Electrochemical Biosensors for Monitoring of Drug-DNA Interactions.

The binding of the therapeutic agents to the nucleic acids is one of the paramount issues in the drug development area that is studied by various techniques. Electrochemical studies have a big portion in this area due to the fact that they allow designing of novel monitoring systems that have superior properties such as being feasible and sustainable. These electrochemical monitoring tools analyze these interactions in in vitro conditions and give the results precisely and rapidly. In the scope of this manuscript, the electrochemical monitoring platforms developed for the determination of DNA-drug interactions were under the spotlight. The electrode types mostly used for the electrochemical monitoring of drug-DNA interactions were described. The binding mechanisms of the drugs to the DNA structure were explained, and the evaluation strategies of the interactions using electrochemical techniques were stated. Most of the reports of the last 25 years were given, and some of the electrochemical biosensor applications including both voltammetric and impedimetric studies were explained in detail. Furthermore, it is possible to reach nanomaterials/biomaterials-based biosensor platforms for the monitoring of DNA-drug interactions, and these applications were in the scope of this manuscript. The future aspects of these areas were also stated.

Electrochemical investigation of the interaction of 2,4-D and double stranded DNA using pencil graphite electrodes.

2,4-dichlorophenoxyacetic acid (2,4-D) is an auxinic herbicide used to control broadleaf weeds. It is also a threatening factor for not only aquatic life but also human health due to its genotoxicity and endocrine disruptive property. Herein, the interaction between 2,4-D and double stranded DNA was investigated by using single-use pencil graphite electrodes (PGE) in combination with electrochemical techniques. The detection mechanism was based on the monitoring of the changes at the guanine oxidation signal obtained before/after surface-confined interaction of 2,4-D and DNA at the surface of PGE. The electrochemical characterization of the interaction was studied by using microscopic and electrochemical techniques. The response obtained by interaction in the presence of another herbicide, glyphosate, which is widely used with 2,4-D for weed control, was compared to the one occurred in the presence of 2,4-D. Electrochemical monitoring of the interaction between the herbicide whose active molecule was 2,4-D and DNA was also investigated. The detection (LOD) and quantification limits (LOQ) for 2,4-D and the herbicide could be obtained in the linear concentration ranges of 30-70 µg/mL and 10-30 µg/mL, respectively and LOD and LOQ values were found to be 2.85 and 9.50 µg/mL for both 2,4-D and the herbicide. The sensitivity of the biosensor was calculated as 0.087 µA.mL / µg.cm2 .This is the first study in literature by means of not only voltammetric detection of 2,4-D and DNA interaction but also the herbicide-DNA interaction at the surface of PGE based on the changes at the guanine signal.

Monitoring of glyphosate-DNA interaction and synergistic genotoxic effect of glyphosate and 2,4-dichlorophenoxyacetic acid using an electrochemical biosensor.

Glyphosate (GLY) is a broad-spectrum herbicide used worldwide to control broadleaf sedge, and grass weeds to control non-specific vegetation. Although it was evaluated as non-toxic agent in 20th century, its carcinogenic and genotoxic potential has being intensively investigated all over the world in the last decade. Moreover, the combination of GLY and 2,4-dichlorophenoxyacetic acid (2,4-D) has been widely applied. Although genotoxicity of GLY has been evaluated in vivo studies, there is no report in the literature for the monitoring of in vitro biointeraction of GLY and double stranded DNA, or how effect the combination of GLY and 2,4-D onto DNA. Herein, an electrochemical biosensor platform was developed for detection of the pesticide-DNA interaction by using disposable pencil graphite electrodes (PGEs). First, voltammetric detection of the interaction between GLY and DNA was investigated and the electrochemical characterization of the interaction was achieved. Taking a step further, the synergistic genotoxic effect of the mixture of GLY and 2,4-dichlorophenoxyacetic acid (2,4-D) or the mixture of their herbicide forms onto DNA could be monitored. This effect was concentration dependent, and the herbicide of GLY or the use of mixture of herbicides of GLY and 2,4-D had more genotoxic effect than analytical grade of the active molecules, GLY and 2,4-D. The single-use PGEs provided to fabricate robust, eco-friendly and time saver recognition platform for monitoring of herbicide-DNA interaction with the sensitive and reliable results. It is expected that this study will lead to be designed miniaturized lab-on-a chip platforms for on-line analysis of the pesticide-nucleic acid interactions.

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.

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.

Voltammetric Aptasensor Based on Magnetic Beads Assay for Detection of Human Activated Protein C.

Aptamers are defined as new generation of nucleic acids, which has recently presented promising specifications over to antibodies. An increasing number of electrochemical studies related to aptamer-based sensors, so-called aptasensors have been introduced in the literature. Herein, the interaction between human activated protein C (APC) and its cognate DNA aptamer (DNA APT) was performed at the surface of magnetic beads (MBs), followed by voltammetric detection using disposable graphite electrodes (PGEs).

