CN109253999A - A kind of preparation and application of pesticide quick measuring card - Google Patents
A kind of preparation and application of pesticide quick measuring card Download PDFInfo
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- CN109253999A CN109253999A CN201811092703.6A CN201811092703A CN109253999A CN 109253999 A CN109253999 A CN 109253999A CN 201811092703 A CN201811092703 A CN 201811092703A CN 109253999 A CN109253999 A CN 109253999A
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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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Abstract
The present invention relates to nano material, catalysis and analytical chemistry fields, specifically include the preparation and application of a kind of pesticide quick measuring card.This method utilizes and obtains the quick measuring card quickly detected on cobaltosic oxide nano ball, acetylcholinesterase, choline oxidase and developer-modified filter paper with imitative peroxidase activity, realizes the detection of simple, quick acetylcholine and organophosphorus pesticide.This novel colorimetric Pesticides Testing method is practical, and therefore, this method has broad application prospects in food inspection, analytical chemistry, environmental project and agriculture field.
Description
Technical field
The present invention relates to nano material, catalysis and analytical chemistry field, specifically include a kind of pesticide quick measuring card preparation and
Using.
Technical background
With agrotechnical development, pesticide is widely used.And pesticide residue becomes threat human food's peace
Complete important hidden danger, therefore, the detection of pesticide become particularly important, and what is be subject to allows people greatly to pay close attention to.There are many about
The article of Pesticides Testing, for example, Jung et al. is in " M13 bacteriophage/silver nanowire surface-
enhanced raman scattering sensor for sensitive and selective pesticide
Detection " using M13 bacteriophage functionalization silver nanowires by Raman come sensitive and selective enumeration method pesticide (ACS Applied Materials & Interfaces2018,10,10388-10397).Pumera et al. is in " Two-
dimensional 1T-phase transition metal dichalcogenides as nanocarriers to
enhance and stabilize enzyme activity for electrochemical pesticide
Detection " in utilize two-dimensional transient metal sulfide as nano-carrier to enhance the activity of enzyme and be used for Electrochemical Detection
Pesticide (ACS Nano2017,11,5774-5784).Mukherjee et al. is in " Acetylcholinesterase
(AChE)-mediated immobilization of silver nanoparticles for the detection of
Organophosphorus pesticides " acetylcholinesterase is fixed on Nano silver grain in a text and is realized to organic
Phosphorus pesticide colorimetric detection (RSC Advances2016,6,64769-64777).Although these reports are all realized to agriculture
The detection of medicine, but detection process is more complicated, needs to develop quick detection of the new method realization of one kind to pesticide.
Test strips have been widely used in life as a kind of portable rapid detection method, such as blood sugar test paper
Item, urinalysis test strips etc..Therefore, many documents have also done certain research to test strips.Yan et al. exists
By the colloid in traditional test strip in " Nanozyme-strip for rapid local diagnosis of Ebola " text
Gold replaces with magnetic Nano enzyme, detects Ebola virus by the method for colour developing, and the sensitivity of test strips is made to improve 100
Times (Biosensors and Bioelectronics2015,74,134-141).Cai et al. is in " A simple and
rapid colloidal gold-based immunochromatogarpic striptest for detection of
FMDV derotype A " immunoassay combined with test strips in a text for detect foot and mouth disease virus (ACS Applied Materials & Interfaces2014,6,21624-21631).Although test strips have a degree of application,
Existing method manufacturing process is more complicated and higher cost, and the above-mentioned test strips mechanism of action and pesticide molecule detection
The mechanism of action has essential distinction.Therefore, it is necessary to develop the new method of one kind to realize that simple, quickly preparation and pesticide molecule are examined
It surveys.
Summary of the invention
The object of the present invention is to provide the preparations and application of a kind of pesticide quick measuring card.
To achieve the above object, the technical solution adopted by the present invention are as follows:
A kind of preparation of pesticide quick measuring card, it is characterised in that the preparation process of the pesticide quick measuring card is as follows:
(1) bovine serum albumin (BSA) solution that mass concentration is 1% is dripped on cellulose membrane, stands 1 hour and at room temperature
It is dry；
(2) the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N- hydroxyl amber for being 2% by mass concentration
Amber acid imide (NHS) drip respectively it is described in step (1) be modified on the cellulose membrane of BSA, stand 5 hours, wash and in room
The lower drying of temperature；
(3) it by cellulose membrane obtained in the uniform drop coating of the solution of composite catalyzing material to step (2), at 4 DEG C overnight, washes
And it is dry；
(4) it will be modified on the cellulose membrane of composite catalyzing material described in organic chromogenic agent solution drop coating to step (3), 4 DEG C
Lower drying, obtains quick measuring card.
