Electrochemical Detection Of DNA Hybridization Research Articles

Genosensing on a 3D-printed nanocarbon electrode

In this paper we present the characterization of 3D-printed nanocarbon electrodes (3DnCes) and their application in electrochemical enzyme-linked detection of DNA hybridization. The approach takes advantage of a facile procedure based on adsorption of target DNA on the electrode surface followed by hybridization with a biotinylated probe and binding of streptavidin–alkaline phosphatase conjugate. The alkaline phosphatase converts 1-naphthyl phosphate in the background electrolyte into electrochemically oxidizable 1-naphthol, which is subsequently detected using linear sweep voltammetry. The preparation, characterization, and analytical performance of the 3DnCes are reported. The results show the applicability of such 3DnCes in detection of target DNA hybridization specifically with the complementary biotinylated probe, and indicate the potential of 3D printed electrodes for use in various bioanalytical approaches.

Label-Free Electrochemical Detection of DNA Hybridization: A Method for COVID-19 Diagnosis.

This paper presents label-free electrochemical transduction as a suitable scheme for COVID-19-specific viral RNA/c-DNA detection, with an aim to facilitate point of care diagnosis. In lieu of this, we discuss the proposed electrochemical biosensing scheme, based on electrodeposited gold nanoparticles as the transducing elements. Specific to this approach, here, the protocols associated with the immobilization of the single-stranded probe nucleotide on to the biosensor, have also been laid out. This paper also discusses the methods of electrochemical analysis, to be used for data acquisition and subsequent calibration, in relation to target analyte detection. Towards facilitating portable diagnosis, development of miniaturized sensors and their integration with readout units have also been discussed.

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.

Electrochemical detection of DNA hybridization based on three-dimensional ZnO nanowires/graphite hybrid microfiber structure

In the present work, zinc oxide (ZnO) nanowire (NWs)/graphite microfiber hybrid electrodes were used for the detection of DNA hybridization. ZnO NWs were in-situ synthesized onto graphite fibers using the hydrothermal technique, and the result was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM) as well as transmission electron microscope (TEM). An electrochemical sensor for the analysis of DNA hybridization was developed based on immobilizing DNA probes onto the ZnO NWs/graphite microfiber electrodes surface via electrostatic interaction. Cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and X-ray photoelectron spectroscopy (XPS) were used to corroborate the fabrication of the sensor. Based on the results, ZnO NWs/graphite microfiber hybrid electrodes showed a great hybridization capacity for the determination of target DNA. A wide dynamic detection range from 1.0 × 10−14 M to 1.0 × 10−7 M with an ultralow detection limit of 3.3 × 10−15 M was achieved for detecting the target DNA. Moreover, it showed good selectivity and stability during the detection process.

Silver-dendrimer nanocomposites as oligonucleotide labels for electrochemical stripping detection of DNA hybridization

Silver-dendrimer nanocomposites were synthesized and used as oligonucleotide labels for electrochemical stripping detection of DNA hybridization. The synthesized silver-dendrimer nanocomposites were characterized by UV–vis spectrophotometry, X-ray photoelectron spectroscopy (XPS) and transmission electron microscopy (TEM). Ratios of silver/dendrimer were optimized in order to obtain stable nanocomposites with maximal silver loading in the interior of a polymeric shell. The silver-dendrimer nanocomposites were attached to sequence-known DNA probes specific to colitoxin, and used to detect probe hybridization by dissolution of the silver nanoparticles in the interior of dendrimer in a diluted nitric acid, followed by measurement of Ag+ ions by anodic stripping voltammetry (ASV). Use of differential pulse voltammetry for the stripping step, along with optimization of the ASV conditions, enabled a detection limit of 0.78 pM. The present strategy, in combination with dendrimer-encapsulated copper labeled oligonucleotides probe reported previously, could potentially be used to detect single or multiple DNA targets in one sample.

