A Dual-Mode Fluorescent and Colorimetric Biosensor Based on AuNPs@RCA Nanogel-Integrated Multipedal DNA Walker for Ultrasensitive miRNA Detection

Abstract The development of highly sensitive and reliable biosensors is essential for the early diagnosis and therapeutic monitoring of cancer. Herein, we report a dual-mode fluorescence–colorimetric biosensing platform for the ultrasensitive detection of miRNA-21, integrating AuNP-supported dual-stage rolling circle amplification (RCA) with an RCA-generated multipedal binding-and-cleavage mechanism. The sensing hierarchy is initiated by target-dependent activation of the primary RCA on the gold nanoparticle (AuNP) surface, producing long DNA strands with multiple repeated recognition domains that function as numerous walker “legs” and form a DNA-rich nanogel matrix. Through these repeated domains, the RCA-generated multipedal walker repeatedly and multivalently binds fluorophore-labeled hairpin substrates on the AuNP surface, thereby facilitating APE1-mediated substrate cleavage and fluorescence recovery. The APE1-generated cleavage fragments subsequently initiate the secondary RCA following the addition of the T2 template. The two successive RCA reactions ultimately produce an Au@RCA DNA nanogel containing abundant G-quadruplex/hemin DNAzymes that catalyze a visually detectable color change. By integrating the multipedal walker-mediated multivalent binding-and-cleavage process with dual-stage RCA, the platform enables dual-mode fluorescence–colorimetric detection with efficient signal amplification and low background, achieving limits of detection of 9.854 and 790 fM for the fluorescence and colorimetric modes, respectively. Furthermore, the two complementary outputs provide independent signal readouts for cross-validation. The biosensor demonstrated excellent specificity and distinguished the endogenous responses of MCF-7, HeLa, and HEK293T cell lysates. These results support the potential of the platform for ultrasensitive tumor-associated biomarker detection, while the cell-lysate experiments represent a preliminary proof-of-concept evaluation in complex biological matrices.