Browsing by Author "Morais, Stephanie L."
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- Assessment of the antioxidant capacity of commercial coffee using conventional optical and chromatographic methods and an innovative electrochemical DNA-based biosensorPublication . Morais, Stephanie L.; Rede, Diana; Ramalhosa, Maria João; Correia, Manuela; Santos, Marlene; Delerue-Matos, Cristina; Moreira, Manuela M.; Soares, Cristina; Barroso, Maria FátimaAs one of the most popular beverages in the world, coffee is a rich source of non-enzymatic bioactive compounds with antioxidant capacity. In this study, twelve commercial coffee beverages found in local Portuguese markets were assessed to determine their total phenolic and flavonoid contents, as well as their antioxidant capacity, by conventional optical procedures, namely, ferric reducing antioxidant power and DPPH-radical scavenging assay, and non-conventional procedures such as a homemade DNA-based biosensor against two reactive radicals: HO• and H2O2. The innovative DNA-based biosensor comprised an adenine-rich oligonucleotide adsorbed onto a carbon paste electrode. This method detects the different peak intensities generated by square-wave voltammetry based on the partial damage to the adenine layer adsorbed on the electrode surface by the free radicals in the presence/absence of antioxidants. The DNA-based biosensor against H2O2 presented a higher DNA layer protection compared with HO• in the presence of the reference gallic acid. Additionally, the phenolic profiles of the twelve coffee samples were assessed by HPLC-DAD, and the main contributors to the exhibited antioxidant capacity properties were caffeine, and chlorogenic, protocatechuic, neochlorogenic and gallic acids. The DNA-based sensor used provides reliable and fast measurements of antioxidant capacity, and is also cheap and easy to construct.
- Authenticating honey origin from Natural Park of Montesinho: innovative electrochemical genosensor devices assembled in paper-based transducersPublication . Pereira, Eduarda; Morais, Stephanie L.; Seguro, Isabel; Silva, Nádia F. D.; Santos, Marlene; Pacheco, João G.; Delerue-Matos, Cristina; Barroso, M. FátimaThe Natural Park of Montesinho (NPM) has a diverse ecosystem that is home to countless species of trees and plants characteristic of this area, including Castanea sativa. The honey produced in this region is highly appreciated for its taste and nutritional value, but it also has a wide range of health benefits. The geographical origin of the honey is a very important aspect in assessing the quality and authentication of the final product. Currently, several approaches are being explored to determinethese parameters, among which deoxyribonucleic acid (DNA)-based methods stand out.
- Challenging invasive fungal infections: development of innovative electrochemical nanogenosensors to detect Candida spp.Publication . Castanheira, Michelle; Morais, Stephanie L.; Seguro, Isabel; Santos, Marlene; Lima, Luís; Pacheco, João; Barroso, M. FátimaDespite the considerable advances in the prevention and treatment of fungal infections, invasive fungus such as Candida spp., continues to be one of the major causes of morbidity and mortality. The Global Action Fund Infections reported that, annually, more than 300 million people are infected with fungal infection, from these, about 1.5 million ends up dying. Candida albicans is the most important fungal 66 opportunistic pathogen, it can cause superficial or invasive infections. Candida, often, causes superficial infections, per example in skin or mucous membranes with simple and effective treatment, however, also can break to the bloodstream and disseminate to internal organs. It has been observed among high-risk patients such as allogeneic stem-cell transplant recipients and with acute leukemia receiving highdose chemotherapy. These patients are at a heightened risk of developing infections due to the suppression of their immune system during the transplantation process. The diagnosis of systemic fungal infections persists as a problematic issue. Therefore, the development of more efficient, sensitive and specific methods for early diagnosis is need. In this study, an easy, rapid, and accurate detection methods for fungal infections in patients undergoing hematopoietic stem cell transplantation (HSCT) was designed. To address this challenge, it was developed an electrochemical nanogenosensor for the detection of Candida albicans.This nanogenosensor was assembled in an innovative low-cost electrochemical paper based analytical devices (ePAD). A sandwich hybridization reaction was used to enhaced the sensitivity of the electrochemical signal. Preliminary results demonstrated that using this nanogenosensors it was possible to detect Candida spp., in synthetic fungus sample. Despite these results, the optimization of the nanogenosensor in terms of quantifying Candida albicans is being carried out, which will be validated in future studies.The applicability in hospital environment relatively to sensitivity, accuracy, quickness response, challenges and opportunities will be discuss in future developments.
