Chemiluminescence assay for uric acid in human serum and urine using flow-injection with immobilized reagents technology

A novel chemiluminescence (CL) flow sensor for the determination of uric acid in human urine and serum has been developed by using controlled-reagent-release technology. The reagents involved in the chemiluminescence (CL) reaction, luminol and periodate, are immobilized on anion-exchange resin packed in a column. After injection of water, chemiluminescence generated by released luminol and periodate in alkaline media is inhibited in presence of uric acid. By measuring the decreased chemiluminescence (CL) intensity the uric acid is sensed. The decreased response is linear in the 5.0-500.0 ng mL(-1) range, with a detection limit of 1.8 ng mL(-1). The flow sensor showed remarkable operational stability and could be easily reused for over 80 h with sampling frequency of 100 h(-1). The proposed sensor was applied to the determination of uric acid in human urine and serum, and monitoring metabolic uric acid in human urine with RSD less than 3.0%.     

Chemiluminescence microfluidic system sensor on a chip for determination of glucose in human serum with immobilized reagents

A chemiluminescence (CL) biosensor on a chip coupled to microfluidic system is described in this paper. The CL biosensor measured 25×45×5 mm in dimension, was readily produced in analytical laboratory. Glucose oxidase (GOD) was immobilized onto controlled-pore glass (CPG) via glutaraldehyde activation and packed into a reservoir. The analytical reagents, including luminol and ferricyanide, were electrostatically co-immobilized on an anion-exchange resin. The most characteristic of the biosensor was to introduce the air as the carrier flow in stead of the common solution carrier for the first. The glucose was sensed by the CL reaction between hydrogen peroxide produced from the enzymatic reaction and CL reagents, which were released from the anion-exchange resin. The proposed method has been successfully applied to the determination of glucose in human serum. The linear range of the glucose concentration was 1.1-110 mM and the detection limit was 0.1 mM (3σ).     

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.     

Spectrofluorimetric method for the determination of uric acid in human serum

A new spectrofluorimetric method is described for the determination of uric acid (UA), that can remarkably reduce the fluorescence intensity of the enoxacin (ENX)-terbium ion (Tb³⁺) complex at 545 nm. The reduced fluorescence intensity of Tb³⁺ ion at pH 5.7 is proportional to the concentration of UA. Optimum conditions for the determination of UA have been investigated. The linear range and detection limit for the determination of UA are 6.0 × 10^?7–3.0 × 10^?5 M and 1 × 10^?7 M, respectively. The relative standard deviation (RSD) was 0.4% for 6 × 10^?6 M UA (n = 11). The method is simple, practical and relatively free of interferences. It has been successfully applied to assess UA in serum at the level of 3 × 10^?4 M with an RSD of 5–7% (n = 3). The results were evaluated by comparison with a common clinical spectrophotometric method using phosphotungstic acid as developer.     

A biosensor based on urate oxidase-peroxidase coupled enzyme system for uric acid determination in urine

A new amperometric biosensor based on urate oxidase-peroxidase coupled enzyme system for the specific and selective determination of uric acid in urine was developed. Commercially available urate oxidase and peroxidase were immobilized with gelatin by using glutaraldehyde and fixed on a pretreated teflon membrane. The method is based on generation of H₂O₂ from urine uric acid by urate oxidase and its consuming by peroxidase and then measurement of the decreasing of dissolved oxygen concentration by the biosensor. The biosensor response depends linearly on uric acid concentration between 0.1 and 0.5 μM. In the optimization studies of the biosensor, phosphate buffer (pH 7.5; 50 mM) and 35 °C were obtained as the optimum working conditions. In addition, the most suitable enzyme activities were found as 64.9×10⁻³ U cm⁻² for urate oxidase and 512.7 U cm⁻² for peroxidase. And also some characteristic studies of the biosensor such as reproducibility, substrate specificity and storage stability were carried out.     

基于氧化还原体系纸芯片法对血清尿酸含量的快速检测

建立了以纸芯片为分析平台的尿酸含量快速检测方法.基于尿酸的还原性,以FeCl3为氧化剂组成氧化还原体系,以邻二氮菲为显色剂对反应产物Fe2+进行显色,根据显色强度对尿酸含量进行测定.结果 表明,当FeCl3与尿酸比例为1.6∶1,反应10 min,显色剂pH 7.4时显色最佳.显色强度(RGB值)与尿酸浓度在1.19～...     

