CN101968448A - Acetylcholinesterase chemiluminescence bioreactor, and preparation method and application thereof - Google Patents
Acetylcholinesterase chemiluminescence bioreactor, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an acetylcholinesterase chemiluminescence bioreactor, and a preparation method and application thereof, and belongs to the technical fields of analysis of pesticide residues and biotechnology. The reactor is a glass tube of which the two ends are sealed by 300-mesh bolting silk, wherein immobilized acetylcholinesterase is held in the glass tube; and the immobilized acetylcholinesterase is purified crucian muscle acetylcholinesterase which is immobilized on cyanogen bromide-activated (CNBr) sepharose 4B. The application of the reactor is characterized by comprising the following steps of: establishing the acetylcholinesterase chemiluminescence bioreactor; and detecting organic phosphorus and carbamate type pesticides. The crucian muscle acetylcholinesterase (AChE) extracted in the invention has high enzyme activity, obvious purification effect and high immobilized enzyme activity recovery rate; the detection limit of the organic phosphorus type pesticides and the carbamate type pesticides is low; the analysis time is short; and the enzyme reactor can be repeatedly used.
Description
Technical field
The invention belongs to pesticide residue analysis field and biological technical field, be specifically related to a kind of acetylcholinesterase chemiluminescence bio-reactor and its production and application.
Background technology
Organophosphorus pesticide is that the mankind are synthetic the earliest and so far still at a widely used insecticides, is that China uses one of topmost agricultural chemicals at present.Since organophosphorus pesticide have the drug effect height, wide in variety, controlling object is many, in environment easy advantage such as degraded, in China's agricultural production, obtain extensive application, account for 77.76% of the total use amount of agricultural chemicals.Since the seventies in 20th century, the organo-chlorine pesticide kind is forbidden or limits use in succession by country variant, and the insect species of anti-organophosphorus pesticide is increasing, and the use amount of carbamate chemicals for agriculture increases year by year.In China, carbamate pesticide also is the very important pesticide of a class, has obtained especially using widely aspect Seed Treatment.Can estimate that within a certain period of time, carbamate insecticides will be an important component part in the field of pesticides.
Studies show that of ecological toxicology, the concentration of organophosphorus and carbamate chemicals for agriculture and cholinesterase (ChE) are active is subjected to exist good linear relationship between the inhibition degree.Therefore, when we can also survey whether there is this two compounds in the sample with the activity of acetylcholinesterase.When having organophosphorus and carbamate chemicals for agriculture in the sample; they can make hydroxyl generation phosphorylation and methylamine acidylate in the serine residue in the acetylcholinesterase catalytic center, make acetylcholinesterase thoroughly lose hydrolysis substrate---the ability of acetylcholine ester.
Crucian (crucian) is a kind of fresh water fingerling of economy, and is distributed more widely in the rivers and lakes of China, and it is not only enjoyed for the mankind as aquatic products, and also plays a role in the purified water source process.Up to now, the property research of the acetylcholinesterase of crucian and to the rarely seen report of the Study of Sensitivity of organophosphorus pesticide.And select for use crucian as AChE enzyme source, the enzyme source is extensive and have the representative meaning, be widely used by the research field of organophosphorus and carbamate chemicals for agriculture pollution level as the monitoring water body environment.
Adopt Sepharose 4B to fix AChE through the CNBr activation.The end of this Sepharose 4B has-NH
2Base, can with the AChE end-COOH base covalent bond, form immobilization AChE.This process for fixation has the higher enzyme recovery alive.
Chemiluminescence (Chemiluminescence, be called for short CL), be chemiluminescent substance after the oxidation of the catalysis of catalyzer or oxygenant, form the intermediate of an excited state, when this excited state intermediate is got back to stable ground state, launch photon simultaneously, can utilize luminous measuring instrument measuring light quantum yield.Owing to have advantages such as sensitivity, easy, quick and low cost, in recent years, the research application of chemiluminometry on pesticide residue analysis receives much concern.
