Preparation method of PANIAuNPs-based enzyme biosensor for rapidly detecting CPF residues

Abstract

The invention discloses a preparation method of a CPF residue rapid detection enzyme biosensor based on PANI / AuNPs. The preparation method comprises the following steps: preparation of an AuNPs / GCE modified electrode, preparation of a PANI / AuNPs / GCE modified electrode and preparation of an AChE / PANI / AuNPs / GCE biosensor. The enzyme biosensor based on the PANI / AuNPs composite material, which is constructed by the invention, is of great importance for medicinal farmers and related institutions to master the development mode of medicinal plant diseases, and is beneficial to realizing a timely early warning mechanism, reducing the cost expenditure in the production process, improving the quality of medicinal materials and increasing economic benefits; and reference and technical support are expected to be provided for development of a novel, portable and on-site pesticide residue law enforcement detection technology.

Description

Technical Field

[0001] The invention belongs to the field of Chinese medicinal material planting, on-site rapid detection of pesticide residues and enzyme biosensors, and specifically relates to a preparation method of a PANI / AuNPs-based CPF residue rapid detection enzyme biosensor. Background Art

[0002] Chinese medicine is a natural medicine and its preparations used to prevent and treat diseases under the guidance of traditional Chinese medicine concepts. In recent years, due to its natural properties, definite efficacy, low toxic side effects and health-preserving characteristics, Chinese medicine has been widely welcomed worldwide, promoting the rapid growth of China's Chinese medicine industry and the continuous increase in the export volume of Chinese medicinal materials. According to statistics from the World Health Organization, 80% of the world's population relies on Chinese medicine to maintain their health. With the growing demand for Chinese medicinal materials, wild resources have become increasingly scarce and it is difficult to meet the growing market demand, so the artificial cultivation of Chinese medicinal materials has become crucial. However, during the planting process, the use of pesticides can increase the yield, but it also brings about the problem of pesticide residues in Chinese medicinal materials, which not only damages the quality of the medicinal materials, but also may become "drug Chinese medicine", posing a threat to human health and causing adverse effects on the export and international image of Chinese medicinal materials. CPF is the most commonly used type of organophosphorus pesticides. It has the effect of inhibiting the activity of acetylcholinesterase AChE or cholinesterase ChE in the body and destroying the normal conduction of nerve impulses. Due to its low water solubility and high adsorption coefficient, CPF is likely to remain on the surface of Chinese medicinal materials for a long time, and then enter the human body through the gastrointestinal tract, skin, respiratory tract, etc. with Chinese medicinal materials, causing neurological defects, developmental and autoimmune diseases, etc., posing a serious threat to human health. Therefore, accurate and convenient detection of residual CPF in Chinese medicinal materials is crucial to ensure the safety of Chinese medicinal materials and human health, as well as the healthy and sustainable development of Chinese medicinal materials in the international market.

[0003] At present, the traditional methods for detecting chlorpyrifos mainly include gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), etc. Although these methods have high sensitivity and accuracy, the equipment is expensive, time-consuming and labor-intensive, and the detection personnel need to have certain knowledge. In addition, the sample processing of the traditional analysis method is complicated and time-consuming, and the compound may decompose before analysis, which is difficult to meet the needs of rapid on-site detection and analysis. Electrochemical enzyme biosensors have the advantages of small detection equipment size, low price, short time consumption, easy operation, high accuracy, etc., and have broad prospects in clinical diagnosis, environmental monitoring and food safety. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing a PANI / AuNPs-based CPF residue rapid detection enzyme biosensor, in order to provide reference and technical support for the development of new, portable and on-site pesticide residue law enforcement detection technology, and promote the practical application development of enzyme-based biosensors in high-quality Chinese medicinal materials planting and production and on-site rapid detection of pesticide residues.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing a PANI / AuNPs-based CPF residue rapid detection enzyme biosensor comprises the following steps: S1. Preparation of AuNPs / GCE modified electrode: Before modification, the glassy carbon electrode was polished on a smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then ultrasonically cleaned with methanol and distilled water, respectively. -1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range was -0.4-0.8 V at 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; S2. Preparation of PANI / AuNPs / GCE modified electrode: The AuNPs / GCE modified electrode obtained in S1 was placed in a solution containing 1-3 mol L -1 of hydrochloric acid and 0.1-0.3 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of , so that aniline was deposited on the AuNPs / GCE modified electrode, and the PANI / AuNPs / GCE modified electrode was obtained; S3. Preparation of AChE / PANI / AuNPs / GCE biosensor: 5-7 μL AChE solution was added dropwise to the surface of the PANI / AuNPs / GCE electrode obtained in S2, and the electrode was placed to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was then stored in a 4°C refrigerator for later use.

