CN111537504B - Siphon type test paper and method for detecting pesticide residue by using same - Google Patents
Siphon type test paper and method for detecting pesticide residue by using same Download PDFInfo
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- CN111537504B CN111537504B CN202010354225.2A CN202010354225A CN111537504B CN 111537504 B CN111537504 B CN 111537504B CN 202010354225 A CN202010354225 A CN 202010354225A CN 111537504 B CN111537504 B CN 111537504B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
- G01N2021/7759—Dipstick; Test strip
Abstract
The invention discloses a siphon-type test paper and a method for detecting pesticide residue by adopting the test paper, the siphon-type test paper comprises a bottom plate, a single-sided adhesive tape, a double-sided adhesive tape and a cover plate, wherein the single-sided adhesive tape is adhered on the top surface of the bottom plate, the lower end of the top surface of the single-sided adhesive tape is respectively provided with a lower notch and an upper notch from bottom to top, acetylcholinesterase and dithionitrobenzoic acid are solidified in the lower notch to form a reaction zone, acetylcholine is solidified in the upper notch to form a detection zone, the double-sided adhesive tape is adhered on the lower end of the top surface of the single-sided adhesive tape, the double-sided adhesive tape is provided with a through groove which is communicated with the lower notch and the upper notch at the same time, the through groove is communicated with the lower end of the double-sided adhesive tape, the cover plate is adhered on the top surface of the double-sided adhesive tape, and the method for detecting pesticide residue by adopting the siphon-type test paper comprises a) sample preparation, a, b) Reaction and c) detection, and the whole operation is convenient and simple.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of pesticide residue detection, in particular to siphon type detection test paper and a method for detecting pesticide residue by using the test paper.
[ background of the invention ]
China is a big agricultural country, and agriculture plays a vital role in national economy and people's life. In modern agricultural production, pesticides are important production materials for preventing and treating plant diseases and insect pests, ensuring agricultural harvest and ensuring food supply. However, the negative effects of the pesticide residue exceeding the standard are gradually shown while the crop yield is improved due to the large amount of the pesticide. The pesticide residue refers to a general term of trace amounts of pesticide original, toxic metabolites, degradation products and impurities which remain in organisms, agricultural and sideline products and environment after the pesticide is used. Generally, the pesticide is not easy to volatilize, is not easy to decompose in the plant body and in the soil, and has higher residue after the pesticide with strong fat solubility is applied. With the continuous improvement of the living standard of people in China, the quality safety problem of agricultural products is more and more concerned, particularly, the problem of pesticide residue in vegetables becomes the focus of public attention, hundreds of acute poisoning events caused by eating agricultural products polluted by pesticides exist in the whole country every year, the body health of consumers is seriously influenced, and therefore the food safety problem caused by the overproof pesticide residue is more and more concerned by governments and the public in various countries.
The majority of pesticides in use today are organic phosphorus and carbamate compounds. The conventional detection method for pesticide residue at home and abroad is mainly a physical and chemical analysis method, namely, the pesticide residue is generally analyzed and detected by using instruments such as a gas chromatograph, a liquid chromatograph, a gas chromatograph-mass spectrometer and a liquid chromatograph-mass spectrometer. The methods have the advantages of qualitative and quantitative detection of the pesticide residue and are national standard detection methods. However, these methods also require complicated pretreatment processes such as extraction and separation of samples, require a large amount of manpower and material resources, and have high detection cost, which are urgently needed to be solved.
[ summary of the invention ]
The invention aims to solve the problems in the prior art, and provides siphon-type test paper and a method for detecting pesticide residues by using the test paper, which are convenient to use and reliable in detection effect.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the siphon-type test paper comprises a bottom plate, a single-sided adhesive tape, a double-sided adhesive tape and a cover plate, wherein the single-sided adhesive tape is pasted on the top surface of the bottom plate, the lower end of the top surface of the single-sided adhesive tape is provided with a lower notch and an upper notch from bottom to top respectively, the lower notch is internally cured with acetylcholinesterase and dithionitrobenzoic acid to form a reaction area, the upper notch is internally cured with acetylcholine to form a detection area, the double-sided adhesive tape is pasted on the lower end of the top surface of the single-sided adhesive tape, the double-sided adhesive tape is provided with a through groove which is communicated with the lower notch and the upper notch simultaneously, the through groove is communicated with the lower end of the double-sided adhesive tape, the cover plate is pasted on the top surface of the double-sided adhesive tape, and the cover plate is provided with an air hole communicated with the through groove.
