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-ni trobenzoic 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 dehydrog enase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase ( cytochrome b(2)) (Cyb(2)), were immobilized onto ODTNB-modified gold electr odes simply by adsorption. The short-length thio acid may provide electrost atic interactions with enzyme surface charges, while the alkanethiolate ena bles hydrophobic interaction with the largely lipophilic, membrane-bound en zymes. The immobilization of FDH, GADH, and Cyb(2) onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM), Spec trophotometric and electrochemical assays indicate that the immobilized enz yme retains its enzymatic activity after immobilization onto the ODTNB-modi fied gold surface. The amount of immobilized (and active) enzyme was estima ted from QCM to be of the order of 2.5 x 10-(12)-5.3 x 10(-12) mol cm(-2). A fructose biosensor was developed, making use of a gold surface modified w ith ODTNB and fructose dehydrogenase, employing hydroxymethyl-ferrocene as a mediator in solution, Calibration curves exhibited a linear relation betw een the biosensor response and the substrate concentration up to 0.7 mM, St atistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM(-1) fructose, The biosensor shows a significant stabl e catalytic current for at least 25 days.