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.     

Direct electrochemistry of DNA,guanine and adenine at a nanostructured film-modified electrode

A nanostructured film electrode, a multi-wall carbon nanotubes (MWNT)-modified glassy carbon electrode (GCE), is described for the simultaneous determination of guanine and adenine. The properties of the MWNT-modified GCE were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The oxidation peak currents of guanine and adenine increased significantly at the MWNT-modified GCE in contrast to those at the bare GCE. The experimental parameters were optimized and a direct electrochemical method for the simultaneous determination of guanine and adenine was proposed. Using the MWNT-modified GCE, a sensitive and direct electrochemical technique for the measurement of native DNA was also developed, and the value of (G+C)/(A+T) of HCl-digested DNA was detected.     

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.     

DNA sensing on glassy carbon electrodes by using hemin as the electrochemical hybridization label

The electrochemical behavior of hemin, an iron complex of porphyrin, on binding to DNA at a glassy carbon electrode (GCE) and in solution, is described. Hemin, which interacts with covalently immobilized calf thymus DNA, was detected by use of a bare GCE, a double-stranded DNA-modified GCE (dsDNA-modified GCE), and a single-stranded DNA-modified GCE (ssDNA-modified GCE), in combination with differential pulse voltammetry (DPV). The structural conformation of DNA was determined from changes in the voltammetric signals acquired on reduction of hemin. As a result of its large steric structure and anionic substitution on its porphyrin plane, hemin intercalates between the base pairs of dsDNA. A scan-rate study for hemin and the dsDNA-hemin complex were also performed to determine the electrochemical behavior of the complex. The partition coefficient was obtained from the peak currents measured when different concentrations of hemin were in the presence of dsDNA. By observing the oxidation signals of guanine, damage to DNA after reaction with hemin at the GCE surface was also detected. The electrochemical detection of hybridization between the covalently immobilized probe and its target sequence was detected by use of hemin. These results demonstrate the use of DNA biosensors in conjunction with hemin for electrochemical detection of hybridization and damage to DNA.     

Novel electrochemical sensor based on functionalized graphene for simultaneous determination of adenine and guanine in DNA

A nano-material carboxylic acid functionalized graphene (graphene-COOH) was prepared and used to construct a novel biosensor for the simultaneous detection of adenine and guanine. The direct electrooxidation behaviors of adenine and guanine on the graphene-COOH modified glassy carbon electrode (graphene-COOH/GCE) were carefully investigated by cyclic voltammetry and differential pulse voltammetry. The results indicated that both adenine and guanine showed the increase of the oxidation peak currents with the negative shift of the oxidation peak potentials in contrast to that on the bare glassy carbon electrode. The electrochemical parameters of adenine and guanine on the graphene-COOH/GCE were calculated and a simple and reliable electroanalytical method was developed for the detection of adenine and guanine, respectively. The modified electrode exhibited good behaviors in the simultaneous detection of adenine and guanine with the peak separation as 0.334V. The detection limit for individual determination of guanine and adenine was 5.0×10(-8)M and 2.5×10(-8)M (S/N=3), respectively. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G+C)/(A+T) of single-stranded DNA was calculated as 0.80. The biosensor exhibited some advantages, such as simplicity, rapidity, high sensitivity, good reproducibility and long-term stability.     

以乙二胺为手臂分子制备的DNA修饰电极及其伏安性能

Carboxyl was formed on the surface of glassy carbon electrode(GCE) by electrochemical oxidation. Ethylenediamine(En) was used as the arm molecule to link carboxyl with dsDNA using 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) as the activators to prepare dsDNA modified electrode(dsDNA/En/GCE). It was shown that dsDNA couM be covalently immobilized on the surface of GCE. ssDNA modified electrode(ssDNA/En/GCE) was obtained via the thermal denaturation of dsDNA/En/GCE. The dsDNA/En/GCE and ssDNA/En/GCE were characterized by voltammetry with methylene blue(MB) as the indicator. The results indicated that the currents of the redox peaks of MB at ssDNA/En/GCE were larger than those at dsDNA/En/GCE, and the currents of the redox peaks at En/GCE were the smallest. The peak-currents of MB at the DNA modified electrode had good reproducibility after multi-denaturation and hybridization cycles.     

