Hakan Karadeniz

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.

Label-free impedimetric aptasensor for lysozyme detection based on carbon nanotube-modified screen-printed electrodes

We report on the direct electrochemical detection of aptamer-protein interactions, namely between a DNA aptamer and lysozyme (LYS) based on electrochemical impedance spectroscopy (EIS) technique. First, the affinity of the aptamer to LYS and control proteins was presented by using filter retention assay. An amino-modified version of the DNA aptamer-recognizing lysozyme was covalently immobilized on the surface of multiwalled carbon nanotube-modified screen-printed electrodes (MWCNT-SPEs), which were employed for measurements and have improved properties compared with bare SPEs. This carbon nanotube setup enabled the reliable monitoring of the interaction of lysozyme with its cognate aptamer by EIS transduction of the resistance to charge transfer (R(ct)) in the presence of 2.5 mM [Fe(CN)₆]³⁻/⁴⁻. This assay system provides a means for the label-free, concentration-dependent, and selective detection of lysozyme with an observed detection limit of 12.09 μg/ml (equal to 862 nM).

Electrochemical monitoring of indicator-free DNA hybridization by carbon nanotubes–chitosan modified disposable graphite sensors

Single walled carbon nanotubes (SWCNT)-chitosan (CHIT) modified pencil graphite electrodes (PGEs) were developed for monitoring of DNA hybridization. SWCNT-chitosan modified PGE (CNT-CHIT-PGE), Chitosan modified PGE (CHIT-PGE) and unmodified PGE (bare-PGE) were firstly characterized by using scanning electron microscopy (SEM), and their electrochemical behaviors were investigated using electrochemical impedance spectroscopy (EIS). The concentrations of CHIT, carbon nanotube (CNT) and also amino linked DNA probe etc. were respectively optimized in order to obtain the better working conditions of CNT-CHIT modified PGE in DNA analysis. The sequence selective DNA hybridization related to Hepatitis B virus (HBV) was then explored in the case of hybridization between amino linked HBV probe and its complementary (target), or noncomplementary (NC), or mismatch (MM) sequences, and also hybridization in mixture sample.

Dendrimer modified graphite sensors for detection of anticancer drug Daunorubicin by voltammetry and electrochemical impedance spectroscopy

The development of amino-terminated G4 PAMAM dendrimer (PDR) modified disposable electrodes were developed as the first time in our study by using the dendrimer modified disposable graphite (PDR-PGE) and multiwalled carbon nanotube based screen-printed graphite (PDR-MWCNT-SPE) electrodes. Firstly, the microscopic characterization of bare PGEs and PDR modified PGEs was performed. These sensors were then applied for electrochemical monitoring of an anticancer drug, Daunorubicin (DNR). The enhanced oxidation signal of DNR was measured at +0.50 V by using differential pulse voltammetry (DPV) in combination with the PDR-PGEs. The detection limit, estimated from S/N = 3, corresponds accordingly to 317 nM and 128 nM for DNR respectively at the PGE and PDR-PGE. The voltammetric results were consistent with electrochemical impedance spectroscopy (EIS) that was used to characterize the successful modification of PDR onto the surface of PGE and MWCNT-SPE.

Echinomycin and cobalt-phenanthroline as redox indicators of DNA hybridization at gold electrodes

A bis-intercalator echinomycin (ECHI) and a simple intercalator [Co(phen)3]3+ were used as a novel electrochemical redox indicators to detect DNA hybridization at gold electrodes (AuE). In order to minimize the nonspecific adsorption of oligonucleotides (ODN), the thiol-derivatized oligonucleotides were immobilized onto AuE in the first step, and the exposition of AuE to 6-mercapto-1-hexanol (MCH) followed in the second step of this procedure. In this arrangement good reproducibility and discrimination between single-stranded (ss) probe and double-stranded (ds) hybrid DNA were obtained. While both redox indicators showed a good ability to discriminate between the ss probe and ds hybrid DNA, the signals of ECHI were by an order of maginitude higher than those of [Co(phen)3]3+ in a good agreement with stronger DNA binding by the bis-intercalator as compared to the simple intercalator. In addition, DNA single-base mismatch (DNA point mutation) was easily detected by means of ECHI.

