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 ammonia in untreated serum with a fiber-optic ammonia gas ensor

A fluorescence-type fiber-optic ammonia gas sensor has been developed for the determination of ammonia in untreated serum. An internal solution has been designed to provide both the selectivity and limit of detection required for this measurement. Selectivity over carbon dioxide is accomplished by adjusting the level of ammonium chloride in the internal solution. A sub-micromolar detection limit is obtained by using a combination of 2′,7′-dichlorofluorescein and 5-carboxy-2′,7′-dichlorofluorescein as the indicator dyes. The limit of detection for the resulting sensor is 0.09 μM and response times range from 2 to 6 min. When applied to the determination of ammonia in eighteen untreated serum samples, the results from the sensor compare favorably with those from the conventional glutamate dehydrogenase assay.     

A Powder Based Polyaniline Carbon Paste Electrode for Urea Analysis

A new polyaniline carbon paste electrode prepared by mixing polyaniline (emeraldine), nafion, graphite powder and urease for urea analysis was exploited. The ratio of polyaniline, nafion, urease and graphite for the construction of the electrodes and the optimal conditions for urea determination were studied. The detection limit of this sensor for urea is 5 μM and the linearity from 5 μM to 7.5 mM is obtained in FIA. This sensor has a response time of 90s and shows good reproducibility and stability (RSD, 6.3%, n = 43). The blood samples from a patient during blood dialysis were taken and analyzed. The urea concentrations in blood obtained from this sensor are comparable with urea test kit method.     

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.     

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.     

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.     

Bioelectrochemical Response of Polypyrrole Modified Urease Sensor

The preparation method of biological ferment electrode is one of decisive factors that affect its bio-electrochemical responding sign. It demands both suitable indicator electrode and antiwater soluble film that can keep catalytic activity. In this paper,urease was doped into polypyrrole(PPy) film while pyrrole(Py) was polymerized by electrochemical method to formed urease biosensor based on PPy film,then,ferment electrode was combined with CO₂ gas-sensing electrode to assemble a urease biosensor responding to urea.     

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.     

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.     

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.     

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.     

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.     

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.     

Studies on urea biosensors based on immobilized corynebacterium glutamicum and their kinetic response processes

Two novel biosensors for urea based on immobilized corynebacterium glutamicum 617 and corynebacterium glutamicum ATCC13032 in calcium alginate gel coupled with an ammonia gas-sensing electrode, were designed and constructed. Calibration plots of measured potential difference (mV) vs. log of urea concentration were linear in the range of 5.6 x 10(-5)-1.4 x 10(-2) and 5.6 x 10(-5)-1.1 x 10(-2) mol l(-1), with slopes of 59.2 and 61.3 mV per decade respectively, in pH 8.0, 0.1 mol l(-1) phosphate buffer solution at 30 degrees C. The relationship between the initial response velocity and the substrate concentration was also discussed. The results indicate that the kinetic response process of the reaction catalyzed by bacteria is similar to that by isolated enzyme. Using an Eadie-Hofstee plot, the apparent Michaelis constant K(m) and the maximum initial response velocity V(m) for urease in the immobilized bacterial membrane were determined. The two urea biosensors were successfully applied for the actual measurement of urea in urine and were relatively stable for 20 and 40 days respectively.     

Novel surface acoustic wave-interdigitated array electrode gas sensor for dissolved ammonia

A novel surface acoustic wave-interdigitated array electrode (SAW-IDA) ammonia gas sensor is proposed. A gas-permeable membrane is employed to separate the buffer solution in the inner cell of the gas-sensing probe from the sample solution in the detection cell. The response of the IDA conductive electrodes is based on the impedance change of the buffer solution during ammonia adsorption. Therefore, this gas sensor overcomes the influence of water vapour in the conventional film-coated SAW gas sensor and can be used for the detection of gases in aqueous solutions. The ammonia sensor exhibits a favourable frequency response to 5 × 10⁻⁷−1 × 10⁻³ mol/1 ammonia. The optimal buffer composition and probe parameters have been determined. Dynamic range, response time, selectivity, and temperature drift are discussed. The ammonia sensor was also applied to the determination of serum ammonia. Results were in good agreement with those from the conventional enzymatic-spectro-photometric method.     

A flow-through detector for simultaneous determination of glucose and urea in serum samples

Glucose and urea electrodes are prepared by immobilizing glucose oxidase and urease on nylon net and fixing the nets on oxygen and ammonia gas sensors. Both enzyme electrodes are fixed in a single flow cell (40 μl volume). Serum is diluted tenfold with 0.1 M Tris buffer (pH 8.3) to fit the calibration graphs for both sensors. Samples are pumped for 1 min, with wash periods of 2 min for recovery to the baseline. Results on serum samples are in good agreement with the results obtained by conventional spectrophotometry.     

