Electrochemical enzyme biosensor based on metal-organic framework material ZIF8 and its application

Abstract

The invention discloses an electrochemical enzyme biosensor based on metal organic framework material ZIF8 and its application. The electrochemical enzyme biosensor uses a glassy carbon electrode as a working electrode, modifies a complex including GR, ZIF8 and AChE on the surface of the glassy carbon electrode, and encapsulates it with CS, and the structure is GCE / GR-ZIF8-AChE-CS. The electrochemical enzyme biosensor of the invention has a simple structure, uses the metal organic framework compound ZIF8 as a load material, has good biological inertness, a large load capacity, can be applied to the detection of isothiocarb, and has high detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical biosensors, and in particular to an electrochemical enzyme biosensor based on a metal organic framework material ZIF8 and applications thereof. Background Art

[0002] Organophosphorus pesticides are mostly phosphate or thiophosphate compounds. They are widely used due to their high insecticide effect. Some commonly used organophosphorus pesticides, such as parathion and methyl parathion, can cause poisoning through the esophagus, skin and respiratory tract, and have acute toxicity and carcinogenicity. Irrational disposal of organophosphorus compounds will cause pollution to the atmosphere, soil and water bodies. Agricultural products with excessive pesticide residues may also cause pesticide poisoning and endanger human health, causing environmental pollution and human health risks. Establishing a simple, sensitive, fast and economical organophosphorus sensing platform is of great significance to alleviating people's concerns about environmental protection and food safety, and can also be used as a means of detecting nerve agents.

[0003] At present, common organophosphorus detection can be roughly divided into two categories. One is the traditional instrumental analysis and detection method based on chromatography and spectroscopy, which has the characteristics of high sensitivity and high accuracy. The other is a new detection method that has been continuously developed in recent years, which takes biosensor technology as the core and combines a variety of analytical technologies. It is fast and efficient.

[0004] Biosensors are detection devices composed of signal sensors and biorecognition elements. According to the different biorecognition elements, biosensors can be divided into nucleic acid aptamer biosensors, enzyme biosensors, immune biosensors and tissue biosensors; according to the different signal conversion methods, biosensors can be divided into electrochemical biosensors, piezoelectric biosensors, optical biosensors, thermosensitive biosensors, etc. Among them, electrochemical biosensors are detection devices that convert the response signal generated by the interaction between the substance to be tested and the recognition element into electrical signals such as voltage or current. Electrochemical biosensors also have significant advantages over other sensors. They can detect analytes at low detection limits and show a wide linear response range and reproducibility. Not only do they have the characteristics of fast analytical response, high specificity and sensitivity, but the instrument is also easy to operate, stable in performance, easy to miniaturize and automate, suitable for on-site detection and online analysis, and have broad development prospects.

[0005] Acetylcholinesterase has a specific binding with organophosphates and can be used for efficient detection of organophosphates. However, enzymes are very fragile in nature. A series of factors can lead to enzyme inactivation, such as extreme pH (strong acid or strong base), chemical reagents, high salt concentrates, mechanical force, surfactants, long-term storage and even self-aggregation. Therefore, it is worth paying attention to improve the stability of enzymes. People have proposed and explored a variety of immobilization matrices such as diatomaceous earth, silica gel, organic polymers, natural polymers, activated carbon, mesoporous silica, and carbon nanotubes, but the specific surface area of ​​these carriers is not enough to immobilize more enzymes, and there are not many types, poor designability, and limited choices. Therefore, metal-organic framework compounds (MOFs) are considered to be a new hope for immobilizing enzymes. Compared with traditional immobilization matrices, MOFs have the advantages of larger specific surface area and higher porosity, diverse types and structures, strong adsorption affinity, good thermal stability, and rich organic ligands and metal nodes. They are very suitable as carriers of biosensitive substances in biosensors, and have attracted more and more attention in the field of enzyme immobilization.

[0006] Metal-Organic Frameworks (MOFs) are highly porous crystalline materials composed of organic ligands and metal ions / metal clusters through coordination bonds. They combine the inherent advantages of flexible organic materials and rigid inorganic systems. The main ones are ZIF-8, UiO-66, MIL-101, PCN-224, etc. ZIF-8 is a representative zeolitic imidazole MOFs material. It is a new type of porous material with a zeolite topological structure formed by the coordination of Zn with N in the bridging ligand imidazole. It is mostly used in gas storage and adsorption or as a drug carrier for drug adsorption and sustained release. Since the internal structure of ZIFs is similar to the skeleton structure of zeolite materials, its structure will not deform or collapse due to boiling treatment in water or some organic solvents. It has excellent chemical stability and hydrothermal stability and can provide good selectivity for target analytes.

