A method for detecting organophosphorus pesticides using a ratiometric fluorescence and photothermal dual-mode probe

Abstract

A method for detecting organophosphorus pesticides using a dual-mode probe of ratio fluorescence and photothermal effect, including photothermal detection, ratio fluorescence detection and data processing. The photothermal detection includes adding acetylcholinesterase and a sample solution to be tested into a PBS buffer solution, irradiating with near-infrared light at 808 nm for 15-25 min after treatment, recording the photothermal signal of the solution using an infrared thermal camera, and substituting it into a linear fitting equation to obtain the photothermal detection concentration value of the organophosphorus pesticide sample. The ratio fluorescence detection includes adding acetylcholinesterase and a sample solution to be tested into a PBS buffer solution, recording the fluorescence spectrum at room temperature after treatment, calculating the fluorescence intensity ratio of two fluorescence signals, and substituting it into the corresponding linear fitting equation to obtain the ratio fluorescence detection value of the organophosphorus pesticide sample. The detection value is equal to (photothermal detection concentration value + ratio fluorescence detection value) / 2. The present invention simultaneously uses two methods to quickly detect organophosphorus pesticide residues, and the two detection values can be mutually verified, improving the reliability of the detection.

Description

Technical Field

[0001] The present invention relates to a method for detecting organophosphorus pesticides by using a ratio fluorescence and photothermal dual-mode probe, belonging to the technical field of pesticide residue detection. Background Art

[0002] Organophosphorus pesticides refer to organic compound pesticides containing phosphorus elements, which are mainly used for preventing and controlling plant diseases, insects and weeds. Most organophosphorus pesticides are phosphoric acid esters or thiophosphoric acid esters, such as commonly used parathion, demeton, malathion, dimethoate, trichlorfon, dichlorvos and chlorpyrifos, etc.

[0003] As common pesticides, organophosphorus (OPs) pesticides are widely used in agricultural production. OPs play a huge role in increasing crop yields and improving the quality of agricultural products. The extensive use of these OPs compounds in fruits, vegetables and other crops has led to OPs pesticide residues. Research shows that almost all OPs can inhibit the activity of acetylcholinesterase (AChE), and the normal operation of the human central nervous system must ensure the activity of AChE. The presence of OPs causes AChE to be unable to normally catalyze acetylcholine (ATCh), resulting in the excessive accumulation of ATCh, thus causing choline poisoning in organisms, and then triggering various health problems such as cancer, diabetes, respiratory diseases, nervous system diseases, and reproductive diseases. In addition, the problem of pesticide residues caused by the long-term improper use of pesticides can lead to groundwater pollution and affect biodiversity, etc. The World Health Organization (WHO) and the Food and Agriculture Organization of the United Nations (FAO) have stipulated, in accordance with good agricultural practice (GAP) norms, that the maximum concentration of pesticide residues formed in food and feed after the direct or indirect use of pesticides is the maximum residue limit (MRL). International organizations and governments of various countries have successively promulgated a series of laws and regulations to clearly stipulate the maximum residue limits of organophosphorus pesticides in plant-derived foods. Among them, the European Union stipulates that the maximum residue limit of acephate in wheat should be ≤0.02 mg / kg, in corn ≤0.02 mg / kg, in solanaceous vegetables ≤0.02 mg / kg, and in watermelon ≤0.02 mg / kg. China stipulates that the maximum residue limit of acephate in wheat should be ≤0.2 mg / kg, in corn ≤0.2 mg / kg, in solanaceous vegetables ≤0.02 mg / kg, and in watermelon ≤0.02 mg / kg. Considering the harmful effects of OPs on human health and the environment, the problem of pesticide residues and its rapid and accurate detection and analysis technology have attracted great attention.

[0004] Traditional analytical methods mainly include gas chromatography, liquid chromatography, mass spectrometry and their combined determination methods. Although these methods can provide high sensitivity and accuracy, the expensive equipment, complex operation and dependence on professional technicians limit their wide application. In recent years, fluorescence detection method has attracted much attention due to its advantages such as high sensitivity, fast reaction, simple operation and low cost. Most fluorescence detections are based on a single signal, and it is difficult to identify the enhancement or fading of color with the naked eye, and its detection performance is easily interfered by probe concentration and environmental factors. The emergence of ratio fluorescence probes brings new hope for solving the above disadvantages, and the two fluorescence signals are easier to visualize and detect, improving the detection sensitivity.

