Preparation method and application of electrochemical biosensor based on neuraminidase

Abstract

The invention discloses a preparation method of an electrochemical biosensor based on neuraminidase, and relates to the field of electrochemical sensors. The method comprises the following steps: preparing N-Gr / MWCNTs, preparing N-Gr / MWCNTs (at) GCE, preparing Ppy / N-Gr / MWCNTs (at) GCE, and preparing NA / Ppy / N-Gr / MWCNTs (at) GCE. According to the electrochemical biosensor based on neuraminidase, MWCNTs and N-Gr are compounded to form an N-Gr / MWCNTs composite material, high-conductivity polypyrrole is polymerized on the N-Gr / MWCNTs composite material through pyrrole, the biosensor without a mediator is prepared, electrons can be directly transmitted between an electrode and a bioactive substance, and the electrochemical biosensor can be used for detecting neuraminidase. The response performance of the sensor is obviously improved; the electrochemical biosensor is based on the NA inhibition principle, is mainly used for rapidly screening antiviral components in traditional Chinese medicines, can realize rapid screening and evaluation of the antiviral components in the traditional Chinese medicines, belongs to a convenient, efficient and low-cost means, and also provides scientific basis and technical support for discovery and evaluation of active components.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and particularly relates to a preparation method and application of an electrochemical biosensor based on neuraminidase. Background Art

[0002] Influenza virus belongs to respiratory viruses, often causing severe influenza (abbreviation: "flu"), and is also one of the important causes leading to severe acute respiratory diseases and even death. After entering the 21st century, the emergence of highly pathogenic avian influenza and swine influenza has continuously threatened human life safety.

[0003] Neuraminidase (NA), as one of the three important proteins distributed on the surface of influenza virus, is a tetrameric protein present on the viral cell membrane and plays an important role in the replication and transmission of influenza virus. Since newly replicated virus particles are easily adhered to host cells by sialic acid (SA) during the process of detaching from host cells, while NA can hydrolyze SA receptors, cut off the connection between virus particles and host cells, and promote the release of progeny viruses to form new infections. Therefore, developing neuraminidase inhibitors (NAIs) to inhibit the activity of NA, prevent the cleavage of glycosidic bonds, and make the virus unable to be released from the original host cells, thereby playing a role in blocking the transfer and infection of influenza virus. Currently, the discovered neuraminidase inhibitors mainly include: SA class, cyclohexene class, and five-membered ring class. Although the above-mentioned neuraminidase inhibitors have good activities, they have many chiral centers in their structures, are relatively complex to synthesize, and in addition, the mutations of viruses and the increasingly obvious signs of influenza outbreaks have forced humans to continuously discover and develop new anti-influenza virus drugs.

[0004] Compared with western medicines, traditional Chinese medicines have advantages such as reducing adverse reactions and lowering costs. Multiple traditional Chinese medicine compound prescriptions have been proven effective and used in the clinical treatment of influenza virus. Therefore, screening out active ingredients that inhibit NA from the rich treasure house of traditional Chinese medicines and developing them into drugs for the prevention and treatment of influenza virus have become the focus of the current screening of active ingredients of traditional Chinese medicine efficacy.

[0005] With the continuous development of drug activity screening and evaluation technologies, in vitro detection technologies based on the principle of enzyme inhibition, as an analytical step before in vivo detection, play an important role in drug clinical research, thus achieving the purpose of cost savings and increased R & D success rate. Enzyme inhibition-based electrochemical sensors immobilize enzymes on the surface of electrode materials, construct a redox electron transfer platform during the enzymatic reaction, and establish the relationship between the change in electrical signal and the concentration of the target analyte through the response degree of the composite enzyme electrode to the target analyte during the reaction, thereby realizing the precise discovery and evaluation of enzyme inhibitor drugs. The parameters such as the enzymatic reaction rate, the kinetic parameters of the enzyme, the half inhibitory concentration (IC 50 50) of the enzyme to the target, the inhibition rate of known drugs or lead compounds to be developed on the enzyme, etc. are often measured by immobilizing enzymes, so as to detect marketed drugs and screen out small molecule compounds that are clinically equivalent to or superior to marketed drugs. Enzyme inhibition-based electrochemical sensors have both the characteristics of miniaturization and high selectivity of biosensors, and also reflect the advantages of rapidity and high sensitivity of electrochemical analysis methods, and have positive significance in drug design, drug detection, molecular screening, etc. Summary of the Invention

