Three immobilized enzymes acting in series in layer by layer assemblies: Exploiting the trehalase-glucose oxidase-horseradish peroxidase cascade reactions for the optical determination of trehalose

A new optical biosensor for trehalose determination has been realized immobilizing three glycoenzymes on a transparent support. Trehalase, glucose oxidase and horseradish peroxidase have been alternated with layers of Concanavalin A by a “layer-by-layer” (LbL) deposition. The driving force of this assembly is the biospecific complexation between Concanavalin A and sugar residues in the glycoenzymes. As confirmed by UV–vis spectroscopy, the LbL deposition allowed a high ordinate architecture with high loading of enzymes. After the assembly, the functionality of immobilized enzymes was spectrophotometrically proven, demonstrating also that they can act in series catalyzing cascade reactions.The prepared biosensor was used to optically detect trehalose, giving a LOD of 10 μM and a linear response up to 4 mM, and it showed also good time stability. The trehalose content in a real sample (eyewash) was successfully determined by the biosensor.