Preparation method of electrochemical sensor for organophosphorus pesticide residues in traditional Chinese medicinal materials based on AuNPs / BiVO4

Abstract

The invention discloses a preparation method of an electrochemical sensor for organophosphorus pesticide residues in traditional Chinese medicinal materials based on AuNPs / BiVO4, and relates to the field of electrochemical sensors, the method comprises preparation of a BiVO4 / GCE modified electrode, preparation of an AuNPs / BiVO4 / GCE modified electrode, and preparation of an AChE / PANI / AuNPs / GCE electrochemical sensor. According to the invention, BiVO4 and AuNPs are used as electrode substrate materials to load acetylcholin esterase (AChE), so that the electrochemical enzyme biosensor which takes AChE as a recognition element and has high sensitivity, high selectivity, recognition and fast detection is constructed, and the electrochemical enzyme biosensor is used for detecting organophosphorus pesticide residues in traditional Chinese medicinal materials. The sensor detects Ops by using an enzyme inhibition principle, shows higher detection sensitivity and wider detection range, shows high stability and reproducibility in detection of organic phosphorus pesticide residues in traditional Chinese medicinal materials, and provides a more effective technical means for safety monitoring of the traditional Chinese medicinal materials.

Description

Technical Field

[0001] The present invention relates to the field of electrochemical sensors, and particularly to a preparation method of an electrochemical sensor for detecting organophosphorus pesticide residues in traditional Chinese medicines based on AuNPs / BiVO4. Background Art

[0003] As a class of early-applied and broad-spectrum insecticides, organophosphorus pesticides are highly toxic or moderately toxic, and a few are low-toxic. The pesticide pollution and excessive residues caused by their abuse seriously affect the quality and safety of traditional Chinese medicines. After being consumed by humans, traditional Chinese medicines contaminated and residual with organophosphorus pesticides will affect myocardial cells and exhibit significant cardiotoxicity. The toxicity is mainly reflected in its inhibitory effect on cholinesterase, which causes acetylcholine to accumulate in the synaptic cleft of nerves, and then over-activates the cholinergic receptors of the heart. This over-stimulation can cause cardiac dysfunction such as arrhythmia, weakened myocardial contractility, and decreased blood pressure. Its toxicity may also cause acute, subacute or chronic damage to myocardial cells, further disturbing cardiac function, breaking the electrolyte balance, and even seriously damaging the cardiac structure, leading to irreversible consequences such as heart failure, shock and even death. Therefore, it is particularly important to identify the presence of organophosphorus and evaluate its cardiotoxicity.

[0004] Currently, traditional methods for detecting organophosphorus pesticide residues include gas chromatography, high-performance liquid chromatography, and colorimetry, etc., which are characterized by high sensitivity and high accuracy. However, they require relatively complex processing steps before analysis and are not suitable for on-site rapid detection. In addition, these traditional analytical methods are complex and time-consuming in sample processing, and there is also a problem that compounds may decompose before analysis, making it difficult to meet the requirements of on-site rapid detection.

[0005] The detection method of electrochemical sensors has attracted extensive research interest of researchers as an effective method for rapidly detecting pesticides in food and the environment due to its advantages such as high sensitivity, simple structure, miniaturization and portability. Especially for electrochemical enzyme biosensors, using cholinesterase as a biorecognition element, they can rapidly detect organophosphorus pesticide residues. Therefore, it is necessary to develop an efficient and convenient electrochemical sensor for detecting organophosphorus pesticide residues in traditional Chinese medicines. Summary of the Invention

[0006] The object of the present invention is to provide a preparation method of an electrochemical sensor for detecting organophosphorus pesticide residues in traditional Chinese medicines based on AuNPs / BiVO4. This method uses bismuth vanadate, which has low toxicity, good chemical stability, cost-effectiveness and ecological friendliness, as the electrode substrate material. At the same time, gold nanoparticles (AuNPs) with high conductivity and large specific surface area are modified on the electrode surface by electrochemical deposition method. The gold-amine bond is formed between AuNPs and the amino group on acetylcholinesterase (AChE), and an electrochemical enzyme biosensor with high sensitivity, high selectivity and fast recognition is constructed for detecting organophosphorus pesticide residues in traditional Chinese medicines. This sensor shows high stability and reproducibility in the detection of organophosphorus pesticide residues in traditional Chinese medicines, providing an effective means for the accurate monitoring of organophosphorus pesticide residues in traditional Chinese medicines.

