Amount Of Horseradish Peroxidase Research Articles

A QCM immunosensor to rapidly detect ractopamine using bio-polymer conjugate and magnetic β-cyclodextrins

An enzyme-amplified magnetic immunosensor was developed for the sensitive and selective detection of ractopamine (RAC) in fodder using a quartz crystal microbalance (QCM). Magnetic capture probes were prepared by immobilizing β-cyclodextrins (β-CD) on Fe3O4 nanoparticles in order to enrich RAC. PowerVision (PV), a kind of enzyme–polymer complex with a high enzyme-to-antibody ratio, served as a signal label. Horseradish peroxidase (HRP) on the immnunocomplex, which arises from sandwich-type immunoreactions, causes 3,3′,5,5′-tetramethylbenzidine (TMB) to produce large amounts of sedimentation with a mass proportional to RAC. The large amounts of HRP on the PV greatly amplify the QCM sensor's signal. Under optimum conditions, the differences of the QCM frequency (Δf) are proportional to the concentration of RAC over the range from 0.03 to 25ngmL−1. The limit of detection was 0.01ngmL−1. More importantly, this assay's results in swine fodder samples were consistent with those of ELISA, with recoveries between 92.3% and 110%. Due to its high sensitivity, portability and selectivity, the immunosensor achieved reliable quantification of RAC in real fodder samples and has the potential to become an on-site screening tool to enhance food safety.

An in-line photonic biosensor for monitoring of glucose concentrations.

This paper presents two PDMS photonic biosensor designs that can be used for continuous monitoring of glucose concentrations. The first design, the internally immobilized sensor, consists of a reactor chamber, micro-lenses and self-alignment structures for fiber optics positioning. This sensor design allows optical detection of glucose concentrations under continuous glucose flow conditions of 33 μL/h based on internal co-immobilization of glucose oxidase (GOX) and horseradish peroxidase (HRP) on the internal PDMS surface of the reactor chamber. For this design, two co-immobilization methods, the simple adsorption and the covalent binding (PEG) methods were tested. Experiments showed successful results when using the covalent binding (PEG) method, where glucose concentrations up to 5 mM with a coefficient of determination (R2) of 0.99 and a limit of detection of 0.26 mM are detectable. The second design is a modified version of the internally immobilized sensor, where a microbead chamber and a beads filling channel are integrated into the sensor. This modification enabled external co-immobilization of enzymes covalently onto functionalized silica microbeads and allows binding a huge amount of HRP and GOX enzymes on the microbeads surfaces which increases the interaction area between immobilized enzymes and the analyte. This has a positive effect on the amount and rate of chemical reactions taking place inside the chamber. The sensor was tested under continuous glucose flow conditions and was found to be able to detect glucose concentrations up to 10 mM with R2 of 0.98 and a limit of detection of 0.7 mM. Such results are very promising for the application in photonic LOC systems used for online analysis.

An Ultrasensitive Electrochemical Immunosensor for Alpha-Fetoprotein Using an Envision Complex-Antibody Copolymer as a Sensitive Label

A novel strategy is presented for sensitive detection of alfa-fetoprotein (AFP), using a horseradish peroxidase (HRP)-functionalized Envision antibody complex (EVC) as the label. The Envision-AFP signal antibody copolymer (EVC-AFP Ab2) was composed of a dextran amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of secondary antibody, and acted as a signal tag in the immunosensor. The sensor was constructed using the following steps: First, gold electrode (GE) was modified with nano-gold (AuNPs) by electro-deposition in HAuCl4 solution. The high affinity of the AuNPs surface facilitates direct formation of a self-assembled thiolated protein G layer. Next, the coated GE was incubated in a solution of AFP capture antibody (AFP Ab1); these antibodies attach to the thiolated protein G layer through their non-antigenic regions, leaving the antigen binding sites for binding of target analyte. Following a sandwich immunoreaction, an EVC-AFP Ab2-AFP-AFP Ab1 immunocomplex was formed on the electrode surface, allowing large amounts of HRP on the complex to produce an amplified electrocatalytic current of hydroquinone (HQ) in the presence of hydrogen peroxide (H2O2). Highly amplified detection was achieved, with a detection limit of 2 pg/mL and a linear range of 0.005–0.2 ng/mL for AFP in 10 μL undiluted serum; this is near or below the normal levels of most cancer biomarker proteins in human serum. Measurements of AFP in the serum of cancer patients correlated strongly with standard enzyme-linked immunosorbent assays. These easily fabricated EVC-modified immunosensors show excellent promise for future fabrication of bioelectronic arrays. By varying the target biomolecules, this technique may be easily extended for use with other immunoassays, and thus represents a versatile design route.

