Electrochemical DNA Biosensor For Detection Research Articles

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.

A novel electrochemical biosensor for DNA detection based on exonuclease III-assisted target recycling and rolling circle amplification

A strategy for electrochemical detection of DNA by exonuclease III-assisted DNA recycling and the rolling circle amplification was developed.

Coupling electrochemical response of a DNA biosensor with PCR for Neisseria gonorrhoeae detection

Early diagnosis of gonococcal infections is important with regard to a patient's health and stage of infection. In this context, we report the development of an opa-gene–based electrochemical DNA biosensor for detection of Neisseria gonorrhoeae by monitoring redox peak of methylene blue indicator. The fabricated biosensor has been shown to be highly sensitive and specific when evaluated with complementary, non-complementary, and 1-base mismatch DNA sequences and polymerase chain reaction (PCR) amplified products (amplicons) of standard strain of N. gonorrhoeae (ATCC49226). The biosensor has been further evaluated using amplicons of known positive and negative clinical samples, and cut-off for positives has been determined using receiver operating characteristic curve. The sensitivity (SN), specificity (SP), positive predictive value, and negative predictive value of the biosensor have been found to be 96.2%, 88.2%, 92.6%, and 93.8%, respectively. We conclude that the combination of PCR amplification with electrochemical detection shows distinct advantage of high SN and increased SP for gonococcal detection.

Graphene oxide-chitosan nanocomposite based electrochemical DNA biosensor for detection of typhoid

Graphene oxide (GO)-Chitosan (CHI) nano-composite is employed for the development of DNA based electrochemical biosensor for diagnosis of typhoid. Biosensor has been prepared by covalent immobilization of Salmonella typhi specific 5′-amine labeled single stranded (ss) DNA probe on GO-CHI/ITO via glutaraldehyde. Differential pulse voltammetry (DPV) studies revealed good specificity and ability of ssDNA/GO-CHI/ITO biosensor to distinguish complementary, non-complementary and one base mismatch sequences. The ssDNA/GO-CHI/ITO biosensor showed detection range of 10fM to 50nM and LOD 10fM within 60s hybridization times for complementary sequence. Further, ssDNA/GO-CHI/ITO bioelectrode is able to detect complementary target present in serum samples with LOD of 100fM at 25°C. The excellent performance of biosensor is attributed to large surface-to-volume ratio and good electrochemical activity of graphene oxide, and good biocompatibility of chitosan, which enhances the DNA immobilization and facilitate electron transfer between DNA and electrode surface (ITO).

Ultrasensitive electrochemical DNA detection based on dual amplification of circular strand-displacement polymerase reaction and hybridization chain reaction

We developed a novel electrochemical strategy for ultrasensitive DNA detection using a dual amplification strategy based on the circular strand-displacement polymerase reaction (CSDPR) and the hybridization chain reaction (HCR). In this assay, hybridization of hairpin-shaped capture DNA to target DNA resulted in a conformational change of the capture DNA with a concomitant exposure of its stem. The primer was then hybridized with the exposed stem and triggered a polymerization reaction, allowing a cyclic reaction comprising release of target DNA, hybridization of target with remaining capture DNA, polymerization initiated by the primer. Furthermore, the free part of the primer propagated a chain reaction of hybridization events between two DNA hairpin probes with biotin labels, enabling an electrochemical reading using the streptavidin-alkaline phosphatase. The proposed biosensor showed to have very high sensitivity and selectivity with a dynamic response range through 10fM to 1nM, and the detect limit was as low as 8fM. The proposed strategy could have the potential for molecular diagnostics in complex biological systems.

Chitosan encapsulated quantum dots platform for leukemia detection

We report results of the studies relating to electrophoretic deposition of nanostructured composite of chitosan (CS)–cadmium-telluride quantum dots (CdTe-QDs) onto indium-tin-oxide coated glass substrate. The high resolution transmission electron microscopic studies of the nanocomposite reveal molecular level coating of the CdTe-QDs with CS molecules in the colloidal dispersion medium. This novel composite platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by immobilizing amine terminated oligonucleotide probe sequence containing 22 base pairs, identified from BCR–ABL fusion gene. The results of differential pulse voltammetry reveal that this nucleic acid sensor can detect as low as 2.56pM concentration of complementary target DNA with a response time of 60s. Further, the response characteristics show that this fabricated bioelectrode has a shelf life of about 6 weeks and can be used for about 5–6 times. The results of experiments conducted using clinical patient samples reveal that this sensor can be used to distinguish CML positive and the negative control samples.

