Nano-Oligo Sensor for RNA Detection via a Two-Fold Amplification Process

This study presents a dual amplification approach for sensitive RNA detection using gold-iron oxide (Au-Fe₃O₄) magnetic superstructures. In the search for alternatives to reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods, many nanotechnology-based cyclic amplification techniques suffer from insufficient sensitivity due to limited signal enhancement. To overcome this limitation, we have developed a 2-fold duplex-specific nuclease (DSN)-assisted amplification process that enhances detection efficiency. This method employs a "Nano-Oligo sensor" based on a bifunctional Au-Fe₃O₄ nanostructure that anchors two single-stranded DNA (ssDNA) probes, enabling enhanced target binding and amplification. The detection mechanism involves the hybridization of probes to target RNA, followed by DSN-mediated cleavage, which releases small RNA fragments that further interact with other probes, triggering continuous amplification cycles. This sensor achieved a detection limit of 1.2 fM with high specificity against nontarget sequences. It successfully detected Dengue RNA without the need for RNA extraction, showing a strong correlation with RT-qPCR results across dilutions. This approach offers a rapid, extraction-free diagnostic platform, making it highly suitable for RNA detection and clinical outbreak surveillance.