Detection of Human Apolipoprotein E Genotypes by DNA Electrochemical Biosensor Coupled with PCR

Abstract

Background: Apolipoprotein E (apoE) is an important constituent of several plasma lipoproteins, mainly VLDL, HDL, and chylomicrons. It is involved in the redistribution of lipids in the liver and is implicated in growth and repair of injured neurons in the nervous system. apoE has also been associated with the risk of developing cardiovascular diseases and in familial type III hyperlipoproteinemia. Methods: We developed a new procedure for detecting genetic polymorphisms of apoE in human blood samples. The procedure is based on coupling of DNA electrochemical sensors with PCR-amplified DNA extracted from human blood. The DNA electrochemical sensor incorporated single-stranded oligonucleotides immobilized on graphite screen-printed electrodes (SPEs) by adsorption at controlled potential. The hybridization reaction on the electrode surface was monitored by chronopotentiometric stripping analysis (PSA), using daunomycin as indicator. Results: With use of two different probes, it was possible to investigate both DNA positions in which the apoE polymorphism takes place and thus to distinguish different genotypes. Real samples containing only complementary sequences gave a good increase in the area of the daunomycin peak (∼600 ms) compared with the peak observed with the buffer. Samples containing 50% complementary sequences gave a much lower increase, and samples containing only mismatch sequences gave a decrease in the daunomycin area. The procedure was validated by comparison with a method based on polyacrylamide gel electrophoresis. Conclusion: The coupling of DNA electrochemical sensors with PCR allowed quick discrimination between the different genotypes of apoE.

Chapter 26 Thick- and thin-film DNA sensors

“…The electrodes obtained with this technology are usually called screenprinted electrodes (SPEs). Most of them are carbon thick film [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35], although gold thick films are also used [36,37]. The screen-printing technology has become increasingly popular recently for the fabrication of electrodes and complete cells for applications where a disposable, one-time-use electrochemical measurement is desired.…”

“…In other cases (carbon films), the electrodes are ready as soon as the solvent has evaporated and the film has dried. The most common substrates used to fabricate these SPEs are aluminium ceramic [16][17][18], polyester flexible film [19][20][21][22][23][24][25][26]36,37] and polycarbonate sheet [27][28][29][30][31].…”

“…Several strategies for the immobilisation of ssDNA have been carried out and the strategy selected to immobilise the ssDNA probe depends, in many cases, on the nature of the electrode surface. The strategies for the immobilisation of ssDNA probes on solid surfaces include adsorption at controlled potential usually on pretreated screen-printed carbon surfaces [16][17][18][19][20][21][22]29]. However, in these cases, the immobilised ssDNA probes are not totally accessible for hybridisation, resulting in poor hybridisation efficiency.…”

“…Other indicators, such as daunomycin, cobalt complex and ferrocenyl-naphtalene diimide (FND), act as intercalators. Shifts in the peak potential or area of the oxidation process of daunomycin on dsDNA when compared to that obtained with ssDNA have been successfully used as electrochemical signals [19,20]. The treading intercalator FND has higher duplex affinity and binding stability and the ferrocene oxidation increases when duplex DNA is formed.…”

“…-Thermal denaturation. It is the most commonly used strategy [6,12,13,15,[20][21][22]29,[34][35][36]42]. It consists of denaturing the dsDNA thermally by using a boiling water bath for sometime and then immersing the tube containing the denaturalised DNA in an icewater bath for a short time in order to retard the re-annealing of the strands.…”