Systematic study of fluorescein-functionalized macrophotoinitiators for colorimetric bioassays

We report a systematic investigation of a set of photoreducible macrophotoinitiators for use in polymerization-based signal amplification. To test the dependence of photopolymerization responses on the number of photoinitiators localized per molecular recognition event, we gradually increased the number of photoinitiator molecules coupled to a constant scaffold macromolecule from an average of 7 per polymer to an average of 168 per polymer. To evaluate the capacity of the macrophotoinitiators to detect molecular recognition, we coupled neutravidin to these molecules to recognize biotin-labeled DNA immobilized on biochip test surfaces. Fluorescein macroinitiators were found to be useful in detecting molecular recognition above a threshold number of initiators per polymer. Above this threshold, increasing the number of initiators per macroinitiator resulted in increased signal strength. These findings demonstrate the feasibility of increasing the number of photoreducible initiators per binding event beyond three, the number used in previous studies, that the initiation reaction remains limiting in the range we investigated, and that the number of initiators per binding event in this system has a clear impact on assay sensitivity and signal strength.