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- Warfarin genetic biomarkers in VKORC1 and CYP2C9*2 genes: Advancing personalized anticoagulant therapy using electrochemical genosensorsPublication . Moreira, Tiago; Pereira, Eduarda; Costa, Inês F.; Sousa, António J.S.F.; Morais, Stephanie L.; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaThe genetic variants of vitamin K epoxide reductase complex (VKORC1) and in the cytochrome CYP2C9*2 genes have been identified to influence the anticoagulant warfarin and influence its plasmatic levels. Therefore, the pharmacogenetic information on these genes is useful for reducing warfarin adverse reaction. This work addresses the development of disposable electrochemical genosensors able of detecting single nucleotide polymorphism (SNP) in the VKORC1 and CYP2C9*2 genes. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear DNA-capture probe and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE). To improve the genosensor’s selectivity and avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the DNA allele was designed using a complementary fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. The developed electrochemical genosensors were able to discriminate between the two synthetic target DNA targets in both SNPs, as well as the targeted denatured genomic DNA. Several analytical parameters, such as DNA capture probe, 6-mercaptohexanol (as spacer) and antibody concentrations, as well as hybridization temperature and incubation time, were optimized. Using the best analytical conditions calibration curves employing increasing DNA target concentractions were ploted. Polymerase Chain Reaction (PCR), will be used for further validation of the electrochemical genosensor. Disposable electrochemical genosensors capable of detecting and distinguishing between two synthetic CYP2C9*2 and VKORC1 polymorphic sequences, with high selectivity and sensibility and in various concentrations, was developed. The functionality of these analytical approaches as alternative to the conventional genotyping methodologies can relieve the public health-care systems and, hopefully, prevent ADRs related to CDV episodes.
- Enhancing the detection of Dinophysis spp. using electrochemical genosensorsPublication . Pereira, Eduarda; Silva, Aurora; Morais, Stephanie L.; Costa-Rama, Estefanía; Moreira, Patrícia R.; Fraga-Corral, M.; Torrado, Ana M.; Rodríguez, Francisco; Barros, Piedade; Cruz, Agostinho; Delerue-Matos, Cristina; Prieto, M. A.; Simal-Gandara, J.; Silva, Nádia F. D.; Santos, Marlene; Barroso, M. FátimaHarmful algal blooms (HABs) pose a significant threat to the environment and public health. These blooms are defined by an accumulation of microscopic algae in water, and they can occur inlakes, rivers, estuaries, orcoastal areas. Factors like the unregulated runoff of agricultural and industrial wastes into the aquatic environment are believed to have transformed these ecosystems into favorable habitats for algae growth and proliferation. As a result, the frequency of these blooms is rising worldwide. Although these blooms are mostly harmless, certain species, namely dinoflagellates from the genus Dinophysis, produce toxins that pose a risk for human health. Therefore, the need for technological developments towards fast and precise detection of these toxin-producing microalgae is critical to prevent socio economical damages, as well as to assess the ecological status of marine ecosystems. In this work, an analytical approach based on an electrochemical genosensor device was developed to create a low-cost platform able to detect two dinoflagellate species from the genus Dinophysis: D.acuminataand D.acuta. The design of the DNA-based sensor involved three key steps: i) Sensing phase: consisted by a mixed self-assembled monolayer composed by a linear DNA capture probe and mercaptohexanol on to the disposable screen-printed gold electrodessurface; ii) Hybridization of complementary DNA sequence by using a sandwich format assay with enzymatic labels and iii) Electrochemical detection by chronoamperometry using an enzymatic scheme to amplify the electrochemical signal. The best analytical conditions used to study the relationship between electrochemical signal and DNA target concentration, to produce the best electrochemical genosensor device. Molecular biology tools, namely Polymerase Chain Reaction (PCR), will be used for further validation of the electrochemical genosensor to confirm its reliability. These advancements in analytical technologies contribute to the on going efforts in environmental management and public health protection by providing effective means for detectingand mitigating the risks associated with HABs. Further research and widespread implementation of these methods are required to ensure the safety and sustainability of aquatic ecosystems, safeguard public health, and facilitate proactive environmental management practices.
- Detecting BDNF gene polymorphisms using genosensors and molecular biology toolsPublication . Caldevilla, Renato; Morais, Stephanie L.; Cruz, Agostinho; Barroso, M. Fátima; Santos, MarleneMajor depressive disorder (MDD) is a complex and highly prevalent psychiatric disorder with a high impact on quality of life and negative effects on mood, behaviour, and cognition. Currently, the main medical treatment for MDD is antidepressant medication. The selective serotonin reuptake inhibitors (SSRIs), including fluoxetine, sertraline, fluvoxamine, paroxetine and citalopram, are the most commonly prescribed drugs. However, as with all antidepressant treatments, about 30–40% of MDD patients do not respond sufficiently to SSRIs. Several factors, including genetic factors, play important roles in antidepressant responses. BDNF is one of the most investigated genes regarding depression and antidepressant response. In fact, the rs6265 (Val66Met) non-synonymous polymorphism, has been demonstrated to decrease pro-BDNF processing, and consequently affect the dependent secretion of BDNF. Curiously, carriers of Met-allele have been described to have smaller hippocampal volume, either in healthy or depressed patients. So, it is likely they can contribute to the interindividual differences in patient´s responses to antidepressants. Therefore, it is crucial to develop methodologies to predict the individual antidepressant response. In this work, two analytical approaches based in molecular biology and electrochemical genosensor techniques are under development to create a low-cost genotyping platform able to genotype BDNF SNPs related with antidepressants therapeutic response.