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

Abstract

This study presents a homogeneous electrochemiluminescence (ECL) biosensor for ultrasensitive detection of HEK293 cells by targeting the hERG potassium channel at the single-cell level. The biosensor integrates multiple signal amplification steps, including photocleavable DNA–antibody conjugates, entropy-driven strand displacement, T7 RNA polymerase-mediated transcription, and CRISPR/Cas12a-mediated trans-cleavage. This cascade enables precise and robust signal enhancement. A key feature of the system is its ability to generate a clearly measurable ECL response from as little as a single HEK293 cell, without the need for signal averaging. Specificity was confirmed using unrelated cell lines and mismatched DNA sequences, with minimal background observed in negative controls. Optimization of key parameters—such as enzyme concentrations, reaction times, and duplex composition—ensured consistent and reproducible performance. ECL measurements were conducted under a defined voltage scan (0–1.3 V) in a three-electrode system, and emission signals were recorded using a photomultiplier tube. This biosensor demonstrates the feasibility of single-cell detection with high sensitivity and specificity, offering a promising platform for future applications in cell-based analysis and molecular diagnostics.