Allele-specific genotype detection of factor V Leiden mutation from polymerase chain reaction amplicons based on label-free electrochemical genosensor

An electrochemical genosensor for the genotype detection of allele-specific factor V Leiden mutation from PCR amplicons using the intrinsic guanine signal is described. The biosensor relies on the immobilization of the 21-mer inosine-substituted oligonucleotide capture probes related to the wild-type or mutant-type amplicons, and these probes are hybridized with their complementary DNA sequences at a carbon paste electrode (CPE). The extent of hybridization between the probe and target sequences was determined by using the oxidation signal of guanine in connection with differential pulse voltammetry (DPV). The guanine signal was monitored as a result of the specific hybridization between the probe and amplicon at the CPE surface. No label-binding step was necessary, and the appearance of the guanine signal shortened the assay time and simplified the detection of the factor V Leiden mutation from polymerase chain reaction (PCR)-amplified amplicons. The discrimination between the homozygous and heterozygous mutations was also established by comparing the peak currents of the guanine signals. Numerous factors affecting the hybridization and nonspecific binding events were optimized to detect down to 51.14 fmol/mL target DNA. With the help of the appearance of the guanine signal, the yes/no system is established for the electrochemical detection of allele-specific mutation on factor V for the first time. Features of this protocol are discussed and optimized.     

Electrogenerated chemiluminescence DNA biosensor based on hairpin DNA probe labeled with ruthenium complex

A highly selective electrogenerated chemiluminescence (ECL) biosensor for the detection of target single-strand DNA (ss-DNA) was developed using hairpin DNA as the recognition element and ruthenium complex as the signal-producing compound. The ECL-based DNA biosensor was fabricated by self-assembling the ECL probe of thiolated hairpin DNA tagged with ruthenium complex on the surface of a gold electrode. In the absence of target ss-DNA, the ECL probe immobilized on the surface of the electrode was in the folded configuration in which its termini were held in close proximity to the electrode, and thus a strong ECL signal could be generated. In the presence of target ss-DNA, a stem-loop of the ECL probe on the electrode was converted into a linear double-helix configuration due to hybridization, resulting in the tag moving away from the electrode surface, which in turn decreased the ECL signal. The ECL intensity of the DNA biosensor generated a "switch off" mode, which decreased with an increase of the concentration of target DNA, and a detection limit of 9 x 10(-11) M complementary target ss-DNA was achieved. Single mismatched target ss-DNA was effectively discriminated from complementary target ss-DNA. The effect of different loop lengths of the hairpin DNA on the selectivity of the ECL DNA biosensor has been investigated. This work demonstrated that the sensitivity and specificity of an ECL DNA biosensor could be greatly improved using a hairpin DNA species which has an appropriate stem and loop length as the recognition element.     

Amplified oligonucleotide sensing in microliter volumes containing copper ions by solution streaming

We present a simple, cost-effective design for amplifying oligodeoxynucleotide (ODN) sensing, in microliter ODN volumes containing copper ions, by solution streaming (bubbling). The inert gas streaming (bubbling) at a constant pressure of 0.04 bar drives the motion of a 30-microL ODN droplet containing a three-electrode circuit (inverted drop microcell), and in the presence of copper ions offers an approximately 50-times improvement in the detection of ODN samples. The detection of ODNs at the carbon paste electrode is based on the enhancement of the oxidation peaks of purine bases (adenine and guanine) by the anodic stripping of the electrochemically accumulated complex of Cu(I) with purine base residues of acid hydrolyzed ODN samples (Cu(I)-ahODN complex). We used the proposed method for (i) the determination of the percentage content of adenine and guanine units within analyzed ODN samples at subnanomolar concentrations (related to monomer content) and (ii) the detection of the (TTC)n triplet expansion using magnetic DNA hybridization with reporter probes containing guanine units (the TTC trinucleotide repeat expansion is associated with serious hereditary diseases, including Friedreich ataxia).     

