S3: Dispersing reduced graphene oxide (RGO) in deionized water and ultrasonicating for 0.5 h to obtain a reduced graphene oxide suspension, dispersing the obtained pure white solid product in anhydrous ethanol solution and ultrasonicating for 0.5 h to obtain a ZIF-8 suspension, and preparing a chitosan solution using glacial acetic acid;

[0010] S4: The reduced graphene oxide suspension and the ZIF-8 suspension were mixed at a volume ratio of 1:1 and ultrasonicated for 0.5 h, and the ultrasonically homogenized mixed solution and the chitosan solution were mixed at a volume ratio of 10:1 and ultrasonicated for 0.5 h to fully mix them to obtain a ZIF-8 / RGO composite modified material;

[0011] S5: adding the obtained ZIF-8 / RGO composite modified material dropwise onto the surface of the glassy carbon electrode, and drying it in an oven at 40 degrees Celsius for 0.5 h to form a ZIF-8 / RGO modified electrode, thereby obtaining a ZIF-8 / RGO / GCE semi-finished product;

[0012] S6: Platinum nanoparticles were electrodeposited onto the ZIF-8 / RGO / GCE semi-finished product using cyclic voltammetry technology. After the electrodeposition was completed, the semi-finished product was washed with a large amount of deionized water and dried with nitrogen to obtain a ZIF-8 / RGO / Pt / GCE sensor.

[0013] The preceding step of step S1 is: accurately weighing 0.2 g of zinc nitrate and completely dissolving it in 5 ml of methanol solution to obtain a methanol solution containing zinc nitrate, accurately weighing 4 g of 2-methylimidazole and dissolving it in another 8 ml of methanol solution to obtain a methanol solution containing 2-methylimidazole.

[0014] Wherein, in step S1, the concentration of 2-methylimidazole is 48.72 mmol / L, and the stirring time at room temperature is 15 minutes.

[0015] Among them, the specific steps of step S3 are: dispersing 2 mg / mL of reduced graphene oxide (RGO) in deionized water and ultrasonicating for 0.5 h to obtain a reduced graphene oxide suspension, dispersing 1 mg / mL of the pure white solid product obtained in anhydrous ethanol solution and ultrasonicating for 0.5 h to obtain a ZIF-8 suspension, and preparing a 1 mg / mL chitosan solution with 4% glacial acetic acid.

[0016] Among them, in step S5, the glassy carbon electrode needs to be pre-treated, and the specific steps for pre-treating the glassy carbon electrode are as follows: before modification, the glassy carbon electrode is polished with 0.3 and 0.05 μm alumina powder respectively until the surface is mirror smooth, and then a bare electrode CV test is performed. After calculation, if it is found that the electrode meets the requirements, it is blown dry with nitrogen.

[0017] Wherein, in step S6, the conditions for electrodeposition are: the electrodeposition of Pt NPs is carried out in 20 mM KCl containing 2 mM K2PtCl6, the potential window is set to -0.4 V to +0.6 V, and the scanning speed is 50 mV / s.

[0018] The invention discloses a preparation method of a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryo cells. The method comprises the following steps: adding a methanol solution containing 2-methylimidazole dropwise into a methanol solution containing zinc nitrate, stirring the solution at room temperature to obtain a white precipitate, washing the white precipitate three times, placing the solution containing the white precipitate into a centrifuge for centrifugation, transferring the solution into a vacuum drying oven after the centrifugation, drying the solution, and obtaining a pure white solid product. The pure white solid product is then dispersed in an anhydrous ethanol solution, subjected to ultrasonic treatment for 0.5 h, and a ZIF-8 suspension is obtained. The reduced graphene oxide suspension and the The ZIF-8 suspension is mixed and ultrasonicated, the ultrasonically homogenized mixed solution and the chitosan solution are mixed and ultrasonicated to fully mix to obtain a ZIF-8 / RGO composite modified material, the obtained ZIF-8 / RGO composite modified material is dropped onto the surface of a glassy carbon electrode, and platinum nanoparticles are electrodeposited onto the ZIF-8 / RGO / GCE semi-finished product using cyclic voltammetry technology to obtain a ZIF-8 / RGO / Pt / GCE sensor. The ZIF-8 / RGO / Pt / GCE sensor using the above steps has excellent characteristics such as fast response, low detection limit, and wide linear range, and can accurately detect H2O2 in complex environments or at the cellular level. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 It is a technical roadmap for the preparation method and application of the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells provided by the present invention.

