Chemiluminescence sequential injection immunoassay for vitellogenin using magnetic microbeads

A rapid and sensitive immunoassay for the determination of carp vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with a chemiluminescence detector and a samarium-cobalt magnet. An anti-Vg monoclonal antibody, immobilized on magnetic beads, was used as a solid support for the immunoassay. The introduction, trapping and release of the magnetic beads in the flow cell were controlled by a samarium-cobalt magnet and the flow of the carrier solution. The immunoassay was based on a sandwich immunoreaction of anti-Vg monoclonal antibody (primary antibody) on the magnetic beads, Vg, and the anti-Vg antibody labeled with horseradish peroxidase (HRP) (secondary antibody), and was based on a subsequent chemiluminescence reaction of HRP with hydrogen peroxide and p-iodophenol, in a luminol solution. The magnetic beads to which the primary antibody was immobilized were prepared by coupling the primary antibody with the magnetic beads after an agarose-layer on the surface of the magnetic beads was epoxidized. The primary antibody-immobilized magnetic beads were introduced, and trapped in the flow cell equipped with the samarium-cobalt magnet, a Vg sample solution, an HRP-labeled secondary antibody solution and the luminol solution were sequentially introduced into the flow cell based on an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photomultiplier located at the upper side of the flow cell. The optimal incubation times both for the first and second immunoreactions were determined to be 20 min. A concave calibration curve was obtained between Vg concentration and chemiluminescence intensity when various concentrations of standard Vg samples (2–100 ng mL⁻¹) were applied to the SIA system under optimal conditions. In spite of a narrow working range, the lower detection limit of the immunoassay was about 2 ng mL⁻¹.     

Sequential injection chemiluminescence immunoassay for nonionic surfactants by using magnetic microbeads

A rapid and sensitive immunoassay based on a sequential injection analysis (SIA) using magnetic microbeads for the determination of alkylphenol polyethoxylates (APnEOs) is described. An SIA system was constructed from a syringe pump, a switching valve, a flow-through type immunoreaction cell equipped with a photon counting unit and a neodymium magnet. Magnetic beads, to which an anti-APnEOs monoclonal antibody was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of the magnetic beads in and from the immunoreaction cell were controlled by means of a neodymium magnet and adjusting the flow of a carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an anti-APnEOs monoclonal antibody immobilized on the magnetic beads with a sample APnEOs and a horseradish peroxidase (HRP)-labeled APnEOs in the same sample solution, and was based on the subsequent chemiluminscence reaction of HRP on the magnetic microbeads with a luminol solution containing hydrogen peroxide and p-iodophenol. The anti-APnEOs antibody was immobilized on the magnetic microbeads by coupling the antibody with the magnetic beads after activation of a carboxylate moiety on the surface of the magnetic beads that had been coated with a polylactic acid film. The antibody immobilized magnetic beads were introduced in the immunoreaction cell and trapped in it by the neodymium magnet, which was equipped beneath the immunoreaction cell. An APnEOs sample solution containing the HRP-labeled APnEOs at a constant concentration, and a luminol solution containing hydrogen peroxide and p-iodophenol were sequentially introduced into the immunoreaction cell, according to an SIA programmed sequence. Chemiluminescence emission was monitored by means of a photon counting unit located at the upper side of the immunoreaction cell by collecting the emitted light with a lens. A typical sigmoidal calibration curve was obtained, when the logarithm of the concentration of APnEOs was plotted against the chemiluminescence intensity as the number of photons in 100 ms using standard APnEOs sample solutions at various concentrations (0–1000 ppb) under optimum conditions. The lower detection limit defined as IC₈₀ is ca 10 ppb. The time required for analysis is less than 15 min per a sample. The present method was successfully applied to the determination of APnEOs in river water.     

Electrochemical immunoassay for vitellogenin based on sequential injection using antigen-immobilized magnetic microbeads.

