Gulsah Congur

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.

Impedimetric detection of in situ interaction between anti-cancer drug bleomycin and DNA

Surface confined interaction of anti-cancer drug bleomycin (BLM) with nucleic acids: single stranded and double stranded DNA was investigated herein by using electrochemical impedance spectroscopy (EIS) technique in combination with a graphite sensor technology. The experimental conditions were optimized: such as, dsDNA concentration, BLM concentration and interaction time. The main features of impedimetric DNA biosensor, such as its detection limit and the repeatability, were also discussed. The in situ interaction of BLM with dsDNA was also tested impedimetrically in the absence or presence of other chemotherapeutic agents, such as mitomycin C (MC) and cis-platin (cis-DDP) for testing the selectivity.

Label-free voltammetric detection of MicroRNAs at multi-channel screen printed array of electrodes comparison to graphite sensors

The multi-channel screen-printed array of electrodes (MUX-SPE16) was used in our study for the first time for electrochemical monitoring of nucleic acid hybridization related to different miRNA sequences (miRNA-16, miRNA-15a and miRNA-660, i.e, the biomarkers for Alzheimer disease). The MUX-SPE16 was also used for the first time herein for the label-free electrochemical detection of nucleic acid hybridization combined magnetic beads (MB) assay in comparison to the disposable pencil graphite electrode (PGE). Under the principle of the magnetic beads assay, the biotinylated inosine substituted DNA probe was firstly immobilized onto streptavidin coated MB, and then, the hybridization process between probe and its complementary miRNA sequence was performed at MB surface. The voltammetric transduction was performed using differential pulse voltammetry (DPV) technique in combination with the single-use graphite sensor technologies; PGE and MUX-SPE16 for miRNA detection by measuring the guanine oxidation signal without using any external indicator. The features of single-use sensor technologies, PGE and MUX-SPE16, were discussed concerning to their reproducibility, detection limit, and selectivity compared to the results in the earlier studies presenting the electrochemical miRNA detection related to different miRNA sequences.

Electrochemical investigation of the interaction between topotecan and DNA at disposable graphite electrodes

Topotecan (TPT) is a semisynthetic, water soluble analog of the plant alkaloid camptothecin which has been widely used for the treatment of ovarian and cervical cancers. To obtain better understanding on how it can affect DNA structure, electrochemical biosensor platforms for the investigation of TPT-double stranded DNA (dsDNA) interaction were developed for the first time in this study. The electrochemical detection of TPT, and TPT-dsDNA interaction were investigated at the surface of pencil graphite electrodes (PGEs) and single-walled carbon nanotube (SWCNT) modified PGEs by using differential pulse voltammetry (DPV). The changes at the oxidation signals of TPT and guanine were evaluated before/after each modification/immobilization step. An enhanced sensor response was obtained by using SWCNT-PGEs compared to unmodified PGEs with resulting limits of detection (LODs) for TPT as 0.51 μg/mL, 0.45 μg/mL, 0.37 μg/mL (130 pmol, 117 pmol, 96.5 pmol in a 110 μL sample, respectively) by using electrochemically pretreated PGE, unmodified PGE and SWCNT modified PGE. In addition, electrochemical impedance spectroscopy (EIS) measurements were performed for the purpose of modification of PGEs by using SWCNTs and the interaction process at the surface of SWCNT-PGEs by evaluating the changes at the charge transfer resistance (Rct).

Voltammetric aptasensor combined with magnetic beads assay developed for detection of human activated protein C

A sensitive and selective label free voltammetric aptasensor based on magnetic beads assay was performed for the first time in our study for monitoring of human activated protein C (APC), which is a serine protease (i.e., key enzyme of the protein C pathway). An amino modified DNA aptamer (DNA APT) was covalently immobilized onto the surface of carboxylated magnetic beads (MBs), and then, the specific interaction between DNA APT and its cognate protein, APC, was performed at the surface of MBs. Similarly a biotinylated DNA APT was immobilized onto the surface of streptavidin coated MBs. Before and after interaction process, the oxidation signal of guanine was measured at disposable pencil graphite electrode (PGE) surface in combination with differential pulse voltammetry (DPV) technique and accordingly, the decrease at the guanine signal was evaluated. The biomolecular recognition of APC was successfully achieved with a low detection limit found as 2.35 µg mL(-1) by using MB-COOH based assay. Moreover, the selectivity of this aptasensor assay was tested in the presence of numerous proteins and other biomolecules: protein C (PC), thrombin (THR), bovine serum albumin (BSA), factor Va (FVa) and chromogenic substrate (KS).

Voltammetric and impedimetric DNA detection at single-use graphite electrodes modified with gold nanorods

In this study, single-use pencil graphite electrodes (PGEs) were modified by gold nanorods (AuNRs) and then single stranded oligonucleotide probe molecules (ssODNs) were immobilized onto the surface of AuNRs modified PGE. The electrochemical characterizations using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) techniques were firstly performed to present the successfull modification of AuNR onto PGE surface. Under the optimum experimental conditions based on enhanced sensor response, single stranded thiol linked DNA probe ssODNs was immobilized onto AuNR modified PGE and accordingly, a sequence-selective DNA hybridization related to Hepatitis B virus (HBV) DNA was performed at the surface of AuNR modified electrodes. The impedimetric sequence-selective HBV DNA hybridization was then explored in the case of complementary (target), or noncomplementary (NC) DNA sequences.