Preferably, the bottom plate is a PET plate.
Preferably, the immobilization of the acetylcholinesterase, the dithionitrobenzoic acid and the acetylcholine on the reaction zone and the detection zone respectively comprises the following steps:
a) preparation of a reagent: weighing acetylcholinesterase, dissolving the acetylcholinesterase into a solution with the enzyme activity of 2200-2400U/ml by using a buffer solution or purified water to obtain an enzyme preparation, weighing dithio nitrobenzoic acid, dissolving the dithio nitrobenzoic acid into a solution with the enzyme activity of 8-10 mg/ml by using the buffer solution or purified water to obtain a color developing agent, weighing acetylcholine, dissolving the acetylcholine into a solution with the enzyme activity of 8-10 mg/ml by using the purified water to obtain a substrate preparation;
b) coating and curing: mixing the enzyme agent and the color developing agent prepared in the step a) according to the volume ratio of 1: 1-1.5, obtaining a reactant, adding 0.10-0.14% of hydroxyethyl cellulose in mass fraction into the reactant and the substrate agent prepared in the step a), sucking 1-1.5 mu L of the reactant mixed with the hydroxyethyl cellulose, dripping the reactant on a reaction area, and then carrying out vacuum freeze-drying, sucking 1-1.5 mu L of the substrate agent mixed with the hydroxyethyl cellulose, dripping the substrate agent on a detection area, and then carrying out vacuum freeze-drying.
Preferably, the buffer solution in step a) is an anhydrous dipotassium hydrogen phosphate and dipotassium hydrogen phosphate buffer solution with pH 8.0.
Preferably, the viscosity of the reactant and the primer mixed with the hydroxyethyl cellulose is 4500 to 6500 cps.
The method for detecting pesticide residue by using the siphon test paper comprises the following steps:
a) preparing a sample: taking a vegetable sample to be tested, wiping soil, cutting into square pieces of 1-2 cm, taking 5g of vegetable pieces, adding 10ml of purified water, stirring for 5-10 min, and standing for 1-2 min to obtain an extracting solution;
b) reaction: adding 1/2 samples of the extract prepared in the step a) from the front side of the test paper along the through groove, wherein the samples are 0.5-1 mu L, allowing the extract to enter a reaction zone to react with acetylcholinesterase and dithionitrobenzoic acid, and standing for 5-10 min;
c) and (3) detection: and (3) adding the residual 1/2 sample adding amount of the extracting solution prepared in the step a) from the front side of the test paper along the through groove, pushing the mixed solution which is fully reacted in the reaction area into a detection area, standing for 5-10 min, detecting absorbance by an instrument, and calculating the enzyme inhibition rate to obtain the pesticide residue.
The invention has the beneficial effects that: according to the invention, the reaction areas with the enzyme and the color developing agent and the test areas with the substrate are respectively arranged on the bottom plate, the extracting solution is dripped on the sample injection sheet in sections, and the reaction time and the reaction amount of each step of reaction are accurately controlled by using the siphon action, so that the pesticide residue is quantitatively detected through the absorbance change caused by the color development condition of the test paper, the whole operation is convenient and simple, no professional technical training is required, the test paper is easy to store, and the carrying is convenient.
The features and advantages of the present invention will be described in detail by embodiments with reference to the accompanying drawings.
[ description of the drawings ]
FIG. 1 is a schematic perspective view of a siphon test strip according to the present invention;
FIG. 2 is a front view of a siphonic test strip of the present invention;
FIG. 3 is a top view of a siphonic test strip of the present invention;
FIG. 4 is a top view of a siphonic test strip of the present invention with the cover removed;
FIG. 5 is a top view of a siphonic test strip of the present invention with the cover plate and double-sided adhesive removed;
FIG. 6 is a top view of the base of a siphonic test strip according to the present invention.
In the figure: 1-bottom plate, 2-single-side adhesive plaster, 21-lower notch, 22-upper notch, 3-double-side adhesive plaster, 31-through groove, 4-cover plate and 41-air hole.