[0021] Figure 2 These are SEM images of ZIF-8, PtNPs, ZIF-8 / RGO and ZIF-8 / RGO / Pt modified glassy carbon electrodes (GCEs) provided by the present invention.

[0022] Figure 3 It is the EDS characterization diagram of ZIF-8 / RGO / Pt / GCE and the distribution diagram of platinum and zinc elements provided by the present invention.

[0023] Figure 4 The present invention provides the EDS characterization diagram of ZIF-8 / RGO / GCE without platinum deposition and the distribution of carbon and zinc elements.

[0024] Figure 5 The present invention provides an optimization result diagram of the ZIF-8 / RGO modification volume and the number of platinum electrodeposition circles.

[0025] Figure 6 The present invention provides a curve diagram for investigating the conductivity and hydrogen peroxide catalytic performance of ZIF-8 / RGO / Pt / GCE.

[0026] Figure 7 The present invention provides a diagram of the optimal working potential optimization results of ZIF-8 / RGO / Pt / GCE.

[0027] Figure 8 The present invention provides a comparison chart of the catalytic curves of ZIF-8 / RGO / Pt / GCE, ZIF-8-Pt / GCE and RGO-Pt / GCE for hydrogen peroxide.

[0028] Fig. 9 The present invention provides a linear range result diagram of ZIF-8 / RGO / Pt / GCE for hydrogen peroxide detection.

[0029] Fig.10 The present invention provides a diagram of the results of investigating the anti-interference performance of ZIF-8 / RGO / Pt / GCE.

[0030] Fig.11 The present invention provides a graph showing the results of investigating the reproducibility and stability of ZIF-8 / RGO / Pt / GCE.

[0031] Fig.12 The present invention provides a curve chart of ZIF-8 / RGO / Pt / GCE used for zebrafish embryo detection. DETAILED DESCRIPTION

[0032] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0033] See also Figures 1 to 12 The present invention provides a method for preparing a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells, comprising the following steps:

[0034] S1: adding a methanol solution containing 2-methylimidazole dropwise into a methanol solution containing zinc nitrate, and stirring at room temperature. After stirring, a white precipitate is obtained;

[0035] S2: After washing the obtained white precipitate three times with methanol solution and deionized water, the solution containing the white precipitate is placed in a centrifuge for centrifugation, and the white solid obtained by centrifugation is transferred to a vacuum drying oven for drying to obtain a pure white solid product;

[0036] S3: Dispersing reduced graphene oxide (RGO) in deionized water and ultrasonicating for 0.5 h to obtain a reduced graphene oxide suspension, dispersing the obtained pure white solid product in anhydrous ethanol solution and ultrasonicating for 0.5 h to obtain a ZIF-8 suspension, and preparing a chitosan solution using glacial acetic acid;

[0037] S4: The reduced graphene oxide suspension and the ZIF-8 suspension were mixed at a volume ratio of 1:1 and ultrasonicated for 0.5 h, and the ultrasonically homogenized mixed solution and the chitosan solution were mixed at a volume ratio of 10:1 and ultrasonicated for 0.5 h to fully mix them to obtain a ZIF-8 / RGO composite modified material;

[0038] S5: adding the obtained ZIF-8 / RGO composite modified material dropwise onto the surface of the glassy carbon electrode, and drying it in an oven at 40 degrees Celsius for 0.5 h to form a ZIF-8 / RGO modified electrode, thereby obtaining a ZIF-8 / RGO / GCE semi-finished product;

[0039] S6: Platinum nanoparticles were electrodeposited onto the ZIF-8 / RGO / GCE semi-finished product using cyclic voltammetry technology. After the electrodeposition was completed, the semi-finished product was washed with a large amount of deionized water and dried with nitrogen to obtain a ZIF-8 / RGO / Pt / GCE sensor.

