摘要
本文将化学发光传感技术、纳米技术相结合,探索构建了高灵敏度化学发光免疫传感器的新方法。基于化学发光、光谱学、电化学等手段对构筑的免疫传感器性能进行研究及优化。金纳米粒子作为固相载体,增加酶标抗体(或抗体)在固体表面的固定量,利用新型化学发光增强剂放大化学发光信号,提高化学发光免疫传感器的灵敏度。结果表明,所建立的检测方法灵敏度高,易制作、简便,在生物分析中将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-4-(1, 2, 4-三氮唑-1-基)苯酚增强化学发光新体系的研究及其应用
研究了一种新型酚类衍生物4-(1,2,4-三氮唑-1-基)苯酚(TRP)对Luminol-H_2O_2-HRP化学发光体系的增强作用。结果表明TRP可以作为化学发光体系Luminol-H_2O_2-HRP的增强剂。建立了一种增强化学发光新体系Luminol-H_2O_2-HRP-TRP,并对新体系的各项影响因素进行了优化。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8.0×10~(-10) g/mL ~ 5.0×10~(-8) g/mL,检测限为5.0×10~(-10) g/mL。与经典增强剂对碘苯酚(PIP)相比,TRP增强的化学发光强度相对要高,发光时间更长。对Luminol-H_2O_2-HRP-TRP增强化学发光体系的机理进行了讨论。利用Luminol-H_2O_2-HRP-TRP增强化学发光体系并结合磁性微球技术对H_2O_2进行了测定。研究表明,磁性微球作为固相载体固定HRP时,本体系对H_2O_2测定的线性范围为2.0×10~(-6) mol/L ~ 1.0×10~(-3)mol/L,检测限为2.0×10~(-6) mol/L。
二、Luminol-H_2O_2-HRP-p-[1,2,4]-三氮唑基苯酚磷酸单酯预增强化学发光新体系的研究
利用三氯氧磷法制备了p-[1,2,4]-三氮唑基苯酚磷酸单酯(TAPM)。在碱性磷酸酯酶(ALP)催化水解下,TAPM对Luminol-H_2O_2-HRP化学发光体系具有明显的预增强作用。考察了这一预增强发光体系中,影响发光强度的各种因素。建立了Luminol-H_2O_2-HRP-TAPM-ALP发光体系测定ALP的化学发光新方法。该方法测定ALP的检出限可达1.0μg/L(S/N=3),ALP浓度在1.0μg/L ~ 100μg/L范围内与化学发光强度呈良好的线性相应关系。
三、Luminol-H_2O_2-HRP-4-羟基-4′-碘-联苯(IPP)增强化学发光新体系的研究及其应用
研究了一种对位具有活性取代基的酚类化合物4-羟基-4′-碘-联苯(IPP)对Luminol-H_2O_2-HRP化学发光体系的增强作用。建立了新型增强化学发光体系Luminol-H_2O_2-HRP-IPP,并对体系的各影响因素进行了优化,确定了反应的最佳条件。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5.0×10~(-11) g/mL ~ 4.0×10~(-8) g/mL,检测限为9.0×10~(-12) g/mL。研究表明IPP的增强效果优于经典增强剂对碘苯酚(PIP)。对Luminol-H_2O_2-HRP-IPP增强化学发光体系的机理进行了讨论。并利用Luminol-H_2O_2-HRP-IPP增强化学发光酶联免疫分析新体系,采用双抗体夹心法,结合免疫磁性微球分离和化学发光分析技术建立了血清中AFP含量的测定方法。该方法操作简单、快速,对AFP测定的线性范围为0.5 ng/mL ~ 5.0 ng/mL,检测限为0.5 ng/mL,比酶联免疫吸附测定光度法(ELSA)低。用所建立的方法对病人血清样品进行了测定,并与ELISA法进行了对照,二者相关性良好。
四、Luminol-H_2O_2-HRP-溴酚兰增强新化学发光体系的研究及其应用
研究了一种新型化学发光增强剂溴酚兰(BPB)对Luminol-H_2O_2-HRP化学发光反应的增强作用。建立了新型增强化学发光体系Luminol-H_2O_2-HRP-BPB,并对体系的各影响因素进行了优化,确定了反应的最佳条件。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5.0×10~(-11) g/mL ~ 1.0×10~(-9) g/mL,检测限为1.0×10~(-11) g/mL。对Luminol-H_2O_2-HRP-BPB增强化学发光体系的机理进行了讨论。利用Luminol-H_2O_2-HRP-BPB增强化学发光酶联免疫分析新体系,采用双抗体夹心法,结合免疫磁性微球分离和化学发光分析技术建立了血清中AFP含量的测定方法。该方法操作简单、快速,对AFP测定的线性范围为1.0 ng/mL ~ 50.0 ng/mL,检测限为0.2 ng/mL。用所建立的方法对病人血清样品进行了测定,并与ELISA法进行了对照,二者相关性良好。
五、基于金纳米粒子作固相载体的增强化学发光免疫传感器的研制
研究了以金纳米粒子作为酶标抗体的固相载体,利用静电吸附作用将HRP标记AFP抗体固定在其表面,结合磁性微球的高效分离技术,采用双抗体夹心法,建立了Luminol-H_2O_2-HRP-BPB测定AFP、Luminol-H_2O_2-HRP-IPP测定AFP的新型化学发光免疫传感器。二者对AFP测定的线性范围分别为0.1 ng/mL ~ 5.0 ng/mL,0.008 ng/mL ~ 0.3 ng/mL,检测限分别为0.01 ng/mL,5.0 pg/mL。以金纳米粒子作为固相载体,利用共价结合作用将发光底物鲁米诺和CEA抗体同时固定在其表面,结合磁性分离技术,采用双抗体夹心法,建立了一种新型的、简便的化学发光标记技术,研究了一种新型的Luminol-H_2O_2-HRP-IPP增强化学发光体系测定CEA的化学发光免疫传感器。对CEA测定的线性范围为0.5 ng/mL ~ 50.0 ng/mL,检测限为0.5 ng/mL。用所建立的方法对病人血清样品进行了测定,并与ELISA法进行了对照,二者相关性良好。
