A GDH / Co-TPC OF electrode modification material, an electrochemical sensor and a preparation method and application thereof

Abstract

The application belongs to the technical field of biosensing and electroanalytical chemistry detection, and discloses a GDH / Co-TPC OF electrode modification material, an electrochemical sensor and a preparation method and application thereof. The electrode modification material takes Co-TPC OF as a carrier, and is loaded with GDH. The Co-TPC OF material is ultrasonically treated in a PBS buffer solution with pH = 7.0-9.0 to obtain a dispersion liquid; the dispersion liquid is subjected to degassing treatment, then GDH is added and stirred at 0-10 DEG C for 12-24 h; and then vacuum drying is performed to obtain the GDH / Co-TPC OF electrode material. An electrochemical sensor prepared by using the GDH / Co-TPC OF electrode modification material. The electrode modification material can realize sensitive detection of NADH and glutamic acid at a low potential, exhibits high sensitivity, a low detection limit and a fast response, and has excellent anti-interference, repeatability, reproducibility and stability.

Description

Technical Field

[0001] This invention belongs to the field of biosensing and electroanalytical chemical detection technology, specifically relating to a GDH / Co-TPCOF electrode material, an electrochemical sensor, its preparation method, and its application. Background Technology

[0002] The rapid aging of the global population has led to an increase in chronic diseases. Alzheimer's disease (AD), a neurodegenerative disease associated with aging, is the most common form of dementia. Its progressive and insidious course places a heavy burden on families and society. Intervening in AD development through biological mechanisms is an effective means of maintaining the quality of life for older adults. Advances in molecular diagnostic technologies allow for the assessment of the disease through biomarker detection. Pathological features of AD include β-amyloid plaques and tau tangles, which accumulate years before the onset of the disease but primarily reflect disease severity rather than initial changes. Glutamate and NADH play crucial roles in cognitive changes; abnormal fluctuations may reflect AD progression. Accurately determining the roles of glutamate and NADH in neurotransmission remains challenging.

[0003] Electrochemical methods, with their advantages of short response time, good reproducibility, ease of operation, and low cost, have made NADH oxidation on electrodes particularly attractive in the field of biosensing. However, NADH oxidation on conventional / unmodified electrode surfaces typically requires high potentials, and the formation of intermediates / products often leads to electrode passivation. Furthermore, at high potentials, detection is highly susceptible to interference from the oxidation of other biomolecules (such as ascorbic acid). Therefore, considerable effort is needed to reduce the oxidation potential. Summary of the Invention

[0004] To address the problems of electrode passivation and susceptibility to interference caused by high detection potential in existing electrochemical NADH detection technologies, the present invention aims to provide a GDH / Co-TPCOF electrode material, an electrochemical sensor, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A GDH / Co-TPCOF electrode modification material, using Co-TPCOF as a carrier, on which GDH is loaded, wherein GDH is glutamate dehydrogenase.

[0006] A method for preparing the GDH / Co-TPCOF electrode modification material comprises the following steps: (1) The Co-TPCOF material was placed in PBS buffer solution with pH=7.0~9.0 and sonicated to obtain a uniform dispersion; (2) Degas the dispersion obtained in step (1), then add GDH and stir at 0~10℃ for 12~24h; then vacuum dry to obtain GDH / Co-TPCOF electrode material. The ratio of raw materials is Co-TPCOF∶PBS buffer∶GDH = (1~3) mg∶(1~3) mL∶(1~3) mg.

[0007] The purpose of degassing is to remove air. The preferred degassing methods are vacuuming or introducing inert / protective gas.

[0008] An application of the GDH / Co-TPCOF electrode modification material in modified electrodes. The method for modifying the electrode is as follows: first, the GDH / Co-TPCOF electrode modification material is prepared into a dispersion, then the dispersion is drop-coated onto the electrode, and finally, a film is formed and sealed using Nafion solution.

[0009] Preferably, the electrode is a glassy carbon electrode or a screen-printed electrode. The glassy carbon electrode, after modification, serves as the working electrode and needs to be combined with a reference electrode and a counter electrode to form a three-electrode system for detecting NADH or glutamate levels in human serum. The screen-printed electrode, because it integrates the working electrode, reference electrode, and counter electrode, is modified directly in the area where the working electrode is located. The modified screen-printed electrode is then directly used to detect NADH or glutamate levels in human serum.

