Construction of a N-Acetyl-L-methionine Electrode and Determination of Acylase with an Ammonia Gas Sensor

Abstract

The N-acetyl-L-methionine electrode is based on a coupled enzymatic system consisting of acylase and L-amino acid oxidase with an ammonia gas sensor; conditions of imobilization are optimized. N-acetyl-L-methionine in the range 4×10^?5–2×10^?3M gives a linear potential vs. log(concentration) plot with a response time of 2–5 min over the range specified. This electrode combined with an L-methionine electrode, based only on L-amino acid oxidase and an ammonia gas sensor, can be used for the determination of both substrates in mixtures, thus extending the feasibility of the method. Acylase (0.1–2.00) is determined in aqueous solutions by adding N-acetyl-L-methionine to the sample, and measuring the ammonia evolved with the gas-sensing electrode.     

A bio-selective membrane electrode prepared with living bacterial cells.

A novel potentiometric sensor has been devised by coupling intact microorganisms (Streptococcus faecium) with an ammonia gas-sensing membrane electrode. The resulting electrode provides a linear response to arginine over the concentration range 5.0 × lO^-5–1. 0 × 10^-3 M in phosphate buffer pH 7.4, with selectivity over other amino acids. The slope of the calibration graph is -40 to -45 mV/decade during the period 2–20 days after preparation. This membrane electrode with living bacterial cells may serve as a model for the development of other new sensing systems.     

Induced bacterial electrode for the potentiometric measurement of tyrosine

A bacterial tyrosine-selective potentiometric electrode is proposed in which the desired biocatalytic activity is biochemically induced during growth of the bacterial cells. As the result of this induction, a normally ineffective biocatalyst, Aeromonas phenologenes ATCC 29063 can be coupled with an ammonia gas-sensing electrode in order to produce a useful tyrosine-selective electrode. The sensor shows excellent response characteristics, having a slope of 50–58 mV/decade, a range of logarithmic response from 8.3 × 10^-5 M to 1.0 × 10^-3 M tyrosine, a lower limit of detection of 3.3 × 10^-5 M tyrosine, response times of 4–6 min, and a useful lifetime in excess of one week. Specific enzyme inhibitors are employed to enhance the selectivity of the electrode while maintaining high biocatalytic activity with respect to tyrosine.     

Selective kinetic determination of pyridoxal-5'-phosphate with an ammonia gas-sensing electrode

The coenzyme pyridoxal-5'-phosphate (PLP) is deiermined by in situ measurement of the rate of ammonia production using a potentiometric gas-sensing membrane electrode. It is shown that the initial rate of ammonia liberation from L-tryptophan by action of tryptophanase apoenzyme and the coenzyme can be linearly related to PLP levels in the nanogram range with minimal interference from related compounds. Calibration data for the 1 × 10^-7–2.5 × 10^-6 M range for PLP yielded a least-squares equation of rate (mV min^-1) = (0.64 ± 0.01) C — 0.01 ± 0.02 with a standard error of 0.02 mV min^-1, where concentration, C, is expressed in units of 10^-7 mol l^-1.     

Tissue- and bacteria-loaded tubular reactors for the automatic determination of glutamine

Porcine kidney tissue or Sarcina Flava bacterial cells are used as biocatalysts for the conversion of glutamine to ammonia, which is monitored with a gas-sensing membrane electrode in an automated flow system. Conversion to ammonia is 100% for ? 10^-4 M glutamine.     

Kinetic determination of l-alanine and l-alanine dehydrogenase with an ammonia gas-sensing electrode

The ammonia gas-sensing electrode is used to assay l-alanine and l-alanine dehydrogenase. Alanine is de-aminated by bacterial alanine dehydrogenase in the presence of β-NAD⁺. The initial rate of ammonia release is proportional to alanine concentration or the enzyme activity. Optimal conditions for the assays are specified. Alanine (1.0 × 10^?4-1.0 × 10^?3 M in a 1-ml sample) and enzyme (0.181-0.181 U in a 0.1-ml sample) can eb determined. Application to the determination of alanine in human serum gave results that compared well with values obtained by the Yoshida method.     