This paper discussed the novel and sensitive chemiluminescence immunoassay biosensors integrating with the chemiluminescence technique and nanotechnique. The CL reaction conditions and the performance of the biosensors were studied by using chemiluminescence assay, spectral analysis, and electrochemical methods. The novel and sensitive chemiluminescence immunoassay (CLIA) has been developed by employing the new chemiluminescence (CL) enhancers based on colloidal gold nanoparticles (AuNPs) modified with HRP-labeled antibody (or antibody) for further signal amplification. In summary, ease of fabrication, a low cost and high sensitivity make the proposed biosensors in this study may provide an interesting alternative tool for detection protein in clinical laboratory.
The main jobs of this thesis can be concluded as follows:
1. The study on the new enhanced CL system of luminol-H_2O_2-horseradish peroxidase -4-(1,2,4-triazol-1-yl)phenol and its applications
This study described the employment of a novel p-phenol derivative 4-(1,2,4-triazol-1-yl)phenol (TRP) as a highly potent signal enhancer of the luminol-H_2O_2-HRP CL system. The CL reaction conditions were optimized and its enhancement characteristics were compared with that of p-iodophenol (PIP). Under the optimized conditions, the dynamic range of HRP by using TRP as an enhancer was 8.0×10~(-10) g/mL-5.0×10~(-8) g/mL, with the detection limit of 5.0×10~(-10) g/mL, compared with 4-iodophenol (PIP). TRP is a more potent enhancer for the luminol chemiluminescence. TRP produced a strong enhancement of the CL with the effect of prolonging the light emission. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of TRP has been discussed. The developed system was then applied to the determination of H_2O_2 with immobilized HRP using magnetic beads as a solid support. The linear range for H_2O_2 was 2.0×10~(-6) mol/L-1.0×10~(-3) mol/L. The detection limit for H_2O_2 was 2.0×10~(-6) mol/L.
2. The studies on the new proenhanced CL system of luminol-H_2O_2-HRP- p-[1,2,4]-triazole-phenol phosphate monoester
In this work, a novel proenhancer, p-[1,2,4]-triazole-phenol phosphate monoester (TAPM) was synthesized by the PClO3 method. Under the enzymatic hydrolysis of the alkaline phosphatase (ALP), TAPM produced a strong proenhancement on the luminol-H_2O_2-HRP chemiluminescence system. The reaction conditions of luminol-H_2O_2-HRP-TAPM-ALP chemiluminescence system were optimized, and the system was applied to the determination of ALP. The chemiluminescence intensity was linear to the concentration of ALP in the range of 1.0μg/L-100μg/L, with a detection limit of 1.0μg/L (S/N=3).