[0007] At present, although electrochemical biosensors have been reported in the literature, there has been no report on electrochemical bioenzyme sensors that use MOFs as immobilization materials to immobilize acetylcholinesterase for detecting isothiocarb. Summary of the invention

[0008] In view of the above technical problems to be solved, the present invention provides an electrochemical enzyme biosensor based on the metal organic framework material ZIF8 and its application.

[0009] In order to achieve the above objectives, the present invention provides an electrochemical enzyme biosensor based on the metal organic framework material ZIF8, wherein the electrochemical enzyme biosensor uses a glassy carbon electrode as a working electrode, a complex including GR, ZIF8 and AChE is modified on the surface of the glassy carbon electrode, and is encapsulated with CS.

[0010] Based on a general technical concept, the present invention also provides an application of the electrochemical enzyme biosensor in detecting isocarbophos.

[0011] The above application, further, the method of the application is:

[0012] S1, performing an electrochemical DPV test on the electrochemical enzyme biosensor in a PBS solution containing 1 mM thioacetylcholine chloride to obtain an initial response signal I0;

[0013] S2, taking out the electrochemical enzyme biosensor and immersing it in a sample solution to be tested;

[0014] S3, taking out, placing in PBS solution containing 1 mM ATCl, and performing electrochemical DPV test to obtain the response curve I1 after inhibition;

[0015] S4. Calculate the inhibition rate according to the inhibition rate calculation formula as follows:

[0016]

[0017] Wherein, I0 is the initial response current value of the sensor catalyzing the substrate, and I1 is the response current value of the sensor catalyzing the substrate after being inhibited by ICP treatment;

[0018] S5. Calculate the linear regression equation between the concentration inhibition rate of isocarbothion in the sample and the logarithm of the ICP concentration according to the linear fitting equation, and calculate the concentration of isocarbothion in the test solution.

[0019] In the above application, further, the linear regression equation after fitting in S5 is:

[0020] Inhibition (%) = 0.1431 gC ICP +0.284.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] 1. The electrochemical biosensor GCE / GR-ZIF8-AChE-CS of the present invention has a simple structure and is prepared by a one-pot method, and the preparation method is simple and convenient.

[0023] 2. The electrochemical biosensor GCE / GR-ZIF8-AChE-CS of the present invention uses the metal organic framework compound ZIF8 as the loading material, which has good biological inertness and large loading capacity.

[0024] 3. The electrochemical biosensor GCE / GR-ZIF8-AChE-CS of the present invention has high sensitivity for isocabophos detection. The linear detection range of isocabophos (ICP, molecular weight 289.3) is 0.5-100 ng / mL (1.73-345.7 nM) and the detection limit is 0.179 ng / mL (0.62 nM). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0026] Figure 1 Schematic diagram of the preparation and detection process of the GCE / GR-ZIF8-AChE-CS sensor of Example 1 of the present invention.

[0027] Figure 2 The DPV response results of the sensor of Example 1 of the present invention after being inhibited by different ICP concentrations in a PBS solution containing ATCl.

[0028] Figure 3 This is a calibration curve diagram of the logarithm of the concentration of isocarbophos and the inhibition rate detected by the sensor in Example 1 of the present invention.

[0029] Figure 4 The electrochemical performance results of the sensors of Example 1 and Comparative Examples 1, 2, and 3 of the present invention are shown in Figure 1, where a is the cyclic voltammetry curves of different modified electrodes and b is the AC impedance diagram.

[0030] Figure 5 DPV response curves of enzyme electrodes modified with different nanomaterials in 0.1 M PBS buffer solution containing 1 mM ATCl. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby.

[0032] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

[0033] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0034] Example

[0035] The materials and instruments used in the following examples are all commercially available.

[0036] Embodiment 1:

[0037] An electrochemical enzyme biosensor for detecting isothiocarb is provided in the present invention. AChE is loaded on a glassy carbon electrode (GCE) using ZIF8, and graphene (GR) is used to improve the conductivity and enhance the affinity of the immobilized enzyme, and chitosan (CS) is used for encapsulation. The structure is as follows: a glassy carbon electrode is used as a working electrode, a complex including GR, ZIF8 and AChE is modified on the surface of the glassy carbon electrode, and CS is used for encapsulation, and is represented by GCE / GR-ZIF8-AChE-CS. The preparation method and detection process thereof are shown in Figure 1 .

