Electrochemical DNA Hybridization Research Articles

Novel hybridization indicator for DNA sequences related to the hepatitis B virus: Electrochemical and in-silico approach

In the present study, esculetin, as a novel electroactive indicator, was used to produce an electrochemical DNA hybridization biosensor for Hepatitis B Virus (HBV) detection. At first, alkanothiol-modified DNA probe (from Hepatitis B) was self-assembled on the surface of a glassy carbon electrode modified by the nanocomposite of polyaniline and Au nanoparticles (PANI-Au). Hybridization conditions were enhanced, including the immobilization of the probe strands, the hybridization process and the indicator accumulation. For evaluating the DNA hybridization, the differential pulse voltammetric response of the accumulated esculetin was used as an electroactive indicator on different electrode surfaces. According to the obtained results, the voltammetric responses of esculetin accumulated on the probes interacted with non-complementary, mismatch and complementary targets were significantly different. Therefore, in optimum conditions, electrochemical signals of esculetin were linear in the concentration range of 0.01–3.00 × 10−6 M of the complementary strands, and a detection limit of 3.80 × 10−9 M was obtained for the target. Subsequently, the modified electrode was utilized for the detection of the HBV in spiked serum and plasma samples. The results obtained from electrochemical technique were confirmed by computational molecular simulation using MOE software. Moreover, the simulation outcomes provided valuable information about quantum chemical properties including the HOMO–LUMO maps and chemical reactivity for esculetin-the DNA target components.

A novel spin-based label-free electrochemical dna hybridization biosensor and its applications for dengue virus detection

In this study, we have rationally constructed a spin-based label-free DNA hybridization sensor employing the spin-polarized electrons as a tool of detection. Recent research, that takes into account chiral-induced spin selectivity, has demonstrated colossal spin filtering by DNA at room temperature. Traditional DNA hybridization sensors use electronic(charge) current to probe the hybridization of double-strand (ds)-DNA. The benefit of the chiral-induced spin selectivity (CISS)-based DNA sensor lies in the direct probe of hybridization through its symmetry present in the secondary structure of ds-DNA. To this end, a self-assembled monolayer of DNA was prepared on Au-coated Ni devices and hybridization phenomena were investigated using differential pulse voltammetry. The accuracy of the CISS-based hybridization sensor was tested using additional complementary techniques such as cyclic voltammetry, electrochemical impedance spectroscopy, FTIR, and XPS, and the same method was successfully employed to detect the Dengue virus as well. With a limit of detection of up to 0.12 picomolar (pM), the results are reliable and repeatable. The current approach opens up new possibilities for developing spin-based point-of-care devices in the future.

Understanding Changes of Both Faradaic and Nonfaradaic Currents with Temperature in DNA Monolayer-Based Sensors on Gold Electrodes

Electrochemical sensors are well studied for detecting DNA, RNA, small molecules, inorganic ions, etc. Using the latest DNA-based sensors, a change in the location of a redox reporter attached to an electrode-bound nucleic acid gives rise to faradaic current changes, which can be monitored by square wave voltammetry (SWV) and used for sensing of various analytes. However, the nonfaradaic current generated along with the faradaic current has often been viewed as a limiting factor in sensor performance and either subtracted or ignored. Nonfaradaic current can be suppressed somewhat by surface blocking and reduced electrode surface area, and our group recently introduced a differential potentiostat (DiffStat) that provided efficient, in-hardware subtraction. On the other hand, removal of nonfaradaic current may lead to loss of important information embedded within. Moreover, a change in temperature can affect both faradaic and nonfaradaic currents by altering the movement of DNA strands and other ions present in the solution. Usually, faradaic current is proportional to the temperature. However, temperature-dependent changes in nonfaradaic current have not been well studied in DNA monolayer-based sensors. In this study, the changes in both faradaic and nonfaradaic currents were monitored in an electrochemical DNA hybridization assay. A thiolated DNA (thio-DNA) monolayer was assembled onto gold electrodes, and current was measured at different square wave frequencies (1 to 1000 Hz). The DNA analyte and the methylene blue tagged DNA (MB-DNA) were hybridized with the thio-DNA separately to compare the signals. Using an in-house built Peltier controller, the effects of temperature were monitored during operation of an analyte-induced proximity assay with SWV readout. In-house written MATLAB code was developed to automatically analyze each set of raw SWV data. Within this code, a polynomial baseline was calculated in the vicinity of the redox potential of MB-DNA (around -200 mV) to allow faradaic and nonfaradaic currents to be separated, post assay. Two-dimensional heatmaps were generated to study the variation of these currents with both temperature and square wave frequency. The faradaic current increased up to a certain temperature and then decreased due to dissociation (or melting) of the DNA from the surface. The nonfaradaic current, on the other hand, increased with temperature in a viscosity-dependent manner and also increased with the square wave frequencies as expected from a capacitive, single exponential time decay. Current changes exhibited by the DNA sensors upon analyte binding and temperature fluctuation suggested that both faradaic and nonfaradaic signals could be used for multidimensional analysis of sensor responses in the future. Figure 1

