Multi-layer core-shell AChE (at) PILs (at) Ag NPs (at) PDEA biosensor with SERS (Surface Enhanced Raman Scattering) amplification function and method for ultrasensitively detecting organophosphorus pesticide by using biosensor

Abstract

The invention provides a multi-layer core-shell AChE (at) PILs (at) Ag NPs (at) PDEA biosensor with an SERS (Surface Enhanced Raman Scattering) amplification function and a method for ultrasensitively detecting organophosphorus pesticides by using the biosensor. Acetylcholinesterase (AChE) is packaged in polymer ionic liquid (PILs) microspheres, silver nanoparticles (Ag NPs) are orderly assembled by utilizing the positive charge characteristic of the PILs to construct a nano array coupling local electromagnetic field, and a surface enhanced Raman scattering (SERS) signal is synergistically enhanced. 2-(pyridyldithio) ethylamine (PDEA) is modified on the surface of the composite structure: when AChE catalyzes acetylthiocholine (ATCL) to generate thiocholine (TCl), TCl cracks a disulfide bond in PDEA to release a pyridine ring, and SERS signal attenuation is initiated; and after the organophosphorus pesticide inhibits the enzyme activity, the generation of TCl is reduced, the signal attenuation degree is reduced, and indirect quantitative analysis of the pesticide is realized. The method has a wide linear range and a low detection limit, has excellent thermal stability, storage stability and anti-interference capability, and provides a novel high-reliability detection platform for on-site rapid analysis of agricultural product safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of food analysis, and in particular relates to a multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor with SERS amplification function and a method for ultrasensitively detecting organophosphorus pesticides using the same. Background Art

[0002] To meet growing consumer demand, nutritional requirements, and industrial processing needs, the global production and cultivation of fresh fruits and vegetables has surged. To maximize yields and extend shelf life, large quantities of pesticides, particularly organophosphorus pesticides (OPs), are used to control pests and diseases. However, the overuse of OPs has caused significant environmental and health problems. Environmental leakage of pesticide residues can persist in ecosystems, disrupting their balance, while human exposure to pesticides through contaminated food can cause neurotoxicity, liver and kidney damage, respiratory failure, and even cancer or infertility.

[0003] Currently, pesticide residue analysis relies primarily on traditional chromatographic and spectroscopic techniques, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and mass spectrometry (MS). While these methods are considered the gold standard for their accuracy, they also have inherent limitations: complex sample pretreatment, reliance on expensive instrumentation, and time-consuming procedures, which hinder their application in rapid on-site detection. Therefore, there is an urgent need to develop simple, rapid, and highly sensitive alternative analytical methods.

[0004] Surface-enhanced Raman spectroscopy (SERS) has emerged as a promising solution due to its exceptional sensitivity, molecular specificity, and non-destructive real-time analytical capabilities. By leveraging unique "fingerprint" spectral signatures, SERS can enable trace detection of analytes in fields such as food safety and environmental monitoring. However, direct SERS-based detection of organophosphorus pesticides (OPs) faces significant challenges. The structural diversity of OPs and spectral overlap in complex matrices complicate unambiguous identification and quantification. Furthermore, the reproducibility of SERS signals depends largely on the molecular orientation and substrate homogeneity, further limiting its practical application in heterogeneous pesticide mixtures.

[0005] To circumvent these limitations, enzyme inhibition strategies have garnered increasing attention. The toxicity of organophosphorus pesticides has been shown to inhibit the activity of acetylcholinesterase (AChE), a key enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine (ACh). This mechanism provides a versatile detection principle: the degree of inhibition of AChE activity is directly correlated with OP concentration, thereby bypassing the need for direct molecular recognition while ensuring specificity. However, enzymes like AChE require a mild immobilization environment to maintain catalytic function. Polymeric ionic liquids (PILs) are ideal immobilization matrices due to their biocompatibility, structural tunability, and stability. PILs not only provide a protective microenvironment for enzymes but can also be synergistically integrated with nanomaterials (such as silver powder) to enhance catalytic efficiency and SERS signal amplification. This unique combination makes PIL-based biosensors a transformative platform for rapid, sensitive, and field-deployable pesticide detection.

