Ascorbic acid electrochemical sensor based on enzyme-like metal organic framework as well as preparation method and application of ascorbic acid electrochemical sensor

Abstract

The invention discloses an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework, which is characterized in that a copper metal organic framework material is used as a precursor to synthesize an amino acid chelating auxiliary etching CuMOF material, and then the CuMOF material is modified on an electrode to obtain the ascorbic acid electrochemical sensor based on the enzyme-like metal organic framework. And high-performance detection of ascorbic acid is realized by using the electrode as a working electrode. The ascorbic acid electrochemical sensor simulates a catalytic center and an active site of natural ascorbic acid oxidase, when the ascorbic acid electrochemical sensor specifically captures and recognizes a target object ascorbic acid, response current is generated, and the current response intensity and the ascorbic acid concentration are in a linear relationship. The ascorbic acid electrochemical sensor solves the problems that existing ascorbic acid detection is poor in selectivity and stability, complex samples are difficult to detect in real time and the like, and ultra-fast, high-specificity and stable ascorbic acid detection in complex biological samples such as the brain is achieved.

Description

Technical Field

[0001] The invention belongs to electrochemical sensing technology and its application in the field of living bodies, and specifically relates to an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework and a preparation method and application thereof. Background Art

[0002] Ascorbic acid, also known as vitamin C, is a small molecule substance that is widely present in organisms and plays a key role. It is of great importance in many fields, which makes the detection of ascorbic acid a key link in related research and applications. In the body, ascorbic acid plays multiple important roles. From the perspective of metabolism, it is involved in the synthesis and metabolism of many substances in the human body. For example, in the synthesis of collagen, ascorbic acid, as a coenzyme of proline hydroxylase and lysine hydroxylase, is essential for maintaining the structural and functional integrity of collagen. In addition, a lack of ascorbic acid can lead to collagen synthesis disorders, which in turn leads to a series of health problems such as scurvy. In the field of neurobiology, ascorbic acid is an important neurochemical substance that actively participates in the synthesis and metabolic regulation of neurotransmitters, and is of great significance for maintaining the normal function of the nervous system. It can effectively remove free radicals in the brain, protect neurons from oxidative stress damage, and has a positive effect on preventing neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. The study also found that the concentration of ascorbic acid will change significantly during the development of neurological diseases such as epilepsy and cerebral edema. Therefore, accurate detection of ascorbic acid can help to gain a deeper understanding of the pathological mechanisms of these diseases and provide a key basis for early diagnosis and treatment.

[0003] Current ascorbic acid (AA) detection technologies mainly include electrochemical method, optical method, chromatography-mass spectrometry, enzyme method and aptamer biosensor technology, but they still face huge challenges: electrochemical method is easily interfered by coexisting oxidizing substances (such as uric acid and dopamine) and the electrode stability is poor; optical method has weak anti-matrix interference ability and relies on complex instruments; chromatography / mass spectrometry is cumbersome to operate and difficult to monitor in real time; natural enzyme method is costly and highly environmentally sensitive; existing aptamer biosensor technology has problems such as difficulty in modification and poor reproducibility, especially in complex biological environments (such as the brain). Therefore, the development of new bionic sensors and detection methods with both specificity, stability and portability has become the key to breaking through the bottleneck of existing technologies. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework in view of the deficiencies in the prior art. The ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework solves the problems of poor specificity, rapid inactivation of natural enzymes, and poor electrode stability in the existing methods for detecting ascorbic acid by simulating the catalytic center and active site of natural ascorbic acid oxidase, and can achieve ultra-fast, highly specific and stable ascorbic acid detection.

[0005] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0006] An ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework comprises the following steps:

[0007] (1) mixing a copper acetate aqueous solution and a pentaethoxyisophthalic acid aqueous solution uniformly to obtain a blue mixture; subjecting the obtained blue mixture to a hydrothermal reaction in a reactor, and after the hydrothermal reaction is completed, separating a blue solid product, which is a copper metal organic framework (CuMOF) material;

[0008] (2) The CuMOF material is used as a precursor and dispersed in solvent water, and then a mixed solution of tryptophan (Trp) and histidine (His) is added, followed by stirring at room temperature for 8 to 16 hours, and the solid product is collected, namely the amino acid chelation-assisted etching CuMOF material (i.e., enzyme-like CuMOF-HT material), and is ground and stored in a dry, light-proof environment for later use;

[0009] (3) ① The enzyme-like CuMOF-HT material is dispersed in solvent water to obtain a CuMOF-HT aqueous dispersion; ② The CuMOF-HT aqueous dispersion is dip-coated onto a gold-based electrode (NEG), and after drying, a CuMOF-HT-modified working electrode (abbreviated as a CuMOF-HT-NEG electrode) is obtained; ③ The CuMOF-HT-NEG electrode is immersed in an electrolyte solution containing sulfobetaine-3,4-ethylenedioxythiophene (SBEDOT) and lithium perchlorate (LiClO4), and in situ polymerization is performed by cyclic voltammetry to form a polymer polysulfobetaine-3,4-ethylenedioxythiophene (PSBEDOT, a zwitterionic polymer, abbreviated as Zw) with both antifouling and conductivity on the surface of the NEG-CuMOF-HT electrode, thereby obtaining a CuMOF-HT-Zw-NEG electrode, i.e., an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework.

[0010] According to the above scheme, in step (1), the concentration of the copper acetate aqueous solution is 5 to 25 mg mL -1 ; The concentration of pentaethoxyisophthalic acid aqueous solution is 7~28mg mL -1When the copper acetate aqueous solution and the pentaethoxyisophthalic acid aqueous solution are mixed, the mass ratio of copper acetate monohydrate to pentaethoxyisophthalic acid is 1: (0.5-2.1).

[0011] According to the above scheme, in step (1), the temperature of the hydrothermal reaction is 100-120°C and the time is 64-80h.

[0012] According to the above scheme, in step (2), the dispersion concentration of CuMOF material in solvent water is 0.3-1 mg mL -1 ; The mass ratio of tryptophan and histidine is (2~3):1; the mass ratio of CuMOF material to tryptophan and histidine is 1:(4~6):(1.5~2.5).

