CN106053454A - Quick sensibilization detecting method of organophosphorus pesticide - Google Patents
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Abstract
The invention relates to the field of chemical analysis, particularly relates to a quick sensibilization detecting method of organophosphorus pesticide, and aims to solve the problems that in quick organophosphorus pesticide detection by the traditional enzymatic inhibition method, because organophosphorus pesticide contains P=S perssad, inhibiting ability to cholinesterase is weak, and sensitivity and accuracy of detection to the organophosphorus pesticide which contains P=S perssad are low. An oxidized sensibilization treatment process which is compatible to enzyme reaction is added in pretreatment of a sample, a carbamate substrate and a color developing agent are added in enzyme reaction, and according to change of absorbance, whether the residual quantity of the organophosphorus pesticide in the sample exceeds a limit value or not can be judged. Compared with an existing organophosphorus pesticide detecting method, the quick sensibilization detecting method has the advantages that through a unique sensibilization treatment method, the P=S perssad in the organophosphorus pesticide is converted into P=O perssad, and therefore, the detectability of the organophosphorus pesticide containing the P=S perssad by the enzymatic inhibition method is improved remarkably.
Description
Technical field
The present invention relates to chemical analysis field, be specifically related to the quick sensitization detection method of a kind of organophosphorus pesticide.
Background technology
Organophosphorus pesticide (OPs) refer to containing C-P, C-O-P, C-S-P or C-N-P key for preventing and treating phytopathy, worm, harmful
Organic compound, majority is oily liquids, and minority is solid (Rogor, metrifonate etc.), develops the color for faint yellow to brown, has big
Bulbus Allii abnormal smells from the patient.Most of OPs are water insoluble, and are dissolved in organic reagent, and facile hydrolysis lost efficacy in the basic conditions.Except minority is used as to remove
Outside grass agent, antibacterial and plant growth regulator, great majority are used as agricultural fungicides.OPs has efficient, quick, wide spectrum
Advantage, is an extremely important compounds in pesticide, but accumulation too much can have certain toxic hazard to humans and animals, makes
With middle needs, safety is paid special attention to.Nineteen forty-one Adrian etc. propose phosphate ester first to inhibited the pushing away of acetylcholine esterase
Disconnected.Subsequently, Balls in 1949 confirms that this inhibitory action is owing to the phosphorylated at acyl group position causes.(organophosphor in vegetable
Insecticide carbamate insecticides is studied) at present, the OPs that OPs mechanism of poisoning the is well recognized as suppression sexology to internal acetylcholine esterase
Say, i.e. OPs and internal acetylcholine esterase (being present in teleneuron synaptic space in a large number, hydrolysis choline carries out neurotransmitter transmission)
In conjunction with forming phosphorylated acetylcholine esterase, making acetylcholine esterase lose activity, neurotransmitter choline accumulates in a large number, causes function of nervous system tight
Heavily damaging, produce acute poisoning phenomenon, severe patient is the most dead.
Current agricultural produce in, the pesticide based on organophosphorus pesticide is widely used in a variety of applications, also result in environment with
Food aspect severe compromise.Select the residual detection method of agriculture efficient, quick, simple, effective, pesticide residues are implemented effectively
Monitoring, there is huge real value and potential economic worth.In the current residual fast detecting method of agriculture, inhibiting AChE is should
With the residual method for quick of widest agriculture, but the method there is also the biggest drawback: such as the basis at tradition pretreatment technology
On the enzyme level detection method that carries out, to part enzyme sensitivity pesticide on the low side (if Rogor, chlopyrifos etc. are containing P=S group
OPs) its detection limit often higher than country's limit standard specification.It is fast that the food that this kind of pesticide is exceeded standard not easily passs through inhibiting AChE
Speed detection, or detected level is well below pesticide actual content, causes sample false negative, so that the product that exceeds standard flows directly into city
In Chang, cause potential food safety risk.Therefore, in order to improve sensitivity and the accuracy of inhibiting AChE, need sample
Pre-treating method and detection method improve, to improve the detection of the pesticide on the low side to this part acetylcholinesterase sensitivity
Sensitivity and accuracy, thus improve the residual content monitoring capacity of the agriculture in food, preferably ensure food safety.
The present invention conscientiously classifies from principle on the basis of being combined with machine phosphorus insecticide structural difference, has probed into containing P=S base
Group and the difference to esterase inhibition ability of the group organophosphorus pesticide Han P=O, will contain P=S base by unique enhanced sensitivity processing method
The organophosphorus pesticide of group is converted into the organophosphorus pesticide containing P=O, to improve the inspection of the organophosphorus pesticide of inhibiting AChE detection P=S
Survey ability.
