Construction method of aggregation-induced emission multi-residue sensing system based on bionic AR-DBD / LBD

Abstract

The invention discloses a construction method of an aggregation-induced emission (AIE) multi-residue sensing system based on a bionic AR-DBD / LBD. Belongs to the technical field of biochemical detection technology and food science, and comprises optimized AR-DBD / LBD, an AIE probe and a PBS buffer solution, and the construction method comprises the following steps: recombining the bionic AR-DBD / LBD into escherichia coli, and obtaining a target protein through induced expression; the structure of the bionic AR-DBD / LBD is represented through circular dichroism spectrum and infrared spectrum technologies; the method comprises the following steps: combining an AIE probe with a bionic AR-DBD / LBD; confirming the successful combination of the probe and the protein by using a two-photon laser confocal microscope; to establish a sensor response to detect different bisphenol analogue (BPs) concentrations; the sensor is applied to the soil matrix effect, and the performance of the sensor in the actual environment is evaluated; and verifying the reliability of sensor data through statistical analysis. According to the present invention, the optimized androgen receptor protein is combined with the AIE probe to construct the high-sensitivity sensor system so as to effectively monitor and evaluate the BPs pollution level in the environment.

Description

Technical Field

[0001] The present invention belongs to the fields of biochemical detection technology and food science technology, and relates to the preparation and application of an AIE multi-residue sensing system based on a bionic AR-DBD / LBD; specifically, it relates to the development of a sensor system based on a bionic androgen receptor and an AIE probe and its application in food safety and environmental pollution detection. Background Art

[0002] With the rapid development of industrialization and the widespread abuse of chemical materials, a large number of environmental endocrine disruptors (EDCs) will be enriched into the human body through the food chain, thus affecting physical health. Therefore, the rapid quantitative detection of endocrine disruptors (EDCs) in the environment has become a key issue that needs to be solved urgently. At present, the detection methods for these EDCs mainly include high performance liquid chromatography, gas chromatography and electrochemical methods. Although these methods have high sensitivity, they are time-consuming and require complex sample pretreatment techniques. Therefore, it is urgent to develop a BPs detection method with high sensitivity, strong specificity, simple operation and suitable for high-throughput detection to evaluate the EDCs pollution in the environment and food. Summary of the invention

[0003] In view of the above problems, the purpose of the present invention is to solve the problem that the detection methods of environmental endocrine disruptors in the prior art are insufficient, and to provide a high-sensitivity sensor system based on optimized androgen receptor protein and AIE probe.

[0004] The technical solution of the present invention is: an AIE multi-residue sensing system based on a bionic AR-DBD / LBD according to the present invention, the sensor system comprises a bionic AR-DBD / LBD optimized by ABACUS and PROSS methods, an AIE probe and a PBS buffer solution;

[0005] Wherein, the concentration of the bionic AR-DBD / LBD is 0.5 μM, and the concentration of the AIE probe is 7.5 μM.

[0006] Furthermore, a method for preparing and applying an AIE multi-residue sensing system based on a bionic AR-DBD / LBD is provided, and the construction steps are as follows:

[0007] Step (1): using gene recombination technology to recombinantly transfer the biomimetic AR-DBD / LBD into Escherichia coli, and obtaining the biomimetic AR-DBD / LBD by inducing expression;

[0008] Step (2): characterizing the structure of the biomimetic AR-DBD / LBD by circular dichroism and infrared spectroscopy;

[0009] Step (3): combining the AIE probe with the biomimetic AR-DBD / LBD, optimizing the concentration ratio of the AIE probe and the biomimetic AR-DBD / LBD, and achieving effective detection;

[0010] Step (4): using two-photon laser confocal microscopy to confirm the successful binding of the AIE probe to the biomimetic AR-DBD / LBD;

[0011] Step (5): performing concentration gradient analysis to establish the sensor response for detecting different BPs concentrations;

[0012] Step (6): Apply this sensor to the soil matrix effect and evaluate its performance in the actual environment;

[0013] Step (7): Verify the reliability of sensor data through statistical analysis to ensure its scientific validity.

