Desenvolvimento e caracterização de uma plataforma aplicada ao diagnóstico molecular através de técnicas eletroquímicas

Abstract

Infectious diseases remain one of the major global public health challenges, especially in low- income regions, where outbreaks significantly impact social and economic indicators. The continuous emergence of new pathogens, driven by globalization, climate change, and demographic shifts, reinforces the need for sensitive, rapid, and accessible diagnostic methods. In this scenario, molecular diagnostics has become an essential tool, enabling accurate pathogen detection, epidemiological monitoring, and support for clinical decision-making. However, the application of these methods in point-of-care (POCT) platforms still faces limitations related to laboratory infrastructure, cost, operational complexity, and the need for regulatory standardization. The first chapter of this thesis presents a comprehensive review (2015–2025) on nucleic acid amplification techniques, detection methods, and miniaturization strategies aimed at developing POCT platforms. The advantages and limitations of approaches such as PCR, LAMP, RPA, and other isothermal techniques, as well as systems based on fluorescence, colorimetry, and electrochemistry, are discussed. The relevance of microfluidics, including reaction chambers, continuous flow systems, and droplet-based platforms, is also emphasized as a critical component for functional integration, reagent reduction, energy savings, and portability. Despite advancements, obstacles persist, such as the complexity of miniaturization, bioreceptor instability, and regulatory barriers imposed by agencies like the FDA and ANVISA. The second chapter presents the experimental development of an integrated molecular diagnostic platform that combines digital thermal control and real-time electrochemical detection. The system utilizes Peltier modules, high-precision sensors, and control algorithms to ensure thermal stability, in addition to an optimized electrochemical cell for monitoring nucleic acid amplification. The platform allows for the execution of PCR, RT-PCR, and isothermal methods (LAMP and RPA), integrating hardware, mechanical design, control electronics, and a software interface developed in Python and React. The results obtained demonstrate adequate thermal performance, electrochemical reproducibility, and sensitivity consistent with recent studies in the literature. As a proof of concept, the amplification of the HA gene of the Influenza A H5N1 virus was used, highlighting the platform's potential as a low-cost solution for detecting nucleic acid amplification. Therefore, this thesis contributes to the advancement of the field by combining engineering, materials science, and biotechnology in the construction of a robust, accessible device compatible with the contemporary demands of epidemiological surveillance and rapid response to emerging outbreaks