Summary of the invention
It is an object of the invention to solve above technical problem, it is provided that the quick enhanced sensitivity detection side of a kind of organophosphorus pesticide
Method, the function utilizing cholinesterase catalysis to hydrolyze is suppressed by organophosphorus pesticide, and its inhibition exists good with the concentration of pesticide
Dependency, in enzyme reaction tested, add carbamates substrate and developer, the change of Observe and measure color, and then sentence
Whether the residual quantity of disconnected organophosphorus pesticide exceedes Limited Doses.Compared with the detection method of existing organophosphorus pesticide, the present invention is led to
Organic agricultural chemicals containing P=S group is converted into the organic agricultural chemicals containing P=O group, to improve enzyme by the enhanced sensitivity processing method crossing uniqueness
The power of test of the suppression method detection organic agricultural chemicals containing P=S group.
For solve the problems referred to above, the present invention by the following technical solutions:
A kind of quick sensitization detection method of organophosphorus pesticide, concrete preparation process is as follows:
(1) sample extraction, takes the sample after homogenate crushes and acetone mixing, vortex concussion extraction, filtering and impurity removing, then blows
Dry acetone filtrate, adds phosphate buffer constant volume, obtains sample extracting solution；
(2) enhanced sensitivity processes, and in sample extracting solution, adds calcium hypochlorite solution, oxidation reaction 10-40min, adds Asia
Sodium nitrate solution, reduction reaction 10-40min, obtain liquid to be measured；
(3) enzyme level detection, takes distilled water and liquid to be measured, adds work enzyme liquid, and 30-40 DEG C of constant temperature suppresses 3-30min, then
Add reaction substrate and developer, 30-40 DEG C of reaction 5-30min, use microplate reader detection absorbance at 410nm, press
Below equation calculating suppression ratio:
Wherein, Δ A0The absorbance of reaction system during for suppressing without pesticide；ΔA1The suction of reaction system during for there being pesticide to suppress
Luminosity.
Further, in described step (1), the consumption of sample is 5-20g, and acetone consumption is 20-30mL, adds phosphate
Buffer is settled to 1000uL；In described step (2), calcium hypochlorite solution consumption is 50-500uL, and sodium nitrite solution consumption is
50-500uL；In described step (3), the consumption of distilled water is 50-200uL, and the consumption of sample extracting solution is 50-100uL, work
The consumption of enzyme liquid is 30-80uL, and the consumption of reaction substrate is 30-80uL, and the consumption of developer is 15-40uL；
Further, described calcium hypochlorite solution is the calcium hypochlorite solution of mass concentration 0.02%-0.10%；Described
Sodium nitrite solution is the sodium nitrite solution of mass concentration 5%-20%；
Further, described calcium hypochlorite solution is the calcium hypochlorite solution of mass concentration 0.05%；Described nitrous acid
Sodium solution is the sodium nitrite solution of mass concentration 10%.
Further, described work enzyme liquid is acetylcholinesterase；Described reaction substrate is mass concentration 0.5%-5% iodine
Change acetylthiocholine；Described developer is by the 5 of 0.03-0.05g, and 5-dithio-2,2-dinitrobenzoic acid, adding pH value is
7.71 phosphate buffers dissolve and to be settled to 100mL formulated.
Further, described phosphate buffer pH value is 7.71.
Further, in described step (1), drying up acetone filtrate is to dry up acetone filtrate by the nitrogen of 30-40 DEG C.
Further, in described step (2), the operation temperature that enhanced sensitivity processes is 20-40 DEG C.
The quick sensitization detection method of a kind of organophosphorus pesticide of the present invention, compared with prior art, its prominent feature and
Excellent effect is:
1. the organic agricultural chemicals containing P=S group is converted into containing P=O group by the present invention by unique enhanced sensitivity processing method
Organic agricultural chemicals, to improve the inhibiting AChE detection power of test of organic agricultural chemicals containing P=S group.
2. the detection method of the present invention is simple to operate, highly sensitive, it is simple to quickly detect.
Accompanying drawing explanation
Fig. 1 is the suppression curve of 12 kinds of organophosphorous pesticides on acetylcholinesteraseands；
Fig. 2 is to process the impact on mixing mark-on sample detection by enhanced sensitivity.
Detailed description of the invention
Below by way of detailed description of the invention and accompanying drawing, the present invention is described in further detail, but should this be interpreted as
The scope of the present invention is only limitted to below example.In the case of without departing from said method thought of the present invention, according to this area
Various replacements that ordinary technical knowledge and customary means are made or change, should be included in the scope of the present invention.
