Flexible Membrane-Integrated Zipper CHA Amplification for Rapid and Sensitive MicroRNA Detection

Abstract

Catalytic hairpin assembly (CHA) is a widely employed signal amplification strategy for developing highly sensitive DNA biosensors, holding great promise for early disease diagnosis. However, conventional CHA systems often suffer from slow reaction kinetics, high nonspecific background signals, and limited stability in complex biological matrices. Inspired by the spatial confinement and fluidity of natural cell membranes, we herein report a red blood cell (RBC) membrane-anchored zipper-CHA system (RBC-ZCHA). In this system, preassembled low-background zipper-CHA probes (ZCHA) are anchored to the RBC membrane via cholesterol-modified DNA strands. This design synergistically enhances local probe concentration and intermolecular collision efficiency through multilevel confinement effects and dynamic membrane fluidity, thereby accelerating the reaction kinetics and improving detection sensitivity. Compared with the free-diffusion CHA system (Free-CHA), the RBC-ZCHA system exhibits lower background signals, accelerated reaction rates (approximately 5-fold increase), enhanced detection sensitivity (approximately 2 orders of magnitude higher), and superior stability in complex matrices. The designed system enables rapid and sensitive detection of microRNA in human serum, establishing a biocompatible and efficient platform for molecular diagnostics.