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.     

Chitosan/Ionic Liquid Composite Electrode for Electrochemical Monitoring of the Surface‐Confined Interaction Between Mitomycin C and DNA

In the present study a chitosan/ionic liquid modified pencil graphite electrode (CHIT‐IL‐PGEs) was developed for the first time for enhanced electrochemical monitoring of nucleic acid, and the interaction of the anticancer drug Mitomycin C (MC) and calf thymus double stranded DNA (dsDNA) by measuring the oxidation signals of MC and guanine in the same voltammetric scale. Differential pulse voltammetry, cyclic voltammetry and electrochemical impedance spectroscopy techniques were used to evaluate the performance of the CHIT‐IL based biosensor on electrochemical monitoring of DNA, and drug‐DNA interaction. The experimental parameters, IL, dsDNA and MC concentration and the interaction time were then optimized.     

Determination of DNA Hypermethylation Using Anti‐cancer Drug‐Temozolomide

An electrochemical drug‐DNA biosensor was developed for the detection of interaction between the anti‐cancer drug, Temozolomide (TMZ), and DNA sequences by using Differential Pulse Voltammetry at the graphite electrode surfaces. TMZ is a pro‐drug and an alkylating agent that crosses the blood‐brain barrier, so it is mainly used for brain cancers treatment. In this study, we aim to develop a‐proof‐of‐concept study to investigate the effect of TMZ on formerly methylated DNA sequences since TMZ shows its anti‐cancer activity by methylating the DNA. Interaction between TMZ and DNA causes localized distortion of DNA away from an idealized B‐form, resulting in a wider major groove and greater steric accessibility of functional groups in the base of the groove. According to the results, TMZ behaves as a ‘hybridization indicator’ because of its different electrochemical behavior to different strands of DNA. After interaction with TMZ, hybrid (double stranded DNA‐dsDNA) signals decreased dramatically whereas probe (single stranded DNA‐ssDNA) and control signals remain almost unchanged. The signal differences enabled us to distinguish ssDNA and dsDNA without using a label or tag. It is the first study to demonstrate the interaction between the TMZ and dsDNA created from probe and target. We use specific oligonucleotides sequences instead of using long dsDNA sequences.     

Square Wave Stripping Voltammetry of Unlabeled Single‐ and Double‐Stranded DNAs

We show that, in difference to previously applied electrochemical methods working with stationary electrodes, square wave voltammetry produces well‐developed peaks II_SW (specific for dsDNA) and III_SW yielded by ssDNA at hanging mercury drop electrode (HMDE) and solid amalgam electrodes (SAEs). Using these peaks various kinds of DNA structural transitions can be studied, including unwinding of dsDNA at negatively charged electrode surfaces. The sensitivity of the DNA analysis is much better than that obtained with guanine oxidation signals at carbon electrodes. Both carbon electrodes and SAEs appear attractive as transducers in label‐free RNA and DNA sensors.     

Sensitive Nucleic Acid Detection at NH2‐MWCNTs Modified Glassy Carbon Electrode and its Application for Monitoring of Gemcitabine‐DNA Interaction

An electrochemical dsDNA nanobiosensor was fabricated using amino‐functionalized multi walled carbon nanotubes modified glassy carbon electrode (NH₂fMWCNTs/GCE) for the sensitive detection of DNA bases and electrochemical monitoring of drug‐DNA interaction. The influence of functional groups on MWCNT was studied by MWCNT functionalized with NH₂ (NH₂fMWCNTs) and COOH (COOHfMWCNT) groups based on the signal of DNA bases. The modified electrodes were characterized by scanning electron microscopy. One layer of calf thymus double stranded deoxyribonucleic acid (ct‐dsDNA) was immobilized onto the NH₂fMWCNTs/GCE (dsDNA/NH₂fMWCNTs/GCE). The dsDNA/NH₂fMWCNTs/GCE were used to investigate the interaction between the dsDNA and the anticancer drug gemcitabine by differential pulse voltammetry in acetate buffer of pH 4.70. For the confirmation of interaction, the lowering in intensity of the current signals of guanine and adenine was considered as an indicator. Electrochemical impedance spectroscopy studies were performed for the comparison of the modified surfaces. In order to define and visualize the interaction mechanism between gemcitabine and dsDNA/NH₂fMWCNTs/GCE at the molecular level, in silico methods including docking and molecular dynamics simulations were employed.     

