| Food safety has become a very serious social and livelihood issues in China, which illegally adding and abuse of veterinary drugs is the main cause of animal food hazards. Estradiol, being a small molecular steroidal hormone,has been widely used in the livestock and poultry breeding for its advantage of whetting appetite, increasing protein conversion rate, improving lean meat ratio, promoting animal growth and weight gaining. U.S. Environmental Protection Agency designated it as one of the Environmental Endocrine Disrupting Chemicals as early as 1996. The limit standard had been formulated by many countries, while Chinese Ministry of Agriculture expressly provided estradiol may not be detected in animal foods. EDCs can produce a series of harm on human and the natural environment, wherein the estrogen residues in milk and dairy products has been a serious threat to the health of our young children. Therefore, strict and effective monitoring of its residues in food is very necessary. Commonly detection technology using for estrogen residues are chromatography-mass spectrometry, which requiring large expensive equipment and professional operation, along with complex and time-consuming sample pretreatment,made them far to meet the food safety site rapid testing requirements.Our study established an efficient, sensitive enzyme thermistor technique for estradiol detecting for the propose of maintaining food safety and controlling veterinary abuse. The method is based on the direct competition immunological principle, shows rapid,convenient, specific features of the thermal biosensor, acquires stable results while abandoning tedious sample preparation work.The main contents of this study contains:1) Carboxyl transformation and coupling of estradiol, purification of monoclonal antibodies.Since estradiol is small molecular substance which does not have immunogenic activity, we conducted carboxyl transformation by adding KOH and bromine acid,using ethyl acetate extraction and methanol chloroform recrystallization, then coupled the products with target enzyme protein through EDC method. Identified E2-3-CME by1 H NMR, while using mass spectrometry affirmed successful coupling. We purified estradiol monoclonal antibodies, then identified products by ELISA, and tentatively identified for the antigen- antibody dilution ratio of further test.2)Optimization of enzyme thermistor detection system.We used TELISA technique in the experiment. Agarose gel particles SPG were applied as enzyme column filler to adsorb estradiol antibody, afterwards free estradiol and β- lactamase labeled estradiol competed the binding sites on the antibody. The concentration of small molecules were estimated by the thermal signal generated by different concentration of substrate. We gradually optimized experimental parameters Such as the reaction substrate, the system flow rate, carrier liquid and regeneration conditions. Final install of the enzyme thermal detection system were as follows:System temperature 37℃, detection sensitivity 500, substrate ampicillin(4mmol / L), the antigen-antibody dilution ratio E2 1: 200 / E2-m Ab 1: 16000, carrier liquid 0.02 mol / LPBS + 2% DMSO + 0.5% glycerol, p H7.0, regeneration conditions:0.1mol / LGly-Hcl + 0.5mol / LNacl + 1% DMSO + 0.1% Triton X-100, p H2.3(shift1200ul / min × 6 min.), system flow rate 400μl / min, sample incubated 30 min /co-injection; injection time: 6min30 sec. Diluent and carrier liquid used above were filtered through 0.22μm membrane, ultrasonic degassing and putting in 37 ℃ water bath.3)Construction of enzyme thermistor method for estradiol detection.We constructed standard curve on the basis of the optimization with the liner range5.1-19.4 ng/ml, IC50 10.2ng/ml and the detection limit of 2.8ng/ml(Y=96.4438+(6.7545-96.4438)/(1+(x/10.57676)^2.5011),R2=0.99021. Procedure asfollows: dissolved E2 standard in DMSO, gradient diluted from 0 to 200 ng / ml,opened enzyme thermal sensor and injected sample sequentially(each injection volume 350μl)- regeneration- record peak signal, repeated the test 3 times under same conditions and drew standard curve by the mean data.The results was stable,reliable and specific, no cross-reaction could be demonstrated with a variety of estrogen analogues and CV% was 3.4%(X±S:80.8±2.8mv,E2 12.5ng/ml,n=6). We established a simple method for milk sample pretreatment:centrifuged liquid milk samples after 16000 rpm for 30 min, extracted the middle whey layer and reserved in-20 ℃.Milk samples obtained can be successfully applied in enzyme sensing equipment, blank samples making no matrix interference signals.Drew standard curve with the liner range 0.24-0.79 ng/ml, IC50 0.45 ng/ml, the detection limit of 0.12ng/ml and recoveries ranged in the vicinity of 100%, Y=97.91128+( 2.91161-97.91128) /1+( X/0.32696) ^2.32955, R2=0.99819. The result was satisfactory as compared with the LC-ESI-MS method.Enzyme thermal technology is still rarely reported being applied on the detection of pesticide and veterinary drug residues, not mentioning the small molecules hormones.In this study we built a fast, efficient, convenient and economic thermal biosensor method services food safety on-site detecting, which extends its application, achieves technical and theoretical innovation and has considerable application and development prospects. We used SPG as immobilized enzyme column for the advantage of omitting immobilized enzyme(or other biological original identification)step, and also can easily change detection target by the replacement of antigen-antibody combination, making this sensor technology has good versatility. We explored a fast and effective method of milk samples before treatment, which provides a guideline for the future food testing works. We obtained a preferable lower limit with the flaw of narrow detection range, which may be owing to the complexity of estradiol’ metabolic profilein milk samples. In the actual operation a dilution stepmay be taken to obtain accurate detection values, while further studies will explore ways to improve. |
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