CN111323405A - Organophosphorus pesticide activity detection method based on fluorescent molecule OliGreen response construction - Google Patents
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Abstract
The invention discloses a method for detecting the activity of organophosphorus pesticide constructed based on fluorescent molecule OliGreen response, which comprises the following specific analysis steps: firstly, mixing organophosphorus pesticide with acetylcholinesterase, then adding acetylcholine chloride solution into the above-mentioned mixed solution, then adding the prepared T-Hg2+And (4) adding OliGreen fluorescent molecules and a Tris-HCl buffer solution into the solution T, reacting for a period of time in a dark environment, recording the fluorescence spectrum of the mixed solution, and obtaining the concentration of the organophosphorus pesticide through the fluorescence intensity. The invention does not need to use expensive precise instruments for detection, simplifies the detection method, greatly reduces the detection cost of the organophosphorus pesticide, and has the advantages of low operation cost, quick, simple and convenient detection, good selectivity and the like.
Description
Technical Field
The invention belongs to the field of detection, and particularly relates to organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response.
Background
Organophosphorus Pesticides (OPs), which are organic compound pesticides containing phosphorus elements, are an important pesticide type, and are widely used in life due to relatively high efficiency and poor durability, but cause serious pollution to agricultural products and environment due to improper treatment and residues. Acetylcholinesterase (AChE) is the major cholinesterase in nervous system function, and it can specifically modulate the level of acetylcholine by hydrolyzing acetylcholine, thereby modulating nerve impulses. Inhibition of acetylcholinesterase activity can lead to accumulation of acetylcholine in synaptic transmission, and is a powerful therapeutic strategy for disease. OPs can inhibit the activity of acetylcholinesterase, but are harmful to human health. Therefore, means for rapidly and sensitively detecting the activity of OPs are important.
The traditional methods for detecting the OPs activity mainly comprise Liquid Chromatography (LC), Gas Chromatography (GC), gas chromatography and mass spectrometry (GC/MS), enzyme-linked immunosorbent assay (ELISA) test and the like. Most of these methods have the disadvantage of being time consuming and complex to operate.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting the activity of organophosphorus pesticide constructed based on fluorescent molecule OliGreen response. The invention does not need complex marking process, simplifies the detection method, greatly reduces the cost of OPs detection, and has the advantages of low operation cost, rapid detection, simplicity, convenience, good selectivity and the like.
The purpose of the invention can be realized by the following technical scheme:
an analysis method for organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response comprises the following steps:
the first step is as follows: firstly, organophosphorus pesticides with different concentrations are respectively mixed with acetylcholinesterase solution according to the volume ratio of 11 mixing, adding an acetylcholine chloride solution into the mixed solution, and then adding T-Hg2+A solution T, adding OliGreen fluorescent molecule solution and Tris-HCl buffer solution, reacting for a period of time in a dark environment, recording the fluorescence spectrum of the mixed solution, and establishing a linear regression method for the fluorescence intensity of different fluorescence spectra and different organophosphorus pesticides corresponding to the fluorescence intensity;
the second step is that: repeating the first step, except that the organophosphorus pesticides with different concentrations are replaced by samples to be detected, recording the fluorescence spectrum of the mixed solution after the samples to be detected react, substituting the fluorescence intensity of the fluorescence spectrum into the linear regression equation, and calculating to obtain the concentration of the organophosphorus pesticides in the samples to be detected;
the technical scheme of the invention is as follows: the concentrations of the organophosphorus pesticides at different concentrations in the first step were 5pg/mL, 50pg/mL, 250pg/mL, 500pg/mL, 5ng/mL, 25ng/mL and 50ng/mL DDVP solutions.
The technical scheme of the invention is as follows: the concentration of the acetylcholinesterase solution is 0.5-12.5 mU/mL, the concentration of the acetylcholine chloride solution is 0.1-5 uM/mL, and the volume ratio of the acetylcholinesterase to the acetylcholine chloride solution is 1: 2.
the technical scheme of the invention is as follows: the T-Hg2+The synthesis of the solution T is as follows: poly (30T) and mercury nitrate solution according to the volume ratio of 1:1, mixing and placing in a constant-temperature water bath at 37 ℃ for reaction for 20-40 minutes.
