流动注射协同增强化学发光免疫分析法的建立与评价

以兔IgG为模型 ,NaTPB与PPP协同增强的Luminol H2 O2 HRP混合液为发光体系 ,HRP标记抗体 ,建立了灵敏度高、特异性强、重现性好的流动注射协同增强化学发光免疫分析法。结果表明 :在 2～ 6 0 μg L范围内兔IgG量与发光强度有良好的线性关系 ,相关系数r=0 .994 1 (P <0 .0 1 ) ;绝对检测限为 0 .6 5fmol;方法精密度为 4 .72 %～ 9.31 % ;回收率为 92 .5 0 %～ 99.4 0 %。免疫柱可反复使用 2 0 0次以上。本法测定不同浓度兔IgG标准溶液的结果与RIA法的测定结果基本一致     

以新亚甲蓝为介体的过氧化氢传感器的电化学行为研究

利用共价键合法,将新亚甲蓝(NMB)与辣根过氧化酶(HRP)修饰于玻碳电极表面,制成一种新型的电流型H2O2传感器。探讨了该传感器在0·1mol/L磷酸缓冲溶液(pH=7·0)中的电化学性质。结果表明,NMB作为介体能够有效地在辣根过氧化酶和电极之间传递电子。测得电子转移系数为0·861,表观反应速率常数为1·27s-1。研究了传感器对H2O2的响应及动力学性质,米氏常数为8·27μmol/L,线性响应范围为2·5~100μmol/L。同时研究了pH、缓冲容量及温度等因素对H2O2传感器的影响。     

癌胚抗原毛细管电泳-化学发光均相免疫分析

设计了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测均相免疫分析新方法。采用四苯硼酸钠增强luminol-H2O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物。测定癌胚抗原的线性范围2.0~80.0μg/L(R=0.9921),检出限为0.1μg/L(绝对检出限为0.75 fg)。     

癌胚抗原毛细管电泳-化学发光均相免疫分析

建立了一种测定人血清中癌胚抗原的毛细管电泳-化学发光检测的均相免疫分析新方法.采用四苯硼钠增强luminol-H2 O2-HRP体系化学发光的原理,化学发光检测经毛细管电泳分离的,用辣根过氧化物酶(HRP)标记的癌胚抗体及免疫复合物.测定癌胚抗原的线性范围2.0～80.0 μg/L(R=0.9921),检出限为0.1 μg/L(绝对检出限为0.75 fg).     

一种基于碳纳米管的安培型过氧化氢生物传感器

利用硫堇(th ion ine,Th i)作为介体结合多壁碳纳米管(MWNTs)、壳聚糖(ch itosan,CH IT)、辣根过氧化酶(HRP)的混合包埋物制作过氧化氢(H2O2)生物传感器。研究结果表明所得传感器对H2O2具有明显的增敏效应,线性范围为0.03~5.5 mmol/L,相关系数为0.9995;检出限为19μmol/L(S/N=3),具有良好的稳定性及工作寿命。     

亚硫酸氢钠-过氧化氢超微弱化学发光体系用于核黄素检测

亚硫酸氢钠(Na HSO3)和过氧化氢(H2O2)反应可产生微弱的化学发光。核黄素对亚硫酸氢钠和过氧化氢的化学发光有极大的增强作用,且化学发光强度在0.02~2.0μg/m L范围内与核黄素浓度呈良好的线性关系。据此,建立了核黄素的高灵敏化学发光分析方法,方法检出限为0.007μg/m L,相对标准偏差(n=11)不大于1.6%。利用荧光光谱、化学发光谱对核黄素增强Na HSO3-H2O2体系化学发光的机理进行探讨,为该体系的应用研究提供了新思路。     

