Double-Flapped Dumbbell Probe Functionalized with Silver Nanoclusters for Sensitive Fluorometric Detection of miRNA

MicroRNA (miRNA) has emerged as a potential diagnostic marker for malignancies, such as prostate cancer; yet, few analytical approaches have been established for the simple and effective identification and quantification of miRNA. We depict here a fluorescent dumbbell probe utilizing DNA-silver nanoclusters (DNA-AgNCs) to facilitate the simple and label-free quantification of miRNA. The fluorescent probe was methodically engineered using a double-flapped dumbbell structure, wherein the 5' flap was synthesized using DNA-AgNCs, and the 3' flap was extended by a G-rich sequence. The DNA scaffold rigidifies the DNA-AgNCs and the G-rich sequence, bringing them into close contact, which leads to enhanced fluorescence due to the activation of DNA-AgNCs by the G-rich sequence. Upon the introduction of miRNA, the 5' flap of the probe unfolds, liberating the G-rich sequence from the probe, disrupting the closeness between DNA-AgNCs and the G-rich region, and resulting in decreased fluorescence. The proposed method, by incorporating target recycling and polymerase/endonuclease-assisted cycles, enables sensitive detection of miRNA with a detection limit of 4.2 fM and demonstrates high specificity for monitoring target miRNA in clinical samples, offering a robust platform for miRNA monitoring and disease diagnosis.