[0006] In this invention, we took advantage of the multifunctional properties of PILs to design and synthesize a sandwich-like core-shell structure composite material (AChE@PILs@Ag NPs). AChE is encapsulated in PILs to form the core, PILs serve as the middle layer, and Ag NPs are uniformly assembled on the outer layer to provide a surface enhancement effect. Traditional SERS substrates often cannot produce consistent "hot spots" due to the limited scattering cross-section of single Ag NPs and insufficient electromagnetic enhancement effect. The AChE@PILs@Ag NPs substrate, based on the adjustable repeating unit charge density and ordered structural framework of PILs, can assemble Ag NPs into uniformly distributed nanoscale arrays, thereby effectively coupling the local electromagnetic field and significantly enhancing the SERS amplification effect. In addition, in order to enhance the specificity of sensing, 2-(pyridyldithio)ethylamine (PDEA) - a multifunctional molecule containing amino groups, disulfide bonds and pyridine rings - was grafted on the surface of the composite material. The amino group promotes the assembly of PDEA on AChE@PILs@Ag NPs@PDEA through Ag-N coordination, while the pyridine ring serves as a strong Raman reporter, generating a high SERS response. Importantly, the disulfide bond is a dynamic recognition site: acetylcholinesterase (AChE) hydrolyzes acetylthiocholine (ATCL) to form thiocholine (TCl), triggering a thiol-disulfide exchange reaction with PDEA. This reaction releases the pyridine ring from the surface, weakening the SERS signal. Conversely, organophosphorus pesticides (OPs) inhibit AChE activity and TCl production, preserving the integrity of the disulfide-pyridine ring of PDEA and maintaining a strong SERS signal. By correlating the SERS signal attenuation with OP concentration, this surface reaction-based strategy enables rapid, sensitive, and indirect quantification of OPs. Furthermore, AChE@PILs@Ag NPs@PDEA exhibits high stability, including long-term and thermal stability. Summary of the Invention

[0007] This paper designs a highly stable pesticide detection composite material based on a multilayer core-shell composite structure (AChE@PILs@Ag NPs@PDEA), and characterizes its structure, detection mechanism, and SERS performance. Specifically:

[0008] (1) The present invention uses emulsion polymerization to encapsulate acetylcholinesterase (AChE) within polyionic liquid (PILs) microspheres, constructing an AChE@PILs@Ag NPs@PDEA multilayer core-shell composite structure. PILs achieve uniform nanoscale arrays of silver nanoparticles (Ag NPs) through tunable repeating unit charge density and an ordered framework, significantly enhancing the local electromagnetic field coupling effect and SERS signal stability.

[0009] (2) The present invention utilizes the positive charge characteristics of PILs to fix negatively charged Ag NPs through electrostatic interaction to enhance the catalytic performance, while providing a biocompatible microenvironment for the enzyme, effectively preventing enzyme leakage and maintaining its activity, and synergistically improving the catalytic performance of the composite structure.

[0010] (3) The detection mechanism of the present invention is based on the specific reaction between the disulfide bond in 2-(pyridyldithio)ethylamine (PDEA) and thiocholine (TCl): when AChE catalyzes the hydrolysis of acetylthiocholine (ATCl) to produce TCl, TCl triggers PDEA to release the pyridine ring, resulting in a decrease in the SERS signal; when organophosphorus pesticides inhibit AChE activity, ATCl hydrolysis is hindered, TCl generation is reduced, and the SERS signal change is suppressed. The change in SERS signal intensity is inversely proportional to the pesticide concentration, thereby realizing indirect quantitative detection of pesticides.

[0011] (4) The synergistic enhancement effect of PILs@Ag NPs was utilized to enhance the SERS sensitivity through electromagnetic field coupling of Ag NPs array; PDEA was used as a signal control unit to achieve quantitative response of SERS signal through dynamic reaction of thiol-disulfide bond. -12 ~3.8×10 -7 It shows excellent linear response in the M range and the detection limit is as low as 1.38×10 -13 M. It has high thermal stability (activity retention of 92% after 7 days at 60°C) and long-term storage stability (signal attenuation <5% after 30 days), and maintains high selectivity (interference response <8.2%) and reproducibility (RSD = 3.7%) in complex matrices such as tomatoes and tap water.

[0012] (5) The present invention achieves rapid on-site detection of organophosphorus pesticides in agricultural products through the synergistic effect of charge-driven assembly and precise structural control. It can be directly used for residue analysis on the surface of fruits and vegetables under a wide range of climatic conditions, and the actual sample spike recovery rate reaches 96.5-103.2%.

