The influence of labelling mechanisms on the fluorescence behaviour of polymers bearing fluorescein labels

The labelling of macromolecules by fluorescent labels is a well established method for studying their motion, location and environment. There are a wide range of labels and methods for attaching these to polymers, and the resulting fluorescence behaviour can depend on both the label and labelling method. Fluorescein is a commonly used label because of its relative ecological safety and its pH dependence. Various methods for attaching fluorescein (via its amine derivative) and eosin to water soluble polymers were investigated, together with the effect on fluorescence behaviour. For fluoresceinamine, it was necessary to prevent the lone pair on the nitrogen from quenching the fluorescence by using an amide (via Woodwards reagent) or isothiocyanate as the linking group between the label and macromolecule. This also largely preserved the well known pH dependence of the fluorescence of fluorescein. Eosin (tetra bromo fluorescein) provides a constant fluorescent intensity over a wider pH range than fluorescein and can be linked to macromolecules via the carboxyl group without significantly influencing this pH stability. (C) 1997 Elsevier Science Ltd.