Nitrate ion-selective electrode in microbial media

A nitrate ion-selective electrode has been used to follow the kinetics of the reduction of nitrate by a bacillus isolated from the rumen of a sheep. The electrode system and the microbial culture were found to be quite compatible, although the formation of nitrite limits the utility and precision of the measurement. The formation of nitrite was followed simultaneously by a colorimetric method. Nitrate was converted quantitatively to nitrite. The rate of the reaction was found to increase to a plateau value, then drop abruptly when the nitrate was exhausted.     

Approaches to KL a measurements in solid state fermentation

Summary A convenient method for measuring K_L a in a solid state medium is proposed. Due to the particular nature of the substrate used in solid state fermentation, different modifications of the sulfite oxidation method have been necessary. This first approach allows to study the influence of air inflow rate and dry matter percentage of the medium on the oxygen volumetric mass transfer coefficient.     

Control of membrane potential by external H+ concentration in Bacillus subtilis as determined by an ion-selective electrode

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly--(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonimum ion. It was found that the tetraphenylphosphonium ion was not countertransported against H+ movement. The membrane potential of Bacillus subtilis was estimated to be 80-120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H+] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H+ equilibrium potential. The addition of N,N'-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K+] did not affect the pH dependence. CN- partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.     

Control of membrane potential by external H concentration in Bacillus subtilis as determined by an ion-selective electrode

The membrane potential of intact bacteria was monitored by measuring the tetraphenylphosphonium ion distribution across the membrane using poly-(vinyl chloride) matrix-type electrode selective to tetraphenylphosphonium ion. It was found that the tetraphenylphosphonium ion was not countertransported against H⁺ movement. The membrane potential of Bacillus subtilis was estimated to be 80–120 mV inside-negative at external pH 7. The effect of the external pH on the membrane potential was studied. It varied from 30 to 40 mV/decade change in the external [H⁺] in the pH region of greater than 6.5, increasing pH making it more inside-negative. The addition of carbonyl cyanide m-chlorophenylhydrazone depolarized the membrane, and the membrane potential approached the H⁺ equilibrium potential. The addition of N,N′-dicyclohexylcarbodiimide did not abolish the pH dependence of the membrane potential. Increasing the external [K⁺] did not affect the pH dependence. CN⁻ partially depolarized the membrane. A parallel conductance model for membrane potential could explain the results qualitatively.     

Development of a multiple-bile-ion-sensing membrane electrode

A multiple-bile-ion-sensing polyvinyl chloride-based membrane electrode capable of monitoring any of the three common bile ions in humans, namely, cholate, deoxycholate, and chenodeoxycholate, was developed and characterized. Compared to single-bile-ion-sensing electrodes, it showed a sub-Nernstian response. All other electrode properties were, however, similar, making this a successful replacement for three individual electrodes. With appropriate conditioning, this electrode could repeatedly change selectivity without losing membrane activity. It was reproducible, was stable for 5 months, had low response time, and could be used to measure critical micelle concentrations. The lower limit of detection was 10 nM. Selectivity coefficients for various anions with respect to bile ions more or less followed the Hoffmeister series. Plots of R ((Nernst equivalent of slope in the presence of primary ion and a fixed amount of interfering ion)/(slope in the presence of only the primary ion)) vs square root of ionic strength for an interfering ion were linear. One major application of this electrode is its use in kinetics. We have tested its ability to monitor continuously changing bile ion concentrations during their interactions with a biocompatible polymer, polyethylene glycol (6000), and determined rate constants.     

Cationic membrane conductances induced by intracellularly elevated cAMP and Ca2+: measurements with ion-selective microelectrodes

Adenosine 3',5'-cyclic monophosphate (cAMP) and CaCl2 were injected by a fast and quantitative pressure injection technique into voltage-clamped, identified Helix neurons. Intracellular elevation of cAMP as well as of Ca2+ activated an inward current (IcAMP and IN). To identify the ionic fluxes during IcAMP and IN changes in [Na+]i, [K+]o, [H+]i, and [Cl-]i were measured with ion-selective microelectrodes (ISMs). Near resting potential, Na+ was the main carrier of IcAMP. K+, and less effectively Ca2+, could substitute for Na+ in carrying IcAMP. H+ and Cl- were excluded as current carriers for IcAMP by means of ISMs. Simultaneous to this action, cAMP decreased a K+ conductance. This decrease was associated with a reduction of the K+ efflux activated by long-lasting depolarizing voltage steps, as directly measured with ISMs located near the external membrane surface. The nearly compensatory increase and decrease of two membrane conductances in the same neuron left the cell input resistance unchanged despite the considerable depolarizing action of intracellularly elevated cAMP. IN was also of nonspecific nature. However, our findings indicate less selectivity for the Ca2+-activated nonspecific channels. Large cations such as choline, TEA, and Tris passed nearly as well as Na+ through the channels. Measurements with ISMs showed that [H+]i and [Cl-]i were unchanged during IN. IN was largest in bursting pacemaker neurons compared with other cells of similar size. It was found to be essential for the burst production in these cells. IcAMP, on the other hand, might be involved in the presynaptic facilitatory action of cAMP, which as yet was attributed solely to a reduction of a K+ conductance.     

