Ultrasensitive Electrochemical DNA Biosensor Research Articles

An ultrasensitive electrochemical DNA biosensor based on graphene/Au nanorod/polythionine for human papillomavirus DNA detection

An ultrasensitive electrochemical DNA biosensor for human papillomavirus (HPV) detection was developed by electrochemical impedance spectroscopy and differential pulse voltammetry. A capture probe was immobilized on a glassy carbon electrode modified with graphene/Au nanorod/polythionine (G/Au NR/PT). Two auxiliary probes were designed and used to long-range self-assemble DNA nanostructure. The target DNA can connect DNA structure to the capture probe on the electrode surface. [Ru(phen)3]2+ was selected as a redox indicator for amplifying electrochemical signal significantly. Enhanced sensitivity was obtained through combining the excellent electric conductivity of G/Au NR/PT architecture and the long-range self-assembly DNA nanostructure with the multi-signal amplification. The DNA biosensor displayed excellent performance for HPV DNA detection over the range from 1.0×10–13 to 1.0×10–10mol/L with a detection limit of 4.03×10–14mol/L. Furthermore, the proposed method can also be used for the detection of HPV DNA in human serum samples and provides a potential application of DNA detection in clinic research.

An electrochemical biosensor for the sensitive detection of specific DNA based on a dual-enzyme assisted amplification

In this work, a simple and ultrasensitive electrochemical DNA biosensor was developed based on the horseradish peroxidase (HRP)-catalyzed electrochemical process and exonuclease III (Exo III)-assisted target recycling amplification strategy. A biotin tag labeled molecular beacon (MB) with hairpin structure was ingeniously designed and assembled on an electrode as a recognition element. The 3’ overhang end of the MB labeled with biotin would bind streptavidin (SA)-HRP to give a strong initial signal. Upon target DNA sensing, the MB probe would be stepwise removed by the Exo III accompanied by the release of the target DNA for the successive hybridization and cleavage process. Simultaneously, numerous biotin labeled mononucleotides were liberated, leading to a decrease in the binding of the biotin and SA-HRP polymer on the electrode. As a result, the amplified electrochemical current significantly decreased. Because of the autocatalytic target recycling amplification and the HRP-catalyzed electrochemical process, the dual enzyme-based strategy provided an ultrasensitive approach (down to the 10fM level) for the electrochemical detection of DNA, and discriminated mismatched DNA from perfectly matched target DNA, which has great potential for early diagnosis in gene-related diseases.

A label-free ultrasensitive electrochemical DNA sensor based on thin-layer MoS2 nanosheets with high electrochemical activity

A label-free and ultrasensitive electrochemical DNA biosensor, based on thin-layer molybdenum disulfide (MoS2) nanosheets sensing platform and differential pulse voltammetry detection, is constructed in this paper. The thin-layer MoS2 nanosheets were prepared via a simple ultrasound exfoliation method from bulk MoS2, which is simpler and no distortion compared with mechanical cleavage and lithium intercalation. Most importantly, this procedure allows the formation of MoS2 with enhanced electrochemical activity. Based on the high electrochemical activity and different affinity toward ssDNA versus dsDNA of the thin-layer MoS2 nanosheets sensing platform, the tlh gene sequence assay can be performed label-freely from 1.0×10−16M to 1.0×10−10M with a detection limit of 1.9×10−17M. Without labeling and the use of amplifiers, the detection method described here not only expands the application of MoS2, but also offers a viable alternative for DNA analysis, which has the priority in sensitivity, simplicity, and costs. Moreover, the proposed sensing platform has good electrocatalytic activity, and can be extended to detect more targets, such as guanine and adenine, which further expands the application of MoS2.

