Lable‐Free Electrochemical DNA Sensor Based on Gold Nanoparticles/Poly(neutral red) Modified Electrode

We present a new strategy for the label‐free electrochemical detection of DNA hybridization based on gold nanoparticles (AuNPs)/poly(neutral red) (PNR) modified electrode. Probe oligonucledotides with thiol groups at the 5‐end were covalently linked onto the surface of AuNPs/PNR modified electrode via S‐Au binding. The hybridization event was monitored by using differential pulse voltammetry (DPV) upon hybridization generates electrochemical changes at the PNR‐solution interface. A significant decrease in the peak current was observed upon hybridization of probe with complementary target ssDNA, whereas no obvious change was observed with noncomplementary target ssDNA. And the DNA sensor also showed a high selectivity for detecting one‐mismatched and three‐mismatched target ssDNA and a high sensitivity for detecting complementary target ssDNA, the detection limit is 4.2×10^?12 M for complementary target ssDNA. In addition, the DNA biosensor showed an excellent reproducibility and stability under the DNA‐hybridization conditions.     

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

We present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.     

Developing a Nano‐Biosensor for DNA Hybridization Using a New Electroactive Label

Development of electrochemical DNA hybridization biosensors based on carbon paste electrode (CPE) and gold nanoparticle modified carbon paste electrode (NGMCPE) as transducers and ethyl green (EG) as a new electroactive label is described. Electrochemical impedance spectroscopy and cyclic voltammetry techniques were applied for the investigation and comparison of bare CPE and NGMCPE surfaces. Our voltammetric and spectroscopic studies showed gold nanoparticles are enable to facilitate electron transfer between the accumulated label on DNA probe modified electrode and electrode surface and enhance the electrical signals and lead to an improved detection limit. The immobilization of a 15‐mer single strand oligonucleotide probe on the working electrodes and hybridization event between the probe and its complementary sequence as a target were investigated by differential pulse voltammetry (DPV) responses of the EG accumulated on the electrodes. The effects of some experimental variables on the performance of the biosensors were investigated and optimum conditions were suggested. The selectivity of the biosensors was studied using some non‐complementary oligonucleotides. Finally the detection limits were calculated as 1.35×10^?10 mol/L and 5.16×10^?11 mol/L on the CPE and NEGCPE, respectively. In addition, the biosensors exhibited a good selectivity, reproducibility and stability for the determination of DNA sequences.     

Electrochemical Detection of DNA Hybridization Based on the Probe Labeled with Carbon‐Nanotubes Loaded with Silver Nanoparticles

A sensitive electrochemical method for the detection of DNA hybridization based on the probe labeled with multiwall carbon‐nanotubes (MWNTs) loaded with silver nanoparticles (Ag‐MWNTs) has been developed. MWNTs were electroless‐plated with a large number of silver nanoparticles to form Ag‐MWNTs. Probe single strand DNA (ss‐DNA) with a thiol group at the 3′‐terminal labeled with Ag‐MWNTs by self‐assembled monolayer (SAM) technique was employed as an electrochemical probe. Target ss‐DNA with a thiol group was immobilized on a gold electrode by SAM technique and then hybridized with the electrochemical probe. Binding events were monitored by differential pulse voltammetric (DPV) signal of silver nanoparticles. The signal difference permitted to distinguish the match of two perfectly complementary DNA strands from the near perfect match where just three base pairs were mismatched. There was a linear relation between the peak current at +120 mV (vs. SCE) and complementary target ss‐DNA concentration over the range from 3.1×10^?14 to 1.0×10^?11 mol/L with a detection limit of 10 fmol/L of complementary target ss‐DNA. The proposed method has been successfully applied to detection of the DNA sequence related to cystic fibrosis. This work demonstrated that the MWNTs loaded with silver nanoparticles offers a great promising approach for sensitive detection of DNA hybridization.     

Nano‐Gold Modified Genosensor for Direct Detection of DNA Hybridization

In this paper, nano‐gold modified carbon paste electrode (NGMCPE) was employed to develop an electrochemical DNA hybridization biosensor. The proposed sensor was made up by immobilization of 15‐mer single stranded oligonucleotide probe for detection of target DNA. Hybridization detection relies on the alternation in guanine oxidation signal following hybridization of the probe with complementary genomic DNA. The guanine oxidation was monitored using differential pulse voltammetry (DPV). Different factors such as activation potential, activation time and probe immobilization conditions were optimized. The selectivity of the sensor was investigated by non‐complementary oligonucleotides. Diagnostic performance of the biosensor was described and the detection limit was found 1.9 × 10^?13 M at the NGMCPE surface. All of the investigations were performed in both CPE and NGMCPE and finally their results were compared.     