The technical scheme of the invention is as follows: the concentration of the p (30T) is 20-30 nM, and the concentration of the mercuric nitrate is 200-400 nM.
The technical scheme of the invention is as follows: the OliGreen fluorescent molecule solution is prepared by diluting OliGreen with water by a factor of 0.5-1 x104And (4) doubling.
The technical scheme of the invention is as follows: the Tris-HCl buffer solution is as follows: 5 to 10mM Tris-HCl, pH 6.5 to 8.5.
The technical scheme of the invention is as follows: acetylcholinesterase solution: T-Hg2+-T solution: OliGreen fluorescent molecule solution: the volume ratio of the Tris-HCl buffer solution is 1: 2: 0.3-0.8: 10 to 20.
In particular toIn turn, the AChE induces hydrolysis of ATCh and changes in fluorescence of the solution: 5mU/mL of AChE (10. mu.L) and 1. mu.M ATCH (20. mu.L) were mixed and incubated at 37 ℃ for 30 minutes. Then, 300nM T-Hg was added thereto2+-T solution (20. mu.L) and incubation continued for 30 min. Finally, 5. mu.L OliGreen (0.005X) and 145. mu.L Tris-HCl buffer solution were added to the above mixed solution to a final volume of 200. mu.L, and left to react in a dark environment for 15 minutes. The fluorescence spectrum of the mixed solution was recorded at room temperature.
Specifically, the organophosphorus pesticide detection step comprises: after mixing the solution containing 10. mu.L of AChE (5mU/mL) and 10. mu.L of DDVP at various concentrations and incubating at 37 ℃ for 30 minutes, 20. mu.L of ATCH solution (1. mu.M) was added thereto and incubated for 30 minutes. Then, 20 mu L T-Hg was added2+-solution in T (300nM) and incubation continued for 30 min. Finally, 5. mu.L of OliGreen (0.005X) and 135. mu.L of Tris-HCl buffer solution were added to the above mixture and reacted in a dark environment for 15 minutes. The fluorescence spectrum of the mixed solution was recorded at room temperature.
The invention has the beneficial effects that:
compared with the prior art, the invention has the following characteristics and advantages: the invention has simple principle, short experimental period and lower cost of the used raw materials, does not need any large-scale instrument and can detect the object to be detected with lower content under the same condition. OliGreen fluorescent molecule pairs of poly (30T) and T-Hg2+-T both structures show different fluorescence intensities. In the absence of organophosphorus pesticides, acetylcholinesterase (AChE) hydrolyzes the substrate acetylcholine chloride (ATCh) into choline (TCh) and acetic acid. T-Hg can be abstracted by TCh2+Hg in solution T2+Large amount of TCh-Hg is generated2+TCh and poly (30T), where poly (30T) in solution binds to OliGreen fluorescent molecules with a weaker fluorescent signal. In the presence of the organophosphorus pesticide DDVP, the activity of AChE is inhibited, TCh generation is inhibited, and only a small amount of Hg is present2+From T-Hg by TCh2+Deprivation in the-T structure, so that a large amount of T-Hg still exists in the solution2+-T, which binds to OliGreen fluorescent molecules with a strong fluorescent signal. The invention does not need to rely on expensive precise instrument for detection, and simplifies the detectionThe detection method greatly reduces the detection cost of the organophosphorus pesticide, and has the advantages of low operation cost, quick and simple detection, good selectivity and the like.
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FIG. 1 shows a flow chart of an analysis method for organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response;
FIG. 2 shows a schematic spectrum diagram of an analysis method for organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response.