[0006] In order to further implement the present invention, the concentration of the AChE solution in S3 is 10 U mL -1 .

[0007] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses polyaniline (PANI) with high conductivity and biocompatibility as the electrode substrate material, and uses electrochemical deposition method to modify gold nanoparticles (AuNPs) with high conductivity and large specific surface area on the electrode surface. AuNPs form gold-ammonia bonds with the amino groups on acetylcholinesterase (AChE), and constructs an electrochemical enzyme biosensor with high sensitivity, high selectivity and rapid detection using acetylcholinesterase (AChE) as the recognition element, which is used for the detection of organophosphorus pesticides in traditional Chinese medicines, chlorpyrifos (CPF). Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were used to optimize the experimental conditions affecting the enzyme biosensor. Under the optimized experimental conditions, the sensor has a high sensitivity and high selectivity at 1×10 -3 ~1×10 1 It has good linear relationship in the ppm range and the detection limit is 7.9×10 -5 When used for actual sample detection, the sensor showed high stability, reproducibility and sensitivity in the detection of CPF pesticide residues in traditional Chinese medicine, providing an effective means for its accurate monitoring. Compared with other detection methods, the enzyme biosensor AChE / PANI / AuNPs / GCE prepared by the present invention has a wider detection range and a lower detection limit, and the sensitivity of the present invention has been greatly improved compared with the prior art.

[0008] The enzyme biosensor based on PANI / AuNPs composite material constructed in the present invention can quickly detect CPF, which is crucial for farmers and related institutions to understand the development pattern of medicinal plant diseases. It helps to realize a timely early warning mechanism, reduce cost expenditure in the production process, improve the quality of medicinal materials, and increase economic benefits. It is also expected to provide reference and technical support for the development of new, portable and on-site pesticide residue law enforcement detection technology.

[0009] The present invention provides an efficient and convenient detection method for the highly sensitive identification and detection of trace chlorpyrifos pesticide residues in traditional Chinese medicinal materials. It is also expected to provide reference and technical support for the development of new, portable and on-site pesticide residue law enforcement detection technologies, and promote the practical application and development of enzyme-based biosensors in the cultivation and production of high-quality traditional Chinese medicinal materials and on-site rapid detection of pesticide residues. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The figure is a flow chart of the preparation of the sensor of the present invention; Figure 2 The images of SEM electron microscopy, EDX spectrum and EDS mapping characterization of the PANI / AuNPs / GCE modified electrode in the experimental example of the present invention, wherein A is the SEM image of AuNPs; B is the SEM image of PANI / AuNPs; C is the EDX spectrum of PANI / AuNPs; D is the corresponding elemental mapping of PANI AuNPs; Figure 3 The electrochemical characterization results of GCE, AuNPs / GCE, PANI / AuNPs / GCE and AChE / PANI / AuNPs / GCE in the experimental examples of the present invention are shown in Table 1. -1 Contains 0.01 mmol L -1 CV curves of KCl in K3[Fe(CN)6] / K4[Fe(CN)6] solution; B is the CV curve of KCl in 4 mmol L -1 Contains 0.01 mmol L -1 EIS curves of KCl in K3[Fe(CN)6] / K4[Fe(CN)6] solution; C is the EIS curves of KCl in K3[Fe(CN)6] / K4[Fe(CN)6] solution at different scan rates (10 mVs -1 -200 mVs -1 )CV curves of PANI / AuNPs / GCE; D is the relationship between the logarithm of the peak current and the logarithm of the scan rate; E is the CV curves of PANI / AuNPs / GCE in the presence of 0.1 M KCl and 4 mM Fe(CN)6 3− / 4− Chronocoulomb curve in the probe solution; F is Qt 1 / 2 curve; Figure 4 The results of the gradient concentration sample detection of the enzyme biosensor AChE / PANI / AuNPs / GCE in the experimental example of the present invention, wherein A is the DPV curve of AChE / PANI / AuNPs / GCE detection at different concentrations; B is the standard curve of AChE / PANI / AuNPs / GCE for different concentrations of CPF; Figure 5 These are the test results of specificity, repeatability and stability of the enzyme biosensor AChE / PANI / AuNPs / GCE in the experimental example of the present invention, wherein A is the result of the test in the presence of various interfering substances; B is the result of measuring the current response on the 5th, 10th, 15th, 20th and 25th days after the modified electrode was prepared and sealed; C is the result of 5 DPV tests on the same electrode in the test solution. DETAILED DESCRIPTION

