CN102043045A - High-flux detection method of various pesticide and veterinary drug residues - Google Patents
High-flux detection method of various pesticide and veterinary drug residues Download PDFInfo
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Abstract
The invention relates to a high-flux detection method of various pesticide and veterinary drug residues. Various pesticide and veterinary drug residues in the environment and food catch the attentions of people, and in order to solve the problem that the traditional technologies cannot be used for quickly, sensitively and accurately detecting the various residues, novel high-flux suspended chip detection technology and method of various pesticide and veterinary drug residues are established, which improve the conventional suspended chip technologies and achieve the purpose of simultaneously detecting seven pesticide and veterinary drug residue targets at most at a time. The technology overcomes the defects of complicated steps and long experiment time of the conventional suspended chip technologies, and through optimizing and screening experiment conditions, the complete reaction of antigens and antibodies is basically achieved, wherein detection limit can reach the pg level at most, and detection intervals comprises 2-4 magnitude orders. The method has the advantages of simplicity for operation, sensitivity, high speed, low cost and stable and reliable result obtaining performance, and the whole detection process takes only 1-2 hours, thus the invention provides a new method for quickly detecting various pesticide and veterinary drug residues.
Description
Technical field
The present invention relates to the residual detection method of many agricultural and veterinary chemicals in a kind of food, particularly relate to the residual high flux multivariate detection method of many agricultural and veterinary chemicals in a kind of food.
Background technology
Food security has become the focus that the whole world is paid close attention to, exists sternness and distinct issues in China.Because the agricultural and veterinary chemicals non-edible material from soybeans of residual, illegal interpolation (as melamine) that the unreasonable use of agricultural and veterinary chemicals causes and abuse adjuvant (essence and flavoring agent), the food-safety problem serious harm human body health such as seasonal food origin disease broken out on a large scale the time, also caused very big psychological fear and mental handicape, seriously influenced the foreign trade of socioeconomic normal development and China to the common people.Wherein, multiple agricultural and veterinary chemicals residue problem is on the rise in environment and the food, and along with international and domestic pay attention to day by day to food security, in the food security index, the agricultural and veterinary chemicals residual quantity has become important detection index.Agricultural and veterinary chemicals residual (pesticide and/or veterinary residue) is meant the general name that remains in agricultural and veterinary chemicals parent, derivant, metabolin, degradation product and impurity in environment, biosome and the food after agricultural and veterinary chemicals uses.
(1) agricultural chemicals, residue of veterinary drug problem have a strong impact on the healthy of people
When the development of modern society brings the agricultural product of abundant, high yield to people, use agricultural chemicals, veterinary drug to cause in food and the agricultural product agricultural chemicals, residue of veterinary drug problem such as exceed standard to work the mischief in agricultural cultivation and the aquaculture production run unreasonably to human health.In the Ministry of Public Health circular food poisoning that reports in all parts of the country in 2005, the death toll of chemical and poisonous of animal or plant nature food poisoning respectively accounts for 50% of total death toll, poisons in the majority with highly toxic pesticide, veterinary drug etc.Zhangbei County, Hebei pesticide-tainted vegetable that exposed successively in these 2 years incident, Xianghe poison leek of going to the capital is gone to the capital residues of pesticides such as incident, Qidong, Hunan poison day lily incident, Langxi, Anhui poison tealeaves incident, Hebei poison Yongnian garlic incident and causes that the food poisoning case occurs repeatedly; The steady chemical structure of " clenbuterol hydrochloride " clenobuterol hydrochloride (beta-stimulants compounds), conventional cooking method does not play destruction to clenobuterol hydrochloride residue in food and the agricultural product, and easily poison in human edible back.Clinical manifestation is for palpitating quickly, and four limbs tremble, suffer from abdominal pain dizzy, and symptoms such as expiratory dyspnea, n and V are arranged simultaneously.Chloromycetin is usually used in the infection of bacterium in the prevention of water product cultivating process, but chloromycetin also exists stronger spinoff and toxicity, and prolonged application can cause agranulocytosis even alpastic anemia; The baby of the milk that the edible for a long time chloromycetin of baby dyes may cause " gray syndrome "; Long-term in addition trace is taken in chloromycetin, not only makes Escherichia coli, salmonella etc. produce drug resistance, and can cause the imbalance of body normal flora, makes people's easy infection various diseases.
