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A high-sensitivity ECL biosensor for single-cell analysis: Integrating CRISPR/Cas12a and entropy-driven

Jiaying Wang1, Yu Tian1, Kai Zhang2

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Bioelectrochemistry (Amsterdam, Netherlands)
|July 15, 2025
PubMed
Summary

This study developed a novel electrochemiluminescence (ECL) biosensor for detecting single HEK293 cells. The biosensor uses multiple amplification steps for ultrasensitive and specific single-cell analysis.

Keywords:
CRISPR/Cas12a biosensor，single-cell detection，electrochemical sensing，biomarker monitoring

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Area of Science:

  • Biomedical Engineering
  • Molecular Diagnostics
  • Cell Biology

Background:

  • Single-cell analysis is crucial for understanding cellular heterogeneity and disease mechanisms.
  • Existing methods often lack the sensitivity or specificity for reliable single-cell detection.
  • Targeting specific biomarkers like the hERG potassium channel offers a route to precise cell identification.

Purpose of the Study:

  • To develop a homogeneous electrochemiluminescence (ECL) biosensor for ultrasensitive, single-cell detection of HEK293 cells.
  • To achieve high sensitivity and specificity by integrating multiple signal amplification strategies.
  • To validate the biosensor's performance in detecting single cells without signal averaging.

Main Methods:

  • Fabrication of a homogeneous ECL biosensor utilizing photocleavable DNA-antibody conjugates.
  • Integration of entropy-driven strand displacement, T7 RNA polymerase transcription, and CRISPR/Cas12a trans-cleavage for signal cascade amplification.
  • Detection of HEK293 cells by targeting the hERG potassium channel at the single-cell level.
  • Optimization of reaction parameters including enzyme concentrations and DNA duplex composition.

Main Results:

  • The biosensor achieved ultrasensitive detection of single HEK293 cells, generating a measurable ECL signal from just one cell.
  • High specificity was demonstrated against unrelated cell lines and mismatched DNA sequences, with negligible background noise.
  • Optimized parameters ensured consistent and reproducible ECL responses, validating the multi-step amplification cascade.

Conclusions:

  • The developed ECL biosensor enables highly sensitive and specific detection of individual HEK293 cells.
  • This platform showcases the potential of integrated amplification strategies for single-cell analysis.
  • The biosensor represents a promising tool for advancing cell-based assays and molecular diagnostics.