Functional nucleic acid-based miniaturized homogeneous electrochemical biosensor for label-free microRNA detection

The development of miniaturized, label-free microRNA (miRNA) sensors is of great significance for early cancer diagnosis, disease monitoring, and point-of-care testing (POCT), which can substantially enhance clinical efficiency and diagnostic accuracy. In this work, we proposed a functional nucleic acid-based miniaturized homogeneous electrochemical biosensor for label-free miRNA detection. The system features an integrated microelectrode (IME) comprising a gold fiber microelectrode paired with carbon rod/Ag/AgCl wires within a three-channel glass microtube, enabling electrochemical monitoring at the microliter scale. A functional nucleic acid probe containing a miRNA-complementary DNA sequence and a G-triplex (G3) motif was synthesized. Leveraging the strong affinity between G3 and methylene blue (MB), the G3/MB complex serves as a signal generator by significantly reducing MB diffusion current upon binding. Using microRNA21 (miR21) as the model target, the biosensor achieved a detection limit of 0.78 nM with excellent specificity. Successful detection of miR21 in diluted human serum and MCF-7 cell lysate samples confirmed its applicability in complex matrices. This work establishes a “Mix and Measure” platform for homogeneous, label-free electrochemical miRNA biosensing.