Uricase based methods for determination of uric acid in serum

The most common methods for determination of uric acid in serum are based on the use of the enzyme uricase. Uric acid is enzymatically oxidized by oxygen to produce hydrogen peroxide, allantoin, and carbon dioxide. The four most often applied uricase methods are critically compared in this review.     

An amperometric uric acid biosensor based on chitosan-carbon nanotubes electrospun nanofiber on silver nanoparticles

A novel amperometric uric acid biosensor was fabricated by immobilizing uricase on an electrospun nanocomposite of chitosan-carbon nanotubes nanofiber (Chi–CNTsNF) covering an electrodeposited layer of silver nanoparticles (AgNPs) on a gold electrode (uricase/Chi–CNTsNF/AgNPs/Au). The uric acid response was determined at an optimum applied potential of ?0.35 V vs Ag/AgCl in a flow-injection system based on the change of the reduction current for dissolved oxygen during oxidation of uric acid by the immobilized uricase. The response was directly proportional to the uric acid concentration. Under the optimum conditions, the fabricated uric acid biosensor had a very wide linear range, 1.0–400 μmol L^?1, with a very low limit of detection of 1.0 μmol L^?1 (s/n?=?3). The operational stability of the uricase/Chi–CNTsNF/AgNPs/Au biosensor (up to 205 injections) was excellent and the storage life was more than six weeks. A low Michaelis–Menten constant of 0.21 mmol L^?1 indicated that the immobilized uricase had high affinity for uric acid. The presence of potential common interfering substances, for example ascorbic acid, glucose, and lactic acid, had negligible effects on the performance of the biosensor. When used for analysis of uric acid in serum samples, the results agreed well with those obtained by use of the standard enzymatic colorimetric method (P?>?0.05).

A Fiber Optic Biosensor for Uric Acid Based on Immobilized Enzymes

Abstract

A simplified enzyme based fiber optic uric acid biosensor is reported. It is constructed by coimmobilizing uricase and horseradish peroxidase [HRP] to bovine albumin via glutaraldehyde. A new fluorimetric substrate, thiamine, is used to indicate the sensing process. Under optimized conditions, the linear range of uric acid concentration is 0.5 to 5.0 ug/mL with a correlation coefficient of 0.997, and the detection limit is 0.15 ug/mL with excellent reversibility and stability. The sensor can be used at least two months at room temperature. It is possibly to determine uric acid directly in serum and urine samples with satisfactory results.     

Flow-coulometric detector for uric acid in human urine

A novel flow-coulometric detector integrating an immobilized uricase reactor and an electrolytic cell was fabricated and used for the determination of uric acid in human urine. The procedure is based on the measurement of the total charge with and without passing the sample through an enzyme reactor which allows the complete conversion of the electro-active uric acid in electro-inactive products. The amount of uric acid is linearly related to the difference between the two total charges. The current efficiencies for 1 × 10^?4-1 × 10^?3 M uric acid were found to be nearly 100% (r.s.d. < 1%).     

Degradation of uric acid and 3-ribosyluric acid during treatment with charcoal

Uric acid and 3-ribosyluric acid at a concentration of 1.5 × 10^?4M were quantitatively adsorbed to charcoal, but were not recovered when the charcoal was washed with ethanol:water:NH₄OH (60:36:4), a solvent which readily eluted a number of other bases and nucleosides. With [2-¹⁴C]uric acid it was shown that the radioactivity was adsorbed to the charcoal and that [¹⁴C]allantoin was the primary product recovered after elution. Incubation of uric acid or 3-ribosyluric acid in the ethanol:water:NH₄OH did not result in any degradation. The elution of uric acid from charcoal with other eluents such as 7% phenol, 0.1 M NaOH, or ethanol:water:pyridine (50:40:10) also resulted in the conversion of uric acid to allantoin. It was concluded that when uric acid and 3-ribosyluric acid are adsorbed to charcoal and then eluted, there is a substantial conversion of these compounds to the corresponding allantoin.     

Highly sensitive flow detection of uric acid based on an intermediate regeneration of uricase

The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow-injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to microM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 ml min-1 and 37 degrees C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10(-10) M (amplification factor; more than 10,000). This chemically amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to microM) of uric acid in highly diluted body fluids in which the concentration of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4 degrees C in 10 mM TRIS-HCl buffer, pH 9.0.     

