A novel glucose biosensor based on immobilization of glucose oxidase into multiwall carbon nanotubes-polyelectrolyte-loaded electrospun nanofibrous membrane

Nanofibrous glucose electrodes were fabricated by the immobilization of glucose oxidase (GOx) into an electrospun composite membrane consisting of polymethylmethacrylate (PMMA) dispersed with multiwall carbon nanotubes (MWCNTs) wrapped by a cationic polymer (poly(diallyldimethylammonium chloride) (PDDA)) and this nanofibrous electrode (NFE) is abbreviated as PMMA-MWCNT(PDDA)/GOx-NFE. The NFE was characterized for morphology and electroactivity by using electron microscopy and cyclic voltammetry, respectively. Field emission transmission electron microscopy (FETEM) image reveals the dispersion of MWCNT(PDDA) within the matrix of PMMA. Cyclic voltammetry informs that NFE is suitable for performing surface-confined electrochemical reactions. PMMA-MWCNT(PDDA)/GOx-NFE exhibits excellent electrocatalytic activity towards hydrogen peroxide (H(2)O(2)) with a pronounced oxidation current at +100 mV. Glucose is amperometrically detected at +100 mV (vs. Ag/AgCl) in 0.1M phosphate buffer solution (PBS, pH 7). The linear response for glucose detection is in the range of 20 microM to 15 mM with a detection limit of 1 microM and a shorter response time of approximately 4 s. The superior performance of PMMA-MWCNT(PDDA)/GOx-NFE is due to the wrapping of PDDA over MWCNTs that binds GOx through electrostatic interactions. As a result, an effective electron mediation is achieved. A layer of nafion is made over PMMA-MWCNT(PDDA)/GOx-NFE that significantly suppressed the electrochemical interference from ascorbic acid or uric acid. In all, PMMA-MWCNT(PDDA)/GOx-nafion-NFE has exhibited excellent properties for the sensitive determination of glucose like high selectivity, good reproducibility, remarkable stability and without interference from other co-existing electroactive species.     

The binding of zinc (II) to a double-stranded oligodeoxyribonucleotide. A voltammetric study

Binding of zinc to a 19 mer double-stranded oligodeoxyribonucleotide was investigated by anodic stripping voltammetry and cyclic voltammetry in order to understand the roles of zinc in DNA cleavage catalyzed by mung bean nuclease. These methods rely on the direct monitoring of zinc oxidation current in the absence and in the presence of the oligo. Zinc titration curves with the ds-oligodeoxyribonucleotide were obtained in concentrations ranging from 3.62 x 10(-9) to 3.62 x 10(-8) M and 4.06 x 10(-10) to 5.25 x 10(-9) M. The acquired data were used to determine the dissociation constant, stoichiometry and zinc binding sites of the complex and to understand the specific changes of ds-oligodeoxyribonucleotide secondary structure by zinc binding. The oxidation-reduction process of zinc was also investigated by cyclic voltammetry through I (oxidation current) versus v(1/2) (square root of scan rate) curves in the absence and in the presence of the double-stranded oligodeoxyribonucleotide.     

Electrochemical DNA biosensor for the study of ciprofloxacin-DNA interaction

The interaction of ciprofloxacin with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of ciprofloxacin was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current at +0.9 V was used as an indicator for the interaction mechanism in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 1.33+/-0.02 x 10(4) and 1.32+/-0.08 x 10(4) M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak currents was observed in the range of 40-80 microM ciprofloxacin, with a detection limit of 24 microM with r=0.995 and 9 microM with r=0.999 by using constant current potentiometry and differential pulse voltammetry, respectively. Moreover, the influence of sodium and calcium ions was also studied to elucidate the mechanism of ciprofloxacin-DNA interaction at different solution conditions, and this proved to be helpful in understanding the ciprofloxacin-DNA interaction.     