Nanopatterned Cadmium Selenide Langmuir–Blodgett Platform for Leukemia Detection

We present results of the studies relating to preparation of Langmuir-Blodgett (LB) monolayers of tri-n-octylphosphine oxide-capped cadmium selenide quantum dots (QCdSe) onto indium-tin oxide (ITO) coated glass substrate. The monolayer behavior has been studied at the air-water interface under various subphase conditions. This nanopatterned platform has been explored to fabricate an electrochemical DNA biosensor for detection of chronic myelogenous leukemia (CML) by covalently immobilizing the thiol-terminated oligonucleotide probe sequence via a displacement reaction. The results of electrochemical response studies reveal that this biosensor can detect target DNA in the range of 10(-6) to 10(-14) M within 120 s, has a shelf life of 2 months, and can be used about 8 times. Further, this nucleic acid sensor has been found to distinguish the CML-positive and the control negative clinical patient samples.

Exonuclease III-based and gold nanoparticle-assisted DNA detection with dual signal amplification

Herein we report a sensitive electrochemical biosensor for DNA detection by making use of exonuclease III and probe DNA functionalized gold nanoparticles. While probe DNA P1 modified on a gold electrode surface can self-hybridize into a stem-loop structure with an exonuclease III-resistant 3′ overhang end, in the presence of target DNA, P1 may also hybridize with the target DNA to form a duplex region. Therefore, exonuclease III may selectively digest P1 from its 3′-hydroxyl termini until the duplex is fully consumed. Since a single target DNA can trigger exonuclease III digestion of numerous P1 strands, the first signal amplification is achieved. On the other hand, since the digested P1, exposing its complementary sequence to probe DNA P2, can further hybridize with P2 that has been previously modified on the surface of gold nanoparticles, many nanoparticles loaded with numerous DNA strands are immobilized onto the electrode surface. Consequently, large amount of electroactive molecules [Ru(NH3)6]3+ can bind with the DNA strands to produce an intense electrochemical response as the second signal amplification. Based on the studies with cyclic voltammetry (CV) and chronocoulometry (CC) techniques, the proposed biosensor can sensitively detect specific target DNA at a picomolar level with high specificity.

A simple and label-free electrochemical biosensor for DNA detection based on the super-sandwich assay

The focus of this work was on designing a label-free DNA biosensor based on a super-sandwich assay using the electrochemical impedance spectroscopy technique. For this purpose, we designed a signal-up configuration whose linker probes could hybridize with two regions of the target DNA. In this configuration, the presented target DNA would effectively decrease the electron transfer, which would improve the sensitivity of the sensor. Ultimately, we employed gel electrophoresis to further confirm the formation of the proposed super-sandwich structure.

An electrochemical DNA biosensor based on the “Y” junction structure and restriction endonuclease-aided target recycling strategy

Based on the "Y" junction structure and restriction endonuclease-aided target recycling strategy, an electrochemical biosensor for DNA detection was developed. This universal biosensor was suitable for detecting different sequences of target DNA by changing the sequence of capture and assistant strands.

A new electrochemical biosensor for DNA detection based on molecular recognition and lead sulfide nanoparticles

In this paper, we constructed a new electrochemical biosensor for DNA detection based on a molecule recognition technique. In this sensing protocol, a novel dual-labeled DNA probe (DLP) in a stem–loop structure was employed, which was designed with dabcyl labeled at the 3′ end as a guest molecule, and with a Pb nanoparticle labeled at the 5′ end as electrochemical tag to indicate hybridization. One α-cyclodextrin-modified electrode (α-CD/MCNT/GCE) was used for capturing the DNA hybridization. Initially, the DLP was in the “closed” state in the absence of the target, which shielded dabcyl from the bulky α-CD/MCNT/GCE conjugate due to a steric effect. After hybridization, the loop sequence (16 bases) formed a rigid duplex with the target, breaking the relatively shorter stem duplex (6 bases). Consequently, dabcyl was forced away from the Pb nanoparticle and became accessible by the electrode. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical reduction current signal of Pb. Using this method, as low as 7.1 × 10 −10 M DNA target had been detected with excellent differentiation ability for even a single mismatch.

