Meiping Zhao

Development and characterization of an immunoaffinity monolith for selective on-line extraction of bisphenol A from environmental water samples

Matrix interference and contamination from analytical procedures are two major issues in the detection of trace level of bisphenol A (BPA) in environmental water. In this paper, we report a highly selective and efficient method for on-line extraction of BPA from water samples followed by quantification with liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI/MS/MS). Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in 50 mm x 4.6mm i.d. stainless steel cartridges and the epoxy-groups on the surface of the monolith were hydrolyzed and oxidized to aldehyde functions. Antibodies against BPA were covalently immobilized onto the monolithic column via Schiff base reaction. The optimum application buffer and elution buffer were found to be pH 5.5 phosphate buffered saline (PBS) and methanol-water (70:30, v/v), respectively. The obtained immunoaffinity monolithic columns were on-line coupled to LC-MS/MS using column-switching valves and the system was applied to analyze BPA in real environmental water samples. The method achieved a detection limit of 0.3ngL(-1) using a sample volume of 100mL. The linear calibration range was 1.0-160ngL(-1). Samples including tap water, lake water and effluent from municipal sewage treatment plant were all measured with satisfactory results.

Development and characterization of an immunoaffinity column for the selective extraction of bisphenol A from serum samples.

An immunoaffinity column (IAC) was developed by covalently coupling polyclonal antibodies against estrogenic bisphenols to CNBr-activated Sepharose 4B. The IAC showed high affinity for bisphenol A, while phenol was barely retained. Proteins in the sample matrix showed little nonspecific adsorption on the column. The best binding solvent for bisphenol A was found to be 0.01 mol l(-1) phosphate-buffered saline (PBS) and the optimal operating temperature was 4 degrees C. The bound bisphenol A could be quantitatively recovered by 1 ml of methanol-water (80:20) with an average recovery of 91.8% and a relative standard deviation of 7.1% (n=6). The immunoaffinity column has been successfully used for the isolation and purification of bisphenol A from serum samples.

Polyethylene glycol diacrylate-based supermacroporous monolithic cryogel as high-performance liquid chromatography stationary phase for protein and polymeric nanoparticle separation

A supermacroporous monolithic cryogel was directly prepared by in situ cryo-copolymerization in a stainless steel cartridge (70mmx5.0mm I.D.) using methacrylic acid (MAA) as functional monomer and polyethylene glycol diacrylate (PEGDA) as crosslinker. The highly crosslinked (90%, molar ratio) poly(MAA-PEGDA) cryogel had more uniform supermacropores with a mean diameter of 25microm compared to the poly(acrylamide)-based cryogels. The viability of poly(MAA-PEGDA) cryogel as a medium was demonstrated for separations of lysozyme from chicken egg white (CEW) and water-soluble poly(N-isopropylacrylamide-co-3-(dimethylamino) propyl methacrylamide) (NIPAM-DMAPMA) nanoparticles from its crude reaction solution. The dynamic binding capacities of lysozyme and the polymeric nanoparticles were 4.51x10(-3)micromol/ml and 33.4microg/ml, respectively. The lysozyme recovered from the above separations had a purity of more than 85%, and retained 90% of its enzymatic activity.

Class-specific immunoaffinity monolith for efficient on-line clean-up of pyrethroids followed by high-performance liquid chromatography analysis.

A class-specific monolithic immunoaffinity column was developed for on-line clean-up of pyrethroid insecticides in complex samples. Deltamethrin was oxidized with ozone to generate the hapten of (RS)-alpha-cyano-3-phenoxybenzyl (RS)-cis,trans-2,2-dimethyl-3-formyl-cyclopropane carboxylate. Class-specific antibodies against pyrethroids were produced using the conjugate of above hapten with bovine serum albumin as the immunogen. Poly(ethylene dimethacrylate-glycidyl methacrylate) monolith was synthesized in a 50mmx4.6mm i.d. stainless steel cartridge with two auxiliary pipette tips. The polymerization method was proved to be economic and reproducible. Antibodies against pyrethroids were covalently immobilized onto the monolithic support via Schiff base reaction. With a column-switching valve system, the immunoaffinity monolith (IAM) could be readily adapted to the reversed-phase high-performance liquid chromatography (HPLC) system. Under the optimum loading, washing and eluting conditions, the IAM specifically retained deltamethrin, flumethrin, flucythrinate and cis/trans permethrin, which were further baseline separated by C18 column using acetonitrile-water (83:17, V/V) as the mobile phase at a flow rate of 1.0mL/min. The established system provides a highly efficient approach for high-throughput on-line clean-up of pyrethroid in various samples.