[0013] According to the above scheme, the gold-based electrode is obtained by etching the substrate electrode with hydrofluoric acid and depositing a gold layer on the surface; wherein the substrate electrode is preferably a microelectrode, such as a micrometer-level electrode with a diameter of 1 to 1000 microns and a length of centimeters or millimeters.

[0014] According to the above scheme, in step (3), the concentration of CuMOF-HT aqueous dispersion is 0.5-4 mg mL -1 , coated on one end of the gold-based electrode (NEG), with a coating volume of 5 to 50 μL mm -2 The electrolyte solution is a mixed aqueous solution of SBEDOT monomer and LiClO4, the concentration of SBEDOT is 40-80mM, the concentration of LiClO4 is 80-120mM, and the electrolyte solution is subjected to 20-40 cycles of in-situ polymerization by cyclic voltammetry in an electrochemical window of -0.6-1.3V.

[0015] The present invention also provides an application of the above-mentioned enzyme-like metal organic framework-based ascorbic acid electrochemical sensor in detecting ascorbic acid, that is, a method for detecting ascorbic acid using the enzyme-like metal organic framework-based ascorbic acid electrochemical sensor, comprising the following steps:

[0016] 1) dissolving an ascorbic acid standard sample in a buffer solution to prepare an ascorbic acid standard solution with a gradient concentration; using the ascorbic acid electrochemical sensor of the present invention as a working electrode, and then immersing it in the ascorbic acid standard solution together with a reference electrode and a counter electrode, the three electrodes are respectively connected to an electrochemical workstation through wires, and current response data is collected under a voltage of 0 V; establishing a standard curve with the ascorbic acid concentration in the ascorbic acid standard solution as the abscissa and the current response data of the corresponding ascorbic acid standard solution as the ordinate;

[0017] 2) Under the same current collection conditions as step 1), the ascorbic acid electrochemical sensor is used as a working electrode, and then inserted into the sample solution to be tested or in vivo together with a reference electrode and a counter electrode, and current response data is collected. Then, combined with the standard curve obtained in step 1), the ascorbic acid concentration in the sample solution to be tested or in vivo is obtained.

[0018] According to the above scheme, the linear range of the standard curve is 5-1000 μM.

[0019] According to the above scheme, the platinum electrode was used as the counter electrode and Ag / AgCl was used as the reference electrode.

[0020] The present invention uses epileptic seizure and brain edema as models to detect the ascorbic acid concentration in a living body, wherein the living body mainly refers to the hippocampus or cerebral cortex of the living body.

[0021] The present invention first synthesizes a copper metal organic framework (CuMOF) material as a precursor, and realizes selective etching by controlling the ratio of tryptophan to histidine and the etching time, as well as the amount and concentration of tryptophan, histidine and the CuMOF material, thereby synthesizing an enzyme-like CuMOF-HT material with a chelation-assisted selective etching strategy, and then integrating it onto an electrode, and further electropolymerizing and modifying a polysulfobetaine-3,4-ethylenedioxythiophene antifouling film to obtain an enzyme-like metal organic framework-based ascorbic acid electrochemical sensor, which is used as a working electrode to realize the detection of ascorbic acid in a three-electrode system. In this electrochemical sensing system, the CuMOF-HT material has a metal catalytic center and a specific recognition site. When the CuMOF-HT material specifically captures and identifies the target ascorbic acid, it accelerates its electrochemical oxidation at the copper site, that is, an efficient and specific redox reaction occurs at the electrode interface, generating a corresponding response current. At this time, the chemical signal is converted into a current signal, and the current response intensity is linearly related to the ascorbic acid concentration, thereby achieving high-performance detection and in-situ monitoring of ascorbic acid in solution samples and ascorbic acid in vivo, which can be used to monitor the dynamic changes of ascorbic acid concentration in the cerebral cortex and other organisms in real time.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] (1) The present invention synthesizes enzyme-like CuMOF-HT material through amino acid chelation-assisted selective etching strategy, modifies it on the electrode surface, and obtains an ascorbic acid electrochemical sensor based on enzyme-like metal organic framework. By using it as the working electrode, ultra-fast, highly specific and stable ascorbic acid detection can be achieved (response time is less than 0.1 s, detection limit is as low as 1.46 μM, and storage stability at room temperature can reach more than 6 months).

[0024] (2) The ascorbic acid electrochemical sensor based on the enzyme-like metal organic framework described in the present invention has an excellent copper catalytic center, a specific amino acid recognition site and a water-stable MOF structure, and exhibits excellent electron transfer rate, enzyme-like reaction activity and stability. In particular, after the surface in situ polymerization of the zwitterionic polymer polysulfobetaine-3,4-ethylenedioxythiophene with both antifouling and conductivity, it is very suitable for in situ monitoring of ascorbic acid in vivo, which solves the difficulty of real-time detection in complex biological samples in the prior art and is successfully used to monitor the ascorbic acid level in the mouse brain in real time, achieving high specificity, high stability and real-time dynamic detection of ascorbic acid in complex biological samples such as the mouse brain in seconds.

[0025] (3) The ascorbic acid electrochemical sensor based on enzyme-like metal organic framework described in the present invention simulates the function of natural enzymes and has instant recognition effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the preparation process of the gold-based electrode (NEG) and CuMOF-HT-NEG electrode used in the examples and field emission scanning electron microscope (FESEM) images, (a) from left to right are: original stainless steel acupuncture needles, i.e., substrate microelectrodes (I), microelectrodes after hydrofluoric acid etching (II), microelectrodes after electrodeposition of gold (III) and CuMOF-HT-NEG electrodes (IV); in (b), (A, F) are original stainless steel acupuncture needles, i.e., substrate microelectrodes; (B, G) are microelectrodes after hydrofluoric acid etching, (C, H) are microelectrodes after electrodeposition of gold, (D, I) are CuMOF-NEG electrodes, and (E, J) are CuMOF-HT-NEG electrodes.

[0027] Figure 2 Characterization of CuMOF materials and enzyme-like CuMOF-HT materials, where (A) is the X-ray diffraction (XRD) spectrum, (B) and (C) are FESEM images; (D) is the attenuated total reflection infrared spectrum (ATR-IR); (E) and (F) are N2 adsorption-desorption isotherms and pore size distribution diagrams.

