Preparation method and application of an enzyme-catalyzed biosensor of AChE@PAH-HOF

Abstract

The application relates to a preparation method of a biosensor. The application relates to a preparation method of a portable pesticide detection biosensor based on acetylcholinesterase (AChE)@poly(allylamine hydrochloride)-hydrogen bond organic framework (PAH-HOF) and application of the biosensor in on-site rapid chlorpyrifos detection, and belongs to the technical field of biosensors. According to the method, AChE is combined with poly(allylamine hydrochloride) (PAH) to change the surface charge and promote self-assembly of the AChE and the hydrogen bond organic framework (HOF), so that an AChE@PAH-HOF composite material with high catalytic activity, high embedding rate and high stability is formed; then the material is integrated into a water-retaining glycerol-sodium alginate hydrogel (G-SA) matrix to construct a disc-shaped sensor. The sensor can be attached to the surface of plants and used for long-term in-situ monitoring of organophosphorus pesticides. In combination with an image processing algorithm, the system realizes accurate tracking of the chlorpyrifos degradation process on the surface of tomato plants, and the detection limit reaches 1 ng / mL. The application provides non-invasive and low-cost technical support for crop health management and sustainable precision agriculture.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for preparing a biosensor composed of an acetylcholinesterase@poly(allylamine hydrochloride)-hydrogen bonded organic framework (AChE@PAH-HOF) composite material and its application in the rapid on-site detection of chlorpyrifos. Background Technology

[0002] Chlorpyrifos is a broad-spectrum, highly effective organophosphate insecticide. Due to its excellent control efficacy against a variety of chewing and sucking insects, it is widely used in agricultural production, storage, and sanitary pest control. The toxic mechanism of chlorpyrifos involves inhibiting the activity of acetylcholinesterase. This mechanism is effective not only against target pests but also poses a significant toxic risk to mammals and humans. Chlorpyrifos and its metabolites have long-lasting residues in the environment and easily accumulate through the food chain, posing a serious threat to the ecological environment and agricultural product safety. Studies have shown that long-term or acute exposure to chlorpyrifos residues may lead to damage to the human nervous system, endocrine disruption, and even developmental toxicity.

[0003] Hydrogen-bonded organic frameworks (HOFs) are a new class of crystalline porous materials with mild synthesis conditions (typically carried out in aqueous phase at room temperature) and excellent biocompatibility, showing broad application potential in the field of enzyme immobilization. HOFs self-assemble through reversible hydrogen bonding, and their hydrogen bonds can dynamically form and break under certain conditions, allowing the material to recover its complete crystal structure after dissolution through a simple recrystallization process, thus exhibiting good purification and recyclability. By rationally designing the geometry and functional groups of the organic building blocks, the sites and orientations of hydrogen bonds can be precisely controlled, thereby achieving the controllable construction of a pre-defined framework structure. In addition, HOFs also exhibit good pH stability, maintaining their structural integrity under harsh environments. In this invention, hydrogen-bonded organic frameworks are used to encapsulate acetylcholinesterase, significantly improving enzyme stability and encapsulation efficiency. Combined with a glycerol-sodium alginate hydrogel system, in-situ, highly sensitive monitoring of chlorpyrifos pesticides is achieved. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing a biosensor made of AChE@PAH-HOF composite material and its application in the rapid on-site detection of chlorpyrifos.

[0005] This invention constructs a novel high-performance enzyme-framework composite material. First, positively charged poly(allylamine hydrochloride) (PAH) is pre-bound to negatively charged acetylcholinesterase (AChE) via electrostatic interactions, forming an AChE–PAH complex. This complex effectively regulates the surface charge and chemical microenvironment of the enzyme molecule. Subsequently, using the AChE–PAH complex as the core, self-assembly with the HOF structural unit 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) is carried out via hydrogen bonding, ultimately constructing the AChE@PAH-HOF composite material. This composite material is used as the recognition and signal amplification unit of a sensor, embedded in a glycerol-sodium alginate hydrogel (G-SA) to fabricate a miniaturized disc-shaped sensor. Its detection mechanism is based on the following principle: chlorpyrifos inhibits the activity of AChE, weakening its ability to catalyze the formation of a yellow product from the substrate, thereby causing a systematic change in the intensity of the colorimetric reaction. Optical images from sensors are captured by a smartphone and converted into digital signals. Image processing software then analyzes the color channel values ​​to establish a linear standard curve of "color coordinates versus chlorpyrifos concentration." In actual testing, only the color coordinate values ​​of the sample corresponding to the sensor test are needed. By substituting these values ​​into the standard curve, the concentration of chlorpyrifos in the sample can be accurately calculated.

