Mushroom Tyrosinase in Polyelectrolyte Multilayers as an Optical Biosensor for o-diphenols

Determination of phenolic derivatives is very important in medical, food and environmental samples because of their relevant significance in health care and pollution monitoring. Tyrosinase-based biosensors are promising tools for this purpose because of several advantages with respect to currently used detection methods. A key aspect in the development of a biosensor is the effective immobilization of the enzyme. In this work, ordered tyrosinase films on an optical transparent support were immobilized by a "layer-by-layer" (LbL) assembly, alternating the enzyme with the polycation polymer poly(dimethyldiallylammonium chloride). As confirmed by UV-vis spectroscopy, the LbL deposition allowed a high loading of enzyme. The immobilized tyrosinase functionality was proven and its kinetic parameters were spectrophotometrically determined. The prepared biosensor was used to optically detect the o-diphenolic compound l-3,4-dihydroxyphenyl-alanine (l-DOPA) and exhibited good repeatability and time stability. The sensing properties of the system were studied by means of both absorption and fluorescence spectroscopy. The bioassay based on the absorbance measurements gave a LOD of 23M and a linear response up to 350M. The bioassay based on the fluorescence measurements gave a LOD of 3Mand a linear response in the range of tens of micromolar (the exact value depends on the number of mushroom tyrosinase layers). Biosensor sensitivity could be modulated varying the number of the immobilized enzyme layers.