过氧亚硝酸根离子诱导DNA断裂机理的研究

在体内NO和超氧阴离子生成的过氧亚硝酸根离子（ONOO^-，peroxynitrite）是一种强氧化性物质，它可以诱导DNA单链断裂使DNA发生损伤。为了探讨ONOO^-断裂DNA的作用机理，以质粒DNA pBR322为研究对象，采用琼脂糖凝胶和硫代巴比妥酸(TBA)显色反应等方法对ONOO^-与DNA的反应进行研究。结果表明ONOO^-能明显使DNA发生断裂，而且随着ONOO^-浓度的增加，DNA断裂的程度增加，在酸性和中性条件下ONOO^-断裂DNA的能力明显高于碱性介质，而CO₂对该反应有显著的抑制作用，TBA显色反应进一步证实该反应为自由基机理，其机理为ONOO^-与H⁺形成ONOOH，然后裂解为二氧化氮自由基（·NO₂）和羟基自由基（·OH），继而对DNA造成损伤。     

Electrochemical DNA biosensor for polycyclic aromatic hydrocarbon detection

Four DNA electrochemical biosensors using four types of DNA (calf thymus ssDNA, calf thymus dsDNA, salmon testis ssDNA and salmon testis dsDNA) were constructed using graphite screen printed electrodes. These biosensors were exploited as analytical tool to detect polycyclic aromatic hydrocarbons-DNA interactions using benzo(a)anthracene and phenantrene as model analytes, the guanine oxidation peak variation being the signal revealing the interaction between PAHs and immobilized DNA. The salmon testis ssDNA biosensor resulted as the most promising device and was further evaluated for benzo(a)anthracene, fluorene, indeno(1,2,3-cd)pyrene, anthracene, and phenanthrene in 5–40 ng mL^?1 solutions, and for benzo(a)pyrene (5–50 ng mL^?1). A concentration dependent variation of the DNA guanine oxidation peak was observed for all compounds. The effect of benzo(a)pyrene ultraviolet (UV) activation on the benzo(a)pyrene (BaP)-DNA interaction was evaluated at concentration levels of 20 and 50 ng mL^?1, and a 3.5- and 2.7-fold increases of the guanine oxidation peak was measured respectively. The salmon testis ssDNA biosensor was examined with PAHs contaminated samples of Mytilus galloprovincialis. Upon UV irradiation of three sample extracts exceeding the BaP maximum level, a positive variation of the DNA guanine oxidation was obtained. An average 2.4-fold increase of the guanine oxidation peak was detected demonstrating that the sensor can be used to detect toxic degradation products of PAHs.     

Electrochemical Oxidation of ds‐DNA on Polypyrrole Nanofiber Modified Pencil Graphite Electrode

Preparation of a polypyrrole nanofiber (PPyNF) modified pencil graphite (PG) electrode and its usage in the electrochemical DNA biosensors was investigated. The electrodes (PPyNF/PG1 and PPyNF/PG2) were prepared from a solution containing 0.1 M pyrrole, 0.1 M Na₂CO₃ and 0.1 M LiCIO₄ by using potentiostatic and potentiodynamic methods. PPyNF/PG2 electrodes which were prepared by potentiostatic procedure showed higher responses for the oxidation of ds‐DNA than the PPyNF/PG1 electrodes prepared by potentiodynamic methods. Immobilization of the ds‐DNA on PG and PPyNF/PG surfaces was performed at a constant potential, +0.5 V, for 300 s in 0.5 M ABS (pH 4.8) containing 15 μg mL^?1 ds‐DNA and 20 mM LiCIO₄. The oxidation peak potentials of the ds‐DNA bases, guanine and adenine, were shifted to more cathodic values by using PPyNF/PG electrodes. The oxidation signal of the guanine base of ds‐DNA was decreased in the presence of methylene blue.     

