Electrochemical DNA Biosensor For Detection Research Articles

Au/NG/SWCNTs/FTO-glass modified electrode based electrochemical biosensor for DNA detection

An easy, quick, and efficient detection of Multidrug Resistance (MDR) of cancer cells at the clinical level is the need of the hour for the effective treatment of leukemia patients. In this study, we reported carbon nanomaterials-based biosensors based on Au nanoparticles (Au-NPs) decorated on Nitrogen-doped Graphene (NG) and Single-Walled Carbon Nanotubes (SWCNTs) for effective, efficient, and sensitive detection of affected DNA. Three Au/NG/SWCNT-based Modified Electrode (ME) were made containing 2, 4, and 6 mg SWCNTs. The thiol group attached at the 5′end was immobilized at ME due to a strong bond between the Au-NPs and thiol group. NG/SWCNTs enhanced the performance of the composite due to its better sensitivity and fast electron transport. Electrochemical Impedance Spectroscopy (EIS), Cyclic Voltammetry (CV), and Differential Potential Voltammetry (DPV) analysis were performed to monitor the performance of the sensor after the hybridization of target DNA onto the capture probe on the modified electrode surface. Different target DNA sequences were used to check the selectivity and sensitivity of the fabricated biosensor. The selectivity performance of prepared DNA sensors was also proved to be good shown by DPV results. The findings of this study provide insights for developing new highly sensitive, selective, and cost-effective portable biosensors for MDR gene detection, which will enable the early diagnosis of cancer more effective at the clinical level. Compared with previous DNA sensors with oligonucleotides directly incorporated on carbon electrodes, this carbon nanotube-based assay with its large surface area and good charge-transport characteristics dramatically increased DNA attachment quantity and complementary DNA detection sensitivity.

The Optimization of a Label-Free Electrochemical DNA Biosensor for Detection of Sus scrofa mtDNA as Food Adulterations

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box–Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5–1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.

An ultrasensitive and specific electrochemical biosensor for DNA detection based on T7 exonuclease-assisted regulatory strand displacement amplification

An electrochemical biosensor for determination of DNA is developed based on T7 exonuclease-assisted regulatory strand displacement dual recycling signal amplification strategy. First, the hairpin probe recognized and bound the target DNA to form a double strand nucleotide structure, and then the T7 exonuclease was introduced. After be digested by T7 exonuclease, the target DNA was released and entered the next cycle of T7 exonuclease-assisted recycle amplification, while accompanied by a large number of mimic targets (output DNAs) into another cycle. Second, the mimic target reacted with double-chain substrated DNA (CP) by a regulated toehold exchange mechanism, yielding the product complex of detection probes with the help of assisted DNA (S). Finally, after many cycles, a large number of detection probes were produced for binding numerous streptavidin-alkaline phosphatases. The electrochemical biosensor showed very high sensitivity and selectivity with a dynamic response ranged from 0.1 fM to 10 pM with a detection limit of 31.6 aM. Furthermore, this proposed biosensor was successfully applied to the detection of target DNA in 20% diluted serum. The developed strategy has been demonstrated to have the potential for application in molecular diagnostics.

A Miniaturized Device for Performing PCR, Integrated with an Electrochemical DNA Biosensor for Detection of Corynebacterium Diphtheriae

