Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

Conversion of methanol to formic acid through the coupling of the enzyme reactions of alcohol oxidase,catalase and formaldehyde dismutase

Summary A novel process for the production of formic acid from methanol has been developed that involves the coupled reactions of the three enzymes, alcohol oxidase, catalase and formaldehyde dismutase. In this process, methanol is oxidized to formaldehyde by alcohol oxidase and catalase, followed by the formaldehyde dismutase reaction that leads to the formation of methanol and formic acid. Ultimately, the substrate methanol (100 to 200 mM) is completely converted to formic acid, by the recycling of the consecutive enzyme reactions.     

Quinoprotein glucose dehydrogenase and its application in an amperometric glucose sensor

Glucose dehydrogenase (GDH), one of the recently discovered NAD(P)⁺-independent ‘quinoprotein’ class of oxidoreductase enzymes, was purified from Acinetobacter calcoaceticus LMD 79.41 and immobilised on a 1,1'-dimethylferrocene-modified graphite foil electrode.The second-order rate constant (k_s) for the transfer of electrons between GDH and ferrocenemonocarboxylic acid (FMCA) in a homogeneous system, determined using direct current (DC) cyclic voltammetry, was found to be 9.4 × 10⁶ litres mol⁻¹ s⁻¹. This value of k_s for GDH was more than 40 times greater than that for the flavoprotein glucose oxidase (GOD) under identical conditions. Such high catalytic activities were also observed when GDH was immobilised in the presence of an insoluble ferrocene derivative; a biosensor based on GDH was found to produce more than twice the current density of similar GOD-based electrodes. The steady-state current produced by the GDH-based electrode was limited by the enzymic reaction since methods which increased the enzyme loadings elevated the upper limit of glucose detection from 5 mM to 15 mM.The temperature, pH, stability and response characteristics of the GDH-based glucose sensor illustrate its potential usefulness for a variety of practical applications. In particular, the high catalytic activity and oxygen insensitivity of this biosensor make it suitable for in vivo blood glucose monitoring in the management of diabetes.     

A preliminary study of a biosensor based on flow injection of the recognition element

A biosensor based on flow injection of the recognition element has been developed. As a model a pH-transducer was used, and urease was chosen as the recognition element. The pH-transducer was immersed in an internal flow-through chamber which was in contact with the sample solution via a semi-permeable membrane. The recognition element, urease, was injected into the buffer solution passing through the biosensor. The enzyme catalysed the hydrolysis of urea and the concomitant increase in pH was recorded. The biosensor response time was about three minutes at a constant flow rate of 0·05 ml/min. The linear range of the calibration curve of the biosensor was 0–5 mM. The observed detection limit was approximately 0.1 mM. The sample throughput was 6–12 per hour. The pH-response of the biosensor, for a sample solution containing urea (3·26 mM), showed a reproducibility (r.s.d) of 28% (n = 5) and a repeatability (r.s.d.) of 8% (n = 5). Operation at elevated temperatures (up to 50°C) was demonstrated. The presence of glucose (28 mM), acetone (6·7 mM), citric acid (0·2 mM) or sodium acetate (0·6 mM) in the sample solution did not interfere with the sensor response. A lowering of the biosensor response which was observed in the presence of copper ions (due to urease inhibition) could be completely eliminated by adding EDTA to the urease solution. Thus, this work demonstrates a new type of biosensors, based on SIRE-technology (Sensors with Injectable Recognition Elements), which show high accuracy and stability, quick response and high sample throughput. These features suggest the suitability of the system for automation. Such sensors should readily be combined with other enzymes or enzyme systems. The enzyme (urease) cost per analysis (injection) for the biosensor was estimated to be approximately US$0·02. This could be substantially reduced by further optimisation and miniaturisation.     

Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T)

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L⁻¹ day⁻¹ even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.     

Study of real-time lectin-carbohydrate interactions on the surface of a quartz crystal microbalance

A quartz crystal microbalance (QCM) biosensor system for lectin-carbohydrate interactions has been developed. Yeast mannan was immobilised on polystyrene-coated quartz crystals, and interactions tested with the lectin concanavalin A (Con A). The biosensor could be easily operated, where mannan immobilisation and all binding analyses were performed in real-time using a flow-through system. The apparent binding constant for yeast mannan to Con A was estimated to be 0.4 microM, well in accordance to reported literature values. In addition, the effective concentration values (EC50-values) for a series of mannose/mannoside ligands, acting as competitors to the mannan/Con A interaction, were determined to range from 0.18 to 5.3 mM, in good correlation with a related enzyme-labelled lectin assay (ELLA) protocol.     

