Voltammetric determination of DNA hybridization using methylene blue and self-assembled alkanethiol monolayer on gold electrodes

An electrochemical DNA biosensor based on the recognition of single stranded DNA (ssDNA) by hybridization detection with immobilized complementary DNA oligonucleotides is presented. DNA and oligonucleotides were covalently attached through free amines on the DNA bases using N-hydroxysulfosuccinimide (NHS) and N-(3-dimethylamino)propyl-N′-ethylcarbodiimide hydrochloride (EDC) onto a carboxylate terminated alkanethiol self-assembled monolayers (SAM) preformed on a gold electrode (AuE). Differential pulse voltammetry (DPV) was used to investigate the surface coverage and molecular orientation of the immobilized DNA molecules. The covalently immobilized probe could selectively hybridize with the target DNA to form a hybrid on the surface despite the bases being attached to the SAM. The changes in the peak currents of methylene blue (MB), an electroactive label, were observed upon hybridization of probe with the target. Peak currents were found to increase in the following order: hybrid-modified AuE, mismatched hybrid-modified AuE, and the probe-modified AuE which indicates the MB signal is determined by the extent of exposed bases. Control experiments were performed using a non-complementary DNA sequence. The effect of the DNA target concentration on the hybridization signal was also studied. The interaction of MB with inosine substituted probes was investigated. Performance characteristics of the sensor are described.     

Electrochemical detection of human papilloma virus (HPV) target DNA using MB on pencil graphite electrode

The present paper describes the use of methylene blue (MB) as an electroactive label on a pencil graphite (lead) electrode (PGE) to provide a well-defined recognition interface for the detection of HPV target DNA. In order to construct the sensor, a 20-mer single strand oligonucleotide probe related to human papilloma virus (HPV) major capsid protein L1 gene was immobilized on the PGE electrode. Hybridization event between the probe and its complementary sequence was studied by measurement of MB signal accumulated on the PGE using square wave voltammetry (SWV) method. Some hybridization experiments with noncomplementary oligonucleotides were carried out to examine the selectively of the sensor to the target DNA from other DNAs related to Hepatitis C virus (HCV), fungi, and bacterial genes. Moreover, some factors affecting the function of sensor including electrode activation and probe immobilization condition were also investigated. The data showed that the constructed electrode detects the target DNA with detection limit of 1.2 ng μl^?1 and discriminates it from various DNAs originated from a wide variety of organisms.     

Label-Free Detection of DNA Hybridization Based on MnO2 Nanoparticles

Abstract

Nano-MnO₂/chitosan composite film modified glassy carbon electrode (MnO₂/CHIT/GCE) was fabricated and a DNA probe was immobilized on the electrode surface. The immobilization and hybridization events of DNA were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The EIS was applied to the label-free detection of the target DNA. The human immunodeficiency virus (HIV) gene fragment was successfully detected by this DNA electrochemical sensor. The dynamic detection range was from 2.0 × 10^?11 to 2.0 × 10^?6 mol/L, with a detection limit of 1.0 × 10^?12 mol/L.     

Gold Nanoparticle-based Layer-by-Layer Enhancement of DNA Hybridization Electrochemical Signal at Carbon Nanotube Modified Carbon Paste Electrode

Nanoporous materials have been widely applied to biosensor investigation. Recently, Guo et al. have investigated the mesoporous materials modified carbon paste electrode for rapid cTnI (cardiac troponin I) detection with enhanced sensitivity1-3. However, …     

DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes

A novel approach to construct an electrochemical DNA sensor based on immobilization of a 25 base single-stranded probe, specific to E. coli lac Z gene, onto a gold disk electrode is described. The capture probe is covalently attached using a self-assembled monolayer of 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) and mercaptohexanol (MCH) as spacer. Hybridization of the immobilized probe with the target DNA at the electrode surface was monitored by square wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. Variables involved in the sensor performance, such as the DTSP concentration in the modification solution, the self-assembled monolayers (SAM) formation time, the DNA probe drying time atop the electrode surface and the amount of probe immobilized, were optimized.