A rapid and sensitive immunoassay for the determination of vitellogenin (Vg) is described. The method involves a sequential injection analysis (SIA) system equipped with an amperometric detector and a neodymium magnet. Magnetic beads, onto which an antigen (Vg) was immobilized, were used as a solid support in an immunoassay. The introduction, trapping and release of magnetic beads in an immunoreaction cell were controlled by means of the neodymium magnet and by adjusting the flow of the carrier solution. The immunoassay was based on an indirect competitive immunoreaction of an alkaline phosphatase (ALP) labeled anti-Vg monoclonal antibody between the fraction of Vg immobilized on the magnetic beads and Vg in the sample solution. The immobilization of Vg on the beads involved coupling an amino group moiety of Vg with the magnetic beads after activation of a carboxylate moiety on the surface of magnetic beads that had been coated with a polylactate film. The Vg-immobilized magnetic beads were introduced and trapped in the immunoreaction cell equipped with the neodymium magnet; a Vg sample solution containing an ALP labeled anti-Vg antibody at a constant concentration and a p-aminophenyl phosphate (PAPP) solution were sequentially introduced into the immunoreaction cell. The product of the enzyme reaction of PAPP with ALP on the antibody, paminophenol, was transported to an amperometric detector, the applied voltage of which was set at +0.2 V vs. an Ag/AgCl reference electrode. A sigmoid calibration curve was obtained when the logarithm of the concentration of Vg was plotted against the peak current of the amperometric detector using various concentrations of standard Vg sample solutions (0-500 ppb). The time required for the analysis is less than 15 min.     

Magnetic beads-based immunoassay as a sensitive alternative for atrazine analysis

A new immunoassay strategy for sensitive atrazine determination based on magnetic beads is reported. The immuno-method is a competitive solid-phase immunoassay where the anti-atrazine antibody is immobilized on the magnetic beads surface and fixed at the reaction cell bottom using a simple magnet, which generates a magnetic field. Analyte and HRP (horseradish peroxidase) tracer compete for active sites of antibody. After the immunointeractions antibody-analyte and antibody-tracer, atrazine quantification from the sample is performed by injection of the chemiluminescence substrate (luminol, hydrogen peroxide and p-iodophenol). Different antibodies (polyIgG anti-atrazine Ab I and affinity purified polyIgG anti-atrazine AbI) were tested in this configuration. Also, optimum concentration of antibody-covered magnetic beads was set up (8 mg/l Ab II). Finally, the performance of magnetic beads-based immunoassay for atrazine determination was evaluated demonstrating that the magnetic beads-based immunoassay is one of the most sensitive method for atrazine determination (LoD = 3 pg/l, IC₅₀ = 37 pg/l, DR = 10-1000 pg/l).     

Sequential injection/electrochemical immunoassay for quantifying the pesticide metabolite 3,5,6-trichloro-2-pyridinol

An automated and sensitive sequential injection electrochemical immunoassay was developed to monitor a potential insecticide biomarker, 3,5,6-trichloro-2-pyridinol. The current method involved a sequential injection analysis (SIA) system equipped with a thin-layer electrochemical flow cell and permanent magnet, which was used to fix 3,5,6-trichloro-2-pyridinol (TCP) antibody coated magnetic beads (TCP-Ab-MBs) in the reaction zone. After competitive immunoreactions among TCP-Ab-MBs, TCP analyte, and horseradish peroxidase (HRP) labeled TCP, a 3,3′,5,5′-tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB-H₂O₂) substrate solution was injected to produce an electroactive enzymatic product. The activity of HRP tracers was monitored by a square wave voltammetric scanning electroactive enzymatic product in the thin-layer flow cell. The voltammetric characteristics of the substrate and the enzymatic product were investigated under batch conditions, and the parameters of the immunoassay were optimized in the SIA system. Under the optimal conditions, the system was used to measure as low as 6 ng L⁻¹(ppt) TCP, which is around 50-fold lower than the value indicated by the manufacturer of the TCP RaPID Assay^® kit (0.25 μg/L, colorimetric detection). The performance of the developed immunoassay system was successfully evaluated on tap water and river water samples spiked with TCP. This technique could be readily used for detecting other environmental contaminants by developing specific antibodies against contaminants and is expected to open new opportunities for environmental and biological monitoring.     

