Immobilization and characterization of alcohol dehydrogenase on single-walled carbon nanotubes and its application in sensing ethanol

Abstract

The negatively charged (at pH 8.2) alcohol dehydrogenase (ADH, pI ∼ 6.8) was assembled onto the surface of single-walled carbon nanotubes (SWNTs), which was covered (or wrapped) by a layer of positively charged polyelectrolyte poly(dimethyldiallylammonium chloride) (PDDA), via the negative–positive charge interaction forming ADH–PDDA–SWNTs nanocomposites. The atomic force microscopy (AFM), Fourier transform infrared (FTIR), Raman, ultraviolet–visible (UV–Vis), and electrochemical impedance spectroscopy (EIS) were used to characterize the growth processes of the nanocomposites. The results indicated that ADH retained its native secondary conformational structure after it was immobilized on the surface of PDDA–SWNTs. A biosensor, Nafion/ADH–PDDA–SWNTs/GC, was developed by immobilization ADH–PDDA–SWNTs nanocomposites on the surface of glassy carbon (GC) electrode using Nafion (5%) as a binder. The biosensor showed the electrocatalytic activity toward the oxidation of ethanol with a good stability, reproducibility and higher biological affinity. Under an optimal condition, the biosensor could be used to detection ethanol, representing a typical characteristic of Michaelis–Menten kinetics with the apparent Michaelis–Menten constant of KMapp∼5.0mM, with a linear range span the concentration of ethanol from 0.5 to 5.0 mM (with correlation coefficient of 0.998) and the detection limit of ∼90 μM (at a signal-to-noise ratio of 3). The facile procedure of immobilizing ADH used in present work would promote the developments of electrochemical research for enzymes (proteins), biosensors, biofuel cells and other bioelectrochemical devices.