A method for colorimetric / fluorescent dual-mode detection of acetylcholinesterase and / or organophosphorus pesticides

Abstract

This invention discloses a bifunctional nanomaterial, BSA-CeO2NCs, which is made by dissolving bovine serum albumin in deionized water and adding Ce. 3+ The solution was adjusted to pH 7-12, stirred, and dried to obtain the product. This invention discloses a colorimetric / fluorescence dual-mode detection method for acetylcholinesterase and / or organophosphorus pesticides, comprising the following steps: Step (a) synthesizing bifunctional nanomaterials BSA-CeO2NCs; Step (b) constructing an acetylcholinesterase colorimetric sensor and establishing an AChE UV standard curve; Step (c) constructing an acetylcholinesterase fluorescence sensor and establishing an AChE fluorescence standard curve; Step (d) sample detection to obtain the acetylcholinesterase concentration in the sample. The bifunctional nanomaterials BSA-CeO2NCs of this invention increase the practicality of the dual-mode sensor and enable simple, rapid, and sensitive detection of AChE and OPs.

Description

Technical Field

[0001] This invention belongs to the field of analytical detection and relates to a method for detecting acetylcholinesterase and / or organophosphorus pesticides based on colorimetric / fluorescence dual-mode detection. More specifically, it relates to a method for detecting acetylcholinesterase and / or organophosphorus pesticides based on a highly sensitive colorimetric / fluorescence dual-mode sensor of nanozymes BSA-CeO2 NCs with high oxidase activity. Background Technology

[0002] Acetylcholinesterase (AChE) hydrolyzes acetylthiocholine (ATCh) to thiocholine (TCh), playing a crucial role in controlling ATCh levels. Studies have shown that ATCh imbalance can lead to various neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and myasthenia gravis. Therefore, monitoring acetylcholinesterase levels and screening for potential acetylcholinesterase inhibitors are essential.

[0003] In recent years, the overuse of organophosphorus pesticides (OPs) has caused serious ecological and human health problems. Furthermore, OPs irreversibly inhibit acetylcholinesterase, leading to abnormal accumulation of ATCh, resulting in various diseases and ultimately death. Currently, most pesticide residue detection methods (such as high-performance liquid chromatography, surface-enhanced Raman spectroscopy, and electrochemical methods) require expensive instruments and complex operations, making rapid real-time detection impossible. Therefore, designing a reliable method for on-site detection of organophosphorus pesticide residues in food and the environment is imperative.

[0004] In recent years, nanozymes have attracted increasing attention due to their high stability, modifiable catalytic activity, and low cost. However, most nanozyme-based sensors are currently limited to single-mode detection, which suffers from false positives. Dual-mode sensors not only possess inherent self-calibration and self-verification capabilities but can also meet the requirements of different detection conditions, providing more reliable detection results. Nanoclusters, as common fluorescent materials, have been reported to exhibit enzyme-like catalytic activity in recent years, becoming potential bifunctional nanozymes. However, few studies have combined the enzymatic catalytic properties of nanoclusters with their fluorescence properties to achieve multimodal sensing of analytes. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing detection methods, such as low sensitivity, time-consuming detection, high cost, and cumbersome procedures. A novel nanozyme, BSA-CeO2 NCs, was synthesized using a method doped with bovine serum albumin (BSA). Surprisingly, BSA-CeO2 NCs exhibit excellent fluorescence properties and oxidase-like properties, meeting the requirements for both fluorescence and ultraviolet (UV) detection. The inventors established a colorimetric and fluorescence sensing system using the bifunctional nanozyme BSA-CeO2 NCs and applied this novel dual-mode analysis system to detect acetylcholinesterase and organophosphorus pesticides. This invention provides a method for detecting acetylcholinesterase and / or organophosphorus pesticides using a highly sensitive colorimetric / fluorescence dual-mode sensor based on BSA-CeO2 NCs with oxidase activity. This method enables simple, rapid, intuitive, and highly sensitive detection of acetylcholinesterase and / or organophosphorus pesticides.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A bifunctional nanomaterial, BSA-CeO2 NCs, was synthesized using a BSA incubation strategy, specifically: bovine serum albumin (BSA) was dissolved in deionized water, and CeO2 was added. 3+ The solution was prepared by adjusting the pH of the reaction system to 7-12, stirring the reaction mixture, and then drying it.

[0008] The mass-to-volume ratio of bovine serum albumin (BSA) to deionized water is 50:50 to 300:50 mg / mL, preferably 100:50 to 150:50 mg / mL.

[0009] The Ce 3+ The solution is a (CH3COO)3Ce·4H2O solution prepared with deionized water; the concentration of (CH3COO)3Ce·xH2O is 10-100 mg / mL, preferably 20-50 mg / mL.

[0010] The mass ratio of BSA to (CH3COO)3Ce·4H2O is 1:1 to 6:1, preferably 2:1 to 4:1, and most preferably 3:1.

[0011] Preferably, the pH of the reaction system is adjusted to 10-11.

[0012] The reaction time is 3 to 10 hours, preferably 5 to 7 hours.

[0013] The drying process described is freeze drying. The freeze drying temperature is generally -30 to -50°C.

[0014] A method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase and / or organophosphorus pesticides, comprising the following steps:

[0015] Step (a): Synthesis of bifunctional nanomaterials BSA-CeO2 NCs;

[0016] Step (b) Construction of acetylcholinesterase (AChE) colorimetric sensor: Thioacetylcholine (ATCh) and different concentrations of AChE, and the first reaction solvent were subjected to the first incubation reaction to obtain reaction solution A; the bifunctional nanomaterial BSA-CeO2NCs, o-phenylenediamine (OPD), reaction solution A, and the second reaction solvent were mixed and subjected to the second incubation reaction. After the reaction was completed, the ultraviolet absorption curve of the sample was measured at a wavelength of 350-550 nm. The final concentration of AChE in the sample was used as the abscissa and the maximum ultraviolet absorption value at 450 nm was used as the ordinate to establish the AChE ultraviolet standard curve.

[0017] Step (c) Construction of an acetylcholinesterase (AChE) fluorescence sensor: Thioacetylcholine (ATCh) and different concentrations of AChE, along with the first reaction solvent, are subjected to a first incubation reaction to obtain reaction solution A; bifunctional nanomaterials BSA-CeO2NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent are mixed and subjected to a second incubation reaction. After the reaction, the fluorescence curve of the sample is measured at a wavelength of 400–650 nm. The final concentration of AChE in the sample is plotted on the x-axis, and the ratio F of the fluorescence value at 440 nm to the fluorescence value at 570 nm is plotted on the y-axis. 440 / F 570 Establish an AChE fluorescence standard curve with the vertical axis as the ordinate;

[0018] Step (d), Sample detection: Measure the maximum UV absorbance of the sample with unknown AChE concentration according to step (b), and substitute it into the AChE UV standard curve in step (b) to obtain the AChE concentration in the sample.

[0019] The fluorescence ratio F of the test sample with unknown AChE concentration was measured according to step (c). 440 / F 570 Substitute the AChE fluorescence standard curve from step (c) to obtain the AChE concentration in the sample to be tested.

[0020] In step (b), the first reaction solvent is a Tris-HCl buffer solution with pH 6.0 to 9.0, preferably a 50 mM Tris-HCl buffer solution with pH 7.4.

[0021] The temperature of the first incubation reaction is 30–55°C, preferably 37°C; the time of the first incubation reaction is 10–60 minutes, preferably 30 minutes.

[0022] After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent, the final concentration of OPD is 1.5–2 mM, preferably 1.67 nM; the final concentration of ATCh is 0.2 mM; the final concentration of AChE is 0.1–25 mU / mL, specifically, the final concentrations of AChE are 0.1 mU / mL, 0.5 mU / mL, 1 mU / mL, 2 mU / mL, 3 mU / mL, 5 mU / mL, 10 mU / mL, 15 mU / mL, 20 mU / mL, and 25 mU / mL; and the final concentration of BSA-CeO2 NCs is 1–50 μg / mL, preferably 5 μg / mL.

