Ultrasensitive detection of miRNA based on efficient immobilization of probe and electrochemiluminescent quenching of Ru(bpy)32+ by methylene blue.

A high performance miRNA biosensor based on effective click chemistry assembly of a Ru(bpy)32+ labeled DNA probe and efficient electrochemiluminescence (ECL) quenching of the Ru(bpy)32+/BDEA (BDEA = N-butyldiethanolamine) system by surface-confined electroactive methylene blue (MB) dye is reported. When the target miRNA was present, the ECL signal instantly changed from "light off" to "light on" status. Using the specific miRNA let-7d as the target analyte, this biosensor provided sensitive detection over approximately six orders of magnitude (10 fM-10 nM), with a limit of detection of 10 fM (S/N = 3). Detailed study of the ECL quenching behavior of the Ru(bpy)32+/BDEA system by MB in solution suggested that the ECL quenching involves a combination of photoluminescence dynamic quenching and quenching processes directly associated with the redox reactions, as well as resonance energy transfer. A large binding constant of 4.7 × 1011 M-1 between let-7d and the DNA hairpin was estimated using an ECL-based extended Langmuir isotherm model, suggesting remarkably strong binding of the target to the probe. Furthermore, our biosensor exhibited excellent specificity and reproducibility. Using the developed system, the concentration of the target miRNA extracted from the A549 cell line could be obtained, demonstrating the potential application of the developed biosensor to practical biological sample analysis.