Potentiometric determination of urea by sequential injection using Jack bean meal crude extract as a source of urease

A sequential injection system was proposed to accomplish the potentiometric determination of urea. This procedure used an ammonium tubular selective electrode to assess ammonium concentration produced by enzymatic hydrolysis of urea from Jack bean meal (Canavalia ensiformis DC) crude extract. A gaseous diffusion device was coupled to the flow set-up allowing on-line sampling and suitable selectivity for determinations. A detection limit of 6.0x10(-4) mol urea l(-1), a relative standard deviation of 1.9% (n=10) and a sampling rate of 20 samples h(-1) were observed when 172 Sumner units (SU) of urease and 900 mul of sample were used. Results agreeing with a comparative method were obtained by the proposed procedure and the use of the crude extract solution combined with the sequential injection approach improved the performance, producing reproducible results and low costs in comparison with procedures using commercial enzymes.     

Optical Urea Biosensor Based On Ammonium Ion Selective Membrane

ABSTRACT

An optical urea biosensor was developed by immobilizing an urease enzyme layer on a thin ammonium-selective polymer membrane. The ammonium optical membrane utilized dichlorofluorescein octadecyl ester (DCFOE) as anionic chromophore and nonactin as neutral ionophore. The urease layer was coated on the top of the ammonium layer by gelatin entrapment combined with glutaradehyde cross-linking. Hydrolysis of urea catalyzed by urease produced ammonium ion, which was extracted into the-polymer film to form complexes with nonactin. A proton was released which resulted in a color change of the optical membrane due to charge neutrality principle. The biosensor     

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.     

Gas phase derivatization of ammonia with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and its application to urease assay.

An ammonia-specific and rapid fluorometric method for determination of ammonia and urease activity was developed. The method is designed to assay ammonia levels or urease activity for the rapid diagnosis of Helicobacter pylori infection. 4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole was used to derivatize ammonia and 4-amino-7-nitrobenzo-2-oxa-1,3-diazole was analysed by high performance liquid chromatography at an excitation wavelength of 455 nm and an emission wavelength of 520 nm. Derivatization was designed to react with ammonia gas produced in a strong alkaline pH sample. The fluorescent intensity was linear in the range of 0.1-10 mM ammonia per tube when the reaction was carried out for 15 min at 37 degrees C. Urease activity, judged as the amount of ammonia production from urea, could be measured at 25 ng per tube (S/N = 1.5) with Jack bean meal urease. Because of its rapidity, this assay is potentially superior to the current standard method in use in clinical settings.     

A Novel Bioelectrochemical Sensor Based on Immobilized Urease on the Surface of Nickel Oxide Nanoparticle and Polypyrrole Composite Modified Pt Electrode

Nickel oxide nanoparticle (NiO?NP) and polypyrrole (PPy) composite were deposited on a Pt electrode for fabrication of a urea biosensor. To develop the sensor, a thin film of PPy?NiO composite was deposited on a Pt substrate that serves as a matrix for the immobilization of enzyme. Urease was immobilized on the surface of Pt/PPy?NiO by a physical adsorption. The response of the fabricated electrode (Pt/PPy?NiO/Urs) towards urea was analyzed by chronoamperometry and cyclic voltammetry (CV) techniques. Electrochemical response of the bio‐electrode was significantly enhanced. This is due to electron transfer between Ni²⁺ and Ni³⁺ as the electro‐catalytic group and the reaction between polypyrrole and the urease‐liberated ammonium. The fabricated electrode showed reliable and demonstrated perfectly linear response (0.7–26.7 mM of urea concentration, R²= 0.993), with high sensitivity (0.153 mA mM^?1 cm^?2), low detection of limit (1.6 μM), long stability (10 weeks), and low response time (～5 s). The developed biosensor was highly selective and obtained data were repeatable and reproduced using PPy‐NiO composite loaded with immobilized urease as urea biosensors.     

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.     

