Novel fluorescent C₂-symmetric sequential on-off-on switch for Cu²⁺ and pyrophosphate and its application in monitoring of endogenous alkaline phosphatase activity

A doubly armed hydrazone-based FLRHYDDFP probe selectively detects Cu²⁺ and pyrophosphate (PPi) ions through an colorimetric response-“colorless → yellow → colorless”- as well as “on-off-on” photonic switching response under physiological conditions in a sequential manner. The binding stoichiometries of the analytes Cu²⁺ and PPi were 1:2 and 2:4 for FLRHYDDFP-Cu²⁺ and Cu²⁺/PPi, respectively. The sequential sensing ability of FLRHYDDFP toward Cu²⁺ and PPi, attributed to effective complexation-aided d→π* electron transfer (ET) from Cu²⁺ to FLRHYDDFP and intramolecular charge/electron transfer from FLRHYDDFP to FLRHYDDFP⁺, resulted in the formation of a non-symmetric Cu²⁺ chelate that provided a yellow-colored solution with a significant bathochromic shift from 376 to 446 nm in the UV–vis spectrum and quenching in the emission spectrum. Upon addition of PPi, Cu²⁺ was extruded from the complex, resulting in a revival of the fluorescence centered at 572 nm. Thus, sequential addition of Cu²⁺ and PPi yielded a colorless–yellow–colorless transition under visible light and on-off-on switching under 365-nm light (fluorescence). The lowest detection limits for Cu²⁺ and PPi, when using colorimetric and fluorimetric methods, were in the sub-micromolar and nanomolar levels, respectively. By exploiting this PPi sensing strategy, invitro as well as endogenous alkaline phosphatase activity could be monitored effectively, as demonstrated by exploiting the intracellular production or residual PPi in human salivary glands (normal) and cancer cell lines.