Preparation of a potentiometric immobilized urease electrode and urea determination in serum

Polyvinylalcohol was activated with 2-fluoro-1-methylpyridiniumtoluene-4-sulphonate and urease (EC.3.5.1.5) was covalently linked to the activated matrix. PVA-urease was then immobilized on the surface of a pH glass electrode with gelatine gel and it was cross-linked using glutaraldehyde. This potentiometric membrane electrode provides a linearity to urea in the 8.910⁻⁵ to 1.110⁻³ M concentration range, but by changing the buffer concentration can be studied in the range of 10⁻⁴ to 10⁻² M urea concentration. Reproducibility experiments (n:20) were carried out with the urease enzyme electrode and with photometric methods for pooled serum sample. Average values for the two methods were 5.96 and 5.86 mM, variation coefficients were 2.5 and 3.5% respectively.     

Enzymatic determination of choline in milk using a FIA system with potentiometric detection

The development of a FIA system for the determination of total choline content in several types of milk is described. The samples were submitted to hydrochloric acid digestion before injection into the system and passed through an enzymatic reactor containing choline oxidase immobilised on glass beads. This enzymatic reaction releases hydrogen peroxide which then reacts with a solution of iodide. The decrease in the concentration of iodide ion is quantified using an iodide ion selective tubular electrode based on a homogeneous crystalline membrane. Validation of the results obtained with this system was performed by comparison with results from a method described in the literature and applied to the determination of total choline in milks. The relative deviation was always < 5%. The repeatability of the method developed was assessed by calculation of the relative standard deviation (RSD) for 12 consecutive injections of one sample. The RSD obtained was < 0.6%.     

Enzymatic flow-injection determination of l-phenylalanine using the stopped-flow and merging-zones techniques

A flow injection enzymatic method for the spectrophotometric determination of l-phenylalanine has been developed. l-phenylalanine is deaminated in the presence of l-amino acid oxidase and the keto acid formed is made to react with borate to give a coloured enol-borate complex that can be detected at 282 nm. Catalase is added to the catalyzed reaction to prevent the keto acid being destroyed by the hydrogen peroxide generated. Kinetic determinations are performed by measuring the change in absorbance between 2 and 4 min. The proposed procedure, involving both merging-zones and stopped-flow techniques, can be applied to the quantitation of l-phenylalanine between 10 and 260 mg l(-1). Detection limit and R.S.D. are 1.1 mg l(-1) and 3.0%, respectively.     

Indirect potentiometric flow-injection determination of silver in photographic fixing solutions

The thiosulfate present in fixing solutions was destroyed on-line by acidification and the undissolved gaseous products were separated from the flow stream by use of polytetrafluoroethylene microporous tubing. The silver was precipitated with an excess of sulfide, and the excess was measured with a silver/silver sulfide electrode. Silver was determined in several ranges from 1 mg 1^?1 to 10 g l^?1.     

Oxidative removal of interferences in flow-injection potentiometric determination of chloride

Direct potentiometric determination of chloride in a flow-injection system can be performed in the presence of excess bromide, iodide, sulphide and cyanide, when potassium bromate in nitric acid is used as the carrier solution. The hydrodynamics and temperature of such a system have been examined and various oxidants and indicating electrodes investigated. The analysis can be performed at a maximum rate of 120 samples per hour.     

Fully automated potentiometric determination of ionized magnesium in blood serum

Basic observations leading to the successful application of magnesium ion-selective electrodes for the automated determination of ionized magnesium in undiluted serum are discussed. The principles of the method are described and the first numerical results are reported.     

Miniaturized module with biosensors for potentiometric determination of urea

We describe the construction of a miniaturized module which allows carrying out potentiometric urea determination with the usage of biosensors. The module was fabricated using new hybrid technologies developed in our group which combine ceramic and polymeric materials. Its simplicity and easy way of preparation makes the system very useful for analytical measurements in a flow mode. Application of urease-modified polymer membranes deposited on silver screen-printed electrodes in the module allows to determine urea concentration in clinical range. Moreover, it is a very promising construction for other applications in that other enzymes can be immobilized and various bioanalytes can be determined using this module.     

Spectrophotometric flow-injection determination of urea in body fluids by using an immobilized urease reactor

A flow system involving a packed-bed enzyme reactor (volume 180 μl) with urease immobilized covalently on poly(glycidyl methacrylate)-coated porous glass is used for determining urea in blood serum and urine. Enzymatically produced ammonia is converted to an indophenolate dye (by oxidative coupling with hypochlorite and sodium salicylate), which is detected spectrophotometrically at 700 nm. The calibration graph is rectilinear for 25–500 μM urea when injecting samples (75 μl) diluted 1:50 for serum or 1:1000 for urine at a frequency of 60 h^?1; the relative standard deviation is 1.1% for ten injections of 300 μM urea. The immobilized urease is stabilized by the addition of disodium EDTA, sodium azide and 2-mercaptoethanol to a 0.2 M phosphate buffer (pH 6.9) used as the carrier stream, which serves also as a preservative for longterm storage of the urease reactor packing at 4°C.     

Kinetic determination of lactate dehydrogenase in blood serum by multi-detection with a cyclic flow-injection system

The use of a cyclic flow-injection system for the determination of lactate dehydrogenase (LDH) is proposed. This configuration allows the repeated passage of the reacting plug through the detector resulting in multiple peak recordings. From the data obtained, which correspond to a typical kinetic curve, the required sensitivity can be selected by using procedures based on fixed-time measurements (peak maxima or minima) or reaction-rate measurements (signal increment between two successive maxima or minima). The methods were applied successfully to the determination of LDH in blood sera; the average recovery was 100.9%.     

