Přehled o publikaci

The improved highly sensitive piezoelectric immunosensor has been developed. The gold electrodes of the piezoelectric quartz crystals were modified with self-assembled layer of cysteamine and carboxyderivative of atrazine was covalently attached after activation with O-(N-succinimidyl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TSTU). The competitive immunoassay for atrazine was developed using anti-atrazine monoclonal antibody (MAb) D6F3. The limit of detection for atrazine was 0.025 ng/ml, the total time of analysis being 25 min. The repeated use of the immunosensors was possible after regeneration using pepsin at pH 2.0. The direct immunosensor for atrazine was constructed by oriented immobilization of anti-atrazine MAb to Protein A covalently attached to the gold surface activated with 3,3'-dithio-bis(propionic acid N-hydroxysuccinimide ester) (DTSP). The MAb-Protein A complex was stabilized by cross-linking with dimethyl pimelimidate. Thus developed immunosensor was able to specifically respond to atrazine present in solution, providing limit of detection of 1.5 ng/ml, one assay was completed within 10 min (5 min binding, 5 min washing of surface). The observed changes of frequency are expected to result from conformational changes of the immobilized antibody after formation of the immunocomplex with atrazine. Furthermore, kinetic studies were carried out in different arrangements, the obtained kinetic association equilibrium constant was 2.3x10^7 l mol-1 for interaction between immobilized antibody and atrazine in solution.