Preparation of controllable multi-enzyme cascade ache-hrp-cho@hnf composite material and its application in plant wearable sensor

Abstract

The present application relates to a controllable multi-enzyme cascade AChE-HRP-CHO@HNF composite material preparation and plant wearable sensor application, belong to biosensing, enzyme immobilization material and plant in-situ rapid detection field. The composite material is prepared by biomimetic mineralization and layer-by-layer protein crown assembly, takes enzyme-inorganic hybrid nanoflower as carrier, realizes spatial ordered fixation of choline oxidase (CHO), horseradish peroxidase (HRP) and acetylcholinesterase (AChE) in nanostructure, and forms hierarchical distribution multi-enzyme cascade system. The structure can shorten the transmission distance of intermediate product in cascade reaction, improve the cascade catalytic efficiency and pesticide inhibition response sensitivity. Further embedding it in double network hydrogel, a hydrogel sensing disc suitable for plant surface attachment can be prepared, the hydrogel sensing disc has good flexibility, surface adaptability and storage stability, can be used for on-site visual detection of organophosphorus pesticides such as chlorpyrifos, has the characteristics of simple operation and non-invasive detection.

Description

Technical Field

[0001] This invention belongs to the fields of biosensors, enzyme immobilization materials and in-situ rapid detection of plants, specifically involving the preparation of a controllable multi-enzyme cascade AChE-HRP-CHO@HNF composite material and its application as a wearable plant sensor. Background Technology

[0002] Pesticides play a vital role in ensuring crop yields and controlling pests and diseases. However, excessive or improper use can lead to pesticide residues, threatening the quality and safety of agricultural products, the ecological environment, and human health. Therefore, establishing rapid, sensitive, and on-site pesticide residue detection methods is crucial for precision application, crop health management, and food safety supervision. Currently, pesticide residue detection mainly relies on chromatography, mass spectrometry, spectral imaging, and photoelectric sensing. While these methods offer high accuracy and sensitivity, they typically suffer from problems such as expensive instruments, complex operation, cumbersome sample pretreatment, and long detection cycles. Furthermore, most methods require destructive sampling, making it difficult to meet the needs for in-situ, real-time, and non-destructive monitoring of pesticide residues on living plant surfaces.

[0003] Enzyme-catalyzed biosensing methods offer advantages such as mild reaction conditions, good recognition specificity, intuitive signal output, and ease of on-site detection. Multi-enzyme cascade systems, in particular, can achieve signal recognition and amplification through continuous catalytic reactions, which is beneficial for improving pesticide detection sensitivity. However, the performance of multi-enzyme cascade reactions is highly dependent on the spatial distance, arrangement order, and local microenvironment between different enzymes. If multiple enzymes are randomly distributed or disorderly aggregated in the carrier, it can easily lead to decreased substrate transfer efficiency, intermediate product diffusion loss, and weakened inter-enzyme synergy, thereby reducing cascade catalytic efficiency and pesticide inhibition response sensitivity. Existing enzyme immobilization strategies mainly include physical adsorption, covalent bonding, gel embedding, and framework encapsulation. These methods struggle to achieve ordered localization of multiple enzymes, limiting the catalytic performance and detection sensitivity of multi-enzyme cascade biosensor materials. Enzyme-inorganic hybrid nanoflowers (HNFs) possess characteristics such as large specific surface area, strong enzyme loading capacity, good structural stability, and microenvironment protection, providing a favorable carrier for constructing spatially controllable multi-enzyme cascade systems. By combining biomimetic mineralization with layer-by-layer protein crown assembly, it is hoped that multiple enzymes can be orderly integrated into the nanoflower structure, forming a multi-enzyme cascade composite material with spatial distribution characteristics, thereby improving cascade catalytic efficiency and signal amplification capability. Therefore, developing a spatially controllable acetylcholinesterase-horseradish peroxidase-choline oxidase hybrid nanoflower composite material (AChE-HRP-CHO@HNF) and applying it to the rapid in-situ detection of pesticide residues on plant surfaces has significant application value. Summary of the Invention

