Direct electrochemical sensor for label-free DNA detection based on zero current potentiometry

A direct electrochemical DNA biosensor based on zero current potentiometry was fabricated by immobilization of ssDNA onto gold nanoparticles (AuNPs) coated pencil graphite electrode (PGE). One ssDNA/AuNPs/PGE was connected in series between clips of working and counter electrodes of a potentiostat, and then immersed into the solution together with a reference electrode, establishing a novel DNA biosensor for specific DNA detection. The variation of zero current potential difference (ΔE(zcp)) before and after hybridization of the self-assembled probe DNA with the target DNA was used as a signal to characterize and quantify the target DNA sequence. The whole DNA biosensor fabrication process was characterized by cyclic voltammetry and electrochemical impedance spectroscopy with the use of ferricyanide as an electrochemical redox indicator. Under the optimized conditions, ΔE(zcp) was linear with the concentrations of the complementary target DNA in the range from 10nM to 1μM, with a detection limit of 6.9nM. The DNA biosensor showed a good reproducibility and selectivity. Prepared DNA biosensor is facile and sensitive, and it eliminates the need of using exogenous reagents to monitor the oligonucleotides hybridization.     

Disposable DNA electrochemical sensor for hybridization detection

A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 microgram/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.     

A label-free electrochemical DNA sensor based on exonuclease III-aided target recycling strategy for sequence-specific detection of femtomolar DNA

The present work demonstrates a rapid, single-step and ultrasensitive label-free and signal-off electrochemical sensor for specific DNA detection with excellent discrimination ability for single-nucleotide polymorphisms, taking advantage of Exonuclease III (Exo III)-aided target recycling strategy to achieve signal amplification. Exo III has a specifical exo-deoxyribonuclease activity for duplex DNAs in the direction from 3' to 5' terminus, however its activity on the duplex DNAs with 3'-overhang and single-strand DNA is limited. In response to the specific features of Exo III, the proposed E-DNA sensor is designed such that, in the presence of target DNA, the electrode self-assembled signaling probe hybridizes with the target DNA to form a duplex in the form of a 3'-blunt end at signaling probe and a 3'-overhang end at target DNA. In this way, Exo III specifically recognizes this structure and selectively digests the signaling probe. As a result, the target DNA dissociates from the duplex and recycles to hybridize with a new signaling probe, leading to the digestion of a large amount of signaling probes gradually. A redox mediator, Ru(NH(3))(6)(3+) (RuHex) is employed to electrostatically adsorbed onto signaling probes, which is directly related to the amount and the length of the signaling probes remaining in the electrode, and provides a quantitative measure of sequence-specific DNA with the experimentally measured (not extrapolated) detection limit as low as 20 fM. Moreover, this E-DNA sensor has an excellent differentiation ability for single mismatches with fairly good stability.     

Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film

We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp² and sp³ mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (∼24mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5′-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60mers) with different methylation ratios.     

DNA hybridization electrochemical sensor using conducting polymer

We report the use of poly(thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (PTAE) for application to electrochemical hybridization sensor. A synthetic route for the thiophen-3-yl-acetic acid 1,3-dioxo-1,3-dihydro-isoindol-2-yl ester (TAE) is described, which is used as a monomer of conducting polymer sensor. A direct chemical substitution of probe oligonucleotide to good leaving group site in the PTAE is carried out on the conducting polymer film. A biological recognition can be monitored by comparison with the electrochemical signal (cyclic voltammogram) of single and double strand state oligonucleotide. The sensitivity of the electrochemical sensor is 0.62 microA/nmole and the detection limit is 1 nmole. The oxidation current of double strand state oligonucleotide is a half of that of single strand, that is corresponding to the decrease of electrochemical activity of conducting polymer with increase of stiffness of side group of the polymer. The oxidation current decreasing ratios of perfect matched and single nucleotide mismatched samples are 52 and 25-30%, respectively. The more decreasing ratio is attributable to the more steric hindrance of single nucleotide mismatched sample.     

