In situ electrochemical evaluation of anticancer drug temozolomide and its metabolites–DNA interaction

Temozolomide (TMZ) is an antineoplastic alkylating agent with activity against serious and aggressive types of brain tumours. It has been postulated that TMZ exerts its antitumor activity via its spontaneous degradation at physiological pH. The in vitro evaluation of the interaction of TMZ and its final metabolites, 5-aminoimidazole-4-carboxamide (AIC) and methyldiazonium ion, with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine residues. The results obtained revealed the decrease of the dsDNA oxidation peaks with incubation time, showing that TMZ and AIC/methyldiazonium ion interact with dsDNA causing its condensation. Furthermore, the experiments of the in situ TMZ and AIC/methyldiazonium ion–dsDNA interaction using the multilayer dsDNA-electrochemical biosensor confirmed the condensation of dsDNA caused by these species and showed evidence for a specific interaction between the guanosine residues and TMZ metabolites, since free guanine oxidation peak was detected. The oxidative damage caused to DNA bases by TMZ metabolites was also detected electrochemically by monitoring the appearance of the 8-oxoguanine/2,8-dyhydroxyadenine oxidation peaks. Nondenaturing agarose gel electrophoresis of AIC/methyldiazonium ion–dsDNA samples confirmed the occurrence of dsDNA condensation and oxidative damage observed in the electrochemical results. The importance of the dsDNA-electrochemical biosensor in the in situ evaluation of TMZ–dsDNA interactions is clearly demonstrated.     

In situ dsDNA-bevacizumab anticancer monoclonal antibody interaction electrochemical evaluation

The interaction of the anticancer monoclonal antibody bevacizumab (BEVA) with double-stranded DNA (dsDNA) was studied by voltammetry and gel-electrophoresis in incubated samples and using the dsDNA-electrochemical biosensor. The voltammetric results revealed a decrease and disappearance of the dsDNA oxidation peaks with increasing incubation time, showing that BEVA binds to the dsDNA but no DNA oxidative damage was detected electrochemically. Non denaturing agarose gel-electrophoresis experiments were in agreement with the voltammetric results showing the formation of compact BEVA-dsDNA adduct. The dsDNA-electrochemical biosensor in incubated solutions showed that BEVA also undergoes structural modification upon binding dsDNA, and BEVA electroactive amino acid residues oxidation peaks were detected.     

Electrochemical study of quercetin-DNA interactions: part II. In situ sensing with DNA biosensors

Quercetin interaction with dsDNA was investigated electrochemically using two types of DNA biosensor in order to evaluate the occurrence of DNA damage caused by oxidized quercetin. The results showed that quercetin binds to dsDNA where it can undergo oxidation. The radicals formed during quercetin oxidation cause breaks of the hydrogen bonds in the dsDNA finally giving rise to 8-oxoguanine since the DNA guanosine and adenosine nucleotides in contact with the electrode surface can easily be oxidized. A mechanism for oxidized quercetin-induced damage to dsDNA immobilized onto a glassy carbon electrode surface is proposed and the formation of 8-oxoguanine is explained. The importance of DNA-electrochemical biosensors in the determination of the interaction mechanism between DNA and quercetin is clearly demonstrated.     

In situ evaluation of heavy metal-DNA interactions using an electrochemical DNA biosensor

Heavy metal ions, lead, cadmium and nickel, are well known carcinogens with natural different origins and their direct mode of action is still not fully understood. A dsDNA-electrochemical biosensor, employing differential pulse voltammetry, was used for the in situ evaluation of Pb2+, Cd2+ and Ni2+ interaction with dsDNA. The results confirm that Pb2+, Cd2+ and Ni2+ bind to dsDNA, and that this interaction leads to different modifications in the dsDNA structure. These modifications were electrochemically recognized as changes in the oxidation peaks of guanosine and adenosine bases. Using homopolynucleotides of guanine and adenine it has been proved that the interaction between Pb2+ and DNA causes oxidative damage and preferentially takes place at adenine-containing segments, with the formation of 2,8-dihydroxyadenine, the oxidation product of adenine residues and a biomarker of DNA oxidative damage. The Pb2+ bound to dsDNA can still undergo oxidation. The interaction of Cd2+ and Ni2+ causes conformational changes, destabilizing the double helix, which can enable the action of other oxidative agents on DNA.     