[0040] In this embodiment, a methanol solution containing 2-methylimidazole is first added dropwise to a methanol solution containing zinc nitrate, and stirred at room temperature to obtain a white precipitate. After washing the white precipitate three times, the solution containing the white precipitate is placed in a centrifuge for centrifugation. After the centrifugation is completed, it is transferred to a vacuum drying oven for drying to obtain a pure white solid product. The obtained pure white solid product is then dispersed in an anhydrous ethanol solution and ultrasonicated for 0.5 h to obtain a ZIF-8 suspension. The reduced graphene oxide suspension and the ZIF-8 suspension are mixed and ultrasonicated to uniformly precipitate the mixture. The mixed solution and the chitosan solution are mixed and ultrasonicated to fully mix to obtain a ZIF-8 / RGO composite modified material. The obtained ZIF-8 / RGO composite modified material is dropped onto the surface of a glassy carbon electrode. Platinum nanoparticles are electrodeposited onto the ZIF-8 / RGO / GCE semi-finished product using cyclic voltammetry technology to obtain a ZIF-8 / RGO / Pt / GCE sensor. The ZIF-8 / RGO / Pt / GCE sensor using the above steps has excellent characteristics such as fast response, low detection limit, and wide linear range, and can accurately detect H2O2 in complex environments or at the cellular level.

[0041] Furthermore, the preceding step of step S1 is: accurately weighing 0.2 g of zinc nitrate and completely dissolving it in 5 ml of methanol solution to obtain a methanol solution containing zinc nitrate, accurately weighing 4 g of 2-methylimidazole and dissolving it in another 8 ml of methanol solution to obtain a methanol solution containing 2-methylimidazole.

[0042] Further, in step S1, the concentration of 2-methylimidazole is 48.72 mmol / L, and the stirring time at room temperature is 15 minutes.

[0043] Furthermore, the specific steps of step S3 are as follows: dispersing 2 mg / mL of reduced graphene oxide (RGO) in deionized water and ultrasonicating for 0.5 h to obtain a reduced graphene oxide suspension, dispersing 1 mg / mL of the obtained pure white solid product in anhydrous ethanol solution and ultrasonicating for 0.5 h to obtain a ZIF-8 suspension, and preparing a 1 mg / mL chitosan solution with 4% glacial acetic acid.

[0044] Furthermore, in step S5, the glassy carbon electrode needs to be pre-treated. The specific steps for pre-treating the glassy carbon electrode are as follows: before modification, the glassy carbon electrode is polished with 0.3 and 0.05 μm alumina powders respectively until the surface is mirror smooth, and then a bare electrode CV test is performed. After calculation, if it is found that the electrode meets the requirements, it is blown dry with nitrogen.

[0045] Further, in step S6, the conditions for electrodeposition are as follows: the electrodeposition of PtNPs is carried out in 20 mM KCl containing 2 mM K2PtCl6, the potential window is set to -0.4 V to +0.6 V, and the scanning speed is 50 mV / s.