[0028] Figure 3 FESEM image of cubic defects on enzyme-like CuMOF-HT material and the corresponding element C, O, N, and Cu distribution map.

[0029] Figure 4 (A) Overall XPS spectra of CuMOF material and enzyme-like CuMOF-HT material; (B), (C), and (D) are XPS spectra of Cu 2p, C 1s, and O1s of these two materials, respectively; (E) is the XPS spectrum of N1s of the enzyme-like CuMOF-HT material.

[0030] Figure 5 (A) shows the cyclic voltammetry (CV) curves of the substrate microelectrode (a), the microelectrode after hydrofluoric acid etching (b), the NEG electrode (c), the CuMOF-NEG electrode (d), and the CuMOF-HT-NEG electrode (e) in 0.1 M KCl solution (containing 5 mM K3 / K4[Fe(CN)6]), with a scan rate of 50 mV s - 1; (B) is the Nyquist plot of these electrodes in 0.1M KCl (containing 5mM K3 / K4[Fe(CN)6]), frequency range: 0.1Hz to 100kHz, amplitude: 5mV; (C) is the chronoamperometric (it) curve of these electrodes in 0.1M PBS (pH=7.4) at 0V for 200μM ascorbic acid; (D) is the relationship between the capacitive current and the scan rate of these electrodes at 0.20V.

[0031] Figure 6 (A) is the density of states (DOS) diagram of CuMOF and CuMOF-HT; (B), (C), and (D) show the adsorption sites of ascorbic acid on NEG, CuMOF-NEG, and CuMOF-HT-NEG electrodes, respectively; (E) is the corresponding binding energy of ascorbic acid on NEG, CuMOF-NEG, and CuMOF-HT-NEG electrodes; (F) is the Gibbs free energy diagram of ascorbic acid oxidation on NEG, CuMOF-NEG, and CuMOF-HT-NEG electrodes.

[0032] Figure 7 (A) Current responses of ascorbic acid solutions with different concentrations (5 μM to 1000 μM). Inset: Calibration curve of CuMOF-HT-Zw-NEG to ascorbic acid. Error bars indicate relative standard deviations (RSD) based on three measurements; (B) Comparison of the current response stability of CuMOF, ascorbate oxidase (AAO) and CuMOF-HT to ascorbic acid solution; (C) Reproducibility test of five independent CuMOF-HT-Zw-NEG microelectrodes for 200 μM ascorbic acid solution; (D) Selectivity evaluation of CuMOF, AAO and CuMOF-HT when ascorbic acid and interfering substances are continuously added; (E) Comparison of normalized currents of CuMOF, AAO and CuMOF-HT electrodes to ascorbic acid and interfering substances; (F) Schematic diagram of nonspecific capture of ascorbic acid on CuMOF; (G) Schematic diagram of specific capture of ascorbic acid by Trp / His residues on CuMOF-HT; (H) Confocal fluorescence imaging of HUVEC cultured on CuMOF-HT-Zw-NEG electrode for 48 hours, stained with Calcein-AM (live cell stain) and propidium iodide (dead cell stain). Scale bar: 50 μm.

[0033] Figure 8 (A) and (B) are the XRD spectra and Ampere response curves of the freshly prepared CuMOF-HT material in Example (red line) and the CuMOF-HT material after being immersed in water for 6 months (blue line), respectively.

[0034] Fig. 9 The feasibility of the microelectrode sensing system for in vivo analysis. (A) HE-stained partial images of the hippocampus of the control group and the epilepsy group. (B) Changes in Evans blue content in the brain of C57 mice before and after brain edema; error bars indicate the relative standard deviation of three measurements (n=3). (C) Mouse brain water content-BWC (%), x-axis: BWC (%), y-axis: bar graph of mean ± standard deviation, aCSF = control group (healthy animals); NMDA (NMDA group-edema induced by NMDA); Inset: Actual images of the aCSF group and NMDA group before and after drying; error bars indicate the relative standard deviation of three measurements (n=3). (D) Current responses recorded by CuMOF-HT-Zw-NEG after exogenous injection of 2.5 μL KA in the hippocampus of C57 mice. (E) Microinjection (1 μLmin) in the cortex of C57 mice - 1, lasting 60 s, red line indicates) ascorbic acid release at 0, 100, 200, 500 and 1000 μM NMDA. (F) Ascorbic acid release at 500 μM NMDA (1 μL min - 1, lasting 60 s) (top) and co-injection of 500 μM NMDA and 5 mM DIDS (1 μL min - 1, 300 s) (bottom). (Each in vivo experiment was repeated three times independently with consistent results. Only representative curves are shown.)

[0035] In the figure, CuMOF and CuMOF-HT represent CuMOF materials and enzyme-like CuMOF-HT materials, respectively. DETAILED DESCRIPTION

[0036] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with the embodiments, but the present invention is not limited to the following embodiments.

[0037] In the following examples, the gold-based electrode (NEG) used is prepared as follows:

[0038] (a) A stainless steel acupuncture needle (purchased from Yunlong Medical Instrument Co., Ltd., diameter 100 μm, length 5 cm) was used as the substrate microelectrode;

[0039] (b) adding 0.5 mL of 40% hydrofluoric acid solution to a 2 mL centrifuge tube, and then immersing the stainless steel acupuncture needle in the hydrofluoric acid solution with the needle tip downward for 45 min, followed by ultrasonic cleaning with deionized water and ethanol in sequence, and repeating the above process twice to obtain an etched microelectrode;

[0040] (c) The etched microelectrode was immersed in a 0.1 M KCl solution containing 0.5 wt % HAuCl4, with a platinum electrode as the counter electrode and Ag / AgCl as the reference electrode, and deposited at a potential of -0.2 V for 90 s to obtain a microelectrode after electrodeposition of gold, i.e., a gold-based electrode (NEG), which was then rinsed with distilled water and dried naturally ( Figure 1 ). In this step, gold nanoparticles (Au NPs) are modified on the surface of the microelectrode after hydrofluoric acid etching by electrodeposition to enhance its conductivity.