[0005] However, as a single detection mode, the detection performance of fluorescence detection is easily interfered by environmental factors, resulting in false signals. In contrast, the dual-mode detection method can provide multiple information, and the detection results can be mutually verified, significantly improving the accuracy and reliability. In this case, dual-mode probes have recently been developed for real-time detection to provide more comprehensive and accurate results. For example, colorimetric / fluorescent probes have been used to detect organophosphorus pesticide residues. These biosensors can well achieve dual-signal and sensitivity detection of analytes, but are affected by the autofluorescence and background color of biological samples. Due to the photothermal effect, photothermal materials can be precisely heated under near-infrared (NIR) light irradiation and have been applied in biosensing. In photothermal biosensing, the photothermal signal can be read by a simple thermometer or an infrared camera, which can not only eliminate the need for precision instruments, but also improve the sensitivity of photothermal sensing. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe.

[0007] To achieve the above purpose and other related purposes, the technical solution provided by the present invention is: A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe, including photothermal detection, ratio fluorescence detection and data processing:

[0008] The photothermal detection includes: adding acetylcholinesterase and the sample solution to be tested into PBS buffer, incubating after mixing at 36 - 38 °C to obtain a first solution system; next, adding acetylcholine, manganese dioxide nanosheet solution and ruthenium tris(bipyridine) solution into the first solution system, reacting for 25 - 35 min to obtain a reaction solution; then transferring the reaction solution into a multi-well plate, irradiating with 808 nm near-infrared light for 15 - 25 min, and using an infrared thermal camera to record the photothermal signal of the solution, substituting it into a linear fitting equation to obtain the photothermal detection value of the organophosphorus pesticide sample;

[0009] The ratio fluorescence detection includes: adding acetylcholinesterase and the sample solution to be measured into PBS buffer, mixing and incubating at 36 - 38 °C to obtain a second solution system; next, adding acetylcholine, manganese dioxide nanosheet solution and ruthenium tris(bipyridine) solution into the second solution system, reacting for 25 - 35 min to obtain a reaction solution; adding thiamine solution to the reaction solution and incubating at 36 - 38 °C for 25 - 35 min, recording the fluorescence spectrum at room temperature, calculating the fluorescence intensity ratio of two fluorescence signals, substituting into the corresponding linear fitting equation to obtain the ratio fluorescence detection value of the organophosphorus pesticide sample;

[0010] Data processing includes: calculating the detection value of the organophosphorus pesticide sample;

[0011] Detection value = (photothermal detection value + ratio fluorescence detection value) / 2.

[0012] The preferred technical solution is:

[0013] Linear fitting equation for photothermal detection:

[0014] For dichlorvos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.731×logx + 8.668, R 2 = 0.993, S / N = 3, and the detection limit is 1.01 ng / mL;

[0015] For chlorpyrifos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.478×logx + 8.594, R 2 = 0.996, S / N = 3), and the detection limit is 1.02 ng / mL;

[0016] Linear fitting equation for ratio fluorescence detection:

[0017] For dichlorvos, in the range of 0.1 - 50 ng / mL, the fitting equation is y = 0.136×logx + 0.054, R 2 = 0.999, S / N = 3, in the range of 50 - 8000 ng / mL, the fitting equation is y = 0.114x + 0.852, R 2 = 0.992, S / N = 3, and the detection limit is 1.13 pg / mL;

[0018] For chlorpyrifos, in the range of 1 - 8000 ng / mL, the fitting equation is y = 0.098x + 0.302, R 2 = 0.996, S / N = 3, and the detection limit is 0.86 ng / mL.

[0019] The preferred technical solution is as follows: The preparation method of the manganese dioxide nanosheet solution includes: adding a KMnO4 solution to an MES buffer solution, then ultrasonically treating the mixture, centrifuging, washing the precipitate with ultrapure water, vacuum-drying the washed precipitate to obtain a manganese dioxide nanosheet solid, and dispersing it in water to prepare a 0.5 mg / mL manganese dioxide nanosheet solution; the concentration of the KMnO4 solution is 0.1 M; the concentration of the MES buffer solution is 0.1 M, and the pH value is 6.

[0020] Due to the application of the above technical solution, the advantages of the present invention compared with the prior art are as follows:

[0021] 1. The present invention simultaneously uses two methods to rapidly detect organophosphorus pesticide residues, and the two detection values can be mutually verified, improving the reliability of detection.

