Arzum Erdem

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.

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.

Nanomaterial-based electrochemical DNA sensing strategies.

DNA sensing strategies have recently been varieted with the number of attempts at the development of different biosensor devices based on nanomaterials, which will further become DNA microchip systems. The investigations at the side of material science in connection with electrochemical biosensors open new directions for detection of specific gene sequences, and nucleic acid-ligand interactions. An overview is reported here about nanomaterial-based electrochemical DNA sensing strategies principally performed for the analysis of specific DNA sequences and the quantification of nucleic acids. Important features of electrochemical DNA sensing strategies, along with new developments based on nanomaterials 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.

Magnetic bead-based label-free electrochemical detection of DNA hybridization

Magnetic bead capture has been used for eliminating non-specific adsorption effects hampering label-free detection of DNA hybridization based on stripping potentiometric measurements of the target guanine at graphite electrodes. In particular, the efficient magnetic separation has been extremely useful for discriminating against unwanted constituents, including a large excess of co-existing mismatched and non-complementary oligomers, chromosomal DNA, RNA and proteins. The new protocol involves the attachment of biotinylated oligonucleotide probes onto streptavidin-coated magnetic beads, followed by the hybridization event, dissociation of the DNA hybrid from the beads, and potentiometric stripping measurements at a renewable graphite pencil electrode. Such coupling of magnetic hybridization surfaces with renewable graphite electrode transducers and label-free electrical detection results in a greatly simplified protocol and offers great promise for centralized and decentralized genetic testing. A new magnetic carbon-paste transducer, combining the solution-phase magnetic separation with an instantaneous magnetic collection of the bead-captured hybrid, is also described. The characterization, optimization and advantages of the genomagnetic label-free electrical protocol are illustrated below for assays of DNA sequences related to the breast-cancer BRCA1 gene.

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.

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.

Genomagnetic electrochemical assays of DNA hybridization

An electrochemical genomagnetic hybridization assay has been developed to take advantage of a new and efficient magnetic separation/mixing process, the amplification feature of enzyme labels, and single-use thick-film carbon transducers operated in the pulse-voltammetric mode. It represents the first example of coupling a magnetic isolation with electrochemical detection of DNA hybridization. The new protocol employs an enzyme-linked sandwich solution hybridization, with a magnetic-particle labeled probe hybridizing to a biotinylated DNA target that captures a streptavidin-alkaline phosphatase (AP). The alpha-naphthol product of the enzymatic reaction is quantitated through its well-defined, low-potential (+0.1 V vs. Ag/AgCl) differential pulse-voltammetric peak at the disposable screen-printed electrode. The efficient magnetic isolation is particularly attractive for electrical detection of DNA hybridization which is commonly affected by the presence of non-hybridized nucleic acid adsorbates. The new biomagnetic processing combines such magnetic separation with a low-volume magnetic mixing, and allows simultaneous handling of 12 samples. The attractive bioanalytical behavior of the new enzyme-linked genomagnetic electrical assay is illustrated for the detection of DNA segments related to the breast-cancer BRCA1 gene.

Disposable electrochemical biosensor for the detection of the interaction between DNA and lycorine based on guanine and adenine signals

The interaction of lycorine (LYC) with calf thymus double stranded DNA (dsDNA) and calf thymus single stranded DNA (ssDNA) was studied electrochemically based on the oxidation signals of guanine and adenine by using differential pulse voltammetry (DPV) at carbon paste electrode (CPE) and pencil graphite electrode (PGE). As a result of the interaction of LYC with DNA, the voltammetric signal of guanine and adenine greatly decreased. The changes in the experimental parameters such as the concentration of LYC, and the accumulation time of LYC were studied by using DPV with PGE. The interaction of LYC with synthetic polynucleotides, such as poly[G] was also observed. The interaction of LYC with dsDNA was also observed at PGE in solution phase. In addition, the detection limit and the reproducibility was determined by using both electrochemical transducers. The application of electrochemical methods on the interactions between DNA and DNA targeted agent were explored.

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.