Biosensors Based on Membrane-Bound Enzymes Immobilized in a 5-(Octyldithio)-2-nitrobenzoic Acid Layer on Gold Electrodes
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- 作者：M. Darder ; E. Casero ; F. Pariente ; and E. Lorenzo
- 刊名：Analytical Chemistry
- 出版年：2000
- 出版时间：August 15, 2000
- 年：2000
- 卷：72
- 期：16
- 页码：3784 - 3792
- 全文大小：182K
- 年卷期：v.72,no.16(August 15, 2000)
- ISSN：1520-6882
文摘
Gold electrodes were modified through chemisorption of5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providinga heterogeneous-like alkanethiol layer after adsorption ongold electrodes. Membrane-bound enzymes, in particularD-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase (cytochromeb2) (Cyb2), were immobilized onto ODTNB-modified goldelectrodes simply by adsorption. The short-length thioacid may provide electrostatic interactions with enzymesurface charges, while the alkanethiolate enables hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, andCyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that theimmobilized enzyme retains its enzymatic activity afterimmobilization onto the ODTNB-modified gold surface.The amount of immobilized (and active) enzyme wasestimated from QCM to be of the order of 2.5 × 10-12-5.3 × 10-12 mol·cm-2. A fructose biosensor was developed, making use of a gold surface modified with ODTNBand fructose dehydrogenase, employing hydroxymethylferrocene as a mediator in solution. Calibration curvesexhibited a linear relation between the biosensor responseand the substrate concentration up to 0.7 mM. Statisticalanalysis gave an excellent linear correlation (r = 0.9993)and a sensitivity of 6.1 mM-1 fructose. The biosensorshows a significant stable catalytic current for at least 25days.