Monitoring Of DNA Hybridization Research Articles

Electrochemical DNA hybridization signal amplification system using methylene blue and ascorbic acid

This study presents a method for real-time monitoring of heterogeneous DNA hybridization using chronoamperometry (CA). The electrochemical assay is based on methylene blue (MB)-labeled ssDNA target (tMB) hybridization with a complementary ssDNA capture probe (p) chemisorbed to a gold electrode through the Au-S linkage. Real-time monitoring of nucleic acid binding is enabled by introducing ascorbic acid (AA) during CA measurements at oxidative potential. After hybridization, the newly formed MB-labeled double stranded DNA (ptMB) oxidized AA and, as a result, shuttled the electrons directly to the electrode. AA-ptMB electrocatalytic cycle allows amplifying the signal from hybridized MB-labeled DNA through oxidation of AA. The process consists three particular processes: hybridization of tMB to the surface probe, reaction between AA and ptMB, and (c) the electron transfer from ptMB to the electrode. We have demonstrated that system is limited by the AA-ptMB redox reaction (bimolecular constant, k = 8 ± 0.1 M–1 s–1). AA-ptMB cycle is sequence-specific, thus fully developed current saturation curves can be used to calculate hybridization parameters and evaluate binding kinetics under various conditions. In addition, evaluating the initial hybridization rate allows quantifying the concentration of labeled ssDNA (tMB) in several minutes. This study provides the first report of a simple electrocatalytic cycle between MB-labeled DNA and a reducer AA for monitoring DNA hybridization in real time.

Monitoring DNA Hybridization with Organic Electrochemical Transistors Functionalized with Polydopamine

AbstractOrganic electrochemical transistors (OECTs) are finding widespread application in biosensing, thanks to their high sensitivity, broad dynamic range, and low limit of detection. An OECT biosensor requires the immobilization of a biorecognition probe on the gate, or else on the channel, through several, often lengthy, chemical steps. In this work, a fast and straightforward way to functionalize the carbon gate of a fully screen‐printed OECT by means of a polydopamine (PDA) film is presented. By chemical immobilization of an amine‐terminated single‐stranded oligonucleotide, containing the HSP70 promoter CCAAT sequence, on the PDA film, the detection of the complementary DNA strand is demonstrated. Furthermore, the specificity of the developed genosensor is assessed by comparing its response to the fully complementary strand with the one to partially complementary and noncomplementary oligonucleotides. The developed sensor shows a theoretical limit of detection (LOD) of 100 × 10−15 m and a dynamic range over four orders of magnitude.

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.

Highly sensitive electrochemical DNA sensor based on the use of three-dimensional nitrogen-doped graphene.

This paper describes a voltammetric method for sensitive determination of specific sequences of DNA. The assay is based on three-dimensional nitrogen-doped graphene (3D-NG) which, due to its excellent electrical conductivity, provides a favorable microenvironment to retain the activity of immobilized probe single-stranded DNA and also facilitates electron transfer. The free-standing 3D-NG electrode was characterized by scanning electron microscopy, Raman and X-ray photoelectron spectroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. Differential pulse voltammetry was applied to monitor DNA hybridization using Methylene Blue as an electrochemical indicator. Under optimal conditions, the peak currents (best measured at 0.28V vs. Ag/AgCl) increase linearly with the logarithm of the concentrations of ssDNA in the 10 f. to 10nM concentrations range, with a 3.5 f. detection limit (at a signal/noise ratio of 3). The biosensor exhibits good selectivity for ssDNA and can distinguish even single-base mismatches. The capability of the method was tested with spiked serum samples, and excellent reproducibility and stability is found. This indicates that the strategy is promising for use in clinical applications. Graphical abstract Three-dimensional nitrogen-doped graphene as an innovative and simple electrochemical DNA biosensor was fabricated and used in a biosensor that shows high sensitivity and good performance in the determination of target DNA in human serum samples.

An electrochemical DNA biosensor based on Oracet Blue as a label for detection of Helicobacter pylori

An innovative method of a DNA electrochemical biosensor based on Oracet Blue (OB) as an electroactive label and gold electrode (AuE) for detection of Helicobacter pylori, was offered. A single–stranded DNA probe with a thiol modification was covalently immobilized on the surface of the AuE by forming an Au–S bond. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of reduction of the OB binding to double–stranded DNA (ds–DNA). Our results showed that OB–based DNA biosensor has a decent potential for detection of single–base mismatch in target DNA. Selectivity of the proposed DNA biosensor was further confirmed in the presence of non–complementary and complementary DNA strands. Under optimum conditions, the electrochemical signal had a linear relationship with the concentration of the target DNA ranging from 0.3nmolL−1 to 240.0nmolL−1, and the detection limit was 0.17nmolL−1, whit a promising reproducibility and repeatability.

