Preparation method and application of multistage through hole carbon powder molecular imprinting sensor

Abstract

The invention discloses a preparation method and application of a multistage through hole carbon powder molecular imprinting sensor, belongs to the technical field of rapid detection, and aims to greatly improve the high sensitivity, stability and response speed of the molecular imprinting sensor. The preparation method comprises the following steps: preparing radix astragali straw carbon powder with multistage through holes, preparing a solution A, preparing a sensor electrode, electrically polymerizing a molecularly imprinted polymer, and eluting a molecular template. The application comprises the field of pesticide residue rapid detection and the field of pesticide residue rapid detection in different periods after pesticide spraying during traditional Chinese medicine planting field management. According to the invention, a new material and a new application are found, and a new way is provided for waste treatment of astragalus membranaceus straws in a traditional Chinese medicine planting area.

Description

Technical Field

[0001] This invention belongs to the field of rapid detection technology, specifically relating to the preparation method and application of a multi-level through-pore carbon powder molecular imprint sensor. Background Technology

[0002] Biochar is a carbonaceous material produced by high-temperature pyrolysis of biomass materials under oxygen-deficient or low-oxygen conditions. It has a highly porous structure and abundant surface functional groups, and possesses high specific surface area, excellent conductivity, good adsorption performance and tunable surface chemical properties. It has potential application value in many fields. In recent years, its application has gradually expanded to electrode materials for molecularly imprinted electrochemical sensors.

[0003] Molecularly imprinted electrochemical sensors are a novel technology based on the principle of molecular recognition. Their core lies in the specific interaction between template molecules and functional monomers to form recognition sites with specific spatial structures, enabling the specific recognition of target molecules and their analogues. They are commonly used for rapid detection. In the construction of electrochemical sensors, the electrode materials determine the sensor's response performance.

[0004] In the existing technology, the main sources of biochar materials are common biomass such as wheat, coconut shells, wood, and tea. These biochar materials are used as electrode materials in fields such as electrochemical sensors, supercapacitors, lithium-ion batteries, catalytic reactions, and biosensors. However, there is a lack of records on their application as electrode materials for molecularly imprinted electrochemical sensors.

[0005] Compared to general biological stems such as corn stalks and wheat stalks, which have scientifically mature disposal methods, astragalus stems are currently subject to severe waste. In its planting areas, due to environmental protection requirements, they cannot be burned and are often piled up in large quantities in the fields, where natural degradation takes many years, affecting the planting area of ​​farmers. On the other hand, astragalus stems have significant advantages as a source of biochar: they are naturally rich in complex organic matter containing nitrogen and oxygen, such as amino acids, polysaccharides, flavonoids, and saponins, far more than the single components such as cellulose and hemicellulose that account for more than 80% of general biological stems. In-depth research on these characteristics has found that they can be converted into various active functional groups such as pyrrole, pyridine, and phenolic hydroxyl groups during pyrolysis, which makes the surface active sites of the prepared biochar more abundant. At the same time, the pyrolysis of polysaccharides and saponins easily forms multi-level pores with high specific surface area, better pore structure, and adsorption capacity. These preliminary research results have led researchers to link them with molecular imprinted sensors, because these characteristics are very important for constructing high-performance molecular imprinted sensors, which can effectively improve the stability and recognition efficiency of molecular imprinted polymers.

[0006] However, how to obtain biochar powder that is more compatible with molecular imprinted sensors, and further combine the two for rapid detection of pesticide residues, has become a technical challenge for researchers.

[0007] Based on the above research and development ideas, the researchers proposed a method for preparing a multi-level through-pore carbon powder molecular imprint sensor and its application. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing a multi-level through-pore carbon powder molecular imprinted sensor, so as to solve the problem of significantly improving the high sensitivity, stability and response speed of the molecular imprinted sensor.

[0009] Another objective of this invention is to provide an application of a multi-level through-pore carbon powder molecular imprint sensor to solve the problem of the inability to quickly detect pesticide residues during field management and raw material acquisition of traditional Chinese medicine.

[0010] To solve the above problems, the technical solution of the present invention is as follows: A method for preparing a multi-level through-pore carbon powder molecularly imprinted sensor, comprising the following steps: S1. Preparation of Astragalus straw charcoal powder with multi-level through holes; Astragalus straw was treated by a one-step carbonization method to prepare Astragalus straw charcoal powder D with multi-level through pores; S2, Preparation of solution A; Take 3±0.1 mg of Astragalus straw charcoal powder D prepared by S1, dissolve it in 1±0.1 ml of deionized water, and sonicate the mixed solution for 30 s-1 min to obtain solution A.

[0011] S3. Fabrication of sensor electrodes: Take 10 μL of solution A and coat it onto the surface of an initial empty electrode with a diameter of 4 mm. Blow it dry with nitrogen gas. Solution A is uniformly distributed on the surface of the glassy carbon electrode as an electrode material, thus obtaining electrode A.

[0012] S4, electropolymerized molecularly imprinted polymer: Electrode A, prepared by S3, was used as the working electrode in the three-electrode system of the electrochemical workstation. The target analyte and its compatible monomer were polymerized on the electrode surface to form a molecularly imprinted polymer, thus obtaining electrode B.

[0013] S5, Elution of molecular template: Electrode B prepared in S4 was immersed in the eluent and eluted. After elution, the electrode was removed and the surface of the electrode was rinsed with a small amount of deionized water to obtain molecularly imprinted sensor electrode C, which can be used to detect target analytes.

