[Show abstract][Hide abstract] ABSTRACT: Urease thin films were produced by Matrix Assisted Pulsed Laser Evaporation (MAPLE) and Pulsed Laser Deposition from two types of targets: frozen water solutions of urease with different concentrations (1–10% m/v) and pure urease pellets. The fluence of the ablating KrF excimer laser was varied between 300 and 2200 mJ/cm2. Fourier transform infrared spectra of the deposited films showed no difference as compared to the original urease. Morphologic studies proved that the films consist of a smooth “base” layer with embedded micrometer-sized droplets. Absorption-coefficient measurements contradicted the traditional “absorptive matrix” model for MAPLE deposition. The laser energy was absorbed by urease clusters leading to a local heating-up and evaporation of the frozen matrix from the uppermost layer accompanied by the release of dissolved urease molecules. Significant enzymatic activity of urease was preserved only during matrix assisted transfer.
[Show abstract][Hide abstract] ABSTRACT: 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.
No preview · Article · Jan 2008 · Journal of Biomedical Materials Research Part A