Preparation method of double-recognition-element electrochemical sensor for detecting azamethiphos

Abstract

According to the invention, a sensor with double recognition elements for detecting azamethiphos is constructed by utilizing the enhancement effect of a composite material (AuNPs (at) SnS2 (at) ZnCo-MOF) prepared from a zinc-cobalt bimetal organic framework (ZnCo-MOF), tin disulfide nanoflowers (SnS2) and gold nanoparticles (AuNPs) on an electrochemical signal. The specific preparation process is as follows: dropwise adding the composite material on the surface of a treated glassy carbon electrode, then fixing an aptamer (Apt) by using a gold-sulfur bond, and synthesizing a molecularly imprinted polymer (MIPs) by using an electric polymerization mode. The composite material can promote the transfer rate of electrons in a system and enhance a current signal. Meanwhile, due to the large specific surface area of the composite material, more imprinting cavities can be generated. In addition, the double recognition elements endow the sensor with higher specificity. The dual-recognition sensor is high in specificity and good in stability, and can be used for high-specificity detection of azamethiphos residues in vegetables.

Description

Technical Field

[0001] The present invention provides a preparation method of an electrochemical sensor with dual recognition elements for the detection of azamethiphos, belonging to the technical field of food safety detection. Background Art

[0002] To ensure the quality and yield of agricultural products, a large number of pesticides are widely used, including organophosphorus pesticides, carbamate pesticides, pyrethroid pesticides, etc. Among them, organophosphorus pesticides (OPs) achieve insecticidal effects by inhibiting the activity of acetylcholinesterase in pests. Due to their low cost, excellent insecticidal effects, and wide application scope, they are widely used in agricultural production.

[0003] Azamethiphos (AZA) is an organophosphorus nerve agent widely used in the prevention and control of parasites in aquaculture and the killing of mites and cockroaches in the public health field. However, the residues of AZA in the environment not only act on target pests but may also cause ecological harm to non-target organisms (such as microorganisms, earthworms, and crustaceans). In addition, AZA may enter the human body through food chain enrichment, posing a potential risk to human health.

[0004] The detection methods of OPs mainly include large-scale instrument detection methods, enzyme-linked immunosorbent assay (ELISA), colorimetry, and electrochemical sensors. Among them, large-scale instrument detection methods have high sensitivity and accuracy, but the equipment is expensive, the operation is complex, and professional technical personnel are required for operation. While ELISA and colorimetry have low costs, they have disadvantages such as poor anti-interference ability and limited detection range. In contrast, electrochemical sensors have received extensive attention due to their advantages such as simple operation, high sensitivity, and low detection cost.

[0005] Aptamer (Apt) is a single-stranded DNA or RNA molecule screened by systematic evolution of ligands by exponential enrichment (SELEX) technology, which can specifically recognize specific targets. Apt has the advantages of short synthesis cycle, low preparation cost, in vitro synthesis, and easy chemical modification. However, Apt is easily affected by environmental factors and is easily degraded by nucleases, which poses a major challenge in actual detection. Therefore, there is an urgent need for a strategy to improve the stability of Apt and reduce the influence of the external environment on its recognition ability.

[0006] As a kind of polymer material with specific recognition ability, molecularly imprinted polymers (MIPs) can form stable recognition sites by simulating antigen-antibody interactions. MIPs not only have high chemical stability and can maintain strong recognition ability in complex environments, but also can encapsulate Apt to keep it in the optimal recognition conformation and protect Apt from nuclease degradation. More importantly, the preparation of MIPs-Apt dual recognition elements by binding Apt and MIPs can achieve a synergistic recognition effect, improving the specificity and detection sensitivity of the sensor. Summary of the Invention

[0007] The purpose of the present invention is to establish a highly specific and sensitive detection technology for detecting AZA residues in vegetables.

[0008] The technical solution of the present invention is as follows: The composite material, Apt@AZA complex, MIPs are successively modified on the electrode surface through the layer-by-layer self-assembly technology. The specific preparation process is as follows: (1) Prepare the AuNPs@SnS2@ZnCo-MOF composite material and drop-coat it on the electrode surface; (2) Preparation of the Apt@AZA complex; (3) Modification of the Apt@AZA complex; (4) Electro-polymerization to prepare MIPs; (5) Elution of the template molecule AZA; (6) Detection of AZA.