[0014] Furthermore, the specific steps for preparing Astragalus stalk charcoal powder D1 with multi-level through pores by the one-step carbonization method described in S1 are as follows: S1.1 Preparation of phosphoric acid solution; Add 31.3-38.1 mL of phosphoric acid to 55-60 mL of deionized water with stirring, and bring the volume to 100 mL to obtain a phosphoric acid solution.

[0015] S1.2 Preparation of Astragalus straw powder: Rinse the astragalus stalks with deionized water, dry them at 50°C for at least 8 hours, pulverize them, and pass them through a 50-mesh sieve to obtain astragalus stalk powder A with uniform particle size.

[0016] S1.3, Chemical activation: Add 2g of Astragalus stalk powder prepared in S2 to 20mL of phosphoric acid solution and stir well. Soak for 24 hours, filter to obtain filter residue, wash twice with deionized water, and dry in an oven at 80℃ for 12 hours or more to obtain activated Astragalus stalk powder B.

[0017] S1.4, Pyrolysis and Carbonization: The activated Astragalus straw powder prepared in S3 was placed in a tube furnace and heated to 500±5℃ at a heating rate of 5°C / min under a nitrogen atmosphere. It was then held at this temperature for 2 hours for pyrolysis and carbonization. The final product was Astragalus straw carbon powder C.

[0018] S1.5 Washing and drying: The Astragalus straw carbon powder prepared in S4 was washed with deionized water until the pH reached 7±0.1. The powder was then collected by centrifugation and dried at 80±5℃ for 12 hours or more to obtain the final product – Astragalus straw carbon powder D with multi-level through pores.

[0019] Furthermore, in S4, the target analyte is selected as the pesticide chlorpyrifos, and the compatible polymerizable monomer is dopamine. The specific polymerization process is as follows: Electrode A prepared by S3 was used as the working electrode in the three-electrode system of the electrochemical workstation. The three electrodes were immersed in a PBS solution with pH=6 containing dopamine and chlorpyrifos (molar concentration ratio of 3:2). The parameters of the electrochemical workstation were adjusted, and CV scan was performed at a rate of 100mV / s for 20 cycles to form a molecularly imprinted polymer on the electrode surface, thus obtaining electrode B.

[0020] Furthermore, the eluent for electrode B in S5 is DES, and the mass ratio of choline chloride to urea in the mixed solution is 1:2. The solution is prepared by stirring at 80°C for 30 minutes. During elution, soak the electrode for 10 minutes before removing it and then rinse the electrode surface with a small amount of deionized water.

[0021] Furthermore, this sensor is applied in the field of rapid detection of pesticide residues.

[0022] Furthermore, it can be applied to the rapid detection of pesticide residues at different stages after pesticide spraying during the field management of Chinese medicinal herb cultivation.

[0023] The beneficial effects of this invention are as follows: (1) Astragalus membranaceus was chosen as the raw material for the multi-level interconnected pore carbon powder of this invention. This was chosen because, on the one hand, it was to dispose of a large amount of waste from Astragalus membranaceus straw, and on the other hand, because the properties of its characteristic components in the carbon powder after pyrolysis are very important for constructing high-performance molecularly imprinted sensors, which can effectively improve the stability and recognition efficiency of molecularly imprinted polymers. Therefore, the two were combined in the research and development. This invention uses a chemical activation-one-step carbonization method to treat Astragalus membranaceus straw. By controlling the concentration of H3PO4 and the carbonization temperature, Astragalus membranaceus straw biomass carbon powder material with multi-level interconnected pores and a highly active surface was prepared.

[0024] This carbon powder material has a multi-level porous structure. Micropores provide a high specific surface area, mesopores retain the directional channels of plant fibers, and macropores maintain the three-dimensional skeleton of the cell cavity. Structurally, the three-dimensional skeleton directional channels provide a stable construction platform for molecularly imprinted polymer recognition sites. The high specific surface area can provide more molecularly imprinted recognition sites, improve the recognition efficiency of the sensor, and overcome the limitations of traditional straw in terms of "poor structural support and few active sites".

[0025] This carbon powder material has an ultra-high specific surface area and heteroatom doping active sites that synergistically increase the reaction interface, improve charge mobility, reduce charge transfer resistance, and have excellent electrical conductivity. It can be used as an ideal substrate material for highly sensitive electrochemical sensors. In molecular imprinted directional detection, it can significantly improve the sensitivity and response efficiency of the sensor and enhance the application efficiency of rapid detection technology.

[0026] (2) The multi-level through-pore carbon powder molecular imprinted sensor prepared in this invention uses Astragalus straw biomass carbon powder as a carrier. Due to the excellent compatibility between the carbon powder and the molecular imprinted electrochemical sensor, the sensor performance can be greatly improved. The active groups enriched on the surface of Astragalus straw biomass carbon powder can significantly enhance the structural stability and construction efficiency of the molecular imprinted polymer, laying the foundation for the accurate anchoring of recognition sites; its unique multi-level porous structure can efficiently expand the specific surface area, further increasing the number and accessibility of molecular imprinted recognition sites, and improving the capture ability of target substances; its inherent high conductivity can accelerate the electron transfer rate on the electrode surface, greatly optimizing the sensor's sensing response efficiency. The synergistic effect of multiple aspects can ultimately achieve high-sensitivity detection of target analytes by the sensor, effectively breaking through the limitations of traditional sensors in terms of stability, response speed and detection limit, which is of great significance in the field of rapid detection.