[0041] Example

[0042] An ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework, the specific preparation steps are as follows:

[0043] (1) Preparation of CuMOF material: 0.4 g of copper acetate monohydrate (Cu(CH3COO)2·H2O) was dissolved in 30 mL of ultrapure water to obtain a copper acetate aqueous solution; 0.42 g of pentaethoxyisophthalic acid was dissolved in 30 mL of ultrapure water to obtain a ligand solution; the ligand solution was added dropwise into the copper acetate aqueous solution while stirring to form a blue suspension, which was stirred evenly to obtain a blue mixture; the obtained blue mixture was transferred to a reactor and heated at 110°C for 72 h, and a blue solid product, namely the CuMOF material, was separated and washed and dried for later use.

[0044] (2) Preparation of enzyme-like CuMOF-HT material: 20 mg of CuMOF material was dispersed in 40 mL of ultrapure water by stirring, and then a mixed solution of tryptophan and histidine (102 mg tryptophan and 38.75 mg histidine) was added, followed by stirring at room temperature for 12 h. The solid product, i.e., the copper metal organic framework material assisted by amino acid chelation etching (i.e., the enzyme-like CuMOF-HT material), was collected, and after grinding, it was stored in a dry, light-proof environment for later use.

[0045] (3)① Preparation of enzyme-like CuMOF-HT material aqueous dispersion: 2 mg of enzyme-like CuMOF-HT material was dispersed in 1 mL of ultrapure water, stirred and dispersed, and ultrasonicated for 20 min to obtain a concentration of 2 mg mL -1 ② The CuMOF-HT material dispersion was dip-coated onto the tip of the NEG electrode (the amount was 32.25 μL mm -2), dried in an oven at 60°C for 3h to obtain a CuMOF-HT-NEG electrode; ③ SBEDOT monomer and LiClO4 electrolyte were mixed in ultrapure water, wherein the concentrations of SBEDOT and LiClO4 were 60mM and 100mM, respectively, as an electrolyte solution; the CuMOF-HT-NEG electrode was immersed in the electrolyte solution, and 30 cycles of in situ polymerization were performed by cyclic voltammetry in the electrochemical window of -0.6V to 1.3V to form a PSBEDOT antifouling film on the surface of the NEG-CuMOF-HT electrode, which was recorded as the CuMOF-HT-ZW-NEG electrode, i.e., an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework (CuMOF-HT-ZW-NEG microsensor).

[0046] Comparative Example 1

[0047] A metal organic framework ascorbic acid sensor, which differs from the embodiment only in that: step (2) is omitted, the enzyme-like CuMOF-HT material in step (3) is replaced by an equal amount of CuMOF material, and the rest is the same as step (1) and step (3), to obtain a CuMOF-Zw-NEG electrode.

[0048] Comparative Example 2

[0049] The natural ascorbate oxidase sensor differs from the embodiment only in that commercial AAO and bovine serum albumin (BSA) are mixed in 0.1 M phosphate buffered saline (PBS), and the concentration of both substances is 4 mg mL -1 , the concentration of AAO was 250 U mg -1 , used for dip coating on the NEG working electrode, and dried at room temperature to obtain an AAO-based electrode. Among them, AAO is used as a recognition element and BSA is used as a stabilizer. Subsequently, 5μL of 1wt% chitosan-acetic acid solution was taken and dip coated on the AAO-based electrode, and then 0.5μL of 0.5% glutaraldehyde solution was taken and dip coated on the AAO-based electrode to cross-link the chitosan to form an AAO enzyme fixed layer to obtain an AAO-NEG electrode, which was refrigerated at 4°C overnight. In addition, SBEDOT monomer and LiClO4 electrolyte were mixed in ultrapure water, wherein the concentrations of SBEDOT and LiClO4 were 60mM and 100mM, respectively, as an electrolyte solution; the AAO-NEG electrode was immersed in the electrolyte solution, and 30 cycles of in-situ polymerization were performed by cyclic voltammetry in the electrochemical window of -0.6V to 1.3V to form a PSBEDOT anti-fouling film on the surface of the AAO-NEG electrode, recorded as AAO-Zw-NEG electrode.

[0050] Performance Characterization

[0051] (1) Characterization of CuMOF materials and enzyme-like CuMOF-HT materials

[0052] like Figure 2 As shown, CuMOF materials and enzyme-like CuMOF-HT materials were successfully prepared. Figure 2 As shown in A, compared with the CuMOF material, the enzyme-like CuMOF-HT material has no new XRD diffraction peaks, indicating that after selective etching with a suitable concentration of amino acid mixed solution, the original crystal structure of CuMOF is not destroyed and no ligand exchange reaction occurs; Figure 2 FESEM images of B and C show that CuMOF has a hexagonal prism structure with a smooth surface and a layered stacking structure. However, there are a large number of irregular pores on the surface of CuMOF-HT, but the hexagonal prism structure remains intact. Figure 2 The infrared spectrum of D shows the successful modification of various functional groups of enzyme-like CuMOF-HT after amino acid etching; in addition, Figure 2 As shown in E and F, the specific surface area of ​​enzyme-like CuMOF-HT material (7.59 m 2 g -1 ) is significantly higher than that of CuMOF materials (3.32m 2 g -1 The improvement of the BET value of the enzyme-like CuMOF-HT material can be attributed to the selective etching effect assisted by tryptophan and histidine chelation. Pore size distribution curve ( Figure 2 E, F insets) show that the pores of CuMOF materials and CuMOF-HT materials are mainly concentrated around 3.78nm and 22.97nm, indicating that they both have rich mesoporous structures. In addition, the average pore sizes calculated for CuMOF materials and CuMOF-HT materials are 25.89nm and 46.64nm, respectively, further confirming that amino acid treatment can promote the formation of larger pores in MOF structures. In summary, enzyme-like CuMOF-HT materials have a large specific surface area and rich pore structure, which helps to enhance the exposure of active sites and promote interface mass diffusion and electron transfer.

[0053] Depend on Figure 3 The element distribution map shows that tryptophan and histidine residues are successfully incorporated into the surface of CuMOF-HT crystals.