[0009] Preferably, the preparation method of zinc-cobalt bimetallic organic framework (ZnCo-MOF): Dissolve Co(NO3)2·6H2O (1.0 g) and Zn(NO3)2·6H2O (1.0 g) in 30 mL of methanol to form solution A. Dissolve 2-methylimidazole (1.2 g) in 30 mL of methanol to form solution B. Slowly add solution B to solution A under stirring. At 25°C, continuously stir the mixed solution for 6 h, and then centrifuge (8000 rpm, 10 min) to obtain a precipitate. Wash the precipitate with methanol 3-5 times and then dry it in vacuum at 60°C for 10 h. The obtained purple powder is ZnCo-MOF.

[0010] Preferably, the preparation method of the composite material of tin disulfide (SnS2) and ZnCo-MOF (SnS2@ZnCo-MOF): Add 10 mg of ZnCo-MOF to 25 mL of ultrapure water and ultrasonically treat for 30 min. Add SnCl2·2H2O (112.5 mg) and L-cysteine (181.5 mg) to the above solution and continuously stir for 1 h. Add the stirred mixed solution to a hydrothermal reaction kettle and react at 180°C for 12 h. After the reaction, centrifuge (8000 rpm, 10 min) to obtain a precipitate, and wash it with ultrapure water and ethanol 3-5 times. Dry the precipitate in vacuum at 65°C for 10 h. The obtained dark brown powder is SnS2@ZnCo-MOF.

[0011] Preferably, the preparation method of the composite material of gold nanoparticles (AuNPs) and SnS2@ZnCo-MOF (AuNPs@SnS2@ZnCo-MOF) is as follows: Disperse the obtained SnS2@ZnCo-MOF (60 mg) in 20 mL of water. Add 5 mL of 10 mM L-cysteine (stir for 30 min), 5 mL of 3 mM HAuCl4·3H2O (stir for 30 min), and 10 mL of 5 mM ascorbic acid (stir for 3 h) to the suspension in sequence. Centrifuge (6000 rpm, 5 min) to collect the precipitate, wash it with ethanol 3-5 times, and then dry it in vacuum at 60 °C for 10 h to obtain AuNPs@SnS2@ZnCo-MOF. Drop 10 μL of 5 mg / mL AuNPs@SnS2@ZnCo-MOF solution onto the electrode surface for modification.

[0012] Preferably, the preparation and modification method of the Apt@AZA complex: Treat the thiolated aptamer with tris(2-carboxyethyl)phosphine hydrochloride (10 mM) to cleave the disulfide bond. Mix the treated aptamer (20 μM) with AZA (15 μM). Incubate the mixture at 37 °C for 40 min to promote the formation of the Apt@AZA complex. Drop the Apt@AZA complex onto the surface of the AuNPs@SnS2@ZnCo-MOF modified electrode and incubate it at 25 °C for 2 h to immobilize the Apt@AZA complex on the electrode surface through gold-sulfur bonds.

[0013] Preferably, the preparation method of MIPs: Place Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE in an o-phenylenediamine solution (4 mM). Perform cyclic voltammetry scanning at a scanning rate of 50 mV / s in the voltage range from -0.5 V to 0.5 V (20 cycles) to obtain MIPs / Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE.

[0014] Preferably, the elution method of the template molecule AZA: Insert MIPs / Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE into an eluent composed of methanol / acetic acid (8 / 2, V / V), and the elution time is 25 min.

[0015] Preferably, the detection process of AZA: Drop the vegetable sample to be tested containing AZA onto the sensor surface and perform DPV measurement in a mixture containing 5 mM 3- / 4- [Fe(CN)6] and 0.1 M KCl. Analyze the change in peak current to reflect the concentration of AZA.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention prepares an AuNPs@SnS2@ZnCo-MOF composite material with good conductivity and a large specific surface area, which should be used to improve the sensitivity of the sensor. At the same time, an MIPs-Apt dual recognition element is prepared to achieve a synergistic enhancement of the specific recognition ability. Description of the Drawings

[0017] Figure 1 Preparation process of the dual recognition sensor.

[0018] Figure 2 Characterization diagrams of the composite material.

[0019] Figure 3 Characterization diagrams of the sensor assembly process.

[0020] Figure 4 Feasibility analysis diagrams of the sensor.

[0021] Figure 5 Experimental condition optimization diagrams.

[0022] Figure 6 Standard curve diagrams of the sensor for detecting AZA.

[0023] Figure 7 Diagrams of the stability, reproducibility, and specificity of the sensor. Detailed Description of the Invention

[0024] The present invention will be further described in detail below with reference to the drawings and embodiments, but the embodiments do not impose any form of limitation on the present invention.

