ИСТИНА

Layer-by-layer technology (LBL technology) is proving to be a new promising and versatile way to form interpolyelectrolyte multilayered thin films with wide range of physical, mechanical and chemical properties. The technology is based on the consequent adsorption of positively and negatively charged polyelectrolytes on a solid substrate and gives broad possibilities to control surface properties by inserting functional elements like conductive nanoparticles, enzymes, mediators, etc. into polyelectrolyte assembly. Due to the LBL technology gives an ability to create surfaces with prescribed and controlled characteristics it has high potential for biosensor design. The aim of this work was to develo a new amperometric phenol biosensor using LBL technology. The components of layer-by-layer assembly were: polydimethyldiallylammonium chloride (as a polycation), sodium polyanethole sulfonate (as a polyanion), tyrosinase (oxidoreductase, producing the oxidation of phenol to electroactive o-quinone), and metoxymethylphenazonium methyl sulfate (mediator of electroreduction of o-quinone). The phenol biosensor includes enzymatic oxidation of phenol via catechol into o-quinone by tyrosinase and subsequent electrochemical reduction of o-quinone to catechol directly at the electrode when the required potential is applied. Inclusion the mediator into electrochemical reduction cycle gives rise to more efficient electron transfer and so noticeably increases the sensitivity of biosensor to phenol in comparison with unmediated transfer. A number of aspects concerning the electrode construction and operation have been studied and optimized. Different consequences and combinations of LBL assembly were examined and the optimum layer's number deposited on the graphite electrode surface was found. The topography, surface mechanical and adhesive properties of tyrosinase-polyelectrolyte films was investigated with Atomic Force Microscopy. The regular complete LBL film found to form after four cycles of polyelectrolyte deposition. The optimal conditions of tyrosinase modified electrodes constructing were investigated. The influence of tyrosinase, polyelectrolyte and mediator concentrations on the electrode characteristics was studied. The efficiency of both the mediator immobilization and its electron transfer action was studied for several variants of LBL assemblies using Cyclic Voltammetry. The analytical parameters of the tyrosinase modified electrodes were obtained. The minimum detectable concentration of phenol found to be 80 nM. Use the LBL technology for forming mediator modified tyrosinase based electrodes results in visible decreasing enzyme expense, background noise of detection system and signal output time. The possibility to monitor phenol concentration in kinetic mode was shown for butyrylcholinesterase activity analysis with phenyl valerate as the substrate. This biosensor method, therefore, provides sensitive, simple, rapid and economical bioanalytical system that could be applied for esterase activity assay in clinical medicine and toxicology.