One-step coelectrodeposition-assisted layer-by-layer assembly of gold nanoparticles and reduced graphene oxide and its self-healing three-dimensional nanohybrid for an ultrasensitive DNA sensor.

A layer-by-layer (LBL) assembly was employed for preparing multilayer thin films with a controlled architecture and composition. In this study, we report the one-step coelectrodeposition-assisted LBL assembly of both gold nanoparticles (AuNPs) and reduced graphene oxide (rGO) on the surface of a glassy carbon electrode (GCE) for the ultrasensitive electrochemical impedance sensing of DNA hybridization. A self-healable nanohybrid thin film with a three-dimensional (3D) alternate-layered nanoarchitecture was obtained by the one-step simultaneous electro-reduction of both graphene oxide and gold chloride in a high acidic medium of H2SO4 using cyclic voltammetry and was confirmed by different characterization techniques. The DNA bioelectrode was prepared by immobilizing the capture DNA onto the surface of the as-obtained self-healable AuNP/rGO/AuNP/GCE with a 3D LBL nanoarchitecture via gold-thiol interactions, which then served as an impedance sensing platform for the label-free ultrasensitive electrochemical detection of DNA hybridization over a wide range from 1.0 × 10-9 to 1.0 × 10-13 g ml-1, a low limit of detection of 3.9 × 10-14 g ml-1 (S/N = 3), ultrahigh sensitivity, and excellent selectivity. This study presents a promising electrochemical sensing platform for the label-free ultrasensitive detection of DNA hybridization with potential application in cancer diagnostics and the preparation of a self-healable nanohybrid thin film with a 3D alternate-layered nanoarchitecture via a one-step coelectrodeposition-assisted LBL assembly.

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.

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.

Chitosan‐carbon Nanofiber Modified Single‐use Graphite Electrodes Developed for Electrochemical Detection of DNA Hybridization Related to Hepatitis B Virus

AbstractChitosan (CHIT) and carbon nanofiber (CNF) modified pencil graphite electrodes (PGEs) were developed for the first time in the present study for enhanced monitoring of DNA hybridization. CHIT‐CNF modified PGE, CHIT modified PGE and unmodified PGE were firstly characterized by scanning electrone microscopy (SEM), and the electrochemical behaviour of each electrode was investigated by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The optimization of experimental studies; CNF concentration, or DNA probe concentration was performed by differential pulse voltammetry (DPV) technique. The sequence‐selective DNA hybridization related to Hepatitis B virus (HBV) was then performed in the case of hybridization between HBV probe and its target DNA, or different non‐complementary DNA sequences (NC1 and NC2), and also the hybridization case in the mixture samples containing both target and NC sequences in the ratio of 1 : 1. CHIT‐CNF‐PGEs presented very effective discrimination of HBV DNA hybridization, which also presented high sensitivity and better reproducibility due to the unique chemical and structural properties of CNF.

Applying Mesoporous Silica SBA‐15 in Electrochemical Detection of DNA Hybridization

AbstractMesoporous silica SBA‐15 powder was used as the separation surface for hybridization purposes and it has proved appropriate and effective. By help of a simple procedure any oligodeoxynucleotide (ODN) modified by NH2‐group can be covalently bound at the surface of SiO2 particles, what significantly extends the capabilities of the experimenter. Use of cathodic stripping voltammetry for electrochemical detection of ODN/DNA hybridization ensures high sensitivity of the analyses. Only ordinary laboratory equipment is needed for the implementation of these experiments. Due to the very small consumption of SBA‐15, commonly sold quantity (5 g) may be sufficient for many years of work.