[0014] Furthermore, in step (1), the specific process of obtaining the bionic AR-DBD / LBD is: optimizing the amino acid sequence of the wild-type AR-DBD / LBD by using the ABACUS and PROSS methods to enhance its expression efficiency and solubility in Escherichia coli, and synthesizing the optimized bionic AR-DBD / LBD sequence by gene synthesis and cloning it into an expression vector;

[0015] The recombinant expression vector was then transformed into Escherichia coli, and the expression of bionic AR-DBD / LBD was induced by isopropyl-β-D-thiogalactoside (IPTG). The cells were disrupted by ultrasound, and the bionic AR-DBD / LBD was obtained by dialysis and centrifugation.

[0016] Furthermore, in step (1), the induction concentration of isopropyl-β-D-thiogalactoside (IPTG) used in the expression of the bionic AR-DBD / LBD is 0.5 mM, and ultrasonic disruption is performed after expression to extract soluble bionic AR-DBD / LBD.

[0017] Furthermore, in step (2), the analysis of the circular dichroism spectrum and the infrared spectrum is used to confirm the α-helix and β-sheet content of the biomimetic AR-DBD / LBD, thereby ensuring the activity and structural integrity of the biomimetic AR-DBD / LBD.

[0018] Furthermore, in step (3), the optimized AIE probe and biomimetic AR-DBD / LBD mixture is used to detect BPs concentrations ranging from 0.5 μM to 50 μM, and incubated, and its fluorescence response is detected by a spectrometer to ensure the binding of the AIE probe to the biomimetic AR-DBD / LBD.

[0019] Furthermore, in step (4), the process of confirming the successful binding of the AIE probe to the biomimetic AR-DBD / LBD using a two-photon laser confocal microscope is:

[0020] The complex formed by the combination of the AIE probe and the biomimetic AR-DBD / LBD was imaged and analyzed under a laser confocal microscope to visually confirm the binding of the AIE probe and the biomimetic AR-DBD / LBD.

[0021] 0.5 μM biomimetic AR-DBD / LBD was dissolved in PBS buffer solution, mixed with 7.5 μM AIE probe, and incubated at room temperature for 20 min to form the sensor complex;

[0022] The complex sample was placed under a laser confocal microscope to observe its binding status and confirm the successful binding of the AIE probe to the biomimetic AR-DBD / LBD.

[0023] Furthermore, in step (5), the establishment of the response curve and sensitivity of the sensor to BPs is specifically:

[0024] In the configured sensor system, BPs standard solutions with concentrations ranging from 0.5 μM to 50 μM were introduced, and their fluorescence intensity was measured at an excitation wavelength of 360 nm to evaluate its detection sensitivity to BPs.

[0025] Furthermore, in step (6), the process of detecting the matrix effect of the soil-affected sensor by BPs analysis is: introducing a soil matrix into the sensor system, adding a BPs standard solution with a concentration of 0.5 μM to 50 μM for detection to evaluate the performance and accuracy of the sensor in the soil matrix.

[0026] Furthermore, the step (7) specifically includes: performing a statistical analysis of the spiked recovery rate by adding a standard BPs solution to the soil sample and then using the sensor system for detection to calculate its recovery rate, so as to verify the accuracy and reliability of the sensor data and ensure its application in environmental monitoring and risk assessment.

[0027] The beneficial effect of the present invention is that by combining the optimized androgen receptor protein with the AIE probe, a highly sensitive sensor system is constructed to effectively monitor and evaluate the pollution level of BPs in the environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the prediction of the solubility of wild-type and biomimetic AR-DBD / LBD by CamSol Intrinsic in the embodiment of the present invention;

[0029] Figure 2Schematic diagram of the analysis of the wild-type and biomimetic AR-DBD / LBD Ramachandran plots in the embodiments of the present invention;

[0030] Figure 3 Schematic diagram of electrostatic potential analysis of wild-type and biomimetic AR-DBD / LBD in an embodiment of the present invention;

[0031] Figure 4 Schematic diagram of the binding site analysis between pollutants and biomimetic AR-DBD / LBD in an embodiment of the present invention;

[0032] Figure 5 is a heat map of the binding affinity between pollutants and biomimetic AR-DBD / LBD in the embodiments of the present invention;

[0033] Figure 6 Schematic diagram of expression and Western blot analysis of biomimetic AR-DBD / LBD in an embodiment of the present invention;

