Simultaneous Monitoring of Cytochrome c and Caspase-3 Dynamics Using a Dual-Channel Nanoprobe for Deciphering Single-Cell Apoptosis

Unraveling the precise kinetics of the intrinsic apoptotic pathway requires resolution of the temporal lag between mitochondrial permeabilization and downstream proteolytic execution. However, dissecting these distinct molecular events within the crowded cytosolic environment of a single live cell remains limited by the lack of nonperturbative, multiplexed sensing tools. Here, we report a dual-channel nanoprobe capable of label-free, simultaneous monitoring of Cytochrome c (Cyt c) translocation and Caspase-3 activation. The nanoprobe features physically isolated nanochannels allowing for orthogonal functionalization: one channel acts as an aptamer-based sensor for Cyt c, while the second functions via the enzymatic cleavage of a specific peptide substrate by Caspase-3. This design enables the synchronous transduction of protein binding and enzymatic activity into independent current signals. The probe exhibited high sensitivity, selectivity, and stability in complex biological fluids. We applied this platform to monitor the dynamics of Cyt c and caspase-3 in breast cancer cells, revealing significant heterogeneity in apoptosis and a spatiotemporal delay between Cyt c release and caspase-3 activation, consistent with the intrinsic apoptosis pathway. This dual-detection methodology provides a minimally invasive approach with high spatial and temporal resolution for probing apoptotic signaling at the single-cell level and enables quantitative assessment of heterogeneous cellular responses to apoptotic stimuli.