Nanopore sensor for fast label-free detection of short double-stranded DNAs

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with γ-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.     

Selective detection of silver ions using mushroom-like polyaniline and gold nanoparticle nanocomposite-based electrochemical DNA sensor

A highly sensitive electrochemical DNA biosensor made of polyaniline (PANI) and gold nanoparticles (AuNPs) nanocomposite (AuNPs@PANI) has been used for the detection of trace concentration of Ag⁺. In the presence of Ag⁺, with the interaction of cytosine–Ag⁺–cytosine (C–Ag⁺–C), cytosine-rich DNA sequence immobilized onto the surface of AuNPs@PANI has a self-hybridization and then forms a duplex-like structure. The whole detection procedure of Ag⁺ based on the developed biosensor was evaluated by electrochemical impedance spectroscopy. On semi-logarithmic plots of the log Ag⁺ concentration versus peak current, the results show that the prepared biosensor can detect silver ions at a wide linear range of 0.01–100 nM (R = 0.9828) with a detection limit of 10 pM (signal/noise = 3). Moreover, the fabricated sensor exhibits good selectivity and repeatability. The detection of Ag⁺ was determined by Ag⁺ self-induced conformational change of DNA scaffold that involved only one oligonucleotide, showing its convenience and availability.     

A gold nanoparticle-based chronocoulometric DNA sensor for amplified detection of DNA

We report a protocol for the amplified detection of target DNA by using a chronocoulometric DNA sensor (CDS). Electrochemistry is known to be rapid, sensitive and cost-effective; it thus offers a promising approach for DNA detection. Our CDS protocol is based on a 'sandwich' detection strategy, involving a capture probe DNA immobilized on a gold electrode and a reporter probe DNA loaded on gold nanoparticles (AuNPs). Each probe flanks one of two fragments of the target sequence. A single DNA hybridization event brings AuNPs, along with hundreds of reporter probes, in the proximity of the electrode. We then employ chronocoulometry to interrogate [Ru(NH3)6]3+ electrostatically bound to the captured DNA strands. This AuNP-amplified DNA sensor can selectively detect as low as femtomolar (zeptomoles) concentrations of DNA targets and conveniently analyze a breast cancer-associated BRCA-1 mutant DNA. The time range for the entire protocol is approximately 3 d, whereas the DNA sensing takes less than 2 h to complete.     

Chemiluminescent DNA optical fibre sensor for Brettanomyces bruxellensis detection

Food and beverage industries require rapid tests to limit economic losses and one way to do so is via molecular tests. In the present work, DNA capture and secondary probes, were designed to target the ITS (Internal Transcribed) sequences of Brettanomyces bruxellensis, a yeast responsible for the production of off flavours in both wine and beer. ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. The dot blot technique was used to determine the sensitivity and specificity of the capture probe. Both probes were, thereafter, adapted to construct an optical fibre genosensor, which produced neither false positives nor false negatives, and was both repeatable and faster with respect to traditional methods, the latter requiring at least one week to detect B. bruxellensis.     

双链DNA 膜效应在序列点突变电化学检测中的应用

报道一种基于金表面的双链DNA膜效应检测DNA点突变的新方法。致密的双链DNA分子层可以将电化学信号分子禁闭在金表面和双链DNA之间,或者将信号分子与金表面隔离开,使其无法接触裸金面．在实验系统中采用Fe(CN）6^3-为信号分子,在升高温度时,双链DNA膜被破坏,信号分子离开或接触金表面,解链曲线会出现一个陡峭的电化学信号变化。在特定的温度下,完全互补的序列和单碱基点突变的序列的信号比达到了100：1。这种方法简单而且灵敏,同时避免了复杂的共价修饰信号分子的过程。     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations. The method is based on the formation and deformation of double-stranded DNA (dsDNA) membranes on a gold surface. It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface. In these systems, Fe(CN)₆ ³⁻ was used as the reporter. As the temperature increases, a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears. At a special temperature, the method gives 100:1 selectivity for the perfect complement and single base mutation target. Thus, the system provides a simple and sensitive method to detect DNA point mutations without labeling targets. __________ Translated from Acta Biophysica Sinica 2005, 21 (2) [译 自: 生物物理学报, 2005,21(2)]     

