CN103278622A - Preparation method of 96-well enzyme label plate chloramphenicol molecularly imprinted polymer film - Google Patents
Preparation method of 96-well enzyme label plate chloramphenicol molecularly imprinted polymer film Download PDFInfo
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- CN103278622A CN103278622A CN2013101522466A CN201310152246A CN103278622A CN 103278622 A CN103278622 A CN 103278622A CN 2013101522466 A CN2013101522466 A CN 2013101522466A CN 201310152246 A CN201310152246 A CN 201310152246A CN 103278622 A CN103278622 A CN 103278622A
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- chloromycetin
- chloramphenicol
- enzyme label
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Abstract
The invention discloses a preparation method of a 96-well enzyme label plate chloramphenicol molecularly imprinted polymer film. The preparation method comprises the following steps of a, dissolving chloramphenicol as a template molecule, N,N-diethylaminoethyl methacrylate as a functional monomer and ethylene glycol dimethacrylate as a cross-linking agent in tetrahydrofuran as a pore forming agent according to a mole ratio of 1: 4: 0.2, and carrying out ultrasonic treatment for 30min, wherein a use amount ratio of tetrahydrofuran to chloramphenicol is 5ml/0.25mmol, b, adding azodiisobutyronitrile as an initiator into the mixture, and carrying out ultrasonic treatment for 15min, wherein a use amount ratio of azodiisobutyronitrile to chloramphenicol is 5mg/0.25mmol, c, dropwisely adding the solution obtained by the step b on a 96-well enzyme label plate, putting the 96-well enzyme label plate into a sealing bag, filling nitrogen into the sealing bag, carrying out sealing, and carrying out ultraviolet lamp irradiation to initiate polymerization lasting for 6h, d, putting the 96-well enzyme label plate into a methanol-acetic acid mixed solution having a volume ratio of 3: 1, and carrying out ultrasonic elution for 48h, and e, drying the 96-well enzyme label plate at a temperature of 50 DEG C for 2h to obtain the chloramphenicol molecularly imprinted polymer film.
Description
Technical field
The present invention relates to a kind of preparation method of molecular imprinted polymer membrane, especially a kind of preparation method of 96 hole ELISA Plate chloramphenicol molecular imprinting polymeric membrane to chloromycetin high selectivity absorption.
Background technology
(Chloramphenicol is nineteen forty-seven to separate the microbiotic that obtains first from microbe metabolite CAP) to chloromycetin, owing to its good antibiotic property, the stable property of medicine, cheap price, extensively as feed addictive, is used for the treatment of bacteriosis.Yet owing to be with nitro on its phenyl ring, its half life of decomposition is long and have serious toxic and side effect, easily causes the human body blood poisoning, causes serious toxic and side effects such as irreversible alpastic anemia, and human beings'health is constituted huge potential threat.Therefore, European Union, the U.S. all in rules the residual limit standard of regulation CAP be " zero tolerance ", China Ministry of Agriculture also regulation CAP and salt, fat etc. must not detect in all edible tissues of food animal.At present, mainly contain microbial method, immunoassay, vapor-phase chromatography, combined gas chromatography mass spectrometry, supercritical fluid chromatography and immunofluorescence capillary electrophoresis technique etc. for CAP detection method residual in the sample.Existing detection method is for the sample pre-treatments complex steps, and sample matrix complicated component, CAP residual concentration are low in addition, are difficult to realize effectively detecting fast.
Advantage such as that immunoassay has is simple to operate, quick, highly sensitive, high specificity and testing cost are low is classified as the Analytical Techniques of Pesticide Residues of preferential research, development and utilization by external many countries.The immuno analytical method of using mainly is enzyme linked immunological (ELISA) analytical approach at present, but detection limit sometimes is difficult to reach the residual examination criteria requirement of medicine at 0.1 μ g/L.Chemiluminescent immunoassay(CLIA) is a kind of emerging immunoassay that develops behind enzyme immune technology, is the immune analysis method with the direct labelled antigen of chemiluminescence agent or antibody.Chemical illumination immunity analysis instrument comprises two parts, i.e. immune response system and chemiluminescence analysis system.Immune response system is that luminescent substance (exciting generation excited state intermediate down at reactant) directly is marked on antigen (chemiluminescence immune assay) or the antibody (immunochemiluminometry), or enzyme acts on luminous substrate.Chemiluminescence analysis system is to utilize chemiluminescent substance through the catalysis of catalyzer and the oxidation of oxygenant, form the intermediate of an excited state, when this excited state intermediate is got back to stable ground state, launch photon (hM) simultaneously, utilize luminous signal surveying instrument measuring light quantum yield.Though the sensitivity of chemiluminescence immunoassay detection technique is easy to reach 0.01 μ g/L level, but preparation process very complicated, expense height, the used instrument costliness of antibody are not suitable for the detection of batch samples medicament residue in the used kit of immune response system.
