Cavaliere, Erica (2026) NUCLEIC ACID-BASED COLORIMETRIC BIOSENSORS FOR ENVIRONMENTAL MONITORING AND LIQUID BIOPSY. [Tesi di dottorato]

Documento PDF Cavaliere_Erica_38.pdf Download (6MB)

Abstract

Early cancer diagnosis and environmental monitoring of agricultural pathogens represent two critical challenges of our time, with profound implications for human health and global food security. In oncology, breast cancer remains the most prevalent malignancy among women, and its prognosis is heavily dependent on the ability to detect it at an early stage, ideally through non-invasive methods. Concurrently, in agriculture, phytopathogenic fungi such as Fusarium oxysporum cause devastating economic losses in high-value crops, often without visible symptoms until irreversible damage has occurred. In both domains, conventional diagnostic methods are slow, expensive, and require specialized infrastructure, which limits their use in point-of-care or field settings. To overcome these limitations, this doctoral thesis shows the development of two innovative colorimetric biosensors based on gold nanoparticles (AuNPs) and nucleic acids (NAs) as biorecognition elements, capable of delivering a direct visual signal without complex instrumentation. Both systems exploit localized surface plasmon resonance (LSPR) but employ different types of NAs and detection strategies, thereby demonstrating the platform’s versatility. The first biosensor was designed for the detection of breast cancer-derived exosomes in biological fluids. It utilizes an RNA aptamer, Ex.50.T, which specifically targets GREM1, a protein expressed on the surface of tumor-derived exosomes. The detection scheme employs aptamer-functionalized gold nanoparticles that are induced to aggregate through controlled salt addition, yielding a blue-colored solution. Upon binding to target exosomes, the specific aptamer–exosome interaction triggers nanoparticle disaggregation, resulting in a visible color shift from blue to red. This target-induced disaggregation mechanism enables direct detection in diluted serum samples without signal amplification, paving the way for rapid liquid biopsy tests. The second biosensor was developed for the detection of environmental DNA (eDNA), specifically genomic DNA from F. oxysporum in environmental samples. This sensor relies on single-stranded DNA (ssDNA) probes and an anti-aggregation mechanism that uses two distinct batches of gold nanoparticles: one functionalized with a target-complementary probe, referred to as Probe, and the other with its complementary sequence, referred to as C_Probe. In the absence of the target, Probe and C_Probe hybridize, leading to AuNPs and a red-to-blue color change. When F. oxysporum DNA is present, it binds to the Probe during an initial hybridization step, preventing interaction with C_Probe and preserving the red color. Both hybridization steps are accelerated by freezing, enabling detection in just five minutes. This approach allows for rapid and instrument-free target identification following appropriate sample preparation. Together, these two biosensors embody an emerging paradigm: one platform, multiple missions. The key to this adaptability lies in the remarkable versatility of nucleic acids as biorecognition elements. Aptamers, selected to bind protein targets with high affinity, and ssDNA probes, engineered to recognize specific genetic sequences through hybridization, are both synthetic, stable, easily modifiable, and fully sequence-programmable molecules. This dual capability, targeting either proteins or nucleic acids, enables the same plasmonic platform to address vastly different biological contexts simply by changing the nucleic acid probe immobilized on the AuNPs surface. Although both biosensors share the same gold nanoparticle–localized surface plasmon resonance transduction core, they tackle fundamentally distinct challenges, one clinical and the other environmental, demonstrating how the programmability of nucleic acids can be harnessed to design highly specific and adaptive optical detection strategies. This doctoral work demonstrates that a single plasmonic platform, when appropriately functionalized with different nucleic acids, can address diagnostic challenges across seemingly disparate fields, from liquid biopsy for breast cancer to phytosanitary pathogen monitoring, offering rapid, low-cost, and instrument-free solutions. The exosome biosensor has already been validated in real clinical matrices, confirming its applicability in biomedical contexts, whereas the F. oxysporum system provides a solid foundation for future field-deployable diagnostic tools, with implementation further enhanced by validation in real agricultural soil samples. Future perspectives include integration with smartphone-based readers for colorimetric signal quantification and extension of the platform to additional clinically or environmentally relevant targets, thereby reinforcing the vision of versatile, decentralized, and accessible diagnostics.

Downloads

Actions (login required)

Modifica documento