Photoelectrochemical Sensor for the Rapid Detection of in Situ DNA Damage Induced by Enzyme-Catalyzed Fenton Reaction

Abstract

Photoelectrochemical sensors were developed for the rapid detection of oxidative DNA damage induced by Fe2+ and H2O2 generated in situ by the enzyme glucose oxidase. The sensor is a multilayer film prepared on a tin oxide nanoparticle electrode by layer-by-layer self-assembly and is composed of separate layers of a photoelectrochemical indicator, DNA, and glucose oxidase. The enzyme catalyzes the formation of H2O2 in the presence of glucose, which then reacts with Fe2+ and generates hydroxyl radicals by the Fenton reaction. The radicals attack DNA in the sensor film, mimicking metal toxicity pathways in vivo. The DNA damage is detected by monitoring the change of photocurrent of the indicator. In one sensor configuration, a DNA intercalator, Ru(bpy)2(dppz)2+ (bpy = 2,2'-bipyridine, dppz = dipyrido[3,2-a:2',3'-c]phenazine), was employed as the photoelectrochemical indicator. The damaged DNA on the sensor bound less Ru(bpy)2(dppz)2+ than the intact DNA, resulting in a drop in photocurrent. In another configuration, ruthenium tris(bipyridine) was used as the indicator and was immobilized on the electrode underneath the DNA layer. After oxidative damage, the DNA bases became more accessible to photoelectrochemical oxidation than the intact DNA, producing a rise in photocurrent. Both sensors displayed substantial photocurrent change after incubation in Fe2+/glucose in a time-dependent manner. And the detection limit of the first sensor was less than 50 microM. The results were verified independently by fluorescence and gel electrophoresis experiments. When fully integrated with cell-mimicking components, the photoelectrochemical DNA sensor has the potential to become a rapid, high-throughput, and inexpensive screening tool for chemical genotoxicity.

A chemical toxicity sensor based on the electrochemiluminescence quantification of apurinic/apyrimidinic sites in double-stranded DNA monolayer

“…In a different approach, osmium tetroxide-2,2 -bipyridine was employed as an electrochemical probe of AP sites, which reacts covalently with the unpaired thymine bases in damaged DNA [23]. Furthermore, although a few chemical toxicity sensors based on the detection of oxidized or methylated DNA bases have been developed [24][25][26][27][28][29][30][31][32][33][34][35][36], so far there has been no report on the study of toxicity sensors based on AP site detection.…”

Development of microplate-based photoelectrochemical DNA biosensor array for high throughput detection of DNA damage

“…For instance, DNA damage was investigated with electrochemical and electrochemiluminescent sensors and sensor arrays by a number of research groups [10][11][12][13][14][15]. We also developed a photoelectrochemical DNA sensor for the rapid detection of chemical-induced DNA damage [16][17][18][19]. The sensor was fabricated by assembling a double-stranded DNA film on SnO 2 electrode surface.…”

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

“…For DNA adduct identification and quantification, 32 P post-labeling is the method of choice, although the procedure is very time-consuming, including enzymatic digestion of DNA sample, purification, labeling with 32 P, and TLC or PAGE analysis [20]. Recently, we developed a photoelectrochemical DNA sensor for the rapid detection of structural DNA damage [21][22][23]. The sensor surface was composed of a double-stranded DNA (ds-DNA) film assembled on a SnO 2 semiconductor electrode.…”