3. The study on the new enhanced CL system of luminol-H_2O_2-HRP-4-(4′-iodo)- phenyl-phenol and its applications
In this work, a novel enhancer, 4-(4′-iodo)phenylphenol (IPP) was employed in the luminol-H_2O_2-HRP CL system. The optimized CL reaction conditions and the evaluation of its enhancing capabilities in the luminol-H_2O_2-HRP CL system were described. Under the optimized conditions, the dynamic range of HRP by using PIP as an enhancer was 5.0×10-11 g/mL-4.0×10-8 g/mL, with the detection limit of 9.0 pg /mL. The results showed that IPP would be a more potent enhancer than PIP, which was the most widely used enhancer. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of IPP has been discussed. AFP was chosen to prove the novel CL immunoassay as a typical model. The new CL immunoassay involved a binding event between carboxylic acid coated MBs and anti-AFP, and the formation of sandwich immunocomplexes between the MBs and anti-AFP-HRP. The concentration of AFP was monitored based on the HRP label activity toward the oxidation of luminol, which was quantified by CL method. The corresponding calibration plot of relative CL intensity versus the concentration of human AFP was linear over the range from 0.5 ng/mL to 5.0 ng/mL, and the detection limit was 0.5 ng/mL, which was lower than that in the ELISA. The feasibility of the immunoassay system for clinical applications was investigated by analyzing several real samples, in comparison with the ELISA method. The calibration curve indicated that there was no significant difference between the results given by two methods. 4. The study on the new enhanced CL system of luminol-H_2O_2-HRP-bromophenol blue and its applications
In this work, a novel enhancer, bromophenol blue (BPB) was employed in the luminol-H_2O_2-HRP CL system. The optimized CL reaction conditions and the evaluation of its enhancing capabilities in the luminol-H_2O_2-HRP CL system were described. Under the optimized conditions, the dynamic range of HRP by using BPB as an enhancer was 5.0×10~(-11) g/mL-1.0×10~(-9) g/mL, with the detection limit of 1.0×10~(-11) g/mL. The results showed that BPB would be a more potent enhancer than PIP, which was the most widely used enhancer. The precise mechanism of the HRP-catalyzed CL oxidation of luminol in the presence of BPB has been studied. After optimizing the CL reaction conditions, this new luminol-H_2O_2-HRP-BPB CL system was applied to a sandwich-type CLIA based on the magnetic separation. A linear range was obtained when the concentrations of AFP were from 1.0 ng/mL to 50.0 ng/mL, with the detection limit of 0.2 ng/mL. The present method was successfully applied to the determination of AFP in human serum samples. The results indicated that this proposed protocol could be quite promising for the application in immunoassay.
5. The studies on the enhanced chemiluminescence immunoassay biosensor by using gold nanoparticles as solid supports
In this work, we described a sensitive and promising analytical mehtod employing new CL enhancers, BPB or IPP, based on MBs separation and gold nanoparticles (AuNPs) labeling technique. HRP-labeled AFP antibody was first bound on the surface of AuNPs by electrostatic, then the new luminol-H_2O_2-HRP-BPB (or IPP) CL systems were applied to a sandwich-type CLIA for the determination of AFP based on the magnetic separation. This novel strategy takes advantage of easy magnetic separation by MBs and amplification feature of colloidal gold label along with CL intensity enhanced by BPB (or IPP) for luminol-H_2O_2-HRP system. A linear ranges of AFP standard solution were 0.1 ng/mL-5.0 ng/mL and 0.008 ng/mL-0.3 ng/mL,respectively. And the detection limits of AFP were 0.01 ng/mL and 5.0 pg/mL, respectivrly. An enhanced chemiluminescence immunoassay method for the sensitive and rapid determination of CEA was proposed in this paper. Firstly, luminol and anti-CEA-antibody were simultaneously bound on the surface of AuNPs by covalence. Secondly, the new luminol-H_2O_2-HRP-IPP CL systems were applied to a sandwich-type CLIA for the determination of CEA based on the magnetic separation. A linear range of CEA standard solution was 0.5 ng/mL-50.0 ng/mL, with detection limit of 0.5 ng/mL.
The proposed methods were also used to determine AFP (or CEA) in human serum samples, and the results were well corresponding to those of conventional ELISA.
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