- Cytochrome P450 polymorphisms with impact in cardiovascular drugs metabolisms in European populationsPublication . Morais, Stephanie L.; Gonçalves, Tiago F.C.; Delerue-Matos, Cristina; Ferrreira-Fernandes, Hygor; Pinto, Giovanny R.; Domingues, Valentina F.; Barroso, M. FátimaThe cytochrome P450 (CYP) enzymes constitute a large polymorphic family that play a huge role in the metabolism of endogenous compounds and in the metabolization of 70–80% of all clinically prescribed medications. Among them, the CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genes are of clinical relevance, as they are highly polymorphic and implicated in the metabolism of several drugs. These genetic polymorphisms which induce variability in CYPs expression present qualitative and quantitative differences between ethnic groups and geographic regions. This review aims to evaluate the allele frequencies, genotypic distribution and predicted CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genetic variants in the European countries. Therefore, a PubMed and a Web of Science search from 1989 to 2021 on the data on the polymorphic prevalence among European countries of the CYP2C9, CYP2C19, CYP2D6 and CYP4F2 genes was performed. After excluding the duplicates, a total of 1179 studies were found. The results were structured and presented in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The present paper is an overview on the frequency CYP genetic variations, facilitating the prediction of a patient's response to medication and, consequently, enabling the selection of personalized medicine
- 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.
- Detection of alexandrium minutum dinoflagellate in environmental samples using electrochemPublication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia C.; Barroso, M. FátimaDinoflagellates are aquatic microorganisms that inhabit both salt and fresh waters. These microorganisms are mostly harmless, however, under certain conditions, some species rapidly re produce forming water blooms that not only discolor the waters but also compromise the health of every organism in the vicinity, as some dinoflagellates produce potent toxins deemed unsafe for human health (e.g. Alexandrium minutum). In this work, a disposable electrochemical genosensor for the detection of the toxic dinoflagellate Alexandrium minutum was developed. The analytical plat form methodology consisted in a sandwich format heterogeneous hybridization of complementary DNA sequences assay. The 70 bp A. minutum-specific targeting probe, the 45 bp fluorescein isothi ocyanate-labelled signaling DNA probe and the 25 bp thiolated-DNA-capture probe were designed, after analyzing public databases. To maximize the complementary DNA hybridization and to avoid the formation of strong secondary structures, a mixed mercaptohexanol (MCH) and self-assembled monolayer (SAM) A. minutum-specific DNA-capture probe was immobilized onto disposable screen-printed gold electrodes (SPGE). Using chronoamperometric measurements, the enzymatic amplification of the electrochemical signal was obtained with a concentration range from 0.12 to 1.0 nM, a LD of 24.78 pM with a RSD < 5.2 %. This electrochemical genosensor was successfully applied to the selective analysis of the targeted A. minutum specific region of denatured genomic DNA, ex tracted from toxic dinoflagellates present in the Atlantic Ocean.
- Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3Publication . Morais, Stephanie L.; Magalhães, Júlia M.C. S.; Domingues, Valentina F.; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaCardiovascular diseases are among the major causes of mortality and morbidity. Warfarin is often prescribed for these disorders, an anticoagulant with inter and intra-dosage variability dose required to achieve the target international normalized ratio. Warfarin presents a narrow therapeutic index, and due to its variability, it can often be associated with the risk of hemorrhage, or in other patients, thromboembolism. Single-nucleotide polymorphisms are included in the causes that contribute to this variability. The Cytochrome P450 (CYP) 2C9*3 genetic polymorphism modifies its enzymatic activity, and hence warfarin's plasmatic concentration. Thus, the need for a selective, rapid, low-cost, and real-time detection device is crucial before prescribing warfarin. In this work, a disposable electrochemical DNA-based biosensor capable of detecting CYP2C9*3 polymorphism was developed. By analyzing genomic databases, two specific 78 base pairs DNA probes; one with the wild-type adenine (Target-A) and another with the cytosine (Target-C) single-nucleotide genetic variation were designed. The biosensor implied the immobilization on screen-printed gold electrodes of a self-assembled monolayer composed by mercaptohexanol and a linear CYP2C9*3 DNA-capture probe. To improve the selectivity and avoid secondary structures a sandwich format of the CYP2C9*3 allele was designed using complementary fluorescein isothiocyanate-labeled signaling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometric measurements were performed at a range of 0.015–1.00 nM for both DNA targets achieving limit of detection of 42 p.m. The developed DNA-based biosensor was able to discriminate between the two synthetic target DNA targets, as well as the targeted denatured genomic DNA, extracted from volunteers genotyped as non-variant homozygous (A/A) and heterozygous (A/C) of the CYP2C9*3 polymorphism.
