An electrochemical biosensor based on copper-based conductive metal-organic framework modification, its preparation method and application

Abstract

This invention belongs to the field of biosensors, and particularly relates to an electrochemical biosensor based on a copper-based conductive metal-organic framework (MOF), its preparation method, and its application. The enzyme is immobilized on a copper-based conductive MOF. The preparation method includes the following steps: (1) adding a copper-based conductive MOF dispersion to the surface of a glassy carbon electrode and allowing it to air dry at room temperature; (2) adding an aqueous solution of glutaraldehyde to the surface of the electrode prepared in step (1) and allowing it to air dry at room temperature; (3) adding a phosphate buffer solution of the model enzyme to the surface of the electrode prepared in step (2) and drying it to obtain the copper-based conductive MOF-modified electrochemical biosensor. The sensor of this invention has a simple preparation process, low cost, and is easy to carry. The detection time is short, the detection potential is 0.4V lower than that of the glassy carbon electrode, and the electrochemical signal is increased by about 5 times. It can effectively avoid interference from potential coexisting substances in the environment.

Description

Technical Field

[0001] This invention belongs to the field of biosensors, and particularly relates to an electrochemical biosensor based on copper-based conductive metal-organic framework modification, its preparation method, and its application. Background Technology

[0002] Organophosphorus pesticides have been widely used to prevent crop diseases and pests, playing a significant role in increasing grain yields. However, organophosphorus pesticides are highly toxic to humans and animals. Their mechanism of action involves the irreversible inhibition of acetylcholinesterase activity, damaging the nervous system and leading to harm or even death. Therefore, a simple, rapid, and efficient method for detecting organophosphorus pesticides in fruits, vegetables, and environmental samples is an urgent problem to be solved.

[0003] Traditional methods for detecting organophosphorus pesticides, such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS), suffer from drawbacks such as complex pretreatment, long processing times, and high costs, making them unsuitable for rapid on-site detection. Electrochemical enzyme biosensors, which have seen some development, can effectively address these shortcomings. Electrochemical enzyme sensors often rely on the construction of nanomaterials to immobilize enzyme molecules and improve sensitivity. However, commonly used nanomaterials such as graphene, carbon nanotubes, MXene, black phosphorus, and TMDs have complex and environmentally unfriendly preparation processes, are expensive, unstable, easily oxidized by air, and require further post-modification to achieve gain effects, making them difficult to meet practical needs. Summary of the Invention

[0004] To address the shortcomings of the above technologies, the present invention aims to provide the fabrication and application of an electrochemical biosensor based on a copper-based conductive metal-organic framework.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] In one aspect, the present invention provides an electrochemical biosensor, which includes an enzyme immobilization substrate, wherein the enzyme immobilization substrate is a copper-based conductive metal-organic framework.

[0007] Another aspect of the present invention provides a method for preparing an electrochemical biosensor based on a copper-based conductive metal-organic framework, the method comprising the following steps:

[0008] (1) A dispersion of copper-based conductive metal-organic framework was dropped onto the surface of a glassy carbon electrode and allowed to stand and dry at room temperature to obtain an electrode modified with copper-based conductive metal-organic framework.

[0009] (2) Add an aqueous solution of glutaraldehyde to the surface of the electrode prepared in step (1), let it stand at room temperature to dry, and obtain the glutaraldehyde-modified electrode.

[0010] (3) The phosphate buffer solution of the model enzyme is added dropwise to the surface of the electrode prepared in step (2), and dried to obtain an electrochemical biosensor modified with a copper-based conductive metal-organic framework.

[0011] Preferably, in step (1), the copper-based conductive metal-organic framework dispersion is obtained by mixing a copper-based conductive metal-organic framework with water, and the concentration of the copper-based conductive metal-organic framework in the dispersion is 0.5-2.0 mg / mL. -1 The volume is 2-10 μL.

[0012] Preferably, the preparation method of the copper-based conductive metal-organic framework in step (1) is as follows: using degassed water as a solvent, tetrahydroxy-1,4-benzoquinone hydrate, copper nitrate, and ethylenediamine are added under nitrogen protection to obtain a mixed solution A; the concentration of tetrahydroxy-1,4-benzoquinone hydrate in mixed solution A is 1.5 mg / mL. -1 The concentration of copper nitrate was 2.5 mg / mL. -1 The volume ratio of ethylenediamine to water was 1:1000; the reaction was then carried out in a sealed container at 85°C with continuous stirring for 24 hours. After the reaction was completed, the sample was collected by centrifugation, washed with deionized water, and dried under vacuum to obtain a copper-based conductive metal-organic framework.

[0013] Preferably, the concentration of the aqueous solution of glutaraldehyde in step (2) is 0.1-0.5%, and the volume is 2-10 μL.

[0014] Preferably, the model enzyme in step (3) includes, but is not limited to, acetylcholinesterase and butyrylcholinesterase, with an enzyme activity of 0.005U-0.4U.

[0015] Preferably, in step (3), the phosphate buffer solution is a mixed aqueous solution of Na2HPO4 and NaH2PO4 with an equimolar concentration of 20-100 mmol / L and a pH of 6-8.