Antigen synthetic strategy and immunoassay development for detection of acrylamide in foods

Acrylamide, a toxic and carcinogenic compound, has been found to be present in a range of processed starchy foods. To prepare an effective immunogen compound for acrylamide, N-acryloxysuccinimide (NAS) was conjugated to bovine serum albumin (BSA) at a high molar ratio of 21.2:1. Antisera were obtained by immunization of rabbits with additional booster injections of the NAS-BSA conjugate after the regular process. The IgGs purified by an ammonium sulfate precipitation method were further fractionated with a BSA-immobilized immunoaffinity column. The affinity constant between the collected antibody and coated antigen (NAS-ovalbumin) is found to be 6.7 x 10(7) L mol(-1). Asparagine, the key precursor of acrylamide formation in foods, showed negligible cross-reactivity to the antibody. A biotin-avidin enzyme-linked immunosorbent assay (BA-ELISA) was developed and the optimum assay medium was found to be 0.1 mol L(-1) NaHCO(3) (pH 8.3, containing 0.5 mol L(-1) NaCl). The BA-ELISA afforded a practical sensitivity with a working range of 10-100,000 ng mL(-1) and a detection limit of 6 ng mL(-1). The assay was applied to detect acrylamide in potato fries and biscuits and the quantitative results were in good agreement with those obtained by the high-performance liquid chromatography method. This immunoassay will be very useful for monitoring acrylamide in food samples.

Simultaneous discrimination of jasmonic acid stereoisomers by CE‐QTOF‐MS employing the partial filling technique

Jasmonic acid (JA), an essential plant hormone controlling the plant defense signaling system and developmental processes, has stereospecific bioactivities that have not been well understood mainly due to the limitation in separation and detection methodologies. In this work, a fast CE‐UV method based on short‐end injection technique and a sensitive CE‐QTOF‐MS method based on partial filling technique were successfully developed for the enantioseparation of racemic JA. The successive coating technique was also involved by modifying the capillary with multiple ionic polymer layers of polybrene‐dextran sulfate‐polybrene. This was the first report on the direct resolution of both pairs of JA enantiomers, including two naturally occurring JA stereoisomers. Although no pure JA stereoisomers were commercially available, all the separated JA stereoisomers were identified indirectly by comparing the difference between the racemic standard and plant samples based on the presence and the ratio of each stereoisomer. Satisfactory results were obtained in terms of sensitivity (LOD, 24u2009ng/mL or 0.7u2009fmol for single JA stereoisomer) using 45u2009mmol/L ammonium acetate at pH 4.5 containing 70u2009mmol/L α‐CD as the buffer system. This established CE‐QTOF‐MS method was later successfully applied for the study of the naturally occurring JA stereoisomers in wounded tobacco leaves.

Immunochemical Analysis of Endogenous and Exogenous Estrogens

Endogenous estrogens, namely estradiol, estriol and estrone are essential substances for the sexual determination, growth and reproduction of human beings. Their levels in the human sera are important index in monitoring pregnancy, evaluating menstrual dysfunctions and diagnosing many other diseases. On the other hand, many exoestrogens, including the phytoestrogen such as isoflavones, the synthetic estrogens such as diethylstilbestrol and chemicals of industry origin with suspected estrogenic activity such as bisphenol A and 4-nonylphenol, are drawing increasing attention in recent years. This paper reviews the main immunochemical analytical methods for these important estrogenic substances. The immunizing hapten design and its influence on the specificity of produced antibody were discussed. The typical available immunoassays including enzyme immunoassay (EIA), fluoroimmunoassay (FIA) and chemiluminescence immunoassay (CLIA) for the detection of estrogens were compared with respect to their features (especially dynamic range and limit of detection). The performance of different types of immunosensors with respect to their advantages and disadvantages were evaluated. The future developing trend in this field was also briefly discussed.