DNA sensor based on an Escherichia coli lac Z gene probe immobilization at self-assembled monolayers-modified gold electrodes

A novel approach to construct an electrochemical DNA sensor based on immobilization of a 25 base single-stranded probe, specific to E. coli lac Z gene, onto a gold disk electrode is described. The capture probe is covalently attached using a self-assembled monolayer of 3,3′-dithiodipropionic acid di(N-succinimidyl ester) (DTSP) and mercaptohexanol (MCH) as spacer. Hybridization of the immobilized probe with the target DNA at the electrode surface was monitored by square wave voltammetry (SWV), using methylene blue (MB) as electrochemical indicator. Variables involved in the sensor performance, such as the DTSP concentration in the modification solution, the self-assembled monolayers (SAM) formation time, the DNA probe drying time atop the electrode surface and the amount of probe immobilized, were optimized.

A good stability of the single- and double-stranded oligonucleotides immobilized on the DTSP-modified electrode was demonstrated, and a target DNA detection limit of 45 nM was achieved without signal amplification. Hybridization specificity was checked with non-complementary and mismatch oligonucleotides. A single-base mismatch oligonucleotide gave a hybridization response only 7 ± 3%, higher than the signal obtained for the capture probe before hybridization. The possibility of reusing the electrochemical genosensor was also tested.     

A reagentless signal-on architecture for electronic, aptamer-based sensors via target-induced strand displacement

Thrombin binding stabilizes the alternative G-quadruplex conformation of the aptamer, liberating the methylene blue (MB)-tagged oligonucleotide to produce a flexible, single-stranded DNA element. This allows the MB tag to collide with the gold electrode surface, producing a readily detectable Faradaic current at thrombin concentrations as low as approximately 3 nM.     

Modification of Escherichia coli single-stranded DNA binding protein with gold nanoparticles for electrochemical detection of DNA hybridization

The unique binding event between Escherichia coli single-stranded DNA binding protein (SSB) and single-stranded oligonucleotides conjugated to gold (Au) nanoparticles is utilized for the electrochemical detection of DNA hybridization. SSB was attached onto a self-assembled monolayer (SAM) of single-stranded oligonucleotide modified Au nanoparticle, and the resulting Au-tagged SSB was used as the hybridization label. Changes in the Au oxidation signal was monitored upon binding of Au tagged SSB to probe and hybrid on the electrode surface. The amplified oxidation signal of Au nanoparticles provided a detection limit of 2.17 pM target DNA, which can be applied to genetic diagnosis applications. This work presented here has important implications with regard to combining a biological binding event between a protein and DNA with a solid transducer and metal nanoparticles.     

Methylene blue as an electrochemical discriminator of single- and double-stranded oligonucleotides immobilised on gold substrates.

Self-assembly of thiol-terminated oligonucleotides on gold substrates provides a convenient and versatile route to DNA-functionalised surfaces. Here we show that the square-wave voltammetric peak position of methylene blue complexed to thiol-terminated single-stranded oligonucleotides immobilised on gold electrodes differs from that of methylene blue complexed to thiol-terminated double-stranded oligonucleotides immobilised on gold electrodes. The peak potential of methylene blue at the single-stranded oligonucleotide array was consistently found to occur at potentials ca. 10-15 mV more positive than that at double-stranded oligonucleotide arrays, the precise difference being dependent on the direction of the voltammetry. This voltammetric behaviour mirrors that found for methylene blue bound to freely diffusing single- and double-stranded calf thymus DNA and suggests that the immobilised oligonucleotides retain the methylene blue binding properties of their freely diffusing counterparts. Thus methylene blue provides a simple electrochemical indicator for the status of oligonucleotide-functionalised gold surfaces.     

