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- Label free profiling of biomolecular Interactions with coupled acoustic wave biosensor and heat conduction calorimetryPublication . Águas, Ana Catarina Pina; Santos, Rui; Ferreira, GuilhermeUnderstanding 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.
- Deteção de proteínas virais com nanossensores óticosPublication . Águas, Ana Catarina Pina; Ferreira, Guilherme Nuno de Passos MatosO desenvolvimento de tecnologias para a deteção de vírus é um tema de grande interesse em medicina de diagnóstico. Neste contexto, os biossensores têm-se revelado importantes ferramentas bioanalíticas, na medida em que permitem efetuar deteções moleculares de forma rápida, específica, reprodutível e com baixo custo associado. No âmbito desta dissertação, estabeleceram-se metodologias para a deteção fluorescente do fator de infetividade viral (Vif) do VIH através de um nanossensor ótico funcionalizado com anticorpos recombinantes anti-Vif, fragmentos variáveis de cadeia simples 4BL. Quantum dots carboxílicos conjugados a proteínas Vif são excitados a um comprimento de onda de 405 nm, transmitindo luz fluorescente a 605 nm a um fotodetetor de silício amorfo hidrogenado com um filtro de fluorescência integrado. Para implementar um sistema de deteção sensível e eficiente, estudaram-se estratégias de ativação de superfície em chips de vidro. A variabilidade das condições experimentais de um protocolo de silanização com (3-mercaptopropil)-trimetoxisilano (MPTS) foi avaliada através da análise de ângulos de contacto e densidades moleculares de fluoresceína funcionalizada com maleimida. Estabeleceu-se que, uma incubação dos substratos por 4 h com 5% (w/v) MPTS, seguida de cura a 110 ºC por 2 h, e redução com 10 mM ditiotreitol por 30 min, promove a formação de camadas de organosilanos de qualidade com grupos tiol reativos bem orientados. Estratégias de imobilização do elemento recetor foram testadas em substratos de vidro ativados e em sistemas de microfluídica vidro/PDMS, tomando por base o tag de afinidade, glutationa S-transferase (GST), do anticorpo recombinante. Vidros funcionalizados com glutationa e anti-GST mostraram-se igualmente reativos ao anticorpo GST-4BL, resultando uma capacidade de ligação do antigénio de aproximadamente 4.76 x 1010 moléculas/cm2 de QD-Vif, o que corresponde a 15% da cobertura máxima de superfície. Dos ensaios em sistemas de microfluídica resultaram sinais fracos e poucos reprodutíveis, destacando a necessidade de desenvolver metodologias de funcionalização mais apropriadas.
- Microfluidic Organ/Body-on-a-Chip Devices at the Convergence of Biology and MicroengineeringPublication . Perestrelo, Ana Rubina; Águas, Ana C. P.; Rainer, Alberto; Forte, GiancarloRecent advances in biomedical technologies are mostly related to the convergence of biology with microengineering. For instance, microfluidic devices are now commonly found in most research centers, clinics and hospitals, contributing to more accurate studies and therapies as powerful tools for drug delivery, monitoring of specific analytes, and medical diagnostics. Most remarkably, integration of cellularized constructs within microengineered platforms has enabled the recapitulation of the physiological and pathological conditions of complex tissues and organs. The so-called organ-on-a-chip technology, which represents a new avenue in the field of advanced in vitro models, with the potential to revolutionize current approaches to drug screening and toxicology studies. This review aims to highlight recent advances of microfluidic-based devices towards a body-on-a-chip concept, exploring their technology and broad applications in the biomedical field.