A kind of preparation method of acetylcholinesterasefluorescence fluorescence biosensor is disclosed in the patented claim of Chinese invention patent publication number 101581670A.This method is at first extracted separation, purifying to the crucian carp brain acetylcholinesterase, adopts shitosan that the crucian carp brain acetylcholinesterase is carried out immobilization then, prepares the immobilization acetylcholinesteraseelectrochemistry film, promptly gets acetylcholinesterasefluorescence fluorescence biosensor.But this patent has the following disadvantages: patent utilization crucian carp brain is the enzyme source, but the crucian brain volume very little, through amount behind the SephadexG-200 purifying still less.Patent adopts fixedly AChE of glutaraldehyde covalent cross-linking method, though combination degree is more firm, the association reaction between enzyme and the carrier also can be finished fast, and glutaraldehyde itself has toxic action to AChE, can very big influence be arranged to the enzyme recovery alive.
Summary of the invention
The objective of the invention is to overcome the shortcoming and defect that prior art exists, a kind of acetylcholinesterase chemiluminescence bio-reactor and its production and application is provided.
The object of the present invention is achieved like this:
One, acetylcholinesterase chemiluminescence bio-reactor
Reactor be a kind of two ends with 300 mesh sieve thin,tough silk sealed glass pipes, immobilization acetylcholinesteraseelectrochemistry (AChE) is housed in it;
Described immobilization acetylcholinesteraseelectrochemistry is the purifying crucian muscle acetylcholinesterase that is fixed on the Sepharose 4B of CNBr activation.
Two, the preparation method of immobilization acetylcholinesteraseelectrochemistry (AChE)
This preparation method comprises the following steps:
1. selecting the enzyme source is crucian muscle AChE
The work of crucian muscle AChE enzyme is 119.61 μ molmin
-1G
-1
Crucian muscle AChE to the susceptibility of two class agricultural chemicals relatively and time-the inhibiting rate curve plotting
Crucian muscle acetylcholinesterase (AChE) is the enzyme source, is inhibitor with phoxim, Rogor, DDVP, chlopyrifos, carbosulfan, carbofuran, analyzes the activity influence of the concentration of these several agricultural chemicals to crucian muscle AChE.In addition, analyze these several agricultural chemicals to the inhibition time of crucian muscle AChE and the activity influence of enzyme.Is horizontal ordinate with agricultural chemicals to inhibition time of enzyme, and the inhibiting rate of AChE is the ordinate curve plotting.
2. polyglycol (PEG) fractional precipitation is in conjunction with ammonium sulfate precipitation purifying crucian muscle AChE
Behind the PEG fractional precipitation, the AChE of 8%PEG precipitation has enzyme work after dialyse 48h, the freeze drying to be: 1253.74 μ molmin through ammonium sulfate precipitation again
-1G
-1
3. fixing condition is chosen as: the initial enzyme amount of immobilization 7U, the immobilization time is 10h
Adopt the Sepharose 4B through the CNBr activation to fix AChE, immobilised enzymes in contrast to resolvase, and it has stability preferably, and the immobilised enzymes recovery alive is 75%, immobilised enzymes Michaelis constant Km=0.3774mmol/L.
Three, the application of acetylcholinesterase chemiluminescence biosensor
The application of acetylcholinesterase chemiluminescence bio-reactor mainly is to make up acetylcholinesterase chemiluminescence biosensor (abbreviation sensor).This sensor can detect organophosphorus and carbamate chemicals for agriculture.The detection limit of using this sensor DDVP, chlopyrifos and carbosulfan is respectively 0.279mg/kg, 0.009mg/kg, 0.068mg/kg, and analysis efficiency reaches 50min/ time.As enzyme restorative alive, immobilised enzymes is reused 15 times with 2-PAM.