Electrochemical DNA biosensor for the determination of benzo[a]pyrene–DNA adducts

The metabolites of the environmental pollutant, benzo[a]pyrene (BaP) are carcinogenic and mutagenic agents. Thus, the determination of additional products (adducts) of the interaction between DNA and BaP, attracts great interest in cancer research.

In this study, the determination of interaction between BaP and calf thymus double-stranded DNA (dsDNA) was performed by using differential pulse voltammetry (DPV) and constant current chronopotentiometric stripping analysis (PSA) in connection with carbon paste electrode (CPE) or glassy carbon electrode (GCE). As a result of interaction of BaP with dsDNA, the signal obtained from the oxidation of guanine decreased and a new adduct signal at a more positive potential appeared. This new peak is attributed to the formation of an adduct from the interaction of guanine with BaP. The chemically prepared anti-7,8,9,10-tetrahydrobenzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) adduct by using iodine oxidation was analyzed and the electrochemical signal of the adduct was observed. When the dsDNA modified GCE was immersed into various concentrations of BaP solution, the oxidation peak of guanine decreased and the adduct peak increased with the increasing BaP concentration. The partition coefficient was also obtained from the peak of BaP with dsDNA. The results revealed that the formation of adducts could be determined by using electrochemical DNA biosensors, which are fast, simple and cost-effective devices. Furthermore, this study promises that the analysis of other important adducts would benefit from the introduction of electrochemical methods.     

Direct electrochemistry of double stranded DNA on ionic liquid modified carbon paste electrode

The direct electrochemistry of herring sperm double-stranded DNA (dsDNA) on an ionic liquid N-butylpyridinium hexafluorophosphate modified carbon paste electrode was investigated. The cyclic voltammogram showed two irreversible oxidation peaks at 0.868 V and 1.188 V (vs. SCE), which corresponded to the oxidation of guanine and adenine residues, respectively. Compared to the common carbon paste electrode the electrochemical response was greatly improved. The electrochemical behavior of dsDNA on the modified electrode was carefully investigated with the electrochemical parameters were calculated. Under optimal conditions the dsDNA can be directly determined in the concentration range from 50 to 600 μg mL^?1 with a detection limit of 17 μg mL^?1 (3σ).     

Antidiabetic Drug Metformin Oxidation and in situ Interaction with dsDNA Using a dsDNA‐electrochemical Biosensor

The antidiabetic drug metformin (MET) is one of a group of emerging pharmaceutical drug contaminants in the wastewater treatment plants. The electrochemical behaviour of MET?Cu(II) complex by differential pulse and square wave voltammetry, in a wide pH range, at a glassy carbon electrode modified with a carbon black dihexadecylphosphate film (CB?DHP/GCE), was investigated. The MET?Cu(II) complex oxidation showed one pH‐dependent process, which leads to the formation of an oxidation product, being oxidized at a lower potential. The electroanalytical MET?Cu(II) complex detection limit, LOD=0.63 μM, and quantification limit, LOQ=2.09 μM, were obtained, and the MET?Cu(II) complex determination in wastewater samples collected from a senior residence effluent, using the CB?DHP/GCE, was achieved. Considering MET toxicity, the electrochemical evaluation of MET?dsDNA interaction, in incubated solutions and using dsDNA‐electrochemical biosensors, following the changes in the oxidation peaks of guanosine and adenosine residues electrochemical currents, was also investigated. The MET?dsDNA interaction mechanism, for shorter times, occurs by the binding of MET molecules in the minor grooves of the dsDNA, and for long times, the stabilization of the MET?dsDNA complex, causing a local distortion and/or unwinding of dsDNA morphology, is described. However, MET did not promote DNA oxidative damage.     

