Bi-enzyme biosensor based on NAD+- and glutathione-dependent recombinant formaldehyde dehydrogenase and diaphorase for formaldehyde assay

The present work reports on the development of a bi-enzyme biosensor using diaphorase from Bacillus stearothermophilus, nicotinamide adenine dinucleotide (NAD+)- and glutathione (GSH)-dependent formaldehyde dehydrogenase (FDH) from the genetically-engineered methylotrophic yeast Hansenula polymorpha as bio-recognition elements. The sensor architecture comprises a first layer containing diaphorase cross-linked with an osmium complex-modified redox polymer (poly(vinylpyridine)-[osmium-(N,N′-methylated-2,2′-biimidalzole)3]3+/2+ complex). On its top, a second layer was formed by additional cross-linking of FDH with poly(ethylene glycol)(400)diglycidyl ether. The sensor's architecture was optimized with respect to efficient electron transfer and stability of the enzyme(s). The characteristics of the optimized formaldehyde biosensor were: sensitivity 22 A m−2 M−1, detection limit 32 μM, and linear dynamic range 50–500 μM.