Purification of a recombinant plant peroxidase produced in Pichia pastoris by a simple 2-step strategy

The enzyme horseradish peroxidase (HRP), which is frequently applied in industry and medicine, is primarily isolated from plant. This purification procedure is costly and the obtainable amount of HRP from the horseradish root is low. However, recombinant HRP (rHRP) produced in yeast is hyperglycosylated rendering the subsequent purification cumbersome and the recombinant production of HRP in yeast not competitive.In this study, we screened different common techniques to develop a fast and efficient purification strategy for hyperglycosylated rHRP expressed in Pichia pastoris. We demonstrated that the extensive glycosylation pattern on the surface of rHRP masked its physico-chemical properties, which is why standard purification strategies were rather unsuccessful. Only switching the strategies to a flowthrough mode gave satisfactory results. After determining the optimal operation conditions in a multivariate Design of Experiments approach, we present a simple 2-step strategy for the purification of hyperglycosylated rHRP. Combining a hydrophobic charge induction chromatography operated in flowthrough mode and a size-exclusion chromatography, we were able to purify rHRP more than 12-fold from a specific activity of 80U/mg to more than 1000U/mg.

Fluid Outflow in a Large-Animal Model of Posttraumatic Syringomyelia

Posttraumatic syringomyelia affects approximately 28% of spinal cord injury patients, and current treatments are often ineffective. The pathogenesis of this condition remains poorly understood. Previous reports have focused on pathways and mechanisms of fluid inflow; however, disturbances of fluid outflow mechanisms and pathways may be important in syrinx formation and enlargement. To determine the route of fluid outflow from a syrinx in an animal model of posttraumatic syringomyelia. A model of posttraumatic syringomyelia using excitotoxic amino acid and kaolin-induced arachnoiditis was created in 12 Merino wethers. Six weeks after syrinx induction, the cavities were localized and a cerebrospinal fluid tracer, horseradish peroxidase (HRP), was injected into the syrinx under ultrasonic guidance. After 10 minutes, the animals were killed and the spinal cords harvested for microscopy. An extracanalicular syrinx developed in 6 of the 12 sheep. HRP was successfully injected into 5 of the 6 syrinx cavities. HRP reaction product was observed in gray and white matter adjacent to the syrinx in a diffuse pattern. There were moderate amounts of HRP around the central canal and perivascular spaces and minimal amounts in the dorsal subarachnoid space. In this model of posttraumatic syringomyelia, fluid outflow occurred in a diffuse manner into the surrounding extracellular space and toward the central canal and perivascular spaces. Fluid outflow may be an important consideration in the pathogenesis of syringomyelia and the development of new therapies.

Sensitive Chemiluminescent Imaging for Chemoselective Analysis of Glycan Expression on Living Cells Using a Multifunctional Nanoprobe