Determination of Electrochemical Interaction between 2‐(1H‐benzimidazol‐2‐yl) Phenol and DNA Sequences

A benzimidazole derivate, 2‐(1H‐benzimidazol‐2‐yl) phenol (2‐Bip) and its interaction mechanism with sequence specific DNA was examined with Differential Pulse Voltammetry (DPV). We, for the first time, investigated the effect of 2‐Bip on sequence specific DNA with electrochemical methods by evaluating both guanine and 2‐Bip oxidation signal changes. In the study, probe sequences were immobilized to the surface of the electrodes and then hybridization was achieved by sending the complementary target onto the probe modified electrodes. Following the hybridization, 2‐Bip solution was interacted with probe and hybrid sequences to see the effect of 2‐Bip on different DNA sequences. The binding constant (K), toxicity (S%) and thermodynamic parameters, i. e., Gibbs free energy (ΔG°) of 2‐Bip‐DNA complexes were evaluated. K was calculated as 5×10⁵ and the change in the ΔG° was found as ?32.50 kJ mol^?1, which are consistent well with the literature. Furthermore, S% showed that 2‐Bip is moderately toxic to single stranded DNA (ssDNA) and toxic to double stranded DNA (dsDNA). From our experimental data, we made four conclusions (i) 2‐Bip affects both ssDNA and dsDNA, (ii) 2‐Bip interaction mode with DNA could be non‐covalent interactions, (iii) 2‐Bip could be used as new DNA hybridization indicator due to its distinct effects on ssDNA and dsDNA, (iv) 2‐Bip could be used as a drug molecule for its DNA effect.     

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.     

Electrochemical detection of DNA damage induced by in situ generated bisphenol A radicals through electro-oxidation

A glassy carbon electrode was modified with dsDNA and a nanocomposite composed of multi-walled carbon nanotubes and chitosan (MWNT-chit). The electrode was applied to the electrochemical detection of DNA damage as induced by in situ generated bisphenol A (BPA) radicals through electro-oxidation. The modified electrode was characterized by cyclic voltammetry and electrochemical impedance spectroscopy. The results indicate that MWNT-chit nanocomposite represents a viable platform for the immobilization of DNA that effectively promotes electron transfer between DNA and the electrode. The mode of interaction between DNA and BPA was investigated by differential pulse voltammetry and UV-vis spectrophotometry, indicating that the dominant interaction is intercalation. In order to explore the mechanism of damage caused by BPA radicals, the electro-oxidation of BPA at the modified glass electrode was investigated. Based on the signal for guanine without any other external indicator, DNA damage was investigated through the electro-oxidation of BPA.     

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.     

Multiwalled Carbon Nanotubes‐Chitosan Modified Single‐Use Biosensors for Electrochemical Monitoring of Drug‐DNA Interactions

A multiwalled carbon nanotubes (CNT)‐chitosan (CHIT) modified pencil graphite electrode (CNT‐CHIT/PGE) was developed for the first time herein for electrochemical monitoring of the interaction of an anticancer drug, mitomycin C (MC) and DNA. The characterization of unmodified PGE, CHIT/PGE, CNT/PGE and CHIT‐CNT/PGE were performed by scanning electron microscopy and cyclic voltammetry techniques. The oxidation signals of MC and guanine were measured before and after interaction at the surface of CNT‐CHIT/PGEs using differential pulse voltammetry. Electrochemical impedance spectroscopy technique was also successfully utilized for monitoring of the interaction process at the surface of CNT‐CHIT/PGEs in different interaction times.     

Electrochemical Study of the Interaction of the Alkylating Agent Busulfan with Double Strand DNA

Damage of salmon sperm double strand ss dsDNA in solution or immobilized on screen‐printed carbon electrode (SPCE) induced by incubation of DNA with the antineoplastic alkylating agent busulfan (BUS) at various conditions was detected for the first time by simple electrochemical methods. Chemical changes in DNA bases can be detected through the altered electroactivity of the DNA. Electrochemical voltammetric sensing of damage caused by BUS to dsDNA in solution was monitored by the appearance of peaks diagnostic of the oxidation of guanine and adenine. Moreover, crystal violet, which interacts with the DNA immobilized on SPCEs, was used as an effective electroactive indicator, in combination with cyclic voltammetry and differential pulse voltammetry techniques to monitor the cross‐links or damage to DNA. The interaction between BUS and DNA were determined by the changes in the voltammetric peak of crystal violet. The effects of various conditions upon the crystal violet signal were investigated.     