Highly sensitive and label-free electrochemical biosensor based on gold nanostructures for studying the interaction of prostate cancer gene sequence with epirubicin anti-cancer drug

A label-free electrochemical DNA hybridization biosensor was developed based on gold nanocubes (AuNCs) modified graphite screen-printed electrodes (GSPEs) for investigating the epirubicin (EPI) interaction with the short sequence DNA of prostate cancer gene. The electrochemical behavior of the genosensor was assessed in different steps of construction using cyclic voltammetry and electrochemical impedance spectroscopy methods. The changes in the biosensor responses versus the DNA interactions with the various concentrations of EPI were monitored by the differential pulse voltammetry method. The reduction peak current of EPI linearly increased with the increasing concentration of EPI. Under optimal conditions, two linear ranges were obtained from 0.04 to 0.80 μM and 0.8 to 20.0 μM, respectively with a limit of detection 0.01 μM. The prepared biosensor exhibited a proper selectivity toward the target drugs. The applicability of the sensor was demonstrated by the explored of epirubicin in a human blood serum sample. In addition, the prepared biosensor was used as a reproducible and stable device for EPI determination.

Nature Inspired Anti-Biofouling Strategy for the Detection of Protein Biomarkers Directly in a Drop of Blood

Electrode biofouling in complex biological fluids is one of the major problems in analytical electrochemistry. Direct electrochemical detection in human blood remains challenging due to quick surface passivation by proteins present in blood, degrading sensor’s sensitivity. This challenge has dramatically slowed down commercialization efforts of point-of-care devices. Here we have developed a simple nature-inspired electrochemical DNA hybridization (e-hyb) assay based on steric hindrance which enable electrodes to detect nanomolar concentration of proteins (i.e. antibody) directlywithin a drop ofblood without any sample preparation. This e-hyb assay involves two DNA strands; one-captured DNA immobilized on electrode surface and another complementary reporter DNA labelled with an electroactive molecule (methylene blue) at one end and a specific recognition element at the another end. In presence of a specific large analyte binding to the small recognition element, the reporter DNA cannot reach to the electrode surface due to steric hindrance. This simple strategy enables us to quantify different protein biomarkers (HIV antibody, anti-DNP antibody, anti-DIG antibodyetc) in human blood without being limited affected by biofouling.Figure 1: e-DNA hybridization based anti-biofouling strategy for the detection of antibody in human blood Figure 1

Fast enzyme-linked electrochemical sensing of DNA hybridization at pencil graphite electrodes. Application to detect gene deletion in a human cell culture

In this paper we present a rapid electrochemical enzyme-linked DNA hybridization assay using disposable pencil graphite electrodes (PeGE) to detect target DNA (tDNA) sequences in DNA fragments amplified by polymerase chain reaction. The procedure consists of several short (1–2 min) incubation steps, including adsorption of the tDNA at unpretreated PeGE from denaturing medium, surface blocking with milk proteins, hybridization with a biotinylated oligonucleotide probe and binding of streptavidin-alkaline phosphatase conjugate to the biotin tags. Then the PeGE is transferred into background electrolyte solution containing 1-naphthyl phosphate, which is enzymatically dephosphorylated to give electrochemically oxidizable indicator 1-naphthol. The assay, which can be performed within 7–8 min, offers a perfect discrimination between specific and nonspecific DNA amplicons and easy detection of about ~40 femtomoles of tDNA in large excesses of non-complementary DNA. An application on the detection of p53 gene deletion in a human cell culture, featuring a real biological model, is presented. The designed setup has a potential to be applied as one of simple, fast, robust ultralow cost “do-it-yourself “instruments recently introduced as diagnostic tools for third world countries.