[0034] Figure 7 Schematic diagram of the circular dichroic spectrum and Fourier transform infrared spectrum of the bionic AR-DBD / LBD in the embodiment of the present invention;

[0035] Figure 8 1 is a comparison diagram of the AIE probe screening (A) and the fluorescence analysis of the biomimetic AR-DBD / LBD combined with the AIE probe in the embodiment of the present invention;

[0036] Fig. 9 is an optimized schematic diagram of a sensor system in an embodiment of the present invention;

[0037] Fig.10 is a schematic diagram of the competition binding curve of BPs in an embodiment of the present invention;

[0038] Fig.11 is a comparison diagram of the influence of soil matrix effect on the sensor in the embodiment of the present invention;

[0039] Fig.12 It is an operation flow chart of the present invention. DETAILED DESCRIPTION

[0040] The specific technical solutions of the present invention are further described in detail below in conjunction with specific examples.

[0041] As shown in the figure, the present invention proposes a novel sensor system (AIE multi-residue sensing system based on biomimetic AR-DBD / LBD), the main components of which are AR-DBD / LBD optimized by ABACUS and PROSS methods, AIE probe and PBS buffer solution;

[0042] The concentrations of the proteins and AIE probes in the sensor system are as follows: the concentration of AR-DBD / LBD is 0.5 μM, and the concentration of the AIE probe is 7.5 μM.

[0043] For this sensor system, it discloses a construction method thereof, comprising the following steps:

[0044] (1) The amino acid sequence of AR-DBD / LBD was optimized using the ABACUS and PROSS methods to enhance its expression efficiency and solubility in Escherichia coli. The optimized sequence was synthesized and cloned into an expression vector;

[0045] (2) Transforming the recombinant expression vector into Escherichia coli, inducing the expression of the target protein by IPTG, disrupting the cells by ultrasound, and obtaining the target protein by dialysis and centrifugation;

[0046] (3) Mix the purified protein and the AIE probe in a preset ratio, incubate in a suitable buffer solution, and detect the fluorescence response using a spectrometer to ensure effective binding of the probe to the protein;

[0047] (4) Perform imaging analysis of the combined complex under a laser confocal microscope to visually confirm the binding of the AIE probe to the receptor protein;

[0048] (5) 0.5 μM of the optimized androgen receptor protein DBD / LBD was dissolved in PBS buffer solution, mixed with 7.5 μM of the AIE probe, and incubated at room temperature for 20 min to form a sensor complex;

[0049] (6) Place the complex sample under a laser confocal microscope to observe its binding status and confirm the successful binding of the probe to the receptor protein;

[0050] (7) Into the configured sensor system, different concentrations of BPs standard solutions (0.5 μM to 50 μM) were introduced, and their fluorescence intensity was measured at an excitation wavelength of 360 nm to evaluate its detection sensitivity to BPs;

[0051] (8) Introducing soil matrix into the sensor system, adding different concentrations of BPs standard solution (0.5 μM to 50 μM) for detection, and observing the influence of soil matrix effect on sensor sensitivity;

[0052] (9) A spike recovery experiment was conducted by adding a standard BPs solution to the soil sample and then using the sensor system of the present invention for detection, thereby calculating its recovery rate (between 95% and 105%) to verify the accuracy and applicability of the sensor.

[0053] Example

[0054] (1) Homology modeling and solubility design of AR-DBD / LBD wild-type protein sequence:

[0055] (1) Homology modeling and solubility design of wild-type AR-DBD / LBD sequence:

[0056] The wild-type AR-DBD / LBD amino acid sequence was obtained by searching for Androgen receptor in NCBI (https: / / www.ncbi.nlm.nih.gov / ) with Gene as the category and Homo sapiens as the species. The wild-type AR-DBD / LBD amino acid sequence was searched for as a template in the Swiss model (https: / / swissmodel.expasy.org / ) with Androgen receptor as the species. receptor(Q6QT55.1.A) was used as a template for homology modeling to obtain the PDB structure file of the wild-type AR-DBD / LBD; PROSS (https: / / pross.weizmann.ac.il / step / pross-terms / ) and ABACUS (http: / / biocomp.ustc.edu.cn / servers / software.php) were used to design the wild-type AR-DBD / LBD: for PROSS, the PDB file of the initial wild-type AR-DBD / LBD was uploaded to PROSS, and the design was performed using the default parameter settings of the website; for ABACUS, the PDB file of the wild-type AR-DBD / LBD was uploaded to ABACUS, and the PDB structure of the wild-type AR-DBD / LBD was used as the main chain. The consistency between the sequences was set to 0.5, and the consistency with the input sequence was set to 0.75 for design, and the designed AR-DBD / LBD sequence and PDB structure file were obtained;