Enzymatic immunoassay of α-fetoprotein,insulin and 17-α-hydroxyprogesterone base on chemiluminescence in a flow-injection system

Highly-sensitive enzymatic immunoassay procedures based on a chemiluminescent reaction are described. Glucose oxidase was used as the labelling enzyme conjugated with anti-α-fetoprotein IgG, insulin or 17-α-hydroxyprogesterone. Free and bound fractions present after the immune reaction were separated by an immobilized antibody or a second antibody. The enzyme activity was measured by the chemiluminescence produced by luminol and hydrogen peroxide, catalyzed by potassium hexacyanoferrate(III) after incubation with glucose. The chemiluminescence was measured in a flow-injection system, with a home-made luminescence detector equipped with a spiral flow cell. The detection limits for each substance were 10^?15–10^?17 mol. Recoveries of added α-fetoprotein from diluted serum were quantitative.     

Sequential injection immunoassay for environmental measurements

Sequential injection immunoassay systems for environmental measurements based on the selective immunoreaction between antigen and antibody were described. A sequential injection analysis (SIA) technique is suitable to be applied for the procedure of enzyme-linked immunosorbent assay (ELISA), because the washing and the addition of reagent solutions can be automated by using a computer-controlled syringe pump and switching valve. We selected vitellogenin (Vg), which is a biomarker for evaluating environmental risk caused by endocrine-disrupting chemicals in the hydrosphere, and linear alkylbenzene sulfonates (LAS) and alkylphenol polyethoxylates (APEO), which are versatile surfactants, as target analytes in the flow immunoassay systems. For Vg monitoring, SIA systems based on spectrophotometric, chemiluminescence, and electrochemical determinations were constructed. On the other hand, chemiluminescence determination was applied to the detection of LAS and APEO. For APEO, an SIA system combined with surface plasmon resonance (SPR) sensor was also developed. These new sequential injection immunoassay systems are expected to be useful systems for environmental analysis.     

Development of a heterogeneous chemiluminescent flow immunoassay for DDT and related compounds

A heterogeneous chemiluminescent (CL) flow immunoassay for DDT was optimized comparing different types of immunoaffinity supports: beads, nylon coils and membranes (membranes HyBondN+). In order to characterize solid immunoaffinity supports two basic immunoassay formats were performed, using (1) enzyme-labeled secondary and (2) enzyme-labeled specific monoclonal antibodies (MAbs). In both formats, hapten DDT5 conjugated to ovalbumin immobilized on solid supports according to the appropriate immobilization procedure, enzyme label (horseradish peroxidase, HRP) and luminescent detection (luminol/H₂O₂/p-iodophenol) were used. The lowest limit of detection (LOD), 1 nM p,p-DDT, was obtained with a membrane-based flow immunoassay with HRP-labeled specific antibody. Beads and packed tubing were discarded as appropriate supports because of the difficulties encountered for reproducible packing and the occurrence of light scatterring (beads), which seriously compromised the performance and reproducibility of the flow immunoassay.     

