An electrostatic driving-based double-enzyme cascade amplification fluorescence sensor, a preparation method and application thereof

Abstract

The application discloses a double-enzyme cascade amplification fluorescence sensor based on electrostatic driving and a preparation method and application thereof, and the nano-enzyme fluorescence sensor is composed of a probe Azo-Bodipy 685, choline oxidase (CHO) and acetylcholine chloride (ACh). Based on the electrostatic adsorption principle, the application proposes an electrostatic complex co-immobilization-enzyme cascade reaction synergistic strategy, constructs a new electrostatic driving fluorescence sensor, utilizes the electrostatic effect of the positive charge of the quaternary ammonium salt of ACh and the negative charge of the surface of CHO to construct a nano-scale space limited enzyme reaction microenvironment on the surface of a specific fluorescent probe Azo-Bodipy 685. When an organophosphorus pesticide inhibits acetylcholine esterase (AChE), the decrease of the generation of choline chloride leads to the decrease of the amount of hydrogen peroxide generated by CHO catalysis, and the fluorescence signal intensity is weakened, and a series of fluorescence intensity changes can realize the detection of AChE activity and the determination of the content of organophosphorus pesticide residues.

Description

Technical Field

[0001] This invention belongs to the field of biological detection sensors, specifically relating to an electrostatically driven dual-enzyme cascade amplified fluorescence sensor, its preparation method, and its application. Background Technology

[0002] Organophosphorus pesticides (OPs) are phosphorus-containing agricultural chemicals that account for 40% of global pesticide use. However, OP residues pose serious health risks, as they can enter the body through the food chain, causing the accumulation of the neurotransmitter acetylcholine, leading to symptoms such as dizziness and nausea, and even pulmonary edema and cerebral edema, resulting in death. Therefore, detecting whether OP residues in traditional Chinese medicine exceed the standards is crucial for protecting human health. Currently, various analytical methods for detecting OPs have been established, including chromatographic methods such as gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), immunoassay methods such as enzyme-linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA), and capillary electrophoresis (CE). Unfortunately, the various drawbacks of these methods have hampered the rapid detection of pesticide residues. For example, their detection range is limited; GC is only suitable for the determination of volatile pesticides. The high cost of these methods means that LC-MS cannot be used for routine analysis of OP residues. Extreme conditions can lead to antibody inactivation, preventing the widespread use of immunoassays. Furthermore, the complexity of these techniques and the skill level required for operation further limit the rapid detection of pesticides, significantly restricting their widespread application. These limitations have also encouraged the development of new rapid detection analytical methods.

[0003] Currently, fluorescence methods have attracted much attention due to their unique advantages of rapid response, simple operation, high sensitivity, and compatibility with high-throughput screening, and are expected to become an alternative method for detecting pesticide residues (OPs). However, the core challenge of fluorescence technology for pesticide residue detection lies in optimizing the trade-off between detection efficiency, cost-effectiveness, and analytical performance. While existing fluorescent probes targeting AChE can achieve rapid response and high signal transduction rates, they face inherent limitations in practical applications. For example, gold nanoprobes (AuNPs) based on thiocholine derivatives are costly to synthesize and have a narrow linear response range, severely limiting their field applications. Similarly, most fluorescent sensors reflect enzyme activity and pesticide content by indirectly detecting AChE substrates. Although the preparation cost is considerable and signal transduction is diversified, the diverse preparation materials lead to low output efficiency and make it impossible to screen complex samples. Ultimately, most fluorescent sensors suffer from the following problems due to the introduction of heterogeneous components: (1) Diffusion restriction: Brownian dynamics simulations have confirmed that excessive interlayer spacing in two-dimensional nanosheets leads to an extension of the substrate diffusion path, resulting in a decrease in the collision frequency of CHO-AChE; (2) Steric hindrance effect: The CHO immobilized by nanoparticles exhibits a high activity loss due to the blockage and masking of active sites caused by the introduction of heterogeneous components. Summary of the Invention

[0004] Purpose of the invention: To address the problems existing in the prior art, the present invention provides an electrostatically driven dual-enzyme cascade amplification fluorescence sensor, which can rapidly and easily detect AChE activity and determine organophosphorus pesticide residue content by means of changes in fluorescence intensity.

[0005] The present invention also provides a method for preparing and applying the electrostatically driven dual-enzyme cascade amplified fluorescence sensor.

[0006] Technical Solution: To achieve the above objectives, the present invention provides an electrostatically driven dual-enzyme cascade amplified fluorescence sensor, wherein the nanoenzyme fluorescence sensor comprises a probe Azo-Bodipy 685, CHO, and ACh; the structure of the probe Azo-Bodipy 685 is shown below:

[0007] .