[0038] Its preparation method is:

[0039] (1) Synthesis of GR-ZIF8-AChE-CS complex: The GR-ZIF8-AChE-CS complex was prepared by a one-pot method. The specific steps are as follows:

[0040] 1.1. Disperse the metal organic framework compound ZIF-8 powder evenly in PBS buffer and ultrasonicate for 30 min to obtain a ZIF-8 dispersion with a concentration of 3.5 mg / mL.

[0041] 1.2. Use a pipette to measure 2 μL of ZIF-8 dispersion and 2 μL of 4 μg / mL graphene dispersion, and then add 2 μL of 0.2 U / μL acetylcholinesterase (AChE) buffer solution and 1 μL of 0.2% chitosan (CS) solution at pH 5.0 into a 0.5 mL centrifuge tube. Use a oscillating mixer to completely mix the liquid in the centrifuge tube (oscillation time 30 s) to obtain a GR-ZIF8-AChE-CS complex.

[0042] (2) Construction of electrochemical enzyme sensor:

[0043] 2.1. After pretreatment by polishing and cleaning, the glassy carbon electrode (GCE) was cleaned and dried with nitrogen.

[0044] 2.2. Use a pipette to take 7 μL of the above GR-ZIF8-AChE-CS complex and evenly apply it on the clean GCE surface. Store it in a 4°C refrigerator for 4 hours to dry, and then you can get a GCE / GR-ZIF8-AChE-CS sensor.

[0045] An application of the electrochemical enzyme biosensor of Example 1 in detecting isocarbophos, and its application method is: analyzing and characterizing the electrochemical properties of the electrode and the response current to the concentration of the substrate acetylthiocholine chloride (ATCl) by electrochemical testing methods such as electrochemical differential pulse voltammetry (DPV), and using the enzyme inhibition method to achieve efficient and sensitive detection of isocarbophos (ICP).

[0046] The specific detection steps are:

[0047] (1) The stock solution of carbofuran pesticide was diluted into a standard solution with gradient concentration (0.5-100 ng / mL), and the electrochemical enzyme biosensor of Example 1 was subjected to electrochemical DPV test in a PBS solution containing 1 mM acetylthiocholine chloride (ATCl) (potential range of 0.3-1.1 V, pulse width of 0.05 s, pulse period of 0.02 s, modulation amplitude of 0.05 V, and standing time of 2 s) to obtain an initial response signal I0. Figure 2 The DPV response results of the sensor in a PBS solution (pH = 7.4) containing 1 mM ATCl after being inhibited by different ICP concentrations (0.5, 1, 5, 10, 50, 100 ng / mL).

[0048] (2) Take out the sensor, clean it with deionized water, and immerse it in the sample solution for 15 minutes.

[0049] (3) After the sensor surface was taken out and cleaned with deionized water, it was placed in a PBS solution containing 1 mM ATCl for electrochemical DPV testing to obtain the response curve I1 after inhibition.

[0050] (4) The inhibition rate is calculated according to the inhibition rate calculation formula as follows:

[0051]

[0052] Wherein, I0 is the initial response current value of the sensor catalyzing the substrate, and I1 is the response current value of the sensor catalyzing the substrate after being inhibited by ICP treatment.

[0053] (5) According to the linear fitting equation, the concentration inhibition rate of isocarbophos in the sample is calculated to be linearly related to the logarithm of the ICP concentration. Figure 3 is the calibration curve of the logarithm of carbofuran concentration and inhibition rate, and the fitted equation is:

[0054] Inhibition (%) = 0.1431 gC ICP +0.284

[0055] Test principle: Organic phosphorus detection is carried out by competitive method. Acetylcholinesterase combines with isocarbothion in the sample, and the remaining acetylcholinesterase catalyzes acetylcholine thiochloride ATCl to obtain a current signal. The peak current has a positive correspondence with the remaining acetylcholinesterase. Quantitative detection of isocarbothion is achieved according to the degree of inhibition of the signal by different isocarbothion concentrations.

[0056] From the above results, it can be seen that the linear detection range of the electrochemical enzyme biosensor of Example 1 for isocarbophos is 0.5-100 ng / mL (1.73-345.7 nM), and the detection limit is 0.179 ng / mL (0.62 nM).