Label-Free Electrochemical Detection of DNA Hybridization Related to Anthrax Lethal Factor by using Carbon Nanotube Modified Sensors

Background: Anthrax Lethal Factor (ANT) is the dominant virulence factor produced by B. anthracis and is the major cause of death of infected animals. In this paper, pencil graphite electrodes GE were modified with single-walled and multi-walled carbon nanotubes (CNTs) for the detection of hybridization related to the ANT DNA for the first time in the literature. Methods: The electrochemical monitoring of label-free DNA hybridization related to ANT DNA was explored using both SCNT and MCNT modified PGEs with differential pulse voltammetry (DPV). The performance characteristics of ANT-DNA hybridization on disposable GEs were explored by measuring the guanine signal in terms of optimum analytical conditions; the concentration of SCNT and MCNT, the concentrations of probe and target, and also the hybridization time. Under the optimum conditions, the selectivity of probe modified electrodes was tested and the detection limit was calculated. Results: The selectivity of ANT probes immobilized onto MCNT-GEs was tested in the presence of hybridization of probe with NC no response was observed and with MM, smaller responses were observed in comparison to full-match DNA hybridization case. Even though there are unwanted substituents in the mixture samples containing both the target and NC in the ratio 1:1 and both the target and MM in the ratio 1:1, it has been found that ANT probe immobilized CNT modified graphite sensor can also select its target by resulting with 20.9% decreased response in comparison to the one measured in the case of full-match DNA hybridization case Therefore, it was concluded that the detection of direct DNA hybridization was performed by using MCNT-GEs with an acceptable selectivity. Conclusion: Disposable SCNT/MCNT modified GEs bring some important advantages to our assay including easy use, cost-effectiveness and giving a response in a shorter time compared to unmodified PGE, carbon paste electrode and glassy carbon electrode developed for electrochemical monitoring of DNA hybridization. Consequently, the detection of DNA hybridization related to the ANT DNA by MCNT modified sensors was performed by using lower CNT, probe and target concentrations, in a shorter hybridization time and resulting in a lower detection limit according to the SCNT modified sensors. In conclusion, MCNT modified sensors can yield the possibilities leading to the development of nucleic acid sensors platforms for the improvement of fast and cost-effective detection systems with respect to DNA chip technology.

Poly-L-lysine Coated Surfaces for Ultrasensitive Nucleic Acid Detection.

Poly‐L‐lysine is one of the biocompatible polymers having amino and carboxyl groups in its structure. This attractive feature of poly‐L‐lysine makes it very convenient for bioactive molecule attachment. This study details the preparation of poly‐L‐lysine‐based pencil graphite electrodes (PLL/PGEs) and use of the coated electrodes for direct ultrasensitive DNA hybridization detection. In the first part of this study, poly‐L‐lysine coated electrodes were prepared using L‐lysine as the monomer by cyclic voltammetry (CV) with different cyclic scans. The effect of these cyclic scans during the electropolymerization was investigated. Coated electrodes were characterized by cyclic voltammetry, electrochemical impedance spectroscopy (EIS) and scanning electron microscopy (SEM). Then, one‐pot preparation of poly‐L‐lysine composites with graphene (GN) and multi‐walled carbon nanotubes (MWCNTs) onto the pencil graphite electrodes were achieved. Electrochemical responses of these 3 electrodes were compared. After all, electrochemical DNA hybridization was performed using the poly‐L‐lysine‐based electrodes prepared at optimum polymerization condition. The PLL/PGE coated electrode presented a good linear response in the target concentration range of 1.0×10−13 to 1.0×10−6 with a detection limit of 2.25×10−14 using differential pulse voltammetry as the detection method. We believe that poly‐L‐lysine‐based surfaces will be useful for further clinical applications.