[0004] The purpose of this invention is to provide a controllable method for preparing a multi-enzyme cascade AChE-HRP-CHO@HNF composite material and its application in plant wearable sensors, to solve the problems of difficult spatial positioning of enzymes, insufficient cascade catalytic efficiency, and limited sensitivity of in-situ pesticide detection in existing multi-enzyme immobilization systems. This invention constructs a spatially controllable AChE-HRP-CHO@HNF three-enzyme cascade composite material through a biomimetic mineralization and protein canopy layer-by-layer assembly strategy. First, CHO undergoes a biomimetic mineralization reaction with Ca²⁺ and phosphate to form a CHO@HNF core structure. Then, HRP and AChE are sequentially immobilized through a protein canopy layer-by-layer assembly method, achieving a spatially ordered distribution of the three enzymes in the inorganic nanoflower, forming a multi-enzyme cascade system with a radial hierarchical structure. This spatially ordered structure can shorten the transport path between substrates and intermediates in the cascade reaction, improving enzyme cascade catalytic efficiency and pesticide response sensitivity. Furthermore, this invention embeds the AChE-HRP-CHO@HNF composite material into a dual-network hydrogel to construct a plant wearable sensor. Among them, bovine serum albumin forms a protein cross-linking network with glutaraldehyde, and sodium alginate forms a cross-linking network with Ca. 2+ An ion-crosslinked network is formed, with two networks interpenetrating to form a dual-network hydrogel structure. The resulting hydrogel exhibits good flexibility, transparency, adhesion, and surface adaptability, enabling effective collection of pesticide residue information from plant surfaces. The sensing mechanism is based on AChE catalyzing the formation of choline from acetylcholine, CHO further catalyzing the formation of hydrogen peroxide from choline, and HRP utilizing hydrogen peroxide (H2O2) to catalyze a color reaction in the chromogenic substrate. When organophosphorus pesticides are present, they inhibit AChE activity, leading to a weakening of the cascade reaction and thus a change in color signal. Color images are acquired via a smartphone and combined with RGB digital analysis to achieve rapid quantitative detection of organophosphorus pesticide residues. The plant wearable sensor constructed in this invention possesses high sensitivity, high stability, good plant surface adaptability, and on-site visual detection capabilities, enabling in-situ, non-destructive, and dynamic monitoring of organophosphorus pesticides such as chlorpyrifos on plant surfaces. This technology is applicable to fields such as rapid screening of pesticide residues in agricultural products, precision application management, crop health monitoring, and food safety supervision.

[0005] The preparation of a controllable multi-enzyme cascade AChE-HRP-CHO@HNF composite material and its application as a wearable sensor in plants, as described in this invention, includes the following steps:

[0006] A. Preparation of controllable multi-enzyme cascade AChE-HRP-CHO@HNF composite material

[0007] 30 μL, 0.20 mol·L -1 CaCl2 solution and 100 μL, 5 mg·mL -1Mix the CHO solution and vortex for 1 min; then add 870 μL of 0.01 mol·L⁻¹ solution. -1 The mixture was vortexed for 1 min in PBS buffer (pH 8.0) and incubated at 4 °C for 12 h. The product was collected by centrifugation and washed twice with deionized water to obtain CHO@HNF. Then, 100 μL of 1 mg·mL⁻¹ PBS buffer was added to the CHO@HNF suspension. -1 HRP solution and 900 μL PBS buffer were incubated at 4 °C for 12 h. After centrifugation and washing, HRP-CHO@HNF was obtained. Finally, 100 μL of 2.5 mg·mL⁻¹ PBS buffer was added. -1 The AChE solution and 900 μL PBS buffer were incubated at 4 °C for 12 h. After centrifugation and washing, the mixture was resuspended in 1 mL PBS buffer to obtain the AChE-HRP-CHO@HNF composite material.