[0022] 2. The present invention takes the average value of the photothermal detection concentration value and the ratio fluorescence detection value of the organophosphorus pesticide sample as the detection value, improving the accuracy of detection.

[0023] 3. The present invention integrates the ratio fluorescence and photothermal detection modes to form a dual-mode probe, which can reduce the generation of false positive signals, and the two detection results can be mutually verified, thereby improving the sensitivity and reliability of detection. This dual-mode probe with a self-verification function is expected to provide more accurate and robust results than a single-mode probe and has a broader application prospect. Description of the Drawings

[0024] Figure 1 Shows the schematic diagram of the analysis method for detecting organophosphorus pesticides by a ratio fluorescence and photothermal dual-mode probe;

[0025] Figure 2 A of shows the TEM image of manganese dioxide nanosheets, Figure 2 B of shows the HADDF-STEM image of manganese dioxide nanosheets;

[0026] Figure 3 Shows the fluorescence spectra of thiamine, thiamine and manganese dioxide nanosheet mixed solution, and the inset is a photo under 365 nm ultraviolet light;

[0027] Figure 4 Shows the fluorescence spectra of ruthenium pyridine, ruthenium pyridine and manganese dioxide nanosheet mixed solution;

[0028] Figure 5 Shows the photothermal heating curves (808 nm laser, 2 W / cm 2 ) at manganese dioxide nanosheet concentrations of 0, 4, 12, 20 μg / mL.

[0029] Figure 6 A of shows the photothermal photos at different organophosphorus concentrations,Figure 6 B shows the fluorescence spectra at different organophosphorus concentrations (the inset is a photo under 365 nm ultraviolet light).

[0030] Figure 7 A shows the fitting relationship between temperature change and dichlorvos concentration, Figure 7 B shows the fitting relationship between temperature change and chlorpyrifos concentration, Figure 7 C shows the fitting relationship between fluorescence intensity ratio and dichlorvos concentration, Figure 7 D shows the fitting relationship between fluorescence intensity ratio and chlorpyrifos concentration. Detailed implementation manners

[0031] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this embodiment.

[0032] Please refer to Figure 1-7 . It should be noted that the structures, ratios, sizes, etc. shown in the drawings of this specification are only used to cooperate with the content disclosed in the specification for those skilled in the art to understand and read, and are not used to limit the limited conditions for the implementation of the present invention. Therefore, they do not have technical essence. Any modification of the structure, change of the proportional relationship or adjustment of the size. The following embodiments are provided to better understand the present invention, rather than to limit the present invention. The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The experimental materials used in the following embodiments are all obtained from conventional biochemical reagent stores unless otherwise specified.

[0033] Reagents and instruments used in the present invention:

[0034] Potassium permanganate (KMnO4) comes from Kelong Chemical Co., Ltd. (Chengdu, China). 4-Morpholineethanesulfonic acid (MES), thiamine (TH), and tris(2,2'-bipyridine)ruthenium(II) hexahydrate chloride (Ru(bpy)3Cl2·6H2O) are all provided by Macklin Biochemical Technology Co., Ltd. (Shanghai, China). Acetylcholinesterase (AChE from fly heads) and acetylcholine (ATCh) are provided by Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Organophosphorus pesticide standard solutions are purchased from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China).

[0035] In the embodiments of the present invention, dichlorvos and chlorpyrifos are uniformly selected as the organophosphorus pesticides. However, the solution of the present application is not limited to dichlorvos and chlorpyrifos in specific applications, and can also be used for organophosphorus pesticides such as quinalphos and methyl parathion.

[0036] The PBS buffer solution in the present invention is: 0.1M NaH2PO4·2H2O, 0.1M Na2HPO4·12H2O, pH 7.

[0037] Example 1: A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe

[0038] I. Synthesis of manganese dioxide nanosheets (MnO2 NSs)

[0039] Add 9 mL of KMnO4 solution (0.1 M) to 12 mL of MES buffer (0.1 M, pH 6.0). Then ultrasonically treat the mixture for 5 min to form a dark brown colloid. Centrifuge the resulting solution at 8000 rpm / min for 10 min, wash it 3 times with ultrapure water, and after drying the precipitate in a vacuum at 60 °C for 24 h, disperse it in water with a concentration of 1 mg / mL for subsequent experiments.