基于固定化纳米金增强化学发光双酶传感器测定葡萄糖

研制了一种新型流动注射化学发光（CL）双酶传感器,用于葡萄糖的检测．该传感器将掺杂金纳米粒子（GNPs）的壳聚糖膜包覆在硅烷化试剂预处理的玻璃微珠上,用于吸附固定葡萄糖氧化酶（GOD）和辣根过氧化物酶（HRP）．葡萄糖在GOD的催化下发生氧化反应生成H2O2,生成的H2O2在HRP的催化作用下与鲁米诺发生反应,并产生化学发光信号．实验表明,壳聚糖中掺杂的GNPs不仅能够有效的吸附酶分子并保持其生物活性,还对Luminol-H2O2-HRP化学发光体系具有增敏作用．通过化学发光光谱和紫外光谱表征,详细研究了固定化GNPs增强Luminol—H2O2-HRP体系的化学发光机理．在优化的实验条件下,该传感器对葡萄糖检测的线性范围为0．01～6．0mmol／L,检测限为5．0μmol／L（3σ）．将所建立的方法用于临床血清样品中葡萄糖含量的测定,获得了满意的结果．     

The electrochemical behavior of hydrogen peroxide sensor based on new methylene blue as mediator

利用共价键合法,将新亚甲蓝(NMB)与辣根过氧化酶(HRP)修饰于玻碳电极表面,制成一种新型的电流型H2O2传感器。探讨了该传感器在0·1mol/L磷酸缓冲溶液(pH=7·0)中的电化学性质。结果表明,NMB作为介体能够有效地在辣根过氧化酶和电极之间传递电子。测得电子转移系数为0·861,表观反应速率常数为1·27s-1。研究了传感器对H2O2的响应及动力学性质,米氏常数为8·27μmol/L,线性响应范围为2·5~100μmol/L。同时研究了pH、缓冲容量及温度等因素对H2O2传感器的影响。     

辣根过氧化酶在磁性纳米修饰的离子注入电极上的电化学和电催化行为

辣根过氧化酶(HRP)在Co/NH2/ITO离子注入电极上有一对良好的氧化还原峰,峰电位分别为Epc=-0.2 V,Epa=-0.01 V(vsAg/AgCl)。该修饰电极对H2O2具有催化作用,可以用作H2O2的生物传感器,峰电流与H2O2的浓度分别在1.0×10-10~2.0×10-8mol/L和2.0×10-8~1.0×10-7mol/L范围内呈线性关系,线性回归方程分别为Ip(mA)=2.2986+0.06632c(nmol/L)和Ip(mA)=3.5788+7.3053E-4c(nmol/L),相关系数分别为0.9972和0.9688。检出限为1.0×10-10mol/L。     

流动注射化学发光法测定醋氯芬酸

基于酸性条件下醋氯芬酸对高锰酸钾氧化甲醛产生微弱化学发光反应体系具有很强的增敏作用,且增强的发光强度在一定范围内与醋氯芬酸浓度呈良好的线性关系,结合流动注射分析技术,建立了测定醋氯芬酸的化学发光新方法。该法测定醋氯芬酸的线性范围为0.08~5.0 mg/L,检出限(3σ)为3.0×10-2mg/L,相对标准偏差(RSD)为1.7%(n=11,c=0.50 mg/L),回收率为98.1%~104.6%。该法已用于片剂和胶囊中醋氯芬酸含量的测定。     

基于酶辅助靶标循环的适体传感器灵敏检测中药中黄曲霉毒素B1

该文基于酶辅助靶标循环信号放大策略构建了用于黄曲霉毒素B1(AFB1)高灵敏检测的化学发光适体传感器。以G-四链体/氯化血红素DNA酶为信号分子设计了免标记的适体探针H1-S1和发夹探针H2。适体探针结合目标AFB1,在核酸外切酶I辅助下,触发靶标循环反应产生发夹H1。发夹H1与H2杂交,释放出完整的G-四链体序列,并进一步与氯化血红素结合形成G-四链体/氯化血红素DNA酶。DNA酶通过催化氧化鲁米诺-H2O2化学发光体系产生化学发光信号,实现AFB1的放大检测。在最优实验条件下,化学发光强度与AFB1质量浓度的对数在0.001~100 ng/mL范围内呈良好的线性关系,相关系数(r2)为0.9955,检出限为0.93 pg/mL,回收率为93.7%~107%。该适体传感器操作简单、灵敏度高、特异性好,在黄曲霉毒素污染检测方面具有良好的应用前景。     