Determination of glutamine in cerebrospinal fluid with a tissue-based membrane electrode

A glutamine-selective sensor consisting of porcine kidney tissue immobilized at an ammonia gas electrode is utilized. It yields good precision and accuracy over the clinically important range of glutamine concentrations (10^-4–10^-2 M).     

Novel surface acoustic wave-interdigitated array electrode gas sensor for dissolved ammonia

 A novel surface acoustic wave-interdigitated array electrode (SAW-IDA) ammonia gas sensor is proposed. A gas-permeable membrane is employed to separate the buffer solution in the inner cell of the gas-sensing probe from the sample solution in the detection cell. The response of the IDA conductive electrodes is based on the impedance change of the buffer solution during ammonia adsorption. Therefore, this gas sensor overcomes the influence of water vapour in the conventional film-coated SAW gas sensor and can be used for the detection of gases in aqueous solutions. The ammonia sensor exhibits a favourable frequency response to 5×10^-7–1×10^-3 mol/l ammonia. The optimal buffer composition and probe parameters have been determined. Dynamic range, response time, selectivity, and temperature drift are discussed. The ammonia sensor was also applied to the determination of serum ammonia. Results were in good agreement with those from the conventional enzymatic-spectrophotometric method. Received: 1 December 1995/Revised: 9 April 1996/Accepted: 14 April 1996     

Enzyme electrode system for oxalate determination utilizing oxalate decarboxylase immobilized on a carbon dioxide sensor

A highly selective enzyme electrode system for oxalate is described in which the enzyme oxalate decarboxylase is immobilized on a carbon dioxide gas-sensing electrode. The response of the system is linear with the logarithm of the oxalate concentration between 2 × 10^-4 and I × 10^-2 M with a slope of 57–60 mV/decade. The oxalate detection limit is 4 × 10^-5 M. Electrodes used with chemically immobilized enzyme are not affected by phosphate and sulfate at levels normally found in urine and are very stable showing no decrease in response after one month of operation. The enzyme electrode system functions well in urine, requiring minimal sample pretreatment. The recovery of oxalate added to five aliquots of a human control urine sample averaged 97.7% with an average relative standard deviation of 4.5%.     

Novel surface acoustic wave-interdigitated array electrode gas sensor for dissolved ammonia

A novel surface acoustic wave-interdigitated array electrode (SAW-IDA) ammonia gas sensor is proposed. A gas-permeable membrane is employed to separate the buffer solution in the inner cell of the gas-sensing probe from the sample solution in the detection cell. The response of the IDA conductive electrodes is based on the impedance change of the buffer solution during ammonia adsorption. Therefore, this gas sensor overcomes the influence of water vapour in the conventional film-coated SAW gas sensor and can be used for the detection of gases in aqueous solutions. The ammonia sensor exhibits a favourable frequency response to 5 × 10⁻⁷−1 × 10⁻³ mol/1 ammonia. The optimal buffer composition and probe parameters have been determined. Dynamic range, response time, selectivity, and temperature drift are discussed. The ammonia sensor was also applied to the determination of serum ammonia. Results were in good agreement with those from the conventional enzymatic-spectro-photometric method.     

An enzymatic method for the kinetic measurement of L-asparaginase activity and L-asparagine with an ammonia gas-sensing electrode

A simple kinetic method to assay L-asparaginase and L-asparagine with an ammonia gas-sensing electrode is described. The method is based upon the de-amination of L-asparagine by L-asparaginase from Escherichia coli, resulting in the production of ammonia. The initial rate (mV/min) of ammonia release is proportional to the activity of L-asparaginase and also to the concentration of L-asparagine in the presence of a large amount of the enzyme. Optimal temperature, buffer composition and pH for the assays are specified. L-Asparaginase was determined in the range of 0.4-1.6 U in a 0.1 ml sample; the recovery was 98.1-103.8% for 16 determinations and sigma n was 1.59. L-Asparagine was determined in the concentration range of 1 x 10(-4)--1 x 10(-3) M with sigma n-1 1.92. The method was applied to the determination of 1-5 x 10(-4) M asparagine added to human serum with sigma n-1 1.96 for 5 determinations.     

