功能化纳米材料电致化学发光新体系的建立及其生物分析应用

发布时间：2017-12-27 23:14

本文关键词：功能化纳米材料电致化学发光新体系的建立及其生物分析应用　出处：《西南大学》2017年博士论文　论文类型：学位论文

【摘要】：电致化学发光(electrogenerated chemiluminescence或electrochemiluminescence,ECL)分析技术是电化学和化学发光技术相结合的产物,兼具高可控性和高灵敏度等优点,在临床检验诊断、食品安全检测和环境污染监测等领域具有广阔的应用前景。在ECL研究过程中,我们发现当仅存发光物质时,其ECL强度相对较低;当加入共反应试剂时,可以显著地提高发光物质的ECL强度,因此,发展高效的ECL共反应体系是获得高的ECL强度进而提高检测灵敏度的主要途径之一。纳米材料的小尺寸效应和表面效应使其具有比表面积大和表面活性位点多等优点,能有效提高界面电化学的反应活性,从而提高ECL强度;另一方面,纳米材料的量子限域效应使其本身可以作为发光物质(例如,量子点、金属纳米簇等),在ECL领域也展现出优异的发光效率。因此,纳米材料在ECL分析方法中得到了广泛的应用。基于此,本文主要从利用纳米材料提高共反应试剂的作用效率以及开发成本低廉、生物相容性好和ECL性能优异的纳米材料类发光物质这两方面出发,发展高效的新型ECL体系,并构建ECL生物传感器应用于蛋白质和生物小分子的灵敏检测。具体研究工作分为以下几部分:1.基于纳米金功能化的磁性石墨烯构建高灵敏电致化学发光免疫传感器的研究共反应试剂直接置于检测底液中存在试剂用量大、操作复杂和增加测量误差等问题,因此,寻求一种有效的方法将共反应试剂固载在电极界面以解决上述问题。碳纳米材料因其大的比表面积、优异的导电性和良好的生物相容性等特点,被广泛地用作纳米载体应用于生物传感器领域。为进一步增加碳纳米材料表面的活性位点,通常需要对碳纳米材料进行表面功能化处理。首先,通过一锅法合成了纳米金功能化的磁性石墨烯(Au@Fe_3O_4-rGO)复合材料,该复合纳米材料具有更大的比表面积、更多的活性位点和易分离等优点,可以显著地提高生物分子的固载量。再利用Au-N键作用将联吡啶钌(Ru(bpy)32+)的共反应试剂精氨酸二肽(bi-Arg)修饰在Au@Fe_3O_4-rGO复合材料表面,然后继续组装脱嘌呤/脱嘧啶核酸内切酶1(APE-1)二抗,制得增强型二抗耦合物探针。基于夹心免疫反应,构建了高灵敏的免疫传感器用于APE-1的定量分析。该传感器对1.0 fg m L-1~5.0 pg m L-1浓度范围内的APE-1呈现出良好的线性响应,检测限低至0.3 fg m L-1。2.基于纳米金功能化的C60纳米复合物构建免标记电致化学发光适体传感器的研究在ECL研究中,纳米材料通常作为纳米载体用于固载共反应试剂,但该方式固载共反应试剂的量十分有限。因此,为拓展共反应试剂的类型,本文研究了纳米材料本身作为共反应试剂的可能性。采用溶剂交换法合成了C60纳米颗粒(nano-C60),然后通过疏水作用将牛血清白蛋白(BSA)修饰到nano-C60表面,使其表面富含氨基和巯基。然后再将纳米金原位还原到nano-C60表面获得纳米金功能化的C60纳米复合物(Au@nano-C60),该复合纳米材料具有比表面大、表面活性位点多和易于成膜等优点,是一种理想的电极修饰材料。值得注意的是,Au@nano-C60复合材料能够有效地增强S_2O_8~(2-)-O_2体系的ECL信号,并结合聚组氨酸共反应试剂和金纳米颗粒的促进作用,构建“on-off-on”信号转换模式的免标记ECL适体传感器用于卡那霉素的灵敏检测。该制备的适体传感器对浓度在0.15 nmol L-1~170 mmol L-1范围内的卡那霉素呈现良好的线性关系,检测限低至45 pmol L-1。该工作扩宽了纳米材料在ECL生物传感领域的应用空间。3.基于hemin/G-四分体脱氧核酶对自增强钌(Ⅱ)复合物的三重猝灭作用构建信号减小型电致化学发光适体传感器的研究传统的ECL反应中,共反应试剂对发光物质的ECL增强作用均是通过分子间的电子传递实现的。然而,这种分子间的电子传递存在较大的能量损失,因此限制了共反应试剂增强ECL的效率。鉴于此,本文将发光物质联吡啶钌衍生物和共反应试剂聚乙烯亚胺(PEI)通过共价交联在有机半导体纳米材料傒四甲酸(PTCA)表面上,制得自增强钌(Ⅱ)复合物。该自增强钌(Ⅱ)复合物中共反应试剂与发光物质间的相互作用通过分子内的电子传递实现的,缩短了电子传递的距离,从而极大地增强发光效率。将该自增强钌(Ⅱ)复合物滴涂在电极界面成膜,实现了有效地固载共反应试剂和发光物质。利用hemin/G-四分体脱氧核酶(hemin/G-quadruplex DNAzymes)对自增强钌(Ⅱ)复合物的三重ECL猝灭作用,构建信号减小型ECL适体传感器用于凝血酶的检测。