Enzyme-iron nanoparticle direct interactions: hybrid formation and application for a coulometric biosensor for aminoaldehydes

Recombinant aminoaldehyde dehydrogenase from tomato (LeAMADH1) was used as a model protein for studying the intimate interaction at the interface between proteins and surface active maghemite nanoparticles (SAMNs). SAMNs represent a new class of maghemite nanoparticles, displaying great colloidal behavior and specific surface chemical properties, as well as, peculiar electrochemical characteristics and specificity toward protein binding [1-2]. A potential anchor zone was individuated in a carboxylic rich surface area of the protein structure by molecular modeling. LeAMADH1 was successfully bound to the surface of bare SAMNs and its enzymatic activity was preserved, leading to a magnetic drivable enzymatic hybrid (SAMN@LeAMADH1). The catalytic parameters of SAMN@LeAMADH1 were determined, and exploited for the construction of a coulometric biosensor for the determination of aminoaldehydes in alcoholic beverages. A suspension of SAMN@LeAMADH1, used inside a low volume (1 µL) electrochemical flow cell, fabricated in-house, led to the complete oxidation of aminoaldehydes, producing a correspondent amount of NADH. The hybrid nanomaterial was magnetically removed after the enzymatic reaction allowing its reutilization. At the same time, a SAMN modified carbon paste electrode, inserted in the microcell was used for the direct electro-oxidation of NADH, leading to the coulometric determination of NADH produced during the enzymatic process. The novel biosensor showed a series of peculiarities: a) SAMN modified carbon paste electrode was used for NADH electro-oxidation; b) the complete enzymatic oxidation of sample aminoaldehydes, producing NADH, was carried out in a colloidal suspension, inside a low volume electrochemical flow cell, optimizing reactant diffusion; c) NADH, produced during the complete enzymatic oxidation of aminoaldehydes, was coulometrically determined at the SAMN modified lectrode; d) the capture of the hybrid, by the application of an external magnet, makes it reusable. 1)Baratella et al., Biosensors and Bioelectronics, 2013, 45, 13–18. 2)Magro et al., Biosensors and Bioelectronics, 2014, 52, 159–165.