Indicator-based and indicator-free magnetic assays connected with disposable electrochemical nucleic acid sensor system

An indicator-based and indicator-free magnetic assays connected with a disposable pencil graphite electrode (PGE) were successfully developed, and also compared for the electrochemical detection of DNA hybridization. The oxidation signals of echinomycin (ECHI) and electroactive DNA bases, guanine and adenine, respectively were monitored in the presence of DNA hybridization by using differential pulse voltammetry (DPV) technique. The biotinylated probe was immobilized onto the magnetic beads (magnetic particles, microspheres) and hybridization with its complementary target at the surface of particles within the medium was exhibited successfully using electrochemical sensor system. For the selectivity studies, the results represent that both indicator-based and indicator-free magnetic assays provide a better discrimination for DNA hybridization compared to duplex with one-base or more mismatches. The detection limits (S/N=3) of the magnetic assays based on indicator or indicator-free were found in nM concentration level of target using disposable sensor technology with good reproducibility. The characterization and advantages of both proposed magnetic assays connected with a disposable electrochemical sensor are also discussed and compared with those methods previously reported in the literature.

Electrochemical Determination of Glutathione in Plasma at Carbon Nanotubes Based Screen Printed Electrodes

Glutathione (GSH) is a major endogenous antioxidant highly active in human tissues and plays a key role in controlling cellular thiol redox system, maintaining the immune and detoxification system. The determination of GSH levels in tissue is important to estimate endogenous defenses against oxidative stress. In our study, the multi-walled carbon nanotube modified screen-printed electrodes (MWCNT-SPEs) were used to determine the levels of GSH in trichloroacetic acid (TCA)-treated or untreated samples of rat plasma. It was found that the deproteinization of samples with TCA improved the electrochemical detection of GSH particularly in plasma. The oxidation of GSH was measured by using differential pulse voltammetry (DPV) method in combination with MWCNT-SPE (n=3), and the detection limit of GSH was found to be 0.47 µM (S/N=3). The GSH levels in plasma samples were also measured spectrophotometrically in order to compare the effectiveness of electrochemical method and we obtained a high correlation between the two methods (R(2)=0.976).

Synthesis and characterization of water-insoluble statistical copolymer and its application in the development of electrochemical DNA sensor

Water-insoluble statistical copolymer was synthesized by copolymerization of methyl methacrylate (MMA) with 2-(dimethylamino)ethyl methacrylate (DMA) via group transfer polymerization (GTP). The DMA residues of the precursor P(MMA-co-DMA) statistical copolymer were then quaternized by reacting with methyl iodide under mild conditions to get well-defined P(MMA-co-QDMA) cationic copolymer. Then, P(MMA-co-QDMA) copolymer was successfully used for surface modification of pencil graphite electrode (PGE) to develop a disposable DNA sensor. This P(MMA-co-QDMA) copolymer modified electrode (q-PGE) was examined for electrochemical monitoring of DNA by using differential pulse voltammetry (DPV) in contrast to unmodified one. The effect of both DNA concentration and sonication time was also examined based on the response of q-PGE. The detection limit was calculated to be 8.06 μg/mL at q-PGE. Electrochemical impedance spectroscopy (EIS) was used for the characterization of the surface modification of q-PGE and consequently, the results were found to be in good agreement with the voltammetric measurements.

Procedure 27 Electrochemical detection of calf thymus double-stranded DNA and single-stranded DNA by using a disposable graphite sensor

Publisher Summary This chapter presents a method to immobilize double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) onto pretreated pencil graphite electrodes (PGEs) by dip coating procedure. It also performs an electrochemical detection of calf thymus dsDNA and ssDNA by using differential pulse voltammetry (DPV) with a disposable sensor: PGE, and to measure the oxidation signal of guanine. The advantages electrochemical sensing assay of DNA include inexpensive cost preparation of disposable graphite sensors and easy-modification of DNA by dipcoating procedure to detect DNA in lower DL and better reproducibility. Such a use of electrochemical sensing assay for dsDNA/ssDNA can decrease the assay time and cost of screening for any DNA fragment. While the concept has been demonstrated in connection with different types of calf thymus DNA, the further improvements may be achieved by using several DNA probes from different regions of specific sequences related to inherited or infectious diseases, in connection with multielectrode array and multiple hybridization events.