Dissertação para obtenção do Grau de Doutor em
BiotecnologiaThe main objective of this thesis was to study the fluorescence modulation induced by gold nanoparticles (AuNPs) on fluorophores nearby and/or bonded to the AuNPs’ surface through nuclei acid molecules. The understanding of the effect of distance in the spectral properties of fluorophores would allow the development of a biosensor for the characterisation of DNA and/or RNA sequences.
To study the photophysics involved in the fluorescence modulation by AuNPs it was necessary to develop an experimental approach that removed the effect of the optical interference caused by the presence of AuNPs. By comparing the samples with controlled reference solutions it was possible determine the fluorescence quantum yield and fluorescence decay time of the fluorophores in the vicinity of AuNPs. During the characterisation several non-photophysical phenomena involving nanoparticles were unveiled, such as a local pH effect, coupling of the plasmonic oscillator with transition moments of the fluorophore or AuNP-induced fluorophore aggregation.
The developed experimental method was applied to the study of the effect of distance in the modulation of fluorescence caused by AuNPs. Using DNA molecules as spacer, the photophysical properties of fluorophores at different distances of the surface of the AuNPs showed a distance-dependence fitting into a 1/r6 dependence.
The knowledge gathered on AuNP-DNA-fluorophore systems allowed for a successful semi-quantitative detection of RNA in solution. The same system showed to be useful for the simultaneous quantification and control RNA synthesis in vitro. In situ detection and gene silencing was demonstrated by targeting EGFP mRNA as proof-of-concept. A similar approach was successfully achieved in siRNA and endogenous miRNA targets. The application of this system to micro-deletions and RNA isoforms analysis was also demonstrated in synthetic targets.Fundação para a Ciência e Tecnologia - (SFRH/BD/43320/2008