Effect of molecular crowding on the response of an electrochemical DNA sensor

E-DNA sensors, the electrochemical equivalent of molecular beacons, appear to be a promising means of detecting oligonucleotides. E-DNA sensors are comprised of a redox-modified (here, methylene blue or ferrocene) DNA stem-loop covalently attached to an interrogating electrode. Because E-DNA signaling arises due to binding-induced changes in the conformation of the stem-loop probe, it is likely sensitive to the nature of the molecular packing on the electrode surface. Here we detail the effects of probe density, target length, and other aspects of molecular crowding on the signaling properties, specificity, and response time of a model E-DNA sensor. We find that the highest signal suppression is obtained at the highest probe densities investigated, and that greater suppression is observed with longer and bulkier targets. In contrast, sensor equilibration time slows monotonically with increasing probe density, and the specificity of hybridization is not significantly affected. In addition to providing insight into the optimization of electrochemical DNA sensors, these results suggest that E-DNA signaling arises due to hybridization-linked changes in the rate, and thus efficiency, with which the redox moiety collides with the electrode and transfers electrons.     

Fraying and electron autodetachment dynamics of trapped gas phase oligonucleotides

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double- and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double- and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed.     

Homogeneous Electrochemical Assay for Real‐time Monitoring of Exonuclease III Activity Based on a Graphene Monolayer

A homogeneous electrochemical assay based on a graphene monolayer electrode was developed for simple, sensitive, rapid and quantitative analysis of the exonuclease III (Exo III) activity. The method utilized a methylene blue (MB) tagged DNA substrate with hairpin structure, and a graphene monolayer attached on the working electrode. Before digestion, the hairpin structure prevents the adsorption of the DNA substrate to the graphene surface. Degradation of the substrate by the 3′–5′ Exo III, however, yields single‐stranded DNA (ssDNA), resulting in its subsequent binding to the graphene surface through π‐π stacking, which produces the voltammetric current from electrochemical reduction of the MB tag anchoring at the end of ssDNA. A direct quantification of the Exo III activity can be achieved by measuring the reductive peak current of the MB tag under easily attainable potential (∼ −0.1 V vs Ag/AgCl) range comparably sensitive to the conventional methods such as a gel‐based or fluorescence‐based assays. Our approach can be applied to measure various exonucleases activity by adjusting the structure of DNA substrate suggesting a new assay method in drug screening and basic research related to the enzymes.     

Studying electron transfer through alkanethiol self-assembled monolayers on a hanging mercury drop electrode using potentiometric measurements

A new approach based on measuring the change of the open-circuit potential (OCP) of a hanging mercury drop electrode (HMDE), modified with alkanethiols of different chain length conducted in a solution containing a mixture of Ru(NH3)6(2+) and Ru(NH3)6(3+) is used for studying electron transfer across the monolayer. Following the time dependence of the OCP allowed the extraction of the kinetic parameters, such as the charge transfer resistance (R(ct)) and the electron transfer rate constant (k(et)), for different alkanethiol monolayers. An electron tunneling coefficient, beta, of 0.9 A(-1) was calculated for the monolayers on Hg.     

Electron transfer rates in DNA films as a function of tether length

A homologous series of DNA-modified electrodes has been investigated in which the molecular tether length varies. Using intercalated, covalently bound daunomycin as a redox probe, an exponential dependence of electron transfer rates on the number of intervening methylene groups in the sigma-bonded tether is observed. In contrast, variation in DM position within DNA yields no detectable change in rate. These data confirm that overall electron transfer rates in DNA films are limited by the tether, not the DNA.     

亚甲蓝修饰电极推动的血红素蛋白质直接电子转移反应

本文研究了几种血红素蛋白质包括牛血红蛋白, 人肌红蛋白和马心细胞色素C在亚甲蓝修饰电极上的非均相电子转移反应, 采用光透薄层光谱电化学法监测了血红素蛋白的直接电化学反应过程, 并进行了动力学研究。     

Dynamics of electron transport by elastic bending of short DNA duplexes. Experimental study and quantitative modeling of the cyclic voltammetric behavior of 3'-ferrocenyl DNA end-grafted on gold