[0013] The present invention constructs a highly stable biosensor based on the multilayer core-shell structure AChE@PILs@Ag NPs@PDEA. Acetylcholinesterase (AChE) is encapsulated in polyionic liquid (PILs) microspheres by emulsion polymerization. The positive charge characteristics of PILs are utilized to immobilize Ag nanoparticles (Ag NPs) to form a uniform nanoarray, enhancing local electromagnetic field coupling and SERS signals. The surface-modified 2-(pyridyldithio)ethylamine (PDEA) achieves pesticide response through a thiocholine-mediated molecular replacement reaction: when organophosphorus pesticides inhibit AChE activity, thiocholine production decreases, resulting in a decrease in the release of pyridine rings on the PDEA surface and a weakening of the SERS signal. The sensor has a detection limit of 1.38×10 -13 M, showing excellent selectivity and reproducibility in complex matrices such as tomato and tap water.

[0014] The technical solution adopted in the present invention is:

[0015] A multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, the preparation method of which comprises the following steps:

[0016] 1) Acetylcholinesterase (AChE) was encapsulated into polymeric ionic liquid (PILs) microspheres by emulsion polymerization to form the core;

[0017] 2) PILs are used as an intermediate layer to uniformly load silver nanoparticles (Ag NPs) on their surface to construct an isotropic composite material;

[0018] 3) The surface of the composite material was modified with 2-(pyridyldithio)ethylamine PDEA containing a pyridine ring and a disulfide bond.

[0019] Furthermore, in the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, in step 1), the ionic liquid is 1-vinyl-3-ethylimidazolium bromide ViEtIm + Br - .

[0020] Furthermore, in the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, the specific operation steps of step 1) are: preparing 1-vinyl-3-ethylimidazolium bromide poly (ViEtIm + Br -) microspheres; the dispersed aqueous phase contains 0.125g monomer ViEtIm + Br - , 0.8-2.4U acetylcholinesterase AChE, 0.004-0.01g cross-linker N,N′-methylenebisacrylamide and 0.0025g initiator ammonium persulfate, with 20mM Tris-HCl as dispersant; the oil phase consists of 75μL dodecane and 25μL Span 80; the dispersed aqueous phase is slowly added dropwise to the oil phase to form an emulsion, after deoxygenation by nitrogen, 10μL TEMED is added to initiate the polymerization reaction. After the reaction is completed, the precipitated polymer is washed three times with acetone and PBS in sequence and centrifuged at 4000rpm for 10 minutes, and then freeze-dried to obtain AChE@PILs composite microgel.

[0021] Furthermore, in the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, the specific operation steps of step 2) are as follows: take 8 mg of AChE@PILs composite microgel in a centrifuge tube, add 5 mL of Ag NPs solution, and ultrasonicate for 30 minutes until the color of the solution changes from yellow-green to brown. Centrifuge at 4000 rpm for 10 minutes to separate the organic-inorganic composite material AChE@PILs@Ag NPs.

[0022] Furthermore, in the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, the step 3) is to modify PDEA on the material surface by N-Ag bond self-assembly method, and the specific operation steps are as follows: 200 μL 10 -2 The M PDEA solution was added dropwise onto the surface of AChE@PILs@Ag NPs. After incubation for 2 h, the nonspecifically adsorbed PDEA was washed with pure water to remove the nonspecifically adsorbed PDEA. The composite material AChE@PILs@Ag NPs@PDEA was obtained by centrifugation at 8000 rpm for 10 min.

[0023] Furthermore, in the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, in step 2), the preparation method of the Ag NPs includes the following steps: 18 mg of AgNO3 is dissolved in 100 mL of deionized water, and then the solution is heated to reflux under vigorous stirring, and then 2.0 mL of 1 wt% trisodium citrate solution is quickly added, and heating is continued for 15 minutes until the color of the solution changes from colorless to gray-green. After cooling to room temperature, the solution is stored at 4°C.

[0024] Application of any of the above-mentioned multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensors in the detection of organophosphorus pesticides.

[0025] Furthermore, in the above application, the detection method is as follows: 200 μL of a sample solution containing organophosphorus pesticides was mixed with 500 μL of AChE@PILs@Ag NPs@PDEA solution and incubated for 15 minutes, followed by the addition of 200 μL of 6 M ATCl solution and continued incubation for 1 hour. The unreacted products were removed by centrifugation at 8000 rpm, and the supernatant was added dropwise to a glass slide. After drying at 40°C, surface enhanced Raman spectroscopy (SERS) detection and analysis was performed.

[0026] The beneficial effects of the present invention are:

[0027] 1. The present invention adopts a multilayer core-shell composite structure AChE@PILs@Ag NPs@PDEA. Acetylcholinesterase (AChE) is encapsulated by PILs microspheres. While providing a biocompatible microenvironment, stabilizing enzyme activity and preventing leakage, the PILs utilize their tunable charge density characteristics to achieve uniform nanoarray assembly of silver nanoparticles (Ag NPs), significantly enhancing the local electromagnetic field coupling effect and SERS signal amplification.