[0010] An electrochemical sensor prepared using the GDH / Co-TPCOF electrode modification material is described below: (1) Dissolve the GDH / Co-TPCOF electrode modification material in PBS buffer at a solid-liquid ratio of 1 mg: (1~3) mL; (2) Take the solution obtained in step (1) and drop it onto the working electrode area of ​​the screen-printed electrode. After drying, add Nafion solution to form a thin film. After rinsing with PBS buffer, the electrochemical sensor is obtained and stored at 0~4℃ for later use.

[0011] Preferably, in steps (1) and (2), the pH of the PBS buffer is 7.0 to 8.0.

[0012] Preferably, in step (2), the drying is carried out by nitrogen blowing.

[0013] Preferably, in step (2), the concentration of the Nafion solution is 0.01 wt%; for a screen-printed electrode with a working electrode diameter of 3 mm, the amount of solution obtained in step (1) is 5~8 µL, and the amount of Nafion solution added is 5~8 µL.

[0014] An application of the electrochemical sensor in the detection of NADH or glutamic acid levels in human serum.

[0015] In this invention, Co-TPCOF is prepared according to existing technologies, including but not limited to M. Liu et al. Porphyrin-Based COF 2D Materials: Variable Modification of Sensing Performances by Post-Metallization. Angew, chsm. Int. Ed 2022, 61, e202115308(5 of 7).

[0016] Beneficial effects: The GDH / Co-TPCOF electrode modification material prepared in this invention can achieve sensitive detection of NADH and glutamate at low potentials, exhibiting high sensitivity, low detection limit and fast response. It also has excellent anti-interference, repeatability, reproducibility and stability, providing a specific reference for promoting biomedical research on Alzheimer's disease and advancing early clinical diagnosis. Attached Figure Description

[0017] Figure 1 The figures show the current-time response curve (A) of the sensor GDH / Co-TPCOF / SPE in PBS buffer (10mM, pH 7.0) with continuous addition of NADH, and the calibration curve (B) of current change versus NADH concentration; the inset shows the response time.

[0018] Figure 2 The figures show the current-time response curve (A) of the sensor GDH / Co-TPCOF / SPE in PBS buffer (50mM, pH 8.0) with continuous addition of glutamate, and the calibration curve (B) of current change versus glutamate concentration; the inset shows the response time.

[0019] Figure 3 The results show the anti-interference detection results for glutamate (A) and NADH (B).

[0020] Figure 4 The results are for the reproducibility assessment of NADH and glutamate.

[0021] Figure 5 Results of repeatability assessment for NADH and glutamate.

[0022] Figure 6 The results show the stability assessment of NADH (a) and glutamic acid (b). Detailed Implementation

[0023] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0024] Example 1

[0025] A method for preparing a GDH / Co-TPCOF electrode modification material, comprising the following steps: (1) Place 2 mg of Co-TPCOF material in 2 mL of PBS buffer (pH=8.0) and sonicate for 10 min to obtain a uniform dispersion; (2) The dispersion obtained in step (1) is vacuum degassed three times to remove all the air. Then, 2 mg of GDH (glutamate dehydrogenase) is added and stirred at 4°C for 24 h. Subsequently, it is dried under vacuum at 60°C for 6 h to obtain the GDH / Co-TPCOF electrode modification material.

[0026] Example 2

[0027] Preparation method of electrochemical sensor (GDH / Co-TPCOF / SPE): Take 1 mg of the GDH / Co-TPCOF electrode modification material prepared in Example 1 and disperse it in 1 mL of PBS buffer (pH=8.0). Take 7 µL of this dispersion and drop it directly onto the working electrode surface of a screen-printed electrode (SPE) (electrode leads: Pt; working electrode: Pt, diameter 3 mm; counter electrode: Pt; reference electrode: Ag / AgCl). After drying with nitrogen, add 7 µL of 0.01 wt% Nafion solution to form a film. After rinsing with PBS buffer (pH=8.0), store at 4 °C to prevent enzyme denaturation for later use.

[0028] Multiple electrochemical sensors (GDH / Co-TPCOF / SPE) were prepared according to the above method for use in the following experiments.

