Potentiometric determination of proteins with an ammonia sensor

A flow system is described for the cleavage of proteins with immobilized protease enzyme to L-amino acids which are then converted to ammonia with glass-immobilized L-amino acid oxidase. An ammonia gas electrode is used as detector. Immobilization techniques are discussed, as are optimum conditions for L-phenylalanine. Bovine serum albumin was determined in the range 0.1–100 μg ml^-1. Human blood sera required dilution for analysis.     

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.     

Part I. A specific electrode for urea.

A specific simple enzyme stirrer electrode is described for the assay of urea in blood serum. The enzyme is placed directly on a magnetic stirrer and held in place with a nylon net. The enzyme stirrer both stirs the solution and effects an enzymatic transformation, permitting the direct assay of a substrate such as urea. Potassium, Na⁺ , NH₄⁺ and other organic and inorganic species present in blood do not interfere. Linear curves are obtained from 5· 10^-2M to 1· 10^-4M urea with slopes close to Nernstian, 0.95 pH/decade. Urea in blood was assayed with an accuracy of 1.8% and a precision of 2.0% with immobilized urease in the stirrer. The stirrers were used for 15 weeks and over 500 assays with excellent results.     

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.     

An enzyme reactor electrode for determination of amino acids

L-Leucine can be determined with an enzyme reactor electrode containing L-amino acid oxidase immobilized with glutaraldehyde to glass. The reactor also contains immobilized catalase which splits the hydrogen peroxide formed. Oxygen for the reaction is also supplied by adding hydrogen peroxide to the samples. The electrode is an ammonia gas sensor. The calibration curve is strictly linear with Nernstian slope between 3·10^-5 and 10^-3 M leucine.     

Molecular lego for the assembly of biosensing layers

We propose a procedure to assemble monolayers of redox mediator, coenzyme, enzyme and stabilizing polyelectrolyte on an electrode surface using essentially electrostatic and complexing interactions. In a first step a monolayer of redox mediator, substituted nitrofluorenones, is adsorbed. In a second step, a layer of calcium cations is immobilized at the interface. It establishes a bridge between the redox mediator and the subsequently adsorbed coenzyme NAD⁺. In the next step we use the intrinsic affinity of the NAD⁺ monolayer for dehydrogenases to build up a multilayer composed of mediator/Ca²⁺/NAD⁺/dehydrogenase. The so obtained modified electrode can be used as a biosensor. Quartz crystal microbalance measurements allowed us to better understand the different parameters responsible for the adsorption. A more detailed investigation of the system made it possible to finally stabilize the assembly sufficiently by the adsorption of a polyelectrolyte layer in order to perform rotating disk electrode measurements with the whole supramolecular architecture on the electrode surface.     

Construction of a guanine enzyme electrode and determination of guanase in human blood serum with an ammonia gas sensor

The guanine electrode is based on guanase used with an ammonia gas-sensing membrane electrode; immobilization of the enzyme is optimized. Guanine in the range 10^-4–10^-2 M gives a linear potential vs. log(concentration) plot with a response time of 4–1.5 min over the range specified. Guanase (0.12–12 I.U. I^-1) is determined in serum by adding guanine to the sample, and measuring the ammonia evolved with the gas-sensing electrode. Results compare favourably with the xanthine oxidase method.     

Ammonia oxidation to nitrogen mediated by electrogenerated active chlorine on Ti/PtOx-IrO2

The electrochemical oxidation of ammonia (NH₃ and/or NH₄⁺) in the presence of chloride was investigated on a Ti/PtO_x–IrO₂ electrode. It was shown that ammonia is effectively removed from solution via electrogenerated active chlorine. Based on mass balances, nitrogen is postulated to be the main product of ammonia electrolysis. In the bulk, the concentration of chloramines was low. This could be explained by the fact that the oxidation of ammonia takes place close to the electrode surface where an excess of chlorine relative to ammonia is ensured during the process. This results in the oxidation of ammonia to N₂ and in a local pH decrease. As a result, chloramines were decomposed in the proximity of the electrode prior to diffusing into the bulk.     

An improved electrode for the assay of urea in blood

An improved urea enzyme electrode is applied for the determination of urea in blood samples. The electrode is based on the enzymatic hydrolysis of urea, and potentiometric detection of the ammonium ion produced. A silicone rubber-based nonactin ammonium ion-selective electrode serves as the sensor. The selectivity coefficients of this electrode were 6.5 for NH₄⁺/K⁺; 750 for NH₄⁺/Na⁺, and much higher for other cations. The reaction layer of the electrode was made of urease enzyme chemically immobilized on polyacrylic gel. The prepared gel was stable at 4° for over four months. The electrodes retained their activity for over one month. A three-electrode system, which allowed dilution to a constant interference level, was applied to avoid interfering effects in blood samples. Analyses of blood sera showed good agreement with a standard spectrophotometric method. Routine clinical assays of blood urea are feasible.     

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.     