[0054] like Figure 4As shown, the elemental composition and valence state of CuMOF and CuMOF-HT materials were analyzed by XPS technology. The total spectrum clearly shows the peaks corresponding to Cu2p, C1s, O1s and N1s. It is worth noting that the obvious N peak confirms the successful functionalization of amino acid residues on the CuMOF surface. In addition, the valence state distribution shows that the binding energy of Cu 2p in CuMOF-HT is shifted by 0.5eV. These findings indicate that CuMOF-HT has a different surface Cu coordination structure compared to CuMOF, confirming the coordination force between tryptophan and histidine and Cu.

[0055] (2) The electrochemical properties of the substrate microelectrode and the microelectrode after hydrofluoric acid etching as well as the NEG, CuMOF-NEG and CuMOF-HT-NEG electrodes were evaluated by cyclic voltammetry combined with a redox probe (K3 / K4[Fe(CN)6]). Cyclic voltammetry was performed on the different electrodes in 0.1 M KCl solution with 5 mM K3 / K4[Fe(CN)6] at a scan rate of 50 mV s -1 ,like Figure 5 A. Subsequently, the electronic dynamics of the above electrodes were studied by electrochemical impedance spectroscopy. The Nyquist plots of these electrodes in 5 mM K3 / K4[Fe(CN)6] (frequency range 0.1 Hz to 100 kHz, amplitude 5 mV) are shown as follows: Figure 5 B. The electrochemical performance of these electrodes was further evaluated by chronoamperometry by continuous addition of ascorbic acid in 0.1 M PBS. Specifically, in 0.1 M PBS (pH = 7.4) buffer, under the action of 200 μM ascorbic acid, the it curve was obtained, as shown in Figure 5 C. To further verify the excellent electrochemical activity of the CuMOF-HT-NEG electrode, the electrochemical surface area (ECSA) of each electrode material was evaluated. To further verify the excellent electrochemical activity of the CuMOF-HT-NEG electrode, the ECSA values ​​of the above electrodes were evaluated at different scan rates (25, 50, 75, 100, 125, 150, 175, and 200 mV s -1 )The functional relationship between the capacitance current and the scan rate is as follows: Figure 5 D. In the above tests, different test data were collected by connecting to an electrochemical workstation, and the responding electrode to be tested was used as the working electrode, and the reference electrode Ag / AgCl and the counter electrode Pt were connected to the electrochemical workstation through external wires.

[0056] Depend on Figure 5As shown in Figure 4, the peak currents of the electrodes NEG and CuMOF-NEG were significantly higher than those of the substrate microelectrode and the microelectrode after hydrofluoric acid etching, indicating that the introduction of gold and CuMOF effectively increased the electrochemical active sites and accelerated the electron transfer rate. In addition, the CuMOF-HT-NEG electrode material showed the smallest anode-cathode peak potential separation (ΔE p ) and the highest oxidation peak current value (I pa ), reflecting its excellent electron transfer ability. Figure 5 As shown in B, the semicircle diameter corresponds to the charge transfer resistance (R ct ), R ct The smaller the value, the faster the electron transfer rate. Figure 5 The Randles equivalent circuit fitting in the inset of B shows the R of the substrate microelectrode, the microelectrode after HF etching, and the NEG, CuMOF-NEG, and CuMOF-HT-NEG electrodes. ct The values ​​are 16490.08, 13953.24, 2.62, 1.89 and 0.92 KΩ, respectively, indicating that the charge transfer ability of the CuMOF-HT-NEG electrode is significantly enhanced. From 5C, it can be seen that the steady-state current response of the CuMOF-HT-NEG electrode to ascorbic acid is 2040 times and 3 times that of the NEG electrode and the CuMOF-NEG electrode, respectively, indicating that it has excellent electrochemical activity as an ascorbic acid sensing material. Figure 5 D shows that the current density (Δj 0.20 V) increases significantly with the increase of scan rate. The double layer capacitance (C dl = k / 2), the substrate microelectrode, the microelectrode after hydrofluoric acid etching, and the NEG, CuMOF-NEG, and CuMOF-HT-NEG electrodes were 0.00041, 0.0012, 0.0054, 0.007, and 0.0094 μF cm - 2. Since ECSA and C dl These results confirm that the CuMOF-HT-NEG electrode has more active sites and higher electrochemical activity.

[0057] Density functional theory (DFT) calculations were performed to gain a deeper understanding of the excellent electrocatalytic activity of CuMOF-HT materials for ascorbic acid oxidation. Density of states (DOS) analysis showed that the peak of CuMOF-HT materials near the Fermi level was more significant, indicating that it had a higher reactivity (e.g. Figure 6In addition, the binding energies of ascorbic acid on the electrode materials NEG, CuMOF-NEG and CuMOF-HT-NEG are -0.86, -0.91 and -1.2 eV, respectively. The significant enhancement of the binding energy on the CuMOF-HT surface is conducive to the interaction between ascorbic acid and the active sites of amino acids (as shown in Figure 2A). Figure 6 BE), thereby improving the electrochemical sensing performance. In addition, the reaction pathways of ascorbic acid oxidation catalyzed by NEG, CuMOF-NEG and CuMOF-HT-NEG electrode materials were further studied (as shown in Figure 6 F). The formation steps of dehydroascorbic acid and 2,3-diketo-L-glutamic acid and the desorption step of 2,3-diketo-L-glutamic acid on the CuMOF-HT-NEG electrode are endothermic processes with energy barriers of 0.75, 0.64, and 0.12 eV, respectively. The rate-determining step (RDS) of CuMOF-HT-NEG is the formation of dehydroascorbic acid, and the energy barrier (0.75 eV) is significantly lower than that of NEG (1.06 eV) and CuMOF-NEG (0.97 eV), which explains its excellent electrocatalytic activity. The above experiments are consistent with theoretical calculations, indicating that CuMOF-HT-NEG works through a specific catalytic mechanism similar to natural enzymes: amino acids specifically capture and recognize ascorbic acid, followed by rapid oxidation on Cu catalytic sites.