Amplified electrochemical DNA sensor based on hemin/G-quadruplex DNAzyme as electrocatalyst at gold particles modified heated gold disk electrode

A new method for electrochemical detection of DNA hybridization with elevated electrode temperature at gold particles modified heated gold disk electrode (Au-HAuDE) is demonstrated. This method employs a hairpin DNA probe for target DNA capture and incorporates a specific DNAzyme sequence into the probe as electrocatalytic label. The hairpin DNA probes are immobilized on the Au-HAuDE. The presence of target DNA opens the hairpin structure, releases the DNAzyme sequence, and forms the hemin/G-quadruplex peroxidase-mimicking DNAzyme (HRP-DNAzyme) with hemin addition. With elevated electrode temperature, the electrocatalytic activity of HRP-DNAzyme for H2O2 reduction by oxidation of hydroquinone (used as an electron transfer mediator) can be significantly improved, resulting in an enhanced electrochemical reduction current of benzoquinone for DNA detection. With an electrode temperature of 50°C, the detection limit for target DNA measurements can be decreased ca. one magnitude compared with that at 0°C.

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.

A novel electrochemical DNA-sensing nanoplatform based on supramolecular ionic liquids grafted on nitrogen-doped graphene aerogels

In this paper, the application of supramolecular ionic liquids grafted on nitrogen-doped graphene aerogels (SIL-g-(N)GAs), a novel electrode system, for the preparation of electrochemical DNA sensing platform is proposed. The super dispersion of SIL-g-(N)GAs in water makes it an ideal candidate for biological purposes such as gene delivery. In fact, SIL-g-(N)GAs/glassy carbon working electrode (GCE) can realize the simultaneous detection of all four DNA bases in double-stranded DNA without a prehydrolysis step. On the SIL-g-(N)GAs/GCE, due to the presence of SIL on the surface of three-dimensional nitrogen-doped graphene, the anchoring of the DNA probe can be achieved by electrostatic association of SIL cations with the DNA backbone readily. So, herein we report a novel strategy for DNA hybridization without any electroactive tags or intercalators and suggest the potential applications of SIL-g-(N)GAs/GCE in the label-free electrochemical detection of DNA hybridization or DNA damage for further research. Moreover, the influence of potentially interfering substances on the determination of DNA is investigated.

Cu(phen)2]2+ acts as electrochemical indicator and anchor to immobilize probe DNA in electrochemical DNA biosensor

We demonstrate a novel protocol for sensitive in situ label-free electrochemical detection of DNA hybridization based on copper complex ([Cu(phen)2]2+, where phen = 1,10-phenanthroline) and graphene (GR) modified glassy carbon electrode. Here, [Cu(phen)2]2+ acted advantageously as both the electrochemical indicator and the anchor for probe DNA immobilization via intercalative interactions between the partial double helix structure of probe DNA and the vertical aromatic groups of phen. GR provided large density of docking site for probe DNA immobilization and increased the electrical conductivity ability of the electrode. The modification procedure was monitored by electrochemical impedance spectroscopy (EIS). Square-wave voltammetry (SWV) was used to explore the hybridization events. Under the optimal conditions, the designed electrochemical DNA biosensor could effectively distinguish different mismatch degrees of complementary DNA from one-base mismatch to noncomplementary, indicating that the biosensor had high selectivity. It also exhibited a reasonable linear relationship. The oxidation peak currents of [Cu(phen)2]2+ were linear with the logarithm of the concentrations of complementary target DNA ranging from 1 × 10−12 to 1 × 10−6 M with a detection limit of 1.99 × 10−13 M (signal/noise = 3). Moreover, the stability of the electrochemical DNA biosensor was also studied.

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).

E-DNA sensor of Mycobacterium tuberculosis based on electrochemical assembly of nanomaterials (MWCNTs/PPy/PAMAM).

