l-Proline sensor based on layer-by-layer immobilization of thermostable dye-linked l-proline dehydrogenase and polymerized mediator

l-Proline sensor has been fabricated through multilayer assembly of recombinant dye-linked l-proline dehydrogenase (l-proDH) originally from hyperthermophilic archaeon (Therococcus profundus) and polymerized mediator, poly(allylamine) ferrocene (PAA-Fc), on a gold electrode by electrostatic layer-by-layer alternate adsorption method. The characteristic redox peaks of ferrocene groups were obvious exhibited in cyclic voltammograms, and both anodic and cathodic peaks increased with PAA-Fc/l-proDH bilayer number which confirmed the formation of a multilayer structure. Further experiments showed that an electrode fabricated in this way catalyzed the oxidation of l-proline. It suggested that polymerized mediator could transfer electrons between electrode surface and immobilized l-proDH. Ampeormetric experiments showed that the current response to l-proline increased with PAA-Fc/l-proDH bilayer number. The stability of the sensor was measured, and it kept a 40% relative response after 30 days storage in buffer under 4 °C.