[0006] The preparation method of a pesticide biosensor made of AChE@PAH-HOF according to the present invention comprises the following steps: A. Synthesis of AChE@PAH-HOF composite material 1-5 mg PAH and 1-5 mg AChE were dissolved separately in 4 mL Tris-HCl buffer (50 mM, pH = 8.0) at a volume ratio of 1:1 and reacted with the solution at 3-5 °C for 5-10 minutes. Then, 2-20 mg H4TBAPy (dissolved in 1 mL DMF) was added at a volume ratio of 8:1 to the above solutions, and the mixture was stirred at 3-5 °C for another 10-20 minutes. After centrifugation, a yellow precipitate was obtained, which was washed three times with deionized water to obtain the AChE@PAH-HOF composite material.

[0007] B. Preparation of the chlorpyrifos detection sensor: A master mold for casting a hydrogel disc was fabricated using a quartz slider. The master mold had two rows of holes with a diameter of 7 mm and a depth of 0.9 mm to form the hydrogel disc. Sodium alginate was dissolved in a mixture of glycerol and water (mass ratio 6:4) at a concentration of 10-30 mg / mL. AChE@PAH-HOF was dissolved in the same mixture at a concentration of 2-5 mg / mL. 25 μL of the AChE@PAH-HOF solution was pipetted into the holes of the mold, and then immersed in a 0.02-0.1 M CaCl2 solution (glycerol and water mixture) for 10-30 min to obtain the hydrogel disc. The hydrogel disc was removed from the holes of the master mold using tweezers and placed on a glass slide, thus obtaining a portable biosensor for detecting chlorpyrifos.

[0008] C. Construct a standard relationship curve between color coordinates and chlorpyrifos concentration. Chlorpyrifos standard solution was diluted with ultrapure water to a concentration gradient of 1 ng / mL to 800 ng / mL. 25 μL of each concentration solution was added dropwise to the sensor and incubated at room temperature for 30–40 minutes. Subsequently, a colorimetric reaction was performed: 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was dissolved in PBS buffer (10 mM, pH = 8.0) to prepare solution A at a concentration of 0.1–0.5 mg / mL. 25 μL of thioacetylcholine (ATCh, 25–35 mmol / L) aqueous solution was mixed with solution A at a 1:1 volume ratio and immediately added dropwise to the incubated biosensor, reacting at room temperature for 20–40 minutes. The sensor was then placed in a camera box, and images were captured using a smartphone. The ImageJ commercial software was used to directly analyze the color coordinates (including red, green, and blue channels) of the image to obtain the color coordinate values ​​corresponding to different concentrations of chlorpyrifos. Furthermore, an ED algorithm was used to construct a standard relationship curve between color coordinates and chlorpyrifos concentration. In actual testing, the portable device was used to acquire images of the sensor in samples with unknown concentrations of chlorpyrifos. The color coordinate values ​​were extracted using the same method and substituted into the aforementioned standard curve to calculate the concentration of chlorpyrifos in the actual sample.

[0009] D. Testing of actual samples: Apples and oranges were juiced separately and diluted 5-10 times with ultrapure water to obtain actual sample solutions (samples purchased from a farmers' market, free of chlorpyrifos pesticide). Tap water was also filtered and used as a sample. Chlorpyrifos standard solution was then added to the above actual sample solutions to achieve final concentrations of 0.005, 0.05, and 0.5 μg / mL, respectively. The actual sample solutions containing chlorpyrifos were then mixed with acetonitrile (sample extract) at a volume ratio of 1:10, sonicated for 5-10 min, shaken for 20-30 min, and centrifuged at 3000-5000 rpm for 5-10 min. The resulting organic phase was diluted 1-10 times with PBS buffer (10.0 mmol / L, pH = 8.0). 25 μg / mL of the solution was then used as the final sample solution. After diluting the solution by μL, it is added dropwise to the sensor prepared in step B. The self-made portable device described in step C is used to acquire the sensor image and obtain the corresponding color coordinate value. Substitute the color coordinate - chlorpyrifos concentration standard relationship curve obtained in step C to calculate the chlorpyrifos concentration in the actual sample solution. Then compare it with the added amount to verify the feasibility of the device of the present invention.