化学发光免疫分析法检测血清中前列腺特异抗原复合物

采用杂交瘤技术构建抗前列腺特异抗原-α1-抗胰凝乳蛋白酶( Prostate specific antigen-α1-antichymot-rypsin complex,PSA-ACT)单克隆抗体(单抗)细胞株,共获得8株稳定分泌PSA-ACT单抗的细胞株,纯化后进行免疫特性鉴定并做单抗配对。选择其中一对单抗,采用辣根过氧化物酶( HRP)催化鲁米诺( lumino)-H2 O2化学发光体系,建立了测定人血清中PSA-ACT浓度的化学发光酶免疫分析方法( CLEIA)。对包被缓冲液、包被抗体浓度、酶标抗体稀释度、温育时间、发光反应时间和免疫反应步骤等实验参数进行了优化。在最佳实验条件下, PSA-ACT浓度在5~40 ng/mL范围内线性关系良好(R2=0.9943),检出限为0.53 ng/mL,批内相对标准偏差(RSD)为4.6%~6.6%,批间RSD为5.7%~8.0%,回收率为95.4%~104.2%,与游离前列腺特异性抗原F-PSA的交叉反应率为0.6%。本方法简单、稳定、灵敏、快速,为开发检测PSA-ACT的CLEIA试剂盒奠定了基础。     

偶合反应化学发光酶免疫分析研究: I. HRP及其标记物的化学发 光测定

本文将辣根过氧化物酶(HRP)催化过氧化氢氧化KI生成I~2的反应与Luminol-I~2的化学发光反应相偶合,提出了测定HRP及其抗原、抗体标记物的高灵敏度的化学发光法。检测下限为7pg,线性范围为10-6000pg。通常使用的HRP直接催化Luminol-H~2O~2化学发光法灵敏度高10-100倍;克服了固相吸附法直接测定HRP标记物的缺陷,提高了测定的选择性。文中还对该偶合反应的机理及其动力学特性进行了研究。     

Development of a rapid and sensitivity magnetic chemiluminescence immunoassay for DNA methyltransferase 1 in human serum

DNA methyltransferase 1(DNMT1) is a useful biomarker for lung cancer in early clinical diagnosis. A rapid magnetic chemiluminescence immunoassay(MCLIA) for DNMT1 in human serum has been developed.Horseradish peroxidase(HRP)-second-Ab was used to labeled polyclonal antibodies of anti-DNMT1. DNMT1 in sample integrates with specific immunomagnetic beads and can constitute a supersandwiched immunoreaction. In magnetic field, nonspecific materials can be separated. After luminescent substrate luminol-H_2O_2-BIP was added, the relative light unit(RLU) of HRP was detected and was discovered to be directly proportional to the content of DNMT1 in sample. The correlative variables involved in the MCLIA value were optimized and the methodological evaluation was carried out. After optimization, in the range of0.5–128 ng/mL, the linear regression equation was y = 0.5014 x + 1.769(x was logCDNMT1, y was relative luminescence units(RLU)/RLU0), and the limit of detection was 0.01 ng/mL. The RSD of intra-and interassays were 15.8%–16.9% and 14.3%–18.1%, respectively. The recovery was from 70.0% to 106.2%.Furthermore, paralleled with purchasable enzyme-linked immunosorbent assay(ELISA) kits, MCLEIA had lower detection limit, wider linear range and shorter detection time. Therefore, the MCLEIA established in this study could be used for the sensitive detection of DNMT1 in serum sample.     

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%.     

