Preparation method of AChE (at) aCOFs-DB photoenzyme integrated biosensor

Abstract

The invention relates to a preparation method of a biosensor, in particular to a construction method of an acetylcholin esterase (at) amorphous covalent organic framework (AChE (at) aCOFs-DB) photoenzyme integrated biosensor, and belongs to the technical field of biosensors. According to the method, an in-situ growth mineralization strategy is adopted, effective packaging of AChE in aCOFs-DB is achieved, the fluorescence performance of aCOFs is enhanced through a hydrogen bond auxiliary intramolecular rotation limitation design, and meanwhile the structural stability and catalytic activity of AChE are improved; the biosensor with high sensitivity and high selectivity is constructed based on the synergistic effect of fluorescence characteristics and enzyme catalytic performance; the sensor can be used for sensitive detection of organophosphorus pesticides such as chlorpyrifos, and the detection limit can reach a nanogram / milliliter level; the detection principle is as follows: AChE catalyzes a substrate to generate a product, and fluorescence quenching is caused by an inner filtering effect; when the organophosphorus pesticide (such as chlorpyrifos) exists, the AChE activity is inhibited, the catalytic capability is reduced, and a fluorescence signal is recovered, so that quantitative detection of the pesticide is realized; the preparation method is simple and convenient to operate and controllable in structure, is suitable for rapid detection of organophosphorus pesticides in agricultural products and environmental samples, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for preparing AChE@aCOFs-DB composite material and its application in the detection of chlorpyrifos. Background Technology

[0002] In modern agricultural production, pesticides play a crucial role in improving crop yield and quality, but they also pose serious environmental and health risks. Chlorpyrifos, a typical and widely used organophosphorus pesticide, specifically binds to acetylcholinesterase (AChE) in the nervous system to form a phosphorylase complex, posing a serious threat to food safety, the ecological environment, and human health. Therefore, achieving rapid, highly sensitive, and selective on-site detection of chlorpyrifos is of great significance for ensuring food safety, environmental protection, and human health. Fluorescent biosensors, with their rapid, label-free, multifunctional trace analysis, and real-time monitoring capabilities, have shown great potential in environmental monitoring, food safety, and biomedicine. However, ensuring high specificity and sensitivity of sensors in complex real-world detection environments remains a significant challenge. Enzymes, as biological catalysts, possess the dual capabilities of specific recognition and signal amplification. Combining them with fluorescence detection technology is an effective strategy for improving the selectivity and sensitivity of biosensors. Unfortunately, the inherent stability of enzymes limits their practical application; they are easily inactivated under harsh conditions. In addition, how to efficiently transduce the recognition of biological events by enzymes into detectable fluorescent signals is another core challenge in building high-performance sensors.

[0003] Immobilizing enzymes in porous supports, utilizing the physical and chemical properties of the supports to protect the enzymes, is an effective way to improve enzyme stability and sensor performance. Covalent organic frameworks (COFs) are a class of porous materials formed by organic structural units linked by covalent bonds. Their unique pore structure, tunable pore environment, and flexible functionalization make them ideal candidate materials for enzyme immobilization. Based on stable covalent bonds, COFs exhibit excellent physicochemical stability and environmental adaptability, maintaining structural integrity under extreme conditions. Furthermore, encapsulating enzymes with fluorescent COFs holds promise for simultaneously achieving enzyme structural stabilization and real-time fluorescence reporter function. However, this strategy is fundamentally limited: most COFs have low fluorescence efficiency. Simultaneously, the synthesis of fluorescent COFs often requires harsh conditions such as continuous heating, prolonged stirring, or acidic catalysts, all of which can lead to enzyme denaturation during immobilization. Here, we developed a photoenzyme integrated biosensor by encapsulating AChE in amorphous fluorescent COFs. Leveraging the integrated advantages of fluorescence properties and catalytic activity in the enzyme@aCOFs system, a highly sensitive and selective biosensor was constructed. The strong synergistic effect of photoenzymes significantly enhances the biorecognition to signal transduction function, enabling sensitive detection of chlorpyrifos pesticide at the nanogram per milliliter level. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing AChE@aCOFs-DB composite material and its application in the detection of chlorpyrifos.