Introduction Corynebacterium diphtheriae by secreting harmful toxin causes lethal disease called diphtheria. It occurs mainly in underdeveloped areas of the world with low vaccination rate [1]. Rapid detection of this pathogen is crucial to prevent serious health complications of the patient and spreading of the epidemic. As an alternative to time-consuming phenotypic analysis, a device has been designed, which allows the fast detection of the pathogen based on biological interactions at the molecular level, which reduces the risk of error. This device consists of a microfluidic system for performing a fast polymerase chain reaction (PCR), which is integrated with a DNA biosensor. It enables fast and unambiguous detection of bacteria in the sample, therefore it can be potentially used as a point-of-care device. Microfluidic PCR device The concept of rapid bacterial analysis is based on the amplification of a DNA fragment specific for a pathogen followed by its fast detection with e.g. biosensor. A miniaturized system for carrying out PCR was developed. The geometry of the microchannels was designed using CAD software and then micromilled in poly(methyl methacrylate). The microsystem was connected with miniaturized peristaltic pump and with a valve to enable automatic introduction, circulation and removal of the solution from the system. Moreover, a set of six heaters was used to divide the microsystem into three temperature zones. One cycle of the solution in a microchip corresponded with one reaction cycle. Temperatures of three zones were measured and controlled by resistance temperature sensor and set to the suitable values for following reaction steps: denaturation, annealing and elongation. Using the microsystem, any number of reaction cycles could be carried out and peristaltic pump enabled the medium flow in wide range (4 - 150µL/min). A number of both standard and asymmetric PCR reactions were carried out in the microchip using various polymerases, of which the Phire polymerase gave the most promising results. The products were analyzed by electrophoretic techniques and compared with the results of reactions carried out in thermocyclers. The ultimate goal is direct detection of the reaction product using a DNA biosensor. DNA biosensor An electrochemical DNA biosensor has been developed as detection part of a device. Its receptor layer was based on a hairpin like DNA probe labeled with methylene blue (MB). After the asymmetric PCR reaction the hybridization process took place which results in formation of double stranded DNA. The changes in hairpin like structure influenced the registered current and the same the biosensor response, proportional to PCR product concentration [2]. Results and Conclusions The miniaturized device consisting of a microchip with designed channel geometry, connected to the pump and valve was constructed. It enabled to automatically control the flow of a solution in a microchip and to carry out any number of reaction cycles. The reaction mixture changed its temperature rapidly when it flowed between different zones of the microchip, which contributed to a significant reduction in the reaction time. Asymmetric PCR was successfully carried out using the designed device in just 15 minutes. The electrochemical genosensor enabled detection of ssDNA, and its electrochemical signal was directly proportional to the concentration of the analyte in 0.0125-0.25µmol/L range. Studies on the effects of different factors, such as probe sequence, temperature, magnesium ion concentration and addition of dimethyl sulfoxide on electrochemical signal of a biosensor have also been performed.The constructed microchip allowed to significantly shorten the PCR reaction time, when compared with the thermocycler. Integration of microPCR with the genosensor might results in construction of fully automatized portable device enabling rapid and reliable pathogen detection. This type of instrument could be used in medical diagnostics as the point-of-care device contributing to the rapid detection of the disease, hence preventing the spread of the epidemic [3].

Electrochemical DNA biosensor for detection of pork (Sus scrof) using screen printed carbon-reduced graphene oxide electrode

Electrochemical DNA biosensor for detection of pork (Sus scrof) using screen printed carbon-reduced graphene oxide electrode

Influence of Graphene Oxide Concentration when Fabricating an Electrochemical Biosensor for DNA Detection.

We have investigated the influence exerted by the concentration of graphene oxide (GO) dispersion as a modifier for screen printed carbon electrodes (SPCEs) on the fabrication of an electrochemical biosensor to detect DNA hybridization. A new pretreatment protocol for SPCEs, involving two successive steps in order to achieve a reproducible deposition of GO, is also proposed. Aqueous GO dispersions of different concentrations (0.05, 0.1, 0.15, and 0.2 mg/mL) were first drop-cast on the SPCE substrates and then electrochemically reduced. The electrochemical properties of the modified electrodes were investigated after each modification step by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS), while physicochemical characterization was performed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), and Raman spectroscopy. Finally, the sensing platform was obtained by the simple adsorption of the single-stranded DNA probe onto the electrochemically reduced GO (RGO)-modified SPCEs under optimized conditions. The hybridization was achieved by incubating the functionalized SPCEs with complementary DNA target and detected by measuring the change in the electrochemical response of [Fe(CN)6]3–/4– redox reporter in CV and EIS measurements induced by the release of the newly formed double-stranded DNA from the electrode surface. Our results showed that a higher GO concentration generated a more sensitive response towards DNA detection.

