A potentiometric rapid method of determining the level of phenylacetic acid during the production of beta-lactam antibiotics

A potentiometric rapid method for control of phenylacetic acid (PAA) concentration in production of ++beta-lactam antibiotics is described. The method is based on ion selective electrodes with a film membrane. The results of the theoretical and experimental studies on estimation of the electrode selectivity specific of PAA in the presence of various interfering ions are presented. It was shown possible to use the electrodes for PAA control in the media containing nitrates, bicarbonates and chlorides. Recommendations how to use the ion selective electrodes at various stages in production of ++beta-lactam antibiotics are given. Prospects for improving the method and designing an instrument for rapid assay of phenyl acetate ion activity are discussed.     

Fabrication and Evaluation of Membranes as Specific Electrodes for Calcium ions

Membranes fabricated from an inert polyvinyl chloride matrix impregnated with tributyl phosphate and with tributyl phosphate plus thenoyl trifluoroacetone were tested as electrodes specific for calcium ions. Both types of membrane exhibited a high specificity for calcium ions in the presence of sodium, magnesium, and barium ions. In the presence of perturbing ions, the usefulness of membranes is limited by both the transport of current and development of potentials by the interfering ion. An experimental method for evaluating these interfering quantities is presented.     

The standard electrode potential (Eθ) predicts the prooxidant activity and the acute toxicity of metal ions

The standard electrode potential (E^θ) has been known for many decades to predict the toxicity of metal ions. We have compiled acute toxicity data from fifteen studies and find that the toxicity of thirty metal ions correlates with E^θ at r² = 0.868 when toxicity is expressed as log concentration of comparably effective doses. We have discovered an even stronger relationship between the prooxidant activity (PA) of metal ions and E^θ (and electronegativity, χ). Data compiled from thirty-four studies demonstrate that the PA of twenty-five metal ions correlates with E^θ at r² = 0.983 (and χ at r² = 0.968). PA was commonly measured as metal-induced peroxidation of cell membranes or accumulation of reactive oxygen species. None of the redox metals (capable of Fenton-like reactions) in our studies (i.e., Mn, Fe, Co, Ni, and Cu) was prooxidative or toxic beyond what was expected from E^θ or χ. We propose that the formation of superoxide-metal ion complexes is greater at greater E^θ or χ values and that these complexes, whether free or enzyme-bound, function in PA without redox cycling of the complexed ion.     

Electrochemical assay for deoxyribonuclease I activity

A thiolated oligonucleotide having three ferrocenes was immobilized on a gold electrode through the sulfur-gold linkage. This electrode showed a current response based on the redox reaction of the ferrocene moieties and this response was decreased after treatment with deoxyribonuclease I (DNase I), suggesting the disappearance of the ferrocene moieties on the electrode by the DNase I digestion. A linear correlation between i₀ and i, which are current peaks before and after DNase I treatment, respectively, was observed and this slope was decreased with increase in the amount of DNase I. No current decrease was observed in the presence of EDTA or RNase A instead of DNase I. These results suggested that the current decrease responded specifically to the amount of DNase I and this electrode could be used for an electrochemical DNase I assay. Under the optimum conditions of DNase I digestion at 37 °C for 30 min, a quantitative analysis could be achieved in the range of 10⁻⁴-10⁻² units/μl of DNase I.     

Electrochemical assay for the determination of nitric oxide metabolites using copper(II) chlorophyllin modified screen printed electrodes