[0027] The multi-level through-pore carbon powder molecular imprint sensor prepared by this invention has significant breakthrough implications for the quality control of Chinese medicinal materials. On the one hand, it can determine the pesticide spraying cycle during field management of Chinese medicinal material cultivation, changing the current situation where growers spray pesticides based on experience and planting enterprises spray pesticides according to a set cycle. These extensive drug administration methods are an important reason for excessive pesticide residues in raw materials for medicinal slices. With the rapid detection technology of the sensor of this invention, the amount of pesticide residues on the roots, stems and leaves of medicinal materials can be quickly determined to determine whether pesticide spraying is necessary. On the other hand, it can quickly detect pesticide residues in raw materials such as flowers, leaves, roots, stems and seeds of medicinal materials at the time of harvest. For growers and planting enterprises, they can choose to harvest after the pesticides have been basically metabolized. For medicinal slice processing enterprises, they can use the sensor of this invention to quickly detect pesticide residues in raw materials at the raw material procurement stage, and control the quality from the source of procurement to avoid purchasing raw materials with excessive pesticide residues. Naturally, the problem of excessive pesticide residues can be controlled in the processing, production and inspection of medicinal slices.

[0028] (3) Through in-depth research on Astragalus membranaceus straw, this invention has discovered the special properties of its carbon powder material. From the preparation of molecular imprinted sensors to their application in rapid detection of pesticide residues, the entire process is a utilization of a large amount of Astragalus membranaceus straw waste in existing technologies. In addition to discovering new materials, this invention also provides a new way out for the waste treatment of Astragalus membranaceus straw in Chinese medicinal herb planting areas, and its environmentally friendly effect is also quite prominent. Attached Figure Description

[0029] Figure 1 This is an electron microscope image of Astragalus straw charcoal powder D1 in Example 1 at 1000x magnification; Figure 2 EDS spectrum of Astragalus straw charcoal powder D1 in Example 1; Figure 3 The Fourier transform infrared spectrum (FT-TR) of Astragalus straw charcoal powder D1 in Example 1 is shown below. Figure 4 This is a pore size analysis diagram of Astragalus straw charcoal powder D1 in Example 1; Figure 5 This is a pore size analysis diagram of wheat straw biochar used for comparison in Example 1; Figure 6 Electron micrograph of Astragalus straw charcoal powder D2 prepared in Example 2 at 5058x magnification; Figure 7 This is a comparison of the electrochemical impedance spectroscopy (EIS) spectra before and after electrode coating in Example 3; Figure 8 The standard curve for detecting the organophosphorus pesticide chlorpyrifos is shown for the molecularly imprinted sensor electrode C prepared in Example 4. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Therefore, the following detailed description of embodiments of the invention provided in the accompanying drawings is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention.

[0032] Example 1 A method for preparing multi-level through-pore carbon powder, comprising the following steps: S1, Preparation of phosphoric acid solution; Add 31.3 mL of phosphoric acid to 60 mL of deionized water with stirring, and then bring the volume to 100 mL to obtain a phosphoric acid solution.

[0033] S2. Preparation of Astragalus straw powder: Astragalus straw was rinsed with deionized water, dried at 50°C for 8 hours, pulverized and passed through a 50-mesh sieve to obtain Astragalus straw powder A1 with uniform particle size.

[0034] S3, Chemical Activation: Add 2g of Astragalus stalk powder prepared in S2 to 20mL of phosphoric acid solution and stir well. Soak for 24 hours, filter to obtain filter residue, wash twice with deionized water, and dry in an oven at 80℃ for 12 hours to obtain activated Astragalus stalk powder B1.

[0035] S4, Pyrolytic Carbonization: The activated Astragalus straw powder prepared in S3 was placed in a tube furnace and heated to 500°C at a heating rate of 5°C / min under a nitrogen atmosphere. The temperature was maintained at this temperature for 2 hours for pyrolysis and carbonization. The final product was Astragalus straw carbon powder C1.

[0036] S5. Washing and drying: The Astragalus straw carbon powder prepared in S4 was washed with deionized water until the pH reached 7. The powder was then collected by centrifugation and dried at 80°C for 12 hours to obtain the final product—Astragalus straw carbon powder D1 with multi-level through pores.

[0037] The final product, Astragalus straw charcoal powder D1, was divided into multiple samples and subjected to the following tests in sequence: 1. Scanning electron microscopy; The SEM images of the Astragalus membranaceus straw charcoal powder D1 sample were observed under 1000x electron microscopy. Figure 1 As shown, the material in the sample has a hierarchical porous structure.

[0038] 2. Energy dispersive spectroscopy (EDS) analysis; EDS (Energy Dispersive Spectroscopy) enables qualitative and quantitative analysis of sample composition by detecting the characteristic X-rays of elements. The figure shows the raw data, and the table is a quantitative analysis report of the signals in the figure. The EDS results of the Astragalus straw charcoal powder D1 sample are shown in the table below: As can be seen, the sample contains elements such as C, O, N, P, and S. The high carbon content of 71.25% and the low oxygen ratio of 15.88% construct a dense and low-defect carbon framework, significantly reducing electron transport resistance and enabling rapid response to the electrochemical signals of the target analyte, thus significantly improving the sensor's response speed and detection sensitivity. Uniform doping with 11.62% P, 0.96% N, and 0.29% S forms numerous doped active centers in the carbon matrix, providing abundant electrochemical active sites and addressing the problem of insufficient active sites in traditional biomass carbon electrodes. The high carbon structure endows the material with excellent chemical stability and durability. These superior electrochemical sensing properties are the key elemental basis for this material to meet the requirements of high sensitivity, high selectivity, and high stability in electrochemical sensing. The accompanying EDS spectrum is shown below. Figure 2 As shown.