A ratiometric electrochemical DNA biosensor for detection of exosomal MicroRNA

The detection of exosomal microRNAs (miRNAs) derived from cancer cells with sensitive and selective methods has stimulated increasing interest due to its potential utility in the application of tumor diagnosis. Here, we developed a ratiometric electrochemical DNA biosensor based on a locked nucleic acid (LNA)-modified “Y” shape-like structure for the detection of exosomal miRNA-21 (miR-21). When miR-21 is present, the LNA-assisted strand displacement reaction on the “Y” shape-like structure is activated, leading to a structure change and augmentation of the signal ratio, which reflects the different distances between the electrode surface and two electroactive molecules labeled on the “Y” shape-like structure. With this dual signal ratiometric method, the biosensor shows high accuracy and sensitivity with a limit of detection as low as 2.3 fM. Moreover, because of the logarithm of the signal ratio displays a linear relationship with the logarithm of the miR-21 concentration, the biosensor is stable enough to be used in the detection of miR-21 in MCF-7 cell-derived exosomes. In addition, the biosensor shows good selectivity even in the detection of even a single base-mismatched target due to the LNA-assisted strand displacement reaction. Notably, the sensor is both regenerative and robust. In brief, the high sensitivity and selectivity, combined with the low cost of the glassy carbon electrode, make this biosensor a promising tool for the development of point-of-care testing in cancer.

A triple-amplification differential pulse voltammetry for sensitive detection of DNA based on exonuclease III, strand displacement reaction and terminal deoxynucleotidyl transferase

In this research, a sensitive and specific electrochemical biosensor for DNA detection was constructed. The highly sensitivity of this biosensor is due to the exploitation of exonuclease III-assisted double recycling and toehold-mediated strand displacement recycling to achieve the target triple recycling amplification, thus generating a large amount of Y-shaped DNA structures. Combination with a terminal deoxynucleotidyl transferase (TDT)-mediated cascaded signal amplification strategy can catalyze the repetitive incorporation of biotin-dUTP to the 3'-OH of the Y-shaped DNA. Via biotin-streptavidin interaction, multiple streptavidin-alkaline phosphatases were conjugated to the surface of an Au electrode and generated a sharply increasing electrochemical signal in a 1-naphthyl phosphate (1-NP) solution. In this method, an impressive detection limit of 0.05 fM was obtained, presenting outstanding selectivity with a dynamic response scope between 0.1 fM and 1 nΜ. Thus, the designed biosensor opens an avenue for DNA detection in clinical molecular diagnostics, pathogen detection, gene therapy, food safety and environmental monitoring.

A new genosensor for meningococcal meningitis diagnosis using biological samples

In this work, a new electrochemical biosensor for DNA detection of bacterial meningitis is proposed. The system is based on specific DNA fragments from the Neisseria meningitidis genome as a probe incorporated on graphite electrodes modified with poly(4-aminophenol). Detection of a complementary oligonucleotide sequence, a specific 710-base pair amplicon, and the genomic DNA of bacteria was carried out by differential pulse voltammetry, using ethidium bromide as an electroactive indicator of hybridization. The complementary oligonucleotide and the genomic DNA of Neisseria meningitidis were quantified by the genosensor, showing detection limits of 0.6 ng μL−1 and about 6 ng μL−1, respectively. Morphological differences were observed between hybridized and unhybridized surfaces by atomic force microscopy. The biosensor showed high selectivity, discriminating non-specific targets, and high stability retaining over 98% of its original activity after 120 days of storage. The bioelectrode was effective in discriminating the genomic DNA in samples with human serum without significant interference, proving to be an interesting platform for meningococcal meningitis diagnosis.