[0023] Preferably, a bifunctional nanomaterial BSA-CeO2 NCs solution and an o-phenylenediamine solution are prepared separately using ultrapure water, and 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine solution, 50 μL of reaction solution A, and 150 μL of the second reaction solvent are mixed.

[0024] The second reaction solvent is an acetate-sodium acetate buffer solution with a pH of 3.0 to 4.0, preferably a 0.2M acetate-sodium acetate buffer solution with a pH of 4.0.

[0025] The temperature of the second incubation reaction is 30–55°C, preferably 37°C; the time of the second incubation reaction is 10–60 minutes, preferably 30 minutes.

[0026] In step (c), the first reaction solvent is a Tris-HCl buffer solution with pH 6.0 to 9.0, preferably a 50 mM Tris-HCl buffer solution with pH 7.4.

[0027] The temperature of the first incubation reaction is 30–55°C, preferably 37°C; the time of the first incubation reaction is 10–60 minutes, preferably 30 minutes.

[0028] After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent, the final concentration of OPD is 1.5–2 mM, preferably 1.67 nM; the final concentration of ATCh is 0.2 mM; the final concentration of AChE is 0.1–25 mU / mL, specifically, the final concentrations of AChE are 0.1 mU / mL, 0.5 mU / mL, 1 mU / mL, 2 mU / mL, 3 mU / mL, 5 mU / mL, 10 mU / mL, 15 mU / mL, 20 mU / mL, and 25 mU / mL; and the final concentration of BSA-CeO2 NCs is 1–50 μg / mL, preferably 5 μg / mL.

[0029] Preferably, a bifunctional nanomaterial BSA-CeO2 NCs solution and an o-phenylenediamine solution are prepared separately using ultrapure water, and 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine solution, 50 μL of reaction solution A, and 150 μL of the second reaction solvent are mixed.

[0030] The second reaction solvent is an acetate-sodium acetate buffer solution with a pH of 3.0 to 4.0, preferably a 0.2M acetate-sodium acetate buffer solution with a pH of 4.0.

[0031] The temperature of the second incubation reaction is 30–55°C, preferably 37°C; the time of the second incubation reaction is 10–60 minutes, preferably 30 minutes.

[0032] The first reaction solvent in steps (b) and (c) can be the same, the second reaction solvent in steps (b) and (c) can be the same, the conditions for the first incubation reaction in steps (b) and (c) can be the same, and the conditions for the second incubation reaction in steps (b) and (c) can be the same.

[0033] In step (d), the maximum UV absorption value of the sample to be tested is measured according to step (b): Thioacetylcholine (ATCh) and the sample to be tested, and the first reaction solvent are subjected to the first incubation reaction to obtain reaction solution A; the bifunctional nanomaterial BSA-CeO2NCs, o-phenylenediamine, reaction solution A, and the second reaction solvent are mixed and subjected to the second incubation reaction. After the reaction is completed, the UV absorption curve of the sample is measured at a wavelength of 350-550 nm to obtain the maximum UV absorption value at 450 nm.

[0034] Preferably, the maximum UV absorbance at 450 nm is substituted into the AChE UV standard curve in step (b), and the AChE concentration in the sample is calculated based on the dilution factor of the sample.

[0035] The fluorescence ratio F of the sample to be tested was measured according to step (c). 440 / F 570 The first step of the reaction involves incubating thioacetylcholine (ATCh) with the test sample and the first reaction solvent to obtain reaction solution A. The second step involves mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and the second reaction solvent, and incubating the mixture. After the reaction is complete, the fluorescence curve of the sample is measured at a wavelength of 400–650 nm to obtain the fluorescence ratio F. 440 / F 570 .

[0036] Preferably, F 440 / F 570Substitute the AChE fluorescence standard curve from step (c) into the sample and then calculate the AChE concentration in the sample based on the dilution factor of the sample.

[0037] As a preferred embodiment of the colorimetric / fluorescence dual-mode detection method for acetylcholinesterase and / or organophosphorus pesticides according to the present invention, it further includes colorimetric / fluorescence detection of organophosphorus pesticides, comprising:

[0038] Step (e) Construction of organophosphorus pesticide (OPs) colorimetric sensor: ATCh, AChE and different concentrations of OPs and the first reaction solvent are subjected to the first incubation reaction to obtain reaction solution B; the bifunctional nanomaterial BSA-CeO2 NCs, o-phenylenediamine, reaction solution B and the second reaction solvent are mixed and subjected to the second incubation reaction. After the reaction is completed, the ultraviolet absorption curve of the sample is measured at a wavelength of 350-550 nm. The final concentration of OPs in the sample is used as the x-axis and the maximum ultraviolet absorption value at 450 nm is used as the y-axis to establish the Ops ultraviolet standard curve.

[0039] Step (f) Construction of organophosphorus pesticide (OPs) fluorescence sensor: ATCh, AChE, and different concentrations of OPs, along with the first reaction solvent, are subjected to a first incubation reaction to obtain reaction solution B; bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and a second reaction solvent are mixed and subjected to a second incubation reaction. After the reaction, the fluorescence curve of the sample is measured at wavelengths of 400–650 nm. The final concentration of OPs in the sample is plotted on the x-axis as ln, and the ratio of the fluorescence value at 570 nm to the fluorescence value at 440 nm is plotted as F. 570 / F 440 Establish a standard fluorescence curve for OPs with the ordinate as the ordinate;

[0040] Step (g), Sample detection: Measure the maximum UV absorbance of the sample with unknown OPs concentration according to step (e), and substitute it into the OPs UV standard curve of step (e) to obtain the OPs concentration in the sample.

[0041] The fluorescence ratio F of the test sample with unknown OPs concentration was measured according to step (f). 570 / F 440 Substitute the OPs fluorescence standard curve from step (f) to obtain the OPs concentration in the sample to be tested.

[0042] In step (e), the first reaction solvent is a pH 7.4, 50 mM Tris-HCl buffer; the temperature of the first incubation reaction is 37°C, and the incubation time is 30 minutes.

[0043] After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and a second reaction solvent, the final concentration of OPs is 0.001–6 μg / mL. Specifically, the final concentrations of OPs are 0.001 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, and 6 μg / mL, respectively; the final concentration of AChE is 25 mU / mL; the final concentration of OPD is 1.5–2 mM, preferably 1.67 mM; the final concentration of ATCh is 0.2 mM; the final concentration of AChE is 0.1–25 mU / mL, preferably 25 mU / mL; and the final concentration of BSA-CeO2 NCs is 1–50 μg / mL, preferably 5 μg / mL.

[0044] Preferably, a bifunctional nanomaterial BSA-CeO2 NCs solution and an o-phenylenediamine solution are prepared separately using ultrapure water, and 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine solution, 50 μL of reaction solution B, and 150 μL of the second reaction solvent are mixed.

[0045] The solvent for the second reaction was a pH 4.0, 0.2M acetate-sodium acetate buffer solution; the temperature for the second incubation reaction was 37°C, and the incubation time was 30 minutes.

[0046] In step (f), the first reaction solvent is a pH 7.4, 50mM Tris-HCl buffer, the first incubation temperature is 37°C, and the first incubation time is 30 minutes.

[0047] After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and a second reaction solvent, the final concentration of OPs is 0.001–6 μg / mL. Specifically, the final concentrations of OPs are 0.001 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, and 6 μg / mL, respectively; the final concentration of AChE is 25 mU / mL; the final concentration of OPD is 1.5–2 mM, preferably 1.67 mM; the final concentration of ATCh is 0.2 mM; the final concentration of AChE is 0.1–25 mU / mL, preferably 25 mU / mL; and the final concentration of BSA-CeO2 NCs is 1–50 μg / mL, preferably 5 μg / mL.

[0048] Preferably, a bifunctional nanomaterial BSA-CeO2 NCs solution and an o-phenylenediamine solution are prepared separately using ultrapure water, and 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine solution, 50 μL of reaction solution B, and 150 μL of the second reaction solvent are mixed.