Electrochemical Study on DNA Damage Based on the Direct Oxidation of 8‐Hydroxydeoxyguanosine at an Electrochemically Modified Glassy Carbon Electrode

The study of DNA damage induced by Fenton reaction (Fe²⁺/H₂O₂) in vitro was performed based on the direct electrochemical oxidation of 8‐hydroxydeoxyguanosine (8‐OH‐dG), the biomarker of DNA oxidative damage, at an electrochemically modified glassy carbon electrode (GCE). The effects of antioxidants, such as ascorbic acid, and hydroxyl‐radical scavenger (mannitol) on the DNA damage were also investigated. 8‐OH‐dG, the oxidation product of guanine residues in DNA, has shown significantly oxidative peak on the electrochemically modified GCE. The oxidative peak current of 8‐OH‐dG was linear with the damaged DNA concentration in the range of 10–200 mg/L. The experimental results demonstrate that ascorbic acid has ambivalent effect on DNA oxidative stress. It can promote DNA oxidative damage when ascorbic acid concentration is below 1.5 mM and protect DNA from damage in the range of 1.5–2.5 mM. As a hydroxyl‐radical scavenger, mannitol inhibits significantly DNA oxidative damage. The influence of Fe²⁺, as reactant, and EDTA as iron chelator in the system were also studied. The proposed electrochemical method can be used for the estimation of DNA oxidative damage from new point of view.     

A boronate-based ratiometric fluorescent probe for fast selective detection of peroxynitrite

Given that peroxynitrite (ONOO^?) is profoundly associated with health and diseases, a new fluorescent probe ABT was designed and synthesized for detection of ONOO^?. ABT manifested not only ratiometric fluorescence signals simultaneously in response to concentrations of ONOO^? (within 10?s), but high selectivity and sensitivity towards ONOO^? over other physiological relevant species (detection limit?=?26.3?nM). Moreover, ABT worked in a broad pH range with biological relevance. Thus, ABT could be used to quantitative detection of ONOO^? concentration and has the potential to efficiently monitor ONOO^? in living organisms.     

Formation of peroxynitrite induced by spark plasma radiation

The formation of peroxynitrite (ONOO^?) in water by the action of plasma UV radiation from spark discharge in air has been studied. Solutions with pH₀ 3.11–12.86 have been treated. In all cases, the pH has been observed to decrease immediately after the treatment and for the following 14 days. An absorption line characteristic of peroxynitrite (λ = 302 nm) appears in the spectrum by the fourth to fifth day after the treatment and reaches a maximum by the 10th–12th day. The appearance of peroxynitrite a few days after the treatment can be caused by the formation of a substance X during a radiation pulse at a high density of active species, which decomposes according to the scheme X → ONOO^? → NO ₃ ^? and has a lifetime of about 10 days. The maximal yield of peroxynitrite is (1.2 ± 0.5) × 10^?2 mol/L.     

Electrochemical approach to detect the presence of peroxynitrite in aerobic neutral solution

This work describes the conditions of use of bare gold electrode to detect electrochemically the presence of peroxynitrite ONOO^? in phosphate buffer solution at pH 7.1. As ONOO^? is extremely unstable in neutral solution, current–potential curve was reconstructed between ?0.5 and 0.7 V vs SCE by amperometry experiments at rotating disk electrode at different potentials. Comparison of this reconstructed curve with voltammograms of the common interfering species (dopamine, hydrogen peroxide, nitrite, ascorbic acid and glutamate) shows that the presence of ONOO^? can be selectively determined at ?0.1 V vs SCE. This detection occurs through the electrochemical reduction of peroxynitrous acid ONOOH, the conjugated acid of ONOO^?. Detection of ONOO^? produced in situ by the reaction of nitric oxide with superoxide was also achieved.     

Study of the Interaction Between Peroxynitrite and Hemoglobin

Abstract

Peroxynitrite (ONOO^?), which is usually generated as a response of the immune system or in inflammatory processes, is a powerful oxidant species, while hemoglobin is an important peroxynitrite scavenger in vivo. In this work, we have studied the interaction between peroxynitrite and hemoglobin through an electrochemical method and UV-Vis spectroscopy. It is found that peroxynitrite, at a relatively high concentration level, may make the protein exhibit a concentration-dependent increase of its catalytic activity towards hydrogen peroxide, whereas peroxynitrite at low concentration will result in the slight decrease of the catalytic activity. Further studies reveal that the diversification of the enzymatic activity is ascribed to the different extent of tyrosine nitration and, accordingly, the spatial conformation of the protein.     

