Direct-Electron-Transfer Bio-Nanoink with Single-Walled Carbon Nanotube and Aspergillus terreus var. aureus Flavin Adenine Dinucleotide Glucose Dehydrogenase

This pioneering study contains the fabrication of a novel concept of direct-electron-transfer (DET) bio-nanoink for a mediator-free and oxygen-insensitive glucose sensing. The DET bio-nanoink is composed of glycan chain-rich flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), single-walled carbon nanotubes (SWCNTs), and surfactant sodium cholate in aqueous solution. We discovered FAD-GDH genes in various fungi and characterized the enzymatic properties of the recombinant enzymes produced by Pichia pastoris as a host. Glycan-chain-rich Aspergillus terreus var. aureus FAD-GDH is screened and is the most suitable for the bio-nanoink compared to conventional FAD-GDHs from Aspergillus species in terms of chemical stability and activity. The amperometric biosensor is fabricated with piezoelectric inkjet printing, where the small fraction (several pL) of the bio-nanoink can be erupted without clogging of the nozzle. The biosensor with the bio-nanoink showed a large (1 mA cm–2 at +0.6 V 48 mM glucose) and a glucose-concentration-dependent current, indicating that the DET between FAD-GDH and SWCNTs in the bio-nanoink is accomplished. The DET is supported by the observation of the atomic force and transmission microscopies, where debundled SWCNTs are connected to FAD-GDH molecules. The mediator-free and oxygen-insensitive biosensor fabricated by the DET bio-nanoink revealed a high sensitivity of 70 μA mM–1 cm–2, a broad linear dynamic range of 0.0025–3.2 mM, and selectivity toward an interferent, and a low detection limit of 1.1 μm, which are superior to those of any other glucose biosensor.