Layer-by-layer assembly of enzymes and polymerized mediator on electrode surface by electrostatic adsorption

Through layer-by-layer adsorption (LBL) technique, multilayer film was prepared from enzymes, diaphorase (DI) and glucose-6-phosphate dehydrogenase (G6PDH) and polyelectrolytes. The adsorption interface morphology was directly observed by atomic force microscopy, and the immobilization amount and layer thickness were characterized from quartz crystal microbalance which showed formation of nanoscale multilayer structure and linear mass increase which depended on alternate adsorption cycles. In order to construct a new mediated bi-enzyme biosensor system, polymerized mediator, DI and G6PDH were immobilized on carbon electrode surface by using LBL method. Electrochemical experiments indicated highly efficient electron transfer by the polymerized mediator. Two enzymes kept their activities after immobilization, and the electrode immobilized by mediator and enzymes showed sensitive response to glucose-6-phosphate in the presence of free NAD⁺, and high stability during long period of storage.