Enzymatic determination of urea in undiluted whole blood by flow injection analysis using an ammonium ion-selective electrode

A flow-injection system is described for the assay of urea in undiluted whole blood. Urea is quantified by means of an ammonium ion-selective electrode covered with a membrane with covalently immobilized urease. The enzymatically generated ammonium ion is directly related to the urea concentration. Interference from potassium is reduced by adjusting the potassium ion concentration in the carrier stream and in the aqueous calibration solutions to 4.0 mM; it can be eliminated by measuring the potassium ion concentration in the sample separately and applying a mathematical correction for the K⁺ contribution to the signal. The linear measuring range is 1–40 mM urea, with an injection frequency of 40 h^?1 and a standard deviation of 1% for whole blood samples. The result of the measurement is obtained within 25 s from the time of injection. Vatiations in the hematocrit level of the sample have no effect on the measurement. The results obtained by the flow-injection method are in excellent agreement with those found routinely at a local hospital. The sensor is stable for more than 25 days.     

Enzymatic analysis by means of the air-gap electrode determination of urea in blood

The newly developed air-gap electrode has been used for enzymatic assay of urea in serum and whole blood; analyses can be done accurately, reliably, simply and quickly. The determination is highly selective because the electrode senses only the ammonium ion, which is selectively released from urea by urease in a preliminary rapid incubation step. The reproducibility of the determination (standard deviation less than 2.4%) is sufficient for clinical purposes; the linear range of the method is 10^-2–lO^-4M urea. Since the electrode actually never touches the sample solution, the problems caused by the presence of proteins, blood cells etc. do not arise.     

Enzymatic methods for the determination of ethanol based on linear and cyclif flow-injection systems

Linear and cyclic systems are described for the determination of ethanol (ca. 0.17–30×10^?3 M). In the linear system, the solution passes either through a minicolumn of yeast alcohol dehydrogenase (YADH) immobilized on controlled-pore glass or through minicolumns of the immobilized YADH and of yeast aldehyde dehydrogenase immobilized on cyanogen bromide-activated Sepharose-4B. The NADH formed is monitored either spectrophotometrically or spectrofluorimetrically. In the cyclic system, the solution passes through the same enzyme columns, and the NADH produced is monitored similarly before reconversion to NAD⁺ in a minicolumn of glutamate dehydrogenase immobilized on cyanogen bromie-activated Sepharose-4B in the presence of α-ketoglutarate and ammonium ions also present in the flow system. the sample throughout for both systems is ca. 40 h^?1 and 50 h^?1 for spectrophotometric and spectrofluorimetric detection, respectively. An on-line double-injection technique is described as an alternative to the cyclic system for limiting the consumption of NAD⁺.     

A flow-injection system based on immobilized penicillinase and a linear pH-buffer for potentiometric determination of penicillin

Penicillin is determined in a flow-injection manifold by hydrolysis of the β-lactam ring by means of an on-line reactor containing immobilized penicillinase with detection of the produced acid by a glass electrode. The penicillin concentration is calculated as the difference in response between a sample passing through the enzyme reactor and a sample flowing directly to the glass electrode. The pH signal is made linearly dependent on the acid concentration by using a buffer mixture of constant buffer capacity and the reactor is designed so that hydrolysis of the penicillin is essentially complete in the reactor. The linear range is 0.1–15 mM penicillin and the sensitivity is 0.056 pH mM^?1. The sample throughput is 60 h^?1 and the relative standard deviation < 1%. The proposed method is primarily intended for the analysis of purified potassium salts of penicillins in pharmaceutical preparations.     

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.     

Enzymatic fluorimetric determination of the individual bile acids in blood serum

An enzymatic fluorimetric method is described for the determination of total bile acids (cholic acid and deoxycholic acid), primary bile acids (cholic and chen acids and individual bile acids in serum without prior separation of the acids. Total and primary bile acids are determined by equilibrium procedures by conver of the 3α- and 7α-hydroxy bile acids to 3-oxo and 7-oxo bile acids by α-NAD⁺, in the presence of 3α- and 7α-hydroxysteroid dehydrogenase (HSD), respectively, and measurement of the generated NADH fluorimetrically. Chenodeoxycholic acid is determined with 7α-HSD in the presence of cholic and deoxycholic acids by a differential kinetic procedure, and cholic and deoxycholic acids are calculated by difference. Interferents are removed by treatment of serum with Sachrom rein. Only 1.00 ml of serum is required. Low cost, simplicity and reliability are the main features of the method. The recovery of bile acids added to serum averaged 103% (range 83–122%). The method is suitable for routine use in small clinical laboratories.     

Simultaneous determination of ammonia and urea with an asymmetric merging zones/flow-injection configuration

A method is proposed for the simultaneous determination of urea and ammonia using a reagent-injection configuration that includes a dual injection valve (for insertion of Nessler's reagent and for accommodating the enzyme reactor). The resolution of the two peaks obtained on each injection allows the determination of both analytes in mixtures. The determination range is 1–5 μg ml^?1 for ammonia and 1–6 μg ml^?1 for urea, with relative standard deviations of 1.13% and 2.31% for ammonia (first and second peaks) and 1.86% for urea.     

Simultaneous determination of lithium, sodium and potassium in blood serum by flame photometric flow-injection analysis

A simultaneous flow-injection analysis (FIA) manifold that could analyse three ions from a single injection was designed, constructed, calibrated and used successfully to analyse Li(+), Na(+) and K(+). This FIA method was 10 times faster than the batch technique. The sample volume required was a fraction of about 1/110 to 1/75 that of the batch technique. The outputs were quite reproducible and calibration curves were linear. Results obtained for artificial sera compared favourably with the actual known concentrations of ions and results obtained in the analysis of eight natural human blood sera compared well with those obtained by the traditional batch technique.