[0008] B. Preparation of AChE-HRP-CHO@HNF dual-network hydrogel

[0009] A hydrogel precursor solution containing 4.5% bovine serum albumin, 1% sodium alginate, and 0.5% glutaraldehyde was prepared. The AChE-HRP-CHO@HNF composite material was then added to the precursor solution and thoroughly mixed. The mixture was then poured into a mold with a pore size of 7 mm and a depth of 0.22 mm, and the mold was immersed in 0.01 mol·L⁻¹ water. -1 Cross-linking in CaCl2 solution for 3 min allows bovine serum albumin to form a protein cross-linking network with glutaraldehyde, while sodium alginate reacts with Ca... 2+ An ionic cross-linked network is formed. After gelation, the gel is removed to obtain a dual-network hydrogel loaded with AChE-HRP-CHO@HNF.

[0010] C. Plant wearable sensors for digital detection of chlorpyrifos pesticide images

[0011] A dual-network hydrogel plant wearable sensor loaded with AChE-HRP-CHO@HNF was attached to the surface of the plant to be tested, allowing it to contact the area with chlorpyrifos residue for 20 min. Then, 40 μL of a mixed solution containing acetylcholine, PBS buffer, and 3,3',5,5'-tetramethylbenzidine (TMB) was added to the hydrogel surface, and the mixture was incubated at 37 °C for 30 min. Finally, 20 μL of 0.2 mol·L⁻¹ PBS buffer was added. -1The reaction was continued for 10 min using an acetate buffer solution at pH 4.0. After the reaction, hydrogel color images were acquired using a smartphone under fixed light source, distance, and angle conditions. The red, green, and blue channel values ​​of the images were extracted using ImageJ software, and the red / (green+blue) response parameter was calculated to establish a linear relationship between it and the concentration of chlorpyrifos, thus realizing the digital quantitative detection of chlorpyrifos pesticide residues.

[0012] The mechanism of this invention is as follows:

[0013] This invention leverages the specific inhibitory effect of organophosphorus pesticides on AChE, combined with a spatially controllable AChE-HRP-CHO@HNF three-enzyme cascade catalytic system, to achieve highly sensitive visual detection and digital image analysis of chlorpyrifos. AChE first catalyzes the formation of choline from acetylcholine, followed by CHO further catalyzing the formation of H₂O₂ from choline. HRP utilizes H₂O₂ to catalyze the oxidation and colorimetric reaction of TMB, resulting in a blue color signal. In the presence of chlorpyrifos, AChE activity is inhibited, reducing the efficiency of the cascade reaction and decreasing the formation of colorimetric products, leading to a weakened color signal that responds systematically to changes in chlorpyrifos concentration.

[0014] This invention employs a biomimetic mineralization and protein canopy-based stepwise assembly strategy to spatially and orderly immobilize CHO, HRP, and AChE within CaHPO4 hybrid nanoflowers, forming a three-enzyme cascade system with a radial hierarchical structure. This shortens the diffusion path between substrates and intermediates, improving cascade catalytic efficiency and pesticide response sensitivity. Furthermore, this invention embeds an AChE-HRP-CHO@HNF composite material into a dual-network hydrogel, enabling the hydrogel to simultaneously function as an enzyme immobilization carrier, a flexible attachment interface, and a visual signal output platform. This hydrogel sensor can conform to the curvature, undulations, and microscopic roughness of plant leaf and fruit surfaces, achieving stable conformal contact with plant organ surfaces. It can perform contact sampling, in-situ reaction, and color reading of pesticide residues without damaging plant tissue, thus demonstrating the application characteristics of a plant wearable sensor.

[0015] Compared with existing technologies, the present invention has the following characteristics:

[0016] (1) A spatially controllable AChE-HRP-CHO@HNF three-enzyme cascade composite material was constructed by using a biomimetic mineralization and protein canopy stepwise assembly strategy to achieve spatially ordered fixation of multiple enzymes, thereby improving enzyme stability, resistance to environmental interference, long-term storage performance and cascade catalytic efficiency.

