CN103278622A - Preparation method of 96-well enzyme label plate chloramphenicol molecularly imprinted polymer film - Google Patents

Abstract

The invention discloses a method for preparing a chloramphenicol molecularly imprinted polymer film on a 96-well microplate plate. Diol dimethacrylate was dissolved in porogen tetrahydrofuran at a molar ratio of 1:4:0.2, and ultrasonicated for 30 minutes; the amount of tetrahydrofuran was 5ml/0.25mmol chloramphenicol; the initiator azobisisobutyronitrile was added, Sonicate for 15 minutes, add 5mg/0.25mmol chloramphenicol; drop the solution obtained in step b onto a 96-well microplate, then put the 96-well microplate into a sealed bag, fill it with nitrogen, seal it and put it under the ultraviolet light Under the condition of irradiation, polymerization was initiated for 6 hours; then the 96-well microplate was placed in a methanol-acetic acid mixture with a volume ratio of 3:1, and ultrasonically eluted for 48 hours; the 96-well microplate was dried at 50°C for 2 hours to obtain Chloramphenicol Molecularly Imprinted Polymeric Membrane.

Description

Preparation method of 96-well ELISA plate chloramphenicol molecularly imprinted polymer membrane

technical field

The invention relates to a method for preparing a molecularly imprinted polymer film, in particular to a method for preparing a chloramphenicol molecularly imprinted polymer film on a 96-hole microplate plate with high selective adsorption of chloramphenicol.

Background technique

Chloramphenicol (Chloramphenicol, CAP) is an antibiotic isolated from microbial metabolites for the first time in 1947. Due to its excellent antibacterial properties, stable drug properties, and low price, it has been widely used as a feed additive to treat bacterial diseases. However, due to the nitro group on its benzene ring, its decomposition half-life is long and has serious toxic side effects, which can easily cause human blood poisoning, lead to serious toxic side effects such as irreversible aplastic anemia, and constitute a huge potential for human health. threaten. Therefore, the European Union and the United States have stipulated in the regulations that the CAP residue limit standard is "zero tolerance". The Ministry of Agriculture of my country also stipulates that CAP and its salt, fat, etc. should not be detected in all edible tissues of food animals. At present, the detection methods for CAP residues in samples mainly include microbiological methods, immunoassay methods, gas chromatography, chromatography-mass spectrometry, supercritical fluid chromatography, and immunofluorescence capillary electrophoresis techniques. Existing detection methods are cumbersome for sample pretreatment steps, coupled with complex sample matrix components and low CAP residual concentration, it is difficult to achieve rapid and effective detection.

Immunoassay has the advantages of simple operation, rapidity, high sensitivity, strong specificity and low detection cost, and has been listed as a priority research, development and utilization of pesticide residue analysis technology by many foreign countries. The currently used immunoassay technique is mainly enzyme-linked immunosorbent assay (ELISA) analysis method, but the detection limit is 0.1 μg/L, and sometimes it is difficult to meet the requirements of the drug residue detection standard. Chemiluminescent immunoassay is a new immunoassay technology developed after enzyme immunoassay technology. It is an immunoassay method that uses chemiluminescent agents to directly label antigens or antibodies. The chemiluminescence immunoassay analyzer consists of two parts, the immune reaction system and the chemiluminescence analysis system. The immune reaction system is to directly label the luminescent substance (excited state intermediate generated under the excitation of the reactant) on the antigen (chemiluminescence immunoassay) or antibody (immunochemiluminescence assay), or the enzyme acts on the luminescent substrate. The chemiluminescent analysis system uses chemiluminescent substances to form an excited state intermediate through the catalysis of the catalyst and the oxidation of the oxidant. When the excited state intermediate returns to the stable ground state, it emits photons (hM) at the same time. The signal measurement instrument measures the photon quantum yield. Although the sensitivity of chemiluminescence immunoassay technology can easily reach the level of 0.01 μg/L, the preparation process of the antibody in the kit used in the immune reaction system is cumbersome and complicated, the cost is high, and the equipment used is expensive, which is not suitable for the detection of drug residues in large batches of samples .

