Luminol-H_2O_2-HRP-BPB增强化学发光新体系的研究及其在磁酶免疫分析中的应用
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摘要
化学发光分析法由于仪器设备简单,分析速度快,灵敏度高,线性范围宽等优点,越来越受到科研工作者的广泛关注。本工作研究了新型增强剂溴酚兰(BPB)对Luminol-H_2O_2-HRP化学发光反应的增强作用,提出了Luminol-H_2O_2-HRP-BPB化学发光新体系。在最佳实验条件下,分别测定了游离HRP和HRP标记物的线性范围及检测限。将此化学发光增强体系与磁性分离技术和酶联免疫分析相结合,对癌胚抗原(CEA)、甲状腺素(T4)等肿瘤标记物进行了测定。此外,采用纳米金作为酶标抗体标记物,利用化学发光增强体系对甲胎蛋白(AFP)进行检测。结果表明,该方法灵敏度高,分析快速、简便,在生物分析中具有广阔的应用前景。
本文主要开展了以下几个方面的工作:
一、Luminol-H_2O_2-HRP-BPB增强化学发光新体系的研究
研究了Luminol-H_2O_2-HRP-BPB增强化学发光反应体系的增强作用。在最佳实验条件下,对游离HRP进行了测定,测定游离HRP的线性范围为1.0×10-11～5.0×10-10 g/mL,检测限为1.25×10-12 g/mL;并对HRP标记物进行了测定,检测限比Luminol-H_2O_2-HRP直接化学发光体系低1000倍,比四甲基联苯胺ELISA显色法低10倍。并对Luminol-H_2O_2-HRP-BPB增强化学发光体系机理进行了初步探讨。
二、基于Luminol-H_2O_2-HRP-BPB增强化学发光磁酶免疫体系测定CEA和T4
采用羧基修饰的磁性微球,经EDC/NHS活化处理后得到活化磁性微球,将AFP单克隆抗体固定于活化磁性微球表面,所制备的免疫磁性微球具有识别和捕获相应抗原的能力。将磁珠分离技术与Luminol-H_2O_2-HRP-BPB增强化学发光体系相结合,分别对癌胚抗原(CEA)和血清总甲状腺素(T4)进行了测定。采用双抗体夹心法对CEA进行了测定,测得CEA的线性范围为1.0～40.0 ng/mL,检测限为1.0 ng/mL,比四甲基联苯胺ELISA显色光度法低5倍;采用竞争法对T4进行了测定,测得T4的线性范围为1.0～30.0 ng/mL,检测限为1.0 ng/mL,比四甲基联苯胺ELISA光度法低5倍。该方法操作简单、快速,用所建立的方法对病人血清样品进行了测定,并与酶联免疫吸附测定光度法(ELISA)进行了对照,二者相关性良好。
三、基于纳米粒子标记的Luminol-H_2O_2-HRP-BPB增强化学发光磁免疫体系测定AFP
采用柠檬酸三钠还原法制备纳米金粒子,通过控制还原剂柠檬酸三钠的加入量,制得不同粒径的纳米金粒子。通过静电作用将HRP标记的甲胎蛋白(AFP)抗体连接到纳米金粒子上。利用紫外-可见吸收光谱和荧光光谱分析确定纳米金粒子与抗体连接时所需抗体的最佳浓度为0.6μg/mL。
以纳米金作为酶标记物,将磁分离技术与Luminol-H_2O_2-HRP-BPB增强化学发光体系相结合,采用双抗体夹心法,对AFP进行了测定,并与直接加入酶标抗体的方法相比较。酶标抗体标记方法测定AFP的线性范围为1.0～50.0 ng/mL,检测限为1.0 ng/mL;纳米金作为酶标抗体标记物测定AFP的线性范围为0.1～50.0 ng/mL,检测限为0.1 ng/mL,比四甲基联苯胺ELISA显色光度法低50倍。用所建立的方法对病人血清样品进行了测定,并与酶联免疫吸附测定光度法(ELISA)进行了对照,相关性良好。
Chemiluminescence (CL) has become a more and more popular analytical technique owing to its simple instrumentation, high sensitivity, wide dynamic range, reproducibility, simplicity and rapidity. This thesis studied a novel CL enhancer, bromophenol blue (BPB), in the system of Luminol-H_2O_2-HRP, and proposed a new enhanced CL system of Luminol-H_2O_2-HRP-BPB. Under the optimum conditions, the linear ranges and detection limits of free horseradish peroxidase (HRP) and labeled HRP were determined. This established enhanced CL system was also used to detect carcino enbryonic antigen (CEA) and T4 which integrates this method with magnetic separation technique. Furthermore, this new enhanced CL system was also applied to detect aloha-fetal protein (AFP) using Au nanoparticles (AuNPs) as the HRP-antibody (HRP-Ab) labels. The results showed that this method is of high sensitivity, rapidity and simplicity, and could be widely used in the field of biological analysis.