[ detailed description ] embodiments
Referring to fig. 1 to 6, the siphon-type test paper comprises a bottom plate 1, a single-sided adhesive 2, a double-sided adhesive 3 and a cover plate 4, wherein the single-sided adhesive 2 is attached to the top surface of the bottom plate 1, the lower end of the top surface of the single-sided adhesive 2 is provided with a lower notch 21 and an upper notch 22 from bottom to top, acetylcholinesterase and dithionitrobenzoic acid are cured in the lower notch 21 to form a reaction zone, acetylcholine is cured in the upper notch 22 to form a detection zone, the double-sided adhesive 3 is attached to the lower end of the top surface of the single-sided adhesive 2, a through groove 31 which is communicated with the lower notch 21 and the upper notch 22 is arranged on the double-sided adhesive 3, the through groove 31 is communicated with the lower end of the double-sided adhesive 3, the cover plate 4 is attached to the top surface of the double-sided adhesive 3, and an air hole 41 which is communicated with the through groove 31 is arranged on the cover plate 4.
The bottom plate 1 is a PET plate.
The method for respectively solidifying the acetylcholinesterase, the dithionitrobenzoic acid and the acetylcholine on the reaction area and the detection area comprises the following steps:
a) preparation of a reagent: weighing acetylcholinesterase, dissolving the acetylcholinesterase into a solution with the enzyme activity of 2200-2400U/ml by using a buffer solution or purified water to obtain an enzyme preparation, weighing dithio nitrobenzoic acid, dissolving the dithio nitrobenzoic acid into a solution with the enzyme activity of 8-10 mg/ml by using the buffer solution or purified water to obtain a color developing agent, weighing acetylcholine, dissolving the acetylcholine into a solution with the enzyme activity of 8-10 mg/ml by using the purified water to obtain a substrate preparation;
b) coating and curing: mixing the enzyme agent and the color developing agent prepared in the step a) according to the volume ratio of 1: 1-1.5, obtaining a reactant, adding 0.12 mass percent of hydroxyethyl cellulose into the reactant and the substrate agent prepared in the step a), sucking 1-1.5 mu L of the reactant mixed with the hydroxyethyl cellulose, dripping the reactant on a reaction area, performing vacuum freeze-drying, sucking 1-1.5 mu L of the substrate agent mixed with the hydroxyethyl cellulose, dripping the substrate agent on a detection area, and performing vacuum freeze-drying.
The buffer solution in the step a) is anhydrous dipotassium hydrogen phosphate and dipotassium hydrogen phosphate buffer solution with the pH value of 8.0.
The viscosity ranges of the reactant and the primer mixed with the hydroxyethyl cellulose are 4500-6500 cps.
The method for detecting the pesticide residue by adopting the siphon-type detection test paper comprises the following steps:
a) preparing a sample: taking a vegetable sample to be tested, wiping soil, cutting into square pieces of 1-2 cm, taking 5g of vegetable pieces, adding 10ml of purified water, stirring for 5-10 min, and standing for 1-2 min to obtain an extracting solution;
b) reaction: adding 1/2 samples of the extract prepared in the step a) from the front side of the test paper along the through groove 31, wherein the samples are 0.5-1 mu L, allowing the extract to enter a reaction zone to react with acetylcholinesterase and dithionitrobenzoic acid, and standing for 5-10 min;
c) and (3) detection: and (3) adding the residual 1/2 sample adding amount of the extracting solution prepared in the step a) from the front side of the test paper along the through groove 31, pushing the mixed solution which is fully reacted in the reaction area into the detection area, standing for 5-10 min, detecting the absorbance by an instrument, and calculating the enzyme inhibition rate to obtain the pesticide residue.
In the testing process, if the vegetable sample does not contain organophosphorus pesticides or carbamate pesticides, the acetylcholine is hydrolyzed into choline and acetic acid under the catalytic action of acetylcholinesterase, and the choline reacts with dithionitrobenzoic acid to generate yellow substances; if the vegetable sample contains the organophosphorus pesticide or the carbamate pesticide, the pesticide can inhibit the activity of enzyme, so that the hydrolysis and color development of the acetylcholine are inhibited, and the more the pesticide content is, the lighter the color development is until the color development is colorless. The coloration change brings the change of absorbance, and the enzyme inhibition rate can be calculated through the absorbance so as to obtain the pesticide residue.