[0040] Material characterization and properties

[0041] (1) Structural morphology

[0042] Figure 2 A is the TEM image of MnO2 NSs in aqueous solution. It can be seen that MnO2 NSs exhibit a two-dimensional sheet structure with an average lateral size close to 200 nm; Figure 2 The HR-TEM image of B shows that the lattice spacing of MnO2 NSs is 0.56 nm, and the energy dispersive spectroscopy (EDS) elemental mapping shows that manganese and oxygen elements are evenly distributed on MnO2 NSs.

[0043] (2) Peroxidase-like activity of MnO2 NSs

[0044] Figure 3 is the fluorescence spectrum of the thiamine, MnO2 NSs, and thiamine mixed solution and the image under a 365 nm ultraviolet lamp. It can be seen that thiamine itself has no fluorescence. After adding MnO2 NSs, strong blue fluorescence is shown at 440 nm. This indicates that MnO2 NSs can oxidize thiamine to strong fluorescence thiochrome.

[0045] (3) Fluorescence quenching effect of MnO2 NSs

[0046] Figure 4 is the fluorescence spectrum of the ruthenium bipyridine, MnO2 NSs, and ruthenium bipyridine mixed solution. It can be seen that after adding MnO2 NSs, the fluorescence at 612 nm decreases. This indicates that MnO2 NSs can quench the fluorescence of ruthenium bipyridine.

[0047] (4) Photothermal performance of MnO2 NSs

[0048] Figure 5It is the heating curve of water and aqueous dispersions of MnO2 NSs with different concentrations. It can be seen that the temperature of the MnO2 NSs dispersion increases with the increase of concentration, while the water temperature in the control group does not increase significantly, indicating that MnO2 NSs can effectively perform photothermal conversion under 808 nm laser irradiation and increase the local temperature.

[0049] Therefore, the peroxidase-like activity, quenching effect, and good photothermal effect of MnO2 NSs can be utilized for ratio fluorescence detection and photothermal detection.

[0050] II. Construction of the dual-mode probe

[0051] Disperse 4 mg of manganese dioxide nanosheets in 8 mL of ultrapure water to prepare a 0.5 mg / mL manganese dioxide nanosheet solution. Mix 50 μL of the manganese dioxide nanosheet solution with ATCh (50 μL, 6 mg / mL) solution and ruthenium terpyridine (50 μL, 0.4 mg / mL) solution, and mix the composite solution evenly to form a dual-mode probe. Incubate the organophosphorus pesticide solution with AChE in PBS buffer to obtain a mixed solution, and add the dual-mode probe to the mixed solution. After irradiating with 808 nm near-infrared light for 20 min, use an infrared thermal camera to record the photothermal signal of the solution, record the rising temperature of the solution, substitute it into the corresponding linear fitting equation to obtain the concentration of the organophosphorus pesticide, and complete the photothermal detection of the organophosphorus pesticide. After incubating with thiamine solution for 40 min, record the fluorescence spectrum of the probe solution in the range of 390 - 700 nm with 370 nm excitation light, calculate the fluorescence intensity ratio of the two fluorescence signals, substitute it into the corresponding linear fitting equation to obtain the concentration of the organophosphorus pesticide, and complete the fluorescence detection of the organophosphorus pesticide. Finally, take the average value of the two organophosphorus concentrations calculated by photothermal detection and fluorescence detection respectively as the result measured by the dual-mode probe. Photothermal photos, fluorescence spectra, and visualization photos are shown in Figure 6 。

[0052] III. Detection of organophosphorus pesticides

[0053] (i) Photothermal detection

[0054] Mix 50 μL of AChE (1 U / mL) and 50 μL of OPs solutions with different concentrations in 1 mL of PBS buffer and incubate at 37 °C for 30 min. Next, add 50 μL of ATCh (6 mg / mL), 50 μL of manganese dioxide nanosheets (0.5 mg / mL), and 50 μL of ruthenium terpyridine (0.4 mg / mL) solution to the above solution at 37 °C and react for 30 min. Next, introduce the reaction solution into a 48-well plate. After irradiating with 808 nm near-infrared light for 20 min, use an infrared thermal camera to record the photothermal signal of the solution, record the rising temperature of the solution, substitute it into the corresponding linear fitting equation to obtain the concentration of the organophosphorus pesticide.

