Temperature Influence on the Kinetics of a f-BPM Oligonucleotide Sandwich Assay

• Autor(a) principal: Macedo, Manuel José Pimentel
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/162019
• topic: continuously monitoring; low-concentrations; single-molecule resolution; affinity interactions; temperature influence; Domínio/Área Científica::Engenharia e Tecnologia::Nanotecnologia

The need for sensing techniques capable of continuously monitoring biomolecules in fields such as healthcare, food industry, and ecological systems has led to the development of a method that uses biofunctionalized particles moving freely over a sensing surface. This method offers high sensitivity and specificity for detecting low-concentration biomolecules at single-molecule resolution. It relies on monitoring changes in particle Brownian motion, recorded through brightfield microscopy, as they bind and unbind from the surface due to reversible interactions. Experiments using this affinity-based technique are usually conducted at room temperature, therefore this thesis investigates the influence of temperature changes on the kinetics of a DNA sandwich assay. Measurements spanning temperatures from 22 °C to 37 °C reveal a temperature-dependent increase in particle diffusivity, rising from 0.27739 µm2/s to 0.43890 µm2/s with increasing temperature. An assay optimization was performed where several particle blockers were tested to obtain good stability at different temperatures. Biotin-polyT outperformed biotin-mPEG as it yielded a lower background bound fraction, thus it was used as a blocker. Additionally, testing Bovine Serum Albumin (BSA) on particles resulted in a significant bound fraction increase over time. The study highlights a dependence of the bound fraction with temperature, with values ranging from 25% to nearly 100% across temperatures from 12 °C to 36 °C. Furthermore, temperature changes can influence the frequency of transition between bound and unbound states. However, the investigation into state lifetimes of the DNA 9-base-pair interaction encountered challenges, yielding unreliable data mainly due to possible unspecific and multivalent interactions. This aspect warrants further exploration. Overall, these results show that temperature is a tunable parameter and can be used to improve the Biosensing by Particle Motion (BPM) technique.