[0025] Example 1: Preparation process of the dual recognition sensor. As Figure 1As shown, 10 μL of AuNPs@SnS2@ZnCo-MOF solution was dropped onto the surface of GCE, and AuNPs@SnS2@ZnCo-MOF / GCE was obtained after natural drying. Thiolated aptamer was treated with TCEP (10 mM) to cleave disulfide bonds. The treated aptamer (20 μM) was mixed with AZA (15 μM). The mixture was incubated at 37 °C for 40 min to promote the formation of Apt@AZA complex. 10 μL of Apt@AZA complex was dropped onto the electrode surface, and the complex was immobilized on the electrode surface through gold-sulfur bonds, thus forming Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE. Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE was placed in o-phenylenediamine solution (4 mM). Cyclic voltammetry scanning was performed at a scanning rate of 50 mV / s in the voltage range from -0.5 V to 0.5 V (20 cycles) to obtain MIPs / Apt@AZA / AuNPs@SnS2@ZnCo-MOF / GCE. Subsequently, AZA on the electrode surface was removed using the eluent (methanol / acetic acid = 8 / 2, V / V), and the MIPs / Apt / AuNPs@SnS2@ZnCo-MOF / GCE sensor was obtained.

[0026] Example 2: Characterization diagrams of composite materials. The morphologies of ZnCo-MOF, SnS2@ZnCo-MOF, and AuNPs@SnS2@ZnCo-MOF were characterized using scanning electron microscopy (SEM). As Figure 2 shown in A, ZnCo-MOF presents a uniform dodecahedral structure with an edge length of approximately 300 nm. SnS2@ZnCo-MOF shows a three-dimensional nanoflower-like structure ( Figure 2 B). AuNPs@SnS2@ZnCo-MOF still maintains the nanoflower-like structure ( Figure 2 C), and the modification of AuNPs does not affect the structure of SnS2@ZnCo-MOF. The crystal structures of the nanomaterials were studied using X-ray diffraction (XRD) ( Figure 2D). The diffraction peaks in the XRD pattern of ZnCo-MOF at 7.33°, 10.36°, 12.73°, 14.68°, 16.46°, 18.03°, 24.43° and 26.68° correspond to the lattice planes (011), (002), (112), (022), (013), (222), (233) and (134), respectively. The six characteristic diffraction peaks (15.37°, 28.89°, 32.47°, 42.14°, 50.37° and 52.71°) in the XRD pattern of SnS2@ZnCo-MOF correspond to the crystal planes (001), (100), (101), (102), (110) and (111), respectively. Compared with the XRD pattern of SnS2@ZnCo-MOF, the diffraction peaks in the XRD pattern of AuNPs@SnS2@ZnCo-MOF at 38.77°, 44.52°, 64.17° and 77.41° correspond to the (111), (200), (220) and (311) crystal planes of gold, respectively. The chemical states and elemental compositions of the nanomaterials were further analyzed using X-ray photoelectron spectroscopy (XPS). As Figure 2 shown in E, the XPS spectrum of ZnCo-MOF has characteristic peaks of C1s, O 1s, N 1s, Co 2p and Zn 2p. After wrapping the SnS2 nanosheets, characteristic peaks of S2p, Sn 3d and Sn 3p appeared. After modifying AuNPs, Au4f appeared.

[0027] Example 3: Characterization diagram of the sensor assembly process. The electrochemical properties of electrodes modified with different modified materials were characterized using differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS). As Figure 3 shown in A, after modifying with the AuNPs@SnS2@ZnCo-MOF composite material, the peak current increased significantly. When the Apt@AZA complex was modified, the peak current decreased. After electro-polymerizing MIPs on the electrode surface, the peak current decreased further. After eluting the template molecules, an imprinted cavity was formed on the electrode surface, so the peak current increased significantly. The construction process of the sensor was further characterized using EIS. As Figure 3 shown in B, compared with the bare electrode, after modifying with AuNPs@SnS2@ZnCo-MOF with excellent electrical conductivity, the impedance value of the electrode decreased significantly. When the Apt@AZA complex and MIPs were modified, the impedance value of the electrode gradually increased. After eluting the template molecules, the impedance value of the electrode decreased again.