Colorimetric detection of peroxynitrite-induced DNA damage using gold nanoparticles, and on the scavenging effects of antioxidants

We describe a rapid and convenient colorimetric method for the detection of oxidative DNA damage caused by peroxynitrite (ONOO^?) using unmodified gold nanoparticles (AuNPs). AuNPs are stable in the presence of single-stranded DNA (ssDNA) against the aggregation induced by a high ionic strength. If adsorbed ssDNA are cleaved by ONOO^? to form smaller fragments, the AuNPs rapidly aggregate due to electrostatic attraction. As a result, the color of the solution changes from red to blue, and this can be seen with bare eyes. We also have evaluated the activity of the antioxidants gallic acid, ascorbic acid and caffeic acid to scavenge ONOO^?. This method therefore also can be applied to screen for anti-oxidation drugs and agents.

Nucleic Acid Biosensor for Detection of Human Immunodeficiency Virus Using Aquabis(1,10‐phenanthroline)copper(II) Perchlorate as Electrochemical Indicator

The electrochemical behavior of aquabis(1,10‐phenanthroline)copper(II) perchlorate [Cu(H₂O)(phen)₂]·2ClO₄, where phen=1,10‐phenanthroline, on binding to DNA at a glassy carbon electrode (GCE) and in solution, was described. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) results showed that [Cu(H₂O)(phen)₂]²⁺ had excellent electrochemical activity on the GCE with a couple of quasi‐reversible redox peaks. The interaction mode between [Cu(H₂O)(phen)₂]²⁺ and double‐strand DNA (dsDNA) was identified to be intercalative binding. An electrochemical DNA biosensor was developed with covalent immobilization of human immunodeficiency virus (HIV) probe for single‐strand DNA (ssDNA) on the modified GCE. Numerous factors affecting the probe immobilization, target hybridization, and indicator binding reactions were optimized to maximize the sensitivity and speed of the assay. With this approach, a sequence of the HIV could be quantified over the range from 7.8×10^?9 to 3.1×10^?7 mol·L^?1 with a linear correlation of γ=0.9987 and a detection limit of 1.3×10^?9 mol·L^?1.     

A novel poly(cyanocobalamin) modified glassy carbon electrode as electrochemical sensor for voltammetric determination of peroxynitrite

This report described the direct voltammetric detection of peroxynitrite (ONOO⁻) at a novel cyanocobalamin modified glassy carbon electrode prepared by electropolymeriation method. The electrochemical behaviors of peroxynitrite at the modified electrode were studied by cyclic voltammetry. The results showed that this new electrochemical sensor exhibited an excellent electrocatalytic activity to oxidation of peroxynitrite. The mechanism of catalysis was discussed. Based on electrocatalytic oxidation of peroxynitrite at the poly(cyanocobalamin) modified electrode, peroxynitrite was sensitively detected by differential pulse voltammetry. Under optimum conditions, the anodic peak current was linear to concentration of peroxynitrite in the range of 2.0 × 10⁻⁶ to 3.0 × 10⁻⁴ mol L⁻¹ with a detection limit of 1.0 × 10⁻⁷ mol L⁻¹ (S/N of 3). The proposed method has been applied to determination of peroxynitrite in human serum with satisfactory results. This poly(cyanocobalamin) modified electrode showed high selectivity and sensitivity to peroxynitrite determination, which could be used in quantitative detection of peroxynitrite in vivo and in vitro.     

Human Cytochrome P450 (CYP1A2)‐dsDNA Interaction in situ Evaluation Using a dsDNA‐electrochemical Biosensor

Human cytochrome CYP1A2 is one of the major hepatic cytochrome P450s involved in many drugs metabolism, and chemical carcinogens activation. The CYP1A2‐dsDNA interaction in situ evaluation using a DNA‐electrochemical biosensor and differential pulse voltammetry was investigated. A dsDNA‐electrochemical biosensor showed that CYP1A2 interacted with dsDNA causing conformational changes in the double helix chain and DNA oxidative damage. A preferential interaction between the dsDNA guanosine residues and CYP1A2 was found, as free guanine and 8‐oxoguanine, a DNA oxidative damage biomarker, oxidation peaks were detected. This was confirmed using guanine and adenine homopolynucleotides‐electrochemical biosensors. The CYP1A2‐dsDNA interaction and dsDNA conformation changes was also confirmed by UV‐Vis spectrophotometry.     