This work presents a novel electrochemical assay for the collective measurement of nitric oxide (NO) and its metabolites nitrite (NO₂⁻) and nitrate (NO₃⁻) in volume miniaturized sample at low cost using copper(II) chlorophyllin (CuCP) modified sensor electrode. Zinc oxide (ZnO) incorporated screen printed carbon electrode (SPCE) was used as a host matrix for the immobilization of CuCP. The morphological changes of the ZnO and CuCP modified electrodes were investigated using scanning electron microscopy. The electrochemical characterization of CuCP–ZnO–SPCE exhibited the characteristic quasi-reversible redox peaks at the potential +0.06 V versus Ag/AgCl. This biosensor electrode showed a wide linear range of response over NO concentrations from 200 nM to 500 μM with a detection limit of 100 nM and sensitivity of 85.4 nA μM⁻¹. Furthermore, NO₂⁻ measurement showed linearity of 100 nM to 1 mM with a detection limit of 100 nM for NO₂⁻ and sensitivity of 96.4 nA μM⁻¹. Then, the concentration of NO₃⁻ was measured after its enzymatic conversion into NO₂⁻. Using this assay, the concentrations of NO, NO₂⁻, and NO₃⁻ present in human plasma samples before and after beetroot supplement were estimated using suitable membrane coated CuCP–ZnO–SPCE and validated with the standard Griess method.     

Electrochemical behavior of neomycin at DNA-modified gold electrodes

Deoxyribonucleic acid (DNA) modified gold electrodes are prepared by the dry adsorptive method and the electrochemical behavior of neomycin and the influence of Pb(II) are studied by cyclic voltammetry, chronocoulometry, differential pulse voltammetry. It is found that in 0.01 M phosphate-buffered saline (PBS) buffer solutions (pH 7.3) at DNA/Au electrode neomycin exhibits an irreversible cathodic peak (E_p = 0.489 V), which is more positive and less sensitive compared with that at bare gold electrodes (E_p = 0.423 V). In the presence of Pb(II) the peak shifts toward positive with its height increasing. Moreover, the peak height is linear to neomycin concentration over the range of 0.15-57 μM. The interaction of Pb(II)-neomycin complex with calf thymus DNA is also studied by calculating the binding constants (K) of the Pb(II)-neomycin complex to DNA and binding site size (s) from voltammetric data (1.0 × 10⁷ M⁻¹ and 4 bp, respectively).     

Application of screen-printed microband biosensors to end-point measurements of glucose and cell numbers in HepG2 cell culture

Microband glucose biosensors were produced by insulating and sectioning through a screen-printed, water-based carbon electrode containing cobalt phthalocyanine redox mediator and glucose oxidase enzyme. Under quiescent conditions at 37 °C, at an operating potential of +0.4 V, they produced an amperometric response to glucose in buffer solutions with a sensitivity of 26.4 nA/mM and a linear range of 0.45 to 9.0 mM. An optimal pH value of 8.5 was obtained under these conditions, and a value for activation energy of 40.55 kJ mol⁻¹ was calculated. In culture medium (pH 7.3), a sensitivity of 13 nA/mM was obtained and the response was linear up to 5 mM with a detection limit of 0.5 mM. The working concentration was up to 20 mM glucose with a precision of 11.3% for replicate biosensors (n = 4). The microband biosensors were applied to determine end-point glucose concentrations in culture medium by monitoring steady-state current responses 400 s after transfer of the biosensors into different sample solutions. In conjunction with cultures of HepG2 (human Caucasian hepatocyte carcinoma) cells, current responses obtained in 24-h supernatants showed an inverse correlation (R² = 0.98) with cell number, indicating that the biosensors were applicable for monitoring glucose metabolism by cells and of quantifying cell number. Glucose concentrations determined using the biosensor assay were in good agreement, for concentrations up to 20 mM, with those determined spectrophotometrically (R² = 0.99). This method of end-point glucose determination was used to provide an estimated rate of glucose uptake for HepG2 cells of 7.9 nmol/(10⁶ cells min) based on a 24-h period in culture.     