[0057] Subsequently, the designed biomimetic AR-DBD / LBD and wild-type AR-DBD / LBD sequences were subjected to homology comparison analysis by ESPript 3.0 (https: / / espript.ibcp.fr / ESPript / cgi-bin / ESPript.cgi) to evaluate the sequence conservation and mutation changes before and after the design;

[0058] (2) Analysis of the physical and chemical properties of wild-type and biomimetic AR-DBD / LBD:

[0059] First, the solubility of wild-type and biomimetic AR-DBD / LBD was predicted: the wild-type and biomimetic AR-DBD / LBD sequences were used to predict their corresponding solubility through CamSol Intrinsic; secondly, ProtParam (https: / / web.expasy.org / protparam / ) was used to calculate their instability index, molecular weight, theoretical isoelectric point, amino acid composition, etc.

[0060] (3) Comparison of the Ramachandran plots, surface electrostatic potential, solvent accessible surface area and homology of wild-type and biomimetic AR-DBD / LBD:

[0061] The wild-type and biomimetic AR-DBD / LBD were constructed into Ramachandran plots on UCLA-DOE LAB (https: / / saves.mbi.ucla.edu / ) to analyze the rationality of their protein amino acid residues. The electrostatic potential surface and solvent accessible surface area (SASA) were calculated in Pymol to further explore the main factors for solubility enhancement.

[0062] (4) Affinity analysis of wild-type and biomimetic AR-DBD / LBD for androgen disruptors:

[0063] 43 androgen disruptors including Testosterone, TBECH, and BPA were selected to analyze the changes in the binding ability of AR-DBD / LBD before and after the design;

[0064] First, the 2D structure SDF file of the interferent was downloaded from the PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ) database. The 2D file of the interferent was converted into a 3D structure using Chem3D software, and its structure was optimized to the lowest energy, and finally output as the mol2 file of the corresponding interferent. The PDB files of the wild-type and biomimetic AR-DBD / LBD were dehydrated and hydrogenated using AutoDockTools-1.5.7 and set as binding receptors. At the same time, the interferent was set as a binding ligand after hydrogenation, and finally molecular docking was performed using AutoDock Vina.

[0065] (5) Expression of biomimetic AR-DBD / LBD:

[0066] According to the comparative analysis in the above chapters, the optimally designed biomimetic AR-DBD / LBD was selected for recombinant expression experiments; the expression steps are as follows:

[0067] 1. After the sterilized solid culture medium has cooled, add Amp antibiotic (final concentration 100ug / mL), pour into a plate and cool to solidify (liquid: 1mL; solid: 50uL);

[0068] 2. Take out the glycerol-preserved bacteria from the -80℃ refrigerator, burn the inoculation loop with an alcohol lamp, pick out the bacteria and spread them on the surface of the solid culture medium on the plate, invert and culture overnight;

[0069] 3. The next day, use a sterilized pipette tip to pick up a single colony from the plate, put it into 10 mL of liquid culture medium (containing 100 ug / mL of Amp antibiotic), place it in a 37°C constant temperature shaker at 220 rpm, and culture until the OD value reaches 0.6;

[0070] 4. According to the ratio of 1:100, pipette 1mL of culture solution with OD value of 0.6 into 100mL of liquid culture medium containing 100ug / mL of Amp antibiotic, and place it in a constant temperature shaker at 37℃ and the speed is 220rpm;

[0071] 5. When the OD value is 0.6-0.8, take out 1 mL of culture, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the bacterial pellet with 80 uL PBS and 20 uL 5× loading buffer; add IPTG with a final concentration of 1 mM to the remaining culture, and adjust the shaker temperature to 15°C and the speed to 220 rpm to induce the expression of the fusion protein;

[0072] 6. After culturing for 12 hours, take 1 mL of the culture into a centrifuge tube, centrifuge at 8000 rpm for 10 minutes, discard the supernatant, and resuspend the bacterial pellet with 80 uL PBS and 20 uL 5× loading buffer;

