CN110849857B - Organophosphorus pesticide detection method based on magnetic nano cholinesterase fluorescent carbon dots - Google Patents
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Abstract
The invention discloses a method for detecting organophosphorus pesticide based on magnetic nanometer cholinesterase fluorescence carbon dots, which adopts sucrose as a carbon source, enriches the magnetic nanometer cholinesterase combined with organophosphorus pesticide, fixes the excitation wavelength of 400-.
Description
Technical Field
The invention relates to the technical field of pesticide detection, in particular to a method for detecting organophosphorus pesticide based on magnetic nano cholinesterase fluorescent carbon dots.
Background
Organophosphorus pesticides are a class of high-efficiency broad-spectrum insecticides, and are widely used worldwide. However, organophosphorus pesticides work by inhibiting the acetylcholinesterase activity in animals and are therefore highly toxic to non-target organisms, particularly humans. According to statistics, the number of organophosphorus pesticide poisoning people worldwide is hundreds of thousands of people every year. Strict maximum limit of organophosphorus pesticide residues in food species is established in all countries of the world, and risk monitoring and detection are carried out on organophosphorus pesticide residues in food.
The traditional organophosphorus pesticide detection method mainly comprises a Gas Chromatography (GC), a High Performance Liquid Chromatography (HPLC), a gas chromatography/mass spectrometry combined method (GC/MS) and the like. Despite the high accuracy and sensitivity of the above methods, the required equipment is expensive, the pretreatment is cumbersome, the analysis period is long, and specialized operators are required. Aiming at the current situation that agricultural product production main bodies in China are mostly small farmers, a technical means for rapidly detecting and screening organophosphorus pesticides on site with rapidness, high efficiency, low cost and high sensitivity is urgently needed to be developed.
Aiming at organophosphorus pesticides, the main rapid screening technology at present is an enzyme inhibition chromogenic method, but the detection sensitivity of the method is mostly between 0.5 ppm and 2ppm at present, and has a difference with the maximum residual limit of 0.01 ppm to 0.2ppm, so a more accurate detection method is needed.
Disclosure of Invention
The invention aims to provide a method for detecting organophosphorus pesticide based on magnetic nano cholinesterase fluorescent carbon dots.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the invention comprises the following steps:
step one, respectively preparing 20mL of the solution to be detected with the concentration of 25ppb, 100ppb, 500ppb, 1ppm and 5ppm for preparing a standard calibration curve.
And step two, preparing the fluorescent carbon dots and the magnetic acetylcholinesterase, weighing 0.5g of sucrose as a carbon source, dissolving the sucrose in 50mL of deionized water, and carrying out hydrothermal reaction for 12 hours at 180 ℃ in a high-pressure reaction kettle to obtain the fluorescent carbon dots with the maximum excitation wavelength of 410nm and the maximum emission wavelength of 565 nm.
And step three, taking 5mL of the standard solution with each concentration, respectively adding 25 mu L of magnetic acetylcholinesterase, fully performing vortex dispersion, performing oscillation incubation at 37 ℃ for 20min, and separating and enriching the magnetic AChE combined with the organophosphorus pesticide by adopting a strong magnet and an external magnetic field.
Step four, adding 100 muL of iodoacetylcholine sulfide (ATCHI 10mg/mL), 100 muL of DTNB (8mg/mL) color development solution and 50 muL of carbon dots into the standard solution, standing for 3min, fixing the excitation wavelength of 400-460nm and the emission wavelength of 560-600nm for detection, and carrying out fluorescence spectrum detection on the standard solution.
Specifically, it is preferable that the excitation wavelength be 412nm and the emission wavelength be 565nm for detection.
A preparation method of magnetic nano cholinesterase is characterized by comprising the following steps:
comprises the following steps
A, synthesizing magnetic nanoparticles by a hydrothermal method;
b, performing amination on the surface of the magnetic nano particle to obtain magnetic nano Fe 3 O 4 Particles;
c, adopting a glutaraldehyde method to mix acetylcholinesterase (AChE) with the magnetic nano Fe 3 O 4 Coupling the particles to obtain the magnetic acetylcholinesterase.
Specifically, the hydrothermal method comprises weighing FeCl2 & 4H under the protection of nitrogen 2 O and FeCl 3 ·6H 2 O, and the mass ratio of 1: 1 preparing a mixed solution, stirring at a constant temperature of 65 ℃, dropwise adding a concentrated NaOH solution until the pH value is 7 and brown particle solids are generated, continuously stirring and adding absolute ethyl alcohol, standing for 10min, raising the temperature to 70 for curing, standing for 30min, settling in a strong magnetic field, separating supernatant, washing precipitates to be neutral, drying at 75 ℃ for 5h to obtain magnetic nano Fe 3 O 4 And (3) particles.
