Determination of Surface-Immobilized Double-Stranded DNA Using a Metallointercalator and Catalytic Voltammetry

Wei, Ming-Yuan; Guo, Liang-Hong; Chen, Hao

doi:10.1007/s00604-006-0596-8

Cited by 20 publications

(22 citation statements)

References 39 publications

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“…It can be estimated using the Sauerbrey equation that 318 ng/cm 2 nucleic acid was immobilized on the avidin-coated gold electrode. 21 The value is very close to that measured by Rusling et al on myoglobin-coated surface. 18 The dependence of QCM frequency change on DNA concentration displayed a plateau when the deposition solution was higher than 200 µg/mL, suggesting surface saturation.…”

Section: Resultssupporting

confidence: 89%

Multiple DNA Binding Modes of a Metallointercalator Revealed by DNA Film Voltammetry

2006

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Binding and the redox reaction of the metallointercalator Ru(bpy) 2 (dppz) 2+ (bpy ) 2,2′-bipyridine, dppz ) dipyrido[3,2-a:2′,3′-c]phenazine) with DNA was investigated by DNA film voltammetry. Calf-thymus DNA (CT-DNA) was assembled on a tin-doped indium oxide electrode by layer-by-layer electrostatic adsorption. Voltammetry of Ru(bpy) 2 (dppz) 2+ (Ru-dppz) bound to the DNA film was measured in a redox-free electrolyte and showed strong dependence on the concentration of the metallointercalator. At low Ru-dppz concentrations, a single oxidation peak was observed, the potential of which shifted from 1.25 to 1.1 V with increasing Ru-dppz concentration (peak 1). At high metal chelate concentrations, an additional oxidation peak emerged with a potential of 1.25 V which was unaffected by the Ru-dppz concentration (peak 2). Three experiments were performed to investigate the mechanism and structural basis of the multiple peaks. First, voltammetry of Os(bpy) 2 (dppz) 2+ bound to the CT-DNA film displayed only one peak at its oxidation potential of about 0.75 V. Second, the concentration dependence of Ru-dppz bound to a poly-(AU) film (which does not contain any guanine bases) exhibited only one oxidation peak at about 1.22 V that was independent of the Ru-dppz concentration. Third, when the guanine concentration in a mixed film of CT-DNA and poly-(AU) was changed and the bound Ru-dppz was kept constant, a pre-peak emerged and shifted to 1.1 V with increasing guanines. Based on these results, the appearance of two peaks in the voltammetric measurements of CT-DNA was rationalized by invoking two different DNA binding modes for the Ru-dppz complex: intercalation and electrostatic association. Peak 2 arises from slow oxidation of guanines catalyzed by Ru-dppz electrostatically associated with the DNA film, since the addition of Mg 2+ decreases the magnitude of peak 2. Peak 1 was not affected by Mg 2+ ions, leading us to conclude that it is due to intercalated Ru-dppz. The intercalation positions the metal complex in close contact with the guanines inside DNA resulting in fast electrocatalytic reaction, giving rise to a catalytic pre-peak.

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Section: Resultssupporting

confidence: 89%

Multiple DNA Binding Modes of a Metallointercalator Revealed by DNA Film Voltammetry

2006

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“…Secondly, DNA usage in the sensor method is very low. It has been determined that the amount of DNA immobilized in a film is 3.2 ng mm −2 [27]. This is much less than the amount (micrograms) needed for 32 P post-labeling and gel electrophoresis experiments.…”

Section: Resultsmentioning

confidence: 96%

Detection and mechanistic investigation of halogenated benzoquinone induced DNA damage by photoelectrochemical DNA sensor

2010

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Halogenated phenols are widely used as biocides and are considered to be possibly carcinogenic to humans. In this report, a previously developed photoelectrochemical DNA sensor was employed to investigate DNA damage induced by tetra-halogenated quinones, the in vivo metabolites of halogenated phenols. The sensor surface was composed of a double-stranded DNA film assembled on a SnO 2 semiconductor electrode. A DNA intercalator, Ru (bpy) 2 (dppz) 2+ , was allowed to bind to the DNA film and produce photocurrent upon light irradiation. After the DNA film was exposed to 300 μM tetrafluoro-1,4-benzoquinone (TFBQ), the photocurrent dropped by 20%. In a mixture of 300 μM TFBQ and 2 mM H 2 O 2 , the signal dropped by 40%. The signal reduction indicates less binding of Ru (bpy) 2 (dppz) 2+ due to structural damage of ds-DNA in the film. Similar results were obtained with tetra-1,4-chlorobenzoquinone (TCBQ), although the signal was not reduced as much as TFBQ. Fluorescence measurement showed that TFBQ/H 2 O 2 generated more hydroxyl radicals than TCBQ/H 2 O 2 . Gel electrophoresis proved that the two benzoquinones produced DNA strand breaks together with H 2 O 2 , but not by themselves. Using the photoelectrochemical sensor, it was also found that TCBQ covalently bound with DNA did not produce additional oxidative damage in the presence of H 2 O 2 . The combined photoelectrochemistry, gel electrophoresis, and fluorescence data revealed distinctive differences between TFBQ and TCBQ in terms of DNA adduct formation and hydroxyl radical generation.

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“…To conduct ECL displacement experiments, DNA films were first prepared on ITO electrodes by layer-by-layer electrostatic selfassembly [23]. Due to the presence of surface oxide groups, ITO is negatively charged in neutral pH.…”

Section: Resultsmentioning

confidence: 99%

Highly sensitive electrochemiluminescence displacement method for the study of DNA/small molecule binding interactions

Huang¹,

Wang²,

Guo³

2010

Analytica Chimica Acta

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a b s t r a c tNon-covalent binding interactions of small molecules with DNA play important roles in regulating gene expression and gene function. In this work, a highly sensitive electrochemiluminescence (ECL) displacement method has been developed to investigate such interactions, particularly for weak DNA binders. This ECL method relies on a double-stranded DNA film deposited on an indium tin oxide electrode (ITO) surface by layer-by-layer self-assembly. A DNA intercalator, [Ru(bpy) 2 (dppz)] 2+ (bpy = 2,2 -bipyridine, dppz = dipyrido[3,2-a:2 3 -c]phenazine), is employed as the ECL signal indicator. If a test compound competes with the indicator for the same binding sites in DNA, it would displace the indicator from the film and reduce ECL signal. The new method was validated by measuring five well-known DNA-binding organic molecules including quinacrine, H33258, thiazole orange, ethidium bromide and 4,6-diamidine-2-phenylindole dihydrochloride. Due to high ECL sensitivity, only 0.4 mol L −1 [Ru(bpy) 2 (dppz)] 2+ was needed in the ECL displacement measurement, which is about 75-fold less than the concentration in the voltammetric measurement. The lowered concentration permitted direct measurement of IC 50 values of eight hydroxylated polycyclic aromatic hydrocarbons in their ECL displacement curves and subsequent calculation of their binding constants with DNA. The ECL displacement method is particularly useful for investigating weak DNA binders with limited aqueous solubility.

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Determination of Surface-Immobilized Double-Stranded DNA Using a Metallointercalator and Catalytic Voltammetry

Cited by 20 publications

References 39 publications

Multiple DNA Binding Modes of a Metallointercalator Revealed by DNA Film Voltammetry

Multiple DNA Binding Modes of a Metallointercalator Revealed by DNA Film Voltammetry

Detection and mechanistic investigation of halogenated benzoquinone induced DNA damage by photoelectrochemical DNA sensor

Highly sensitive electrochemiluminescence displacement method for the study of DNA/small molecule binding interactions

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