The dynamics of electron transport within a molecular monolayer of 3'-ferrocenylated-(dT)(20) strands, 5'-thiol end-grafted onto gold electrode surfaces via a short C2-alkyl linker, is analyzed using cyclic voltammetry as the excitation/measurement technique. It is shown that the single-stranded DNA layer behaves as a diffusionless system, due to the high flexibility of the ss-DNA chain. Upon hybridization by the fully complementary (dA)(20) target, the DNA-modified gold electrode displays a highly unusual voltammetric behavior, the faradaic signal even ultimately switching off at a high enough potential scan rate. This remarkable extinction phenomenon is qualitatively and quantitatively justified by the model of elastic bending diffusion developed in the present work which describes the motion of the DNA-borne ferrocene moiety as resulting from the elastic bending of the duplex DNA toward and away from the electrode surface. Its use allows us to demonstrate that the dynamics of electron transport within the hybridized DNA layer is solely controlled by the intrinsic bending elasticity of ds-DNA. Fast scan rate cyclic voltammetry of end-grafted, redox-labeled DNA layers is shown to be an extremely efficient method to probe the bending dynamics of short-DNA fragments in the submillisecond time range. The persistence length of the end-anchored ds-DNA, a parameter quantifying the flexibility of the nanometer-long duplex, can then be straightforwardly and accurately determined from the voltammetry data.     

A new peptide nucleotide acid biosensor for electrochemical detection of single nucleotide polymorphism in duplex DNA via triplex structure formation

In this paper, we report a new PNA biosensor for electrochemical detection of point mutation or single nucleotide polymorphism (SNP) in p53 gene corresponding oligonucleotide based on PNA/ds-DNA triplex formation following hybridization of PNA probe with double-stranded DNA (ds-DNA) sample without denaturing the ds-DNA into single-stranded DNA (ss-DNA). As p53 gene is mutated in many human tumors, this research is useful for cancer therapy and genomic study. In this approach, methylene blue (MB) is used for electrochemical signal generation and the interaction between MB and oligonucleotides is studied by differential pulse voltammety (DPV). Probe-modified electrode is prepared by self-assembled monolayer (SAM) formation of thiolated PNA molecules on the surface of Au electrode. A significant increase in the reduction signal of MB following hybridization of the probe with the complementary double-stranded oligonucleotide (ds-oligonucleotide) confirms the function of the biosensor. The selectivity of the PNA sensor is investigated by non-complementary ds-oligonucleotides and the results support the ability of the sensor to detect single-base mismatch directly on ds-oligonucleotide. The influence of probe and ds-DNA concentrations on the effective discrimination against complementary sequence and point mutation is studied and the concentration of 10^?6 M is selected as appropriate concentration. Diagnostic performance of the biosensor is described and the detection limit is found to be 4.15 × 10^?12 M.     

Effect of the Length of the Hydrocarbon Chain of Adsorbed Aliphatic Acids and Alcohols on the Electron Transfer Probability

The kinetics of redox reactions of hemin adsorbed over a monolayer of long-chain aliphatic alcohols, such as cetyl alcohol (C₁₆) and stearyl alcohol (C₁₈), and stearic (C₁₈), behenic (C₂₂), and melissic (C₃₀) acids is studied. The reaction inhibition on an alcohol monolayer (0.34 orders of magnitude per methylene group) is close to that reported in the literature. The inhibition on an acid monolayer is substantially smaller (0.2 to 0.07 orders), which is attributed to irregularity of the structure of the monolayer of carboxylic acids caused by the dissociation of carboxyl groups, especially in alkaline solutions. Violation of a regular structure of an acid monolayer is confirmed by a considerable irreversible increase in the electrode capacitance in alkaline solutions and by a decrease in the ohmic resistance of a monolayer of the longest saturated acid, i.e. triacontanoic acid. For a regular monolayer structure, when the length of an extended hydrocarbon chain defines the barrier thickness (alcohols), experimental data accord with predictions of the superexchange theory, which holds that the probability of a long-distance electron transfer depends on the length of the chain of covalent bonds over which the superexchange occurs. If the film structure is irregular (film thickness decreases with increasing hydrocarbon chain length (acids)), the packing of these bonds becomes essential, which allows the electron not to pass over all the bonds but to hop onto a nonbonded chain link nearby. The obtained data indicate that the electron transfer probability is defined by the electron's path, rather than by the barrier's geometrical thickness.     