Ultrasensitive electrochemical biosensing platform based on spherical silicon dioxide/molybdenum selenide nanohybrids and triggered Hybridization Chain Reaction

An ultrasensitive sandwich-type electrochemical biosensor for DNA detection is developed based on spherical silicon dioxide/molybdenum selenide (SiO2@MoSe2) and graphene oxide–gold nanoparticles (GO–AuNPs) hybrids as carrier triggered Hybridization Chain Reaction (HCR) coupling with multi-signal amplification. The proposed sensoring assay utilizes a spherical SiO2@MoSe2/AuNPs as sensing platform and GO–AuNPs hybrids as carriers to supply vast binding sites. H2O2+HQ system is used for DNA detection and HCR as the signal and selectivity enhancer. The sensor is designed in sandwich type to increase the specificity. As a result, the present biosensor exhibits a good dynamic range from 0.1fM to 100pM with a low detection limit of 0.068fM (S/N=3). This work shows a considerable potential for quantitative detection of DNA in early clinical diagnostics.

Electrochemical DNA biosensor for detection of DNA damage induced by hydroxyl radicals

A simple electrochemical DNA biosensor based on a glassy carbon electrode (GCE) was prepared by adsorbing double-stranded DNA (dsDNA) onto the GCE surface and subsequently used for the detection of dsDNA damage induced by hydroxyl radicals. Investigation of the mutual interaction between hydroxyl radicals and dsDNA was conducted using a combination of several electrochemical detection techniques: square-wave voltammetry for direct monitoring the oxidation of dsDNA bases, and cyclic voltammetry and electrochemical impedance spectroscopy as indirect electrochemical methods making use of the redox-active indicator [Fe(CN)6]4−/3−. Hydroxyl radicals were generated electrochemically on the surface of a boron-doped diamond electrode and chemically (via the Fenton's reaction or the auto-oxidation of Fe(II)). The extent of dsDNA damage by electrochemically generated hydroxyl radicals depended on the current density applied to the generating electrode: by applying 5, 10, and 50mAcm−2, selected relative biosensor responses decreased after 3min incubation from 100% to 38%, 27%, and 3%, respectively. Chemically generated hydroxyl radicals caused less pronounced dsDNA damage, and their damaging activity depended on the form of Fe(II) ions: decreases to 49% (Fenton's reaction; Fe(II) complexed with EDTA) and 33% (auto-oxidation of Fe(II); Fe(II) complexed with dsDNA) were observed after 10min incubation.

An ultrasensitive and label-free electrochemical DNA biosensor for detection of DNase I activity

An ultrasensitive and label-free DNA biosensor was developed to detect deoxyribonuclease I activity based on electrochemical method.

Electrochemical biosensor for silver ions based on amplification of DNA–Au bio–bar codes and silver enhancement

A highly sensitive and selective electrochemical DNA biosensor for detection of Ag+ was developed based on amplification of DNA–Au bio–bar codes and silver enhancement. Cytosine-rich single stranded DNA oligonucleotide (sub–DNA) was immobilized on the surface of the gold electrode via Au–S chemical interaction. In the presence of Ag+, with the interaction of cytosine–Ag+–cytosine (C–Ag+–C), cytosine-rich DNA oligonucleotide probes-labeled AuNPs have a hybridization with sub–DNA and then form a duplex-like structure, following the AuNPs-catalyzed silver enhancement. The whole detection procedure of Ag+ based on the developed biosensor was evaluated by electrochemical techniques. Owing to the hybridization of sandwich assay format and distinct electrochemical signal amplification of silver enhancement, the proposed biosensors exhibited a good analytical performance for sensitive transduction of Ag+ recognition with a wide detection linear range from 5pM to 50μM, and a low detection limit of 2pM. The developed biosensor was highly selective and its practical applicability in real water samples was also tested with satisfactory results.

