Acoustic "wall-less" test tubes for cell biology in type 2 diabetes related research

• Autor(a) principal: Lemos, Sandra
• Data de Publicação: 2008
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/3901
• topic: Levitação acústica; Insulina; Diabetes tipo 2; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

New insights into biomedicine and related areas require the parallel development of new analytical methods. A technique for chemical analysis based on the use of acoustically levitated droplets, suitable for the study of intra and extracellular reactions at single or few cells level has been developed. Microenvironments suited for specific cell types and cell reactions can be created in levitated droplets to serve as biomimetic systems. The applicability of the Airborne System is broad and circumvents limitations associated with chip-based analytical systems, such as adsorption of the analytes or optical interferences at the walls of the sample containers. Furthermore, the low attomole detection limit highlights the potential of the method. Dysfunctional adipocytes and insulin-producing pancreatic β-cells are dynamic parts in the development of type 2 diabetes. The mechanisms involved are however not yet fully understood. With this project we aim to give our contribution for the understanding of the phenomena associated with the development of the disease and at the same time provide science with new tools which can be useful in the pursuit of new knowledge, as well as in the development of novel drugs, both in type 2 diabetes or in other metabolic diseases. The system was here applied in cellular studies at the single/few cell level. Reagents are added to the droplet using flow-through dispensers, with no physical contact with the droplet. After exposure of the cells to drugs, activators or inhibitors, the cell response (or lack of response) is monitored using fluorescence imaging detection and MALDI-TOF-MS. Adipocyte lipolysis was followed. Stimulation of lipolysis with isoprenaline resulted in a pH decrease detectable by this methodology. Moreover, the addition of insulin resulted in the inhibition of isoprenaline stimulated lipolysis in adipocytes in the levitated droplet. The addition of glycated insulin to an adipocyte containing droplet revealed not to be as effective to stop lipolysis as non-glycated insulin, which is na important finding in the search for new targets in type 2 diabetes related research. Additionally, we are now attempting to develop techniques which can interrogate and monitor analytes of interest in a levitated droplet, non-destructively, ondemand, and over extended periods of time and in real-time by MS. In current work droplet fission is induced by polarization in a containerless environment. Various electrode configurations are attempted varying parameters such as number and shape, voltage, pulse length and frequency for the applied electric field in order to get the proper droplet response. Miniaturization and single-cell analysis could ultimately result in a decreasing of the number of animal trials necessary in medical research.