Design of a sandwich-mode amperometric biosensor for detection of PML/RARα fusion gene using locked nucleic acids on gold electrode

In this study, a novel DNA electrochemical probe (locked nucleic acid, LNA) was designed and involved in constructing an electrochemical DNA biosensor for detection of promyelocytic leukemia/retinoic acid receptor alpha (PML/RARα) fusion gene in acute promyelocytic leukemia for the first time. This biosensor was based on a ‘sandwich’ sensing mode, which involved a pair of LNA probes (capture probe immobilized at electrode surface and biotinyl reporter probe as an affinity tag for streptavidin–horseradish peroxidase (streptavidin–HRP). Since biotin can be connected with streptavidin–HRP, this biosensor offered an enzymatically amplified electrochemical current signal for the detection of target DNA. In the simple hybridization system, DNA fragment with its complementary DNA fragment was evidenced by amperometric detection, with a detection limit of 74fM and a linear response range of 0.1–10pM for synthetic PML/RARα fusion gene in acute promyelocytic leukemia (APL). Otherwise, the biosensor showed an excellent specificity to distinguish the complementary sequence and different mismatch sequences. The new pattern also exhibited high sensitivity and selectivity in mixed hybridization system.

Locked nucleic acids biosensor for detection of BCR/ABL fusion gene using benzoate binuclear copper (II) complex as hybridization indicator

Benzoate binuclear copper (II) complex, [Cu 2(C 7H 5O 2) 4(C 2H 6O) 2] (abbreviated as CuR 2) was prepared and its interaction with double-stranded salmon sperm DNA (dsDNA) in pH 7.4 phosphate buffer solution was studied by electrochemical experiments at the Au electrode (AuE). It was revealed that CuR 2 presented an excellent electrochemical activity on AuE and could bind with dsDNA by intercalation mode. The CuR 2 was further utilized as a new indicator in the fabrication of an electrochemical DNA biosensor for detection of BCR/ABL fusion gene. The biosensor based on nanogold (NG) modified AuE was developed by using thiolated-hairpin locked nucleic acids (LNA) as the capture probe for hybridization with BCR/ABL fusion gene. The results indicated this new method has excellent specificity for single-base mismatch and complementary after hybridization. The constructed electrochemical DNA biosensor achieved a detection limit of 1.0 × 10 −10 M for complementary target DNA with a good stability.

Electrochemical detection of DNA hybridization using a water-soluble branched polyethyleneimine–cobalt(III)–phenanthroline indicator and PNA probe on Au electrodes

An electrochemical method for the detection of DNA hybridization using a novel electroactive, cationic, and water-soluble branched polyethyleneimine (BPEI)–cobalt(III)–phenanthroline(phen) polymeric indicator and single-stranded neutral peptide nucleic acid (PNA) probe on the Au electrode was developed. The indicator possesses some free amine groups, as well as cationic cobalt complexes in the polymer chain. It does not bind to neutral PNA capture probe alone. However, the indicator strongly interacts with the negatively charged backbone of the complementary oligonucleotide bound to the PNA probe through electrostatic interactions. The coordination spherical moieties also interact with the probe by embedding into the double-helix structure of PNA–DNA. These two interactions enable transduction of hybridization, producing a clear electrical signal in differential pulse voltammetry (DPV). The correlation against non-complementary DNA, three-base and one-base mismatch DNA was sharp, and the signal of indicator for the target DNA demonstrated a linear relationship within the concentration range of 5.0 × 10 −9 to 2.5 × 10 −7 M ( R = 0.9940) with a detection limit of 5.6 × 10 −10 M. These studies showed that the novel polymeric indicator and single-stranded PNA probe could be used to fabricate an electrochemical biosensor for DNA detection. This technique can provide an alternative route for expanding the range of detection methods available for DNA hybridization.

Nanostructured electrochemical DNA biosensors for detection of the effect of berberine on DNA from cancer cells

Multi walled carbon nanotubes (MWNT) in dimethylformamide (DMF) or aqueous sodium dodecyl sulfate (SDS) solution, colloidal gold nanoparticles (GNP) in phosphate buffer solution (PBS), and a GNP-MWNT mixture in aqueous SDS solution have been investigated for chemical modification of a screen-printed carbon electrode used as the signal transducer of a dsDNA-based biosensor. Differential pulse voltammetry of the DNA redox marker Co[(phen)3]3+ and the guanine moiety anodic oxidation and cyclic voltammetry with K3[Fe(CN)6] as indicator revealed substantial enhancement of the response of the biosensor, particularly when MWNT in SDS solution was used. The biosensor was used in testing of berberine, an isoquinoline plant alkaloid with significant antimicrobial and anticancer activity. Berberine had a very strong, concentration-dependent, effect on the structural stability of DNA from the human cancer cells (U937 cells) whereas non-cancer cells were changed only when berberine concentrations were relatively high 75 and 50 microg mL(-1).