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Projeto de investigação
Novos biomarcadores de doenças cardiovasculares: deteção rápida de Homoscisteína-tiolactona e implicações bioquímicas em amostras de sangue humano e no diagnóstico clínico
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Microelectrode sensor for real-time measurements of nitrite in the living brain, in the presence of ascorbate
Publication . Monteiro, Tiago; Dias, Cândida; Lourenço, Cátia F.; Ledo, Ana; Barbosa, Rui M.; Almeida, M. Gabriela; UCIBIO - Applied Molecular Biosciences Unit; MDPI - Multidisciplinary Digital Publishing Institute
The impaired blood flow to the brain causes a decrease in the supply of oxygen that can result in cerebral ischemia; if the blood flow is not restored quickly, neuronal injury or death will occur. Under hypoxic conditions, the production of nitric oxide (• NO), via the classical L-arginine–• NO synthase pathway, is reduced, which can compromise• NO-dependent vasodilation. However, the alternative nitrite (NO2− ) reduction to• NO, under neuronal hypoxia and ischemia conditions, has been viewed as an in vivo storage pool of• NO, complementing its enzymatic synthesis. Brain research is thus demanding suitable tools to probe nitrite’s temporal and spatial dynamics in vivo. In this work, we propose a new method for the real-time measurement of nitrite concentration in the brain extracellular space, using fast-scan cyclic voltammetry (FSCV) and carbon microfiber electrodes as sensing probes. In this way, nitrite was detected anodically and in vitro, in the 5–500 µM range, in the presence of increasing physiological concentrations of ascorbate (100–500 µM). These sensors were then tested for real-time and in vivo recordings in the anesthetized rat hippocampus; using fast electrochemical techniques, local and reproducible transients of nitrite oxidation signals were observed, upon pressure ejection of an exogenous nitrite solution into the brain tissue. Nitrite microsensors are thus a valuable tool for investigating the role of this inorganic anion in brain redox signaling.
Development of point-of-care tests using enzyme (NiR & PON) based electrochemical biosensors
Publication . Monteiro, Tiago Carvalho; Almeida, Maria Gabriela; Macedo, Maria dos Anjos
The present thesis focuses on the application of two distinct enzymes – cytochrome c nitrite reductase (ccNiR) and paraoxonase 1 (PON1) – as the key elements in the development of two electrochemical analytical devices, namely a 3rd generation miniaturized biosensor for nitrite quantification, and a 1st generation biosensor for the detection of homocysteine thiolactone (HTL). The analytical surveillance of nitrite is crucial in the management of public health and environmental safety. Additionally, it is considered an important marker of proper endothelial function and a common indicator of urinary tract infection. The endogenously produced HTL has been implied in the pathophysiology of the vascular system, making the detection and quantification of this metabolite necessary in the study and diagnosis of processes related with cardiovascular diseases.
The biocompatibility of ccNiR with carbon conductive inks and low-temperature curing processes was previously demonstrated on standard pyrolytic graphite electrodes. In this work, the next step in the development of a disposable miniaturized nitrite biosensor was performed, by manually applying a carbon ink and ccNiR composite on carbon-screen printed electrodes. The analytical performance was evaluated by cyclic voltammetry, and the bioelectrodes obtained in this way showed good catalytic response towards nitrite, although with poor reproducibility. Future automated fabrication processes could improve this. Nonetheless, the successful incorporation of the catalytically competent and stable enzyme, indicate a putative application for future co-printing with the transducing electrode. Due to the need of an oxygen-free environment, two enzymatic deoxygenation systems were integrated with the transducer: one based on the well-known couple glucose oxidase/catalase, while the other was a novel system based on multicopper oxidases. Overall, the combination of these enzymes with ccNiR on carbon screen-printed electrodes resulted in miniaturized nitrite voltammetric biosensors capable of operating at low potentials, under ambient air.
Additionally, for the measurement of nitrite dynamics in cerebral tissue, carbon fiber microelectrodes and ceramic-based platinum microelectrodes arrays were employed as transducers in the development of microscale (bio)sensing platforms. Preliminary data showed that exogenous nitrite real-time clearance could thus be detected in situ and in vivo.
Owing to the putative relation between lower PON1 activity and the greater risk of developing diseases with an inflammatory component, a novel electrochemical protocol for PON1 status determination was developed. Additionally, the potential role of the enzyme as the bioreceptor in an HTL voltammetric biosensor was also addressed.
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Fundação para a Ciência e a Tecnologia
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OE
Número da atribuição
PD/BD/109687/2015