[0055] (ii) Fluorescence detection

[0056] Mix 50 μL of AChE (1 U / mL) and 50 μL of OPs solutions with different concentrations in 1 mL of PBS buffer and incubate at 37 °C for 30 min. Next, add 50 μL of ATCh (6 mg / mL), 50 μL of manganese dioxide nanosheets (0.5 mg / mL), and 50 μL of ruthenium terpyridine (0.4 mg / mL) solutions to the above solution at 37 °C and react for 30 min. Finally, add 50 μL of thiamine (4 mg / mL) solution and incubate at 37 °C for 40 min. Record the fluorescence spectrum at room temperature, calculate the fluorescence intensity ratio of the two fluorescence signals, substitute it into the corresponding linear fitting equation to obtain the concentration of organophosphorus pesticides.

[0057] According to the photothermal signal obtained by method (i), record the temperature rise of the solution; according to the fluorescence spectrum obtained by method (ii), calculate the fluorescence intensity ratio (I 440 / I 612 ). Respectively plot the concentration of organophosphorus pesticides against the temperature rise of the solution and the fluorescence intensity ratio (I 440 / I 612 ) in origin software for linear fitting to obtain the corresponding linear fitting regression equation. Substitute the photothermal signal obtained by photothermal detection and the fluorescence signal obtained by fluorescence detection into the corresponding fitting equation to calculate the corresponding organophosphorus concentration, and take its average value as the detection result of the final organophosphorus concentration.

[0058] Specifically, the linear fitting results are as Figure 7 shown. As Figure 7 shown in FIGS. 7A and 7B, the results show that there is a good linear relationship between the photothermal signal and the concentrations of dichlorvos and chlorpyrifos. Among them, for dichlorvos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.731×logx + 8.668 (R 2 = 0.993, S / N = 3), and the detection limit is 1.01 ng / mL; for chlorpyrifos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.478×logx + 8.594 (R 2 = 0.996, S / N = 3), and the detection limit is 1.02 ng / mL. As Figure 7 shown in FIGS. 7C and 7D, the results show that there is a good linear relationship between the fluorescence signal and the concentrations of dichlorvos and chlorpyrifos. Among them, for dichlorvos, in the range of 0.1 - 50 ng / mL, the fitting equation is y = 0.136×logx + 0.054 (R 2 = 0.999, S / N = 3), and in the range of 50 - 8000 ng / mL, the fitting equation is y = 0.114x + 0.852 (R 2= 0.992, S / N = 3), and the detection limit was 1.13 pg / mL; for chlorpyrifos, in the range of 1 - 8000 ng / mL, the fitting equation was y = 0.098x + 0.302 (R 2 = 0.996, S / N = 3), and the detection limit was 0.86 ng / mL.

[0059] IV. Application of the dual-mode probe in tea samples

[0060] To evaluate the application potential and feasibility of this method in actual detection, the standard addition method was used to analyze the residues of dichlorvos and chlorpyrifos in tea. Standard solutions of dichlorvos and chlorpyrifos at different concentrations were added to the tea, left at room temperature for 1 hour, and stored in the refrigerator overnight to allow the analytes to be fully absorbed. The tea samples were purchased from a local supermarket and pretreated according to the literature: 0.5 g of the tea sample was immersed in 50 mL of water, ultrasonicated for 20 min, centrifuged at 4000 rpm / min for 10 min, the supernatant was transferred to another centrifuge tube, and the residual liquid was extracted by adding 20 mL of water. The upper aqueous solutions were combined and filtered through a 0.45 μm filter. Finally, the constructed dual-mode probe was used for detection.

[0061] V. Application of the dual-mode probe in brown rice samples

[0062] To evaluate the application potential and feasibility of this method in actual detection, the standard addition method was used to analyze the residues of dichlorvos and chlorpyrifos in brown rice. Standard solutions of dichlorvos and chlorpyrifos at different concentrations were added to the brown rice, left at room temperature for 1 hour, and stored in the refrigerator overnight to allow the analytes to be fully absorbed. The brown rice samples were pretreated as follows: 10 g of the well-ground sample was weighed into a conical flask containing 50 mL of distilled water, ultrasonicated for 30 min, and then centrifuged at 4500 rpm / min for 10 min in a centrifuge to remove insoluble impurities. 15 mL of the supernatant was transferred to a centrifuge tube containing 15 mL of dichloromethane, vortexed for 1 min and then centrifuged for 10 min. Then, 10 mL of the supernatant was taken and filtered through a 0.22 μm microporous membrane. Finally, the constructed dual-mode probe was used for detection.

