Alginate Microspheres Comprising Multilayered Assemblies of Cresol Red and Polyelectrolytes Towards an Optical Urea Biosensor

An absorbance based enzymatic sensor for the determination of urea has been described. The sensing principle of the present device is based upon the measurement (spectrophotometrically) of the pH change produced in the aqueous environment by the products of the enzyme-catalyzed hydrolysis of urea. Cresol red (CR) dye has been used in the present work to observe the change in the pH produced by the formation of ammonia in the aqueous medium. Immobilization of urease enzyme has been accomplished in the alginate microspheres and multilayer nanofilms of poly (allylamine) hydrochloride (PAH) and sodium polystyrene sulfonate (PSS) polyelectrolytes pre-mixed with the CR dye molecules were assembled on the microspheres using Layer-by-Layer (LbL) self-assembly technique. The size of the microspheres was determined to be in the range of 50-80 mum and the immobilized dye molecules were characterized using fluorescence microscopy. The enzyme activity experiments have been performed with urease in solution as well as in the immobilized form in the microspheres. Thereafter, a calibration curve for different concentrations of urea (0.16-6.7 mM) was prepared and compared with the validation curve obtained for urease immobilized particles along with cresol red in the multilayers. Urea sensing results demonstrate that per mM increase in the urea concentration corresponds to an increase of ~1.38 absorbance ratio units.