Nano-silver modified covalent organic framework composite material, preparation method and application thereof, and electrochemical aptamer sensor

Abstract

The invention belongs to the technical field of sensor materials and malathion detection, and relates to a nano-silver modified covalent organic framework composite material, a preparation method and application thereof, and an electrochemical aptamer sensor. The preparation method comprises the following steps: S1, dissolving 1, 3, 5-tri (4-aminophenyl) benzene and 2, 5-divinyl-1, 4-phthalaldehyde in acetonitrile, adding a catalyst to carry out condensation reaction, standing, separating, washing and drying to obtain a covalent organic framework material; s2, dispersing the covalent organic framework material and silver nitrate in water, adding sodium citrate after ultrasonic dispersion, and washing and drying to obtain the nano-silver modified covalent organic framework composite material. The nano-silver modified covalent organic framework composite material prepared by the invention shows excellent crystallinity, extremely high porosity and good conductivity, and realizes high-sensitivity, rapid and accurate quantitative analysis of malathion in traditional Chinese medicinal materials.

Description

Technical Field

[0001] The invention belongs to the technical field of sensor materials and malathion detection, and relates to a nanosilver-modified covalent organic framework composite material, a preparation method and application thereof, and an electrochemical aptamer sensor. Background Art

[0002] Malathion is a broad-spectrum organophosphorus insecticide widely used in pest control of traditional Chinese medicines. However, its potential residual toxicity poses a significant threat to human health and ecological balance. Therefore, it is necessary to detect the malathion content in traditional Chinese medicines.

[0003] Traditional detection methods for organophosphorus pesticides include chromatography, mass spectrometry, and a combination of the two. Although these methods have a wide range of applications and high specificity, the instruments are expensive and the pre-treatment process is lengthy, which leads to certain limitations in actual detection. Although the immunoassay method disclosed in the prior art can circumvent the use of the above-mentioned large instruments to a certain extent, it is difficult to overcome the defects such as the complex detection preparation process and the difficulty in long-term storage of antibodies, and it is still difficult to meet the detection requirements of organophosphorus. Aptamer sensors have the advantages of easy modification, easy synthesis, stable properties, strong specificity and high affinity, and can be used for the detection of organophosphorus. The methods of aptamer sensors for pesticide detection include colorimetry, electrochemical method and fluorescence method. Among them, electrochemical aptamer sensors not only significantly shorten the detection time required by traditional laboratory techniques, but also provide a platform for multi-analyte detection and high-throughput analysis in clinical diagnosis, food safety and environmental monitoring.

[0004] However, existing electrochemical aptamer sensors are mainly based on metal-organic frameworks (MOFs) and hydrogen-bonded organic frameworks (HOFs). However, when these electrochemical aptamer sensors are used to detect malathion, due to the structural limitations of the materials, they have poor conductivity and low electron transfer efficiency, resulting in low malathion detection sensitivity and slow detection speed.

[0005] Based on this, how to develop an electrochemical aptamer sensor with high sensitivity and rapid detection is a major issue currently facing malathion detection. Summary of the Invention

[0006] The silver nanoparticles modified covalent organic framework composite material (AgNPs@COFs) prepared by room temperature standing and in situ reduction technology in the present invention exhibits excellent crystallinity, extremely high porosity and good conductivity, and realizes high-sensitivity, rapid and accurate quantitative analysis of malathion in traditional Chinese medicine, thereby solving the problems of low detection sensitivity and slow detection speed of malathion in traditional Chinese medicine.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A method for preparing a nanosilver modified covalent organic framework composite material comprises the following steps:

[0009] S1. Preparation of covalent organic framework materials

[0010] 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde are dissolved in acetonitrile, a catalyst is added to carry out a condensation reaction, and then the reaction is allowed to stand, separated, washed and dried to obtain a covalent organic framework material.

[0011] S2. Preparation of nanosilver modified covalent organic framework composites

[0012] The covalent organic framework material and silver nitrate are dispersed in water, sodium citrate is added after ultrasonic dispersion, and the nanosilver modified covalent organic framework composite material is obtained through washing and drying.

[0013] It is further defined that in step S1, the mass ratio of 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde in every 5 mL of acetonitrile is (10-15) mg:(11-11.5) mg; and the volume ratio of acetonitrile to catalyst is (3-5) mL:(800-1000) μL.

