Uricase-immobilization on radiation grafted polymer support for detection of uric acid using Ag-nanoparticle based optical biosensor

A functional polymer support was designed for covalent immobilization of enzyme uricase, which was further utilized in a single enzyme based silver nanoparticles (Ag-NPs)-optical biosensor system for estimation of uric acid. A carboxylic acid functionalized polymer support was fabricated via gamma radiation induced mutual-irradiation grafting of polyacrylic acid (PAA) on to non-woven polypropylene (PP) matrix. Grafting parameters, such as dose, monomer concentration, homopolymer inhibitor and inorganic acid concentration were studied to optimize the grafting process. PAA-g-PP samples were characterized by grafting yield measurement, water uptake, FTIR, TGA and SEM techniques. Uricase enzyme was immobilized on to the PAA-g-PP support via covalent amide linkage using coupling agents, namely N-hydroxysuccinimide (NHS) and N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC). The catalytic activity of uricase was investigated spectrophotometrically using uric acid as a substrate. Study of kinetic parameters (Km. V,,,x) revealed the value of Km for covalently immobilized uricase to be similar to 1.2-fold higher than that of free enzyme. The uricase-immobilized-PAA-g-PP samples could be repeatedly used for 20 cycles over a period of 30 days when stored in phosphate buffer (pH similar to 7.4) at 4 C without significant decrease in the enzyme activity. The uricase immobilized samples were successfully used in conjunction with the radiolytically synthesized Ag-NPs as a LSPR-optical biosensor for estimation of uric acid. (C) 2014 Elsevier Ltd. All rights reserved.