[0028] 2. The synergistic effect of the pyridine ring and disulfide bond in the PDEA molecule, through the cleavage of the disulfide bond mediated by thiocholine (TCl), releases the pyridine ring, establishes a dynamic response mechanism of the SERS signal, and combines the AChE activity inhibition effect to achieve highly sensitive indirect detection of organophosphorus pesticides.

[0029] 3. The biosensor has an ultra-low detection limit (1.38×10 -13 M) with a wide linear range (3.8×10 -7 ~3.8×10 -12 M), and has excellent thermal stability and storage stability. It can achieve accurate detection in complex matrices (such as tomatoes and tap water) and is suitable for rapid on-site analysis of pesticide residues in agricultural products under multiple climatic conditions.

[0030] 4. The synergistic effect of the structural precision of PILs and charge-driven assembly improves the catalytic performance and SERS signal repeatability of Ag NPs, while enhancing the anti-interference ability and selectivity of biosensors, providing a new strategy for trace small molecule detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the SEM image of AgNPs in Example 1.

[0032] Figure 2 Scanning electron microscope images of (A) PILs, (B) AChE@PILs, and (C) AChE@PILs@Ag NPs in Example 1, and (D) EDS elemental image and mapping of C, N, and Ag elements on AChE@PILs@Ag NPs.

[0033] Figure 3 (a) AChE, (b) monomer ViEtIm in Example 1 + Br - , (c) AChE@PILs, (d) Ag NPs, and (e) zeta potential diagrams of AChE@PILs@Ag NPs.

[0034] Figure 4 XPS spectra of (a) AChE@PILs and (b) AChE@PILs@Ag NPs in Example 1.

[0035] Figure 5 This is the Ag 3d XPS spectrum of AChE@PILs@Ag NPs in Example 1.

[0036] Figure 6 A is (a) AChE@PILs@Ag NPs, (b) PDEA powder, (c) 10 -2 SERS spectra of M PDEA and Ag NPs and (d) 10 -2 SERS spectra of M PDEA on AChE@PILs@Ag NPs; Figure 6 B is the 10 -2 SERS signal of (a) AChE@PILs@Ag NPs@PDEA and 10 mM ATCl, (b) SERS signal of AChE@PILs@Ag NPs@PDEA and 10 ng mL -1 SERS signal after MP reaction.

[0037] Figure 7 (A) is the SERS spectra obtained at different ATCl concentrations in Example 2; (B) is based on 998 cm -1 The standard curve of the relationship between the intensity at 400 nm and the concentration of ATCl.

[0038] Figure 8 (A) is the SERS spectra obtained at different MP concentrations in Example 2; (B) is the SERS spectra at 998 cm -1 The corresponding SERS intensity at 998 cm -1 The standard curve of the relationship between the intensity at 400 nm and the concentration of MP is shown.

[0039] Figure 9 A is the AChE@PILs@Ag NPs@PDEA at 15 different positions and 10 -9 mol L -1 After MP reaction, the-1 SERS signal at ; Figure 9 B is the six different batches of AChE@PILs@Ag NPs@PDEA and 10 -9 mol L - 1 After MP reaction, the -1 SERS signal at ; Figure 9 C is AChE@PILs@Ag NPs@PDEA and 10 -9 mol L -1 SERS signals of MP after storage for 0, 5, 10, 15, 20, 25, and 30 days after reaction; Figure 9 D is the AChE@PILs@Ag NPs@PDEA and 10 -9 mol L -1 SERS signals of MP after reaction at 25, 30, 35, 40, 45, and 50 °C.

[0040] Figure 10 (A) AChE@PILs@Ag NPs@PDEA and 10 -8 M MP, 8.0 mM glucose, 8.0 mM PO4 3- , 8.0mM ascorbic acid, 8.0mM SO4 2- , 8.0 mM citric acid, 8.0 mM K + 、8.0mM NO3 - SERS spectra after reaction with 8.0 mM oxalic acid; (B) 998 cm -1 The peak SERS intensity at .

[0041] Figure 11 (A) is the preparation flow chart of AChE@PILs@Ag NPs@PDEA; (B) is the schematic diagram of the detection of organophosphorus pesticides based on AChE@PILs@Ag NPs@PDEA biosensor. DETAILED DESCRIPTION

[0042] In order to better understand the technical solution of the present invention, a specific embodiment is given to further illustrate it in detail, but the technical solution is not limited thereto.