An ultrasensitive chemiluminescence immunosensor for PSA based on the enzyme encapsulated liposome

A highly sensitive chemiluminescence immunosensor for the detection of prostate-specific antigen (PSA) was developed based on a novel amplification procedure with the application of enzyme encapsulated liposome. Horseradish peroxidase (HRP) encapsulated and antibody-modified liposome acts as the carrier of a large number of markers and specific recognition label for the amplified detection of PSA. In the detection of PSA, the analyte was first bound to the specific capture antibody immobilized on the microwell plates, and then sandwiched by the antibody-modified liposomes encapsulating HRP. The encapsulated markers, HRP molecules were released by the lysis of the specifically bound liposomes in the microwell with Triton X-100 solution. Then, the analyte PSA could be determined via the chemiluminescence signal of HRP-catalyzed luminol/peroxide/enhancer system. The “sandwich-type” immunoassay provides the amplification route for the PSA detection in ultratrace levels. The CL emission intensity exhibits dynamic correlation to PSA concentration in the range from 0.74 pg/ml to 0.74 μg/ml with readily achievable detection limit of 0.7 pg/ml.     

Spectrophotometric determination of carp vitellogenin using a sequential injection analysis technique equipped with a jet ring cell

A sequential injection analysis (SIA) technique, in which antibody-immobilized microbeads were transferred to a jet ring (JR) cell, was used in determination of carp vitellogenin (Vg). The determination is based on a sandwich immunoassay in which two types of reactions between anti carp Vg antibodies and carp Vg are used. Namely, the antibody for the first reaction step was immobilized on microbeads (Sephadex beads), and an antibody labeled with a horseradish peroxidase (HRP) was used in the second step of the reaction. A mixed solution of hydrogen peroxide and o-phenylenediamine (OPD) was used as the source of the chromophore in the reaction. The microbeads-immobilized antibody, Vg analyte, HRP-labeled anitbody and the color developing solution were introduced automatically into the JR cell of the SIA system in a programmed sequence, and the absorbance of the oxidized OPD product was used to determine the amount of Vg present. The optimal incubation times for the immuno-raction for the first and the second steps were determined at 120 and 60 min, respectively, taking into account the sensitivity to the Vg determination. Under these conditions, a good linear correlation was obtained between Vg concentration and the absorbance of the oxidized OPD. The lower detection limit for the determination of Vg was about 5 ng ml⁻¹ in this system. The method developed here represents a simple, accurate method for the determination method of Vg.     

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/ H₂O₂/enhancer). The second immunoassay is based on the use of bovine serum albumin–daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu³⁺-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/ well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.     

Surface Plasmon Resonance Immunosensor for Anionic Surfactants Based on an Indirect Competitive Immunoreaction

Abstract

A highly sensitive surface plasmon resonance immunosensor for the determination of linear alkylbenzene sulfonate (LAS) was fabricated. The method is based on an indirect competitive reaction of an anti‐LAS antibody in a sample solution with LAS immobilized on a sensor chip and with LAS in the sample solution. A sensor chip immobilized with LAS was prepared by utilizing an electrostatic interaction between an LAS conjugate, LAS–horseradish peroxidase (LAS‐HRP), and a self‐assembled monolayer of 11‐amino‐1‐undecanethiol hydrochloride, which was preliminary prepared on a gold thin film of the sensor chip. The quantitative determination of LAS in the concentration range 10–1000 ppb was achieved by using the proposed immunosensor.     

Analytical performance of luminescent immunoassays of different format for serum daidzein analysis

Two sensitive competitive-type solid-phase immunoassays for serum daidzein analysis have been developed and optimized. The first is a chemiluminescent enzyme immunoassay that uses black polystyrene microtiter wells in which daidzein-specific antibodies raised in rabbits are immobilized and a daidzein derivative is coupled to horseradish peroxidase (HRP) as a label. The HRP activity of the antibody-bound tracer is measured with an enhanced chemiluminescent system (luminol/H2O2/enhancer). The second immunoassay is based on the use of bovine serum albumin-daidzein derivative immobilized on microtiter plates and a secondary anti-rabbit IgG-Fc fragment conjugated with 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). Formation of the complex Eu3+-BCPDA enables time-resolved fluorescence-mode detection of the amount of antibody bound to the immobilized antigen. Both methods fulfilled all the requirements of accuracy and precision. The detection limit was the same for each method, 10 pg/well; this is better than that of other immunoassays. The specificity of the two methods was different, because of their competitive-type mechanisms. The performance of the chemiluminescence method is better, because the cross-reactivity of the main interfering compound (genistein) was 5%, compared with 25% for the time-resolved fluoroimmunoassay.     

Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of α-fetoprotein

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with γ-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The α-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-100 ng mL⁻¹. The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.     

Bioelectrochemical Magnetic Immunosensing of Trichloropyridinol: A Potential Insecticide Biomarker

A fast, simple and sensitive bioelectrochemical magnetic immunosensing method is developed to monitor a potential insecticide biomarker, trichloropyridinol (TCP), in environmental sample. A magnet/glassy carbon (MGC) working electrode was used to accumulate immunocomplex associated magnetic beads and separate free and unbound reagents after liquid phase competitive immunoreaction among TCP antibody coated magnetic beads, TCP analyte and horseradish peroxidase (HRP) labeled TCP. The activity of HRP tracers was monitored by square‐wave voltammetry (SWV) by scanning electrocactive enzymatic product in the presence of 3,3′,5,5′‐tetramethylbenzidine dihydrochloride and hydrogen peroxide (TMB‐H₂O₂) substrate solution. The electrochemical signal of enzymatic product was greatly enhanced by dual accumulation events: magnetic accumulation of enzyme tracers bound magnetic beads and constant potential accumulation of enzymatic product. The voltammetric characteristics of substrate and enzymatic product were investigated, and the parameters of SWV analysis and immunoassay were optimized. Under the optimal conditions the immunosensor was used to measure as low as 5 ng L^?1 (ppt) TCP, which is 50‐fold lower than the value indicated by the manufacture of the TCP RaPID Assay kit (0.25 μg/L, colorimetric detection). The performance of the developed immunosensing system was successfully evaluated with river water samples spiked with TCP, indicating this convenient and sensitive technique offers great promise for decentralized environmental application. This technique could be readily used for detection of other environmental contaminants by developing specific antibodies against the contaminants and are expected to open new opportunities for environmental monitoring and public health.     

Flow-Injection Enzyme Immunoassay for Human IgG by Using Enhanced Chemiluminescence Reaction for Horseradish Peroxidase Label Quantitation

Abstract

A flow-injection sandwich enzyme immunoassay for human IgG as model antigen by using horseradish peroxidase as label, polystyrene beads as solid support, and the enhanced chemiluminescence reaction for peroxidase quantitation is described. the kinetics of antigen—immobilized antibody interaction has been studied and the quantitative time-concentration ranges of reactions have been estimated. Each of the two immunochemical steps of analysis have been pursued in the kinetic regime. the time for each immunochemical step was reduced to 2–3 min. the enhanced luminescent reaction involving luminol and p-iodophenol as substrates was used to detect the peroxidase label. the conditions for chemiluminescent reaction were optimized. the detection limit for peroxidase in a 3 min assay was 5–10^?16 moles/tube. the detection limit for IgG, in the developed immunoassay, is 10^?9 M, the overall time of the assay being 5–10 min.     

Spectral and fluorescent kinetics features of Nd3+ ion in Nb2O5, Ta2O5 and La2O3 mixed lithium zirconium silicate glasses

A sensitive competitive flow injection chemiluminescence (CL-FIA) immunoassay for immunoglobulin G (IgG) was developed using gold nanoparticle as CL label. In the configuration, anti-IgG antibody was immobilized on a glass capillary column surface by 3-(aminopropyl)-triethoxysilane and glutaraldehyde to form immunoaffinity column. Analyte IgG and gold nanoparticle labeled IgG were passed through the immunoaffinity column mounted in a flow system and competed for the surface-confined anti-IgG antibody. CL emission was generated from the reaction between luminol and hydrogen peroxide in the presence of Au (III), generated from chemically oxidative dissolution of gold nanoparticle by an injection of 0.10 mol L⁻¹ HCl–0.10 mol L⁻¹ NaCl solution containing 0.10 mmol L⁻¹ Br₂. The concentration of analyte IgG was inversely related to the amount of bound gold nanoparticle labeled IgG and the CL intensity was linear with the concentration of analyte IgG from 1.0 ng mL⁻¹ to 40 ng mL⁻¹ with a detection limit of 5.2 × 10⁻¹⁰ g mL⁻¹. The whole assay time including the injections and washing steps was only 30 min for one sample, which was competitive with CL immunoassays based on a gold nanoparticle label and magnetic separation. This work demonstrates that the CL immunoassay incorporation of nanoparticle label and flow injection is promising for clinical assay with sensitivity and high-speed.     