The detection embodiment comprises the following steps:
and (3) testing a sample: taking 5 groups of vegetable leaves, and then respectively and uniformly spraying 2mg/Kg, 20mg/Kg, 40mg/Kg, 80mg/Kg and 120mg/Kg of metoclopramide, phosphorus dibromide and aldicarb on the 5 groups of vegetable leaves. After standing for 1-2 days, cutting a vegetable sample to be tested into square pieces of 1-2 cm, taking 5g of the vegetable pieces, adding 10ml of purified water, stirring for 5-10 min, and standing for 1-2 min to obtain an extracting solution. And adding 0.5 mu L of extracting solution along the through groove 31 from the front side of the test paper, enabling the extracting solution to enter a reaction zone to react with acetylcholinesterase and dithionitrobenzoic acid, and standing for 5-10 min. Then, the remaining 0.5 μ L of the extracting solution is added from the front side of the test paper along the through groove 31, so that the mixed solution which is fully reacted in the reaction area is pushed into the detection area, and then the test paper is kept still for 5-10 min. Then, the mixture was subjected to colorimetry at a wavelength of 412nm using a spectrophotometer, and the change value Δ A of absorbance within 3min of the reaction was recorded 1 。
Control test: changing the extracting solution into purified water, testing the sample in other operations, and recording the absorbance change value delta A in 3min of reaction 0 。
Calculation of enzyme inhibition:
enzyme inhibition rate (%) ═ delta A 0 -△A 1 )/△A 0 ]×100％
In the formula:
△A 1 -the change in absorbance of the sample set after 3min of reaction
△A 0 -the change value of absorbance of the control group after reacting for 3min
The results are shown in the following table:
on 5 groups of vegetable leaves, the test paper detects two pesticides, and the enzyme inhibition rate is gradually increased and almost completely inactivated along with the increase of the pesticide concentration, so that the test paper is sensitive to the detection of metoclopramide, phosphorus dibromide and aldicarb.
According to the invention, the reaction areas with the enzyme and the color developing agent and the test areas with the substrate are respectively arranged on the bottom plate, the extracting solution is dripped on the sample injection sheet in sections, and the reaction time and the reaction amount of each step of reaction are accurately controlled by using the siphon action, so that the pesticide residue is quantitatively detected through the absorbance change caused by the color development condition of the test paper, the whole operation is convenient and simple, no professional technical training is required, the test paper is easy to store, and the carrying is convenient.
The above embodiments are illustrative of the present invention, and are not intended to limit the present invention, and any simple modifications of the present invention are within the scope of the present invention.
Claims (6)
1. A hydrocone type test paper which characterized in that: comprises a bottom plate (1), a single-sided adhesive tape (2), a double-sided adhesive tape (3) and a cover plate (4), the single-sided adhesive tape (2) is adhered on the top surface of the bottom plate (1), the lower end of the top surface of the single-sided adhesive tape (2) is respectively provided with a lower notch (21) and an upper notch (22) from bottom to top, the lower gap (21) is internally solidified with acetylcholine enzyme and dithio-nitrobenzoic acid so as to form a reaction zone, acetylcholine is solidified in the upper gap (22) to form a detection area, the double-sided adhesive tape (3) is stuck on the lower end of the top surface of the single-sided adhesive tape (2), a through groove (31) which is communicated with the lower notch (21) and the upper notch (22) is arranged on the double-sided adhesive tape (3), the through groove (31) runs through the lower end of the double-sided adhesive tape (3), the cover plate (4) is adhered on the top surface of the double-sided adhesive tape (3), and the cover plate (4) is provided with an air hole (41) communicated with the through groove (31).
2. The siphonic test strip according to claim 1, wherein: the bottom plate (1) is a PET plate.
3. The siphonic test strip according to claim 1, wherein: the method for respectively solidifying the acetylcholinesterase, the dithionitrobenzoic acid and the acetylcholine on the reaction area and the detection area comprises the following steps:
a) preparation of a reagent: weighing acetylcholinesterase, dissolving the acetylcholinesterase into a solution with the enzyme activity of 2200-2400U/ml by using a buffer solution or purified water to obtain an enzyme preparation, weighing dithio nitrobenzoic acid, dissolving the dithio nitrobenzoic acid into a solution with the enzyme activity of 8-10 mg/ml by using the buffer solution or purified water to obtain a color developing agent, weighing acetylcholine, dissolving the acetylcholine into a solution with the enzyme activity of 8-10 mg/ml by using the purified water to obtain a substrate preparation;
b) coating and curing: mixing the enzyme agent and the color developing agent prepared in the step a) according to the volume ratio of 1: 1-1.5, obtaining a reactant, adding 0.10-0.14% of hydroxyethyl cellulose in mass fraction into the reactant and the substrate agent prepared in the step a), sucking 1-1.5 mu L of the reactant mixed with the hydroxyethyl cellulose, dripping the reactant on a reaction area, and then carrying out vacuum freeze-drying, sucking 1-1.5 mu L of the substrate agent mixed with the hydroxyethyl cellulose, dripping the substrate agent on a detection area, and then carrying out vacuum freeze-drying.
