Immobilization of urease by laser techniques: synthesis and application to urea biosensors

Urease thin films have been immobilized using matrix-assisted pulsed laser evaporation for biosensor applications in clinical diagnostics. The targets exposed to laser radiation were made of frozen composites that had been manufactured by dissolving urease in distilled water. An UV KrF* (lambda = 248 nm, tauFWHM congruent with 30 ns, nu = 10 Hz) excimer source was used for the multipulse laser irradiation of the targets that were cooled down to solidification using Peltier elements. The incident laser fluence was set at 0.4 J/cm2. The surface morphology and chemical bonding states of the laser immobilized urease thin films were investigated by atomic force microscopy and Fourier transform infrared spectroscopy. The enzymatic activity and kinetics of the immobilized urease were assayed by the Worthington method, which monitors urea hydrolysis by coupling ammonia production to a glutamate dehydrogenase reaction. Decreased absorbance was found at 340 nm and correlated with the enzymatic activity of urease.