Simultaneous enzymatic/electrochemical determination of glucose and L-glutamine in hybridoma media by flow-injection analysis

A split-stream flow-injection analysis system is described for simultaneous determination of glucose and L-glutamine in serum-free hybridoma bioprocesses media. Amperometric measurement of glucose is based on anodic oxidation of hydrogen peroxide produced by immobilized glucose oxidase within a triple layer membrane of an integrated flow-through glucose-selective biosensor. Determination of L-glutamine is based on quantitating ammonium ions produced in a flow-through enzymes reactor containing immobilized glutaminase enzyme, and subsequent downstream potentiometric detection of these ions by a nonacting-based ion-selective polymer membrane electrode. Endogenous potassium and ammonium ion interference in the L-glutamine determination are eliminated by using a novel in-line tubular cation-exchange membrane unit to exchange these interferent species for cations undetectable by the membrane electrode. The first generation split-steam flow-injections system can assay 12 samples/h using direct injections of 50 muL of media samples, with linear responses to glucose in the range of 0.03 to 30mM, and log-linear response to L-glutamine from 0.1 to 10 mM. (c) 1993 Wiley & Sons, Inc.     

A novel urea sensitive biosensor with extended dynamic range based on recombinant urease and ISFETs

A novel urea biosensor based on immobilised recombinant urease as sensitive element and ion sensitive field effect transistor as transducer was developed. Recombinant urease from E. coli with an increased Km was photoimmobilised in PVA/SbQ (poly(vinyl alcohol) containing styrylpyridinium) membrane and has demonstrated quite good performance as biosensitive element. Enzymatic field effect transistors based on such a bioselective element were studied in model buffer solutions. This biosensor demonstrated an extended dynamic range up to 80 mM, a quite good reproducibility (standard deviation of the sensor responses was approximately 2.5%, n= 20 for urea concentration 10 mM) and a high stability. Such characteristics fit with the analytical requirements needed for urea control in plasma and liquids used during renal dialysis.     

Integrated processing and multiple re-use of immobilised lipase by magnetic separation technology

High-gradient magnetic separation based processing is demonstrated for the semi-continuous multicycle re-use of a lipase immobilised on magnetic microparticles. The lipase of Candida antarctica A-type (CALA) was immobilised on polyvinyl alcohol coated magnetic particles (1-2mum diameter) with epoxy functionalisation. The immobilised CALA was used to hydrolyse a model oil-water 2-phase-system composed of a phosphate buffer with tributyrin at up to 3l scale. The immobilised enzyme was subsequently recovered in a magnetic filter using high-gradient magnetic separation and reapplied in repeated cycles of hydrolysis and recovery. Two different temperatures of 30 and 50 degrees C, tributyrin concentrations (0.12 and 35gl(-1)) and reaction times were tested. In each case the reaction was followed by pH titration using NaOH, as well as by HPLC analysis. Consecutive cycles were conducted for each reaction condition and in total the immobilised CALA was subjected to 20 recovery and re-use cycles, after which approximately 14% of the initial specific activity still remained.     

Alpha-chymotrypsin immobilized on ferromagnetic particles coated with titanium oxide: production and catalytic properties

Immobilization of alpha-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction. X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification. The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed by immobilized alpha-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The catalytic constant (25 s-1) and KM (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding characteristics for the free enzyme.     

The dye-linked alcohol dehydrogenase of Rhodopseudomonas acidophila. Comparison with dye-linked methanol dehydrogenases.