[0014] It is further defined that the catalyst is acetic acid, lactic acid or butyric acid.

[0015] It is further defined that in step S2, the mass ratio of the covalent organic framework material, silver nitrate and sodium citrate in every 5 mL of water is (10-15) mg: (2-5) mg: (50-80) mg.

[0016] It is further defined that in both step S1 and step S2, washing is first performed with tetrahydrofuran and then with ethanol; and the drying temperature is both 60°C-100°C.

[0017] The nano-silver modified covalent organic framework composite material is prepared by utilizing the preparation method of the nano-silver modified covalent organic framework composite material.

[0018] The nanosilver modified covalent organic framework composite material is used as a sensor in the electrochemical detection of malathion.

[0019] An electrochemical aptamer sensor comprises an electrode matrix and an electrode material modified on the electrode matrix, wherein the electrode material is the nano-silver modified covalent organic framework composite material.

[0020] Application of electrochemical aptasensor in quantitative detection of malathion.

[0021] It is further defined that in the quantitative detection of malathion, a thiol-modified and ferrocene-labeled hairpin electrochemical aptamer sensor is used as a molecular recognition element, and differential pulse voltammetry is used for the quantitative detection of malathion.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The present invention provides a nanosilver modified covalent organic framework composite material. The porous structure of the covalent organic framework (COFs) gives it a large specific surface area and can provide abundant active sites. At the same time, the stable strong covalent bonds such as imine bonds and amide bonds in COFs ensure that it can still maintain structural integrity under extreme conditions such as strong acid and strong base. Nanosilver (AgNPs) has excellent conductivity and can enhance the electrocatalytic performance of porous COFs. After the nanosilver and covalent organic framework are composited, they have excellent crystallinity, extremely high porosity and good conductivity, which is conducive to the high-sensitivity, rapid and accurate quantitative analysis of malathion in traditional Chinese medicine.

[0024] 2. The present invention prepares a new type of nanosilver-modified covalent organic framework composite material through simple room-temperature synthesis and in situ growth technology, which significantly improves the electron transfer efficiency; combined with thiol-modified and ferrocene (Fc)-labeled hairpin electrochemical aptamers, it achieves specific recognition of the target analyte malathion and effective amplification of the electrochemical signal, realizing the accurate quantitative detection of malathion residues in traditional Chinese medicine.

[0025] 3. Through optimization, the present invention has achieved a linear detection range of 10-800 ng / L for malathion using an electrochemical aptamer sensor, with a detection limit as low as 8.237 ng / L (signal-to-noise ratio, S / N = 3). This sensor exhibits high precision, excellent repeatability (relative standard deviation, RSD <2.0%), and high selectivity. Testing also demonstrated that the electrochemical aptamer sensor was successfully applied to the quantitative detection of malathion in actual Chinese medicinal herb samples (such as ginseng, astragalus, wolfberry, and honeysuckle), with recoveries ranging from 98.32% to 101.92%. This provides a new strategy for constructing electrochemical aptamer sensors based on silver nanoparticle-modified covalent organic framework composites, which can be applied to the rapid screening of pesticides, offering a new direction for the field of pesticide detection.

[0026] 4. The electrochemical aptasensor constructed in this invention exhibits excellent stability and anti-interference capabilities, making it suitable for the precise determination of malathion in complex real-world samples. This invention not only provides a simple, economical, rapid, and highly sensitive method for malathion detection, but also offers new insights for monitoring contaminants in other Chinese medicinal materials or foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1TEM images of COFs and AgNPs@COFs composites; AB is the TEM image of COFs; CF is the TEM image of AgNPs@COFs;

[0028] Figure 2 XPS spectrum of AgNPs@COFs (A), high-resolution Ag 3d XPS spectrum of AgNPs@COFs composite material (B);

[0029] Figure 3 The schematic diagram of the preparation of electrochemical aptamer sensor;

[0030] Figure 4 For 5mM [Fe(CN)6] 3- / 4- Cyclic voltammetry curve of a bare glassy carbon electrode in solution;

[0031] Figure 5 The cyclic voltammetry curves of the assembled electrode were tested in 0.1 M PBS;

[0032] Figure 6 DPV graphs of different concentrations of malathion in 0.1 M phosphate buffer (PBS, pH = 7.4);