A good stability of the single- and double-stranded oligonucleotides immobilized on the DTSP-modified electrode was demonstrated, and a target DNA detection limit of 45 nM was achieved without signal amplification. Hybridization specificity was checked with non-complementary and mismatch oligonucleotides. A single-base mismatch oligonucleotide gave a hybridization response only 7 ± 3%, higher than the signal obtained for the capture probe before hybridization. The possibility of reusing the electrochemical genosensor was also tested.     

rmpM Genosensor for Detection of Human Brain Bacterial Meningitis in Cerebrospinal Fluid

Human brain bacterial meningitis is a life-threatening disease caused mainly by Neisseria meningitidis, lead to damage of the outer membrane covering (meninges) of brain or even death. The usual methods of diagnosis are either time-consuming or have some limitations. The specific rmpM (reduction-modifiable protein M) virulent gene based genosensor is more sensitive, specific, and can detect N. meningitidis directly from the patient cerebrospinal fluid in 30 min including 1-min response time. 5′-Thiol-labeled single-stranded DNA (ssDNA) probe was immobilized onto screen-printed gold electrode (SPGE) and hybridized with denatured (95 °C) single-stranded genomic DNA (ssG-DNA) for 10 min at 25 °C. The electrochemical response was measured by cyclic voltammetry, differential pulse voltammetry (DPV) and electrochemical impedance using redox indicators. The sensitivity of the genosensor was 9.5087?(μA/cm²)/ng with DPV and limit of detection was 3 ng/6 μL ssG-DNA. The immobilization of the ssDNA probe and hybridization with ssG-DNA from N. meningitidis was characterized by atomic force microscopy and Fourier transform infrared spectroscopy. The rmpM genosensor was stable for 6 months at 4 °C with 10 % loss in initial DPV current. The advantage of rmpM genosensor is to detect bacterial meningitis simultaneously in multiple patients using SPGE array during an outbreak of the disease.     

A nanocomposite consisting of plasma-polymerized propargylamine and graphene for use in DNA sensing

We report on a novel graphene-based nanoarchitecture modified with plasma-polymerized propargylamine (G-PpPG) and its application in electrochemical sensors for DNA. Films of G-PpPG were characterized by X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. The presence of graphene enhances the electrochemical activity of the films, and the high density of amino groups (deposited at a low plasma input power) on their surface assists in the immobilization of probe DNA on the water-swollen polymeric network. By contrast, the degree of hybridization of the total complementary target DNA to the probe DNA remains unchanged when G-PpPG nanofilms prepared at higher input power. No substantial non-specific adsorption of totally mismatched target DNA on the polymer films is observed because of the complete coverage of the probe DNA. The detection limit for total complementary target DNA is approximately 1.84 nmol · L⁻¹. The dynamic range extends from 0.1 to 1,000 nmol · L⁻¹. The new nanocomposite may also be used to immobilize other probe DNA sequences, and this makes the approach potentially applicable to the detection of other oligomers.

Preparing the DNA sensor made from the graphene-based nanoarchitecture modified by using PpPG (G-PpPG) includes the following processes: (a) Modifying the Au electrode with the graphene nanosheet, (b) depositing the PpPG film onto the Au electrode coated with graphene, (c) immobilizing the probe DNA onto the G-PpPG film, and (d) hybridizing the MM0 target with the G-PpPG film immobilized with P1

     

DNA/Single-Walled Carbon Nanotubes Based Fluorescence Detection of Hg2+

A new, highly selective, and sensitive technique has been developed for the detection of Hg²⁺ using singled-wall carbon nanotubes (SWNTs) and two kinds of oligonucleotides. The fluorescence of the thymine-rich single stranded DNA labeled with dye (the probe ssDNA) was effectively quenched by the SWNTs. In the presence of a target DNA (rich T-T mismatched with probe), the tightness of the DNA wrapping around the SWNTs was loosened. Since binding of Hg²⁺ turned the T-T mismatches to stable T-Hg²⁺-T base pairs, and the binding rate of DNA and the nanotube was lower than that of DNA hybridization, it induced the release of DNA molecules from the SWNTs, and this resulted in a remarkable increase of fluorescence compared to that of the DNA-SWNTs. The assay exhibited a dynamic response range for Hg²⁺ from 4.52 × 10^?8 M to 7.21 × 10^?7 M with a detection limit of 10 nM.     

