Development of a Field Effect Transistor biosensor (bioFET) for the detection of clinically relevant microRNAs

MicroRNAs (miRNAs) are small non-coding RNA molecules, approximately 19-25 nucleotides long, that play a crucial role in the post-transcriptional regulation of gene expression. Since alterations in their expression profiles are correlated with the onset of oncological, cardiovascular, and neurological pathologies, specific miRNAs have emerged as promising circulating biomarkers of extreme interest. In particular, the monitoring of specific sequences such as miR-155, involved in inflammatory processes, and miR-141, associated with prostate cancer, represents today a fundamental frontier for early diagnosis and personalized medicine. Despite their clinical relevance, miRNA detection presents analytical challenges due to their low concentration in biological fluids and high sequence homology. Established diagnostic techniques, such as RT-qPCR or Next-Generation Sequencing (NGS), while accurate, show significant limitations in terms of costs, response times, and the need for centralized laboratories with specialized personnel. These disadvantages make these methods difficult to apply in rapid or decentralized screening contexts. In this scenario, biosensors emerge as an extremely promising technological solution. Defined as compact analytical devices that integrate a biological recognition element (probe) with a physicochemical transducer, biosensors can transform a molecular binding event into a measurable signal, ensuring speed, selectivity, and precision. Among the various platforms, field-effect transistors (bioFETs) represent a technology of choice for next-generation molecular diagnostics. Their operating principle is based on the modulation of the conductance of a semiconductor channel in response to variations in the surface electrostatic potential induced by analyte binding. The ability of bioFETs to operate in label-free mode, combined with high intrinsic sensitivity and scalability towards portable devices, makes them ideal tools for overcoming current technological limits in miRNA detection. However, the implementation of bioFETs for nucleic acid detection presents significant challenges, mainly related to ionic shielding (Debye) in physiological environments and the need to optimize the bio-recognition interface to maximize the signal. Therefore, in this thesis work, a customized biosensor based on FET technology was developed for the detection of clinically relevant miRNAs. The research was divided into several phases: Development of an Extended Gate configuration platform: An architecture was engineered in which a commercial MOSFET, acting as a transducer, was interfaced with an external and disposable Screen-Printed Electrode (SPE). This system, immersed in the analytical solution containing the target miRNA, was thoroughly characterized to evaluate its • electrical performance and effective applicability in biosensing, optimizing a versatile and low-cost setup. • Study and validation of capture probes: A comparative analysis was conducted between canonical probes (antisense RNA) and non-canonical probes, such as peptide nucleic acids (PNAs). Both types were evaluated for their molecular recognition capability by systematically studying the interaction kinetics with the target miRNA. These tests were performed under experimental conditions designed to mimic the bioFET sensing environment, allowing for the determination of the affinity and stability of the probe-target duplex at the transducer interface. • Evaluation of ionic strength as a critical parameter: The ionic strength of the medium was investigated not only as a determining factor for molecular hybridization efficiency but also for its crucial role in electrical signal transduction. In this phase, specific strategies were proposed and tested to balance the need for correct hybridization with the requirement to minimize the ionic shielding effect (Debye), thus optimizing the device's response in potentially real matrices. • Engineering and optimization of self-assembled monolayers (SAMs): Part of the research concerned the molecular organization on the sensor surface. Different strategies were studied to optimize the packing and order of the SAM, which are fundamental parameters for ensuring signal reproducibility. • Molecular design of the probe and linker: Closely connected to the SAM organization, the impact of alternative probe designs was evaluated, analyzing how sequence length and linker size influence target accessibility. The careful choice of the linker combined with the shortening of the probe proved decisive in modulating the distance of the miRNA charge from the sensing surface, maximizing the detected potential variation. To support the development of the device, the work was integrated with functional characterizations using various techniques. Surface Plasmon Resonance (SPR) was employed to validate the interaction kinetic constants between the different probes and the target miRNA in real-time, while Atomic Force Microscopy (AFM) and Atomic Force Spectroscopy (AFS) allowed for mapping the surface morphology and quantifying binding forces at the single-molecule level. In parallel, Electrochemical Impedance Spectroscopy (EIS) was used as a complementary technique to monitor variations in interfacial resistance and capacitance during the functionalization and SAM optimization phases. In conclusion, this work demonstrates how the strategies proposed and implemented in our bioFET platform allow for competitive performance, ensuring sensitive detection even in complex matrices. Prospectively, the results obtained lay the groundwork for the development of decentralized diagnostic systems; future miniaturization and validation on real clinical samples will allow for the consolidation of the versatility and applicability of these platforms in the biomedical field