1. A dye-linked alcohol dehydrogenase was purified 20-fold from extracts of Rhodopseudomonas acidophila 10050 grown anaerobically in the light on methanol/HCO3-. 2. The enzyme resembled many previously reported methanol dehydrogenases from other methylotrophic organisms in coupling to phenazine methosulphate, requiring ammonia as an activator, possessing a pH optimum of 9 and a mol.wt. of approx. 116000. In many other respects the enzyme showed singular properties. 3. The stability of the enzyme under various conditions of temperature and pH was studied. 4. Primary aliphatic amines containing up to nine carbon atoms (the longest tested) were better activators than ammonia. 5. A wide range of primary alcohols and aldehydes served as substrates, with apparent Km values ranging from 57 mM for methanol to 6 micron for ethanol. 6. O2 was an inhibitor competitive with respect to the alcohol substrate. In the presence of O2, apparent Km values of 145 mM were recorded for methanol. 6. Cyanide and alphaalpha'-bipyridine were inhibitors competitive with respect to the amine activator. 7. The properties of the enzyme from Rhodopseudomonas acidophila are compared with those of similar enzymes from other organisms, and implications of the salient differences are discussed.     

Use of competitive inhibition for driving sensitivity and dynamic range of urea ENFETs

An urea biosensor based on urease-BSA (bovine serum albumin) membrane immobilised on the surface of an ion-sensitive field effect transistor (ISFET) has been studied in a mix buffer solution composed of potassium phosphate, Tris, citric acid and sodium tetraborate. In this mix buffer, the biosensor showed a dynamic larger than the one observed in a phosphate or Tris buffer. Investigation of the individual effect of each component of the buffer solution on the biosensor response has shown that tetraborate anion acts as a strong competitive inhibitor for the hydrolysis reaction of urea catalysed by urease. The biosensor response was investigated in a phosphate buffer with different concentrations of tetraborate anion. The results showed that the apparent constant of Michaelis-Menten, K(m(app)), increases from 4.3 to 79.3 mM, for experiments realised without and with 0.5 mM sodium tetraborate, respectively. The mean value, determined graphically, for the inhibition constant, K(i), was 29 microM. The graphical representation of biosensor calibration curves in semilogarithmic co-ordinates showed that the linear range of the biosensor can be extended up to three orders of magnitude, allowing an urea detection in a concentration range 0-100 mM.     

Alcohol oxidases of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures.

1. Alcohol oxidase (alcohol: oxygen oxidoreductase) of a thermophilic methanol-utilizing yeast, Hansenula polymorpha DL-1, was isolated in crystalline form. 2. This alcohol oxidase of H. polymorpha was more stable to heat than was the enzyme of Kloeckera sp. This difference in heat stability is compatible with the difference in growth temperatures for both yeasts. 3. The crystalline alcohol oxidases of both yeast oxidized the lower primary alcohols (C-2 to C-4) as well as methanol. The apparent Km values for the methanol of Kloeckera and H. polymorpha enzymes were 0.44 and 0.23 mM, respectively. The enzymes could also oxidize formaldehyde to formate, and were inactivated by relatively low concentrations of hydrogen peroxide. 4. The molecular weight for both enzymes was calculated to be about 670000. Each enzyme is composed of eight identical subunits (molecular weight 83000) and contains eight moles of FAD as the prosthetic group. The NH2-terminal and COOH-terminal amino acids of H. polymorpha enzyme were identified as alanine and phenylalanine, respectively. The octameric subunits model of each enzyme was confirmed by electron micrographs, which showed an octad aggregate, composed of two tetragons face to face.     

Simultaneous monitoring of glucose and lactate by an interference and cross-talk free dual electrode amperometric biosensor based on electropolymerized thin films.

An interference and cross-talk free dual electrode amperometric biosensor integrated with a microdialysis sampling system is described, for simultaneous monitoring of glucose and lactate by flow injection analysis. The biosensor is based on a conventional thin layer flow-through cell equipped with a Pt dual electrode (parallel configuration). Each Pt disk was modified by a composite bilayer consisting of an electrosynthesised overoxidized polypyrrole (PPYox) anti-interference membrane covered by an enzyme entrapping gel, obtained by glutaraldehyde co-crosslinking of glucose oxidase or lactate oxidase with bovine serum albumin. The advantages of covalent immobilization techniques were coupled with the excellent interference-rejection capabilities of PPYox. Ascorbate, cysteine, urate and paracetamol produced lactate or glucose bias in the low micromolar range; their responses were, however, completely suppressed when the sample was injected through the microdialysis unit. Under these operational conditions the flow injection responses for glucose and lactate were linear up to 100 and 20 mM with typical sensitivities of 9.9 (+/- 0.1) and 7.2 (+/- 0.1) nA/mM. respectively. The shelf-lifetime of the biosensor was at least 2 months. The potential of the described biosensor was demonstrated by the simultaneous determination of lactate and glucose in untreated tomato juice samples; results were in good agreement with those of a reference method.     

