Sensitivity enhancement of fluorescent pH indicators using pH-dependent energy transfer

The concept of energy transfer is examined, in view of its application to enhance pH sensitivity of fluorescent dyes. Two fluorescent pH sensitive indicators are chosen such that they have nearly the same pKa values and have maximum spectral overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor. In this work we studied the pH dependent energy transfer between 7-hydroxy-4-methylcoumarin-3-acetic acid (HCA) as the donor with fluorescein and 5- (and 6-) carboxy-4ʹ,5ʹ-dimethyl fluorescein (CDF) as the acceptors. The extent of energy transfer was measured experimentally by following the increase in acceptor emission at different pH values. The sensitivity of the acceptor dye is almost double when excited via energy transfer, provided the donor emission itself exhibits a similar dependence on pH. The pH sensitivity of fluorescein (ΔIΔPH), was observed to increase from 0.28 when excited directly, to 0.71 when excited via energy transfer. CDF also shows a similar increase from 0.49 to 0.71.