Enhanced electrochemical detection of DNA hybridization with carbon nanotube modified paste electrode

A novel electrochemical genesensor using twice hybridization enhancement of gold nanoparticles based on carbon paste modified electrode is described. The carbon nanotube modified carbon paste electrode (CNTPE) and mesoporous molecular sieve SBA-15 modified carbon paste electrode (MSCPE) were investigated. The assay relies on the immobilization of streptavidin-biotin labeled target oligonucleotides onto the electrode surface and its hybridization to the gold nanoparticle-labeled DNA probe. After twice hybridization enhanced connection of gold nanoparticles to the hybridized system, the differential pulse voltammetry (DPV) signal of total gold nanoparticles was monitored. It was found that the adsorption of oligonucleotide and hybridized DPV signal on CNTPE were both enhanced in comparison with that of pure carbon paste electrode (CPE). But this trend was reverse on MSCPE. The DPV detection of twice hybridized gold nanoparticles indicated that the sensitivity of the genesensor enhanced about one order of magnitude compared with one-layer hybridization. One-base mismatched DNA and complementary DNA could be distinguished clearly. However, no distinct advantage of MSCPE over CPE was found.     

Investigation of Electrochemical Properties of Carbon Nanofibers Prepared by CCVD Method

Carbon nanofibers, with diameters between 80 and 290 nm and specific area of 242 m²g^-1, have been prepared by the catalytic chemical vapor deposition method. After preparation, the powder was mixed with silicon oil in order to create a paste electrode. The electrochemical behavior of this type of electrode was investigated by cyclic voltammetry, using a solution of 10^-3 M ferrocenecarboxylic acid as mediator. The redox process is quasi-reversible, and it involves the transfer of electrons between Fe(II) and Fe(III).

The same mediator was used for the construction of a second-generation glucose biosensor. The mediator was co-immobilized with the enzyme in the carbon nanofibers paste. The sensor linearly responded to glucose, in the concentration range of 1.7 to 7 mM. A time of around 30 seconds was required to reach 95% of the maximum steady-state current.

Also, the oxidation of calf thymus DNA at the carbon nanofiber paste electrode was investigated by differential pulse voltammetry (DPV). A clear signal, due to guanine oxidation, was obtained in the case of single-stranded DNA.     

Synthesis of MCM-41 nanoparticles from stem of common reed ash silica and their application as substrate in electrooxidation of methanol

In this work, stem of common reed ash (SCRA) is introduced as a new source of silica in the preparation of mesoporous materials. Mesoporous silicate MCM-41 nanoparticles were synthesized hydrothermally using sodium silicate prepared from SCRA as a silica source. The characterization of MCM-41was carried out by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), N\(_{2}\) adsorption/desorption (BET) and transmission electron microscopy (TEM). SEM shows that MCM-41 nanoparticles are sphere-like with size in the range of 30–50 nm with some degree of agglomeration. TEM image of the synthesized sample shows the open framework structure of MCM-41. A type IV isotherm can be observed from adsorption/desorption curves, which is the characteristic of mesoporous materials. The prepared MCM-41 nanoparticles were used as substrate to facilitate the oxidation of methanol through the modification with an electroactive species. The modification was achieved by impregnation of MCM-41 pores with \(\hbox {Ni}^{2+}\) ions (Ni-doped MCM-41). A modified carbon paste electrode (CPE) was prepared by mixing Ni-doped MCM-41 with carbon paste (NiMCM-41CPE). Cyclic voltammetry of NiMCM-41CPE shows an increment in current density of methanol oxidation in comparison with CPE in alkaline solution. Moreover, a decrease in the overpotential of methanol oxidation occurred on the surface of modified electrode. The effects of some parameters such as scan rate and methanol concentration are also investigated on the behaviour of NiMCM-41CPE. Also, the heterogeneous electron transfer rate for the catalytic reaction (k) of methanol is calculated.     

Investigation of Electrochemical Properties of Carbon Nanofibers Prepared by CCVD Method

Carbon nanofibers, with diameters between 80 and 290 nm and specific area of 242 m²g^?1, have been prepared by the catalytic chemical vapor deposition method. After preparation, the powder was mixed with silicon oil in order to create a paste electrode. The electrochemical behavior of this type of electrode was investigated by cyclic voltammetry, using a solution of 10^?3 M ferrocenecarboxylic acid as mediator. The redox process is quasi-reversible, and it involves the transfer of electrons between Fe(II) and Fe(III).

The same mediator was used for the construction of a second-generation glucose biosensor. The mediator was co-immobilized with the enzyme in the carbon nanofibers paste. The sensor linearly responded to glucose, in the concentration range of 1.7 to 7 mM. A time of around 30 seconds was required to reach 95% of the maximum steady-state current.