[0006] The purpose of the present invention is to propose a preparation method and application of an electrochemical biosensor based on neuraminidase. This electrochemical biosensor has excellent electrical conductivity, high specific surface area, and efficient electron transfer ability, and is used for rapid screening of antiviral components in traditional Chinese medicine (TCM).

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: In the first aspect, a preparation method of an electrochemical biosensor based on neuraminidase proposed by the present invention includes the following steps: S1. Preparation of N-Gr / MWCNTs Add multi-walled carbon nanotubes (MWCNTs) and nitrogen-doped graphene (N-Gr) to N,N-dimethylformamide (DMF) solution, and ultrasonicate for 2 h to disperse them evenly to obtain N-Gr / MWCNTs composite material, and store it at 4 °C for later use; S2. Preparation of N-Gr / MWCNTs@GCE Polish a glassy carbon electrode (GCE) on suede successively with 0.3 μm and 0.05 μm alumina suspensions, then ultrasonically clean it with methanol and secondary distilled water for 10 min respectively, and then dry it at room temperature for later use; perform 20 cycles by cyclic voltammetry (CV) in 0.5 M H2SO4 to generate -COOH on the surface of GCE, take 9 μL of N-Gr-MWCNTs composite material and drop-coat it on GCE, and dry it under an infrared lamp to prepare N-Gr-MWCNTs@GCE; S3. Preparation of Ppy / N-Gr / MWCNTs@GCE Take 100 μL of pyrrole (py) with a purity of 99%, add 9.57 mL of methanol as the electropolymerization solution. Place N-Gr / MWCNTs@GCE as the base electrode in the electropolymerization solution, and scan 6 cycles using the CV method. Dry the electrode under an infrared lamp to obtain the Ppy / N-Gr / MWCNTs modified electrode; S4. Preparation of NA / Ppy / N-Gr / MWCNTs@GCE Prepare a 50 mM EDH / NHS mixture in a 50 mM MES buffer solution with a pH of 5.5 to obtain the EDC / NHS activation buffer. Take 2 μL of the EDC / NHS activation buffer and drop it on Ppy / N-Gr / MWCNTs@GCE for reaction for 1 h. Then rinse the electrode surface with a MES buffer solution with a pH of 5.5 and a PBS buffer solution with a pH of 7.4. Drop 8 μL of the NA solution on Ppy / N-Gr / MWCNTs@GCE to prepare the electrochemical biosensor NA / Ppy / N-Gr / MWCNTs@GCE.

[0008] As a preferred embodiment of the present invention, in step S1, the dosage ratio of MWCNTs, N-Gr, and DMF is 10 mg: 10 mg: 5 mL.

[0009] As a preferred embodiment of the present invention, in step S2, the scanning rate of the CV method is 150 mV·s -1 , and the scanning potential range is 0.3 V to 1.5 V.

[0010] As a preferred embodiment of the present invention, in step S3, the scanning rate of the CV method is 50 mV·s -1 , and the scanning potential range is 1.2 V to -0.1 V.

[0011] As a preferred embodiment of the present invention, in step S4, the preparation method of the NA solution is to first weigh 1.0 mg of NA dry powder with 250 U / mg and dilute it with deionized water to 1 mg·mL -1 .