Developing a Nano‐Biosensor for DNA Hybridization Using a New Electroactive Label

Development of electrochemical DNA hybridization biosensors based on carbon paste electrode (CPE) and gold nanoparticle modified carbon paste electrode (NGMCPE) as transducers and ethyl green (EG) as a new electroactive label is described. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were applied for the investigation and comparison of bare CPE and NGMCPE surfaces. Our voltammetric and spectroscopic studies showed gold nanoparticles are enable to facilitate electron transfer between the accumulated label on DNA probe modified electrode and electrode surface and enhance the electrical signals and lead to an improved detection limit. The immobilization of a 15‐mer single strand oligonucleotide probe on the working electrodes and hybridization event between the probe and its complementary sequence as a target were investigated by differential pulse voltammetry (DPV) responses of the EG accumulated on the electrodes. The effects of some experimental variables on the performance of the biosensors were investigated and optimum conditions were suggested. The selectivity of the biosensors was studied using some non‐complementary oligonucleotides. Finally the detection limits were calculated as 1.35×10^?10 mol/L and 5.16×10^?11 mol/L on the CPE and NEGCPE, respectively. In addition, the biosensors exhibited a good selectivity, reproducibility and stability for the determination of DNA sequences.     

Adsorption and Electrooxidation of Nucleic Acids at Glassy Carbon Electrodes Modified with Multiwalled Carbon Nanotubes Dispersed In Polylysine

We report the advantages of the adsorption and electrooxidation of oligonucleotides and calf‐thymus double stranded DNA (dsDNA) at glassy carbon electrodes (GCE) modified with a dispersion of multiwalled carbon nanotubes (MWCNT) in poly‐L ‐lysine (Plys) (GCE/MWCNT‐Plys). Important enhancement in the guanine oxidation signal was obtained by adsorptive stripping voltammetry (AdSV) due to a most favorable interaction between the negatively charged DNAs and the positively charged Plys that support the MWCNT. The layer of oligo(dG)₁₁ immobilized at GCE/MWCNT‐Plys was successfully used for the selective detection of the hybridization event using oligo(dG)₁₁/oligo(dC)₁₁ as model.     

Tin oxide nanoparticles-polymer modified single-use sensors for electrochemical monitoring of label-free DNA hybridization

In this study, SnO₂ nanoparticles (SNPs)-poly(vinylferrocenium) (PVF⁺) modified single-use graphite electrodes were developed for electrochemical monitoring of DNA hybridization. The surfaces of polymer modified and polymer-SNP modified pencil graphite electrodes (PGEs) were firstly characterized by using SEM analysis. The electrochemical behaviours of these electrodes were also investigated using the differential pulse voltammetry (DPV) and electrochemical impedance spectroscopy (EIS) techniques. The polymer-SNP modified PGEs were then tested for the electrochemical sensing of DNA based on the changes at the guanine oxidation signals. Experimental parameters, such as; different modifications in DNA oligonucleotides, DNA probe concentrations were examined to obtain more sensitive and selective electrochemical signals for nucleic acid hybridization. After optimization studies, DNA hybridization was investigated in the case of complementary of hepatitis B virus (HBV) probe, mismatch (MM), and noncomplementary (NC) sequences.     

Interaction of Mitomycin C with DNA Immobilized onto Single‐walled Carbon Nanotube/Polymer Modified Pencil Graphite Electrode

The electrochemical investigation of the interaction between the anticancer drug mitomycin C (MC) and DNA was described using a single‐walled carbon nanotube (SWCNT)/poly(vinylferrocenium) (PVF⁺) modified pencil graphite electrode (PGE). The electrochemical oxidation signals of guanine were monitored before and after the interaction between MC and DNA by using differential pulse voltammetry. The effects of DNA and MC concentration and MC interaction time were examined based on the electrode response. Cyclic voltammetry and electrochemical impedance spectroscopy were used for the characterization of SWCNT/PVF⁺ modified and PVF⁺ modified PGEs. The detection limit corresponded to 625 ng/mL for MC using calf thymus double‐stranded DNA immobilized SWCNT/PVF⁺ modified PGE.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