The basic principle of molecular imprinting technology (MIT) is to interact template molecules (molecules to be separated and identified) with functional monomers with suitable functional groups, and form macropores, For network polymers, template molecules are removed by solvent elution or hydrolysis under certain conditions, leaving a "memory" hole in the polymer that matches the template molecule in size, shape, and functional groups. It can carry out specific affinity with the template molecules to be separated in the mixture, so as to achieve separation, purification, pre-enrichment of template molecules, etc., and then use chemiluminescence methods such as enzyme-linked immunosorbent immunoassays to realize rapid and accurate detection of template molecules in samples. effective detection. However, so far there is no relevant report on the preparation of molecularly imprinted polymer membranes of chloramphenicol.

Contents of the invention

The present invention aims to solve the above-mentioned technical problems existing in the prior art, and provides a method for preparing a chloramphenicol molecularly imprinted polymer membrane on a 96-well enzyme plate with high selective adsorption of chloramphenicol.

The technical solution of the present invention is: a kind of preparation method of chloramphenicol molecularly imprinted polymembrane of 96 well microplate plate, it is characterized in that carry out according to following steps:

a. Dissolve the template molecule chloramphenicol, the functional monomer diethylaminoethyl methacrylate and the cross-linking agent ethylene glycol dimethacrylate in the porogen tetrahydrofuran at a molar ratio of 1:4:0.2, and ultrasonically 30min; the consumption of described tetrahydrofuran is 5ml/0.25mmol chloramphenicol;

b. Add the initiator azobisisobutyronitrile, ultrasonic for 15min, and the addition amount is 5mg/0.25mmol chloramphenicol;

c. Add the solution obtained in step b to a 96-well microtiter plate dropwise, then put the 96-well microtiter plate into a sealed bag, fill it with nitrogen gas, seal it and irradiate it with a UV lamp, and initiate polymerization for 6 hours;

d. Then place the 96-well ELISA plate in a methanol-acetic acid mixture with a volume ratio of 3:1, and ultrasonically elute for 48 hours;

e. Dry the 96-well ELISA plate at 50°C for 2 hours to obtain a chloramphenicol molecularly imprinted polymer membrane.

The a step is to weigh 0.25mmol chloramphenicol in a flask, add 5ml tetrahydrofuran to it, and ultrasonicate for 5min; Add 0.05mmol ethylene glycol dimethacrylate into the flask, and ultrasonicate for 5min.

The present invention uses diethylaminoethyl methacrylate as a functional monomer, ethylene glycol dimethacrylate as a crosslinking agent, tetrahydrofuran as a porogen, and azobisisobutyronitrile as an initiator, and according to Specific ratios and production conditions, etc., produce chloramphenicol molecularly imprinted polymer membranes with very high selectivity and specificity, which have high stability, long service life and strong resistance to harsh environments. The invention has the advantages of low cost, simple operation and easy control of reaction conditions.

Description of drawings

Fig. 1 is a standard curve diagram for the detection of chloramphenicol by direct competition ELISA according to the embodiment of the present invention.

Detailed ways

a. Weigh 0.25mmol of chloramphenicol into a flask, add 5ml of tetrahydrofuran to it, and sonicate for 5 minutes; Diol dimethacrylate was added into the flask, ultrasonicated for 5min;

b. Add 5mg of initiator azobisisobutyronitrile, ultrasonic for 15min;

c. Add the solution obtained in step b to the 96-well ELISA plate dropwise, then put the 96-well ELISA plate into a sealed bag, fill it with nitrogen gas, seal it and irradiate it with a 100W UV lamp (λ=365nm) to initiate polymerization 6 hours;

d. Then place the 96-well ELISA plate in a methanol-acetic acid mixture with a volume ratio of 3:1, ultrasonically elute for 48 hours, and change the eluent every four hours to remove template molecules;

e. Dry the 96-well ELISA plate at 50°C for 2 hours to obtain a chloramphenicol molecularly imprinted polymer membrane.

