Cyclic voltammetry of DNA at a mercury electrode: an anodic peak specific for guanine

Synthetic homopolyribonucleotides poly(A), poly(U), poly(C), and poly(G), poly(A, G, U), apurinic acid and native and denatured DNA from calf thymus were analyzed by means of cyclic voltammetry (CV) using a hanging mercury drop electrode. It was shown that guanine containing polynucleotides, i.e. poly(G), poly(A, G, U) and DNA yield an anodic peak of guanine in the vicinity of a potential of -0.3 V (against a saturated calomel electrode). The guanine peak appeared only at a sufficiently negative switching potential (about -2 V). The appearance of the guanine peak was conditioned by a reduction of guanine residues in the region of the switching potential and reoxidation of the reduction product in the vicinity of -0.3 V. Native and thermally denatured DNAs were investigated under the conditions of both complete and incomplete coverage of the electrode in various background electrolytes. Both DNA forms yielded anodic CV peaks of guanine with the peak of denatured DNA being always higher than that of native DNA. Irradiation of native DNA with relatively small doses of gamma radiation (5-120 Gy) resulted in an increase of the anodic peak. A comparison of changes induced by gamma radiation in the anodic (guanine) and cathodic (reduction of adenine and cytosine) peaks showed a steeper increase of the cathodic peak as compared to that of the anodic one. It has been concluded that in the given dose range the DNA double-helical structure is mainly damaged in the adenine-thymine rich regions.     

尿酸在聚6-甲基香豆素修饰的玻碳电极上的电化学行为

制备了聚6-甲基香豆素修饰玻碳电极,研究了尿酸(UA)在该修饰电极上的电化学行为。实验结果表明:在pH=5.0的磷酸盐缓冲溶液中,扫描速率为50mV/s时,尿酸在修饰电极上于0.352V处产生一个灵敏的氧化峰,在0.278V处有一弱的还原峰。经线性扫描伏安法测定,氧化峰电流与尿酸浓度在2.5×10-6～1.0×10-5mol/L范围内表现出良好的线性关系,检出限为1.0×10-6mol/L。将修饰电极在常温下放置50d及将体系温度升高到75℃时,修饰电极对尿酸的响应电流大体不变,结果满意。     

A novel method to construct a third-generation biosensor: self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) nanospheres

A novel method for fabrication of horseradish peroxidase (HRP) biosensor has been developed by self-assembling gold nanoparticles on thiol-functionalized poly(styrene-co-acrylic acid) (St-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in thiol-functionalized poly(St-co-AA) nanosphere latex prepared by emulsifier-free emulsion polymerization of St with AA and function with dithioglycol to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups. Finally, horseradish peroxidase was immobilized on the surface of the gold nanoparticles. The sensor displayed an excellent electrocatalytical response to reduction of H2O2 without the aid of an electron mediator. The sensor was highly sensitive to hydrogen peroxide with a detection limit of 4.0 micromoll(-1), and the linear range was from 10.0 micromoll(-1) to 7.0 mmoll(-1). The biosensor retained more than 97.8% of its original activity after 60 days of use. Moreover, the studied biosensor exhibited good current repeatability and good fabrication reproducibility.     

Characterization and electrocatalytic properties of Prussian blue electrochemically deposited on nano-Au/PAMAM dendrimer-modified gold electrode

Gold electrode was modified with 3-mercaptopropionic acid (MPA) and further reacted with poly(amidoamine) (PAMAM) dendrimer (generation 4.0) then attached the nano-Au to obtain films on which Prussian blue (PB) was electrochemically deposited to afford much wider pH adaptive range, much better electrochemical stability and excellent electrochemical response. The microstructure and electrochemical behavior of Au/MPA/PAMAM/nano-Au/PB electrode were investigated by scanning electron microscopy (SEM) and cyclic voltammetry. The electrochemical response of the Au/MPA/PAMAM/nano-Au/PB-modified electrode for the electrocatalytic reduction of hydrogen peroxide was investigated, and it was found that the sensitivity as well as the corresponding detection limits were improved as compared to the voltammetric response of a Au/PB-modified electrode and Au/MPA/PAMAM/PB electrode. Based on this, a new electrochemical sensor for determination of hydrogen peroxide has been developed.     