[0063] The results are shown in Table 1. The recoveries of dichlorvos and chlorpyrifos in the actual samples were 95% - 107.5% and 96% - 106.5% respectively, and the relative standard deviation ranged from 0.07% to 2.34%. These results showed good recoveries and relative standard deviations, indicating that the provided method performed well in the detection of OPs. Therefore, this analytical method will have excellent application potential in the detection of organophosphorus in actual samples.

[0064] Table 1. Detection of dichlorvos and chlorpyrifos in real samples.

[0065]

[0066]

[0067] Example 2: A method for detecting organophosphorus pesticides using a dual-mode fluorescence and photothermal probe

[0068] The dual-mode probe mentioned above refers to integrating the ratio fluorescence and photothermal detection modes to jointly detect organophosphorus pesticide residues.

[0069] Probe definition: A fluorescence probe uses a fluorescent substance as an indicator, and under the excitation of light with a certain wavelength, the indicator generates fluorescence. Qualitative or quantitative analysis of the substance to be detected is achieved by detecting the generated fluorescence. A photothermal probe uses a substance with a photothermal effect as an indicator. Under laser irradiation, it absorbs light energy and converts it into heat energy, causing the temperature to rise.

[0070] The solution reaction system of the present invention is the probe because this solution reaction system contains both a fluorescent substance and a substance with a photothermal effect, and can simultaneously perform fluorescence detection and photothermal detection, that is, it has two detection methods, so it is called a dual-mode probe.

[0071] The entire solution system for detecting OPs:

[0072]

[0073]

[0074] Photothermal detection

[0075] 1 mL PBS + 50 μL AChE + 50 μL OPs at 37 °C for 30 min;

[0076] + 50 μL MnO2 NSs + 50 μL Ru + 50 μL ATCh at 37 °C for 30 min;

[0077] Irradiate with laser for 20 min, record the rising temperature, substitute it into the fitting equation, and calculate the OPs concentration;

[0078] Fluorescence detection

[0079] 1 mL PBS + 50 μL AChE + 50 μL OPs at 37 °C for 30 min;

[0080] + 50 μL MnO2 NSs + 50 μL Ru + 50 μL ATCh at 37 °C for 30 min;

[0081] + 50 μL TH at 37 °C for 40 min.

[0082] The fluorescence spectrometer measures the fluorescence spectrum, records the fluorescence intensity ratio of two fluorescence signals, substitutes it into the fitting equation, and calculates the OPs concentration;

[0083] The detected value of OPs = (the value of photothermal detection concentration + the value of ratio fluorescence detection) / 2

[0084] A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe, the content range of the detected organophosphorus pesticides is: 0.1 ng / mL - 8000 ng / mL (dichlorvos), 1 ng / mL - 8000 ng / mL (chlorpyrifos), and the detection accuracy reaches: 1.13 pg / mL (dichlorvos), 0.86 ng / mL (chlorpyrifos).

[0085] A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe, specifically including the following technical steps.

[0086] (1) Prepare a manganese dioxide nanosheet solution

[0087] Add 9 mL of KMnO4 solution to 12 mL of MES buffer. Then ultrasonicate the mixture for 5 minutes to form a dark brown colloid. Centrifuge the obtained solution, then wash it 3 times with ultrapure water, and dry the precipitate in a vacuum at 60 °C for 24 h to obtain manganese dioxide nanosheet solid. Among them, the concentration of KMnO4 is 0.1 M, the concentration of MES buffer is 0.1 M, and pH = 6.

[0088] (2) Prepare an organophosphorus pesticide sample solution

[0089] React the organophosphorus pesticide sample with acetylcholinesterase in PBS buffer to obtain a mixed solution. The volume ratio of the organophosphorus pesticide to acetylcholinesterase is 1:1. The concentration of acetylcholinesterase is 0.1 U / mL - 1 U / mL. Among them, the PBS buffer solution is: 0.1 M NaH2PO4·2H2O, 0.1 M Na2HPO4·12H2O, pH 5 - 10.

