Sequence-Specific Gene Detection with a Gold Electrode Modified with DNA Probes and an Electrochemically Active Dye

Abstract

A synthesized 20-mer DNA probe complementary to a part of an oncogene v-myc region having a mercaptohexyl group at the 5'-phosphate end was immobilized on a gold electrode by chemisorption. The immobilized DNA was detected voltammetrically using Hoechst 33258 with a DNA minor groove binder and an electrochemically active dye. The modified electrode was immersed into a 100 mumol/L Hoechst 33258 solution and washed with a phosphate buffer (pH 7.0). The anodic peak current (ipa) of Hoechst 33258 on the modified electrode was higher than that on a bare gold electrode (128 and 75 nA, respectively). It was considered that Hoechst 33258 was concentrated on the electrode surface due to its association with DNA. When the modified electrode was hybridized in a solution of a model targeted gene (10(-7) g/mL), single-stranded pVM623 containing the PstI fragment of a 1.5-kilobase pair of oncogene v-myc, the ipa was 192 nA. On the other hand, the ipa was 128 nA when the modified electrode was reacted in a solution of single-stranded pUC119 without a region complementary to v-myc in pVM623. The ipa was related to the concentration of the targeted DNA in the hybridization reaction. The use of Hoechst 33258 resulted in a sequence-specific detection of the targeted DNA quantitatively ranging from 10(-7) to 10(-13) g/mL in a buffer solution.

Carbon and gold electrodes as electrochemical transducers for DNA hybridisation sensors

“…This treatment is also important to produce a hydrophilic gold surface. Residual contaminants on bare gold can be removed by repeated cycling in diluted H 2 SO 4 (Hashimoto et al, 1994b) or diluted KOH (Aoki et al, 2000) until stable voltammograms are obtained. Clean gold shows a characteristic anodic peak current near +1.1 V (versus SCE) and a single cathodic peak near +0.9 V in dilute sulfuric acid solutions (Finklea et al, 1987).…”

“…Hashimoto and co-workers pioneered the use of thiolmodified DNA probes for electrochemical sequence-specific gene detection almost 10 years ago (Hashimoto et al, 1994b). Nevertheless, most of recent publications follow Herne and Tarlov "guidelines".…”

“…The most widely used is a method based on the cyclic voltammetric measurement of the [Fe(CN) 6 ] 3− /[Fe(CN) 6 ] 4− faradic response (Hashimoto et al, 1994b;Steel et al, 1998;Kelley and Barton, 1997;Xu et al, 2001;Nakayama et al, 2002). The peak currents of the redox couple decreased and the peak-to-peak separation ( E p ) increased when probe-modified electrodes were used and compared with bare electrodes.…”

“…The use of faradic impedance spectroscopy for probe biorecognition, however, was a major innovation. Hashimoto et al (1994b) reported the detection of a 4.2 kbp linearised plasmid carrying the PstI fragment of oncogene v-myc. The linear-sweep voltammetric measurement of the redox-active, minor groove binder, Hoechst 33258, allowed a detection limit of 10 −13 g/ml of target sequence.…”

A new chemically amplified electrochemical system for DNA detection in solution

“…A variety of approaches have been proposed and tested for electrochemical detection: (a) detecting the oxidative or reductive current of DNA bases directly [3,4]; (b) detecting the anodic current of DNA bases with the aid of a mediator [5][6][7]; (c) detecting the oxidation of DNA sugar moieties [8]; (d) detecting the electrochemical reaction of a non-covalent DNA binder [9][10][11][12][13][14][15][16][17]; (e) detecting the current generated from a covalently linked label inorganic and organic molecules, enzymes, and nanoparticles [18][19][20][21][22][23]. The advantages of electrochemical devices are their low-cost, fast response, simple design, small dimension and low power requirement.…”