[0028] Example 4: Feasibility analysis diagram of the sensor. Chlorpyrifos CPF with a structure similar to AZA was selected as the interfering substance to test the feasibility of the experimental scheme. As Figure 4As shown in A, the sensor detects different ΔI values (13.9 and 33.8 μA) for AZA solutions with different concentrations. At the same time, when the sensor detects CPF solutions with the same concentration, the ΔI value is only 3.7 μA, which is much smaller than that of the AZA solution with the same concentration (33.8 μA). In summary, the experimental scheme for preparing the sensor is feasible. As Figure 4 As shown in B, after adding o-PD, the positive and negative peaks of the Apt@AZA complex did not change significantly. This indicates that the addition of o-PD does not affect the interaction between Apt and AZA. Therefore, o-PD and Apt may bind to different binding sites of AZA respectively to form a stable ternary complex.

[0029] Example 5: Experimental condition optimization diagram. To improve the stability, repeatability, and detection efficiency of the sensor, multiple experimental conditions that may affect the detection results were optimized, including the complexation time of the aptamer and the target, the concentration of the functional monomer, the number of cycles and scanning rate of electropolymerization, the elution time, and the incubation time. As Figure 5 As shown in A, the ΔI value gradually increases with the increase of the complexation time of the aptamer and the target. The ΔI value reaches the maximum at 40 min and then tends to be stable. Therefore, 40 min is selected as the optimal complexation time. As Figure 5 As shown in B, the ΔI value increases with the increase of the functional monomer concentration and reaches the maximum at 4 mM. When the functional monomer concentration further increases, it may cause self-crosslinking of the functional monomer. Figure 5 As shown in C, the optimal scanning rate is 50 mV / s. To control the thickness of the polymer film and improve the accessibility of the recognition sites, the number of cycles of electropolymerization was optimized. As Figure 5 As shown in D, the ΔI value reaches the maximum at 20 cycles. The elution time affects the efficiency of template molecule removal and the structural integrity of the imprinted cavity. Figure 5 As shown in E, the optimal elution time is 25 min. The incubation time determines the sufficiency of the binding between the target and the recognition site and the signal intensity. If the incubation time is too short, the binding will be incomplete. Figure 5 As shown in F, 30 min is selected as the optimal incubation time.

[0030] Example 6: Standard curve graph of the sensor for detecting AZA. The sensor was used to detect AZA solutions with different concentrations (10 -2 -10 4 ng / mL) and the corresponding DPV curves were obtained. As Figure 6 shown, as the AZA concentration increases, the peak current of DPV gradually decreases, and the ΔI value gradually increases. This is attributed to the gradual increase in the number of imprinted cavities occupied by AZA, blocking the electron transfer channels in the system. As Figure 6As shown in the illustration, there is a good linear relationship between the ΔI value and the logarithm of the AZA concentration. The linear equation is ΔI = 7.260 + 20.902Lgc (R 2 = 0.998), and the detection limit is 3.33×10 -3 ng / mL (S / N = 3).

[0031] Example 7: Sensor stability, reproducibility and specificity graphs. The stability, reproducibility and specificity of a sensor are important indicators for evaluating its performance. The sensors prepared under the optimal conditions were evenly divided into 6 groups and stored in a 4°C refrigerator. One group of sensors was taken out every 2 days to detect an AZA solution with a concentration of 10 ng / mL, and the corresponding ΔI values were calculated. As Figure 7 shown in A, the ΔI values of the last group of sensors still remained at 90.3% of the initial values respectively. Six sensors prepared under the optimal conditions were used to detect an AZA solution with a concentration of 10 ng / mL, and the ΔI values were calculated. As Figure 7 shown in B, the relative standard deviation (RSD) of the six sensors was 4.55%. As Figure 7 shown in C, the recognition ability of the sensor for AZA was significantly higher than that of the interfering substances. At the same time, the mixture of AZA and the interfering substances did not affect the recognition ability of the sensor. (Fenthion (FEN), Methyl parathion (MP), Chlorpyrifos (CPF), Dimethoate (DMT), Malathion (MAL)).