[0039] 3. Fourier transform infrared spectroscopy analysis; Energy dispersive spectroscopy (EDS) analysis was performed on sample D1 of Astragalus membranaceus straw charcoal powder. The Fourier transform infrared (FT-TR) spectrum of the sample is shown below. Figure 3 ,visible: 3437cm -1 The broad peak at that location reflects the hydroxyl structure present on the surface of the carbon material; 2920 / 2850cm -1 The absence of peaks indicates high-temperature carbonization and complete decomposition of aliphatic chains. 2354cm -1 The peak at this point represents the stretching vibration peak of P-OH, which is introduced by chemical activation of P atoms; 1546cm -1 The peak may originate from aromatic sp. 2 Hybrid C=C structure; 1156cm -1 The peak may originate from the stretching vibration of the POC; 1081cm -1 It originates from the CO structure in the material.

[0040] 4. Comparison of biochar pore size analysis; Pore ​​size analysis was performed on samples D1 of Astragalus membranaceus straw charcoal powder and wheat straw biomass charcoal powder prepared using the same method. The results are compared in the table below: The specific surface area and pore size analysis diagrams of the two were obtained as follows: Figure 4 and Figure 5 : in, Figure 4 This is a pore size analysis diagram of Astragalus membranaceus straw biochar. Figure 5 This is a pore size analysis diagram of wheat straw biochar. The comparison shows: The specific surface area of ​​Astragalus straw biochar prepared by the method of this invention is as high as 1655.5398 m². 2 / g, nearly an order of magnitude higher than the control wheat straw biochar. The high specific surface area provides ample space for grafting of molecularly imprinted polymers and recognition of target molecules. A larger surface area can accommodate more molecularly imprinted sites, significantly improving the sensor's adsorption capacity and recognition probability for target molecules. Simultaneously, the high specific surface area increases the contact area between the material and the electrode substrate, reducing interfacial resistance and laying the foundation for efficient electrochemical signal transmission, ensuring that the identified target molecules can quickly convert the recognition signal into a detectable electrochemical signal.

[0041] The high pore volume of Astragalus straw charcoal powder can accommodate more molecularly imprinted polymer segments, avoiding the aggregation of imprinted sites due to insufficient space, which reduces recognition efficiency and ensures that mass transfer is not easily hindered by "space congestion" during molecular transport within the pores. In contrast, wheat straw biochar powder has a low pore volume (0.1009 cm³). 3 / g) cannot load a sufficient amount of molecularly imprinted polymer, nor can it meet the diffusion requirements of the target molecule, and is prone to problems such as "waste of imprinted sites and slow mass transfer".

[0042] From the perspective of pore structure characteristics, the average pore size of Astragalus straw charcoal powder is 21.3899 nm, which is in the mesoporous range (2-50 nm). The aspect ratio of Astragalus natural fibers allows it to retain the gap structure between fibers after carbonization, forming slit pores. Combined with the conclusion of nitrogen adsorption isotherm, it further confirms its multi-level structure of "mesoporous + slit pore". Mesopores can serve as dedicated transport channels for "target molecules - imprinted sites", ensuring that molecules diffuse rapidly to the recognition sites. At the same time, slit pores can help screen impurities. Wheat straw biomass charcoal powder has an average pore size of only 1.7057 nm, which is in the microporous range (<2 nm). This structure makes it difficult for molecularly imprinted polymers to enter the pores for loading. Target molecules cannot diffuse smoothly through the micropores and are easily blocked on the material surface, resulting in extremely slow mass transfer rate and sluggish sensor response.

[0043] In summary, the high specific surface area and hierarchical porous structure of this Astragalus straw biochar precisely match the core requirements of molecularly imprinted electrochemical sensors for "high recognition efficiency, high signal sensitivity, and high anti-interference ability," and can serve as a high-quality carrier material for sensors, providing key structural support for accurate detection.

[0044] Example 2 A method for preparing multi-level through-pore carbon powder, comprising the following steps: S1, Preparation of phosphoric acid solution; Add 38.1 mL of phosphoric acid to 55 mL of deionized water with stirring, and then bring the volume to 100 mL to obtain a phosphoric acid solution.

[0045] S2. Preparation of Astragalus straw powder: Astragalus straw was rinsed with deionized water, dried at 50°C for 8 hours, pulverized and passed through a 50-mesh sieve to obtain Astragalus straw powder A2 with uniform particle size.

[0046] S3, Chemical Activation: Add 2g of Astragalus stalk powder prepared in S2 to 20mL of phosphoric acid solution and stir well. Soak for 24 hours, filter to obtain filter residue, wash twice with deionized water, and dry in an oven at 80℃ for 12 hours to obtain activated Astragalus stalk powder B2.

[0047] S4, Pyrolytic Carbonization: The activated Astragalus straw powder prepared in S3 was placed in a tube furnace and heated to 500°C at a heating rate of 5°C / min under a nitrogen atmosphere. The temperature was maintained at this temperature for 2 hours for pyrolysis and carbonization. The final product was Astragalus straw carbon powder C2.

[0048] S5. Washing and drying: The Astragalus straw carbon powder prepared in S4 was washed with deionized water until the pH reached 7. The powder was then collected by centrifugation and dried at 80°C for 12 hours to obtain the final product, Astragalus straw carbon powder D2 with multi-level through pores.

