Cardiovascular pharmacogenomics-related genetic biomarkers: electrochemical genosensors for personalized medicine

Barbosa, Tiago Manuel Moreira

http://hdl.handle.net/10400.22/29538

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Authors

Barbosa, Tiago Manuel Moreira

Advisor(s)

Barroso, M. Fátima

Santos, Marlene

Vieira, Mónica

Abstract(s)

Cardiovascular diseases (CVDs) are one of the leading causes of death worldwide, and the anticoagulant warfarin is routinely used to treat and control CVDs. However, due to several individual factors, including genetic variations, as singles nucleotide polymorphisms (SNPs), drug’s efficacy varies between individuals. Moreover, given warfarin’s narrow therapeutic index, genotype-guided approaches have become critical in establishing personalized doses. Since conventional technologies for SNP detection such as Polymerase Chain Reaction or Real-Time Polymerase Chain Reaction (PCR or RT-PCR) are costly, time-consuming and demand skilled specialists, novel disposable electrochemical genosensors have been developed and optimized to facilitate SNP detection. In this project, two electrochemical genosensors were designed to detect both the -1639 G>ASNP in the vitamin K epoxide reductase complex (VKORC1) gene, important in the anticoagulant effect, and the CYP2C9 g.9133C>T gene variation involved in the metabolization of warfarin. The construction of these genosensors employed the formation of binary mixed self-assembled monolayers (SAMs) composed by DNA capture probes and mercaptohexanol (MCH), immobilized onto gold screen-printed electrodes (AuSPE). Furthermore, these genosensors employed a sandwich format hybridization technique (to increase sensitivity) that included the use of a fluorescein-labeled signaling probe and the enzymatic amplification of the electrochemical signal, measured via chronoamperometry. To obtain genomic DNA useful to be used in the genosensors, PCR technique was used to amplify genomic DNA from biological samples. Then, these samples were tested in the genosensors. The genosensors were able to detect genomic DNA associated to the CYP2C9 and VKORC1 genes, but only distinguished SNPs existent in the CYP2C9 gene displaying the ability to help in establishing the optimal warfarin dosage, providing a cost-effective alternative to the conventional procedures. This technique has the potential to improve patient care in cardiovascular medicine by tackling the problems associated with CVD therapy and the genetic heterogeneity of medication response.