Characterization of biosensors based on membranes containing a conducting polymer

A new type of biosensor based on the coupling of an enzyme to an ion-selective membrane containing a conducting polymer is evaluated. The results obtained with the enzyme field- effect transistor (ENFET) and the ion-selective electrode (ISE) for the determination of creatinine and urea are compared. The presence of the conducting polymer significantly lowers the detection limit for creatinine by one decade to 10^?7 and 10^?4 M for the ENFET and ISE, respectively. The determination of urea in urine and serum with the ENFET was carried out, and the results correlated well with those obtained by spectrophotometry.     

Renewable urea sensor based on a self-assembled polyelectrolyte layer

A renewable urea sensor based on a carboxylic poly(vinyl chloride) (PVC-COOH) matrix pH-sensitive membrane has been proposed, in which a positively charged polyelectrolyte layer is first constructed by using a self-assembly technique on the surface of a PVC-COOH membrane, and urease, with negative charges, is then immobilized through electrostatic adsorption onto the PVC-COOH membrane, by controlling the pH of the urease solution below its isoelectric point. The response characteristics of the PVC-COOH pH-sensitive membrane and the effects of experimental conditions have been investigated in detail. Compared with conventional covalent immobilization, the urea sensor made with this self-assembly immobilization shows significant advantage in terms of sensitivity and ease of regeneration. The potential responses of the urea sensor with self-assembly immobilization increase with the urea concentration over the concentration range 10(-5) - 10(-1) mol l(-1), and the detection limit is 0.028 mmol(-1). Moreover, this type of urea sensor can be repeatedly regenerated by using a simple washing treatment with 0.01 mol l(-1) NaOH (containing 0.5 mol l(-1) NaCl) and 0.01 mol l(-1) HCl. The urease layers and the polyelectrolyte layers on the PVC-COOH membrane are removed, the potential response of the sensor to urea solutions of different concentrations returns nearly to zero, and another assembly cycle of urease and polyelectrolyte can then be carried out.     

Use of CdSe/ZnS luminescent quantum dots incorporated within sol-gel matrix for urea detection

In this work, urea detection techniques based on the pH sensitivity of CdSe/ZnS QDs were developed using three types of sol-gel membranes: a QD-entrapped membrane, urease-immobilized membrane and double layer consisting of a QD-entrapped membrane and urease-immobilized membrane. The surface morphology of the sol-gel membranes deposited on the wells in a 24-well microtiter plate was investigated. The linear detection range of urea was in the range of 0-10 mM with the three types of sol-gel membranes. The urea detection technique based on the double layer consisting of the QD-entrapped membrane and urease-immobilized membrane resulted in the highest sensitivity to urea due to the Michaelis-Menten kinetic parameters. That is, the Michaelis-Menten constant (K_m =2.0745 mM) of the free urease in the QD-entrapped membrane was about 4-fold higher than that (K_m =0.549 mM) of the immobilized urease in the urease-immobilized membrane and about 12-fold higher than that (K_m =0.1698 mM) of the immobilized urease in the double layer. The good stability of the three sol-gel membranes for urea sensing over 2 months showed that the use of sol-gel membranes immobilized with QDs or an enzyme is suitable for biomedical and environmental applications.     

Trace Level Analysis of Mercury Using Urease in Combination with an Ammonia Gas Sensitive Semiconductor Structure

Abstract

A method for the determination of mercury(II) ions at trace levels is described. The method is based on the profound inhibitory effect of mercury on the enzyme urease. The activity of the enzyme was determined by the rate of ammonia produced from urea as followed by an ammonia gas sensitive iridium thin metalfilm-oxide-semiconductor (IrTMOS) structure. Two systems were investigated. For the initial urease activity studies, a simple microcell was used. Also, a test plate, containing dry reagent strips with all necessary chemicals was developed, making the analytical procedure very simple to perform. The test volume applied was 2 μl and the sensitivity to standards of mercury(II) ions is at least 0.005 μM (1.0 ng/ml). One sample could be analyzed in less than 8 minutes. Furthermore, the kinetics of sensor response versus enzyme activity is discussed.     

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.     

Preparation and Response Properties of Selective Bio-Electrodes Utilizing Polymer Membrane Electrode-Based Ammonia Gas Sensors

Abstract

A new type of potentiometric ammonia gas sensor is employed in the preparation of selective bio-electrodes for urea and glutamine. The bio-electrodes are constructed by immobilizing the enzyme urease and intact porcine kidney cells, respectively, at the surface of a disposable ammonium selective polymer membrane electrode-based ammonia gas sensor. The resulting electrodes have favorable response properties when compared to corresponding devices previously assembled with costly commercial gas sensors. Preliminary studies with the urea electrode demonstrate its usefulness for the rapid determination of urea in serum samples.     

