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- Construction and optimization of an electrochemical genosensor for the detection of BDNF gene Val66Met polymorphismPublication . Caldevilla, Renato; Santos, Marlene; Barroso, M. FátimaMajor depression disorder (MDD) is the most prevalent psychiatric condition worldwide. Currently, antidepressants constitute the main pharmacological treatment for this condition. Despite the growing arsenal of antidepressant medications, almost half of MDD patients have insufficient remission rates during their treatments. A growing body of scientific evidence points to genetic factors as having a crucial role in patients’ response to antidepressant treatments. One of these genetic components is the BDNF gene, that codes for the BDNF neurotrophin, an important player in neuroplasticity and neurogenesis.
- Design of an electrochemical genosensor for the BDNF gene polymorphism sequence detection using an enzymatic labelled DNA probePublication . Caldevilla, Renato; Morais, Stephanie; Carvalho, Serafim; Medeiros, Rui; Delerue-Matos, Cristina; Cruz, Agostinho; Santos, Marlene; Barroso, M. FátimaThe BDNF gene is associated with high degrees of variability in antidepressant treatments. The Val66Met polymorphism is widely known as a source of this variability, warranting growing interest in genotyping patients that undergo antidepressant treatment to better suit their needs. This paper reports on an electrochemical genosensing platform, based on gold electrodes, capable of detecting this polymorphism, through the use of synthetic enzymatic labelled DNA-probes for 2 different BDNF alleles. The sensor showed promising results, and its applicability to real samples is currently being tested.
- Is there a link between NRF2 and depression?Publication . Santos, Marlene; Fonseca, Débora; Caldevilla, Renato; Barroso, M. Fátima; Cruz, AgostinhoDepression is a common mental health disorder that affects millions of people worldwide. Recent studies have highlighted the role of oxidative stress and inflammation in the pathogenesis of depression. NRF2 is a transcription factor that plays a crucial role in cellular defense against oxidative stress by binding to antioxidant response elements (AREs) located in the promoter region of various phase II antioxidant enzymes and stress-responsive enzymes. Decreased Keap1-Nrf2 signaling has been implicated in the development of mood disorders, such as Major Depressive Disorder. Therefore, this review aims to evaluate the in vitro and in vivo evidence of the involvement of Nrf2 in depression. A review was conducted on the PubMed database for articles published until March 8, 2022 Papers that evaluated NRF2 in animals and/or cell lines with depression and were published in English were included in the review. Studies that addressed other diseases/topics, systematic reviews, and those that did not address NRF2 were excluded. Quality assessment was performed according to Koch et al., 2022. Out of the 203 possibly relevant abstracts found through the PubMed search, 45 papers were included in the review. The results suggest that Nrf2 levels tend to decrease in animals exposed to oxidative stress or depressive behavior. When animals were treated with antidepressants or anti-inflammatory drugs, Nrf2 levels increased. Additionally, the study found that IL-10 and BDNF were key elements that were positively influenced by Nrf2 levels, protecting against oxidative stress through Keap1/Nrf2. The findings suggest that Nrf2 activation may play a crucial role in controlling oxidative stress and inflammation during depression. Furthermore, it provides evidence of the involvement of Nrf2 in depression and highlights its potential as a therapeutic target. However, further studies on clinical samples are necessary to evaluate NRF2’s putative effect in depression and antidepressant response.
- 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.
- Design and optimization of an electrochemical genosensing platform for BDNF Val66Met polymorphism detectionPublication . Caldevilla, Renato; Santos, Marlene; Barroso, M. FátimaMajor depressive disorder (MDD) is a debilitating and highly prevalent psychiatric illness. Antidepressant drugs (AD) have remained the main pharmacological treatment for this condition, and since their discovery and despite their high efficacy, insufficient remission rates and treatment-resistant depression remain a cause of concern for clinicians. The BDNF gene is an extensively studied gene regarding depression and AD response rates. Moreover, the rs6265 (Val66Met) non-synonymous single nucleotide polymorphism (SNP) has been linked to variable remission rates to ADs. Therefore, there is a growing interest in genotyping approaches to detect SNPs, such as the Val66Met, to better suit patients’ needs. Current SNP identification procedures are based on the polymerase-chain reaction (PCR) technique. This methodology, although extremely efficacious, is time-consuming, requires expensive equipment and highly trained personnel. Thus, the development of cheaper, faster and lower-cost genotyping tools, such as electrochemical genosensors, capable of detecting an electrochemical signal from a hybridization event between DNA probes, is warranted. To develop a genotyping platform based on the electrochemical biosensing principles, capable of distinguishing Val66Met genotypes. 2 specific target DNA sequences of interest from the Val66Met SNP were selected and designed. Employing screen-printed gold electrodes (SPGE) as transducers, the genosensor development protocol included four stages: pre-treatment; sensing phase; sandwich DNA hybridization and electrochemical detection. The electrochemical detection was carried out through chronoamperometry techniques. Several experimental conditions, such as capture probe and antibody concentrations, were successfully optimized. Furthermore, a calibration curve employing different target concentrations was obtained. The DNA sequence complementary to the capture probe showed greater current signals than the non-complementary, as expected. The developed methodology showed consistent results, with the genosensor exhibiting the ability to distinguish between both DNA targets. A linear relationship between DNA target concentration and current intensity was achieved between 0.10 nmolL-1 to 2.0 nmolL-1.
- 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.
- 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.
- 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.
- Electrochemical genosensor for the detection of Alexandrium minutum dinoflagellatesPublication . Morais, Stephanie L.; Barros, Piedade; Santos, Marlene; Delerue-Matos, Cristina; Gomes, Andreia C.; Barroso, M. FátimaThis work addresses the development of a disposable electrochemical genosensor for the detection of the toxic dinoflagellate, Alexandrium minutum. Analyzing public databases, a specific 70 bp DNA probe, targeting A. minutum, was selected and designed. The genosensor methodology implied the immobilization of a A. minutum-specific DNA-capture probe onto screen-printed gold electrodes (SPGE). To improve both the selectivity and to avoid strong secondary structures, that could hinder the hybridization efficiency, a sandwich format of the A. minutum gene was designed using a fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. Using this electrochemical genosensor, a oncentration range from 0.12 to 1.0 nM, a LD of 24.78 pM with a RSD <5.2% was determined. The genosensor was successfully applied to the selective analysis of the targeted A. minutum specific region denatured genomic DNA extracted from toxic dinoflagellates present in the Atlantic Ocean.
- 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.
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