[0012] In the second aspect, the present invention proposes the application of the above-mentioned electrochemical biosensor prepared by the preparation method in the rapid screening of antiviral active ingredients in traditional Chinese medicine. Compared with the prior art, the beneficial technical effects of the present invention: 1. In the electrochemical biosensor based on neuraminidase constructed in the present invention, MWCNTs and N-Gr are combined to form an N-Gr / MWCNTs composite material, which overcomes the problem that the single MWCNTs material affects the loading of modified substances on its surface due to its relatively small radius of curvature. It also overcomes the problem that the single N-Gr material, due to its single-layer or few-layer carbon atom thickness and large specific surface area, is prone to stacking and agglomeration in solution, reducing its specific surface area and also affecting its performance as a catalyst or other applications. The N-Gr / MWCNTs composite material effectively prevents the stacking between N-Gr lamellae and also builds a bridge for electron transfer between N-Gr lamellae, greatly increasing the specific surface area and conductivity, thereby improving its electrocatalytic ability. By polymerizing highly conductive polypyrrole (Ppy) on the N-Gr / MWCNTs composite material using pyrrole (py), a mediator-free biosensor is fabricated, enabling direct electron transfer between the electrode and bioactive substances, thus significantly improving the response performance of the sensor.

[0013] 2. The electrochemical biosensor proposed in the present invention is based on the NA inhibition principle and is mainly used for rapid screening of antiviral components in traditional Chinese medicine (TCM). It can achieve rapid screening and evaluation of components with antiviral effects in traditional Chinese medicine, which is a convenient, efficient and low-cost method, and also provides a scientific basis and technical support for the discovery and evaluation of active components. Description of the Drawings

[0014] Figure 1 It is a physical and chemical structure characterization diagram of the materials in the embodiments of the present invention. Among them, A is the infrared spectra of (a) MWCNTs, (b) N-Gr, (c) N-Gr / MWCNTs, (d) Ppy / N-Gr / MWCNTs; B is the Raman spectra of (a) MWCNTs, (b) N-Gr / MWCNTs, (c) Ppy / N-Gr / MWCNTs, (d) N-Gr; C is the XRD diagrams of (a) N-Gr, (b) MWCNTs, (c) N-Gr / MWCNTs, (d) Ppy / N-Gr / MWCNTs; Figure D is the SEM diagram of MWCNTs@GCE; Figure E is the SEM diagram of N-Gr@GCE; Figure F is the SEM diagram of N-Gr / MWCNTs@GCE; G is the SEM diagram of Ppy / N-Gr / MWCNTs@GCE.

[0015] Figure 2It is the electrochemical characterization diagram of the materials in the embodiments of the present invention. Among them, A are the CV curves of (a) GCE, (b) MWCNTs@GCE, (c) N-Gr@GCE, (d) N-Gr / MWCNTs@GCE, (e) Ppy / N-Gr / MWCNTs@GCE; B are the EIS curves of Ppy / N-Gr / MWCNTs@GCE, N-Gr / MWCNTs@GCE, MWCNTs@GCE, N-Gr@GCE, GCE; C are the CV curves of Ppy / N-Gr / MWCNTs@GCE at scanning rates of 20 - 400 mV·s-1; D is the relationship between the logarithm of the peak current and the logarithm of the scanning rate; E are the chronocoulometry curves of (a) GCE, (b) N-Gr@GCE, (c) MWCNTs@GCE, (d) N-Gr / MWCNTs@GCE, (e) Ppy / N-Gr / MWCNTs@GCE; F are the Q-t1 / 2 curves of (a) GCE, (b) N-Gr@GCE, (c) MWCNTs@GCE, (d) N-Gr / MWCNTs@GCE, (e) Ppy / N-Gr / MWCNTs@GCE.

[0016] Figure 3 It is the practicality test result of NA / Ppy / N-Gr / MWCNTs@GCE in the embodiments of the present invention. Among them, A are the verification experiment results with and without OP, B are the repeatability experiment results of NA / Ppy / N-Gr / MWCNTs@GCE, C are the reproducibility experiment results of NA / Ppy / N-Gr / MWCNTs@GCE, and D are the stability detection results of NA / Ppy / N-Gr / MWCNTs@GCE.

[0017] Figure 4 It is the current response of five compounds, namely (A) baicalein, (B) baicalin, (C) rutin, (D) matrine, and (E) imperatorin A, to NA at different concentrations in the embodiments of the present invention.

[0018] Figure 5 It is the inhibition rate of five compounds, namely baicalein, baicalin, rutin, matrine, and imperatorin A, to NA at the same concentration in the embodiments of the present invention.