Also, the oxidation of calf thymus DNA at the carbon nanofiber paste electrode was investigated by differential pulse voltammetry (DPV). A clear signal, due to guanine oxidation, was obtained in the case of single-stranded DNA.     

DNA adsorption characteristics of hollow spherule allophane nano-particles

To understand the propensity of natural allophane to adsorb the DNA molecules, the adsorption characteristics were assessed against natural allophane (AK70), using single-stranded DNA (ss-DNA) and adenosine 5′-monophosphate (5′-AMP) as a reference molecule. The adsorption capacity of ss-DNA on AK70 exhibited one order of magnitude lower value as compared with that of 5′-AMP. The adsorption capacity of ss-DNA decreased with increasing pH due to the interaction generated between phosphate groups of ss-DNA and functional Al–OH groups on the wall perforations through deprotonating, associated with higher energy barrier for the adsorption of ss-DNA. The adsorption morphologies consisting of the individual ss-DNA with mono-layer coverage of the clustered allophane particle were observed successfully through transmission electron microscopy analysis.     

A glucose biosensor employing a stable artificial peroxidase based on ruthenium purple anchored cinder

Iron-enriched industrial waste cinder (CFe*) has been recycled for efficient and stable anchoring of Ru(CN)6(4-) to the formation of a hybrid ruthenium purple complex. The cinder/ruthenium purple hybrid-modified carbon paste electrode (designated as CPE/CFe*-RP) was worked out for hydrodynamic analysis of H2O2 at a low detecting potential of 0.0 V versus Ag/AgCl in pH 7 ammonium buffer solution. The highly active, selective, and stable electrocatalytic system with a function similar to peroxidase enzyme shows a linear calibration curve up to 0.8 mM H2O2 at a rotation rate of 3600 rpm with slope and detection limit (S/N = 3) of 0.11 microA/microM and 33 nM, respectively. Interference by direct electrochemical oxidation of easily oxidizable substances can be prevented as a result of the low detecting potential of the working system. A glucose biosensor was further constructed by coating with glucose oxidase and Tosflex on the CPE/CFe*-RP (denoted as CPE/CFe*-RP/GOx/Ts). The proposed CPE/CFe*-RP/GOx/Ts with a two-layer configuration, that is, enzyme and protecting layers, exhibits good operational performance in terms of response time, linearity, detection limit, and lifetime.     

Application of avidin-biotin technology and adsorptive transfer stripping square-wave voltammetry for detection of DNA hybridization and avidin in transgenic avidin maize

The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.     

Selective voltammetric determination of norepinephrine in the presence of acetaminophen and tryptophan on the surface of a modified carbon nanotube paste electrode

A new electrochemical sensor for the determination of norepinephrine (NE), acetaminophen (AC) and tryptophan (TRP) is described. The sensor is based on carbon paste electrode (CPE) modified with 5-mino-3′,4′-dimethyl-biphenyl-2-ol (5ADB) and takes the advantages of carbon nanotubes (CNTs), which makes the modified electrode highly sensitive for the electrochemical detection of these compounds. Under the optimum pH of 7.0, the oxidation of NE occurs at a potential about 170 mV less positive than that of the unmodified CPE. Also, square wave voltammetry (SWV) was used for the simultaneous determination of NE, AC and TRP at the modified electrode.     

Monitoring DNA immobilization and hybridization on surfaces by atomic force microscopy force measurements

DNA immobilization and hybridization was carried out on Au substrates that were modified with mercaptopropanoic acid and then treated with aluminum(III) solution. The positively charged AI(III) film can be used to immobilize both ds-DNA and ss-DNA. Atomic force microscopy (AFM) was used to monitor the process by force measurements between a negatively charged silica tip and the substrates while immersed in dilute electrolyte. Surface hybridization of ss-DNA produces an increase in the surface charge and surface potential of the substrates, which is reflected by the increasing repulsive force as determined from AFM force-separation curves. A single-base mismatch was detectable in surface hybridization. The AFM force measuring technique was also employed to investigate the interaction of Ru(phen)3(2+) with ss-DNA and ds-DNA. The force measurement results showed that there is a small interaction between Ru(phen)3(2+) and ss-DNA, which was ascribed to the electrostatic binding of Ru(phen)3(2+) to the ss-DNA surface. For ds-DNA, there is a strong interaction which is believed to be due to the association or intercalation of Ru(phen)3(2+) with ds-DNA.     