Preferably, the surface amination of the magnetic nanoparticles comprises weighing 0.1g of magnetic nanoparticles, dispersing the magnetic nanoparticles in 150mL PBS buffer solution, performing ultrasonic treatment for 30min, dropwise adding 0.4mL of 3-Aminopropyltriethoxysilane (APTES) into the buffer solution to improve the dispersion phase uniformity of the magnetic nanoparticles, stirring for 7h at room temperature, centrifuging at a rotation speed of 10000r/min for 30mm, and mixingFe 3 O 4 Separating the nanoparticles, washing the nanoparticles with ethanol solution, and dispersing the nanoparticles in ethanol solution with the concentration of 1 g/L.
Specifically, the glutaraldehyde method comprises separating magnetic nanoparticles from 10mL of an ethanol solution of amino-modified magnetic nanoparticles by applying a strong ferromagnetic field, transferring the magnetic nanoparticles to 10mL of PBS (0.05M pH 7.5) buffer, adding 1mL of 50% glutaraldehyde, performing shaking table oscillation activation for 10 hours, magnetically separating the supernatant, washing the supernatant with deionized water for 2 times, transferring the supernatant to 2mL of PBS (0.05M pH 7.5) buffer, adding 2mL of acetylcholinesterase PBS solution (250U/mL) into the system, and coupling the solution at 37 ℃ for 2 hours. And (3) washing the successfully coupled magnetic acetylcholinesterase in a strong magnetic separation manner, and dispersing the obtained magnetic acetylcholinesterase in a 5mLPBS solution.
The magnetic nano cholinesterase can be applied to detection of organophosphorus pesticides.
The invention has the beneficial effects that:
the method can quickly and effectively detect the phosphorus content in the organophosphorus pesticide by the fluorescence detection of the carbon dots after the organophosphorus pesticide is enriched by the magnetic nano cholinesterase, has higher selectivity and sensitivity, is simple to operate, and has high light stability and good repeatability of the prepared fluorescent dots.
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FIG. 1 is a flow chart of the method for detecting organophosphorus pesticide based on magnetic nano cholinesterase fluorescent carbon dots;
FIG. 2 is a graph showing a calibration standard curve prepared during the detection of organophosphorus pesticides based on magnetic nano cholinesterase fluorescent carbon dots;
Detailed Description
The invention is further described below with reference to the accompanying drawings:
as shown in figure 1, the preparation of the magnetic nano cholinesterase of the invention comprises the following steps:
a, synthesizing the magnetic nanoparticles by a hydrothermal method.
Weighing FeCl 2 ·4H 2 O and FeCl 3 ·6H 2 O, as 1: 1 the amount ratio of the substances to prepare a mixed solution250mL of the solution is put into a three-necked bottle and placed into a thermostat water bath kettle at 65 ℃, the mixture is rapidly stirred, and concentrated NaOH solution is dropwise added until the pH value is 7, and brown particles are generated. Then adding diluted NaOH solution dropwise to a specified pH value, continuously stirring, adding absolute ethyl alcohol, standing for 10min, raising the temperature for curing, and adjusting the acidity. The whole reaction process is carried out under the protection of nitrogen. After 30min, settling with strong magnet, separating supernatant, and repeatedly washing precipitate with distilled water and anhydrous alcohol until pH of washing water is about 7. Putting the precipitate into a vacuum drying oven, and drying at 75 deg.C for 5h to obtain magnetic nanometer Fe 3 O 4 Particles.
b surface amination of magnetic nanoparticles
0.1g of magnetic nanoparticles were weighed out and dispersed in 150ml PBS buffer, for better dispersion of Fe 3 O 4 Subjecting the solution to ultrasonic treatment for 30min, adding dropwise 0.4mL of mixed solution of 3-Aminopropyltriethoxysilane (APTES), stirring at room temperature for 7 hr, centrifuging at 10000r/min by use of centrifuge for 30mm, and mixing with Fe modified by APTES 3 O 4 The nanoparticles were separated from the reaction medium and washed 5 times with ethanol solution. APTES modified Fe before the next reaction 3 O 4 The nanoparticles were formulated in 1g/L ethanol solution. The solid sample is obtained by vacuum drying at 70 ℃.
And c, coupling acetylcholinesterase (AChE) with the magnetic nanoparticles by adopting a glutaraldehyde method.