[0019] Figure 6 It is the inhibition rate of five compounds, namely baicalein, baicalin, rutin, matrine, and imperatorin A, to NA at different concentrations in the embodiments of the present invention.

[0020] Figure 7 It is the dose-effect curves of five compounds, namely baicalein, baicalin, rutin, matrine, and imperatorin A, at different concentration logarithms in the embodiments of the present invention. Detailed implementation mode

[0021] The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0022] In the present invention, unless otherwise specified, the raw materials involved are commercially available products well-known in the art.

[0023] Embodiment A preparation method of an electrochemical biosensor based on neuraminidase includes the following steps: S1. Preparation of N-Gr / MWCNTs Add 10 mg of multi-walled carbon nanotubes (MWCNTs) and 10 mg of nitrogen-doped graphene (N-Gr) to 5 mL of N,N-dimethylformamide (DMF) solution, and ultrasonicate for 2 h to disperse evenly to obtain the N-Gr / MWCNTs composite material, store it at 4 °C for later use; S2. Preparation of N-Gr / MWCNTs@GCE Polish the glassy carbon electrode (GCE) on suede successively with 0.3 μm and 0.05 μm alumina suspensions, then ultrasonically clean it with methanol and secondary distilled water for 10 min respectively, and then dry it at room temperature for later use; perform 20 cycles by cyclic voltammetry (CV) in 0.5 M H2SO4, the scanning rate of CV scanning is 150 mV·s -1 , the scanning potential range is 0.3 V to 1.5 V to generate -COOH on the surface of GCE, take 9 μL of the N-Gr-MWCNTs composite material and drop-coat it on GCE, and dry it under an infrared lamp to obtain N-Gr-MWCNTs@GCE; S3. Preparation of Ppy / N-Gr / MWCNTs@GCE Take 100 μL of pyrrole (py) with a purity of 99%, add 9.57 mL of methanol as the electropolymerization solution, place the N-Gr / MWCNTs@GCE as the working electrode in the electropolymerization solution, and scan 6 cycles by CV method, the scanning rate of CV scanning is 50 mV·s -1 , the scanning potential range is 1.2 V to -0.1 V, and dry the electrode under an infrared lamp to obtain the Ppy / N-Gr / MWCNTs modified electrode; S4. Preparation of NA / Ppy / N-Gr / MWCNTs@GCE Prepare a 50 mM EDH / NHS mixture in a 50 mM MES buffer solution with pH = 5.5 to obtain an EDC / NHS activation buffer. Take 2 μL of the EDC / NHS activation buffer and drop it on Ppy / N-Gr / MWCNTs@GCE for reaction for 1 h. Then rinse the electrode surface with MES buffer at pH = 5.5 and PBS buffer at pH = 7.4. Weigh 1.0 mg of NA dry powder with an activity of 250 U / mg and dilute it with deionized water to 1 mg·mL -1， to obtain an NA solution. Drop 8 μL of the NA solution on Ppy / N-Gr / MWCNTs@GCE to fabricate an electrochemical biosensor NA / Ppy / N-Gr / MWCNTs@GCE.

[0024] In this example, after constructing Ppy / N-Gr / MWCNTs@GCE, infrared spectroscopy, Raman spectroscopy, XRD, and SEM electron microscopy characterization were carried out on it, and the results are as Figure 1 shown.

[0025] Figure 1 The test results show that the N-Gr / MWCNTs composite material, N-Gr / MWCNTs@GCE, and Ppy / N-Gr / MWCNTs@GCE were all successfully prepared.

[0026] Subsequently, electrochemical characterization was carried out to characterize the preparation and performance of the electrode through electrochemical testing methods. Electrochemical testing methods include cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and differential pulse voltammetry (DPV). The measurement of CV was carried out at a sensitivity of 10 -4 , a potential range of -0.4 V to 0.8 V, and a scan rate of 0.10 V·s -1 . The measurement frequency parameter of EIS was 1 Hz to 1×10 -6 Hz, the rest time was 2 s, and the voltage amplitude was 0.005 V. The sensitivity of DPV was 10-4, the potential range was 0.4 V to -0.4 V, the rest time was 2 s, the voltage amplitude was 0.05 V, the pulse period was 0.5 s, and the sampling width was 0.0167 s. The test bottom solution was a mixed solution containing 2.0 mM [Fe(CN)6] 3- / 4- and 0.1 M KCl. The test results are as Figure 2 shown.