(2) residue problem such as agricultural chemicals, veterinary drug is the main bottleneck that influences China's food and the sound development of agricultural product industry
In recent years in the frequent clenbuterol hydrochloride poisoning that takes place of China, milk antibiotic residue exceed standard, problem such as residual chloromycetin in the shrimp, not only consumer's health has been caused to have a strong impact on, and the food and the Agricultural Products Trade of China have been brought totally unfavorable negative effect.The melamine milk powder case makes just China's dairy industry that steps into " golden period of development " be inflicted heavy losses on, and brings disaster to several related industries; Support fowl Da Sheng Shandong because the residue of veterinary drug problem in the chicken falls sharply the chicken export volume; Shrimp be because residual chloromycetin almost can not export European Union because European Union, the U.S. etc. all in rules the residual limit standard of regulation chloromycetin be " zero tolerance ", promptly must not detect.In January, 2002, U.S. agricultural product drug administration emphasizes to ban use of 11 kinds of medicines such as chloromycetin once more in animal derived agricultural product, and in March, 2002, Japanese health ministry has also been announced 11 kinds of medicine maximum residue limit(MRL) that comprise chloromycetin." public health＜animal and the bird〉(chemicals remnants) regulations " of Government of the Hong Kong Special Administrative Region promulgation also clearly stipulated to ban use of 7 kinds of medicines that comprise chloromycetin in edible animal.The U.S., Japan and European Union have also formulated physical and chemical indexs such as more, stricter organoleptic indicator and agricultural and veterinary chemicals be residual, and as garlic, the detection index of European Union has 111, and Japan has 61, and China has only 37.Because the agricultural and veterinary chemicals residual quantity exceeds standard, cause that agricultural exports suffers external rejection repeatly, incident such as detain, return goods, claim damages, cancel a contract, annual about 9,500,000,000 dollars of the foreign trade economy loss that causes thus, agricultural and veterinary chemicals residue problem have become influences agricultural products in China producing and selling and export trade principal element.Appear at the domestic various objectionable impurities residual components that do not detect time and again owing to factors such as objectionable impurities Fast Detection Technique in food and the agricultural product fall behind, but detected residual phenomenon abroad, the security of food and agricultural product can not get guaranteeing, its competitive power on market must descend, and has influenced normal sale and China's food and the export of farm produce trade of food and agricultural product.One of effective way of dealing with problems is exactly to set up the high-flux quick detection technique of agricultural chemicals, residue of veterinary drug, strengthens the residual regular monitoring of agricultural and veterinary chemicals.
(3) the suspending chip technology is that agricultural chemicals in food and the agricultural product, residue of veterinary drug are quick, the hi-tech regulatory measure of detection by quantitative
For the detection of agricultural chemicals, residue of veterinary drug in food and the agricultural product, National Standard Method and industry standard are many based on chromatography, application of gas chromatorgraphy/mass and euzymelinked immunosorbent assay (ELISA) (ELISA) etc., and external conclusive evidence method is also many based on these methods.Though look-matter coupling and chromatography are highly sensitive, laboratory condition requires high, and the detection method complexity wastes time and energy, the cost height, and difficulty satisfies rig-site utilization in practice; ELISA method stability and repeatability are relatively poor, and sensing range is narrow, the false positive rate height.Because residual agricultural and veterinary chemicals and pollutant are unknown often in food and the agricultural product, multiple sometimes pollutant is used as object of suspicion, and several sometimes pollutants can exist simultaneously, and above method is felt simply helpless often for polynary trace detection.Therefore still can not satisfy the actual detected requirements of one's work.The suspending chip technology combines the advantage of low cytometric analysis and fluorescent microsphere technology.It organically whole and coloured microballoon, laser technology, fluidics, up-to-date high speed digital signal processor and computer algorithm, improved detection specificity and sensitivity greatly.This system can detect the most nearly 100 kinds of different test items simultaneously in the sample of one 25 μ L, have repeatability and good stability, high flux, detection index plurality of advantages such as selection and high sensitivity and high s/n ratio flexibly; Compare with traditional solid phase chip, overcome solid phase chip and when big Molecular Detection, be subjected to the interference to reaction kinetics such as surface tension, steric effect; After the optimization experiment condition, the reaction system of approximate complete liquid phase almost can be ignored the influence of specific biological reaction, and the stability and the repeatability of testing result also are greatly improved.
At above problem, do not reach and detect fast, sensitively and accurately for solving some existing routine techniquess how residual, set up a kind of novel residual high flux suspending chip detection technique and method of many agricultural and veterinary chemicals, realize quick, accurate, special and detection by quantitative efficiently, lay the foundation for setting up the high-throughout many agricultural and veterinary chemicals residue detection of suspending chip technology platform.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, provide a kind of many agricultural and veterinary chemicals residual high throughput testing method.
