Interaction of the mutagen ethidium bromide with DNA, using a carbon paste electrode and a hanging mercury drop electrode

The interaction of ethidium bromide (2,7-diamino-10-ethyl-9-phenylphenanthridinium bromide; EB) with double stranded (ds) calf thymus DNA and thermally denatured single stranded (ss) DNA was studied in solution and at the electrode surface by means of transfer voltammetry using a carbon paste electrode (CPE) as working electrode in 0.2 M acetate buffer, pH 5.0. As a result of intercalation of this dye between the base pairs of dsDNA, the characteristic peak of dsDNA, due to the oxidation of guanine residues, decreased and after a particular concentration of EB a new peak at +0.81 V appeared, probably due to the formation of a complex between dsDNA and EB. The non-intercalated EB gives another peak, but at an increased concentration of the dye. A similar behaviour was observed during the interaction of the dye with ssDNA.Furthermore, the interaction of EB with ds, ss and supercoiled (sc) DNA was studied at the hanging mercury drop electrode (HMDE) surface by means of alternating current voltammetry in 0.3 M NaCl and 50 mM sodium phosphate buffer (pH 8.5) as supporting electrolyte. dsDNA yields a smaller peak at −1.42 V (peak III) compared to the one yielded by ssDNA, since the latter is a relaxed and more accessible form. By addition of EB into the buffer solution an increase of peak III was observed in the dsDNA form as well as in ssDNA resulting from their interaction with EB. Furthermore, the appearance of peak III in covalently closed circular scDNA after exposure to increasing concentrations of EB is a result of the introduction of ‘free ends’ in DNA affecting its structural integrity.     

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.     

ssDNA/十八酸修饰碳糊电极的制备及伏安法表征

将石墨粉与十八酸在80 ℃下混合制成表面富含—COOH的基底碳糊电极(SA/CPE), 然后在活化剂N-羟基琥珀酰亚胺(NHS)和1-乙基-3-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)存在下将ssDNA固定到电极表面制备ssDNA修饰电极(ssDNA/SA/CPE). 以亚甲基蓝(MB)为指示剂, 用循环伏安法对SA/CPE和ssDNA/SA/CPE进行电化学表征, 发现其在ssDNA/SA/CPE上较在SA/CPE上的氧化峰电流(i_pa)和还原峰电流(i_pc)分别增大1.9倍和1.7倍, 式电势(E_f)负移8 mV. 把ssDNA/SA/CPE放在互补ssDNA溶液中杂交后, MB的i_pa 和i_pc较在SA/CPE上分别增大1.0倍和0.8倍, E_f负移18 mV. 用0.5 mol/L 的NaOH溶液冲洗使电极表面杂交而成的dsDNA变性洗脱, MB的伏安信号几乎与在ssDNA/SA/CPE上一样. i_pc与SA/CPE上固定的ssDNA质量在1.0×10^－7～5.0×10^－6 g范围内成线性关系, 检测限为2.0×10^－9 g (S/N＝3). 这种既廉价又灵敏的电化学生物传感器有望在转基因植物产品检测研究中得到应用.     

Electroanalytical Study of the Interaction Between Double Stranded DNA and Antitumor Agent Curcumin

Curcumin, the major active component of the spice turmeric, which is considered to be a very useful compound in health matters, is recognized as a safe component with great potential for cancer chemoprevention and cancer therapy. For the first time, an interaction between the non-toxic agent curcumin and double stranded (ds) calf thymus DNA has been demonstrated by using voltammetry. The interaction of curcumin (CU) with dsDNA was studied using a carbon paste electrode (CPE) and a hanging mercury drop electrode (HMDE). Significant changes in the characteristic peaks of dsDNA were observed after addition of curcumin to a solution containing dsDNA.     

Electroanalytical study of SYBR Green I and ethidium bromide intercalation in methylated and unmethylated amplicons

This work involves the electrochemical study of the interaction of SYBR Green I (SG) with native DNA using differential pulse voltammetry at a carbon paste electrode (CPE) and alternating current voltammetry at a hanging mercury drop electrode (HMDE). At the CPE the peak current intensity at 1.0 V decreased by increasing the concentration of SG. At the HMDE, a decrease in the current intensity of the DNA peak at −1.2 V was also observed by increasing the concentration of SG. These results electrochemically confirmed that SG intercalates within the DNA double helix and changes its conformation. Through the present work the differentiation of differently methylated analytes was achieved by application of alternative current and differential pulse voltammetric techniques. Amplicons (PCR products) corresponding to the GC-rich p53 exon 5 containing cytosine and its methylated analogue, synthesized by substituting 60% of cytosine by 5-methyl-cytosine, were amplified and investigated electrochemically in the presence of SG and ethidium bromide (EtBr) by differential pulse voltammetry. Considerable peak current differences were observed in the presence of SG and EtBr for unmethylated exon 5 vs. methylated. Therefore, both SG and EtBr could serve as electrochemical probes for identifying different DNA conformations.     

