Light-induced activation of an inert surface for covalent immobilization of a protein ligand

A simple and mild procedure is developed for the preparation of an activated polymer surface, used for immobilization of a protein ligand through a covalent linkage. Activation of the polymer surface is carried out by attaching an active functional group through 1-fluoro-2-nitro-4-azidobenzene (FNAB). UV irradiation of FNAB transforms its azido group into a highly reactive nitrene, which binds with the inert polymer surface, whereas the active fluoro group of FNAB, now part of the polymer, remains intact. Covalent linkage between the ligand and the inert surface is established through this active fluoro group in a thermochemical reaction. The photochemical step is carried out under dry conditions to exclude the possibility of undesirable reactions between the solvent and the highly reactive nitrene. The method can be used for activation of different inert polymer surfaces having carbon hydrogen bonds. The efficacy of our method is demonstrated by immobilizing horseradish peroxidase on an activated polystyrene surface. The enzyme, immobilized through the photolinker, is found to give a twofold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. The method may have many applications in the preparation of bioreactors, biostrips, and biosensors, and in diagnostic tests involving the ELISA technique.