The present invention has following advantage and good effect:
1, the crucian muscle AChE of Ti Quing has bigger enzymatic activity, to organophosphorus and carbamate chemicals for agriculture sensitivity;
2, the PEG fractional precipitation is obvious in conjunction with ammonium sulfate precipitation purifying purifying crucian muscle AChE purification effect;
3, immobilised enzymes recovery height alive has stability preferably;
4, detect at the bottom of organophosphorus and the carbamate pesticide residue detection limit, analysis time is short, enzyme reactor is reusable.
Description of drawings
Fig. 1 is the structural representation of acetylcholinesterase chemiluminescence biosensor;
Fig. 2 is these Fundamentals of Sensors reaction equation figure;
Fig. 3 is muscle AChE inhibiting rate and concentration logarithmic relationship curve;
Fig. 4 is muscle AChE time-inhibiting rate curve;
Fig. 5 variable concentrations DDVP is to the inhibiting rate figure of immobilised enzymes;
Fig. 6 variable concentrations chlopyrifos is to the inhibiting rate figure of immobilised enzymes;
Fig. 7 variable concentrations carbosulfan is to the inhibiting rate figure of immobilised enzymes.
Among the figure:
1,2,3-the 1st, 2,3 T-valve; The 4-reactor; 5,6-the 1st, 2 constant flow pumps;
7,8-the 1st, the threeway of 2Y type; The 9-spiral pipe; The 10-camera bellows; The 11-photomultiplier;
The 12-computer; The outlet of 13-waste liquid;
The a-phosphate buffered solution; The b-ATCh substrate solution; The c-2-PAM restorative; The d-sample;
The e-potassium ferricyanide solution; The f-luminol solution;
The A-phoxim; B-Rogor; C-DDVP; The D-chlopyrifos;
The E-carbosulfan; The F-carbofuran.
Embodiment
Describe in detail below in conjunction with drawings and Examples:
One, acetylcholinesterase chemiluminescence biosensor (abbreviation sensor)
1, overall
As Fig. 1, this sensor comprises the 1st, 2,3 T- valve 1,2,3, reactor 4, the 1,2 constant flow pumps 5,6, the 1,2Y type threeway 7,8, spiral pipe 9, camera bellows 10, the outlet 13 of photomultiplier 11, computer 12 and waste liquid;
Also comprise phosphate buffered solution a, ATCh substrate solution b, 2-PAM restorative c, sample d, potassium ferricyanide solution e and luminol solution f;
Phosphate buffered solution a and ATCh substrate solution b are communicated with the 1st T-valve 1 respectively;
2-PAM restorative c and sample d are communicated with the 2nd T-valve 2 respectively;
The 1st T-valve 1 and the 2nd T-valve 2 are communicated with the 3rd T-valve 3 respectively;
Potassium ferricyanide solution e, the 2nd constant flow pump 6 and 1Y type threeway 7 front and back are communicated with successively;
Luminol solution f, the 2nd constant flow pump 6 and 2Y type threeway 8 front and back are communicated with successively;
In camera bellows 10, be provided with the outlet 13 of spiral pipe 9, photomultiplier 11 and waste liquid;
2, functional part
1) the 1st, 2,3 T-valve
T-valve the 1,2, the 3rd, a kind of common components.
2) reactor 4
Aforementioned, reactor 4 be a kind of two ends with 300 mesh sieve thin,tough silk sealed glass pipes, immobilization acetylcholinesteraseelectrochemistry (AChE) is housed in it.
3) the 1st, 2 constant flow pumps 5,6
1st, 2 constant flow pumps the 5, the 6th, a kind of common components.
4) the 1st, 2Y type threeway 7,8
1st, the threeway of 2Y type the 5, the 6th, a kind of common components.
5) spiral pipe 9
6) camera bellows 10
Camera bellows 10 is canisters of a kind of lucifuge.
7) photomultiplier 11
8) computer 12
9) phosphate buffered solution a
Phosphate buffered solution a is 0.01mol/L, the buffer solution of pH 7.5.
10) ATCh substrate solution b
ATCh substrate solution b is the phosphate buffered solution that contains 1mM ATCh.