- Development of electrochemical genosensors for the CYPC*2 gene polymorphism detectionPublication . Sousa, António J. S. F.; Costa, Inês; Banegas, Rodrigo S.; Morais, Stephanie L.; Magalhães, Júlia; Rodrigues, Valentina; Delerue-Matos, Cristina; Ramos-Jesus, Joilson; Ferreira-Fernandes, Hygor; Pinto, Giovanny R.; Santos, Marlene; Barroso, M. FátimaPharmacogenetic studies search for heritable genetic polymorphisms that influence responses to drug therapy. Pharmacogenetics has many possible applications in cardiovascular pharmacotherapy including screening for polymorphisms to choose agents with the greatest potential for efficacy and least risk of toxicity. Pharmacogenetics also informs dose adaptations for specific drugs in patients with aberrant metabolism. Cardiovascular diseases (CVD) are considered one of the leading causes of death worldwide. To prevent cardiovascular complications and further loss of life oral anticoagulants (e.g., warfarin) are frequently prescribed to patients. Nevertheless, warfarin therapeutic agent presents narrow therapeutic windows with well-documented health risks. Some of these dose-responses are a result of specific single-nucleotide polymorphism (SNP) genetic variations present in a patient´s DNA. Among them, determined SNP in the cytochrome P4502C9 (CYP2C9), namely the CYP2C9*2, gene has been identified as dose-response altering SNP. Therefore, the need for a rapid, selective, low-cost and in real time detection device is crucial before prescribing any anticoagulant. In this work an analytical approach based on electrochemical genosensor technique is under development to create a low-cost genotyping platform able to genotype SNPs related with the therapeutic response of warfarin. Analyzing public databases, two specific 71 bp DNA probes, one with adenine (TA) and other with guanine (TG) SNP genetic variation were selected and designed. The design of this electrochemical genosensor consists of ssDNA immobilization onto gold surfaces that act as the SNPs complementary probes. The hybridization reaction is performed in a sandwich format of the complementary ssDNA, using an enzymatic scheme to amplify the electrochemical signal. The electrochemical signal was performed by using chronoamperometric technique.
- Development of electrochemical genosensors for the detection of toxic dinoflagellate alexandrium spp.Publication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia Castro; Barroso, M. FátimaMicroalgae represent a photosynthetic microorganisms’ group that inhabit both salt and fresh waters. These microorganisms, namely several species of dinoflagellates (e.g. Alexandrium spp.)1 , are mostly harmless; however, some species produce toxins classified as unsafe to human health. The uncontrolled proliferation of such species results in a hazardous occurring event designated harmful algal blooms (HAB). The effects of these episodes can lead to severe ecological and socio-economic impacts (e.g. decrease of the local tourism, fishing and port activities, the contamination or death of the nearby wildlife, discoloration of the beach coasts). Therefore, the need for a rapid, selective and in real time detection device that can monitor the presence of these microalgae in aquaculture waters is critical to prevent human, ecological and economical losses. In this work, an analytical approach based on electrochemical genosensor techniques was developed to create a low cost platform able to detect the dinoflagellates: Alexandrium minutum and Alexandrium ostenfledii. The design of this genosensor consisted of several steps including: i) Sensing phase: Creation of a mixed self assembled monolayer (SAM) composed by a linear DNA capture probe (DNA-CP) and mercaptohexanol (MCH) onto screen-printed gold electrodes (SPGE) surface; ii) Heterogenous hybridization of complementary DNA sequence (DNA target) by using a sandwich format assay with enzymatic labels and iii) Electrochemical detection by chronoamperometry using an enzymatic scheme to amplify the electrochemical signal (Figure 1). The best analytical conditions were used to study the relationship between electrochemical signal and DNA target concentration.
- Electrochemical chemically based sensors and emerging enzymatic biosensors for antidepressant drug detection: a reviewPublication . Caldevilla, Renato; Morais, Stephanie L.; Cruz, Agostinho; Delerue-Matos, Cristina; Moreira, Fernando; Pacheco, João G.; Santos, Marlene; Barroso, Maria FátimaMajor depressive disorder is a widespread condition with antidepressants as the main pharmacological treatment. However, some patients experience concerning adverse reactions or have an inadequate response to treatment. Analytical chromatographic techniques, among other techniques, are valuable tools for investigating medication complications, including those associated with antidepressants. Nevertheless, there is a growing need to address the limitations associated with these techniques. In recent years, electrochemical (bio)sensors have garnered significant attention due to their lower cost, portability, and precision. Electrochemical (bio)sensors can be used for various applications related to depression, such as monitoring the levels of antidepressants in biological and in environmental samples. They can provide accurate and rapid results, which could facilitate personalized treatment and improve patient outcomes. This state-of-the-art literature review aims to explore the latest advancements in the electrochemical detection of antidepressants. The review focuses on two types of electrochemical sensors: Chemically modified sensors and enzyme-based biosensors. The referred papers are carefully categorized according to their respective sensor type. The review examines the differences between the two sensing methods, highlights their unique features and limitations, and provides an in-depth analysis of each sensor.