FIG. 2A: T-Hg2+-T/OliGreen(a),poly(30T)/OliGreen(b),Hg2+OliGreen (c) and OliGreen (d); FIG. 2B: T-Hg2+-T/OliGreen(a),ATCh/T-Hg2+-T/OliGreen(b),AChE/T-Hg2+-T/OliGreen(c)and OPs/AChE/ATCh/T-Hg2+-T/OliGreen(d)and AChE/ATCh/T-Hg2+-T/OliGreen(e)；
FIG. 3A shows an optimized plot of poly (30T) concentration; FIG. 3B shows an optimization chart of ATCH concentration; FIG. 3C shows an optimization plot of ATCH versus AChE reaction time;
FIG. 4A shows a graph of the change in fluorescence intensity of the quantitatively detected DDVP; FIG. 4B shows a calibration curve of fluorescence values versus different concentrations of DDVP. (inset: linear relationship between the value of fluorescence intensity and the log concentration of DDVP, ranging from 5pg/mL to 25 ng/mL).
Detailed Description
The invention is further illustrated by the following examples, without limiting the scope of the invention:
reagents and instruments used in this experiment:
mercury nitrate (Hg (NO)3)2) Purchased from Nanjing lattice chemical Limited (Nanjing, China). Hydrochloric acid, tris (hydroxymethyl) aminomethane, was purchased from national pharmaceutical chemicals, Inc. (Shanghai, China). AChE (acetylcholinesterase from electrophohorus electroscus) and ATCh were purchased from Sigma-Aldrich (shanghai, china). Organophosphorus pesticide standards were purchased from the national center for standards (beijing, china). DNA strand (poly (30T) is supplied by living organisms (Shanghai, China) DNA strand (poly (30T): 5'-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3'
OliGreen was purchased from ThermoFisher Scientific (www.thermofisher.com).
The fluorescence spectrum was collected by a fluorescence spectrometer (FluoroMax-4, HORIBA Jobin Yvon, Japan).
In the embodiment of the invention, the organophosphorus pesticide is dichlorvos (DDVP) uniformly.
The Tris-HCl buffer solution in the embodiment of the invention is 5mM Tris-HCl and the pH value is 8.
Example 1T-Hg2+Synthesis of-T Structure
10 μ L of 25nM poly (30T) and 10 μ L of 300nM Hg (NO)3)2The solution is mixed and then is put at 37 ℃ for 30 minutes to generate the solution containing T-Hg2+-a solution of the structure T.
FIG. 2A shows poly (30T), T-Hg2+-fluorescence signal values after binding of both structures to OliGreen. From the fluorescence intensity values of the above two structures, T-Hg is known to be comparable to poly (30T)2+The structure of-T generates stronger fluorescence signal after being combined with OliGreen, T-Hg2+-T/OliGreen(a),poly(30T)/OliGreen(b),Hg2+/OliGreen(c)and OliGreen(d)。
Example 2T-Hg2+Synthesis of-T Structure
10 μ L of 50nM poly (30T) and 10 μ L of 300nM Hg (NO)3)2The solution is mixed and then is put at 37 ℃ for 30 minutes to generate the solution containing T-Hg2+-a solution of the structure T.
Example 3 Acetylcholinesterase (AChE) hydrolysis of acetylcholine chloride (ATCH) and change in fluorescence of solution
FIG. 2B, after 0.5mU/mL of AChE (10. mu.L) and 1. mu.M of ATCH (20. mu.L) were mixed and incubated at 37 ℃ for 30 minutes, 20. mu.L of T-Hg prepared in example 1 was added thereto2+-T solution, incubation is continued for 30 minutes. Finally, 5. mu.L of 0.005XOliGreen fluorescent molecule and 145. mu.L of Tris-HCl buffer were added to the above mixed solution to a final volume of 200. mu.L, and reacted in a dark environment for 15 minutes. The fluorescence spectrum of the mixed solution was recorded at room temperature. FIG. 2B is a graph showing the fluorescence spectrum, ATCH/T-Hg, of the mixed solution2+-T/OliGreen(b)，AChE/ATCh/T-Hg2+-T/OliGreen(e)。
Example 4 hydrolysis of acetylcholine by acetylcholinesterase and change in fluorescence of solution
AChE (10. mu.L) and 1. mu.M ATCH (20. mu.L) at 1mU/mL were mixed and incubated at 37 ℃ for 30 minutes, and then 20. mu.L of T-Hg prepared in example 1 was added thereto2+-T solution, incubation is continued for 30 minutes. Finally, 5. mu.L of 0.005XOliGreen fluorescent molecule and 145. mu.L of Tris-HCl buffer were added to the above mixed solution to a final volume of 200. mu.L, and reacted in a dark environment for 15 minutes. The fluorescence spectrum of the mixed solution was recorded at room temperature.