Hybridization of mismatched or partially matched DNA at surfaces

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.     

DNA and PNA sensing on mercury and carbon electrodes by using methylene blue as an electrochemical label

Described here are the electrochemical parameters for MB on binding to DNA at hanging mercury drop electrode (HMDE), glassy carbon electrode (GCE), and carbon paste electrode (CPE) in the solution and at the electrode surface. MB, which interacts with the immobilized calf thymus DNA, was detected by using single-stranded DNA-modified HMDE or CPE (ssDNA-modified HMDE or CPE), bare HMDE or CPE, and double-stranded DNA-modified HMDE or CPE (dsDNA-modified HMDE or CPE) in combination with adsorptive transfer stripping voltammetry (AdTSV), differential pulse voltammetry (DPV), and alternating current voltammetry (ACV) techniques. The structural conformation of DNA and hybridization between synthetic peptide nucleic acid (PNA) and DNA oligonucleotides were determined by the changes in the voltammetric peak of MB. The PNA and DNA probes were also challenged with excessive and equal amount of noncomplementary DNA and a mixture that contained one-base mismatched and target DNA. The partition coefficient was also obtained from the signal of MB with probe, hybrid, and ssDNA-modified GCEs. The effect of probe, target, and ssDNA concentration upon the MB signal was investigated. These results demonstrated that MB could be used as an effective electroactive hybridization indicator for DNA biosensors. Performance characteristics of the sensor are described, along with future prospects.     

A nanocomposite consisting of plasma-polymerized propargylamine and graphene for use in DNA sensing

We report on a novel graphene-based nanoarchitecture modified with plasma-polymerized propargylamine (G-PpPG) and its application in electrochemical sensors for DNA. Films of G-PpPG were characterized by X-ray photoelectron spectroscopy and electrochemical impedance spectroscopy. The presence of graphene enhances the electrochemical activity of the films, and the high density of amino groups (deposited at a low plasma input power) on their surface assists in the immobilization of probe DNA on the water-swollen polymeric network. By contrast, the degree of hybridization of the total complementary target DNA to the probe DNA remains unchanged when G-PpPG nanofilms prepared at higher input power. No substantial non-specific adsorption of totally mismatched target DNA on the polymer films is observed because of the complete coverage of the probe DNA. The detection limit for total complementary target DNA is approximately 1.84 nmol?·?L^?1. The dynamic range extends from 0.1 to 1,000 nmol?·?L^?1. The new nanocomposite may also be used to immobilize other probe DNA sequences, and this makes the approach potentially applicable to the detection of other oligomers. Figure

Preparing the DNA sensor made from the graphene-based nanoarchitecture modified by using PpPG (G-PpPG) includes the following processes: (a) Modifying the Au electrode with the graphene nanosheet, (b) depositing the PpPG film onto the Au electrode coated with graphene, (c) immobilizing the probe DNA onto the G-PpPG film, and (d) hybridizing the MM0 target with the G-PpPG film immobilized with P1     

Electrochemical enzyme-linked immunoassay in a DNA hybridization sensor

In most of the currently developed electrochemical DNA hybridization sensors short single-stranded probe DNA is immobilized on an electrode and both the hybridization and detection steps are carried out on the electrode surface. Here we use a new technology in which DNA hybridization is performed on commercially available magnetic beads and detection on solid electrodes. Paramagnetic Dynabeads Oligo(dT)₂₅ (DBT) with covalently bound (dT)₂₅ probe are used for the hybridization with target DNA containing adenine stretches. Target DNA is modified with osmium tetroxide,2,2′-bipyridine (Os,bipy) and the immunogenic DNA-Os,bipy adduct is determined by the enzyme-linked immunoassay with electrochemical detection. Electroinactive 1-naphthyl phosphate is used as a substrate and the electroactive product (1-naphthol) is measured on the carbon electrodes. Alternatively Os,bipy-modified target DNA can be determined directly by measuring the osmium signal on the pyrolytic graphite electrode (PGE). A comparison between determinations of the 67-mer oligodeoxynucleotide on carbon electrodes using (a) the guanine oxidation signal, (b) direct determination of the DNA-Os,bipy adduct and (c) its electrochemical immunoassay showed immunoassay to be the most sensitive method. In combination with DBT, the DNA hybridization of long target deoxyoligonucleotides (such as 67- and 97-mers) and a DNA PCR product (226-base pairs) have been detected by immunoassay at high sensitivity and specificity.     

