A carregar...
Publicação
Sensor LSPR para uma rápida deteção da COVID-19
| Nome: | Descrição: | Tamanho: | Formato: | |
|---|---|---|---|---|
| 2.38 MB | Adobe PDF |
Autores
Orientador(es)
Resumo(s)
O presente trabalho teve como objetivo o desenvolvimento de um teste de antigénio (Ag) baseado em Ressonância do Plasmão de Superfície Localizada (LSPR) que combinasse a elevada especificidade dos anticorpos monoclonais (Ab) com a elevada sensibilidade das nanopartículas de ouro (AuNPs). A dissertação teve como subobjetivos estudar a influência de diferentes parâmetros na formação e estabilidade de bionanoconjugados assim como realizar uma meta-análise sobre amostras fisiológicas alternativas. Os ensaios foram realizados com AuNPs em solução e em placas de ITO com ouro depositado. O primeiro passo consistiu na preparação do ouro que inclui a sua funcionalização com diferentes tióis (MUA, MPA ou mistura de ambos) e a adição de agentes de acoplamento (EDC e NHS). A formação dos bionanoconjugados compreendeu a imobilização de anticorpos com diferentes especificidades e a imobilização de diferentes antigénios ou do vírus SARS-CoV-2. O fenómeno LSPR permitiu detetar um desvio da banda plasmónica após a imobilização das biomoléculas e os bionanoconjugados resultantes foram caraterizados por espectrofotometria de UV-Visível, eletroforese em gel de agarose e microscopia eletrónica de transmissão (TEM). A funcionalização com MUA e a imobilização das biomoléculas da GenScript permitiram obter os melhores resultados com um desvio após estas etapas. A meta-análise consistiu na pesquisa em diferentes bases de dados e à seleção dos artigos que cumpriam o objetivo da meta-análise e, por fim, procedeu-se à avaliação metodológica dos artigos assim como a meta-análise com vista à avaliação da sensibilidade, especificidade e precisão de amostras alternativas na deteção do SARS-CoV-2. A saliva apresentou os melhores resultados com uma precisão de 92.1%, indicando a possibilidade de ser utilizada como amostra alternativa para a deteção da COVID-19. O presente trabalho permitiu otimizar a estabilidade dos bionanoconjugados e estudar a possibilidade da utilização de amostras alternativas para detetar esta patologia. Contudo, não foi possível concluir o objetivo de desenvolver o teste rápido de antigénio, uma vez que o desvio da banda plasmónica obtido encontrava-se dentro do erro do aparelho de medida.
Abstract The present work aimed to develop an antigen (Ag) test based on Localized Surface Plasmon Resonance (LSPR) that combines the high specificity of monoclonal antibodies (Ab) with the high sensitivity of gold nanoparticles (AuNPs). The dissertation had the sub-goals to study the influence of different parameters on the formation and stability of bionanoconjugates and perform a meta-analysis on alternative physiological samples. The assays included AuNPs in solution and ITO plates with gold deposited. The first step consisted of the preparation of AuNPs, which implies the functionalization of the gold with different thiols (MUA, MPA or a mixture of both) and the addition of coupling agents (EDC and NHS). The formation of the bionanoconjugates includes the immobilization of antibodies with different specificities and the immobilization of different antigens or the SARS-CoV-2 virus. The LSPR phenomenon allowed the detection of a plasmon band shift after the immobilization of the biomolecules. The resulting bionanoconjugates were characterized by UV-Visible spectrophotometry, agarose gel electrophoresis and transmission electron microscopy (TEM). The functionalization with MUA and the immobilization of GenScript biomolecules allowed the obtention of the best results with a shift after these steps. The meta-analysis consisted of a search in different databases and the selection of articles that fulfilled the goal of the meta-analysis, finally, the methodological evaluation of the articles was conducted, as well as the meta-analysis to assess the sensitivity, specificity, and precision of alternative samples in the detection of SARS-CoV-2. Saliva showed the best results with an accuracy of 92.1%, indicating the possibility of being used as an alternative sample for the detection of COVID-19. The present work allowed the optimization of the stability of the bionanoconjugates and to study the possibility of using alternative samples to detect this pathology. However, it was not possible to complete the aim of developing the rapid antigen test, since the plasmonic band deviation obtained was within the error of the measurement device.
Abstract The present work aimed to develop an antigen (Ag) test based on Localized Surface Plasmon Resonance (LSPR) that combines the high specificity of monoclonal antibodies (Ab) with the high sensitivity of gold nanoparticles (AuNPs). The dissertation had the sub-goals to study the influence of different parameters on the formation and stability of bionanoconjugates and perform a meta-analysis on alternative physiological samples. The assays included AuNPs in solution and ITO plates with gold deposited. The first step consisted of the preparation of AuNPs, which implies the functionalization of the gold with different thiols (MUA, MPA or a mixture of both) and the addition of coupling agents (EDC and NHS). The formation of the bionanoconjugates includes the immobilization of antibodies with different specificities and the immobilization of different antigens or the SARS-CoV-2 virus. The LSPR phenomenon allowed the detection of a plasmon band shift after the immobilization of the biomolecules. The resulting bionanoconjugates were characterized by UV-Visible spectrophotometry, agarose gel electrophoresis and transmission electron microscopy (TEM). The functionalization with MUA and the immobilization of GenScript biomolecules allowed the obtention of the best results with a shift after these steps. The meta-analysis consisted of a search in different databases and the selection of articles that fulfilled the goal of the meta-analysis, finally, the methodological evaluation of the articles was conducted, as well as the meta-analysis to assess the sensitivity, specificity, and precision of alternative samples in the detection of SARS-CoV-2. Saliva showed the best results with an accuracy of 92.1%, indicating the possibility of being used as an alternative sample for the detection of COVID-19. The present work allowed the optimization of the stability of the bionanoconjugates and to study the possibility of using alternative samples to detect this pathology. However, it was not possible to complete the aim of developing the rapid antigen test, since the plasmonic band deviation obtained was within the error of the measurement device.
Descrição
Palavras-chave
COVID-19 SARS-CoV-2 Nanopartículas de ouro LSPR Anticorpo Antigénio Bionanoconjugados TRAg Gold nanoparticles Antibody Antigen Bionanoconjugates RAT