DETERMINATION OF ORGANONITRILES USING ENZYME-BASED SELECTIVITY MECHANISMS .2. A NITRILASE-MODIFIED GLASSY-CARBON MICROELECTRODE SENSOR FOR BENZONITRILE

A nitrilase-modified glassy carbon microelectrode for the detection of benzonitrile in water was developed and analytically characterized, T he detection scheme uses Rhodococcus sp, nitrilase to catalyze the hyd rolysis of benzonitrile derivatives directly into carboxylic acid and ammonia, The glassy carbon microelectrode surface modification was acc omplished by attaching a hydrophilic tether to the microelectrode surf ace and then, using the free terminal amine at its other end, biotinyl ating with sulfo-NHS-biotin, Afterward, avidin was attached to the spa cer arm, followed by the biotinylated nitrilase through avidin-biotin coupling, Benzoic acid, an enzymatic reaction product, is electrochemi cally reduced at the glassy carbon microelectrode, producing a steady- state reduction current proportional to the concentration of benzonitr ile in the sample, In the temperature range of 25 to -20 degrees C, th e biotinylated nitrilase was found to be stable up to 3 times longer t han the native enzyme, The activity of the biotinylated nitrilase was optimum over a pH range of 7-11, while that of the native enzyme was o ptimum only between pH 7 and 9. The nitrile sensor was stable for at l east 7 days at 25 degrees C. It was demonstrated for the detection of- benzonitrile at concentrations of 0.1-5 mM in both simple and benzene/ chloroform/nitrobenzene containing samples.