Percorrer por autor "Almeida, M. Gabriela"
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- Construction of effective disposable biosensors for point of care testing of nitritePublication . Monteiro, Tiago; Rodrigues, Patrícia R.; Gonçalves, Ana Luisa; Moura, José J.G.; Jubete, Elena; Añorga, Larraitz; Piknova, Barbora Piknova; Schechter, Alan N.; Silveira, Célia M.; Almeida, M. Gabriela"In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes’ system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40 °C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR’s robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca. -0.4 V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1 – 6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M-1 cm-2 with a linear response range 0.7 – 370 μM."
- Current clinical framework on nitric oxide role in periodontal disease and blood pressurePublication . Lima, Leonel; Gaspar, Sara; Rocha, Bárbara S.; Alves, Ricardo; Almeida, M. GabrielaObjectives: In this review, we explored potential associations between NO and its derivatives, nitrite and nitrate, with periodontal and cardiovascular diseases, with special emphasis on the former. By providing a state-of-the-art and integrative understanding of this topic, we aimed to shed light on the potential role of these three nitrogen oxides in the periodontitis-hypertension nexus, identify knowledge gaps, and point out critical aspects of the experimental methodologies. Materials and methods: A comprehensive literature review was conducted on human salivary and plasma concentrations of nitrate and nitrite, and their impact on periodontal and cardiovascular health. Results: A nitrate-rich diet increases nitrate and nitrite levels in saliva and plasma, promoting oral health by favorably altering the oral microbiome. Chlorhexidine (CHX) mouthrinses disrupt the nitrate-nitrite-NO pathway, reducing NO bioavailability, and potentially affecting blood pressure. This is because CHX eliminates nitrate-reducing bacteria, which are essential for NO production. Although endogenous NO production may be insufficient, the nitrate-nitrite-NO pathway plays a critical role in maintaining appropriate endothelial function, which is balanced by the microbiome and dietary nitrate intake. Dietary nitrate supplementation may lead to beneficial changes in the oral microbiome, thereby increasing the NO bioavailability. However, NO bioavailability can be compromised by reactive oxygen species (ROS) and the uncoupling of endothelial nitric oxide synthase (eNOS), leading to further ROS generation and creating a detrimental cycle. Studies on NO and periodontal disease have shown increased nitrite concentrations in patients with periodontal disease, although these studies have some methodological limitations. In terms of blood pressure, literature suggests that CHX mouthrinses may reduce the capacity of nitrate-reducing bacteria, potentially leading to an increase in blood pressure. Conclusions: Several studies have suggested an association between NO levels and the development of cardiovascular and periodontal diseases. However, the exact mechanisms linking these diseases remains to be fully elucidated. Clinical relevance: Nitric oxide (NO) is a signaling molecule that plays a crucial role in several physiological processes such as vascular homeostasis, inflammation, immune cell activity, and pathologies such as hypertension and periodontitis.
- Development of new analytical tools for monitoring of cardiovascular disease markers – towards the detection of homocysteine-thiolactonePublication . Monteiro, Tiago; Oliveira, Francisco; Silveira, Célia M.; Pereira, Sofia A.; Almeida, M. Gabriela
- Diagnosis of SARS-Cov-2 infection by RT-PCR using specimens other than naso- and oropharyngeal swabs : a systematic review and meta-analysisPublication . Moreira, Vânia M.; Mascarenhas, Paulo; Machado, Vanessa; Botelho, João; Mendes, José João; Taveira, Nuno; Almeida, M. GabrielaThe rapid and accurate testing of SARS-CoV-2 infection is still crucial to mitigate, and eventually halt, the spread of this disease. Currently, nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the recommended standard sampling techniques, yet, these have some limitations such as the complexity of collection. Hence, several other types of specimens that are easier to obtain are being tested as alternatives to nasal/throat swabs in nucleic acid assays for SARS-CoV-2 detection. This study aims to critically appraise and compare the clinical performance of RT-PCR tests using oral saliva, deep-throat saliva/posterior oropharyngeal saliva (DTS/POS), sputum, urine, feces, and tears/conjunctival swab (CS) against standard specimens (NPS, OPS, or a combination of both). In this systematic review and meta-analysis, five databases (PubMed, Scopus, Web of Science, ClinicalTrial.gov and NIPH Clinical Trial) were searched up to the 30th of December, 2020. Case-control and cohort studies on the detection of SARS-CoV-2 were included. The methodological quality was assessed using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS 2). We identified 1560 entries, 33 of which (1.1%) met all required criteria and were included for the quantitative data analysis. Saliva presented the higher accuracy, 92.1% (95% CI: 70.0–98.3), with an estimated sensitivity of 83.9% (95% CI: 77.4–88.8) and specificity of 96.4% (95% CI: 89.5–98.8). DTS/POS samples had an overall accuracy of 79.7% (95% CI: 43.3–95.3), with an estimated sensitivity of 90.1% (95% CI: 83.3–96.9) and specificity of 63.1% (95% CI: 36.8–89.3). The remaining index specimens could not be adequately assessed given the lack of studies available. Our meta-analysis shows that saliva samples from the oral region provide a high sensitivity and specificity; therefore, these appear to be the best candidates for alternative specimens to NPS/OPS in SARS-CoV-2 detection, with suitable protocols for swab-free sample collection to be determined and validated in the future. The distinction between oral and extra-oral salivary samples will be crucial, since DTS/POS samples may induce a higher rate of false positives. Urine, feces, tears/CS and sputum seem unreliable for diagnosis. Saliva testing may increase testing capacity, ultimately promoting the implementation of truly deployable COVID-19 tests, which could either work at the point-of-care (e.g. hospitals, clinics) or at outbreak control spots (e.g., schools, airports, and nursing homes).
