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Estudo da aplicabilidade de biossensores piezoelécticos para a detecção de lipopolissacáridos bacterianos
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Advisor(s)
Abstract(s)
A detecção precoce de endotoxinas (lipopolissacáridos, LPS) é de somais importância para combater eficazmente casos de bactericemia, uma das principais causas de mortalidade em unidades de cuidados intensivos por todo o mundo. Os métodos actuais são limitados no tempo de resposta ou nas condições em que terá de ocorrer a detecção.
O presente trabalho consiste no desenvolvimento de um biossensor de microbalança de cristal de quartzo (QCM) para detecção de LPS. Polimixina B (PmB), um antibiótico catiónico com elevada afinidade por endotoxinas de bactérias Gram-negativas, foi imobilizada em cristais de quarzto de 5MHz com corte AT com a ajuda de monocamadas auto-organizadas (SAM) de alcanotióis. A variação de frequência resultante da adsorção de endotoxina foi registada, bem como a de vários controlos negativos. Os resultados demonstraram a selectividade do biosensor a endotoxinas, numa gama de concentrações de 20-50 g/mL com uma SAM de ditiobis (succinimidil) propionato (DSP). A primeira iteração do sistema é selectiva, 6 vezes mais rápida em comparação a outros processos e quantitativa, mas tem uma baixa reprodutibilidade e sensibilidade.
The timely detection of endotoxins (lipopolysaccharide, LPS) is of major importance to the effective treatment of sepsis, one of the major causes of death in intensive care units around the world. The current detection methods are limited by their response time or the conditions where said detection has to occur. The present work leads to the development of a quartz crystal microbalance (QCM) biosensor for the detection of LPS. Polymyxin B (PMB), which binds to endotoxins of gram-negative bacteria, was immobilized on the alcanethiol self-assembled monolayer (SAM) modified 5 MHz AT-cut quartz crystal. The frequency shift due to endotoxin adsorption was measured, as well as that of several negative controls. The results showed the biosensor’s selectivity to endotoxins, in a range of 20-50 g/mL, using a dithiobis (succinimidill) propionate (DSP) SAM. The first iteration of this system is selective, 6 times faster than alternative methods and quantitative in nature, although it has low reprodutibility and sensitivity.
The timely detection of endotoxins (lipopolysaccharide, LPS) is of major importance to the effective treatment of sepsis, one of the major causes of death in intensive care units around the world. The current detection methods are limited by their response time or the conditions where said detection has to occur. The present work leads to the development of a quartz crystal microbalance (QCM) biosensor for the detection of LPS. Polymyxin B (PMB), which binds to endotoxins of gram-negative bacteria, was immobilized on the alcanethiol self-assembled monolayer (SAM) modified 5 MHz AT-cut quartz crystal. The frequency shift due to endotoxin adsorption was measured, as well as that of several negative controls. The results showed the biosensor’s selectivity to endotoxins, in a range of 20-50 g/mL, using a dithiobis (succinimidill) propionate (DSP) SAM. The first iteration of this system is selective, 6 times faster than alternative methods and quantitative in nature, although it has low reprodutibility and sensitivity.
Description
Dissertação de Mestrado, Engenharia Biológica, Faculdade de Engenharia de Recursos Naturais, Universidade do Algarve, 2009
Keywords
Teses Engenharia biológica Biossensor Endotoxinas Microbalança de cristal de quartzo Polimixina B