11) 2-PAM restorative c
2-PAM restorative c is the phosphate buffered solution that contains 1mM 2-PAM.
12) sample d
Sample d is a solution to be checked
13) potassium ferricyanide solution e
Potassium ferricyanide solution e is the 3mM potassium ferricyanide aqueous solution.
14) and luminol solution f
Luminol solution c is sodium carbonate-sodium bicarbonate (pH=9.52) buffer solution that contains the 0.6mM luminol.
3, its principle of work is:
This sensor is in order to improve the selectivity that chemiluminometry detects agricultural chemicals, and under the prerequisite of giving full play to the chemiluminescence advantage, the utilization enzyme suppresses ratio juris, as Fig. 2, has introduced AChE and substrate thereof.Enzyme inhibition activity changes, and is because the variation of the thiocholine concentration that generates, when substrate (acetylthiocholine) and agricultural chemicals with pump by after being loaded with fixedly the acetylcholinesterase reactor.Thiocholine is with regard to because of luminous and detected in the chemical luminous system of luminol-potassium ferricyanide.Agricultural chemicals is relevant by the front and back difference between the chemiluminescence intensity of reactor with pesticide concentration.Thereby can enlarge the residual range of application of chemiluminescence detection farming.For under the situation of not changing immobilised enzymes, make the sensor continuous working, the immobilised enzymes of inactivation recovered after this sensor will use revivifier 2-PAM to sample determination.
By the front and back difference between the chemiluminescence intensity of reactor, its inhibiting rate can use formula to calculate according to agricultural chemicals:
Wherein I is the baseline luminous intensity; I
0Be the luminous intensity when the agricultural chemicals not; I
1For by the luminous intensity behind the agricultural chemicals;
Negative logarithm concentration with the standard items agricultural chemicals is done horizontal ordinate, and inhibiting rate is done ordinate, the drawing standard curve, and the inhibiting rate substitution typical curve equation with sample obtains sample concentration then.With the concentration of inhibiting rate 5% correspondence detection limit as sensor.
4, the application result of sensor
The detection limit of using this sensor DDVP, chlopyrifos and carbosulfan is respectively 0.279mg/kg, 0.009mg/kg, 0.068mg/kg, and analysis efficiency reaches 50min/ time.As enzyme restorative alive, immobilised enzymes is reused 15 times with 2-PAM.
5, the method for operating of sensor
1. open the 1st, 2 constant flow pumps 5,6, adjust the 1st, 3 T- valve 1,3, make phosphate buffered solution a, 3mM potassium ferricyanide solution e and 0.6mM luminol solution f respectively by pipeline, the negative high voltage of regulating photomultiplier 11 is 400V, baseline opening entry, luminous intensity are I;
2. adjust the 1st T-valve 1, phosphate buffered solution a is replaced by the phosphate buffered solution b that contains 1mM ATCh, the substrate injection length is 70s, refills phosphate buffered solution a then.The chemiluminescence intensity minimum of record will be equivalent to the luminous intensity I of non-inhibitory reaction
0
3. stop the 2nd constant flow pump 6, adjust the 2nd, 3 T- valve 2,3, make phosphate buffered solution a be replaced by sample d, injection length is 20min, the 1. adjust the 1st, 3 T- valve 1,3 then, repeat, 2. step, the chemiluminescence intensity minimum of record will be equivalent to the luminous intensity I of inhibitory reaction
1
4. recover enzyme and live, stop the 2nd constant flow pump 6, adjust the 2nd, 3 T- valve 2,3, make 2-PAM restorative c, continue 15min by reactor 4.
5. detect next sample;
6. end to be detected back flushing with clean water pipeline.