[0029] (a) Laboratory testing (1.1) NADH detection The sensor GDH / Co-TPCOF / SPE of this invention was connected to a CHI760C electrochemical workstation. The GDH / Co-TPCOF / SPE was then placed in 10 mM PBS buffer (pH 7.0), and a voltage of +0.52 V (referring to the potential difference between the working electrode and the reference electrode, the same applies below) was applied. The steady-state current was recorded after 0.1 s. After the current reached steady state, NADH aqueous solution was added to the PBS buffer solution to bring the final concentration of NADH in the system to 10 nM. This process was repeated until steady state was reached. NADH aqueous solution was then added again to bring the final concentration of NADH in the system to 1 µM. After steady state was reached again, NADH aqueous solution was added again to bring the final concentration of NADH in the system to 50 µM. This process was repeated until the final concentrations of NADH in the system were 0 nM, 10 nM, 1 µM, 50 µM, 200 µM, 600 µM, 1 mM, 3 mM, and 5 mM, respectively. A standard curve was obtained based on the relationship between the steady-state current and the NADH concentration.

[0030] Figure 1 The figures show the current-time response curve (A) of the sensor GDH / Co-TPCOF / SPE in PBS buffer (10 mM, pH 7.0) with continuous addition of NADH, and the calibration curve (B) of current change versus NADH concentration; the inset shows the response time. Figure 1 It can be seen that in the range of 10 nM to 5 mM, ΔI (the difference between the signal and blank current) is directly proportional to the logarithmic concentration of NADH, and the linear relationship conforms to the regression equation ΔI = 2.83logC + 0.04(R). 2 =0.997), the detection limit is calculated to be 7.07 nM (signal-to-noise ratio = 3); GDH / Co-TPCOF / SPE has a fast response to analytes, and steady state can be achieved within 1 s.

[0031] Table 1 compares the NADH detection performance of the sensor GDH / Co-TPCOF / SPE of this invention with that of existing sensors. As shown in Table 1, the GDH / Co-TPCOF / SPE of this invention has a competitive advantage in both detection range and detection limit. This may be due to the high catalytic activity of the COF material, which leads to the superior performance of GDH / Co-TPCOF / SPE compared to other reported sensors. Furthermore, it can be seen that the sensor GDH / Co-TPCOF / SPE of this invention can achieve NADH detection at a relatively low potential.

[0032]

[0033] (1.2) Glutamic acid detection The sensor GDH / Co-TPCOF / SPE of this invention was connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in a solution containing 10 mM NAD. + In PBS buffer (50 mM, pH 8.0), a voltage of +0.65 V was applied, and the steady-state current was recorded after 0.1 s. After the current reached steady state, 40 mM of glutamate aqueous solution was added to the PBS buffer solution to make the final concentration of glutamate in the system 50 µM. This process was repeated until steady state was reached. Glutamate aqueous solution was added again to make the final concentration of glutamate in the system 200 µM. After steady state was reached again, glutamate aqueous solution was added again to make the final concentration of glutamate in the system 400 µM. This process was repeated to make the final concentration of glutamate in the system 0 µM, 50 µM, 200 µM, 400 µM, 1 mM, 2 mM, 3 mM, 4 mM, and 5 mM. A standard curve was obtained based on the relationship between steady-state current and glutamate concentration.

[0034] Figure 2 The figures show the current-time response curve (A) and the calibration curve (B) of the current change versus glutamate concentration for the sensor GDH / Co-TPCOF / SPE of this invention when glutamate is continuously added to PBS buffer (50 mM, pH 8.0); the inset shows the response time. Figure 2 It can be seen that in the range of 50μM to 5mM, Δ The difference between the signal and blank current (I) is directly proportional to the logarithmic concentration of glutamate, and the linear relationship conforms to the regression equation ΔI = 2.94logC + 0.14(R²). 2 =0.996), with a detection limit as low as 3.74μM (signal-to-noise ratio = 3).

[0035] Table 2 compares the detection performance of the sensor GDH / Co-TPCOF / SPE of this invention with that of existing sensors for glutamic acid. As shown in Table 2, the sensor GDH / Co-TPCOF / SPE of this invention has a higher detection range and detection limit than other materials; the reasons are the same as in Table 1. Furthermore, it can be seen that the sensor GDH / Co-TPCOF / SPE of this invention can achieve glutamic acid detection at a lower potential.

[0036]

[0037] (1.3) Anti-interference test The following interference tests were conducted to detect redox-active interfering substances (ascorbic acid, uric acid, glucose and other amino acids) that may be present in actual biological samples (such as AD cerebrospinal fluid and serum).

[0038] NADH interference immunity detection: The sensor GDH / Co-TPCOF / SPE of this invention was connected to a CHI760C electrochemical workstation. Then, GDH / Co-TPCOF / SPE was placed in 10mM PBS buffer (pH 7.0), and NADH aqueous solution and interfering substances (vitamin A, glucose, lactate, ascorbic acid, glycated albumin, uric acid, anhydrous ethanol) were added to make the final concentration of NADH in the system 3mM and the final concentration of interfering substances in the system 1mM. Then, a voltage of +0.52V was applied, and the steady-state current was recorded after 0.1s.