[0033] Figure 7 is the relationship between the current change (ΔI) and the malathion concentration (C Mal ) Linear graph (B);

[0034] Figure 8 The results of the test condition optimization are as follows: A is the amount of silver added; B is the concentration optimization result of AgNPs@COFs-CS; C is the concentration optimization result of aptamer; D is the incubation time result of aptamer and AgNPs@COFs;

[0035] Figure 9 The reproducibility results of the electrochemical aptasensor based on AgNPs@COFs;

[0036] Figure 10 The stability results of the electrochemical aptasensor based on AgNPs@COFs;

[0037] Figure 11 The specific results of the AgNPs@COFs-based electrochemical aptasensor. DETAILED DESCRIPTION

[0038] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention are considered to be within the scope of the present invention by making some conventional modifications to the reaction conditions of the present invention.

[0039] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0040] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, should be covered by the scope of protection of the present invention.

[0041] In a technical solution provided by the present invention, a method for preparing a nanosilver modified covalent organic framework composite material comprises the following steps:

[0042] S1. Preparation of covalent organic framework materials

[0043] 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde are dissolved in acetonitrile, a catalyst is added to carry out a condensation reaction, and then the reaction is allowed to stand, separated, washed and dried to obtain a covalent organic framework material.

[0044] Preferably, in step S1, the mass ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinyl-1,4-benzenedicarboxaldehyde in every 5 mL of acetonitrile is (10-15) mg:(11-11.5) mg; and the volume ratio of the acetonitrile to the catalyst is (3-5) mL:(800-1000) μL.

[0045] Illustratively, the mass ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinyl-1,4-benzenedicarboxaldehyde per 5 mL of acetonitrile is 10 mg:11 mg, 12 mg:11 mg, 15 mg:11 mg, 10 mg:11.5 mg, 12 mg:11.5 mg, or 15 mg:11.5 mg.

[0046] Illustratively, the volume ratio of acetonitrile to catalyst is 3 mL:800 μL, 3 mL:900 μL, 3 mL:1000 μL, 4 mL:800 μL, 4 mL:900 μL, 4 mL:1000 μL, 5 mL:800 μL, 5 mL:900 μL or 5 mL:1000 μL.

[0047] Preferably, the catalyst is acetic acid, lactic acid or butyric acid.

[0048] S2. Preparation of Nanosilver Modified Covalent Organic Framework Composites

[0049] The covalent organic framework material and silver nitrate are dispersed in water, and sodium citrate is added for in-situ reduction after ultrasonic dispersion, and then washed and dried to obtain a nanosilver modified covalent organic framework composite material.

[0050] Preferably, in step S2, the mass ratio of the covalent organic framework material, silver nitrate and sodium citrate in every 5 mL of water is (10-15) mg: (2-5) mg: (50-80) mg.

[0051] Exemplarily, in every 5 mL of water, the mass ratio of the covalent organic framework material, silver nitrate and sodium citrate is 10 mg:2 mg:50 mg, 10 mg:2 mg:80 mg, 10 mg:5 mg:50 mg, 10 mg:5 mg:80 mg, 15 mg:2 mg:50 mg, 15 mg:2 mg:80 mg, 15 mg:5 mg:50 mg or 15 mg:5 mg:80 mg.

[0052] Further defined, in both step S1 and step S2, washing is first performed with tetrahydrofuran and then with ethanol, and the drying temperature is 60° C. to 100° C. Exemplarily, the drying temperature is 60° C., 70° C., 80° C., 90° C. or 100° C.

[0053] In another technical solution provided by the present invention, a nanosilver-modified covalent organic framework composite material is prepared using the above-mentioned method for preparing a nanosilver-modified covalent organic framework composite material; the nanosilver-modified covalent organic framework composite material can be used as a sensor for the detection of malathion.

[0054] In another technical solution provided by the present invention, a nanosilver-modified covalent organic framework composite material is used as an electrode material, and the electrode material is modified on an electrode substrate to form an electrochemical aptamer sensor for the quantitative detection of malathion.

[0055] The technical solutions protected by the present invention are described in detail below with reference to several groups of embodiments.

[0056] The materials and reagents used in the following examples are as follows.

