Browsing by Author "Pereira, Eduarda"
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- 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.
- Comparison between anthropometric equipment and scanners in hand measurementPublication . Filho, P. C. Anacleto; Silva, Lincoln da; Castellucci, H. I.; Rodrigues, Matilde; Pereira, Eduarda; Pombeiro, Ana; Colim, Ana; Carneiro, Paula; Arezes, PedroAnthropometric studies have influenced the design of apparel, accessories, medical prostheses, equipment, workstations, and tools. Particularly, hand anthropometry is related to safety and adequacy of hand tools and devices. Different equipment and methods can be used to obtain body measurements with different precision and reliability levels. However, precision and reliability are not the only aspects to be considered. The present study aims to evaluate the agreement of four different pieces of equipment for anthropometric measurement of the hand, namely, anthropometer, anthropometric tape, 2D scanner, and 3D scanner. These measurement methods were compared in terms of required time, precision, complexity, and cost, for the case of two-dimensional hand measurements. Data was collected on hand length and breadth from 25 workers in North Portugal. Among the main findings, we can highlight a relatively low accuracy and higher measurement times for 2D and 3D scanners due to scanning, processing, digitalization, and calibration steps. Traditional direct measurement methods were considered the most appropriate to obtain hand length and breadth measures, as they required less time and were more accurate, less costly and complex than 2D and 3D scanner methods. These results emphasize the caution required when selecting anthropometric methods.
- Detecting toxic dinoflagellates (Dinophysis spp.) using electrochemical genosensorsPublication . Pereira, Eduarda; Barros, Piedade; Cruz, Agostinho; Santos, MarleneAquatic environments are important economic and ecological sources for human activities (e.g. fisheries, tourism, agriculture and aquaculture). However, the increase in those practices has, over the years, compromised the integrity of these ecosystems. Runoffs of terrestrial nutrients (from, for example, agricultural and industrial waste) and higher surface temperatures are believed to have transformed these ecosystems into favourable habitats for algae growth and proliferation. As a result, the frequency in phytoplankton microalgae blooms is rising worldwide. These microorganisms are mostly harmless, however certain species, namely belonging to dinoflagellates (e.g., Dinophysis spp.) produce toxins that pose a potential risk for human health. Therefore, the need for technological developments towards fast and precise detection of these toxin-producing microalgae is critical to prevent socioeconomical damages and 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. acuminata and D. acuta, which are lipophilic toxin producers responsible of diarrhetic shellfish poisoning (DSP) in humans. The design of this DNA-based sensor consists of several steps including: i) Sensing phase: consisted by a mixed self-assembled monolayer (SAM) composed by a linear DNA capture probe(DNA-CP) and mercaptohexanol (MCH) onto disposable screen-printed gold electrodes (SPGE) surface; ii) 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. The best analytical conditions were used to study the relationship between electrochemical signal and DNA target concentration, to produce the best electrochemical genosensor device.
- 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.
- 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.
- Is it possible to prevent Harmful Algal Blooms? An electrochemical genosensor that detects dinoflagellates could be the answerPublication . 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) are a very common phenomenon in the last few years that results from the accumulation of microalgae, such as dinoflagellates of the Dinophysis genus. Hence, the development of analytical technologies capable of detecting these microorganisms and thereby avoiding environmental and public health crisis, has become a major priority. Biosensors have been gaining recognition lately given their notable assets: they are fast, sensitive and allow in situ analysis.
- Plant honey origin authentication: Use of electrochemical genosensors for food safety and quality controlPublication . Morais, Stephanie; Pereira, Eduarda; Castanheira, Michelle; Santos, Marlene; Domingues, Valentina; Delerue-Matos, Cristina; Barroso, M. FátimaHoney is a natural high-quality food product consumed worldwide due to its diverse nutritional profile and beneficial medical properties. These factors, along with honey’s unique sweet taste and odour, make it one of the most popular products for a healthy diet. Nevertheless, food fraud is an increasing problem with various impacts on the economy, health, and in the environment, as honey prices are established based on its botanical origin and nutritional composition. In the European Union, honey is one of the most adulterated products found in the market. Mislabeling of a honey’s geographic origin and unethical mixing with low-grade honeys, sugars, and other substances are some of the common fraudulent practices. Hence, it is imperative to develop analytical tools to quickly, cheaply, and successfully identify fraudulent products. In this work, an electrochemical genosensor for the detection of two different plant species, Calluna vulgaris (the heather flower) and Castanea sativa (the chestnut tree), was developed and optimized. Analyzing public database platforms, a 98 base pair DNA target probe for Calluna vulgaris and a 103 base pair DNA target probe for Castanea sativa were selected and designed. The developed genosensor resulted from a linear self-assembled monolayer of the DNA capture probe of each respective species immobilized onto screen-printed gold electrodes and mercaptohexanol. To improve the genosensor’s selectivity and avoid strong secondary structures, a sandwich format for both DNA target probes was designed using a complementary fluorescein isothiocyanate-labelled DNA signaling probe. Chronoamperometry measurements were performed in a 0.13 to 2.00 nM range for both species. The developed genosensor was able to detect the hybridization reaction between the synthetic strands of each plant. Therefore, electrochemical genosensors offer a promising and cost-effective analytical tool to authenticate the botanical origin of honey, guaranteeing honey safety, quality control, and authenticity for both industries and consumers and showing potential for application in combatting fraud.
- Toxic dinoflagellates (dinophysis spp.) detection by genosensors and molecular biology approachesPublication . Pereira, Eduarda; Barros, Piedade; Cruz, Agostinho; Santos, MarleneOver the years, the marine ecosystems integrity has been compromised, due to multiple factors that disrupt the natural balance of phytoplankton. Factors such as the unregulated runoff of agricultural and industrial wastes into the aquatic environment and higher surface temperatures are believed to have transformed these ecosystems into favorable habitats for algae growth and proliferation. As a result, multiple species may produce harmful toxins that significantly affect the integrity of rivers, lakes, estuaries, and coastal areas. Although these microorganisms are mostly harmless, certain species, namely belonging to dinoflagellates (e.g. Dinophysis spp.) 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 socioeconomical damages, as well as to assess the ecological status of marine ecosystems. The goal of this work was to develop analytical approaches based on electrochemical genosensors devices in order to create a low-cost platform able to detect two dinoflagellate species from the genus Dinophysis: D. acuminata and D. acuta, which are lipophilic toxin producers responsible for diarrhetic shellfish poisoning (DSP) in humans. The design of this DNA-based sensor consists of three steps: i) Sensing phase: consisted by a mixed self-assembled monolayer composed by a linear DNA capture probe and mercaptohexanol onto disposable screen-printed gold electrodes surface; ii) 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. The best analytical conditions used to study the relationship between electrochemical signal and DNA target concentration, to produce the best electrochemical genosensor device are described in Table 1. Molecular biology tools were used to validate the electrochemical genosensor.
- 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.
- 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.