Sequential Turn-on/off Aggregation-Induced Emission Nanoprobe Based on Salicylaldehyde Azine for Detection of Copper(II) and Sulfide Ions

To address the limitations of conventional fluorescent probes, including aggregation-caused quenching (ACQ) and poor water solubility, an intelligent nanoprobe (LPNPs@SA) with tumor microenvironment responsiveness and aggregation-induced emission (AIE) properties was developed. This nanoprobe enables sequential, reversible, and highly sensitive detection of Cu²⁺ and S^2-. The AIE-active fluorophore salicylaldehyde azine (SA) was designed and synthesized, exhibiting intense emission in aggregated states but poor stability under non-neutral pH conditions. To overcome this limitation, SA was encapsulated into lipid-polymer hybrid nanoparticles (LPNPs) via self-assembly using an amphiphilic copolymer (C₁₆-SS-PEG/DMMA), polycaprolactone (PCL), and phosphatidylcholine (PC). The resulting LPNPs@SA nanoprobe possessed favorable storage stability, broad pH tolerance, effective surface charge conversion, and good hemocompatibility, with a hemolysis rate below 5%. In terms of sensing performance, the nanoprobe showed selective fluorescence quenching ("turn-off" mode) toward Cu²⁺. Stern-Volmer analysis revealed a quenching constant (<i>K</i>_sv) of 2.9 × 10⁵ L/mol. Further fluorescence titration and Job's plot analyses confirmed a 1:1 binding stoichiometry between the nanoprobe and Cu²⁺, with an association constant (<i>K</i>_a) of 2.2 × 10⁴ L/mol. The detection limit (LOD) for Cu²⁺ was determined to be 1.2 μmol/L, demonstrating high sensitivity. Remarkably, upon introducing S^2- into the copper-loaded nanoprobe system, substantial fluorescence recovery was observed, yielding a distinct "turn-on" signal for S^2- detection. In summary, this work presents a biocompatible nanoprobe with excellent optical properties that functions as an effective reversible fluorescence-switching platform. It offers a promising analytical tool for dynamically monitoring Cu²⁺ and S^2- in biological environments, facilitating studies of related pathological processes and potential diagnostic applications.