[0016] Preferably, in step (3), the drying is performed in a low-temperature oven at a temperature of 30°C for 35 minutes.

[0017] In another aspect, the present invention provides an application of the electrochemical sensor for detecting organophosphorus pesticides in fruit and vegetable, water and soil samples.

[0018] Preferably, the organophosphorus pesticide includes paraoxon, parathion, dichlorvos, and chlorpyrifos.

[0019] Preferably, for the detection of organophosphorus pesticides, the content of organophosphorus pesticides in the sample is determined by the correlation curve between the inhibition rate obtained from the current intensity detected by the electrochemical biosensor and the organophosphorus pesticide concentration. The detection specifically includes the following steps:

[0020] (1) Immerse the electrochemical biosensor in a phosphate buffer solution, add acetylthiocholine chloride phosphate buffer solution at a working voltage of 0.25V, perform electrochemical scanning, record the current-time relationship curve, and record the current i1 after stabilization.

[0021] (2) After the electrodes are removed, they are rinsed with phosphate buffer solution and then immersed in standard solutions of organophosphorus pesticides of different concentrations for 10-30 min.

[0022] (3) After the electrode is removed, it is rinsed with phosphate buffer solution and then immersed in phosphate buffer solution. At a working voltage of 0.25V, the same phosphate buffer solution of acetylthiocholine chloride as in step (1) is added. The current i2 is recorded through the current-time relationship curve. Then the inhibition rate I can be expressed as (i1-i2) / i1, so as to obtain the linear correlation curve between the inhibition rate and the concentration of organophosphorus pesticide.

[0023] (4) The content of organophosphorus pesticides in the sample was calculated by the linear correlation curve between the inhibition rate and the concentration of organophosphorus pesticides.

[0024] The same phosphate buffer solution as the above preparation method is used in steps (1), (2), and (3).

[0025] The beneficial effects of this invention are as follows:

[0026] 1. The sensor prepared by this invention achieves low-potential detection, which is 0.4V lower than the detection potential of glassy carbon electrode, while the electrochemical signal is improved by about 5 times, which can effectively avoid interference from potential coexisting substances in the environment.

[0027] 2. The sensor of this invention has a simple manufacturing process, low cost, is easy to carry, and has a short detection time, making it suitable for rapid on-site detection.

[0028] 3. Copper-based conductive metal-organic frameworks have a wide range of raw material sources, low cost, and simple and green synthesis process. The coordination and conjugation effect between ligands and copper can endow them with strong stability and conductivity.

[0029] 4. The high-density copper in the copper-based conductive metal-organic framework can effectively catalyze the oxidation of enzyme products, thereby improving the electrochemical signal while effectively reducing the detection potential. Attached Figure Description

[0030] Figure 1 Transmission electron microscopy (TEM) image of the copper-based conductive metal-organic framework composite material prepared in Example 1;

[0031] Figure 2The current-voltage relationship curves of acetylthiocholine chloride response for different modified electrodes in Example 2 are shown.

[0032] Figure 3 The curve showing the linear correlation between the inhibition rate and the concentration of paraoxon in Example 3 is shown. Detailed Implementation

[0033] The following examples are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0034] Example 1

[0035] Copper-based conductive metal-organic frameworks were synthesized via a solvothermal method. Specifically, degassed water was used as the solvent, and the final concentration of each reactant was 1.5 mg / mL of tetrahydroxy-1,4-benzoquinone hydrate. -1 Copper nitrate 2.5 mg mL -1 The mixture was reacted with 0.1% ethylenediamine in a sealed round-bottom flask at 85°C for 24 hours. After the reaction was completed, the sample was collected by centrifugation, washed with deionized water, and dried under vacuum to obtain a copper-based conductive metal-organic framework.

[0036] Example 2

[0037] (1) Polish the surface of glassy carbon electrode (GC) with aluminum oxide powder, then ultrasonically clean it in deionized water and anhydrous ethanol and dry it under nitrogen.

[0038] (2) The liquid composed of the copper-based conductive metal-organic framework prepared in Example 1 and deionized water was sonicated for 30 minutes to obtain a uniformly dispersed 1 mg mL solution. -1 A copper-based conductive metal-organic framework dispersion was prepared by dropping 5 μL of the dispersion onto the electrode surface and allowing it to stand and dry at room temperature.

[0039] (3) Take 2 μL of 0.2% glutaraldehyde aqueous solution and add it dropwise to the electrode surface, and let it stand at room temperature to dry;

[0040] (4) Add 2 μL of 0.02 U / μL -1 Acetylcholinesterase (containing 1.5% bovine serum albumin) phosphate buffer (50 mmol / L) -1 Na2HPO4 / NaH2PO4 solution (pH=7.4) was added dropwise to the electrode surface and dried in an oven at 35℃ for 30 min to obtain a glassy carbon electrode-copper-based conductive metal-organic framework-acetylcholinesterase biosensor (GC / Cu2(THQ)3 / AChE).