[0017] (2) AChE-HRP-CHO@HNF was embedded in a dual-network hydrogel to construct a plant wearable sensor with flexibility, adhesion and surface adaptability. It can be stably attached to the surface of leaves and stems to realize in-situ, non-destructive detection of pesticide residues on plant surfaces.

[0018] (3) Combining smartphone image acquisition and RGB digital analysis, the enzyme inhibition reaction caused by chlorpyrifos is converted into a quantifiable color signal, enabling rapid, visual and quantitative detection, which is suitable for on-site screening of pesticide residues and dynamic degradation monitoring. Attached Figure Description

[0019] Figure 1 The preparation and characterization of AChE-HRP-CHO@HNF are shown in Figure 1. (a) is a schematic diagram of the stepwise assembly of AChE-HRP-CHO@HNF as described in Example 1. (b) is a thermogravimetric analysis diagram of CaHPO4, CHO@HNF, HRP-CHO@HNF and AChE-HRP-CHO@HNF as described in Example 1. (c) is a confocal fluorescence micrograph of AChE-HRP-CHO@HNF as described in Example 1. (d) is a radial fluorescence intensity distribution diagram of AChE, HRP and CHO in HNF as described in Example 1.

[0020] Figure 2 The following diagrams illustrate the spatially controllable assembly mechanism of AChE-HRP-CHO@HNF: (a) Transmission electron microscopy images of CaHPO4 and CHO@HNF at different reaction times as described in Example 2; (b) Particle size distribution of AChE-HRP-CHO@HNF under different protein concentrations as described in Example 2; (c) Schematic diagram of the synthesis of the protein-protein@HNF composite material as described in Example 2; and (d) Comparison of the relative activities of CHO@HNF and CHO-CHO@HNF under the same CHO concentration as described in Example 2, wherein CHO-CHO@HNF is synthesized at 0.5 mg / mL. -1 The product was prepared by adding CHO to CHO@HNF.

[0021] Figure 3 The diagram shows the cascade catalytic performance of the spatially controllable AChE-HRP-CHO@HNF composite material; (a) shows the half-inhibitory concentration (IC50) under different enzyme loading modes described in Example 3. 50 (a) Comparison diagram; (b) Relative enzyme activity changes of AChE-HRP-CHO@HNF and free enzyme during 30 days of storage as described in Example 3.

[0022] Figure 4The following are excerpts from the study on the construction and properties of the dual-network hydrogel discs: (a) a schematic diagram of the preparation of the dual-network hydrogel described in Example 4; (b) a SEM image of the cross-section of the hydrogel disc described in Example 4, with an inset showing the actual hydrogel disc; (c) the effective adhesion of the hydrogel disc described in Example 4 to the surface of a tomato plant; (d) a schematic diagram of the image digitization process described in Example 4, including the process of separating the true color image into RGB channels and calculating the red / (green+blue) value to generate a pseudo-color image; and (e) a graph showing the relationship between the red / (green+blue) response value and the concentration of chlorpyrifos described in Example 4.

[0023] Figure 5 The following are performance characteristics of the plant wearable sensor in practical applications: (a) is a state diagram of the plant wearable sensor described in Example 5 attached to the surface of plant leaves in the absence of exposure and exposure to chlorpyrifos; (b) is a color signal response diagram of the plant wearable sensor described in Example 5 under the presence and absence of chlorpyrifos; (c) is a monitoring result diagram of the degradation process of chlorpyrifos residue on the surface of plant leaves within 11 days by the plant wearable sensor described in Example 5. Detailed Implementation

[0024] Example 1: Preparation and characterization of AChE-HRP-CHO@HNF composite material

