A kind of electrochemical biosensor based on graphdiyne and copper nanoparticle composite material modification and its preparation method and application

Abstract

The application relates to a preparation method and application of an electrochemical biosensor based on a graphdiyne and copper nanoparticle composite material, and belongs to the field of biosensors. The biosensor is an acetylcholinesterase sensor for detecting organophosphorus pesticides. The first step of the preparation is to drop and coat a graphdiyne and copper nanoparticle composite material on the surface of a working electrode, the second step is to drop and coat a chemical crosslinking agent glutaraldehyde, and the third step is to drop and coat an enzyme sensitive unit of the biosensor. The graphdiyne has a large specific surface area and good biocompatibility, is beneficial to the stability of copper nanoparticles and enzymes, realizes low-potential super-sensitive detection of organophosphorus pesticides, avoids the interference of common electroactive substances, and can be used for the detection of actual samples.

Description

Technical Field

[0001] This invention belongs to the field of biosensors, and particularly relates to an electrochemical biosensor modified with a composite material of graphdiyne and copper nanoparticles, its preparation method and 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 compensate for these shortcomings; however, their high detection potentials make them susceptible to severe interference from electrochemically active substances in fruit, vegetable, and environmental samples, such as vitamin C, phenol, and aniline. Although electron mediators such as Prussian blue and cobalt phthalocyanine can achieve low-potential detection, they cannot effectively amplify the electrical signal. 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 composite material of graphdiyne and copper nanoparticles.

[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 composite material of graphyne and copper nanoparticles.

[0007] Preferably, the graphyne and copper nanoparticle composite material is synthesized by catalytic coupling of copper nanowires and hexaethynylbenzene.

[0008] Another aspect of the present invention provides a method for preparing an electrochemical biosensor modified with a composite material of graphdiene and copper nanoparticles, the method comprising the following steps:

[0009] (1) The dispersion of the graphyne and copper nanoparticle composite material was dropped onto the electrode surface and allowed to stand at room temperature to dry, thus obtaining the electrode modified by the graphyne and copper nanoparticle composite material.

[0010] (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.

[0011] (3) The buffer solution of the model enzyme was added dropwise to the surface of the electrode prepared in step (2), and dried to obtain an electrochemical biosensor modified with graphdiyne and copper nanoparticle composite material.

[0012] Preferably, the preparation method of the graphyne and copper nanoparticle composite material in step (1) is as follows: copper nanowires, hexaethynylbenzene, and pyridine are added and mixed with diethyl ether as a solvent to obtain a mixed solution A; the concentration of hexaethynylbenzene in mixed solution A is 0.5 mg / mL. -1 The concentration of copper nanowires was 0.1-1 mg / mL. -1 The volume ratio of pyridine to diethyl ether was 1:100; the reaction was then carried out in an autoclave at 30-50℃ for 20-60 h, preferably at 40℃ for 48 h; after the reaction was completed, the sample was collected by centrifugation, washed with ethanol, and dried under vacuum to obtain the graphyne and copper nanoparticle composite material.

[0013] Preferably, the electrodes in step (1) include, but are not limited to, glassy carbon electrodes, gold electrodes, paper electrodes, and polymer electrodes.

[0014] Preferably, the model enzyme in step (3) includes, but is not limited to, acetylcholinesterase and butyrylcholinesterase, with an enzyme activity of 0.002-0.2U.

[0015] Preferably, the concentration of the dispersion of the graphdiyne and copper nanoparticle composite material in step (1) is 0.5-4.0 mg / mL. -1 The volume is 2-10 μL.

[0016] Preferably, the aqueous solution of glutaraldehyde has a mass concentration of 0.1-0.5% and a volume of 2-10 μL.

[0017] Preferably, the 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.

[0018] Preferably, the drying in step (3) is low-temperature drying in an oven at a temperature of 30-35°C for 20-40 minutes.

[0019] In another aspect, the present invention provides an application of the electrochemical sensor for detecting organophosphorus pesticides in fruit and vegetable or water samples.

[0020] Preferably, for the detection of organophosphorus pesticides, the content of organophosphorus pesticides in the sample is determined by a standard curve obtained from the relationship between the inhibition rate obtained by the current intensity detected by the electrochemical biosensor and the concentration of organophosphorus pesticides.

[0021] Preferably, the detection specifically includes the following steps:

[0022] (1) Immerse the biosensor in a blank detection solution, add acetylthiocholine chloride solution to the above detection solution under working voltage, and simultaneously perform electrochemical scanning to record the current-time relationship curve. After stabilization, record the current i1.

[0023] (2) After the electrode is removed, it is rinsed with buffer solution and then immersed in the sample to be tested for incubation.