[0057] Materials: 2,5-Divinyl-1,4-benzenedicarboxaldehyde (DVA) and 1,3,5-tris(4-aminophenyl)benzene (TAPB) were purchased from Jilin Zhongke Kaisheng Technology Co., Ltd. (Jilin, China). Analytical-grade reagents, including acetonitrile, acetic acid, sodium citrate, malathion, ethanol (EtOH), tetrahydrofuran (THF), tris(2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl)aminomethane (Tris-HCl), and mercaptoethanol (MCH), were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Silver nitrate (AgNO3) and chitosan (CS, viscosity: 100–200 cP, deacetylation degree ≥90%) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Beijing, China) and Xi'an Guoan Biotechnology Co., Ltd. (Xi'an, China), respectively.

[0058] All reagents used were of analytical grade, and aqueous solutions were prepared with ultrapure water. Oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequences of the ferrocene (Fc)-labeled and thiol (SH)-modified malathion aptamers are as follows: Aptamer probe:

[0059] 5'-SH-(CH2)6-ATCCGTCACACACCTGCTTACTTACAATTGTTTTTCTTAACTTCTTGACTGCTGGTGTGTTGGCT-Fc-3'.

[0060] The instruments used in the following examples are as follows.

[0061] The sample morphology was observed using a Hitachi H-7500 transmission electron microscope (TEM). The elemental composition and chemical state of the AgNPs@COFs composites were characterized using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectrometer (XPS). The electrochemical performance of the materials was evaluated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) using a Multi-EmStat 4 electrochemical workstation and a CHI 660 electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd., China). The electrochemical experiments used a conventional three-electrode system: a glassy carbon electrode (GCE, geometric surface area = 0.071 cm 2 ) was the working electrode, saturated calomel electrode (SCE) was the reference electrode, and platinum wire was the counter electrode.

[0062] It should be noted that in the following examples, mM refers to mmol / L, and μM refers to μmol / L.

[0063] Example 1

[0064] This embodiment provides a method for preparing a nanosilver modified covalent organic framework composite material, comprising the following steps:

[0065] S1. Preparation of covalent organic framework materials TAPB-DVACOFs

[0066] 1,3,5-Tris(4-aminophenyl)benzene (TAPB) (14 mg, 0.04 mM) and 2,5-divinyl-1,4-benzenedicarbaldehyde (DVA) (11.1 mg, 0.06 mM) were dissolved in 5 mL of acetonitrile and sonicated for 1 minute to completely dissolve the monomers. 12 M acetic acid (800 μL) was then added as a catalyst, and the condensation reaction was initiated by vortexing for 15 seconds. The reaction mixture was allowed to stand for 72 hours and then centrifuged to obtain a yellow precipitate. This was washed sequentially with tetrahydrofuran and ethanol, and finally dried at 60°C to yield a spherical covalent organic framework material, designated TAPB-DVACOFs.

[0067] S2. Nanosilver modified covalent organic framework composites

[0068] At room temperature, 10 mg of the TAPB-DVA-COFs and 2 mg (0.4 mM) of silver nitrate were dispersed in 5 mL of deionized water. Ultrasonic treatment was performed for 5 minutes to achieve uniform dispersion. Subsequently, 50 mg of sodium citrate was added and stirred for 30 minutes to promote silver ion reduction and AgNP formation. The resulting material was washed sequentially with THF and EtOH, centrifuged, and dried at 60°C to obtain a silver nanoparticle-modified covalent organic framework composite, designated AgNPs@TAPB-DVACOFs.

[0069] The properties of the silver nanoparticles modified covalent organic framework composite material AgNPs@COFs prepared above were characterized.

[0070] (1) Morphological characterization

[0071] Transmission electron microscopy (TEM) was used to characterize the morphology of TAPB-DVA-COFs and AgNPs@COFs.

[0072] Figure 1 TEM images show that the synthesized COFs have a uniform spherical morphology with an average diameter of about 300 nm and a clear convex structure on the surface. This hierarchical structure provides abundant anchoring sites (AB) for the loading of nanoparticles. Figure 1 The CF further confirmed that AgNPs were successfully modified on the surface of COFs spheres. The loaded composite material still maintained its original spherical structure, and AgNPs (about 10 nm) were evenly distributed on the COFs surface.

