以葛根素为辣根过氧化物酶底物的新体系及其分析应用

以一种天然活性成分葛根素(Puerarin)为辣根过氧化物酶(HRP)底物建立了葛根素-辣根过氧化物酶-过氧化氢反应新体系. 在反应体系中 HRP 催化H₂O₂ 氧化葛根素(弱荧光)形成二聚体产物(强荧光), 该产物在315 nm 的激发光下能发射波长为478 nm的强荧光, 并且反应体系荧光强度增加与HRP量在一定浓度范围内呈线性相关. 根据此关系和竞争型免疫定量原理, 以兔布氏杆菌抗体为分析对象建立了基于葛根素的酶联荧光免疫传感分析新方法. 对葛根素性质的研究结果证实, 葛根素在空气中稳定、对温度稳定, 对H₂O₂＋HRP 敏感性优于传统底物如对羟基苯乙酸、Amplex Red和高香草酸. 优化了酶联荧光免疫传感分析方法的实验条件如HRP-BrAb 用量、温度等. 运用新体系测定了兔血清样品的布氏杆菌抗体, 该方法线性范围为1.3～120 ng/mL, 检测限为1.3 ng/mL (3σ), 相对标准偏差为3.8%.     

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.     

Immunosensor based on fluorescence quenching matrix of the conducting polymer polypyrrole

In this study, the combination of autofluorescent proteins and fluorescence quenching polymers was shown to be a design which can increase the selectivity and sensitivity of immunosensors. With this objective, the conducting polymer polypyrrole (Ppy) was used as a matrix for immobilization of proteins, which enables biological recognition of the analyte, and as a fluorescence quencher, which increases the selectivity of fluorescence-based detection. In this study, bovine leukemia virus proteins gp51 were immobilized within the Ppy matrix and formed a polymeric layer with affinity for antibodies against protein gp51 (anti-gp51). The anti-gp51 antibodies are present at high levels in the blood serum of cattle infected by bovine leukemia virus. Secondary antibodies labeled with horseradish peroxidase (HRP) were used as specific fluorescent probes for detection of a particular target, because the fluorescence of HRP was readily detectable at the required sensitivity. The Ppy was used as fluorescent background, because its fluorescence was almost undetectable when excited by near UV light at 325 nm. Moreover the Ppy quenched the fluorescence of some fluorescent agents including fluorescein-5(6)-isothiocyanate (fluorescein), rhodamine B, and HRP by almost 100% when these fluorescent agents were adsorbed on the surface of Ppy. It is predicted that Ppy-induced fluorescence quenching could be used in the design of immunosensors to increase selectivity and sensitivity.     

Superior performance of liposomes over enzymatic amplification in a high-throughput assay for myoglobin in human serum

Myoglobin is one of several cardiac markers which become elevated in the blood following an acute myocardial infarction and can aid in the diagnosis of a heart attack. Here, a sandwich immunoassay for myoglobin was developed, including a thorough optimization of fluorescent dye-encapsulating liposomes versus enzymatic amplification (alkaline phosphatase and horseradish peroxidase) at each step. The optimized microtiter plate-based assay was capable of detecting as low as 11.3 pg/mL myoglobin and was successfully applied for the quantification of myoglobin in human serum. In comparison to enzymatic approaches, the liposomes demonstrated lower limits of detection, significantly reduced limits of quantification, improved signal discrimination through substantial signal enhancement, and reduced assay time. Liposomes were stable and functional at ambient temperatures for over 400 days. Finally, ease of use was greater due to lack of reliance on additional reagents, non-time-based signal enhancement, and excellent photostability. Optimal conditions identified for enzymatic approaches can also be used for liposome amplification, which makes substitution of these liposomes into existing assays straightforward. Thus, the extensive studies carried out here suggest that liposomes may be incorporated into formats currently utilizing enzymatic enhanced fluorescence with a potential for increased performance on various levels.