[0007] To achieve the above object, the present invention adopts the following technical solutions: A preparation method of an electrochemical sensor for detecting organophosphorus pesticide residues in traditional Chinese medicines based on AuNPs / BiVO4 proposed by the present invention includes: (1) Preparation of BiVO4 / GCE modified electrode Weigh 0.2 - 0.4 g of bismuth vanadate, add it to 100 mL of 1 wt% hydrochloric acid solution, and continuously stir until completely dissolved to obtain a BiVO4 dispersion solution, which is stored at room temperature for standby; use 0.30 μm and 0.05 μm alumina powder to polish the glassy carbon electrode (GCE) successively on a smooth suede, then ultrasonically clean the electrode with methanol and distilled water respectively. Drop 6 μL of the prepared BiVO4 dispersion solution onto the cleaned GCE, and after infrared drying, the BiVO4 / GCE modified electrode is obtained; (2) Preparation of AuNPs / BiVO4 / GCE modified electrode Place the BiVO4 / GCE modified electrode in 0.5 - 0.7 mol·L -1 H2SO4 solution containing 5 - 7 mmol·L -1 HAuCl4, and use the cyclic voltammetry (CV) method to scan at a rate of 50 mV·s -1 in the potential range of -0.4 - 0.8 V for 20 cycles to deposit AuNPs, and the AuNPs / BiVO4 / GCE modified electrode is obtained; (3) Preparation of AChE / PANI / AuNPs / GCE biosensor Drop 8 - 10 μL of 10 U·mL -1 AChE solution onto the surface of the AuNPs / BiVO4 / GCE modified electrode, and leave it to dry at room temperature to obtain the AChE / PANI / AuNPs / GCE electrochemical sensor, which is placed in a refrigerator at 4 °C for storage and standby.

[0008] Compared with the prior art, the beneficial technical effects of the present invention are as follows: By using BiVO4 and AuNPs as the electrode substrate materials, an electrochemical enzyme biosensor with high sensitivity, high selectivity, and fast detection for the detection of organophosphorus pesticide residues in traditional Chinese medicines is constructed with acetylcholinesterase (AChE) as the recognition element. The sensor uses the enzyme inhibition principle to detect Ops, showing higher detection sensitivity and a wider detection range, and exhibiting high stability and reproducibility in the detection of organophosphorus pesticide residues in traditional Chinese medicines, providing a more effective technical means for the safety monitoring of traditional Chinese medicines. Description of the Drawings

[0009] Figure 1 It is the material physical and chemical structure characterization diagram in the test example of the present invention. Among them, A is the infrared diagram of BiVO4, B is the Raman diagram of BiVO4, C is the SEM diagram of BiVO4, D is the SEM diagram of AuNPs / BiVO4, E is the EDX spectrum of AuNPs / BiVO4, F is the mapping diagram of V element in AuNPs / BiVO4, G is the mapping diagram of Bi element in AuNPs / BiVO4, H is the mapping diagram of Au element in AuNPs / BiVO4, and I is the mapping diagram of O element in AuNPs / BiVO4; Figure 2 It is the electrochemical characterization diagram of the relevant electrodes in the test example of the present invention. Among them, A is the CV curves of GCE (a), BiVO4 / GCE (b), AuNPs / BiVO4 / GCE (c), and AChE / AuNPs / BiVO4 / GCE (d), B is the EIS curves of AChE / AuNPs / BiVO4 / GCE (a), GCE (b), AuNPs / BiVO4 / GCE (c), and BiVO4 / GCE (d), C is the CV curve of AuNPs / BiVO4 / GCE at a scanning rate of 10 mV·s -1 ~200 mV·s -1 ; D is the relationship between the logarithm of the peak current and the logarithm of the scanning rate; E is the chronocoulometry curves of GCE (a), BiVO4 / GCE (b), and AuNPs / BiVO4 / GCE (c), and F is the Q-t 1 / 2 curve.

[0010] Figure 3 It is the DPC curves of different organophosphorus pesticides in the test example of the present invention. Among them, A is the DPV curve of CPF, B is the DPV curve of Rogor, C is the DPV curve of Phoxim, D is the DPV curve of DP, E is the DPV curve of FTHN, and F is the dose-effect curve fitting half-inhibition concentration curve.

[0011] Figure 4 shows the practicality test results of AChE / AuNPs / BiVO4 / GCE in the test of the present invention. Among them, A is the result of 5 DPV tests of the same AChE / AuNPs / BiVO4 / GCE in the test solution, and B is the result of measuring the solution containing OPs by the DPV method on the 5th, 10th, 15th, 20th, and 25th days with AChE / AuNPs / BiVO4 / GCE respectively. Detailed implementation mode

[0012] 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 the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0013] In the present invention, unless otherwise specified, the raw materials involved are well-known commercially available products in the art.