A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer

Electrochemical assays potentially offer a highly cheap, automated, portable and sensitive method for a great deal of disease biomarker detections, and minimization of nonspecific adsorption is a critical issue for their application in natural complex media. We present herein, the development and application of a highly sensitive and selective electrochemical DNA hybridization biosensor for breast cancer marker BRCA1, based on a zwitterionic peptide self-assembled monolayer (SAM) support serving as the low fouling substrate, the 19-mer BRCA1 related sequence-specific oligonucleotides, and the sensitively label-free sensing technique of electrochemical impedance spectroscopy (EIS). The capability of such peptide SAM in resisting nonspecific protein adsorption was surveyed using EIS, and the biosensor fabrication process and sensing performance were also investigated with X-Ray photoelectron spectroscopy (XPS) and EIS. Significantly, this developed biosensing strategy achieved a linear detection range from 1.0fM to 10.0pM with a limit of detection of 0.3fM, associated with satisfactory sensing properties of facile fabrication, desired sensitivity, high selectivity, and favourable reproducibility and antifouling ability, manifesting its promising potential in practical application.

Electrospun manganese (III) oxide nanofiber based electrochemical DNA-nanobiosensor for zeptomolar detection of dengue consensus primer

Nanoscale biosensors, owing to their high-sensitivity and extremely low limits-of-detection, have enabled the realization of highly complex and sophisticated miniaturized platforms for several important healthcare applications, the most predominant one being disease diagnosis. In particular, nanomaterial facilitated electrochemical detection of DNA hybridization has had an exceptional impact on fields such as genetics and cancerous mutation detection Here we report an ultrasensitive electrochemical platform using electrospun semi-conducting Manganese (III) Oxide (Mn2O3) nanofibers for DNA Hybridization detection. The proposed platform coalesces the inherent advantages of metal-oxide nanofibers and electrochemical transduction techniques, resulting in label-free zeptomolar detection of DNA hybridization. As proof of concept, we demonstrate zeptomolar detection of Dengue consensus primer (limit of detection: 120×10–21M) both in control as well as spiked serum samples. Our reported detection limit is superior in comparison with previously reported electrochemical DNA hybridization sensors for Dengue virus detection, spanning both labeled and label-free transductions. This ultra-sensitivity, we believe, is a result of synthesizing a low bandgap electrospun metal-oxide nanomaterial corresponding to a specific oxidation state of Manganese. This methodology can be extended for detection of any hybridization of interest by simply adapting an appropriate functionalization protocol and thus is very generic in nature.

Graphene quantum dot-based on-chip electrochemical DNA hybridization sensor for pancreatic cancer

Graphene quantum dot-based on-chip electrochemical DNA hybridization sensor for pancreatic cancer

Introducing curcumin as an electrochemical DNA hybridization indicator and its application for detection of human interleukin-2 gene

In this paper, the application of curcumin (CU) as a non-toxic electrochemical DNA hybridization indicator was described. Hybridization investigations on a pencil graphite electrode surface as a transducer using oligonucleotides containing only one base type, including poly A, poly T, poly C, and poly G as probe and as related complementary/non-complementary sequences, showed that CU has no specific interaction with each of the oligonucleotides of DNA. Furthermore, results showed good interaction between CU and the hybridized form of oligonucleotides; thus, the extent of hybridization was evaluated based on the difference between differential pulse voltammetry (DPV) signals of CU accumulated on the probe-pencil graphite electrode (PGE) and CU accumulated on the probe-target-PGE. Then, the developed biosensor was successfully applied for the detection of short sequences of human interleukin-2 (hIL-2) gene as a model. A hybridization experiment with non-complementary oligonucleotide showed that the suggested DNA sensor responds selectively to the target. At optimized conditions, two linear ranges were obtained for hIL-2 gene, first from 50 to 1000 pM and second from 0.01 to 1 μM with a detection limit of 12 pM. 7.0) containing 20 mM NaCl.