A biosensor based on the self-entrapment of glucose oxidase within biomimetic silica nanoparticles induced by a fusion enzyme

We constructed a fusion protein (GOx-R5) consisting of R5 (a polypeptide component of silaffin) and glucose oxidase (GOx) that was expressed in Pichia pastoris. Silaffin proteins are responsible for the formation of a silica-based cell matrix of diatoms, and synthetic variants of the R5 protein can perform silicification in vitro[1]. GOx secreted by P. pastoris was self-immobilized (biosilicification) in a pH 5 citric buffer using 0.1 M tetramethoxysilane as a silica source. This self-entrapment property of GOx-R5 was used to immobilize GOx on a graphite rod electrode. An electric cell designed as a biosensor was prepared to monitor the glucose concentrations. The electric cell consisted of an Ag/AgCl reference electrode, a platinum counter electrode, and a working electrode modified with poly(neutral red) (PNR)/GOx/Nafion. Glucose oxidase was immobilized by fused protein on poly(neutral red) and covered by Nafion to protect diffusion to the solution. The morphology of the resulting composite PNR/GOx/Nafion material was analyzed by scanning electron microscopy (SEM). This amperometric transducer was characterized electrochemically using cyclic voltammetry and amperometry in the presence of glucose. An image produced by scanning electron microscopy supported the formation of a PNR/GOx complex and the current was increased to 1.58 μA cm⁻¹ by adding 1 mM glucose at an applied potential of −0.5 V. The current was detected by way of PNR-reduced hydrogen peroxide, a product of the glucose oxidation by GOx. The detection limit was 0.67 mM (S/N = 3). The biosensor containing the graphite rod/PNR/GOx/Nafion detected glucose at various concentrations in mixed samples, which contained interfering molecules. In this study, we report the first expression of R5 fused to glucose oxidase in eukaryotic cells and demonstrate an application of self-entrapped GOx to a glucose biosensor.     

Evaluation and elimination of inhibitory effects of salts and heavy metal ions on biodegradation of Congo red by Pseudomonas sp. mutant

In this study, it was attempted to evaluate the influences and also recommended some elimination methods for inhibitory effects offered by salts and heavy metal ions. Congo red dye solution treated with mutant Pseudomonas sp. was taken as a model system for study. The salts used in this study are NaCl, CaCl₂ and MgSO₄·7H₂O. Though the growth was inhibited at concentrations above 4 g/l, toleration was achieved by acclimatization process. In case of heavy metal ions, Cr (VI) showed low inhibition up to 500 mg/l of concentration, compared to Zn (II) and Cu (II). It was due to the presence of chromium reductase enzyme which was confirmed by SDS-PAGE. Zn (II) and Cu (II) ion inhibitions were eliminated by chelation with EDTA. The critical ion concentrations obtained as per Han-Levenspiel model for Cr (VI), Zn (II) and Cu (II) were 0.8958, 0.3028 and 0.204 g/l respectively.     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Stoichiometry of electrocatalytic cycle of cytochrome P450 2B4

Stoichiometry of the electrocatalytical cycle of cytochrome P450 2B4 was studied in kinetic mode according to bielectrode scheme. Graphite screen-printed electrodes with immobilized cytochrome P450 2B4 were used as the operating electrode (at the potential E^0′ = −450 mV) and electrodes, modified with cytochrome c (E^0′ = −50 mV) or Prussian Blue (E^0′ = 0), as measuring electrodes (for H₂O₂) and Clark-type electrode (for O₂). Benzphetamine N-demethylation rate was 17 ± 3 nmol/nmol of enzyme/min, peroxide production was 4.8 ± 0.7 nmol/nmol of enzyme/min (substrate-free system), 3.3 ± 0.6 nmol/nmol of enzyme/min (0.5 mM benzphetamine), the oxygen consumption rate by Р450 2В4 was 19.4 ± 0.6 nmol/nmol of enzyme/min (in the presence of benzphetamine), 4.8 ± 0.4 nmol/nmol of enzyme/min (without substrate). Based on stoichiometry of P450 electrocatalysis adequacy of electrochemical reduction and P450-monooxygenase system was revealed.     

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA > LCA > CDCA > GDCA > TDCA > CA > UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.     