In situ evaluation of chromium–DNA damage using a DNA-electrochemical biosensor

The in situ evaluation of the direct interaction of chromium species with double-stranded DNA (dsDNA) was studied using differential pulse voltammetry at a glassy carbon electrode. The DNA damage was electrochemically detected following the changes in the oxidation peaks of guanosine and adenosine bases. The results obtained revealed the interaction with dsDNA of the Cr(IV) and Cr(V) reactive intermediates of Cr(III) oxidation by O₂ dissolved in the solution bound to dsDNA. This interaction leads to different modifications and causes oxidative damage in the B-DNA structure. Using polyhomonucleotides of guanine and adenine, it was shown that the interaction between reactive intermediates Cr(IV) and Cr(V)–DNA causes oxidative damage and preferentially takes place at guanine-rich segments, leading to the formation of 8-oxoguanine, the oxidation product of guanine residues and a biomarker of DNA oxidative damage. The interaction of Cr(VI) with dsDNA causes breaking of hydrogen bonds, conformational changes, and unfolding of the double helix, which enables easier access of other oxidative agents to interact with DNA, and the occurrence of oxidative damage to DNA.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Hydroxyl radicals electrochemically generated in situ on a boron-doped diamond electrode

The hydroxyl radicals electrochemically generated in situ on a boron-doped diamond (BDD) electrode have been investigated for the first time in different electrolyte media, over the whole pH range between 1 and 11. A more extensive characterisation of BDD electrochemical properties is very important to understand the reactivity of organic compounds towards electrochemical oxidation on the BDD electrode, which is related to their interaction with adsorbed hydroxyl radicals due to water oxidation on the electrode surface. An oxidation peak corresponding to the transfer of one electron and one proton was observed in pH <9 electrolytes, associated with the water discharge process and electrochemical generation of hydroxyl radicals, which can interact and enhance the electro-oxidation of organic compounds. In pH >9 electrolytes the electrochemical generation of hydroxyl radicals was not observed; ammonia buffer electrolyte gave a pH-independent peak corresponding to the ammonia oxidation reaction. Additionally, for most pH values studied, a few small peaks associated with the electrochemical interaction between non-diamond carbon species on the doped diamond electrode surface and the electrolyte were also seen, which suggests that the doped diamond is relatively unreactive, but not completely inert, and the electrogenerated hydroxyl radicals play a role as mediator in the oxidation of organics.     

Ferrocenecarboxaldehyde labeled DNA probe for the study on DNA damage and protection

A ferrocenecarboxaldehyde (FCA) labeled DNA probe is used for the first time in the study of DNA damage and protection. The electrochemically active reagent FCA was labeled successfully on to a denatured calf-thymus DNA by ¶1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide (EDC). The FCA labeled DNA probe was used to hybridize with the sample DNA sequence accumulated on the surface of a graphite electrode. The anodic peaks of the FCA bound to the double-stranded DNA (dsDNA) by differential pulse voltammetry (DPV) were used for the detection of DNA damage and protection. Thiourea, sodium benzoic acid and isopropanol can decrease DNA damage by hydroxyl radicals, and their protection efficiencies are discussed.     

Voltammetric DNA Biosensor Based on a Microcrystalline Natural Graphite–Polystyrene Composite Transducer

New voltammetric DNA biosensor based on a microcrystalline natural graphite–polystyrene composite film in the role of a transducer was used for the investigation of the interaction between model carcinogenic substance (2-aminofluorene; one of the most extensively studied examples of the aromatic amine class of carcinogens) and calf thymus double-stranded DNA (dsDNA). The layer of dsDNA immobilized at the electrode surface was utilized as a biocomponent responsive interface. The biosensor was characterized regarding the detection of DNA damage (induced by direct interaction with 2-aminofluorene) using square wave voltammetric responses of the guanine and adenine moieties and cyclic voltammetric responses of the anionic redox indicator [Fe(CN)₆]^4–/3– present in the solution.     