[0014] Example 1 Preparation of BiVO4 / GCE modified electrode: Weigh 0.2 g of bismuth vanadate, add it to 100 mL of 1 wt% hydrochloric acid solution, and continuously stir until completely dissolved to obtain a BiVO4 dispersion, which is stored at room temperature for later use; Use 0.30 μm and 0.05 μm alumina powder to polish the GCE continuously on a smooth suede, then ultrasonically clean the electrode with methanol and distilled water respectively, drop 6 μL of the prepared BiVO4 dispersion onto the cleaned GCE, and obtain the BiVO4 / GCE modified electrode after infrared drying; Preparation of AuNPs / BiVO4 / GCE modified electrode: Place the BiVO4 / GCE modified electrode in 0.5 mol·L - 1 H2SO4 solution containing 5 mmol·L -1 HAuCl4, and use the CV method to scan at a rate of 50 mV·s in the potential range of -0.4 to 0.8 V for 20 cycles to deposit AuNPs, thus obtaining the AuNPs / BiVO4 / GCE modified electrode; -1 Preparation of AChE / PANI / AuNPs / GCE biosensor: Drop 8 μL of 10 U·mL -1 AChE solution onto the surface of the AuNPs / BiVO4 / GCE modified electrode, leave it to dry at room temperature, obtain the AChE / PANI / AuNPs / GCE electrochemical sensor, and store it in a refrigerator at 4 °C for later use. ​

[0015] Example 2 Preparation of BiVO4 / GCE modified electrode: Weigh 0.3 g of bismuth vanadate and add it to 100 mL of 1 wt% hydrochloric acid solution. Continuously stir until it is completely dissolved to obtain a BiVO4 dispersion, which is stored at room temperature for later use. Use 0.30 μm and 0.05 μm alumina powder to polish the glassy carbon electrode (GCE) successively on a smooth suede, and then ultrasonically clean the electrode with methanol and distilled water respectively. Drop 6 μL of the prepared BiVO4 dispersion onto the cleaned GCE, and after infrared drying, the BiVO4 / GCE modified electrode is obtained; Preparation of AuNPs / BiVO4 / GCE modified electrode: Place the BiVO4 / GCE modified electrode in a 0.6 mol·L - 1 HAuCl4-containing 0.6 mol· L -1 H2SO4 solution, and use cyclic voltammetry (CV) to scan at a rate of 50 mV·s -1 in the potential range of -0.4 to 0.8 V for 20 cycles to deposit AuNPs, and the AuNPs / BiVO4 / GCE modified electrode is obtained; Preparation of AChE / PANI / AuNPs / GCE biosensor: Drop 9 μL of 10 U·mL -1 AChE solution onto the surface of the AuNPs / BiVO4 / GCE modified electrode, and leave it to dry at room temperature to obtain the AChE / PANI / AuNPs / GCE electrochemical sensor, which is placed in a refrigerator at 4 °C for storage and later use.

[0016] Example 3 Preparation of BiVO4 / GCE modified electrode: Weigh 0.4 g of bismuth vanadate and add it to 100 mL of 1 wt% hydrochloric acid solution. Continuously stir until it is completely dissolved to obtain a BiVO4 dispersion, which is stored at room temperature for later use. Use 0.30 μm and 0.05 μm alumina powder to polish the glassy carbon electrode (GCE) successively on a smooth suede, and then ultrasonically clean the electrode with methanol and distilled water respectively. Drop 6 μL of the prepared BiVO4 dispersion onto the cleaned GCE, and after infrared drying, the BiVO4 / GCE modified electrode is obtained; Preparation of AuNPs / BiVO4 / GCE modified electrode: Place the BiVO4 / GCE modified electrode in a 0.7 mol·L - 1 HAuCl4-containing 0.7 mol· L -1 H2SO4 solution, and use cyclic voltammetry (CV) to scan at a rate of 50 mV·s -1Perform 20 cycles of rate scanning to deposit AuNPs, thus obtaining the AuNPs / BiVO4 / GCE modified electrode; Preparation of AChE / PANI / AuNPs / GCE biosensor: Drop 10 μL of 10 U·mL -1 AChE solution onto the surface of the AuNPs / BiVO4 / GCE modified electrode, leave it to dry at room temperature, obtain the AChE / PANI / AuNPs / GCE electrochemical sensor, and store it in a refrigerator at 4 °C for standby.