[0005] This invention constructs an AChE@aCOFs-DB photoenzyme-integrated biosensor with excellent fluorescence performance and good stability. Utilizing the specific inhibitory effect of chlorpyrifos on AChE and its efficient signal transduction capability, a fluorescent biosensing platform targeting chlorpyrifos was established. Both the enzyme catalysis and photoresponse processes are completed within the assembled composite material; this design, integrating biorecognition and signal transduction functions, effectively improves detection sensitivity. Compared with single colorimetric detection methods, this AChE@aCOFs-DB photoenzyme-integrated biosensor significantly improves detection sensitivity, providing a novel technological approach for pesticide detection and food safety assurance.

[0006] The preparation method of the AChE@aCOFs-DB composite material described in this invention and its application in the detection of chlorpyrifos include the following steps: (1) Preparation of AChE@aCOFs-DB: Dissolve 0.15–0.09 mM trimesaldehyde in 500 µL of DMF, then transfer to 5.5 mL of deionized water. Dissolve 0.15–0.06 mM 2,5-dihydroxyterephthalamide in 1 mL of deionized water, then add to the trimesaldehyde solution. Sonicate the mixture for 10 minutes until thoroughly mixed. Then add 2 mL of AChE aqueous solution (1–5 mg / mL) and 1 mL of scandium trifluoromethanesulfonate aqueous solution (0.5–5 mg / mL). After reacting for 1 hour, wash the precipitate three times with deionized water, and suspend the precipitate in 2 mL of deionized water for later use.

[0007] (2) Activity study of AChE@aCOFs-DB: Take 10-50 μL of AChE@aCOFs-DB aqueous solution, then add 300 μL of deionized water, 50 μL of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) solution (0.1-0.6 mg / mL), 50 μL of PBS buffer (10 mM, pH = 7.4), and 25 μL of acetylthiocholine iodide (ATCh) (5-15 mM). After incubating at 37℃ for 20-40 minutes, monitor the absorbance change of the solution at 405 nm.

[0008] (3) Construction of the AChE@aCOFs-DB-based chlorpyrifos sensing platform: Mix 10-50 μL of AChE@aCOFs-DB aqueous solution with 25 μL of chlorpyrifos at different concentrations and incubate at 37 ℃ for 20-40 minutes. Then add 300 μL of deionized water, 50 μL of DTNB solution (0.1-0.6 mg / mL), 50 μL of PBS buffer (10 mM, pH = 7.4), and 25 μL of ATCh (5-15 mM). Monitor the change in fluorescence intensity after incubation at 37 ℃ for 30 minutes.

[0009] (4) Performance testing of the AChE@aCOFs-DB-based chlorpyrifos sensing platform: Fenphos, defoliant, glyphosate, cypermethrin, oxychlorpyrifos, piperazine, deltamethrin, benzthiamethoxam, ovalbumin, bovine serum albumin, sodium chloride, potassium chloride, barium chloride, serine, and ascorbic acid were used as negative controls to determine the specific recognition of AChE@aCOFs-DB against chlorpyrifos. To assess its resistance to interference, the response of AChE@aCOFs-DB to chlorpyrifos was collected in the presence of the aforementioned coexisting interfering substances. The fluorescence changes after the completion of the biochemical colorimetric reaction were monitored for evaluation. The mechanism of this invention is as follows: The core mechanism of the AChE@aCOFs-DB composite material for detecting chlorpyrifos is a sensitive switching system based on "enzyme inhibition-internal filtration effect-fluorescence recovery". The principle is as follows: the immobilized AChE can normally catalyze the substrate reaction to generate the yellow product 5-thio-2-nitrobenzoic acid (TNB). This product quenches the fluorescence of the carrier aCOFs-DB through the internal filtration effect, putting the system in a "fluorescence off" state. When chlorpyrifos is present, it irreversibly inhibits the activity of acetylcholinesterase, leading to a reduction in TNB production and a weakening of the internal filtration effect, thus allowing the fluorescence signal of aCOFs-DB to "recover". Ultimately, the higher the concentration of chlorpyrifos and the deeper the enzyme inhibition, the stronger the fluorescence signal recovery, thereby enabling accurate and quantitative detection of chlorpyrifos.

[0010] The photoenzyme integrated sensing platform based on AChE@aCOFs-DB prepared in this invention can reliably acquire pesticide concentration information. Compared with single enzyme-based sensors and fluorescence sensors, it has superior stability, sensitivity and specificity.

[0011] Compared with existing technologies, the present invention has the following characteristics: (1) Efficient encapsulation of AChE using an amorphous covalent organic framework.