Nucleotide sequence-dependent opening of double-stranded DNA at an electrically charged surface

It has been shown earlier that the DNA double helix is opened due to a prolonged contact of the DNA molecule with the surface of the mercury electrode. At neutral pH, the opening process is relatively slow (around 100 s), and it is limited to potentials close to -1.2 V (against SCE). The opening of the double helix has been explained by strains in the DNA molecule due to strong repulsion of the negatively charged phosphate residues from the electrode surface where the polynucleotide chain is anchored via hydrophobic bases. Interaction of the synthetic ds polynucleotides with alternating nucleotide sequences/poly(dA-dT).poly (dA-dT), poly (dA-dU).poly (dA-dU), poly (dG-dC).poly (dG-dC)/ and homopolymer pairs/poly (dA).poly (dT), poly (rA).poly (rU) and poly (dG).poly (dC)/ with the hanging mercury drop electrode has been studied. Changes in reducibility of the polynucleotides were exploited to indicate opening of the double helix. A marked difference in the behaviour was observed between polynucleotides with alternating nucleotide sequence and homopolymer pairs: opening of the double-helical structures of the former polynucleotides occurs at a very narrow potential range (less than 100 mV) (region U), while with the homopolymer pairs containing A X T or A X U pairs, the width of this region is comparable to that of natural DNA (greater than 200 mV). In contrast to natural DNA, the region U of homopolymer pairs is composed of two distinct phases. No region U was observed with poly (dG).poly (dC). In polynucleotides with alternating nucleotide sequence, the rate of opening of the double helix is strongly dependent on the electrode potential in region U, while in homopolymer pairs, this rate is less potential-dependent. It has been assumed that the difference in the behaviour between homopolymer pairs and polynucleotides with alternating nucleotide sequence is due to differences in absorbability of the two polynucleotide chains in the molecule of a homopolymer pair (resulting from different absorbability of purine and pyrimidine bases) in contrast to equal adsorbability of both chains in a polynucleotide molecule with alternating nucleotide sequence. It has been shown that the mercury electrode is a good model of biological surfaces (e.g. membranes), and that the nucleotide sequence-dependent opening (unwinding) of the DNA double helix at electrically charged surfaces may play an important role in many biological processes.     

Preparation of poly(thionine) modified screen-printed carbon electrode and its application to determine NADH in flow injection analysis system

A poly(thionine) modified screen-printed carbon electrode has been prepared by an electrooxidative polymerization of thionine in neutral phosphate buffer. The modified electrodes are found to give stable and reproducible electrocatlytic responses to NADH and exhibit good stability. Several techniques, including cyclic voltammetry, X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM), have been employed to characterize the poly(thionine) film. Further, the modified screen-printed carbon electrode was found to be promising as an amperometric detector for the flow injection analysis (FIA) of NADH, typically with a dynamic range of 5-100 microM.     

Preparation of polyaniline-modified electrodes containing sulfonated polyelectrolytes using layer-by-layer techniques

Polyaniline (PAni) has been used frequently for the construction of biosensors. However, a prime limitation is its instability at basic or neutral pH because of the loss of its electrochemical activity and conductivity. In this study, three available sulfonated polyanions: Nafion, poly(vinyl sulfonate) (PVS), and poly(styrene sulfonate) (PSS) serving as the counterion and providing an acidic microenvironment to stabilize PAni, are used to fabricate a sensor for ammonium ion detection. Nafion used to be a common ion-sensitive membrane due to its high proton conductivity. However, its high cost and limited solubility has constrained its uses. PVS and PSS are water-soluble polymers, easily incorporating with PAni to form the composites. Surface analysis by electron spectroscopy for chemical analysis (ESCA) and scanning electron microscope (SEM), and the electrochromic property for the PAni composites provided the convenient tools to characterize the electrode fabrication. On the aspect of sensing the ammonium ions, the modified electrodes exhibited electroactivity of PAni in ammonium ion detection and also showed the linear dependence of reduction current on the ammonium ion concentration. The pH effect on the sensing response was also evaluated and found insignificant to the response (ranging from pH 6.9-7.6). For increasing the stability of the electrodes, the diazo-resin (DAR) was introduced to the coat on the outmost layer and then cured by UV irradiation, giving the covalent network between the layers of polyelectrolytes. The PSS-doped PAni electrode was found to perform detection sensitivity in the linear range of 0-100mM of ammonium ion concentration.     