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.     

Impedimetic biosensor for the DNA of the human papilloma virus based on the use of gold nanosheets

The authors describe an impedimetric method for the quantitation of the DNA of the human papilloma virus (HPV) type 16. A glassy carbon electrode (GCE) was modified with gold nanosheets and is shown to be superior to a common gold disk electrode. A single-stranded 25mer oligonucleotide (ssDNA) acting as the probe DNA was immobilized via its thiolated 5′ end on both electrodes. After hybridization with target (analyte) DNA, electrochemical impedance spectra were acquired in the presence of hexacyanoferrate as a redox marker. The sensor can distinguish between complementary, non-complementary and single base pair mismatches of HPV ssDNA. At a 1 mM hexacyanoferrate concentration, the biosensors respond to target DNA in the 1 μM to 1 pM concentration range, and the detection limit is 0.15 pM. The results illustrate that the use of gold nanosheets on a GCE distinctly improves the detection and differentiation of HPV compared to using bare gold.

Open image in new window

Graphical abstract Schematic of exploiting gold nanosheets as a platform for HPV detection. The method works in the 1 μM to 1 pM HPV concentration range and has a 0.15 pM detection limit..

     

Fabrication of a Sensitive Impedance Biosensor of DNA Hybridization Based on Gold Nanoparticles Modified Gold Electrode

In this work, we report on the preparation of a simple, sensitive DNA impedance sensor. Firstly gold nanoparticles were electrodeposited on the surface of a gold electrode, and then probe DNA was immobilized on the surface of gold nanoparticles through a 5′‐thiol‐linker. Electrochemical impedance spectroscopy (EIS) was used to investigate probe DNA immobilization and hybridization. Compared to the bare gold electrode, the gold nanoparticles modified electrode could improve the density of probe DNA attachment and the sensitivity of DNA sensor greatly. The difference of electron transfer resistance (ΔR_et) was linear with the logarithm of complementary oligonucleotides sequence concentrations in the range of 2.0×10^?12 to 9.0×10^?8 M, and the detection limit was 6.7×10^?13 M. In addition, the DNA sensor showed a fairly good reproducibility and stability during repeated regeneration and hybridization cycles.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.     

DNA Hydrolysis and Voltammetric Determination of Guanine and Adenine Using Different Electrodes

Abstract

This work reports the development of a biosensor method for the label‐free detection of specific DNA sequences. In the initial phase, square wave voltammetry (SWV) was used in a comparative investigation into the electrochemical oxidation of purines (guanine and adenine) and DNA fragments at various electrode surfaces: carbon paste (CPE), glassy carbon electrode (GCE), and gold (AuE). Relative to the carbon electrodes, an approximate 4.0‐fold, 6.0‐fold, and 3.25‐fold increase in the anodic response was observed when guanine, adenine, and hydrolyzed DNA, respectively, were measured on the AuE. It was shown that the guanine and adenine bases could be successfully determined by use of SWV for a deoxyribonucleic acid sample following acid hydrolysis. This label‐free detection of hydrolyzed DNA on gold electrodes has significant advantages over methods using existing carbon electrode materials because of its higher sensitivity and the potential applicability of microfabrication techniques for the production of the requisite gold electrodes.

In another phase of development, the times and conditions for DNA hydrolysis and purine release were investigated. It was shown that under optimal conditions, trace levels of the purine bases could be readily detected following 20 min of hydrolysis at room temperature. The proposed method can be used to estimate the guanine and adenine contents in DNA with in a linear range of 5–30 ng ml^?1.

