A fluorescent nanoprobe based on copper nanoclusters (CuNCs) has been developed for ratiometric detection of hydroxyl radicals (•OH) and superoxide anion radicals (O2•-). Two differently luminescent CuNCs, namely cyan-emissive poly(methacrylic acid)-protected copper nanoclusters (PCuNCs) and orange-emissive bovine serum albumin-protected CuNCs (BCuNCs), were conjugated to obtain a hybrid, dual-emission nanoprobe (PCuNCs-BCuNCs) with the corresponding peaks at 445 nm and 652 nm at an excitation wavelength of 360 nm. In particular, the fluorescence peak at 445 nm gradually enhanced with the incremental addition of •OH and O2•-. However, the fluorescence emission at 652 nm was greatly quenched in the presence of •OH, while in case of O2•-, the fluorescence intensity remained constant. The differential response of the PCuNCs-BCuNCs towards •OH and O2•- formed the basis of ratiometric detection. Under optimal conditions, the PCuNCs-BCuNCs exhibited good sensitivity and linearity towards •OH and O2•- with limits of detection of 0.15 μM and 1.8 μM, respectively. Moreover, the nanoprobe exhibited high selectivity for •OH and O2•- over other potential ROS interferences. Besides, PCuNCs-BCuNCs were eventually applied for qualitative and quantitative ratiometric assessment of intracellular •OH and O2•- in L-132 cells. Therefore, this strategy unveils a new potential for copper nanocluster-based sensing of ROS.
Keywords: Copper nanoclusters; Fluorescence; Intracellular cell analysis; Nanoprobe; ROS; Ratiometric detection.