Thermodynamics of ATP hydrolysis from membrane electrode measurements of metal-ion ATP and ADP complexation

Free energies for the ATP-ADP hydrolysis reaction have been recalculated on the basis of new thermodynamic formation constants for metal-ion ATP and ADP complexes as determined by potentiometric measurements with ion-selective membrane electrodes. It is shown that free-energy maps are sharply altered at metal-ion levels greater than 10^?3m because of the effect of previously unrecognized 2:1 complexes of divalent metal ions with ATP and ADP. New formation constants for ADP complexes with sodium, potassium, calcium, and magnesium are also reported.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Kinetic study of the reaction between trinitrobenzenesulfonic acid and amino acids with a trinitrobenzenesulfonate ion-selective electrode

Potentiometric studies of the reaction between trinitrobenzenesulfonic acid (TNBS) and several amino acids with the TNBS electrode indicate that the reaction is first-order with respect to TNBS and amino acid concentration. The reaction is zero-order with respect to hydroxide concentration at pH greater than 10.5, indicating that the nonprotonated amino group is the reactive species. Rate constants were calculated for each amino acid and a simple mechanism of the reaction is proposed.     

Polymer membrane ion-selective electrodes as a convenient tool for lipases and esterases assays

We have proposed a novel assay for lipases and esterases activity determination based on potentiometry with ion-selective electrodes (ISEs). Enzyme preparations, obtained from the living cells, are complex mixtures of various proteins, short peptides, lipids, carbohydrates, and other compounds. The most commonly used quantitative methods in enzyme studies are based on spectrophotometric or spectroflourimetric protocols which has significant limitations. They are not valid for samples that are turbid or strongly colored. To overcome those drawbacks we have proposed an assay based on potentiometry with ISEs for lipases and esterases activity determination. This electrochemical methodology represents an attractive tool for enzyme analysis, because of its low detection limit, independence from sample volume and from sample turbidity. The usefulness of this assay has been proven by the determination of the activity of various raw enzymes “acetone powders” isolated from animal tissues. Moreover, activities of fractions obtained during purification of one of those raw biocatalysts were also determined that way. The reliability of determination enzyme activity with ISE assay was proven by comparison with a classical spectrophotometric method.     

Ag-AgCl electrode noise in high-resolution ECG measurements

The authors measured the noise and impedance from face-to-face Ag-AgCl electrode pairs, as well as the noise from Ag-AgCl electrodes placed on the human body surface, in the frequency band from 0.5 Hz to 500 Hz, which corresponds to high-resolution ECG measurements. Electrode noise and electrode impedance were measured simultaneously to compare electrode noise with the thermal noise from the real part of electrode impedance. The results show that electrode noise depends on electrode area, electrolytic gel, the patient, and the placement site. In the frequency band from 0.5 Hz to 500 Hz, root-mean-square electrode noise is typically less than 1 microV for electrodes placed face-to-face and ranges from 1 microV to 15 microV for electrodes on the body surface. The noise spectral density increases at low frequencies as 1/fa and it is always higher than the thermal noise from the real part of the electrode impedance. There is a high correlation between electrode dc offset voltage and electrode noise. Thus, offset voltage measurements allow identification of noise from low-noise electrodes.     

Bacterial oxidation of polythionates: determination of tetrathionate with an ion-selective electrode.

A commercially available ion-selective electrode for nitrate was used to continuously monitor tetrathionate oxidation by Thiobacillus dentrificans. The electrode was much more sensitive to tetrathionate than to nitrate. The same electrode could also be used for the determination of trithionate.     

Chlorophyll a fluorescence transients: a fast data acquisition system to facilitate in vivo measurements

Characteristics of primary phases in chlorophyll a fluorescence transients based on room temperature in vivo measurement with a Plant Productivity Fluorometer (Brancker Model SF-10) can be greatly facilitated by coupling the instrument to a fast data acquisition system. The SF-10 was linked to a Multitech Industrial Corporation Microprofessor Microcomputer and further modified to ensure simultaneous onset of light activation and signal capture. Circuit diagrams and program listings are given in detail. This microprocessor system is capable of capturing signal changes over a minimum period of 200 milliseconds to a maximum of 6 seconds. Accuracy of recorded data is dependent on rate of change of the input signal and the recording time period. Acquisition and storage of 5000 points from zero to 300 milliseconds ensured clear resolution of F_o, I and D when played back over 120 seconds on a chart recorder. For routine use, the primary transient can be captured over 0–2 seconds and then replayed as an accompaniment to standard slower presentation of primary plus secondary transients. Coincidence of signal amplitude for F_p on both systems can then be ascertained while retaining adequate resolution of F_o and I.     

