Two-surface strategy in electrochemical DNA hybridization assays: Detection of osmium-labeled target DNA at carbon electrodes

Target DNAs, including a 71-mer oligonucleotide, a PCR product and a plasmid DNA, all containing oligo(A) stretches,were hybridized at magnetic Dynabeads oligo(dT)(25) (DBT). The hybridization events were detected using a technique based on chemical modification of the target DNA with a complex of osmium tetroxide with 2,2'-bipyridine (Os, bipy) and voltammetric detection at carbon electrodes. DNA was modified with Os, bipy prior to capture at DBT, at the beads, or after release from the beads. In the latter case, DNA-Os, bipy was detected in the reaction mixture using adsorptive transfer stripping voltammetry involving extraction of unreacted Os, bipy from the electrode by organic solvents. Pre-labeling of the target plasmid DNA and the PCR product with Os, bipy significantly increased the yield of DNA captured at the beads. Tens of femtomoles of both short (the 71-mer oligonucleotide) and long (the 3-kilobase plasmid) target DNAs in a 20-microliter hybridization sample can be easily detected by means of these techniques: Various carbon electrode materials, including pyrolytic graphite (PGE), highly oriented pyrolytic graphite (HOPGE), carbon paste (CPE), glassy carbon and pencil graphite, were tested regarding their suitability for the detection of osmium-labeled DNA. Among them, PGE and HOPGE appeared usable in the measurements of both purified DNA-Os, bipy and its mixtures with unreacted Os, bipy while CPE was suitable for the detection purified osmium-labeled DNA.