Structural transitions of polyadenylic acid due to protonation: the influence of the length of single strands on the polarographic behaviour of the double-helical form

Transition of single-stranded poly(A) into its double-helical protonated form was followed by means of derivative pulse polarography, spectrophotometry, and other methods. It was found that properties of protonated poly(A) depended on the length of single strands from which the protonated double helix was formed. In contrary to longer poly(A) transition of short single-stranded molecules (s₂₀,w lower than about 3) caused practically no decrease in the pulse-polarographic current. It was concluded that the formation of the protonated double helix of poly(A) did not result in the inaccesibility of the reduction sites (located in the vicinity of the surface of the molecule) for the electrode process, as it was in DNA-like double-helical polynucleotides. The current changes observed in the course of transition of longer poly(A) were explained as due to slower transport of long double-stranded molecules to the electrode.     

A novel nitric oxide biosensor based on electropolymerization poly(toluidine blue) film electrode and its application to nitric oxide released in liver homogenate

A novel poly(toluidine blue)-modified electrode has been constructed for the determination of nitric oxide in biological sample. The electrochemical behavior of poly(toluidine blue) film electrode and its electrocatalytic activity toward NO were studied in detail by cyclic voltammetry. Possible interferences were tested and evaluated after further coated with Nafion. The poly(toluidine blue) and Nafion composite film-modified electrode exhibits a good linear relationship over a NO concentration of 1.8 x 10(-7) to 8.6 x 10(-5)mol/L, and the detection limit is 1.8 x 10(-8)mol/L (S/N=3). NO release from the rat liver homogenate stimulated by l-arginine was studied, and the responses were decreased by the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine.     

Amperometric biosensor for the detection of hydrogen peroxide using catalase modified electrodes in polyacrylamide

A simple biosensor for the detection of hydrogen peroxide in organic solvents has been developed and coupled to a flow injection analysis (FIA) system. Catalase was entrapped in polyacrylamide gel and placed on the surface of platinum (working electrode) fixed in a Teflon holder with Ag-wire (auxiliary electrode), followed by addition of filter paper soaked in KCl. The entrapped catalase gel was held on the electrode using membranes. The effects of cellulose and polytetrafluroethylene (PTFE) membranes on the electrode response towards hydrogen peroxide have been studied. The modified electrode has been used to study the detection of hydrogen peroxide in solvents like water, dimethyl sulfoxide (DMSO), and 1,4-dioxane using amperometric techniques like cyclic voltammetry (CV) and FIA. The CV of modified catalase electrode showed a broad oxidation peak at -150 mV and a clear reduction peak at -212 mV in the presence of hydrogen peroxide. Comparison of CV with hydrogen peroxide in various solvents has been carried out. The electrode showed an irreversible kinetics with DMSO as the solvent. A flow cell has been designed in order to carry on FIA studies to obtain calibration plots for hydrogen peroxide with the modified electrode. The calibration plots in several solvents such as water, dimethyl sulfoxide, 1,4-dioxane have been obtained. The throughput of the enzyme electrode was 10 injections per hour. Due to the presence of membrane the response time of the electrode is concentration dependent.     

Fundamental aspects of antimony thin film electrodes for pH measurement.