[0057] Comparative Example 1: Glassy Carbon Electrode (GCE)

[0058] Comparative Example 2: ZIF8 was evenly dropped on the clean GCE surface, and the GCE / ZIF8 sensor was obtained by storing it in a 4°C refrigerator for 4 hours to dry it.

[0059] Comparative Example 3: The ZIF8 and graphene (GR) composite was evenly drop-coated on the clean GCE surface, and then stored in a 4°C refrigerator for 4 hours to dry, thereby obtaining a GCE / ZIF8-GR sensor.

[0060] Experiment 1: The electrochemical properties of the modified electrode surfaces of Example 1 and Comparative Examples 1, 2, and 3 were studied and analyzed by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

[0061] The electrodes of Example 1 and Comparative Examples 1, 2 and 3 were respectively tested in a 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] (1:1) mixed solution containing 0.1 M KCl to obtain cyclic voltammetry curves.

[0062] Figure 4The electrochemical performance results of the sensor of Example 1 of the present invention and Comparative Examples 1, 2, and 3. As can be seen from Figure a, the cyclic voltammetry curve results measured by different material modified electrodes all show a pair of reversible redox peaks, and the bare glassy carbon electrode has a strong redox peak. After adding ZIF8 nanomaterials, the redox current obtained is reduced, which is due to the poor conductivity of ZIF-8. After further adding graphene to modify the electrode, the redox current value increases, because the graphene with good conductivity can not only improve the properties of the electrode surface, but also enhance electron transfer. However, after further using AChE-CS mixed solution and the above-mentioned material composite modified electrode, the enzyme electrode appears to have a phenomenon of significantly reduced redox current, and the reversibility of the cyclic voltammetry curve also deteriorates. This is mainly because AChE is a protein molecule that does not have conductive properties, and the conductivity of CS is also weak, so the diffusion and electron transfer of the redox probe are hindered, so that the redox current is reduced, and the result also illustrates the successful fixation of AChE on the electrode surface.

[0063] The results of the above cyclic voltammetry were further verified by EIS in Figure b. The electron transfer resistance of the electrode can be represented by the semicircle diameter of the high-frequency region of the Nyquist plot. The bare glassy carbon electrode has a low resistance of 174.5Ω, while the resistance of GCE / ZIF8 increases to 677Ω. When GR is added, the resistance drops to 470.9Ω, indicating that the GR nanomaterial with good conductivity can not only effectively promote the transfer of electrons, but also reduce the interface resistance. When the GR-ZIF8-AChE-CS / GR composite material is fixed to the GCE surface, the resistance increases to 981.2Ω. This is because AChE is a non-conductive biomacromolecule that hinders electron transfer, thereby increasing the resistance of the electrode surface. In summary, the conclusions of the AC impedance test are consistent with those of the cyclic voltammetry test, proving the effective fixation of each material on the electrode surface.

[0064] Experiment 2: Study on the response of different enzyme electrodes to substrates:

[0065] GCE / AChE-CS, GCE / ZIF8-AChE-CS and GCE / GR-ZIF8-AChE-CS were constructed according to the method of Example 1, and the responses of different enzyme electrodes to the substrate ATCl were investigated by differential pulse voltammetry (DPV) in a 0.1 M PBS buffer solution containing 1 mM ATCl.

[0066] Figure 5The DPV response curves of GCE / AChE-CS, GCE / ZIF8-AChE-CS and GCE / GR-ZIF8-AChE-CS in 0.1M PBS buffer solution containing 1mM ATCl are shown in the figure. As can be seen from the figure, we used phosphate buffer containing a certain amount of substrate ATCl as the electrolyte solution, and all three electrodes showed obvious oxidation currents, which were generated by the oxidation of thiocholine, the catalytic hydrolysis product of the substrate ATCl. By comparing the differences between the three response currents, it can be found that when only AChE-CS is used to construct the enzyme sensor, the oxidation current generated is the smallest, and the oxidation current of the enzyme sensor constructed with the ZIF8-AChE-CS composite material is significantly increased. When graphene (GR) is added, the oxidation current is further increased, indicating that it can effectively enhance the catalytic activity of AChE.

[0067] The above experimental results fully demonstrate that compared with using ZIF-8 or ZIF8-GR composite materials alone to construct enzyme sensors, they can better maintain the activity of AChE and amplify the response electrical signals, which can effectively increase the electron transfer rate and improve the detection sensitivity of the sensor.