[0008] The method for preparing the electrostatically driven dual-enzyme cascade amplified fluorescence sensor described in this invention involves the electrostatic complexation of CHO and ACh to form CA NPs, which encapsulate Azo-Bodipy 685 to form a condensation-driven fluorescence sensor CA-B NPs.

[0009] In this process, Tris-HCl buffer solution was mixed with probe Azo-Bodipy 685, and then CHO and ACh were added to obtain a mixed solution. The mixture was stirred and allowed to stand in the dark to obtain an electrostatically driven dual-enzyme cascade amplified fluorescent sensor CA-B NPs.

[0010] This invention utilizes an electrostatic co-assembly strategy to synthesize CA NPs, and optimizes the synthesis conditions using a controlled variable method to obtain optimal process parameters. Based on this, the CA NPs are further encapsulated with Azo-Bodipy 685 NPs (B NPs) to ultimately prepare nanoscale electrostatically driven dual-enzyme cascade amplified fluorescence sensors, CA-B NPs. B NPs, CHO, and ACh are stirred at room temperature. After the reaction is complete, the solution is stored at 4°C in the dark to obtain CA-B NPs.

[0011] The material ratio of CHO to ACh is 2-6 U: 2.5-20 μmol.

[0012] The concentration of the probe Azo-Bodipy 685 in the mixed solution is 1-3 mg / mL, and the enzyme activity of CHO is 2-6 U.

[0013] The stirring speed is 1000-1500 rpm, and the stirring time is 2-2.5 h.

[0014] Preferably, the stirring speed is 1000 rpm.

[0015] Preferably, the stirring reaction time of component ACh in the CA NPs is 0.75-2.0 h.

[0016] More preferably, the stirring reaction time of component ACh in the CA NPs is 1.0-1.5 h.

[0017] More preferably, the stirring reaction time of component ACh in the CA NPs is 1.5 h.

[0018] Preferably, the stirring reaction time of component Azo-Bodipy 685 in the CA-B NPs is 0.75-2.0 h.

[0019] More preferably, the stirring reaction time of component Azo-Bodipy 685 in the CA-B NPs is 1-1.5 h.

[0020] More preferably, the stirring reaction time of component Azo-Bodipy 685 in the CA-B NPs is 1.0 h.

[0021] Preferably, the material ratio of CHO and ACh in CA NPs in the mixed solution is 2 U:2.5 μmol to 6 U:20 μmol.

[0022] More preferably, the material ratio of CHO and ACh in CA NPs in the mixed solution is 4 U: 5 μmol.

[0023] As an embodiment of the present invention, the concentration of Azo-Bodipy 685 in the mixed solution is 1 mg / mL.

[0024] Preferably, the stirring speed is 1000 rpm, the stirring time for component ACh in the CA NPs is 1.5 h, and the stirring time for component Azo-Bodipy 685 in the CA-B NPs is 1.0 h. The concentration of probe Azo-Bodipy 685 in the mixed solution is 1 mg / mL, and the material ratio of CHO to ACh is 4 U:5 μmol.

[0025] The present invention relates to the application of the electrostatically driven dual-enzyme cascade amplification fluorescence sensor in detecting AChE activity and determining organophosphorus pesticide residues.

[0026] The method for detecting AChE activity includes the following steps:

[0027] S1. Dissolve AChE in Tris-HCl buffer solution to obtain AChE solution;

[0028] S2. Add the AChE solution obtained in step S1 to the well plate, incubate once, add CA-B NPs, incubate a second time, add DMSO, and measure the fluorescence response intensity.

[0029] As an embodiment of the present invention, the concentration of AChE in the mixed solution is 0.01 U / μL.

[0030] The method for determining organophosphorus pesticide residues using the electrostatically driven dual-enzyme cascade amplified fluorescence sensor includes the following steps:

[0031] T1, Measure the fluorescence intensity F0 of the blank buffer solution;

[0032] T2, Measure the fluorescence intensity F of the control group containing AChE buffer;

[0033] T3. Incubate organophosphorus pesticides and AChE together in buffer solution and measure the fluorescence intensity F1 after co-incubation.

[0034] T4. Based on F0 obtained in step T1, F obtained in step T2, and F1 obtained in step T3, calculate the AChE inhibition rate. The formula for calculating the AChE inhibition rate is as follows: AChE inhibition rate = (F-F1) ÷ F × 100%. As the concentration of organophosphorus pesticides increases, the fluorescence signal continues to increase. After conversion to a logarithmic scale, the (F-F1) ÷ F × 100% value of the curve shows a linear response with the triazophos concentration, thus obtaining the organophosphorus pesticide content.

