Browsing by Author "Rodrigues, Valentina"
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- 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.