A novel sensitive chemiluminescent (CL) imaging method was developed for in situ monitoring of cell surface glycan expression through chemoselective labeling of carbohydrate motifs and then binding to a multifunctional nanoprobe. The nanoprobe was fabricated by assembling biotin-DNA and a large amount of horseradish peroxidase (HRP) on gold nanoparticles (AuNPs). The chemoselective labeling was performed by selective oxidization of the hydroxyl sites of sialyl and galactosyl groups on cell surfaces into aldehydes by periodate and galactose oxidase, respectively, and then aniline-catalyzed hydrazone ligation with biotin hydrazide for specific recognition to avidin. With the biotin-avidin system, the multifunctional nanoprobe could conveniently be bound to the glycan sites on the cell surface. The DNA chain presenting between the AuNPs and biotin assembled on the nanoprobe could obviate the steric effect, and HRP acted to trigger the CL emission of the luminal-H(2)O(2) system. Therefore the expression of both sialyl and galactosyl groups could be selectively monitored by CL imaging with high sensitivity due to the high amount of HRP. Using human liver cancer HCCC-9810 cells as a model, this CL imaging strategy could detect HCCC cells ranging from 6 × 10(2) to 1 × 10(7) cells mL(-1) with a detection limit down to 12 cells. More importantly, this method could be used for distinguishing cancer cells from normal cells and monitoring of dynamic carbohydrate expression on living cells, providing promising application in clinical diagnosis and treatment of cancer.

Study on Synthesis and Properties of a Starch-Polyacrylamide Graft Copolymer by HRP

A starch-polyacrylamide graft copolymer has been synthesized in water by means of horseradish peroxidase (HRP)/H2O2/acetylacetone (ACAC). It is discussed through single factor experiment that the effects of system pH, reaction temperature and amount of HRP on the residual acrylamide (AAM) content. The results show that the ideal copolymer was synthesized when mass ratio of starch and AAM was 2:1, and catalyst was HRP/H2O2 at 30 °C for 3.5 h. The residual AAM content was about 1.0%. The structure and properties of the graft copolymer are characterized by FTIR, NMR. The graft copolymer is used as retannage.

Enzymatic miniemulsion polymerization of styrene with a polymerizable surfactant

Enzymatic polymerization is a powerful tool for synthesizing useful polymeric materials. However, the preparation of polymer particles by enzymatic polymerization has been reported only rarely. We demonstrated herein the preparation of polystyrene particles by enzyme-mediated miniemulsion polymerization with a polymerizable surfactant, i.e., N,N-dimethyl-N-n-dodecyl-N-2-methacryloyloxyethylammonium bromide (C12-DMAEMA), using a horseradish peroxidase (HRP)/H2O2/β-diketone system in water. Effects of various amounts of HRP, H2O2, and β-diketone on styrene conversion and particle size were investigated. The obtained polystyrene particles were characterized using DLS, GPC, and SEM measurements, and the polymerization kinetics was examined. Comparison of five β-diketones was also conducted: acetylacetone (ACAC), ethylacetoacetate (EAA), 1,3-cyclopentanedione (CP), tetronic acid (TA), and dibenzoylmethane (DM), for enzymatic miniemulsion polymerization.

Study on Synthesis and Properties of a Starch-Polyacrylamide Graft Copolymer by HRP

A starch-polyacrylamide graft copolymer has been synthesized in water by means of horseradish peroxidase (HRP)/H2O2/acetylacetone (ACAC). It is discussed through single factor experiment that the effects of system pH, reaction temperature and amount of HRP on the residual acrylamide (AAM) content. The results show that the ideal copolymer was synthesized when mass ratio of starch and AAM was 2:1, and catalyst was HRP/H2O2 at 30 °C for 3.5 h. The residual AAM content was about 1.0%. The structure and properties of the graft copolymer are characterized by FTIR, NMR. The graft copolymer is used as retannage.

A New Dual Immunoassay for Tumor Markers Based on Chemiluminescence Signal Amplification by Magnetic Mesoporous Silica and Enzyme Modified Gold Nanoparticles

A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies immobilized on magnetic mesoporous silica particles (Fe(3)O(4)/SiO(2)) were prepared as the primary probe. Horseradish peroxidase (HRP) labeled antibodies co-coated with HRP on gold nanoparticles (AuNPs) were used as the secondary probe to achieve signal amplification. HRP tags were retained in the flow cells after a sandwich immunoassay. By controlling two switches on the two channels, chemiluminescent substrates were injected orderly man way, and then signals for CEA and AFP were sequentially detected by HRP-luminol-H(2)O(2). Due to the increased amount of HRP on AuNPs and the increased amount of monoclonal antibodies on Fe(3)O(4)/SiO(2), the signals were largely amplified. Under the optimal conditions, CEA and AFP could be detected in the linear ranges of 1.0 - 80 and 1.0 - 75 ng mL(-1) with detection limits of 0.25 and 0.5 ng mL(-1), respectively.

