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Dissertation, Universidade Nova de Lisboa, 2018; 224 pp. (2018). = Dissertation, Universidade Nova de Lisboa, 2018, The use of gold nanoparticles (AuNPs) to develop sensing platforms for the detection of nucleic acids has gained momentum for the past decade. This is due to unique physical properties of AuNPs, such as intense optical modulation, coupled to the possibility of tailoring their reactivity through surface modification. The main objective of this thesis was to develop a biosensor based on the gold nanobeacons technology coupled to wavelength shift mediated by Förster resonance energy transfer (FRET). As a proof-of-concept, the molecular hallmark of Chronic Myeloid Leukemia (CML) - the BCR-ABL fusion gene - was chosen as target. The detection is based on a spectral shift of the donor signal to the acceptor, which allows the corroboration of the hybridization event. The Au-nanobeacon acts as a scaffold for detection of the target in a homogenous format whose output capability (i.e. additional layers of information/NP) is potentiated via the specific FRET signal. To do so, the design of the conventional molecular beacon was changed into a two-component system comprising 1) a donor labeled single-strand DNA with a hairpin structure and 2) an acceptor labeled oligonucleotide that hybridizes to the open state of the hairpin. This system was optimized to allow the specific detection of the fusion sequences and not be triggered by the partial non-fused sequence and, subsequently, hybridize to an acceptor-labeled oligo that identifies which hairpin was triggered in solution. The spectral coded Au-nanobeacons (BioCode) allowed the detection of synthetic pathogenic fusion sequences (with high specificity towards partial complementary sequences), using two distinct fluorescence signals (FRET signal and a partial recovery of the donor emission) over the same hybridization event. Translation of this BioCode strategy to intracellular signaling was also attempted in a theranostic approach. Here, the specific gene silencing and tracking the silencing events via the beacon’s emission. Conceptually, silencing of the enhanced green fluorescence protein (EGFP) mRNA in zebrafish embryos, followed by application to CML cell lines.

The authors acknowledge Fundacao para a Ciencia e Tecnologia (FCT/MEC) for funding: REQUIMTE (Pest-C/EQB/LA0006/2011); UCIBIO (UID/Multi/04378/2013); SFRH/BD/87836/2012 for MC; PD/BD/105734/2014 for PP. WaterJPI/0003/2013-TRACE for FFC. The remarkable physicochemical properties of gold nanoparticles (AuNPs) have prompted developments in the exploration of biomolecular interactions with AuNP-containing systems, in particular for biomedical applications in diagnostics. These systems show great promise in improving sensitivity, ease of operation and portability. Despite this endeavor, most platforms have yet to reach maturity and make their way into clinics or points of care (POC). Here, we present an overview of emerging and available molecular diagnostics using AuNPs for biomedical sensing that are currently being translated to the clinical setting. publishersversion published

Raquel Vinhas,1,2* Milton Cordeiro,1,3* Fábio Ferreira Carlos,1,4 Soraia Mendo,1,2 Alexandra R Fernandes,2 Sara Figueiredo,1 Pedro V Baptista1 1Nanomedicine@FCT, CIGMH (Centro de Investigação em Genética Molecular Humana), UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Caparica, Portugal; 2UCIBIO (Unidade de Ciências Biomoleculares Aplicadas), Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Caparica, Portugal; 3REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus da Caparica, Caparica, Portugal; 4STABVIDA, Investigação e Serviços em Ciências Biológicas, Lda, Madan Parque, Caparica, Portugal *These authors contributed equally to this work Abstract: Nanotheranostics takes advantage of nanotechnology-based systems in order to diagnose and treat a specific disease. This approach is particularly relevant for personalized medicine, allowing the detection of a disease at an early stage, to direct a suitable therapy toward the target tissue based on the molecular profile of the altered phenotype, subsequently facilitating disease monitoring and following treatment. A tailored strategy also enables to reduce the off-target effects associated with universal treatments and improve the safety profile of a given treatment. The unique optical properties of gold nanoparticles, their ease of surface modification, and high surface-to-volume ratio have made them central players in this area. By combining imaging, targeting, and therapeutic agents in a single vehicle, these nanoconjugates are (ought to be) an important tool in the clinics. In this review, the multifunctionality of gold nanoparticles as theranostics agents will be highlighted, as well as the requirements before the translation of these nanoplatforms into routine clinical practice. Keywords: nanotheranostics, nanomaterials, gold nanoparticle, theranostics, cancer, nanomedicine