DNA duplex membrane effect for the electrochemical detection of single-base DNA mutations

Here we report a new method to detect DNA point mutations.The method is based on the formation and deformation of double-stranded DNA(dsDNA)membranes on a gold surface.It can encage reporter molecules between the gold surface and the double-stranded DNA or keep them away from the gold surface.In these systems,Fe(CN)63- was used as the reporter.As the temperature increases,a sharp electrochemical signal change in the melting curve of wild-type dsDNA appears.At a special temperature,the and single base mutation target.Thus,the system provides a simple and sensitive method to detect DNA point mutations without labeling targets.     

Label-free electrochemical DNA sensor based on functionalised conducting copolymer

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.     

Ferrocene conjugated oligonucleotide for electrochemical detection of DNA base mismatch

We describe the synthesis, binding, and electrochemical properties of ferrocene-conjugated oligonucleotides (Fc-oligos). The key step for the preparation of Fc-oligos contains the coupling of vinylferrocene to 5-iododeoxyuridine via Heck reaction. The Fc-conjugated deoxyuridine phosphoramidite was used in the Fc-oligonucleotide synthesis. We show that thiol-modified Fc-oligos deposited onto gold electrodes possess potential ability in electrochemical detection of DNA base mismatch.     

Ligase-based multiple DNA analysis by using an electrochemical sensor array

We herein report an electrochemical biosensor for the sequence-specific detection of DNA with high discrimination ability for single-nucleotide polymorphisms (SNPs). This DNA sensor was constructed by a pair of flanking probes that "sandwiched" the target. A 16-electrode electrochemical sensor array was employed, each having one individual DNA capture probe immobilized at gold electrodes via gold-thiol chemistry. By coupling with a biotin-tagged detection probe, we were able to detect multiple DNA targets with a single array. In order to realize SNP detection, a ligase-based approach was employed. In this method, both the capture probe and the detection probe were in tandem upon being hybridized with the target. Importantly, we employed a ligase that specifically could ligate tandem sequences only in the absence of mismatches. As a result, when both probes were complementary to the target, they were ligated in the presence of the ligase, thus being retained at the surface during the subsequent stringent washing steps. In contrast, if there existed 1-base mismatch, which could be efficiently recognized by the ligase, the detection probe was not ligated and subsequently washed away. A conjugate of avidin-horseradish peroxidase was then attached to the biotin label at the end of the detection probe via the biotin-avidin bridge. We then electrochemically interrogated the electrical current for the peroxidase-catalyzed reduction of hydrogen peroxide. We demonstrated that the electrochemical signal for the wild-type DNA was significantly larger than that for the sequence harboring the SNP.     

电化学生物传感器快速检测DNA研究进展

本简要地介绍了DNA电化学生物传感器研究的最新进展,重点讨论了改善生物传感器选择性和灵敏度的技术和方法。     

Direct detection of methylation in genomic DNA

The identification of methylated sites on bacterial genomic DNA would be a useful tool to study the major roles of DNA methylation in prokaryotes: distinction of self and nonself DNA, direction of post-replicative mismatch repair, control of DNA replication and cell cycle, and regulation of gene expression. Three types of methylated nucleobases are known: N⁶-methyladenine, 5-methylcytosine and N⁴-methylcytosine. The aim of this study was to develop a method to detect all three types of DNA methylation in complete genomic DNA. It was previously shown that N⁶-methyladenine and 5-methylcytosine in plasmid and viral DNA can be detected by intersequence trace comparison of methylated and unmethylated DNA. We extended this method to include N⁴-methylcytosine detection in both in vitro and in vivo methylated DNA. Furthermore, application of intersequence trace comparison was extended to bacterial genomic DNA. Finally, we present evidence that intrasequence comparison suffices to detect methylated sites in genomic DNA. In conclusion, we present a method to detect all three natural types of DNA methylation in bacterial genomic DNA. This provides the possibility to define the complete methylome of any prokaryote.     

