A benzocoumarin based two-photon fluorescent probe for ratiometric detection of bisulfite

Bisulfite, an equilibrium species with sulfite, is generated from endogenous sulfur dioxide in cell from biothiols. At its elevated level, bisulfite can cause several physiological disorder and diseases. To study such "bisulfite biology" in cell and in tissue, a two-photon fluorescent probe with ratiometric imaging capability is in great demand. To this end, we investigated an aryl aldehyde-type probe derived from a dipolar benzocoumarin dye. The probe sensed bisulfite through formation of the corresponding aldehyde-bisulfite adduct in aqueous buffer at pH 7.4, with excellent selectivity over other biologically relevant species and with well-resolved ratiometric fluorescence changes from 625 nm to 552 nm. The high sensitivity (LOD=0.08 mu M) and reactivity (within 30 s) of the probe towards bisulfite enabled us to quantify an endogenous bisulfite level present in human serum. The promising in vitro ratiometric behavior, however, was deteriorated in the cellular environment, owing to large red-edge and blue-shift effects, observed for both the probe and its bisulfite adduct in cell, which occurred into the unfavorable direction. Thus the cellular imaging with the probe was confined to observe the bisulfite level changes, using one-photon as well as two-photon microscopy.