Browsing by Author "Ferreira-Fernandes, Hygor"
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- 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.
- VKORC1 gene polymorphism as cardiovascular biomarker: Detection by electrochemical genosensorsPublication . Costa, Inês; J. S. F. Sousa, António; 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. FátimaWarfarin is an anticoagulant generally used to prevent cardiovascular diseases. Since of the low therapeutic index of warfarin and frequent complications of prevention or treatment, significant differences in individual doses of warfarin are needed to achieve prophylactic and therapeutic ranges. Recent studies have been reporting that genetic variants of vitamin K epoxide reductase complex (VKORC1) influence the response to warfarin and doses [9]. So, the genetic and pharmacogenetic information of the major cardiovascular diseases plays an important role in the identification of the cardiovascular risk factors and in the diagnosis and treatment of these conditions. This work addresses the development of a disposable electrochemical genosensor able of detecting single nucleotide polymorphism (SNP) in the VKORC1 gene. Analysing public databases, two specific 52 bp DNA probes, one with adenine (TA) and another with guanine (TG) SNP genetic variation were selected and selected and designed. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear VKORC1 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 VKORC1 allele was designed using a complementary fluorescein isothiocyanate-labelled signaling DNA probe and enzymatic amplification of the electrochemical signal. Preliminary studies indicate that differences in the electrochemical answers were obtained depending of the hybridization reaction format. In fact, higher electrochemical intensities were measured when the hybridization reaction was performed with a complementary DNA (without SNPs). These results suggested that the sensor is able to discriminate between the complementary DNA and single base mismatch targets having a great potential for the DNA polymorphism analysis.
- 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.