[0024] (3) After the electrode is removed, it is rinsed with buffer solution and then immersed in blank detection solution. At the working voltage, the same concentration of acetylthiocholine chloride solution 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. The correlation curve between the inhibition rate and the concentration of organophosphorus pesticide can be obtained.

[0025] (4) The content of organophosphorus pesticides in the sample was calculated and analyzed by the obtained inhibition rate and correlation curve.

[0026] Preferably, the organophosphorus pesticide includes paraoxon, parathion, dichlorvos, and malathion.

[0027] Preferably, the incubation time in step (2) is 15 minutes.

[0028] The beneficial effects of this invention are as follows:

[0029] 1. The high specific surface area of ​​graphyne is beneficial for increasing the enzyme loading capacity, its porosity is beneficial for the diffusion and mass transfer of electroactive substances, and its good conductivity can enhance the current response. These characteristics can effectively improve the sensitivity of the sensor.

[0030] 2. Copper nanoparticles are generated by etching copper nanowires with graphyne and are simultaneously encapsulated by graphyne, which can effectively improve the stability of copper nanoparticles, thereby not only improving the electrochemical signal but also reducing the detection potential.

[0031] 3. The sensor prepared by this invention achieves low-potential detection, which is 0.55V lower than the detection potential of glassy carbon electrode, while the electrochemical signal is improved by about 5 times. It can effectively avoid interference from vitamin C, phenol and aniline in fruits, vegetables and environmental water samples.

[0032] 4. The preparation process of this invention is simple, inexpensive, portable, and quick to detect. It does not require complex pretreatment of actual samples and is suitable for on-site testing. Attached Figure Description

[0033] Figure 1 This is a scanning electron microscope (SEM) image of the graphdiyne and copper nanoparticle composite material prepared in Example 1 of the present invention.

[0034] Figure 2 This is a transmission electron microscope (TEM) image of the graphdiyne and copper nanoparticle composite material prepared in Example 1 of the present invention.

[0035] Figure 3 The current-voltage relationship curves of acetylthiocholine chloride response for different modified electrodes in Example 2 are shown.

[0036] Figure 4 The curve showing the linear correlation between the inhibition rate of the acetylcholinesterase biosensor in Example 3 and the concentration of paraoxon is shown.

[0037] Figure 5 This is a comparison chart of the current curves of Example 2 with Comparative Examples 1 and 2. Detailed Implementation

[0038] 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.

[0039] Example 1: Preparation of graphdiyne and copper nanoparticle composite materials

[0040] The graphdiyne and copper nanoparticle composite material of the present invention is synthesized by catalytic coupling of copper nanowires and hexaethynylbenzene. Specifically, the preparation method is as follows: using diethyl ether as a solvent, the final concentration of each reactant is 0.5 mg / mL of copper nanowires. -1 0.5 mg mL of hexaethynylbenzene -1 The sample was reacted with 1% pyridine in an autoclave at 40°C for 48 h. After the reaction was completed, the sample was collected by centrifugation, washed with ethanol, and dried under vacuum to obtain a composite material of graphyne and copper nanoparticles.

[0041] Example 2: Preparation of an acetylcholinesterase biosensor

[0042] (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.

[0043] (2) The liquid composed of graphdiyne copper nanoparticle composite material and deionized water was sonicated for 30 min to obtain a uniformly dispersed 1 mg / mL solution. -1 A 3 μL dispersion of graphyne and copper nanoparticle composite material was dropped onto the electrode surface and allowed to air dry.

[0044] (3) Add 2 μL of 0.2% glutaraldehyde aqueous solution to the electrode surface and allow it to dry naturally;

[0045] (4) Add 2 μL of 0.02 U / μL -1 Acetylcholinesterase (containing 2% 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-NGDY-acetylcholinesterase biosensor (GC / GDY-Cu / AChE).

[0046] Example 3: Standard solution for detecting organophosphorus pesticides using an acetylcholinesterase biosensor

[0047] (1) Add 5 mL of phosphate buffer solution to the detection solution of the three-electrode system consisting of acetylcholinesterase-modified glassy carbon electrode (GC / GDY-Cu / AChE) prepared in Example 2, and measure the time-current curve at a constant voltage of 0.25 V. The current value is recorded as i1.

[0048] (2) After the electrode is removed, it is rinsed with phosphate buffer and then immersed in para-phosphorus phosphate buffer of different concentrations for 15 min.

[0049] (3) After the electrode is removed, it is rinsed with phosphate buffer and then immersed in acetylthiocholine chloride phosphate buffer to measure the current value i2. The inhibition rate I can be expressed as (i1-i2) / i1. The relationship between the inhibition rate and the pesticide concentration is as follows: Figure 4 As shown.