A conductometric biosensor for the estimation of the number of cleaving sites in peptides and proteins

A conductometric enzyme biosensor using proteinase K was developed and then tested to relate its electrical signal to the number of proteinase K hydrolysis sites in bovine serum albumin (BSA) and angiotensin, a ten amino acid peptide, with one cleaving site.The conductometric sensor presents a large linear range of response for BSA and angiotensin ranging from 0.5 to 8 mg/l and from 4 to 8 mg/l, respectively. For a same tested concentration (mg/l), the response for native BSA is 12 times higher than for angiotensin. Aspartam was used as negative test and no response was obtained with the proteinase K biosensor. The conductometric sensor permitted also to detect difference on enzyme activity on native and non-native BSA, a response three times higher was obtained for non-native BSA.     

Nanohybrid Comprising Gold Nanoparticles – MoS2 Nanosheets for Electrochemical Sensing of Folic Acid in Serum Samples

Folic acid (FA) deficiency is associated with several clinical conditions such as megaloblastic anemia, neuropsychiatric, and pregnancy-related syndromes, this makes FA an important metabolite to be monitored. We have fabricated an electrochemical biosensor based on gold nanoparticles decorated molybdenum disulfide nanosheets (AuNPs−MoS₂NSs) nanocomposite as a transducer matrix for specific and rapid electrochemical detection of FA. Differential pulse voltammetry (DPV) studies displayed a rapid analytical response of the fabricated AuNPs−MoS₂NSs/GCE sensor probe towards FA in a wide concentration range of 0.001–100 μM with a very low detection limit of 0.72±0.03 nM. The selectivity of the fabricated sensor probe has been examined in the presence of interferents such as dopamine, uric acid, ascorbic acid, glucose, and urea. The clinical potential of the fabricated biosensor was established by monitoring FA in human serum samples. The developed AuNPs−MoS₂NSs/GCE sensor probe showed high reproducibility and stability, indicating its promise for FA detection in clinical settings.     

Novel surface acoustic wave-interdigitated array electrode gas sensor for dissolved ammonia

 A novel surface acoustic wave-interdigitated array electrode (SAW-IDA) ammonia gas sensor is proposed. A gas-permeable membrane is employed to separate the buffer solution in the inner cell of the gas-sensing probe from the sample solution in the detection cell. The response of the IDA conductive electrodes is based on the impedance change of the buffer solution during ammonia adsorption. Therefore, this gas sensor overcomes the influence of water vapour in the conventional film-coated SAW gas sensor and can be used for the detection of gases in aqueous solutions. The ammonia sensor exhibits a favourable frequency response to 5×10^-7–1×10^-3 mol/l ammonia. The optimal buffer composition and probe parameters have been determined. Dynamic range, response time, selectivity, and temperature drift are discussed. The ammonia sensor was also applied to the determination of serum ammonia. Results were in good agreement with those from the conventional enzymatic-spectrophotometric method. Received: 1 December 1995/Revised: 9 April 1996/Accepted: 14 April 1996     

Potentiometric Biosensor System Based on Recombinant Urease and Creatinine Deiminase for Urea and Creatinine Determination in Blood Dialysate and Serum

A biosensor system for simultaneous determination of creatinine and urea in blood serum and dialysate samples was developed. It consisted of creatinine and urea biosensors based on a potentiometric transducers with two identical pH‐sensitive field‐effect transistors. In creatinine biosensor, creatinine deiminase immobilized via photopolymerization in PVA/SbQ polymer on one transistor served as a biorecognition element, while bovine serum albumin in PVA/SbQ polymer placed on the second transistor was used for reference. The urea biosensor was created in the same way but recombinant urease was used instead of creatinine deiminase. The linear ranges of creatinine and urea measurement were 0.02–2 mM and 0.5–15 mM, correspondingly, which allowed simultaneous determination of the metabolites. Response time of the biosensor system was 2–3 min; RSD of responses did not exceeded 5 %. The biosensors demonstrated absence of non‐selective response towards components of blood dialysate and serum. Urea and creatinine concentrations were determined in 20 samples of blood dialysate and serum. The results correlated well with traditional methods of analysis. Creatinine and urea biosensors were stable during five months of storage (during this time the responses decreased by about 10 %). The proposed biosensor system can be effectively used for analysis of serum samples and for hemodialysis control.