A Fluorene based Fluorogenic "Turn-off" Chemosensor for the Recognition of Cu²⁺ and Fe²⁺: Computational Modeling and Living-cell Application

Background: The traditional methods for the detection and quantification of Cu²⁺ and Fe³⁺ heavy metal ions are usually troublesome in terms of high-cost, non-portable, time-consuming, specialized personnel and complicated tools, so their applications in practical analyses is limited. Therefore, the development of cheap, fast and simple-use techniques/instruments with high sensitivity/selectivity for the detection of heavy metal ions is highly demanded and studied. Methods: In this study, a fluorene-based fluorescent ''turn-off'' sensor, methyl 2-(2-((((9H-fluoren-9- yl)methoxy)carbonyl)amino)-3- phenylpropanamido) acetate (probe FLPG) was synthesized via onepot reaction and characterized by ¹H-NMR, ¹³C-APT-NMR, HETCOR, ATR-FTIR and elemental analysis in detailed. All emission spectral studies of the probe FLPG have been performed in CH3CN/HEPES (9/1, v/v, pH=7.4) media at rt. The quantum (φ) yield of probe FLPG decreased considerably in the presence of Cu²⁺ and Fe³⁺. The theoretical computation of probe FLPG and its complexes were also performed using density functional theory (DFT). Furthermore, bio-imaging experiments of the probe FLPG was successfully carried out for Cu²⁺ and Fe³⁺ monitoring in living-cells. Results: The probe FLPG could sense Cu²⁺ and Fe³⁺ with high selectivity and sensitivity, and quantitative correlations (R2>0.9000) between the Cu²⁺/Fe³⁺ concentrations (0.0−10.0 equiv). The limits of detection for Cu²⁺ and Fe³⁺ were found as 25.07 nM and 37.80 nM, respectively. The fluorescence quenching in the sensor is managed by ligand-to-metal charge transfer (LMCT) mechanism. Job’s plot was used to determine the binding stoichiometry (1:2) of the probe FLPG towards Cu²⁺ and Fe³⁺. The binding constants with strongly interacting Cu²⁺ and Fe³⁺ were determined as 4.56×10⁸ M^-2 and 2.02×10³⁺ M^-2, respectively, via the fluorescence titration experiments. The outcomes of the computational study supported the fluorescence data. Moreover, the practical application of the probe FLPG was successfully performed for living cells. Conclusion: This simple chemosensor system offers a highly selective and sensitive sensing platform for the routine detection of Cu²⁺ and Fe³⁺, and it keeps away from the usage of costly and sophisticated analysis systems.