[0050] Comparative Example 1

[0051] The glassy carbon electrode-acetylcholinesterase (GC / AChE) was prepared according to the preparation method in Example 2.

[0052] Comparative Example 2

[0053] Following the preparation method in Example 2, a glassy carbon electrode-graphyne-acetylcholinesterase (GC / GDY / AChE) was prepared. The copper nanoparticles were dissolved by stirring the composite material of graphyne and copper nanoparticles overnight in a 5% (molar concentration) hydrochloric acid and a 5% (mass concentration) ferric chloride solution. The copper nanoparticles were then removed by centrifugation and washing with deionized water.

[0054] 10 μM (final concentration of the detection solution after addition) acetylthiocholine chloride (ATCh) phosphate buffer was added dropwise (five times consecutively) to the detection solution (5 mL phosphate buffer) of the three-electrode systems composed of electrodes prepared in Example 2, Comparative Example 1, and Comparative Example 2. The time-current curves were measured at a constant voltage of 0.25 V, resulting in a comparison graph of the current curves of Example 2 with those of Comparative Examples 1 and 2. Figure 5 As shown.

[0055] 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. The application of an electrochemical biosensor in detecting organophosphorus pesticides in fruit, vegetable, and water samples, characterized in that, The sensor includes an enzyme-supported substrate, which is a composite material of graphyne and copper nanoparticles. The method for preparing the sensor includes the following steps: (1) The dispersion of graphyne and copper nanoparticle composite material was dropped onto the electrode surface and allowed to stand at room temperature to dry, thus obtaining an electrode modified with graphyne and copper nanoparticle composite material. (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; (3) The buffer solution of the model enzyme was added dropwise to the electrode surface prepared in step (2), and dried to obtain an electrochemical biosensor modified with graphdiyne and copper nanoparticle composite material. In step (3), the model enzyme is one of acetylcholinesterase or butyrylcholinesterase.

2. The application according to claim 1, characterized in that, The preparation method of the graphylene and copper nanoparticle composite material in step (1) is as follows: copper nanowires, hexaethynylbenzene, and pyridine are added and mixed with diethyl ether as solvent to obtain mixed solution A; the concentration of hexaethynylbenzene in mixed solution A is 0.5 mg / mL. -1 The concentration of copper nanowires was 0.1-1 mg / mL. -1 The volume ratio of pyridine to diethyl ether was 1:100; the reaction was then carried out in an autoclave at 30-50 °C for 20-60 h. After the reaction was completed, the sample was collected by centrifugation, washed with ethanol, and dried under vacuum to obtain a composite material of graphyne and copper nanoparticles.

3. The application according to claim 2, characterized in that, The electrodes in step (1) include glassy carbon electrodes, gold electrodes, paper electrodes, and polymer electrodes; the enzyme activity of the model enzyme in step (3) is 0.002-0.2 U.

4. The application according to claim 2, characterized in that, In step (1), the concentration of the graphylene and copper nanoparticle composite material dispersion is 0.5-4.0 mg / mL. -1 The volume of the aqueous solution of glutaraldehyde in step (2) is 2-10 µL.

5. The application according to claim 2, characterized in that, In step (3), the 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; the drying temperature is 30-35 ℃ and the drying time is 20-40 min.

6. The application according to claim 1, characterized in that: For the detection of organophosphorus pesticides, a standard curve is obtained by comparing the inhibition rate (obtained from the current intensity detected by the sensor) with the concentration of organophosphorus pesticides to determine the content of organophosphorus pesticides in the sample.

7. The application according to claim 1, characterized in that, The detection specifically includes the following steps: (1) Immerse the sensor in a blank detection solution, add acetylthiocholine chloride solution to the above detection solution under working voltage, and simultaneously perform electrochemical scanning to record the current-time relationship curve. After stabilization, record the current i1. (2) After the electrode is removed, it is rinsed with buffer solution and then immersed in the sample to be tested for incubation; (3) After the electrode is removed, it is rinsed with buffer solution and then immersed in blank detection solution. At the working voltage, the same concentration of acetylthiocholine chloride solution is added. The current i2 is recorded through the current-time relationship curve. The inhibition rate I is expressed as (i1-i2) / i1. The correlation curve between the inhibition rate and the concentration of organophosphorus pesticide is obtained. (4) The content of organophosphorus pesticides in the sample was calculated and analyzed by the obtained inhibition rate and correlation curve.

8. The application according to claim 1, characterized in that, The organophosphorus pesticides include paraoxon, parathion, dichlorvos, and malathion.

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