Embodiment 1
A kind of quick sensitization detection method of organophosphorus pesticide:
Sample extraction, weighs the sample being mixed with machine phosphorus insecticide after 10g homogenate crushes and the mixing of 25mL acetone, whirlpool respectively
Rotation concussion extraction 10min, then filtering and impurity removing, dry up acetone with the nitrogen of 35 DEG C, add phosphate buffer and be settled to
1000uL；
Enhanced sensitivity processes, and in 1000uL sample extracting solution, adds 0.05% calcium hypochlorite solution of 300uL, and 30 DEG C of oxidations are anti-
Answer 15min, add 10% sodium nitrite solution of 300uL, 30 DEG C of reduction reactions 15min, obtain liquid to be measured；
Enzyme level detects, and in ELISA Plate, takes the distilled water of 100uL and the liquid to be measured of 75uL, adds the work of 50uL
Enzyme liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 15min, add reaction substrate ATChI's and 25uL of 50uL
Developer 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 10min, adopt microplate reader and detect at 410nm
The absorbance at place.
Embodiment 2
A kind of quick sensitization detection method of organophosphorus pesticide:
Sample extraction, weighs the sample being mixed with machine phosphorus insecticide after 5g homogenate crushes and the mixing of 20mL acetone, whirlpool respectively
Rotation concussion extraction 5min, then filtering and impurity removing, dry up acetone with the nitrogen of 35 DEG C, add phosphate buffer and be settled to
1000uL；
Enhanced sensitivity processes, and in 1000uL sample extracting solution, adds 0.05% calcium hypochlorite solution of 50uL, 30 DEG C of oxidation reactions
10min, adds 10% sodium nitrite solution of 50uL, 30 DEG C of reduction reactions 10min, obtains liquid to be measured；
Enzyme level detects, and in ELISA Plate, takes the distilled water of 50uL and the liquid to be measured of 50uL, adds the work enzyme of 30uL
Liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 3min, add reaction substrate ATChI and 15uL aobvious of 30uL
Toner 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 5min, use microplate reader to detect at 410nm
Absorbance.
Embodiment 3
A kind of quick sensitization detection method of organophosphorus pesticide:
Sample extraction, weighs the sample being mixed with machine phosphorus insecticide after 20g homogenate crushes and the mixing of 30mL acetone, whirlpool respectively
Rotation concussion extraction 30min, then filtering and impurity removing, dry up acetone with the nitrogen of 35 DEG C, add phosphate buffer and be settled to
1000uL；
Enhanced sensitivity processes, and in 1000uL sample extracting solution, adds 0.05% calcium hypochlorite solution of 500uL, and 30 DEG C of oxidations are anti-
Answer 40min, add 10% sodium nitrite solution of 500uL, 30 DEG C of reduction reactions 40min, obtain liquid to be measured；
Enzyme level detects, and in ELISA Plate, takes the distilled water of 200uL and the liquid to be measured of 100uL, adds the work of 80uL
Enzyme liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 30min, add reaction substrate ATChI's and 40uL of 80uL
Developer 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 30min, use microplate reader detection to exist
Absorbance at 410nm.
Embodiment 4
A kind of quick sensitization detection method of organophosphorus pesticide:
Sample extraction, weighs the sample being mixed with machine phosphorus insecticide after 15g homogenate crushes and the mixing of 24mL acetone, whirlpool respectively
Rotation concussion extraction 15min, then filtering and impurity removing, dry up acetone with the nitrogen of 35 DEG C, add phosphate buffer and be settled to
1000uL；
Enhanced sensitivity processes, and in 1000uL sample extracting solution, adds 0.05% calcium hypochlorite solution of 200uL, and 30 DEG C of oxidations are anti-
Answer 20min, add 10% sodium nitrite solution of 200uL, 30 DEG C of reduction reactions 20min, obtain liquid to be measured；
Enzyme level detects, and in ELISA Plate, takes the distilled water of 120uL and the liquid to be measured of 80uL, adds the work of 55uL
Enzyme liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 20min, add reaction substrate ATChI's and 28uL of 55uL
Developer 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 15min, use microplate reader detection to exist
Absorbance at 410nm.
