Energy Transfer Between i-Motif DNA Encapsulated Silver Nanoclusters and Fluorescein Amidite Efficiently Visualizes the Redox State of Live Cells

The redox regulation, maintaining a balance between oxidation and reduction in living cells, is vital for cellular homeostasis, intricate signaling networks, and appropriate responses to physiological and environmental cues. Here, a novel redox sensor, based on DNA-encapsulated silver nanoclusters (DNA/AgNCs) and well-defined chemical fluorophores, effectively illustrating cellular redox states in live cells is introduced. Among various i-motif DNAs, the photophysical property of poly-cytosines (C₂₀)-encapsulated AgNCs that sense reactive oxygen species (ROS) is adopted. However, the sensitivity of C₂₀/AgNCs is insufficient for evaluating ROS levels in live cells. To overcome this drawback, the ROS sensing mechanism of C₂₀/AgNCs through gel electrophoresis, mass spectrometry, and small-angle X-ray scattering is primarily defined. Then, by tethering fluorescein amidite (FAM) and Cyanine 5 (Cy5) dyes to each end of the C₂₀/AgNCs sensor, an Energy Transfer (ET) between AgNCs and FAM is achieved, resulting in intensified green fluorescence upon ROS detection. Taken together, the FAM-C₂₀/AgNCs-Cy5 redox sensor enables dynamic visualization of intracellular redox states, yielding insights into oxidative stress-related processes in live cells.