Biosensor with Protective Membrane for the Detection of DNA Damage and Antioxidant Properties of Fruit Juices

With the purpose to prepare a DNA biosensor protected with an outer‐sphere membrane against high molecular weight interferences, a carbon film electrode was layer‐by‐layer modified with dsDNA and chitosan. Using cyclic and square‐wave voltammetry and impedance spectroscopy, the oxidative damage of DNA by the hydroxyl and superoxide anion radicals was detected which consists of opening of the helix structure followed by deep DNA chain degradation. The biosensor has been applied to the detection of the antioxidant effect of apple and orange juices. The investigation of the novel biosensor with a protective membrane represents a significant contribution to the field of DNA biosensors utilization.     

In situ evaluation of anticancer drug methotrexate-DNA interaction using a DNA-electrochemical biosensor and AFM characterization

An in situ evaluation of the dsDNA-methotrexate (MTX) interaction was performed by voltammetry using a DNA-electrochemical biosensor and characterized by atomic force microscopy (AFM) at a highly oriented pyrolytic graphite (HOPG) surface. Electrochemical experiments in incubated solutions showed that the interaction of MTX with dsDNA leads to modifications to the dsDNA structure in a time-dependent manner. The AFM images show reorganization of the DNA self-assembled network on the surface of the HOPG electrode upon binding methotrexate and the formation of a more densely packed and slightly thicker MTX-dsDNA lattice with a large number of aggregates embedded into the network film. The intercalation of MTX between complementary base pairs of dsDNA lead to the increase of purine oxidation peaks due to the unwinding of the dsDNA. The dsDNA-electrochemical biosensor and the purinic homo-polynucleotide single stranded sequences of guanosine and adenosine, poly[G] and poly[A]-electrochemical biosensors, were used to investigate and understand the interaction between MTX and dsDNA.     

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.     

Electrochemical biosensing for dsDNA damage induced by PbSe quantum dots under UV irradiation

<正>An electrochemical sensor for the detection of the natural double-stranded DNA(dsDNA) damage induced by PbSe quantum dots(QDs) under UV irradiation was developed.The biosensing membranes were prepared by successively assembling 3- mercaptopropionic acid,polycationic poly(diallyldimethyl ammonium) and dsDNA on the surface of the gold electrode.Damage of dsDNA was fulfilled by immersing the sensing membrane electrode in PbSe QDs suspension and illuminating it with an UV lamp. Cyclic voltammetry was utilized to detect dsDNA damage with Co(phen)_3~(3+) as the electroactive probe.The UV irradiation,Pb~(2+) ions liberated from the PbSe QDs under the UV irradiation and the reactive oxygen species(ROS) generated in the presence of the PbSe QDs also under the UV irradiation were the three factors of inducing the dsDNA damage.The synergistic effect of the three factors might dramatically enhance the damage of dsDNA.This electrochemical sensor provided a simple method for detecting DNA damage,and may be used for investigating the DNA damage induced by other QDs.     

Evaluation of DNA damage and antioxidant capacity of sericin by a DNA electrochemical biosensor based on dendrimer-encapsulated Au-Pd/chitosan composite

Au-Pd bimetallic nanoparticles were prepared in the presence of an amine-terminated dendrimer of a fourth generation poly(amidoamine) type. A biosensor was fabricated by immobilizing dsDNA on a thin layer of a dendrimer-encapsulated bimetallic nanoparticles (Au-Pd) in a chitosan composite on a glassy carbon electrode. The biosensor was evaluated by square wave voltammetry for the determination of the oxidative damage of immobilized DNA and the antioxidant capacity of sericin. The biosensor is shown to be suitable for the rapid detection of DNA damage and the assessment of the antioxidant capacity of sericin.     

Adsorptive transfer voltammetry applied to the study of chromium-induced DNA damage in the presence of curcumin

The interaction between double-stranded (ds) calf-thymus DNA and chromium in the presence of curcumin (CC) was studied by differential pulse adsorptive transfer voltammetry using carbon paste electrode (CPE). Curcumin–Cr complex generated changes in calf thymus DNA. The mechanism for DNA cleavage by curcumin–Cr complex appears to involve both the hydroxyl radical as well as singlet oxygen. The characteristic peak of dsDNA, due to the oxidation of guanine residues, drastically decreased. The increased DNA damage by curcumin–Cr complex was observed in the presence of various concentrations of chromium(VI).     