Direct detection of double-stranded DNA: Molecular methods and applications for DNA diagnostics

Methodologies to detect DNA sequences with high sensitivity and specificity have tremendous potential as molecular diagnostic agents. Most current methods exploit the ability of single-stranded DNA (ssDNA) to base pair with high specificity to a complementary molecule. However, recent advances in robust techniques for recognition of DNA in the major and minor groove have made possible the direct detection of double-stranded DNA (dsDNA), without the need for denaturation, renaturation, or hybridization. This review will describe the progress in adapting polyamides, triplex DNA, and engineered zinc finger DNA-binding proteins as dsDNA diagnostic systems. In particular, the sequence-enabled reassembly (SEER) method, involving the use of custom zinc finger proteins, offers the potential for direct detection of dsDNA in cells, with implications for cell-based diagnostics and therapeutics.     

Telemetric electrochemical sensor

A telemetric system was designed and constructed to sense pH and ethanol variation in aqueous solutions. The measured signals were transferred by software digitally and transmitted wirelessly by the telemeter, personal digital assistant (PDA), through the General Packet Radio Service (GPRS) protocol. The pH sensing electrode was designed to measure a chemical potential induced by a proton concentration gradient on the electrode's surface which exhibits internal Donnon diffusion behavior, and a linear relationship between the electrical potential and pH was found. The result shows that the wireless sensing system allowed not only long-term usage and long-distance transmission but also with high accuracy (e.g. S.D. less than +/-2%). The telemetric system can also be modified to measure ethanol concentration in aqueous solution amperometrically. It was found that the sensitivity of that ex situ measurements matched those of in field measurements with negligible deviation, less than 4%.     

Zinc oxide/redox mediator composite films-based sensor for electrochemical detection of important biomolecules

Electrochemical oxidation of serotonin (SN) onto zinc oxide (ZnO)-coated glassy carbon electrode (GCE) results in the generation of redox mediators (RMs) that are strongly adsorbed on electrode surface. The electrochemical properties of zinc oxide-electrogenerated redox mediator (ZnO/RM) (inorganic/organic) hybrid film-coated electrode has been studied using cyclic voltammetry (CV). The scanning electron microscope (SEM), atomic force microscope (AFM), and electrochemical techniques proved the immobilization of ZnO/RM core/shell microparticles on the electrode surface. The GCE modified with ZnO/RM hybrid film showed two reversible redox peaks in acidic solution, and the redox peaks were found to be pH dependent with slopes of −62 and −60 mV/pH, which are very close to the Nernst behavior. The GCE/ZnO/RM-modified electrode exhibited excellent electrocatalytic activity toward the oxidations of ascorbic acid (AA), dopamine (DA), and uric acid (UA) in 0.1 M phosphate buffer solution (PBS, pH 7.0). Indeed, ZnO/RM-coated GCE separated the anodic oxidation waves of DA, AA, and UA with well-defined peak separations in their mixture solution. Consequently, the GCE/ZnO/RMs were used for simultaneous detection of DA, AA, and UA in their mixture solution. Using CV, calibration curves for DA, AA, and UA were obtained over the range of 6.0 × 10⁻⁶ to 9.6 × 10⁻⁴ M, 1.5 × 10⁻⁵ to 2.4 × 10⁻⁴ M, and 5.0 × 10⁻⁵ to 8 × 10⁻⁴ M with correlation coefficients of 0.992, 0.991, and 0.989, respectively. Moreover, ZnO/RM-modified GCE had good stability and antifouling properties.