Biosensors Based on Membrane-Bound Enzymes Immobilized in a 5-(Octyldithio)-2-nitrobenzoic Acid Layer on Gold Electrodes

摘要

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB in-cludes a long chain in a short-length thio acid, providing a heterogeneous-like alkanetbiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDII), D-gluconate dehydro-genase (GADH), and L-lactlc dehydrogenase (cytochrome b2) (Cyb2), were immobilized onto ODTNB-modified gold electrodes simply by adsorption. The short-length thio acid may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate enables hydro-phobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been stud-ied with the quartz czystal microbalance (QCM). Spectro-photnmelric and elecirochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modifled gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10-l2~ 5.3 x lO~~ molcm2. A fructose biosensor was devel-oped, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethyl-ferrocene as a mediator in solution. Calibration curves exhibited a linear relation between the biosensor response and the substrate concentration up to 0.7 mM. Statistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM~ fructose. The biosensor shows a significant stable catalytic current for at least 25 days.