[0073] (2) X-ray photoelectron spectroscopy (XPS) analysis

[0074] like Figure 2 As shown in Figure 1, the characteristic peaks of core elements such as C, N, O, and Ag can be clearly observed in the XPS spectrum of AgNPs@COFs. Figure 2 As shown in Figure B, the high-resolution Ag 3d XPS spectrum of the AgNPs@COFs composite material is fitted with two obvious characteristic peaks, located at 373.4eV (Ag 3d 3 / 2 ) and 367.2eV(Ag 3d 5 / 2 ), indicating the presence of Ag in the sample + (Ag2O). The successful synthesis of AgNPs@COFs was further verified.

[0075] Example 2

[0076] An electrochemical aptamer sensor was constructed using the silver nanoparticle-modified covalent organic framework composite material AgNPs@TAPB-DVA COFs prepared in Example 1.

[0077] In this embodiment, the electrochemical aptamer sensor is constructed as follows:

[0078] The nanocomposite-modified glassy carbon electrode (GCE) was prepared as follows: 1 mg of AgNPs@COFs was dispersed in 1 mL of 0.5% (w / v) chitosan solution and sonicated for 10 minutes to form a homogeneous suspension. Prior to modification, a bare GCE (3 mm diameter) was polished with 0.3 μm and then 0.05 μm alumina slurries, followed by ultrasonic cleaning in a 1:1 (v / v) ethanol / water mixture for 5 minutes to remove adsorbed contaminants. A 7 μL drop of the suspension was applied to the GCE surface and allowed to dry at room temperature.

[0079] To immobilize the thiol-modified aptamer on the AgNPs@COFs / GCE surface, the electrode was incubated with 5 μL of a 3.0 μM aptamer mixture (containing 100 μM thiol-modified aptamer, 15 mM PBS, 1 mM TCEP, pH 7.4) at room temperature for 1.5 hours to obtain an aptamer nanoconjugate-modified electrode. The aptamer nanoconjugate-modified electrode was then rinsed with ultrapure water and blocked with 1 mM mercaptoethanol (MCH) for 1 hour to block unmodified sites. The electrode was then gently dried with nitrogen gas. Finally, the aptamer nanoconjugate-modified electrode was incubated in a range of malathion solution concentrations for subsequent detection.

[0080] In this embodiment, the sensing mechanism and performance of the electrochemical aptamer sensor are verified.

[0081] (1) Synthesis of electrochemical aptasensors

[0082] like Figure 3 As shown, uniformly sized COF spheres were first synthesized and loaded with AgNPs via an in situ growth method to enhance electron transfer efficiency. Subsequently, a ferrocene (Fc)-labeled hairpin aptamer was attached to the COF surface via an Ag-S bond. Subsequently, unbound sites were blocked with mercaptoethanol (MCH) to prevent nonspecific adsorption. In the presence of the target analyte, the high-affinity interaction between the aptamer and malathion triggered specific recognition and binding, resulting in a conformational change in the hairpin aptamer. This structural rearrangement shifted the Fc tag away from the electrode surface, reducing the electrochemical signal intensity monitored by differential pulse voltammetry (DPV). Based on this principle, the DPV signal gradually decreased with increasing malathion concentration. The constructed aptamer sensor was applied to the analysis of real samples. The results demonstrated that this sensing strategy provides a new approach for the development of efficient aptamer sensors and has broad application prospects in the analysis of traditional Chinese medicines and foods.

[0083] (2) Verification of the successful construction of electrochemical aptasensors

[0084] Cyclic voltammetry (CV) was used to study the effects of different modified electrodes on the 3- / 4- Sensing kinetics in solution.

[0085] See also Figure 4 The cyclic voltammetry curve of the bare glassy carbon electrode exhibited a pair of reversible redox peaks. When COFs were modified alone on the electrode surface, the redox peak signal was lower than that of the bare electrode due to their large steric hindrance. The introduction of AgNPs significantly enhanced the current signal, indicating that the excellent conductivity of AgNPs facilitates electron transport. The current signal further increased after the assembly of an Fc-labeled hairpin aptamer, as the hairpin structure of the aptamer brought the Fc group closer to the electrode surface, promoting its redox process. After the electrode was blocked with MCH, the interfacial impedance increased due to its insulating properties, further reducing the current signal. These results confirm the successful construction of the electrochemical aptamer sensor.

[0086] In addition, the cyclic voltammetry (CV) curves of the step-by-step assembled electrodes were tested in 0.1 M PBS to verify the applicability of the prepared electrodes.