[0058] Application Examples

[0059] The method for detecting ascorbic acid using the enzyme-like metal organic framework-based ascorbic acid electrochemical sensor prepared in the above embodiment comprises the following steps:

[0060] ① Prepare gradient concentrations of ascorbic acid standard solutions: dissolve the ascorbic acid standard sample in PBS buffer solution (0.1 M, pH 7.4) to prepare a series of ascorbic acid standard solutions with concentrations of 5 μM, 10 μM, 50 μM, 100 μM, 200 μM, 400 μM, 600 μM, 800 μM, and 1000 μM;

[0061] ② Connect the electrochemical workstation to collect current data of ascorbic acid standard solutions of different concentrations and fit the linearity: The ascorbic acid electrochemical sensor is used as the working electrode, and the reference electrode Ag / AgCl and the counter electrode Pt are connected to the electrochemical workstation through external wires to collect current data. The ascorbic acid concentration C in the standard solution is used as the abscissa, and the current value I of the corresponding ascorbic acid standard solution is used as the ordinate to establish a linear regression equation I=1.295C-30.93(R 2 =0.9908). Figure 7As shown in Figure A, at a working potential of 0 V, the response current is linearly related to the ascorbic acid concentration, and the lowest detection concentration is 1.46 μM. Moreover, a signal is generated after adding the ascorbic acid standard solution, which has the advantages of ultra-fast response (response time is less than 0.1 s) and instant recognition. At the same time, compared with CuMOF-Zw-NEG and AAO-Zw-NEG, the CuMOF-HT-Zw-NEG electrode maintains excellent stability within the test time of 1 h for ascorbic acid (e.g. Figure 7 B).

[0062] Repeatability:

[0063] Repeatability is crucial to the detection capability of the ascorbic acid electrochemical sensor (i.e., CuMOF-HT-Zw-NEG electrode). Under the conditions of Example 1, five CuMOF-HT-Zw-NEG electrode samples were prepared in parallel. Figure 7 C shows the reproducibility of five independent CuMOF-HT-Zw-NEG microelectrodes tested for 200 μM ascorbic acid, with an RSD of only 1.7%.

[0064] Specificity:

[0065] Specificity is the top priority in evaluating whether the CuMOF-HT-Zw-NEG electrode can be adapted to in vivo detection, because there are many electrochemically active substances in the brain, such as dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT), Ca 2+ Mg 2+ 、Zn 2+ , Fe 2+ , leucine (Leu), histidine (His), threonine (Thr) and L-cysteine ​​(Cys), which may significantly affect the specificity of the sensor. The neurotransmitters, ions and amino acids with high content and representativeness in the brain were used as interferents to test the anti-interference ability of the prepared microelectrochemical sensor. The standard of the interferent was dissolved in the buffer solution and prepared into 50μM DA, 20μM NE, 10μM 5-HT, 1mM CaCl2, 1mM MgCl2, 1mM ZnSO4, 1mMFeCl2, 1mM Leu, 1mM His, 1mM Thr and 1mM Cys, respectively, and the current size of 200μM ascorbic acid solution was compared.

[0066] like Figure 7As shown in D, in the presence of 200 μM ascorbic acid, the current of the CuMOF-HT-Zw-NEG electrode increased significantly, while other interfering substances had little effect on the current, indicating that it has excellent specificity for monitoring ascorbic acid in the brain. Comparative tests of the CuMOF-HT-Zw-NEG electrode with CuMOF-Zw-NEG and AAO-Zw-NEG electrodes further confirmed that the CuMOF lacking active sites has insufficient selectivity and inaccurate response signals; while natural ascorbate oxidase has good selectivity, it is easily inactivated and the response signal is unstable.

[0067] Figure 7 The radar diagram of E intuitively shows the specific response of the CuMOF-HT-Zw-NEG electrode to ascorbic acid, verifying the specific recognition ability of CuMOF-HT. The tryptophan and histidine residue pockets on the surface of CuMOF-HT ensure the specific capture of ascorbic acid while excluding the interference of other biomolecules ( Figure 7 F, G).

[0068] Biocompatibility:

[0069] Considering that the prepared electrochemical microsensor is in direct contact with the living brain, the cytotoxicity of the CuMOF-HT-Zw-NEG electrode was studied using human umbilical vein endothelial cells (HUVEC). After the CuMOF-HT-Zw-NEG electrode was co-cultured with HUVEC cells for 48 hours, the living cells were stained with calcein (Calcein-AM) and the dead cells were stained with propidium iodide (PI). The control group (without CuMOF-HT-Zw-NEG electrode) and the experimental group (with CuMOF-HT-Zw-NEG electrode) were observed using a confocal microscope to evaluate its biocompatibility for real-time monitoring of ascorbic acid in vivo.

[0070] Figure 7 The confocal fluorescence images of H showed that the survival rate of HUVECs exceeded 99% after immersion for 48 h, indicating its excellent biosafety.

[0071] stability:

[0072] like Figure 8 As shown, by comparing the newly prepared CuMOF-HT material with the CuMOF-HT material stored for more than 6 months, the XRD pattern of its crystal structure and the electrochemical sensing performance to ascorbic acid remain basically the same, indicating that this type of enzyme CuMOF-HT material has ultra-long-term storage stability.

[0073] Feasibility of in vivo analysis:

[0074] As previously demonstrated, the enzyme-like metal organic framework-based ascorbic acid electrochemical sensor of the present invention has high specificity and stability, and can be used to monitor ascorbic acid levels in the living brain, as well as to track the dynamic changes of ascorbic acid in the complex brain environment. Clinical studies have shown that abnormal ascorbic acid levels are pathological features of epileptic seizures and cytotoxic edema. Epilepsy and cytotoxic edema models were established by exogenous injection of kainic acid (KA) and N-methyl-D-aspartate (NMDA), respectively, to verify the feasibility of using such enzyme metal organic framework-based ascorbic acid electrochemical sensors for analysis in the living brain.

[0075] Determination of brain water content: The experimental mice were euthanized after isoflurane anesthesia, and the whole brain tissue was quickly removed and placed in a pre-weighed aluminum foil. The wet weight was recorded by a precision electronic balance, and then the sample was transferred to an 85°C constant temperature drying oven for continuous dehydration for 6 days to constant weight. After measuring the dry weight, the brain water content (BWC) was calculated in percentage using the following formula: BWC (%) = {(wet weight-dry weight) / wet weight} × 100%. Under the same operating conditions, 1 μL of 1000 μM NMDA was exogenously injected into the experimental group; 1 μL of artificial cerebrospinal fluid (aCSF) was exogenously injected into the control group, and then the experimental mice were euthanized after waiting for one minute, and the brain wet weight was weighed and recorded, and then placed in an 85°C oven for drying. After measuring the dry weight, the water loss was calculated using the formula.