[0046] The present invention also includes the morphological characterization of the ZIF-8 / RGO / Pt / GCE sensor:

[0047] The ZIF-8 / RGO composite modified material was modified onto a detachable glassy carbon electrode for morphological characterization, and the material was magnified to a suitable multiple for photographic observation. The appearance and shape of the prepared ZIF-8 / RGO / Pt / GCE sensor were observed using the S-3700 scanning electron microscope developed and produced by Hitachi Co., Ltd. The detachable electrode was placed under a scanning electron microscope, the focus and magnification were adjusted, the object was magnified 10,000 times and 20,000 times, and then photographed to obtain a clear and detailed image. Then an energy dispersive spectrometer (EDS) was used to chemically characterize the elements of ZIF-8 / RGO / Pt / GCE. The sample was placed under a microscope, a suitable acceleration voltage was selected, the surface of the material was scanned with an electron beam, and the X-ray signal was measured. For X-rays of different elements, EDS can measure their energy and intensity, thereby determining which elements and the content of the elements are contained in the sample.

[0048] In order to observe the morphology of nanomaterials on the electrode surface and the element distribution, SEM and EDS characterizations were carried out on different modified electrodes. Figure 2 SEM images of ZIF-8, PtNPs, ZIF-8 / RGO and ZIF-8 / RGO / Pt modified glassy carbon electrode (GCE) are shown. It can be shown that RGO is wrinkled, and ZIF-8 and Pt are evenly distributed on the surface of RGO. The average particle size of ZIF-8 is close to 200nm, showing an octahedral structure. The average particle size of PtNPs is about 100nm, the shape is spherical, and it is evenly distributed on the surface. Figure 2 (d) shows that a large number of ZIF-8 and Pt particles are distributed on the GCE surface, the larger particles are ZIF-8, and the smaller particles are Pt nanoparticles.

[0049] In addition, EDS line scan facets can further explain what types of elements are contained in these nanomaterials. The presence of these nanoparticles leads to an increase in the surface coverage of the sensing platform. The EDS spectrum of the sample shows characteristic peaks for the presence of Pt, C, and Zn ( Figure 3 ). The EDS line scan image further proves the presence of C, O, and Pt in the nanocomposite. The EDS surface scan image shows the distribution of Pt and Zn elements (respectively Figure 3 b, c).

[0050] Figure 4 (a) is the EDS line characterization diagram of ZIF-8 / RGO / GCE without electrodeposition of Pt particles. It can be seen that the prepared sensor has characteristic peaks of C and Zn elements, proving that ZIF-8 / RGO has been successfully modified on the GCE surface. Figure 4 (b, c) The distribution of RGO and ZIF-8 on the electrode surface can be observed.

[0051] The present invention also includes the optimization of the preparation conditions of the ZIF-8 / RGO / Pt / GCE sensor:

[0052] The amount of nanomaterials modified on the electrode surface directly affects the detection performance of the prepared sensor, so the modified volume of ZIF-8 / RGO and the modified amount of platinum nanoparticles were optimized. First, cyclic voltammetry was used to detect ZIF-8 / RGO / GCE with modified volumes of 3μL, 5μL, 6μL, 8μL, and 10μL. To control the variables, the treatment methods of the glassy carbon electrode before modification were the same. The test results are shown in Figure 2. Figure 5 As shown in (a), compared with the bare electrode, the currents of the oxidation peak and reduction peak of ZIF-8 / RGO / GCE modified with different volumes increased significantly. When 6μL of ZIF-8 / RGO was modified on the electrode, the current increased the most, which was about 4.5 times that of the bare electrode. Since ZIF-8 itself is not conductive, too much or too little modification volume is not conducive to improving the sensor performance. Therefore, 6μL of ZIF-8 / RGO composite material was selected as the optimal modification volume. Next, the electrodeposited PtNPs were controlled by changing the number of CV electrodeposition cycles in chloroplatinic acid solution (2mM), as shown in FIG. Figure 5 As shown in (b), the response sensitivity of the modified electrode with different number of PtNPs electrodeposited to hydrogen peroxide was evaluated by chronoamperometry (IT). It can be observed from the figure that the sensitivity is highest when the number of CV scans of electrodeposited PtNPs is 10. Therefore, the modified ZIF-8 / RGO with 6 μL and the PtNPs electrodeposited with 10 cycles were selected as the optimal modification conditions.