[0049] The final product, Astragalus straw charcoal powder D2, was divided into multiple samples and subjected to the following tests in sequence: 1. Scanning electron microscopy; Electron microscopy was performed on sample D2 of Astragalus membranaceus straw charcoal powder. The SEM images of the sample are shown below. Figure 6 As shown, the material in the sample retains the porous carbon skeleton structure of Astragalus membranaceus straw.

[0050] Example 3 The product obtained in Example 1, Astragalus straw charcoal powder D1, was used as an electrode material to prepare a molecularly imprinted electrochemical sensor.

[0051] A method for preparing a multi-level through-pore carbon powder molecular imprinted sensor, comprising the following steps: S6. Preparation of solution A: Take 3 mg of the product obtained in Example 1, dissolve it in 1 ml of deionized water, and sonicate the mixed solution for 30 s to 1 min to obtain solution A.

[0052] S7. Fabrication of sensor electrodes: Take 10 μL of solution A and coat it onto the surface of a glassy carbon electrode (initial empty electrode) with a diameter of 4 mm. Blow it dry with nitrogen gas. Solution A is uniformly distributed on the surface of the glassy carbon electrode as an electrode material, thus obtaining electrode A.

[0053] Compare the electrochemical impedance of the glassy carbon electrode before and after coating with that of electrode A, such as... Figure 7 As shown in the figure, this graph compares the resistance of the electrodes before and after coating. The diameter of the simulated semicircle in the high-frequency region directly corresponds to the charge transfer resistance (Rct). The smaller the semicircle, the lower the resistance to charge transfer at the electrode / electrolyte interface, and the higher the electron transport efficiency. The semicircle of the electrode coated with Astragalus straw char material (red dot) almost disappears in the high-frequency region, and Rct is significantly reduced compared to the semicircle of the empty electrode (blue triangle), indicating that the coated material has good conductivity and low resistance. The slope of the red line in the low-frequency region is significantly larger than that of the blue line, indicating that the diffusion impedance is reduced and the mass transfer efficiency is high. Compared to the empty electrode, the electrode coated with the material can improve the detection sensitivity of the sensor, providing core performance support for molecularly imprinted electrochemical sensors.

[0054] S8, Electropolymerized Molecularly Imprinted Polymer: Electrode A prepared by S7 was used as the working electrode in the three-electrode system of the electrochemical workstation. The three electrodes were immersed in a PBS solution with pH=6 containing dopamine and chlorpyrifos (molar concentration ratio of 3:2). The parameters of the electrochemical workstation were adjusted, and CV scan was performed at a rate of 100mV / s for 20 cycles to form a molecularly imprinted polymer on the electrode surface, thus obtaining electrode B.

[0055] S9, Elution of molecular template: Electrode B prepared by S8 was immersed in DES, a mixed solution of choline chloride and urea (mass ratio of 1:2, prepared by stirring at 80°C for 30 minutes). After 10 minutes, the electrode was removed and the surface of the electrode was rinsed with a small amount of deionized water to obtain a molecularly imprinted sensor electrode C that can be used to detect the organophosphorus pesticide chlorpyrifos.

[0056] The aforementioned deep eutectic solvents (DES), also known as eutectic solvents, are mixtures formed by two or more components through hydrogen bonding. They are currently used in fields such as chemical synthesis and catalysis, separation and purification, and materials science. Experiments have shown that they possess excellent molecular template elution capabilities, with short elution times and significant effects.

[0057] Example 4 The application of a multi-level through-pore carbon powder molecular imprint sensor is specifically to apply the sensor to the field of rapid detection of pesticide residues in medicinal slices.

[0058] The detection effect of the molecularly imprinted sensor electrode C prepared in Example 3 on the organophosphorus pesticide chlorpyrifos was verified, and a synchronous comparative experiment was carried out using the initial empty electrode without any treatment or coating.

[0059] The detection experimental method consists of the following steps: S1. Preparation of standard solutions for the pesticides to be tested; In this embodiment, the pesticide to be tested is chlorpyrifos.

[0060] Chlorpyrifos 1×10 -3 Preparation of mol / L standard solution: Accurately weigh 0.0350±0.0001g of chlorpyrifos using a 0.01% balance, completely dissolve in 5 mL of methanol, transfer to a 100 mL volumetric flask, and dilute to the mark with PBS (pH=6). Shake well to obtain 1×10⁻⁶ mol / L standard solution. -3 mol / L stock solution. A stepwise dilution method was used, with the stock solution being diluted sequentially with PBS to 1×10⁻⁶. -4 - 1×10 -10 A gradient of mol / L standard solutions was prepared, and all solutions were stored in brown reagent bottles and refrigerated at 2-8°C.

[0061] S2. Preparation of simulated pesticide residue solution of Angelica sinensis; Angelica sinensis slices were selected as the actual sample. After being pulverized into powder, the Chinese herb Angelica sinensis was passed through an 80-mesh sieve. 0.1g of the fine powder of Chinese herb Angelica sinensis was added to 9 ml of a mixed solution of 0.1 mol / L PBS (pH=6.0) containing 25% methanol (volume ratio, methanol accounts for 25%).

[0062] Add 1×10 -5 mol / L-1×10 -10 1 ml of chlorpyrifos standard solution at various mol / L concentrations was added and vortexed for 1 min to ensure uniform dispersion of the spiked solution.

