Detection of human apolipoprotein E genotypes by DNA biosensors coupled with PCR

Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins and has been associated with the risk of developing cardiovascular diseases and in familiar type III hyperlipoproteinemia. We developed new procedures for the detection of apolipoprotein E polymorphism in human blood based on coupling DNA electrochemical or piezoelectric sensors with polymerase chain reaction (PCR). The electrochemical sensor was obtained by immobilizing single-stranded oligonucleotides onto graphite screen-printed electrodes by adsorption at controlled potential. The hybridization reaction that occurred on the electrode surface was evidenced by chronopotentiometric stripping analysis-using daunomycin as indicator. In the piezoelectric sensor, biotinylated 23-mer probes were immobilized on the streptavidin-coated gold surface of a quartz crystal; streptavidin was covalently bound to the thiol/dextran modified gold surface. The hybridization of the immobilized probes with complementary and mismatched DNA was investigated. With the use of two different probes, it was possible to investigate both positions in which apoE polymorphism takes place and consequently, to distinguish the different genotypes. The procedure was validated with both kinds of biosensor with a reference method based on polyacrilamide gel electrophoresis.