[0035] Further, step T1 includes the following steps: adding Tris-HCl blank buffer solution to the well plate, incubating once, adding CA-B NPs, incubating a second time, adding DMSO, measuring the fluorescence intensity, and obtaining the blank fluorescence intensity F0;

[0036] Step T2 includes the following steps: adding AChE solution to the well plate, incubating once, adding CA-B NPs, incubating a second time, adding DMSO, measuring the fluorescence intensity, and obtaining the fluorescence intensity F of the AChE control group;

[0037] Step T3 includes the following steps: adding AChE solution to the well plate, incubating once, adding organophosphorus pesticide triazophos or traditional Chinese medicine tangerine peel sample, incubating twice, adding CA-B NPs, incubating three times, adding DMSO, measuring fluorescence intensity, and obtaining the fluorescence intensity F1 after co-incubation.

[0038] The electrostatically driven dual-enzyme cascade amplified fluorescence sensor is used in the determination of different concentrations of the organophosphorus pesticide triazophos.

[0039] The organophosphorus pesticide residues in the traditional Chinese medicine tangerine peel are obtained by extracting the tangerine peel extract, and the organophosphorus pesticide residues in the tangerine peel have an inhibitory effect on AChE.

[0040] Among them, the electrostatically driven dual-enzyme cascade amplified fluorescence sensor is used in the screening and determination of tangerine peel extract containing the organophosphorus pesticide triazophos.

[0041] The preparation method of the tangerine peel decoction includes the following steps: Tangerine peel is pulverized into powder (passed through a No. 3 sieve), approximately 5 g is accurately weighed, 5 g of anhydrous sodium sulfate is added, and 50-100 mL of ethyl acetate is added. The mixture is ultrasonically treated in an ice bath for 3 minutes, allowed to stand, and the supernatant is filtered. The residue is added to 30-50 mL of ethyl acetate, ultrasonically treated in an ice bath for 2 minutes, allowed to stand, and filtered. The two filtrates are combined, and the filter paper and residue are washed with a small amount of ethyl acetate and combined with the above filtrate. The filtrate is concentrated under reduced pressure below 40℃ to near dryness, transferred to a 5 mL volumetric flask with ethyl acetate, and diluted to the mark. 1 mL of the above solution is accurately pipetted onto a graphitized carbon column (250 mg / 3 mL pre-washed with 5 mL of ethyl acetate), and eluted with 5 mL of a hexane-ethyl acetate (1:1) mixed solution. The eluent is collected, concentrated to near dryness on a nitrogen blower, and diluted to 1 mL with ethyl acetate. The solution is then vortexed to dissolve, yielding the tangerine peel decoction.

[0042] This invention proposes a "static complexation-driven cascade reaction chain" strategy based on the synergistic effect of AChE and CHO. This invention integrates a fluorescent probe with a sensor, utilizing charge-complementary self-assembly technology to embed it into a complete reaction chain (AChE-CHO-H2O2) to form an integrated sensing design. By optimizing the spatial distribution of the enzyme-substrate through electrostatic complexation, diffusion and steric hindrance effects are overcome, interference from heterogeneous components is avoided, and detection efficiency and specificity are improved, enabling rapid detection of AChE activity and OPs content in complex matrices.

[0043] This invention, based on the principle of electrostatic adsorption, provides a synergistic strategy of "electrostatic complexation co-immobilization-enzyme cascade reaction" to construct a novel aggregation-driven fluorescence sensor. Utilizing the electrostatic interaction between the positive charge of the quaternary ammonium salt of ACh (Zeta potential = +27.94 mV) and the negative charge on the surface of CHO (Zeta potential = -29.5 mV), a nanoscale spatially confined enzyme reaction microenvironment is constructed on the surface of Azo-Bodipy 685, stabilizing the enzyme conformation and increasing the nanoparticle loading density. This nanoscale spacing leads to a localized increase in the concentration of choline chloride, the hydrolysis product of the CHO-AChE coupling reaction, significantly accelerating the catalytic cycle of CHO to produce H2O2. This allows Azo-Bodipy 685 to recognize dynamic fluorescence signals in the form of absorption or emission band changes generated by H2O2. When organophosphorus pesticides inhibit AChE, the reduced generation of choline chloride leads to a decrease in the amount of H2O2 produced by CHO catalysis, resulting in a weakening fluorescence signal. A series of fluorescence intensity changes can be used to detect AChE activity and determine the content of organophosphorus pesticide residues (e.g., ...). Figure 1 and Figure 2This design achieves dual optimization, effectively controlling the spatiotemporal range, improving reaction volume utilization, and enhancing environmental robustness. Furthermore, it lowers the detection limit through cascade amplification. This design pioneers a "reaction-diffusion synergy" cascade paradigm, bridging the gap between enzyme kinetics and nanoscale limitations, and laying the foundation for future development of multi-target detection platforms.