The potential application of a poly(3,4-ethylenedioxythiopene) modified platinum DNA biosensor in mutation analysis

In this study, we have fabricated a label free DNA biosensor by modifying the platinum wire with electrochemically synthesized poly(3,4-ethylenedioxythiopene) and poly(p-aminobenzoic acid). A designed single-strand DNA oligo was immobilized with the carboxyl group of poly(p-aminobenzoic acid) and served as the probe, a target DNA was then hybridized with the probe under a proper condition. Differential pulse voltammetry was performed to characterize the hybridization efficiency in the presence of daunorubicin hydrochloride that was able to be intercalated into the hybridized double-strand DNA and possessed the redox activity. Our results revealed a satisfied linear correlation between the peak current of differential pulse voltammetry and the concentration of complementary target DNA. On the other hand, the mismatches between the target- and probe-DNA caused a significant reduction of electrochemical response, in which was correlated with the amount of mismatched base pairs, therefore the current DNA biosensor had potential applications not only in DNA quantification but also in mutation detection for clinical diagnostics and laboratory applications.     

An ascorbic acid amperometric sensor using over-oxidized polypyrrole and palladium nanoparticles composites

We constructed a highly responsive ascorbic acid (AA) sensor utilizing over-oxidized polypyrrole (OPPy) and Palladium nanoparticles (PdNPs) composites (OPPy-PdNPs). In the presence of PdNPs, polypyrrole (PPy) was coated on a gold (Au) electrode through cyclic voltammetry (CV) and over-oxidized at a fixed potential in NaOH solution. The PdNPs were characterized using ultraviolet-visible (UV-vis) spectrum and transmission electron microscopy (TEM). The surface of OPPy-PdNPs on the Au electrode was investigated using field-emission scanning electron microscopy (FE-SEM). Results revealed that the OPPy-PdNPs-modified Au electrode (OPPy-PdNPs/Au) has the capacity to catalyze the oxidation of AA by lowering its oxidation potential to 0 V. The OPPy-PdNPs/Au electrode exhibited 2 different linear concentration ranges. In the low concentration range (1-520 μM), OPPy-PdNPs/Au exhibited a direct linear relation with current responses and had high sensitivity (570 μA mM(-1)cm(-2)) and a high correlation coefficient (0.995). In contrast, in the higher concentration range (120-1600 μM), the relationship between current responses and concentration of AA can be represented by a two-parameter sigmoidal equation. In addition, the sensor exhibited a short response time (less than 2s) and a very low limit of detection of 1 μM. The electrochemical AA sensor constructed in this study was simple, inexpensive, reproducible, sensitive, and resistant to interference. Thus, the proposed sensor has great potential for detecting AA in complex biosystems and can be applied in various fields, particularly neuroscience.     

Fentanyl decreases catecholamine metabolism measured by in vivo voltammetry in the rat locus coeruleus

The objective of this study was to investigate under controlled conditions the effects of fentanyl on the rat locus coeruleus catechol oxidation current. Using differential normal pulse voltammetry combined with electrochemically treated carbon fiber electrodes to measure the catechol oxidation current, catecholamine metabolism can be reliably monitored. Male Sprague-Dawley rats weighing 500-600 g had carbon fiber electrodes implanted into the locus coeruleus under halothane - O2 - air anesthesia with controlled ventilation and muscle relaxation. Experiments consisted of four groups of rats given the following treatments: (A) saline (n = 6); (B) fentanyl, 10 micrograms.kg-1 i.v. (n = 6); (C) naloxone, 800 micrograms.kg-1 i.v. followed 2 min later by fentanyl, 10 micrograms.kg-1 (n = 5); (D) clonidine, 200 micrograms.kg-1 i.p. (n = 6). There was no significant change in the catechol oxidation current following saline. Fentanyl produced a significant (ANOVA, p less than 0.05) decrease in the catechol oxidation current (maximum 32 min postinjection was 75.8 +/- 4.6% of baseline). This decrease was prevented by a prior injection of naloxone. Clonidine produced a significant decrease in catechol oxidation current (maximum 40 min postinjection was 54.1 +/- 7.0% of baseline). Systolic blood pressure was significantly decreased following clonidine and there were no significant changes in arterial blood gases throughout the experiments. The alpha 2-adrenergic agonist clonidine and the opioid fentanyl produced a decrease in locus coeruleus catechol oxidation current measured by in vivo voltammetry, which monitors catecholamine turnover.     

