Development of electrochemical genosensors applied to cardiovascular pharmacogenetics

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 and clopidogrel) are frequently prescribed to patients. Nevertheless, both therapeutic agents present 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 CYPC9*2 and CYP2C*3, and the SNP 1639G>A in the vitamin K epoxide reductase complex subunit 1 (VKORC1) genes have both 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 anticoagulants.

This work addresses the development of a disposable electrochemical genosensor capable of detecting SNP in the CYPC9*3 allele. Analysing public databases, two specific 78 bp DNA probes; one with the adenine (TA) and another with the cytosine (TC) SNP genetic variation were selected and designed. The genosensor methodology implied the immobilization of a mixed self-assembled monolayer (SAM) linear CYPC9*3 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 CYPC9*3 allele was designed using a complementary fluorescein isothiocyanate-labelled signalling DNA probe and enzymatic amplification of the electrochemical signal. Chronoamperometry measurements were carried out obtaining a concentration range from 0.015 to 1.0 nM for both TA and TC SNP target probes. Analysing the results, the developed genosensor could discriminate between the two SNP probes.