The preparation process of composite catalyzing material, it is characterised in that this method comprises the following steps:
(1) it is added a certain amount of sodium borohydride into the mixed solution containing BSA and divalent cobalt ion, it is reaction, centrifugation, dry
To Co3O4NPs；
(2) by a certain amount of cobaltosic oxide nano ball (Co3O4NPs) it is added to the t-butyl carbamate (BOC) of 0.2 mM
In solution, washing, drying after standing 2 hours；
(3) by step (2) resulting Co3O4NPs is added in EDC the and NHS mixed solution that mass concentration is 2%, and it is small to stand 5
When, washing, drying；
(4) by step (3) resulting Co3O4NPs is added to acetylcholinesterase (AchE) and choline oxidase (ChOx) plus mixes
It closes in liquid, reaction overnight at 4 DEG C, washing, dry, obtained composite catalyzing material.
A kind of preparation and application of pesticide quick measuring card, the composite catalyzing material can be used for the detection of pesticide.
Preferably, the pesticide quick measuring card is made of substrate, composite catalyzing material and color developing agent, and composite catalyzing material is to repair
It is decorated with the Co of AchE and ChOx3O4NPs, composite catalyzing material and color developing agent are fixed in substrate.
More excellent to be, the substrate can be nitrocellulose filter, cellulose acetate membrane, polyester fiber, glass fibre, nonwoven
Cloth etc.；The organicvisualization reagent be 2,2 '-connection nitrogen-bis- (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) diamine salts, 3,3 ', 5,5 '-four
Methyl biphenyl amine, dopamine or phenol.
It is further preferred that, the detection application of a kind of pesticide quick measuring card, it is characterised in that application process is as follows:
(1) two quick measuring cards are taken, one adds a certain amount of organic phosphorus or carbamate chemicals for agriculture solution on quick measuring card, another
It is a that pesticide is not added, it places 5 minutes, acts on organic phosphorus or carbamate chemicals for agriculture sufficiently with composite catalyzing material；
(2) then add a certain amount of acetylcholine on quick measuring card, stand 5 minutes, react it sufficiently with composite catalyzing material；
(3) color change of quick measuring card is observed to realize to organic phosphorus or carbamate chemicals for agriculture qualitative detection, if with not
Organic phosphorus or carbamate chemicals for agriculture quick measuring card comparison lighter is added then to illustrate there is organic phosphorus or carbamic acid in system
Ester pesticides；
(4) taking photograph of intelligent mobile phone is used, can be carried out when taking pictures under LED light irradiation to prevent blurring of photos.By using hand
The color identification software of machine reads the specific rgb value of quick measuring card in photo, by BNB is calculated in=B/ (R+G+B)N, substitute into BN-
Concentration working curve equation is realized with calculating the concentration of pesticide to organic phosphorus or carbamate chemicals for agriculture quantitative detection.
More excellent to be, the organophosphorus pesticide can be the organophosphorus pesticides such as paraoxon, parathion, 2-dichloroethylk dimethyl phosphate, basudin；
The carbamate chemicals for agriculture can be the amino such as MTMC, sevin, Aldicarb, carbofuran, isoprocarb and Aphox
Formate ester pesticide.
Effect of the invention is:
1. the present invention utilizes the cobaltosic oxide nano ball and acetylcholinesterase and choline oxygen with imitative peroxidase activity
Change the tandem reaction of enzyme, constructs the sensor of quickly detection organophosphorus pesticide.
2. composite catalyzing material and prepared test strips that the present invention synthesizes realize one kettle way Pesticides Testing, avoid point
The progress of step reaction.
3. the present invention have certain practical value in actual life, and extend to food fermentation, biological medicine,
Chemical industry, environment, the quantitative analysis of biotechnology field and catalyst etc..
Detailed description of the invention
Fig. 1 is the preparation flow figure of quick measuring card provided in an embodiment of the present invention.
Fig. 2 is the detection principle diagram of quick measuring card provided in an embodiment of the present invention.
Fig. 3 is the feasibility of quick measuring card provided in an embodiment of the present invention detection.
Fig. 4 is that the one kettle way pH of composite catalyzing material provided in an embodiment of the present invention optimizes.