Embodiment 5
A kind of quick sensitization detection method of organophosphorus pesticide:
Sample extraction, weighs the sample being mixed with machine phosphorus insecticide after 12g homogenate crushes and the mixing of 23mL acetone, whirlpool respectively
Rotation concussion extraction 10min, then filtering and impurity removing, dry up acetone with the nitrogen of 35 DEG C, add phosphate buffer and be settled to
1000uL；
Enhanced sensitivity processes, and in 1000uL sample extracting solution, adds 0.05% calcium hypochlorite solution of 400uL, and 30 DEG C of oxidations are anti-
Answer 25min, add 10% sodium nitrite solution of 400uL, 30 DEG C of reduction reactions 25min, obtain liquid to be measured；
Enzyme level detects, and in ELISA Plate, takes the distilled water of 150uL and the liquid to be measured of 80uL, adds the work of 60uL
Enzyme liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 10min, add reaction substrate ATChI's and 33uL of 60uL
Developer 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 12min, use microplate reader detection to exist
Absorbance at 410nm.
Embodiment 6
Sample extraction, weighs the sample after 10g homogenate crushes respectively and (is separately added into same concentrations Rogor, chlopyrifos, enemy hundred
Worm, phoxim, Malathion, fenifrothion, triazophos, omethoate, dichlorvos, orthene, Profenofos, malaoxon
In the sample of organophosphorus pesticide, and a sample without pesticide) and the mixing of 25mL acetone, vortex concussion extraction 10min, then
Filtering and impurity removing, dries up acetone with the nitrogen of 35 DEG C, adds phosphate buffer and is settled to 1000uL；
Sample processes the detection of direct enzyme level without enhanced sensitivity, in ELISA Plate, takes the distilled water of 100uL and the sample of 75uL
Extracting solution, adds the work enzyme liquid acetylcholinesterase of 50uL, mix homogeneously, 35 DEG C of constant temperature suppression 15min, adds 50uL
The developer 5 of reaction substrate ATChI and 25uL, 5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reactions
10min, uses microplate reader detection absorbance at 410nm, calculates its suppression ratio.
Laboratory test results such as Fig. 1, wherein, the organophosphorus pesticide containing P=S group includes: Rogor, chlopyrifos, metrifonate,
Phoxim, Malathion, fenifrothion, triazophos, the organophosphorus pesticide containing P=O group includes: omethoate, dichlorvos, second
Acyl Bayer 71628, Profenofos, malaoxon.Test result indicate that the organophosphorus pesticide containing P=O group under same concentrations is to acetyl
The suppression ratio of acetylcholine esterase is significantly higher than the organophosphorus pesticide containing P=S group, if not taking corrective measure to there is part containing P
=S group organophosphorus pesticide is difficult to the possibility being detected.
Embodiment 7
Sample extraction, weighs the sample after 10g homogenate crushes respectively and is separately added into the Rogor of variable concentrations, chlopyrifos, enemy
The organophosphorus pesticide containing P=S group such as hundred worms, phoxim, Malathion, fenifrothion, triazophos, mixes with 25mL acetone,
Vortex concussion extraction 10min, then filtering and impurity removing, merge acetone filtrate, dry up acetone with the nitrogen of 35 DEG C, add phosphate and delay
Rush liquid and be settled to 1000uL；
Sample aliquot is two parts, and portion carries out enhanced sensitivity process, i.e., in 1000uL sample extracting solution, add 400uL's
0.05% calcium hypochlorite solution, 30 DEG C of oxidation reactions 25min, add 10% sodium nitrite solution of 400uL, 30 DEG C of reduction are anti-
Answer 25min；Another part does not carry out enhanced sensitivity process, as comparison.
Enzyme level detects, and in ELISA Plate, takes the distilled water of 100uL and the liquid to be measured of 75uL, adds the work of 50uL
Enzyme liquid acetylcholinesterase, mix homogeneously, 35 DEG C of constant temperature suppression 15min, add reaction substrate ATChI's and 25uL of 50uL
Developer 5,5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 10min, use microplate reader detection to exist
Absorbance at 410nm.By calculating its suppression ratio curvilinear equation and IC50Value.The relatively organophosphorus pesticide containing P=S group
After enhanced sensitivity processes, the change to acetylcholinesterase sensitivity.
Experimental result is shown in Table 1, shows the oxidation-treated rear organophosphorous pesticides on acetylcholinesteraseand containing P=S group
Sensitivity generally improves 2～4 orders of magnitude, significantly improves the inhibiting AChE detection organophosphorus pesticide containing P=S group
Sensitivity.
Table 1 is containing suppressing curvilinear equation and IC before and after P=S group organophosphorus pesticide oxidation processes50Value
Wherein y is suppression ratio, and x is the suppression time, R2For correlation coefficient, IC50(M) it is semi-inhibit rate.