Advantage of the present invention:
Method of the present invention has been abandoned the deficiency that conventional suspending chip technical step is loaded down with trivial details, test length consuming time, by optimizing and the screening experiment condition, substantially reach the complete reaction of antigen-antibody, in experiment, need not filter membrane plate and suction filter pump that conventional Luminex tests necessary costliness, only need common 96 porocyte culture plates or ELISA Plate to get final product, also need not to carry out the suction filtration step of repeated multiple times; Therefore, detect easy, quick, the shortening time, saved experimental cost, the result obtains reliable and stable, 1～2 hour only consuming time of whole testing process.This method is simple to operate, and fast sensitive, with low cost, the fast detecting residual for multiple agricultural and veterinary chemicals provides new method, lays the foundation for setting up the high-throughout many agricultural and veterinary chemicals residue detection of suspending chip technology platform.
Description of drawings
Fig. 1 detects the typical curve of 3 kinds of veterinary drugs simultaneously for suspending chip.
Fig. 2 detects 6 kinds of typical curves that agricultural and veterinary chemicals is residual simultaneously for suspension chip system.
Fig. 3 is the microballoon distribution plan that the suspending chip of 7 kinds of agricultural and veterinary chemicals detects.
Fig. 4 suspension chip system detects the typical curve of 7 kinds of agricultural and veterinary chemicals.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
The present invention with 4 kinds of veterinary drugs (chloromycetin, Clenbuterol, estradiol and tylosin) commonly used and 3 kinds of agricultural chemicals (Atrazine, Imidacloprid and carbaryl) totally 7 kinds of agricultural and veterinary chemicals be target, immunology detection principle based on the indirect competition method, improved conventional suspending chip technology methods for immobilizing antibodies on microballoon, realized that one-time detection can detect 7 kinds of residual targets of agricultural and veterinary chemicals at most simultaneously; This technology has also been abandoned the deficiency that conventional suspending chip technical step is loaded down with trivial details, test length consuming time, by optimizing and the screening experiment condition, substantially reach the complete reaction of antigen-antibody, in experiment, need not the filter membrane plate and the suction filter pump of the necessary costliness of Luminex normal experiment, only need common 96 porocyte culture plates or ELISA Plate to get final product, need not to carry out the suction filtration step of repeated multiple times; Therefore, detect easy, quick, the shortening time, saved experimental cost, the result obtains reliable and stable.
Technical scheme of the present invention is summarized as follows:
(1) preparation and the evaluation of various agricultural and veterinary chemicals target molecule protein conjugates to be measured;
(2) coupling and the conclusive evidence of agricultural and veterinary chemicals protein conjugates on the carboxyl fluorescent microsphere;
(3) the residual suspending chip single channel detection method of agricultural and veterinary chemicals is set up;
(4) the high flux suspending chip technical research of many agricultural and veterinary chemicals residue detection;
(5) methodology of agricultural and veterinary chemicals residue detection comparison research.
The preparation of the various agricultural and veterinary chemicals target molecule protein conjugates to be measured of step (1) and evaluation are the protein conjugates that adopts synthetic various agricultural and veterinary chemicals such as active ester method, diazotising method, mixed anhydride method and carbodlimide method; Behind the purifying, synthetic product is carried out ultraviolet and mass spectrum evaluation; With UV scanning method and mass spectroscopy the protein conjugates of 7 kinds of agricultural and veterinary chemicals of acquisition is identified, wavelength generation red shift or blue shift when UV scanning, the molecular weight that mass spectroscopy obtains can think that greater than the molecular weight of BSA or OVA synthetic protein conjugates can be used for the coupling of microballoon.By the detection of ultraviolet and mass spectroscopy to 7 kinds of agricultural and veterinary chemicals protein conjugates, confirmed that the success of various agricultural and veterinary chemicals antigen bonds is synthetic, obtained accurate coupling ratio, for the calculating of bond coupling amount on microballoon provides foundation.
The coupling of step (2) agricultural and veterinary chemicals protein conjugates on the carboxyl fluorescent microsphere and conclusive evidence are whether the agricultural and veterinary chemicals protein conjugates is carried out the well secured check of carrying out on fluorescent microsphere, to guarantee the drafting of next step typical curve;
Step (3) is drawn out 7 kinds of agricultural and veterinary chemicals target single channel examination criteria curves by 7 kinds of concentration gradients for the treatment of agricultural and veterinary chemicals, sets up the residual suspending chip single channel detection method of agricultural and veterinary chemicals.Drawn out 7 kinds of Logistic recurrence typical curve equations that the residual single channel suspending chip of agricultural and veterinary chemicals detects respectively, curved line relation is good, coefficient of determination R
2＞0.99, obtain between the minimum detectable concentration and detection zone of every kind of target.The result shows, simple, the sensitivity, quick of single channel detection method that various agricultural and veterinary chemicals are residual.