Two, the experiment of immobilization acetylcholinesteraseelectrochemistry (AChE)
1, experiment reagent
In the following embodiments if not otherwise specified, test used reagent all from following dated information and manufacturer:
Bio-engineering research institute is built up in Coomassie brilliant blue kit (G-250) Nanjing;
Acetylthiocholine iodide (ATCh) Sigma company;
5,5-dithio-bis-nitrobenzoic acid (DTNB) Sigma company;
The import packing of bovine serum albumin freeze-dried powder Roche company;
Triton X-100 Amresco company;
The import packing of CNBr Activated Sepharose 4B Amresco company;
80% DDT EC Hubei sand swell reaches incorporated company;
40% phoxim emulsifiable concentrate Anhui U.S. Kodak farmingization company limited;
40% dimethoate emulsifiable concentrate Jiangsu Tenglong Biological Pharmaceutical Co., Ltd.;
Benefit Hai'an, 40% chlorpyrifos ec Hebei Ge Nuonongization company limited;
10% carbosulfan Qingdao Hansen Biologic Science Co., Ltd.;
3% carbofuran Zhejiang Tianyi Agricultural Chemical Co., Ltd.;
Chemical reagent company limited of sodium carbonate group of nations
Chemical reagent company limited of sodium bicarbonate group of nations
Potassium ferricyanide Tianjin City Tanggu district moral China chemical reagent factory
Luminol Sigma company;
Pralidoxime (2-PAM) Sigma company;
Chemical reagent company limited of ascorbic acid group of nations
Chemical reagent company limited of n-bromo-succinimide (NBS) group of nations
2, experimentation
1) selection in AChE enzyme source
(1) extraction of enzyme
Get 10 of the live crucians raised and train, choose muscle of back, inhale a part of dehematizing with filter paper, weigh in surface plate, the back of weighing is 1: 5 ratio and the 0.1molL that contains 1%Triton X-100 in W/V
-1Phosphate buffer (pH value 8.0) mixes.Under condition of ice bath, after the abundant homogenate of glass homogenizer, place high speed freezing centrifuge 10000rmin under 4 ℃ of conditions in the centrifuge tube of packing into
-1Centrifugal 30min gets supernatant, promptly gets crude enzyme liquid, and recording volume is also preserved standby down at 4 ℃.
What measure crucian muscle AChE crude enzyme liquid is 119.61 μ molmin than enzyme work
-1G
-1
(2) crucian muscle AChE compares the susceptibility of two class agricultural chemicals
Use liquid to join in the enzyme liquid test tube in the agricultural chemicals of variable concentrations, the method for living according to the survey enzyme is measured absorbance then, and the calculating enzyme is lived and inhibiting rate (comparing with the zero-dose processing).As seen from Figure 3: crucian muscle AChE has susceptibility preferably to the variety classes agricultural chemicals.
(3) crucian muscle AChE to two class agricultural chemicals time-the inhibiting rate curve plotting
Use liquid to join in the enzyme liquid test tube in the different agricultural chemicals of 0.5mL 10 μ g/mL, measure enzyme and live and also calculate inhibiting rate, make then time-the inhibiting rate curve.From Fig. 4 as seen, along with the prolongation that suppresses the time, these 6 kinds of agricultural chemicals increase gradually to muscle AChE maximum inhibition; And it is 12min that the inhibiting rate of muscle AChE is reached the peaked time.
According to crucian muscle AChE to agricultural chemicals susceptibility and time-inhibiting rate curve result, explanation can use crucian muscle AChE as the enzyme source, and muscle in contrast to brain, raw material is sufficient more.Therefore final selected crucian muscle AChE is as the enzyme source in the research later on.
2) the PEG fractional precipitation is in conjunction with ammonium sulfate precipitation purifying purifying crucian muscle AChE
Get muscle AChE crude enzyme liquid, add 50% (w/v) PEG storing solution, progressively making the PEG final concentration is 3%, 8%, 16% and 24%, and ice bath leaves standstill 30min successively.4 ℃ of centrifugal 10min of 10000g, the precipitation part is a component 1,2,3 and 4.With the ammonium sulfate of each component adding 55%, the centrifugal 10min of 10000g behind the placement 3h will precipitate with a spot of damping fluid dissolving, put into bag filter, 4 ℃ of dialysis 48h, and freeze drying is again measured respectively than enzyme and is lived.The result shows that component 2 has the highest enzyme and lives, and can reach 1253.74 μ molmin
-1G
-1This has also established good basis for being further purified AChE.