Electrochemical DNA Biosensors Applicable to the Study of Interactions Between DNA and DNA Intercalators

Electrochemical DNA biosensors, based either on carbon paste electrode (CPE) or hanging mercury drop electrode (HMDE) were prepared. These biosensors were used in the study of interaction between double stranded DNA (dsDNA) and single stranded DNA (ssDNA) and acridine orange, a well known DNA intercalator. The different electrochemical behaviors were compared in the article.     

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

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.     

Electrochemical genosensor for the detection of interaction between methylene blue and DNA

Described here are the chronocoulometric and voltammetric parameters for methylene blue [3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB] on binding to DNA at carbon paste electrode (CPE) surface. MB, which interacts with the immobilized calf thymus DNA was detected by using single stranded DNA modified CPE (ssDNA modified CPE), bare CPE and double stranded DNA modified CPE (dsDNA modified CPE) in combination with chronocoulometry and differential pulse voltammetry (DPV) techniques. The effect of ionic strength to the behavior of MB with dsDNA and ssDNA was also studied by means of voltammetry. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

An Electrochemical DNA Sensor Based on Electrodepositing Aluminum Ion Films on Stearic Acid‐Modified Carbon Paste Electrode and Its Application for the Detection of Specific Sequences Related to Bar Gene and CP4 Epsps Gene

An electrochemical DNA biosensor was fabricated by immobilizing DNA probe on aluminum ion films that were electrodeposited on the surface of the stearic acid‐modified carbon paste electrode (CPE). DNA immobilization and hybridization were characterized with cyclic voltammetry (CV) by using methylene blue (MB) as indicator. MB has a couple of well‐defined voltammetric redox peaks at the CPE. The currents of redox peaks of MB decreased after depositing aluminum ion films on the CPE (Al(III)/CPE) and increased dramatically after immobilizing DNA probe (ssDNA/Al(III)/CPE). Hybridization of DNA probe led to a marked decrease of the peak currents of MB, which can be used to detect the target single‐stranded DNA. The conditions for the preparation of Al(III)/CPE, and DNA immobilization and hybridization were optimized. The specific sequences related to bar transgene in the transgenic corn and the PCR amplification of CP4 epsps gene from the sample of transgenic roundup ready soybean were detected by differential pulse voltammetry (DPV) with this new electrochemical DNA biosensor. The difference between the peak currents of MB at ssDNA/Al(III)/CPE and that at hybridization DNA modified electrode (dsDNA/Al(III)/CPE) was applied to determine the specific sequence related to the target bar gene with the dynamic range comprised between 1.0×10^?7 mol/L to 1.0×10^?4 mol/L. A detection limit of 2.25×10^?8 mol/L of oligonucleotides can be estimated.     

Electrochemical Study of Interaction Between Clozapine and DNA and Its Analytical Application

Abstract

The interaction between clozapine (CLZ) as an orally administrated antipsychotic drug with double stranded calf thymus DNA (dsDNA) was investigated at electrode surface using differential pulse voltammetry (DPV). Activated carbon paste electrode (CPE) was modified with dsDNA and used for monitoring the changes of the characteristics peak of CLZ in 0.05 M acetate buffer (pH 4.3). The adsorptive stripping voltammetry on dsDNA‐modified carbon paste electrode (dsDNA‐CPE) was used for determination of very low concentration of CLZ. Under optimal conditions, the oxidation peak current is proportional to CLZ concentration in the range of 7×10^?9?1.2×10^?6 mol l^?1 with a detection limit of 1.5×10^?9 mol l^?1 for 180 s accumulation time by DPV. The proposed dsDNA‐CPE was successfully used for determination of CLZ in human serum samples with recovery of 97.0±2.5%.     

Electrochemical Study of the Interaction Mechanism of Proflavine (PF) with DNA Using Carbon Paste (CPE) and Hanging Mercury Drop (HMDE) Electrode

Abstract

Proflavine binds with DNA in a complicated manner. This work involves the electrochemical study of this interaction using differential pulse voltammetry at a carbon paste electrode (CPE) and alternating current voltammetry at a hanging mercury drop electrode (HMDE). At the CPE the peak current intensity at 1.0 V (corresponding to the oxidation of the guanine residues) decreased by increasing the concentration of proflavine. At the HMDE, a decrease in the current intensity of the DNA peak at ? 1.2 V (corresponding to segmental desorption) was also observed by increasing the concentration of proflavine. These results confirmed, electrochemically, that proflavine intercalates within the DNA double helix and changes its conformation.     

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.     