The main jobs of this thesis can be concluded as follows:
1. The studies on the new enhanced CL system of Luminol-H_2O_2-HRP-BPB
This thesis studies a new enhanced CL system of Luminol-H_2O_2-HRP-BPB. Under the optimum conditions, the free HRP was detected with a linear range of 1.0×10-11～5.0×10-10 g/mL and a detection limit of 1.25×10-12 g/mL. The labeled HRP was also detected with the detection limit 1000 folds lower than that of direct CL system of Luminol-H_2O_2-HRP and 10 folds of the traditional ELISA absorptionmetric method using 3,3’,5,5’-Tetramethylbenzidine (TMB) as the chromogenic agent. The mechanism of this new enhanced CL system of Luminol-H_2O_2-HRP-BPB was discussed preliminarily.
2. The detection of CEA and T4 based on the system of Luminol-H_2O_2-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassay
Carboxyl-modified magnetic beads were activated by EDC/NHS, and then conjugated monocloning antibody which were endowed with the capacity to capture and detect target antigens. CEA was detected by double antibody sandwish immunoassay method by integrating the magnetic separation technique with this new enhanced CL system of Luminol-H_2O_2-HRP-BPB with the linear range of 1.0～40.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. T4 was detected by competitive immunoassay method with the linear range of 1.0～30.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. Under the optimum conditions, the human serum samples were detected, and the results showed good corresponding relationship with those of ELISA absorptionmetric method.
3. The detection of AFP based on the system of Luminol-H_2O_2-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassay using AuNPs as HRP labels
Uniformly and excellent dispersivity AuNPs were prepared by water phase synthetical method. Different preparation conditions were chosen, and different size AuNPs were gotten. HRP-labeled AFP antibody were bound on the surface of AuNPs by electrostatic at special pH condition. The optimum concentration of HRP-labeled antibody was 0.6μg/mL based on UV-Vis and fluorescence spectrum. Using AuNPs as HRP labels, AFP was detected by integrating the system of Luminol-H_2O_2-HRP-BPB with magnetic separation. Based on the double antibody sandwich immunoassay method, the linear range of AFP was 0.1～50.0 ng/mL and the detection limit was 0.1 ng/mL, which was 10 folds lower than that of the system without using AuNPs as the labels and 50 folds of ELISA.
本文主要开展了以下几个方面的工作:
一、Luminol-H_2O_2-HRP-BPB增强化学发光新体系的研究
研究了Luminol-H_2O_2-HRP-BPB增强化学发光反应体系的增强作用。在最佳实验条件下,对游离HRP进行了测定,测定游离HRP的线性范围为1.0×10-11～5.0×10-10 g/mL,检测限为1.25×10-12 g/mL;并对HRP标记物进行了测定,检测限比Luminol-H_2O_2-HRP直接化学发光体系低1000倍,比四甲基联苯胺ELISA显色法低10倍。并对Luminol-H_2O_2-HRP-BPB增强化学发光体系机理进行了初步探讨。
二、基于Luminol-H_2O_2-HRP-BPB增强化学发光磁酶免疫体系测定CEA和T4
采用羧基修饰的磁性微球,经EDC/NHS活化处理后得到活化磁性微球,将AFP单克隆抗体固定于活化磁性微球表面,所制备的免疫磁性微球具有识别和捕获相应抗原的能力。将磁珠分离技术与Luminol-H_2O_2-HRP-BPB增强化学发光体系相结合,分别对癌胚抗原(CEA)和血清总甲状腺素(T4)进行了测定。采用双抗体夹心法对CEA进行了测定,测得CEA的线性范围为1.0～40.0 ng/mL,检测限为1.0 ng/mL,比四甲基联苯胺ELISA显色光度法低5倍;采用竞争法对T4进行了测定,测得T4的线性范围为1.0～30.0 ng/mL,检测限为1.0 ng/mL,比四甲基联苯胺ELISA光度法低5倍。该方法操作简单、快速,用所建立的方法对病人血清样品进行了测定,并与酶联免疫吸附测定光度法(ELISA)进行了对照,二者相关性良好。
三、基于纳米粒子标记的Luminol-H_2O_2-HRP-BPB增强化学发光磁免疫体系测定AFP
采用柠檬酸三钠还原法制备纳米金粒子,通过控制还原剂柠檬酸三钠的加入量,制得不同粒径的纳米金粒子。通过静电作用将HRP标记的甲胎蛋白(AFP)抗体连接到纳米金粒子上。利用紫外-可见吸收光谱和荧光光谱分析确定纳米金粒子与抗体连接时所需抗体的最佳浓度为0.6μg/mL。
以纳米金作为酶标记物,将磁分离技术与Luminol-H_2O_2-HRP-BPB增强化学发光体系相结合,采用双抗体夹心法,对AFP进行了测定,并与直接加入酶标抗体的方法相比较。酶标抗体标记方法测定AFP的线性范围为1.0～50.0 ng/mL,检测限为1.0 ng/mL;纳米金作为酶标抗体标记物测定AFP的线性范围为0.1～50.0 ng/mL,检测限为0.1 ng/mL,比四甲基联苯胺ELISA显色光度法低50倍。用所建立的方法对病人血清样品进行了测定,并与酶联免疫吸附测定光度法(ELISA)进行了对照,相关性良好。
