Tracker Nanocatalyst for Screening of Intracellular Copper-Catalyzed Azide-Alkyne Cycloadditions.

Intracellular copper-catalyzed azide-alkyne cycloaddition (CuAAC) offers immense potential for bioorthogonal chemistry, but its application is severely hindered by copper toxicity and the challenge of controlling catalysis within the complex cellular environment. Heterogeneous copper catalysts can reduce toxicity by minimizing free copper exposure and enabling localized activity, yet optimizing their performance in situ within living cells remains a significant hurdle. Here, the development of a novel dual-functional nanocatalyst, Cu@BTTAA-Cy5-NPs, that combines robust heterogeneous CuAAC catalytic activity with intrinsic fluorescence tracking is reported. The successful synthesis and characterization of these monodispersed nanoparticles is demonstrated, confirming efficient copper loading stabilized by BTTAA and the nanoparticle matrix, and critically, the retention of Cy5 fluorescence for tracking. This unique dual functionality allows for real-time monitoring of nanoparticle localization and correlation with catalytic product formation via distinct fluorescence channels, enabling, for the first time to our knowledge, comprehensive in situ screening and optimization of CuAAC reaction conditions directly within living cells using fluorescence feedback. The nanoparticles exhibit excellent biocompatibility and cellular uptake, showing no significant toxicity, apoptosis, or oxidative stress at active concentrations.