Label-Free DNA Hybridization Detection by Poly(Thionine)-Gold Nanocomposite on Indium Tin Oxide Electrode

A simple and label-free electrochemical DNA hybridization sensor was developed based on a poly(thionine)/gold nanoparticle composite-modified indium tin oxide (PTH/GNPs/ITO) electrode. The PTH/GNPs/ITO electrode was prepared by the electrodeposition of GNPs on ITO followed by the electropolymerization of thionine (TH). Probe DNA (pDNA) was immobilized on this substrate via covalent linkage between the -NH2 group of PTH and the PO43− group at the 5′ end of pDNA. The immobilized single-strand pDNA increased the stiffness of the PTH film and decreased the PTH oxidation current, whereas the current was reduced to a greater extent after the formation of double-strand DNA by the hybridization of target complementary DNA (cDNA). The pDNA/PTH/GNPs/ITO sensor exhibited excellent stability, and selectivity to discriminate DNA base pair mismatches. The semi-log plot of the [cDNA] vs. oxidation current of PTH was linear in the concentration range of 1 μM − 1 fM with the detection limit (S/N = 3) and sensitivity of ca. 0.3 fM and ca. 2.0 μA·cm−2·fM−1, respectively.

Electrochemical Biosensor for Detection of Target DNA Sequence and Single-Base Mismatch Related to Helicobacter Pylori Using Chlorogenic Acid as Hybridization Indicator

An electrochemical DNA hybridization biosensor was developed using a novel electroactive indicator. The DNA hybridization was detected based on the interaction of chlorogenic acid, CGA, with an 18-mer alkanethiol DNA probe (from Helicobacter pylori) self-assembled gold electrode (S1/MCH/AuE) and its hybridization form (S2-S1/MCH/AuE). The conditions were optimized for the immobilization of probe DNA, the exposure of MCH, the accumulation of indicator and the hybridization process. The differential pulse voltammetric response of the accumulated CGA, as an electroactive indicator, on different electrode surfaces was used to assess the DNA hybridization. The results indicate that there is a significant difference between the voltammetric responses of CGA accumulated on the probes hybridized with non-complementary, mismatch and complementary targets. The potential of the developed biosensor is, thus, confirmed in detecting and discriminating the target complementary DNA from non-complementary and mismatch oligonucleotides. In optimum conditions, CGA electrochemical signals had a linear dependence on the concentration of the complementary DNA within the range of 20.0 to 410.0 nM. Finally, a detection limit of 7.2 nM was obtained for the target DNA.

Poly(indole-5-carboxylic acid)-functionalized ZnO nanocomposite for electrochemical DNA hybridization detection

In this paper, a novel direct DNA electrochemical biosensor was developed for detection of breakpoint cluster region gene and the cellular abl (BCR/ABL) fusion gene from chronic myelogenous leukemia. Nanosized ZnO was first immobilized on the surface of a carbon paste electrode, and then poly(indole-5-carboxylic acid) (PICA), a new electroactive material, was electropolymerized on the ZnO substrate by using the potentiostatic method to form a unique nanocomposite structure. The obtained PICA/ZnO nanocomposite not only served as a substrate for DNA immobilization but also reflected the electrochemical transduction originating from DNA immobilization and hybridization without any outer indicators or complicated labeling. The immobilization of probe DNA was successfully conducted via covalent bonding between the abundant carboxyl groups of PICA/ZnO nanocomposite film and amino groups at the 5′-end of probe DNA. After the hybridization of probe DNA with target DNA, the formation of double-helix structure induced the “signal-on” of the nanocomposite. The dynamic range of this DNA biosensor for detecting the complementary target sequence of BCR/ABL fusion gene was from 1.0 × 10−15 to 1.0 × 10−9 mol/L using electrochemical impedance spectroscopy, and the detection limit was estimated to be 2.2 × 10−16 mol/L.