[0039] Glutamic acid interference resistance detection: The sensor GDH / Co-TPCOF / SPE of this invention was connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in a solution containing 10 mM NAD. + Glutamic acid aqueous solution and interfering substances (ascorbic acid, urea, uric acid, triglycerides, glycated albumin, glucose, bilirubin) were added to PBS buffer (50mM, pH 8.0) to make the final concentration of glutamate in the system 3mM and the final concentration of interfering substances in the system 1mM. Then, a voltage of +0.65V was applied and the steady-state current was recorded after 0.1s.

[0040] Figure 3 Results of interference resistance detection for glutamate (A) and NADH (B). Figure 3 It can be seen that GDH / Co-TPCOF / SPE exhibits excellent selectivity, with current response changes not exceeding 7% even in the presence of high concentrations of interfering substances. Experiments show that GDH / Co-TPCOF / SPE has high selectivity for common interfering substances and maintains long-term stability at room temperature.

[0041] (1.4) Reproducibility assessment The same sensor GDH / Co-TPCOF / SPE was used to perform six repeated it tests on NADH at a concentration of 3 mM: The sensor GDH / Co-TPCOF / SPE was connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in 10 mM PBS buffer (pH 7.0), and NADH aqueous solution was added to make the final concentration of NADH in the system 3 mM. Then a voltage of +0.52 V was applied, and the steady-state current was recorded after 0.1 s.

[0042] Six repeated it-tests were performed on 3 mM glutamate using the same sensor GDH / Co-TPCOF / SPE: The sensor GDH / Co-TPCOF / SPE was connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in a solution containing 10 mM NAD.+ Glutamic acid aqueous solution was added to PBS buffer (50mM, pH 8.0) to make the final concentration of glutamate in the system 3mM. Then, a voltage of +0.65V was applied and the steady-state current was recorded after 0.1s.

[0043] Figure 4 For the reproducibility assessment of NADH and glutamate. (By...) Figure 4 It can be seen that the relative standard deviations of the current response are 0.8% (NADH) and 1.3% (glutamate), respectively, indicating that the sensor GDH / Co-TPCOF / SPE of the present invention has good reproducibility.

[0044] (1.5) Repeatability assessment Six different sensors, GDH / Co-TPCOF / SPE, were used to perform six it tests on NADH at a concentration of 3 mM: The GDH / Co-TPCOF / SPE sensors were connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE sensors were placed in 10 mM PBS buffer (pH 7.0). NADH aqueous solution was added to make the final concentration of NADH in the system 3 mM. Subsequently, a voltage of +0.52 V was applied, and the steady-state current was recorded after 0.1 s.

[0045] Six it tests were performed on 3 mM glutamate using six different GDH / Co-TPCOF / SPE sensors: The GDH / Co-TPCOF / SPE sensors were connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE sensors were placed in a solution containing 10 mM NAD. + Glutamic acid aqueous solution was added to PBS buffer (50mM, pH 8.0) to make the final concentration of glutamate in the system 3mM. Then, a voltage of +0.65V was applied and the steady-state current was recorded after 0.1s.

[0046] Figure 5 The results are for the reproducibility assessment of NADH and glutamate. (From...) Figure 5 It can be seen that when six different sensors were used to test samples of the same concentration, the relative standard deviations of the current response were 8.9% (NADH) and 7.1% (glutamate), respectively, indicating that the sensor GDH / Co-TPCOF / SPE of the present invention has good repeatability.

[0047] (1.6) Stability assessment Using the same sensor GDH / Co-TPCOF / SPE, it was stored at room temperature for 0d, 7d, 14d, 21d, 28d and 35d respectively, and the 5mM concentration of NADH was tested: The sensor GDH / Co-TPCOF / SPE was connected to the CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in 10mM PBS buffer (pH 7.0), and NADH aqueous solution was added to make the final concentration of NADH in the system 5mM. Then a voltage of +0.52V was applied and the steady-state current was recorded after 0.1s.