Claims

1. A method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridinium phosphide, characterized in that: AuNPs@SnS2@ZnCo-MOF composites with good conductivity and Apt-MIPs dual recognition elements with high specificity were synthesized, and a dual recognition element sensor was constructed by combining it with an electrochemical sensor. The specific preparation process is as follows: (1) Preparation of AuNPs@SnS2@ZnCo-MOF composites: S1: Synthesis of zinc-cobalt bimetallic organic framework (ZnCo-MOF) by wet method. Weigh 1.0 g Co(NO3)2·6H2O and 1.0 g Zn(NO3)2·6H2O and dissolve them in 30 mL methanol to form solution A. Dissolve 1.2 g dimethylimidazole in 30 mL methanol to form solution B. Slowly add solution B to the stirred solution A. The mixture is stirred, centrifuged, washed and vacuum dried to obtain ZnCo-MOF. S2: A composite material of tin disulfide (SnS2) and ZnCo-MOF (SnS2@ZnCo-MOF) was synthesized by hydrothermal method. 10 mg ZnCo-MOF was added to 25 mL ultrapure water and treated with ultrasound. SnCl2·2H2O (112.5 mg) and L-cysteine ​​(181.5 mg) were added to the above solution and stirred continuously. The stirred mixed solution was added to a hydrothermal reactor for reaction. After the reaction, the precipitate was obtained by centrifugation and washed several times with ultrapure water and ethanol. The dark brown powder obtained after vacuum drying is SnS2@ZnCo-MOF; S3: A composite material of gold nanoparticles (AuNPs) and SnS2@ZnCo-MOF (AuNPs@SnS2@ZnCo-MOF) was synthesized by reduction method. The obtained SnS2@ZnCo-MOF (60 mg) was dispersed in 20 mL of water. 5 mL of 10 mM L-cysteine, 5 mL of 3 mM HAuCl4·3H2O and 10 mL of 5 mM ascorbic acid were added to the suspension in sequence, with continuous stirring. The precipitate was collected by centrifugation, washed several times with ethanol and dried under vacuum; (2) Preparation of Apt@AZA complex: The thiolated aptamer was treated with tris(2-carbonylethyl)phosphine hydrochloride (10 mM) to cleave the disulfide bond. The treated aptamer was mixed with AZA and incubated to promote the formation of Apt@AZA complex; (3) Construction of dual recognition sensor: A three-electrode detection system was constructed with a saturated calomel electrode as the reference electrode, a platinum wire electrode as the counter electrode, and a glassy carbon electrode as the working electrode. First, 10 μL of AuNPs@SnS2@ZnCo-MOF was modified on the electrode surface. After natural drying, 10 μL of Apt@AZA complex was dropped on the electrode surface and fixed using a gold-sulfur bond. Subsequently, the material-modified electrode was inserted into an o-phenylenediamine solution (4 mM) for electropolymerization to form MIPs, which wrapped the electrode surface. Then, AZA was eluted from the electrode surface using an eluent, thereby generating an imprinted cavity that can specifically recognize AZA. When AZA in the detection system reoccupies the imprinted cavity, it will hinder the electron transfer in the system, thereby causing a change in the current signal. The intensity of the signal change can indicate the content of AZA in an unknown concentration.

2. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: In step (1) S1, the stirring temperature is 25°C and the stirring time is 6 h. The centrifugal speed is 8000 rpm and the centrifugal time is 10 min. The washing solution is methanol, the centrifugal speed during washing is 8000 rpm, the time is 10 min, and the number of washing times is 3-5 times. The vacuum drying temperature is 60°C and the drying time is 10 h.

3. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: In step (1) S2, the ultrasonic treatment time is 30 min. The continuous stirring time is 1 h. The hydrothermal reaction temperature is 180 °C and the heating time is 10 h. The centrifugal speed is 8000 rpm and the centrifugal time is 10 min. The vacuum drying temperature is 65 °C and the drying time is 10 h.

4. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: Step (1) Add cysteine ​​to S3 and stir for 30 min, then add HAuCl4·3H2O, stir for another 30 min, then add ascorbic acid, and continue stirring for 3 h. The centrifugal speed is 6000 rpm, and the centrifugal time is 5 min. The vacuum drying temperature is 60°C, and the drying time is 10 h.

5. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: In step (2), the mixture is incubated at 37°C for 40 min.

6. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: In step (3), the incubation time required for the Apt@AZA complex to be fixed on the electrode surface via gold-sulfur bonds is 2 h, and the incubation temperature is 25°C.

7. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: The relevant conditions of electropolymerization in step (3) are: voltage range of -0.5 V to 0.5 V, scan rate of 50 mV / s, and number of cycles of 20 times.

8. The method for preparing a dual recognition element electrochemical sensor for detecting methyl pyridophos according to claim 1, characterized in that: The composition of the eluent in step (3) is methanol / acetic acid = 8 / 2 (V / V), and the elution time is 20-30 min.

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