Electrofluorochromic Detection of Cyanide Anions Using a Nanoporous Polymer Electrode and the Detection Mechanism

An electrofluorochromic (EFC) conjugated copolymer ( P_EFC ) containing carbazole and benzothiadiazole (BTD) moieties is synthesized through Suzuki coupling followed by electrochemical polymerization, resulting in a nanoporous EFC polymer electrode. The electrode exhibits high sensitivity and selectivity in the EFC detection of cyanide anions (CN^?) in largely aqueous electrolyte (67 vol % water) because electrochemical oxidation of P_EFC leads to significant fluorescence quenching, and the presence of different concentrations (1 to 100 μM ) of CN^? in the electrolyte can weaken the oxidative quenching to substantially different extents. Although P_EFC is hydrophobic in the neutral state, it is converted to radical cation/dication states upon oxidation, rendering the P_EFC some hydrophilicity. Moreover, its nanoporous morphology provides a large surface area and short diffusion distance, facilitating the movement of CN^? in the electrolyte into the P_EFC film to interact with receptors. Density functional theory calculations show that the noncovalent interaction between electron‐deficient BTD and nucleophilic CN^? is energy favorable in the oxidized states in both aqueous and organic media, suggesting that the specific π^?–π⁺ interaction plays the main role in the CN^? detection.     

Electrochemical strategy for sensing DNA methylation and DNA methyltransferase activity

The present work demonstrates a novel signal-off electrochemical method for the determination of DNA methylation and the assay of methyltransferase activity using the electroactive complex [Ru(NH₃)₆]³⁺ (RuHex) as a signal transducer. The assay exploits the electrostatic interactions between RuHex and DNA strands. Thiolated single strand DNA1 was firstly self-assembled on a gold electrode via Au–S bonding, followed by hybridization with single strand DNA2 to form double strand DNA containing specific recognition sequence of DNA adenine methylation MTase and methylation-responsive restriction endonuclease Dpn I. The double strand DNA may adsorb lots of electrochemical species ([Ru(NH₃)₆]³⁺) via the electrostatic interaction, thus resulting in a high electrochemical signal. In the presence of DNA adenine methylation methyltransferase and S-adenosyl-l-methionine, the formed double strand DNA was methylated by DNA adenine methylation methyltransferase, then the double strand DNA can be cleaved by methylation-responsive restriction endonuclease Dpn I, leading to the dissociation of a large amount of signaling probes from the electrode. As a result, the adsorption amount of RuHex reduced, resulting in a decrease in electrochemical signal. Thus, a sensitive electrochemical method for detection of DNA methylation is proposed. The proposed method yielded a linear response to concentration of Dam MTase ranging from 0.25 to 10 U mL⁻¹ with a detection limit of 0.18 U mL⁻¹ (S/N = 3), which might promise this method as a good candidate for monitoring DNA methylation in the future.     

Selective voltammetric and flow-injection determination of guanine and adenine on a glassy carbon electrode modified by a ruthenium hexachloroplatinate film

An inorganic film of ruthenium hexachloroplatinate (RuPtCl₆), deposited on the surface of a glassy carbon electrode, exhibits electrocatalytic activity in the oxidation of purine bases, such as adenine and guanine. Appropriate working conditions are found for fabricating a polymer film on the surface of glassy carbon and for recording the maximum electrocatalytic effect for the modified electrode. A method is developed for the selective voltammetric determination of guanine and adenine in their simultaneous presence on an electrode modified by a RuPtCl₆ film. A procedure is proposed for the amperometric detection of purine bases with this modified electrode under the conditions of flow-injection analysis. The dependence of the analytical signal on the concentration of guanine and adenine is linear up to 5 × 10^?6 and 5 × 10^?7 M in the stationary mode and to 5 × 10^?7 and 5 × 10^?8 M under flow conditions, respectively. The proposed method was tested in the analysis of calf thymus DNA for the concentrations of guanine and adenine.