Electrochemical performance of 8-hydroxy-2'-deoxyguanosine and its detection at poly(3-methylthiophene) modified glassy carbon electrode

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) has attracted enormous attention in recent years because it has been acknowledged as a typical biomarker of oxidative DNA damage. In this paper, the electrochemical performance of 8-OH-dG at the poly(3-methylthiophene) (P3MT) modified glassy carbon electrode (GCE) was investigated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The conducting polymer P3MT can effectively decrease the oxidation peak potential of 8-OH-dG and greatly enhance its peak current. In 0.1 M pH 7.0 phosphate buffer solution (PBS), the anodic peak currents of cyclic voltammograms are linear with the 8-OH-dG concentration in two intervals, viz. 0.700-35.0 microM and 35.0-70.0 microM, with the correlative coefficients of 0.9992 and 0.9995, respectively. The detection limit of 8-OH-dG can be estimated to be 0.100 microM (S/N=3). This modified electrode can be used to detect the amount of 8-OH-dG in human urine. Furthermore, the effects of scan rate, pH, and interference of uric acid (UA) for the voltammetric behavior and detection of 8-OH-dG are also discussed. This proposed modified electrode also shows excellent reproducibility and stability that makes it an ideal candidate for amperometric detection of 8-OH-dG in flow injection analysis (FIA) and high performance liquid chromatography (HPLC).     

Effect of pH on malonyl-CoA inhibition of carnitine palmitoyltransferase I.

Malonyl-CoA inhibition of carnitine palmitoyltransferase I was found to be very pH-dependent. Malonyl-CoA concentrations causing 50% inhibition (I50) at pH 6.0, 6.5, 7.0, 7.5 and 8.0 were 0.04, 1, 9, 40 and 200 microM respectively. It is suggested that a lowering of intracellular pH, such as might occur in ketoacidosis, may attenuate hepatic fatty acid oxidation by increasing malonyl-CoA sensitivity of carnitine palmitoyltransferase I.     

Peroxidase-catalyzed co-oxidation of 3,3',5,5'-tetramethylbenzidine in the presence of substituted phenols and their polydisulfides

The steady-state kinetics of the horseradish peroxidase (HRP)-catalyzed oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) has been studied in the presence of 2-amino-4-nitrophenol (ANP), gallic acid (GA) or 4,4'-dihydroxydiphenylsulfone (DDS) and their polydisulfides poly(ADSNP), poly(DSGA), poly(DSDDS) at 20 degrees C in 10 mM phosphate buffer, pH 6.4, supplemented with 5-10% dimethylformamide. The second-order rate constants for the reactions of ANP, GA, poly(DSGA) and poly(DSDDS) with HRP-Compound I (k2) and Compound II (k3) have been determined at 25 degrees C in 10 mM phosphate buffer, pH 6.0 by stopped-flow spectrophotometry. ANP, GA and their polydisulfides strongly inhibited HRP-catalyzed TMB oxidation. Inhibition constants (Ki) and stoichiometric coefficients of inhibition (f) have been determined for these reactions. The most effective inhibitor was poly(DSGA) (Ki=1.3 microM, f=35.6). The oxidation of substrate pairs by HRP, i.e., TMB-DDS and TMB-poly(DSDDS) at pH 7.2 resulted in a approximately 8- and approximately 12-fold stimulation of TMB oxidation rates, respectively. The mechanisms of the HRP-catalyzed co-oxidation of TMB-phenol pairs are discussed.     