[0048] Using the same GDH / Co-TPCOF / SPE electrode, it was stored at room temperature for 0, 7, 14, 21, 28, and 35 days, and six it tests were performed on 5 mM glutamate: The sensor GDH / Co-TPCOF / SPE was connected to a CHI760C electrochemical workstation, and then the GDH / Co-TPCOF / SPE was placed in a solution containing 10 mM NAD. + Glutamic acid aqueous solution was added to PBS buffer (50mM, pH 8.0) to make the final concentration of glutamate in the system 5mM. Then, a voltage of +0.65V was applied and the steady-state current was recorded after 0.1s.

[0049] Figure 6 The results show the stability assessment of NADH (a) and glutamate (b). Figure 6 It can be seen that the sensor GDH / Co-TPCOF / SPE has good long-term stability, and the current response decreases by less than 20% after 35 days.

[0050] (II) Detection of NADH and glutamate in serum samples To evaluate the clinical analytical capabilities of the constructed sensor in real samples and to reveal its value in AD diagnosis, this work collected clinical serum samples from healthy individuals and AD patients. Before use, 20 μL of vitamin A solution and 15 μL of coenzyme Q10 solution (both vitamin A and coenzyme Q10 solutions were 1 mg / mL, and the solvents were composed of 1:1 volumes of anhydrous ethanol and dimethyl sulfoxide) were added to each milliliter of serum to stabilize NADH and prevent its oxidation in serum. Subsequently, EDTA was added to the serum to achieve a concentration of 1 mmol / L to minimize the reduction of metal ions (such as Cu). 2+ To mitigate interference, the levels of NADH and glutamate in human serum were then measured using the methods described below: (i) Dilute serum samples with PBS buffer (10 mM, pH 7.0) at a dilution ratio of 1:1000 (volume ratio). Take 5 mL of the diluted serum sample and insert it into the sensor GDH / Co-TPCOF / SPE prepared in Example 2 of this invention. Connect the sensor GDH / Co-TPCOF / SPE to a CHI760C electrochemical workstation, and then apply a working voltage of +0.52 V. Record the NADH it response in human serum and the steady-state current after 0.1 s. Substitute the steady-state current (signal current) value into the formula ΔI=2.83logC+0.04(R) 2 =0.997), the concentration of NADH in serum was calculated; (ii) Through a solution containing 10 mmol / L NAD + Serum samples were diluted with PBS buffer (50 mmol / L, pH 8.0) at a dilution ratio of 1:1000 (volume ratio). 5 mL of the diluted serum sample was inserted into the GDH / Co-TPCOF / SPE sensor prepared in Example 2 of this invention. The GDH / Co-TPCOF / SPE sensor was connected to a CHI760C electrochemical workstation. A working voltage of +0.65 V was applied, and the it response of glutamate was recorded. The steady-state current after 0.1 s was recorded. The steady-state current (signal current) value was substituted into the formula ΔI=2.94logC+0.14(R) 2 =0.996), the concentration of glutamate in serum was calculated.

[0051] All analytical tests on samples were conducted at room temperature.

[0052] The results are shown in Table 3.

[0053]

[0054] According to reports, the concentration range of NADH in the human body is 0.05-500 μmol / L. The NADH concentration range analyzed using this sensor is consistent with previous reports. Analysis of serum glutamate and NADH levels using GDH / Co-TPCOF / SPE revealed that serum glutamate levels in AD patients were significantly higher than in non-AD patients, but NADH levels were not significantly increased compared to non-AD patients. In conclusion, the data from this study preliminarily confirm that blood small molecule metabolite analysis holds promise for precise metabolomics research in AD, providing valuable reference for a deeper understanding of the neurometabolic mechanisms of AD and for conducting longitudinal studies.

