Gold nanoprobes for molecular detection of SNPs related to lactose intolerance

• Autor(a) principal: Dias, André Teixeira
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/135869
• topic: Gold nanoparticles; Single nucleotide polymorphism; Lactose intolerance; Point-of-care detection system; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Lactose intolerance is a condition that affects millions of people around the world. The current diagnosis methods have shortcomings, namely high costs and long processing times. As such, innovative detection mechanisms should be developed to allow fast, reliable, and inexpensive diagnosis in a point-of-care format. The utilization of gold nanoparticles could overcome these limitations due to their aggregation-dependent colorimetric properties. The developed work focused on their use for the diagnostic of lactose intolerance through the single nucleotide polymorphism associated with European populations (C/T -13910 LCT). Gold nanoparticles with different diameters (15 or 40 nm) were synthesized, characterized and functionalized (salt-aging or pH method). The functionalization was performed using a 5’ or 3’ thiol-modified oligonucleotide. The salt-aging method yielded better results for 15 nm gold nanoparticles, while the pH method worked better for 40 nm gold nanoparticles. The evaluation of several DNA targets through non-cross-linking method showed that anti-parallel hybridization resulted in DNA duplexes with greater stability and stronger colorimetric distinction, when compared to parallel hybridization. Cross-linking and non-cross-linking methodologies were then applied to the optimized conditions. The cross-linking method revealed that nanoprobes obtained using the 3’ thiol-modified oligonucleotide had problems with hybridization, requiring further optimization. From the non-cross-linking method, it was possible to conclude that the 40 nm nanoprobes led to better colorimetric distinction between the complementary and mutated samples (upwards of 60%) and improved statistical significance in comparison to 15 nm nanoprobes. For the tested conditions, smaller DNA targets (40 b.p.) resulted in better colorimetric distinction compared to larger DNA targets (120 b.p.). The differences seen also improved with the increase of DNA target concentration (from 0.25 up to 100 ng/μL). These results demonstrate the capabilities of 40 nm nanoprobes for detection of lactose intolerance and highlight the importance of optimizing the length and concentration of the DNA target.