Enzymatic amplification detection of peanut allergen Ara h1 using a stem-loop DNA biosensor modified with a chitosan-mutiwalled carbon nanotube nanocomposite and spongy gold film

In this paper, a highly sensitive biosensor was constructed for peanut allergen Ara h1 detection. The biosensor was constructed by coating a glassy carbon electrode with a chitosan-mutiwalled carbon nanotube nanocomposite and then adding a spongy gold film via electro-deposition to increase the effective area. The probe switched from an “on” to an “off” state in the presence of target DNA, which detached biotin from the electrode surface. This also detached streptavidin–horseradish peroxidase (HRP-SA), which was bound to the electrode via specific interaction with biotin. The HRP-SA catalyzed chemical oxidation of hydroquinone by H2O2 to form benzoquinone, and when it was detached, electrochemical reduction of the signal of benzoquinone could be used to monitor DNA hybridization via chronoamperometry. Under optimum conditions, a wide dynamic detection range (3.91×10−17–1.25×10−15molL−1) and a low detection limit (1.3×10−17molL−1) were achieved for the complementary sequence. Furthermore, the DNA biosensor exhibited an excellent ability to discriminate between a complementary target and a one-base mismatch or non-complementary sequence. The sensor was successfully applied to Ara h1 analysis in peanuts.

Real-time monitoring of DNA hybridization for rapid detection of Vibrio cholerae O1

Effective control and treatment of the severe diarrheal disease caused by Vibrio cholerae requires rapid detection of the bacteria. For this purpose, an optical DNA sensor has been developed to detect real-time DNA hybridization after amplifying the bacterium's ctx gene. This sensor was prepared by immobilizing the 5′-biotinylated oligonucleotide probe containing the ctx sequence on the surface of a poly[methyl methacrylate] (PMMA) prism, using neutravidin. A Cy5-labelled primer for the ctx gene was used for PCR amplification. The PCR amplicon was applied to the active surface of the ctx gene chip to detect hybridization, and evanescent field light of 635 nm revealed 680 nm emission light from the Cy5-labelled DNA target hybridized to the DNA probe. It was estimated that the lower limit of detection of the Cy5-labelled DNA target was 0.2 nM, and the fluorescence signal detected by this sensor chip showed that all 12 V. cholerae O1 were positive for the ctx gene. Of the 12 bacterial strains not belonging to the cholera strains, none produced amplicons with a ctx-specific primer. Within minutes after PCR, this optical biosensor system can detect the ctx gene of V. cholerae O1 with high specificity and sensitivity.

Monitoring DNA Hybridization and Thermal Dissociation at the Silica/Water Interface Using Resonantly Enhanced Second Harmonic Generation Spectroscopy

The immobilization of oligonucleotide sequences onto glass supports is central to the field of biodiagnostics and molecular biology with the widespread use of DNA microarrays. However, the influence of confinement on the behavior of DNA immobilized on silica is not well understood owing to the difficulties associated with monitoring this buried interface. Second harmonic generation (SHG) is an inherently surface specific technique making it well suited to observe processes at insulator interfaces like silica. Using a universal 3-nitropyrolle nucleotide as an SHG-active label, we monitored the hybridization rate and thermal dissociation of a 15-mer of DNA immobilized at the silica/aqueous interface. The immobilized DNA exhibits hybridization rates on the minute time scale, which is much slower than hybridization kinetics in solution but on par with hybridization behavior observed at electrochemical interfaces. In contrast, the thermal dissociation temperature of the DNA immobilized on silica is on average 12 °C lower than the analogous duplex in solution, which is more significant than that observed on other surfaces like gold. We attribute the destabilizing affect of silica to its negatively charged surface at neutral pH that repels the hybridizing complementary DNA.