Example 5 detection of DDVP at various concentrations
After 10. mu.L of AChE (5mU/mL) was mixed with 10. mu.L of 5pg/mL, 10. mu.L of 50pg/mL, 10. mu.L of 250pg/mL, 10. mu.L of 500pg/mL, 10. mu.L of 5ng/mL, 10. mu.L of 25ng/mL, 10. mu.L of 50ng/mL DDVP solution, respectively, and incubated at 37 ℃ for 30 minutes, 20. mu.L of ATCH solution (1nM) was added thereto and incubated for 30 minutes. Then, 20. mu.L of T-Hg prepared in example 1 was added2+-solution T (300nM) and incubation continued for 30 min. Finally, 5. mu.L of 0.005XOliGreen fluorescent molecule solution and 135. mu.L of Tris-HCl buffer (5mM Tris, pH 8) were added to the above mixture to a final volume of 200. mu.L, respectively, and reacted in a dark environment for 15 minutes. The fluorescence spectrum of the mixed solution was recorded at room temperature. FIG. 4A shows the fluorescence spectra of the system in the presence of various concentrations of DDVP. (a)0, (b)5pg/mL, (c)50pg/mL, (d)250pg/mL, (e)500pg/mL, (f)5ng/mL, (g)25ng/mL, and (h)50 ng/mL. As shown in fig. 4B, the curve fitted to the log values of the fluorescence intensity and DDVP concentration of the system: in the range of 5.0 pg/mL-25 ng/mL, the fluorescence intensity and the logarithmic concentration value of DDVP present a good linear relation, and the linear regression equation is as follows: f/106＝0.99+0.235log CDDVP(R20.9978), the limit of detection is 2.9 pg/mL.
Example 6
To further explore the practical applicability of this assay, samples of water were taken from the jiulong lake under the designations 1, 2, 3, 4, 5 and centrifuged at 13000rpm for 30 minutes to remove insoluble material. Subsequently, filtration was performed using a 0.22 micron nitrocellulose membrane filter. Then, 5 water samples were sampledAdding known concentrations of DDVP: 0.005ng/mL, 0.05ng/mL, 0.5ng/mL, 5ng/mL, 25ng/mL for further assay experiments. After 10. mu.L of AChE (5mU/mL) was mixed with 10. mu.L of water samples containing DDVP at various concentrations and incubated at 37 ℃ for 30 minutes, 20. mu.L of ATCH solution (1nM) was added thereto and incubated for 30 minutes. Then, 20. mu.L of T-Hg prepared in example 1 was added2+-solution T (300nM) and incubation continued for 30 min. Finally, 5. mu.L of 0.005XOliGreen fluorescent molecule and 135. mu.L of Tris-HCl buffer were added to the above mixed solution to a final volume of 200. mu.L, and reacted in a dark environment for 15 minutes, and the fluorescence spectrum of the mixed solution was recorded at room temperature. And comparing the detection result with a standard curve by using the fluorescence intensity value at the maximum fluorescence intensity position of the fluorescence spectrum to obtain the residual amount of the DDVP in the lake water. The experimental results are shown in table 1, and these results indicate acceptable recovery rates and relative standard deviations, indicating that the prepared method has good performance and can be used for detecting DDVP in actual water samples.