Novel electrochemical DNA hybridization biosensors for selective determination of silver ions

In this work, novel electrochemical biosensors for Ag+ determination based on Ag+-induced DNA hybridization, using Ethyl green (EG) as an electroactive label on the surface of bare carbon paste electrode (CPE) and gold nanoparticles-modified carbon paste electrode (GN-CPE) are reported. Two single-strand poly-C (100% cytosine bases) DNAs are used as oligonucleotide probe and target. In the presence of Ag+, the target DNA with full cytosine–cytosine (C–C) mismatches could hybridize with the probe DNA by forming C–Ag+–C complex. The induced hybridization of the two oligonucleotides leads to the decrease in the reduction peak currents of EG, which could be used for electrochemical determination of Ag+. This difference in the values of the reduction peak current of EG before and after DNA hybridization (∆I) was linear with the concentration of Ag+ in the ranges from 3.0×10−10 to 1.0×10−9molL−1 and 9.0×10−11 to 1.0×10−9molL−1, for the biosensor and nanoparticles modified-biosensor, respectively. Calculated detection limits were 1.04×10−10 and 2.64×10−11molL−1 for biosensor and nanoparticles modified-biosensor, respectively. The biosensors demonstrated good selectivity towards Ag+ ions in the presence of some metal ions such as Pb2+, Cu2+, Ca2+, Zn2+, Fe2+ and Hg2+. The proposed biosensors were applied successfully to the voltammetric determination of Ag+ in real samples.

Detection of DNA Hybridization by Methylene Blue Electrochemistry at Activated Nanoelectrode Ensembles.

Nanoelectrode ensembles (NEEs) obtained by electroless gold deposition in track-etched poly-carbonate (PC) membranes are functionalized and applied for DNA hybridization detection, using methylene blue (MB) as electroactive probe. To this aim, an amine terminated (ss)DNA probe is immobilized on the PC surface of the NEE by reaction via carbodiimide and N-hydroxysulfosuccinimide. In order to increase the number of carboxylic groups present on PC and suitable for the functionalization, the surface of NEEs is oxidized with potassium permanganate. The presence of carboxylic functionalities is verified by spectrochemical titration with thionin acetate (THA) and the effect of the activation treatment on the electrode performances is evaluated by cyclic voltammetry (CV). After activation and functionalization with the probes, the NEE-based sensor is hybridized with complementary target sequences. The effect of the functionalization of the NEEs both with the (ss)DNA probe alone and after hybridization with the target, is studied by measuring the changes in the MB reduction signal by square wave voltammetry (SWV), after incubation in a suitable MB solution, rinsing and transfer to the measurement cell. It was observed that this peak signal decreases significantly after hybridization of the probe with the complementary target. Experimental evidences suggest that the interaction between MB and the guanines of (ss)DNA and (ds)DNA is at the basis of the development of the here observed analytical signal. The proposed approach allows the easy preparation and testing of NEE-based sensors for the electrochemical DNA hybridization detection.

Electrochemical DNA hybridization sensors based on conducting polymers.

Conducting polymers (CPs) are a group of polymeric materials that have attracted considerable attention because of their unique electronic, chemical, and biochemical properties. This is reflected in their use in a wide range of potential applications, including light-emitting diodes, anti-static coating, electrochromic materials, solar cells, chemical sensors, biosensors, and drug-release systems. Electrochemical DNA sensors based on CPs can be used in numerous areas related to human health. This review summarizes the recent progress made in the development and use of CP-based electrochemical DNA hybridization sensors. We discuss the distinct properties of CPs with respect to their use in the immobilization of probe DNA on electrode surfaces, and we describe the immobilization techniques used for developing DNA hybridization sensors together with the various transduction methods employed. In the concluding part of this review, we present some of the challenges faced in the use of CP-based DNA hybridization sensors, as well as a future perspective.

Label and indicator free electrochemical nanobiosensing of DNA hybridization based on MnO2 nanomaterial modified GCPE

A label and indicator free electrochemical DNA hybridization detection method based on MnO2-Nps/GCPE was developed. Compared to plain GCPE, a very robust and sensitive genosensor was obtained.

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

AbstractIn this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10−13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.