[0044] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0045] The sensor prepared in this invention features (1) optimized dual-enzyme reaction through spatial confinement effect: a nanoscale reaction microenvironment was established on the surface of Azo-Bodipy 685 by utilizing the electrostatic self-assembly of ACh and CHO. This spatial confinement and co-localization structure significantly shortens the substrate diffusion distance at the nanoscale and increases the local concentration of choline chloride, which may help improve the diffusion confinement and steric hindrance effect caused by heterogeneous system components in traditional sensors. Therefore, an optimized "reaction-diffusion balance" was achieved, greatly improving the enzyme efficiency in the confined space. (2) Integrated cascade signal amplification mechanism: the complete biocatalytic cascade (AChE-CHO-H2O2) was embedded into the sensor architecture through electrostatically driven self-assembly, achieving a simplified design that allows for in-situ reaction and signal transduction. This cascade system utilizes the inherent amplification characteristics of enzyme cascades, in which the H2O2 generated by the cascade directly modulates the fluorescence intensity. Notably, the fluorescence output is negatively correlated with the AChE inhibition caused by OPs, thus enabling sensitive and rapid quantification of OPs through single-step measurement. (3) Practical Application and Field Capability: The sensor simplifies the detection procedure, enabling one-step OPs detection without complex sample pretreatment. Even in complex matrices such as dried tangerine peel, the system maintains a recovery rate of approximately 88.13-113.09%, demonstrating excellent matrix tolerance and anti-interference capabilities. Furthermore, the entire detection process can be completed within 20 minutes, meeting the urgent requirements for high-throughput, on-site OPs screening in the cultivation and processing environment of traditional Chinese medicine.

[0046] This invention pioneers a novel "reaction-diffusion synergy" paradigm, overcoming the inherent mass transfer limitations of traditional sensor systems and providing a generalizable strategy for spatially controlled nanoscale cascade reactions. This approach may offer valuable insights for the future design of efficient biosensing platforms in pesticide residue analysis and other fields. Attached Figure Description

[0047] Figure 1 This is a schematic diagram illustrating the detection principle of the present invention;

[0048] Figure 2 This is a flowchart of the screening process in this invention;

[0049] Figure 3The fluorescence emission spectra of Azo-Bodipy 685 in different solvents obtained in Exercise 1 are shown below.

[0050] Figure 4 Fluorescence imaging images of Azo-Bodipy 685 in different solvents obtained in Exercise 1;

[0051] Figure 5 The fluorescence excitation and emission spectra of Azo-Bodipy 685 obtained in Implementation 1 are shown below;

[0052] Figure 6 The UV absorption spectrum of Azo-Bodipy 685 obtained in Implementation 1;

[0053] Figure 7 To investigate the effect of different CHO and ACh ratios obtained in step 2 on the synthesis of CA-B NPs;

[0054] Figure 8 To investigate the effect of ACh stirring time on the synthesis of CA-B NPs obtained in step 3;

[0055] Figure 9 To investigate the effect of stirring time of Azo-Bodipy 685 on the synthesis of CA-B NPs obtained in step 4;

[0056] Figure 10 Transmission electron microscope image of the CA-B NPs sensor obtained from implementation 5;

[0057] Figure 11 The hydration particle size and zeta potential of the CA-B NPs sensor obtained from implementation 5;

[0058] Figure 12 To implement the CA NPs sensor obtained in step 6, fluorescence standard curves of AChE at different concentrations were detected;

[0059] Figure 13 To implement the CA-B NPs sensor obtained in step 6, a fluorescence standard curve for AChE at concentrations of 0.5–50 U / L was obtained.

[0060] Figure 14 To implement the CA-B NPs sensor obtained in step 6, fluorescence standard curves for detecting 50-500 U / L AChE were obtained.

[0061] Figure 15 Fluorescence standard curves for detecting different concentrations of triazophos pesticides using the CA-B NPs sensor obtained in step 7 were obtained.

[0062] Figure 16 To implement the CA-B NPs sensor obtained in 8 for pesticide residue detection of different dried tangerine peel samples. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent companies.