Step (5) has been carried out the multivariate analysis that some kinds of agricultural and veterinary chemicals (3 kinds, 6 kinds and 7 kinds) detect simultaneously on the basis that the single channel detection method is set up, experimental result shows, each typical curve equation and corresponding coefficient of determination R
2Performance is good; To being lower than GB with minimum detectability between the detection zone of various targets; Aspect the specificity of suspending chip multivariate analysis, the test of the detection specificity of suspending chip is good, does not have obvious the intersection and disturbance reponse to other structural similarities with non-similar multiple medicine; In to the detection that contains the residual different blind samples of multiple agricultural and veterinary chemicals, the relative deviation of detectable concentration value and actual concentrations is less.All things considered, suspension chip system can be used for the Preliminary detection to the residual actual sample of many agricultural and veterinary chemicals, and whole testing process 1～2h only consuming time has satisfied the sensitivity of many agricultural and veterinary chemicals residue detection, special, demand fast and efficiently basically.
It is residual that step (6) adopts traditional conventional indirect competitive ELISA method and LC-MS method to detect various agricultural and veterinary chemicals, with the research of comparing of suspending chip method, two kinds of classic method height of remolding sensitivity that the suspending chip method of most of targets detects, and between wide detection zone, quick and high-throughout advantage be conventional indirect competitive ELISA method and GB LC-MS method can not compare.
Summary of the invention
Experiment material is as follows:
Atrazine (Artrazine, Ar),
Clenbuterol (Clenbuterol, CL),
Estradiol (17-beta-estradiol, E
2),
Carbaryl (Carbaryl, Cbl),
Imidacloprid (Imidacloprid, Im),
N-hydroxy-succinamide (N-hydroxysuccinimide, NHS),
Tylosin (Tylosin, Tyl),
(Biotinyl-N-Hydroxysuccinimide Ester is BNHS) available from Sigma company, the U.S. for N-hydroxy-succinamide biotin ester;
BSA and OVA are available from Roche company, Germany;
Chloromycetin (Chloramphenicol is CAP) with the 2-[morpholine] and ethylsulfonic acid (2-[N-Morpholino] ethanesulfonic acid hydrate, MES) available from Ameresco company, the U.S..
CAP monoclonal antibody (CAP-mAb), E
2Monoclonal antibody (E
2-mAb) available from Biodesign company, the U.S.;
CL monoclonal antibody (CL-mAb) is available from Abcam company, Britain;
Ar monoclonal antibody (Ar-mAb), Im monoclonal antibody (Im-mAb) are available from Beijing Wanhuapuman Bioengineering Co Ltd, and be homemade;
How grand antibody (Cbl-pAb) is available from China Agricultural University for Cbl, and is homemade.
Antibody protective agent is available from Galaxy bioengineering company limited, and is homemade.
Bio-Plex
TMThe blank carboxyl fluorescent microsphere of albumen amino coupled kit (comprise and activate damping fluid and deposit damping fluid) and various codings, Bio-Bad company, the U.S..
Experiment prepares with the deionized water preparing instrument of deionized water by Millipore company, and resistivity is 18M Ω cm.
Other chemical reagent is homemade analytical reagent.
It is as follows to test used instrument:
Bio-Plex
TMSuspension chip system, Bio-Rad company, the U.S.;
LC-MS instrument: Waters Acquity UPLC-Quattro Premier XE MS, Waters company, the U.S.;
Ultra Performance Liquid Chromatography post: Waters ACQUITY UPLC
TMBEH C18 post (2.1mm * 50mm, 1.7 μ m), Waters company, the U.S.;
Protein nucleic acid analyser: UV-2450 type, SHIMADZU company, Japan;
Salt dialysis cartridge: YM-10 type, Millipore company, the U.S.;
Concrete technical scheme of the present invention is as follows:
(1) preparation and the evaluation of various agricultural and veterinary chemicals target molecule protein conjugates to be measured
Because most of agricultural and veterinary chemicals are small-molecule substance (molecular weight is less than 1000), the site, space of itself is limited, can't be coupled on the fluorescent microsphere effectively.Therefore, must prepare artificial antigen, the agricultural and veterinary chemicals micromolecule is marked on the microballoon with the macromolecular carrier coupling.Adopt be the BSA that uses always and OVA as carrier protein, adopt methods such as diazotising method, carbodlimide method, active ester method and mixed anhydride method that 7 kinds of agricultural and veterinary chemicals micromolecule are coupled on the carrier protein.The protein conjugates to 7 kinds of agricultural and veterinary chemicals obtaining with UV scanning and mass spectroscopy identifies that the protein conjugates of accreditation can be used for the coupling of microballoon.(7 kinds of agricultural and veterinary chemicals)
(2) coupling and the conclusive evidence of agricultural and veterinary chemicals protein conjugates on the carboxyl fluorescent microsphere
Carry out the coupling of agricultural and veterinary chemicals protein conjugates on the carboxyl fluorescent microsphere with reference to the method for Luminex official website, prove conclusively then.Whether successfully conclusive evidence is in order to detect albumen mark on fluorescent microsphere.With reference to the method for Luminex official website, and done certain improvement.Concrete experimental procedure is as follows:
1) every kind is detected thing and gets two Eppendorf centrifuge tubes, the negative control on the marking pen mark of drawing, and another puts on experiment tube.
