Mehmet Ozsoz

Novel hybridization indicator methylene blue for the electrochemical detection of short DNA sequences related to the hepatitis B virus

Abstract A novel assay for the voltammetric detection of DNA sequences related to the hepatitis B virus (HBV), using MB as the hybridization indicator was performed. The voltammetric signals of MB have been investigated at bare CPE, double stranded DNA (dsDNA)-modified CPE and single stranded DNA (ssDNA)-modified CPE by means of differential pulse voltammetry and cyclic voltammetry and the increased peak currents were observed, in respect to the order of electrodes. The extent of hybridization between the 21-mer oligonucleotides of complementary sequences was determined by the enhancement of the voltammetric signal of MB. The response of the hybridization of the probe with the single-base mismatch oligonucleotide at CPE was detected by MB. In this case, the unbound guanine bases increased the voltammetric signal obtained with the hybrid-modified CPE. Control experiments with the noncomplementary oligonucleotides were performed to assess whether the DNA biosensor responds selectively, via hybridization, to the target. Numerous factors, affecting the probe immobilization, target hybridization and indicator binding reactions are optimized to maximize the sensitivity and reduce the assay time. The new indicator MB holds great promise for rapid screening of HBV infection.

DNA electrochemical biosensors for environmental monitoring. A review

Abstract DNA sensing protocols, based on different modes of nucleic acid interaction, possess an enormous potential for environmental monitoring. This review describes recent efforts aimed at coupling nucleic acid recognition layers with electrochemical transducers. It considers DNA hybridization sensors for sequences related to microbial or viral pathogens, and DNA-modified carbon electrodes for monitoring low molecular weight priority pollutants interacting with the surfaceconfined DNA. Carbon strip or paste electrode transducers, supporting the DNA recognition layer, are used with a highly sensitive chronopotentiometric transduction of the DNA analyte recognition event. Factors influencing the performance of these new environmental biosensors are discussed, and their environmental utility is illustrated. While the use of DNA biosensors is at a very early stage, these and similar developments are expected to have a profound effect on environmental analysis.

Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.

Electrochemical DNA Biosensors Based on DNA-Drug Interactions

The world of drug designing is ever changing. The investigations of drug-DNA interactions would provide new compounds to be tested for an effect on a biochemical target, and also to be used as promising hybridization indicators for the design of DNA biosensors, which will further become DNA microchip systems. An overview is reported here about DNA biosensors based primarily on drugs interacting with DNA and shows how to determine this interaction electrochemically, the quantification of drug and/or DNA, and the promising applications of these drugs as DNA hybridization indicator. The applications of these electrochemical DNA biosensors are described and discussed.

Methylene Blue as a Novel Electrochemical Hybridization Indicator

The interaction of double stranded DNA (dsDNA) and single stranded DNA (ssDNA) with Ru(II) complex with 2,2′-bipyridine ligand (Ru(bpy)3]2+) and methylene blue (MB) were explored by using voltammetric and spectrophotometric methods. The electrochemistry of [Ru(bpy)3]2+ and MB was also investigated at a dsDNA-modified carbon paste electrode (CPE), a ssDNA-modified CPE and a bare CPE by means of differential pulse voltammetry (DPV), and cyclic voltammetry (CV) and the increased peak currents were observed, in respect to the order of electrodes. The structure of DNA was determined by the magnitudes of the voltammetric peaks of [Ru(bpy)3]2+ and MB. The hypochromicity of the visible absorption bands of [Ru(bpy)3]2+ and MB upon the interaction with dsDNA and ssDNA was observed. Numerous factors affecting the DNA immobilization, indicator binding reactions and ionic strength were optimized to maximize the sensitivity and reduce the assay time.

Evidence for the Direct Interaction Between Methylene Blue and Guanine Bases Using DNA-Modified Carbon Paste Electrodes

It is explored that methylene blue interacts with the guanine bases specifically, rather than the bases of ss-DNA in general. This interaction can be used as a method of quantifying the amount of oligonucleotide that is immobilized onto an electrode surface.