[0043] Example 1 Preparation method of AChE@PILs@Ag NPs@PDEA

[0044] 1) Synthesis of AuNPs

[0045] Dissolve 18 mg of AgNO₃ in 100 mL of deionized water. Heat the solution to reflux with vigorous stirring. Rapidly add 2.0 mL of a 1 wt% trisodium citrate solution and continue heating for 15 minutes, until the solution changes color from colorless to gray-green. After cooling to room temperature, store the solution at 4°C for further use.

[0046] 2) Poly(ViEtIm + Br - Preparation of ionic liquid microgels encapsulating acetylcholinesterase (AChE@PILs)

[0047] Preparation of 1-vinyl-3-ethylimidazolium bromide (poly(ViEtIm)) by concentrated emulsion polymerization + Br - )) microspheres. The dispersed aqueous phase contains monomer ViEtIm + Br - The mixture was prepared by mixing 1% paraformaldehyde (0.125 g), acetylcholinesterase (AChE) (0.8-2.4 U), crosslinker N,N′-methylenebisacrylamide (0.004-0.01 g), and initiator ammonium persulfate (0.0025 g), with 20 mM Tris-HCl as the dispersant. The oil phase consisted of dodecane (75 μL) and Span 80 (25 μL). The dispersed aqueous phase was slowly added dropwise to the oil phase to form an emulsion. After deoxygenation with nitrogen, 10 μL of TEMED was added to initiate the polymerization reaction. After the reaction was completed, the precipitated polymer was washed three times with acetone and PBS (centrifugation at 4000 rpm for 10 minutes) and freeze-dried to obtain AChE@PILs composite microgels.

[0048] 3) Preparation of AChE@PILs@Ag NPs

[0049] 8 mg of AChE@PILs composite microgel was placed in a centrifuge tube, 5 mL of silver nanoparticle (Ag NPs) solution was added, and the mixture was sonicated for 30 minutes (the solution color changed from yellow-green to brown). The mixture was then centrifuged at 4000 rpm for 10 minutes to obtain the organic-inorganic composite material AChE@PILs@Ag NPs.

[0050] 4) Preparation of AChE@PILs@Ag NPs@PDEA

[0051] PDEA was modified on the surface of the material by N-Ag bond self-assembly method. -2 The PDEA solution was added dropwise to the surface of AChE@PILs@Ag NPs. After incubation for 2 hours, the nonspecifically adsorbed PDEA was removed by rinsing with pure water. The composite material AChE@PILs@Ag NPs@PDEA was obtained by centrifugation at 8000 rpm for 10 minutes.

[0052] The composition and properties of related AChE@PILs@Ag NPs@PDEA are shown in Figure 1-5 .

[0053] Figure 1 is the scanning electron microscope image of Ag NPs. Figure 1 It can be clearly seen that the monodisperse and uniform morphology of the prepared Ag NPs lays the foundation for providing uniform Raman signals.

[0054] Figure 2 Figure 3 is the scanning electron microscopy images of PILs, AChE@PILs, AChE@PILs@Ag NPs and the EDS elemental images of AChE@PILs@AgNPs. Spherical structures were observed in both PILs and AChE@PILs, indicating that the encapsulation of AChE by emulsion polymerization did not change the morphology of the microspheres. Figure 2 A and Figure 2 As shown in Figure 2B, comparative analysis revealed that the average diameter of the single-component PIL (4.2 μm) was slightly larger than that of the two-component AChE@PIL (3.4 μm). Figure 2 C shows the scanning electron microscope image of AChE@PILs@Ag NPs, in which the surface of AChE@PILs is densely covered with granular particles, which may correspond to Ag NPs. At the same time, EDS spectroscopy was also used to verify the elemental composition of the composite material. Figure 2 As shown in Figure 4D, the presence of key elements (C, N, and Ag) confirms the successful and uniform distribution of Ag on AChE@PILs.

[0055] Figure 3 AChE, monomeric ViEtIm + Br - The zeta potential of AChE@PILs, Ag NPs, and AChE@PILs@Ag NPs. It can be seen from the figure that the zeta potential of AChE is -3.42mV, which is similar to the positively charged ViEtIm + Br - The electrostatic interaction between monomers was also observed. Meanwhile, zeta potential measurements showed that the effective binding of AChE@PILs (+32.23 mV) to Ag NPs (-37.1 mV) was also driven by the electrostatic interaction between the two components.