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.

Label-Free DNA Biosensor for Electrochemical Detection of Short DNA Sequences Related to Human Papilloma Virus

Abstract

Highly sensitive label-free techniques of DNA determination are particularly interesting in relation to the present development of an electrochemical hybridization biosensor for the detection of short DNA fragments specific to the human papilloma virus (HPV). Unlabeled DNA probes have been immobilized by spontaneous coadsorption of thiolated single-stranded oligonucleotides (HS-ssDNA) onto the sensing surface of a screen-printed gold electrode (SPGE). The covalently immobilized single-stranded DNA probe (HS-ssDNA) could selectively hybridize with its complementary DNA (cDNA) in solution to form double-stranded DNA (dsDNA) on the surface. DNA is treated with acid (e.g., 0.5 M chloridric acid), and the acid-released purine bases are directly determined by square wave voltammetry (SWV).

Variables of the probe-immobilization and hybridization steps are optimized to offer convenient quantitation of HPV DNA target, in connection with a short hybridization time. Peak currents were found to increase in the following order: hybrid-modified SPGE, 11-base mismatched modified SPGE, 18-base mismatched SPGE, and the probe modified SPGE. Control experiments with noncomplementary oligonucleotides were carried out to assess whether the suggested DNA sensor responds selectively to the target. The effect of the target DNA concentration on the hybridization signal was also studied. Under optimal conditions, this sensor has a good calibration range with HPV DNA sequence detection limit of 2 pg · ml^?1 (S/N = 3).     

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, …     

Application of diazo-thiourea and gold nano-particles in the design of a highly sensitive and selective DNA biosensor

An effective procedure for constructing a DNA biosensor is developed based on covalent immobilization of NH_2 labeled,single strand DNA(NH_2-ssDNA) onto a self-assembled diazo-thiourea and gold nanoparticles modified Au electrode(diazo-thiourea/GNM/Au).Gold nano-particles expand the electrode surface area and increase the amount of immobilized thiourea and single stranded DNA(ssDNA) onto the electrode surface.Diazo-thiourea film provides a surface with high conductibility for electron transfer and a bed for the covalent coupling of NH_2-ssDNA onto the electrode surface.The immobilization and hybridization of the probe DNA on the modified electrode is studied by differential pulse voltammetry(DPV) using methylene blue(MB) as a well-known electrochemical hybridization indicator.The linear range for the determination of complementary target ssDNA is from 9.5(±0.1) × 10~(-13) mol/L to1.2(±0.2) x 10~(-9) mol/L with a detection limit of 1.2(±0.1) 10~(-13) mol/L.     

The detection of DNA deamination by electrocatalysis at DNA-modified electrodes

The method of electrocatalysis based on using a methylene blue (MB) as an electrochemical indicator and ferricyanide ions [Fe(CN)6]3- as an electron acceptor was applied in screening DNA for lesions caused by deamination of nucleobases. The damaged DNA was modeled by short 18-mer oligonucleotides containing the different number of mismatched target bases (uracil instead of cytosine residues). The hybridization capacity of these oligomers with complementary probes (immobilized on gold electrodes or free) was investigated by both electrochemical methods and UV spectroscopy. We have shown that the amplitude of the reduction signal corresponding to ferricyanide ions considerably increases in the presence of MB. This electrocatalytic effect allowed us to detect the changes in electrochemical properties of DNA caused by dU.dG mismatches. Using differential pulse voltammetry and cyclic voltammetry, we showed that the electron transport from the electrode through the double-stranded DNA to MB and then to ferricyanide ions is suppressed by the mismatches in duplex structure. According to UV-monitored melting data, single or multiple wobble dU.dG base pairs destabilize 18-mer DNA duplex by 9-27 degrees C.     

High Sensitive Electrochemical Detection of Sequence‐Specific DNA Using Low Current Voltammetry

In this article, we report on efforts to construct a high sensitive electrochemical sensor with immobilized sandwich‐type DNA borne ferrocene (Fc) head for sequence‐specific DNA detection using ultramicroelectrode and low current voltammetry. Based on the difference in deformability between the bending rigid complementary DNA double helix and its anomalous flexile mismatches, the fully complementary target can be distinguished from mismatched targets including the single‐base mismatched target. Detection limit estimated as the amount of DNA is observed to be 100 fM via low current voltammetry. The method offers great promise of high sensitivity and selectivity simultaneously for effective gene identification.     