[0011] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0012] like Figure 1 As shown, a method for preparing a CPF residue rapid detection enzyme biosensor based on PANI / AuNPs comprises the following steps: S1. Preparation of AuNPs / GCE modified electrode: Before modification, the glassy carbon electrode was polished on a smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then ultrasonically cleaned with methanol and distilled water, respectively. -1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range is -0.4-0.8V at 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; S2. Preparation of PANI / AuNPs / GCE modified electrode: The AuNPs / GCE modified electrode obtained in S1 was placed in a solution containing 1-3 mol L -1 of hydrochloric acid and 0.1-0.3 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of 1.5 % to deposit aniline on the AuNPs / GCE modified electrode, thus obtaining the PANI / AuNPs / GCE modified electrode. S3. Preparation of AChE / PANI / AuNPs / GCE biosensor: Add 5-7 μL of AChE solution to the surface of the PANI / AuNPs / GCE electrode obtained in S2. The concentration of AChE solution is 10 UmL -1 , and placed in the air to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was stored in a 4°C refrigerator for future use.

[0013] Embodiment 1: 1. Preparation of AuNPs / GCE modified electrodes Before modification, the glassy carbon electrode (GCE) was polished on smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then ultrasonically cleaned with methanol and distilled water, respectively. - 1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range of -0.4-0.8 V was 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; 2. Preparation of PANI / AuNPs / GCE modified electrode The AuNPs / GCE modified electrode was placed in a solution containing 1 mol L-1 of hydrochloric acid and 0.1 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of , so that aniline was deposited on the AuNPs / GCE modified electrode, and the PANI / AuNPs / GCE modified electrode was obtained; 3. Preparation of AChE / PANI / AuNPs / GCE biosensor Add 5 μL of 10 UmL -1 AChE solution was added to the surface of PANI / AuNPs / GCE electrode and allowed to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was then stored in a refrigerator at 4°C for future use.

[0014] Embodiment 2: 1. Preparation of AuNPs / GCE modified electrodes Before modification, the glassy carbon electrode (GCE) was polished on smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then the electrode was ultrasonically cleaned with methanol and distilled water, respectively. -1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range is -0.4-0.8V at 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; 2. Preparation of PANI / AuNPs / GCE modified electrode The AuNPs / GCE modified electrode was placed in a solution containing 2 mol L -1 of hydrochloric acid and 0.2 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of , so that aniline was deposited on the AuNPs / GCE modified electrode, and the PANI / AuNPs / GCE modified electrode was obtained; 3. Preparation of AChE / PANI / AuNPs / GCE biosensor Add 6 μL of 10 UmL -1 AChE solution was added to the surface of PANI / AuNPs / GCE electrode and allowed to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was then stored in a refrigerator at 4°C for future use.

[0015] Embodiment 3: 1. Preparation of AuNPs / GCE modified electrodes Before modification, the glassy carbon electrode (GCE) was polished on smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then ultrasonically cleaned with methanol and distilled water, respectively. - 1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range is -0.4-0.8V at 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; 2. Preparation of PANI / AuNPs / GCE modified electrode The AuNPs / GCE modified electrode was placed in a solution containing 3 mol L -1 of hydrochloric acid and 0.3 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of , so that aniline was deposited on the AuNPs / GCE modified electrode, and the PANI / AuNPs / GCE modified electrode was obtained; 3. Preparation of AChE / PANI / AuNPs / GCE biosensor Add 7 μL of 10 UmL -1 AChE solution was added to the surface of PANI / AuNPs / GCE electrode and allowed to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was then stored in a refrigerator at 4°C for future use.

[0016] Experimental examples, electrochemical measurements and reaction processes: 1. Electrochemical characterization of basic electrodes like Figure 2 As shown, after constructing the PANI / AuNPs / GCE modified electrode, the present invention conducted SEM electron microscopy characterization, and the test results showed that the composite material and the modified electrode were successfully prepared.