[0065] Morphological evaluation of the CA-BNPs sensor was performed using a Talos transmission electron microscope (Thermo Fisher, Talos F200X G2, Czech Republic); hydrodynamic dimensions and zeta potentials were measured at room temperature using a dynamic light scattering apparatus (Malvern Instruments, Zetasizer Nano 90, UK); UV-Vis absorption spectra were measured using a UV-Vis spectrophotometer (Shimadzu Company, UV-2450, Japan); fluorescence data were determined using a microplate reader (Thermo Scientific Varioskan Flash, Finland); and the enzyme was incubated in a biochemical incubator (KB53) to reach maximum activity for detection. Azo-Bodipy 685 (ABDP685) was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; acetylcholinesterase (AChE) (210 U / g, 100 mg, HY-P2860), choline oxidase (CHO) (100 U, HY-P2743), and acetylcholine chloride (Ach, HY-B0282) (500 mg) were purchased from MCE Ltd.; triazophos solution (100 μg / mL, 1 mL) was purchased from Tianjin Alta Technology Co., Ltd.; dimethyl sulfoxide was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; acetonitrile and methanol, both chromatographic grade, were purchased from Merck Co., Ltd. (Germany); hydrogen peroxide (H2O2) (content: 30%) was purchased from Guangzhou Guangshi Reagent Technology Co., Ltd.; and water for experiments was treated by deionization ultrafiltration using a Milli-Q IQ 7000 system.

[0066] The Tris-HCl buffer solutions used in the examples were all 1M Tris-HCl buffer solutions with pH=8.0.

[0067] Example 1

[0068] Luminescence performance testing of Azo-Bodipy 685:

[0069] To investigate the UV and fluorescence data of Azo-Bodipy 685 in solutions with different DMSO volume fractions (10%, 30%, 50%, 70%, 90%, and 100%).

[0070] Figure 3 The fluorescence intensity of Azo-Bodipy 685 in DMSO / Tris-HCl mixed solutions with different ratios is given. Figure 4 The results indicate that Azo-Bodipy 685 exhibits excellent complete dissolution and a large spiking range in fluorescence response under 50% DMSO conditions. Therefore, 50% DMSO was selected as the detection environment for AChE in the following reactions. The excitation and emission wavelengths of Azo-Bodipy 685 were measured using a microplate reader to obtain the optimal excitation and emission wavelengths. Figure 5 The excitation wavelength of Azo-Bodipy 685 is shown to be 706 nm (Ex = 706 nm), and the emission wavelength is 746 nm (Em = 746 nm). The UV-Vis absorption spectrum of Azo-Bodipy 685 is shown below. Figure 6 As shown.

[0071] Example 2

[0072] Synthesis of electrostatically driven dual-enzyme cascade amplified fluorescence sensor CA-B NPs

[0073] Take 60 µL of probe Azo-Bodipy 685 (solvent acetonitrile, 1 mg / mL) and slowly add it dropwise to 900 µL of Tris-HCl buffer solution. During this process, the solution is stirred at 1000 rpm for 1 h to obtain a mixed solution of fluorescent nanoparticles (B NPs) containing probe Azo-Bodipy 685.

[0074] 20 µL of CHO (0.2 U / μL Tris-HCl buffer solution) was added to the above mixture and stirred at 1000 rpm for 1 hour at room temperature. Then, 80 µL of ACh (5 μmol ultrapure water solution) was added and stirring continued for 1.5 hours. After the reaction was complete, the mixture was left to stand in the dark overnight to allow the acetonitrile to evaporate completely, yielding the final CA-B NPs, which were stored in a refrigerator at 4°C protected from light for later use.

[0075] Example 3

[0076] The effect of the ACh component ratio in CA NPs on the synthesis of CA NPs:

[0077] With a fixed CHO concentration, the effects of different amounts (2.5, 5, 10, 15, and 20 μmol) of ACh on the synthesis of CA NPs were investigated.

[0078] Accurately weigh five different amounts of ACh (2.5, 5, 10, 15 and 20 μmol) and dissolve them in 80 µL of ultrapure water.

[0079] Add 20 µL of Tris-HCl solution containing CHO (0.2 U / μL) to 900 µL of Tris-HCl solution and mix. Stir at 1000 r / min for 1 h at room temperature.

[0080] Add 80 µL of different amounts of ACh solution to the mixed solution and stir at 1000 r / min for 1.5 h at room temperature to obtain different nanozymes CA NPs.

[0081] Add 5 µL of Tris-HCl buffer solution and 5 µL of AChE (0.01 U / μL, in Tris-HCl buffer solution) to an EP tube and incubate at 37 °C for 5 minutes. Add 40 µL of CA NPs, mix thoroughly, and incubate at 37 °C for another 20 minutes. Finally, add 50 µL of DMSO solution containing Azo-Bodipy 685 (47 µL DMSO, 3 µL 1 mg / mL Azo-Bodipy 685) to terminate the enzyme reaction.