[0053] The present invention also includes the electrocatalytic reduction of hydrogen peroxide on ZIF-8 / RGO / Pt / GCE:

[0054] The electrochemical performance of ZIF-8 / RGO / Pt / GCE was investigated, including the effective area of ​​the modified electrode and the electrocatalytic reduction performance of hydrogen peroxide. First, in the presence of a standard electrochemical probe molecule [Fe(CN)6] 3- In the solution, the effective area of ​​the electrode was calculated through the CV curve of the modified electrode. Figure 6(a) CV curves of five different modified electrodes in K3[Fe(CN)6] solution are shown. Compared with the bare electrode GCE, the peak currents of Pt / GCE, RGO / Pt / GCE and ZIF-8 / RGO / Pt / GCE increased. The reduction peak potential gradually moved toward the positive potential direction. This may be attributed to the excellent electrocatalytic performance and conductivity of ZIF-8, PtNPs and RGO. Among them, ZIF-8 / RGO / Pt / GCE has the largest peak current, which may be due to the fact that ZIF-8 is a three-dimensional porous metal organic framework material with a large surface area and pores of adjustable size, on which weak polar groups can be adsorbed and improve the redox reaction of reagents on the electrode surface.

[0055] Based on the Randles-Sevcik equation, Ip = 2.69 × 10 5 AD 1 / 2 n 3 / 2 γ 1 / 2 C. The effective areas of four different modified electrodes were successfully calculated. This equation describes the relationship between the redox peak current value (Ip) and the effective area of ​​the electrode (A), which also involves the diffusion coefficient of potassium ferrocyanide solution (D), the number of electron transfers (n), the scan rate of the cyclic voltammetry curve (γ), and the concentration of the redox probe molecule in the solution (C). Specifically, using the known parameters D = (6.70 ± 0.02) × 10 -6 cm 2 s -1 (diffusion coefficient of potassium ferrocyanide solution), n = 1 (for [Fe(CN)6] 3- / 4- The effective areas of the four electrodes were calculated based on the number of electron transfers in the redox reaction), γ = 100 mV / s (specific scanning speed) and C = 1 mM (concentration of potassium ferrocyanide solution).

[0056] The results showed that the effective area of ​​the unmodified bare glassy carbon electrode (GCE) was 0.03121 cm 2 , while the effective area of ​​Pt_GCE is 0.07068 cm 2 Furthermore, when reduced graphene oxide (RGO) was modified on the electrode, i.e., RGO / Pt / GCE, its effective area increased significantly to 0.09110 cm 2 Most notably, when zeolite imidazolate framework-8 (ZIF-8) was further compounded in the modified material, i.e., the effective area of ​​the ZIF-8 / RGO / Pt modified electrode was further increased to 0.09487 cm 2 .

[0057] This result clearly shows that the specific surface area can be significantly increased by modifying the electrode, especially the ZIF-8 / RGO / Pt / GCE electrode shows a very high effective area. This increased specific surface area provides a larger reaction interface for the ZIF-8 / RGO / Pt / GCE electrode as a nanozyme sensor, thus providing strong support for its application in the field of high-sensitivity detection. Figure 6 (b) shows the situation of gradually adding different concentrations of hydrogen peroxide to the ZIF-8 / RGO / Pt / GCE sensor in deoxygenated PBS. With the successive addition of H2O2, the reduction peak potential gradually moves toward the negative potential direction, and the current change is the largest near 0.1V. The current increases with the increase of hydrogen peroxide concentration, indicating that hydrogen peroxide can be easily reduced by ZIF-8 / RGO / Pt / GCE in a wide concentration range (80-2000mM). The excellent electrocatalytic reduction performance of ZIF-8 / RGO / Pt / GCE for hydrogen peroxide is mainly due to the use of RGO with good conductivity as the substrate, ZIF-8 adsorbed on it, and platinum nanoparticles further dispersed on the surface of ZIF-8 / RGO, forming a three-dimensional structure, which fully exerts the advantages of the three. Next, the IT technology is used to determine the optimal reduction potential of ZIF-8 / RGO / Pt / GCE for hydrogen peroxide.