Antiviral activity of poly(A)-poly(U) duplexes modified with cis-diammine-dichloroplatinum (II)]

Polyribonucleotide duplex poly(A).poly(U) was modified with cis-diammine dichloroplatinum (II) (cis-DDP). It was shown that the antiinfluenza protective activity of the modified duplex in mice increased with the degree of modification (rb) rising up to 0.2. The effect was different from that for poly(I).poly(C) and poly(G).poly(C). The interferon titers in the murine brain increased in parallel with increasing of the antiviral activity. It was assumed that the structural specificity of the poly(A).poly(U) duplex was responsible for the phenomenon and that cis-DDP interaction with N(7) atoms of the adenine heterocycles blocked the "abnormal" Hoogsteen pairing of adenines with uracils. As a result the antiviral activity increased because of lowering the quantity of the intramolecular defects and increasing the length of the regular double-stranded regions.     

Effect of acute alcohol treatment on dopamine concentration in corpus striatum of rats: a voltammetric study.

A voltammetry technique has been used to determine changes in dopamine release in the rat corpus striatum after two doses of ethanol administration. The dopamine oxidation current reached a maximal value at 30 min after the first alcohol dose with a subsequent decrease towards the initial level at 60 min and kept to the basal level with a statistically insignificant oscillation. When a second dose of alcohol was applied at 60 min, it was followed by a decrease of the dopamine oxidation current peak to 50% of the initial value after another 60 min observation. The results resemble the known effect of alcohol on human behaviour (excitation followed by depression).     

Electrochemical monitoring of malondialdehyde biomarker in biological samples via electropolymerized amino acid/chitosan nanocomposite

This study reports on the electropolymerization of a low toxic and biocompatible nanopolymer with entitle poly arginine‐graphene quantum dots‐chitosan (PARG‐GQDs‐CS) as a novel strategy for surface modification of glassy carbon surface and preparation of a new interface for measurement of malondialdehyde (MDA) in exhaled breath condensate. Electrochemical deposition, as a well‐controlled synthesis procedure, has been used for subsequently layer‐by‐layer preparation of GQDs‐CS nanostructures on a PARG prepolymerized on the surface of glassy carbon electrode using cyclic voltammetry techniques in the regime of ?1.5 to 2 V. The modified electrode appeared as an effective electroactivity for detection of MDA by using cyclic voltammetry, linear sweep voltammetry, and differential pulse voltammetry. The prepared modified electrode demonstrated a noticeably good activity for electrooxidation of MDA than PARG. Enhancement of peak currents is ascribed to the fast heterogeneous electron transfer kinetics that arise from the synergistic coupling between the excellent properties of PARG and semiconducting polymer, GQDs as high density of edge plane sites and subtle electronic characteristics and unique properties of CS such as excellent film‐forming ability, high permeability, good adhesion, nontoxicity, cheapness, and a susceptibility to chemical modification. The prepared sensor showed 1 oxidation processes for MDA at potentials about 1 V with a low limit of quantification 5.94 nM. Finally, application of new sensor for determination of MDA in exhaled breath condensate was suited. In general, the simultaneous attachment of GQDs and CS to structure of poly amino acids provides new opportunities within the personal healthcare.     

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.     

8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase

The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed.     

Cobalt hydroxide nanoparticles modified glassy carbon electrode as a biosensor for electrooxidation and determination of some amino acids

The electrochemical behavior of some amino acids was investigated on cobalt hydroxide nanoparticles modified glassy carbon (CHM-GC) electrode in alkaline solution. The process of oxidation and its kinetics were established by using cyclic voltammetry, chronoamperometry techniques, and steady-state polarization measurements. The results revealed that cobalt hydroxide promotes the rate of oxidation by increasing the peak current, so these bimolecular reactions are oxidized at lower potentials. Cyclic voltammograms and chronoamperometry indicate a catalytic EC′ mechanism to be operative with electrogeneration of Co(IV) as the electrochemical process. Also, the process is diffusion controlled and the current-time responses follow Cottrellian behavior. This result was confirmed by steady-state measurements. The rate constants of the catalytic oxidation of amino acids and the electron transfer coefficients are reported.     