Piezoelectric enzyme sensor with improved temperature characteristics for determining urea in urine

Based on the conductivity change produced by the urea/urease reaction, a piezoelectric enzyme sensor is applied to the detection of urea in urine. The influence of the variation in solution temperature on frequency measurement is greatly reduced by using a buffer giving solution conditions with a near-zero frequency temperature coefficient for the sensor. The sum of the frequency shifts over the reaction time was used to increase the sensitivity of the determination. The linear range of the method is 1–30 g/ml for urea, with RSDs of 1–2.6%. The detection limit is 0.01 g/ml.     

Purification,Properties, and Application of a Novel Acid Urease from <Emphasis Type="Italic">Enterobacter</Emphasis> sp.

It has been demonstrated that acid urease is capable of decomposing urea in fermented beverage and foods. As urea is a precursor of ethylcarbamate, a potential carcinogenic compound, measures must be taken to control the level of urea. We herein describe the purification and characterization of a novel acid urease from Enterobacter sp. R-SYB082 and its application to the removal of urea in Chinese rice wine. The enzyme was purified to electrophoretic homogeneity using ethanol precipitation, Superdex 200 and Mono Q with a fold purification of 21.1 and a recovery of 49%. The molecular weight of the enzyme was 430,000 Da by gel filtration and 72,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it was a hexamer. The activity of this purified enzyme was optimal at pH 4.5 and 35 °C. The temperature stability was under 55 °C, and the pH stability was 4.0~5.0. The enzyme exhibited an apparent K _m of 19.5 μmol/l and a V _max of 109 μmol urea/mg·min at 35 °C and pH 4.5. When incubating two different kinds of Chinese rice wine with the enzyme (0.08 U/ml) at 35 °C for 7 days, over 85% of urea was decomposed, and at 20 °C, above 78% was removed. The result showed that the enzyme is applicable to elimination of urea in Chinese rice wine.     

Cockle shell-derived nanoparticles for optical urea biosensor development based on reflectance transduction

An optical biosensor for urea based on urease enzyme immobilised on functionalised calcium carbonate nanoparticles (CaCO₃-NPs) was successfully developed in this study. CaCO₃-NPs were synthesised from discarded cockle shells via a simple and eco-friendly approach, followed by surface functionalisation with succinimide ester groups. The fabricated biosensor is comprised of two layers. The first (bottom layer) contained functionalised NPs covalently immobilised to urease, and the second (uppermost layer) was alginate hydrogel physically immobilised to the pH indicator phenolphthalein. The biosensor provided a colorimetric indication of increasing urea concentrations by changing from colourless to pink. Quantitative urea analysis was performed by measuring the reflectance intensity of the colour change at a wavelength of 633.16 nm. The determination of urea concentration using this biosensor yielded a linear response range of 30–1000 mM (R² = 0.9901) with a detection limit of 17.74 mM at pH 7.5. The relative standard deviation of reproducibility was 1.14%, with no signs of interference by major cations, such as K⁺, Na⁺, NH?⁺, and Mg²⁺. The fabricated biosensor showed no significant difference with the standard method for the determination of urea in urine samples.     

Fiber-Optic biosensor for urea based on sensing of ammonia gas

A fiber-optic biosensor for urea is described. This biosensor is based on the immobilization of urease at the sensing tip of a fluorescence-based ammonia gas-sensing fiber-optic chemical sensor. Urease is immobilized on a Teflon membrane by the well known bovine serum albumin (BSA)/glutaraldehyde cross-linking method. The indicator solution for this biosensor is composed of 0.145 M sodium chloride, 5.00 mM ammonium chloride, 9.4 μM 2′,7′-bis(carboxyethyl)-5 (and 6)-carboxyfluorescein and 0.9 μM 5 (and 6)-carboxyfluorescein. The steady-state and dynamic response properties of the sensor have been established. Results show that the urease/BSA protein layer has a significant effect on sensor response and recovery times. Also, the fluorescence-based sensor has been found to be faster than a conventional potentiometric ammonia gas-sensing electrode. In addition, the fluorescence sensor responds significantly quicker than a similar absorbance-based fiber-optic urea biosensor. The utility of the resulting urea biosensor for the determination of urea in diluted serum samples is demonstrated.     