[0010] The mechanism of this invention is as follows: By altering the surface charge of acetylcholinesterase (AChE), converting it from negative to positive, a hydrogen-bonded organic framework is formed between AChE and the negatively charged HOF building unit (H4TBAPy). This composite material, used as the sensor's recognition and signal amplification unit, is embedded in a glycerol-sodium alginate hydrogel (G-SA) to prepare a biosensor. Its detection mechanism is based on the specific inhibition of AChE activity by chlorpyrifos: AChE normally catalyzes the reaction of substrates to produce a yellow product; when its activity is inhibited, the color intensity systematically decreases. Sensor images are captured by a smartphone and converted into digital signals. Image processing software is then used to analyze the color channel values, establishing a linear standard curve of "color coordinates versus chlorpyrifos concentration." In actual detection, only the color coordinate values ​​of the sensor corresponding to the sample need to be obtained and substituted into the standard curve to accurately calculate the concentration of chlorpyrifos in the sample.

[0011] The AChE@PAH-HOF biosensor prepared in this invention can sensitively acquire chlorpyrifos residue information. Based on the high affinity of acetylcholinesterase, this biosensor exhibits good linearity in the chlorpyrifos pesticide range of 1-800 ng / mL. This study utilized a smartphone to collect sensor information, successfully converting the sensor's optical images into data information, achieving accurate quantification of chlorpyrifos pesticide with a detection limit as low as 1 ng / mL. By improving bioanalytical parameters, particularly enhancing detection sensitivity and stability, this biosensor not only shortens the overall analysis time and reduces reagent consumption but also simplifies the readout equipment required for on-site detection. This portable device was further applied to monitor the dynamic degradation process of chlorpyrifos in tomato leaves, demonstrating good on-site applicability.

[0012] Compared with existing technologies, the present invention has the following characteristics: (1) In this invention, PAH successfully encapsulates AChE in a hydrogen-bonded organic framework mediated by H4TBAPy by changing the surface charge of AChE. This framework has a high enzyme encapsulation capacity and improves the stability of the encapsulated enzyme.

[0013] (2) By introducing glycerol to functionalize the traditional sodium alginate hydrogel, the polyhydroxy structure of glycerol is used to make it water-retaining, thus overcoming the problem of water loss in traditional hydrogel sensors during monitoring.

[0014] (3) The AChE@PAH-HOF biosensor prepared by the present invention has good sensitivity, selectivity and stability, and the construction process is low cost and simple to operate. Attached Figure Description

[0015] Figure 1 (a) is a transmission electron microscope image of AChE@PAH-HOF described in Example 1; Figure 1 (b) is a scanning electron microscope image of AChE@PAH-HOF prepared in Example 1.

[0016] Figure 2 (a) is the optimal PAH concentration for the synthesis of AChE@PAH-HOF described in Example 2; Figure 2 (b) is the optimal H4TBAPy concentration for the synthesis of AChE@PAH-HOF described in Example 2; Figure 2 (c) is the optimal reaction time optimization diagram of PAH and AChE in AChE@PAH-HOF described in Example 2; Figure 2 (d) is the optimal reaction time optimization diagram of the PAH and AChE complex of AChE@PAH-HOF described in Example 2 with H4TBAPy.

[0017] Figure 3 (a) is the UV spectrum of AChE and the mixture of AChE and PAH described in Example 3. (b) is the fluorescence spectrum of AChE and the mixture of AChE and PAH described in Example 3.

[0018] Figure 4 (a) Chemical stability of AChE@PAH-HOF as described in Example 4 compared to AChE. (b) Day stability of AChE@PAH-HOF as described in Example 4 compared to AChE. Detailed Implementation

[0019] Example 1: Synthesis and Characterization of AChE@PAH-HOF Material Poly(allylamine hydrochloride) (PAH, 1-5 mg) was dissolved in 4 mL Tris-HCl (50 mM, pH = 8). Acetylcholinesterase (AChE, 1-5 mg) was dissolved in 4 mL Tris-HCl (50 mM, pH = 8). The two solutions were mixed at a volume ratio of 1:1 and stirred at 3-5 °C for 5-10 minutes. Then, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy, 2-20 mg) was dissolved in N,N-dimethylformamide (DMF, 1 mL) and mixed with the above solutions at a volume ratio of 8:1. The mixture was stirred at 3-5 °C for 10-20 minutes. A yellow precipitate (AChE@PAH-HOF) was subsequently obtained. AChE@PAH-HOF was washed three times with Tris-HCl buffer (50 mM, pH = 8.0) and recovered by centrifugation (12,000 g, 5–10 min).