Ultrasensitive electrochemical DNA biosensor by exploiting hematin as efficient biomimetic catalyst toward in situ metallization

In this work, we presented a novel signal amplification approach to construct an electrochemical DNA biosensor for the ultrasensitive determination of sequence-specific DNA by exploiting hematin as biomimetic catalyst toward in situ metallization. Briefly, thiolated peptide nucleic acid (PNA) probes were firstly immobilized onto gold electrode through the formation of self-assembled monolayer (SAM) and then hybridization was accomplished in the ensuing step. After that, hematin molecules were introduced to the hybridized PNA/DNA heteroduplexes by employing phosphate–zirconium–carboxylate coordination chemistry. Next, the attached hematin molecules acted as catalyst in accelerating the reduction of silver ions in the presence of catechol, leading to the in situ deposition of silver particles onto the electrode. Finally, the deposited silver particles were electrochemically stripped into KCl solution and measured by square wave voltammetry (SWV). Under optimal conditions, the hematin-based electrochemical DNA biosensor presented a good linear relationship between the stripping peak currents and logarithm of single-stranded DNA (ssDNA) concentrations in the range from 0.1fM to 0.1nM with a low detection limit of 62.41aM, and it rendered satisfactory analytical performance for the determination of ssDNA in serum samples. Furthermore, it exhibited good reproducibility and stability, meanwhile, it also showed excellent specificity toward single-nucleotide polymorphism (SNP). Therefore, the hematin-based signal amplification approach has great potential in clinical applications and is also suitable for quantification of biomarkers at ultralow level.

Ultra-sensitive electrochemical DNA biosensor based on signal amplification using gold nanoparticles modified with molybdenum disulfide, graphene and horseradish peroxidase

We have developed an ultra-sensitive electrochemical DNA biosensor by assembling probe ssDNA on a glassy carbon electrode modified with a composite made from molybdenum disulfide, graphene, chitosan and gold nanoparticles. A thiol-tagged DNA strand coupled to horseradish peroxidase conjugated to AuNP served as a tracer. The nanocomposite on the surface acts as relatively good electrical conductor for accelerating the electron transfer, while the enzyme tagged gold nanoparticles provide signal amplification. Hybridization with the target DNA was studied by measuring the electrochemical signal response of horseradish peroxidase using differential pulse voltammetry. The calibration plot is linear in the 5.0 × 10−14 and 5.0 × 10−9 M concentration range, and the limit of detection is 2.2 × 10−15 M. The biosensor displays high selectivity and can differentiate between single-base mismatched and three-base mismatched sequences of DNA. The approach is deemed to provide a sensitive and reliable tool for highly specific detection of DNA.

Ultrasensitive electrochemical supersandwich DNA biosensor using a glassy carbon electrode modified with gold particle-decorated sheets of graphene oxide

We describe a supersandwich type of electrochemical DNA biosensor based on the use of a glassy carbon electrode (GCE) modified with reduced graphene oxide (rGO) sheets that are decorated with gold nanoparticles (Au NPs). Thiolated capture DNA (probe DNA) was covalently linked to the Au NPs on the surface of the modified GCE via formation of Au-S bonds. In presence of target DNA, its 3′ terminus hybridizes with capture probe and the 5′ terminus hybridizes with signal probe labeled with Methylene Blue (MB). On increasing the concentration of target DNA, hybridization between signal probe and target DNA results in the formation of three different DNA sequences that form a supersandwich structure. The signal intensity of MB improves distinctly with increasing concentrations of target DNA in the sample solution. The assembling process on the surface of the electrode was studied by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS). Differential pulse voltammetry (DPV) was used to monitor the hybridization event by measuring the changes in the peak current for MB. Under optimal conditions, the peak currents in DPV for MB linearly increase with the logarithm of target DNA concentration in the range from 0.1 μM to1.0 fM, with a detection limit of 0.35 fM (at an signal/noise ratio of 3). This biosensor exhibits good selectivity, even over single-base mismatched target DNA.

