Peptide Nucleic Acid Probes for MicroRNA Detection: Mg ²⁺ Ion Effect, Surface Hybridization, and Surface Plasmon Resonance Biosensing

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 <i>T</i> _m 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.