Peptide Nucleic Acid Probes for MicroRNA Detection: Mg2+Ion Effect, Surface Hybridization, and Surface Plasmon Resonance Biosensing

Abstract

This study examines how Mg2+ ions affect the hybridization between surface-immobilized peptide nucleic acid (PNA) probes and microRNA targets (miR125 and miR141), which is important for the development of nucleic acid-based biosensors utilizing surface plasmon resonance (SPR). The results show that appropriate concentrations of Mg2+ significantly enhance microRNA hybridization with PNA probes, whereas Na+ does not yield similar results. Kinetic analysis demonstrated that 30 and 100 mM concentrations of Mg2+ facilitate the interaction between the PNA probe and its microRNA target by effectively screening the negative charges of the microRNA molecules as they approach the surface. These Mg2+ levels also stabilize the heteroduplexes formed on the surface by reducing the dissociation rate. However, a higher Mg2+ concentration (300 mM) was found to hinder the surface-confined hybridization. In comparison, Na+ showed a considerably smaller role in improving the hybridization. Melting curve analysis in solution indicated that the increase in Tm of PNA/miRNA heteroduplexes in the presence of Mg2+ does not fully explain the enhanced surface interaction, underscoring the role of surface confinement. These findings demonstrate that optimizing the Mg2+ concentration can significantly improve the sensitivity and efficiency of PNA- and SPR-based microRNA biosensors. This optimization is particularly relevant for diagnostic and research applications involving the analysis of low concentrations of microRNAs in biofluids.