Abstract
Accurate detection of low-abundance microRNA (miRNA) as tumor biomarkers in body fluids and cells is critical due to its strong association with tumor initiation, progression, invasion, and metastasis. DNA walkers enable signal amplification through cyclic enzymatic reactions, offering powerful strategies for the highly sensitive detection of low-abundance miRNAs. However, fluctuations in the cellular microenvironment─such as variations in ion concentration, enzyme activity, temperature, and pH across subcellular compartments─can disrupt amplification efficiency and compromise detection accuracy. To address this challenge, we developed a symmetric bipedal DNA walker design in which one "leg" generates a detection signal while the other "reference leg" synchronously amplifies a dynamic (environment-responsive) internal reference signal. Unlike traditional static internal reference strategies, this design introduces a real-time, synchronized internal reference during the DNA walking process to correct for system errors. When combined with ratiometric fluorescence-based error compensation, this approach effectively eliminates signal variation caused by nanoparticle loading, strand assembly, and environmental fluctuations, thereby improving the detection precision and reproducibility. The system, constructed on gold nanoparticles using an inverted hairpin scaffold, demonstrated robust and consistent performance under varied experimental conditions. Confocal ratiometric fluorescence imaging enabled the high-contrast quantification of intracellular miRNA-21 (miR-21) levels across different cell lines. Moreover, spatially resolved fluorescence signals revealed heterogeneous miR-21 distribution within individual cells, providing valuable insights into its subcellular localization and functional relevance. This strategy represents a reliable platform for accurate miRNA quantification even in complex biological environments.