Employing Cluster Transfer Strategy in Hybridisation Chain Reaction-Silver Nanoclusters Hybrid Sensor for Nucleic Acid Detection

Abstract

Hybridisation chain reaction (HCR) is a robust isothermal amplification technique widely used for nucleic acid detection, often paired with silver nanoclusters (AgNCs) for fluorescence-based readouts. However, conventional HCR-AgNCs assays are prone to circuit leakage and unpredictable AgNCs formation, which can compromise assay reliability. In this study, we present a modular HCR-AgNCs hybrid sensor employing a cluster transfer strategy, where pre-formed AgNCs are introduced separately to the HCR system. This design minimises interference between AgNCs and HCR components, enhancing signal specificity and predictability. Using a DNA analogue of miRNA-141 (DNA-141), a potential prostate cancer biomarker, we demonstrate selective red fluorescence activation upon target recognition. The sensor design was guided by NUPACK simulations, reducing trial-and-error and lowering assay costs. Gel electrophoresis and fluorescence spectroscopy confirmed the system's specificity and sensitivity, achieving a limit of detection of 46 nM. This work establishes a foundational framework for modular and adaptable nucleic acid biosensing, with potential for future sensitivity enhancements and multiplexed detection.