Urea potentiometric biosensor based on urease immobilized on chitosan membranes

Potentiometric biosensors based on urease (E.C. 3.5.1.5.) immobilized on chitosan membranes coupled to all-solid-state nonactin ammonium ion selective electrodes are described. The enzyme was immobilized on the chitosan membranes by four procedures: (A) adsorption; (B) adsorption followed by reticulation with dilute aqueous glutaraldehyde solution; (C) activation with glutaraldehyde followed by contact with the enzyme solution; and (D) activation with glutaraldehyde, contact with the enzyme solution and reduction of the Schiff base with sodium borohydride. The response characteristics of the biosensors obtained with these enzymatic membranes were determined and compared. The biosensor with best response characteristics, obtained by procedure (B), showed the following characteristics of response to urea: (i) linearity in the 10(-4) to 10(-2) M range; (ii) slope of up to 56 mV per decade; (iii) response time between 30 s and 2 min; and (iv) lifetime of 2 months. This biosensor was tested in the determination of urea in blood serum samples.     

Urea Biosensor Based on Electrochromic Properties of Prussian Blue

Prussian blue (PB) is an electrochromic material, which can be used as a signal transducer in the formation of optical urea biosensors. The previous researches in electrochromic properties of PB demonstrated the optical PB response to ammonium ions, which occurs when ammonium ions are interacting with PB layer at a constant 0.2 V vs Ag|AgCl|KCl_sat potential. In this work PB optical dependence on ammonium ions concentration was applied in the formation of electrochromic urea biosensor. Biosensor was formed by modifying the optically transparent indium tin oxide (ITO) coated glass electrode (glass/ITO) with Prussian blue layer and immobilizing urease (glass/ITO/PB‐urease). Calibration curve showed the linear dependency (R²=0.995) between the change of maximal absorbance (ΔA) and urea concentration in concentration range varying from 3 mM to 30 mM. The highest sensitivity (4 ΔA M^?1) of glass/ITO/PB‐urease biosensor is in the concentration range from 7 mM to 30 mM. It was determined that working principle of the glass/ITO/PB‐urease biosensor is not related to pH changes occurring during enzymatic hydrolysis of urea.     

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.     

Mathematical Model and Numerical Simulation of Conductometric Biosensor of Urea

In this article, a mathematical model was developed to describe and optimize the configuration of the urea biosensor. The biosensor is based on interdigitated gold microelectrodes modified with a urease enzyme membrane. The model presented here focuses on the enzymatic reaction and/or diffusion phenomena that occur in the enzyme membrane and in the diffusion layer. Numerical resolution of differential equations was performed using the finite difference technique. The mathematical model was validated using experimental biosensor data. The responses of the biosensor to various conditions were simulated to guide experiments, improve analytical performance, and reduce development costs.     

Photo-switchable ion and enzyme sensors. Photoinduced potentiometric response of glassy carbon electrode coated with polymer or polymer/enzyme dual membrane

Photo-switchable ion and enzyme sensors were fabricated by the use of glassy carbon electrode coated with nonactindoped or enzyme modified poly(vinyl chloride) (PVC) membranes. The ion sensor with nonactin-doped PVC membrane, which contained spirobenzopyran as the photosensitive dye, exhibited a potentiometric photoresponse to NH4+ ion in the solution. The dynamic range of the NH4+ ion sensor was 10(-7)--10(-3) M. Urea, adenosine, and asparagine sensors were prepared by coating the surface of the NH4+-ion sensor with urease, adenosine deaminase, and asparaginase membranes, respectively. These enzyme sensors could be used for determining the substrates at the micro mole level. The performance characteristics of these sensors were compared with those previously prepared membrane electrode sensors.     

Amperometric determination of urea using an NADH-dependent coupled enzyme

Enzyme electrodes for the amperometric measurement of urea were prepared by co-immobilizing l-glutamate dehydrogenase and urease onto an Immobilon-AV affinity membrane with attachment to a glassy carbon electrode. Reduced nicotinamide adenine dinucleotide (NADH) was used as the electroactive species. The electrochemical oxidation of NADH was monitored at +1.0 V vs. Ag/AgCl. The enzyme immobilized electrode was linear over the range of 2.0 x 10(-5) to 2 x 10(-4)M. The response time of the electrode was 3 min and the optimum pH of enzyme immobilized membrane was pH 7.4-7.6 (Dulbecco's buffer solution). It was stable for at least two weeks and 50 assays. There were no interferences from other physiological material, except for high levels of ascorbic acid.     