[0087] like Figure 5 As shown, the bare electrode surface exhibits a smooth closed curve (black line) due to the lack of electrochemically active species. After modification with AgNPs@COFs, a clear silver oxidation peak appears at 0.015 V (red line), indicating successful modification of the AgNPs@COFs onto the electrode surface. Immobilization of the Fc-functionalized hairpin aptamer onto the AgNPs@COFs-modified electrode via an Ag-S bond yields a stable and distinct Fc redox peak (blue line). Incubation of the electrode with 500 ng / L malathion significantly reduces the Fc redox peak current (green line). This is attributed to the specific binding of the aptamer to malathion on the electrode surface, which results in the opening of the hairpin structure and the removal of Fc from the electrode surface, leading to a decrease in the CV peak current. These analytical results demonstrate that the prepared electrochemical aptamer sensor electrode is suitable for malathion detection.

[0088] Example 3: Detection of Malathion

[0089] This example mainly uses the electrochemical aptamer sensor constructed in Example 2 to detect malathion in traditional Chinese medicine, thereby evaluating the quantitative detection capability of the electrochemical aptamer sensor.

[0090] The electrochemical aptasensor constructed in Example 2 was incubated with different concentrations (10–800 ng / L) of malathion in 0.1 M phosphate buffer (PBS, pH = 7.4), and the DPV response of the ferrocene (Fc) redox probe was recorded in the potential range of -0.1–0.5 V. Its signal intensity was negatively correlated with the malathion concentration.

[0091] like Figure 6 As shown in the figure, as the malathion concentration increased from 10.0 ng / L to 800.0 ng / L, the DPV signal gradually decreased. This is because after different concentrations of malathion bind to the Fc-labeled aptamer, the aptamer changes conformation and moves away from the electrode surface, resulting in a weakened Fc signal.

[0092] In this detection range, the current change (ΔI) is related to the malathion concentration (C Mal ) is linearly related, such as Figure 7 As shown, the linear regression equation is:

[0093] ΔI=0.01255–0.000774C Mal

[0094] R 2 =0.995, the detection limit was 8.237 ng / L, and the signal-to-noise ratio (S / N) was 3. Compared with similar aptasensors (Table 1), the electrochemical aptasensor based on AgNPs@COFs electrode has a wider linear range.

[0095] Table 1. Comparison of analytical performance of this method with other reported pesticide detection techniques

[0096] method Detection limit (ng / L) Detection range (ng / L) Colorimetry 7350 <![CDATA[2.5×10 4 –2.5×10 5 ]]> Colorimetry 166.7 <![CDATA[5×10 2 –5×10 6 ]]> Fluorescence method 1930 <![CDATA[0.1–10 5 ]]> Surface-enhanced Raman spectroscopy 66 <![CDATA[0.1–3.16×10 2 ]]> Photoelectrochemical method 0.167 <![CDATA[5×10 -4 –3.0]]> Gas chromatography-flame photometric detection <![CDATA[10 7 ]]> <![CDATA[10 5 –10 7 ]]> Gas chromatography-electron capture detection <![CDATA[10 8 ]]> <![CDATA[4×10 5 ]]> Electrochemical method 63.4 <![CDATA[0.1–10 4 ]]> Electrochemical method 28 <![CDATA[0.05–10 3 ]]> Electrochemical method 8.237 10–800

[0097] The above results indicate that the electrochemical aptamer sensor constructed in the present invention can be used for the quantitative detection of malathion, and the formed aptamer Apt / AgNPs@COFs / GCE complex serves as an amplification carrier of the Fc signal and has high sensitivity.

[0098] Furthermore, taking actual Chinese medicinal materials as an example, the applicability of the electrochemical aptamer sensor to the actual detection of malathion was evaluated.

[0099] In this example, spiked samples of four Chinese medicinal herbs (TCMs)—ginseng, astragalus, wolfberry, and honeysuckle—were used for the experiment. Preferably, the TCM samples were purchased from a local market. The specific processing steps were as follows: 1.0 g of the TCM sample was weighed, minced, and homogenized; methanol was added to extract the pesticide residues, the mixture was centrifuged at 6000 rpm for 10 minutes, and the supernatant was collected; the extraction was repeated once, and the two supernatants were combined; the combined supernatant was filtered through a 0.22 μm filter to remove particulate matter; finally, the filtrate was brought to volume with PBS buffer, mixed thoroughly, and stored at 4°C.