[0056] Figure 4 are the XPS spectra of AChE@PILs and AChE@PILs@Ag NPs. Figure 4Figure a shows that the synthesized AChE@PILs are mainly composed of C, O, and N elements. After further functionalization, the characteristic peak of Ag appears in AChE@PILs@Ag NPs compared with the AChE@PILs spectrum.

[0057] Figure 5 The figure shows the Ag 3d XPS spectrum of AChE@PILs@Ag NPs. The binding energy peaks at 373.7 eV and 367.8 eV correspond to Ag 3d 3 / 2 and Ag 3d 5 / 2, respectively, confirming the presence of Ag NPs and demonstrating the successful construction of the AChE@PILs@Ag NPs ternary system.

[0058] Example 2 Performance testing of AChE@PILs@Ag NPs@PDEA

[0059] SERS detection of ATCl

[0060] ATCl was dissolved in deionized water to prepare solutions of varying concentrations. 200 μL of each ATCl solution was mixed with 500 μL of a ChE@PILs@Ag NPs@PDEA solution. After incubation for 1 hour, the mixture was centrifuged at 8000 rpm for 10 minutes to remove unreacted ATCl. The supernatant was then dripped onto a glass slide, dried at 40°C, and analyzed by surface-enhanced Raman spectroscopy (SERS).

[0061] SERS detection of MPs

[0062] Methyl parathion (MP) was used as an organophosphorus model for detection. -3 The MMP ethanol stock solution was diluted to the desired concentration. 200 μL of MP solution at various concentrations was mixed with 500 μL of AChE@PILs@Ag NPs@PDEA solution and incubated for 15 minutes. 200 μL of 6 M ATCl solution was then added and incubated for another hour. Unreacted products were removed by centrifugation at 8000 rpm, and the supernatant was dripped onto a glass slide and dried at 40°C before SERS analysis.

[0063] Related performance tests see Figure 6-10 .

[0064] Figure 6 The corresponding SERS spectra are shown in Figure 2. After PDEA was modified onto the surface of AChE@PILs@Ag NPs, the pyridine rings showed strong peaks at 1000, 1050, 1082, and 1116 cm -1 The main vibrations are attributed to this. Figure 6In A, curve b represents the Raman signal of solid PDEA powder, while curve c corresponds to the signal obtained after adding PDEA to silver nanoparticles. Compared with curve b, the SERS signal in curve c is significantly enhanced. In addition, when PDEA is introduced into the AChE@PILs@Ag NPs system (curve d), the observed SERS signal is even higher than the signal when PDEA is added to silver nanoparticles alone. The addition of PILs to the composite structure helps to significantly enhance the SERS signal. Because PILs have adjustable repeating unit charge density and an ordered structural framework, the AChE@PILs@AgNPs substrate is able to assemble Ag NPs into uniformly distributed nanoscale arrays. This ordered structure can effectively couple local electromagnetic fields to produce consistent "hot spots", thereby significantly amplifying the SERS enhancement effect. This unique structural arrangement solves the limitations of traditional SERS substrates by providing stronger and more uniform electromagnetic enhancement effects. As Figure 6 As shown in Figure B, after the addition of ATCl, ATCl is hydrolyzed to TCl under the catalysis of AChE. TCl then destroys the disulfide bond, resulting in the release of the pyridine ring on the surface of AChE@PILs@Ag NPs@PDEA, which leads to a decrease in the SERS signal (curve b). However, when MP is added, the activity of AChE is inhibited, thereby reducing the generation of TCl. This inhibition alleviates the decrease in the SERS signal (curve c), indicating that MP has an inhibitory effect on AChE activity. These findings highlight the sensitivity of this system to MP and its ability to effectively monitor AChE activity through SERS signal modulation.

[0065] Figure 7 The AChE@PILs@Ag NPs@PDEA in Example 1 of the present invention is used for the detection of different concentrations of ATCl. Figure 7 As shown in Figure 2, the SERS spectra of AChE@PILs@Ag NPs@PDEA changed significantly with the increase of ATCl concentration. Specifically, at 998 cm -1 The peak intensity at the peak decreases. The linear regression equation obtained from the data is: 998cm-1 =20331.3-3085.9C, where “I 998cm-1 " stands for 998cm -1 The SERS intensity at , “C” represents the ATCl concentration, and the correlation coefficient is 0.995 in the concentration range of 1 mM to 6 mM.