Flow injection analysis for glucose and urea with enzyme reactors and on-line dialysis

A flow injection system for glucose and urea determination is described. The glucose determination uses immobilized glucose oxidase in a reactor designed to give 100% substrate conversion. The hydrogen peroxide formed is converted to a coloured complex with 4-aminophenazone and N,N-dimethylaniline. The coupling is catalysed by a reactor containing immobilized peroxidase. The coloured complex is measured in a flow-through spectrophotometric cell. Urea is converted to ammonia in a reactor with immobilized urease and detected with an ammonia gas membrane electrode. Proteins and other interfering species from serum samples are removed in an on-line dialyzer. Calibration curves are linear for glucose in the range 1.6 × 10^-4–1.6 × 10^-2 M and for urea in the range 10^-4–10^-1 M. The samples are 25 μl for glucose determination and 100 μl for urea determination. Linear ranges can be changed by varying the sample sizes. The effects of the dialyser, enzyme reactors and detectors on dispersion are evaluated.     

An Enzyme microsensor for urea based on an ammonia gas electrode

A urea microsensor was fabricated by immobilizing urease at the tip (10-μm diameter) of a rapidly responding ammonia gas microelectrode based on antimony. The construction and evaluation of both the urea senson and the ammonia electrode are described in detail. The urea sensor responds to 10^?2?10^?4 M urea in 30–45 s.     

Electrochemical behavior and detection of guanine using a sodium montmorillonite-modified carbon paste electrode

Herein, a sodium montmorillonite-modified carbon paste electrode is described for the electrochemical determination of guanine. Guanine yields a well-defined and very sensitive oxidation peak at the sodium montmorillonite-modified carbon paste electrode. Compared with the unmodified carbon paste electrode, the modified electrode facilitates the electron transfer of guanine, since it notably increases the oxidation peak current and lowers the oxidation overpotential of guanine. Based on this, a simple sensitive reliable electrochemical method is proposed for the detection of guanine after all the experimental parameters, such as solution pH value, sodium montmorillonite content in the carbon paste electrode, accumulation potential, and time, are optimized. Under the optimized conditions, the oxidation peak current of guanine varies linearly with its concentration in the range 5.0×10⁻⁸ to 2.0×10⁻⁵ M and the detection limit (signal-to-noise=3) is 2.0×10⁻⁸ M after 4-min accumulation. This method is successfully demonstrated with urine samples. Published in Russian in Elektrokhimiya, 2006, Vol. 42, No. 2, pp. 178–182. The text was submitted by the authors in English.     

An enzyme electrode based on immobilized arylsulfatase for the selective assay of sulfate ion

An enzyme electrode has been constructed for the assay of sulfate ion based on inhibition of the reaction

The steady-state current arising from oxidation of the product, 4-nitrocatechol, is measured at +0.8 V vs. S. C. E. The competitive inhibition of this reaction by added sulfate ion causes a decrease in this steady-state current in a linear relationship to pSO₄ in the range 2–4. The enzyme arylsulfatase (arylsulfate sulfohydrolase, EC 3.1·6.1) is chemically immobilized in a layer on a platinum electrode. This enzyme electrode also gives linear calibration plots for phosphate ion (10^-2–10^-4 M) based on its competitive inhibition of the above reaction, and for fluoride ion (10^-2–10^-4 M) based on its activation of the reaction. The assay of 4-nitrocatechol sulfate (NCS) in the range 10^-6–10^-4 M is possible. By proper control of the NCS concentration the electrode can be made almost completely specific for sulfate: only molybdate interferes. To establish the best operating conditions for the electrode, the effect of pH on the V_m and K_m were determined.     