[0100] The actual samples were analyzed using the spiked method (Table 2) to verify the reliability of the electrochemical aptasensor in the detection of actual samples.

[0101] Table 2 Detection results of malathion in Chinese herbal medicine samples (n=3)

[0102]

[0103] The results in Table 2 show that the recoveries of malathion were 98.32%–101.92%, with relative standard deviations (RSDs) ranging from 1.25% to 3.28%. These results demonstrate that the electrochemical aptasensor has good reliability and is suitable for the detection of malathion in real samples.

[0104] Optimization Example

[0105] The conditions for malathion detection were optimized through the following optimization experiments.

[0106] To achieve highly sensitive and rapid detection of malathion, several key experimental parameters were investigated, including the amount of silver added, the concentration of AgNPs@COFs, the aptamer concentration, and the hybridization time between the aptamer and the AgNPs@COFs composite material. By varying these parameters, the conditions for optimal sensitivity and reproducibility in malathion detection were determined.

[0107] (1) Optimization of silver addition

[0108] The amount of silver nitrate added directly affects the electron transfer efficiency of the material ( Figure 8 Middle A): When the addition amount is too low, the amount of silver attached to the COF surface is low, resulting in a weak current signal. As the amount of silver nitrate increases, at 2 mg, the silver nanoparticles are evenly distributed and the amount of silver attached is maximized, producing the strongest current signal in a 5 mM solution. When the addition amount is too high, AgNPs may aggregate, which in turn reduces the current signal. Therefore, adding 2 mg of silver nitrate to 10 mg of COF material was selected as the optimal ratio for subsequent experiments.

[0109] (2) Concentration optimization of AgNPs@COFs dispersion suspension

[0110] The concentration of AgNPs@COFs dispersion suspension was then optimized. Figure 8 As shown in Figure B, when the COFs concentration increases from 0.5 mg / mL to 1 mg / mL, the current response of the AgNPs@COFs material significantly increases. However, further increasing the suspension concentration leads to a decrease in the peak current, likely due to the high concentration of the modifier covering the electrode surface and hindering the electron transfer process. Therefore, the optimal concentration of the AgNPs@COFs dispersion suspension was determined to be 1 mg / mL.

[0111] (3) Optimization of aptamer concentration

[0112] The concentration of the aptamer (i.e., Fc-functionalized hairpin aptamer) directly affects the electrochemical aptasensor signal. Figure 8As shown in Figure C, when the aptamer concentration increases from 0.5 μM to 5 μM, the Fc oxidation peak current first increases and then decreases. This may be because after the AgNPs@COFs composite reaches saturation with the aptamer, the steric hindrance caused by excess binding leads to a slight decrease in the Fc current signal. Therefore, 3 μM was selected as the optimal aptamer concentration.

[0113] (4) Hybridization time between aptamer and AgNPs@COFs composite

[0114] In order to optimize the sensing performance, the hybridization time (15–150 min) of the aptamer (i.e., Fc-functionalized hairpin aptamer) and the AgNPs@COFs composite was investigated. Figure 8 As shown in Figure D, the oxidation peak current of Fc gradually increases with the extension of hybridization time and reaches a peak value at 90 minutes. Therefore, the optimal hybridization time of the aptamer and the AgNPs@COFs composite material is determined to be 90 minutes.

[0115] Verification Example

[0116] The selectivity, repeatability and stability of the electrochemical aptamer sensor were verified.

[0117] (1) Five electrodes were prepared using the same method to test the electrochemical aptasensor based on AgNPs@COFs. Differential pulse voltammetry (DPV) detection was performed in 300 ng / L malathion solution to verify the repeatability of the electrodes.

[0118] like Figure 9 As shown in the figure, the relative standard deviation (RSD) is 1.04%, indicating that the electrode has good repeatability.

[0119] (2) After the electrochemical aptamer sensor prepared in Example 2 was stored in a refrigerator at 4°C for 7 days, the response current was still 96.34% of the initial current, and the relative standard deviation (RSD) was 1.69% ( Figure 10 There was no significant change, indicating that the prepared electrochemical aptasensor had good stability.