[0058] The detection sensitivity of several different modified electrodes to hydrogen peroxide was compared by IT technology to investigate the performance indicators of the constructed ZIF-8 / RGO / Pt / GCE as a hydrogen peroxide sensor. Figure 6 (b) The experimental results show that the current changes significantly around -0.1V, so four potentials of -0.2, -0.15, -0.1V and -0.05V were selected for the experiment. Figure 7 As shown in (a), the slope of the it curve of ZIF-8 / RGO / Pt / GCE for hydrogen peroxide is the largest at -0.1 V. By calculation, Figure 7 (b) It can be seen that the detection sensitivity at -0.1V is about 22.3mAM -1 cm -2 , which is significantly higher than the other three potentials, so -0.1 V is selected as the working potential of the hydrogen peroxide sensor.

[0059] Furthermore, Figure 8 (a) shows the it response curves of three modified electrodes, ZIF-8 / RGO / Pt / GCE, ZIF-8-Pt / GCE and RGO-Pt / GCE, when H2O2 was added dropwise at a working voltage of -0.1V and other conditions were the same. It can be seen that with the increase of H2O2 concentration, the slope of the it curve of ZIF-8 / RGO / Pt / GCE is significantly better than that of ZIF-8-Pt / GCE and RGO-Pt / GCE. Figure 8 As shown in (b), the current response and hydrogen peroxide concentration are fitted. ZIF-8 / RGO / Pt / GCE (sensitivity: 16.512μA / mM, linear correlation coefficient: 0.9998) is more sensitive to H2O2 than ZIF-8Pt / GCE (sensitivity: 9.391μA / mM, linear correlation coefficient: 0.9983) and RGO-Pt / GCE (sensitivity: 9.853μA / mM, linear correlation coefficient: 0.9983) and has a good linear relationship. This shows that ZIF-8 / RGO / Pt composite nanozyme material is an excellent material for detecting hydrogen peroxide.

[0060] Fig. 9 (b) shows the linear range of the ZIF-8 / RGO / Pt / GCE sensor for hydrogen peroxide detection. In the concentration range of 62nM to 13.07mM, the current intensity and hydrogen peroxide concentration were fitted, and the regression linear fitting equation was: I = 6.893C + 0.383, and the linear correlation coefficient R 2 =0.9997. When the signal-to-noise ratio is 3 (S / N=3), it is calculated that the minimum detection limit of the hydrogen peroxide sensor prepared in this experiment is about 62nM. Compared with the same type of sensors, refer to Table 1:

[0061] Table 1. Performance comparison of the prepared sensor and different electrochemical sensors for detecting H2O2

[0062]

[0063] It was found that the sensor has a wide linear range while having a low detection limit. This is attributed to the large specific surface area provided by ZIF-8 / RGO, which promotes the adsorption and conductivity of H2O2 on the modified electrode, and the excellent electrocatalytic properties of Pt NPs, which greatly enhances the electroactivity of the sensor. Compared with other similar sensors, the prepared sensor exhibits excellent electrochemical performance.

[0064] The present invention also provides an anti-interference experiment of the ZIF-8 / RGO / Pt / GCE sensor:

[0065] Evaluating the anti-interference ability of the sensor is of great significance for actual sample detection. The electroactive molecules that may exist in the biological system, such as ascorbic acid (AA), glucose (GLU), uric acid (UA), glutathione (Gln), glutamic acid (Glu), etc., are tested by chrono-amperometric response. Fig.10As shown in the figure, when 5 μL of 50 mM H2O2 is added, the current increases rapidly and quickly reaches a stable state. Then, when 5 μL of 100 mM Glu, UA, AA, GLU, and Gln are added, it can be seen that the current does not change, indicating that these substances have almost no interference with the determination of H2O2. When 5 μL of 50 mM H2O2 is added again, the current still increases rapidly. This shows that the ZIF-8 / RGO / Pt / GCE nanozyme sensor is almost not interfered by these substances and has good selectivity.