[0063] The sample was sonicated at room temperature for 20 min, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected and filtered through a 0.22 μm filter membrane to remove interfering substances from the matrix, thus obtaining spiked sample solution groups with different concentrations, which were used to simulate the pesticide residue detection solution groups of Angelica sinensis slices.

[0064] S3, rapid detection of pesticide residues; The sensor electrodes prepared in Example 3 were left to stand in the solution to be detected for 10 minutes each, and then [Fe(CN)6] was used. 3− / 4− As a redox probe, the current response of the modified electrode after adsorbing different concentrations of chlorpyrifos was recorded using DPV. The current values ​​were recorded and plotted using a standard curve. Figure 8 .

[0065] Figure 8 It can be seen that the peak current value after the modified electrode adsorbs chlorpyrifos molecules is linearly correlated with its concentration.

[0066] The linear range is 10. −10 -10 −6 mol / L; detection limit is 2.45 × 10⁻⁶. −10 mol / L (S / N=3). ΔI (μA) = 81.2 + 6.52 × logC (mol / L), linear correlation coefficient is R. 2 =0.9915.

[0067] The sensor has a linear detection range of 10 for chlorpyrifos. −10 -10 −6 The concentration is mol / L, which means that within this concentration range, the sensor can accurately reflect changes in chlorpyrifos concentration. Whether the concentration of chlorpyrifos is low or relatively high, quantitative analysis can be performed through changes in peak current values. This makes it suitable for detecting chlorpyrifos in different scenarios and expands the application range of the sensor.

[0068] The detection limit is as low as 2.45 × 10⁻⁶. −10 The concentration of chlorpyrifos was approximately 0.086 μg / L (S / N=3), indicating that the sensor has extremely high sensitivity and can detect trace amounts of chlorpyrifos. In contrast, the detection limit for chlorpyrifos using conventional HPLC methods is approximately 800 μg / L, corresponding to a molar concentration of 2.3 × 10⁻⁶ mol / L. -6mol / L (Sun Zhigao, Sheng Ran, Hao Jingmei, et al. QuEChERS-High Performance Liquid Chromatography for the Detection of Chlorpyrifos, Imidacloprid and Vermiculin Residues in Citrus [J]. Food and Machinery, 2017, 33(06):68-72. DOI:10.13652 / j.issn.1003-5788.2017.06.014. This document is attached in the Substantive Examination References); It can be seen that the detection limit of the sensor of the present invention is lower and the detection ability of trace chlorpyrifos residues in Chinese herbal medicine pieces is better.

[0069] Linear correlation coefficient R 2 =0.9915, which is close to the value, indicating that there is a high linear correlation between the peak current value after the modified electrode adsorbs chlorpyrifos molecules and the concentration of chlorpyrifos.

[0070] This means that the sensor's detection results have high reliability and repeatability, and the concentration of chlorpyrifos can be accurately calculated by measuring the peak current value, providing accurate quantitative data in rapid detection scenarios at the application end.

[0071] S4. Perform actual sample spike recovery; According to existing technology, the recovery rate of this sensor in actual sample spike recovery experiments was 91.2–109.4%, with a relative standard deviation of 3.25–4.45%.

[0072] The recovery rates of 91.2%–109.4% fall within the generally accepted acceptable range of 80%–120% in the field of trace pesticide residue detection, and are even closer to 100%.

[0073] The results indicate that impurities such as polysaccharides, saponins, and organic acids in the Angelica sinensis matrix did not interfere with the specific interaction between the sensor and chlorpyrifos. The sensor detection values ​​were highly consistent with the actual content of the target substance, demonstrating its accuracy.

[0074] Meanwhile, the relative standard deviation of 3.25%–4.45% is lower than the industry-recognized 5% precision threshold, indicating that even with minor differences in operation or slight fluctuations in environmental conditions, the sensor's signal response to the same spiked sample remains stable, and the detection results have good repeatability, highlighting its precision. In summary, the sensor of this invention can simultaneously meet the requirements of accuracy and precision when detecting chlorpyrifos residues in complex Chinese herbal medicine matrices such as Angelica sinensis. It can be adapted to the detection scenario of trace pesticide residues in actual Chinese herbal medicine samples, providing a stable and reliable technical solution for the quality and safety management of Chinese herbal medicine. Example 5 The application of a multi-level through-pore carbon powder molecular imprint sensor is specifically to apply the sensor to the field of rapid detection of pesticide residues at different cycles after pesticide spraying during the field management of Angelica sinensis.

[0075] S1, Sampling; Select functional leaves (healthy leaves from the middle of the plant, avoiding withered, yellowed, or diseased leaves) from the field within the angelica planting area. Randomly select 3 angelica plants, and collect 3-5 functional leaves from each plant. Mix them together as one sample, ensuring the total sample weight is ≥10g. Use sterile scissors to cut the target part, remove visible impurities, and do not wash with water. Place the mixed sample into a numbered sampling bag and then into a portable ice box (2-8℃), avoiding room temperature storage.

[0076] Note that rapid testing must be completed within 2 hours after sample collection.

[0077] S2, Preparation of rapid detection solution; Weigh 5.0g of Angelica sinensis sample from the sampling bag, repeatedly and finely cut it into 0.5-1mm fragments, place it in a 50mL centrifuge tube, add 35mL of PBS (pH=6) extraction buffer containing 25% methanol, tighten the centrifuge tube cap, and vortex to mix. Then sonicate for 20 minutes, manually inverting the centrifuge tube every 5 minutes to ensure uniform extraction. After sonication, place the centrifuge tube in a high-speed centrifuge and centrifuge at 10000rpm for 10min. Collect the supernatant and filter it using a disposable syringe filter (0.22μm organic phase filter membrane) to obtain the test solution.