Chlorophyll a fluorescence transients: a fast data acquisition system to facilitate in vivo measurements

Characteristics of primary phases in chlorophyll a fluorescence transients based on room temperature in vivo measurement with a Plant Productivity Fluorometer (Brancker Model SF-10) can be greatly facilitated by coupling the instrument to a fast data acquisition system. The SF-10 was linked to a Multitech Industrial Corporation Microprofessor Microcomputer and further modified to ensure simultaneous onset of light activation and signal capture. Circuit diagrams and program listings are given in detail. This microprocessor system is capable of capturing signal changes over a minimum period of 200 milliseconds to a maximum of 6 seconds. Accuracy of recorded data is dependent on rate of change of the input signal and the recording time period. Acquisition and storage of 5000 points from zero to 300 milliseconds ensured clear resolution of F_o, I and D when played back over 120 seconds on a chart recorder. For routine use, the primary transient can be captured over 0–2 seconds and then replayed as an accompaniment to standard slower presentation of primary plus secondary transients. Coincidence of signal amplitude for F_p on both systems can then be ascertained while retaining adequate resolution of F_o and I.     

Quantitative measurements of NO reaction kinetics with a Clark-type electrode.

Nitric oxide (NO) plays important physiological roles in the body. Knowledge regarding the kinetics of NO catabolism is important for understanding the biological functions of NO. Clark-type NO electrodes have been frequently employed in measuring the kinetics of NO reactions; however, the slow response time of these electrodes can cause measurement errors and limit the application of the electrode in measurements of fast NO reactions. In this study, a simplified diffusion model is given for describing the response process of the NO electrode to the change of NO concentration. The least-square method is used in fitting the currents calculated from the diffusion equation to the experimental curves for determining the diffusion parameters and rate constants. The calculated currents are in excellent accordance with the experimental curves for different NO reaction kinetics. It has been demonstrated that when using an NO electrode with a response time of approximately 6 s to measure fast NO reactions with a half-life of approximately 1s, the response currents of the electrode have large differences compared to the curve of actual NO concentration in the solution; however, the rate constant of NO decay can still be accurately determined by computer simulations with the simplified diffusion model. Theoretical analysis shows that an NO electrode with a response time of 6 s (D/L2=0.06 s-1) and the lowest detection limit of 1 nM NO can be used in measuring kinetics of extremely rapid NO reactions with a half-life below 10 ms.     

Microelectrode of the Thomas type using a liquid membrane electrode

By replacing the glass-based pH electrode (L. R. Pucacco, S. K. Corona, H. R. Jacobson, and N. W. Carter (1986) Anal. Biochem. 153, 251-261) with a liquid membrane-based pH electrode, a relatively easy-to-manufacture modified Thomas electrode has been developed. The liquid membrane-based modified Thomas electrode can be manufactured without the special equipment (forge) and materials (glass) required to make the glass membrane pH microelectrode (L. R. Pucacco and N. W. Carter (1976) Anal. Biochem. 73, 501-512). The sensitivity (57.4 +/- 0.22 mV/pH unit), response time (20.0 +/- 2.67 s), and electrical resistance (3.48 +/- 0.67 X 10(11) ohm) of this electrode are similar to those of the glass-based version.     

Estimation with an ion-selective electrode of the membrane potential in cells of Paracoccus denitrificans from the uptake of the butyltriphenylphosphonium cation during aerobic and anaerobic respiration.

1. Aerobic respiration by cells of Paracoccus dentrificans drives the uptake of the lipophilic cation butyltriphenylphosphonium. Anaerobiosis or addition of an uncoupler of oxidative phosphorylation (carbonyl cyanide p-trifluoromethoxyphenylhydrazone) results in efflux of the cation. Changes in the concentration of butyltriphenylphosphonium in the suspension medium were measured by using an ion-selective electrode, the construction of which is described. 2. If the uptake of butyltriphenylphosphonium is used as an indicator of membrane potential, then at pH 7.3 an estimate of about 160 mV is obtained for cells of P. dentrificans respiring aerobically in 100 mM-Hepes [4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid/NaOH or 100mM-NaH2PO4/NaOH. This potential, however, is decreased by more than 20 mV in reaction media containing a high concentration of phosphate (100 mM) together with at least 1 mM-K+. 3. Anaerobic electron transport with NO3-, NO2- or N2O as terminal electron acceptor generates a membrane potential of about 150mV in described suspension media. The presence of these species under aerobic conditions, moreover, has negligible effect upon the extent of uptake of butyltriphenylphosphonium normally driven by aerobic respiration. These data indicate that none of these molecules exert a significant uncoupling effect on the protonmotive force. 4. No 204Tl+ uptake into respiring cells was detected. This adds to the evidence that 204Tl+ is not a freely permeable cation in bacterial cells and therefore not an indicator of membrane potential as has been proposed. The absence of respiration-driven 204Tl+ uptake indicates that P. denitrificans cells grown under the conditions specified in the present work do not possess K+-transport systems of either the Kdp or TrkA types that have been described in Escherichia coli.