[0041] Example 3

[0042] (1) Add dropwise a phosphate buffer solution of acetylthiocholine chloride to the detection solution of the three-electrode system consisting of the acetylcholinesterase-modified glassy carbon electrode (GC / Cu2(THQ)3 / AChE) prepared in Example 2 (5 mL phosphate buffer solution, 50 mmol / L). -1 A Na2HPO4 / NaH2PO4 solution (pH = 7.4) was used to measure the time-current curve at a constant voltage of 0.25V. The current value was recorded as i1.

[0043] (2) After the electrode is removed, it is rinsed with phosphate buffer solution and then immersed in para-phosphorus phosphate buffer solution of different concentrations for 15 min.

[0044] (3) After the electrode is removed, it is rinsed with phosphate buffer solution and then immersed in acetylthiocholine chloride phosphate buffer solution to measure the current value i2. The inhibition rate I can be expressed as (i1-i2) / i1, and the linear correlation curve between the inhibition rate and the paraoxon concentration is obtained, such as Figure 3 As shown.

[0045] Example 4

[0046] (1) Take 1g each of fresh apples, oranges, cucumbers and lettuce and add them to 4mL of phosphate buffer solution. Vortex for 5min and filter with a 0.45um filter to remove particulate matter. Set the filtrate aside.

[0047] (2) A 100 ng / mL solution of paraoxonium was added to the filtrate to test the recovery rate. The biosensor prepared in Example 2 was used to detect the filtrate containing 100 ng / mL paraoxonium. Under the same detection method and conditions as in Example 3, the recovery rate was 94.2%. This shows that the biosensor prepared in this invention can be applied to the detection of actual samples.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an electrochemical biosensor, characterized in that, The preparation method includes the following steps: (1) Add copper-based conductive metal-organic framework dispersion to the surface of glassy carbon electrode and let it stand at room temperature to dry; (2) Add the aqueous solution of glutaraldehyde dropwise to the surface of the electrode prepared in step (1) and let it stand at room temperature to dry. (3) The phosphate buffer solution of the model enzyme is added dropwise to the electrode surface prepared in step (2), and dried to obtain an electrochemical biosensor modified with a copper-based conductive metal-organic framework. The preparation method of the copper-based conductive metal-organic framework in step (1) is as follows: (1) Using degassed water as a solvent, tetrahydroxy-1,4-benzoquinone hydrate, copper nitrate, and ethylenediamine were added under nitrogen protection to obtain mixed solution A; the concentration of tetrahydroxy-1,4-benzoquinone hydrate in mixed solution A was 0.5-3 mg / mL. -1 The concentration of copper nitrate is 1-5 mg / mL. -1 The volume ratio of ethylenediamine to water is (0.5-2):1000; (2) Stir at 25-90℃ for 24h in a sealed container. After the reaction is complete, collect the sample by centrifugation, wash and vacuum dry to obtain a copper-based conductive metal-organic framework.

2. The preparation method according to claim 1, characterized in that, In step (1), the copper-based conductive metal-organic framework dispersion is obtained by mixing a copper-based conductive metal-organic framework with water, and the concentration of the copper-based conductive metal-organic framework in the dispersion is 0.5-2.0 mg / mL. -1 .

3. The preparation method according to claim 1, characterized in that, The mass concentration of the glutaraldehyde aqueous solution in step (2) is 0.1-0.5%.

4. The preparation method according to claim 1, characterized in that, In step (3), the model enzymes are acetylcholinesterase and butyrylcholinesterase, with enzyme activities of 0.005-0.4 U. The phosphate buffer solution is an aqueous solution of Na2HPO4 and NaH2PO4 with equimolar concentrations, the concentration of the buffer solution is 20-100 mmol / L, and the pH is 6-8.

5. The preparation method according to claim 1, characterized in that, In step (3), the drying temperature is 30-35℃ and the drying time is 20-40min.

6. The application of an electrochemical sensor prepared by the method according to any one of claims 1-5, characterized in that, Used to detect organophosphorus pesticides in fruit and vegetable, water or soil samples.

7. The application according to claim 6, characterized in that, The organophosphorus pesticides mentioned are paraoxon, parathion, dichlorvos, and chlorpyrifos.

8. The application according to claim 6, characterized in that: The detection method is as follows: (1) Immerse the electrochemical biosensor in phosphate buffer solution, add acetylthiocholine chloride phosphate buffer solution at a working voltage of 0.25V, perform electrochemical scanning at the same time, record the current-time relationship curve, and record the current i1 after stabilization; (2) After the electrode is removed, rinse it with phosphate buffer solution, and then immerse it in standard solutions of organophosphorus pesticides of different concentrations for 10-30 min. (3) After the electrode is removed, it is rinsed with phosphate buffer solution and then immersed in phosphate buffer solution. At a working voltage of 0.25V, the same phosphate buffer solution of acetylthiocholine chloride as in step (1) is added. The current i2 is recorded through the current-time relationship curve. Then the inhibition rate I can be expressed as (i1-i2) / i1, so as to obtain the linear correlation curve between the inhibition rate and the concentration of organophosphorus pesticide. (4) The content of organophosphorus pesticides in the sample was calculated by using the linear correlation curve between the inhibition rate and the concentration of organophosphorus pesticides.

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