Its ultimate principle of molecular imprinting (molecular imprinting technology MIT) is that template molecule is (to be separated, the molecule of identification) interacts with the function monomer with suitable functional group, under crosslinking chemical and action of evocating, form and have macropore, netted polymkeric substance, remove template molecule by solvent elution or hydrolysis under certain condition, just stayed size in the polymkeric substance, " memory " hole of shape and functional group and template molecule coupling, specific affinity interaction just can be carried out with template molecule to be separated in the potpourri in such hole, thereby reach separation, purifying, preenrichment template molecule etc., then with chemiluminescence methods such as enzyme linked immunologicals, can realize to template molecule in the sample fast, effectively detect.But, up to now also not about preparing the relevant report of chloramphenicol molecularly imprinted polymeric film.
Summary of the invention
The present invention is in order to solve the above-mentioned technical matters of existing in prior technology, and a kind of preparation method of 96 hole ELISA Plate chloramphenicol molecular imprinting polymeric membrane to the absorption of chloromycetin high selectivity is provided.
Technical solution of the present invention is: a kind of preparation method of 96 hole ELISA Plate chloramphenicol molecular imprinting polymeric membrane is characterized in that carrying out as follows:
A. template molecule chloromycetin, function monomer diethyl aminoethyl methacrylate and crosslinking chemical ethylene glycol dimethacrylate are dissolved in the pore-foaming agent tetrahydrofuran ultrasonic 30min according to mol ratio 1:4:0.2; The consumption of described tetrahydrofuran is 5ml/0.25mmol chloromycetin;
B. add the initiating agent azoisobutyronitrile, ultrasonic 15min, addition is 5mg/0.25mmol chloromycetin;
C. b step gained drips of solution is added on the 96 hole ELISA Plate, afterwards 96 hole ELISA Plate is put into sealing bag, charge into nitrogen, under sealing and the ultra violet lamp condition, initiated polymerization 6 hours;
D. 96 hole ELISA Plate being placed volume ratio is methyl alcohol-acetic acid mixed liquor of 3:1 again, ultrasonic wash-out 48 hours;
E. 50 ℃ of drying 96 hole ELISA Plate are 2 hours, obtain the chloramphenicol molecular imprinting polymeric membrane.
Described a step is to take by weighing 0.25mmol chloromycetin in flask, to wherein adding the 5ml tetrahydrofuran, ultrasonic 5min; After treating the chloromycetin dissolving, add people 1mmol diethyl aminoethyl methacrylate, ultrasonic 20min; Again the 0.05mmol ethylene glycol dimethacrylate is joined in the flask ultrasonic 5min.
The present invention is to be function monomer with the diethyl aminoethyl methacrylate, be that crosslinking chemical, tetrahydrofuran are that pore-foaming agent, azoisobutyronitrile are initiating agent with the ethylene glycol dimethacrylate, and according to special ratios and working condition etc., produce and have very high selectivity and specific chloramphenicol molecularly imprinted polymeric film, advantages of higher stability, long serviceable life and stronger anti-adverse environment ability are arranged.The present invention is with low cost, simple to operate, easy control of reaction conditions.
Description of drawings
Fig. 1 is the canonical plotting of embodiment of the invention direct competitive ELISA chlorine detection mycin.
Embodiment
A. take by weighing 0.25mmol chloromycetin in flask, to wherein adding the 5ml tetrahydrofuran, ultrasonic 5min; After treating the chloromycetin dissolving, add people 1mmol diethyl aminoethyl methacrylate, ultrasonic 20min; Again the 0.05mmol ethylene glycol dimethacrylate is joined in the flask ultrasonic 5min;
B. add 5mg initiating agent azoisobutyronitrile, ultrasonic 15min;
C. b step gained drips of solution is added on the 96 hole ELISA Plate, afterwards 96 hole ELISA Plate is put into sealing bag, charge into nitrogen, and sealing and 100W uviol lamp (under the illuminate condition of λ=365nm), initiated polymerization 6 hours;
D. 96 hole ELISA Plate being placed volume ratio is methyl alcohol-acetic acid mixed liquor of 3:1 again, and ultrasonic wash-out 48 hours changed eluent one time, and removed template molecule in per four hours;
