| Diethylstilbestrol (DES), a synthetic hormone, has been used worldwide since 1938 as a growth-promoting agent for increasing feed conversion efficiency in aquaculture and stockbreeding and as a treatment for estrogen-deficiency disorders in veterinary medicine. But it has been banned for fattening edible animal in most countries for its carcinogenic and toxicological properties since 1980's. So far, several techniques have been applied during last twenty years to quantitatively monitor DES residues in tissues, organs and urine,including gas chromatography (GC), high-performance liquid chromatography (HPLC), GC-mass spectrometry (GC-MS), diode array-MS (DAD-MS), chemiluminescence (CL), thin layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), fluorescence immunoassay (FIA), radioimmunoassays (RIA), radioreceptor assay (RRA), electrochemical immunoassay (ECIA) and so on. DES is a stable and high boiling point compound, and thus derivation is required in GC analysis. HPLC is a suitable technique producing satisfactory results, but this method requires relatively complex sample extraction, concentration and clean-up procedures. HPLC instruments are not available in many analytical laboratories. Compared to RIA and FIA, ELISA has the advantages of safety, speed, reliability, sensitivity, and low-cost.Recently, electrochemical immunosensors have gained growing attention since they combine the high specificity of traditional immunoassay methods with the low detection limits and low expenses of electrochemical measurement system, and the fact that they are fast, accurate, simple, portable, and inexpensive. The method has been used by a number of investigators for determination of various analytes with great sensitivity and specificity. It has been reported that the crucial aspect of electrochemical immunosensor is the immobilization of immunological sensitivity compounds on the electrodes. The biological macromolecules immobilized in films on electrode surfaces should exhibit their specific immunoactivity,retain their native structure and demonstrate excellent stability. For these reasons a number of different type films, such as conducting polymer, titania sol-gel, and self-assemble film, have been used to immobilize immunogenic molecule and achieve their immune response. Among them, casting films, for example, gold nanoparticles films and agarose hydrogel films, have been widely used due to their simplicity, versatility, and biocompatibility. However, these casting films are fragile and fabricated complicatedly. In this paper, we report the employment of nitrocellulose membrane, a high mechanical strength and commercially available membrane ideal for blotting of proteins, as a novel immobilization matrix for development of electrochemical immunosensors.A novel and simple label-free electrochemical immunosensor for determination of diethylstilbestrol was constructed by electrically enhanced adsorption of diethylstilbestrol-bovine serum albumin into nitrocellulose membrane modified on a gold electrode. The electrochemical characteristics of the immunosensor were investigated by cyclic voltammetry and differential pulse voltammetry in a redox probe system of K3Fe(CN)6. The redox peak current of K3[Fe(CN)6] was reduced with the binding of the antibody to the diethylstilbestrol-bovine serum albumin immobilized on the gold electrode. The effect of amount of monoclonal antibody was explored to provide optimum analytical performance. Competition for antibody binding between the free diethylstilbestrol and the diethylstilbestrol-bovine serum albumin on the electrode was applied to detect free diethylstilbestrol. The immunosensor had very high sensitivity and specificity, and was used for the quantitative determination of trace amounts of diethylstilbestrol in spiked samples. |
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