Fabrication of Porcine DNA Biosensor Based on Ruthenium Bipyridine Complex

Electrochemical DNA biosensor for detection of porcine oligonucleotides based on ruthenium (II) as label redox complex has been developed. The system of biosensor is based on the modified screen printed carbon electrode (SPE) with goldnanoparticles (AuNPs) as transducer immobilized with poly(n-butylacrylate-N-acryloxysuccinimide) microsphere and porcine DNA probe sequences was attached onto it via the covalent bond. The ruthenium(II) complex, [Ru(bpy)2PIP]2+ PIP = 2-phenylimidazo[4,5-f[1,10-phenanthroline] intercalator has been been used to determine prcine DNA. The biosensor was measured and optimized by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The interaction of [Ru(bpy)2PIP]2+ with various DNA with different sequences, i) single stranded probe DNA, ii) after hybridization with its complementary DNA and iii) after hybridization with mismatch complementary DNA were studied. The results indicated that the interaction of [Ru(bpy)2PIP]2+ with hybridized complementary DNA gave the highest response. Thus, development of porcine DNA biosensor and influences of many factors such as [Ru(bpy)2PIP]2+ complex and DNA probe concentration, DNA probe immobilization and hybridization time, pH, ionic strength, buffer concentration and temperatures were also studied to evaluate the performance of biosensor. The concentration of [Ru(bpy)2PIP]2+ complex and DNA probe were found to be optimal at 50 µM and 2 µM, respectively. The optimal time for DNA probe immobilization and hybridization were 7 hours and 60 minutes, accordingly. The optimal condition of pH, ionic strength, buffer concentration and temperatures were at pH 7.0, 1.0 M NaCl, 0.05 M of Na-phosphate buffer and 25 °C, respectively. The linear range of different concentration complementary DNA was within the range between 1.0 x 10-13 M to 1.0 x 10-8 M. This study was first reported the used of [Ru(bpy)2PIP]2+ for detection of porcine oligonucleotides.

Development of an electrochemical DNA biosensor for detection of specific Mycobacterium tuberculosis sequence based on poly(L-glutamic acid) modified electrode

An electrochemical DNA biosensor was developed by avidin-biotin interaction of a biotinylated probe and avidin-attached, poly(L-glutamic) acid coated pencil graphite electrode (PGA/PGE) for detection of specific Mycobacterium tuberculosis DNA sequence. The discrimination of fully complementary hybridization and mismatch hybridization was carried out by electrochemical reduction current of Meldola’s Blue (MDB) in square wave voltammetry (SWV). The calibration graph of the DNA biosensor was linear between 1.5–12.5 nM and the detection limit was calculated as 1.3 nM. The proposed biosensor successfully discriminated short and long oligonucleotides related to DNA sequence of Mycobacterium tuberculosis in optimal condition.

An electrochemical biosensor for DNA detection based on tungsten disulfide/multi-walled carbon nanotube composites and hybridization chain reaction amplification

We report the preparation and characterization of tungsten disulfide-multi-walled carbon nanotubes (WS2-MWCNTs) composite via a simple hydrothermal process and demonstrate its application as a biosensing platform for ultrasensitive determination of hepatitis B virus genomic DNA coupled with hybridization chain reaction (HCR) signal amplification. The sensing platform includes capture DNA probe (cDNA), auxiliary DNA, two DNA hairpins, and detection signal is amplified by horse radish peroxidase (HRP) on the DNA duplex. The biosensor is fabricated with the cDNA labeled at 3′ end using thiol immobilized on the WS2-MWCNTs/Au nanoparticels modified electrode. cDNA fixed onto electrode surface then reacts with target DNA through the 3′ end of auxiliary DNA. The remaining segment on 5′ end of auxiliary DNA then takes turns to open two alternating DNA hairpins. This leads to a HCR events to produce a nicked double-helix. A large amount of HRP is then combined on DNA helix by affinity interaction of biotin-avidin, each of which amplifies the final electrochemical signal through the catalytic reaction of H2O2 and hydroquinol. Under optimal conditions, a dynamic range of 10fM–0.1nM is obtained with a detection limit of 2.5fM. Synergistic effect of WS2-MWCNTs composite and HCR have proven to greatly improve sensitivity for DNA detection.