This report concerns the investigation of the sensitivity, temperature dependence, accuracy, and the standard electrode potential EO of an antimony thin film pH electrode which was prepared with electron beam evaporation techniques. The air-formed oxide film on antimony thin film electrodes has been proved by both the cathodic reduction method and electron spectroscopy for chemical analysis (ESCA). The antimony thin film electrode responded rapidly to pH changes and its sensitivity was slightly changed depending on the buffer composition. The accuracy of this electrode was compared with that of the glass electrode. Temperature had some influence on the function of this electrode. The standard electrode potential of this electrode was discussed together with that of other forms of antimony electrodes. The structure and thickness of the surface oxide on antimony thin film electrodes was confirmed by cathodic reduction and ESCA. It was clear that the surface oxide governs the electrode reactions. Possible applications of the antimony thin film electrode are discussed stating some limitations in the use.     

Researches on chemically modified electrode. XIX. preparation of iron protoporphyrin film electrode by electrochemical polymerization and its catalysis

Using cyclic voltammetry the electrochemical polymerization of protoporphyrin (PP) and iron protoporphyrin (FePP) was investigated as radical cationic vinyl polymerization. A number of factors, such as electrode potential, temperature, and concentration of monomers in solution, may affect the rate of polymerization. The polymerized porphyrin film on electrode surface was analyzed with electronic scanning microscope, UV-visible spectroscope, and ESCA. The electrochemically polymerized FePP film glassy carbon electrode (poly(FePP)/GC) exhibits very high catalytic activity for dioxygen reduction in aqueous solution to water as a four-electron irreversible process.     

Determination of the absolute redox potential of Rutin: experimental and theoretical studies

The conditional formal potential, E degrees', of Rutin has been studied by cyclic voltammetry using a Rutin film deposited at the multi-wall carbon nanotubes modified glassy carbon electrode (GCE) as the working electrode in different pH phosphate buffered solutions. The experimental standard redox potential, E degrees, of Rutin is obtained to be 0.88 V versus SHE (Standard Hydrogen Electrode). High-level ab initio calculations have been also performed on a chemical model of Rutin and the absolute reduction potential has been calculated. The theoretical standard reduction potential relative to SHE (0.83 V) is in relatively good agreement with experiment.     

Enzyme electrode for aromatic compounds exploiting the catalytic activities of microperoxidase-11

Microperoxidase-11 (MP-11) which has been immobilised in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode catalyzes the conversion of aromatic substances. This peroxide-dependent catalysis of microperoxidase has been applied in an enzyme electrode for the first time to indicate aromatic compounds such as aniline, 4-fluoroaniline, catechol and p-aminophenol. The electrode signal is generated by the cathodic reduction of the quinone or quinoneimine which is formed in the presence of both MP-11 and peroxide from the substrate. The same sensor principle will be extended to aromatic drugs.     

Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

Impedimetric immunosensor for the specific label free detection of ciprofloxacin antibiotic

Impedance spectroscopy approaches combined with the immunosensor technology have been used for the determination of trace amounts of ciprofloxacin antibiotic belonging to the fluoroquinolone family. The sensor electrode was based on the immobilization of anti-ciprofloxacin antibodies by chemical binding onto a poly(pyrrole-NHS) film electrogenerated on a solid gold substrate. The electrode surface was modified by electropolymerization of pyrrole-NHS, antibody grafting and ciprofloxacin immunoreaction. The sensitive steps of surface modification, cyclic voltammetry (CV) and atomic force microscopy (AFM) imaging have been used for electrode surface characterization. The immunoreaction of ciprofloxacin on the grafted anti-ciprofloxacin antibody directly triggers a signal via impedance spectroscopy measurements which allows the detection of extremely low concentration of 10 pg/ml ciprofloxacin.     

Laccase electrode for direct electrocatalytic reduction of O2 to H2O with high-operational stability and resistance to chloride inhibition

Laccase from Trametes hirsuta basidiomycete has been covalently bound to graphite electrodes electrochemically modified with phenyl derivatives as a way to attach the enzyme molecules with an adequate orientation for direct electron transfer (DET). Current densities up to 0.5mA/cm(2) of electrocatalytic reduction of O(2) to H(2)O were obtained in absence of redox mediators, suggesting preferential orientation of the T1 Cu centre of the laccase towards the electrode. The covalent attachment of the laccase molecules to the functionalized electrodes permitted remarkable operational stability. Moreover, O(2) bioelectroreduction based on DET between the laccase and the electrode was not inhibited by chloride ions, whereas mediated bioelectrocatalysis was. In contrast, fluoride ions inhibited both direct and mediated electron transfers-based bioelectrocatalytic reduction of O(2). Thus, two different modes of laccase inhibition by halides are discussed.     