Intracellular uptake study of radiolabeled anticancer drug and impedimetric detection of its interaction with DNA

Topoisomerase I inhibitor topotecan (TPT) is the only single-agent therapy certified for the remedy of repetitive small cell lung cancer (SCLC). In this study, TPT was labeled with (131)I via iodogen method and its quality control was determined using thin layer radiochromatography and paper electrophoresis methods. Intracellular uptake study was carried out with human lung adenocarcinoma cell line (A-549) and human lung fibroblast cell line (WI-38). The interaction of (131)I-TPT with healthy DNA and cancer DNA was also investigated using single-use sensor technology combined with electrochemical impedance spectroscopy (EIS). The change at the charge transfer resistance (Rct) obtained before/after interaction was evaluated. Similar to the results of intracellular uptake study, it was found that (131)I-TPT could more interact with the cancer DNA than healthy DNA according to the impedimetric results. (131)I-TPT is promising in terms of a new nuclear imaging agent for lung cancer.

Iron(III) and nickel(II) complexes as potential anticancer agents: synthesis, physicochemical and structural properties, cytotoxic activity and DNA interactions

Template reactions of 2-hydroxy-R-benzaldehyde-S-methylisothiosemicarbazones (R = 3-methoxy or 4-hydroxy) with the corresponding aldehydes in the presence of FeCl3 and NiCl2 yielded N1,N4-disalicylidene chelate complexes. The compounds were characterized by means of elemental and spectroscopic methods. The structure of complex 1 was determined by X-ray single crystal diffraction. Crystal data (Mo Kα; 296 K) are as follows: monoclinic space group P21/c, a = 12.9857(8) A, b = 7.8019(4) A, c = 19.1976(12) A, β = 101.655(5)°, Z = 4. Cytotoxic effects of the compounds were evaluated by the MTT assay in K562 leukemia, ECV304 endothelial and normal mononuclear cells, and DNA fragmentation analysis using the diphenylamine reaction was performed. The DNA binding capacity of thiosemicarbazones at IC50 and different concentrations was investigated. The DNA fragmentation percentage of compound treated cells was higher than that of non-treated control cells but was higher for compound 3 (84%) compared to the others. The interaction of compounds 1–4 and DNA was investigated voltammetrically by using nucleic acid modified electrodes after the double stranded fish sperm DNA (fsDNA), or poly(dA)·poly(dT), was immobilized onto the surface of pencil graphite electrodes (PGEs). Accordingly, the oxidation signals of DNA bases, guanine and adenine, were measured by using differential pulse voltammetry (DPV). The changes in the signals of guanine and adenine were evaluated before and after the interaction process. The results indicated that compound 3 was cytotoxic at very low concentrations in K562 leukemia cells unlike other cells and that could damage the DNA double stranded form, specifically the adenine base. Therefore, it may have a selective antileukemic effect and drug potential.

Dendrimer enriched single-use aptasensor for impedimetric detection of activated protein C

A novel impedimetric aptasensor for detection of human activated protein C (APC) was introduced for the first time in the present study. An enhanced sensor response was obtained using poly(amidoamine) (PAMAM) dendrimer having 16 succinamic acid surface groups (generation 2, G2-PS), that was modified onto the surface of screen printed graphite electrode (G2-PS/SPE). An amino modified DNA aptamer was then immobilized onto the surface of G2-PS modified SPE. The selective interaction of APT with its cognate protein, APC was investigated using different electrochemical techniques; differential pulse voltammetry (DPV), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The microscopic characterization was consecutively performed before/after each modification/interaction step using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The selectivity of aptasensor was tested in the presence of numerous proteins; protein C, thrombin, bovine serum albumin, factor Va and chromogenic substrate in different buffer mediums. The APC detection in the artificial serum; fetal bovine serum (FBS) was also performed impedimetrically. This dendrimer modified aptasensor technology brings several advantages: being single-use, fast screening with low-cost per measurement and resulting in sensitive detection of APC with the detection limits of 0.74 μg/mL (0.46 pmol in 35 μL sample) in buffer medium, and 2.03 μg/mL (1.27 pmol in 35 μL sample) in serum.

Development of amino functionalized carbon coated magnetic nanoparticles and their application to electrochemical detection of hybridization of nucleic acids

In our study, the development of amino functionalized carbon coated magnetic nanoparticles (NH2-CC-MNPs) and their usage for electrochemical detection of hybridization of nucleic acids have been aimed. Firstly, NH2-CC-MNPs were prepared by coating of pristine Fe3O4 nanoparticles with two layers via caramelization and silanization processes respectively. After the morphological characterization with scanning electron microscopy (SEM) it was seen that NH2-CC-MNPs was spherical shaped and in 28nm sized. Investigation of chemical composition with the help of scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDX) and fourier transform infrared spectroscopy (FTIR) was showed incorporation of carbon and APTES to the structure of NH2-CC-MNPs. Magnetic property of NH2-CC-MNPs after two layered coatings was demonstrated with electron spin resonance (ESR) technique and g factor was calculated as 2.6. In the second part of this study, optimization studies have carried out onto the surface of NH2-CC-MNPs prepared in saltless phosphate-tween 20 buffer (PBTw) for the analysis of DNA hybridization. The thiol linked DNA probe sequence representing to the Hepatitis B virus (HBV) concentration, target DNA sequence concentration, the most productive hybridization time and the selection of the nanoparticle surfaces have been researched. The electrochemical detection of DNA hybridization was investigated using PGE in combination with differential pulse voltammetry (DPV) technique by measuring the guanine oxidation signal. The detection limit was calculated in the linear target DNA concentration range of 5-25µg/mL and it was found to be 1.15µg/mL (20pmol in 110µL solution). It has been intended to be more reproducible, more sensitive and faster results with developed biosensor technology.