[0076] Determination of Evans blue (EB) content in brain tissue: 10 μL of 0.5% EB dye was injected into mice through the tail vein, and their eyes and skin appeared blue. After 0.5 to 1 h, the mice were euthanized and their brain tissues were collected. The brain tissue was then placed in a 1.5 mL centrifuge tube, 1 mL of PBS was added, and the brain tissue was quickly homogenized with a tissue homogenizer, then centrifuged, the supernatant was removed, an equal volume of 60% trichloroacetic acid was added, and then incubated at 4 ° C for 15 min. Take the solution obtained after incubation and measure the absorbance value (OD value) at 620 nm with a spectrophotometer. At the same time, the OD values ​​of standard EB samples with known different gradients were measured and a standard curve was drawn. Finally, the EB content of the solution obtained after incubation was calculated according to the standard curve.

[0077] The epilepsy model was achieved by exogenously injecting KA using a microsyringe with an inner diameter of 50 μm, an outer diameter of 100 μm, and a length of 4 cm to achieve local drug delivery. The microsyringe was used in conjunction with the CuMOF-HT-Zw-NEG electrode to construct a local drug delivery system. The infusion solution was provided by a microsyringe and pumped through a polytetrafluoroethylene hexafluoropropylene (FEP) tube by a microsyringe pump (USA kdScientific LEGATO130) at a rate of 2.5 μL min -1The perfusion rate of KA was used for local microinfusion; the CuMOF-HT-Zw-NEG electrode prepared in the example was used for real-time dynamic detection of ascorbic acid in the hippocampus of C57 mice at a constant potential of 0 V.

[0078] The brain edema model was achieved by exogenously injecting NMDA using a microsyringe with an inner diameter of 50 μm, an outer diameter of 100 μm, and a length of 4 cm. The microsyringe was used in combination with the CuMOF-HT-Zw-NEG electrode to construct a local drug delivery system. The infusion solution was provided by the microsyringe and pumped through the FEP tube by a microsyringe pump (USA kdScientific LEGATO130) at a rate of 1.0 μL min -1 NMDA was locally microinfused at a perfusion rate of 1.0 μL min -1 A mixed solution containing 500 μM NMDA and 5 mM 4,4'-diisothiocyanate octadecene-2,2'-disulfonic acid disodium salt hydrate (DIDS) was applied at a perfusion rate of 1.5 % for 5 min. The CuMOF-HT-Zw-NEG electrode prepared in the example was used to perform real-time dynamic detection of ascorbic acid in the cerebral cortex of C57 mice at a constant potential of 0 V.

[0079] Hematoxylin-eosin (H&E) staining confirmed that KA injection caused hippocampal damage (e.g. Fig. 9 As shown in A, compared with the control group (aCSF), the pyramidal cells in the CA1 region of the hippocampus of the epilepsy model mice were disordered, the ring structure of the synaptic glomerulus of the olfactory bulb was damaged, and the number of cells in the frontal cortex gradually decreased, indicating that the KA-induced epilepsy model was successfully established. Activation of NMDA glutamate receptors can lead to Na+ in neurons and astrocytes. + and Cl - The concentration increased significantly, triggering osmotic cell swelling and ultimately leading to cytotoxic edema. The OD values ​​of the brain homogenate supernatant of the control group (exogenously injected aCSF) and the experimental group (exogenously injected NMDA) were measured using a spectrophotometer at a wavelength of 620nm, and the EB content in the two groups was determined with reference to the EB standard curve. It is worth noting that the amount of EB dye leakage in the NMDA group was approximately three times that of the control group. This is because EB binds to serum proteins (such as albumin), and under normal physiological conditions, these conjugates do not cross the blood-brain barrier, indicating that a mouse model of brain edema (such as Fig. 9 To further verify this, the BWC of C57 mice was measured using the dry and wet weighing method. Fig. 9As shown in C, the BWC of the experimental group (exogenous injection of NMDA) was significantly higher than that of the control group (exogenous injection of aCSF), which also showed that NMDA successfully induced a brain edema model. In addition, the dry and wet photos of mouse brain tissue more intuitively demonstrated the morphological changes associated with brain edema (such as Fig. 9 C illustration).

[0080] The CuMOF-HT-Zw-NEG microsensor prepared in the example was implanted into the mouse brain, and a significant response signal (such as Fig. 9 D), further demonstrating the feasibility of real-time monitoring of dynamic changes in ascorbic acid levels in vivo. In addition, it was confirmed whether the release of ascorbic acid was indeed triggered by cytotoxic edema, and the effect of NMDA concentration on ascorbic acid efflux in vivo was further explored. 100 μM, 200 μM, 500 μM, and 1000 μM NMDA were further injected into the cortex of C57 mice at a rate of 1 μL min - A constant flow rate of 1 was maintained for 60 s, and the release of ascorbic acid was monitored in situ using the CuMOF-HT-Zw-NEG microsensor. Fig. 9 E Experimental data showed that the group injected with 100μM NMDA showed a clear ascorbic acid signal response compared with the artificial cerebrospinal fluid control group (0μM NMDA), and showed a significant dose-dependent enhancement effect with the increase of NMDA concentration. This phenomenon not only confirms the direct correlation between the release of ascorbic acid and the activation level of NMDA receptors, but also reveals the specific change law of ascorbic acid as a real-time response molecule in the state of cytotoxic edema stress in brain tissue, providing a key experimental basis for its use as a biological monitoring indicator of neurotoxic damage.