Oxidation kinetics of guanine in DNA molecules adsorbed onto indium tin oxide electrodes

Oligonucleotides containing the guanine nucleobase were adsorbed onto ITO electrodes from mixtures of DMF and acetate buffer. Chronocoulometry and chronoamperometry were performed on the modified electrodes in both phosphate buffer and buffer containing low concentrations of the inorganic complex Ru(bpy)3(2+) (bpy = 2,2' bipyridine), which catalyzes guanine oxidation. The charge and current evolution with and without the catalyst were compared to the charge and current evolution for electrodes that were treated with identical oligonucleotides that were substituted at every guanine with the electrochemically inert nucleobase hypoxanthine. Chronocoulometry over 2.5 s shows that roughly 2 electrons per guanine were transferred to the electrode in both the presence and absence of Ru(bpy)3(2+), although at a slower rate for the uncatalyzed process. Chronoamperograms measured over 250 ms can be fit to a double exponential decay, with the intensity of the fast component roughly 6-20 times greater than that of the slow component. First- and second-order rate constants for catalytic and direct guanine oxidation were determined from the fast component. The maximum catalytic enhancement for immobilized guanine was found to be i(cat)/i(d) = 4 at 25 microM Ru(bpy)3(2+). The second-order rate constant for the catalyzed reaction was 1.3 x 10(7) M(-1) s(-1), with an apparent dissociation constant of 8.8 microM. When compared to parallel studies in solution, a smaller value of the dissociation constant and a larger value of the second-order rate constant are observed, probably due to distortion of the immobilized DNA, an increase in the local negative charge due to the oxygen sites on the ITO surface, and redox cycling of the catalyst, which maintains the surface concentration of the active form.     

Electronic detection of target nucleic acids by a 2,6-disulfonic acid anthraquinone intercalator

A DNA hybridization biosensor based on long-range electron transfer that is capable of detecting DNA single-base mismatch is presented. A mixed self-assembled monolayer of single-stranded DNA (ss-DNA), thiolated at the 3' end, and 6-mercapto-1-hexanol was formed on a gold surface. This probe ss-DNA-modified gold surface was incubated in 2,6-disulfonic acid anthraquinone (AQDS) intercalator solution, rinsed, and placed in an AQDS-free buffer solution, whereupon voltammetric experiments were performed. No voltammetric peaks were observed for probe ss-DNA-modified gold electrodes. Upon DNA hybridization and incubation in AQDS, clear voltammetric peaks, consistent with the oxidation and reduction of AQDS, were observed. The absence of AQDS electrochemistry for ss-DNA-modified surfaces clearly shows the electrochemistry is due to long-range electron transfer through the DNA duplex. No peak currents were observed when the probe ss-DNA-modified surface was exposed to noncomplementary target DNA, but there was a diminution in current signal upon hybridization with C-A mismatched and a G-A mismatched targets.     

DNA-decorated carbon nanotubes for chemical sensing

We demonstrate a new, versatile class of nanoscale chemical sensors based on single-stranded DNA (ss-DNA) as the chemical recognition site and single-walled carbon nanotube field effect transistors (swCN-FETs) as the electronic read-out component. swCN-FETs with a nanoscale coating of ss-DNA respond to gas odors that do not cause a detectable conductivity change in bare devices. Responses of ss-DNA/swCN-FETs differ in sign and magnitude for different gases and can be tuned by choosing the base sequence of the ss-DNA. ss-DNA/swCN-FET sensors detect a variety of odors, with rapid response and recovery times on the scale of seconds. The sensor surface is self-regenerating: samples maintain a constant response with no need for sensor refreshing through at least 50 gas exposure cycles. This remarkable set of attributes makes sensors based on ss-DNA decorated nanotubes very promising for "electronic nose" and "electronic tongue" applications ranging from homeland security to disease diagnosis.     