Modelling a biosensor based on the heterogeneous microreactor

Modelling of the amperometric biosensors based on carbon paste electrodes encrusted with a single heterogeneous microreactor is analyzed. The microreactor was constructed from CPCsilica carrier and was loaded with glucose oxidase. The model is based on nonstationary diffusion–reaction equations containing a nonlinear term related to the enzymatic reaction. A homogenization process having an effective algorithm for the digital modelling of the operation of the microreactor is proposed. The influence of the size, geometrical form, and the position of a microreactor on the operation of biosensors are investigated.     

Direct determination of uric acid in serum by a fluorometric-enzymatic method based on uricase

The present paper describes a method for the fluorometric determination of uric acid in blood serum by its reaction with uricase (UOx). The procedure is based on the changes in fluorescence that take place during the enzymatic reaction of UOx with uric acid when the solution is excited at 287 nm and the emission is measured at 330 nm. A mathematical model which relates the analytical signal to the analyte concentration was developed and the model also served to obtain some of the thermodynamic constants of the system (the Michaelis constant and the turnover number). The optimum reaction conditions and its analytical characteristics were studied, linear response range (3x10(-5)-6x10(-4) M) and reproducibility (4%, n=7). The method was applied to the determination of uric acid in three blood serum samples. The results were compared with those obtained by a commercial clinical analyzer and no systematic errors were observed.     

Micro flow sensor on a chip for the determination of terbutaline in human serum based on chemiluminescence and a molecularly imprinted polymer

Based on a molecularly imprinted polymer (MIP) as the recognition element, a novel chemiluminescence (CL) micro flow sensor on a chip for the determination of terbutaline in human serum is described. The MIP was prepared by using terbutaline as the template, methacrylic acid (MAA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the cross-linking monomer, and acetonitrile as the solvent. The chip was fabricated from two 50 mm × 40 mm × 5 mm transparent poly (methylmethacrylate) (PMMA) slices. The microchannels on the chip etched by CO₂ laser were 200 μm wide and 150 μm deep. The microsensor cell filled with 2 mg MIP for selectively on line adsorbing terbutaline was 10 mm long, 1 mm wide, and 0.5 mm deep. All reagents were controlled by the syringe pump with an accurate timer. The on line adsorbed terbutaline by the MIP can enhance the CL intensity of the reaction of luminol with ferricyanide. The enhanced CL intensity is linear with terbutaline concentration from 8.0 to 100 ng/mL with a detection limit of 4.0 ng/mL (3σ). The micro flow sensor provides for good reproducibility with the relative standard deviation of 3.6% (n = 7) for 20 ng/mL terbutaline.     

A quercetin-modified biosensor for amperometric determination of uric acid in the presence of ascorbic acid

The present work reports a quercetin-modified wax-impregnated graphite electrode (Qu/WGE) prepared through an electrochemical oxidation procedure in quercetin-containing phosphate buffer solution (PBS), for the purpose of detecting uric acid (UA) in the presence of ascorbic acid (AA). During modification quercetin was oxidized to the corresponding quinonic structure, and in the blank buffer solution the electrodeposited film exhibits a voltammetric response anticipated for the surface-immobilized quercetin. Retarding effect of the film towards the reaction of anionic species was found; therefore the pH of sample solutions was selected to ensure the analyte in molecular form. At suitable pHs the Qu/WGE shows excellent electrocatalytic effect towards the oxidation of both AA and UA, and separates the voltammetric signal of UA from AA by about 280 mV, allowing simultaneous detection of these two species. A linear relation between the peak current and concentration was obtained for UA in the range of 1-50 μM in the presence of 0.5 mM AA, with a detection limit 1.0 μM (S/N = 3). This sensor was stable, reproducible and outstanding for long-term use.     

Microfluidic paper-based chemiluminescence biosensor for simultaneous determination of glucose and uric acid

In this study, a novel microfluidic paper-based chemiluminescence analytical device (μPCAD) with a simultaneous, rapid, sensitive and quantitative response for glucose and uric acid was designed. This novel lab-on-paper biosensor is based on oxidase enzyme reactions (glucose oxidase and urate oxidase, respectively) and the chemiluminescence reaction between a rhodanine derivative and generated hydrogen peroxide in an acid medium. The possible chemiluminescence assay principle of this μPCAD is explained. We found that the simultaneous determination of glucose and uric acid could be achieved by differing the distances that the glucose and uric acid samples traveled. This lab-on-paper biosensor could provide reproducible results upon storage at 4 °C for at least 10 weeks. The application test of our μPCAD was then successfully performed with the simultaneous determination of glucose and uric acid in artificial urine. This study shows the successful integration of the μPCAD and the chemiluminescence method will be an easy-to-use, inexpensive, and portable alternative for point-of-care monitoring.     