In situ gellable sugar beet pectin via enzyme-catalyzed coupling reaction of feruloyl groups for biomedical applications

In situ gellable hydrogels are more attractive in many biomedical and biopharmaceutical applications than pre-formed hydrogels because they can be implanted simply by injection and allow homogeneous incorporation of bioactive materials. In this study, the potential suitability of in situ gellable sugar beet pectin (SBP) for biomedical and biopharmaceutical applications was investigated. SBP aqueous solution gelled within 1 min after addition of appropriate amounts of horseradish peroxidase (HRP) and H₂O₂ via HRP-catalyzed oxidative coupling reaction of feruloyl groups on SBP molecules. The resultant gels gradually degraded under simulated physiological condition. L929 fibroblast cells encapsulated in the gels were scarcely damaged during the gelation process. A subcutaneously injected mixture of SBP, HRP and H₂O₂ solutions successfully gelled, and the gel did not induce necrosis in the surrounding tissue 1 week after implantation. These results demonstrate that the in situ gellable SBP gels are useful for biomedical and biopharmaceutical applications.

An enhanced ELISA based on modified colloidal gold nanoparticles for the detection of Pb(II)

A novel probe based on colloidal gold nanoparticles (AuNPs) modified with goat anti-mouse IgG and horseradish peroxidase (HRP) was synthesized and an enhanced enzyme-linked immunosorbent assay (ELISA) based on the probe was developed. In the assay, the synthesized probe is bound with a monoclonal antibody (McAb) which is competitively bound by coated BSA–ITCBE–Pb(II) on plate and Pb(II) in samples. The HRP, used here for signal amplification catalytically oxidize the substrate and generate optical signals that is related to the concentration of Pb(II) and can be measured spectrophotometrically. For the monodisperse AuNPs having high surface areas, it can be conjugated with more amount of HRP than that of IgG. Therefore, compared with traditional ELISA, the signal amplification of catalytically oxidized substrate was enhanced. The detection limit for this novel modified AuNPs probe-based assay was 9 pg mL −1. The recoveries obtained by standard Pb(II) addition to real samples, including a commercial mineral water, tap water, and lake water were all from 94.9% to 102.9%. And the coefficient of variation (CV) value of all samples was less than 10%. The results indicated that the enhanced assay gave higher sensitivity and reliable reproducibility. It could provide a general detection format for low-molecular weight contaminants.

Preparation and Properties of Modified Starch Tannage

Modified starch tannage was synthesized by two steps. First step, a graft starch was synthesized by graft copolymerization of degraded starch with p-aminobenzene sulfonic acid and nekal using horseradish peroxidase (HRP)/H2O2 catalyze system. The effects of system pH, reaction temperature and amount of HRP on the graft efficient (GE) and graft percent (GP) were investigated and the maximum GE and GP of modified starch were obtained using 4 mg HRP/2.24 g H2O2 as catalyst at 35 for 3h under pH 7. The second step, the graft starch was oxidized by periodic acid to prepare modified starch tannage with dial dehyde.The effect of periodic acid on aldehyde groups content of graft starch was studied and it was found that the maximum content was at periodic acid concentration of 4 mol/L. The structure of modified starch tannage was characterized by FTIR, NMR and GPC. The shrink temperature (Ts)of tanned leather is 80.6 °C and the retanned leather has distinctive style of softness and flexibility.