2) cross the microballoon 15s of agricultural and veterinary chemicals bond with the coupling of middling speed vortex oscillation, in two arms, add about 2000 coupling microballoons in every pipe.
3) in each experiment tube, add corresponding excessive biotinylation monoclonal antibody, add the PBS of same volume in the negative control pipe; Middling speed oscillating reactions 1h in 37 ℃ of incubators.
4) the centrifugal 4min of 14000g abandons supernatant.
The SA-PE of 5) adding 100 * dilution, middling speed oscillating reactions 30min in 37 ℃ of incubators.
6) open the suspending chip instrument according to normal procedure, and preheating 30min (if indoor temperature changes, need proofread and correct).
7) reacted solution is moved in the 96 hole ELISA Plate, put detection platform into, choose corresponding microballoon coding by supporting Bio-Manager4.0 software, go up machine-readable number, obtain meta fluorescence intensity level (Mean Fluorescent Intensity, MFI), the coupling success can be proved conclusively in MFI＞1000.
(3) the residual suspending chip single channel detection method of agricultural and veterinary chemicals is set up
Various agricultural and veterinary chemicals target waters to be measured are diluted, and other establishes a positive control that does not add standard items, makes 7 kinds of agricultural and veterinary chemicals target final concentrations to be measured reach the concentration shown in the table 1; Add an amount of corresponding antibody 3～10 μ g more respectively in each hole, each hole adds 1800～2300 of corresponding fluorescent microspheres respectively, reaction 1～2h, add an amount of SA-PE to every hole, 37 ℃ of incubation 20～40min, suspending chip read 100 microballoons, by software kit Bio-Manager
TM4.0 analyzing and processing obtains MFI, and adopts the Logistic regression equation to obtain between the minimum detectable concentration and detection zone of 7 kinds of agricultural and veterinary chemicals to be measured.Because each microballoon quantity that detects is 100 beads, each bead has an independently detected value, also is that each sample (comprising standard items) is repeated to have tested 100 times, and sample size has enough representativenesses.
Suspending chip single channel location parameter that 7 kinds of agricultural and veterinary chemicals of table 1 are residual and summary sheet as a result
(4) the high flux suspending chip technical research of many agricultural and veterinary chemicals residue detection.
Add the multiple biotinylated antibody after optimizing in 96 hole reaction plates, the multiple standards product dilute according to different concentration gradients successively, multiple microballoon equal proportion are mixed 2000 in every hole.100 μ L are supplied in last every hole, and every kind of detection thing is prepared a positive control simultaneously.Reaction is 37 ℃ of middling speed vibration 1h on the vortex oscillation device, and every then hole adds 1: 100 SA-PE, 25 ℃ of middling speed oscillating reactions 0.5h.Suspending chip reads 100 microballoons, obtains MFI, and analyzing and processing is also drawn the examination criteria curve.
(5) check and analysis of agricultural and veterinary chemicals residual sample.
The researcher prepares the blind sample of agricultural and veterinary chemicals of some kinds of variable concentrations, transfers to the experimenter who does not know concentration and tests, each blind sample parallel testing 3 times (n=3).Bio-Plex suspending chip detection system detects MFI, calculates the test concentrations value by software kit according to the typical curve of drawing out, and test result and actual concentrations value are compared.