3) immobilization of crucian muscle AChE
Take by weighing the Sepharose 4B of 0.1g through CNBr activation, be the hydrochloric acid solution swelling 20min of 1mmol/L with 10mL concentration after, phosphate buffer (pH=8.0) the wash-out hydrochloric acid of usefulness 0.1mol/L.The gel that swelling is good joins in the enzyme liquid of total enzyme 7U alive, and phosphate buffer (pH=8.0) constant volume of using 0.1mol/L then is to 4mL.4 ℃ down vibration (150r/min) if after 10 hours,, clean for several times with 3.5% NaCl solution and distilled water successively, add at last 3mL contain 0.02%NaN3 phosphate buffer (0.1mol/L, pH=8.0), in 4 ℃ of preservations down.
The immobilised enzymes recovery alive can reach 75%, immobilised enzymes Michaelis constant Km=0.3774mmol/L.
4) foundation of typical curve
(1) foundation of DDVP pesticide standard curve
Dispose 0.2,0.6,1,5,10,50 μ gmL respectively
-1DDVP titer 50mL, measure respectively by the method for operating of sensor, do horizontal ordinate with the negative logarithm concentration of agricultural chemicals, inhibiting rate is done ordinate, the drawing standard curve, as Fig. 5, the typical curve equation be y=18.33652x+15.15948, coefficient R
2=0.9886.
(2) foundation of chlopyrifos pesticides typical curve
Dispose 0.01,0.05,0.1,0.5,1,5,10,20 μ gmL respectively
-1Chlopyrifos titer 50mL, add 0.5mLNBS (0.4g/L), add 0.5mL ascorbic acid (4g/L) again behind the reaction 5min, measure respectively by the method for operating of sensor then, do horizontal ordinate with the negative logarithm concentration of agricultural chemicals, inhibiting rate is done ordinate, the drawing standard curve, as Fig. 6, getting the typical curve equation is y=16.72842x+39.16171, coefficient R
2=0.9829.
(3) foundation of carbosulfan pesticide standard curve
Dispose 0.05,0.2,0.6,1,5,10 μ gmL respectively
-1Carbosulfan titer 50mL, measure respectively by the method for operating of sensor, do horizontal ordinate with the negative logarithm concentration of agricultural chemicals, inhibiting rate is done ordinate, and the drawing standard curve is as Fig. 7, getting the typical curve equation is y=14.16133x+21.5044, coefficient R
2=0.9851.
Claims (4)
1. acetylcholinesterase chemiluminescence bio-reactor is characterized in that:
Reactor be a kind of two ends with 300 mesh sieve thin,tough silk sealed glass pipes, immobilization acetylcholinesteraseelectrochemistry is housed in it;
Described immobilization acetylcholinesteraseelectrochemistry is the purifying crucian muscle acetylcholinesterase that is fixed on the Sepharose 4B of CNBr activation.
2. by the preparation method of the described immobilization acetylcholinesteraseelectrochemistry of claim 1, it is characterized in that comprising the following steps:
1. selecting the enzyme source is crucian muscle AChE;
2. polyglycol (PEG) fractional precipitation is in conjunction with ammonium sulfate precipitation purifying crucian muscle AChE;
3. fixing condition is chosen as: the initial enzyme amount of immobilization 7U, the immobilization time is 10h.
3. by the application of the described acetylcholinesterase chemiluminescence of claim 1 bio-reactor, it is characterized in that:
Make up the acetylcholinesterase chemiluminescence biosensor, detect organophosphorus and carbamate chemicals for agriculture.
