Target-Responsive Template Structure Switching-Mediated Exponential Rolling Circle Amplification for the Direct and Sensitive Detection of MicroRNA

This study reports a fast and straightforward detection of cancer-specific microRNA (miRNA) via isothermal toehold-mediated rolling circle amplification (TRCA) with a dumbbell (DB) template and DNA-tethered gold nanoparticles (GNP-DNA). A preassembled DB template with a specific structure switching to target miRNA and fine-tuning of the surface coverage of GNP-DNA allows the development of a universal and direct miRNA detection platform without the need for washing, labeling, or isolation. This study demonstrates the successful development of the TRCA and visualization method of amplified products with the naked eye that can precisely and rapidly detect the presence of miRNA-21 with a detection limit of 0.87 nM within 1 h using a standard laboratory plate reader. This approach is further extended to initiate subsequent rounds of exponential amplification with the addition of primers (termed hyperbranched TRCA or HTRCA), providing a 1.6-fold improvement in assay sensitivity with a detection limit of 0.55 nM. Fluorescent detection signals are also used to quantify the amplified products, providing a detection limit of 33 pM (for TRCA) and 31 pM (for HTRCA). Thus, this colorimetric and fluorescent assay can be used as an effective molecular diagnostic tool for target biomolecules with a combination of additional signal amplification strategies.