[0020] The AChE@PAH-HOF composite material aqueous solution was diluted 100-fold with ultrapure water, and 8-10 μL was dropped onto a 325-mesh carbon support membrane and a silicon wafer. After drying, it was characterized using transmission electron microscopy and scanning electron microscopy. The results are as follows: Figure 1 a and Figure 1 As shown in b, the AChE@PAH-HOF material has a typical rod-like structure with an average width of 750 ± 50 nm.

[0021] Example 2: Optimization of the synthesis conditions of AChE@PAH-HOF The catalytic activity of the material for the substrate (ATCh) under different synthetic methods was tested by adjusting the concentration of PAH, ligand, and synthesis time using single-factor analysis. The material exhibited high catalytic selectivity for the substrate (ATCh), converting it to TCh, which was then reacted with added DTNB. The absorbance at 412 nm was measured to compare the activity. Figure 2Different concentrations of PAH (0.1, 0.2, 0.5, 1, 1.5, 2 mg / mL) were mixed with 1 mg / mL AChE at a volume ratio of 1:1. Then, H4TBAPy (8 mg / mL, DMF) was added and mixed with the above solution at a volume ratio of 8:1 to synthesize AChE@PAH-HOF. The absorbance at different concentrations was compared and normalized, and the optimal concentration was found to be 1 mg / mL. Figure 2 b. Different concentrations of H4TBAPy (1, 2, 4, 6, 8, 10, 12 mg / mL) were reacted with a mixed solution of 1 mg / mL PAH and 1 mg / mL AChE in a volume ratio of 1:4:4. The absorbance at different concentrations was compared and normalized. Finally, the optimal concentration was selected as 8 mg / mL. Figure 2 c. 1-5 mg / mL PAH and 1-5 mg / mL AChE (Tris-HCl, 50 mM, pH = 8) were mixed at a volume ratio of 1:1 and reacted at different reaction times (0, 5, 10, 20, 30, 45, 60, 90 min). Then, H4TBAPy (2-20 mg / mL, DMF) was added and mixed with the above solution at a volume ratio of 8:1. The absorbance at different reaction times was compared and normalized. Finally, the optimal reaction time was selected as 5 min. Figure 2 The PAH-AChE complex was mixed with H4TBAPy solution (8 mg / mL, DMF) at a volume ratio of 8:1. The activity of the synthesized material under different reaction conditions (0, 1, 5, 10, 20, 35, 60, 90 min) was tested. The absorbance was compared and normalized. Finally, the optimal reaction time was selected as 10 min.

[0022] Example 3: UV and fluorescence spectra of AChE and PAH mixture This is to verify whether PAH will affect the structure of AChE. Figure 3 A. AChE (0.5-2 mg / mL) and PAH (0.5-2 mg / mL) were mixed at a volume ratio of 1:1 and reacted for 5-10 minutes. The visible light absorption spectra of the mixture and AChE (0.5-2 mg / mL) were then measured separately. Figure 3 b. Mix AChE (0.5-2 mg / mL) and PAH (0.5-2 mg / mL) at a volume ratio of 1:1 and react for 5-10 minutes. Then, measure the fluorescence spectra of the mixture and AChE (0.5-2 mg / mL) separately.

[0023] Example 4: Chemical stability and day-long stability of AChE@PAH-HOF A colorimetric biochemical reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and thioacetylcholine (ATCh) substrates was used as a model system. AChE@PAH-HOF catalyzed the conversion of the substrate to 2-nitro-5-thiobenzoic acid (TNB), which exhibited a novel absorption peak at 412 nm. The activity of AChE@PAH-HOF and AChE was tested by reacting them with 1-10 mM hydrochloric acid, sodium hydroxide, 0.1-1 mg / mL proteinase K, and ultrapure water at a 1:1 volume ratio for 20-40 minutes, followed by the addition of ATCh and DTNB. The relative activity was calculated by comparing the absorbance at 412 nm. The formula for calculating the relative activity is: Relative activity (%) = (A2-A0) / (A1-A0) × 100% Where A0 is the absorbance without the addition of enzyme and AChE@PAH-HOF, A1 is the absorbance with the addition of AChE@PAH-HOF and water, and A2 is the absorbance of the reaction after adding AChE@PAH-HOF and other substances.