Shape-dependent surface-enhanced Raman scattering in gold–Raman-probe–silica sandwiched nanoparticles for biocompatible applications

To meet the requirement of Raman probes (labels) for biocompatible applications, a synthetic approach has been developed to sandwich the Raman-probe (malachite green isothiocyanate, MGITC) molecules between the gold core and the silica shell in gold–SiO2 composite nanoparticles. The gold–MGITC–SiO2 sandwiched structure not only prevents the Raman probe from leaking out but also improves the solubility of the nanoparticles in organic solvents and in aqueous solutions even with high ionic strength. To amplify the Raman signal, three types of core, gold nanospheres, nanorods and nanostars, have been chosen as the substrates of the Raman probe. The effect of the core shape on the surface-enhanced Raman scattering (SERS) has been investigated. The colloidal nanostars showed the highest SERS enhancement factor while the nanospheres possessed the lowest SERS activity under excitation with 532 and 785 nm lasers. Three-dimensional finite-difference time domain (FDTD) simulation showed significant differences in the local electromagnetic field distributions surrounding the nanospheres, nanorods, and nanostars, which were induced by the localized surface plasmon resonance (LSPR). The electromagnetic field was enhanced remarkably around the two ends of the nanorods and around the sharp tips of the nanostars. This local electromagnetic enhancement made the dominant contribution to the SERS enhancement. Both the experiments and the simulation revealed the order nanostars > nanorods > nanospheres in terms of the enhancement factor. Finally, the biological application of the nanostar–MGITC–SiO2 nanoparticles has been demonstrated in the monitoring of DNA hybridization. In short, the gold–MGITC–SiO2 sandwiched nanoparticles can be used as a Raman probe that features high sensitivity, good water solubility and stability, low-background fluorescence, and the absence of photobleaching for future biological applications.

Surface-modified ZnSe waveguides for label-free infrared attenuated total reflection detection of DNA hybridization

This communication presents a novel label-free biosensing method to monitor DNA hybridization via infrared attenuated total reflection (IR-ATR) spectroscopy using surface-modified ZnSe waveguides. Well-defined carboxyl-terminated monolayers were formed at H-terminated ZnSe by direct photochemical activation. Chemical activation of the acidic function was obtained by using succinimide/carbodiimide linkers. The sequential surface modification reactions were characterized by XPS and IR-ATR spectroscopy. Finally, a single stranded DNA probe with a C6-NH(2) 5' modifier was coupled to the ester-terminated surface via peptide bonding, and the hybridization of the immobilized DNA sequence with its complementary strand was directly evaluated by IR-ATR spectroscopy in the mid-infrared (MIR) spectral regime (3-20 μm) without requiring an additional label. A shift of the vibrational modes corresponding to the phosphodiester and deoxyribose structures of the DNA backbone was observed. Hence, this approach substantiates a novel strategy for label-free DNA detection utilizing mid-infrared spectroscopy as the optical sensing platform.

Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer

We report a label-free optical detection technique, called total internal reflection ellipsometry (TIRE), which can be applied to study the interactions between biomolecules and a functionalized polymer surface. Zeonor (ZR), a cycloolefin polymer with low autofluorescence, high optical transmittance and excellent chemical resistance, is a highly suitable material for optical biosensor platforms owing to the ease of fabrication. It can also be modified with a range of reactive chemical groups for surface functionalization. We demonstrate the applications of TIRE in monitoring DNA hybridization assays and human chorionic gonadotrophin sandwich immunoassays on the ZR surface functionalized with carboxyl groups. The Ψ and Δ spectra obtained after the binding of each layer of analyte have been fitted to a four-layer ellipsometric model to quantitatively determine the amount of analytes bound specifically to the functionalized ZR surface. Our proposed TIRE technique with its very low analyte consumption and its microfluidic array format could be a useful tool for evaluating several crucial parameters in immunoassays, DNA interactions, adsorption of biomolecules to solid surfaces, or assessment of the reactivity of a functionalized polymer surface towards a specific analyte.

A sensitive DNA biosensor fabricated from gold nanoparticles, carbon nanotubes, and zinc oxide nanowires on a glassy carbon electrode

We outline here the fabrication of a sensitive electrochemical DNA biosensor for the detection of sequence-specific target DNA. Zinc oxide nanowires (ZnONWs) were first immobilized on the surface of a glassy carbon electrode. Multi-walled carbon nanotubes (MWCNTs) with carboxyl groups were then dropped onto the surface of the ZnONWs. Gold nanoparticles (AuNPs) were subsequently introduced to the surface of the MWNTs/ZnONWs by electrochemical deposition. A single-stranded DNA probe with a thiol group at the end (HS–ssDNA) was covalently immobilized on the surface of the AuNPs by forming an Au–S bond. Scanning electron microscopy (SEM) and cyclic voltammetry (CV) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization by measuring the electrochemical signals of [Ru(NH 3 ) 6 ] 3+ bounding to double-stranded DNA (dsDNA). The incorporation of ZnONWs and MWCNTs in this sensor design significantly enhances the sensitivity and the selectivity. This DNA biosensor can detect the target DNA quantitatively in the range of 1.0 × 10 −13 to 1.0 × 10 −7 M, with a detection limit of 3.5 × 10 −14 M (S/N = 3). In addition, the DNA biosensor exhibits excellent selectivity, even for single-mismatched DNA detection.

