Development and Experimentation of New Tools for Bioassays with Red Blood Cells

• Autor(a) principal: Coutinho, Mariana Antunes
• Data de Publicação: 2016
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/24080
• topic: Erythrocytes; Quantum Dots; Oxidative Stress; Microfluidics; Babesia ovis; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

The aim of this work was to develop new tools for handling and studying of erythrocytes. In this context, biological nanoprobes were developed, combining Quantum Dots nanoparticles with specific antibodies to detect proteins in erythrocytes. Also, a microfluidic platform was designed and constructed to evaluate the cytotoxic effect in single-cell analysis of used QDs. To determine the experimental conditions for bioconjugation, preliminary studies were performed to analyse the electrophoretic behaviour of QDs and their pH stability. It was demonstrated that the mobility depends on the surface charge of QDs and it is not affected in pH range between 5 and 8. To evaluate the cytotoxic effect of non-conjugated nanoparticles, QDs functionalized with three different groups were used: PDDA, PEG and COOH, at concentrations of 10, 50 and 100nM. The results showed that the interaction between QDs and cells induces a general oxidative stress, which increases with the concentration of QDs. The conjugation reaction between COOH-QDs and monoclonal antibodies against antigenic protein AMA1 and transmembrane Glycophorin A protein was performed by EDC/NHS chemistry. The QDs-Ab complexes were characterized by agarose gel, comparing their molecular weight and electrophoretic mobility with non-conjugated nanoparticles. Using SDS-PAGE and Immunoblotting techniques, the samples were analysed in order to evaluate their ligation properties, as well as their biological activity. The final bioconjugates were then biological tested, namely in detection of GPA protein in erythrocytes and antigenic AMA1 protein in infected B.ovis erythrocytes, through the immunofluorescence assays on slide and in solution. Comparing the results obtained with QDs and the traditional assays with FITC conjugated antibodies, QDs-Ab complexes appear to be more resistant during large periods of excitation.