Selective coulometric determination of uric acid in human urine using uricase

The coulometric determination of uric acid in human urine is done using porous carbon felt electrodes containing electrolyte. The diluted human urine is dropped on the carbon felt surface and uric acid is completely electrically oxidized in a few minutes. The coulombs consumed by interferences contained in human urine are determined by measuring the electrolytic oxidation of the same diluted human urine to which uricase is added. The current efficiencies of uric acid are nearly 100% (RSD < 1%). The results are in fairly good agreement with those obtained by sepectrophotometry.     

Identification of allantoin, uric acid, and indoxyl sulfate as biochemical indicators of filth in food packaging by LC.

A liquid chromatographic (LC) method was developed for the determination of allantoin, uric acid, and indoxyl sulfate in mammalian urine contaminated packaging material including paper bagging, corrugated cardboard, grayboard, and burlap bagging. The procedure involves solvent extraction and isolation of the 3 analytes by reversed-phase LC with ultraviolet detection at 225 nm for allantoin and 286 nm for uric acid and indoxyl sulfate. The composition of authentic mammalian urine such as mouse, rat, cat, dog, and human were also determined with regard to the 3 compounds of interest. A linear concentration range of 0.11-20.4, 0.02-10.0, and 0.04-30.0 microg/mL was obtained for allantoin, uric acid, and indoxyl sulfate, respectively. Limits of detection (LOD) and quantitation (LOQ) were 0.0104 and 0.0345 microg/mL for allantoin; 0.0018 and 0.0060 microg/mL for uric acid; and 0.0049 and 0.0165 microg/mL for indoxyl sulfate, respectively. Interday relative standard deviation values for a mixture of standard allantoin, uric acid, and indoxyl sulfate (n = 5) were 0.97, 0.80, and 0.94%, respectively. Analyte composition for 5 types of authentic mammalian urine varied from 0.19-6.88 mg/mL allantoin; 0.08-0.57 mg/mL uric acid; and 0.03-0.78 mg/mL indoxyl sulfate. Analyte content for 8 samples including 2 samples each for paper, cardboard, grayboard, and burlap bagging each contaminated with mouse or rat urine ranged from 相似文献   

Immobilization of uricase within polystyrene using polymaleimidostyrene as a stabilizer and its application to uric acid sensor

A novel amperometric uric acid (UA) sensor has been developed by coating the surface of a gold electrode with a polystyrene (PS) membrane formed by 30 μL of a 30 mg mL⁻¹ PS chloroform solution combined with 30 μL of a 5 mg mL⁻¹ polymaleimidostyrene (PMS) solution as a dispersant for enzyme, uricase; this membrane has been successfully employed as an immobilization support for uricase. In the PS membrane, PMS forms micelle-like structures containing uricase in an active state. This immobilized uricase membrane permits the permeation of oxygen, which is consumed by the uricase reaction. A good linear relationship is obtained over the concentration range of 5-105 μM. The concentration of uric acid was determined at a negative potential based on the decrease in the reduction current of oxygen and the interference of l-ascorbic acid can be completely eliminated.     

Simultaneous detection of uric acid and glucose on a dual-enzyme chip using scanning electrochemical microscopy/scanning chemiluminescence microscopy

Scanning electrochemical microscopy (SECM) and scanning chemiluminescence microscopy (SCLM) were used for imaging an enzyme chip with spatially-addressed spots for glucose oxidase (GOD) and uricase microspots. For the SECM imaging, hydrogen peroxide generated from the GOD and/or uricase spots was directly oxidized at the tip microelectrode in a solution containing glucose and/or uric acid (electrochemical (EC) detection). For the SCLM imaging, a tapered glass capillary (i.d. of 1∼2 μm) filled with luminol and horseradish peroxidase (HRP) was used as the scanning probe for generating the chemiluminescence (CL). The inner solution was injected from the capillary tip at 78 pl s⁻¹ while scanning above the enzyme-immobilized chip. The CL generated when the capillary tip was scanned above the enzyme spots was detected using a photon-counter (CL detection). Two-dimensional mapping of the oxidation current and photon-counting intensity against the tip position affords images of which their contrast reflects the activity of the immobilized GOD and uricase. For both the EC and CL detections, the signal responses were plotted as a function of the glucose and uric acid concentrations in solution. The sensitivities for the EC and CL detection were found to be comparable.     

