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Author(s) |
Kumar, V.; Misra, N.; Paul, J.; Dhanawade, B. R.; Varshney, L. (RTDD)
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Source |
Polymer, 2014. Vol. 55 (11): pp. 2652-2660 |
ABSTRACT
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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, Vmax) revealed the value of Km for covalently immobilized uricase to be ~1.2-fold higher than that of free enzyme. The uricase-immobilized-PAA-g-PP samples could be repeatedly used for w20 cycles over a period of 30 days when stored in phosphate buffer (pH~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. |
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