[0049] The solvent for the second reaction was a pH 4.0, 0.2M acetate-sodium acetate buffer solution; the temperature for the second incubation reaction was 37°C, and the incubation time was 30 minutes.

[0050] As a preferred embodiment of the colorimetric / fluorescence dual-mode detection method for acetylcholinesterase and / or organophosphorus pesticides according to the present invention, it further includes:

[0051] Step (h), Construction of hydrogel sensor: Mix bifunctional nanomaterial BSA-CeO2 NCs solution, TMB solution, 0.2M acetate-sodium acetate buffer solution at pH=4 and sodium alginate, and incubate at 37℃ for 10 min; then add a mixed solution of CaCl2 and polyacrylic acid, and shake at 37℃ to obtain hydrogel sensor;

[0052] Step (i): AChE, ATCh, different concentrations of organophosphorus pesticides, and 50mM Tris-HCl buffer at pH 7.4 were mixed and incubated at 37°C for 30 minutes to obtain a reaction solution. The reaction solution was dropped into a hydrogel sensor and sonicated at room temperature for 10 minutes. The RGB values ​​were recorded using the color recognition application "RGB color detector" on a smartphone. An OPs standard curve was established with the concentration of organophosphorus pesticides on the x-axis and the ratio of blue channel to red channel (B / R) on the y-axis.

[0053] Step (j): Process the test sample with unknown organophosphorus pesticide concentration according to step (i). If the hydrogel sensor is blue, it indicates that the test sample contains organophosphorus pesticide. Measure the RGB values ​​of the test sample with unknown organophosphorus pesticide concentration according to step (i) to obtain the ratio B / R of the blue channel to the red channel. Substitute B / R into the OPs standard curve of step (i) to obtain the concentration of organophosphorus pesticide in the test sample.

[0054] In step (h), preferably, the bifunctional nanomaterial BSA-CeO2 NCs solution is prepared with ultrapure water and has a concentration of 30 μg / mL.

[0055] Preferably, the TMB solution is prepared with ultrapure water and has a concentration of 10 mM.

[0056] Preferably, the CaCl2 solution is prepared with ultrapure water and has a concentration of 60 mM.

[0057] Preferably, the polyacrylic acid is prepared with ultrapure water at a concentration of 5 mM.

[0058] Preferably, a solution of bifunctional nanomaterials BSA-CeO2 NCs, a TMB solution, a 0.2M acetate-sodium acetate buffer solution at pH=4, and sodium alginate are mixed to a total volume of 200 μL. The final concentration of TMB is 2.5 mM, the final concentration of BSA-CeO2 NCs is 5 μg / mL, and the final concentration of sodium alginate is 50 mg / mL. The mixture is incubated at 37°C for 10 min. Then, a mixture of 10 μL of CaCl2 solution and 10 μL of polyacrylic acid solution is added, and the mixture is shaken at 37°C to obtain a hydrogel sensor.

[0059] In step (i), after mixing AChE, ATCh, different concentrations of organophosphorus pesticides and a 50mM Tris-HCl buffer at pH 7.4, the final concentration of AChE is 25mU / mL, the final concentration of ATCh is 0.2mM, and the final concentration of organophosphorus pesticides is 0.1–40μg / mL. Specifically, the organophosphorus pesticide concentrations are 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, and 40μg / mL, respectively.

[0060] In step (j), AChE, ATCh, the sample to be tested with an unknown concentration of organophosphorus pesticide, and a 50mM Tris-HCl buffer solution at pH 7.4 are mixed according to step (i) and incubated at 37°C for 30 minutes to obtain a reaction solution. The reaction solution is then dropped into the hydrogel sensor and sonicated at room temperature for 10 minutes. If the hydrogel sensor turns blue, it indicates that the sample to be tested contains organophosphorus pesticide. As the concentration of organophosphorus pesticide increases, the color of the hydrogel changes from light blue to dark blue.

[0061] The organophosphorus pesticides mentioned are chlorpyrifos, ethoprophos, dichlorvos, and acephate.

[0062] The samples to be tested are food samples and environmental samples; the food samples can be fruits; the environmental samples can be river water or lake water.

[0063] The samples to be tested are first pretreated before being tested.

[0064] When the sample to be tested is a food sample, the pretreatment is as follows: the food sample is washed with distilled water to remove external impurities, chopped, and mixed with anhydrous ethanol at a ratio of 1:5 to 1:40 g / mL. Ultrasonic extraction is performed for 2 to 6 hours. The extract is centrifuged at 8000 to 16000 rpm for 5 to 20 minutes. The supernatant is collected and filtered through a 0.45 μm membrane to obtain the sample solution to be tested. The ultrasonic power during ultrasonic extraction is 300 W.

[0065] When the sample to be tested is an environmental sample, the pretreatment is as follows: the environmental sample is centrifuged at 8000-16000 rpm for 5-20 minutes, the supernatant is collected, and filtered through a 0.45 μm membrane to obtain the sample solution to be tested.

[0066] The detection mechanism of the method of this invention is ( Figure 1 BSA-CeO2 NCs possess both fluorescence properties and oxidase activity, catalyzing the oxidation of colorless o-phenylenediamine (OPD) to generate yellow 2,3-diaminophenazine (DAP), producing a UV signal at 450 nm. Meanwhile, AChE catalyzes the formation of ATCh with a thiol group, TCh. The reducing TCh inhibits the formation of DAP, weakening the UV signal. However, in the presence of OPs, the activity of AChE enzyme is inhibited, and the UV signal of DAP is restored, thus enabling colorimetric detection. BSA-CeO2 NCs themselves exhibit fluorescence (emission peak at 440 nm). Their fluorescence emission overlaps with the ultraviolet absorption of DAP, producing a FRET effect, which weakens the fluorescence of the NCs themselves while enhancing the fluorescence of DAP (emission peak at 570 nm). When AChE and ATCh are present, as DAP generation decreases, F570 decreases and F440 increases. When OPs are present, DAP generation recovers, which means that the F570 of DAP increases and the F440 of the fluorescence of the NCs themselves decreases, thus enabling ratiometric fluorescence detection.

[0067] Compared with the prior art, the present invention has the following significant advantages:

[0068] 1. The synthesis method of nanomaterial BSA-CeO2 NCs is simple and green.

[0069] 2. The nanomaterial BSA-CeO2 NCs not only has fluorescent properties, but also has good oxidase activity.

[0070] 3. This invention utilizes bifunctional nanomaterials BSA-CeO2 NCs to achieve dual-mode detection of two targets. The dual-mode strategy not only provides multiple methods for practical detection needs but also enables mutual verification of quantitative measurement results, improving data reliability.

[0071] 4. The bifunctional nanomaterial BSA-CeO2 NCs, due to its excellent stability, enhances the practicality of dual-mode sensors and enables simple, rapid, and sensitive detection of AChE and OPs. This invention provides a promising strategy for dual-mode quantification of analytes in complex matrices, broadening the application of nanozymes in point-of-care detection. Attached Figure Description

[0072] Figure 1This is a schematic diagram illustrating the principle of the colorimetric / fluorescence dual-mode detection of acetylcholinesterase and organophosphorus pesticides based on BSA-CeO2 NCs according to the present invention.

[0073] Figure 2 The images are high-resolution transmission electron microscopy (HRTEM) images of the nanomaterial BSA-CeO2 NCs. The scale bar on the left image is 20 nm, and the scale bar on the right image is 10 nm.

[0074] Figure 3 The image shows the X-ray diffraction (XRD) pattern of the nanomaterial BSA-CeO2NCs.

[0075] Figure 4 X-ray photoelectron spectroscopy (XPS) results for BSA-CeO2 NCs.

[0076] Figure 5 XPS spectra of Ce element in BSA-CeO2 NCs.

[0077] Figure 6 Thermogravimetric (TGA) analysis of BSA-CeO2 NCs and BSA.