Chemiluminescence (CL) has become a more and more popular analytical technique owing to its simple instrumentation, high sensitivity, wide dynamic range, reproducibility, simplicity and rapidity. This thesis studied a novel CL enhancer, bromophenol blue (BPB), in the system of Luminol-H_2O_2-HRP, and proposed a new enhanced CL system of Luminol-H_2O_2-HRP-BPB. Under the optimum conditions, the linear ranges and detection limits of free horseradish peroxidase (HRP) and labeled HRP were determined. This established enhanced CL system was also used to detect carcino enbryonic antigen (CEA) and T4 which integrates this method with magnetic separation technique. Furthermore, this new enhanced CL system was also applied to detect aloha-fetal protein (AFP) using Au nanoparticles (AuNPs) as the HRP-antibody (HRP-Ab) labels. The results showed that this method is of high sensitivity, rapidity and simplicity, and could be widely used in the field of biological analysis.
The main jobs of this thesis can be concluded as follows:
1. The studies on the new enhanced CL system of Luminol-H_2O_2-HRP-BPB
This thesis studies a new enhanced CL system of Luminol-H_2O_2-HRP-BPB. Under the optimum conditions, the free HRP was detected with a linear range of 1.0×10-11～5.0×10-10 g/mL and a detection limit of 1.25×10-12 g/mL. The labeled HRP was also detected with the detection limit 1000 folds lower than that of direct CL system of Luminol-H_2O_2-HRP and 10 folds of the traditional ELISA absorptionmetric method using 3,3’,5,5’-Tetramethylbenzidine (TMB) as the chromogenic agent. The mechanism of this new enhanced CL system of Luminol-H_2O_2-HRP-BPB was discussed preliminarily.
2. The detection of CEA and T4 based on the system of Luminol-H_2O_2-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassay
Carboxyl-modified magnetic beads were activated by EDC/NHS, and then conjugated monocloning antibody which were endowed with the capacity to capture and detect target antigens. CEA was detected by double antibody sandwish immunoassay method by integrating the magnetic separation technique with this new enhanced CL system of Luminol-H_2O_2-HRP-BPB with the linear range of 1.0～40.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. T4 was detected by competitive immunoassay method with the linear range of 1.0～30.0 ng/mL and the detection limit of 1.0 ng/mL, which was 5 folds lower than that of ELISA. Under the optimum conditions, the human serum samples were detected, and the results showed good corresponding relationship with those of ELISA absorptionmetric method.
3. The detection of AFP based on the system of Luminol-H_2O_2-HRP-BPB chemiluminescent magnetic enzyme-linked immunoassay using AuNPs as HRP labels
Uniformly and excellent dispersivity AuNPs were prepared by water phase synthetical method. Different preparation conditions were chosen, and different size AuNPs were gotten. HRP-labeled AFP antibody were bound on the surface of AuNPs by electrostatic at special pH condition. The optimum concentration of HRP-labeled antibody was 0.6μg/mL based on UV-Vis and fluorescence spectrum. Using AuNPs as HRP labels, AFP was detected by integrating the system of Luminol-H_2O_2-HRP-BPB with magnetic separation. Based on the double antibody sandwich immunoassay method, the linear range of AFP was 0.1～50.0 ng/mL and the detection limit was 0.1 ng/mL, which was 10 folds lower than that of the system without using AuNPs as the labels and 50 folds of ELISA.
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