氯化血红素模拟过氧化物酶高效液相色谱柱后衍生法测定过氧化氢和水溶性有机过氧化物

研究了氯化血红素（Ｈｅｍｉｎ）模拟辣根过氧化物酶（ＨＲＰ），对羟基苯乙酸（ＰＨＰＡＡ）作 底物，高效液相色谱（ＨＰＬＣ）柱后衍生荧光法测定Ｈ２Ｏ２和水溶性有机过氧化物的方法。采用 Ｈｅｍｉｎ作催化剂柱后衍生反应的最佳ｐＨ值约为１１，与荧光检测ｐＨ值一致，使得以往ＨＰＬＣ 柱后衍生方法所需的高压泵从３台减少到２台，而一些不能作为ＨＲＰ底物的羟烷基过氧化 物在ｐＨ值≥１０的溶液中迅速水解为Ｈ２Ｏ２从而得到测定。优化了测定Ｈ２Ｏ２和甲基过氧化 氢（ＣＨ３ ＯＯＨ， ＭＨＰ）的条件。最佳条件下 Ｈｅｍｉｎ方法测定 Ｈ２Ｏ２的检测限为 ９．０ × １０－９ ｍｏｌ／Ｌ， 测定 ＭＨＰ的检测限为 ２．０ × １０－７ ｍｏｌ／Ｌ。     

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.     

Aromatic azo compounds as spectrophotometric kinetic assay substrate for HRP

Aromatic azo compounds were efficiently cleaved by the horseradish peroxidase (HRP) into aromicdiazonium ions, during which an intense special absorption of the substrate disappeared completely. This furnished a sensitive spectrophotometric detection of 0.025-6.0 nm peroxidase. Kinetic characteristics of enzyme reaction were investigated with 14 different chemical structures of aromatic azo compounds as the substrates for HRP. Then the structure requirements for substrates were elucidated. PH dependence of enzymatic catalysis was studied with pK'(a)of 6.54 and 8.40 for Eriochrome black T, and 3.22 and 3.83 for Nitrosulfophenol S as the substrate of HRP, respectively.     

板式磁颗粒化学发光免疫分析测定人血清中糖类抗原125

在传统的板式化学发光免疫分析法和管式磁颗粒化学发光免疫分析法基础上,建立了人血清中糖类抗原125(CA125)的板式磁颗粒化学发光免疫分析方法.该方法以磁性微粒子作为分离固相,96孔板为反应容器,辣根过氧化物酶(HRP)催化H2O2-luminol化学发光体系作为检测体系.本法测定CA125的检测灵敏度可达2.0U/mL,线性范围为0～400U/mL.与常用的包被板化学发光免疫分析方法对比,该方法检测范围宽.与管式磁颗粒化化学发光法比较,其分析灵敏度与精密度高、线性范围、分析通量以及分析成本方面均显示了很好的优越性.采用该方法对人血清中CA125进行测定并与罗氏全自动电化学发光系统的测值结果进行了比对,两者显示了良好的相关性.     

Highly Sensitive Chemiluminescent Analysis of Residual Bovine Serum Albumin (BSA) Based on a Pair of Specific Monoclonal Antibodies and Peroxyoxalate–glyoxaline–PHPPA Dimer Chemiluminescent System in Vaccines

Enzyme-linked immunosorbent assay (ELISA), horseradish peroxidase (HRP)-catalyzed fluorescent reaction, and oxalate chemiluminescence analysis have been combined to develop a highly sensitive, simple, and rapid method for analysis of bovine serum albumin (BSA) based on a pair of specific monoclonal antibodies in vaccines. A typical ??sandwich type?? immunoassay was used. Reaction of 3-(4-hydroxyphenyl propionate) (PHPPA) with hydrogen peroxide-urea, catalyzed by HRP, produced fluorescence of 3-(4-hydroxyphenyl propionate) dimer, which was detected by chemiluminescence analysis with the bis(2,4,6-trichlorophenyl)oxalate (TCPO)?CH₂O₂?Cglyoxaline?CPHPPA dimer chemiluminescent system. This method exhibited high performance with a linear correlation between response and amount of bovine serum albumin (BSA) in the range 0.1 to 100.0?ng?mL^?1 (r?=?0.9988), and the detection limit was 0.03?ng?mL^?1 (S/N?=?3). Intra- and interassay coefficient variations were all lower than 9.0% at three concentrations (1.0, 20.0, and 80.0?ng?mL^?1). The proposed method has been used for successful analysis of the amount of residual BSA in vaccines. The results obtained compared well with those obtained by conventional colorimetric ELISA and luminol chemiluminescent ELISA.     

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.     