Fabrication of a Sensitive Electrochemical Biosensor for Detection of DNA Hybridization Based on Gold Nanoparticles/CuO Nanospindles Modified Glassy Carbon Electrode

In this work, a sensitive electrochemical DNA biosensor for the detection of sequence‐specific target DNA was reported. Firstly, CuO nanospindles (CuO NS) were immobilized on the surface of a glassy carbon electrode (GCE). Subsequently, gold nanoparticles (Au NPs) were introduced to the surface of CuO NS by the electrochemical deposition mode. Probe DNA with SH (HS‐DNA) at the 5′‐phosphate end was covalently immobilized on the surface of the Au NPs through Au? S bond. Scanning electron microscopy (SEM) was used to elucidate the morphology of the assembled film, and electrochemical impedance spectroscopy technique (EIS) was used to investigate the DNA sensor assembly process. Hybridization detection of DNA was performed with differential pulse voltammetry (DPV) and the methylene blue (MB) was hybridization indicator. Under the optimal conditions, the decline of reduction peak current of MB (ΔI) was linear with the logarithm of the concentration of complementary DNA from 1.0×10^?13 to 1.0×10^?6 mol·L^?1 with a detection limit of 3.5×10^?14 mol·L^?1 (S/N=3). In addition, this DNA biosensor has good selectivity, and even can distinguish single‐mismatched target DNA.     

Electrochemical DNA Hybridization Assay: Enzyme‐Labeled Detection of Mutation in p53 Gene

This paper discusses a new electrochemical DNA hybridization sensing approach based on the detection of a linked enzyme label. In this method we employ enzyme that is attached to a tethered ssDNA oligomer on the surface and the target analyte is a complementary ssDNA oligomer that does not require any pre‐treatment. The advantage of using of enzyme label is in its amplification of the registration of the hybridization event due to the catalytic reaction facilitated in the process. One particular novelty is associated with the use of enzymes that directly communicate with the electrode surface thus allowing for minimizing the need of additional reagents in the assay. The electrochemical assay was demonstrated when using mixed self‐assembled monolayers from thiolated oligonucleotide and 6‐mercapto 1‐hexanol on gold surfaces. Horseradish peroxidase (HRP) is attached to the surface tethered oligonucleotide using streptavidin‐biotin chemistry, and the enzyme successfully established direct electron transfer (DET) with the electrode or mediated electron transfer (MET) using a mediator. Hybridization results in increasing the angle of contact between electrode and DNA and also the stiffness of the ds DNA, which results in displacing the enzyme away from the electrode surface, and thereby reducing the occurrence of direct electron transfer between the enzyme and the electrode. The cyclic voltammetry showed a clear distinction in response between the complete complementary sequence and the two‐base mismatch sequence. Ellipsometric measurements show that the thickness of the thiol modified oligonucleotide on gold surfaces changes before and after hybridization for the complementary sequence, where as a minimal change in thickness was observed for the noncomplementary sequence. The model target analyte in this study was TP53 gene where a specific mutation is a marker for a list of cancers. Mutations of the TP53 gene have been demonstrated in tumors of the colon, breast, lung, ovary, bladder, and many other organs. Analysis of p53 mutations may provide useful information for the diagnosis, prognosis and therapy of cancer.     

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.     

Detection of Human Interleukine‐2 Gene Using a Label‐Free Electrochemical DNA Hybridization Biosensor on the Basis of a Non‐Inosine Substituted Probe

The human interleukine‐2 gene (hIL‐2) is detected with a label‐free DNA hybridization biosensor using a non‐inosine substituted probe. The sensor relies on the immobilization of a 20‐mer antisense single strand oligonucleotide (chIL‐2) related to the human interleukine‐2 gene on the pencil graphite electrode (PGE) as a probe. The guanine oxidation signal was monitored using anodic differential pulse voltammetry (ADPV). The electrochemical pretreatment of the polished PGE at 1.80 V for 5 min is suggested. Then, 5 min immobilization at 0.50 V was found as the optimum condition for immobilization of the probe. The electrochemical detection of hybridization between chIL‐2 and hIL‐2 as a target was accomplished. The selectivity of the biosensor was studied using noncomplementary oligonucleotides. Diagnostic performance of the biosensor is described and the detection limit is found 36 pg/μL.     

