Abstract
miRNAs are short noncoding RNA single strands, with a crucial role in several biological processes. miRNAs are dysregulated in several human diseases, and their detection is an important goal for diagnosis and screening. Innovative biosensors for miRNAs are commonly based on the hybridization process between a miRNA and its corresponding complementary strand (or suitable aptamers) immobilized onto an electrode surface forming a duplex. A detailed description of the hybridization kinetics in working conditions deserves a great deal of interest for the optimization of the biosensing process. Surface plasmon resonance (SPR) and atomic force spectroscopy (AFS) were applied to investigate the hybridization process between miR-155, a multifunctional miRNA that constitutes an important marker overexpressed in several diseases, and its complementary strand (antimiR-155), immobilized on the gold-coated surface of a commercial electrode. Under well-adjusted pH, ionic strength, surface coverage, and concentration, we found that miR-155 has a high affinity for antimiR-155 with kinetics well described by the 1:1 Langmuir model. Both techniques provided an association rate of about 104 M-1 s-1, while a dissociation rate of 10-5 and 10-4 s-1 was assessed by SPR and AFS, respectively. These results allowed us to establish optimized measurement running times for applications in biosensing. An analysis of AFS data also led us to evaluate the binding free energy for the duplex, which was found to be close to that of free molecules in solution. These results could guide in the implementation of fine-tuned working conditions of a biosensor for detecting miRNAs based on correspondent complementary strands.