[0082] 100 μL of the above optimized group was added to each of the 96-well black plates to determine its fluorescence intensity F value.

[0083] Depend on Figure 7 It is known that both excessively high and low substrate concentrations will have a significant impact on fluorescence intensity. The optimal synthesis conditions are a CHO:ACh material ratio of 4 U:5 μmol, which results in the best fluorescence intensity of CA NPs.

[0084] Example 4

[0085] Effect of stirring time on the synthesis of CA-B NPs:

[0086] Sufficient reaction time also affects the success of nanoparticle synthesis. The effects of different reaction times (0.75 h, 1 h, 1.5 h and 2 h) on the synthesis of CA NPs were investigated.

[0087] Add 20 µL of Tris-HCl solution containing CHO (0.2 U / μL) to 900 µL of Tris-HCl solution and mix. Stir at 1000 r / min for 1 h at room temperature.

[0088] Add 80 µL of ACh (5 μmol) aqueous solution to the mixed solution.

[0089] Different nanozymes CA NPs were obtained by stirring at 1000 rpm at room temperature for 0.75 h, 1 h, 1.5 h and 2 h.

[0090] Add 5 µL of Tris-HCl buffer solution and 5 µL of LAChE (0.01 U / μL, in Tris-HCl buffer solution) to an EP tube and incubate at 37°C for 5 minutes. Add 40 µL of CA NPs, mix thoroughly, and incubate at 37°C for another 20 minutes. Finally, add 50 µL of DMSO solution containing Azo-Bodipy 685 (47 µL DMSO, 3 µL 1 mg / mL Azo-Bodipy 685) to terminate the enzyme reaction.

[0091] 100 μL of the above optimized group was added to each of the 96-well black plates to determine its fluorescence intensity F-value. Figure 8 It can be seen that the fluorescence intensity of CA NPs is optimal when the stirring time of ACh is 1.5 h.

[0092] Example 5

[0093] Effect of stirring time on the synthesis of CA-B NPs by component Azo-Bodipy 685:

[0094] Sufficient reaction time also affects the success of nanoparticle synthesis. This study investigated the effects of different reaction times (0.75 h, 1 h, 1.5 h and 2 h) on the synthesis of CA-B NPs.

[0095] 60 µL of probe Azo-Bodipy 685 (solvent acetonitrile, 1 mg / mL) was slowly added dropwise to 900 µL of Tris-HCl buffer solution. The mixture was stirred at 1000 rpm at room temperature for 0.75 h, 1 h, 1.5 h, and 2 h. 20 µL of CHO (solvent Tris-HCl buffer solution at pH 8, 0.2 U / μL) was added to the mixture and stirred at 1000 rpm at room temperature for 1 h. Then, 80 µL of ACh (solvent ultrapure water, 5 μmol) was added and the mixture was stirred for another 1.5 h. Different aggregation-driven sensor CA-B NPs were obtained from the reaction.

[0096] Place 5 µL of Tris-HCl buffer solution and 5 µL of LAChE (0.01 U / μL, in Tris-HCl buffer solution) into an EP tube and incubate at 37°C for 5 minutes. Then add 40 µL of different CA-B NPs, mix thoroughly, and incubate at 37°C for another 20 minutes. Finally, add 50 µL of DMSO solution to terminate the enzyme reaction.

[0097] 100 μL of the above optimized group was added to each of the 96-well black plates to determine its fluorescence intensity F value.

[0098] Depend on Figure 9 It can be seen that the fluorescence intensity of CA-B NPs is optimal when the stirring time of Azo-Bodipy 685 is 1.0 h.

[0099] Example 6

[0100] The morphology of CA-B NPs was observed using transmission electron microscopy.

[0101] Take 200 µL of the freshly prepared CA-B NPs solution from Example 2 and use a Malvern Instruments Zetasizer Nano 90 laser particle size analyzer to determine the hydrated particle size of the nanoparticles.

[0102] Take 200 µL of freshly prepared CA-B NPs solution and add it to the potential sample cell. Detect the zeta potential using a Malvern Instruments Zetasizer Nano 90 laser particle size analyzer.

[0103] like Figure 10 As shown, CA-B NPs exhibit a single, uniformly dispersed, nearly spherical shape, indicating the successful preparation of CA-B NPs. Figure 11 As shown, the particle size of CA-B NPs is around 167 nm, and the potential is around -21.06 mV, proving that CA-B NPs can exist stably in Tris-HCl solution.

