English
DNA origami-based biosensors provide a powerful and programmable platform for sensitive and specific molecular diagnostics. In this study, a dynamic DNA origami book biosensor is validated for direct detection of microRNA21 (miR-21) in complex biological fluids, including human serum and plasma. Two optical readouts are engineered and tested: Förster resonance energy transfer (FRET) and fluorescence quenching using precisely arranged donor-acceptor fluorophore pairs. To improve sensitivity, detection occurs under varied conditions, including modifications to the lock mechanism (fluorine-modified bases and locked nucleic acid) and the addition of a biocompatible polymer, diethylene glycol (DEG). The biosensor maintains specificity in spiked serum, plasma, and clinical plasma samples from breast cancer patients. Single origami nanostructure detection experiments show that target miRNAs are identified within 10 min, with limits of detection (LoD) of 0.6% pm in buffer with LNA, (.(6 pm in 100% serum, and 2).24 pm in 100% plasma, without enzymatic amplification. Importantly, simultaneous detection of miR-21 and miR-7a in clinical plasma samples is demonstrated, underscoring the platform’s potential for personalized diagnostics and liquid biopsy applications. These results show that tuning the lock mechanism and adding polymers enhances sensitivity and enables adaptable performance, advancing nanotechnology-based biomarker profiling, including diverse microRNA detection.