A glucose biosensor based on electrodeposition of palladium nanoparticles and glucose oxidase onto Nafion-solubilized carbon nanotube electrode

Electrodeposition was used for the co-deposition of glucose oxidase (GOx) enzymes and palladium nanoparticles onto a Nafion-solubilized carbon nanotube (CNT) film. The co-deposited Pd-GOx-Nafion CNT bioelectrode retains its biocatalytic activity and offers an efficient oxidation and reduction of the enzymatically liberated H2O2, allowing for fast and sensitive glucose quantification. The combination of Pd-GOx electrodeposition with Nafion-solubilized CNTs enhances the storage time and performance of the sensor. An extra Nafion coating was used to eliminate common interferents such as uric and ascorbic acids. The fabricated Pd-GOx-Nafion CNT glucose biosensor exhibits a linear response up to 12 mM glucose and a detection limit of 0.15 mM (S/N = 3).     

Application of a biosensor for monitoring of ethanol

An alcohol biosensor for the measurement of ethanol has been developed. It comprises an alcohol oxidase/chitosan immobilized eggshell membrane and a commercial oxygen sensor. Ethanol determination is based on the depletion of dissolved oxygen content upon exposure to ethanol solution. The decrease in oxygen level was monitored and related to the ethanol concentration. The biosensor response depends linearly on ethanol concentration between 60 microM and 0.80 mM with a detection limit of 30 microM (S/N=3) and 1 min response time. In the optimization studies of the enzyme biosensor the most suitable enzyme and chitosan amounts were found to be 1.0 mg and 0.30% (w/v), respectively. The phosphate buffer (pH 7.4, 25 mM) and room temperature (20-25 degrees C) were chosen as the optimum working conditions. In the characterization studies of the ethanol biosensor some parameters such as interference effects, operational and storage stability were studied in detail. The biosensor was also tested with various wine samples. The results of this newly developed biosensor were comparable to the results obtained by a gas chromatographic method.     

An assay method for ganglioside synthase using anion-exchange chromatography

A rapid procedure which is based on combined ion-exchange chromatography and solubility was established for determination of the activity of ganglioside synthases and cerebroside sulfotransferase. The procedure consists of selective elution of radiolabeled reaction products (acidic glycolipids) freed from labeled precursors and breakdown products on a DEAE-Sephadex column and of direct radioassay of the products in the eluate. Monosialogangliosides were eluted from the column with 40 mM ammonium acetate (AcONH4) in methanol, cerebroside sulfate with 90 mM AcONH4 in methanol, and disialogangliosides with 40 mM AcONH4 in isopropanol/n-hexane/water (55/20/19, v/v/v). The established procedure is simple, reproducible, and economical. Using rat Golgi membrane as enzyme source the recovery rate of the products was over 95%.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

A microelectronic conductimetric biosensor

The fabrication and operation of a microelectronic conductimetric biosensor is described. The device monitors the change in solution conductance occasioned by the catalytic action of enzymes immobilised over a planar conductance cell comprising serpentined and interdigitated metal conductor tracks. The output of the instrument was linear over a 3 min period on addition of urea to a sample cell overlaid with immobilised urease. The responses to any given urea concentration were reproducible to within approximately ±1%. The device responds to urea present in serum samples.     

α-Chymotrypsin immobilized on ferromagnetic particles coated with titanium oxide: Production and catalytic characterization

Immobilization of α-chymotrypsin on magnetic particles with stable coat with titanium oxides as a main constituent allowed the biocatalytic system to be quickly and qualitatively separated into the components after completion of the enzymatic reaction. X-ray phase analysis demonstrated that the coat of magnetic particles is composed mainly of titanium dioxide in brookite modification. The maximal capacity of the particles amounted to 0.3 mg protein/mg particles. It was demonstrated that the reaction catalyzed by immobilized α-chymotrypsin proceeds in a kinetic mechanism due to a high dispersion of the ferromagnetic particles. The catalytic constant (25 s⁻¹) andK _M (0.17 mM) for the immobilized enzyme for the hydrolysis of N-acetyl-L-tyrosine ethyl ester are comparable to the corresponding characteristics for the free enzyme.