Determination of trace amounts of nitrite by single-sweep polarography

A single-sweep polarographic determination of nitrite in 0.2 M sulphuric acid medium containing nickel(II) sulphate and ammonium thiocyanate is described. The ternary complex (NiSCNNO)⁺ which is formed in the solution is strongly adsorbed on the surface of the mercury electrode and an adsorptive polarographic wave at ?0.57 V (vs. SCE) is related to the concentration of nitrite in the range 2.0 × 10^?8-1.0 × 10^?6 M. The detection limit is 8 × 10^?9 M. The relative standard deviation is 1.5% and the regression coefficient is 0.998. Most common anions and cations do not interfere. The mechanism of the electrode process was studied by several electrochemical methods. The polarographic wave is attributed to the reduction of nitrogen monoxide in the adsorbed (NiSCNNO)⁺ complex to hydroxylamine. The procedure was applied to the determination of trace amounts of nitrite in sausage, water and nitrate.     

Thermodynamics of the complex formation of copper(II) with L-phenylalanine in aqueous ethanol solutions

Constants of the acid dissociation and complexation of L-phenylalanine (HPhe) with copper(II) ions are determined by potentiometry in aqueous ethanol solutions containing 0 to 0.7 molar fraction of alcohol. Changes in the Gibbs energy for the transfer from water to a binary solvent of L-phenylalanine, Phe^? anion, and [CuPhe]⁺ complex are calculated. It is found that the weakening of solvation of the ligand donor groups in solvents with high ethanol contents is accompanied by an increase in the stability of [CuPhe]⁺ complex.     

A Urea Electrode Based on the Ammonia Probe

Abstract

An enzyme electrode specific for the substrate urea is described. The electrode consists of a layer of urease polymerized directly on to the surface of the gas diffusion membrane of an ammonia probe by means of glutaraldehyde. The calibration curves obtained when the electrode is allowed to measure in buffered solutions are discussed in terms of enzyme activity and pH-gradients in the membrane.     

Arginine Determination with Ion-Selective Membrane Electrodes

Abstract

A new method for the determination of the amino acid arginine utilizes a dual enzyme catalyzed reaction monitored with an ammonium ion selective membrane electrode of the antibiotic type. The technique gives a linear correlation between arginine concentration and electrode response over the 3 × 10^?3 M to 3 × 10^?5 M range and a useful, but non-linear, response over a wider range. Major interferences are urea, K⁺, and NH₄ ⁺, but Na⁺ does not interfere in the physiological concentration range.     

A specific enzyme electrode for urea

A truly specific, simple enzyme electrode is described for the assay of urea in blood serum. The sensor used is the newly developed air-gap electrode of R??i?ka and Hansen, and has advantages of speed of response and specificity over earlier enzyme electrodes for urea. Potassium, sodium and ammonium ions and other organic and inorganic species present in blood do not interfere. Linear curves are obtained from 2 · 10^-2M to 1 · 10^-4M urea with slopes close to Nernstian (about 0.90 pH/decade). Urea in blood was assayed with an accuracy of 2.2% and a precision of 2.0% with immobilized urease; only 3–5 min is required per assay. The electrode was used for a month and almost 500 assays with excellent results. Since the sensor never touches the sample solution, problems caused by blood components which block membrane pores are avoided.     

A novel enzyme electrode method for the determination of nitrite based on nitrite reductase

The enzyme, nitrite reductase, can be extracted and purified from spinach leaves; the freeze-dried preparation is completely stable for at least 4 months if kept in a freezer. The enzyme catalyzes the reduction of nitrite to ammonia in the presence of reduced methyl viologen as electron donor. An assay of nitrite can be based on the measurement of the ammonia formation, with an air-gap electrode as sensor. Nitrite in the 10⁻⁴ M—5 · lO⁻² M range can be accurately determined with either soluble or immobilized enzyme, but the latter is stable for at least 3 weeks, is less susceptible to interferences during assay, and can be used repeatedly for about a hundred runs. These advantages make the method very simple, valuable and economical for the routine analysis of nitrite ion.     

Direct and mediated electrochemical oxidation of ammonia on boron-doped diamond electrode

Direct (non-mediated) electrochemical oxidation of ammonia on boron-doped diamond (BDD) electrode proceeds mainly at high pH (> 8) via free ammonia (NH₃) oxidation. To enhance ammonia oxidation on BDD at low pH (< 8), where mainly ammonium (NH₄⁺) is present, oxidation of ammonia was mediated by active free chlorine. In this process, electro-generated in situ active chlorine rapidly reacts with ammonia instead of being further electro-oxidized to chlorate at the electrode surface. Thus, active chlorine effectively removes ammonia from an acidic solution, while the formation of by-products such as chlorate and possibly perchlorate is minimized.     

Electrocatalytic Volleyball: Rapid Nanoconfined Nicotinamide Cycling for Organic Synthesis in Electrode Pores

In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP⁺ are cycled rapidly between ferredoxin–NADP⁺ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.     

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 Creatinine Specific Enzyme Electrode

Abstract

An extremely specific creatinine electrode was successfully developed for selective analysis of aqueous creatinine with a linear response over the range from 5 mg/l to 100 mg/l. The endogenous and exogenous species generally present in the serum do not interfere in the assay with the exception of ammonia. With the use of the electrode serum samples containing high creatinine can be assayed directly after pre-treatment. However, the electrode is not sensitive enough to detect the normal level serum creatinine due to the dilution of the serum after treatment.