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.     

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.     

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.     

Disposable luminol copolymer-based biosensor for uric acid in urine

A new electrochemiluminescent (ECL) disposable biosensor for uric acid was manufactured by immobilization in a double-layer design of luminol as a copolymer with 3,3′,5,5′-tetramethylbenzidine (TMB) and the enzyme uricase in chitosan on gold screen-printed cells. The good mechanical and improved electroluminescent characteristics of the new copolymer poly(luminol–TMB) make it possible to determine uric acid by measuring the growing ECL emission with the analyte concentration. The combination of enzymatic selectivity with ECL sensitivity results in a disposable analytical device with a linear range for uric acid from 1.5 × 10⁻⁶ to 1.0 × 10⁻⁴ M, a limit of detection of 4.4 × 10⁻⁷ M and a precision of 13.1% (1.0 × 10⁻⁵ M, n = 10) as relative standard deviation. Satisfactory results were obtained for uric acid determination in 24 h-urine samples compared to a reference procedure. This uric acid biosensor can be used as a low-cost alternative to conventional methods.     

HPLC法测定人尿中尿酸含量

建立了一种人体尿液中尿酸含量的高效液相色谱测定方法.采用ACE5 AQ亲水色谱柱,pH3.2的乙酸水溶液为流动相,检测波长280nm.尿酸含量在7.1～224.6μg/mL范围内线性关系良好,平均加样回收率为99.7%～100.5%,RSD小于1.4%.将该法用于健康人和肝硬化病人尿液样本的测定,两类样本中尿酸含量无显...     

Chemiluminescence flow sensor for the determination of ascorbic acid with immobilized reagents

A novel flow sensor based on chemiluminescence (CL) for the determination of ascorbic acid has been proposed. The analytical reagents, luminol and ferricyanide, were both immobilized on an anion-exchange resin column. The CL signal produced by the reaction between luminol and ferricyanide, which were eluted from the column through sodium phosphate injection, was decreased in the presence of ascorbic acid. The CL emission intensity was linear with ascorbic acid concentration in the range 0.01-0.8 mug ml(-1); the detection limit was 5.5 x 10(-3) mug ml(-1). The whole process, including sampling and washing, could be completed in 1 min with a relative standard deviation of less than 5%. The sensor could be reused more than 100 times and has been applied successfully to the analysis of ascorbic acid in pills and vegetables.     

Quantitative analysis of human serum leptin using a nanoarray protein chip based on single-molecule sandwich immunoassay

We report a method for the quantitative analysis of human serum leptin, which is a protein hormone associated with obesity, using a nanoarray protein chip based on a single-molecule sandwich immunoassay. The nanoarray patterning of a biotin-probe with a spot diameter of 150 nm on a self-assembled monolayer functionalized by MPTMS on a glass substrate was successfully accomplished using atomic force microscopy (AFM)-based dip-pen nanolithography (DPN). Unlabeled leptin protein molecules in human serum were detected based on the sandwich fluorescence immunoassay by total internal reflection fluorescence microscopy (TIRFM). The linear regression equation for leptin in the range of 100 zM-400 aM was determined to be y = 456.35x + 80,382 (R = 0.9901). The accuracy and sensitivity of the chip assay were clinically validated by comparing the leptin level in adult serum obtained by this method with those measured using the enzyme-linked immunosorbent assay (ELISA) performed with the same leptin standards and serum samples. In contrast to conventional ELISA techniques, the proposed chip methodology exhibited the advantages of ultra-sensitivity, a smaller sample volume and faster analysis time.     

Highly sensitive uric acid biosensor based on individual zinc oxide micro/nanowires

We describe the use of individual zinc oxide (ZnO) micro/nanowires in an electrochemical biosensor for uric acid. The wires were synthesized by chemical vapor deposition and possess uniform morphology and high crystallinity as revealed by scanning electron microscopy, X-ray diffraction, and photoluminescence studies. The enzyme uricase was then immobilized on the surface of the ZnO micro/nanowires by physical adsorption, and this was proven by Raman spectroscopy and fluorescence microscopy. The resulting uric acid biosensor undergoes fast electron transfer between the active site of the enzyme and the surface of the electrode. It displays high sensitivity (89.74 μA cm^?2 mM^?1) and a wide linear analytical range (between 0.1 mM and 0.59 mM concentrations of uric acid). This study also demonstrates the potential of the use of individual ZnO micro/nanowires for the construction of highly sensitive nano-sized biosensors.