Fig. 5 is the qualitative detection photo of quick measuring card provided in an embodiment of the present invention.
Fig. 6 is the standard working curve of pesticide provided in an embodiment of the present invention quantitative determination.
Specific embodiment
In order in depth illustrate the contents of the present invention, some embodiments will be further enumerated below, but the present invention does not limit to
In cited embodiment.Specific experiment condition or method are such as not specified in the following example, press the normal condition of this field
Or method carries out.
Embodiment 1
The preparation process of composite catalyzing material, it is characterised in that this method comprises the following steps:
(1) a certain amount of sodium borohydride is added into the mixed solution containing BSA and divalent cobalt ion, passes through going back for sodium borohydride
Former and oxygen oxidation obtains Co3O4NPs is centrifuged, is dried to obtain solid；
(2) by a certain amount of Co3O4NPs is added to the BOC solution of 0.2 mM to protect Co3O4It is small to stand 2 for amino on NPs
When after washing, dry；
(3) by step (2) resulting Co3O4NPs is added in EDC the and NHS mixed solution that mass concentration is 2% with activation
Co3O4Carboxyl on NPs stands 5 hours, washing, drying；
(4) by the Co after activated carboxyl obtained by step (3)3O4NPs is added to AchE and ChOx adds in mixed liquor, AchE and
Amino meeting and Co on ChOx3O4Amide reaction, which occurs, for the carboxyl on NPs makes it be fixed to Co3O4It is anti-overnight at 4 DEG C on NPs
It answers, washes, dries, composite catalyzing material is made.
Embodiment 2
The preparation flow (Fig. 1) of quick measuring card:
(1) the BSA solution that mass concentration is 1% is dripped on cellulose membrane, stands 1 hour and is dried at room temperature for；
(2) EDC and NHS that mass concentration is 2% cellulose membrane for being modified with BSA in step (1) is dripped respectively (to select
The nitrocellulose filter of good water absorption) on the carboxyl of activated b SA, stand 5 hours, wash and be dried at room temperature for；
(3) by cellulose membrane obtained in the uniform drop coating of the solution of composite catalyzing material to step (2), on composite catalyzing material
Amino can with the carboxyl on BSA occur amide react make its be fixed to cellulose membrane on, at 4 DEG C overnight, wash simultaneously drying；
(4) it will be modified on the cellulose membrane of composite catalyzing material described in organic chromogenic agent solution drop coating to step (3), 4 DEG C
Lower drying, obtains quick measuring card.
The testing principle (Fig. 2) of quick measuring card:
Pesticide quick measuring card is made of substrate, enzyme and color developing agent, and substrate uses nitrocellulose filter paper, will be connected with AchE and ChOx
Co3O4NPs and color developing agent are fixed in substrate, the basic principle of detection are as follows: when acetylcholine is added, acetylcholine is first
Choline is first generated under the catalytic action of AchE, the choline continuation of generation is reacted with ChOx generates H2O2, there is imitative peroxide later
The Co of compound enzymatic activity3O4NPs can be catalyzed H2O2ABTS is set to change colour, the card that tests the speed becomes green from colourless；Organic phosphorus or amino first
Esters of gallic acid pesticide can inhibit the activity of AchE, cause a series of catalysis, oxidation reaction that can not be normally carried out, the color of quick measuring card
Variation is smaller or non-discolouring, and the quantitative analysis of pesticide can be realized according to the discoloration of quick measuring card.
Embodiment 3
The feasibility of quick measuring card detection:
Catalystic converter system (a), which shreds colorimetric card, to be put into acetylcholine (1 mmol/L) solution (pH=5.0), is reacted 5 minutes
After extract reaction solution carry out full wavelength scanner.Separately do one group of control experiment (b) colorimetric card shredded be put into buffer solution (pH=
5.0), reaction extracts reaction solution middle carry out full wavelength scanner after five minutes.
As shown in figure 3, catalystic converter system (a) has apparent ultraviolet absorption peak, catalystic converter system (b) is without UV absorption
Peak, it was demonstrated that catalysis reaction just can be carried out under the conditions of only existing for the acetylcholine.
Embodiment 4
The one kettle way pH of composite catalyzing material optimizes:
Catalystic converter system includes the buffer of composite catalyzing material (30 μ g/mL), acetylcholine (1 mmol/L) and different pH
(pH 1.0 2.0, glycine-HCI buffer；PH 3.0 6.0, acetic acid-acetic acid acid sodium buffer；PH 7.0 8.0, phosphoric acid
Salt buffer；PH 9.0, Tris- hydrochloride buffer).It is reacted under room temperature (25 °C) after five minutes, surveys reaction solution in 650 nm
The absorbance at place.As shown in Figure 4 (a), optimal pH is 5.0 or so.