[0078] Figure 7 A dual-mode detection standard curve with different concentrations of AChE was added to the detection system constructed in Example 1; wherein, Figure 7 A and B are colorimetric detection channels. Figure 7 C and D are fluorescence detection channels.

[0079] Figure 8 This is a comparison chart of the selectivity of the detection system constructed in Example 1 for AChE.

[0080] Figure 9 A dual-mode detection standard curve with different concentrations of OPs was added to the detection system constructed in Example 3; wherein, Figure 9 A and B are colorimetric detection channels. Figure 9 C and D are fluorescence detection channels.

[0081] Figure 10 This is a comparison chart of the selectivity of the detection system constructed in Example 3 for OPs.

[0082] Figure 11 A comparative graph showing the enzyme activities of nanomaterials prepared with different mass ratios of BSA and (CH3COO)3Ce·4H2O; where, Figure 11 A, B, C, and D are the double reciprocal curves for the mass ratios of BSA to (CH3COO)3Ce·4H2O of 1:1, 3:1, 5:1, and 6:1, respectively.

[0083] Figure 12The results of screening alkaloids with AChE inhibitory activity from natural alkaloids using the method of the present invention are shown.

[0084] Figure 13 To investigate the long-term stability of the dual-mode sensor.

[0085] Figure 14 RGB standard curves of different concentrations of OPs were added to the water-cell-assisted gel detection platform constructed in Example 8. Detailed Implementation

[0086] The technical solution of the present invention will be described in more detail with reference to the accompanying drawings and specific embodiments. Although the following are preferred embodiments of the present invention, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein.

[0087] The inventors studied the properties of BSA-CeO2 NCs using fluorescence and ultraviolet methods. Furthermore, the Michaelis-Menton constant (Km) of the synthesized BSA-CeO2 NCs was compared with that of HRP, indicating that BSA-CeO2 NCs possess strong oxidase activity.

[0088] Example 1

[0089] A highly sensitive colorimetric / fluorescence dual-mode method for detecting acetylcholinesterase based on BSA-CeO2 NCs with oxidase activity comprises the following steps:

[0090] Step (a) Synthesis of bifunctional nanomaterials BSA-CeO2 NCs: BSA-CeO2 NCs were synthesized using a BSA incubation strategy. At 37°C, 125 mg of bovine serum albumin (BSA) was dissolved in 50 mL of deionized water with magnetic stirring. Then, 1 mL of (CH3COO)3Ce·4H2O solution (concentration of (CH3COO)3Ce·4H2O was 42 mg / mL, prepared with deionized water) was gradually added. NaOH solution was then added to the mixed solution to adjust the pH to 10. The reaction was carried out at 37°C for 6 h, followed by freeze-drying at -30 to -50°C to obtain BSA-CeO2 NCs.

[0091] The morphology and lattice structure of the nanomaterial BSA-CeO2 NCs were studied using HRTEM, see [link to HRTEM]. Figure 2 As can be seen, the nanomaterials are uniformly distributed, with a diameter of approximately 2 nm and a lattice spacing of 0.33 nm, belonging to the (111) plane of CeO2. The XRD pattern is shown below. Figure 3 The diffraction peaks for several CeO2 structures were shown, including (111), (200), (220), (311), and (222), consistent with the HRTEM image results. X-ray photoelectron spectroscopy (XPS) is shown below. Figure 4 This study revealed the elemental composition and chemical state of the nanomaterial BSA-CeO2 NCs, showing that the nanomaterial contains carbon, nitrogen, oxygen, and cerium. The Ce3d spectrum (…) Figure 5 In the sample, the peaks at 888 eV, 9001 eV, and 906.5 eV are attributed to Ce. 3+ The peaks at 881.5 eV, 897.5 eV, and 916.2 eV are related to Ce. 4+ Relevant. Based on thermogravimetric analysis results ( Figure 6 It can be seen that CeO2 NCs account for 16.05% of the mass of BSA-CeO2 NCs. This indicates that the nanomaterial BSA-CeO2 NCs was successfully prepared.

[0092] Step (b) Construction of acetylcholinesterase (AChE) colorimetric sensor: Thioacetylcholine (ATCh) and different concentrations of AChE, Tris-HCl buffer (pH 7.4, 50 mM) were used for the first incubation reaction. The temperature of the first incubation reaction was 37℃ and the incubation time was 30 minutes, resulting in reaction solution A. A 30 μg / mL solution of bifunctional nanomaterial BSA-CeO2 NCs and a 10 mM solution of o-phenylenediamine (OPD) were prepared using ultrapure water. 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine (OPD) solution, 50 μL of reaction solution A, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, and AChE was 0.1 mU / mL, 0.5 mU / mL, 1 mU / mL, 2 mU / mL, 3 mU / mL, 5 mU / mL, 10 mU / mL, 15 mU / mL, 20 mU / mL, and 25 mU / mL, respectively. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, colorimetric analysis was performed on the reaction systems with different AChE concentrations. The UV absorption curves of samples with different AChE concentrations were measured using a UV spectrophotometer at wavelengths of 350–550 nm. Figure 7 A) Construct an AChE UV standard curve with the final concentration of AChE in the sample as the x-axis and the maximum UV absorbance at 450 nm as the y-axis: A = -0.0026C AChE +0.3719, R 2 =0.9982 ( Figure 7 B);

[0093] Step (c) Construction of acetylcholinesterase (AChE) fluorescent sensor: Thioacetylcholine (ATCh) and different concentrations of AChE, Tris-HCl buffer (pH 7.4, 50 mM) were used for the first incubation reaction. The temperature of the first incubation reaction was 37℃ and the incubation time was 30 minutes to obtain reaction solution A. 50 μL of bifunctional nanomaterial BSA-CeO2 NCs solution (concentration 30 μg / mL), 50 μL of o-phenylenediamine (OPD) solution (concentration 10 mM), 50 μL of reaction solution A, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, and AChE was 0.1 mU / mL, 0.5 mU / mL, 1 mU / mL, 2 mU / mL, 3 mU / mL, 5 mU / mL, 10 mU / mL, 15 mU / mL, 20 mU / mL, and 25 mU / mL, respectively. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, fluorescence analysis was performed on samples with different AChE contents, and the fluorescence curves were measured at wavelengths from 400 to 650 nm. Figure 7 C) Plot the final concentration of AChE in the sample on the x-axis, and calculate the fluorescence ratio F between the fluorescence value at 440 nm and the fluorescence value at 570 nm. 440 / F 570 Establish an AChE fluorescence standard curve with F as the ordinate: 440 / F 570 =0.0115C AChE +0.5262, R 2 =0.9936 ( Figure 7 D);

[0094] Step (d), Sample Detection: Following step (b), the maximum UV absorbance of the test sample with unknown AChE concentration was measured. Thioacetylcholine (ATCh) and the test sample with unknown AChE concentration, along with Tris-HCl buffer (pH 7.4, 50 mM), were subjected to a first-step incubation reaction at 37°C for 30 minutes to obtain reaction solution A. 50 μL of bifunctional nanomaterial BSA-CeO2 NCs solution (concentration 30 μg / mL), 50 μL of o-phenylenediamine (OPD) solution (concentration 10 mM), 50 μL of reaction solution A, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentration of NCs was 5 μg / mL, the final concentration of OPD was 1.67 mM, and the final concentration of ATCh was 0.2 mM. The second incubation reaction was carried out at a temperature of 37℃ for 30 minutes. After the reaction, the UV absorption curve was measured at a wavelength of 350–550 nm using a UV spectrophotometer. The maximum UV absorbance value at 450 nm was substituted into the AChE UV standard curve in step (b), and the AChE concentration in the sample was calculated based on the dilution factor of the sample.