[0090] (3) Photothermal detection

[0091] Mix 50 μL of AChE (1 U / mL) and 50 μL of OPs solutions with different concentrations in 1 mL of PBS buffer and incubate at 37 °C for 30 min. Next, add 50 μL of ATCh (6 mg / mL), 50 μL of manganese dioxide nanosheets (0.5 mg / mL), and 50 μL of ruthenium tris(bipyridine) (0.4 mg / mL) solution to the above solution at 37 °C and react for 30 min. Next, introduce the reaction solution into a 48-well plate, irradiate it with 808 nm near-infrared light for 20 min, and then use an infrared thermal camera to record the photothermal signal of the solution, and substitute it into the corresponding linear fitting equation to obtain the photothermal detection concentration value of the organophosphorus pesticide sample.

[0092] The concentration of the thiamine solution is 0.1 mg / mL to 6 mg / mL.

[0093] The concentration of the ruthenium terpyridine solution is 0.4 mg / mL.

[0094] (4) Ratio fluorescence detection

[0095] Mix 50 μL of AChE (1 U / mL) and 50 μL of OPs solutions with different concentrations in 1 mL of PBS buffer, and incubate at 37 °C for 30 min. Next, add 50 μL of ATCh (6 mg / mL), 50 μL of manganese dioxide nanosheets (0.5 mg / mL), and 50 μL of ruthenium terpyridine (0.4 mg / mL) solution to the above solution and react at 37 °C for another 30 min. Finally, add 50 μL of thiamine (4 mg / mL) solution and incubate at 37 °C for 30 min. Record the fluorescence spectrum at room temperature, calculate the fluorescence intensity ratio of the two fluorescence signals, and substitute it into the corresponding linear fitting equation to obtain the ratio fluorescence detection value of the organophosphorus pesticide sample.

[0096] The concentration of the thiamine is 4 mg / mL; the concentration of the ruthenium terpyridine is 0.4 mg / mL.

[0097] (5) Calculate the detection value of the organophosphorus pesticide sample

[0098] Calculate the average value of the photothermal detection concentration value of the organophosphorus pesticide sample in step (3) and the ratio fluorescence detection value of the organophosphorus pesticide sample in step (4), which is the detection value of the organophosphorus pesticide sample.

[0099] Detection value = (Photothermal detection concentration value + Ratio fluorescence detection value) / 2.

[0100] Example 3: A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe

[0101] A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe, including photothermal detection, ratio fluorescence detection, and data processing:

[0102] The photothermal detection includes: adding acetylcholinesterase and the sample solution to be detected to PBS buffer, mixing and incubating under the condition of 36 - 38 °C to obtain a first solution system; next, adding acetylcholine, manganese dioxide nanosheet solution, and ruthenium terpyridine solution to the first solution system, reacting for 25 min to obtain a reaction solution; then transferring the reaction solution into a multi-well plate, irradiating with 808 nm near-infrared light for 15 min, and using an infrared thermal camera to record the photothermal signal of the solution, and substituting it into the linear fitting equation to obtain the photothermal detection value of the organophosphorus pesticide sample;

[0103] The ratio fluorescence detection includes: adding acetylcholinesterase and the sample solution to be detected into PBS buffer, mixing and incubating at 36 °C to obtain a second solution system; next, adding acetylcholine, manganese dioxide nanosheet solution and ruthenium tris(bipyridine) solution into the second solution system, reacting for 25 min to obtain a reaction solution; adding thiamine solution into the reaction solution, incubating at 36 °C for 25 min, recording the fluorescence spectrum at room temperature, calculating the fluorescence intensity ratio of two fluorescence signals, substituting it into the corresponding linear fitting equation to obtain the ratio fluorescence detection value of the organophosphorus pesticide sample;

[0104] Data processing includes: calculating the detection value of the organophosphorus pesticide sample;

[0105] Detection value = (photothermal detection value + ratio fluorescence detection value) / 2.

[0106] The preferred technical solution is:

[0107] Linear fitting equation for photothermal detection:

[0108] For dichlorvos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.731×logx + 8.668, R 2 = 0.993, S / N = 3, and the detection limit is 1.01 ng / mL;

[0109] For chlorpyrifos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.478×logx + 8.594, R 2 = 0.996, S / N = 3), and the detection limit is 1.02 ng / mL;

[0110] Linear fitting equation for ratio fluorescence detection:

[0111] For dichlorvos, in the range of 0.1 - 50 ng / mL, the fitting equation is y = 0.136×logx + 0.054, R 2 = 0.999, S / N = 3, in the range of 50 - 8000 ng / mL, the fitting equation is y = 0.114x + 0.852, R 2 = 0.992, S / N = 3, and the detection limit is 1.13 pg / mL;

[0112] For chlorpyrifos, in the range of 1 - 8000 ng / mL, the fitting equation is y = 0.098x + 0.302, R 2 = 0.996, S / N = 3, and the detection limit is 0.86 ng / mL.