Finally, when appropriate probe sequences were first adsorbed on the surface of the screen‐printed gold electrode (SPGE), this electrochemical biosensor could be used to specifically detect sequences from ss corona virus aviair following hybridization and hydrolysis reactions on the sensor surface. No enhancement of the voltammetric response was observed when the sensor was challenged with a non‐complementary DNA sequence.     

One Mismatch Detection of DNA Hybridization Using Fluorescence Polarization

Abstract

The potential of fluorescent polarization analysis as a method for detection of mismatch DNA hybridization was investigated. The dependency of DNA hybridization rate on salt concentration was surveyed. In greater than 0.1 M NaCl, the hybridization of probe and target DNA proceeds rapidly and the reaction is complete within 3 min. Furthermore, the hybridization of probe DNA and one mismatch target DNAs was investigated. It was successfully shown that even one mismatch could be detected using fluorescence polarization analysis if the mismatch position was on the base that pairs with the probe DNA at the 5′ terminus where fluorescein isothiocyanate (FITC) is attached.     

Direct detection and discrimination of double-stranded oligonucleotide corresponding to hepatitis C virus genotype 3a using an electrochemical DNA biosensor based on peptide nucleic acid and double-stranded DNA hybridization

Development of an electrochemical DNA biosensor for the direct detection and discrimination of double-stranded oligonucleotide (dsDNA) corresponding to hepatitis C virus genotype 3a, without its denaturation, using a gold electrode is described. The electrochemical DNA sensor relies on the modification of the gold electrode with 6-mercapto-1-hexanol and a self-assembled monolayer of 14-mer peptide nucleic acid probe, related to the hepatitis C virus genotype 3a core/E1 region. The increase of differential pulse voltammetric responses of methylene blue, upon hybridization of the self-assembled probe with the target ds-DNA to form a triplex is the principle behind the detection and discrimination. Some hybridization experiments with non-complementary oligonucleotides were carried out to assess whether the developed DNA sensor responds selectively to the ds-DNA target. Diagnostic performance of the biosensor is described and the detection limit was found to be 1.8 × 10⁻¹² M in phosphate buffer solution, pH 7.0. The relative standard deviation of measurements of 100 pM of target ds-DNA performed with three independent probe-modified electrodes was 3.1%, indicating a remarkable reproducibility of the detection method.     

Label-Free Electrochemiluminescent Determination of DNA Using Luminol and Hemin Functionalized Nanoparticles

Luminol and hemin dual-functionalized silica nanoparticles were synthesized using a typical reverse water-in-oil microemulsion protocol. The obtained nanoparticles were further characterized by transmission electron microscopy, scanning electron microscopy, atomic absorption spectrometry, chemiluminescence, and electrochemiluminescence. The results indicated that the luminol and hemin dual-functionalized silica nanoparticles exhibited significantly higher chemiluminescence and electrochemiluminescence intensities than those of luminol functionalized silica nanoparticles due to the catalytic effect of hemin on the chemiluminescence and electrochemiluminescence of luminol. Furthermore, a simple and sensitive label-free electrochemiluminescence DNA biosensor was developed based on the chitosan modified luminol and hemin dual-functionalized silica nanoparticles and a single-stranded DNA probe. The chitosan modified luminol and hemin dual-functionalized silica nanoparticles were immobilized on the surface of an indium-doped tin oxide electrode and the single-stranded DNA probe was immobilized on the surface of the nanoparticles through electrostatic interactions between single-stranded DNA and chitosan, which allowed hybridization with the target DNA sequences. The hybridization events were evaluated by electrochemiluminescence, and only the complementary sequence formed double-stranded DNA with the DNA probe to give strong electrochemiluminescence signals. Finally, the electrochemiluminescence intensity was found to be linearly related to the concentration of the complementary sequence at concentrations from 1.0?×?10^?12 to 1.0?×?10^?6?mol·L^?1 with a detection limit of 5.0?×?10^?13?mol·L^?1.