[0025] The preparation process of AChE-HRP-CHO@HNF composite material is as follows: Figure 1 As shown in Figure a. First, CHO, CaCl2, and PBS buffer were mixed to prepare CHO@HNF via a biomimetic mineralization method. Then, utilizing nanoparticle-protein interactions, HRP and AChE protein crowns were sequentially assembled on the surface of CHO@HNF, resulting in a layer-by-layer assembled AChE-HRP-CHO@HNF composite material. To evaluate the layer-by-layer enzyme loading process, thermogravimetric analysis was performed on the materials at different assembly stages. The results showed that the mass loss related to biomacromolecules gradually increased from 13.9% in CHO@HNF to 18.4% in HRP-CHO@HNF and 21.1% in AChE-HRP-CHO@HNF, indicating that the HRP and AChE protein crowns were gradually assembled onto the surface of CHO@HNF. Figure 1 b). The spatial distribution of the three enzymes in HNF was further observed using confocal fluorescence microscopy. The results showed that RhB-labeled CHO was mainly distributed in the core region of HNF, MCA-labeled HRP was mainly located in the middle region, and FITC-labeled AChE was mainly distributed in the outer region. Figure 1 c). Radial fluorescence intensity analysis further confirmed that AChE, HRP, and CHO exhibit a radial hierarchical distribution in HNF, indicating that this method can achieve spatially ordered assembly of multi-enzyme systems. Figure 1 d).

[0026] Example 2: Spatially Controllable Assembly Mechanism of AChE-HRP-CHO@HNF

[0027] Using CHO as a model protein, its effect on the formation of CaHPO4-based CHO@HNF was investigated. Without CHO, CaHPO4 gradually aggregated from amorphous nanoparticles to form a flower-like structure; after the addition of CHO, CHO participated in the nucleation and assembly of the CaHPO4 precursor, forming protein-inorganic hybrid CHO@HNF. Figure 2 a). The particle size distribution of HNF changes under different protein concentrations, indicating that protein concentration affects the structural size and assembly state of HNF. Excessively high protein concentrations may cause local crowding of enzyme molecules, thereby affecting substrate transport and catalytic efficiency. Figure 2 (b) Therefore, relying solely on mineralization to achieve protein incorporation is insufficient to fully meet the demands of multi-enzyme systems for high loading and high catalytic efficiency. To address this, the present invention further introduces a stepwise protein canopy assembly strategy. For example... Figure 2 As shown in Figure c, protein@HNF is first formed through biomimetic mineralization, and then the outer protein is further assembled using the interfacial interaction between the protein and the HNF surface to form a protein-protein@HNF structure. This method can provide additional protein loading sites on the HNF surface, which helps to alleviate internal protein crowding and achieve a stratified distribution of different proteins in the HNF. Under the same CHO concentration conditions, CHO-CHO@HNF obtained by further assembling CHO on the CHO@HNF surface has higher activity (…). Figure 2 (d) This demonstrates that protein canopy assembly can effectively improve protein loading capacity. Therefore, the stepwise protein canopy assembly strategy can overcome the limitations of protein loading and spatial distribution during single-step mineralization.

[0028] Example 3: Evaluation of the catalytic performance and stability of AChE-HRP-CHO@HNF composite material

[0029] To investigate the effect of spatially ordered enzyme assembly on sensing performance, the response of composite materials to chlorpyrifos was compared under different enzyme loading methods. The results showed that AChE-HRP-CHO@HNF exhibited higher detection sensitivity for chlorpyrifos, with a lower half-inhibition concentration (IC50) than other materials. 50 It is 0.042 μg·mL -1 The concentration of enzymes significantly decreased compared to partially ordered assembly systems and disordered co-immobilization systems, indicating that the ordered three-enzyme assembly structure can improve the recognition and signal response capabilities of chlorpyrifos. Figure 3 a). Long-term stability results showed that AChE-HRP-CHO@HNF retained approximately 80% of its initial activity after 30 days of storage in a water environment at room temperature, while the free enzyme activity decreased significantly, indicating that this composite material can significantly improve the long-term catalytic stability of the three-enzyme cascade system. Figure 3 (b) The above results indicate that AChE-HRP-CHO@HNF improves the enzyme cascade catalytic performance, chlorpyrifos response sensitivity, and long-term stability through spatially ordered assembly and HNF protection.

