DNA Sensing on a DNA Probe-Modified Electrode Using Ferrocenylnaphthalene Diimide as the Electrochemically Active Ligand

Abstract

Naphthalene diimide derivative 1 carrying ferrocenyl moieties at the termini of imide substituents binds intact calf thymus DNA 4 times more strongly than the denatured DNA, and its complex with the intact DNA dissociates 80 times more slowly than that with the denatured DNA. On the basis of these observations, ligand 1 was applied to a probe of electrochemical DNA sensing. A thiol-linked single-stranded DNA probe was immobilized through the S-Au bonding to 20-30 pmol/mm2 on a gold electrode. Following hybridization with the complementary DNA, the electrode was soaked in a solution containing 1 (intercalation step) and then washed with buffer for 5 s. The cyclic voltammogram and differential pulse voltammogram for this electrode gave an electrochemical signal due to the redox reaction of 1 that was bound to the double-stranded DNA on the electrode. Thus, dA20 and the yeast choline transport gene were quantitated at the subpicomole level. The sensitivity of DNA detection was improved to 10 zmol by reducing the amount of immobilized DNA probe and protecting the uncovered surface of the electrode with 2-mercaptoethanol.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…Hashimoto et al, proposed the immobilisation of a synthetic 20-mer dsDNA and the subsequent regeneration of a free DNA probe by duplex alkaline denaturation (Hashimoto et al, 1998). The immobilisation of a 20-mer probe, with the subsequent masking of the unmodified gold regions with mercaptoethanol spacers, has been reported (Takenaka et al, 2000;Kertesz et al, 2000). A mixed monolayer of 10-mer cysteine-conjugated PNA probe and 6-mercapto-1-hexanol was used by Umezawa and co-workers to modified gold disk electrodes (Aoki et al, 2000).…”

“…The shielding of the ferro-ferri-cyanide ions from the DNA-modified surface was attributed to a combination of physical coverage by the ssDNA and the electrostatic repulsion between the negatively charged redox couple ions and the DNA phosphate backbone (Yang et al, 1998). Takenaka et al (2000) reported an indirect quantification method based on the HPLC analysis of the excess of DNA remained unadsorbed.…”

“…The substituents serve as anchors to prevent the dissociation of the ligand from double-stranded DNA; this compound displays, on the other hand, a negligible affinity for the DNA probe, since the threading mode of interaction is not advantageous for single-stranded DNA. The differential pulse voltammetric detection of FND allowed a detection limit of 10 zmol of 20-mer target sequence and 1 fmol of a linearised plasmid DNA carrying a part of the yeast choline transport gene (Takenaka et al, 2000). The method was also applied for the detection of DNA extracted for the peripheral whole blood and amplified by PCR (Miyahara et al, 2002).…”

Intercalative Stacking: A Critical Feature of DNA Charge-Transport Electrochemistry

“…56,58,59 DNA-based electrochemical methods have also been developed primarily to detect nucleic acid hybridization. 44,45,[60][61][62][63][64][65][66][67][68][69][70][71][72][73] Notably, those methods do not rely on an intercalator as the redox probe. General principles governing electron transport through modified films involving percolation or the physical displacement of associated ions are also well understood.…”

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

“…These techniques employ species exhibiting i) well-pronounced electrochemistry at less extreme potentials, involving reversible or catalytic electrode processes, and/or ii) non-covalent or covalent structure-selective interactions with DNA. Electroactive non-covalent binders including DNA intercalators (metal chelates such as [Co(phen) 3 ] 3 [11], daunomycin [12], a bis-intercalator bis-9-acridinyl derivative with a viologen linker [13] or a threading intercalator ferrocenyl-coupled naphthalene diimide [14]) or groove binders (Hoechst 33258 [15]) selectively recognizing duplex DNA have been used as redox indicators of DNA hybridization at the electrode surface. Other authors used covalent labeling of target DNA or hybridization (reporter) probes with electroactive markers (such as ferrocene derivatives [16]) or enzymes taking part in electrocatalytic processes (peroxidase [17]).…”