Pyrene-Based Fluorescent Probe for “Off-on-Off” Sequential Detection of Cu²⁺ and CN⁻ with HeLa Cells Imaging

The novel pyrene-appended Schiff base probe L with aggregation-induced emissions (AIE) relevant to an increase in water fractions (0–90%) is synthesized and applied in sequentially detecting Cu²⁺ and CN⁻. The pyrene-based probe L firstly induces the excimer formation in the presence of Cu²⁺. However, the process can be reversed by sequentially adding CN⁻, which is demonstrated using the fluorescence “Off-On-Off” response in semi-aqueous media ethanol water (v/v = 7/3) under physiological pH (5 mM HEPES, pH 7.0). The Job’s plot, mass analysis, ¹H NMR titrations, and density functional theory (DFT) interrogations confirm the 2:1 stoichiometry of excimer complex L–Cu²⁺-L*, preferential binding atoms, and CN⁻ tuned complex reversibility. Based on the photoluminescence (PL) titration, the association constant of L to Cu²⁺ is determined as 4.95 × 10⁶ M⁻¹. From standard deviation and linear fittings, the detection limits (LODs) of Cu²⁺ and CN⁻ are estimated as 219 nM and 580 nM, respectively. The practicality of Cu²⁺ and CN⁻ detection is demonstrated using a TLC plate and a blended polymer membrane through which significant color changes under a UV lamp can be monitored. Moreover, utility of the designed probe L towards biological application with low toxicity is demonstrated by detecting Cu²⁺ and CN⁻ inside HeLa cells. The responses of the probe to Cu(II) ions were also verified using living HeLa cells imaging.