Electrochemical monitoring of the interaction between Temozolamide and nucleic acids by using disposable pencil graphite electrodes.

Temozolomide (TMZ) is an anticancer drug used for the treatment of adult brain tumour and skin cancer. The biomolecular interaction between TMZ and DNA was investigated for the first time in this study using disposable pencil graphite electrodes (PGEs) in combination with electrochemical techniques. The surface confined interactions between TMZ and different type of nucleic acids were performed. Before/after surface confined interaction process, the oxidation signals of TMZ, guanine and adenine were measured using differential pulse voltammetry (DPV) and PGE and accordingly, the changes at the oxidation signals were evaluated. The detection limit (DL) was also estimated based on the oxidation signal of TMZ. The interaction of TMZ with single stranded poly [A], poly [G], or double stranded poly [A]-poly[T] and poly [G]-poly[C] was also explored. Moreover, cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) techniques were utilized for detection the interaction between TMZ and DNA. The features of this single-use electrochemical sensor was discussed in comparison to other reports that were developed for TMZ detection.

Development of Ionic Liquid Modified Disposable Graphite Electrodes for Label-Free Electrochemical Detection of DNA Hybridization Related to Microcystis spp.

In this present study, ionic liquid (1-butyl-3-methylimidazolium hexafluorophosphate (IL)) modified pencil graphite electrode (IL-PGEs) was developed for electrochemical monitoring of DNA hybridization related to Microcystis spp. (MYC). The characterization of IL-PGEs was performed using microscopic and electrochemical techniques. DNA hybridization related to MYC was then explored at the surface of IL-PGEs using differential pulse voltammetry (DPV) technique. After the experimental parameters were optimized, the sequence-selective DNA hybridization related to MYC was performed in the case of hybridization between MYC probe and its complementary DNA target, noncomplementary (NC) or mismatched DNA sequence (MM), or and in the presence of mixture of DNA target: NC (1:1) and DNA target: MM (1:1).

Electrochemical assay for determination of gluten in flour samples.

Herein, an electrochemical assay developed by differential pulse voltammetry (DPV) in combination with the disposable pencil graphite electrode (PGE) was progressed for sensitive and selective detection of gluten. Under the optimum experimental conditions, a linear concentration range of gluten was obtained from 20 to 100 µg/mL and consequently the detection limit was found to be 7.11 µg/mL. In addition, this electrochemical assay was successfully employed for the detection of gluten in different flour samples. Applicability of our method was also tested in the commercial samples of vinegar and baker's yeast.

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).

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.

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.

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.

Genomagnetic assay for electrochemical detection of osteogenic differentiation in mesenchymal stem cells.

The osteogenic differentiation of mesenchymal stem cells (MSCs) was assessed by determining the gene expression levels of proteins; osteocalcin (OSC), osteonectin (OSN) and osteopontin (OSP) based on electrochemical detection protocol combined with genomagnetic assay in parallel to real-time PCR analysis. Genomagnetic assay was performed using streptavidin coated commercial magnetic particles (magnetic beads, MBs) in combination with single-use electrochemical sensor technology. A biotinylated DNA probe was immobilized onto streptavidin coated magnetic particles, and then the hybridization process of the probe with its complementary DNA was performed. The oxidation signals of DNA electroactive bases guanine and adenine were measured voltammetrically using a pencil graphite electrode (PGE) before and after the hybridization process of OSC/OSN/OSP probe sequences with their complementary target sequences. The selectivity of the genomagnetic assay was also tested using each DNA probe individually related to osteogenic differentiations. The voltammetric detection of osteogenic differentiations was confirmed selectively by real-time PCR analysis.

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.

Estrone specific molecularly imprinted polymeric nanospheres: synthesis, characterization and applications for electrochemical sensor development.

The aim of this study is (i) to prepare estrone-imprinted nanospheres (nano-EST-MIPs) and (ii) to integrate them into the electrochemical sensor as a recognition layer. N-methacryloyl-(l)-phenylalanine (MAPA) was chosen as the complexing monomer. Firstly, estrone (EST) was complexed with MAPA and the EST-imprinted poly(2-hyroxyethylmethacrylate-co-N-methacryloyl-(l)-phenylalanine) [EST-imprinted poly(HEMA-MAPA)] nanospheres were synthesized by surfactant- free emulsion polymerization method. The specific surface area of the EST-imprinted poly(HEMA-MAPA) nanospheres was found to be 1275 m2/g with a size of 163.2 nm in diameter. According to the elemental analysis results, the nanospheres contained 95.3 mmole MAPA/g nanosphere. The application of EST specific MIP nanospheres for the development of an electrochemical biosensor was introduced for the first time in our study by using electrochemical impedance spectroscopy (EIS) technique. This nano-MIP based sensor presented a great specificity and selectivity for EST.

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.