[0055] Note: The specific references mentioned in Tables 1 and 2 of this invention are as follows: [1] Erarkc E, Bayndr O, Alanyalolu M. Amperometric quantification ofNADH based on graphene / methylene blue nanocomposite thin films on Au(111)[J].Polymer Composites, 2017, 38, E118-E127. [2] Da Silva L V , Lopes C B , Da Silva W C ,et al.Electropolymerisation of ferulic acid on multi-walled carbon nanotubesmodified glassy carbon electrode as a versatile platform for NADH, dopamineand epinephrine separate detection[J]. Microchemical Journal, 2017, 133: 460-467. [3] Shan C, Yang H, Han D, et al. Graphene / AuNPs / chitosannanocomposites film for glucose biosensing[J]. Biosensors & Bioelectronics,2010, 25: 1070-1074. [4] Amanda L, Tomasz R, Robert F, Teofil J, Grzegorz M,Electrochemical generation of 1-amino-pyrene-4,5,9,10 tetrol on the MWCNTsurface for low potential electrocatalytic NADH oxidation[J]. ElectrochimicaActa, 2023, 463: 142822. [5] Gao L, Zhou Y, Cao L, Cao Y, Zhang H, Photoelectrochemical sensorfor histone deacetylase Sirt1 detection based on Z-scheme heterojunction ofCuS-BiVO4 photoactive material and the cyclic etching of MnO2 by NADH[J].Talanta, 2024, 268: 125307. [6] Chu M, Bai Z, Zhu D, Chen W, Yang G, Xin J, Ma H, Aβ-nicotinamideadenine dinucleotide electrochemical sensor based on polyoxometalate built bythe combination of electrodeposition and self-assembly[J]. ElectroanalyticalChemistry, 2022, 907: 116083. [7] Vusa C, Gokhale N, Panda S, Electro-Structured Cu distortednanopyramids for superior sweat glucose sensing[J]. Food Chemistry 2023, 426:136609. [8] Liang B, Zhang S, Lang Q, Song J, Han L and Liu A, AmperometricL-glutamate biosensor based on bacterial cell-surface displayed glutamatedehydrogenase[J]. Anal. Chim. Acta, 2015, 884: 83-89. [9] Hughes G, Pemberton R M, Fielden P R and Hart J P, A Reagentless,Screen-Printed amperometric biosensor for the determination of glutamate infood and clinical applications[J]. Sens. Actuators, B, 2015, 216: 614-621.

[10] Scoggin J L, Tan C, Nguyen N H, Kansakar U, Madadi M, SiddiquiS, Arumugam P U, DeCoster M A and Murray T A, An enzyme-based electrochemicalbiosensor probe with sensitivity to detect astrocytic versus glioma uptake ofglutamate in real-time in vitro[J]. Biosens. Bioelectron, 2019, 126: 751-757.

[11] Ganesan M, Trikantzopoulos E, Maniar Y, Lee S T and Venton B J,Development of a novel micro biosensor for in vivo monitoring of glutamaterelease in the brain[J]. Biosens. Bioelectron., 2019, 130: 103-109.

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Claims

1. A GDH / Co-TPCOF electrode modification material, characterized in that: Co-TPCOF was used as a carrier, on which GDH was loaded, wherein GDH is glutamate dehydrogenase.

2. A method for preparing the GDH / Co-TPCOF electrode modification material as described in claim 1, characterized in that, The steps are as follows: (1) The Co-TPCOF material was placed in PBS buffer solution with pH=7.0~9.0 and sonicated to obtain a uniform dispersion; (2) Degas the dispersion obtained in step (1), then add GDH and stir at 0~10℃ for 12~24h; then vacuum dry to obtain GDH / Co-TPCOF electrode material. The ratio of raw materials is Co-TPCOF∶PBS buffer∶GDH = (1~3) mg∶(1~3) mL∶(1~3) mg.

3. The method for preparing the GDH / Co-TPCOF electrode material as described in claim 2, characterized in that: The degassing process is performed by vacuuming or by introducing an inert gas / protective gas.

4. The application of the GDH / Co-TPCOF electrode modification material as described in claim 1 in a modified electrode.

5. The application of GDH / Co-TPCOF as described in claim 4, characterized in that: The electrode is a glassy carbon electrode or a screen-printed electrode.

6. An electrochemical sensor prepared using the GDH / Co-TPCOF electrode modification material as described in claim 1, characterized in that, The preparation steps are as follows: (1) Dissolve the GDH / Co-TPCOF electrode modification material in PBS buffer at a solid-liquid ratio of 1 mg: (1~3) mL; (2) Take the solution obtained in step (1) and drop it onto the working electrode area of ​​the screen-printed electrode. After drying, add Nafion solution to form a thin film. After rinsing with PBS buffer, the electrochemical sensor is obtained and stored at 0~4℃ for later use.

7. The electrochemical sensor as described in claim 6, characterized in that: In steps (1) and (2), the pH of the PBS buffer is 7.0 to 8.

0.

8. The electrochemical sensor as described in claim 6, characterized in that: In step (2), the drying is carried out by nitrogen blowing.

9. The electrochemical sensor as described in claim 6, characterized in that: In step (2), the concentration of Nafion solution is 0.01wt%; for a screen-printed electrode with a working electrode diameter of 3mm, the amount of solution obtained in step (1) is 5~8µL, and the amount of Nafion solution added is 5~8µL.

10. The application of the electrochemical sensor as described in claim 6 in the detection of NADH or glutamic acid content in human serum.

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