Embodiment
Embodiment 1:3 kind veterinary drug chloromycetin, Clenbuterol and the residual suspending chip multivariate analysis of estradiol
Experimental technique is as follows:
1) CAP, CL and E after adding is optimized respectively in each hole of 96 orifice plates
2The biotinylation monoclonal antibody 5,1 of (below relate to these 3 kinds of veterinary drugs all in this order) veterinary drug target correspondence, 16ng, respectively with 3 kinds of standard items according to 5 *, 5 * and 3 * gradient be diluted to 7 gradients respectively and add;
2) 3 kinds of microballoon equal proportions are mixed, every hole adds 6000 (2000/ kind of beads), be to include 3 kinds of biotinylation monoclonal antibodies, 3 kinds of target standard items and 3 kinds of microballoons in each reacting hole, 50 μ L are supplied with PBS in every hole, prepare a positive control hole that does not add any standard items simultaneously;
3) 37 ℃ of middling speeds of vortex oscillation vibration 1h makes in veterinary drug BSA bond and the solution on the microballoon corresponding standard product compete biotinylated monoclonal antibody jointly, and every then hole adds an amount of SA-PE, 37 ℃ of middling speed oscillating reactions 30min;
4) suspending chip reads 100 microballoons, obtains MFI.Concentration with various target standard items is X-axis, and the MFIs that obtains with detection is the suspending chip high throughput testing typical curve that Y-axis is made 3 kinds of veterinary drugs, shown in Fig. 1 and table 2.
Table 2 suspending chip detects the typical curve equation of 3 kinds of veterinary drugs simultaneously
5) detection of 3 kinds of blind samples of veterinary drug
The blind sample of 3 kinds of residues of veterinary drug of table 3 detects
The testing result such as the table 3 of 3 kinds of blind samples of veterinary drug.By this table as seen, suspending chip detectable concentration value and actual concentrations deviation do not wait 8.09%～17.03%, can think that deviation is less relatively.
The suspending chip multivariate analysis that embodiment 2:6 kind agricultural and veterinary chemicals is residual
1) the biotinylation monoclonal antibody of 6 kinds of agricultural and veterinary chemicals target correspondences after in each hole of 96 hole reaction plates, add optimizing respectively, respectively with 6 kinds of agricultural and veterinary chemicals standard items according to a certain percentage or gradient dilution add.
2) 6 kinds of microballoon equal proportions are mixed, every hole adds 1.2 * 10
4Individual (2000/ kind of beads, totally 6 kinds) promptly include 6 kinds of biotinylation monoclonal antibodies, 6 kinds of target standard items and 6 kinds of probe microballoons in each reacting hole, 50 μ L are supplied with PBS in every hole, prepare a positive control hole that does not add any standard items simultaneously;
3) 37 ℃ of middling speeds of vortex oscillation vibration 1h makes in agricultural and veterinary chemicals protein conjugates and the solution on the microballoon corresponding standard product compete biotinylated monoclonal antibody jointly, and every then hole adds an amount of SA-PE, 37 ℃ of middling speed oscillating reactions 30min;
4) suspending chip reads 100 microballoons, obtains MFI.Concentration with various target standard items is X-axis, and the MFIs that obtains with detection is the suspending chip high throughput testing typical curve that Y-axis is made 6 kinds of agricultural and veterinary chemicals, shown in Fig. 2 and table 4.
6 kinds of agricultural and veterinary chemicals hyperchannels of table 4 mixing examination criteria curvilinear equation
6 kinds of agricultural and veterinary chemicals standard items carry out the mensuration and the drafting of typical curve equation according to optimizing good concentration gradient by experimental procedure.By the indirect competition method, obtain the typical curve (as shown in Figure 2) of multivariate analysis, between typical curve equation, detection zone and MinDC as shown in table 5.As seen from Figure 2, the microballoon probe of 6 kinds of agricultural and veterinary chemicals all can accurately be discerned by suspension chip system.In detecting interval range, the typical curve equation obtains good response, R
2＞0.99.The MinDC of 6 kinds of detection things is lower than the minimum detectability of national standard method, does not have the E of detection at present
2GB, but this sensing range and detection limit can satisfy daily agricultural and veterinary chemicals residue detection needs in actual applications, also can be used for clinically to E
2Detection, but to carry out necessary serum pre-treatment.
5) detection of 6 kinds of blind samples of agricultural and veterinary chemicals
Table 5 mixes 6 kinds of detections that agricultural and veterinary chemicals is residual in the blind sample
On the basis that 3 kinds of blind samples of veterinary drug detect, carried out the test of 6 kinds of blind samples of agricultural and veterinary chemicals.As shown in Figure 6, realized the suspending chip high flux multivariate analysis of 6 kinds of agricultural and veterinary chemicals, whole testing process 2～3h only consuming time.As can be seen from Table 5, in the residual mixing of multiple agricultural and veterinary chemicals detects, obtained effective MFI, except the testing result of Cbl and actual concentrations deviation big (25.12%), the deviation that other various agricultural and veterinary chemicals detect does not wait between 0.75%～17.38%, can think that deviation is less relatively, the RSD that detects with the high flux suspending chip of 3 kinds of blind samples of veterinary drug is suitable.Because how anti-what Cbl used is, though the purifying mistake is crosslinked serious than other agricultural and veterinary chemicals monoclonal antibodies between the non-specific adsorption of antibody and microballoon, make that detection gained result is bigger than other various target deviations, but on the whole, say it is acceptable for high-throughout detection.