4. based on the sensor of the described acetylcholinesterase chemiluminescence of claim 1 bio-reactor, it is characterized in that:
This sensor comprises the 1st, 2,3 T-valve (1,2,3), reactor (4), the 1st, 2 constant flow pumps (5,6), the 1st, 2Y type threeway (7,8), spiral pipe (9), camera bellows (10), the outlet (13) of photomultiplier (11), computer (12) and waste liquid;
Phosphate buffered solution (a), ATCh substrate solution (b), 2-PAM restorative (c), sample (d), potassium ferricyanide solution (e) and luminol solution (f);
Phosphate buffered solution (a) and ATCh substrate solution (b) are communicated with the 1st T-valve 1 respectively;
2-PAM restorative (c) and sample (d) are communicated with the 2nd T-valve (2) respectively;
The 1st T-valve (1) and the 2nd T-valve (2) are communicated with the 3rd T-valve (3) respectively;
Be communicated with successively before and after potassium ferricyanide solution (e), (2) constant flow pump (6) and the 1Y type threeway (7);
Be communicated with successively before and after luminol solution (f), the 2nd constant flow pump (6) and the 2Y type threeway (8);
Be communicated with successively before and after the outlet (13) of the 3rd T-valve (3), reactor (4), the 1st constant flow pump (5), 1Y type threeway (7), 2Y type threeway (8), spiral pipe (9) and waste liquid;
In camera bellows (10), be provided with the outlet (13) of spiral pipe (9), photomultiplier (11) and waste liquid; Photomultiplier (11) is connected with computer (12).
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| CN102796803A (en) * | 2012-08-24 | 2012-11-28 | 赖智勇 | Method for excluding background interference of fruit and vegetable in rapid detection of fruit and vegetable pesticide residue and application thereof |
| CN102816691A (en) * | 2012-08-24 | 2012-12-12 | 赖智勇 | Device for quickly detecting pesticide residues in fruits and vegetables, and application thereof |
| CN106323950A (en) * | 2016-08-10 | 2017-01-11 | 济南大学 | Preparing method for sensor for detecting organophosphorus pesticide residues |
| CN106680222A (en) * | 2016-12-23 | 2017-05-17 | 郑州科欣生物技术有限公司 | Reagent for determining cholinesterase and kit |
| CN109358043A (en) * | 2018-11-01 | 2019-02-19 | 中国农业科学院茶叶研究所 | A method of sample fast detecting pesticide residue is extracted with organic solvent |
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| CN102816691A (en) * | 2012-08-24 | 2012-12-12 | 赖智勇 | Device for quickly detecting pesticide residues in fruits and vegetables, and application thereof |
| CN102816691B (en) * | 2012-08-24 | 2014-05-21 | 上海林默精密仪器有限公司 | Device for quickly detecting pesticide residues in fruits and vegetables, and application thereof |
| CN106323950A (en) * | 2016-08-10 | 2017-01-11 | 济南大学 | Preparing method for sensor for detecting organophosphorus pesticide residues |
| CN106323950B (en) * | 2016-08-10 | 2018-09-07 | 济南大学 | A kind of preparation method of the sensor of detection organophosphorus pesticide object |
| CN106680222A (en) * | 2016-12-23 | 2017-05-17 | 郑州科欣生物技术有限公司 | Reagent for determining cholinesterase and kit |
| CN106680222B (en) * | 2016-12-23 | 2019-05-28 | 郑州科欣生物技术有限公司 | Cholinesterase measures reagent and kit |
| CN109358043A (en) * | 2018-11-01 | 2019-02-19 | 中国农业科学院茶叶研究所 | A method of sample fast detecting pesticide residue is extracted with organic solvent |
| CN109358043B (en) * | 2018-11-01 | 2022-01-14 | 中国农业科学院茶叶研究所 | Method for rapidly detecting pesticide residues by using organic solvent extraction sample |
| CN112268895A (en) * | 2020-09-04 | 2021-01-26 | 中国能源建设集团安徽省电力设计院有限公司 | Device and method for detecting concentration of trace superoxide radical free radicals in water by adopting chemiluminescence reagent |
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| CN101968448B (en) | 2012-08-22 |
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