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.
Darder
M
M
Departamento de Química Analítica y Análisis Instrumental, Universidad Autónoma de Madrid, Spain.
Casero
E
E
Pariente
F
F
Lorenzo
E
E
D015374:Biosensing Techniques; D004566:Electrodes; D004800:Enzymes, Immobilized / Q000737:chemistry*; D006046:Gold / Q000737:chemistry*; D008567:Membranes, Artificial; D009579:Nitrobenzoates / Q000737:chemistry*
C413244:5-(octyldithio)-2-nitrobenzoic acid; D004800:Enzymes, Immobilized; D008567:Membranes, Artificial; D009579:Nitrobenzoates; D006046:Gold