- Effect of saccharomycin, a natural Saccharomyces cerevisiae biocide, on Hanseniaspora guilliermondii cells surfacePublication . Calvário, Joana; Silva, Nelly; Almeida, M. Gabriela; Albergaria, Helena; Eaton, Peter; Macedo, Anjos; Caldeira, Jorge
- Inhibition-based biosensor for cyanide detection – a preliminary studyPublication . Coelho, Ana R.; Monteiro, Tiago; Viana, Ana S.; Almeida, M. Gabriela
- Microelectrode sensor for real-time measurements of nitrite in the living brain, in the presence of ascorbatePublication . Monteiro, Tiago; Dias, Cândida; Lourenço, Cátia F.; Ledo, Ana; Barbosa, Rui M.; Almeida, M. GabrielaThe 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.
- New PON1-based biosensor for the detection of homocysteine-thiolactone in human plasmaPublication . Monteiro, Tiago; Oliveira, Francisco; Silveira, Célia M.; Pereira, Sofia A.; Almeida, M. Gabriela
- Nitrite quantification in processed meat using an enzyme biosensor, a portable reader and a smartphone : a facile and accurate approachPublication . Surace, Luca; Zumpano, Rosaceleste; Polli, Francesca; Gajdar, Július; Mazzei, Franco; Almeida, M. GabrielaThe control of nitrite levels in foodstuff is essential for ensuring human safety. However, the protocols are long and require lab equipment (spectrophotometer and centrifuge) not available in industrial or commercial settings. Herein, we adapted an electrochemical nitrite biosensor utilizing the enzyme cytochrome c nitrite reductase (ccNiR) previously developed in our lab, to meet the analytical requirements of cured meats, and validated it using real samples from different brands. The enzyme was immobilized on a disposable carbon screen-printed electrode (SPE) modified with multi-walled carbon nanotubes (MWCNTs) and connected to a hand-held potentiostat coupled to a smartphone. The carbon nanotubes suspension was optimized using chemically friendly solvents and the SPE surface was electrochemically characterized using ferricyanide as a redox probe, to fine-tune the modification formulation. The nitrite biosensor delivered a sensitivity of 0.0103 μA/μM, a linear range from 5 to 100 μM, and a lower detection limit (LOD) of 3.2 μM. Subsequently, the biosensor was tested for its ability to detect nitrites in various processed meat samples, considering factors such as type and origin. The validation of the electrochemical biosensor was conducted using the gold standard Griess method and HPLC, although the first one failed to detect nitrites in some samples due to matrix interferences. Accordingly, the nitrite biosensor performed well, providing highly accurate and precise results (smaller SD), even in non-centrifuged samples, making it possible to control the meat quality at any point of the chain distribution.
- Nitrite reduction in bacteria : a comprehensive view of nitrite reductasesPublication . Besson, Stéphane; Almeida, M. Gabriela; Silveira, Célia M.The last years have witnessed a steady increase of social and political awareness for the need of studying, monitoring, and controlling several anthropological activities that are dramatically impacting the environment and human health. The increasing turnover rates of the nitrogen cycle across the Planet are of major concern, so the understanding of the biological, chemical, and physical processes associated with the biogeochemical nitrogen cycle has been attracting the attention of several scientific disciplines. For many years, the primary focus has been the so-called “dissimilatory reduction of nitrate”, which refers to the stepwise conversion of nitrate into molecular nitrogen, closely followed by the assimilatory nitrate reduction pathway, which allow nitrogen incorporation into biomolecules. The contribution of bioinorganic chemists to better understand the enzymology underlying these two branches of the N-cycle has been remarkable. The constant development of mechanistic, structural, and biological tools has been keeping this bioinorganic chemistry field very active, making it a highly relevant research area still today. In this paper, we review the state-of-the-art in both dissimilatory and assimilatory nitrite reducing enzymes, highlighting the structural peculiarities of the different metalloenzymes involved in this step.