One group of control experiment separately is done, catalystic converter system includes Co3O4NPs(30 μ g/mL), AchE(3.75 μ g/mL),
ChOx(1.25 μ g/mL), (pH 1.0 2.0, glycine-HCI are slow for the buffer of acetylcholine (1 mmol/L) and different pH
Fliud flushing；PH 3.0 6.0, acetic acid-acetic acid acid sodium buffer；PH 7.0 8.0, phosphate buffer；PH 9.0 10.0,
Tris- hydrochloride buffer).It is reacted under room temperature (25 °C) after five minutes, surveys absorbance of the reaction solution at 650 nm.Such as Fig. 4
(b) shown in, there are greater activity, but the activity of its activity less than 3(a) in pH for 3.0 6.0 or so.
Embodiment 5
The qualitative detection of quick measuring card:
(1) three quick measuring cards are taken, pesticide is not added in quick measuring card (a), and quick measuring card (b) adds 10 ng/L organic phosphorus or carbamates
The organic phosphorus or carbamate chemicals for agriculture of pesticide, quick measuring card (c) plus 100 ng/L are placed 5 minutes, make organic phosphorus or amino first
Esters of gallic acid pesticide is sufficiently acted on composite catalyzing material；
(2) then add the acetylcholine of 1 mmol/L on quick measuring card, stand 5 minutes, keep it sufficiently anti-with composite catalyzing material
It answers；
(3) taking photograph of intelligent mobile phone is used, can be carried out when taking pictures under LED light irradiation to prevent blurring of photos.As shown in figure 5,
Quick measuring card (a) greening of pesticide is not added, adds the quick measuring card (b) of smaller concentration pesticide compared with quick measuring card (a) lighter, adds larger dense
Degree pesticide quick measuring card (c) is non-discolouring, illustrates the pesticide concentration being added dropwise on quick measuring card (c) maximum, and quick measuring card (b) takes second place, quick measuring card
(a) do not add pesticide, this again demonstrate the feasibilities of this pesticide quick measuring card detection pesticide, and therefore, this pesticide quick measuring card can be real
Now to the detection of pesticide.
Embodiment 6
The standard working curve of pesticide quantitative determination:
(1) eight quick measuring cards are taken, the parathion pesticide (0-50 ng/mL) of various concentration is added dropwise on quick measuring card, places 5 minutes, makes
There is parathion pesticide sufficiently to act on composite catalyzing material；
(2) then add the acetylcholine of 10 μ L, 10 mM on quick measuring card, stand 5 minutes, keep it abundant with composite catalyzing material
Reaction；
(3) taking photograph of intelligent mobile phone is used, can be carried out when taking pictures under LED light irradiation to prevent blurring of photos.By using hand
The color identification software of machine reads the specific rgb value of quick measuring card in photo, by BNB is calculated in=B/ (R+G+B)N, marked
Directrix curve, for specific works curve as shown in fig. 6, the range of linearity is 0-20ng/L, equation is y=- 0.0194x+0.7975(R2=
0.9981).
Claims (7)
1. a kind of preparation of pesticide quick measuring card, it is characterised in that: the pesticide quick measuring card is by substrate, composite catalyzing material and colour developing
Agent is constituted, and composite catalyzing material is to be modified with acetylcholinesterase (AchE) and the cobaltosic oxide of choline oxidase (ChOx) is received
Rice ball (Co3O4NPs), composite catalyzing material and color developing agent are fixed in substrate.
2. a kind of preparation of pesticide quick measuring card according to claim 1, it is characterised in that the preparation of the pesticide quick measuring card
Journey is as follows:
(1) bovine serum albumin (BSA) solution that mass concentration is 1% is dripped on cellulose membrane, stands 1 hour and at room temperature
It is dry；
(2) the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N- hydroxyl amber for being 2% by mass concentration
Amber acid imide (NHS) drip respectively it is described in step (1) be modified on the cellulose membrane of BSA, stand 5 hours, wash and in room
The lower drying of temperature；
(3) it by cellulose membrane obtained in the uniform drop coating of the solution of composite catalyzing material to step (2), is stayed overnight at 4 DEG C, it is dry
And it washes；
(4) it will be modified on the cellulose membrane of composite catalyzing material described in organic chromogenic agent solution drop coating to step (3), 4 DEG C
Lower drying, obtains quick measuring card.
