Dissertação apresentada para obtenção do Grau de Doutor em Engenharia Biológica – especialidade Engenharia Genética, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaThe main objective of this thesis was to develop a simple and inexpensive method for nucleic acid detection in biological samples based on the colorimetric properties of gold nanoparticles. Gold nanoparticles were synthesized and functionalized with thiol-modified DNA oligonucleotides (Au-nanoprobes) able to recognize a target sequence of interest. These Au-nanoprobes were characterized and then used in a colorimetric method for DNA detection with a single base resolution based on a non-cross-linking approach - upon increasing ionic strength, Au-nanoprobes aggregate and the solution changes color from the original red to blue, due to the red-shift of the typical Surface Plasmon Resonance (SPR) band of gold nanoparticles; the presence of a complementary target to the probe sequence, prevents aggregation of the Au-nanoprobes and the solution remains red. The use of gold-silver alloy nanoparticles functionalized with thiol-modified DNA oligonucleotides (AuAg-nanoprobes) to further develop a multiplex non-cross-linking method was also assessed. To carry out the proof-of-concept for single base mutation/Single nucleotide polymorphism (SNP) detection and further develop this non-cross-linking method, three different targets were selected considering clinical samples availability and the commercial interests of STAB VIDA, Lda. Single point mutations associated to β-thalassemia were chosen for the proof-of-concept. Later, to further evaluate the versatility of the method, two other targets were chosen: an SNP within the Diabetes and Obesity Regulated (DOR) gene (i.e. DOR1C/G), associated with diabetes and obesity; and a tag SNP within the Cytochrome P450 2D6 (CYP2D6) gene (i.e. CYP1846G/A), associated to a non-functional CYP2D6, which is responsible for the metabolism of xenobiotics. Studies were also conducted to better understand the underlying mechanisms involved in the non-cross-linking detection, towards the optimization of the method. The use of Atomic Force Microscopy (AFM), fluorescent spectroscopy and electrophoretic mobility measurements (Ferguson analysis) allowed clarifying the nature of the forces involved in the differential colorimetric non-cross-linking aggregation and further optimize the Au-nanoprobe design to SNP/single point mutation discrimination at room temperature. It has been demonstrated that the non-cross-linking method can be used for detection of SNP/single point mutations at room temperature. The detection sensitivity of the non-cross-linking method using Au-nanoprobes was determined to be 73 and 75 nM for ssDNA/dsDNA amplicons and ssDNA oligonucleotide targets, respectively.Fundação para a Ciência e Tecnologia (SFRH/BDE/15544/2005); Fundação Luso-Americana para o Desenvolvimento; STAB VIDA, Lda., especially the CEO, Orfeu Flore