[0066] Figure 8 The AChE@PILs@Ag NPs@PDEA in Example 1 of the present invention is used for the detection of MP at different concentrations. Figure 8As shown in Figure A, the SERS signal intensity of AChE@PILs@Ag NPs@PDEA gradually weakened with the decrease of MP concentration. Figure 8 As shown in B, 998cm -1 The SERS intensity at is used as a quantitative characteristic peak to establish a linear relationship between the SERS signal and the logarithm of the MP concentration. 2 is 0.994. The linear regression equation is I 998cm-1 =32675.5+2519.4log C, where “I 998cm-1 " stands for 998cm -1 The SERS intensity at , “C” represents the MP concentration. Using the linear regression equation and a signal-to-noise ratio of 3, the theoretical detection limit (LOD) of MP was calculated to be 1.38×10 -13 M.

[0067] Figure 9 This is a study on the reproducibility of AChE@PILs@Ag NPs@PDEA. Figure 9 As shown in A, the AChE@PILs@Ag NPs@PDEA and 10 -9 mol L -1 After MP reaction, the - The SERS signal at 1 was observed to be as low as 4.0%, indicating that AChE@PILs@Ag NPs@PDEA has excellent uniformity. Figure 9 B depicts six different batches of AChE@PILs@Ag NPs@PDEA with 10 -9 mol L -1 After MP reaction, the -1 The SERS signal at 4°C was recorded with an RSD of 4.35%, confirming the reproducibility of the prepared system. The stability of AChE@PILs@Ag NPs@PDEA after storage at 4°C was evaluated. Figure 9 As shown in C, the reaction retention rate after storage for 30 days is 92.32%, indicating that the material has good long-term stability. The thermal stability of AChE@PILs@Ag NPs@PDEA was further evaluated by measuring the SERS signal values ​​after reaction with MP at different temperatures ( Figure 9 D) Previous studies have reported that the introduction of PILs can significantly improve the thermal stability of enzymes. Figure 9As shown in Figure D, the residual activity of AChE@PILs@Ag NPs@PDEA gradually decreased after 15 minutes of reaction at temperatures of 35°C, 40°C, 45°C, and 55°C. Notably, after incubation at 50°C for 15 minutes, the encapsulated AChE retained 85% of its initial activity, whereas unprotected AChE is easily inactivated at high temperatures. In comparison, unencapsulated AChE has been reported to lose 60% of its activity within the temperature range of 42–48°C. These results clearly demonstrate that encapsulating AChE in PILs significantly improves its thermal stability. The PILs layer effectively reduces the impact of microenvironmental fluctuations on the enzyme, preserving its activity under harsh conditions. This enhanced thermal stability enables the direct detection of pesticides in real samples at high temperatures without the need for a cooling step, thereby improving analytical efficiency. These findings highlight the potential use of AChE@PILs@Ag NPs@PDEA in practical applications requiring robust and reliable enzyme-based SERS detection systems.

[0068] Figure 10 The specific detection results of AChE@PILs@Ag NPs@PDEA in Example 1 of the present invention are shown in FIG. -8 M) and various potential interfering substances, including glucose, citric acid, oxalic acid, ascorbic acid, SO4 2- PO4 3- 、NO3 - and K + (both 8.0 mM). Figure 10 A shows the interaction between AChE@PILs@Ag NPs@PDEA and MP(10 -8 SERS spectra obtained after reaction of M) with various interfering substances. Figure 10 B shows 998cm -1 It is observed that when these interfering substances are added alone, the SERS signal intensity is still relatively low. In contrast, when there are 10 -8 In the presence of MMPs, the SERS signal intensity was significantly higher. These findings clearly demonstrate that the proposed AChE-based AChE@PILs@Ag NPs@PDEA has excellent anti-interference ability and can reliably detect MPs even in the presence of potential interfering substances, thus supporting its applicability for practical applications.

[0069] Example 3 Actual sample detection

[0070] Detection of MP in actual samples

[0071] The standard addition method was used to evaluate the performance of the composite material. Tomato and tap water were used as actual samples:

[0072] Tomato sample: Apply the MP solution to the tomato epidermis, dry it at room temperature, cut the epidermis fragments, add 30 mL of ethanol and ultrasonically extract for 30 minutes. Centrifuge at 8000 rpm for 15 minutes, and filter the supernatant through a 0.45 μm filter membrane to obtain the tomato sample solution to be tested.

[0073] Tap water sample: Add MP standard solution to the tap water sample, mix well, and dilute to 200 μL to obtain the tap water sample solution to be tested.

