Combined fluorophore and phosphor conjugation: a new design concept for simultaneous and spatially localized dual lifetime intracellular sensing of oxygen and pH.

In this communication, we propose a new strategy for double-parametric biosensing and present a dual pH/O lifetime sensor based on the covalent conjugation of fluorescein (pH sensor) and an orthometalated iridium complex (O sensor) to human serum albumin (HSA). The resulting conjugate demonstrates biocompatibility, low toxicity, and fast cellular uptake, and displays independent lifetime responses towards variations in media acidity and oxygen concentration that makes it suitable for application as an effective pH/O probe in luminescence microscopy using the FLIM/PLIM detection mode. The concept applicability has been exemplified using the dual spatially and temporally localized intracellular sensing of pH and O concentration in living cells.