DNA sensor for o-dianisidine

o-Dianisidine (3,3'-dimethoxybenzidine) is applied in the production of some dyes and also used in analytical tests. However, this compound is anticipated to be a human carcinogen. An analytical strategy utilizing square wave voltammetry for the determination of o-dianisidine is presented. An electrochemical system was consisted of three electrodes: carbon paste working electrode, platinum wire counter electrode and silver-silver chloride (Ag/AgCl) reference electrode. However, square wave voltammograms of direct measurements of o-dianisidine were found to be hardly reproducible, exhibiting few peaks due to some labile short-lived intermediates with the only exception of a quite stable peak at +0.7 V vs. Ag/AgCl. Quantitative determination of o-dianisidine gave satisfactory results only when the carbon paste working electrode was replaced by deoxyribonucleic acids (DNA) electrode obtained by immobilization of double-stranded (ds) DNA on carbon electrode. Square wave voltammogram of DNA showed two peaks attributed to adenine and guanine and the latter was used as analytical signal. After interaction with o-dianisidine, guanine oxidation peak was reduced to the extent related to the concentration of the analyte. Initial reduction of guanine peak took place already at the concentration of o-dianisidine equal to 0.4 microM; high concentrations (above 100 microM) of the analyte quenched completely a guanine response. The presented electrochemical system enables a specific detection of o-dianisidine by the presence of an oxidation peak at +0.7 V and its quantitative determination by measuring a reduction of guanine peak by means of a DNA sensor.     

Electrochemical and Spectroscopic Studies on the Interaction of Gatifloxacin,Moxifloxacin and Sparfloxacin with DNA and Their Analytical Applications

The electrochemical oxidation of the three fluoroquinolone drugs FQs: gatifloxacin GTF, moxifloxacin MXF and sparfloxacin SPF, at the bare and DNA‐modified glassy carbon electrodes has been studied by voltammetric techniques. The three FQs showed one irreversible oxidation peak at potential range 0.85–0.91 V vs. Ag‐AgCl, in phosphate buffer of pH 7.0. Differential pulse voltammetry (DPV) and UV‐absorption spectroscopic techniques were employed to probe the interaction between the FQs and calf thymus double stranded deoxyribonucleic acid (ds CT‐DNA). From electrochemical data, the binding constant between DNA and the gatifloxacin, moxifloxacin and sparfloxacin are calculated to be 3228, 2596 and 2857 M^?1 respectively. Based on electrochemical and spectroscopic results, the mode of binding of fluoroquinolone to DNA through combined effect of intercalation and electrostatic interaction was concluded. A detection scheme based on a preconcentration and differential pulse voltammetric (DPV) determination at dsDNA modified glassy carbon electrode (DNA/GCE) was proposed for the trace determination of the studied analytes. The developed method was successfully applied to the determination of the FQs in pharmaceutical formulations.     

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.     

Studies of an Alkaline Activated Glassy Carbon Electrode on the Determination of Purines and DNA

Performance of glassy carbon electrode on the determination of purines and DNA was found to be improved dramatically by activating the GCE with a simple but effective electrochemical pretreatment. Characteristics such as lowering of oxidation potential, enhancement of peak current and elimination of fouling effect were found for the activated GCE. By flow injection analysis, good reproducibility with relative standard deviations of 0.59 and 2.01% (n = 11) and rather low detection limits of 0.6 + 0.1 and 3.0 ± 0.4 nM can be obtained for the analysis of guanine and adenine. Solutions of denatured calf thymus DNA were analyzed by differential pulse voltammetry with the activated GCE. Good agreement between the obtained results and the known values confirms the feasibility of the activated GCE for DNA analysis.     