[0012] (2) The structural characteristics of the amorphous covalent organic framework endow it with excellent fluorescence properties, and the excellent microenvironment is conducive to its mass transfer process and preserves enzyme activity.

[0013] (3) Both the enzyme catalysis process and the photoresponse process are completed inside the assembled composite material. This design, which integrates bio-recognition and signal transduction functions, effectively improves the detection sensitivity.

[0014] (4) Compared with single enzyme-based sensors and fluorescence sensors, it has improved in terms of stability, sensitivity and specificity. Attached Figure Description

[0015] Figure 1 A schematic diagram of the synthesis of AChE@aCOFs-DB composite material; Figure 2 Transmission electron microscope image and energy dispersive spectroscopy (EDS) spectrum of the AChE@aCOFs-DB composite material obtained in Example 1; Figure 3 The stability of the AChE@aCOFs-DB composite material was studied; (a) the time stability of the AChE@aCOFs-DB composite material described in Example 2; (b) the recyclability of the catalytic ability of the AChE@aCOFs-DB composite material described in Example 2. Figure 4Performance study of the AChE@aCOFs-DB detection platform for chlorpyrifos detection; (a) Schematic diagram of the mechanism of the AChE@aCOFs-DB detection platform described in Example 3; (b) Feasibility analysis of the AChE@aCOFs-DB detection platform; (c) Linear relationship between chlorpyrifos concentration and fluorescence intensity of the AChE@aCOFs-DB detection platform described in Example 3; Figure 5 This demonstrates the selectivity and anti-interference capability of the AChE@aCOFs-DB detection platform described in Example 4. Detailed Implementation

[0016] Example 1: Preparation of AChE@aCOFs-DB material In a typical procedure, 0.09 mM pyromellitic aldehyde was dissolved in 500 µL of DMF solution, and 0.045 mM 2,5-dihydroxyhydrazide was dissolved in 1 mL of deionized water. Both solutions were then transferred to 5.5 mL of deionized water. The mixture was sonicated for 10 minutes to ensure thorough mixing. Finally, 2 mL of AChE aqueous solution (1 mg / mL) and 1 mL of scandium trifluoromethanesulfonate aqueous solution (0.5 mg / mL) were added. The mixture was allowed to stand for 1 hour, and then centrifuged at 8000 rpm for 10 min. The resulting precipitate was washed three times with ultrapure water to obtain the AChE@aCOFs-DB complex. The precipitate was then suspended in 2 mL of deionized water for later use.

[0017] Transmission electron microscopy (TEM) images revealed that AChE@aCOFs-DB exhibits a rod-like structure with lengths reaching the micrometer scale. Energy dispersive spectroscopy showed the distribution of C, N, O, and S elements within the AChE@aCOFs-DB structure, confirming the successful synthesis of the composite material. Figure 2 ).

[0018] Example 2: Activity and stability studies of AChE@aCOFs-DB The colorimetric biochemical reaction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) with acetylthiocholine (ATCh) substrate was used as a model system. AChE@aCOFs-DB catalyzed the conversion of the substrate to 2-nitro-5-thiobenzoic acid (TNB), which exhibited a new absorption peak at 405 nm. Relative activity was defined as the ratio of absorbance measured at 405 nm in the ATCh reaction system. The relative activity value was calculated using the following formula: Relative activity (%) = (A1-A0) / (A2-A0) × 100% Where A0 is the absorbance without enzyme addition, A1 is the absorbance after adding AChE@aCOFs-DB, and A2 is the absorbance after adding AChE.

[0019] The temporal stability and recyclability of the material were studied by testing the activity changes of AChE@aCOFs-DB. Long-term stability studies showed that AChE@aCOFs-DB maintained superior catalytic activity compared to the free enzyme within 30 days, confirming the protective effect of aCOFs-DB. Figure 3 a). AChE@aCOFs-DB can be recovered by simple centrifugation and retains more than 90% of its relative activity after 10 cycles of reuse. Figure 3 b).

[0020] Example 3: Detection of pesticide residue concentration using an immunosensing platform 25 μL of AChE@aCOFs-DB aqueous solution was mixed with 25 μL of chlorpyrifos solutions of different concentrations and incubated at 37 ℃ for 30 minutes. Then, 300 μL of deionized water, 50 μL of DTNB solution (0.3 mg / mL), 50 μL of PBS buffer (10 mM, pH = 7.4), and 25 μL of ATCh (10 mM) were added. The fluorescence intensity was monitored after incubation at 37 ℃ for 30 minutes.