Embodiment 8
Sample extraction, (being separately added into total concentration is 0.02mg/kg and 2mg/ to weigh the sample after 10g homogenate crushes respectively
The organophosphor agriculture containing P=S group such as the Rogor of kg, chlopyrifos, metrifonate, phoxim, Malathion, fenifrothion, triazophos
The hybrid standard product of medicine) and the mixing of 25mL acetone, vortex concussion extraction 10min, then filtering and impurity removing, dry up with the nitrogen of 35 DEG C
Acetone, adds phosphate buffer and is settled to 1000uL；
Sample aliquot is two parts, and portion carries out enhanced sensitivity process, i.e., in 1000uL sample extracting solution, add 400uL's
0.05% calcium hypochlorite solution, 30 DEG C of oxidation reactions 25min, add 10% sodium nitrite solution of 400uL, 30 DEG C of reduction are anti-
Answer 25min；Another part does not carry out enhanced sensitivity process, as comparison；
Again sample is carried out enzyme level detection, in ELISA Plate, take the distilled water of 100uL and the liquid to be measured of 75uL, then add
Enter the work enzyme liquid acetylcholinesterase of 50uL, mix homogeneously, 35 DEG C of constant temperature suppression 15min, add the reaction substrate of 50uL
The developer 5 of ATChI and 25uL, 5-dithio-2,2-dinitrobenzoic acid, mix homogeneously, 35 DEG C of reaction 10min, use enzyme
Mark instrument detection absorbance at 410nm, calculates suppression ratio.
Testing result such as Fig. 2, no matter result shows under high concentration (2mg/kg) or low concentration (0.02mg/kg), warp
After oxidation processes, the suppression ratio containing the organophosphorous pesticides on acetylcholinesteraseand of P=S group is all significantly increased.
Claims (8)
1. the quick sensitization detection method of an organophosphorus pesticide, it is characterised in that concrete detecting step is as follows:
(1) sample extraction, takes the sample after homogenate crushes and acetone mixing, vortex concussion extraction, filtering and impurity removing, then dries up third
Ketone filtrate, adds phosphate buffer constant volume, obtains sample extracting solution；
(2) enhanced sensitivity processes, and in sample extracting solution, adds calcium hypochlorite solution, oxidation reaction 10-40min, adds nitrous acid
Sodium solution, reduction reaction 10-40min, obtain liquid to be measured；
(3) enzyme level detection, takes distilled water and liquid to be measured, adds work enzyme liquid, 30-40 DEG C of constant temperature suppression 3-30min, adds
Reaction substrate and developer, 30-40 DEG C of reaction 5-30min, use microplate reader detection absorbance at 410nm, by following
Formula calculating suppression ratio:
Wherein, Δ A0The absorbance of reaction system during for suppressing without pesticide；ΔA1The extinction of reaction system during for there being pesticide to suppress
Degree.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 1, it is characterised in that: described step
(1) in, the consumption of sample is 5-20g, and acetone consumption is 20-30mL, adds phosphate buffer and is settled to 1000uL；Described step
Suddenly in (2), calcium hypochlorite solution consumption is 50-500uL, and sodium nitrite solution consumption is 50-500uL；In described step (3), steam
The consumption of distilled water is 50-200uL, and the consumption of sample extracting solution is 50-100uL, and the consumption of work enzyme liquid is 30-80uL, reaction
The consumption of substrate is 30-80uL, and the consumption of developer is 15-40uL.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 2, it is characterised in that: described chlorine
Acid calcium solution is the calcium hypochlorite solution of mass concentration 0.02%-0.10%；Described sodium nitrite solution is mass concentration 5%-
The sodium nitrite solution of 20%.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 3, it is characterised in that: described chlorine
Acid calcium solution is the calcium hypochlorite solution of mass concentration 0.05%；Described sodium nitrite solution is the nitrous acid of mass concentration 10%
Sodium solution.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 1 and 2, it is characterised in that: described
Work enzyme liquid is acetylcholinesterase；Described reaction substrate is mass concentration 0.5%-5% acetylthiocholine iodide；Described aobvious
Toner is by the 5 of 0.03-0.05g, and 5-dithio-2,2-dinitrobenzoic acid, adding pH value is that 7.71 phosphate buffers dissolve and determine
Hold formulated to 100mL.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 1 and 2, it is characterised in that: described
Phosphate buffer pH value is 7.71.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 1, it is characterised in that: described step
(1), in, drying up acetone filtrate is to dry up acetone filtrate by the nitrogen of 30-40 DEG C.
The quick sensitization detection method of a kind of organophosphorus pesticide the most according to claim 1, it is characterised in that: described step
(2), in, the operation temperature that enhanced sensitivity processes is 20-40 DEG C.
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