Sensitive DNA biosensor improved by Luteolin copper(II) as indicator based on silver nanoparticles and carbon nanotubes modified electrode

A novel and sensitive electrochemical DNA biosensor has been developed for the detection of DNA hybridization. The biosensor was proposed by using copper(II) complex of Luteolin C₃₀H₁₈CuO₁₂ (CuL₂) as an electroactive indicator based on silver nanoparticles and multi-walled carbon nanotubes (Ag/MWCNTs) modified glassy carbon electrode (GCE). In this method, the 4-aminobenzoic acid (4-ABA) and Ag nanoparticles were covalently grafted on MWCNTs to form Ag/4-ABA/MWCNTs. The proposed method dramatically increased DNA attachment quantity and complementary ssDNA detection sensitivity for its large surface area and good charge-transport characteristics. DNA hybridization detection was performed using CuL₂ as an electroactive indicator. The CuL₂ was synthesized and characterized using elemental analysis (EA) and IR spectroscopy. Cyclic voltammetry (CV) and fluorescence spectroscopy were used to investigate the interaction between CuL₂ and ds-oligonucleotides (dsDNA). It was revealed that CuL₂ presented high electrochemical activity on GCE, and it could be intercalated into the double helices of dsDNA. The target ssDNA of the human hepatitis B virus (HBV) was quantified in a linear range from 3.23 × 10⁻¹² to 5.31 × 10⁻⁹ M (r = 0.9983). A detection limit of 6.46 × 10⁻¹³ M (3σ, n = 11) was achieved.     

Immobilization and voltammetric detection of human interleukine-2 gene on the pencil graphite electrode

The immobilization and differential pulse anodic voltammetry (DPAV) of a 20-mer oligonucleotide related to the human interleukine-2 (hIL-2) using renewable pencil graphite electrode (PGE) is described. The influences of electrochemical pretreatment of PGE on the ability of the electrode in hIL-2 adsorption, and conditions of hiIL-2 immobilization on PGE including immobilization potential and time, sodium chloride concentration as well as stirring of the solution were studied and optimum conditions were suggested. Accordingly, the electrochemical pretreatment of the polished PGE by electrostatic procedure at 1.80 V for 5 min in 0.50 M acetate buffer solution of pH 4.8 is proposed as the optimum pre-treatment procedure. Similarly, the obtained optimum conditions for immobilization of hIL-2 on the activated PGE was an immobilization duration of 5 min at applied potential of 0.50 V. Trace levels of hIL-2 was readily detected following only 5 min immobilization period with detection limit of 6 nM.     

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).     

Cyclic voltammetry of echinomycin and its interaction with double-stranded and single-stranded DNA adsorbed at the electrode.

Interactions of echinomycin (Echi) with DNA was studied by cyclic voltammetry (CV) with hanging mercury drop electrode (HMDE). Echinomycin was electrochemically active, yielding several signals. Interaction of Echi with dsDNA attached to a hanging mercury drop electrode resulted in high Echi signals, suggesting a strong binding of Echi to dsDNA by bis-intercalation at the electrode surface. Under the same conditions, interaction of Echi with ssDNA produced almost no Echi signal. This behavior is in agreement with a strong binding of Echi to dsDNA and a very weak binding of Echi to ssDNA observed earlier in solution. Echi, thus, appears to be a good candidate for redox indicator in electrochemical DNA hybridization sensors.     

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.     

灿烂甲酚蓝在DNA修饰金电极上的电化学行为

利用自组装技术将巯基乙醇固定在金电极表面形成巯基乙醇自组装膜修饰金电极, 用乙基-(3-二甲基氨丙基)碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为偶联试剂, 分别将鲱鱼精单链DNA(ssDNA)和双链DNA(dsDNA)固定于金电极表面形成ssDNA和dsDNA 修饰电极. 考察了灿烂甲酚蓝(BCB)在不同DNA 修饰电极上的电化学行为,结果表明, BCB 在ssDNA 和dsDNA 修饰电极上的吸附常数分别为1.67×10^4和3.22×10^4 L·mol-1, BCB 与ssDNA 主要以静电作用结合, 而与dsDNA作用存在静电和嵌插两种模式. dsDNA 对BCB 具有更高的亲和力, 使BCB 可以作为一种有效的电化学杂交指示剂.     

The electrocatalytic examination of cephalosporins at carbon paste electrode modified with CoSalophen

The electrocatalytic oxidation of cephalexin and cefazolin has been studied at a carbon paste electrode modified with cobalt salophen (CoSal) by cyclic voltammetry. The selectivity of the carbon paste modified with CoSal in detecting cephalexin and cefazolin was examined. To suggest the electrocatalytic mechanism for electro-oxidation of cefazolin, the electrochemical behavior of ceftriaxone was investigated which has a thiol group out of the beta lactam ring. The electrocatalytic oxidation of these antibiotics is shown to be irreversible at the CoSal modified electrode. Scan rate dependence of cefazolin, which is a sulfur-containing compound, has been examined. The results indicated that the electrocatalytic oxidation of the compounds is diffusion controlled. The responses of the modified electrode were compared with those of unmodified electrode and it has shown that the modified electrode has better sensitivity than unmodified electrode to the detection of cefazolin. The overall number of electrons contributed to the oxidation of cefazolin is obtained 1 by chronoamperometry; the number of electron involved in the rate-determining step was 1. The results of differential pulse voltammetry (DPV) using the modified electrode with high sensitivity were applied for the determination of cefazolin in human synthetic serum samples. The linear range was obtained from 1 × 10⁻⁵ to 1 × 10⁻³ M for DPV determination of cefazolin in buffered solutions (pH 3.0).