Studies of the interaction between Aloe-emodin and DNA and preparation of DNA biosensor for detection of PML-RARalpha fusion gene in acute promyelocytic leukemia

The interaction of Aloe-emodin (AE) with salmon sperm DNA in 0.1M Tris-HCl buffer (pH 4.4) and at the DNA-modified glassy carbon electrode (GCE) was systemically studied with voltammetry and ultraviolet-visible (UV-vis) spectroscopy. AE had excellent electrochemical activity on the GCE with a couple of redox peaks. We propose that AE can intercalate into DNA strands forming a nonelectroactive complex, which results in the decrease of the reduction peak current of AE. The Langmuir adsorption constants of AE at ss- and dsDNA/GCE were (2.1+/-0.4)x10(5) and (2.7+/-0.2)x10(5)M(-1), respectively. The difference between AE at ss- and dsDNA has been used for the preparation of a sequence-specific DNA electrochemical biosensor for detection of PML-RARalpha fusion gene in acute promyelocytic leukemia (APL) with a detection limit of 6.7x10(-8)M and a linear range from 1.5x10(-8) to 1.5x10(-7)M. The selectivity of ssDNA-modified electrode was also described.     

Spectroscopic Electrochemical Studies of Interaction Between Fuchsin Basic DNA

Visible spectroscopic and electrochemical methods were used to study the interactions between DNA and fuchsin basic(FB). FB has an irreversible electro-oxidation peak in 5 mmol/L Tris-HCl buffer solution at pH = 7.4 on a glassy carbon electrode(GCE). After adding certain concentration of dsDNA, the oxidation peak current of FB decreases, but the peak potential hardly changs. The visible absorption spectroscopic study shows that the binding mode of FB to dsDNA is intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is smaller than 0. 2, and a new substance, which produces a new absorption peak, is obtained via a covalent binding between dsDNA and FB apart from intercalative binding and electrostatic binding when the ratio of the concentration of dsDNA to FB is larger than 0. 2. The visible absorption spectra varies no longer when the ratio of the concentration of dsDNA to FB is larger than 1.5. A mean binding ratio of dsDNA to FB was determined to be 1.4: 1,suggesting that two complexes FB-dsDNA and FB-2dsDNA be formed. The interaction between FB and ssDNA was only electrostatic binding. The more powerful interaction of FB with dsDNA than with ssDNA may be applied for the recognition of dsDNA and ssDNA, and in DNA biosensor as hybridization indicator.     

纳米金修饰玻碳电极对鸟嘌呤的催化氧化

采用电化学沉积法制备了纳米金修饰玻碳电极,并用循环伏安法和电化学阻抗法进行了表征,以此建立了一种直接测定鸟嘌呤的电分析方法。在磷酸盐缓冲溶液(pH 6.0)中,研究了鸟嘌呤在纳米金修饰电极上的电化学行为,实验结果表明,纳米金修饰电极可以增强鸟嘌呤在电极表面的吸附,并加快鸟嘌呤在电极表面的电子传输,使其电化学信号明显增大,检测灵敏度大大提高,该修饰电极对鸟嘌呤表现出良好的电催化性能。在优化实验条件下对鸟嘌呤进行测定,方法的线性范围为8.0×10-7~6.0×10-5mol/L,检出限为1.0×10-8mol/L,在鸟嘌呤浓度为1.0×10-5mol/L时测得RSD(n=10)为2.5%。     

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.     