An Nb-doped Nickel Oxide-Carbon Nanotubes Composite- Enhanced Electrochemical DNA Biosensor for Detection of Lead(II) Ion

An Nb-doped Nickel Oxide-Carbon Nanotubes Composite- Enhanced Electrochemical DNA Biosensor for Detection of Lead(II) Ion

A Highly Sensitive Oligonucleotide Hybridization Assay for Klebsiella pneumoniae Carbapenemase with the Probes on a Gold Nanoparticles Modified Glassy Carbon Electrode.

To develop a new electrochemical DNA biosensor for determination of Klebsiella pneumoniae carbapenemase, a highly sensitive and selective electrochemical biosensor for DNA detection was constructed based on a glassy carbon electrode (GCE) modified with gold nanoparticles (Au-nano). The Au-nano/GCE was characterized by scanning electromicroscopy, cyclic voltammetry, and electrochemical impedance spectroscopy. The hybridization detection was measured by differential pulse voltammetry using methylene blue as the hybridization indicator. The dynamic range of detection of the sensor for the target DNA sequences was from 1 × 10(-11) to 1 × 10(-8) M, with an LOD of 1 × 10(-12) M. The DNA biosensor had excellent specificity for distinguishing complementary DNA sequence in the presence of non-complementary and mismatched DNA sequence. The Au-nano/GCE showed significant improvement in electrochemical characteristics, and this biosensor was successfully applied for determination of K. pneumoniae.

Electrochemical DNA biosensor based on a glassy carbon electrode modified with gold nanoparticles and graphene for sensitive determination of Klebsiella pneumoniae carbapenemase

We describe the fabrication of a sensitive electrochemical DNA biosensor for determination of Klebsiella pneumoniae carbapenemase (KPC). The highly sensitive and selective electrochemical biosensor for DNA detection was constructed based on a glassy carbon electrode (GCE) modified with gold nanoparticles (Au-NPs) and graphene (Gr). Then Au-NPs/Gr/GCE was characterized by scanning electro miroscope (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The hybridization detection was measured by diffierential pulse voltammetry (DPV) using methylene blue (MB) as the hybridization indicator. The dynamic range of detection of the sensor for the target DNA sequences was from 1×10−12 to 1×10−7mol/L, with a detection limit of 2×10−13mol/L. The DNA biosensor had excellent specificity for distinguishing complementary DNA sequence in the presence of non-complementary and mismatched DNA sequence. The results demonstrated that the Au-NPs/Gr nanocomposite was a promising substrate for the development of high-performance electrocatalysts for determination of KPC.

Simple in situ functionalizing of magnetite nanoparticles by 4-nitrobenzenediazonium for construction of a sensitive electrochemical DNA biosensor for detection of a DNA sequence related to Hepatitis B virus

An in situ method for direct attachment of 4-nitrobenzenediazonium onto the surface of magnetite nanoparticles to develop an efficient template for construction of DNA electrochemical biosensor was proposed. For functionalization of magnetic nanoparticles, firstly azo salt of nitroaniline (4-nitrobenzenediazonium) was prepared and was reacted directly with magnetite nanoparticles in basic solution followed by electrochemical reduction of the terminal nitro group to amine. This NH2 group can interact with an activated phosphate group of non-labeled ss DNA related to Hepatitis B virus. Activated non-labeled ss DNA was prepared using N-(3 dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride and N-hydroxy-succinimide. The DNA hybridization events were monitored by differential pulse voltammetry measurement of the intercalated methylene blue as a well-known electrochemical indicator. Under optimal conditions, the reduction peak currents were linearly decreased with the increasing concentration of HBV target DNA from 7.8 (±0.2) × 10−13 mol L−1 to 8.8 (±0.3) × 10−9 mol L−1 with a detection limit of 3.3 (±0.2) × 10−13 mol L−1. This DNA biosensor exhibited excellent selectivity, reproducibility, and stability in the HBV DNA hybridization assay.