[0104] Example 7

[0105] CA-B NPs are used to monitor AChE activity:

[0106] AChE was dissolved in Tris-HCl buffer at pH 8 and shaken with a turbo mixer to obtain a series of AChE stock solutions of different concentrations. Each AChE solution (10 μL) was added to an EP tube and incubated at 37°C for 5 minutes. Then, 40 μL of the CA-B NPs solution prepared in Example 2 was added, mixed, and incubated in a 37°C oven for 20 minutes in the dark. Finally, 50 μL of DMSO was added to terminate the enzyme cascade reaction. 100 μL of the above solution was introduced into a 96-well black plate to collect the fluorescence intensity F value. The fluorescence intensity was measured using a microplate reader at an excitation wavelength of 706 nm and an emission wavelength of 746 nm. Measurements were repeated three times for each sample. The growth rate (%) was calculated as (F-F0) / F0, where F0 is the fluorescence intensity of CA-B NPs measured without AChE, F is the fluorescence intensity in the presence of AChE, and ΔF is (F-F0).

[0107] like Figure 12As shown, the fluorescence intensity of the aggregation-driven CA-B NPs sensor increases with increasing AChE concentration, and when the linear relationship between ΔF / F0 and AChE concentration is in the range of 0.5-50 U / L, it satisfies Y = 0.787X + 2.107 and R... 2 The regression equation is 0.99, where X is AChE (U / L) and Y is the ΔF / F0 value. Figure 13 When the linear relationship between ΔF / F0 and AChE concentration is in the range of 50-500 U / L, converting the AChE concentration to a logarithmic scale satisfies Y = 118.784X -163.332 and R... 2 The regression equation is 0.99, where X is lg[AChE(U / L)] and Y is the ΔF / F0 value. Figure 14 The detection limit is 0.17 U / L. When the AChE activity reaches 500 U / L, CHO and ACh in the enzyme cascade reaction system can be completely reacted. Therefore, in subsequent experiments, 500 U / L was chosen as the detection limit for the organophosphorus pesticide triazophos.

[0108] Example 8

[0109] CA-B NPs are used to detect the organophosphorus pesticide triazophos.

[0110] A series of triazophos standard solutions with concentrations ranging from 0.0075 to 1 μg / mL (0.0075, 0.01, 0.05, 0.075, 0.1, 0.5, 0.75, and 1 μg / mL) were prepared to establish a standard curve for the organophosphorus pesticide triazophos. First, 5 μL of AChE stock solution (0.01 U / μL) and 5 μL of triazophos methanol solution were incubated at 37°C for 20 min. Then, 40 μL of CA-B NPs prepared in Example 2 were added, mixed well, and incubated in a 37°C oven for another 20 min in the dark. Finally, 50 μL of DMSO was added to terminate the enzyme cascade reaction. 100 μL of the above solution was introduced into a 96-well black plate to collect its F1 value. The fluorescence intensity was measured using a microplate reader at an excitation wavelength of 706 nm and an emission wavelength of 746 nm. Each sample was measured three times. The inhibition rate (%) was calculated as (F-F1) / F, where F is the fluorescence intensity of CA-B NPs measured in the presence of AChE but without organophosphorus pesticides, F1 is the fluorescence intensity of CA-B NPs in the presence of both AChE and organophosphorus pesticides, and ΔF is (F-F1).

[0111] like Figure 15As the triazophos concentration increases, the ΔF fluorescence signal continuously increases. After conversion to a logarithmic scale, the (F-F1) / F value of the curve shows a linear response with respect to the triazophos concentration. The fitted linear equation is: Y = 15.418X + 36.691, R² = 0.99, where X is lg[triazophos (μg / mL)] and Y is ΔF / F. The limit of detection (LOD) is calculated to be 0.0041 μg / mL. This indicates that the CA-B NPs aggregation-driven sensor has excellent sensitivity and a wide detection range for the organophosphorus pesticide triazophos.

[0112] Example 9

[0113] Detection of triazophos in real samples:

[0114] Fifteen batches of dried tangerine peel samples were selected for testing, numbered sequentially as follows: S2410050, S2409650, S2407290, S2406550, S2406090, S2405170, S2404720, S2404830, S2404160, S2403480, S2403340, S2403100, S2402270, S2402010, and S2402020. For actual sample testing, pesticide residues were extracted from the entire dried tangerine peel according to the preparation of the test solution for pesticide residue determination method in the Chinese Pharmacopoeia, and the detection method was the same as that for the triazophos standard in Example 8. The samples were then incubated with AChE and CA-B NPs for further detection of pesticide residues in the 15 batches of samples.