Electrochemical immunosensor for the milk allergen β-lactoglobulin based on electrografting of organic film on graphene modified screen-printed carbon electrodes

A novel label-free voltammetric immunosensor for sensitive detection of β-lactoglobulin using graphene modified screen printed electrodes has been developed. The derivatization of the graphene electrode surface was achieved by electrochemical reduction of in situ generated 4-nitrophenyl diazonium cations in aqueous acidic solution, followed by electrochemical reduction of the terminal nitro groups to amines. The electrochemical modification protocol was optimized in order to generate monolayer of nitrophenyl groups on the graphene surface without complete passivation of the electrode. Unlike the reported method for graphene functionalization, we demonstrated here the ability of the electrografting of aryl diazonium salt to attach an organic film to the graphene surface in a controlled manner by choosing the suitable grafting protocol. Next, the amine groups on the graphene surface were activated using glutaraldehyde and used for the covalent immobilization of β-lactoglobulin antibodies. Cyclic and differential pulse voltammetry carried out in an aqueous solution containing [Fe(CN)(6)](3-/4-) redox pair have been used for the immunosensor characterization. The results demonstrated that the DPV reduction peak current of [Fe(CN)(6)](3-/4-) decreased linearly with increasing the concentration of β-lactoglobulin due to the formation of antibody-antigen complex on the modified electrode surface. The immunosensor obtained using this novel approach enabled a detection limit of 0.85pgmL(-1) and a dynamic range from 1pgmL(-1) to 100ngmL(-1) of β-lactoglobulin in PBS buffer. In addition, the immunosensor evaluated in different samples including cake, cheese snacks, a sweet biscuit, showing excellent correlation with the results obtained from commercially enzyme-linked immunosorbent assay (ELISA) method.     

A third-generation H2O2 biosensor based on horseradish peroxidase-labeled Au nanoparticles self-assembled to hollow porous polymeric nanopheres

A novel third-generation biosensor for hydrogen peroxide (H2O2) was developed by self-assembling gold nanoparticles to hollow porous thiol-functionalized poly(divinylbenzene-co-acrylic acid) (DVB-co-AA) nanospheres. At first, a cleaned gold electrode was immersed in hollow porous thiol-functionalized poly(DVB-co-AA) nanosphere latex to assemble the nanospheres, then gold nanoparticles were chemisorbed onto the thiol groups of the nanospheres. Finally, horseradish peroxidase (HRP) was immobilized on the surface of the gold nanoparticles. The immobilized horseradish peroxidase exhibited direct electrochemical behavior toward the reduction of hydrogen peroxide. The resulting biosensor showed a wide linear range of 1.0 microM-8.0mM and a detection limit of 0.5 microM estimated at a signal-to-noise ratio of 3. Moreover, the studied biosensor exhibited high sensitivity, good reproducibility, and long-term stability.     

The effect of age on the properties of poly(A)-containing messenger RNA in Physarum polycephalum

The effect of ageing on the properties of polysomal poly(A)-containing messenger RNA [poly(A)+ mRNA] of Physarum polycephalum has been investigated. Using poly(U)--Sepharose affinity chromatography it was shown that shortening of the poly(A) tract occurred as the age of the mRNA population increased. Analysis of the poly(A) segments by use of polyacrylamide gel electrophoresis, after digestion of polysomal poly(A)+ mRNA molecules with RNAase A and RNAase T1, revealed that their lengths ranged from 140 to 220 nucleotide residues. A reduction in the efficiency of utilization of mRNA for translation as the age of the mRNA population increased was demonstrated by measuring the proportion of poly(A)+ mRNA present in the polysomal fraction as compared with post-polysomal material.