[0024] The time-stability of the material was studied by testing the activity changes of AChE@PAH-HOF. Long-term stability studies showed that AChE@PAH-HOF maintained superior catalytic activity compared to the free enzyme within 13 days, confirming the protective effect of PAH-HOF. Example 5: AChE@PAH-HOF-based biosensor for chlorpyrifos detection The activity of AChE was inhibited by chlorpyrifos, preventing it from catalyzing the formation of thiocholine (TCh) from thioacetylcholine (ATCh), thus interrupting the production of the yellow product 2-nitro-5-thiobenzoic acid (TNB), which caused a change in the sensor color. Based on this, a fitting curve was established according to the relationship between chlorpyrifos concentration and sensor color.

[0025] Chlorpyrifos standard solution was diluted with ultrapure water to obtain chlorpyrifos solutions of different concentrations (25 μL, concentrations of 1, 2, 20, 50, 200, 500, and 800 ng / mL). These solutions were then added dropwise to the sensor dish and incubated for 20–40 min. Next, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) was dissolved in PBS solution (pH = 8, 10 mM) to obtain solution A at 0.1–0.5 mg / mL. 25 μL of thioacetylcholine aqueous solution (ATCh, 25–35 mmol / L) was mixed with solution A at a volume ratio of 1:1, and the mixture was added dropwise to the incubated sensor and incubated at room temperature for 20–40 min. The incubated sensor is placed in a photography environment and photographed by a smartphone. The sensor image is acquired by the smartphone, and the color coordinates (including the red channel R, green channel G, and blue channel B) of the image are directly analyzed using the commercial software ImageJ. This allows the color coordinates corresponding to different concentrations of chlorpyrifos to be obtained, thereby establishing a standard relationship curve between "color coordinates and chlorpyrifos concentration".

[0026] The fitted curve equation between chromatographic coordinates and chlorpyrifos concentrations (1–800 ng / mL) is Y = 6.377 - 0.00527X. X represents the concentration of chlorpyrifos, Y represents the chromatographic coordinate response value, and the detection limit is 1 ng / mL. The sensor was stored at 4 °C, and its catalytic activity against ATCh was tested at the same time each day. The results showed that its activity remained good for 13 days, indicating that it is suitable for storage and transportation.

[0027] Example 6: Selectivity, anti-interference ability, and application of the sensor in detecting chlorpyrifos in real samples. To test the selectivity of the sensor, we added 25 μL of ions, proteins, and non-organophosphorus pesticides at concentrations of 2-10 μg / mL to the sensor and reacted for 20-40 min. Then, we added a mixed solution of ATCh (25-35 mmol / L) and DTNB (0.1-0.5 mg / mL) (volume ratio 1:1) and reacted. After the reaction, we took a picture of the sensor with a smartphone, processed it with the commercial software ImageJ, and calculated the intensity value for comparison with chlorpyrifos. To verify the sensor's anti-interference capability, we mixed 25 μL of chlorpyrifos solution (0.1-0.5 μg / mL) with 1-5 μg / mL of ions, proteins, and non-organophosphorus pesticides at a volume ratio of 1:1. The mixed solution was then added dropwise to the sensor, and the reaction was allowed to proceed for 20-50 min. A mixed solution of ATCh (25-35 mmol / L) and DTNB (0.1-0.5 mg / mL) (volume ratio of 1:1) was then added for further reaction. After the reaction, the sensor was photographed using a smartphone, and the image was processed using the commercial software ImageJ to calculate the intensity value. We then observed whether the intensity value was consistent with that without the interference substance.