Enzyme coated glass pH-electrode: Its fabrication and applications in the determination of urea in blood samples

A new enzyme coated electrode for the determination of urea in blood samples has been developed. It is based on the encapsulation of urease enzyme in the porous silicate matrix by the sol-gel technique on a glass electrode for the purpose of sensing urea in blood samples. Various parameters like the effect of pH, selection of a suitable buffer of appropriate concentration and interference of common substances in blood samples have been evaluated to optimize the conditions for the determination of urea. The electrode can be used for the determination of urea in the concentration range 0.03-30.0 mM in a solution. The detection limit of the present enzyme-coated electrode is found to be 52 μg/ml of urea. The relative standard deviation for the electrode-to-electrode reproducibility is found to be 2.4% for the determination of 0.1 mM of urea (six replicate electrodes). Sol-gel matrix containing immobilized enzyme was stable for about 25 days at ∼4 °C with 80% urease activity. Urea content in various clinical blood samples has been estimated using this electrode and the results are found to be in good agreement with the standard clinical methods as reported in the literature.     

Determination of urea in serum by a fiber-optic fluorescence biosensor

A new fiber-optic biosensor for urea has been developed, based on immobilized urease coupled to a fluorescence ammonia sensor. The enzymatically generated ammonia diffuses through the membrane into a solution of the fluorescent pH indicator trisodium 8-hydroxypyrene-1,3,6-trisulfonate. The sensor has been successfully used for the determination of urea in serum samples, with results in good agreement with those reported by a local hospital. The proposed sensor is reversible and selective to urea. The ease of construction of the sensor tip offers the possibility of designing disposable tips for use in clinical applications.     

Determination of urea with an ammonia gas-sensitive semiconductor device in combination with urease

An ammonia gas-sensitive Ir/Pd MOS capacitor is used for urea determinations with the aid of urease in two different systems. One combination utilizes a reaction column with immobilized urease in a flow-injection system. The lower limit of urea detection for 150-μl samples was 0.2 μM. Urea in whole blood and blood serum was determined after a 500-fold dilution, and 15 samples per hour could be assayed. The relative standard deviation was 4.6% (n=10). Recovery tests were satisfactory. Values obtained for urea in serum correlated well with those from a spectrophotometric method. The other combination is based on a small flow cell with free urease enclosed between a dialysis membrane and a gas-permeable membrane. Urea was determined in the concentration range 0.01–50 mM. The enzyme probe could be used for up to four days without changes of behaviour.     

Spectrophotometric bioanalytical flow-injection system for control of hemodialysis treatment

A spectrophotometric flow-injection analysis (FIA) system for monitoring clinical hemodialysis is demonstrated. The role of a dialysate urea detector incorporated in this bioanalytical system is played by an optical flow-through biosensor based on Prussian Blue film with chemically linked urease forming a monomolecular layer of the enzyme. This pH-enzyme optode-FIA system is useful for the selective determination of post-dialysate urea in the range of concentration corresponding to its level in real clinical samples (2-16 mmol l(-1)). This bioanalytical system allows the analysis of about 15 samples of spent dialysate per hour. The operational and storage stabilities of the applied biosensor are longer than 2 weeks and 2 months, respectively. Clinical evaluation of the bioanalytical system was performed.     

Urease–glutamic dehydrogenase biosensor for screening heavy metals in water and soil samples

A screen-printed three-electrode amperometric biosensor based on urease and the nicotinamide adenine dinucleotide hydrogen (NADH)–glutamic dehydrogenase system was developed and applied to the screening of heavy metals in environmental samples. The development of an amperometric sensor for the monitoring of urease activity was feasible by coupling the urea breakdown reaction catalysed by urease to the reductive ammination of ketoglutarate catalysed by glutamic dehydrogenase (GLDH). The ammonia provided by the urea conversion is required for the conversion of ketoglutarate to glutamate with the concomitant oxidation of the NADH cofactor. NADH oxidation is monitored amperometrically at 0.3 V (vs. Ag/AgCl) after urease immobilization onto the screen-printed three-electrode configuration. Immobilization of urease on the surface of screen-printed electrodes was performed by entrapment in alginate gel and adsorption on the electrode in a nafion film. Low sensitivity to inactivation by metals was recorded after urease entrapment in alginate gel with detection limits of 2.9 and 29.8 mg L^–1 for Hg(II) and Cu(II), respectively. The use of the negatively charged nafion film created a more concentrated environment of cations in proximity to the enzyme, thus enhancing the urease inhibition when compared to gel entrapment. The calculated detection limits were 63.6 and 55.3 g L^–1 for Hg(II) and Cu(II), respectively, and 4.3 mg L^–1 for Cd(II). A significant urease inactivation was recorded in the presence of trace amounts of metals (g L^–1) when the enzyme was used free in solution. Analysis of water and soil samples with the developed nafion-based sensor produced inhibition on urease activity according to their metal contents. The obtained results were in agreement with the standard methods employed for sample analysis. Nevertheless, the use of the amperometric assay (with free urease) proved more feasible for the screening of trace amounts of metals in polluted samples.     

