A Near-Infrared Fluorescent Probe Based on a FRET Rhodamine Donor Linked to a Cyanine Acceptor for Sensitive Detection of Intracellular pH Alternations.

A fluorescence resonance energy transfer (FRET)-based near-infrared fluorescent probe (B⁺) for double-checked sensitive detection of intracellular pH changes has been synthesized by binding a near-infrared rhodamine donor to a near-infrared cyanine acceptor through robust C-N bonds via a nucleophilic substitution reaction. To demonstrate the double-checked advantages of probe B⁺, a near-infrared probe (A) was also prepared by modification of a near-infrared rhodamine dye with ethylenediamine to produce a closed spirolactam residue. Under basic conditions, probe B⁺ shows only weak fluorescence from the cyanine acceptor while probe A displays nonfluorescence due to retention of the closed spirolactam form of the rhodamine moiety. Upon decrease in solution pH level, probe B⁺ exhibits a gradual fluorescence increase from rhodamine and cyanine constituents at 623 nm and 743 nm respectively, whereas probe A displays fluorescence increase at 623 nm on the rhodamine moiety as acidic conditions leads to the rupture of the probe spirolactam rings. Probes A and B⁺ have successfully been used to monitor intracellular pH alternations and possess pKa values of 5.15 and 7.80, respectively.