4. The siphonic test strip according to claim 3, wherein: the buffer solution in the step a) is anhydrous dipotassium hydrogen phosphate buffer solution and dipotassium hydrogen phosphate buffer solution with the pH value of 8.0.
5. A siphon test strip according to claim 3, wherein: the viscosity ranges of the reactant and the primer mixed with the hydroxyethyl cellulose are 4500-6500 cps.
6. A method of detecting pesticide residues using the siphon test strip of claim 1, comprising the steps of:
a) preparing a sample: taking a vegetable sample to be tested, wiping soil, cutting into square pieces of 1-2 cm, taking 5g of vegetable pieces, adding 10ml of purified water, stirring for 5-10 min, and standing for 1-2 min to obtain an extracting solution;
b) reaction: adding 1/2 samples of the extract prepared in the step a) from the front side of the test paper along the through groove (31), wherein the samples are 0.5-1 mu L, allowing the extract to enter a reaction zone to react with acetylcholinesterase and dithionitrobenzoic acid, and standing for 5-10 min;
c) and (3) detection: and (3) adding the residual 1/2 sample adding amount of the extracting solution prepared in the step a) from the front side of the test paper along the through groove (31), so that the mixed solution which is fully reacted in the reaction area in the past is pushed into a detection area, standing for 5-10 min, detecting the absorbance through an instrument, and calculating the enzyme inhibition rate to obtain the pesticide residue.
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| US6258229B1 (en) * | 1999-06-02 | 2001-07-10 | Handani Winarta | Disposable sub-microliter volume sensor and method of making |
| CN107064123A (en) * | 2017-01-03 | 2017-08-18 | 长沙中生众捷生物技术有限公司 | The detection reagent of triglycerides and the Test paper of triglycerides |
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| US20070062811A1 (en) * | 2005-09-21 | 2007-03-22 | Health & Life Co., Ltd | Bioelectrochemical sensor strip capable of taking trace samples |
| TW200823456A (en) * | 2006-11-24 | 2008-06-01 | Health & Life Co Ltd | Biosensor |
| US20080160556A1 (en) * | 2006-12-13 | 2008-07-03 | Industrial Technology Research Institute | Bioassay element and producing method thereof |
| CN203037583U (en) * | 2013-01-28 | 2013-07-03 | 重庆大学 | Pesticide residue siphon test paper |
| CN203502356U (en) * | 2013-08-20 | 2014-03-26 | 中国计量学院 | Test strip for quickly detecting pesticide residues in fruits and vegetables |
| CN107703125A (en) * | 2016-08-16 | 2018-02-16 | 江南大学 | A kind of preparation method and applications for the intelligent label for judging the critical freshness of pork |
| CN106093031A (en) * | 2016-08-16 | 2016-11-09 | 江南大学 | A kind of preparation method and applications of the intelligent label for judging the critical freshness of aquatic products |
| CN206095954U (en) * | 2016-10-27 | 2017-04-12 | 四川农业大学 | Pesticide residue's card in short -term test fruit vegetables |
| CN107478646A (en) * | 2017-08-14 | 2017-12-15 | 仲恺农业工程学院 | Freshness of meat indicating label, solution and preparation method for the indicating label |
| CN108535249B (en) * | 2018-04-25 | 2021-02-26 | 郑州贝贝生物科技有限公司 | Pesticide residue fast detection point card |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6258229B1 (en) * | 1999-06-02 | 2001-07-10 | Handani Winarta | Disposable sub-microliter volume sensor and method of making |
| CN107064123A (en) * | 2017-01-03 | 2017-08-18 | 长沙中生众捷生物技术有限公司 | The detection reagent of triglycerides and the Test paper of triglycerides |
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