A Reduced Graphene Oxide‐based Electrochemical DNA Biosensor for the Detection of Interaction between Cisplatin and DNA based on Guanine and Adenine Oxidation Signals

A glassy carbon electrode (GCE) was modified by electrochemically reduced graphene oxide (ERGO) for subsequent dsDNA immobilization. The interaction of cisplatin with dsDNA was studied at this modified electrode. Quantitative investigations were performed by adsorptive transfer stripping voltammetry (AdTSV) using differential pulse voltammetry (DPV). The morphology and structure of graphene oxide (GO) and ERGO modified GCEs (GO/GCE and ERGO/GCE, respectively) were characterized by UV‐vis, FT‐IR, Raman spectroscopy and cyclic voltammetry. Compared with the bare GCE and the GO/GCE, the ERGO/GCE exhibited excellent electrocatalytic activity towards the oxidation of dsDNA due to guanine and adenine groups, testified by high oxidation peak currents and decreased oxidation potentials. The interaction of micromolar concentrations of cisplatin with surface confined dsDNA was readily detected as inferred from the decrease of the voltammetric oxidation peaks of guanine and adenine. This trend was significantly greater at the ERGO/GCE compared to the GO/GCE. The interaction of cisplatin with dsDNA was also studied in solution phase by AdTSV with detection at the ERGO/GCE.     

Electrochemical Detection of Abasic Site‐Containing DNA

A simple and rapid approach for detecting apurinic (AP) sites in DNA, based on direct stripping chronopotentiometric measurements of the adenine and guanine nucleobases at a graphite electrode is described. Tetrahydrofuranyl residues, lacking a nucleobase moiety, were utilized for designing the AP sites and were incorporated in 19‐mer oligonucleotides. The change of adenine‐to‐guanine response ratio (A/G) in one‐, two‐ or three‐substituted adenosine residues for stable analogs of AP sites was exploited for electrochemical measurements of the adenine loss. The resulting A/G response ratio decreases linearly upon increasing the number of AP sites in the oligonucleotides; the values of A/G electrochemical signals were slightly enhanced when compared to the actual purine content. HPLC analysis of the released nucleobases confirmed that the sulfuric acid‐induced oligonucleotide cleavage provides complete apurination and dissolution of the released nucleobases in aqueous solution. Additional experiments with mixtures of free nucleobases and purine nucleosides reveal that the larger A/G ratio observed in the electrochemical analysis of AP‐site‐containing oligomers is attributed to the influence of the acid and/or thermal decomposition products (particularly the sugar fragments). This study represents the first step in developing a simple and direct electrochemical assay of AP sites in single‐stranded DNA.     

Electrochemical Study of the Interactions of DNA with Redox‐Active Molecules Based on the Immobilization of dsDNA on the Sol‐Gel Derived Nano Porous Hydroxyapatite‐Polyvinyl Alcohol Hybrid Material Coating

A novel type of sol‐gel inorganic‐organic hybrid material coated on glassy carbon electrode used for immobilization of double‐stranded DNA (dsDNA) and study of dsDNA with redox‐active molecules was developed. The hybrid material coating was produced by sol‐gel method with nano hydroxyapatite (HAp)‐polyvinyl alcohol (PVA). The optimum composition of the hybrid material was first examined, and the morphology of the nano HAp‐PVA coatings was investigated with the help of Scanning Electron Microscope (SEM). DsDNA was immobilized in/on the nano HAp‐PVA hybrid coatings by adsorption and the characteristics of the dsDNA/HAp‐PVA/GCE were studied by cyclic voltammetry (CV) using the probes of Co(phen) and Fe(CN) . The results indicate that the dsDNA can be immobilized on the nano porous HAp‐PVA coating effectively and its stability can satisfy the necessity of study on the interactions of dsDNA with redox‐active molecules on the electrode surface. Co(bpy) and Co(phen) were used as the model molecule to study the interactions of dsDNA with redox‐active molecules. Information such as ratio (K_Ox/K_Red) of the binding constant for the oxidized and reduced forms of a bound species, interaction mode, including change in the mode of interaction, and “limiting” ratio K /K at zero ionic strength (μ) can be obtained using dsDNA/HAp‐PVA/GCE with about 2 μg of DNA samples.     

