Flexible and Reusable Biochip Platform for miRNA Quantification via Dual-Signal Amplification on 3D Au Substrate

Micro ribonucleic acids (miRNAs) are small, noncoding RNA molecules that are pivotal regulators of gene expression and have emerged as critical biomarkers for various diseases and therapeutic targets. Among these, circulating miRNAs in the bloodstream are particularly attractive because of their stability and potential for noninvasive diagnostics. To address the demand for a highly sensitive and straightforward miRNA quantification method, we developed a novel biochip sensor that integrates a three-dimensional (3D) Au nanostructure with a dual-signal amplification strategy. A 3D Au nanostructure was fabricated on a polyvinylidene fluoride membrane using vacuum deposition, which provided a robust and flexible platform with enhanced optical properties. The nanostructure facilitated efficient probe immobilization and supported metal-enhanced fluorescence. Upon target miRNA recognition by the immobilized probes, two sequential signal amplification mechanisms are activated: (1) concatemer-based amplification by multiplex binding of fluorescent probes and (2) enzyme-free target recycling based on toehold-mediated strand displacement reactions. This dual amplification strategy on the 3D Au nanostructure achieved exceptional sensitivity and specificity for miRNA detection, even at lower concentrations. The biochip demonstrated a high-throughput capability, reusability, and practical applicability in molecular diagnostics. This innovative platform represents a significant advancement in miRNA sensing technology and offers a versatile tool for biomarker research and clinical diagnostics.