Label-free and Real-Time Sequence Specific DNA Detection Based on Supramolecular Self-assembly

A new label-free, optical method was developed to detect sequence-specific DNA based on supramolecular self-assembly. A cationic phenylene ethynylene oligomer with two pairs of positively charged side chains (OPE-2) can form a J-dimer or J-aggregate with negatively charged DNA by a combination of electrostatic and hydrophobic interactions. At microM concentrations of dsDNA (number of bases in ssDNA ranges from 8 to 32), the optimum supramolecular self-assembly occurs between OPE-2 and dsDNA and is characterized by a new absorption peak emerging at 418 nm and an increase in fluorescence intensity (about 4.5-fold for dsDNA(1)). In contrast, the self-assembled complexes between OPE-2 and ssDNA are less readily formed under the same conditions. Interestingly, the induced circular dichroism (CD) signal for OPE-2/ssDNA is quite strong, likely owing to the self-assembly onto ssDNA simultaneously templating helix formation. In contrast, the induced CD signal for OPE-2/dsDNA is weak, likely because the dsDNA is in a double helix conformation, and OPE-2 associated with the dsDNA should be outside of the helix. In fact, there is a steady decrease in the induced CD signal for ssDNA with the addition of equimolar complementary ssDNA over time that allows the monitoring of DNA hybridization in real time. Introduction of mismatched bases into the target DNA sequence prevents the full hybridization between ssDNA and the target DNA. For these cases, the decrease in the induced CD signals occurs more slowly and to a lesser extent, as some of the unhybridized portions may remain in helical association with OPE-2. In view of these observed signal changes, sequence specific DNA and single nucleotide mismatch can be detected in a very simple and sensitive manner without any modification of the DNA.

A sensitive DNA biosensor fabricated with gold nanoparticles/ploy ( p-aminobenzoic acid)/carbon nanotubes modified electrode

In this work, we fabricated a sensitive electrochemical DNA biosensor for the detection of target DNA. Aminobenzoic acid (ABA) was firstly electropolymerized on the surface of the glassy carbon electrode (GCE) modified with multi-walled carbon nanotubes with carboxyl groups (MWCNTs) by cyclic voltammetry (CV). Gold nanoparticles (AuNPs) were subsequently introduced to the surface of PABA–MWNTs composite film by electrochemical deposition mode. Probe DNA was immobilized on the surface of AuNPs through Au–S bond. Scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) were used to investigate the film assembly process. Differential pulse voltammetry (DPV) was used to monitor DNA hybridization event by measurement of the intercalated adriamycin. Under the optimal conditions, the increase of reduction peak current of adriamycin was linear with the logarithm of the concentration of the complementary oligonucleotides from 1.0 × 10 −12 to 5.0 × 10 −9 M with a detection limit of 3.5 × 10 −13 M. This DNA biosensor has a good stability and reproducibility.

Potentiometric monitoring DNA hybridization with polyaniline/Nylon-6 working electrode

Electrically conducting polymers are useful in various applications such as transistors and sensors. A potentiometric pH meter has been developed using polyaniline based working electrode and with improved selectivity using ionophores, the polyaniline based potentiometer has been applied to monitor various ions mainly in environmental applications. The polyaniline working electrode can be used to monitor not only the binding of a biological ionic macromolecules on polyaniline surface but also the binding of the adsorbed macromolecule with another macromolecule. We present first how the working electrode was prepared by polymerization of aniline in Nylon-6 matrix to provide the mechanical strength and then how single strand oligonucleotide probe binds with polyaniline surface. We then present how an electrode modification with mercaptoethanol results in a surface protected against non-specific binding and then finally we present the results of monitoring the complimentary strand binding leading to the formation of the double strand DNA.