[0078] S3, rapid detection of pesticide residues; The sensor electrodes prepared in Example 3 were left to stand in the solution to be detected for 10 minutes each, and then removed and placed in [Fe(CN)6]. 3− / 4− The DPV current value was detected in the probe solution, and the results were calculated using a standard curve. The results are summarized in Table 1.

[0079] Simultaneous comparative detection using S4 and HPLC-MS methods; According to reference 1 (Sun Zhigao, Sheng Ran, Hao Jingmei, et al. QuEChERS-High Performance Liquid Chromatography for the Determination of Chlorpyrifos, Imidacloprid and Vermiculin Residue in Citrus [J]. Food and Machinery, 2017, 33(06):68-72. DOI:10.13652 / j.issn.1003-5788.2017.06.014. This reference is attached in the Substantive Examination References), the HPLC used in this embodiment is not precise enough. Referring to the HPLC detection limit of reference 2 ([1] Zhu Zhengwei, Wu Wanqin, Zhu Songsong, et al. Rapid analysis of chlorpyrifos poisoning and its degradation products in fruits and vegetables by high performance liquid chromatography-tandem mass spectrometry [J]. Chinese Journal of Food Hygiene, 2023, 35(01):20-26.DOI:10.13590 / j.cjfh.2023.01.004.), it is not sufficient for synchronous comparison, so HPLC-MS comparative detection is selected. (Does this reference support the claim that the HPLC-MS detection method is correct?)

[0080] After sampling in S1, some samples were used to prepare rapid detection solutions for S2, while the other samples were detected by HPLC-MS.

[0081] Weigh 5.0g of sample and place it in a laboratory high-speed tissue homogenizer. Add 5mL of ultrapure water and 15mL of chromatographic grade acetonitrile containing 0.1% phosphoric acid. Homogenize for 3 minutes to prepare sample slurry. Then, ultrasonic extraction was performed for 30 minutes. Afterwards, add 5g of anhydrous sodium sulfate and 1.5g of sodium chloride, and shake for 20 minutes; Let stand for 15 minutes to allow anhydrous sodium sulfate to settle and the extract to separate into layers, then filter under vacuum. The volume was then concentrated to approximately 2 mL using a rotary evaporator. Take another 3 mL C18 / PSA composite SPE column. After column activation, slowly inject 2 mL of concentrated extract into the SPE column and elute with acetonitrile-water (9:1, V / V). Collect the eluent into a 10 mL eggplant-shaped flask. Concentrate the eluent again to near dryness (0.2-0.3 mL remaining), rinse the bottle wall twice with 1 mL of chromatographic grade acetonitrile, transfer the rinsing solution to a 5 mL centrifuge tube, centrifuge at 10,000 rpm for 8 minutes, and filter the supernatant through a 0.22 μm syringe filter to obtain the test solution.

[0082] A C18 reversed-phase column was used. The mobile phase A was 0.1% formic acid aqueous solution, and the mobile phase B was acetonitrile. The gradient elution program was as follows: 0.0-0.5 min, 15% B; 0.5-1.5 min, 15%-95% B; 1.5-4.0 min, 95% B; 4.0-5.0 min, 95%-15% B; 5.0-7.0 min, 15% B.

[0083] Mass spectrometry: Ion source temperature 120 ℃; desolvation temperature 450 ℃; capillary voltage: 2500 V; acquisition mode: multi-reaction monitoring (MRM).

[0084] Before detection, the column was equilibrated and the mass spectrometer was preheated once. Then, the sample was continuously injected and detected in MRM mode. The detection results of the conventional HPLC-MS method are summarized in Table 2.

[0085] As shown in Tables 1 and 2, two sample numbers were set for each time point (DG-XX-01, DG-XX-02, DG-XX-03, DG-XX-04), and each sample was tested in three parallel tests. The residue level decreased with the extension of time after application, which is consistent with the natural degradation of pesticides in crops.

[0086] HPLC-MS, due to its complex pretreatment process, is prone to loss of chlorpyrifos during operation, naturally leading to relatively low detection results. While the detection results of the sensor of this invention differ from those of HPLC-MS, the residual change trends of both are highly consistent, showing a significant decrease with prolonged post-application time. The residue reduction exceeds 80% after 10 to 30 days, consistent with the objective laws of pesticide residue degradation. The relative deviation (RSD) of parallel samples detected by the sensor is 3.4%-7.5%, with RSD ≤ 5.8% for medium-to-high concentration samples (≥4.5 μg / L) and RSD ≤ 7.5% for trace samples (1.5-2.0 μg / L). All data are better than the general precision requirements for trace detection (RSD ≤ 10%), fully meeting the detection precision standards. Therefore, the sensor of this invention approaches the level of large-scale laboratory instruments in terms of precision, reliability, and data stability in trace detection.

[0087] Finally, comparing the experimental time, taking two groups of samples, with three parallel samples prepared in each group as an example, when the sensor prepared in Example 3 of this invention is detected, due to the significant simplification of the pretreatment steps, ultrasonic extraction only needs to be performed for 30 minutes, centrifugation only needs to be performed for 10 minutes, and membrane filtration only needs to be performed for 5 minutes. There is no need for purification and concentration operations. The total sample preparation time is about 90 minutes, and a single detection only needs to be about 13 minutes. The total time for two groups of six samples is about 170 minutes.

