Fiber-optic ethanol sensor using alcohol dehydrogenase-immobilized Langmuir-Blodgett film

Abstract

A fiber-optic biosensor using alcohol dehydrogenase (ADH)-immobilized Langmuir-Blodgett (LB) films is developed for the measurement of ethanol concentration. The enzyme was immobilized on the lipid monolayers by adsorption from the aqueous subphase solution. A decrease of affinities to ethanol and NAD in ADH-immobilized LB films is observed compared with those of free enzyme based on ordered multisubstrate kinetics. From the measurement of the surface pressure-area isotherm and retained enzyme activity for the four types of lipids, we determined that negatively charged arachidic acid is the most suitable for adsorption of ADH, based on its electrostatic force as well as hydrophobicity, compared to other lipids. The adsorption isotherm pf ADH on the lipid monolayer is a Langmuir isotherm, which indicates that the extent of ADH coverage on the Lipid layer is less than one complete monolayer. The sensor output shows a linear relationship with ethanol concentration up to 40 mM when the ADH-immobilized LB film has 20 layers. As the number of ADH-immobilized LB filmlayers is increased to 20, the linear response range increases. However, the increase in output signal intensity is not proportional to the increase in the number of ADH-immobilized LB films and the increase in the detection range is limited when the ADH-immobilized LB film has 20 layers.