A novel sensitive electrochemical DNA biosensor for assaying of anticancer drug leuprolide and its adsorptive stripping voltammetric determination

The anticancer drug, leuprolide (LPR) bound to double-stranded fish sperm DNA (dsDNA) which was immobilized onto the surface of an anodically activated pencil graphite electrode (PGE), was employed for designing a sensitive biosensor. The interaction of leuprolide (LPR) with double-stranded DNA (dsDNA) immobilized onto pencil graphite electrode (PGE) have been studied by electrochemical methods. The mechanism of the interaction was investigated and confirmed by differential pulse voltammetry using two different interaction methods; at the PGE surface and in the solution phase. The decrease in the guanine oxidation peak current was used as an indicator for the interaction in acetate buffer at pH 4.80. The response was optimized with respect to accumulation time, potential, drug concentration, and reproducibility for both interaction methods. The linear response was obtained in the range of 0.20-6.00 ppm LPR concentration with a detection limit of 0.06 ppm on DNA modified PGE and between 0.20 and 1.00 ppm concentration range with detection limit of 0.04 ppm for interaction in solution phase method. LPR showed an irreversible oxidation behavior at all investigated pH values on a bare PGE. Differential pulse adsorptive stripping (AdSDPV) voltammetric method was developed for the determination of LPR. Under these conditions, the current showed a linear dependence with concentration within a range of 0.005-0.20 ppm with a detection limit of 0.0014 ppm. Each determination method was fully validated and applied for the analysis of LPR in its pharmaceutical dosage form.     

Atomic force microscopy characterization of an electrochemical DNA-biosensor

Electrode surface characteristics represent an important aspect on the construction of sensitive DNA electrochemical biosensors for rapid detection of DNA interaction and damage. Two different immobilization procedures of double-stranded DNA (dsDNA) at the surface of a HOPG electrode were evaluated by MAC mode AFM performed in air. A thin dsDNA adsorbed film forming a network structure with holes exposing the electrode surface and a thick dsDNA film completely covering the electrode surface, presenting a much rougher structure, were investigated. The DNA surface characteristics and structure are discussed with respect to the degree of surface coverage.     

Real-time surface-enhanced Raman spectroscopy monitoring of surface pH during electrochemical melting of double-stranded DNA

The application of a negative potential ramp at a double-stranded DNA (dsDNA) functionalized electrode surface results in the gradual denaturation of the DNA in a process known as electrochemical melting. The underlying physical chemistry behind electrochemically driven DNA denaturation is not well understood, and one possible mechanism is a change in local pH at the electrode surface. We demonstrate that by coimmobilization of p-mercaptobenozic acid at a dsDNA-functionalized electrode surface, it is possible to monitor both DNA denaturation and the local pH simultaneously using surface-enhanced Raman spectroscopy. We find that the local pH at the electrode surface does not change as the applied potential is scanned negative and the dsDNA denatures. We therefore conclude that in these experiments electrochemical melting is not caused by electrochemically driven local pH changes.     

Voltammetric detection of damage to DNA caused by nitro derivatives of fluorene using an electrochemical DNA biosensor

An electrochemical DNA biosensor based on the screen printed carbon paste electrode (SPCPE) with an immobilized layer of calf thymus double-stranded DNA has been used for in vitro investigation of the interaction between genotoxic nitro derivatives of fluorene (namely 2-nitrofluorene and 2,7-dinitrofluorene) and DNA. Two types of DNA damage have been detected at the DNA/SPCPE biosensor: first, that caused by direct association of the nitrofluorenes, for which an intercalation association has been found using the known DNA intercalators [Cu(phen)₂]²⁺ and [Co(phen)₃]³⁺ as competing agents, and, second, that caused by short-lived radicals generated by electrochemical reduction of the nitro group (observable under specific conditions only).     

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.