Determination of Organonitriles Using Enzyme-Based Selectivity Mechanisms. 2. A Nitrilase-Modified Glassy Carbon Microelectrode Sensor for Benzonitrile.

Timothy Z. Liu, Yi Wang, Samuel P. Kounaves,* and Edward J. Brush
Center for Field Analytical Studies & Technology, Department of Chemistry, Tufts University, Medford, Massachusetts 02155

Abstract

A nitrilase-modified glassy carbon microelectrode for the detection of benzonitrile in water was developed and analytically characterized. The detection scheme uses Rhodicoccus sp. nitrilase to catalyze the hydrolysis of benzonitrile derivatives directly into carboxylic acid and ammonia. The glassy carbon microelectrode surface modification was accomplished by attaching a hydrophilic tether to the microelectrode surface and then, using the free terminal amine at its other end, biotinylating with sulfo-NHS-biotin. Afterward, avidin was attached to the spacer arm, followed by the biotinylated nitrilase through avidin-biotin coupling. Benzoic acid, an enzymatic reaction product, is electrochemically reduced at the glassy carbon microelectrode, producing a steady-state reduction current proportional to the concentration of benzonitrile in the sample. In the temperature range of 25 to -20 ºC , the biotinylated nitrilase was found to be stable up to 3 times longer than the native enzyme. The activity of the biotinylated nitilase was optimum over a pH range of 7-11, while that of the native enzyme was optimum only between pH 7 and 9. The nitrile sensor was stable for at least 7 days at 25 ºC. It was demonstrated for the detection of benzonitrile at concentrations of 0.1-5 mM in both simple and benzene/chloroform/nitrobenzene containing samples.