TABLE 1
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Variations or modifications in other variations may occur to those skilled in the art based upon the foregoing description. Not all embodiments need be illustrated or described herein. And obvious variations or modifications of this embodiment may be made without departing from the spirit or scope of the invention.
Claims (8)
1. The analysis method for detecting the activity of the organophosphorus pesticide constructed based on fluorescent molecule OliGreen response is characterized by comprising the following steps:
the first step is as follows: firstly, respectively mixing organophosphorus pesticides with different concentrations with acetylcholinesterase solutionMixing according to the volume ratio of 1:1, then adding an acetylcholine chloride solution into the mixed solution, and then adding T-Hg2+A solution T, adding OliGreen fluorescent molecule solution and Tris-HCl buffer solution, reacting for a period of time in a dark environment, recording the fluorescence spectrum of the mixed solution, and establishing a linear regression method for the fluorescence intensity of different fluorescence spectra and different organophosphorus pesticides corresponding to the fluorescence intensity;
the second step is that: and repeating the first step, except that the organophosphorus pesticides with different concentrations are replaced by samples to be detected, recording the fluorescence spectrum of the reacted mixed solution of the samples to be detected, and substituting the fluorescence intensity of the fluorescence spectrum into the linear regression equation to calculate the concentration of the organophosphorus pesticides in the samples to be detected.
2. The method for analyzing the activity detection of the organophosphorus pesticide constructed based on the fluorescent molecule OliGreen response in claim 1, wherein the concentrations of the organophosphorus pesticides with different concentrations in the first step are 5pg/mL, 50pg/mL, 250pg/mL, 500pg/mL, 5ng/mL, 25ng/mL and 50ng/mL DDVP solutions.
3. The method for analyzing organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response according to claim 1, wherein the concentration of the acetylcholinesterase solution is 0.5-12.5U/mL, the concentration of the acetylcholine chloride solution is 0.1-5 uM/mL, and the volume ratio of acetylcholinesterase to acetylcholine chloride solution is 1: 2.
4. the method for analyzing organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response according to claim 1, wherein T-Hg is used as the fluorescent probe2+The synthesis of the solution T is as follows: poly (30T) and mercury nitrate solution according to the volume ratio of 1:1, mixing and placing in a constant-temperature water bath at 37 ℃ for reaction for 20-40 minutes.
5. The method for analyzing the organophosphorus pesticide activity detection based on fluorescent molecule OliGreen response construction, according to claim 4, wherein the concentration of p (30T) is 20-30 nM, and the concentration of mercuric nitrate is 200-400 nM.
6. The method for analyzing organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response according to claim 1, wherein the OliGreen fluorescent molecule solution is obtained by diluting OliGreen with water, and the dilution factor is 0.5-1 x104And (4) doubling.
7. The method for analyzing the organophosphorus pesticide activity detection based on fluorescent molecule OliGreen response construction according to claim 1, wherein the Tris-HCl buffer solution is: 5 to 10mM Tris-HCl, pH 6.5 to 8.5.
8. The method for analyzing organophosphorus pesticide activity detection constructed based on fluorescent molecule OliGreen response according to claim 1, wherein the acetylcholinesterase solution: T-Hg2+-T solution: OliGreen fluorescent molecule solution: the volume ratio of the Tris-HCl buffer solution is 1: 2: 0.3-0.8: 10 to 20.
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| CN115931786A (en) * | 2022-08-25 | 2023-04-07 | 南京农业大学 | Dual-signal sensor for detecting organophosphorus pesticide and preparation method and application thereof |
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| CN115418221B (en) * | 2022-08-09 | 2023-08-25 | 甘肃中医药大学 | Preparation and detection method of fluorescent sensor for detecting pesticide residues of angelica sinensis |
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| CN115931786B (en) * | 2022-08-25 | 2024-03-15 | 南京农业大学 | Double-signal sensor for detecting organophosphorus pesticide and preparation method and application thereof |
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