The suspending chip multivariate analysis that embodiment 3:7 kind agricultural and veterinary chemicals is residual
1) the biotinylation monoclonal antibody of 7 kinds of agricultural and veterinary chemicals target correspondences after in each hole of 96 hole reaction plates, add optimizing respectively, respectively with 7 kinds of agricultural and veterinary chemicals standard items according to a certain percentage or gradient dilution add.
2) 7 kinds of microballoon equal proportions are mixed, every hole adds 1.4 * 10
4Individual (2000/ kind of beads, totally 7 kinds) promptly include 7 kinds of biotinylation monoclonal antibodies, 7 kinds of target standard items and 7 kinds of probe microballoons in each reacting hole, 50 μ L are supplied with PBS in every hole, prepare a positive control hole that does not add any standard items simultaneously;
3) 37 ℃ of middling speeds of vortex oscillation vibration 1h makes in agricultural and veterinary chemicals protein conjugates and the solution on the microballoon corresponding standard product compete biotinylated monoclonal antibody jointly, and every then hole adds an amount of SA-PE, 37 ℃ of middling speed oscillating reactions 30min;
4) chip reads 100 microballoons, obtains MFI.Concentration with various target standard items is X-axis, and the MFIs that obtains with detection is the suspending chip high throughput testing typical curve that Y-axis is made 7 kinds of agricultural and veterinary chemicals.
7 kinds of agricultural and veterinary chemicals standard items carry out the mensuration and the drafting of typical curve equation according to optimizing good concentration gradient by experimental procedure.As seen from Figure 3, the microballoon probe of 7 kinds of agricultural and veterinary chemicals all can accurately be discerned by the suspending chip instrument.By the indirect competition method, obtain the typical curve equation (as shown in Figure 4) of multivariate analysis, as shown in table 6 between detection zone with minimum detectable concentration.
7 kinds of agricultural and veterinary chemicals high fluxs of table 6 suspending chip examination criteria curvilinear equation
5) detection of 7 kinds of blind samples of agricultural and veterinary chemicals
High flux suspending chip at 3 passage residues of veterinary drug and the residual blind sample of 6 passage agricultural and veterinary chemicals detects on the successful basis, has carried out the test of the blind samples of 7 passage agricultural and veterinary chemicals.High concentration and the residual blind sample of low concentration agricultural and veterinary chemicals are selected in this test respectively for use, the accuracy that the 7 passage suspending chip methods of investigating detect and to the antijamming capability of variable concentrations small-molecule substance.
The testing result that table 7 agricultural and veterinary chemicals low-residual mixes blind sample
As seen from Figure 7, in the blind sample test of agricultural and veterinary chemicals low-residual 7 passages, the deviation of detectable concentration and actual concentrations can think that deviation is less relatively in the test that reaches 7 passages between 2.12%～27.83%; Have only the deviation of Cbl and Tyl bigger, in many agricultural and veterinary chemicals residue detection, do not have obvious phase mutual interference simultaneously low concentration.
The testing result that table 8 agricultural and veterinary chemicals high residue mixes blind sample
Table 8 is testing results that the agricultural and veterinary chemicals high residue mixes blind sample, and the deviation of detectable concentration and actual concentrations can think that deviation is less relatively in the agricultural and veterinary chemicals residue detection that reaches 7 passages between 4.55%～27.78%; Simultaneously, also show the suspending chip method to high density pollution or residual good antijamming capability is arranged, but Preliminary Applications detects in actual sample.
In sum, the suspending chip method is having a clear superiority in to planting in the residual high throughput testing analysis of many agricultural and veterinary chemicals, and whole testing process only needs 1～2h.
This research is by setting up a kind of suspending chip technology and method of high-throughout how agricultural beastly residue detection, realized residual to many agricultural and veterinary chemicals simultaneously high flux multivariate detection analysis, can tentatively be used for the beastly residual high flux test analysis of the many farmings of actual sample, detect sensitive, special, quick and efficient, compare with actual value, error is less, and the fast detecting residual for many agricultural and veterinary chemicals provides new method, lay a good foundation for the foundation of detection platform, have wide application and development prospect.