Taking 10mL of ethanol solution of the amination-modified magnetic nanoparticles, separating the magnetic nanoparticles by adding an intense magnetic field, discarding the supernatant, transferring the nanoparticles to 10mL of PBS (0.05M pH 7.5) buffer solution, and adding 1mL of 50% glutaraldehyde into the system for activation. After 10h activation by shaking the shaker, the supernatant was magnetically separated, washed 2 times with deionized water, transferred to 2mL of PBS (0.05M pH 7.5) buffer, and 2mL of acetylcholinesterase PBS solution (250U/mL) was added to the system and coupled at 37 ℃ for 2 h. And washing the successfully coupled magnetic acetylcholinesterase for 2-3 times under the separation assisting action of strong magnet to remove the uncoupled AChE. The obtained magnetic AChE was dispersed in a 5mLPBS solution.
Preparation of fluorescent carbon dots
Adopting sucrose as a carbon source, weighing 0.5g of sucrose, dissolving the sucrose in 50mL of deionized water, and carrying out hydrothermal reaction in a high-pressure reaction kettle at 180 ℃ for 12h to obtain the fluorescent carbon dots with the maximum excitation wavelength of 410nm and the maximum emission wavelength of 565 nm.
Magnetic fluorescence detection of organophosphorus pesticides
As represented by paraoxon as an organophosphorus pesticide, 20mL of each of solutions having a concentration of 25ppb, 100ppb, 500ppb, 1ppm and 5ppm was prepared and used for preparing a calibration curve as shown in FIG. 2.
And (3) taking 5mL of standard solution with each concentration, adding 25 mu L of magnetic AChE, fully whirling and dispersing, carrying out shaking incubation at 37 ℃ for 20min, and then separating and enriching the magnetic AChE combined with the organophosphorus pesticide by adopting a strong magnet and an external magnetic field. The supernatant was discarded, and 100. mu.L of iodoacetylcholine sulfide (ATCHI 10mg/mL) and 100. mu.L of DTNB (8mg/mL) chromogenic solution were added, as well as 50. mu.L of carbon dots. After 3min, 412nm is used as the excitation wavelength, and 565nm is used as the emission wavelength for detection.
As the ATCHI is hydrolyzed under the action of the AChE to produce the choline sulfide TCh, the TCh and the DTNB generate yellow substances, the maximum absorption wavelength is just 412nm and is the same as the excitation wavelength of the carbon dots, and the carbon dot fluorescence cannot be emitted based on the fluorescence inner filtering effect. The organophosphorus pesticide can inhibit activity of AChE, so that the reaction of hydrolyzing ATCHI to generate TCh is weakened, yellow substances generated by DTNB are reduced, and excitation light with the wavelength of 412nm causes fluorescence emission of carbon dots; therefore, the increase of the concentration of the organic phosphorus in the system can enhance the fluorescence emission of the carbon dots of the system, thereby establishing the positive correlation between the organic phosphorus and the fluorescence signals of the carbon dots.
The method can be used for detecting the organophosphorus pesticide in fruits and vegetables, and has an order of magnitude higher sensitivity than the conventional enzyme-inhibited DTNB color reaction.
The above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, which is not described herein again; the above examples and drawings are only for illustrating the technical solutions of the present invention and are not to be construed as limiting the present invention, the present invention is described in detail with reference to the preferred embodiments, and any modifications, equivalent substitutions, improvements and the like made by those skilled in the art within the spirit of the present invention shall also fall within the protection scope of the claims of the present invention.