[0113] The preferred technical solution is as follows: The preparation method of the manganese dioxide nanosheet solution includes: adding a KMnO4 solution to an MES buffer solution, then ultrasonicating the mixture, centrifuging, washing the precipitate with ultrapure water, vacuum-drying the washed precipitate to obtain a manganese dioxide nanosheet solid, and dispersing it in water to prepare a 0.5 mg / mL manganese dioxide nanosheet solution; the concentration of the KMnO4 solution is 0.1 M; the concentration of the MES buffer solution is 0.1 M, and the pH value is 6.

[0114] The above are only preferred embodiments for explaining the present invention, and are not intended to limit the present invention in any form. Therefore, any modifications or changes to the present invention made under the same inventive spirit should still be included within the scope intended to be protected by the present invention.

Claims

1. A method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe, characterized in that: Including photothermal detection, ratio fluorescence detection and data processing: The photothermal detection includes: adding acetylcholinesterase and the sample solution to be detected into PBS buffer solution, incubating after mixing at 36 - 38 °C to obtain the first solution system; next, adding acetylcholine, manganese dioxide nanosheet solution and ruthenium tris(bipyridine) solution into the first solution system, reacting for 25 - 35 min to obtain the reaction solution; then transferring the reaction solution into a porous plate, irradiating with 808 nm near-infrared light for 15 - 25 min, recording the photothermal signal of the solution with an infrared thermal camera, and substituting it into the linear fitting equation to obtain the photothermal detection concentration value of the organophosphorus pesticide sample; The ratio fluorescence detection includes: adding acetylcholinesterase and the sample solution to be detected into PBS buffer solution, incubating after mixing at 36 - 38 °C to obtain the first solution system; next, adding acetylcholine, manganese dioxide nanosheet solution and ruthenium tris(bipyridine) solution into the first solution system, reacting for 25 - 35 min to obtain the reaction solution; adding thiamine solution to the reaction solution, incubating at 36 - 38 °C for 25 - 35 min, recording the fluorescence spectrum at room temperature, calculating the fluorescence intensity ratio of two fluorescence signals, and substituting it into the corresponding linear fitting equation to obtain the ratio fluorescence detection value of the organophosphorus pesticide sample; Data processing includes: calculating the detection value of the organophosphorus pesticide sample; Detection value = (photothermal detection value + ratio fluorescence detection value) / 2.

2. The method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe according to claim 1, characterized in that: Linear fitting equation for photothermal detection: For dichlorvos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.731×logx + 8.668, R 2 = 0.993, S / N = 3, and the detection limit is 1.01 ng / mL; For chlorpyrifos, in the range of 10 - 10000 ng / mL, the fitting equation is y = 1.478×logx + 8.594, R 2 = 0.996, S / N = 3), and the detection limit is 1.02 ng / mL; Linear fitting equation for ratio fluorescence detection: For dichlorvos, in the range of 0.1 - 50 ng / mL, the fitting equation is y = 0.136×logx + 0.054, R 2 = 0.999, S / N = 3. In the range of 50 - 8000 ng / mL, the fitting equation is y = 0.114x + 0.852, R 2 = 0.992, S / N = 3, and the detection limit is 1.13 pg / mL; For chlorpyrifos, in the range of 1 - 8000 ng / mL, the fitting equation is y = 0.098x + 0.302, R 2 = 0.996, S / N = 3, and the detection limit is 0.86 ng / mL.

3. The method for detecting organophosphorus pesticides using a ratio fluorescence and photothermal dual-mode probe according to claim 1, characterized in that: The preparation method of the manganese dioxide nanosheet solution includes: adding KMnO4 solution to MES buffer solution, then ultrasonically treating the mixture, centrifuging and washing the precipitate with ultrapure water, vacuum-drying the washed precipitate to obtain manganese dioxide nanosheet solid, and dispersing it in water to prepare a 0.5 mg / mL manganese dioxide nanosheet solution; the concentration of the KMnO4 solution is 0.1 M; the concentration of the MES buffer solution is 0.1 M and the pH value is 6.

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