[0017] Test Example 1. Physicochemical structure characterization: After constructing the AuNPs / BiVO4 modified electrode in Example 1, perform SEM electron microscopy characterization on it, and conduct Fourier transform infrared spectroscopy (FTIR) and Raman spectrum tests. The test results are as Figure 1 shown.

[0018] Figure 1 The test results show that the AuNPs / BiVO4 composite material and the AuNPs / BiVO4 / GCE modified electrode in Example 1 are successfully prepared.

[0019] 2. Electrochemical characterization of the base electrode: Use cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and chronocoulometry to characterize the electrochemical behavior of GCE, BiVO4 / GCE, and AuNPs / BiVO4 / GCE in Example 1. Use a 4.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.01 M KCl as the electrolyte. Among them, the measurement conditions for CV are: voltage range is -0.4 V to 0.8 V, scan rate is 60 mV·s -1 , and the measurement conditions for EIS are: frequency range is 1 Hz to 106 Hz, amplitude of the sine wave signal is 5 mV, voltage is 0.22 V. The test results are as Figure 2 shown.

[0020] 3. Cyclic voltammetry characterization of the AChE / AuNPs / BiVO4 / GCE electrochemical sensor: Use CV method in the voltage range of -0.4 V to 0.8 V, with a scan rate of 50 mV·s -1 , in 10 mL of PBS (pH = 7.6) buffer solution containing 1.2 mmol·L -1 ATCHI, conduct CV characterization on the AChE / AuNPs / BiVO4 / GCE electrochemical sensor. The test results are as Figure 2 shown.

[0021] Figure 2The results show that all the composite materials and modified electrodes were successfully prepared, and the A values of GCE, BiVO4 / GCE, and AuNPs / BiVO4 / GCE were 0.05 cm -1 , 0.11 cm -1 , 0.93 cm -1 . Compared with GCE and BiVO4 / GCE, the surface area of the modified AuNPs / BiVO4 / GCE electrode in Example 3 increased significantly, and the electrochemical response signal was significantly enhanced.

[0022] 4. Measurement of OPs: Using differential pulse voltammetry (DPV), the electrochemical response of the AChE / AuNPs / BiVO4 / GCE electrochemical sensor in Example 1 was investigated in a PBS (pH = 7.6) buffer solution containing the corresponding substrate concentration, and the initial current I0 was recorded; then, it was taken out, washed clean with PBS (pH = 7.6) buffer solution, and the electrochemical sensor was placed in organic phosphorus pesticide (OPs) solutions of the same concentration for 10 minutes of inhibition. After the inhibition was completed, DPV scanning was performed again, and the current I i was recorded at this time; according to the inhibition rate = (I0 - I i ) / I0 × 100%, the inhibition rate was calculated. The measurement conditions of DPV were: the voltage range was 0 - 0.416 V, the potential increment was 0.004 V, the amplitude was 0.05 V, the pulse width was 0.05 s, the sampling width was 0.0167 s, the pulse period was 0.5 s, and the standing time was 2 s. Preparation of pesticide standard solutions: Weigh appropriate amounts of chlorpyrifos (CPF), Rogor (Rogor), Fenthion (FTHN), Dipterex (DP), and Phoxim (Phoxim), and dissolve them in anhydrous methanol to obtain their corresponding anhydrous methanol solutions, with a concentration of 10 μg·mL -1 ; then, take the same volume of solution from each standard solution and add it to a PBS buffer solution with a pH of 7.6 for subsequent quantitative analysis. A mixture of PBS (pH = 7.6) containing 1.2 mmol·L -1 ATCHI was used as the supporting electrolyte for DPV testing. Using a pesticide standard solution with an initial concentration of 10 μg·mL -1 , it was serially diluted by the logarithmic dilution method using a phosphate buffer solution with a pH of 7.6 to prepare 0.001, 0.01, 0.1, 1, 10 μg·mL -1Five concentration gradients were set, with three parallel samples for each concentration, and a buffer solution without pesticides was set as a blank control. After incubating the AChE / AuNPs / BiVO4 / GCE electrochemical sensor of Example 3 with each concentration sample, the DPV response current was measured. Each sample was measured 3 times and the average value was taken. Record the current values at different concentrations, analyze the concentration-current relationship curve, calculate the inhibition rate of pesticides on the electrochemical sensor by comparing the current attenuation between the blank and each concentration sample, and fit the half-inhibitory concentration (IC 50 ) based on the dose-effect curve. The test results are as Figure 3 shown.