Designs of Enterobacteriaceae Lac Z Gene DNA Gold Screen Printed Biosensors

This paper describes specific electrochemical enterobacteriaceae lac Z gene DNA sensors based on immobilization of a thiolated 25 base single stranded probe onto disposable screen printed gold electrodes (gold SPEs). Two configurations have been evaluated. In the first one, the capture probe was attached to the electrode surface through its ? SH moiety, while mercaptohexanol (MCH) was used as spacer for the displacement of nonspecifically adsorbed oligonucleotide molecules. The hybridization event between the probe and target DNA sequences was detected at ?0.20 V by square‐wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. The second genosensor configuration involved modification of gold high temperature SPEs with a 3,3′‐dithiodipropionic acid di(N‐succinimidyl ester) (DTSP) self‐assembled monolayer (SAM). Moreover, 2‐aminoethanol was used as blocking agent, and further modification with avidin allowed binding of the biotinylated enterobacteriaceae lac Z gene DNA probe. An enzyme amplified detection scheme was applied, based on the coupling of streptavidin‐peroxidase to the biotinylated complementary target, after the hybridization process, and immobilization of tetrathiafulvalene (TTF) as redox mediator atop the modified electrode. The amperometric response obtained at ?0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. Experimental variables concerning sensors composition and electrochemical transduction were evaluated in both cases. A better precision and reproducibility in the fabrication process, as well as a higher sensitivity were achieved using the biotinylated probe‐based sensor configuration. A limit of detection of 0.002 ng/μL was obtained without any preconcentration step.     

Preparation of Nanogapped Gold Nanoparticle Array for DNA Detection

A novel DNA detection technique using a gold nanoparticle array film electrode has been reported here. The gold nanoparticles molecularly linked with binder molecule (1,10‐decanedithiol) were separated 1.3 nm from each other, and the DNA conductivity change from single to double strand was measured by monitoring a voltage drop across the particles, between which a probe of a 12‐mer oligonucleotide was immobilized. In adding a complementary oligonucleotide on the nanoparticle film chip, an immediate decrease in the film resistance (ca. 1.4 Ω) due to a hybridization event occurred in a reproducible manner with this simple setup. In the paper, we have an interest in the primary sensing properties; effect of the film resistance on the sensor response, dependence of the resistance change on the DNA concentration, and the performance of the system for DNA detection including single nucleotide polymorphisms were described.     

Hybridization Detection of Osmium Tetroxide Bipyridine‐Labeled DNA and RNA on Heated Gold Wire Electrodes

We report about hybridization detection of different nucleic acids on capture probe‐modified heated gold wire electrodes. We have compared three kinds of nucleic acid targets: DNA, uracil‐conjugated DNA, and RNA. All three sorts of nucleic acids targets could be labeled with osmium tetroxide bipyridine, hybridized with immobilized DNA capture probes and then detected by square‐wave voltammetry. Heating the gold electrode instead of the entire bulk hybridization solution leads to improved hybridization efficiency in most cases. The reason could be found in a thermal micro‐stirring effect around the heated wire electrode. Also selectivity was improved. Mismatches could be discriminated for DNA and uracil‐conjugated DNA targets. Mismatches in RNA strands, however, are more difficult to detect due to relatively stable secondary structures.     

基于金纳米粒子的QCM实时检测DNA错配的研究

利用石英晶体微天平（QCM）技术,用双硫醇分子作为连接剂,将金纳米粒子固定于金电极表面,以人类p53基因片断为DNA探针,研究了其在QCM金电极表面的固定、杂交和错配,重点探讨了金纳米粒子修饰的DNA错配碱基个数和错配位点对杂交的影响。在实验条件下,金纳米粒子在QCM金电极表面的修饰使其灵敏度得到了明显提高;而且,错配碱基个数和错配碱基位点的差异都对杂交产生了不同程度的影响。     

基于聚硫堇膜对DNA杂交的直接电化学检测

In this work, the application of a conducting polymer, poly（thionine）, modified electrode as matrix to DNA immobilization as well as transducer to label-free DNA hybridization detection was introduced. The electropolymerization of thionine onto electrode surface was carried out by a simple two-step method, which involved a preanodization of glassy carbon electrode at a constant positive potential in thionine solution following cyclic voltammetry scans in the solution. Electrochemical detection was performed by differential pulse voltammetry in the electroactivity potential domain of poly（thionine）. The resulting poly（thionine） modified electrode showed a good stability and electroactivity in aqueous media during a near neutral pH range. Additionally, the pendant amino groups on the poly（thionine） chains enabled poly（thionine） modified electrode to immobilize phosphate group terminated DNA probe via covalent linkage. Hybridization process induced a clear decrease in poly（thionine） redox current, which was corresponding to the decrease in poly（thionine） electroactivity after double stranded DNA was formed on the polymer film. The detection limit of this electrochemical DNA hybridization sensor was 1.0 × 10^-10mol/L. Compared with complementary sequence, the hybridization signal values of 1-base mismatched and 3-base mismatched samples were 63.9% and 9.2%, respectively.