Electrochemical Characteristics of a Novel Pyridinium Salt as a Candidate Drug Molecule and Its Interaction with DNA

In this article, for the first time, the electrochemical properties of a novel pyridine derivative, 4‐(2‐(2‐hydroxybenzylidene) hydrazinyl)‐1‐(3‐phenylpropyl) pyridinium bromide (abbreviated as 4‐Pyri), and its interaction with double stranded DNA (dsDNA) was investigated. The interaction between candidate drug molecule (4‐Pyri) and dsDNA was analyzed by examining 4‐Pyri (+0.6 V and +0.8 V) and guanine (+1.0 V) oxidation signal changes with Differential Pulse Voltammetry (DPV) and Cyclic Voltammetry (CV). Electrochemical Impedance Spectroscopy (EIS) was used to show the resistance changes before and after the interaction between 4‐Pyri and dsDNA. We showed that after the interaction with 4‐Pyri, the oxidation currents of guanine decreased dramatically, whereas the intrinsic oxidation currents of 4‐Pyri dramatically increased. 4‐Pyri oxidation current differences before and after the interaction with dsDNA enabled us to determine such interaction separately from guanine oxidation signals. In addition, resistance differences were observed at before and after the interaction with each other that confirmed the possible interaction. In addition, toxicity effect (S%) value, which is an important parameter for electrochemical studies indicated 4‐Pyri's toxicity to dsDNA. Our results demonstrated that 4‐Pyri interacts with dsDNA, and could be used as a potential candidate drug molecule due to its remarkable impact on dsDNA.     

Carbon Nanotubes Modified Graphite Electrodes for Monitoring of Biointeraction Between 6‐Thioguanine and DNA

In this present study, single‐walled carbon nanotubes (SWCNT) modified disposable pencil graphite electrodes (SWCNT‐PGEs) were developed for the electrochemical monitoring of anticancer drug, and its interaction with double stranded DNA (dsDNA). Under this aim, SWCNT‐PGEs were applied for the first time in the literature to analyse of 6‐Thioguanine (6‐TG), and also to investigate its interaction with DNA by voltammetric and impedimetric methods. The surface morphologies of PGE and SWCNT‐PGE were explored using scanning electron microscopy (SEM) and electrochemical characterization of unmodified/modified electrodes was performed by cyclic voltammetry (CV). Experimental parameters; such as, the concentration of 6‐TG and its interaction time with dsDNA were optimized by using differential pulse voltammetry (DPV). Additionally, the interaction of 6‐TG with dsDNA was studied in case of different interaction times by electrochemical impedance spectroscopy (EIS) in contrast to voltammetric results. The detection limit of 6‐TG was found to be 0.25 μM by SWCNT‐PGE.     

Electrochemical Biosensor for the Detection of Interaction Between Arsenic Trioxide and DNA Based on Guanine Signal

The interaction of arsenic trioxide (As₂O₃) with calf thymus double‐stranded DNA (dsDNA), calf thymus single‐stranded DNA (ssDNA) and also 17‐mer short oligonucleotide (Probe A) was studied electrochemically by using differential pulse voltammetry (DPV) with carbon paste electrode (CPE) at the surface and also in solution. Potentiometric stripping analysis (PSA) was employed to monitor the interaction of As₂O₃ with dsDNA in solution phase by using a renewable pencil graphite electrode (PGE). The changes in the experimental parameters such as the concentration of As₂O₃, and the accumulation time of As₂O₃ were studied by using DPV; in addition, the reproducibility data for the interaction between DNA and As₂O₃ was determined by using both electrochemical techniques. After the interaction of As₂O₃ with dsDNA, the DPV signal of guanine was found to be decreasing when the accumulation time and the concentration of As₂O₃ were increased. Similar DPV results were also found with ssDNA and oligonucleotide. PSA results observed at a low DNA concentration such as 1 ppm and a different working electrode such as PGE showed that there could be damage to guanine bases. The partition coefficients of As₂O₃ after interaction with dsDNA and ssDNA in solution by using CPE were calculated. Similarly, the partition coefficients (PC) of As₂O₃ after interaction with dsDNA in solution was also calculated by PSA at PGE. The features of this proposed method for the detection of DNA damage by As₂O₃ are discussed and compared with those methods previously reported for the other type of DNA targeted agents in the literature.