[0095] The fluorescence ratio F of the test sample with unknown AChE concentration was measured according to step (c). 440 / F 570 The first incubation reaction was carried out with thioacetylcholine (ATCh), the test sample, and Tris-HCl buffer (pH 7.4, 50 mM) at 37°C for 30 minutes to obtain reaction solution A. Then, 50 μL of bifunctional nanomaterial BSA-CeO2 NCs solution (concentration 30 μg / mL), 50 μL of o-phenylenediamine (OPD) solution (concentration 10 mM), 50 μL of reaction solution A, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, and ATCh was 0.2 mM. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, fluorescence analysis was conducted on the samples. The fluorescence curves were measured at wavelengths of 400–650 nm, and the fluorescence ratio F440 was obtained between the fluorescence values ​​at 440 nm and 570 nm. 440 / F 570 ; F 440 / F 570 Substitute the AChE fluorescence standard curve from step (c) into the sample and then calculate the AChE concentration in the sample based on the dilution factor of the sample.

[0096] Example 2

[0097] AChE Selectivity Experiment

[0098] The selectivity of the sensor system for horseradish peroxidase (HRP), acid phosphatase (ACP), lysozyme (LZ), catalase (CAT), thrombin (Thro), phospholipase D (PLD), alkaline phosphatase (ALP), and glucose oxidase (GOx) was investigated using the colorimetric and fluorescence sensors constructed in Example 1, respectively. The final concentration of AChE was 10 mU / mL, and the final concentration of other interfering substances was 100 mU / mL.

[0099] See results Figure 8 This indicates that the colorimetric / fluorescence dual-mode sensor constructed based on BSA-CeO2 NCs has good selectivity for AChE.

[0100] Example 3

[0101] A highly sensitive colorimetric / fluorescence dual-mode method for detecting organophosphorus pesticides based on BSA-CeO2 NCs with oxidase activity, using chlorpyrifos (CPF) as an example, comprises the following steps:

[0102] Step (a): Synthesize bifunctional nanomaterial BSA-CeO2 NCs (same as step (a) in Example 1);

[0103] Step (b) Construction of organophosphorus pesticide (OPs) colorimetric sensor: ATCh, AChE, and different concentrations of OPs, Tris-HCl buffer (pH 7.4, 50 mM) were used for the first incubation reaction. The temperature of the first incubation reaction was 37℃, and the incubation time was 30 minutes, resulting in reaction solution B. A 30 μg / mL solution of bifunctional nanomaterial BSA-CeO2 NCs and a 10 mM solution of o-phenylenediamine (OPD) were prepared using ultrapure water. 50 μL of the bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of the o-phenylenediamine (OPD) solution, 50 μL of reaction solution B, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, AChE was 25 mU / mL, and chlorpyrifos (CPF) was 0.001 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, and 6 μg / mL, respectively. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, the UV absorption curves of the samples were measured at wavelengths of 350–550 nm. Figure 9 A) Using the final concentration of CPF in the sample (ln value) as the abscissa and the maximum UV absorbance at 450 nm as the apex, construct an Ops UV standard curve: A = 0.007lnC CPF +0.347, R 2 =0.995( Figure 9 B);

[0104] Step (c) Construction of organophosphorus pesticide (OPs) fluorescent sensor and OPs detection: ATCh, AChE, and different concentrations of OPs, Tris-HCl buffer (pH 7.4, 50 mM) were used for the first incubation reaction. The temperature of the first incubation reaction was 37℃, and the incubation time was 30 minutes, yielding reaction solution B. 50 μL of a 30 μg / mL bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of a 10 mM o-phenylenediamine (OPD) solution, 50 μL of reaction solution B, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, AChE was 25 mU / mL, and chlorpyrifos (CPF) was 0.001 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.25 μg / mL, 0.5 μg / mL, 1 μg / mL, 3 μg / mL, 5 μg / mL, and 6 μg / mL, respectively. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, the fluorescence curves of the samples were measured at wavelengths of 400–650 nm. Figure 9 C) Plot the final concentration of CPF in the sample (ln value) on the x-axis, and plot the fluorescence ratio F between the fluorescence value at 570 nm and the fluorescence value at 440 nm on the y-axis. 570 / F 440 Establish an OPs fluorescence standard curve with F as the ordinate: 570 / F 440 =0.0863lnC CPF +1.834, R 2 =0.992( Figure 9 D);

[0105] Step (d), Sample Detection: Following step (b), the maximum UV absorbance of the sample with unknown OPs concentration was measured. ATCh, AChE, the sample, and Tris-HCl buffer (pH 7.4, 50 mM) were used for the first incubation reaction. The temperature of the first incubation reaction was 37℃, and the incubation time was 30 minutes, yielding reaction solution B. 50 μL of a 30 μg / mL bifunctional nanomaterial BSA-CeO2 NCs solution, 50 μL of a 10 mM o-phenylenediamine (OPD) solution, 50 μL of reaction solution B, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, and AChE was 25 mU / mL. A second incubation reaction was carried out at 37°C for 30 minutes. After the reaction, the UV absorption curve of the sample was measured at wavelengths of 350–550 nm. The maximum UV absorption value at 450 nm was substituted into the OPs UV standard curve from step (b), and the OPs concentration in the sample was calculated based on the dilution factor of the sample.

[0106] The fluorescence ratio F of the test sample with unknown OPs concentration was measured according to step (c). 570 / F 440 The first incubation reaction was performed using ATCh, AChE, the test sample, and Tris-HCl buffer (pH 7.4, 50 mM) at 37°C for 30 minutes to obtain reaction solution B. Then, 50 μL of a 30 μg / mL solution of bifunctional nanomaterial BSA-CeO2 NCs, 50 μL of a 10 mM o-phenylenediamine (OPD) solution, 50 μL of reaction solution B, and 150 μL of pH 4.0, 0.2 M acetate-sodium acetate buffer were mixed. In the final reaction system, BSA-CeO2... The final concentrations of NCs were 5 μg / mL, OPD was 1.67 mM, ATCh was 0.2 mM, and AChE was 25 mU / mL. A second incubation reaction was performed at 37℃ for 30 minutes. After the reaction, the fluorescence curves of the samples were measured at wavelengths of 400–650 nm to obtain the fluorescence ratio F. 570 / F 440 ; the fluorescence ratio F 570 / F 440 Substitute the OPs fluorescence standard curve from step (c) into the sample and then calculate the OPs concentration in the sample based on the dilution factor of the sample.

[0107] Example 4

[0108] Selective experiments on OPs

[0109] The colorimetric and fluorescence sensors constructed in Example 3 were used to investigate the sensor system's performance against organophosphorus pesticides: chlorpyrifos (CPF), ethoprophos (ETH), dichlorvos (DDVP), and acephate (ACEP); other pesticides: thiamethoxam (TMX), imidacloprid (IMD), dinotefuran (DTF), and acetamiprid (NPM); and ion interferences: Fe. 3+ Na + K + Ca 2+ The selectivity was determined by the final concentration of OPs being 0.1 μg / mL and the final concentration of other interfering substances being 1 μg / mL.

[0110] See results Figure 10 This indicates that the colorimetric / fluorescence dual-mode sensor constructed based on BSA-CeO2 NCs has good selectivity for OPs-type pesticides.

[0111] Example 5

[0112] To quantify the catalytic efficiency and affinity of the material, the steady-state kinetic parameters, including the Michaelis constant (Km) and the maximum initial rate value Vmax, were calculated using a double reciprocal method. A lower Km value indicates a higher affinity between the simulated enzyme and the matrix. A higher Vmax value indicates a higher saturation reaction rate.

[0113] The preparation method of the BSA-CeO2 NCs nanomaterial is described in step (a) of Example 1. During the synthesis, the amount of BSA was fixed at 125 mg, and the mass ratio of BSA to (CH3COO)3Ce·4H2O was 1:1, 3:1, 5:1, and 6:1, respectively. Nanomaterials of the same concentration were taken, and 50 μL of TMB solutions (prepared with ultrapure water) of different concentrations were added. The solutions were then brought to a final volume of 200 μL using a 0.2 M acetate-sodium acetate buffer solution (pH = 4), resulting in a final concentration of 5 μg / mL for BSA-CeO2 NCs and final TMB concentrations of 0.05, 0.066, 0.1, 0.2, 0.33, 1.0, 2.0, and 3.3 mM, respectively. The mixture was incubated at 37 °C for 5 min, and the absorbance at 650 nm was measured using a microplate reader. The reaction rate is obtained by using the absorption values. Substituting these values ​​into 1 / V=(Km / Vmax)(1 / [S])+1 / Vmax, a double reciprocal curve is obtained. The Michaelis constant Km and the maximum initial velocity Vmax are obtained by using the intercept and slope.