Sub-femtomolar DNA detection based on layered molybdenum disulfide/multi-walled carbon nanotube composites, Au nanoparticle and enzyme multiple signal amplification

A novel 2-dimensional graphene analog molybdenum disulfide/multi-walled carbon nanotube (MoS2/MWCNT) was synthesized by a simple hydrothermal method to achieve excellent electrochemical properties. An ultrasensitive electrochemical DNA biosensor was subsequently constructed by assembling a thiol-tagged DNA probe on a MoS2/MWCNT and gold nanoparticle (AuNP)-modified electrode that has already been coupled with glucose oxidase (GOD). In this work, GOD was used as a redox marker. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the modification of GOD and AuNPs provided multiple signal amplification for electrochemical biosensing. The multiple signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to 0.79fM with a linear range from 10fM to 107fM, and appeared high selectivity to differentiate three-base mismatched DNA and one-base mismatched DNA. The developed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, and would open new opportunities for sensitive detection of other biorecognition events.

Label-Free and Ultrasensitive Electrochemical Detection of Nucleic Acids Based on Autocatalytic and Exonuclease III-Assisted Target Recycling Strategy

In this work, a very simple, label-free, isothermal, and ultrasensitive electrochemical DNA biosensor has been developed on the basis of an autocatalytic and exonuclease III (Exo III)-assisted target recycling amplification strategy. A duplex DNA probe constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon is ingeniously designed and assembled on the electrode as recognition element. Upon sensing of the analyte nucleic acid, the strand of molecular beacon in the duplex DNA probe could be stepwise removed by Exo III accompanied by the releasing of target DNA and autonomous generation of new secondary target DNA fragment for the successive hybridization and cleavage process. Simultaneously, numerous quadruplex-forming oligomers are liberated and folded into G-quadruplex-hemin complexes with the help of K(+) and hemin on the electrode surface to give a remarkable electrochemical response. Because of this autocatalytic target recycling amplification and the specifically catalyzed formation of G-quadruplex-hemin complexes, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the 10 fM level, can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases. It further could be developed as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.

Enzyme-Free and Label-Free Ultrasensitive Electrochemical Detection of Human Immunodeficiency Virus DNA in Biological Samples Based on Long-Range Self-Assembled DNA Nanostructures

Biosensors based on nanomaterials have been used for detection of various biological molecules with high sensitivity and selectivity. Herein, we developed a simple and ultrasensitive electrochemical DNA biosensor using long-range self-assembled DNA nanostructures as carriers for signal amplification, which can achieve an impressive detection limit of 5 aM human immunodeficiency virus (HIV) DNA even in complex biological samples. In this study, we designed two auxiliary probes. A cascade of hybridization events between the two auxiliary probes can lead to long-range self-assembly and form micrometer-long one-dimensional DNA nanostructures. In the presence of target DNA, each copy of the target can act as a trigger to connect a DNA nanostructure to a capture probe on the electrode surface. Then, a great amount of redox indicator [Ru(NH(3))(6)](3+) can be electrostatically bound to the DNA nanostructures and eventually result in significantly amplified electrochemical signals.

Triplex signal amplification for electrochemical DNA biosensing by coupling probe-gold nanoparticles–graphene modified electrode with enzyme functionalized carbon sphere as tracer

An ultrasensitive electrochemical DNA biosensor was constructed by assembling probe labeled gold nanoparticles (ssDNA–AuNP) on electrochemically reduced graphene oxide (ERGO) modified electrode with thiol group tagged (GT) DNA strand (d(GT)29SH) and coupling with horseradish peroxidase (HRP) functionalized carbon sphere (CNS) as tracer. The heteronanostructure formed on the biosensor surface appeared relatively good conductor for accelerating the electron transfer, while the HRP tagged CNS provided dual signal amplification for electrochemical biosensing. The triplex signal amplification strategy produced an ultrasensitive electrochemical detection of DNA down to attomolar level (5aM) with a linear range of 5 orders of magnitude (from 1×10−17M to 1×10−13M), and appeared high selectivity to differentiate single-base mismatched and three-base mismatched sequences of DNA. The proposed approach provided a simple and reliable method for DNA detection with high sensitivity and specificity, indicating promising application in bioanalysis and biomedicine.

A simple and ultrasensitive electrochemical DNA biosensor based on DNA concatamers

A simple, ultrasensitive and selective electrochemical DNA biosensor based on DNA concatamers is described, which can detect as low as 100 aM target DNA even in complex samples.