[0021] This sensing principle is based on the property of TNB (a yellow product) regulating the fluorescence intensity of AChE@aCOFs-DB. Chlorpyrifos reduces TNB production by inhibiting AChE activity, thereby enabling quantitative detection of chlorpyrifos (by monitoring changes in fluorescence signal) Figure 4 a). With increasing chlorpyrifos concentration, the fluorescence intensity of AChE@aCOFs-DB gradually increased ( Figure 4 b). Within the concentration range of 1-1000 ng / mL, the logarithm of the fluorescence intensity ratio (F / F0) showed a good linear relationship with the concentration of chlorpyrifos, where F and F0 represent the fluorescence intensity values ​​with and without chlorpyrifos, respectively. Figure 4 c).

[0022] Example 4: Performance Evaluation of the Chlorpyrifos Pesticide Detection Platform The selectivity and anti-interference ability of the AChE@aCOFs-DB-based chlorpyrifos pesticide detection platform were evaluated. Evaluation was conducted by monitoring fluorescence changes after the completion of the chromogenic biochemical reaction. As negative controls, fenpropathrin, defoliant, glyphosate, cypermethrin, oxychlorpyrifos, imidacloprid, deltamethrin, bensulfuron-methyl, ovalbumin, bovine serum albumin, sodium chloride, potassium chloride, barium chloride, serine, and ascorbic acid were used. Results showed that at the same concentration (1000 ng / mL), only chlorpyrifos could be specifically recognized by AChE@aCOFs-DB. The response of AChE@aCOFs-DB to chlorpyrifos was collected in the presence of the above coexisting interfering substances. Results showed that even in the presence of interfering substances, the sensing platform remained stable in chlorpyrifos detection, exhibiting excellent anti-interference performance. Figure 5 To evaluate the practicality and reliability of the AChE@aCOFs-DB photoenzyme integrated biosensor, chlorpyrifos was detected in different samples (such as tap water, tea, apple juice, and pear juice). The results showed that the recovery rate of chlorpyrifos ranged from 95.47% to 108.98%, with a relative standard deviation (RSD, n=3) of less than 5.14%, indicating that the developed photoenzyme integrated biosensor has broad prospects for practical applications.

[0023] Table 1: Detection of chlorpyrifos in actual samples using the AChE@aCOFs-DB sensing platform prepared according to the present invention.

Claims

1. A method for fabricating an AChE@aCOFs-DB photoenzyme integrated biosensor, characterized in that... The specific steps are as follows: ① Dissolve pyromellitic aldehyde in DMF solution and 2,5-dihydroxyterephthalamide in water. Mix the two in an aqueous solution and sonicate to disperse them evenly. ② Add 2 mL of AChE aqueous solution, and after mixing evenly, add 1 mL of scandium trifluoromethanesulfonate aqueous solution as a catalyst. Let the mixed solution stand for 1 hour at room temperature. ③ The obtained reaction solution was centrifuged at 8000 rpm for 10 min, and the precipitate was washed with ultrapure water to obtain the AChE@aCOFs-DB complex. The precipitate was reconstituted with deionized water and used for the detection of chlorpyrifos pesticide.

2. The preparation method according to claim 1, characterized in that... The aCOFs-DB preparation precursors are pyromellitic methyl ether and 2,5-dihydroxyterephthalohydrazide.

3. The preparation method according to claim 1, characterized in that... The concentration of the pyromellitic methyl ether is 0.09 mM, and the concentration of 2,5-dihydroxyterephthalamide is 0.045 mM.

4. The preparation method according to claim 1, characterized in that... The enzyme used was AChE, with a concentration of 1 mg / mL.

5. The preparation method according to claim 1, characterized in that... The catalyst used is scandium trifluoromethanesulfonate, with a concentration of 0.5 mg / mL.

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

7. The application of the AChE@aCOFs-DB photoenzyme integrated fluorescent biosensor as described in claim 6 in the detection of chlorpyrifos, characterized in that: Different concentrations of chlorpyrifos samples were added to the AChE@aCOFs-DB solution at a volume ratio of 1:1 and incubated for 30 min. AChE catalyzes the formation of products from the substrate, which leads to fluorescence quenching through the internal filtration effect. Chlorpyrifos affects the fluorescence decay effect by directly inhibiting AChE activity. The concentration of chlorpyrifos can be quantitatively analyzed by the degree of fluorescence intensity recovery.

Images