[0030] Example 4: Construction and performance evaluation of AChE-HRP-CHO@HNF-based wearable hydrogel sensor for chlorpyrifos detection

[0031] A two-step interpenetrating network strategy was employed to construct plant-based wearable hydrogel discs loaded with AChE-HRP-CHO@HNF. During preparation, bovine serum albumin (BSA) first cross-linked with glutaraldehyde to form a protein network, while AChE-HRP-CHO@HNF and sodium alginate (SA) were simultaneously introduced into the system. Subsequently, the system was immersed in a Ca²⁺ solution, causing SA to undergo ionic cross-linking, forming a bovine serum albumin / sodium alginate dual-network hydrogel structure. Figure 4 a). The resulting double-network hydrogel was prepared as a disc structure with a thickness of approximately 0.22 mm and a diameter of approximately 7 mm. Figure 4 b). Notably, these hydrogels remained firmly attached to plant tissue even after 50 folding cycles, exhibiting excellent interfacial stability under mechanical deformation, making them suitable for in-situ detection on plant surfaces. Figure 4 c). The detection process showed that the color response of the hydrogel discs gradually changed with increasing chlorpyrifos concentration, and each color channel exhibited a concentration-dependent response. Further analysis revealed that the red / (green + blue) response parameter had the best correlation with chlorpyrifos concentration. Figure 4 d). By digitally fitting the color signal, a linear relationship was established between red / (green+blue) and chlorpyrifos concentration: Y = 0.042 Log[chlorpyrifos] + 0.355. Its linear detection range is 0.5–2000 ng·mL⁻¹. Figure 4 e). The above results demonstrate that this wearable plant sensor, combined with RGB digital analysis from a smartphone, enables rapid, sensitive, and visual detection of chlorpyrifos.

[0032] Example 5: Practical Application Performance of AChE-HRP-CHO@HNF-based Plant Wearable Sensor

[0033] To verify the practical application performance of wearable plant sensors in the on-site detection of pesticide residues on plant surfaces, a dual-network hydrogel sheet loaded with AChE-HRP-CHO@HNF composite material was attached to the surface of tomato leaves, and its detection effect under conditions of chlorpyrifos exposure was investigated. Figure 5As shown in Figure a, the plant wearable sensor can stably adhere to the surface of tomato leaves. In the absence of chlorpyrifos exposure, the plant wearable sensor exhibits a distinct blue color; however, in the presence of chlorpyrifos, the three-enzyme cascade colorimetric reaction is inhibited due to the inhibition of AChE activity by chlorpyrifos, resulting in a transparent or nearly colorless state for the plant wearable sensor. This indicates that the sensor can produce a visual response to chlorpyrifos residues on the plant leaf surface. Further red / (green + blue) signal analysis results show that the plant wearable sensor exhibits a significant difference in signal response under conditions of chlorpyrifos presence or absence. Figure 5 b).

[0034] To verify its detection capability in actual plant samples, chlorpyrifos was sprayed onto the surface of tomato plant leaves, and hydrogel discs were attached to the upper surface of the leaves. Color signals were collected at different time points. The results showed that the degradation process of chlorpyrifos on the leaf surface followed pseudo-first-order kinetics, with a half-life of 1.733 days. Figure 5 c). Monitoring results over 11 consecutive days showed that chlorpyrifos residues on the surface of tomato plants gradually decreased over time, reaching a low level on day 7 after application. These results indicate that the AChE-HRP-CHO@HNF-based wearable plant sensor can achieve in-situ, non-destructive dynamic monitoring of chlorpyrifos residues on plant surfaces.