[0068] Embodiment 2:

[0069] An application of the electrochemical enzyme biosensor of Example 1 in detecting ICP in an environment. Application method: Prepare a 1.0 g / L standard solution of carbofuran with acetone, dilute it with ultrapure water, and obtain a working standard solution with a gradient concentration (0.5-100 ng / mL). Purchase fresh Chinese cabbage from the local market, weigh and take about 20 g of the sample, and shake it with a mixture of 10 mL of water and 10 mL of acetone for 10 minutes. Filter after ultrasonic stirring for 30 minutes. The filtrate is concentrated by rotary evaporation and then diluted to 100 times. Then, different concentrations of ICP are added to the Chinese cabbage or tap water solution to prepare spiked samples for recovery research. The added sample is then detected using a biosensor. The measured results are substituted into the standard curve to calculate the detection concentration, and then divided by the actual concentration to calculate the recovery rate.

[0070] In order to verify the application value of the electrochemical enzyme biosensor GCE / GR-ZIF8-AChE-CS in Example 1 in actual detection, it was used to detect the residual ICP in Chinese cabbage and tap water. The accuracy of the method was evaluated by the recovery experiment of the labeled sample. In order to make the analysis more comprehensive, 0.001, 0.01 and 0.1 μg / mL of ICP were added within the detection range of the sensor, corresponding to low, medium and high concentrations respectively. The test results are listed in Table 1.

[0071] Table 1: Recovery results of GCE / GR-ZIF8-AChE-CS for detecting ICP of Chinese cabbage and tap water

[0072]

[0073] From the results in Table 1, it can be seen that the recovery rate (measured concentration divided by actual concentration) of the diluted cabbage extract sample and tap water is 88.1%-122.4%, and the relative standard deviation is 1.7%-5.6%. The results show that the electrochemical enzyme biosensor of Example 1 is reliable for detecting ICP in actual samples, has high accuracy and reproducibility, and has broad development prospects in the fields of food safety and water monitoring.

[0074] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as above in the preferred embodiment, it is not used to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention, still fall within the scope of protection of the technical solution of the present invention.

Claims

1. An application of an electrochemical enzyme biosensor in detecting isocarbophos, characterized in that: The structure of the electrochemical enzyme biosensor is GCE / GR-ZIF8-AChE-CS, and the preparation method of the electrochemical enzyme biosensor comprises: (1) The GR-ZIF8-AChE-CS complex was prepared by a one-pot method: the metal organic framework compound ZIF-8 powder was evenly dispersed in PBS buffer and ultrasonicated for 30 min to obtain a ZIF-8 dispersion with a concentration of 3.5 mg / mL; 2 μL of ZIF-8 dispersion, 2 μL of graphene dispersion with a concentration of 4 μg / mL, 2 μL of 0.2 U / μL acetylcholinesterase buffer solution and 1 μL of 0.2% chitosan solution at pH 5.0 were added to a 0.5 mL centrifuge tube, and the liquid in the centrifuge tube was completely mixed using a vortex mixer for 30 s to obtain the GR-ZIF8-AChE-CS complex; (2) After the GCE is polished and cleaned, it is cleaned and dried with nitrogen. 7 μL of the GR-ZIF8-AChE-CS complex is evenly drop-coated on the surface of the GCE and stored in a refrigerator at 4°C for 4 h to dry. The GCE / GR-ZIF8-AChE-CS sensor is obtained. The method of application is: S1, performing an electrochemical DPV test on the electrochemical enzyme biosensor in a PBS solution containing thioacetylcholine chloride to obtain an initial response signal I0; S2, taking out the electrochemical enzyme biosensor and immersing it in a sample solution to be tested; S3, taking out, placing in PBS solution containing ATCl to perform electrochemical DPV test to obtain the response curve I1 after inhibition; S4. Calculate the inhibition rate according to the inhibition rate calculation formula as follows: ； Wherein, I0 is the initial response current value of the sensor catalyzing the substrate, and I1 is the response current value of the sensor catalyzing the substrate after being inhibited by ICP treatment; S5. Calculate the linear regression equation between the concentration inhibition rate of isocarbophos in the sample and the logarithm of the ICP concentration according to the linear fitting equation, and calculate the concentration of isocarbophos in the test solution; the linear regression equation after fitting in S5 is: Inhibition%=(0.143lgC ICP + 0.284)×100%。

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