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Piezoelectric biosensors for molecular analysis in biomedical applications
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Abstract(s)
A tecnologia de biossensores label–free trouxe grandes vantagens ao estudo de
interacções biomoleculares, principalmente a biomoléculas sem actividade enzimática,
que requerem métodos experimentais não directos ou complexos. Os sensores
piezoeléctricos são sensores acústicos capazes de detectar eventos biológicos em
tempo real e de forma selectiva.
Este trabalho apresenta algumas das potencialidades destes sensores como sensores
microgravimétricos, nomeadamente na construção de imunosensores. Microbalanças
de cristal de quartzo (QCM), com frequência de ressonância de 10 MHz,
foram utilizadas para detectar o factor de infecciosidade viral (Vif) do HIV–1 através
de um anticorpo recombinante de fragmento variável (scFv) imobilizado. A
cinética de interacção foi analisada e comparada com valores obtidos com sensores
de menor frequência.
As outras aplicações estudadas baseiam-se no estudo de interacções proteína–
DNA através da análise de impedância em tempo real. RXR e Haa1 são factores de
transcrição que regulam a expressão de genes por ligação a sequências específicas
do DNA. As sequências alvo foram imobilizadas nos sensores de 10 MHz. O estudo
cinético da interacção sugere que a ligação de homodímeros de RXR à sequ encia
de DNA específica é dependente da força iónica do meio.
Por outro lado, o estudo das propriedades reológicas da interacção de Haa1
e a sua sequência específica, HRE, demonstrou que uma simples mutação por substituição, no DNA, pode alterar as propriedades físicas do complexo, sugerindo
diferenças conformacionais entre mutantes.
Este tipo de sensores piezoeléctricos demonstrou ser uma ferramenta útil e
versátil no estudo de interacções macromoleculares, principalmente se associado a
técnicas de impedância e modelação.
The study of biomolecular interactions experienced an improvement with label– free biosensor technology development, particularly to the biomolecules with non– enzymatic activity. The piezoelectric sensors are acoustic sensors capable of detecting biological events in a real time and selective way. This thesis presents some of the potential of these sensors such as microgravimetric sensors, namely in immunosensors. The quartz crystal microbalance (QCM) with 10 MHz resonance frequency was used to detect HIV–1 viral infectivity factor (Vif) by a single–chain variable fragment antibody (scFv) immobilized on sensor surface. The interaction kinetics was analyzed and compared with values obtained from lower resonance frequency sensors. Applications based on protein–DNA interactions was studied by real time impedance analysis. The protein models used were RXR and Haa1, transcription factors (TF) that regulate gene expression by binding to specific DNA sequences. The target DNA sequences were immobilized on sensor 10 MHz and the interaction with transcription factor was promoted. The results of kinetic study suggests that the binding of RXR homodimers to the specific DNA sequence is salt dependent. On the other hand, a single base substitution mutation in specific DNA sequence of Haa1 transcription factor changed the rheological properties of the complex TF–DNA, suggesting conformational differences. The QCM proved to be an useful and versatile tool in the study of macromolecular interactions, especially if associated with impedance analysis and physical models techniques.
The study of biomolecular interactions experienced an improvement with label– free biosensor technology development, particularly to the biomolecules with non– enzymatic activity. The piezoelectric sensors are acoustic sensors capable of detecting biological events in a real time and selective way. This thesis presents some of the potential of these sensors such as microgravimetric sensors, namely in immunosensors. The quartz crystal microbalance (QCM) with 10 MHz resonance frequency was used to detect HIV–1 viral infectivity factor (Vif) by a single–chain variable fragment antibody (scFv) immobilized on sensor surface. The interaction kinetics was analyzed and compared with values obtained from lower resonance frequency sensors. Applications based on protein–DNA interactions was studied by real time impedance analysis. The protein models used were RXR and Haa1, transcription factors (TF) that regulate gene expression by binding to specific DNA sequences. The target DNA sequences were immobilized on sensor 10 MHz and the interaction with transcription factor was promoted. The results of kinetic study suggests that the binding of RXR homodimers to the specific DNA sequence is salt dependent. On the other hand, a single base substitution mutation in specific DNA sequence of Haa1 transcription factor changed the rheological properties of the complex TF–DNA, suggesting conformational differences. The QCM proved to be an useful and versatile tool in the study of macromolecular interactions, especially if associated with impedance analysis and physical models techniques.
Description
Keywords
Biosenssores Imunossensor Análise de impedância aInterações proteína-DNA