A Uric Acid Biosensor Based on Langmuir-Blodgett Film as an Enzyme-Immobilizing Matrix

A uric acid biosensor was fabricated by the Langmuir–Blodgett (LB) technique to immobilize the uricase on chitosan/Prussian blue (CS/PB) prefunctionalized indium-tin oxide (ITO) electrode. The effects of ionic strengths, acidity of subphase, and uricase amount on the film were studied. The electrochemical properties of the uricase/n-nonadecanoic acid (UOx/NA) LB film proved that CS/PB was a good electro-catalyst for the reduction of hydrogen peroxide produced by enzymatic reaction of UOx, and protein molecules retained their natural electro-catalytic activity. The linear range of uric acid detection was from 5 × 10^?6 mol/L to 1.15 × 10^?3 mol/L with a detection limit of 1.8 × 10^?7 mol/L.     

Novel Uric Acid Sensor Based on Enzyme Electrode Modified by ZnO Nanoparticles and Multiwall Carbon Nanotubes

Abstract

In this work, we report the development of a highly sensitive and stable uric acid sensor based on the synergic action of multiwalled carbon nanotubes (MWNTs) and ZnO nanoparticles. MWNTs were first cast on pyrolytic graphite (PG) wafers. ZnO nanoparticles were then decorated onto the negatively charged MWNTs via the Vapor Liquid Solid (VLS) growth. Uricase was immobilized on the ZnO nanoparticles surface because of their large differences in the isoelectric point (IEP). Last, a cationic polydiallyldimethylammonium chloride (PDDA) layer was coated onto the uricase-contained ZnO nanoparticle layer and resulted in the PDDA/uricase/ZnO/MWNTs multilayer structure. The unique multilayer structure provides a favorable microenvironment to keep the bioactivity of uricase, which led to rapid amperometric response toward uric acid. Amperometric detection of uric acid was carried out at 320 mV (vs. SCE) in 0.05 mol/L (M) phosphate buffer solution (pH 6.8). For the sensor, a wide linear response range of 5.0 µM to 1 mM with a linear sensitivity of 393 mA cm^?2M^?1, a detection limit of 2.0 µM (3σ), and a long-term stability of 160 days can be obtained by using differential pulse voltammetry (DPV). Testing results in human urine obtained from the sensors were also compared with the data obtained by spectrometry. For five samples with different concentrations of urine, the relative deviations between them were smaller than 3.8%. The recovery was between 96.5 and 104.0%.     

An Electrochemical Biosensor with Uricase Immobilized on Functionalized Gold Coated Copper Wire Electrode for Urinary Uric Acid Assay

A new second generation uricase electrode for urinary uric acid determination has been developed by chemically binding both uricase and redox mediator to inexpensive copper wire through simple electrodeposition of gold on copper surface and subsequent functionalization of the gold with L‐methionine. During a 209‐day testing period, the overall electrode performance exhibits in average a low oxidation potential of 0.33 V, a response time of 5 s, a widest linear calibration concentration range (0–2.38 mM, r²>0.9952), a sensitivity of 50 μA mM^?1, and a detection limit of 2.4 μM. The measurement accuracy and precision for the determination of uric acid in human urine specimens were 85.6–95.5 % and 0.3–2.4 %, respectively. The developed uricase electrode is potential for clinical applications.     

An Electrochemical Determination of Uric Acid in Sera by a Flow-Through Equipment with and Without Uricase

Abstract

A comparison of an enzymeless direct electrochemical oxidation procedure at a platinum electrode for the determination of uric acid, and an enzyme sensor with immobilized urate: oxygen oxidoreductase (uricase), was performed in flow stream systems. The uricase enzyme electrode is based on the H₂O₂ oxidation current. Both amperometric methods were related to the wall-known photometric uricase-catalase-procedure (UCM) as a reference method. The measured values of both methods are of the first derivatives of current change (dI/dt) due to the electrochemical or electrochemical enzymatic reaction, respectively. The analytical quality of the measurements is characterized by: precision s% within run < 2% day to day < 5% accuracy acceptable (control materials) correlation to reference method r >0.93 analysis rate 80 samples/hr     

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.     

