Development of Nanostructured Triggerable Biosensors for Prostate Cancer Early Detection

Cancer remains a leading cause of global mortality, with Prostate Cancer (PCa) ranking as the second most common cancer in men. Early detection poses a significant challenge due to cancer's growth rate and limitations in current diagnostic assays. Despite Prostate Specific Antigen (PSA) being a longstanding biomarker for PCa diagnosis, its lack of specificity leads to a high rate of false positives. In response, alternative biomarkers like Prostate Specific Membrane Antigen (PSMA), detectable in urinary exosomes have emerged. Additionally, PSA in serum facilitates early detection of PCa recurrence post-prostatectomy.  We developed two triggerable oligonucleotide-gold nanoparticle (AuNPs) architectures (AuNPs-Aggregates) that selectively disassembles in the presence of a protein biomarker (PSMA or PSA). The AuNPs-Aggregates were prepared by mixing two batches of AuNPs, functionalised with different ssDNA strands, including a sequence that contains an aptamer responsive to the protein marker. After in silico and in vitro sequences characterisation, I synthetized and functionalised AuNPs with thiolated-ssDNA strands. The PSMA responsive Aggregate is composed by 13 nm AuNPs, and is aimed to ex vivo early detection of PSMA in urinary exosomes. Its response was determined by monitoring changes in its size and optical properties. I obtained a significant specific PSMA response from the high femtomolar range, with purified PSMA protein, and from low picomolar range with the protein extract of PSMA positive exosomes. The second sensor, the AS2-US-AuNPs-Aggregate, is thought for an in vivo application to early detect PSA in the blood of totally resected patients and is composed by ultrasmall AuNPs, a size suitable for renal clearance. This Aggregate has a size of hundreds of nanometres, compatible with prolonged circulation time in bloodstream. In this way, the release of single AuNPs from the Aggregate upon aptamer recognition could permit its analysis in urine. The AS2-AuNPs-Aggregate response to PSA was monitored following its changes in size; its specific response started to be significant in the low fM range. Then, we compared the stability of AS2 annealed sequences to nucleases, both in their free dsDNA form and when integrated into the AS2-AuNPs-Aggregate. This nanostructure has the potential to be tailored for detecting various biomarkers, such as proteins or DNA, by modifying the nucleotide sequences involved in the recognition process. Furthermore, it offers flexibility in selecting the nanoparticles incorporated in the structure, with the possibility of different methods of detection.