Claims

1. A method for preparing a controllable multi-enzyme cascade AChE-HRP-CHO@HNF-based plant wearable sensor, the specific steps of which are as follows: ①Choline oxidase CHO, calcium salt and phosphate buffer were mixed and CHO@HNF composite material was prepared by biomimetic mineralization reaction. The resulting precipitate was collected by centrifugation at 7000 r / min for 10 min, thoroughly rinsed with ultrapure water, and then redispersed for subsequent use. ② Horseradish peroxidase (HRP) is added to the CHO@HNF, allowing HRP to be assembled and immobilized on the surface of CHO@HNF through the protein canopy, resulting in HRP-CHO@HNF. Subsequently, acetylcholinesterase (AChE) is added to the HRP-CHO@HNF, allowing AChE to be further assembled on the surface of HRP-CHO@HNF, resulting in an AChE-HRP-CHO@HNF three-enzyme cascade spatial composite material. ③ The AChE-HRP-CHO@HNF three-enzyme cascade composite material is mixed with a dual-network hydrogel precursor solution and cross-linked to obtain an AChE-HRP-CHO@HNF-based plant wearable sensor.

2. The method for preparing a controllable multi-enzyme cascade AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 1, characterized in that... The calcium salt mentioned in step ① is calcium chloride, and the phosphate buffer is PBS buffer with pH 8.0 and a concentration of 0.01 M. (CHO, Ca) 2+ Through biomimetic mineralization, phosphate groups form an enzyme-inorganic hybrid nanoflower structure with CaHPO4 as the inorganic framework.

3. The method for preparing a controllable multi-enzyme cascade AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 1, characterized in that, In the AChE-HRP-CHO@HNF three-enzyme cascade composite material, CHO is mainly located in the HNF core region, HRP is located in the intermediate protein canopy region, and AChE is located in the outer protein canopy region, forming a three-enzyme cascade structure with spatially ordered distribution.

4. The method for preparing a controllable multi-enzyme cascade AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 1, characterized in that, The concentration of CHO was 0.5 mg / mL. -1 The concentration of AChE was 0.25 mg / mL. -1 And the concentration of HRP was 0.1 mg / mL. -1 .

5. The method for preparing a controllable multi-enzyme cascade AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 1, characterized in that, The sodium alginate concentration of the dual-network hydrogel used is 20.0 mg / mL, the calcium chloride solution concentration is 5.0 mg / mL, and the bovine serum albumin concentration is 4.5%.

6. The application of the AChE-HRP-CHO@HNF-based plant wearable sensor involves the following steps: The plant wearable sensor is attached to the surface of the plant to be tested, bringing it into contact with the area where chlorpyrifos residues are present; a colorimetric reaction solution containing acetylcholine, PBS buffer, and 3,3′,5,5′-tetramethylbenzidine is then added to the surface of the plant wearable sensor for incubation; acetate buffer is then added to continue the reaction; after the reaction is complete, a color image of the plant wearable sensor is acquired, and the color image is subjected to RGB digital analysis to achieve quantitative detection of chlorpyrifos pesticide residues.

7. The application of the AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 6, characterized in that, The contact time between the plant wearable sensor and the surface of the plant to be tested is 20 min; the amount of the colorimetric reaction solution added is preferably 40 μL; after adding the colorimetric reaction solution, incubation is carried out for 40 min; the concentration of the acetate buffer is 0.1 mol·L⁻¹, and the pH is 4.

0.

8. The application of the AChE-HRP-CHO@HNF-based plant wearable sensor according to claim 6, characterized in that, The RGB digital analysis includes extracting the red, green, and blue channel values ​​of the color image and calculating the response parameter obtained by dividing the red channel value by the sum of the green and blue channel values. By establishing a linear relationship between the response parameter and the concentration of chlorpyrifos, the digital quantitative detection of chlorpyrifos pesticide residues on plant surfaces is achieved.

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