3. by the preparation process of composite catalyzing material described in claim 1,2, it is characterised in that:
(1) it is added a certain amount of sodium borohydride into the mixed solution containing BSA and divalent cobalt ion, it is reaction, centrifugation, dry
To Co3O4NPs；
(2) by a certain amount of Co3O4NPs is added in t-butyl carbamate (BOC) solution of 0.2mM, water after standing 2 hours
It washes, dry；
(3) by step (2) resulting Co3O4NPs is added in EDC the and NHS mixed solution that mass concentration is 2%, and it is small to stand 5
When, washing, drying；
(4) by step (3) resulting Co3O4NPs is added to AchE and ChOx adds in mixed liquor, reaction overnight at 4 DEG C, washing,
It is dry, composite catalyzing material is made.
4. a kind of pesticide quick measuring card described in -3 according to claim 1, it is characterised in that:
The substrate can be nitrocellulose filter, cellulose acetate membrane, polyester fiber, glass fibre, non-woven fabrics etc.；It is described to have
Machine color developing agent be 2,2 '-connection nitrogen-bis- (3- ethyl benzo thiazole phenanthroline -6- sulfonic acid) diamine salts, 3,3 ', 5,5 '-tetramethyl benzidines,
Dopamine or phenol.
5. a kind of detection application of pesticide quick measuring card described in -4 according to claim 1, it is characterised in that application process is as follows:
(1) two quick measuring cards are taken, one adds a certain amount of organic phosphorus or carbamate chemicals for agriculture solution on quick measuring card, another
It is a that pesticide is not added, it places 5 minutes, acts on organic phosphorus or carbamate chemicals for agriculture sufficiently with composite catalyzing material；
(2) then add a certain amount of acetylcholine on quick measuring card, stand 5 minutes, react it sufficiently with composite catalyzing material；
(3) color change of quick measuring card is observed to realize to organic phosphorus or carbamate chemicals for agriculture qualitative detection, if with not
Organic phosphorus or carbamate chemicals for agriculture quick measuring card comparison lighter is added then to illustrate there is organic phosphorus or carbamic acid in system
Ester pesticides；
(4) taking photograph of intelligent mobile phone is used, can be carried out when taking pictures under LED light irradiation to prevent blurring of photos.
6. the specific rgb value of quick measuring card in photo is read by using the color identification software of mobile phone, by BN=B/ (R+G+B) meter
Calculation obtains BN, substitute into BNConcentration working curve equation is realized with calculating the concentration of pesticide to organic phosphorus or carbamates agriculture
The quantitative detection of medicine.
7. by the application of quick measuring card described in claim 2-5, it is characterised in that:
The organophosphorus pesticide can be the organophosphorus pesticides such as paraoxon, parathion, 2-dichloroethylk dimethyl phosphate, basudin；The amino
Formate ester pesticide can be the carbamates agriculture such as MTMC, sevin, Aldicarb, carbofuran, isoprocarb and Aphox
Medicine.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811092703.6A CN109253999B (en) | 2018-09-19 | 2018-09-19 | Preparation and application of pesticide quick test card |
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| CN109253999B CN109253999B (en) | 2021-06-04 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
| CN115343280A (en) * | 2022-07-22 | 2022-11-15 | 武汉工程大学 | Colorimetric detection test paper for cellulose membrane-based organophosphorus pesticide, and preparation method and application thereof |
| CN115356336A (en) * | 2022-10-21 | 2022-11-18 | 广东江门中医药职业学院 | Method for rapidly detecting trace dibromophosphate residue in agricultural products |
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|---|---|---|---|---|
| CN113238043A (en) * | 2020-12-14 | 2021-08-10 | 黑龙江大学 | Preparation method and application of furadan test paper based on SERS immunochromatographic technique |
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| CN115356336A (en) * | 2022-10-21 | 2022-11-18 | 广东江门中医药职业学院 | Method for rapidly detecting trace dibromophosphate residue in agricultural products |
| CN115356336B (en) * | 2022-10-21 | 2022-12-13 | 广东江门中医药职业学院 | Method for rapidly detecting trace dibromophosphate residue in agricultural products |
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| Publication number | Publication date |
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| CN109253999B (en) | 2021-06-04 |
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