Biocompatible β-carboline–derived fluorescent probes for nanomolar detection and intracellular Zn²⁺ imaging

Two novel β-carboline–based fluorescent probes (P1 and P2) were synthesized through a concise, high-yielding route for the selective detection of Zn²⁺ ions. Structural and photophysical analyses revealed that both probes exhibit remarkable sensitivity and selectivity toward Zn²⁺ over competing metal ions, showing fluorescence “turn-on” responses of 5.6-fold (P1) and 10.5-fold (P2). The detection limits were determined to be 27.2 nM and 55.1 nM, respectively. Both probes demonstrated excellent anti-interference properties and operated optimally under physiological pH (7.4). DFT and TD-DFT calculations revealed that both probes form stable hexacoordinate Zn²⁺ complexes, with Zn²⁺ binding leading to a reduction in the HOMO–LUMO gap and orbital stabilization. These results support a photoinduced electron transfer (PET) inhibition mechanism, explaining the fluorescence “turn-on” response and the stronger Zn²⁺ affinity of P2. Moreover, the ¹H NMR spectra provided evidence for the formation of probe/Zn²⁺ complexes, as distinct differences were observed between the free probes and their complexed forms. The probes also exhibited low cytotoxicity and high biocompatibility, enabling their successful application in monitoring Zn²⁺ in WiDr cells using confocal fluorescence microscopy.