[0028] To verify the practical application capability of this biosensor, its spiked recovery effect on chlorpyrifos pesticide was investigated. Apples and oranges were juiced, tap water was filtered, and then diluted 5-10 times with ultrapure water. Chlorpyrifos solutions were then added to the actual samples at final concentrations of 0.005, 0.05, and 0.5 μg / mL, respectively. 1 g of the spiked sample was mixed with 10 mL of acetonitrile, and the mixture was ultrasonically extracted for 5-10 min, followed by shaking for 20-30 min, and centrifuged at 3000-4000 rpm for 5-10 min. The resulting organic phase was diluted 1-10 times with PBS buffer (10.0 mmol / L, pH = 8.0). 25 μL of the diluted solution was added dropwise to the sensor obtained in step 2. The sensor image was then acquired using the operating equipment described in Example 5, and the corresponding color coordinate values ​​were obtained. Substituting these values ​​into the standard relationship curve of "color coordinates - chlorpyrifos concentration" obtained in Example 5, the concentration of chlorpyrifos in the actual sample solution could be calculated.

[0029] To evaluate the practicality and reliability of the sensor, chlorpyrifos concentration was analyzed three times in fruit juices (tap water, apples, and oranges) containing different concentrations of chlorpyrifos. As shown in Table 1, the recovery rate (calculated by dividing the measured concentration by the spiked concentration and multiplying by 100%) ranged from 95.9% to 106.78%, with a relative standard deviation of less than 9.72%. This indicates that the biosensor has good accuracy and precision in detecting chlorpyrifos.

[0030] Table 1: Detection of chlorpyrifos in actual samples using the portable biosensor prepared according to the present invention (…). n = 3)

Claims

1. A method for preparing an AChE@PAH-HOF enzyme-catalyzed biosensor, characterized in that... The specific steps are as follows: A. Synthesis of AChE@PAH-HOF composite material 4 mg of poly(allylamine hydrochloride) and 4 mg of AChE were dissolved separately in 4 mL of Tris-HCl buffer (50 mM, pH = 8.0), mixed at a volume ratio of 1:1, and stirred at 3-5 °C for 5-10 minutes. Then, 8 mg of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene (H4TBAPy) (dissolved in 1 mL of DMF) was added at a volume ratio of 8:1 to the above solution, and the mixture was stirred at 4 °C for 10-20 minutes. After centrifugation, a yellow precipitate was obtained. After washing with deionized water three times, the AChE@PAH-HOF composite material was obtained. B. Preparation of the chlorpyrifos detection sensor: A master mold for casting a hydrogel disk was fabricated using a quartz slider. The master mold had two rows of holes with a diameter of 7 mm and a depth of 0.9 mm to form the hydrogel disk. Sodium alginate was dissolved at a concentration of 20 mg / mL in a mixture of glycerol and water (mass ratio 6:4). AChE@PAH-HOF was dissolved at a concentration of 3.5 mg / mL using the same mixture. 25 μL of the AChE@PAH-HOF solution was pipetted into the holes of the mold, and then immersed in a 0.1 M solution of calcium chloride (CaCl2) dissolved in a 6:4 glycerol and water mixture for 10-20 min to obtain the hydrogel disk. The hydrogel disk was removed from the holes of the master mold using tweezers and placed on a glass slide to obtain a portable biosensor for detecting chlorpyrifos.

2. The preparation method according to claim 1, characterized in that... The PAH-HOF preparation precursors are poly(allylamine hydrochloride) and 1,3,6,8 tetrakis(4-carboxyphenyl)pyrene.

3. The preparation method according to claim 1, characterized in that... The concentration of poly(allylamine hydrochloride) is 1 mg / mL, and the concentration of AChE is 1 mg / mL.

4. The preparation method according to claim 1, characterized in that... The ligand used was 1,3,6,8-tetra(4-carboxyphenyl)pyrene at a concentration of 8 mg / mL.

5. The preparation method according to claim 1, characterized in that... The sodium alginate used was at a concentration of 20 mg / mL, and the CaCl2 concentration was 0.1 M.

6. The preparation method according to claim 1, characterized in that... The chlorpyrifos pesticide detection platform is constructed based on AChE@PAH-HOF, and its signal output method is colorimetric signal.

7. The application of the AChE@PAH-HOF enzyme-catalyzed colorimetric biosensor as described in claim 6 in the detection of chlorpyrifos, characterized in that: Add 25 μL to the sensor Different concentrations of chlorpyrifos samples were incubated for 30 minutes; the AChE catalyzed substrate to generate products, and the sensor color changed by adding a chromogenic agent. Chlorpyrifos affects sensor color by directly inhibiting AChE activity, and the concentration of chlorpyrifos can be quantitatively analyzed by the intensity of the sensor color.

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