[0017] The electrochemical behaviors of GCE, AuNPs / GCE, PANI / AuNPs / GCE and AChE / PANI / AuNPs / GCE were characterized by CV, EIS and chronocoulometry. A 4.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.01 M KCl was used as the electrolyte. The CV method was used to characterize the basic electrode under the following measurement conditions: voltage range -0.4 V to 0.8 V; scan rate 60 mVs -1The EIS test setting frequency range is: 1Hz-106Hz, the sine wave signal amplitude is 5mV, and the voltage is 0.22V.

[0018] like Figure 3 As shown in Figure 2, the composite materials and modified electrodes were successfully prepared, and the A values ​​of GCE, AuNPs / GCE, and PANI / AuNPs / GCE were 0.275 cm -1 、0.789 cm -1 、1.257cm -1 ,The results show that compared with other materials, the surface area of ​​the modified PANI / AuNPs / GCE electrode is significantly increased, and the electrochemical response signal is significantly enhanced.

[0019] 2. Cyclic voltammetric characterization of electrochemical enzyme biosensors The CV method was used in the voltage range of -0.4V to 0.8V with a rate of 50mVs -1 Scan rate, 6.0-8.0 mL containing 1.0 mmol L -1 The AChE / PANI / AuNPs / GCE electrochemical enzyme biosensor was characterized by CV in PBS (pH = 7.6) buffer solution of ATCHI. Figure 3 A.

[0020] 3. CPF measurement process AChE / PANI / AuNPs / GCE modified electrodes were prepared by drop coating and electrodeposition. Differential pulse voltammetry (DPV) was used to measure the concentration of 1.0 mmol L -1 The electrochemical response of the AChE / PANI / AuNPs / GCE modified electrode was investigated in ATCHI PBS (pH=7.6) buffer, and the initial current I0 was recorded.

[0021] Preparation of sample standards: Weigh an appropriate amount of CPF and dissolve it in anhydrous methanol to obtain an anhydrous methanol solution of CPF. Then take a certain volume of CPF solution in PBS to prepare 1.0×10 1 , 0.5×10 1 , 0.1×10 1 5.0×10 -2 , 1×10 -2 5.0×10 -3 , 1.0×10 -3 5.0×10 -3 , 1.0×10 -3 The ppm CPF standard was used for quantitative analysis.

[0022] The modified electrode was taken out and washed with PBS (pH=7.6) buffer, then placed in CPF standards of different concentrations for inhibition for 10 min, and then DPV scanning was performed to record the current I n According to the inhibition rate = (I0-I n ) / I0×100%, and the inhibition rate was calculated. The measurement conditions of DPV are: voltage range 0 to 0.416; potential increment 0.004V; amplitude 0.05V; pulse width 0.05s; sampling width 0.0167s; pulse period 0.5s, and standing time 2s. Finally, the linear function relationship between the inhibition rate and the electrochemical response value was obtained by mathematical modeling.

[0023] Perform gradient concentration sample testing under optimized conditions, such as Figure 4 It can be seen that in 1×10 -3 -1×10 1 In the ppm CPF concentration range, the logarithm of CPF concentration and its corresponding enzyme inhibition rate showed a good linear relationship, and the linear equation was Y=3.7708LgC CPF +37.133 (R 2 =0.9948), the detection limit was 7.9×10 -5 ppm (S / N=3).

[0024] To verify the practicality of the sensor, the present invention conducted tests on the specificity, repeatability and stability of the AChE / PANI / AuNPs / GCE sensor. Figure 5 , the AChE / PANI / AuNPs / GCE sensor has excellent specificity, repeatability and stability.

[0025] (1) The specificity of the electrochemical enzyme sensor was evaluated by testing different interfering components, including glycine, glyphosate, soluble starch, urea, and ascorbic acid, and comparing the signal changes in the presence of these interfering substances and CPF pesticide residues. Figure 5 As shown in Figure A, compared with the blank control group, the DPV response signal of the interference group after incubation was almost the same, and the RSD was less than 2.85%, indicating that AChE / PANI / AuNPs / GCE based on the enzyme inhibition principle has great specificity for the detection of CPF pesticide residues.

[0026] (2) If Figure 5 As shown in B, the electrodes were sealed and stored at room temperature (25°C) for 5, 10, 15, 20, and 25 days, respectively, and the CPF content (1×10 -1ppm) solution, the detection results of the electrochemical sensor were 97.84%-88.37% of the initial performance, indicating that the electrochemical sensor has good stability.

