NCPA-templated red-emitting gold nanoclusters: a turn-off–on fluorescent probe for rapid and selective detection of Cu²⁺ ions in live cells and evaluation of anticancer activity

Red-emitting gold nanoclusters (AuNCs) have gained significant attention as promising fluorescent probes for chemical and biomedical applications due to their excellent optical properties and inherent biocompatibility. In this study, a turn-OFF-ON red-emitting AuNC fluorescent probe was synthesized using <i>N</i>-cholyl D-penicillamine (NCPA) as a stabilizing and templating agent above its critical micellar concentration (CMC) under UV light irradiation. The developed synthesis protocol was simple, cost-effective, and produced highly water-soluble AuNCs exhibiting an emission maximum at 664 nm with a large Stokes shift of 294 nm. The average fluorescence lifetime and quantum yield of the AuNCs were determined to be 116.49 ns and 3.26%, respectively. High-resolution transmission electron microscopy (HR-TEM) confirmed the formation of monodispersed nanoclusters with an average core size of 1.94 ± 0.4 nm. The synthesized AuNCs demonstrated excellent selectivity and sensitivity towards copper ions (Cu²⁺), with a detection limit as low as 6.9 nM, based on fluorescence quenching. Notably, the quenched fluorescence of the NCPA@AuNCs-Cu²⁺ complex could be effectively restored (∼94%) upon the addition of ethylenediaminetetraacetic acid (EDTA), enabling a reversible OFF-ON sensing mechanism. Furthermore, the NCPA@AuNCs exhibited potent cytotoxicity against MCF-7 and HeLa cancer cell lines. In an <i>in vitro</i> cellular environment, the quenched fluorescence of the NCPA@AuNCs-Cu²⁺ complex could also be restored by the EDTA treatment, demonstrating the potential of this system for intracellular Cu²⁺ sensing and live-cell imaging. Overall, the developed NCPA-stabilized red-emitting AuNCs offer an efficient, reversible fluorescent sensing platform for Cu²⁺ ions and exhibit significant potential for cancer cell imaging and therapeutic applications.