[0114] Figure 11A, B, C, and D represent the Michaelis-Menten curves of the catalytic oxidation of TMB by BSA-CeO2 NCs nanomaterials synthesized at 37℃ and pH=4 according to the mass ratios of BSA to (CH3COO)3Ce·4H2O of 1:1, 3:1, 5:1, and 6:1, respectively. The Km values ​​are 0.25 mM, 0.54 mM, 0.21 mM, and 1.18 mM, respectively; the Vmax values ​​are 2.08 × 10⁻⁶, respectively. -7 M·s -1 5.64×10 -7 M·s -1 1.93×10 -7 M·s -1 and 87.72×10 -7 M·s -1 Therefore, BSA-CeO2 NCs synthesized at a mass ratio of BSA to (CH3COO)3Ce·4H2O of 3:1 have high enzyme catalytic efficiency.

[0115] Example 6

[0116] Screening of natural alkaloids with acetylcholinesterase inhibitory activity

[0117] Using donepezil as a positive control, the inhibitory activities of four natural alkaloids—berberine hydrochloride, camptothecin, matrine, and evodiamine—on acetylcholinesterase were investigated. First, the alkaloid solution (prepared with ultrapure water), AChE, and ATCh were dispersed in Tris-HCl buffer (pH 7.4, 50 mM) and incubated at 37°C for 30 minutes. Then, the incubated system was mixed with a 30 μg / mL solution of bifunctional nanomaterial BSA-CeO2 NCs (prepared in step (a) of Example 1) and a 10 mM solution of o-phenylenediamine (OPD). In the final reaction system, the final concentrations of OPD, ATCh, AChE, alkaloids, and BSA-CeO2 NCs were 1.67 mM, 0.2 mM, 25 mU / mL, 100 μM, and 5 μg / mL, respectively. An equal volume of Tris-HCl buffer (pH 7.4, 50 mM) was used as a blank control to replace the alkaloid solution. The absorbance value at 450 nm was obtained by incubating the reaction and detecting OPs according to the second step of step (b) of Example 3.

[0118] The effects of four alkaloids on acetylcholine were evaluated at a concentration of 100 μM. The results are shown in [Figure number missing]. Figure 12The results show the inhibitory effects in the presence of various alkaloids, indicating that berberine hydrochloride has a significant inhibitory effect on acetylcholine. These results suggest that this method can be used to screen for AChE inhibitors from natural alkaloids, which is of great significance for research on the diagnosis and treatment of early neurological diseases.

[0119] Example 7

[0120] Long-term stability study

[0121] A BSA-CeO2 NCs nanomaterial (prepared in step (a) of Example 1) was prepared into a BSA-CeO2 NCs solution with a concentration of 30 μg / mL using ultrapure water and stored at 4 °C. 50 μL of the BSA-CeO2 NCs solution after 1, 15, 30, 45, 60, and 90 days of storage was mixed with 50 μL of OPD solution (prepared with ultrapure water, concentration 10 mM), 200 μL of 0.2 M acetate-sodium acetate buffer solution (pH = 4), and 1.67 mM of OPD solution. The final concentration of OPD in the system was 5 μg / mL. The reaction was incubated at 37 °C for 30 minutes, and the UV absorbance and fluorescence ratio F at 450 nm were measured. 440 / F 570 .

[0122] See results Figure 13 This indicates that the dual-mode sensor constructed based on BSA-CeO2 NCs with oxidase activity has good long-term stability.

[0123] Example 8

[0124] Construction of a mobile phone-assisted hydrogel platform

[0125] Step (a): Synthesize bifunctional nanomaterial BSA-CeO2 NCs (same as step (a) in Example 1).

[0126] Step (b), Construction of a real-time hydrogel detection platform: Hydrogel fabrication: Prepare a 30 μg / mL solution of bifunctional nanomaterial BSA-CeO2 NCs and a 10 mM TMB solution using ultrapure water. Prepare a BSA-CeO2 NCs + TMB + ABS (pH = 4, 0.2 M acetate-sodium acetate buffer solution) system in centrifuge tubes. Add sodium alginate (10.0 mg) to the BSA-CeO2 NCs + TMB + ABS system, bringing the final volume to 200 μL. The final concentrations of TMB, BSA-CeO2 NCs, and sodium alginate should be 5 μg / mL and 50 mg / mL respectively. Incubate at 37℃ for 10 min. Then add 10 μL of... A mixed solution was obtained by mixing CaCl2 (60mM, prepared with ultrapure water) and 10μL of polyacrylic acid (PAA, 5mM, prepared with ultrapure water) to form a viscous liquid. The solution was shaken at 37°C for 10 minutes. The centrifuge tube was then inverted, and hydrogel spheres were obtained in the snap-on cap of the centrifuge tube, which are the hydrogel sensors. Different concentrations of CPF were reacted: AChE and ATCh, and different concentrations of CPF solutions (prepared from pH 7.4, 50mM Tris-HCl buffer) were added to Tris-HCl buffer (pH 7.4, 50mM) to make the final concentration of AChE 25mU / mL, the final concentration of ATCh 0.2mM, and the final concentrations of CPF 0, 0.1μg / mL, 0.5μg / mL, 1μg / mL, 5μg / mL, 10μg / mL, 20μg / mL, 30μg / mL, and 40μg / mL, respectively. The mixture was incubated at 37℃ for 30 minutes to obtain the reaction solution. Subsequently, 50μL of the reaction solution was dropped into the hydrogel sensor and reacted at room temperature under ultrasound (ultrasound power 300W) for 10 minutes. The RGB values ​​were recorded using the color recognition application "RGBcolor detector" on a smartphone.

[0127] like Figure 14 As shown, the hydrogel color changes from light blue to dark blue as the concentration of OPs increases, making the detection results visible to the naked eye.

[0128] Quantitative analysis of color changes was performed using a color detector installed on a smartphone: the image was recorded by the color recognition application "RGB color detector" and then converted into specific RGB values; the ratio of the blue channel to the red channel (B / R) was linearly correlated with the OPs concentration, and a standard curve was established with the concentration of chlorpyrifos as the x-axis and the ratio of the blue channel to the red channel (B / R) as the y-axis. Figure 9 B / R = 0.2192C CPF +1.6457, R 2 =0.996;

[0129] Step (c) Sample detection: Measure the RGB values ​​of the sample to be tested with unknown OPs concentration according to step (b), obtain the ratio of blue channel / red channel (B / R), substitute the ratio of blue channel / red channel (B / R) into the standard curve of step (b), and obtain the OPs concentration in the sample to be tested.

[0130] Example 9

[0131] The detection limit (LOD) of the AChE dual-mode sensor:

[0132] The first incubation reaction was carried out with thioacetylcholine (ATCh) and Tris-HCl buffer (pH 7.4, 50mM) at 37°C for 30 minutes to obtain reaction solution A. The bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine (OPD) and reaction solution A were mixed and the second incubation reaction was carried out. The specific operation was the same as step (b) and step (c) of Example 1, and 20 sets were performed in parallel.

[0133] The LOD of the colorimetric sensor was calculated to be 0.081 mU / mL using the formula LOD = 3σ / m; where σ is the standard deviation of the blank, and m is the slope of the linear equation (A = -0.0026C). AChE +0.3719, where A is the UV absorbance at 450 nm.

[0134] The LOD of the fluorescence sensor was calculated to be 0.056 mU / mL using the formula LOD = 3σ / m; where σ is the standard deviation of the blank, and m is the slope of the linear equation (F...). 440 / F 570 =0.0115C AChE +0.5262).