E. 50 ℃ of drying 96 hole ELISA Plate are 2 hours, obtain the chloramphenicol molecular imprinting polymeric membrane.
Test:
1. synthesize enzyme-labelled antigen (enzyme mark chloromycetin):
With glutaraldehyde method chloromycetin is coupled to synthetic enzyme-labelled antigen on the horseradish peroxidase, step is as follows:
(1) gets the chloromycetin of 100mg, join in the hydrochloric acid of 10ml 0.6mol/L;
(2) treat chloromycetin dissolving after, toward the zinc powder that wherein adds 60mg, in 80 ℃ of reaction 30min;
(3) get the supernatant cool to room temperature, under agitation slowly join in the buffer solution that contains horseradish peroxidase, and then drip the glutaraldehyde of 0.2ml 25%, magnetic agitation room temperature reaction 5h;
(4) after stirring finishes, under 4 ℃, with PBS damping fluid (phosphatebuffer buffer system) the dialysis 3d of synthetic product to 0.1mol/L, pH=7.2, change the PBS damping fluid every day 2 times, to remove unreacted small-molecule substance glutaraldehyde, chloromycetin etc.;
(5) measure the volume of enzyme-labelled antigen solution in the bag filter, add isopyknic glycerine again, keep in Dark Place standby in-20 ℃ of refrigerators.
2. the preparation of chloromycetin standard items
Preparation 100mg/L chloromycetin methanol solution is diluted to 30mg/L with it with the PBS buffer solution that contains 5% methyl alcohol then, is diluted to 3mg/L, 0.3mg/L, 0.03mg/L, 0.003mg/L, 0.0003mg/L more successively.Record the luminous value of each concentration, draw the typical curve of chloromycetin.
3. the direct competitive euzymelinked immunosorbent assay (ELISA) detects:
(1) on 96 orifice plates of chloramphenicol molecular imprinting polymeric membrane, every hole adds 100 μ l chloromycetin sample standard solution or chloromycetin earlier and adds sample solution;
(2) adding 100 μ l enzyme-labelled antigen solution toward every hole then, is control wells with the hole that does not add the chloromycetin standard solution;
(3) behind the incubated at room 1h, abandon supernatant, wash plate 4-5 time with PBST;
(4) prepare luminescent solution (luminol and H simultaneously washing plate
2O
21:1 mixes), plate is put into the chemo-immunity analyser, add luminescent solution 250 μ l/ holes, read the luminous intensity luminous value at Chemiluminescence Apparatus;
(5) replication is 6 times, with sample luminous value average substitution chloromycetin typical curve, obtains chloramphenicol residue.
4. the checking of the preparation of testing sample and chloromycetin kit
Get a healthy sea cucumber, detect through liquid phase, there is not chloromycetin in its coelomic fluid,,, get 1ml respectively and in 3 test tubes, (number 1 to test tube behind the membrane filtration with 0.45 μ m from wherein extracting coelomic fluid respectively in test tube with syringe, 2,3), be that the chloromycetin methanol solution of 0.05mg/L is dosed respectively in 1,2, No. 3 test tube with the concentration of 0.02ml, 0.1ml, 0.2ml, be mixed with the testing sample of 1 μ g/L, 5 μ g/L, 10 μ g/L with coelomic fluid.Use chloromycetin quick detection kit and direct competitive ELISA(using artificial antibody) testing result as follows:
Direct competitive ELISA(using artificial antibody) and the chloromycetin kit to adding the testing result of chloromycetin in the sea cucumber coelomic fluid sample
| Add concentration (μ gL -1) | Chloromycetin kit (μ gL -1) | ELISA (μg·L -1) |
| 1 | 0.98±0.01 | 0.90±0.02 |
| 5 | 4.89±0.01 | 4.79±0.02 |
| 10 | 9.87±0.01 | 9.34±0.02 |
5. the typical curve of the ELISA chlorine detection mycin of using artificial antibody
With the inhibiting rate of IC%(chloromycetin to enzyme-labelled antigen and antibody association reaction) be ordinate, the logarithm of chloromycetin standard items is the typical curve that horizontal ordinate is drawn direct competitive ELISA chlorine detection mycin, as shown in Figure 1.
