Biosensors based on membrane-bound enzymes immobilized in a 5-(octyldithio)-2-nitrobenzoic acid layer on gold electrodes

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providing a heterogeneous-like alkanethiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic 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 hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modified gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10(-12)-5.3 x 10(-12) mol x cm(-2). A fructose biosensor was developed, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethylferrocene 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(-1) fructose. The biosensor shows a significant stable catalytic current for at least 25 days.