Optimisation of peptide nucleic acid (PNA) probe immobilisation by EIS for enhanced bioFET detection of miR-155

MicroRNAs (miRNAs) are single-stranded oligonucleotides controlling gene expression whose deregulation is often linked to various human diseases, making them promising biomarkers. Electrochemical genosensors, particularly Field Effect Transistor-based biosensors (bioFETs), offer rapid, label-free methods for miRNA detection. These devices utilise capture probes capable of specifically recognising target miRNAs. Synthetic peptide nucleic acids (PNAs) exhibit high affinity for complementary strands, being then suitable for bioFET-based miRNA detection. The impact of the structural organisation of PNA-based self-assembled monolayers (SAMs) on the sensing performance of bioFETs, despite its critical importance, remains insufficiently understood and requires further investigations. The capture efficiency of PNA probes was optimised through its co-immobilisation with 6-mercapto-1-hexanol (MCH), a small diluent able to finely control the probe density and orientation. Electrochemical impedance spectroscopy (EIS) was employed to systematically characterise how MCH incorporation modulates the structural properties of the SAM and enhances hybridisation efficiency with microRNA-155. The optimised conditions for the PNA:MCH ratio were exploited to develop a custom-made bioFET setup for miRNA detection. We achieved a limit of detection (LoD) of (0.28 ± 0.06) pM with a 1000-fold improvement compared to our previous results. This bioFET platform also demonstrated a high specificity toward miR-155 combined with good recovery rate and reproducibility. The MCH-based surface optimisation strategy, integrated with bioFET technology, offers a simple and scalable solution for enhancing miRNA detection, and then contributing to bridging the gap between biosensor development and clinical application for miRNA-based diagnostics.