Highly Specific Detection of miRNA Mutants Using a Combined Analysis of Functionalized Micropipette and Hybridization Free Energy

Base mutations (mismatches, insertions, or deletions) in microRNAs (miRNAs) are frequently associated with diseases including as cancer, where they significantly alter miRNA structure and function. However, the accurately detection of low-abundance mutants amid highly similar sequences remains a major challenge. In this study, we developed a sensing strategy based on the ionic current and hybridization free energy, which employs glass micropipette channels functionalized with polydopamine nanotubes (PDA-NTs) and peptide nucleic acids (PNA), which enables enzyme-free, high-resolution identification of low-abundance miRNA-21 base mutations. The detection mechanism relies on mutation-induced conformational changes during PNA-miRNA hybridization, which modulate nanopore ion permeability and produce distinct current signatures. By correlating these signals with hybridization Gibbs free energy (ΔG), we established a link between thermodynamic stability and current response. When ΔΔG > 1 kcal/mol, a segmented linear relationship was observed between ΔΔG and the normalized current decrease Δ[(I₀-I_C)/I₀], allowing precise discrimination of mutant types, numbers, and locations based on ionic current response. In contrast, when ΔΔG < 1 kcal/mol, the signal change Δ[(I₀-I_C)/I₀] remained below 5%, making it challenging to differentiate mutants from the wild-type. This portable highly sensitive miRNA mutation sensing strategy offers strong potential for rapid molecular diagnostics and clinical decision-making.