[0088] Conventional HPLC-MS methods are complex in sample preparation and detection, and require equipment rinsing and maintenance after detection. Taking two groups of samples, with three parallel samples prepared for each group as an example, the total operation time is about 7 hours, and a large amount of acetonitrile reagent and solid phase extraction column are consumed.

[0089] It is evident that, compared to the two detection methods, the sensor of this invention has a significant advantage in terms of rapid detection time.

[0090] Especially in practical applications, HPLC-MS, limited by instrument balancing and complex detection procedures, requires 150-200 minutes to batch-detect 10 samples. In contrast, the sensor of this invention, with its rapid response characteristics, takes less than 50 minutes to batch-detect the same number of samples, without requiring additional complex pretreatment or equipment maintenance steps. It is worth noting that as the number of samples increases, the time difference between the two methods widens further, making the sensor's rapid detection advantage increasingly significant. Compared to HPLC-MS, its detection efficiency is improved by at least 60%.

Claims

1. A method for preparing a multi-level through-pore carbon powder molecular imprinted sensor, characterized in that: The method consists of the following steps: S1. Preparation of Astragalus straw charcoal powder with multi-level through holes; Astragalus straw was treated by a one-step carbonization method to prepare Astragalus straw charcoal powder D with multi-level through pores; S2, Preparation of solution A; Take 3±0.1 mg of Astragalus straw charcoal powder D prepared by S1, dissolve it in 1±0.1 ml of deionized water, and sonicate the mixed solution for 30 s-1 min to obtain solution A; S3. Fabrication of sensor electrodes: Take 10 μL of solution A and coat it onto the surface of an initial empty electrode with a diameter of 4 mm. Blow it dry with nitrogen gas. Solution A is uniformly distributed on the surface of the glassy carbon electrode as an electrode material, thus obtaining electrode A. S4, electropolymerized molecularly imprinted polymer: Electrode A, prepared by S3, was used as the working electrode in the three-electrode system of the electrochemical workstation. The target analyte and its compatible monomer were polymerized on the electrode surface to form a molecularly imprinted polymer, thus obtaining electrode B. S5, Elution of molecular template: Electrode B prepared in S4 was immersed in the eluent and eluted. After elution, the electrode was removed and the surface of the electrode was rinsed with a small amount of deionized water to obtain molecularly imprinted sensor electrode C, which can be used to detect target analytes.

2. The method for preparing a multi-level through-pore carbon powder molecular imprinted sensor as described in claim 1, characterized in that: The specific steps for preparing Astragalus stalk charcoal powder D1 with multi-level through pores by the one-step carbonization method described in S1 are as follows: S1.1 Preparation of phosphoric acid solution; Add 31.3-38.1 mL of phosphoric acid to 55-60 mL of deionized water with stirring, and bring the volume to 100 mL to obtain a phosphoric acid solution; S1.2 Preparation of Astragalus straw powder: Rinse the astragalus straw with deionized water, dry it at 50°C for at least 8 hours, pulverize it and pass it through a 50-mesh sieve to obtain astragalus straw powder A with uniform particle size; S1.3, Chemical activation: Add 2g of Astragalus straw powder prepared in S2 to 20mL of phosphoric acid solution and stir well; soak for 24 hours, filter to obtain filter residue, wash twice with deionized water, and dry in an oven at 80℃ for 12 hours or more to obtain activated Astragalus straw powder B. S1.4, Pyrolysis and Carbonization: The activated Astragalus straw powder prepared in S3 was placed in a tube furnace and heated to 500±5℃ at a heating rate of 5°C / min under a nitrogen atmosphere. It was then held at this temperature for 2 hours for pyrolysis and carbonization. The final product was Astragalus straw carbon powder C. S1.5 Washing and drying: The Astragalus straw carbon powder prepared in S4 was washed with deionized water until the pH reached 7±0.

1. The powder was then collected by centrifugation and dried at 80±5℃ for 12 hours or more to obtain the final product – Astragalus straw carbon powder D with multi-level through pores.

3. The method for preparing a multi-level through-pore carbon powder molecular imprint sensor as described in claim 1 or 2, characterized in that: In S4, the target analyte is selected as the pesticide chlorpyrifos, and the suitable monomer for polymerization is dopamine. The specific polymerization process is as follows: Electrode A prepared by S3 was used as the working electrode in the three-electrode system of the electrochemical workstation. The three electrodes were immersed in a PBS solution with pH=6 containing dopamine and chlorpyrifos (molar concentration ratio of 3:2). The parameters of the electrochemical workstation were adjusted, and CV scan was performed at a rate of 100mV / s for 20 cycles to form a molecularly imprinted polymer on the electrode surface, thus obtaining electrode B.

4. The method for preparing a multi-level through-pore carbon powder molecular imprinted sensor as described in claim 3, characterized in that: The eluent for electrode B in S5 is DES. The mass ratio of choline chloride to urea in the mixed solution is 1:

2. The solution is prepared by stirring at 80°C for 30 minutes. During elution, soak the electrode for 10 minutes before removing it and then rinse the electrode surface with a small amount of deionized water.

5. The application of the multi-level through-pore carbon powder molecular imprinted sensor prepared according to claims 1-4, characterized in that, This sensor is used in the field of rapid detection of pesticide residues.

6. The application of the multi-level through-pore carbon powder molecular imprinted sensor prepared according to claims 1-4, characterized in that, It is applied to the field of rapid detection of pesticide residues at different stages after pesticide spraying in the field management of Chinese medicinal plant cultivation.

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