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

Xinhao Li1, Weikang Hu1, Yao Huang1

  • 1Shenzhen Key Laboratory of Biomimetic Robotics and Intelligent Systems, Department of Mechanical and Energy Engineering, Southern University of Science and Technology, Shenzhen 518000, China.

ACS Nano
|March 24, 2026
PubMed
Summary

Researchers developed a novel dual-channel nanoprobe to simultaneously track Cytochrome c (Cyt c) release and Caspase-3 activation, revealing delays in apoptosis signaling within single breast cancer cells.

Keywords:
apoptosiscaspase-3cytochrome csingle-cell analysisspatiotemporal dynamics

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Area of Science:

  • Biotechnology
  • Cell Biology
  • Molecular Biology

Background:

  • Understanding the intrinsic apoptotic pathway is crucial for cancer research.
  • Current methods struggle to monitor key apoptosis events like mitochondrial permeabilization and caspase activation in real-time within single cells.
  • There is a need for nonperturbative, multiplexed sensing tools to dissect the kinetics of apoptosis.

Purpose of the Study:

  • To develop a novel dual-channel nanoprobe for label-free, simultaneous monitoring of Cytochrome c (Cyt c) translocation and Caspase-3 activation.
  • To investigate the spatiotemporal dynamics of apoptosis in breast cancer cells at the single-cell level.
  • To provide a tool for quantitative assessment of heterogeneous cellular responses to apoptotic stimuli.

Main Methods:

  • A dual-channel nanoprobe with physically isolated nanochannels was designed for orthogonal functionalization.
  • One channel utilized aptamers for Cytochrome c sensing; the second used a peptide substrate for Caspase-3 activity detection.
  • The probe transduced protein binding and enzymatic activity into independent current signals for simultaneous monitoring.

Main Results:

  • The nanoprobe demonstrated high sensitivity, selectivity, and stability in biological fluids.
  • Application in breast cancer cells revealed significant heterogeneity in apoptosis.
  • A spatiotemporal delay was observed between Cytochrome c release and Caspase-3 activation, consistent with intrinsic apoptosis.

Conclusions:

  • The developed dual-detection nanoprobe offers a minimally invasive approach for probing apoptotic signaling with high spatial and temporal resolution.
  • This methodology enables quantitative assessment of heterogeneous cellular responses in apoptosis.
  • The findings provide insights into the precise kinetics of the intrinsic apoptotic pathway at the single-cell level.