[0115] The preparation method of the tangerine peel extract includes the following steps: Tangerine peel is pulverized into powder (passed through a No. 3 sieve), approximately 5 g is accurately weighed, 5 g of anhydrous sodium sulfate is added, 50 mL of ethyl acetate is added, and the mixture is sonicated in an ice bath for 3 minutes. After standing, the upper layer is filtered. The residue is added to 30 mL of ethyl acetate, sonicated in an ice bath for 2 minutes, and then filtered. The two filtrates are combined, and the filter paper and residue are washed with a small amount of ethyl acetate and combined with the above filtrate. The filtrate is concentrated under reduced pressure below 40℃ to near dryness, transferred to a 5 mL volumetric flask with ethyl acetate, and diluted to the mark. 1 mL of the above solution is accurately pipetted onto a graphitized carbon column (250 mg / 3 mL pre-washed with 5 mL of ethyl acetate), and eluted with 5 mL of a hexane-ethyl acetate (1:1) mixed solution. The eluent is collected, concentrated to near dryness on a nitrogen blower, and then diluted to 1 mL with ethyl acetate. The solution is vortexed to dissolve, thus obtaining the tangerine peel extract.

[0116] like Figure 16As shown, pesticide residues were found in S2410050, S2407290, and S2404720. The results indicate that the CA-BNPs agglomeration sensor has good accuracy in detecting triazophos residues in traditional Chinese medicine and can be applied to the detection of actual samples.

Claims

1. A dual-enzyme cascade amplified fluorescence sensor based on electrostatic drive, characterized in that, The dual-enzyme cascade amplified fluorescence sensor consists of probe Azo-Bodipy 685, choline oxidase (CHO), and acetylcholine chloride (ACh); choline oxidase (CHO) and acetylcholine chloride (ACh) are electrostatically complexed to form CA NPs, which encapsulate probe Azo-Bodipy 685 to form the dual-enzyme cascade amplified fluorescence sensor CA-B NPs. The structure of the probe Azo-Bodipy 685 is shown below: .

2. A method for preparing the electrostatically driven dual-enzyme cascade amplified fluorescence sensor according to claim 1, characterized in that, The buffer solution was mixed with the probe Azo-Bodipy 685, and then choline oxidase (CHO) and acetylcholine chloride (ACh) were added to obtain a mixed solution. The mixture was stirred and allowed to stand in the dark to obtain the dual-enzyme cascade amplified fluorescent sensor CA-B NPs.

3. The preparation method according to claim 2, characterized in that, The ratio of choline oxidase (CHO) to acetylcholine chloride (ACh) is 2-6 U: 2.5-20 μmol.

4. The preparation method according to claim 2, characterized in that, The concentration of the probe Azo-Bodipy685 in the mixed solution is 1-3 mg / mL, and the enzyme activity of the choline oxidase (CHO) is 2-6 U.

5. The preparation method according to claim 2, characterized in that, The stirring speed is 1000-1500 rpm, and the stirring time is 2-2.5 h.

6. The application of the electrostatically driven dual-enzyme cascade amplification fluorescence sensor as described in claim 1 in detecting acetylcholinesterase (AChE) activity and determining organophosphorus pesticide residue content.

7. The application according to claim 6, characterized in that, The method for detecting acetylcholinesterase activity includes the following steps: S1. Dissolve acetylcholinesterase (AChE) in Tris-HCl buffer solution to obtain acetylcholinesterase (AChE) solution; S2. Add the acetylcholinesterase (AChE) solution obtained in step S1 to the well plate, incubate once, add CA-B NPs, incubate a second time, add DMSO, and measure the fluorescence response intensity.

8. The application according to claim 6, characterized in that, The method for determining organophosphorus pesticide residues using a dual-enzyme cascade amplified fluorescence sensor includes the following steps: T1, Measure the fluorescence intensity F0 of the blank buffer solution; T2, Measure the fluorescence intensity F of the control group containing acetylcholinesterase (AChE) buffer; T3. Organophosphorus pesticides and acetylcholinesterase (AChE) were co-incubated in buffer solution, and the fluorescence intensity F1 after co-incubation was measured. T4. Based on F0 obtained in step T1, F obtained in step T2, and F1 obtained in step T3, calculate the acetylcholinesterase (AChE) inhibition rate. The formula for calculating the acetylcholinesterase (AChE) inhibition rate is as follows: Acetylcholinesterase (AChE) inhibition rate = (F-F1) ÷ F × 100%. As the concentration of organophosphorus pesticides increases, the fluorescence signal continues to increase. After conversion to a logarithmic scale, the (F-F1) ÷ F × 100% value of the curve shows a linear response with the concentration of organophosphorus pesticides, thus obtaining the organophosphorus pesticide content.

9. The application according to claim 6, characterized in that, The application of the dual-enzyme cascade amplified fluorescence sensor in determining the content of organophosphorus pesticide residues in traditional Chinese medicine samples.

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