Affinity assembled multilayers for new dehydrogenase biosensors

We propose a novel approach, which allows the control of the spatial arrangement of redox mediator, coenzyme and enzyme on the electrode at a molecular level, using essentially electrostatic interactions. The first step consists of adsorbing a monolayer of molecules out of a new family of redox mediators, substituted nitrofluorenones. In a second step, a monolayer of calcium cations is immobilized at the interface. It serves as a bridge between the redox mediator and the subsequently adsorbed coenzyme. The weak interaction between a carboxyl group of the redox mediator and the coenzymeʹs phosphate groups, revealed by QCM measurements, allows the coenzyme to keep its natural activity in the adsorbed state. In the last step, we use the intrinsic affinity of this monolayer of NAD+ for dehydrogenases to build up a supramolecular sandwich composed of mediator/Ca2+/NAD+/dehydrogenase. This simple modification procedure, which might constitute a versatile approach for the low cost assembly of well-defined biosensors surfaces, has been successfully applied to the enzymatic detection of glucose, glutamate and alcohol.