Naphthopyran-based photoswitching for simpler chemical sensing and imaging using phase-sensitive mode

Intensity-based fluorescence imaging suffers from spectral overlap and optical background interference. As an alternative, fluorescence lifetime measurements on the nanosecond level are also largely constrained. Herein, we propose phase-sensitive detection of photoswitchable probes containing naphthopyran and fluorescent donors. The method features reaction kinetics in the millisecond-to-second regime, allowing frequency domain detection with cost-effective equipment. A phase shift (Δϕ) in the fluorescence of the probes is extracted by fast Fourier transform, establishing a dependence on the molar ratio of donor to acceptor. Thus, Δϕ is proposed as a self-referencing quantity for selective highlighting of fluorescent probes and a dynamic signal readout in chemical sensing. Phase-sensitive detection of protamine, a polycationic protein often used as a neutralizer of the anticoagulant heparin during surgery, is successfully realized based on the platform.