[0073] In order to extract the biomimetic AR-DBD / LBD, a cell disruptor was used to ultrasonically lyse the E. coli induced for 12 hours; considering that the solubility of the protein is easily affected by the composition of the lysis buffer, the present invention conducted a detailed evaluation of its solubility under various conditions;

[0074] The specific steps are as follows: First, the cell pellet was treated by centrifugation at 8000rpm for 10 minutes, and then resuspended in different 30mL buffer systems; the cell suspension was treated with 350W ultrasound for 10 minutes, with an interval of switching on and off every 5 seconds; after ultrasound treatment, the sample was centrifuged at 8000rpm for 10 minutes at 4°C to separate insoluble fragments, and the supernatant, i.e., the soluble part, was collected; the obtained soluble and insoluble components were subjected to SDS-PAGE analysis to evaluate the protein extraction effect; the quantitative analysis of protein concentration used an enhanced BCA protein assay kit, and BSA was used as a standard sample to determine the total protein concentration in the sample according to the standard curve;

[0075] (6) Western blot

[0076] In order to verify the bionic AR-DBD / LBD, Western blot technology was used to further analyze the bionic AR-DBD / LBD; the Western blot steps are as follows:

[0077] 1. After the transfer is completed, wash the membrane twice with 1X PBS according to the normal washing steps, 5 minutes each time;

[0078] 2. While washing the membrane, prepare the blocking solution and antibody incubation solution: take 5 ml of blocking solution A and 5 ml of blocking solution B, mix them in a centrifuge tube, and vortex to form a uniform suspension, which is the blocking solution C; take another new centrifuge tube, add 5 μl of antibody reaction solution HRP (rabbit) and 5-10 μg of rabbit primary antibody, mix well, let stand for about 5 minutes, and then add 10 ml of antibody diluent and mix well, which is the antibody incubation solution;

[0079] 3. After washing the membrane with 1X PBS in step 1, pour out the washing solution and place the membrane in a container containing the mixed blocking solution C, so that the blocking solution completely immerses the membrane surface, and incubate at room temperature on a shaker at an appropriate speed for about 5 minutes;

[0080] 4. Take the membrane out of blocking solution C, immerse it in distilled water, and rinse it twice on a shaker at a high speed, each time for 1 min;

[0081] 5. Add the mixed antibody incubation solution to the membrane and completely immerse the membrane surface. Incubate on a shaker at an appropriate speed at room temperature for about 40 minutes. At the same time, take 5 ml of 10X washing solution and dilute it to 50 ml with distilled water for later use;

[0082] 6. Recover the antibody incubation solution, rinse the membrane in distilled water for 30 seconds, then rinse with 50 ml of 1X washing solution for 3 minutes, and then rinse with distilled water for 30 seconds;

[0083] 7. Incubate with ECL luminescent reagent at room temperature for 20 minutes, and observe the color development using the 1600 gel imaging analysis system;

[0084] (7) Circular dichroism and Fourier transform infrared characterization:

[0085] Fourier transform infrared: The functional groups of biomimetic AR-DBD / LBD were determined by Fourier transform infrared spectrometer; the specific experimental conditions were as follows: the biomimetic AR-DBD / LBD sample was freeze-dried for 72 hours in advance, and the infrared spectrum of biomimetic AR-DBD / LBD was recorded by reflection method with a wave number range of 4000-400cm-1;

[0086] Circular dichroism: The circular dichroism spectrum of the biomimetic AR-DBD / LBD was measured at a wavelength of 190-250 nm at 25°C; the scanning speed was 60 nm / min, the response time was 1 s, and the bandwidth was 1 nm;

[0087] (8) Screening of AIE probes and establishment of sensing system:

[0088] Sixteen AIE probes were selected for screening in the experiment; the screening process was as follows: Auto Dock Vina was used to perform molecular docking of the AIE probe and the bionic AR-DBD / LBD, the results were visualized by Pymol, and the AIE probe whose docking site was at the bionic AR-DBD / LBD ligand binding site was selected as the preliminary probe screening; the qualified AIE probes were mixed with the bionic AR-DBD / LBD in a ratio of 1:100, and the fluorescence intensity under the corresponding excitation conditions was measured; the blank control was the fluorescence intensity of AIE in PBS buffer; similarly, the ultraviolet absorption spectrum under the corresponding conditions was measured; the probe concentration was fixed at 7.5 μM, and a series of bionic AR-DBD / LBD solutions in the concentration range of 0-5 mM were prepared, and the fluorescence spectrum with an excitation wavelength of 360 nm and an emission wavelength of 400-650 nm was scanned; GraphPad Prism was used to analyze the fluorescence spectrum. 8 software, using a specific binding model to nonlinearly fit the saturation binding analysis data, thereby calculating the dissociation equilibrium constant (Kd) between the biomimetic AR-DBD / LBD and the AIE probe; the protein concentration corresponding to 50% binding time was selected as the optimal concentration of the biomimetic AR-DBD / LBD;

[0089] (9) Laser confocal:

[0090] The fluorescence of AIE probe and biomimetic AR-DBD / LBD-AIE complex was observed by LSM 880NLO two-photon laser confocal microscope. In short, AIE and biomimetic AR-DBD / LBD-AIE solutions were dropped on a glass slide and covered with a coverslip. Before observation, essential oil was added to the coverslip and the sample was observed using the oil objective of the laser confocal microscope.

[0091] (10) Competitive binding:

[0092] The experiment selected the AR antagonist flutamide, bisphenol pollutants TBBPA, BPF and BPS as the detection substances for competitive binding; in order to prevent the bionic AR-DBD / LBD from directly binding to the analyte, the bionic AR-DBD / LBD after concentration optimization was mixed with the AIE probe and incubated at room temperature for 20 minutes to construct a sensor system; then the analyte with a concentration of 0-50mM was added, mixed and incubated for 10 minutes, and the fluorescence spectrum with an excitation wavelength of 360nm and an emission wavelength of 400-650nm was scanned; the concentration of the analyte was used as the horizontal axis and the Y value was used as the vertical axis, and the competitive binding curve was fitted by the formula in the PraphPad software; the Y value calculation equation is:

[0093] Y = (F0-F) / F0 (1)

[0094] Wherein, F0 is the fluorescence intensity before adding the analyte; F is the fluorescence intensity after adding the analyte;

[0095] (11) Matrix effect:

[0096] Soil was selected as a representative matrix to evaluate the matrix effect of the sensor; the sample was processed as follows: the soil was evenly spread on a flat plate and sealed with plastic wrap. Then it was placed in an oven and dried at 60°C for 18 hours; DMSO was used for ultrasonic extraction in an ultrasonic water bath, the extraction time was 2 hours, and the ultrasonic frequency was 50W; the supernatant was taken as the matrix solution by centrifugation at 8000r / min; the matrix solution was mixed with the above sensor system in a ratio of 1:1, and the detection substance with a concentration of 0-50mM was added, mixed and incubated for 10 minutes, and the fluorescence spectrum with an excitation wavelength of 360nm and an emission wavelength of 400-650nm was scanned; the concentration of the test substance was used as the horizontal axis and the Y value was used as the vertical axis, and the competition binding curve was fitted by the formula in the PraphPad software;

[0097] (12) Add recovery experiment:

[0098] Soil was used as the test object, representative BPs were selected, and the recovery rate of addition was determined. The concentrations of BPs added were 1, 3, and 5 μM. The addition amount and recovery rate were calculated through competitive binding curve.

[0099] The present invention proposes an AIE multi-residue sensing system based on bionic AR-DBD / LBD; through protein engineering technology, the wild-type AR-DBD / LBD is sequence optimized and designed using ABACUS and PROSS protein design methods to improve its expression efficiency and solubility in the Escherichia coli prokaryotic expression system. At the same time, combined with the aggregation-induced emission characteristics of the AIE probe, under the interaction between the bionic AR-DBD / LBD and the AIE probe, a highly sensitive and specific EDCs sensor platform is constructed through a competitive binding mechanism. Compared with existing detection methods, the present invention not only has significant improvements in sensitivity and specificity, but also is easy to operate and suitable for high-throughput detection, and has important application value in food safety and environmental pollution detection.

