Biofuel cell and phenolic biosensor based on acid-resistant laccase–glutaraldehyde functionalized chitosan–multiwalled carbon nanotubes nanocomposite film

To immobilize laccase (Lac) from Trametes versicolor that shows its maximum enzymatic activity in acidic aqueous solutions, the biopolymer chitosan (CS) was chemically modified with glutaraldehyde (GA) to form GA functionalized CS (GAfCS), which was then allowed to react with Lac to form a Lac–GAfCS composite that is robust in weakly acidic solutions (two-step protocol), as confirmed by quartz crystal microbalance and durability tests. The Lac–GAfCS–multiwalled carbon nanotubes (MWCNTs)/glassy carbon (GC) electrode exhibited good catalytic activity towards O2 reduction in the presence of 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonate) diammonium salt (ABTS), and the pH-dependent enzymatic activity of the immobilized Lac towards O2 reduction was examined. A glucose/air biofuel cell was fabricated, with the Lac–GAfCS–MWCNTs/GC electrode as the biocathode and a glucose oxidase (GOx)–GAfCS–MWCNTs/GC electrode as the bioanode in a Nafion membrane-separated acetate buffer solution (pH 5.0). The biofuel cell output a maximum power density of 9.6μW/cm2, an open-circuit cell voltage of 0.19V, and a short-circuit current density of 114μA/cm2, respectively, as measured with an electrochemical noise (ECN) apparatus. Furthermore, the Lac–GAfCS–MWCNTs/GC electrode was applied to determine catechol in Britton–Robinson buffer solution (pH 3.0), with a linear range of 0.1–50μM and a limit of detection of 20nM. In comparison with the direct use of GA for one-pot Lac–GA-CS or Lac–GA crosslinking to immobilize Lac, the use of macromolecular GAfCS in the proposed two-step protocol was proven to be less harmful to the enzymatic activity and thus more suitable for immobilizing the enzyme to construct the biofuel cell and biosensor. This work may be helpful for exploiting the popular biocompatible CS as an acid-resistant film matrix for many other biotechnology applications, and the proposed two-step crosslinking protocol is recommended for high-activity immobilization of other biomolecules.