A coated-metal enzyme electrode for urea determinations

A potentiometric enzyme electrode is reported in which an enzyme immobilized in polyvinyl chloride is used to coat an antimony metal electrode to detect changes in pH when the electrode is immersed in a solution of the enzyme substrat. As an example, urea is determined in solution by using immobilized urease on an antimony electrode, giving a linear concentration range of 5.0 × 10^-4–1.0 × 10^-2 M urea with a slope of 44 mV per decade change in urea concentration. The response slope is stable for about 1 week, with response times in the range 1–2 min, but with absolute potential changes occurring from day to day.     

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.     

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 miniature enzyme electrode sensitive to urea

Summary Miniature enzyme electrodes sensitive to urea have been made with tip diameters ranging from 50 to 500m by immobilizing the enzyme urease on the tip of an antimony electrode. These electrodes give a linear response in the concentration range of 1.0×10^–4-1.0×10^–2 M urea with a slope of 40–45 mV per decade change in concentration. The response time of these electrodes is less than 1 min and the wash time is 1–2 min. This type of electrode has a minimum life time of 3 months.

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Eine kleine, harnstoffempfindliche Enzymelektrode

Zusammenfassung Harnstoff-sensitive Enzymelektroden mit Spitzendurchmessern zwischen 50 und 500m wurden durch Immobilisierung des Enzyms Urease auf der Spitze einer Antimon-Elektrode hergestellt. Diese Elektroden verhielten sich linear im Konzentrationsbereich 1,0×10^–4–1,0×10^–2 M, und hatten eine Steigerung von 40–45 mV pro Dekade der Konzentrationsänderung. Die Antwortzeit dieser Elektroden liegt unter 1 min und die Auswaschzeit beträgt l–2 min. Diese Art Elektroden hat eine Lebensdauer von mehr als 3 Monaten.

     

Potentiometric enzyme electrode for urea based on electrochemically prepared polypyrrole membranes

The present study explores the attractiveness of combining flow-injection (FI) with lead hydride generation atomic absorption spectrometry (AAS) to improve the selectivity and sensitivity of analysis. Lead hydride was generated in three acid-oxidant media: HNO₃-(NH₄)₂S₂O₈, lactic acid-K₂Cr₂O₇ and HNO₃-H₂O₂. The effect of chemical parameters (acid-oxidant concentration and NaBH₄ concentration) was investigated and the performance of each generation medium in terms of interferences, sensitivity and detection limits was compared with that obtained in batch mode. In all cases improved sensitivity (HNO₃-H₂O₂, 0.8 ng Pb; lactic acid-K₂Cr₂O₇, 0.2 ng Pb; (NH₄)₂S₂O₈-HNO₃, 4ng Pb) was obtained, most notably in HNO₃-H₂O₂, which provided 12 times higher sensitivity than in batch mode and sharper absorption peaks. Furthermore, interference by Cu and Ni was lower in the proposed FI-HG system. Compared with the batch mode, about 10 to 100 times higher concentrations of interferent are tolerated in the sample. The use of FI also allows work at a lower NaBH₄ concentration. The method was applied to the determination of lead in water samples with a sampling frequency of 180 samples per hour. In terms of both sensitivity and freedom from interferences, lactic acid-K₂Cr₂O₇ was the best of the generation media tested.On leave from the School of Environmental Studies, Jadavpur University, Calcutta 700032, India     

An activated enzyme electrode for creatinine

A highly selective enzyme electrode for creatinine, based on tripolyphosphate-activated creatininase enzyme, is described and evaluated. Kinetic studies comparing purified creatininase enzyme in the activated and non-activated forms show that the activation mechanism involves an increase in V_max but no change in K_m. The analytical effect of enzyme activation is to extend the sensitivity of the electrode to lower limits and to improve the response slope of calibration curves. As a result, this activated creatininase enzyme electrode shows promise as a sensor for urine and serum samples.     

Tungsten electrode for urea

Tungsten electrodes for urea were prepared via covalent linking of urease on oxidized metal surfaces in different ways. The most stable electrodes were obtained when tungsten was silanized and activated by glutaraldehyde or hexamethylene diisocyanate. The electrode with urease coupled via glutaraldehyde was tested for optimum conditions of use. The nature of the buffer and its concentration and ionic strength are particularly important.     

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.     

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.     

Amperometric enzyme electrode for theophylline.

An amperometric biosensor for theophylline, based on the recently isolated enzyme theophylline oxidase, is described. The enzyme is entrapped, together with a ferricytochrome C cofactor, within a polymeric (Nafion) coating. The anodic detection (at +0.4 V versus Ag-AgCl) is facilitated by the addition of a redox-mediating hexacyanoferrate(III) ion. The influence of various experimental variables is described. The limit of detection is 2 x 10(-6) mol dm-3 theophylline, with linearity prevailing up to 3 x 10(-4) mol dm-3. The fast response and wash times permit rapid flow-injection measurements, with a frequency of 180 samples h-1 and a relative standard deviation of 3.0-4.0%. Prospects of using this electrode for clinical diagnostics are discussed.     

