N-hydroxysuccinimide ester functionalized perfluorophenyl azides as novel photoactive heterobifunctional cross-linking reagents. The covalent immobilization of biomolecules to polymer surfaces.

The synthesis of N-hydroxysuccinimide (NHS) functionalized perfluorophenyl azides (PFPAs) 2 and 3 is described together with a general method for the covalent modification of polymer surfaces using heterobifunctional, photoactivable cross-linking reagents 1 and 3. The NHS-active ester group becomes covalently attached to the polymer surface via an efficient CH bond insertion reaction of the photogenerated, highly reactive nitrene intermediate derived from the PFPA. The NHS ester is capable of further reaction with a variety of primary amine-containing reagents including biomolecules by way of amide formation. The method is illustrated as follows. Photolysis of polystyrene (PS) and poly-(3-octylthiophene) (P3OT) thin films spin-coated with NHS PFPA ester 1 or 3 gave films 8 or 9, respectively. Each film was then exposed to an aqueous solution of horseradish peroxidase (HRP), giving films 10 or 11, respectively. The amounts of HRP immobilized on PS and P3OT were calculated from enzyme activity assays to be 0.5 +/- 0.1 ng/mm2 for 10a, 1.0 +/- 0.2 ng/mm2 for 11a, 0.2 +/- 0.1 ng/mm2 for 10b, 0.3 +/- 0.1 ng/mm2 for 11b. Using this surface functionalization methodology, biotin-streptavidin-biotin-HRP was constructed on the PS film. The storage stability of HRP thus immobilized through the extended linker, biotin-streptavidin-biotin, was enhanced as compared to that of HRP directly immobilized on the PS surface.