A flow injection system, comprising a biosensor based on a screen-printed carbon electrode containing Meldola’s Blue-Reinecke salt coated with glucose dehydrogenase, for the measurement of glucose

A biosensor for the measurement of glucose in serum has been developed, based on a screen-printed carbon electrode modified with Meldola’s Blue-Reinecke salt, coated with the enzyme glucose dehydrogenase (from Bacillus sp.), and nicotinamide adenine dinucleotide coenzyme (NAD⁺). A cellulose acetate layer was deposited on top of the device to act as a permselective membrane. The biosensor was incorporated into a commercially available, thin-layer, amperometric flow cell operated at a potential of only +0.05 V versus Ag/AgCl. The mobile phase consisted of 0.2 M phosphate buffer (pH 7.0) containing 0.1 M potassium chloride solution, and a flow rate of 0.8 ml min⁻¹ was used throughout the investigation. The biosensor response was linear over the range of 0.075-30 mM glucose, with the former representing the detection limit. The precision of the system was determined by carrying out 20 repeat injections of a 5-mM glucose standard, and the calculated coefficient of variation was 3.9%. It was demonstrated that this biosensor system could be applied to the direct measurement of glucose in serum without pretreatment. Therefore, this would allow high-throughput analysis, at low cost, for this clinically important analyte.     

Optimization of a polypyrrole glucose oxidase biosensor

An amperometric glucose biosensor was fabricated by the electrochemical polymerization of pyrrole onto a platinum electrode in the presence of the enzyme glucose oxidase in a KCl solution at a potential of + 0·65 V versus SCE. The enzyme was entrapped into the polypyrrole film during the electropolymerization process. Glucose responses were measured by potentio-statting the enzyme electrode at a potential of + 0·7 V versus SCE in order to oxidize the hydrogen generated by the oxidation of glucose by the enzyme in the presence of oxygen. Experiments were performed to determined the optimal conditions of the polypyrrole glucose oxidase film preparation (pyrrole and glucose oxidase concentrations in the plating solution) and the response to glucose from such electrodes was evaluated as a function of film thickness, pH and temperature. It was found that a concentration of 0·3 M pyrrole in the presence of 65 U/ml of glucose oxidase in 0·01 M KCl were the optimal parameters for the fabrication of the biosensor. The optimal response was obtained for a film thickness of 0·17 μm (75 mC/cm²) at pH 6 and at a temperature of 313 K. The temperature dependence of the amperometric response indicated an activation energy of 41 kJ/mole. The linearity of the enzyme electrode response ranged from 1·0 mM to 7·5 mM glucose and kinetic parameters determined for the optimized biosensors were 33·4 mM for the K_m and 7·2 μA for the I_max. It was demonstrated that the internal diffusion of hydrogen peroxide through the polypyrrole layer to the platinum surface was the main limiting factor controlling the magnitude of the response of the biosensor to glucose. The response was directly related to the enzyme loading in the polypyrrole film. The shelf life and the operational stability of the optimized biosensor exceed 500 days and 175 assays, respectively. The substrate specificity of the entrapped glucose oxidase was not altered by the immobilization procedure.     

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (ΔV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.     

Nickel(II)-baicalein complex modified multiwall carbon nanotube paste electrode and its electrocatalytic oxidation toward glycine

A modified electrode, nickel(II)-baicalein complex modified multiwall carbon nanotube paste electrode (Ni(II)-BA-MWCNT-PE), has been fabricated by electrodepositing Ni(II)-BA complex on the surface of MWCNT-PE in alkaline solution. The Ni(II)-BA-MWCNT-PE exhibits the characteristic of improved reversibility and enhanced current responses of the Ni(III)/Ni(II) couple compared with Ni(II)-BA-carbon paste electrode (CPE). It also shows better electrocatalytic activity toward the oxidation of glycine than Ni(II)-MWCNT-PE. Kinetic parameters such as the electron transfer coefficient α, rate constant k_s of the electrode reaction, the diffusion coefficient D of glycine, and the catalytic rate constant k_cat of the catalytic reaction are determined. Moreover, the catalytic currents present linear dependence on the concentration of glycine from 20 μM to 1.0 mM by amperometry. The detection limit and sensitivity are 9.2 μM and 3.92 μA mM⁻¹, respectively. The modified electrode for glycine determination is of the property of simple preparation, fast response, and good stability.     