DNA/protamine complex paste for an injectable dental material

A DNA/protamine complex powder was prepared by reaction between DNA and protamine sulfate solution with stirring in order to develop a new injectable biomaterials for dental therapy. The powder of DNA/protamine complex became paste by kneading the complex powder and distilled water. Complex formation was confirmed by FT-IR measurement. The complex paste had a porous structure and its viscosity was approximately 280.1 Pas. The paste could easily pass through a needle of 0.25 mm internal diameter. It seemed that DNA/protamine complex paste has suitable viscosity for clinical use as an injectable biomaterial. Although, the complex paste delayed the growth speed of Staphylococcus aureus, Pseudomonas aeruginosa, Porphyromonas gingivalis and Prevotella intermedia for limited periods, it cannot kill and inhibit growing bacteria. The complex paste disk showed a mild tissue response and gradually degraded after the implantation into the soft tissue of rats. These results suggested that this DNA/protamine complex paste could be a useful material for a biodegradable biomaterial. In particular, this paste will be applicable as an injectable biomaterial using syringe for the repair of defects of living tissue, GBR treatment and/or GTR treatment in dentistry.     

Modification of indium tin oxide electrodes with nucleic acids: detection of attomole quantities of immobilized DNA by electrocatalysis

Indium tin oxide electrodes were modified with DNA, and the guanines in the immobilized nucleic acid were used as a substrate for electrocatalytic oxidation by Ru(bpy)3(3+) (bpy = 2,2'-bipyridine). Nucleic acids were deposited onto 12.6-mm2 electrodes from 9:1 DMF/water mixtures buffered with sodium acetate. The DNA appeared to denature in the presence of DMF, leading to adsorption of single-stranded DNA. The nucleic acid was not removed by vigorous washing or heating the electrodes in water, although incubation in phosphate buffer overnight liberated the adsorbed biomolecule. Acquisition of cyclic voltammograms or chronoamperomograms of Ru(bpy)3(2+) at the modified electrodes produced catalytic signals indicative of oxidation of the immobilized guanine by Ru(III). The electrocatalytic current was a linear function of the extent of modification with a slope of 0.5 microA/pmol of adsorbed guanine; integration of the current-time traces gave 2.2+/-0.4 electrons/guanine molecule. Use of long DNA strands therefore gave steep responses in terms of the quantity of adsorbed DNA strand. For example, electrodes modified with a 1497-bp PCR product from the HER-2 gene produced detectable catalytic currents when as little as 550 amol of strand was adsorbed, giving a sensitivity of 44 amol/mm2.     

Electrocatalytic determination of dopamine in the presence of uric acid using an indenedione derivative and multiwall carbon nanotubes spiked in carbon paste electrode

In the present study, a modified carbon paste electrode (CPE) containing multi-wall carbon nanotubes and an indenedione derivative(IMWCNT?CPE) was constructed and was successfully used for dopamine(DA) electrocatalytic oxidation and simultaneous determination of DA and uric acid (UA). Cyclic voltammograms of the IMWCNT?CPE show a pair of well-defined and reversible redox. The obtained results indicate that the peak potential of DA oxidation at IMWCNT?CPE shifted by about 65 and 185 mV toward the negative values compared with that at a MWCNT and indenedione modified CPE, respectively. The electron transfer coefficient, α, and the heterogeneous electron transfer rate constant, k′, for the oxidation of DA at IMWCNT?CPE were calculated 0.4 ± 0.01 and (1.13 ± 0.03) × 10^? 3 cm s^? 1, respectively. Furthermore, differential pulse voltammetry (DPV) exhibits two linear dynamic ranges of 1.9–79.4 μM, and 79.4–714.3 μM and a detection limit of 0.52 μM for DA determination. Then IMWCNT?CPE was applied to the simultaneous determination of DA and UA with DPV. Finally, the activity of the modified electrode was also investigated for determination of DA and UA in real samples, such as injection solution of DA and urine, with satisfactory results.     

Electrical properties of cement paste obtained from impedance spectroscopy

The impedance spectra of hydrating Portland cement paste showed a small part of a large depressed arc and a single depressed arc at low- and high-frequency regions, respectively. A new equivalent circuit was proposed for hydrating cement paste, which consists of a serial combination of a resistor and a constant phase element (CPE) with a resistor in parallel for the bulk effects, and a parallel combination of a resistor and a CPE for the electrode effects. The microstructural evolution in hydrating cement paste was associated with the changes in the resistances, capacitance and the critical relaxation time.