Two-step electrochemical patterning methods have been employed to elaborate composite nanomaterials formed with multiwalled carbon nanotubes (MWCNTs) coated with polypyrrole (PPy) and redox PAMAM dendrimers. The nanomaterial has been demonstrated as a molecular transducer for electrochemical DNA detection. The nanocomposite MWCNTs-PPy has been formed by wrapping the PPy film on MWCNTs during electrochemical polymerization of pyrrole on the gold electrode. The MWCNTs-PPy layer was modified with PAMAM dendrimers of fourth generation (PAMAM G4) with covalent bonding by electro-oxidation method. Ferrocenyl groups were then attached to the surface as a redox marker. The electrochemical properties of the nanomaterial (MWCNTs-PPy-PAMAM-Fc) were studied using both square wave voltammetry and cyclic voltammetry to demonstrate efficient electron transfer. The nanomaterial shows high performance in the electrochemical detection of DNA hybridization leading to a variation in the electrochemical signal of ferrocene with a detection limit of 0.3 fM. Furthermore, the biosensor demonstrates ability for sensing DNA of rpoB gene of Mycobacterium tuberculosis in real PCR samples. Developed biosensor was suitable for detection of sequences with a single nucleotide polymorphism (SNP) T (TCG/TTG), responsible for resistance of M. tuberculosis to rifampicin drug, and discriminating them from wild-type samples without such mutation. This shows potential of such systems for further application in pathogens diagnostic and therapeutic purpose.

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.

A novel electrochemical DNA biosensor construction based on layered CuS-graphene composite and Au nanoparticles.

A novel CuS-graphene (CuS-Gr) composite was synthesized to achieve excellent electrochemical properties for application as a DNA electrochemical biosensor. CuS-Gr composite was prepared by a hydrothermal method, in which two-dimensional graphene served as a two-dimensional conductive skeleton to support CuS nanoparticles. A sensitive electrochemical DNA biosensor was fabricated by immobilizing single-stranded DNA (ss-DNA) labeled at the 5' end using 6-mercapto-1-hexane (HS-ssDNA) on the surface of Au nanoparticles (AuNPs) to form ssDNA-S-AuNPs/CuS-Gr, and hybridization sensing was done in phosphate buffer. Cyclic voltammetry and electrochemical impedance spectroscopy were performed for the characterization of the modified electrodes. Differential pulse voltammetry was applied to monitor the DNA hybridization using an [Fe(CN)6](3-/4-) solution as a probe. Under optimum conditions, the biosensor developed exhibited a good linear relationship between the current and the logarithm of the target DNA concentration ranging from 0.001 to 1nM, with a low detection limit of 0.1pM (3σ/S). The biosensor exhibited high selectivity to differentiate one-base-mismatched DNA and three-base-mismatched DNA. The results indicated that the sensing platform based on CuS-Gr provides a stable and conductive interface for electrochemical detection of DNA hybridization, and could easily be extended to the detection of other nucleic acids.

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.

Signal amplification for electrochemical DNA biosensor based on two-dimensional graphene analogue tungsten sulfide–graphene composites and gold nanoparticles

A novel two-dimensional graphene analogue tungsten sulfide–graphene (WS2–Gr) composite was synthesized to achieve excellent electrochemical properties for applications as DNA biosensor. Gr served as a 2D conductive skeleton that supported a highly electrolytic accessible surface area of WS2 nanocomposite which was prepared by a hydrothermal method. A sensitive electrochemical DNA biosensor was fabricated by using the WS2–Gr–chitosan composites modified glassy carbon electrode to anchor Au nanoparticles (AuNPs), which subsequently used to capture ssDNA sequences. Cyclic voltammetry and electrochemical impedance spectroscopy were carried out for the characterization of modified electrodes. Under optimum conditions, the developed biosensor showed a good linear relationship between the current value and logarithm of the target DNA concentration ranging from 0.01 to 500pM with a detection limit of 0.0023pM. The DNA biosensor exhibited excellent discrimination ability to detect one-base mismatched DNA, three-base mismatched DNA and non-complementary DNA sequence. This work described a simple strategy for the preparation of a stable and conductive interface for electrochemical detection of DNA hybridization and opened a path for the application of WS2–Gr nanocomposite in DNA electrochemical biosensing analysis.