Tailoring Electrode/Electrolyte Interfacial Properties in Flexible Supercapacitors by Applying Pressure

Electrode/electrolyte interfacial properties of flexible supercapacitors assembled with nanostructured activated carbon fabric (ACF) electrodes can be tailored by applying a pressure and tuning electrolyte ion size relative to electrode pore size. Experimental results reveal that increasing pressure between the supercapacitor electrodes can significantly improve capacitive performance. The ratio of solvated ion size in the electrolyte to the pore size on the electrodes determines the minimum pressure necessary to achieve an optimum performance. For a specific electrode material, this minimum pressure for optimum performance is primarily governed by the size of the larger solvated ions (either the anions or cations), and is lower (～689 KPa) when the ratio of the solvated ion size to the pore size is higher than 0.6, and is higher (at least 1379 KPa) when the ratio is lower than 0.6. An analytical model capable of predicting the experimental performance data has been developed. These results together provide a fundamental understanding of pressure dependence of electrode/electrolyte interfacial properties and pave the way for practical applications of flexible supercapacitors.     

Interferences of suspended clay fraction in protein quantitation by several determination methods

Seven current methods of protein quantitation, Bradford (standard, micro, and 590/450 nm ratio), Lowry, bicinchoninic acid (BCA), UV spectrophotometry at 280 nm, and Quant-iT fluorescence-based determination, were compared with regard to their susceptibility to interferences due to the presence of suspended and not easily detectable clay particles. Bovine serum albumin (BSA) and Na-Wyoming montmorillonite were selected as model protein and reference clay, respectively. Protein-clay suspension mixtures were freshly prepared for each assay to simulate supernatants not completely centrifuged in batch sorption/kinetic experiments. Seven fixed increasing levels of clay (0.0, 0.00725, 0.0145, 0.029, 0.058, 0.145, 0.435 mg ml⁻¹) were mixed with different levels of BSA in an appropriate range for each assay. To ascertain the interfering effect of different levels of clay, the theoretical concentrations of BSA were plotted against the estimated BSA concentrations of the samples, as obtained from the calibration curve of each method. A correct quantitation of the BSA concentration not influenced by clay would be described by a regression line with slope (b) not significantly different from 1 and an intercept (a) not significantly different from zero. At the lowest clay levels (0.00725 mg ml⁻¹) a significant interference was evident for Bradford micro, Bradford 590/450, UV, and fluorescence. The three methods (Bradford standard, Lowry, and BCA) that seemed to show the better performances in the presence of clay after this first screening step also underwent an ANCOVA analysis, with the measured BSA concentrations as dependent variable and the clay concentrations as covariate. The Bradford standard and BCA methods were affected by a clay-dependent interference on BSA quantitation. The Lowry assay was the only method that gave correct estimates of BSA concentrations in the presence of any of the clay levels tested.     

Study of the biosorption of different heavy metal ions onto Kraft lignin

The ability of Kraft lignin, a waste product of paper production, for removing copper, zinc, cadmium and chromium ions from water was investigated. The studies were conducted by a batch method to determine equilibrium parameters. The adsorbed heavy metal ions followed the order: Cr(VI) ? Cd(II) > Cu(II) > Zn(II). The influence of other ions such as Ni(II), Cd(II) and Pb(II), on Cu(II) adsorption by Kraft lignin was evaluated. Obtained results support the idea that adsorption behaviour of heavy metal ions have to be perceived from the aspect of possible influence of interfering ion species.