Interactions of antitumor drug daunomycin with DNA in solution and at the surface

Abstract The interaction of the antitumor drug daunomycin with double-stranded (ds) calf thymus DNA was studied in solution and at the electrode surface by means of cyclic voltammetry and particularly by constant-current chronopotentiometric stripping analysis (CPSA) with the carbon paste electrodes (CPE). As a result of intercalation of this drug between the base pairs in dsDNA, the CPSA daunomycin peak δ decreased and a new more positive shoulder δb appeared. This shoulder was attributed to the oxidation of the drug intercalated in DNA. Under the same conditions almost no changes in the DNA peak Gox (due to oxidation of guanine residues) were observed. It was shown that daunomycin adheres strongly to the bare CPE (resisting washing) so that a daunomycin-modified electrode can be easily prepared. Daunomycin immobilized at CPE interacted with DNA on immersion of the modified electrode into the dsDNA solution, showing a decrease of peak δ and a well-separated peak δb instead of the shoulder (which resulted from the interaction of DNA with daunomycin in solution). When the DNA-modified CPE was immersed into a daunomycin solution the peak Gox increased in dependence on daunomycin concentration or on the time of interaction of daunomycin with dsDNA at the electrode surface. Such changes in peak Gox were observed only at submicromolar concentrations of daunomycin. At higher daunomycin concentrations or at longer interaction time intervals a daunomycin peak appeared, which was substantially smaller and more positive than the peak of free daunomycin. The increase of the DNA peak Gox was attributed to interaction of daunomycin from the side of the DNA double helix not contacting the electrode surface. Such binding may induce changes in the DNA structure including bending of the DNA molecule which may result in the increase of peak Gox. Our results thus suggest that the interaction of daunomycin with DNA anchored at the surface may significantly differ from that with DNA in solution. The prospects of using of electroanalytical methods in studies of DNA–drug interactions are discussed.

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.

Electrochemical DNA biosensor for the detection of TT and Hepatitis B virus from PCR amplified real samples by using methylene blue.

DNA biosensors based on nucleic acid hybridization processes are rapidly being developed towards the goal of rapid and inexpensive diagnosis of genetic and infectious diseases. Electrochemical transducers are often being used for detecting the DNA hybridization event, due to their high sensitivity, small dimensions, low cost, and compatibility with microfabrication technology. In this study, an electrochemical biosensor for the voltammetric detection of DNA sequences related to the Hepatitis B virus (HBV) and TT virus (TTV) from polymerase chain reaction (PCR) amplified real samples is described for the first time. The biosensor relies on the immobilization of the 21- or 24-mer single stranded oligonucleotides (probe) related to the HBV and TTV sequences and hybridization of these oligonucleotides with their complementary sequences (target) at carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (MB) as the hybridization indicator. As a result of the interaction between MB and the bound guanine bases of hybrid at CPE surface, the MB signal decreased, when it was compared with the MB signal, which was observed with probe modified CPE. The difference between the MB signals, obtained from the hybrid modified and the probe modified CPE is used to detect the DNA sequences of the infectious diseases from PCR amplified real samples. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.

Interaction of the anticancer drug epirubicin with DNA

The interaction of epirubicin (EPR) with calf thymus double-stranded DNA (dsDNA) and calf thymus single-stranded DNA (ssDNA) was studied electrochemically by using differential pulse voltammetry (DPV) and cyclic voltammetry (CV) at carbon paste electrode (CPE). Experimental parameters, such as the concentration of EPR in CV, the concentration of DNA and the accumulation time of EPR, were studied by using DPV; in addition, the detection limit and the reproducibility were determined. It was observed that the signal at a bare electrode was higher than that at the dsDNA-modified CPE. The signals for EPR in CV were found to decrease in the order of bare CPE, ssDNA-modified and dsDNA-modified CPE. The interaction of EPR with smaller oligonucleotides was also evaluated for use as a hybridization indicator.