Delayed luminescence of luminol initiated by a membrane-bound peroxidase

The luminescense of the luminol-H₂O₂ system was initiated by either free or membrane-bound horseradish peroxideae (HRP). The instantaneous luminescene decayed rapidly and was followed by the delayed luminescence in the presence of excess luminol. The delayed luminescence was characterized by a chain reaction, in which luminescence intensity increased exponentially. Membrane-bound HRP demonstrated that the delayed luminescence took place even in the absence of HRP if the instantaneous luminescence was initiated by HRP. A mechanism for the nonenzymatic luminescence is proposed and discussed.     

Investigation of voltammetric enzyme-linked immunoassay based on a new system of HAP-H2O2-HRP

By introducing heterocyclic compound to immunoassay system as an electrochemical substrate for the fist time, a new voltammetric enzyme-linked immunoassay system of 3-hydroxyl-2-aminopyridine (HAP)-H(2)O(2)-horseradish peroxidase (HRP) has been developed. HAP was oxidized with H(2)O(2) catalyzed by HRP, and the resulting electroactive product produced a sensitive voltammetric peak at potential of -0.36 V (vs. SCE) in Britton-Robinson (BR) buffer solution. The process of the enzyme-catalyzed reaction and the electro-reduction of the product have been investigated in detail. The linear range for detection of free HRP was from 4.0x10(-13) to 1.0x10(-9) g/mL with a detection limit of 1.2x10(-13) g/mL. The new system has been successfully applied for the assay of alpha-fetoprotein (alphaFP) in human serum ranging from 0.1 to 200 ng/mL with a detection limit of 0.1 ng/mL, which was 10 times lower than that of traditional spectrophotometric enzyme-linked immunosorbent assay (ELISA) method. HAP-H(2)O(2)-HRP voltammetric enzyme-linked immunoassay showed a promising alternative approach in the detection of alphaFP in clinical diagnosis.     

PAP-H2O2-HRP伏安酶联免疫分析新体系测定人血清总甲状腺素

目前临床检测中测定总甲状腺素(T₄)的常用方法有间接血凝试验、琼脂双扩散及ELISA等方法^[1].其中ELISA法是目前较为流行的检测方法,但灵敏度不高.伏安酶联免疫分析法具有广阔的应用前景^[2,3].     

Capillary electrophoresis enzyme immunoassay for alpha-fetoprotein and thyroxine in human serum with electrochemical detection

A novel CE-based enzyme immunoassay (CE-EIA) method was developed in o-aminophenol (OAP)-H(2)O(2)-horseradish peroxidase (HRP) system and applied to benign liver disease and hyperthyroidism research in the clinical practical field. In the presented method, after the enzyme immunoreaction, the HRP-labeled antibody or HRP-labeled antigen catalyzed the enzyme substrate OAP and H(2)O(2). The product of the enzymatic catalysis reaction 2-aminophenoxazine-3-one (AP) was determined using electrochemical detection on a Pt electrode at the outlet of the reaction capillary. Factors influencing the performance, including running buffer concentration, separation, and detection voltage, were investigated to the optimum conditions. Noncompetitive and competitive models were utilized to detect alpha-fetoprotein (AFP) and thyroxine (T(4)) in human sera, respectively. The linear ranges and the detection limits (S/N = 3) were from 1.5 to 66.6 ng/mL and 0.48 ng/mL for AFP, and from 1.7 to 260.0 ng/mL and 1.0 ng/mL for T(4). The results of this method were linear proportional to those of spectrophotometric ELISA method, giving a good prospect for a new clinical diagnostic instrument.     

OT-H2O2-HRP伏安酶联免疫分析新体系

本文首次提出了邻联甲苯胺(OT)-H2O2-辣根过氧化物酶(HRP)伏安酶联免疫分析新体系。本方法以线性扫描二阶导数伏安法检测HRP催化H2O2氧化OT的产物, 用于游离HRP和各种HRP标记物测定, 灵敏度比经典的ELISA光度法分别高两个至四个数量级。测定游离HRP的检测限达到1.8×10^-^1^2 g/mL, 线性范围为5.0×10^-^1^2-1.0×10^-^8 g/mL。对此伏安酶联免疫分析新体系的偶合反应机理及电极还原过程也进行了详细的研究。