Potentiometric monitoring DNA hybridization

The usual procedure to monitor the ion exchange of small ions utilizes a potentiometer with a selective membrane as part of the working electrode. As the next step, we have applied polyaniline electrodes to the monitoring the activity macromolecular ions during DNA hybridization. Single-strand oligonucleotide (ssODN) probes were immobilized using a nucleophilic substitution reaction of the thiolated ssODN molecules with polyaniline. The anionic phosphate groups of the probe molecules also interacted with the cationic-doped polyaniline surface. Three useful findings were observed with the potentiometric experiments. First, the binding of the complimentary target molecules with the immobilized probes revealed a substantial potential change. Further, potential change was observed neither with the non-complimentary targets nor with the samples with a mutation in the sequence. The last two experiments were important for the future evaluation of the impact of medium and potential interfering compounds: anionic groups and hydrogen bonding groups in the non-complimentary samples did not cause any interactions.

The importance of the photonic mode density in bioassays based on evanescent optical waves

We have determined limits of detection (LODs) of DNA hybridization using an optical detection by two different method, i.e., surface plasmon fluorescence spectroscopy (SPFS) and optical waveguide fluorescence spectroscopy (OWFS), however, an identical sensor. Both techniques have a similar detection principle using the enhanced electromagnetic field to excite fluorophores and to monitor DNA hybridization in real-time with high sensitivity. Firstly, the detection of DNA hybridization was conducted by SPFS and the LOD value was found to be 2.0pM (2.0×10−12mol∕L). The same study was further conducted by OWFS, and a LOD of 170fM (1.7×10−13mol∕L) and 100fM (1×10−13mol∕L) was achieved for TM1 and TE0 modes, respectively, indicating that an improvement of the LOD of one order of magnitude is possible with OWFS. Furthermore, the photonic mode density at resonance of each mode was calculated, and these values are reflected in the LOD values. The present study illustrates the potential of OWFS as biosensor applications being able to detect in real-time even trace amounts of analyte.

Sensitive and selective detection of aspartic acid and glutamic acid based on polythiophene–gold nanoparticles composite

In recent years, gold nanoparticles and water-soluble fluorescent conjugated polymers are promising materials in terms of their potential applications in a variety of fields, ranging from monitoring DNA hybridization to demonstrate the interaction between proteins, or detecting diseased cell, metal ions and small biomolecular. In order to exploit some new properties of the both, many attempts have been devoted to achieve nanoparticle–polymer composite via incorporating metal nanoparticle into polymer or vice versa, however, only few of them are put into practical application. In the present paper, we utilize the “superquenching” property of AuNPs to polythiophene derivatives for detecting aspartic acid (Asp) and glutamic acid (Glu) in pure water, and discuss the factors accounting for fluorescence quenching and recovery via modulating pH. Thus an exceptionally simple, rapid and sensitive method for detecting Asp and Glu is established with a limit of detection (LOD) is 32 nM for Asp and 57 nM for Glu, the linear range of determination for Asp is 7.5 × 10 −8 M to 6 × 10 −6 M and 9.0 × 10 −8 M to 5 × 10 −6 M for Glu. The system is applied to real sample detection and the results are satisfying. Otherwise the composite is very sensitive to pH change of solution, we expect it will be possible to use as pH sensor with wide range in the future.

Gold Nanoparticle Based FRET for DNA Detection

The nanoscience revolution that sprouted throughout the 1990s is having great impact in current and future DNA detection technology around the world. In this review, we report our recent progress on gold nanoparticle based fluorescence resonance energy transfer assay to monitor DNA hybridization as well as the cleavage of DNA by nucleases. We tried to discuss a reasonable account of the science and the important fundamental work carried out in this area. We also report the development of a compact, highly specific, inexpensive and user-friendly optical fiber laser-induced fluorescence sensor based on fluorescence quenching by nanoparticles to detect single-strand DNA hybridization at femtomolar level.

Luminescent chemosensors based on semiconductor quantum dots

Semiconductor quantum dots are inorganic nanoparticles with unique photophysical properties. In particular, their huge one- and two-photon absorption cross sections, tunable emission bands and excellent photobleaching resistances are stimulating the development of luminescent probes for biomedical imaging and sensing applications. Indeed, electron and energy transfer processes can be designed to switch the luminescence of semiconductor quantum dots in response to molecular recognition events. On the basis of these operating principles, the presence of target analytes can be transduced into detectable luminescence signals. In fact, luminescent chemosensors based on semiconductor quantum dots are starting to be developed to detect small molecules, monitor DNA hybridization, assess protein-ligand complementarities, test enzymatic activity and probe pH distributions. Although fundamental research is still very much needed to understand further the fundamental factors regulating the behavior of these systems and refine their performance, it is becoming apparent that sensitive probes based on semiconductor quantum dots will become invaluable analytical tools for a diversity of applications in biomedical research.