Electrochemical DNA Biosensors Based on Palladium Nanoparticles Combined with Carbon Nanotubes

Palladium nanoparticles, in combination with multi‐walled carbon nanotubes (MWCNTs), were used to fabricate a sensitivity‐enhanced electrochemical DNA biosensor. MWCNTs and palladium nanoparticles were dispersed in Nafion, which were used to modify a glassy carbon electrode (GCE). Oligonucleotides with amino groups at the 5′ end were covalently linked onto carboxylic groups of MWCNTs on the electrode. The hybridization events were monitored by differential pulse voltammetry (DPV) measurement using methylene blue (MB) as an indicator. Due to the ability of carbon nanotubes to promote electron‐transfer and the high catalytic activities of palladium nanoparticles for electrochemical reaction of MB, the sensitivity of presented electrochemical DNA biosensors was remarkably improved. The detection limit of the method for target DNA was 1.2×10^?13 M.     

Electrochemical detection of DNA hybridization by using a zirconia modified renewable carbon paste electrode

A simple, polishable and renewable DNA biosensor was fabricated based on a zirconia modified carbon paste electrode. Zirconia was mixed with graphite powder and paraffin wax to produce the paste for the electrode, and response-optimized at 56% graphite powder, 19% ZrO(2) and 25% paraffin wax. An oligonucleotide probe with a terminal 5'-phosphate group was attached to the surface of the electrode via the strong affinity of zirconia for phosphate groups. DNA immobilization and hybridization were characterized by cyclic voltammetry and differential pulse voltammetry, using methylene blue as indicator. Examination of changes in response with complementary or non-complementary DNA sequences showed that the developed biosensor had a high selectivity and sensitivity towards hybridization detection (< or =2x10(-10) M complementary DNA detectable). The surface of the biosensor can be renewed quickly and reproducibly (signal RSD+/-4.6% for five successive renewals) by a simple polishing step.     

Label-free electrochemical detection of DNA hybridization on gold electrode

A label-free electrochemical detection protocol for DNA hybridization is reported for the first time by using a gold electrode (AuE). The oxidation signal of guanine was monitored at +0.73 V by using square wave voltammetry (SWV) on self-assembled l-cysteine monolayer (SAM) modified AuE. The electrochemical determination of hybridization between an inosine substituted capture probe and native target DNA was also accomplished. 6-mer adenine probe was covalently attached to SAM via its amino link at 5^′ end. Then, 6-mer thymine-tag of the capture probe was hybridized with the adenine probe, thus left the rest of the oligonucleotide available for hybridization with the target. The dependence of the guanine signal upon the concentration of the target was observed. Probe modified AuE was also challenged with non-complementary and mismatch containing oligonucletides. Label-free detection of hybridization on AuE is greatly advantageous over the existing carbon and mercury electrode materials, because of its potential applicability to microfabrication techniques. Performance characteristics of the genosensor are described, along with future prospects.     

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.     

Catalytic Au Electrodes Based on SAMs of per(6‐deoxy‐6‐thio‐2,3‐di‐O‐methyl)‐β‐cyclodextrin

A three‐step sequential self‐assembly procedure was applied in preparing gold electrodes modified in a stable and controlled way by a monolayer of thiolated β‐cyclodextrin (β‐CD), with methylene blue (MB) included in its cavity as the active component of the monolayer, and octanethiol as the nonelectroactive spacer blocking the electrode surface not occupied by β‐CD. MB acted as a mediator of electrons with respect to a solution soluble analyte, H₂O₂, and provided electrical contact between the electrode and solution resident enzyme, laccase, catalyzing reduction of oxygen to water.     

光谱电化学法研究亚甲基蓝的电还原反应

用极谱法研究亚甲基蓝(MB)的电极吸附过程早有报道.近几年,用光谱电化学法研究其电化学行为又引起了许多人的兴趣.但在光透薄层电极上,由于光程太短,测试溶液浓度较高,导致亚甲基蓝发生聚合,影响实验结果的准确测定.为此本文利用特制的比色皿型长光程薄层光谱电化学池,在低浓度条件下研究其在SnO_2镀膜玻璃电极上的电还原反应.     

Nafion 膜固定的新亚甲基蓝为介体生物传感器

以Nafion膜固定的新亚甲基蓝为辣根过氧化物酶和玻碳电极间的电子传递介体,制成电流型单酶过氧化氢生物传感器和双酶葡萄糖生物传感器。探讨了工作电位、pH值、温度和干扰物质等对生物传感器的影响。