Glucose and urea biosensors based on all solid-state PVC-NH2 membrane electrodes

Miniaturized urea and glucose sensors prepared by immobilization of glucose oxidase or urease directly onto all solid-state contact PVC-NH₂ membrane ammonium and hydrogen ion selective electrodes are described. The resulting biosensing membranes function equivalently to normal PVC membranes in terms of potentiometric response properties. The most important features of the glucose and urea sensors were high sensitivity, long life-time, easily built at a low cost, micro-construction and short response time. The characteristics of the glucose and urea sensors were examined in several buffer solutions at different concentrations and pH values. The influence of immobilization conditions on the dynamic response properties and life-time of the electrodes was studied. Under optimal conditions, the urea electrode showed a linear response between 5×10⁻² and 5×10⁻⁴ M urea, while the glucose electrode showed a linear response between 5×10⁻² and 1×10⁻⁴ M glucose.     

A miniaturized urea sensor based on the integration of both ammonium based urea enzyme field effect transistor and a reference field effect transistor in a single chip

A urea biosensor prepared by covalent binding of urease directly to the surface of an ammonium-sensitive field effect transistor (FET) is described. Nonactin incorporated in carboxylated polyvinyl chloride was used to obtain the sensitive membrane of the ammonium-sensitive FET. The grafting of urease on the polyvinylchloride-COOH membrane surface was performed through carbodiimide coupling. The activity of the immobilized enzyme was spectrometrically controlled through the time-dependent disappearance of the absorbance of NADH at 340 nm. An apparent activity of 50% was found, compared with free enzyme. The sensitivity of the urea enzyme FET is 50 mV/pUrea working in a differential mode of 2 muM to 1 mM, this sensitivity being constant during 15 days. Finally, in order to test the potentialities of the urea biosensor for the environmental applications, the detection of heavy metal ions such as Cu(II) and Hg(II) in solution was performed by measuring the remaining activity of the inhibited enzyme.     

Enzymatic Biosensor Based on the ISFET and Photopolymeric Membrane for the Determinaion of Urea

An ISFET based enzymatic biosensor was developed for the determination of urea. Immobilization of urease was accomplished by the use of liquid mixture which contained vinylpyrrolidone, oligouretane metacrylate and oligocarbonate metacrylate and which can form a polymer under the influence of ultraviolet. The biosensor has the following characteristics: the linear field of responses is in the range of 0.05–20 mM, curve slope – 38 mV/pC, and response time 5–10 min. The increase of the temperature from 28 to 41 °C leads to 15% increase in the intensity of the response of the biosensor. The maximum response is observed at pH 6.0–6.5. At the increase of the NaCl concentration in solution up to 300 mM the biosensor response drops off and achieves half of its initial level. NH₄Cl causes a stronger inhibition of enzyme activity comparing to NaCl. The results obtained with the developed biosensor correlate with the data of standard calorimetric methods. The intensity of the biosensor response decreases gradually during 40 days up to 80% of the initial level. The biosensors prepared with a fresh membrane or membrane preserved during 46 days at 2 °C gave similar responses in solution with an equal concentration of a substrate. It is concluded that the developed enzymatic biosensor is perspective for its clinical application for the determination of urea in blood and that the proposed method to prepare a selective biological membrane may be in a simple way included in integral technology of the semiconductor transducer manufacturing.     

