Molecular Imprinted Technology And Its Applications In Environmental Estrogens Determination

Environmental Estrogens, a kind of endocrine disrupters, have been shown to induce abnormal differentiation and development of reproductive organs by mimicking or interfering with the action of endogenous gonadal steroid hormones. Unfortunately complex sample matrices in biological and environmental samples often affect the accuracy of BPA determination. The traditional determination methods for this kind of pollutants need tedious and time-consuming pretreatment procedures for its trace or ultra-trace concentration in environment. Solid-phase extraction (SPE) has become a routine sample pretreatment method. It has many attractive features, such as ease of operation, high load abilities, high recoveries, lower consumption of organic solvents, and a wide spectrum of stationary phases available, automation capabilities and so forth. However, SPE is usually based on non-selective interactions between the analyte molecule and the sorbent, which often leads to coextraction of many matrix components.To enhance the extraction efficiency, new extraction sorbents involving reversible and selective antigen-antibody interactions, called immunosorbents (IS), have been synthesized in order to trap target analyte. They give extracts which are almost free of interfering substances. But antibody isolation is expensive, time-consuming, and easily denaturized in the presence of organic solvents. These drawbacks limited the usage of IS in sample pretreatment.Molecularly imprinted polymers (MIPs) are synthetic cross-linked polymers formed by the presence of a target molecule (template). The template is then removed, which produces a cavity with molecular recognition sites that can bind selectivity to the original template. MIPs have advantages such as physical robustness, high strength, resistance to elevated temperatures and pressures, and inertness towards organic solvents, acids or bases. Sellergren first introduced MIPs into SPE procedure, namely molecularly imprinted solid phase extraction (MISPE). Afterward, MISPE has been widely applied in biology, pharmacy and environment fields The application of MIPs in SPE has become the most promising development of selective SPE method for trace analysis.Bisphenol A (BPA) is a known endocrine disrupter. It is the monomer for the production of polycarbonate plastics and epoxy resins, such as baby bottles, foodstuff containers and dental sealants. It is widely used in industry and dentistry. Therefore BPA exists widely in environment and can easily migrate into human body to produce adverse effects on health. To date, liquid chromatography, gas chromatography, and electrophoresis are normally used for BPA identification and quantification. In order to monitor the accurate concentration of trace BPA in biological or environmental samples, sample pre-treatment is necessary. In this research, BPA was chosen as the template. By optimizing the preparing conditions, BPA imprinted polymers were used to determine the trace amount of bisphenol A in biological and environemtal samples (human serum, pig urine, tap water and shrimp) in a offline mode, and also used a MIP column to simultaneously extract, enrich, separate, determine and quantitate bisphenol A in water samples for the first time.1. In this paper, bisphenol A imprinted polymers were synthesized by precipitation polymerization. Effects of the preparing condition on the morphology and imprinted effect of the polymers were investigated. Results showed:(a) the ratio of acetonitrile to toluene may influence the morphology of the polymers. Particles prepared in pure acetonitrile were globular and narrowly dispersible. As the percentage of acetonitrile decrease from 100% to 90%, the particles'shape changed from globular to irregular and the dispersivity increased. Imprinted effect experiments showed the polymer prepared in 100% acetronitrile has the best selectivity through the binding ability was poor.(b) the volume of the acetonitrile used in the synthesis has influence on the morphology of the polymer. The particles prepared in 40ml were irregularly in shape, while particles prepared in 80mlacetonitrile ware too small to be used as sorbents for SPE or chromatography through they were narrow disperse microspheres.(c) the amount of AIBN and the temperature in the preparation have no influence on the imprinted effects of the polymers through they can accelerate the polymerization.(d) Polymers prepared with MAA with a resemble morphology of polymer prepared with 4-VP, but had poor binding ability and selectivity.(e) To examine the effects of the amount of template on the performance of MIP in precipitation polymerization, different amount of template was used in the preparation. Results showed morphology was not influenced by the amount of the template, but binding capability and selectivity increased as the amount of template used increased.2. BPA imprinted MIPs were used as sorbents for MISPE on several biological and environmental samples. Optimal loading, washing and eluting protocols were critical for the superior MISPE procedure. The optimum conditions were: conditioning with 5 mL methanol-acetic acid (3:1), 5 mL methanol,5mL acetonitrile and 5mL water respectively, loading with 5 mL aqueous samples, washing with 1mL acetonitrile, and eluting with 3 mL methanol. MIPs can selectively recognize, effectively trap and pre-concentrate of BPA over a concentration range of 2-20μM. Recoveries ranged from 94.03% to 105.3%, with RSD lower than 7.9%. Under the optimal condition, MISPE recoveries of spiked human serum, pig urine, tap water and shrimp were 65.80%, 82.32%, 76.00% and 75.97% respectively when aqueous samples were applied directly. Compared with C18 SPE, better baseline, better HPLC separation efficiency and higher recoveries were achieved after MISPE.3. A fully simultaneous and direct analysis method for ultra-trace analytes in environmental water samples was established by large injection volume (40mL), direct enrichment and HPLC analysis using one narrowly dispersible MIP column and UV detector. Due to the high retention and specific HPLC characters of BPA imprinted MIP13 to BPA, ultra-trace BPA in 40mL water samples can be simultaneously extracted, enriched, separated, determined and quantitated in one analysis process under optimum conditions. The calibration graph of BPA standard solution was linear in the concentration ranges of 0.1-100 nmol/L with less than 9.6% intra-day and inter-day RSD. For spiked (0.1-10nmol/L) tap water and lake water samples the RSD was less than 8.9% and the recoveries were 96%-101.8%. The enrichment factor for BPA was 10000 as 40 mL water sample was directly injected and analyzed. MIP13 was successful applied to ultra-trace BPA determination in environmental water samples with high accurate and repeatability using commonly used UV detector. This methodology may provide a useful tool for accurate investigation and determination of BPA levels in the environmental and biological matrices.