An ultra-sensitive chemiluminescence immunosensor of carcinoembryonic antigen using HRP-functionalized mesoporous silica nanoparticles as labels

A novel chemiluminescence immunosensor using horseradish peroxidase (HRP)-functionalized mesoporous silica nanoparticles (MSN) as labels was developed, which increases the sensitivity of the chemiluminescence immunoassay. The enzyme-functionalized MSN were fabricated by simultaneous coimmobilization of HRP and the carcinoembryonic antigen antibody (anti-CEA) onto the surface of MSN using 3-aminopropyltriethoxysilane (APTES) as the linkage. Because the large surface area of MSN carriers increased the amount of HRP bound per sandwiched immunoreaction, the conjugates provided a much higher signal and increased sensitivity. This is an improvement over the traditional sandwich immunoassay which often has one or two enzyme molecules per antibody. This approach was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect CEA in practical samples. The analysis showed a linear response within the range of 0.1–40 ng/mL ( r = 0.9912). The relative standard deviation (RSD) for 11 parallel measurements of 20 ng/mL CEA was 3.9%. The sensitivity of the immunosensor using MSN–HRP–Ab2 as labels was about 10-fold higher than that of traditional labels. These labels for immunosensors may provide many potential applications for detection of different biomolecules.

Delivering Horseradish Peroxidase as a Respirable Powder to the Isolated, Perfused, and Ventilated Lung of the Rat: The Pulmonary Disposition of an Inhaled Model Biopharmaceutical

Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 μm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. The total amount of HRP deposited in the lungs was 335 ± 100 μg and 568 ± 47 μg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Electrochemical immunoassay of hepatitis B surface antigen by the amplification of gold nanoparticles based on the nanoporous gold electrode

We describe herein the combination of electrochemical immunoassay using nanoporous gold (NPG) electrode with horseradish peroxidase (HRP) labeled secondary antibody–gold nanoparticles (AuNPs) bioconjugates for highly sensitive detection of protein in serum. The electroactive product of o-phenylenediamine (OPD) oxidized with H 2O 2 catalyzed by HRP was reduced in the Britton–Robinson (BR) buffer and the peak current of which was used to determine the concentration of antigen (Ag) in the analyte. The active surface area of NPG electrode was larger than that of a bare flat one. The presence of AuNPs enhanced the immobilized amount of HRP labeled antibody (Ab), which improved the sensitivity of the immunoassay when used as the secondary antibodies. As a result of these two combined effects, the sensitivity of the immunoassay for the determination of target protein was increased significantly. Using hepatitis B surface antigen (HBsAg) as a model, we demonstrate a dose response in the range of 0.01–1.0 ng/mL with a detection limit of 2.3 pg/mL. Analytical results of several human serum samples obtained using the developing technique are in satisfactory agreement with those given by enzyme-linked immune-absorbent assays (ELISA). In addition, the technique was about 100 times more sensitive in the detection of HBsAg than ELISA. All these demonstrated the feasibility of the present immunoassay method for clinical diagnosis.

Enzyme-Functionalized Silica Nanoparticles as Sensitive Labels in Biosensing

A novel strategy for sensitive detection of biomarkers using horseradish peroxidase (HRP)-functionalized silica nanoparticles as the label is presented. The enzyme-functionalized silica nanoparticles were fabricated by coimmobilization of HRP and alpha-fetoprotein antibody (anti-AFP, the secondary antibody, Ab2), a model protein, onto the surface of SiO(2) nanoparticles using gamma-glycidoxypropyltrimethoxysilane (GPMS) as the linkage. Through "sandwiched" immunoreaction, the enzyme-functionalized silica nanoparticle labels were brought close to the surface of gold substrates, as confirmed by the scanning electron microscopy (SEM) images. Enhanced detection sensitivity was achieved where the large surface area of SiO(2) nanoparticle carriers increased the amount of HRP bound per sandwiched immunoreaction. The electrochemical and chemiluminescence measurement showed 29.5- and 61-fold increases in detection signals, respectively, in comparison with the traditional sandwich immunoassay. The improved particle synthesis using a "seed-particle growth" route yielded particles of narrow size distribution, which allowed consistent loading of HRP and anti-AFP on each microsphere and ensured subsequent immunosensing possessed high sensitivity and reproducibility. This strategy was successfully demonstrated as a simple, cost-effective, specific, and potent method to detect AFP in practical samples.