Claims (2)
1. the preparation method of a hole ELISA Plate chloramphenicol molecular imprinting polymeric membrane is characterized in that carrying out as follows:
A. template molecule chloromycetin, function monomer diethyl aminoethyl methacrylate and crosslinking chemical ethylene glycol dimethacrylate are dissolved in the pore-foaming agent tetrahydrofuran ultrasonic 30min according to mol ratio 1:4:0.2; The consumption of described tetrahydrofuran is 5ml/0.25mmol chloromycetin;
B. add the initiating agent azoisobutyronitrile, ultrasonic 15min, addition is 5mg/0.25mmol chloromycetin;
C. b step gained drips of solution is added on the 96 hole ELISA Plate, afterwards 96 hole ELISA Plate is put into sealing bag, charge into nitrogen, under sealing and the ultra violet lamp condition, initiated polymerization 6 hours;
D. 96 hole ELISA Plate being placed volume ratio is methyl alcohol-acetic acid mixed liquor of 3:1 again, ultrasonic wash-out 48 hours;
E. 50 ℃ of drying 96 hole ELISA Plate are 2 hours, obtain the chloramphenicol molecular imprinting polymeric membrane.
2. the preparation method of 96 hole ELISA Plate chloramphenicol molecular imprinting polymeric membrane according to claim 1, it is characterized in that: described a step is to take by weighing 0.25mmol chloromycetin in flask, to wherein adding the 5ml tetrahydrofuran, ultrasonic 5min; After treating the chloromycetin dissolving, add people 1mmol diethyl aminoethyl methacrylate, ultrasonic 20min; Again the 0.05mmol ethylene glycol dimethacrylate is joined in the flask ultrasonic 5min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111370A (en) * | 2015-09-29 | 2015-12-02 | 徐州工程学院 | Chloramphenicol molecularly imprinted polymer and preparation method thereof |
| CN105732890A (en) * | 2016-04-30 | 2016-07-06 | 中国科学院新疆理化技术研究所 | Preparation method of gossypol molecularly imprinted polymer |
| CN108051594A (en) * | 2017-12-20 | 2018-05-18 | 河南联博生物科技有限公司 | A kind of latex enhancing immune turbidimetry quantitatively detects the reagent of chloramphenicol |
| CN113354818A (en) * | 2021-06-15 | 2021-09-07 | 天津科技大学 | Preparation method and application of microporous plate for high-throughput rapid detection of kaempferol |
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| CN101564681A (en) * | 2009-06-03 | 2009-10-28 | 中国农业科学院农业质量标准与检测技术研究所 | Solid phase extraction column of chloromycetin molecularly imprinted polymer and preparation method thereof |
| CN101650335A (en) * | 2009-07-31 | 2010-02-17 | 宇星科技发展(深圳)有限公司 | Molecularly imprinted membrane detection device for tetracycline and preparation and detection methods thereof |
| CN102288749A (en) * | 2011-07-28 | 2011-12-21 | 山东农业大学 | Dipterex bionic enzyme linked immunosorbent detection method |
| CN102382247A (en) * | 2010-09-03 | 2012-03-21 | 中国科学院过程工程研究所 | Preparation method of molecular imprinting polymer micro-sphere with uniform size and application |
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2013
- 2013-04-27 CN CN2013101522466A patent/CN103278622A/en active Pending
Patent Citations (4)
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| CN101564681A (en) * | 2009-06-03 | 2009-10-28 | 中国农业科学院农业质量标准与检测技术研究所 | Solid phase extraction column of chloromycetin molecularly imprinted polymer and preparation method thereof |
| CN101650335A (en) * | 2009-07-31 | 2010-02-17 | 宇星科技发展(深圳)有限公司 | Molecularly imprinted membrane detection device for tetracycline and preparation and detection methods thereof |
| CN102382247A (en) * | 2010-09-03 | 2012-03-21 | 中国科学院过程工程研究所 | Preparation method of molecular imprinting polymer micro-sphere with uniform size and application |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105111370A (en) * | 2015-09-29 | 2015-12-02 | 徐州工程学院 | Chloramphenicol molecularly imprinted polymer and preparation method thereof |
| CN105732890A (en) * | 2016-04-30 | 2016-07-06 | 中国科学院新疆理化技术研究所 | Preparation method of gossypol molecularly imprinted polymer |
| CN108051594A (en) * | 2017-12-20 | 2018-05-18 | 河南联博生物科技有限公司 | A kind of latex enhancing immune turbidimetry quantitatively detects the reagent of chloramphenicol |
| CN113354818A (en) * | 2021-06-15 | 2021-09-07 | 天津科技大学 | Preparation method and application of microporous plate for high-throughput rapid detection of kaempferol |
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Application publication date: 20130904 |