test:

1. Synthetic enzyme-labeled antigen (enzyme-labeled chloramphenicol):

Use glutaraldehyde method to couple chloramphenicol to horseradish peroxidase to synthesize enzyme-labeled antigen. The steps are as follows:

(1) Take 100mg of chloramphenicol and add it to 10ml of 0.6mol/L hydrochloric acid;

(2) After the chloramphenicol is dissolved, add 60mg of zinc powder to it, and react at 80°C for 30min;

(3) Take the supernatant and cool it to room temperature, slowly add it into the buffer solution containing horseradish peroxidase under stirring, then add 0.2ml of 25% glutaraldehyde dropwise, and react at room temperature for 5 hours with magnetic stirring;

(4) After the stirring, the synthesized product was dialyzed against 0.1mol/L, pH=7.2 PBS buffer (phosphate buffer system) at 4°C for 3 days, and the PBS buffer was changed twice a day to remove untreated Reactive small molecule substances such as glutaraldehyde, chloramphenicol, etc.;

(5) Measure the volume of the enzyme-labeled antigen solution in the dialysis bag, then add an equal volume of glycerol, and store it in a -20°C refrigerator in the dark for future use.

2. Preparation of Chloramphenicol Standards

Prepare 100mg/L chloramphenicol methanol solution, then dilute it to 30mg/L with PBS buffer solution containing 5% methanol, and then dilute to 3mg/L, 0.3mg/L, 0.03mg/L, 0.003mg/L , 0.0003mg/L. The luminescence value of each concentration was measured to draw a standard curve of chloramphenicol.

3. Direct Competitive ELISA Assay:

(1) Add 100 μl chloramphenicol sample standard solution or chloramphenicol-added sample solution to each well on a 96-well plate of chloramphenicol molecularly imprinted polymer membrane;

(2) Then add 100 μl enzyme-labeled antigen solution to each well, and use the wells without chloramphenicol standard solution as control wells;

(3) After incubating at room temperature for 1 hour, discard the supernatant and wash the plate 4-5 times with PBST;

(4) While washing the plate, prepare the luminescence solution (mixed with luminol and H ₂ O ₂ 1:1), put the plate into the chemical immunoassay analyzer, add 250 μl/well of the luminescence solution, and read the luminescence on the chemiluminescence instrument intensity luminous value;

(5) Repeat the measurement 6 times, and substitute the average luminescence value of the sample into the chloramphenicol standard curve to obtain the residual amount of chloramphenicol.

4. Preparation of samples to be tested and validation of the chloramphenicol kit

Take a healthy sea cucumber, and it is detected by liquid phase that there is no chloramphenicol in its body cavity fluid. Use a syringe to extract the body cavity fluid from it into test tubes, filter it with a 0.45 μm filter membrane, and take 1ml into 3 test tubes ( Number the test tubes 1, 2, 3), add 0.02ml, 0.1ml, and 0.2ml of chloramphenicol methanol solution with a concentration of 0.05mg/L into test tubes 1, 2, and 3 respectively, and prepare with body cavity fluid 1μg/L, 5μg/L, 10μg/L samples to be tested. The detection results of the application of chloramphenicol rapid detection kit and direct competition ELISA (application of artificial antibody) are as follows:

Detection results of direct competition ELISA (application of artificial antibody) and chloramphenicol kit for adding chloramphenicol in sea cucumber body cavity fluid samples

Concentration added (μg·L ^-1 ) Chloramphenicol kit (μg·L ^-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. Standard Curve of Chloramphenicol Detection by ELISA Using Artificial Antibody

Take IC% (the inhibition rate of chloramphenicol on the enzyme-labeled antigen and antibody binding reaction) as the ordinate, and the logarithm of the chloramphenicol standard as the abscissa to draw a standard curve for direct competition ELISA detection of chloramphenicol, as shown in Figure 1 Show.

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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