Claims

1. A biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system. It is characterized in that It includes biomimetic AR-DBD / LBD, AIE probe and PBS buffer solution optimized by ABACUS and PROSS methods; Wherein, the concentration of the bionic AR-DBD / LBD is 0.5 μM, and the concentration of the AIE probe is 7.5 μM.

2. A method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 1, It is characterized in that The construction steps are as follows: Step (1): using gene recombination technology to recombinantly transfer the biomimetic AR-DBD / LBD into Escherichia coli, and obtaining the biomimetic AR-DBD / LBD by inducing expression; Step (2): characterizing the structure of the biomimetic AR-DBD / LBD by circular dichroism and infrared spectroscopy; Step (3): combining the AIE probe with the biomimetic AR-DBD / LBD, optimizing the concentration ratio of the AIE probe and the biomimetic AR-DBD / LBD, and realizing detection; Step (4): using two-photon laser confocal microscopy to confirm the successful binding of the AIE probe to the biomimetic AR-DBD / LBD; Step (5): performing concentration gradient analysis to establish the sensor response for detecting different BPs concentrations; Step (6): Apply this sensor to the soil matrix effect and evaluate its performance in the actual environment; Step (7): Verify the reliability of sensor data through statistical analysis to ensure its scientific validity.

3. A method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (1), the specific process of obtaining the bionic AR-DBD / LBD is: optimizing the amino acid sequence of the wild-type AR-DBD / LBD by using the ABACUS and PROSS methods to enhance its expression efficiency and solubility in Escherichia coli, and the optimized bionic AR-DBD / LBD sequence is synthesized by gene synthesis and cloned into an expression vector; The recombinant expression vector was then transformed into Escherichia coli, and the bionic AR-DBD / LBD was expressed by inducing IPTG, and the cells were disrupted by ultrasound, and the bionic AR-DBD / LBD was obtained by dialysis and centrifugation.

4. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (1), the IPTG induction concentration used in the expression of the bionic AR-DBD / LBD is 0.5 mM, and after expression, ultrasonic disruption is performed to extract soluble bionic AR-DBD / LBD.

5. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (2), the analysis of the circular dichroism spectrum and the infrared spectrum is used to confirm the α-helix and β-sheet content of the biomimetic AR-DBD / LBD, thereby ensuring the activity and structural integrity of the biomimetic AR-DBD / LBD.

6. A method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (3), the optimized AIE probe and biomimetic AR-DBD / LBD mixture is used to detect BPs concentrations ranging from 0.5 μM to 50 μM, and incubated, and its fluorescence response is detected by a spectrometer to ensure the binding of the AIE probe to the biomimetic AR-DBD / LBD.

7. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (4), the process of confirming the successful binding of the AIE probe to the biomimetic AR-DBD / LBD using a two-photon laser confocal microscope is: The complex formed by the combination of the AIE probe and the biomimetic AR-DBD / LBD was imaged and analyzed under a laser confocal microscope to visually confirm the binding of the AIE probe and the biomimetic AR-DBD / LBD. 0.5 μM biomimetic AR-DBD / LBD was dissolved in PBS buffer solution, mixed with 7.5 μM AIE probe, and incubated at room temperature for 20 min to form the sensor complex; The complex sample was placed under a laser confocal microscope to observe its binding status and confirm the successful binding of the AIE probe to the biomimetic AR-DBD / LBD.

8. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (5), the establishment of the response curve and sensitivity of the sensor to BPs is specifically: In the configured sensor system, BPs standard solutions with concentrations ranging from 0.5 μM to 50 μM were introduced, and their fluorescence intensity was measured at an excitation wavelength of 360 nm to evaluate its detection sensitivity to BPs.

9. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that In step (6), the process of detecting the matrix effect of the soil-affected sensor by BPs analysis is: introducing a soil matrix into the sensor system, adding a BPs standard solution with a concentration of 0.5 μM to 50 μM for detection to evaluate the performance and accuracy of the sensor in the soil matrix.

10. The method for constructing a biomimetic AR-DBD / LBD-based aggregation-induced emission multi-residue sensing system according to claim 2, It is characterized in that The step (7) specifically includes: performing a statistical analysis of the spiked recovery rate by adding a standard BPs solution to the soil sample and then using the sensor system for detection to calculate its recovery rate, so as to verify the accuracy and reliability of the sensor data and ensure its application in environmental monitoring and risk assessment.

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