Chemiluminescence lateral flow immunoassay based on Pt nanoparticle with peroxidase activity

A lateral flow immunoassay (LF-immunoassay) with an enhanced sensitivity and thermostability was developed by using Pt nanoparticles with a peroxidase activity. The Pt nanoparticles were synthesized by citrate reduction method, and the peroxidase activity of Pt nanoparticles was optimized by adjusting reaction conditions. The peroxidase activity was estimated by using Michaelis–Menten kinetics model with TMB as a chromogenic substrate. The kinetics parameters of K_M and V_max were calculated and compared with horseradish peroxidase (HRP). The thermal stability of the Pt nanoparticles was compared with horseradish peroxidase (HRP) according to the storage temperature and long-term storage period. The feasibility of lateral flow immunoassay with a chemiluminescent signal band was demonstrated by the detection of human chorionic gonadotropin (hCG) as a model analyte, and the sensitivity was determined to be improved by as much as 1000-fold compared to the conventional rapid test based on colored gold-colloids.     

Water quality monitoring using an enhanced chemiluminescent assay based on peroxidase-catalyzed peroxidation of luminol

Enhanced chemiluminescence (ECL) describes the phenomenon of the light output increase in the reaction of oxidation of luminol catalyzed by horseradish peroxidase (HRP) in the presence of certain phenolic compounds. This work summarizes the effects of preincubation of certain substances with HRP on the chemiluminescent reaction intensity. Preincubation of herbicide, detergent, surfactants (Brij-96 and Tween-20), phenol, metal ions (mercury, cobalt, and nickel), and bactericide with HRP had an inhibitory effect on the enzyme activity. HRP-preincubation with metal ions (cadmium, magnesium, and zinc), as well as with some insecticides, stimulated the chemiluminescent intensity. Calibration graphs were obtained to demonstrate the possibility to determine the pollutant concentration. Light emission from the peroxidase catalyzed enhanced chemilum inescence is affected by a wide number of chemicals and, therefore, the method can beused for on-site monitoring of water quality. A rapid and simple assay to detect water contamination has been developed.     

Engineering trienzyme cascade-triggered fluorescent immunosensor platform by sequentially integrating alkaline phosphatase,tyrosinase and horseradish peroxidase

Mulit-enzyme cascades are a major type of chemical transformations and play a crucial role in biological signal transduction and metabolism. Herein, a trienzyme cascade-triggered fluorescent immunosensor platform was constructed by sequentially integrating alkaline phosphatase (ALP), tyrosinase (TYR) and horseradish peroxidase (HRP). The proposed platform was based on HRP-induced a rapid in situ fluorogenic reaction between dopamine (DA) and 1,5-dihydroxynaphthalene (DHA) to produce a strong yellow azamonardine fluorescent compound (AFC). The obtained AFC was clearly characterized by high-resolution mass spectrum, ¹H NMR, ¹³C NMR and theoretical calculations. The integration of the two-enzyme system (TYR and HRP) or three-enzyme system (ALP, TYR and HRP) led to a maximum of 400.0-fold and 250.0-fold fluorescence enhancements, respectively. Using cardiac troponin I (cTnI) as the model antigen, a trienzyme cascade-triggered fluorescent immunosensor platform was developed for quantitative detecting cTnI in a wide linear range from 2 ng/mL to 150 ng/mL with a detection limit of 0.67 ng/mL. In addition, the proposed platform was successfully applied in detection of cTnI in serum of clinical patients. Overall, the developed fluorescent immunosensor performs powerful implications for researching enzyme cascade systems in the field of biomedicine.     

Direct chemiluminescent immunodetection of proteins in agarose gels

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.     

Determination of nitrated pyrenes and their derivatives by high performance liquid chromatography with chemiluminescence detection after online electrochemical reduction

A sensitive and selective high performance liquid chromatographic method for the simultaneous determination of nitrated polycyclic aromatic hydrocarbons and their reduced compounds has been developed. As the model compounds for the proposed method, pyrene, aminopyrene, nitrosopyrene, and several nitrated pyrenes were used. After separation on a reversed phase column, both nitropyrenes and nitrosopyrene which were not fluorescent, were electrochemically reduced to strongly fluorescent aminopyrenes, and detected chemilumigenically by using bis(2,4,6-trichlorophenyl)oxalate and hydrogen peroxide as post column reagents. Their detection limits were all at the fmol levels, which were one or two orders lower than those by fluorescence detection. By the proposed method, nitropyrene, 1,3-, and 1,8-dinitropyrenes were detected in sooty emissions of cars after simple purifications.     

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.     