Chronocoulometric determination of urea in human serum using an inkjet printed biosensor

A biosensor for the determination of urea in human serum was fabricated using a combination of inkjet printed polyaniline nanoparticles and inkjet printed urease enzyme deposited sequentially onto screen-printed carbon paste electrodes. Chronocoulometry was used to measure the decomposition of urea via the doping of ammonium at the polyaniline-modified electrode surface at -0.3 V vs. Ag/AgCl. Ammonium could be measured in the range from 0.1 to 100 mM. Urea could be measured by the sensor in the range of 2-12 mM (r(2)=0.98). The enzyme biosensor was correlated against a spectrophotometric assay for urea in 15 normal human serum samples which yielded a correlation coefficient of 0.85. Bland-Altman plots showed that in the range of 5.8-6.6 mM urea, the developed sensor had an average positive experimental bias of 0.12 mM (<2% RSD) over the reference method.     

Enzyme-octadecylamine Langmuir-Blodgett membranes for ENFET biosensors

Langmuir-Blodgett (LB) films containing butyrylcholinesterase (BuChE) are fabricated to realise an enzymatic field effect transistor (ENFET) for the detection of organophosphorus pesticides in water. Trichlorfon as a common pesticide is examined in our work. The BuChE-immobilised LB films are formed by adsorbing the enzyme molecules onto a stearylamine monolayer using the electrostatic force. Enzyme/stearylamine mixed LB films are immobilized onto a pH-ISFET surface and treated by glutaraldehyde vapour to improve the LB film's stability. The ENFET thus obtained worked as a potentiometric biosensor for trichlorfon detection on the basis of enzyme inhibition. The detection limit for trichlorfon can reach 10(-7) M (26 ppb). The surface characteristics of BuChE/stearylamine LB films obtained under various conditions of the dipping surface pressures are analysed qualitatively by atomic force microscopy (AFM) and analysed quantitatively by FTIR spectroscopy.     

New Urea Biosensor Based on Urease Enzyme Obtained from Helycobacter pylori

The urease enzyme of Helicobacter pylori was isolated from biopsy sample obtained from antrum big curvature cell extracts. A new urea biosensor was prepared by immobilizing urease enzyme isolated from Helicobacter pylori on poly(vinylchloride) (PVC) ammonium membrane electrode by using nonactine as an ammonium ionophore. The effect of pH, buffer concentration, and temperature for the biosensor prepared with urease from H. pylori were obtained as 6.0, 5 mM, and 25 °C, respectively. We also investigated urease concentration, stirring rate, and enzyme immobilization procedures in response to urea of the enzyme electrode. The linear working range of the biosensor extends from 1 × 10(-5) to 1 × 10(-2) M and they showed an apparent Nernstian response within this range. Urea enzyme electrodes prepared with urease enzymes obtained from H. pylori and Jack bean based on PVC membrane ammonium-selective electrode showed very good analytical parameters: high sensitivity, dynamic stability over 2 months with less decrease of sensitivity, response time 1-2 min. The analytical characteristics were investigated and were compared those of the urea biosensor prepared with urease enzyme isolated from Jack bean prepared at the same conditions. It was observed that rapid determinations of human serum urea amounts were also made possible with both biosensors.     

Determination of mercury(II) traces in drinking water by inhibition of an urease reactor in a flow injection analysis (FIA) system

 The integration of an urease reactor into a gas diffusion flow injection system was investigated for the determination of urease inhibitors. The enzyme was immobilized by entrapping in polyacrylamide gel. Besides copper and silver ions mercury ions inhibit the conversion of urea to carbon dioxide and ammonia catalysed by urease. The pH change of the carrier solution caused by the ammonia released was measured potentiometrically with a pH electrode. The inhibition behaviour of Hg(II) ions was investigated. A linear range from 2 to 20 μg L^-1 Hg(II) was obtained after a 90 s inhibition, with a correlation coefficient of r=0.9997. The relative standard deviation was 1.4% for five measurements of 2 μg L^-1Hg(II). A sample frequency of 7 h^-1 was achieved. The inhibited enzyme can be reactivated. The method was applied to the determination of Hg(II) in two drinking water samples. Received: 16 April 1996/Revised: 3 June 1996/Accepted: 11 June 1996     

Characterization of a urea optical sensor based on polypyrrole

A new optical sensor for urea determination is presented. It is based on the enzymatic reaction with urease, which is first photoimmobilized with polyacrylamide onto a chemically polymerized polypyrrole (PPy) film. The main advantage of this sensor is that no indicator dye or pH indicator is needed, because PPy itself acts as the support and the indicator. These PPy films show an absorbance spectrum in the near IR range which is pH dependent. The variation of absorbance is thus directly related to the change of pH caused during the enzymatic reaction, which is also dependent on the urea concentration. The linear range of the sensor is from 0.06 to 1 M of urea, which is the common level of urea concentration found in blood and urine samples.