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.     

Flow-injection analysis for l-glutamate using immobilized l-glutamate oxidase: comparison of an enzyme reactor and enzyme electrode

An enzyme electrode and enzyme based on immobilized l-glutamate oxidase are used for the determination of l-glutamate in a flow-injection system. The hydrogen peroxide produced is monitored amperometrically. The enzyme reactor system surpasses the enzyme electrode system with regard to sensitivity and analytical speed. For both systems, the peak current is linearly related to the l-glutamate concentration in the range 5 × 10^?6-1 × 10^?3 M. l-Glutamate in seasoning can be determined very selectively with < 0.7% r.s.d.     

An enzyme electrode for the amperometric determination of glucose

An amperometric method utilizing a glucose electrode has been developed for the determination of blood glucose. The time of measurement is less than 12 s if a kinetic method is used and 1 min if a steady-state method is used. The long-term stability of the electrode is ca. 0.1% change from maximum response per day when stored at room temperature for over 10 months. The enzyme electrode determination of blood sugar compares favorably with commonly used methods with respect to accuracy, precision, and stability. The only reagent required for blood sugar determinations is a buffer solution. The electrode consists of a metallic sensing layer covered by a thin film of immobilized glucose oxidase held in place by means of cellophane. When poised at the correct potential, the current produced is proportional to the glucose concentration.     

Potentiometric enzyme electrode for uric acid.

An enzyme electrode for uric acid, with immobilized uricase on a pCO₂ membrane electrode, is described. Evaluation studies show that the enzyme electrode attains performance characteristics similar to homogeneous enzymatic conversion of uric acid under optimum solution conditions. Comparison of analyses carried out with the enzyme electrode and classical procedures on urine control samples demonstrates acceptable accuracy and precision for the electrode method.     

An amperometric enzyme electrode for the determination of 3alpha-hydroxysteroids

An enzyme electrode for quantifying the total content of 3alpha-hydroxysteroids is described. 3alpha-Hydroxysteroid dehydrogenase (E.C. 1.1.1.50) was immobilized on the surface of glassy-carbon and low-temperature isotropic-carbon electrodes by intermolecular cross-linking with bovine serum albumin and glutaraldehyde. The effects of the following factors on the response to androsterone were studied: applied potential, the concentration and pH of the pyrophosphate buffer used, and the NAD concentration. The Michaelis-Menten constant for 3alpha-hydroxysteroid dehydrogenase in solution was determined amperometrically with androsterone as substrate (K(m) = 189muM). A preliminary report of the response of the system to serum samples containing a bile acid is presented.     

基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析

将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进行了初步评价。结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平行分析,肽段覆盖率及相对标准偏差分别为44.3%、6.5%(牛血清白蛋白), 65.0%、2.7%(肌红蛋白)和62.0%、5.6%(细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白质组学分析中发挥重要作用。     

Oxygen determinations with the aluminium corrosion electrode

A method for the determination of molecular oxygen in a great variety of liquid and gaseous samples has been developed. The method seems adaptable for control analysis by reason of its sensitivity, rapidity and ease of operation. It gives accurate determinations in the presence of many chemical substances which interfere with other methods. It is based on the variation in corrosion potential of the aluminium electrode, caused by oxygen, and exemplifies use of the corrosion phenomenon in analytical chemistry.     

A specific enzyme electrode for L-phenylalanine

A specific enzyme electrode procedure is described for the rapid assay of L-phenyl- alanine. The enzyme L-phenylalanine ammonia lyase is used, which cleaves L-phenyl-alanine to ammonia. The ammonia liberated is measured with an air-gap electrode. The procedure is specific; L-tyrosine and other amino acids do not interfere, nor do Na⁺ or K⁺ ions. As little as 5 · 10^-5 M L-phenylalanine can be determined.     

Suitable potentiometric enzyme sensors for urea and creatinine

Enzyme sensors for urea and creatinine were developed by coupling an ammonia gas-diffusion electrode with triacetate cellulose membranes entrapping urease or creatinine deiminase enzymes. Satisfactory results were obtained by using these sensors both in standard solutions and in authentic biological matrices.     

Composite liquid membrane for enzyme electrode construction

Microporous polycarbonate membranes were treated with a lipid, isopropyl myristate. The resulting composites were used as external membranes for the construction of hydrogen peroxide-based glucose-detecting enzyme electrodes. The linearity of glucose electrodes was increased to well above the substrate physiological range; for treated membranes of pore size 0.015 μm, linearity was up to 100 mM of glucose. The electrode system had a greatly decreased dependence on temperature, solution pH and background oxygen levels. It also showed reduced interference from electrochemically active species in blood and eliminated the need for sample stirring. Use with whole blood indicated that the treated membrane also had good haemocompatibility, and blood measurements showed an acceptable correlation with a routine spectrophotometric method (y = 0.739x + 1.153; r = 0.969).