DNA Biosensor Based on Carbon Paste Electrodes Modified by Polymer Multilayer

An electrochemical DNA biosensor was developed by DNA immobilization at the electrode surface and its electrochemical behavior was studied in relation with different materials added in the paste. The aim was to study new materials for the development of new electrode surfaces, to be applied in the study of DNA – drug interactions. New electrochemical sensing materials using polymer multilayers were reported for the adsorption of DNA. These materials were prepared by mixing a polymer ion exchanger and graphite powder. The mixture was then used to render the modified carbon paste electrode (CPE), on the surface of which the dsDNA was adsorbed and studied by differential pulse voltammetry (DP voltammetry). The signal of guanine oxidation peak of DNA was followed. This modified biosensor was applied for the study of the interaction between DNA and the known intercalators Ethidium Bromide (EB) and Acridine Orange (AO). The established biosensor exhibited an improvement of its sensitivity and repeatability compared with the conventional CPE DNA biosensor.     

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.     

Voltammetric Behavior of the Alizarin Red S Interaction with DNA and Damage to DNA

The electrochemical behavior and the interaction of alizarin red S (ARS) with calf thymus DNA was investigated on a bare glassy carbon electrode (GCE) and DNA modified GCE (DNA/GCE), respectively. ARS showed a pair of redox peaks at ?0.445 V and ?0.414 V on a bare GCE. On addition of DNA into the ARS solution, the peak current of ARS decreased and the peak potential positively shifted, but without new redox peaks appeared. The ARS reduction peak current increased with immersion time on a DNA/GCE. The results showed that ARS could interact with DNA molecules by intercalative binding mode. The equilibrium constant, binding number and the ratio of binding constant for oxidized and reduced ARS forms were obtained. The DNA damage was directly detected by appearance of guanosine and adenosine bases oxidation signal. The influence of experimental conditions on DNA damage extent was discussed in detail.     

Antileishmanial Drug Miltefosine‐dsDNA Interaction in situ Evaluation with a DNA‐Electrochemical Biosensor

Leishmaniasis is one of the most important parasitic neglected disease. The electrochemical evaluation of the antileishmanial drug miltefosine‐dsDNA interaction was investigated in incubated solutions and using dsDNA‐electrochemical biosensors, following the changes in the oxidation peaks of guanosine and adenosine residues, and the occurrence of the free guanine residues, electrochemical signal. The electrochemical behaviour of miltefosine was also investigated, at a glassy carbon electrode, using cyclic, differential pulse and square wave voltammetry and no electrochemical redox processes were observed. The interaction mechanism of miltefosine‐dsDNA occurs in two ways: independent of the dsDNA sequence, and leading to the condensation/aggregation of DNA strands, producing a rigid miltefosine‐dsDNA complex structure, and a preferential interaction between the guanine hydrogen atoms in the C−G base pair and miltefosine, causing the release of guanine residues detected on the electrode surface. Miltefosine did not induce oxidative damage to DNA in the experimental conditions used.     

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.     

Adsorptive Stripping Voltammetric Detection of Single‐Stranded DNA at Electrochemically Modified Glassy Carbon Electrode

Electrochemically modified glassy carbon electrode (GCE) was used to study the electrochemical oxidation and detection of denatured single‐stranded (ss) DNA by means of adsorptive stripping voltammetry. The modification of GCE, by electrochemical oxidation at +1.75 V (vs.SCE) for 10 min and cyclic sweep between +0.3 V and ?1.3 V for 20 cycles in pH 5.0 phosphate buffer, results in 100‐fold improvement in sensitivity for ssDNA detection. We speculated that the modified GCE has a high affinity to single‐stranded DNA through hydrogen bond (specific static adsorption). Single‐stranded DNA can accumulate at the GCE surface at open circuit and produce a well‐defined oxidation peak corresponding to the guanine residues at about +0.80 V in pH 5.0 phosphate buffer, while the native DNA gives no signal under the same condition. The peak currents are proportional to the ssDNA concentration in the range of 0–18.0 μg mL^?1. The detection limit of denatured ssDNA is ca. 0.2 μg mL^?1 when the accumulation time is 8 min at open circuit. The accumulation mechanism of ssDNA on the modified GCE was discussed.