[0023] Figure 3 The results show that the AChE / AuNPs / BiVO4 / GCE electrochemical sensor of Example 1 can sensitively detect trace amounts of organophosphorus pesticides (OPs) CPF, Rogor, FTHN, DP, and Phoxim, and has a good linear relationship in the concentration range of 31.25 pg·mL -1 ~1.6×10 4 pg·mL -1 , with a detection limit as low as 1.92×10 -2 pg·mL -1 . Compared with other detection methods, it has a wider detection range and a lower detection limit.

[0024] 5. Practicality test: The reproducibility and stability of the AChE / AuNPs / BiVO4 / GCE electrochemical sensor of Example 1 of the present invention were tested.

[0025] (1) Five prepared AChE / AuNPs / BiVO4 / GCE electrochemical sensors were used in parallel, and the DPV method was used to measure the solution containing pesticide residue standards (0.1 μg mL -1 ). Preparation of pesticide standard solutions: Weigh appropriate amounts of chlorpyrifos (CPF), dimethoate (Rogor), fenthion (FTHN), trichlorfon (DP), and phoxim (Phoxim), and dissolve them in anhydrous methanol to obtain their corresponding anhydrous methanol solutions, with a concentration of 0.1 μg·mL -1 . The test results are as Figure 4 shown in A.

[0026] Figure 4The results of A showed that the average RSD of 5 parallel AChE / AuNPs / BiVO4 / GCE electrochemical sensors was 1.17%, indicating that the AChE / AuNPs / BiVO4 / GCE electrochemical sensor of Example 1 had good reproducibility.

[0027] (2) Under the condition of room temperature (25 °C), the prepared AChE / AuNPs / BiVO4 / GCE electrochemical sensors were sealed and stored for 5 days, 10 days, 15 days, 20 days and 25 days respectively, and the solution containing the pesticide residue standard (0.1 μg·mL -1 ) was measured by DPV method. The test results are as Figure 4 shown in B.

[0028] Figure 4 The results of B showed that the stability test results of the AChE / AuNPs / BiVO4 / GCE electrochemical sensor of Example 1 were 98.36% - 89.62% of the initial performance, indicating that the electrochemical sensor had good stability.

[0029] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements 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 an electrochemical sensor for organophosphorus pesticide residues in traditional Chinese medicine based on AuNPs / BiVO4, characterized in that Including: (1) Preparation of BiVO4 / GCE modified electrode Weigh 0.2 - 0.4 g of bismuth vanadate, add it to 100 mL of 1 wt% hydrochloric acid solution, and continuously stir until completely dissolved to obtain a BiVO4 dispersion, which is stored at room temperature for later use; successively polish the GCE on a smooth suede with 0.30 μm and 0.05 μm alumina powder, then ultrasonically clean the electrode with methanol and distilled water respectively. Drop 6 μL of the prepared BiVO4 dispersion onto the cleaned GCE, and after infrared drying, the BiVO4 / GCE modified electrode is obtained; (2) Preparation of AuNPs / BiVO4 / GCE modified electrode The BiVO4 / GCE modified electrode was placed in a 0.5 - 0.7 mol·L -1 HAuCl4 solution containing 5 - 7 mmol·L -1 H2SO4. Using cyclic voltammetry (CV), it was scanned at a rate of 50 mV·s -1 within the potential range of -0.4 - 0.8 V for 20 cycles to deposit AuNPs, thus obtaining the AuNPs / BiVO4 / GCE modified electrode; (3) Preparation of AChE / PANI / AuNPs / GCE biosensor Add 8 - 10 μL of 10 U·mL -1 AChE solution to the surface of the AuNPs / BiVO4 / GCE modified electrode, leave it to dry at room temperature to obtain the AChE / PANI / AuNPs / GCE electrochemical sensor, and store it in a refrigerator at 4 °C for standby.

2. The preparation method of an electrochemical sensor for organophosphorus pesticide residues in traditional Chinese medicine based on AuNPs / BiVO4 according to claim 1, characterized in that: The AChE / PANI / AuNPs / GCE electrochemical sensor is used for the detection of organophosphorus pesticides CPF, Rogor, FTHN, DP, and Phoxim, and the detection concentration range is 31.25 pg·mL -1 ~ 1.6 × 10 4 pg·mL -1 , with a detection limit of 1.92×10 -2 pg·mL -1 .

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