[0135] Detection limit of OPs dual-mode sensor:

[0136] The first incubation reaction was carried out with thioacetylcholine (ATCh), AChE and Tris-HCl buffer (pH 7.4, 50mM) at 37°C for 30 minutes to obtain reaction solution B. The bifunctional nanomaterial BSA-CeO2 NCs, o-phenylenediamine (OPD) and reaction solution B were mixed and the second incubation reaction was carried out. The specific operation was the same as step (b) and step (c) of Example 3, and 20 sets were performed in parallel.

[0137] The LOD of the colorimetric sensor was calculated to be 0.9 ng / mL using the formula LOD = 3σ / m; where σ is the standard deviation of the blank, and m is the slope of the linear equation (A = 0.007lnC). CPF+0.3473, where A is the UV absorbance at 450 nm.

[0138] The LOD of the fluorescence sensor was calculated to be 0.78 ng / mL using the formula LOD = 3σ / m; where σ is the standard deviation of the blank, and m is the slope of the linear equation (F...). 570 / F 440 =0.0863lnC CPF +1.8339).

[0139] Application Example 1

[0140] For testing of whole blood samples from normal individuals, the whole blood sample and a 10% trichloroacetic acid aqueous solution were added at a volume ratio of 1:1. The sample was incubated on ice for 10 minutes, then centrifuged at 12,000 rpm for 15 minutes. The supernatant was then used as the sample to be tested.

[0141] Add 80%, 100%, and 120% of the measured amount of AChE standard to the test samples, respectively, and measure the maximum UV absorbance and fluorescence ratio F at 450 nm according to the method in step (d) of Example 1. 440 / F 570 Substituting the maximum UV absorbance at 450 nm into the AChE UV standard curve established in step (b) of Example 1, the fluorescence ratio F was... 440 / F 570 Substitute the AChE fluorescence standard curve established in step (c) of Example 1 to obtain the AChE concentration in the sample; each sample was measured three times, the average value was taken, and the RSD and recovery rate were calculated. The results are shown in Table 1.

[0142] Table 1. Recovery rate of AChE in whole blood (n=3)

[0143]

[0144] Application Example 2

[0145] The tests were performed on fruit (apple) and river water samples. Sample pretreatment included: washing the fruit samples with distilled water to remove external impurities; chopping the washed fruit samples, adding 1.0 g of the chopped fruit sample to 20 mL of anhydrous ethanol, and extracting by ultrasonication (300 W) for 4 h; centrifuging the extract at 14000 rpm for 10 min; and filtering the supernatant through a 0.45 μm membrane to obtain the test solution. Environmental samples (river and lake water) were centrifuged at 14000 rpm for 10 min, and the supernatant was filtered through a 0.45 μm membrane to obtain the test solution.

[0146] Chlorpyrifos standard was added to the sample solution to achieve final concentrations of 0.1, 1, and 5 μg / mL. The maximum UV absorbance and fluorescence ratio F at 450 nm were measured according to the method in step (d) of Example 3. 570 / F 440 Substituting the maximum UV absorbance at 450 nm into the OPs UV standard curve established in step (b) of Example 3, the fluorescence ratio F 570 / F 440 Substitute the OPs fluorescence standard curve from step (c) of Example 3 to obtain the concentration of chlorpyrifos in the sample; each sample was measured three times, the average value was taken, and the RSD and recovery rate were calculated. The results are shown in Table 2.

[0147] Table 2. Recovery rate of chlorpyrifos in actual samples (n=3)

[0148]

[0149] The embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments.

Claims

1. A method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase, characterized in that: Includes the following steps: Step (a) Synthesis of bifunctional nanomaterials BSA-CeO2 NCs: Bovine serum albumin was dissolved in deionized water, and CeO2 was added. 3+ The solution was prepared by adjusting the pH of the reaction system to 7-12, stirring the reaction mixture, and drying; the mass-to-volume ratio of bovine serum albumin to deionized water was 50:50-300:50 mg / mL; the Ce... 3+ The solution is a (CH3COO)3Ce·4H2O solution prepared with deionized water; the concentration of (CH3COO)3Ce·4H2O is 10-100 mg / mL; the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 1:1-6:

1. Step (b) Construction of acetylcholinesterase colorimetric sensor: Thioacetylcholine and different concentrations of acetylcholinesterase and the first reaction solvent were subjected to the first incubation reaction to obtain reaction solution A; the bifunctional nanomaterial BSA-CeO2 NCs, o-phenylenediamine, reaction solution A and the second reaction solvent were mixed and subjected to the second incubation reaction. After the reaction was completed, the ultraviolet absorption curve of the sample was measured at a wavelength of 350-550 nm. The final concentration of acetylcholinesterase in the sample was used as the abscissa and the maximum ultraviolet absorption value at 450 nm was used as the ordinate to establish the AChE ultraviolet standard curve. Step (c) Construction of an acetylcholinesterase fluorescence sensor: Thioacetylcholine and different concentrations of acetylcholinesterase, along with the first reaction solvent, are subjected to a first incubation reaction to obtain reaction solution A; bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent are mixed and subjected to a second incubation reaction. After the reaction, the fluorescence curve of the sample is measured at a wavelength of 400–650 nm. The final concentration of acetylcholinesterase in the sample is plotted on the x-axis, and the ratio F of the fluorescence value at 440 nm to the fluorescence value at 570 nm is plotted on the y-axis. 440 / F 570 Establish an AChE fluorescence standard curve with the vertical axis as the ordinate; Step (d), Sample detection: Measure the maximum UV absorbance of the sample to be tested according to step (b), and substitute it into the AChE UV standard curve in step (b) to obtain the acetylcholinesterase concentration in the sample to be tested. The fluorescence ratio F of the sample to be tested was measured according to step (c). 440 / F 570 Substitute the AChE fluorescence standard curve from step (c) to obtain the acetylcholinesterase concentration in the sample to be tested.

2. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 1, characterized in that: In step (a), the mass-to-volume ratio of bovine serum albumin to deionized water is 100:50 to 150:50 mg / mL; and the concentration of (CH3COO)3Ce·4H2O is 20 to 50 mg / mL.

3. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 1, characterized in that: In step (a), the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 2:1 to 4:

1.

4. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 3, characterized in that: In step (a), the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 3:

1.

5. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 1, characterized in that: In step (b), the first reaction solvent is a Tris-HCl buffer solution with a pH of 6.0–9.0; The temperature for the first incubation reaction is 30–55°C; the incubation time for the first incubation reaction is 10–60 minutes. After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent, the final concentration of o-phenylenediamine was 1.5–2 mM, the final concentration of thioacetylcholine was 0.2 mM, the final concentration of acetylcholinesterase was 0.1–25 mU / mL, and the final concentration of BSA-CeO2 NCs was 1–50 μg / mL. The solvent for the second reaction is an acetate-sodium acetate buffer solution with a pH of 3.0–4.0; The temperature for the second incubation reaction is 30–55°C; the incubation time for the second incubation reaction is 10–60 minutes.

6. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 5, characterized in that: In step (b), the first reaction solvent is a pH 7.4, 50 mM Tris-HCl buffer.

7. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 5, characterized in that: In step (b), the temperature of the first incubation reaction is 37°C; the time of the first incubation reaction is 30 minutes.

8. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 5, characterized in that: In step (b), the second reaction solvent is a pH 4.0, 0.2 M acetate-sodium acetate buffer solution.

9. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 5, characterized in that: In step (b), the temperature of the second incubation reaction is 37°C; the incubation time is 30 minutes.

10. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 1, characterized in that: In step (c), the first reaction solvent is a Tris-HCl buffer solution with a pH of 6.0–9.0; The temperature for the first incubation reaction is 30–55°C; the incubation time for the first incubation reaction is 10–60 minutes. After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution A, and a second reaction solvent, the final concentration of o-phenylenediamine was 1.5–2 mM, the final concentration of thioacetylcholine was 0.2 mM, the final concentration of acetylcholinesterase was 0.1–25 mU / mL, and the final concentration of BSA-CeO2 NCs was 1–50 μg / mL. The solvent for the second reaction is an acetate-sodium acetate buffer solution with a pH of 3.0–4.0; The temperature for the second incubation reaction is 30–55°C; the incubation time for the second incubation reaction is 10–60 minutes.

11. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 10, characterized in that: In step (c), the first reaction solvent is a pH 7.4, 50 mM Tris-HCl buffer.

12. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 10, characterized in that: In step (c), the temperature of the first incubation reaction is 37°C; the time of the first incubation reaction is 30 minutes.

13. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 10, characterized in that: In step (c), the second reaction solvent is a pH 4.0, 0.2 M acetate-sodium acetate buffer solution.

14. The method for colorimetric / fluorescence dual-mode detection of acetylcholinesterase according to claim 10, characterized in that: In step (c), the temperature of the second incubation reaction is 37°C; the incubation time is 30 minutes.

15. A method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides, characterized in that: The organophosphorus pesticides mentioned are chlorpyrifos, ethoprophos, dichlorvos, and acephate, and the method includes colorimetric / fluorescence detection of organophosphorus pesticides: Step (1) Synthesis of bifunctional nanomaterials BSA-CeO2 NCs: Bovine serum albumin was dissolved in deionized water, and CeO2 was added. 3+ The solution was prepared by adjusting the pH of the reaction system to 7-12, stirring the reaction mixture, and drying; the mass-to-volume ratio of bovine serum albumin to deionized water was 50:50-300:50 mg / mL; the Ce... 3+ The solution is a (CH3COO)3Ce·4H2O solution prepared with deionized water; the concentration of (CH3COO)3Ce·4H2O is 10-100 mg / mL; the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 1:1-6:

1. Step (2): Constructing a colorimetric sensor for organophosphorus pesticides: Thioacetylcholine, acetylcholinesterase, and different concentrations of OPs and the first reaction solvent were subjected to the first incubation reaction to obtain reaction solution B; the bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and the second reaction solvent were mixed and subjected to the second incubation reaction. After the reaction was completed, the ultraviolet absorption curve of the sample was measured at a wavelength of 350-550 nm. The ln value of the final concentration of organophosphorus pesticides in the sample was used as the abscissa, and the maximum ultraviolet absorption value at 450 nm was used as the ordinate to establish the ultraviolet standard curve of OPs. Step (3): Constructing an organophosphorus pesticide fluorescence sensor: Thioacetylcholine, acetylcholinesterase, and different concentrations of OPs and the first reaction solvent were used for the first incubation reaction to obtain reaction solution B; the bifunctional nanomaterial BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and the second reaction solvent were mixed and used for the second incubation reaction. After the reaction, the fluorescence curve of the sample was measured at a wavelength of 400-650 nm. The final concentration of organophosphorus pesticide in the sample was used as the x-axis, and the ratio of the fluorescence value at 570 nm to the fluorescence value at 440 nm was used as the y-axis. 570 / F 440 Establish a standard fluorescence curve for OPs with the ordinate as the ordinate; Step (4), Sample detection: Measure the maximum UV absorbance of the sample to be tested according to step (e), and substitute it into the OPs UV standard curve in step (2) to obtain the concentration of organophosphorus pesticides in the sample to be tested. Measure the fluorescence ratio F of the sample to be tested according to step 3). 570 / F 440 Substitute the OPs fluorescence standard curve from step (3) to obtain the concentration of organophosphorus pesticides in the sample to be tested.

16. The method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides according to claim 15, characterized in that: In step (1), the mass-to-volume ratio of bovine serum albumin to deionized water is 100:50 to 150:50 mg / mL; and the concentration of (CH3COO)3Ce·4H2O is 20 to 50 mg / mL.

17. The method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides according to claim 15, characterized in that: In step (1), the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 2:1 to 4:

1.

18. The method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides according to claim 17, characterized in that: In step (1), the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 3:

1.

19. The method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides according to claim 15, characterized in that: In step (2), the first reaction solvent is a pH 7.4, 50 mM Tris-HCl buffer; the temperature of the first incubation reaction is 37°C, and the incubation time is 30 minutes. After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and a second reaction solvent, the final concentrations of organophosphorus pesticides were 0.001–6 μg / mL, o-phenylenediamine was 1.5–2 mM, thioacetylcholine was 0.2 mM, acetylcholinesterase was 0.1–25 mU / mL, and BSA-CeO2 NCs were 1–50 μg / mL. The solvent for the second reaction was a pH 4.0, 0.2 M acetate-sodium acetate buffer solution; the temperature for the second incubation reaction was 37°C, and the incubation time was 30 minutes. In step (3), the reaction solvent in the first step is pH 7.4, 50 mM Tris-HCl buffer, the incubation temperature in the first step is 37°C, and the incubation time in the first step is 30 minutes. After mixing bifunctional nanomaterials BSA-CeO2 NCs, o-phenylenediamine, reaction solution B, and a second reaction solvent, the final concentrations of organophosphorus pesticides were 0.001–6 μg / mL, o-phenylenediamine was 1.5–2 mM, thioacetylcholine was 0.2 mM, acetylcholinesterase was 0.1–25 mU / mL, and BSA-CeO2 NCs were 1–50 μg / mL. The solvent for the second reaction was a pH 4.0, 0.2 M acetate-sodium acetate buffer solution; the temperature for the second incubation reaction was 37°C, and the incubation time was 30 minutes.

20. A method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides, characterized in that: The organophosphorus pesticides mentioned are chlorpyrifos, ethoprophos, dichlorvos, and acephate, including: Step (1) Constructing a hydrogel sensor: Mix bifunctional nanomaterial BSA-CeO2 NCs solution, TMB solution, 0.2M acetate-sodium acetate buffer solution at pH=4 and sodium alginate, and incubate at 37℃ for 10 min; then add a mixed solution of CaCl2 and polyacrylic acid, and shake at 37℃ to obtain a hydrogel sensor. Among them, the bifunctional nanomaterial BSA-CeO2 NCs was prepared by the following method: bovine serum albumin was dissolved in deionized water, and CeO2 was added. 3+ The solution was prepared by adjusting the pH of the reaction system to 7-12, stirring the reaction mixture, and drying; the mass-to-volume ratio of bovine serum albumin to deionized water was 50:50-300:50 mg / mL; the Ce... 3+ The solution is a (CH3COO)3Ce·4H2O solution prepared with deionized water; the concentration of (CH3COO)3Ce·4H2O is 10-100 mg / mL; the mass ratio of bovine serum albumin to (CH3COO)3Ce·4H2O is 1:1-6:

1. Step (2): Mix acetylcholinesterase, thioacetylcholine, different concentrations of organophosphorus pesticides and pH 7.4, 50 mM Tris-HCl buffer, and incubate at 37°C for 30 minutes to obtain a reaction solution; drop the reaction solution into the hydrogel sensor, sonicate at room temperature for 10 minutes, and record the RGB values ​​using a color recognition application on a smartphone; establish an OPs standard curve with the concentration of organophosphorus pesticides as the x-axis and the ratio of blue channel to red channel (B / R) as the y-axis; Step (3): Process the sample with unknown organophosphorus pesticide concentration according to step (2). If the hydrogel sensor is blue, it means that the sample contains organophosphorus pesticide. Measure the RGB values ​​of the sample with unknown organophosphorus pesticide concentration according to step (2) and obtain the ratio B / R of the blue channel / red channel. Substitute B / R into the OPs standard curve in step (2) to obtain the concentration of organophosphorus pesticide in the sample.

21. The method for colorimetric / fluorescence dual-mode detection of organophosphorus pesticides according to claim 20, characterized in that: In step (2), after mixing acetylcholinesterase, thioacetylcholine, different concentrations of organophosphorus pesticides and a pH 7.4, 50 mM Tris-HCl buffer, the final concentration of acetylcholinesterase is 25 mU / mL, the final concentration of thioacetylcholine is 0.2 mM, and the final concentration of organophosphorus pesticides is 0.1–40 μg / mL.

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