流动注射化学发光免疫分析研究II. 偶合反应测定HRP及其标记 物

本文详细考查了壳质胺,多孔玻璃和粗孔硅胶作为流动注射免疫分析免疫反应器基质的可行性,在此基础上将HRP催化H~2O~2氧化K~4Fe(CN)~6生成K~3Fe(CN)~6的反应,与H~2O~2和K~3Fe(CN)~6对luminol的共氧化化学发光反应相偶合首次提出了一种新的流动注射免疫分析的最终检测手段,由于酶促反应与化学发光反应在检测系统的不同位置进行,因而这种方法克服了已报道的流动注射化学发光免疫分析不能协调酶催化和化学发光反应的最佳pH,底物与酶不能充分接触及载体对光的散射等缺点,具有灵敏度高、精密度好等优点,该方法测定HRP及其标记物检测限均可达fmol级,测定时间为1-2min,相对标准偏差为3.9%。     

Multiplexed In Situ Protein Profiling with High-Performance Cleavable Fluorescent Tyramide

Understanding the composition, function and regulation of complex cellular systems requires tools that quantify the expression of multiple proteins at their native cellular context. Here, we report a highly sensitive and accurate protein in situ profiling approach using off-the-shelf antibodies and cleavable fluorescent tyramide (CFT). In each cycle of this method, protein targets are stained with horseradish peroxidase (HRP) conjugated antibodies and CFT. Subsequently, the fluorophores are efficiently cleaved by mild chemical reagents, which simultaneously deactivate HRP. Through reiterative cycles of protein staining, fluorescence imaging, fluorophore cleavage, and HRP deactivation, multiplexed protein quantification in single cells in situ can be achieved. We designed and synthesized the high-performance CFT, and demonstrated that over 95% of the staining signals can be erased by mild chemical reagents while preserving the integrity of the epitopes on protein targets. Applying this method, we explored the protein expression heterogeneity and correlation in a group of genetically identical cells. With the high signal removal efficiency, this approach also enables us to accurately profile proteins in formalin-fixed paraffin-embedded (FFPE) tissues in the order of low to high and also high to low expression levels.     

High-throughput imaging assay of multiple proteins via target-induced DNA assembly and cleavage

This work integrates target-induced DNA assembly and cleavage on a DNA chip to design a versatile imaging strategy as an assay for multiple proteins. The DNA assembly is achieved via immunological recognition to trigger the proximity hybridization for releasing a DNA sequence, which then hybridizes with FITC-DNA1 immobilized on the chip to induce the enzymatic cleavage of DNA1 and thus decrease the signals. The signal readout is performed with both fluorescent imaging of the left FITC and chemiluminescent (CL) imaging, by adding peroxidase labelled anti-FITC in assembly solution and CL substrates to produce CL emission. This one-step incubation can be completed in 30 min. The imaging method shows wide detection ranges and detection limits down to pg mL^–1 for the simultaneous detection of 4 protein biomarkers. This high-throughput strategy with good practicability can be easily extended to other protein analytes, providing a powerful protocol for protein analysis and clinical diagnosis.     

A rapid and sensitive 384-microtiter wells format chemiluminescent enzyme immunoassay for clenbuterol

A fast and sensitive chemiluminescent (CL) enzyme immunoassay for clenbuterol (CLB) analysis in bovine urine has been developed. Clenbuterol (CLB) specific polyclonal antibodies were raised in rabbit using a CLB azo derivative conjugated with ovalbumin. Horseradish peroxidase (HRP) was used as label and conjugated with the same derivative. In the developed competitive method, antibodies were immobilized on 384-wells black polystyrene microtiter plates; the sample volume was 20 mul and HRP-labeled CLB activity was immediately measured, using different CL substrates, after 10 min incubation time. Emitted light was recorded using a sensitive back-illuminated, cooled CCD camera or a conventional, photomultiplier-based micrtotiter plate reader. The developed method fulfills all the requirements of precision (CV below 10%) and accuracy (mean recovery from 96 to 110%) with a detection limit of 0.08 ppb in urine matrix. The use of 384-wells microtiter plate allows a 5-fold reduction in reagent quantity and the CL detection improves the detectability of the HRP-labeled tracer, thus reducing analysis time. The developed method is therefore suitable for high-throughput screening of CLB in urine samples, with reduced costs as compared with conventional colorimetric enzyme immunoassays, thanks to the possibility to optimize the system in non-equilibrium immunological conditions and with a very fast chemiluminescence detection of the HRP-label activity.