A Proximal-Guaranteed Dual-Marker Electrochemiluminescence System for Accurate Identification of Colorectal Cancer Cell Subpopulations

Accurate identification of cancer cell subgroups is a crucial foundation for targeted therapy and prognosis prediction of cancer. However, existing detection systems lack synergy between spatial proximity assurance and signal resolution, posing challenges of low accuracy in cell subpopulation identification. Herein, we developed a potential-resolved electrochemiluminescence (ECL) system based on an ECL probe integrating spatial proximity assurance and competitive quenching and achieved simultaneous analysis of the dual biomarkers CD133 and CD44 on the surface of colorectal cancer cell subsets. The ECL probe of this system applies double-ended forked DNA as the scaffold, with ECL luminophores (Ru(bpy)₂(mcbpy)²⁺ and <i>N</i>-(4-aminobutyl)-<i>N</i>-ethylisoluminol (ABEI)) and specific recognition modules attached to its termini for simultaneously targeting cell surface biomarkers. Ru(bpy)₂(mcbpy)²⁺ and ABEI generate cathodic and anodic ECL signals using potassium persulfate (K₂S₂O₈) and dissolved oxygen as coreactants, respectively. By specifically quenching the corresponding signals of low-expression biomarkers via the quenching probe, this system enables different phenotypic subpopulations to exhibit distinct ECL signals. Validation using Caco-2 (CD133⁺CD44^-), DLD-1 (CD133^-CD44⁺), and HT-29 (CD133⁺CD44⁺) cells demonstrated high sensitivity and specificity with significantly improved subpopulation discrimination. This design integrates a synergistic mechanism combining proximal recognition of double markers-potential resolution-competitive quenching with a modular DNA-forked scaffold, offering insights into tumor heterogeneity and next-generation cancer diagnosis. The modularity of the strategy allows adaptation to other biomarker combinations via antibody replacement, which provides a flexible platform for tumor heterogeneity research.