Immobilization of streptavidin-horseradish peroxidase onto a biotinylated poly(acrylic acid) backbone that had been radiation-grafted to a PTFE film

Abstract

Films of polytetrafluoroethylene (PTFE) were modified by radiation graft polymerization of acrylic acid (AAc). Optimal conditions for efficient AAc grafting were studied, including pre-irradiation dose in air, monomer concentration, temperature and time of the grafting process. Carboxylic groups of the grafted polyAAc were activated with carbodiimide (EDC) for biotinylation by reaction with 5-(biotinamido) pentylamine. Streptavidin-horseradish peroxidase (SA-HRP) was immobilized by affinity complexation of the SA with the biotin groups on the PTFE surface. The amount of active HRP immobilized on the PTFE films was determined as a function of the extent of polyAAc grafting. This study has demonstrated the utility of combining the processes of (a) radiation grafting of polymers with reactive groups onto inert polymers such as PTFE, (b) biotinylation of the graft polymer reactive groups, (c) immobilization of streptavidin on the biotinylated surface sites, followed by (d) immobilization of biotinylated, biologically active molecules via complexation of their conjugates with streptavidin. In this study, the last two steps were combined by immobilizing the complex of streptavidin and biotinylated HRP onto the biotinylated surface sites. The unique nature of this process is the ability to immobilize biotinylated molecules on an inert surface as PTFE.

Keywords:

• RADIATION GRAFT POLYMERIZATION
• PTFE
• ACRYLIC ACID
• STREPTAVIDIN-HORSERADISH PEROXIDASE