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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.
- 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.
- 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.