Semiconductor gas sensors in bioreactor control

A simple semiconductor gas sensor (TGS 812) is used for the on-line measurement and control of indole during the production of l-tryptophan from indole and l-serine with immobilized E. coli cells. Indole is estimated in the reactor gas space. In combination with an automatic indole supply system, a feed-batch process became possible. The indole concentration was monitored and kept within the optimal range (300–600 mg l^?1). A simple gas-sensing electrode dipped in the reaction medium provides direct measurement of organic solvents and gases in the liquid. Such a system is suitable for on-line determination of ethanol (10–70 g l^?1) during continuous production of ethanol with immobilized yeast cells.     

Rate Study of the Alkline Hydrolysis of Nicotinamide Using an Ammonia Gas-Sensing Electrode

Abstract

The hydrolysis of nicotinamide in alkaline solutions was studied. An ammonia gas-sensing electrode was used to follow the formation of ammonia. A technique making use of simulated reactions has been developed to calibrate the electrode under dynamic conditions overcoming problems arising because of the relatively slow response of the sensor. A general expression has been derived for the pseudo first-order rate constant valid over the concentration range 0.005 to 0.10 M nicotinamide, 0.1 to 0.5 M hydroxide and the temperature range 22° to 31° C, under constant ionic strength (0.5 M NaOH + NaC1O₄).     

An enzyme reactor electrode for urea determinations.

An enzyme reactor electrode system for the determination of urea is described. A buffer is pumped through an enzyme reactor (0.4 ml) containing urease immobilized with glutaraldehyde to glass. The effluent is mixed with sodium hydroxide pumped through a second channel and fed through an ammonia gas electrode. Samples are introduced via a third flow channel and mixed with the buffer. The conversion of urea to ammonia is quantitative for sample concentrations of less than 0.03 M for a flow rate of 40 ml h^-1. The reactor electrode shows a Nernstian slope of 57 mV/decade for 5·10^-5–3·10^-2 M urea. The response is independent of variations in the flow rate, enzyme activity or temperature of the reactor.     

Calcium ion selective membrane electrode

The calcium salts of the mono- and diesters of [4-(1,1,3,3-tetramethylbutyl)phenyl phosphoric acid] have been prepared, and the individual esters as well as mixtures of the esters have been used with several varieties of polyvinyl chloride to construct macro membrane electrodes selective to calcium ions. These electrodes have been calibrated by using solutions of CaCl₂ and Ca ion buffers. The mixed ester electrodes showed Nernstian response in the concentration range 10^-1 to 10^-7M; the diester electrodes showed Nernstian response down to 7.9 x 10^-8M. The detection limit of the mixed ester electrode was 10^-8M, whereas that of the diester electrode was 7.9 x 10^-9M. Contrary to these results, the monoester electrodes showed unsatisfactory behavior. The responses of both the mixed ester and diester electrodes to calcium ions were not affected by the presence of sodium, potassium, or other divalent ions. Only ferric and lanthanum ions showed interferences with the electrode response to calcium ions. p]The electrode response was independent of pH in the approximate range 5–8 at a CaCl₂ concentration of 10^-4M. As the Ca ion concentration was increased, the range of pH independence widened to approximately 4–8. The dynamic response time constant of the mixed ester electrode was in the range 0.7–1.5 sec, whereas that of the diester electrode was in the range 0.5–0.75 sec.     

Determination of creatinine with a sensor based on immobilized glutamate dehydrogenase and creatinine deiminase

Glutamate dehydrogenase and creatinine deiminase are immobilized by adsorption on wet poly(vinyl chloride) membranes. Creatinine is determined by a sensor consisting of the two membranes placed over an ammonia-sensing electrode. Endogenous ammonia is removed as it passes through the glutamate dehydrogenase layer. Creatinine (1–50 mg dl^?1) is converted to ammonia in the inner creatinine deiminase layer and is detected by the ammonia electrode. The assay requires 3 min, the minimum detectable concentration is 1 mg dl^?1 at pH 8.5, and the precision is ca. 5%. Endogenous ammonia can be tolerated up to 2 × 10^?4 M.