[0066] The present invention also provides a test of the reproducibility and stability of the ZIF-8 / RGO / Pt / GCE sensor:

[0067] The sensitivity of three independent ZIF-8 / RGO / Pt / GCE modified electrode sensors was measured, such as Fig.11 (a) shows that the sensitivity is 1 (11.44μAmM -1 ), No. 2 (12.66μAmM -1 ), No. 3 (11.37μAmM -1 ); the relative standard deviation (RSD) is 6.1%, and the sensor shows good reproducibility. The prepared sensor is stored at room temperature and measured every day using IT technology to calculate the sensitivity. Fig.11 (b) It can be seen that the prepared electrode is 60.06% of the initial preparation when measured after 24 hours. This may be because the surface material of ZIF-8 / RGO / Pt nanozyme falls off when it is cleaned after use, resulting in a decrease in specific surface area, electrocatalytic activity, conductivity, etc. However, it can be seen that after 24 hours, the sensitivity of the sensor remains stable and no longer changes.

[0068] The present invention also provides the application of ZIF-8 / RGO / Pt / GCE sensor:

[0069] As a vertebrate model organism, zebrafish has a high degree of homology with the human genome and has been widely used to study apoptosis and cell death during normal development and under cell stress conditions. Its cardiovascular, nervous and digestive systems are similar to those of mammals. Among the many heavy metal pollution, cadmium pollution sources are widely distributed in daily life. For example, it is present in large quantities in cadmium-nickel batteries, plastic stabilizers, fossil fuel combustion, phosphate fertilizers and waste incineration, and even Chinese herbal medicines contain a large amount of cadmium. Existing studies have found that cadmium accumulates most in the liver of zebrafish and causes oxidative damage to tissues such as gills and intestines. Because embryos and larvae are more sensitive to pollutants, many heavy metal toxicity tests on fish are mainly focused on embryos and larvae.

[0070] Therefore, this technical solution uses the chronoamperometry to continuously measure the concentration of hydrogen peroxide produced by zebrafish embryos stimulated by 2mM cadmium ion solution. The experimental group is a solution containing 100 zebrafish embryos, 2.4mL, and 2mM cadmium ions are added three times, 0.2mL each time. The control group is placed with 2.4mL of zebrafish embryo culture solution, and other conditions are the same as the experimental group. Fig.12 As shown in the figure, when zebrafish embryonic cells are stimulated for the first time, the current rises rapidly, and then returns to a lower current value within 150 seconds. Until the next stimulation, after being electrocatalyzed by the ZIF-8 / RGO / Pt / GCE nanozyme sensor, the current value rises rapidly again, and the increase decreases gradually, and is exhausted to the basal level within 150 seconds. It is speculated that the embryonic cells gradually become tolerant to the stimulation after the first stimulation. And many experiments have found that embryonic cells cannot be repeated after one experiment, and the response to subsequent cadmium chloride stimulation is becoming less and less obvious. By consulting the literature, it is speculated that Cd 2+ The fish body is stimulated to produce a large amount of ROS, causing oxidative stress, while the organism is equipped with a powerful antioxidant defense system to inhibit the increase in free radical levels. The control group also responds to the increase in current value every time cadmium ions are added, but each current value is smaller than that of the experimental group and decreases faster, as shown by the black line in the figure. The experiment preliminarily verified the rapid real-time detection of hydrogen peroxide released by zebrafish embryonic cells after oxidative stress by the ZIF-8 / RGO / Pt / GCE nanozyme sensor.

[0071] What is disclosed above is only a preferred embodiment of the present invention, and it certainly cannot be used to limit the scope of rights of the present invention. Ordinary technicians in this field can understand that all or part of the processes of the above embodiment and equivalent changes made according to the claims of the present invention still fall within the scope of the invention.