[0027] Figure 2The test results show that: the N-Gr / MWCNTs composite material, N-Gr / MWCNTs@GCE, and Ppy / N-Gr / MWCNTs@GCE were all successfully prepared, and we calculated that the electrochemically active surface area of Ppy / N-Gr / MWCNTs@GCE was 0.365 cm 2 , the electrochemically active surface area of the bare GCE was 0.096 cm 2 , the electrochemically active surface area of N-Gr@GCE was 0.105 cm 2 , the electrochemically active surface area of MWCNTs@GCE was 0.152 cm 2 , the electrochemically active surface area of N-Gr / MWCNTs@GCE was 0.245 cm 2 . This data confirmed that Ppy / N-Gr / MWCNTs@GCE had more excellent electron transfer ability compared with the bare GCE, N-Gr@GCE, MWCNTs@GCE, and N-Gr / MWCNTs@GCE, and could achieve good current response.

[0028] Under the optimized conditions, the verification, repeatability, reproducibility, and stability of the electrochemical biosensor NA / Ppy / N-Gr / MWCNTs@GCE were detected. During the detection, 5 mg of oseltamivir phosphate (OP) powder was weighed and dissolved in 5 mL of deionized water. 1 mL of the solution system was transferred to an EP tube, and the prepared NA / Ppy / N-Gr / MWCNTs@GCE was inserted into 1 mL of the OP solution and inhibited at 37 °C for 16 min as the control group. After the inhibition ended, its peak current was measured by the CV method; separately, 10 mg of the active ingredient to be tested was dissolved in 1 mL of deionized water, and the prepared NA / Ppy / N-Gr / MWCNTs@GCE was inserted into 1 mL of the solution of the ingredient to be tested and inhibited at 37 °C for 16 min as the experimental group. The test results are as Figure 3 shown.

[0029] Figure 3 The test results show that: NA / Ppy / N-Gr / MWCNTs@GCE was successfully prepared and had good repeatability, reproducibility, and stability.

[0030] Under the optimized conditions, the screening of NAIs was carried out, and the current responses of the NA / Ppy / N-Gr / MWCNTs electrode to NA (1 mg mL -1 ) in the presence of baicalein, baicalin, rutin, matrine, and levistilide A were studied. The concentration of each compound was 1 μg·mL -1, 10 μg·mL -1 , 100 μg·mL -1 , 1000 μg·mL -1 , 10000 μg·mL -1 , the results are as Figure 4 , It can be seen that its peak current decreases with the increase of the inhibitor concentration, which further indicates that this compound is an electroactive component.

[0031] Using NA as the recognition element and OP as the positive control group, 5 compounds were screened by enzyme electrochemical biosensing technology, namely rutin, baicalein, baicalin, matrine, and angelicone A. Each sample was taken at a concentration of 1000 μg·mL -1 for primary screening. As Figure 5 shown, the results indicate that among them, baicalein and baicalin have inhibitory active components on NA to varying degrees, rutin has extremely weak activity, and matrine and angelicone A have no activity.

[0032] To exclude false positive and false negative results, inhibitory rate experiments were carried out on samples with different concentrations of 1 μg·mL -1 , 10 μg·mL -1 , 100 μg·mL -1 , 1000 μg·mL -1 , 10000 μg·mL -1 . The results show that the inhibition rate increases with the increase of the inhibitor concentration. See Figure 6 and Table 1. It can be preliminarily shown that the inhibitory effect is: baicalein > baicalin > rutin > matrine > angelicone A.

