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Exposure of DNA to oxidative stress conditions results in the generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). 8-OxodGuo is genotoxic if left unrepaired. We quantified 8-oxodGuo lesions in double-stranded DNA films by using a photoelectrochemical DNA sensor in conjunction with a specific covalent labeling method. A lesion-containing DNA film was assembled on a SnO(2) nanoparticle modified indium tin oxide electrode through layer-by-layer electrostatic adsorption. The lesions were covalently labeled with a biotin conjugated spermine derivative, and ruthenium tris(bipyridine) labeled streptavidin was introduced as the signal reporter molecule. Photocurrent increased with the number of lesions in the strand and decreased as the film was diluted with intact DNA. Quantification of 8-oxodGuo was achieved with an estimated detection limit of ∼1 lesion in 650 bases or 1.6 fmol of 8-oxodGuo on the electrode. Incubation of the film with a DNA base excision repair enzyme, E. coli formamidopyrimidine-DNA glycosylase (Fpg), resulted in complete loss of the signal, indicating efficient excision of the isolated lesions in the nucleotide. Oxidatively generated DNA damage to a double-stranded calf thymus DNA film by the Fenton reaction was then assessed. One 8-oxodGuo lesion in 520 bases was detected in DNA exposed to 50 μM Fe(2+)/200 μM H(2)O(2). Treatment with Fpg reduced the photocurrent by 50%, indicating only partial excision of 8-oxodGuo. This suggests that tandem lesions, which are resistant to Fpg excision, are generated by the Fenton reaction. Unlike repair enzyme dependent methods, the sensor recognizes 8-oxodGuo in tandem lesions and can avoid underestimating DNA damage.

Reactive oxygen species (ROS) play very important roles in the photocatalytic reactions of semiconductors. Using a continuous flow chemiluminescence (CFCL) system, we developed three methods for the selective, sensitive, and online detection of O2(• -), •OH, and H2O2 generated during ultraviolet (UV) irradiation of nano-TiO2 suspensions. TiO2 nanoparticles were irradiated in a photoreactor and pumped continuously into a detection cell. To detect O2(• -), luminol was mixed with TiO2 before it entered the detection cell. For the detection of short-lived •OH, phthalhydrazide was added into the photoreactor to capture •OH, and then mixed with H2O2/K5Cu(HIO6)2 to produce chemiluminescence (CL). To detect H2O2, an irradiated TiO2 suspension was kept in darkness for 30 min, and then mixed with luminol/K3Fe(CN)6 to produce CL. The selectivity of each method for a particular ROS was verified by using specific ROS scavengers. For a given ROS, a comparison between CL and conventional method showed good agreement for a series of TiO2 concentrations. The sensitivity of CL method was approximately 3-, 1200-, and 5-fold higher than the conventional method for O2(• -), •OH, and H2O2, respectively. To demonstrate the utility of the methods, ROS in three different types of TiO2 suspensions was detected by CFCL. It was found that photodegradation efficiency of Rhodamine B correlated the best (R(2)0.95) with the amount of photogenerated •OH, implying that •OH was the major oxidant in Rhodamine B photodegradation reaction. CFCL may provide a convenient tool for the studies on the reaction kinetics of ROS-participated decomposition of environmental contaminants.

Selective photoelectrochemical oxidation of DNA was achieved by ruthenium tris(bipyridine) immobilized on a tin oxide nanoparticle electrode. The metal complex was covalently attached to a protein, avidin, which adsorbed strongly on the tin oxide electrode by electrostatic interaction. Upon irradiation with 473-nm light, anodic photocurrent was generated in a blank electrolyte and was enhanced significantly after addition of poly(guanadylic acid) (poly-G) into the electrolyte. The current increased progressively with the nucleotide concentration, suggesting the enhancement effect was related to poly-G. The action spectrum indicates that the photocurrent was initiated by light absorption of the ruthenium compound immobilized on the electrode. Among the various polynucleotides examined, poly-G produced the largest photocurrent increase, followed by poly-A, single-stranded DNA, chemically damaged DNA, and double-stranded DNA, whereas poly-C and poly-U showed little effect. The combined experimental data support the hypothesis that the photoexcited Ru2+ species injects an electron into the semiconductor and produces Ru3+, which is then reduced back to Ru2+ by guanine and adenine bases in DNA, resulting in the recycling of the metal complex and enhanced photocurrent. The photoelectrochemical reaction can be employed as a new method for the detection of DNA damage.

The binding interaction of many organic carcinogens such as polycyclic aromatic hydrocarbons with DNA is the key step in their genotoxic effect. In this work, an electrochemical displacement method was developed to study such interaction. In the method, a DNA film is deposited on an indium tin oxide electrode surface by layer-by-layer assembly, and a redox-active DNA intercalator Ru(bpy) 2(dppz)(BF 4) 2 (bpy = 2,2'-bipyridine, dppz = dipyrido [3,2-a:2',3'-c] phenazine) is employed as an electrochemical indicator. If an organic compound competes with the indicator for the same binding site on DNA in the film, it would displace the ruthenium complex from DNA, resulting in a reduction in the measured electrochemical signal. From the titration curve, the binding constant of the organic compound with DNA can be calculated. With the use of oxalate as an electron donor to chemically amplify the oxidation current of the indicator, chemicals can be tested at low micromolar concentrations. Five well-known DNA binding polycyclic organic compounds, thiazole orange, 4,6-diamidine-2-phenylindole, H33258, ethidium bromide, and quinacrine, were investigated by the displacement method. The binding constants obtained in our experiments fall in the range of (4.3 x 10 (5)) to (1.2 x 10 (7)) M (-1), which are generally consistent with those reported in the literature by some established methods. The electrochemical method provides a general tool that complements the commonly used spectroscopic methods for the study of DNA/small molecule interactions.

Quantitative detection of a biological affinity reaction, the biotin/avidin recognition, was achieved using our newly developed photoelectrochemical analytical system. The system is based on the operation mechanism of the well-developed dye-sensitized photoelectrochemical solar cells and comprises a ruthenium tris(2,2'-bipyridine) (Ru-bipy) derivative as the photoelectrochemical signal-generating molecule, oxalate as the sacrificial electron donor, and tin oxide nanoparticle as the semiconductor electrode material. To perform the affinity reaction, avidin was immobilized on SnO(2) electrode by passive adsorption. Biotin-linked bovine serum albumin (BSA) was labeled with an NHS-ester derivative of Ru-bipy. After binding of BSA to the surface-immobilized avidin through biotin, photoelectrochemical measurement was carried out in the presence of oxalate. Anodic photocurrent was turned on and off repeatedly by control of incidental light. The action spectrum of the photocurrent resembled the absorption spectrum of Ru-bipy, proving the photocurrent was generated from the metal complex. A linear relationship between photocurrent and BSA concentration was obtained in the range of 1-100 microg/mL. This is the first case of quantitative photoelectrochemical detection of a biological affinity interaction.