Amphiphilic amylose-g-poly(meth)acrylate copolymers through "click" onto grafting method

Periodate oxidation and subsequent reductive amination with propargylamine was adopted for the controlled functionalization of amylose with alkyne groups, whereas ATRP polymerization was exploited to obtain end-(α)- or end-(ω)-azide functionalized poly(meth)acrylates to be used as "click" reagents in Cu(I) catalyzed azide-alkyne [3 + 2] dipolar cycloaddition. Amylose was effectively grafted with poly(n-butyl acrylate), poly(n-butyl methacrylate), poly(n-hexyl methacrylate), and poly(dimethylaminoethyl methacrylate) with this strategy. Their structure and composition were confirmed by FT-IR, NMR spectroscopies, and thermogravimetric analysis (TGA). Dynamic and static light scattering analyses, as well as TEM microscopy showed that the most amphiphilic among these hybrid graft copolymers self-assembled in water, yielding nanoparticles with ca. 30 nm diameter.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

Antiviral activity of the complexes obtained at different ratios of complementary homopolyribonucleotides

Antiviral activity of the complexes of synthetic polyribonucleotides, i.e. poly (I).poly (C) and poly (G).poly (C) obtained at non-equimolar ratios of homopolymers was studied. The system of chick embryon fibroblasts and horse Venezuellan eguine encephalitis virus served as the model. It was shown that the active and stable complexes poly (I).poly (C) and poly (G).poly (C) were formed at some excess of poly (C), i.e. at the ratio of poly G) or poly (I) to poly (C) equal to 40/60 to 20/80 molar per cent. The role of the excessive poly (C) in formation of the stable secondary structure of the nucleotide complexes and its significance as one of the means for affecting the fine structure of double-stranded RNA were discussed.     

Electrochemical oxidation of water by a cellobiose dehydrogenase from <Emphasis Type="BoldItalic">Phanerochaete chrysosporium</Emphasis>

Cellobiose dehydrogenase (CDH) is a redox protein containing two electron transfer centers; a flavin coenzyme performing a two-electron transfer reaction and an iron-heme coenzyme facilitating single-electron transfer. Purified CDH from Phanerochaete chrysosporium was immobilized on a pyrolytic graphite electrode and electron transfer from cellobiose to the electrode was generated. With cellobiose present during cyclic voltammetry, this novel enzyme/electrode system exhibited sharp, stable oxidation peaks with slower, though equivalent, reduction peaks. During cyclic voltammetry without substrate, the enzyme was rapidly oxidized during the initial scan, with no corresponding enzyme reduction during the reducing half of the cycle. After resting for several hours in aqueous buffer, the full oxidation current appeared again. These results suggest that the CDH is reduced by water splitting, albeit at a slow rate.     

Application of electrochemically prepared polypyrrole-polyvinyl sulphonate films to DNA biosensor

Double stranded calf thymus deoxyribonucleic acid (DNA) was physisorbed onto polypyrrole-polyvinyl sulphonate (PPY-PVS) films electrochemically deposited onto indium-tin-oxide (ITO) coated glass plates. These DNA immobilized PPY-PVS films optimized for various conditions, such as polymerization potential, pH of buffer, DNA concentration and scan rate were characterized using Fourier-transform infrared (FT-IR) spectroscopy, atomic force microscopy (AFM) and cyclic voltammetry (CV) techniques, respectively. The amperometric response studies of these DNA/PPY-PVS electrodes were carried out as a function of 2-aminoantharcene (2-AA, 0.01-20 ppm) and o-chlorophenol (OCP, 0.1-30 ppm) concentration, respectively at 25 degrees C. The observed amperometric current arising due to oxidation of guanine in the DNA/PPY-PVS films decreased linearly with the increase in the concentration of 2-AA and OCP. It has been revealed that 10 ppm of 2-AA is sufficient to reduce the observed guanine oxidation peak current by approximately -95+/-10% as compared to the reported values. A 25 ppm of OCP was capable enough to reduce the guanine oxidation current to zero. These DNA/PPY-PVS electrodes were found to have a shelf life of about 4 months when stored at 25 degrees C.