Claims

1. A method for preparing a ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells, characterized in that: The steps include: S1: adding a methanol solution containing 2-methylimidazole dropwise into a methanol solution containing zinc nitrate, and stirring at room temperature. After stirring, a white precipitate is obtained; S2: After washing the obtained white precipitate three times with methanol solution and deionized water, the solution containing the white precipitate is placed in a centrifuge for centrifugation, and the white solid obtained by centrifugation is transferred to a vacuum drying oven for drying to obtain a pure white solid product; S3: Dispersing reduced graphene oxide in deionized water and ultrasonicating for 0.5 h to obtain a reduced graphene oxide suspension, dispersing the obtained pure white solid product in anhydrous ethanol solution and ultrasonicating for 0.5 h to obtain a ZIF-8 suspension, and preparing a chitosan solution using glacial acetic acid; S4: The reduced graphene oxide suspension and the ZIF-8 suspension were mixed at a volume ratio of 1:1 and ultrasonicated for 0.5 h, and the ultrasonically homogenized mixed solution and the chitosan solution were mixed at a volume ratio of 10:1 and ultrasonicated for 0.5 h to fully mix them to obtain a ZIF-8 / RGO composite modified material; S5: adding the obtained ZIF-8 / RGO composite modified material dropwise onto the surface of the glassy carbon electrode, and drying it in an oven at 40 degrees Celsius for 0.5 h to form a ZIF-8 / RGO modified electrode, thereby obtaining a ZIF-8 / RGO / GCE semi-finished product; S6: Platinum nanoparticles were electrodeposited onto the ZIF-8 / RGO / GCE semi-finished product using cyclic voltammetry technology. After the electrodeposition was completed, the semi-finished product was washed with a large amount of deionized water and dried with nitrogen to obtain a ZIF-8 / RGO / Pt / GCE sensor.

2. The method for preparing the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells according to claim 1, characterized in that: The preceding step of step S1 is: accurately weigh 0.2 g of zinc nitrate and completely dissolve it in 5 ml of methanol solution to obtain a methanol solution containing zinc nitrate, accurately weigh 4 g of 2-methylimidazole and dissolve it in another 8 ml of methanol solution to obtain a methanol solution containing 2-methylimidazole.

3. The method for preparing the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells according to claim 2, characterized in that: In step S1, the concentration of 2-methylimidazole is 48.72 mmol / L, and the stirring time at room temperature is 15 minutes.

4. The method for preparing the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells according to claim 3, characterized in that: The specific steps of step S3 are as follows: 2 mg / mL of reduced graphene oxide (RGO) is dispersed in deionized water and ultrasonicated for 0.5 h to obtain a reduced graphene oxide suspension, 1 mg / mL of the obtained pure white solid product is dispersed in anhydrous ethanol solution and ultrasonicated for 0.5 h to obtain a ZIF-8 suspension, and 1 mg / mL of chitosan solution is prepared with 4% glacial acetic acid.

5. The method for preparing the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells according to claim 3, characterized in that: In step S5, the glassy carbon electrode needs to be pre-treated. The specific steps for pre-treating the glassy carbon electrode are as follows: before modification, the glassy carbon electrode is polished with 0.3 and 0.05 μm alumina powders respectively until the surface is mirror-smooth, and then a bare electrode CV test is performed. After calculation, if it is found that the electrode meets the requirements, it is blown dry with nitrogen.

6. The method for preparing the ZIF-8 / RGO / Pt / GCE sensor for monitoring the release of H2O2 from zebrafish embryonic cells according to claim 3, characterized in that: In step S6, the conditions for electrodeposition are as follows: the electrodeposition of PtNPs is carried out in 20 mM KCl containing 2 mM K2PtCl6, the potential window is set to -0.4 V to +0.6 V, and the scanning speed is 50 mV / s.

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