A novel urea amperometric biosensor based on secretion of carnation petal cells modified on a graphite-epoxy composite electrode

A new kind of biosensor for the detection of urea with a high selectivity, sensitivity and wide detection range was designed based on the secretion of carnation petals cells paste covered over a graphite-epoxy composite basic electrode surface. The carnation petal paste from mashed fresh carnation petals was tightly fixed on the basic electrode surface with Teflon thin film to keep it in contact with the electrode surface. Urea in aqueous solution was detected by differential pulse voltammetry based on the oxidation peak current at 0.316 V (vs. SCE) of the secreted species of carnation petal cells during the mashing process, which interacts with urea molecules and results in the decrease of the oxidation peak current. The oxidation peak current decreases linearly with the logarithm of urea concentration in the range of 1.3 × 10(-16)-4.57 × 10(-8) M and 3.4 × 10(-7)-1.3 × 10(-1) M with a detection limit of 7.5 × 10(-16) M. The biosensor was characterized by electrochemistry and fluorescent spectrometry, and applied to the determination of urea in waste water from a river around Shenyang Normal University campus with a recovery of 104.5% (RSD is 5.00%). The presence of larger amounts of ammonium ion and nitrate ion up to the molar ratio of 10(4) do not interfere with the urea detection.     

Amperometric Urea Biosensor Using Aminated Glassy Carbon Electrode Covered with Urease Immobilized Carbon Sheet,Based on the Electrode Oxidation of Carbamic Acid

A large oxidation current can be observed when ammonium carbamate aqueous solution is electrolyzed using a glassy carbon electrode (GCE) at a potential exceeding 1.0 V vs. Ag/AgCl and amino groups are introduced at the surface of the GCE. Aminated GCE exhibits the electrocatalytic activity of the oxidation of ammonium carbamate that is produced from urea as an intermediate product of urease reaction, and a distinct oxidation current is observed when the aminated GCE is used to oxidize the urea in the urease solution. A novel amperometric determination method to detect urea has been developed. This method is based on the electrooxidation of carbamic acid produced during urease reactions. Urease is immobilized to polymaleimidostyrene (PMS) coated on the insulated amorphous carbon sheet set on the aminated GCE surface. A good linear relationship is observed between urea concentration and the electrolytic current of the urease‐immobilized electrode in the concentration range from 0.5 mM to 21.0 mM. The proposed urea biosensor has an effective merit in that the interference resulting from ammonia and pH change caused by the urease reaction can be eliminated, differing from conventional urea biosensors.     

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.     

Construction and Application of a PVC Membrane Enoxacin Ion－selective Electrode Based on a Needle－shaped Inner Reference Electrode

IntroductionPVC membrane- coated- wire electrodes areused in the analytical field widely. They are pre-pared usually with platinum wire,silver wire orgraphite rod coated with a PVC thin membranecontaining various active components and plasticiz-ers[1— 3 ] .The electrodes have no inner KCl solutionand they are notinfluenced by the sample pressure.Furthermore,they are free from directional selec-tivity when they are installed,and they can be mi-crominiaturized easily. The shortcomings of th…     

Flow injection analysis of sulfite ion with a potentiometric titanium phosphate-epoxy based membrane sensor

A novel pH sensor suitable for use in both aqueous and non-aqueous mediums is reported. The sensor is derived from polymer modified electrode obtained from electrochemical polymerisation of aniline in dry acetonitrile containing 0.5 M tetraphenyl borate at 2.0 V versus Ag/AgCl. The light yellow colour polymer modified electrode obtained under the present experimental condition has been characterised by scanning electron microscopy (SEM). The pH sensing of polymer modified electrode in both aqueous and non-aqueous mediums is examined and reported. As the typical examples, we used weak acid (acetic acid) and weak base (ammonium hydroxide) as analytes. The acetic acid is analysed in both aqueous and dry acetonitrile whereas ammonium hydroxide is analysed only in aqueous medium. The analysis in aqueous medium is conducted in 1 mM Tris-HCl buffer pH 7.0 and also in 0.1 M KCl. The slope of pH sensing is calculated from the data recorded in typical buffers and found to be approximately 86 mV per pH. The application of polymer modified electrode for the construction of urea biosensor is described based on immobilised urease within poly vinyl alcohol (PVA) matrix and also within organically modified sol-gel glass on the surface of polymer-modified electrode. The new urea sensor has shown maximum response of 160 mV at 25 degrees C with a lowest detection limit of 20 muM. The performance of new pH sensor and urea sensor has been studied and reported in this communication.