D'Aponte, Tina (2026) ELECTROCHEMICAL IMPEDANCE BIOSENSING IN COMPLEX BIOLOGICAL SAMPLES. [Tesi di dottorato]

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Abstract

In recent years, the detection of biological targets in complex samples has become increasingly important in clinical diagnostics, environmental monitoring, and food safety. Although conventional analytical methods (microbiological cultures, immunoassays (e.g., ELISA), and nucleic acid amplification (PCR)) ensure high accuracy, they are often time-consuming, resource-intensive, and dependent on specialized equipment and trained personnel. These limitations hinder the performance of rapid, decentralized testing, motivating the development of faster, low-cost analytical platforms that perform reliably in real matrices. Biosensors offer an attractive alternative by integrating selective biorecognition with compact analytical formats. Among them, electrochemical biosensors are particularly promising because they can deliver high sensitivity with short response times, they are readily miniaturized, and compatible with low-cost disposable substrates such as screen-printed electrodes (SPEs). Nevertheless, their performance is strongly affected by the biorecognition layer and analyte transport to the sensing interface. Under static conditions, diffusion-limited delivery—especially at low concentrations and in complex matrices—can increase nonspecific adsorption and signal variability, reducing reproducibility. This PhD work focuses on the development of two impedimetric electrochemical biosensors based on gold-screen-printed electrodes (Au-SPE) integrated with a custom 3D-printed fluidic device. This latter selectively wets the working electrode, thereby improving control and reproducibility by confining all reagents and analytes to the sensing area. The first biosensor is an immunosensor for detecting Candida albicans in urine. The gold working electrode was functionalized with anti-C. albicans IgG antibodies using Photochemical Immobilization Technique (PIT). The immunosensor was initially evaluated in the standard Candida culture medium, achieving a limit of detection (LOD) of 10² CFU/mL.Importantly, translation to urine as a clinically relevant complex matrix yielded a limit of detection of 10 CFU/mL with a total assay time below 90 minutes. The second biosensor is a DNA sensor for single-stranded DNA detection. Building on the same platform and further optimizing the fluidic setup, the working electrode was functionalized with thiolated ssDNA probes. The sensing mechanism relies on sequence-specific hybridization between the immobilized probe and the target DNA. The biosensor achieved a LOD of 0.1 pM in PBS (corresponding to ~0.6 pg/mL for the target sequence). Crucially, performance was validated in a complex biological matrix derived from Saccharomyces cerevisiae culture medium, enabling detection of the secreted target sequence at approximately 30 pg/mL.

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