Bioelectrochemical immunoassay of polychlorinated biphenyl

A simple, rapid, and highly sensitive bioelectrochemical immunoassay method based on magnetic beads (MBs) and disposable screen-printed electrodes (SPE) has been developed to detect polychlorinated biphenyls (PCBs). The principle of this bioassay is based on a direct competitive enzyme-linked immunosorbent assay using PCB-antibody-coated MBs and horseradish peroxidase (HRP)-labeled PCB (HRP–PCB). A magnetic process platform was used to mix and shake the samples during the immunoreactions and to separate free and unbound reagents after the liquid-phase competitive immunoreactions among PCB-antibody-coated MBs, PCB analyte, and HRP–PCB. After a complete immunoassay, the HRP tracers attached to MBs were transferred to a substrate solution containing o-aminophenol and hydrogen peroxide for electrochemical detection. The different parameters, including the amount of HRP–PCB conjugates, immunoreaction time, and the concentration of substrate that governs the analytical performance of the immunoassay have been studied in detail and optimized. The detection limit of 10 pg mL −1 was obtained under optimum experimental conditions. The performance of this bioelectrochemical immunoassay was successfully evaluated with untreated river water spiked with PCBs, and the results were validated by commercial PCB enzyme-linked immunosorbent assay kit, indicating that this convenient and sensitive technique offers great promise for decentralized environmental application and trace PCBs monitoring.

Mesoporous Materials Promoting Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase

AbstractThe direct electron transfer and electrocatalysis of horseradish peroxidase (HRP) immobilized on hexagonal mesoporous silicas (HMS) matrix was studied. The interaction between HRP and HMS was examined by using Fourier transform infrared spectroscopy, nitrogen adsorption isotherms and electrochemical methods. The immobilized HRP at a modified glassy carbon electrode showed a good direct electrochemical behavior, which depended on the specific properties of the HMS. Two couples of redox peaks corresponding to Fe(III) to Fe(II) conversion of the HRP intercalated in the mesopores and adsorbed on the external surface of the HMS were observed with the formal potentials of −0.315 and −0.161 V in 0.1 M pH 7.0 PBS, respectively. The amount of HRP intercalated in the mesopores of HMS proved to be related to the pore size. The HRP intercalated in the mesopores showed a surface controlled electrode process with a single proton transfer. The immobilized HRP displayed an excellent electrocatalytic response to the reduction of hydrogen peroxide (H2O2) without the aid of an electron mediator. The HMS provided a novel matrix for protein immobilization and direct electron transfer study of the immobilized protein.

Renewable fluorometric enzyme immunosensing system using prochlorperazine as the substrate for horseradish peroxidase for the determination of transferrin

A renewable fluorometric enzyme immunosensing system using prochlorperazine (PCP) as the substrate for horseradish peroxidase (HRP) has been developed for the determination of transferrin in serum samples. This system is based on paraffin–graphite–transferrin antibody immunocomposite, which permits its surface to be easily regenerated by simply polishing. The renewed surface served as a platform for the competitive immunoreaction of HRP–transferrin and free transferrin (analyte) with the immobilized antibody bound to the surface and for HRP-catalyzed reaction. By using prochlorperazine as the substrate for HRP, the amount of HRP–transferrin bound was quantified fluorimetrically, which was in turn related to free transferrin content in the samples. Prochlorperazine as the substrate, possesses a satisfactory activity towards HRP as compared to p-hydroxyphenylacetic acid. The analytical conditions, such as pH, the amount of labeled transferrin, incubation time, and temperature were studied in detail. Under the optimized experimental conditions, a detection limit of 7.3 ng/ml and the linear detection ranging from 7.3 to 127 ng/ml were obtained. This approach has been applied to clinical samples with satisfactory results.