Morais, Stephanie L.Magalhães, Júlia M.C. S.Domingues, Valentina F.Delerue-Matos, CristinaRamos-Jesus, JoilsonFerreira-Fernandes, HygorPinto, Giovanny R.Santos, MarleneBarroso, M. Fátima2023-09-112023-09-112023-06-01Morais, S. L., Magalhães, J. M. C. S., Domingues, V. F., Delerue-Matos, C., Ramos-Jesus, J., Ferreira-Fernandes, H., Pinto, G. R., Santos, M., & Barroso, M. F. (2023). Development of an electrochemical DNA-based biosensor for the detection of the cardiovascular pharmacogenetic-altering SNP CYP2C9*3. Talanta, 264, 124692. https://doi.org/10.1016/j.talanta.2023.1246920039-9140http://hdl.handle.net/10400.22/23504Cardiovascular 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.engCardiovascular diseasesChronoamperometryElectrochemical DNA-Based biosensorSandwich format hybridizationSingle-nucleotide polymorphismjournal article10.1016/j.talanta.2023.1246921873-3573