[0033] Table 1 Inhibition rates of samples with different concentrations (%)

[0034] From Figure 6 the experimental data, it was found that the inhibition rates of rutin and matrine did not reach 50%, so their IC 50 values could not be calculated. Further using the dose-effect curve, as shown in Figure 7 and Table 2, the IC 50 values of each inhibitor were calculated and their inhibitory effects were verified. After calculation, the IC 50 values of baicalein and baicalin were respectively: 55 μg·mL -1 , 331 μg·mL -1 . From Figure 7It can be seen that the dose-effect curve of baicalein is steep, indicating that its inhibitory effect increases significantly with the increase of concentration; the curve of baicalin rises gently and its potency is lower than that of baicalein; rutin rises slowly in an almost linear manner and its activity is extremely weak; [the curve of matrine] rises slowly linearly and has no actual inhibitory activity. That is, the order of potency is: baicalein > baicalin > rutin > matrine > ligustilide A, and the 50 smaller the IC value, the stronger the inhibitory ability.

[0035] Table 2 Comprehensive ranking of the inhibitory effects of each active ingredient

[0036] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the technical solutions and concepts of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of a neuraminidase-based electrochemical biosensor, characterized in that, It includes the following steps: S1. Preparation of N-Gr / MWCNTs Add multi-walled carbon nanotubes and nitrogen-doped graphene into N,N-dimethylformamide solution, ultrasonically disperse them evenly for 2 h to obtain N-Gr / MWCNTs composite material, and store it at 4 °C for standby; S2. Preparation of N-Gr / MWCNTs@GCE Polish the glassy carbon electrode on suede successively with 0.3 μm and 0.05 μm Al2O3 suspension, then ultrasonically clean it with methanol and secondary distilled water for 10 min respectively, and then dry it at room temperature for standby; Perform 20 cycles by CV method in 0.5 M H2SO4 to generate -COOH on the surface of GCE. Take 9 μL of N-Gr-MWCNTs composite material and drop-coat it on GCE, and dry it under an infrared lamp to prepare N-Gr-MWCNTs@GCE; S3. Preparation of Ppy / N-Gr / MWCNTs@GCE Take 100 μL of pyrrole with a purity of 99%, add 9.57 mL of methanol as the electropolymerization solution, place N-Gr / MWCNTs@GCE as the base electrode in the electropolymerization solution, scan it 6 circles by CV method, and dry the electrode under an infrared lamp to prepare Ppy / N-Gr / MWCNTs modified electrode; S4. Preparation of NA / Ppy / N-Gr / MWCNTs@GCE Prepare a 50 mM EDH / NHS mixture in a 50 mM MES buffer solution with pH = 5.5 to obtain an EDC / NHS activation buffer solution. Take 2 μL of the EDC / NHS activation buffer solution and drop-coat it on Ppy / N-Gr / MWCNTs@GCE to react for 1 h. Then rinse the electrode surface with MES buffer solution with pH = 5.5 and PBS buffer solution with pH = 7.

4. Drop-coat 8 μL of NA solution on Ppy / N-Gr / MWCNTs@GCE to prepare an electrochemical biosensor NA / Ppy / N-Gr / MWCNTs@GCE.

2. The preparation method of an electrochemical biosensor based on neuraminidase according to claim 1, characterized in that: In step S1, the dosage ratio of MWCNTs, N-Gr, and DMF is 10 mg:10 mg:5 mL.

3. The preparation method of an electrochemical biosensor based on neuraminidase according to claim 1, characterized in that: In step S2, the scanning rate of the CV method is 150 mV·s -1 , and the scanning potential range is 0.3V to 1.5V.

4. The preparation method of an electrochemical biosensor based on neuraminidase according to claim 1, characterized in that: In step S3, the scanning rate of the CV method is 50 mV·s -1 , and the scanning potential range is 1.2 V to -0.1 V.

5. The preparation method of an electrochemical biosensor based on neuraminidase according to claim 1, characterized in that: In step S4, the method for preparing the NA solution is to first weigh 1.0 mg of NA dry powder with a concentration of 250 U / mg and dilute it with deionized water to 1 mg·mL -1 .

6. Application of the electrochemical biosensor prepared by the preparation method according to any one of claims 1-5 in the rapid screening of antiviral active ingredients in traditional Chinese medicine.

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