MicroRNAs (miRNAs) are short non-coding RNAs whose dysregulation is linked to various pathologies, including cancer. Among these, miR-155 represents a promising biomarker with high diagnostic and prognostic potential. However, translating miRNA analysis into clinical practice requires label-free methods that combine high analytical performance with operational simplicity. A custom-assembled Field Effect Transistor biosensor (bioFET) implemented with Peptide Nucleic Acid (PNA) probes was developed for the sensitive detection of miR-155. Characterized by strong binding affinity for complementary targets and reduced ionic screening, PNA represents a highly promising probe for electrochemical miRNA biosensing. We exploited a strategy based on a specific truncation of the PNA capture sequence (10-mer) to ensure that, upon hybridization, the uncoupled miR-155 overhangs are forced into proximity to the electrode surface, leading to a significant enhancement of the bioFET signal. Furthermore, the effects of carbon linkers differing in length as well as the immobilization strategy were systematically investigated using Electrochemical Impedance Spectroscopy (EIS). Results indicate that a C3-linker, combined with a PNA/MCH co-immobilization approach, provides an optimal molecular orientation for efficient target recognition. The protocols were translated into the BioFET platform resulting in a Limit of Detection of ∼0.2 fM, with a significant improvement over previous results based on full-length PNA probes (∼350 fM). Good performance was also achieved in spiked human serum. Furthermore, the sensor maintained remarkable specificity, effectively discriminating against the target isoform. This rational design of the probe-target interface offers a robust and scalable route toward high-performance miRNA diagnostics. • EIS-guided optimization improves short PNA probe immobilization and hybridization. • Short PNA probe creating overhangs enable high sensitivity and specificity for miR-155. • Integration into BioFET allows real-time, label-free detection with LoD of ∼0.2 fM. • Strategy offers a promising platform for clinically relevant miRNA detection.
Tocco et al. (Fri,) studied this question.