Claims (2)
1. An organophosphorus pesticide detection method based on magnetic nano cholinesterase fluorescent carbon dots is characterized in that,
the method comprises the following steps:
step one, respectively preparing 20mL standard solutions with the to-be-detected concentrations of 25ppb, 100ppb, 500ppb, 1ppm and 5ppm for preparing a standard correction curve;
step two, preparing fluorescent carbon dots and magnetic acetylcholinesterase (AChE), taking sucrose as a carbon source, weighing 0.5g of sucrose, dissolving the sucrose in 50mL of deionized water, and carrying out hydrothermal reaction in a high-pressure reaction kettle at 180 ℃ for 12 hours to obtain the fluorescent carbon dots with the maximum excitation wavelength of 412nm and the maximum emission wavelength of 565 nm;
step three, taking 5mL of the standard solution with each concentration, respectively adding 25 mu L of magnetic acetylcholinesterase, fully performing vortex dispersion, performing oscillation incubation at 37 ℃ for 20min, and separating and enriching the magnetic acetylcholinesterase (AChE) combined with the organophosphorus pesticide by adopting a strong magnet and an external magnetic field;
respectively adding 100 mu L, 10mg/mL of iodoacetylcholine sulfide (ATCHI), 100 mu L and 8mg/mL of DTNB chromogenic solution and 50 mu L of fluorescent carbon dots into the magnetic acetylcholinesterase solution combined with the organophosphorus pesticide, standing for 3min, fixing the excitation wavelength to 412nm and the emission wavelength to 565nm, and performing fluorescence spectrum detection on the standard solution;
hydrolyzing iodoacetylcholine ATCHI under the action of acetylcholinesterase AChE to produce choline sulfide TCh, wherein TCh and DTNB generate yellow substances, the maximum absorption wavelength is 412nm, the wavelength is the same as the excitation wavelength of a fluorescent carbon dot, and the carbon dot cannot emit fluorescence based on the fluorescence inner filtering effect; the organophosphorus pesticide can inhibit the activity of acetylcholinesterase AChE, so that the reaction of hydrolyzing iodothiocholine ATCHI to generate TCh is weakened, yellow substances generated by DTNB are reduced, and the excitation light of 412nm enables the fluorescent carbon dots to emit fluorescence;
the preparation method of the magnetic nano cholinesterase comprises the following steps:
A. hydrothermal synthesis of magnetic Fe 3 O 4 Nano particles, the hydrothermal method comprises weighing FeCl under the protection of nitrogen 2 ·4H 2 O and FeCl 3 ·6H 2 O, and the mass ratio of 1: 1 preparing a mixed solution, stirring at a constant temperature of 65 ℃, dropwise adding a concentrated NaOH solution until the pH value is =7 and a brown particle solid is generated, continuously stirring and adding absolute ethyl alcohol, standing for 10min, raising the temperature to 70 ℃ for curing, standing for 30min, settling in a strong magnetic field, separating supernatant, washing precipitate to be neutral, and drying at 75 ℃ for 5h to obtain magnetic Fe 3 O 4 Nanoparticles;
B. for the magnetic Fe 3 O 4 The surface of the nano particle is aminated to obtain aminated modified magnetic Fe 3 O 4 Nanoparticles of said magnetic Fe 3 O 4 The surface amination of the nanoparticles comprises weighing 0.1g of magnetic Fe 3 O 4 Dispersing the nanoparticles in 150mL PBS buffer solution, performing ultrasonic treatment for 30min, and then dropwise adding 0.4mL 3-Aminopropyltriethoxysilane (APTES) in the buffer solution to improve the magnetic Fe 3 O 4 Stirring the dispersed phase uniformity of the nano particles for 7h at room temperature, centrifuging for 30min at the rotating speed of 10000r/min, and carrying out amination modification on the magnetic Fe 3 O 4 Separating nanoparticles, cleaning with ethanol solution, and dispersing in 1g/L ethanol solution;
C. adopting glutaraldehyde method to mix acetylcholinesterase (AChE) with the amino-modified magnetic Fe 3 O 4 Coupling the nanoparticles to obtain magnetic acetylcholinesterase;
the glutaraldehyde method comprises the step of carrying out amination modification on 10mL of magnetic Fe 3 O 4 Separating amino-modified magnetic Fe from ethanol solution of nanoparticles by using strong magnetic field 3 O 4 Nanoparticles of the amino-modified magnetic Fe 3 O 4 After the nanoparticles were transferred to 10mL of 0.05M, pH =7.5 PBS buffer and 1mL of 50% glutaraldehyde was added to activate the nanoparticles by shaking for 10 hours, the supernatant was magnetically separated, washed 2 times with deionized water, and transferred to 2mL of 0.05M, pH =7.5 PBS bufferAdding 2mL and 250U/mL of acetylcholinesterase PBS solution into the system, coupling for 2h at 37 ℃, then carrying out strong magnetic separation and washing on the successfully coupled magnetic acetylcholinesterase, and dispersing the obtained magnetic acetylcholinesterase into 5mL of PBS solution.
2. The method for detecting organophosphorus pesticide based on magnetic nano cholinesterase fluorescent carbon dots according to claim 1 is applied to organophosphorus pesticide detection.
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| CN111323405B (en) * | 2020-04-14 | 2020-12-22 | 南京海关工业产品检测中心 | Organophosphorus pesticide activity detection method based on fluorescent molecule OliGreen response construction |
| CN113219104B (en) * | 2021-05-28 | 2022-04-22 | 中国农业科学院农业质量标准与检测技术研究所 | Method for detecting carbamate pesticide based on dual-magnetic nanoparticles |
| CN116371395A (en) * | 2023-04-13 | 2023-07-04 | 成都理工大学 | Cerium single-atom nano-enzyme, preparation method and method for detecting organic phosphorus |
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