[0074] 200 μL of tomato or tap water sample solution was mixed with 500 μL of AChE@PILs@Ag NPs@PDEA solution and incubated for 15 minutes. Then, 200 μL of 6 M ATCl solution was added and incubated for another hour. Unreacted products were removed by centrifugation at 8000 rpm, and the supernatant was dripped onto a glass slide and dried at 40°C before SERS analysis.

[0075] See Table 1 for relevant data.

[0076] Table 1 Analysis results of MP in actual samples Tomato and tap water samples were added with different amounts of methyl parathion (0.5, 5, and 50 ng mL -1 ) to test the accuracy and reproducibility of the method. Recovery test results are shown in Table 1. The recoveries of methyl parathion in real samples ranged from 97.76% to 101.16%, with relative standard deviations (RSDs) less than 4.22%. These results demonstrate that the AChE@PILs@Ag NPs@PDEA method is feasible for practical applications.

Claims

1. A multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor, characterized in that: The preparation method comprises the following steps: 1) Acetylcholinesterase (AChE) was encapsulated into polymeric ionic liquid (PILs) microspheres by emulsion polymerization to form the core; 2) PILs are used as an intermediate layer to uniformly load silver nanoparticles (Ag NPs) on their surface to construct an isotropic composite material; 3) The surface of the composite material was modified with 2-(pyridyldithio)ethylamine PDEA containing a pyridine ring and a disulfide bond.

2. The multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to claim 1, characterized in that: In step 1), the ionic liquid is 1-vinyl-3-ethylimidazolium bromide ViEtIm + Br - .

3. The multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to claim 2, characterized in that: The specific operation steps of step 1) are: preparing 1-vinyl-3-ethylimidazolium bromide poly (ViEtIm) by concentrated emulsion polymerization + Br - ) microspheres; the dispersed aqueous phase contains 0.125g monomer ViEtIm + Br - , 0.8-2.4U acetylcholinesterase AChE, 0.004-0.01g cross-linker N,N′-methylenebisacrylamide and 0.0025g initiator ammonium persulfate, with 20mMTris-HCl as dispersant; the oil phase consists of 75μL dodecane and 25μL Span 80; the dispersed aqueous phase is slowly added dropwise to the oil phase to form an emulsion, after deoxygenation by nitrogen, 10μL TEMED is added to initiate the polymerization reaction. After the reaction is completed, the precipitated polymer is washed three times with acetone and PBS in sequence and centrifuged at 4000rpm for 10 minutes, and then freeze-dried to obtain AChE@PILs composite microgel.

4. The multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to claim 3, characterized in that: The specific operation steps of step 2) are as follows: take 8 mg of AChE@PILs composite microgel in a centrifuge tube, add 5 mL of AgNPs solution, and ultrasonicate for 30 minutes until the color of the solution changes from yellow-green to brown. Centrifuge at 4000 rpm for 10 minutes to separate the organic-inorganic composite material AChE@PILs@Ag NPs.

5. The multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to claim 4, characterized in that: The step 3) is to modify PDEA on the surface of the material by N-Ag bond self-assembly method, and the specific operation steps are as follows: 200 μL 10 -2 The M PDEA solution was added dropwise onto the surface of AChE@PILs@Ag NPs. After incubation for 2 h, the nonspecifically adsorbed PDEA was washed with pure water to remove the nonspecifically adsorbed PDEA. The composite material AChE@PILs@Ag NPs@PDEA was obtained by centrifugation at 8000 rpm for 10 min.

6. The multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to claim 1, characterized in that: In step 2), the preparation method of Ag NPs comprises the following steps: 18 mg of AgNO3 is dissolved in 100 mL of deionized water, and then the solution is heated to reflux under vigorous stirring, and then 2.0 mL of 1 wt% trisodium citrate solution is quickly added, and heating is continued for 15 minutes until the color of the solution changes from colorless to gray-green. After cooling to room temperature, the solution is stored at 4°C.

7. Use of the multilayer core-shell AChE@PILs@Ag NPs@PDEA biosensor according to any one of claims 1 to 6 in the detection of organophosphorus pesticides.

8. The use according to claim 7, characterized in that The detection method is as follows: 200 μL of a sample solution containing organophosphorus pesticides was mixed with 500 μL of an AChE@PILs@Ag NPs@PDEA solution and incubated for 15 minutes, followed by the addition of 200 μL of a 6MATCl solution and continued incubation for 1 hour. The unreacted products were removed by centrifugation at 8000 rpm, and the supernatant was added dropwise to a glass slide. After drying at 40°C, surface-enhanced Raman spectroscopy (SERS) detection and analysis was performed.

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