Poly(cytosine)‐templated Silver Nanoclusters as Fluorescent Biosensor for Highly Sensitive Detection of Uric Acid

Uric acid (UA) is an important biomarker in urine and serum samples for early diagnosis. This study re‐ ports a fluorescent biosensor based on Poly(cytosine)‐templated silver nanoclusters (C‐Ag NCs) and uricase for the highly sensitive and fast detection of UA. The strong fluorescence of the C‐Ag NCs prepared from poly (cytosine) nucleotides templates could be sensitively quenched by trace amount of H₂O₂, which produced from oxidation reaction of UA catalyzed by uricase. This biosensor exhibits two linear ranges as 50 nM～50 μM and 50 μM～400 μM, with a detection limit of 50 nM. The sensitivity of the biosensor is considerably improved compared with the methods reported in the literature. Furthermore, the detection ability of uric acid in serum samples is confirmed and this C‐Ag NCs‐based uric acid biosensor shows good promise of practical application.     

Disposable biosensor based on cathodic electrochemiluminescence of tris(2,2-bipyridine)ruthenium(II) for uric acid determination

A new method for uric acid (UA) determination based on the quenching of the cathodic ECL of the tris(2,2-bipyridine)ruthenium(II)–uricase system is described. The biosensor is based on a double-layer design containing first tris(2,2-bipyridine)ruthenium(II) (Ru(bpy)₃²⁺) electrochemically immobilized on graphite screen-printed cells and uricase in chitosan as a second layer. The uric acid biosensing is based on the ECL quenching produced by uric acid over the cathodic ECL caused by immobilized Ru(bpy)₃²⁺ in the presence of uricase. The use of a −1.1 V pulse for 1 s with a dwelling time of 10 s makes it possible to estimate the initial enzymatic rate, which is used as the analytical signal. The Stern–Volmer type calibration function shows a dynamic range from 1.0 × 10⁻⁵ to 1.0 × 10⁻³ M with a limit of detection of 3.1 × 10⁻⁶ M and an accuracy of 13.6% (1.0 × 10⁻⁴ M, n = 5) as relative standard deviation. Satisfactory results were obtained for urine samples, creating an affordable alternative for uric acid determination.     

Voltammetric oxidation of some biologically important xanthines at the pyrolytic graphite electrode

Linear and cyclic sweep voltammetry of some biologically important N-methylxanthines has been carried out. The principal and potential determining reaction for oxidation of xanthines is a 2 electron attack at the -N₉C_8⁻ double bond to give the appropriate uric acid, which is then immediately further oxidized to an intermediate 4,5-diol species. In many instances this 4,5-diol is sufficiently stable to be detected by fast sweep cyclic voltammetry as a small cathodic peak formed as a result of reduction of part of the 4,5-diol to the corresponding uric acid. The uric acid so formed can be reoxidized upon subsequent cycles at a potential close to that observed for reduction fo the 4,5-diol, so that most xanthines (and uric acids) show evidence for a reversible redox couple at about 0.4–0.55 V. Methylation at position N₇ decreases somewhat the stability of the intermediate 4,5-diol and dimethylation at N₃ and N₇ results in such a marked decreased in the stability of the diol that it cannot be observed by cyclic voltammetry.     

邻菲啰啉对固定尿酸酶催化反应动力学的影响

The activation energy of the enzyme-catalyzed reaction for uric acid decreases markedly in the presence of o-phenanthroline, which activates the bioelectrochemicla activity of the polypyrrole uricase electrode. The response current of the enzyme electrodeis independent of the concentration of o-phenanthroline. Based on the experimental results, the mechamsm of the enzyme-catalyzed reaction for uric acid in the presence of o-phenanthroline is presented as follows: E+A→EA, EA+S EAS, EAS→EA+P, where E, A, S and P are the enzyme, activator, substrate and product, respectively. The effects of pH value, potential and the uric acid concentration on the response currents of the uricase electrode have been studied in the presence of o-phenanthroline. In the presence of o-phenanthroline, the response current of the enzyme electrode increase linearly with increasing concentration of uric acid in the region of 0.07 to 0.67 mmol·L~(-1), therefore the polypyrrole uricase electrode which has once lost its activity can be activated and used again to determine the substrate concentration.     

A study of uricase biosensor based on a glassy carbon electrode modified with Nafion and methyl viologen

A uricase biosensor was constructed by using bovine serum albumin and glutaraldehyde as cross linker to immobilize uricase on a glassy carbon electrode modified with Nafion and methyl viologen (MV). A linear response of uric acid in serum was observed in a range of 1.0·10^–3 to 5.0·10^–6 mol/1 and the response time is 25 s. This biosensor has the advantage of high sensitivity, fast response as compared with previous sensors and low interferences.