Two‐Surface Strategy in Electrochemical DNA Hybridization Assays: Detection of Osmium‐Labeled Target DNA at Carbon Electrodes

Abstract

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.

Electrochemical DNA sensors

“…reporter molecules. Appearance of the characteristic electrochemical response of the redox reporter therefore signals the hybridization event 29,30 . Using physical separation methods to isolate the labeled sequences, detection limits on the order of ∼10 10 molecules have been reported.…”

A Single-Surface Electrochemical Biosensor for the Detection of DNA Triplet Repeat Expansion

“…Synthetic oligonucleotides (ODNs), including reporter probes (TTC) 12 (12TTC RP) and (GAA) 12 (12GAA RP) biotinylated at their 5'-termini , 97-mer target ODNs (TTC) 24 (A) 25 (F1) and GCGCGCGACACCACGCC-CGGCTAACTTTTCTTTATT(TTC) 12 (A) 25 (F2), a nonspecific 71-mer ODN TTGG-(TTTTTTCTC) 4 TTTTTG(A) 25 , and PCR primers GAA-F and GAA-R were purchased from VBC Genomics (Austria). Plasmids pBSK (-) , pRW3821 and pRW3822 (the latter two kindly donated by Professor Robert Wells [30]) were isolated from chloramphenicol-amplified E. coli TOP10 cells using Qiagen-tip 500 columns and linearized by a restriction endonuclease Eco RI (Takara).…”

“…Commercially available magnetic beads with attached oligonucleotide probes have proven excellent as the surface H in most of the DST applications. Using these techniques, tDNA was detected down to z-mol levels [19] and highly specific detection of long (hundreds to thousands of base pairs) tDNAs has been achieved [9,11,12,14].…”

“…The technique involved multiple hybridization of a labeled reporter probe (RP) with the expanded triplet repeat in tDNA captured at magnetic beads. A biotinylated (TTC) 12 RP (binding to the repeat) was detected using an electrochemical enzyme-linked assay. The target PCR amplicons were labeled with an osmium tetroxide 2,2'-bipyridine complex (Os, bipy) covalently modifying pyrimidine but not purine bases [10 -12, 27, 28] in the tDNA.…”

Determination of Dopamine in Presence of Uric Acid at Poly (Eriochrome Black t) Film Modified Graphite Pencil Electrode

“…The structure of EBT was shown in Scheme 1. The graphite pencil electrode (GPE) has been successfully applied to analyses of certain compounds in recent years [30][31][32][33][34]. The GPE is relatively new type of carbon electrode, it is less expensive, more convenient, and renewable compare to the commonly used CPE or GCE [35].…”