Flow Injection Chemiluminescence for the Determination of Estriol via a Noncompetitive Enzyme Immunoassay

A novel approach to the detection of estriol using a flow injection system coupled to enhanced chemiluminescent immunoassay was developed based on noncompetitive immunoassay formats. A conjugated estriol-ovalbumin immobilized immunoaffinity column was inserted into the flow system to trap the unbound horseradish peroxidase (HRP)-labeled antibody after an off-line incubation of estriol and HRP-labeled anti-estriol antibody. The trapped enzyme conjugate was detected by the injection of chemiluminescent substrates to produce enhanced chemiluminescence. The linear range for the determination of estriol is 10.0 to 400 ng · mL⁻¹ with a correlation coefficient of 0.996 and a detection limit of 5.0 ng · mL⁻¹. The total time for sampling and chemiluminescent detection of one sample is 400 seconds after 30 min of pre-incubation. The results for pregnancy serum samples obtained by this method are in good agreement with those obtained using ELISA.     

Flow-through chemiluminescence sensor using immobilized oxidases for the selective determination of L-glutamate in a flow-injection system.

A selective and sensitive chemiluminometric flow sensor for the determination of L-glutamate in serum, based on immobilized oxidases such as glutamate oxidase (GOD), uricase (UC) and peroxidase (POD), is described herein. The principle for the selective chemiluminometric detection for L-glutamate is based on coupled reactions of four sequentially aligned immobilized oxidases, UC/POD/GOD/POD in a flow cell. The immobilized UC was employed to decompose urate, which is one of the major interfering components in serum for a luminol-H2O2 chemiluminescence reaction. The H2O2 produced from the UC reaction readily reacted with reducing components, such as ascorbate and glutathione, and then the excess H2O2 was decomposed by the immobilized POD. L-Glutamate in the sample plug was enzymatically converted to H2O2 with immobilized GOD. Subsequently, the peroxide reacts with luminol on the immobilized POD to produce chemiluminescence, proportional to glutamate concentration. The enzymes were immobilized on tresylated poly(vinyl alcohol beads). The immobilized enzymes were packed into TPFE tube (1.0 mm i.d. x 60 cm), in turn, and used as a flow cell. The sampling rate was 30 h-1. The calibration graph for L-glutamate is linear for 20 nM-5 microM; the detection limit (signal-to-noise = 3) is 10 nM.     

Chemiluminescence biosensor chip based on a microreactor using carrier air flow for determination of uric acid in human serum

A chemiluminescence biosensor on a chip coupled to a microfluidic system and a microreactor is described in this paper. The chemiluminescence biosensor measured 25 x 75 x 6.5 mm in dimension, and was readily produced in an analytical laboratory. The sol-gel method is introduced to co-immobilize horseradish peroxidase (HRP) and luminol in the microreactor, and to immobilize uricase in the enzymatic reactor. The main characteristic of the biosensor was to introduce air as the carrier flow instead of the more common solution carrier for the first time. The uric acid was determined by a chemiluminescent (CL) reaction between the hydrogen peroxide produced from the enzymatic reactor and luminol under the catalysis of HRP in the microreactor. The linear range of the uric acid concentration was 1 to 100 mg L(-1) and the detection limit was 0.1 mg L(-1) (3sigma).