[0027] (3) If Figure 5 As shown in C, five modified electrodes were prepared in parallel and the DPV method was used to measure the -1 The results showed that the average RSD of 5 parallel prepared electrodes was 1.73%, indicating that the electrochemical platform constructed based on AChE / PANI / AuNPs composite material has good reproducibility.

[0028] In order to verify the feasibility of the present invention in the detection of actual samples, actual samples of Astragalus were analyzed. As shown in Table 1, the results show that the sensor has high sensitivity and can realize the real-time detection of CPF in actual samples.

[0029] In order to study the feasibility of the sensing platform constructed based on AChE / PANI / AuNPs composite materials for sensitive detection of CPF in actual samples. In this study, 10 mL of actual sample solution was selected for 10 min incubation, astragalus was crushed, 5 g of sample was placed in a 100 mL volumetric flask, and PBS solution with pH = 7.6 was added to the scale. Three 10 mL aliquots of the solution were taken and CPF methanol solution was added at concentrations of 25, 50 and 250 ppm, respectively. -1 A mixture of ATCHI and PBS (pH = 7.6) was used as the supporting electrolyte for DPV testing. The recoveries obtained in the three analyses using AChE / PANI / AuNPs / GCE ranged from 96.68% to 102.32%, with relative standard deviations ranging from 1.97% to 2.41% (Table 1), indicating that the sensor is highly sensitive and can be used for rapid detection of CPF in actual samples.

[0030] Compared with the authorized patent "CN104931692A An electrochemical immunosensor for detecting chlorpyrifos and its preparation method and application", the present invention has the following advantages: Optimization of modified materials: Compared with the authorized patents that mainly use naphthol membranes and nano-cobalt tetroxide / polyaniline composite membranes, we use non-toxic, green and easily available gold nanoparticles (AuNPs) and polyaniline (PANI) as substrate materials to enhance the conductivity and stability of the electrode. In particular, AuNPs have better conductivity and larger specific surface area, providing higher electrochemical activity for the sensor.

[0031] Optimization of detection mechanism: Compared with the authorized patent, which uses chlorpyrifos as artificial antigen and blocks nonspecific active sites with bovine serum albumin, we use different concentrations of chlorpyrifos mixed in the reaction buffer to carry out the target recognition reaction. We have developed an electrochemical enzyme biosensor based on the enzyme inhibition principle, which has high sensitivity and excellent selectivity, simple operation and rapid reaction. By optimizing the reaction conditions, we ensure that the specific recognition reaction is more convenient.

[0032] In summary, compared with the authorized patents, the electrode modification material prepared by the present invention is more novel and easily available, and uses the enzyme inhibition principle to detect CPF, showing higher detection sensitivity and wider detection range, which is more suitable for high-sensitivity and rapid detection of poisoned dead ticks in traditional Chinese medicines, and provides a more effective technical means for safety monitoring of traditional Chinese medicines.

Claims

1. A method for preparing a CPF residue rapid detection enzyme biosensor based on PANI / AuNPs, characterized in that The steps include: S1. Preparation of AuNPs / GCE modified electrode: Before modification, the glassy carbon electrode was polished on a smooth chamois leather with 0.3 μm and 0.05 μm alumina powders, and then ultrasonically cleaned with methanol and distilled water, respectively. -1 HAuCl4 0.5 mol L -1 In H2SO4 solution, the potential range is -0.4-0.8 V at 50 mVs -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / GCE modified electrode; S2. Preparation of PANI / AuNPs / GCE modified electrode: The AuNPs / GCE modified electrode obtained in S1 was placed in a solution containing 1-3 mol L -1 of hydrochloric acid and 0.1-0.3 mol L -1 In aniline solution, the potential range of -0.4-0.8 V was 50 mVs -1 The electrochemical deposition was performed by scanning 30 cycles by CV method at a rate of 1.5 % to deposit aniline on the AuNPs / GCE modified electrode, thus obtaining the PANI / AuNPs / GCE modified electrode. S3. Preparation of AChE / PANI / AuNPs / GCE biosensor: 5-7 μL AChE solution was added dropwise to the surface of the PANI / AuNPs / GCE electrode obtained in S2, and the electrode was placed to dry at room temperature to obtain the acetylcholinesterase sensor AChE / PANI / AuNPs / GCE, which was then stored in a 4°C refrigerator for later use.

2. The method for preparing the PANI / AuNPs-based CPF residue rapid detection enzyme biosensor according to claim 1, characterized in that: The concentration of the AChE solution described in S3 is 10 UmL -1 .

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