[0081] Based on the formation mechanism of cytotoxic edema, the above analysis of the release mechanism of ascorbic acid. The above drug intervention experiment showed that chloride ions (Cl - ) channel blocker DIDS inhibits cell swelling-related Cl - Transport not only effectively alleviates cytotoxic edema, but also becomes a key tool for analyzing the dynamic changes of AA related to edema. Injection of 500μM NMDA into the brain region of C57 mice can significantly induce extracellular ascorbic acid accumulation, while the combined administration of 5mM DIDS completely blocks this effect and inhibits the progression of edema ( Fig. 9 F). This result directly established the causal relationship between cell swelling inhibition and AA release inhibition, indicating that the intensity of AA outflow under NMDA stimulation was positively correlated with the degree of cytotoxic edema, and DIDS indirectly inhibited AA release by blocking edema formation. This further clarifies that AA release is a secondary event in the development of cytotoxic edema, and its dynamic changes can specifically reflect the pathological process of neuronal swelling.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and changes without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework, characterized in that: Using copper metal organic framework material as a precursor, amino acid chelate-assisted etching CuMOF material is synthesized through a chelation-assisted selective etching strategy, and then it is modified on an electrode to obtain an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework; the ascorbic acid electrochemical sensor based on the enzyme-like metal organic framework is used as a working electrode to detect ascorbic acid in a three-electrode system, and the current response intensity is linearly related to the ascorbic acid concentration.

2. A method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework, characterized in that: The steps include: (1) mixing a copper acetate aqueous solution and a pentaethoxyisophthalic acid aqueous solution uniformly, and performing a hydrothermal reaction in a reactor. After the hydrothermal reaction is completed, a solid product is separated, which is a CuMOF material; (2) dispersing the CuMOF material into solvent water, then adding a mixed solution of tryptophan and histidine, and stirring the reaction at room temperature to obtain an amino acid chelation-assisted etching CuMOF material, i.e., an enzyme-like CuMOF-HT material; (3) The enzyme-like CuMOF-HT material is dispersed in solvent water to obtain a CuMOF-HT aqueous dispersion, which is then dip-coated on a substrate electrode and dried to obtain a CuMOF-HT modified working electrode, i.e., an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework.

3. The method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework according to claim 2, characterized in that: Step (3) also includes the step of immersing the CuMOF-HT modified working electrode in an electrolyte solution and performing in situ polymerization by cyclic voltammetry to form polysulfobetaine-3,4-ethylenedioxythiophene on the surface of the CuMOF-HT modified working electrode as an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework.

4. The method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework according to claim 2, characterized in that: In step (1), the concentration of the copper acetate aqueous solution is 5 to 25 mg mL -1 ; The concentration of pentaethoxyisophthalic acid aqueous solution is 7~28mg mL -1 When the copper acetate aqueous solution and the pentaethoxyisophthalic acid aqueous solution are mixed, the mass ratio of copper acetate monohydrate to pentaethoxyisophthalic acid is 1: (0.5-2.1); the temperature of the hydrothermal reaction is 100-120° C., and the time is 64-80 hours.

5. The method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework according to claim 2, characterized in that: In step (2), the dispersion concentration of CuMOF material in solvent water is 0.3-1 mg mL -1 ; The mass ratio of tryptophan to histidine is (2-3):1; the mass ratio of CuMOF material to tryptophan to histidine is 1:(4-6):(1.5-2.5); the reaction is stirred at room temperature for 8-16 hours.

6. The method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework according to claim 3, characterized in that: The specific process of step (3) includes: ① The enzyme-like CuMOF-HT material is dispersed in solvent water to obtain a CuMOF-HT aqueous dispersion; ② The CuMOF-HT aqueous dispersion is dip-coated on the modified substrate electrode, and a CuMOF-HT-NEG electrode is obtained after drying; ③ The CuMOF-HT-NEG electrode is immersed in an electrolyte solution containing sulfobetaine-3,4-ethylenedioxythiophene monomer and lithium perchlorate (LiClO4), and in situ polymerization is performed by cyclic voltammetry to form a zwitterionic polymer polysulfobetaine-3,4-ethylenedioxythiophene on the surface of the NEG-CuMOF-HT electrode, thereby obtaining a CuMOF-HT-Zw-NEG electrode, i.e., an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework.

7. The method for preparing an ascorbic acid electrochemical sensor based on an enzyme-like metal organic framework according to claim 6, characterized in that: The substrate electrode is a substrate electrode with a gold layer deposited on the surface after being etched with hydrofluoric acid; the cross-sectional size of the substrate electrode is 1 to 1000 microns, and the length is in the centimeter or millimeter level; The concentration of CuMOF-HT aqueous dispersion is 0.5-4 mg mL -1 The coating amount on the substrate electrode surface is 5 to 50 μL mm -2 The electrolyte solution is a mixed aqueous solution of sulfobetaine-3,4-ethylenedioxythiophene and lithium perchlorate, the concentration of sulfobetaine-3,4-ethylenedioxythiophene is 40-80 mM, the concentration of lithium perchlorate is 80-120 mM, and the electrolyte solution is subjected to 20 to 40 cycles of in-situ polymerization by cyclic voltammetry within the electrochemical window of -0.6 to 1.3 V.

8. Use of the enzyme-like metal organic framework-based ascorbic acid electrochemical sensor according to claim 1 in detecting ascorbic acid, characterized in that: The steps include: 1) dissolving an ascorbic acid standard sample in a buffer solution to prepare an ascorbic acid standard solution with a gradient concentration; using the ascorbic acid electrochemical sensor of the present invention as a working electrode, and then immersing it in the ascorbic acid standard solution together with a reference electrode and a counter electrode; the three electrodes are respectively connected to an electrochemical workstation through wires, and current response data is collected under a voltage of 0 V; A standard curve is established with the ascorbic acid concentration in the ascorbic acid standard solution as the abscissa and the current response data of the corresponding ascorbic acid standard solution as the ordinate; 2) Under the same current collection conditions as step 1), the ascorbic acid electrochemical sensor is used as a working electrode, and then inserted into the sample solution to be tested or in vivo together with a reference electrode and a counter electrode, and current response data is collected. Then, combined with the standard curve obtained in step 1), the ascorbic acid concentration in the sample solution to be tested or in vivo is obtained.

9. Use of the ascorbic acid electrochemical sensor based on enzyme-like metal organic framework in detecting ascorbic acid according to claim 8, characterized in that: The linear range of the standard curve is 5-1000 μM; a platinum electrode is used as the counter electrode and Ag / AgCl is used as the reference electrode.

10. Use of the ascorbic acid electrochemical sensor based on enzyme-like metal organic framework in detecting ascorbic acid according to claim 8, characterized in that: In an organism it is the hippocampus or cerebral cortex of the organism.

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