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Label free profiling of biomolecular Interactions with coupled acoustic wave biosensor and heat conduction calorimetry

Publication . Águas, Ana Catarina Pina; Santos, Rui; Ferreira, Guilherme

Understanding the thermodynamics of molecular interactions can give insights for rational drug design in modern pharmacology. Although a number of methods are available to evaluate the binding of biomolecules, only calorimetry can afford the complete thermodynamic characterization of these interactions, by being able to quantify their binding affinity, enthalpy and entropy. The recent field of biosensor technology provides the approach to meet the specificity and volume requirements that the conventional calorimetric techniques cannot afford. In this thesis we developed an acoustic wave biosensor coupled with a heat conduction calorimeter (HCC), the Microbalance/Calorimeter Flow Sensor (MCFS). The Quartz Cristal Microbalance (QCM) is combined with HCC in order to create a biosensing system able to simultaneous detect the immobilized biological compounds and the heat produced/consumed by the reaction between them. The direct measurement of heat and mass change provides thermodynamic and kinetic information fundamental to understand molecular interactions. During this dissertation, protocol strategies were developed for independent QCM and HCC measurements. The MCFS was built and evaluated through baseline assays, electrical calibration and test reactions. The system proved to be responsive and robust: baseline noises are 0.23 Hz, 30 nV and 4 mΩ, with a temperature control of 0.003 K. Electrical calibration revealed a 10 mV/W sensitivity and 3 μV detection limit of the calorimeter. In a later stage, (1) biotin-streptavidin binding; (2) glucose oxidase-glucose enzymatic reaction; (3) human serum albumin-warfarin interaction; (4) and single stranded DNA hybridization were tested to validate the experimental methodology. Although none of these reactions reveal a measurable calorimetric signal, streptavidin assays confirm the possibility of differential studies with a 4 ng/cm2 QCM sensitivity and a 3 ng precision. MCFS proved to be a potential asset for molecular interactions energetic studies, although its sensitivity needs to be improved. The current MCFS can be placed in the 4th level of technology demonstration, highlighting its potential as platform for innovative early drug discovery.

2020-05-15Doctoral thesis

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