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- 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.
- 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.
- 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.
- Warfarin pharmacogenomics: Designing electrochemical DNA-based sensors to detect CYP2C9*2 gene variationPublication . Barbosa, Tiago; Morais, Stephanie L.; Pereira, Eduarda; Magalhães, Júlia M. C. S.; Domingues, Valentina F.; Ferreira-Fernandes, Hygor; Pinto, Giovanny; Santos, Marlene; Barroso, Maria Fátima; Santos, MarleneThe CYP2C9 enzyme is involved in the metabolism of warfarin. The CYP2C9 gene harbors several single-nucleotide polymorphisms (SNPs), including CYP2C9*2 (rs1799853), which is known to affect warfarin’s therapeutic response. So, it is important to develop analytical tools capable of genotyping these SNPs to adjust warfarin’s therapeutic outcomes. In this work, an electrochemical DNA-based sensor was constructed and optimized for the detection of the CYP2C9*2 polymorphism. Methods: Using bioinformatic database platforms, two 71 base pair DNA target probes with the polymorphic variants A and G were chosen and designed. A DNA-based sensor was composed by mercaptohexanol and the CYP2C9*2 DNA capture probe in a self-assembled monolayer connected to screen-printed gold electrodes. Two independent hybridization events of the CYP2C9*2 allele were designed using complementary fluorescein-labeled DNA signaling to improve selectivity and avoid secondary structures. Three human samples with the homozygous variant (G/G) and non-variant (A/A) and heterozygous (G/A) genotypes were amplified by PCR and then applied to the developed genosensor. Results: Chronoamperometry measurements were performed for both polymorphic probes. A calibration curve in the 0.25 to 2.50 nM (LOD of 13 pM) and another in the 0.15 to 5.00 nM range (LOD of 22.6 pM) were obtained for the homozygous non-variant and variant probes, respectively. This innovative tool was capable of identifying the hybridization reaction between two complementary strands of immobilized DNA, representing a genotyping alternative to the classical PCR methodology. Conclusions: The developed electrochemical DNA-based sensor was able to discriminate two synthetic SNP target sequences (Target-A and Target-G) and detect, with specificity, the three patients’ genotypes (G/G, G/A, and A/A). This tool is therefore a promising, sensitive, and cost-effective analytical way to determine and discriminate an individual’s genotype and predict the appropriate warfarin dose.
- Ensuring food safety: electrochemical genosensors for the authentication of plant honey originPublication . Morais, Stephanie L.; Pereira, Eduarda; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. FátimaHoney is a high-quality and natural ingredient often consumed because of its unique sweet taste and multiple therapeutic and nutritional benefits. These properties are normally intrinsically connected to the regional flora from which the plant pollen is harvested. Hence, the botanical and geographical origins of honeys play a substantial role in the end product's composition. With the recent interest in natural food products many businesses, including the honey industry, have observed a significant expansion in production and market value. However, honey is susceptible to adulteration and, as more and more adulterated honeys are being found on the global market, it is difficult to monitor the safety and quality of all honey products, making honey fraud a serious problem for both consumers and the food industry. Some of the most prevalent fraudulent acts include mislabeling the botanical and geographic origin of honeys and mixing pure honey with inferior honeys, processed sugars, syrups, and other substances. Thus, there is a need to develop an analytical tool that can quickly, cheaply, and easily guarantee the quality and safety of honey. In this study, an electrochemical genosensor, based on a sandwich format DNA hybridization reaction between two complementary probes, for the detection and quantification of two pollen producing plant species: Erica arborea and Castanea sativa were designed and optimized. Analyzing public databases, two synthetic DNA-target sequences capable of unequivocally detecting the pollen from E. arborea and C. sativa were selected and designed. Their complementary oligonucleotide probes were also designed and cut into two distinct sequences: the DNA-capture and DNA-signaling probes. In order to recognize the two plant species in real honey and pollen DNA samples and optimize the hybridization procedure, a mixed selfassembled monolayer of each plant species’ DNA-capture probe and mercaptohexanol was used. Then, the electrochemical signal was enzymatically amplified using chronoamperometric measurements. A concentration range of 0.03 to 2.00 nM for E. arborea and 0.03 to 1.00 nM for C. sativa were obtained. The developed sensors were successfully applied for the detection and quantification of the two plant species in real plant samples and, thus, indicate the botanic origin of honeys. Therefore, the developed electrochemical genosensors are a viable and affordable analytical tool to authenticate the botanical origin of honeys, ensuring honey quality and safety for consumers as well as the industries.
- 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.