Claims (1)
1. the residual high throughput testing method of agricultural and veterinary chemicals more than a kind is characterized in that comprising the steps:
(1) preparation of agricultural and veterinary chemicals target molecule protein conjugates to be measured:
Adopt the diazotising method that chloromycetin, Clenbuterol are made protein conjugates with carrier protein BSA respectively;
Adopt carbodlimide method that Imidacloprid and carrier protein BSA are made protein conjugates;
Adopt active ester method that carbaryl and carrier protein OVA are made protein conjugates;
Adopt mixed anhydride method that Atrazine, estradiol are made protein conjugates with carrier protein BSA respectively;
By carbonyl dimidazoles tylosin and carrier protein BSA are made protein conjugates;
(2) coupling and the conclusive evidence of agricultural and veterinary chemicals target molecule protein conjugates to be measured on the carboxyl fluorescent microsphere
1) the agricultural and veterinary chemicals target molecule protein conjugates to be measured of step (1) preparation being put into seven Eppendorf centrifuge tubes respectively is experiment tube, and the carrier protein that combines with agricultural and veterinary chemicals target molecule to be measured in the described step (1) put in addition seven Eppendorf centrifuge tubes accordingly, negative control pipe for the experiment tube of described correspondence, in described experiment tube and negative control pipe, add 1800～2300 coupling microballoons again, with middling speed vortex oscillation 12-20s;
2) in each experiment tube, add corresponding biotinylated antibody 3～10 μ g; The phosphate buffer of adding same volume in the negative control pipe (Phosphate-buffered saline, PBS); In 37 ℃ of incubator middling speed oscillating reactions 1～2h;
3) 10000～16000g centrefuge experiment pipe and control tube 3～5min abandon supernatant;
Streptavidin-the phycoerythrin of 4) adding 100 * dilution (Strepavidin-phycoerythrin, SA-PE), in 37 ℃ of incubator middling speed oscillating reactions 20～40min;
5) reacted solution is moved in the 96 hole ELISA Plate, put the Bio-Plex of preheating into
TMThe suspending chip instrument is by supporting Bio-Manager
TM4.0 software is chosen corresponding microballoon coding, goes up machine-readable number, and acquisition meta fluorescence intensity level (Mean Fluorescent Intensity, MFI); The coupling success can be proved conclusively in MFI＞1000;
(3) the residual suspending chip single channel detection method of agricultural and veterinary chemicals is set up
Various agricultural and veterinary chemicals standard items chloromycetin, Clenbuterol, Imidacloprid, carbaryl, Atrazine, estradiol and tylosin waters to be measured are carried out gradient dilution (gradient can see the table 1 in the instructions for details) respectively, establish the positive control that does not add any standard items simultaneously; Add in the 96 new hole ELISA Plate, add corresponding biotinylated antibody 3～10 μ g again in each hole respectively; Then, each hole adds 1800～2300 of the fluorescent microspheres that coupling that corresponding described step (2) obtains has protein conjugates, reaction 1～2h respectively, the SA-PE that adds 100 * dilution to every hole, 37 ℃ of incubation 20～40min, suspending chip read 100 microballoons, by supporting Bio-Manager
TM4.0 software analysis is handled, and obtains MFI, and adopts the Logistic regression equation to obtain between the minimum detectable concentration and detection zone of 7 kinds of agricultural and veterinary chemicals to be measured;
(4) residual to the many agricultural and veterinary chemicals simultaneously detection of high flux suspending chip
Again get multiple biotinylated antibody 3～10 μ g that 96 hole ELISA Plate add various agricultural and veterinary chemicals to be measured again, multiple agricultural and veterinary chemicals standard items dilute according to different concentration gradients successively, step (2) is obtained multiple microballoon equal proportion mixes, 1800～2300 in every hole, 50 μ L are supplied with PBS in last every hole, every kind of detection thing is prepared a positive control simultaneously, reaction is 37 ℃ of middling speed vibration 1～2h on the vortex oscillation device, every then hole adds the SA-PE 50 μ L of 50 * dilution, 25 ℃ of middling speed oscillating reactions 0.5～1h, suspending chip reads 100 microballoons, obtains MFI, and analyzing and processing is also made 7 kinds of agricultural and veterinary chemicals examination criteria regression curve equations to be measured;
(5) check and analysis of agricultural and veterinary chemicals residual sample
Prepare the blind sample of agricultural and veterinary chemicals of some kinds of variable concentrations,, use Bio-Plex each blind sample parallel testing 3 times (n=3)
TMSuspension chip system detects the MFI of every kind of agricultural and veterinary chemicals to be measured in the blind sample, by Bio-Manager
TM4.0 the typical curve that software is drawn out according to step (4) calculates the test concentrations value, records agricultural and veterinary chemicals concentration in the sample.And test result and actual concentrations value compared.
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Application publication date: 20110504 |