[0120] (3) To investigate the pesticide selectivity of the electrochemical aptasensor of Example 2, several potential pesticide interfering substances were tested, including pendimethalin, deltamethrin, cypermethrin, cypermethrin, phoxim, and imidacloprid. The malathion concentration was 500 ng / L, the interfering substance concentration was 10 times that of the analyte, and the blank control was a 0.1 M PBS solution (pH = 7.4).

[0121] Figure 11The normalized DPV peak current responses for each interferent, a blank, malathion, and their mixtures from four replicate experiments are shown. The results show only slight changes in the current responses for the non-target and blank groups, while the current response significantly decreases in the presence of the target, malathion. Furthermore, selectivity experiments with the mixed system were designed to verify the cross-sensitivity of the electrochemical aptasensor. The results showed that the response of the mixed system was nearly identical to that of pure malathion. These experimental results confirm the high selectivity of the constructed electrochemical aptasensor for malathion.

[0122] It should be noted that the above performance tests were conducted on the nanosilver-modified covalent organic framework composite material prepared in Example 1 and the electrochemical aptamer sensor in Example 2. When the catalyst type and the ratio of the raw materials in Example 1 were replaced, the nanosilver-modified covalent organic framework composite material also exhibited the same or similar performance, with excellent crystallinity, extremely high porosity and good conductivity, and can achieve high-sensitivity, rapid and accurate quantitative analysis of malathion in traditional Chinese medicine.

[0123] The above are several relatively preferred implementation methods of the preparation method of the present invention, but they cannot be used as limitations on the technical solutions protected by the present invention. Any replacement solutions obtained by ordinary technicians in this field without making creative work based on the technical ideas of the present invention should fall within the scope of protection of the present invention.

Claims

1. A method for preparing a nanosilver modified covalent organic framework composite material, characterized in that: The following steps are involved: S1. Preparation of covalent organic framework materials 1,3,5-tris(4-aminophenyl)benzene and 2,5-divinyl-1,4-benzenedicarboxaldehyde are dissolved in acetonitrile, a catalyst is added to carry out a condensation reaction, and then the reaction is allowed to stand, separated, washed and dried to obtain a covalent organic framework material. S2. Preparation of nanosilver modified covalent organic framework composites The covalent organic framework material and silver nitrate are dispersed in water, sodium citrate is added after ultrasonic dispersion, and the nanosilver modified covalent organic framework composite material is obtained through washing and drying.

2. The method for preparing the nanosilver modified covalent organic framework composite material according to claim 1, characterized in that: In step S1, the mass ratio of 1,3,5-tris(4-aminophenyl)benzene to 2,5-divinyl-1,4-benzenedicarboxaldehyde in every 5 mL of acetonitrile is (10-15) mg:(11-11.5) mg; and the volume ratio of acetonitrile to catalyst is (3-5) mL:(800-1000) μL.

3. The method for preparing the nanosilver modified covalent organic framework composite material according to claim 1, characterized in that: The catalyst is acetic acid, lactic acid or butyric acid.

4. The method for preparing the nanosilver modified covalent organic framework composite material according to claim 1, characterized in that: In step S2, the mass ratio of the covalent organic framework material, silver nitrate and sodium citrate in every 5 mL of water is (10-15) mg: (2-5) mg: (50-80) mg.

5. The method for preparing the nanosilver modified covalent organic framework composite material according to claim 1, characterized in that: In both step S1 and step S2, the product is first washed with tetrahydrofuran and then with ethanol; and the drying temperature is 60° C.-100° C.

6. A nano-silver modified covalent organic framework composite material prepared by the method for preparing a nano-silver modified covalent organic framework composite material according to claim 1.

7. Use of the nanosilver modified covalent organic framework composite material according to claim 6 as a sensor in the detection of malathion.

8. An electrochemical aptamer sensor, characterized in that The invention comprises an electrode matrix and an electrode material modified on the electrode matrix, wherein the electrode material is the nano-silver modified covalent organic framework composite material according to claim 7.

9. Use of the electrochemical aptasensor according to claim 8 in the quantitative detection of malathion.

10. The use according to claim 9, characterized in that In the quantitative detection of malathion, a thiol-modified and ferrocene-labeled hairpin electrochemical aptamer sensor was used as the molecular recognition element, and differential pulse voltammetry was used for the quantitative detection of malathion.

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