该适体传感器实现了凝血酶在1.0×10-14 mol L-1~1.0×10-10 mol L-1范围的高灵敏检测,其检测限达到了飞摩尔水平。4.新型铜纳米簇的电致化学发光及其生物分析应用的研究迄今,由于镉基半导体纳米晶体发射光谱可调控和量子产率高的优点,使其成为最有前途的纳米材料类发光物质,然而,镉离子的存在会带来不可避免的生物毒性,进而引起环境污染和危害健康等问题使其在生物分析中的应用受到限制。因此,寻求无毒、经济、发光效率高的ECL纳米材料类发光物质是ECL技术应用于生物分析的基础。铜纳米簇(CuNCs)作为一类新型荧光和催化的纳米材料,在传感器、生物标记和催化等领域展现出广泛的应用前景。截止目前,CuNCs的ECL现象却还尚未被报道。本文率先研究了CuNCs的阳极ECL信号,并以肼(HZ)作为共反应试剂获得了令人满意的ECL响应。基于该新型的CuNCs/HZ ECL体系,本文构建了信号减小型高灵敏传感平台用于测定多巴胺。实验结果表明CuNCs作为一种新型低成本和优异的发光纳米材料,在ECL生物分析中展现出巨大的应用潜力。5.基于二硫化钼量子点电致化学发光体系和目标物循环同步滚环扩增的放大策略构建高灵敏适体传感器的研究二硫化钼量子点(MoS_2 QDs)是一类具有类似石墨烯独特二维结构的过渡金属二硫化物纳米材料,它不仅具有发射光谱可调控、量子产率高和易功能化等优点,而且由于无重金属离子还展现出良好的生物相容性,现已被广泛地用作催化剂、荧光探针等,迄今,对其ECL的研究尚未报道。本文通过化学剥离法制备了无毒、经济的MoS_2 QDs,并率先研究其ECL性能。在三乙胺(TEA)共反应试剂的存在下,可以观察到强的MoS_2 QDs ECL信号。基于MoS_2 QDs/TEA ECL体系结合适体识别驱动的目标物循环同步滚环扩增(RCA)的放大策略,构建了高灵敏的适体传感器,实现了对内毒素的痕量分析。该传感器对浓度在0.1 fg m L-1~50ng m L-1范围内的内毒素呈现出良好的线性响应,其检测限低至0.07 fg m L-1。实验结果表明,具有稳定发光的新型MoS_2 QDs将成为一种有竞争力的新兴ECL纳米材料类发光物质。
[Abstract]:Electrochemiluminescence (electrogenerated chemiluminescence or electrochemiluminescence ECL) analysis technology is a product of electrochemistry and chemiluminescence technique which combines the advantages of both high controllability and high sensitivity, has broad application prospects in the field of clinical diagnosis, food safety detection and monitoring of environmental pollution etc.. In the process of ECL research, we found that when the only luminescent material, the intensity of ECL is relatively low; when adding co reaction reagent, can significantly improve the strength of ECL, a light emitting substance therefore, development of efficient ECL reaction system is to obtain ECL high strength and one of the main ways to improve the detection sensitivity. Nano size effect and surface effect so that it has a large specific surface area and surface active sites and other advantages, can effectively improve the electrochemical reaction activity of interface, so as to improve the strength of ECL; on the other hand, the quantum confinement effect of nano materials which itself can be used as light emitting material (e.g., quantum dots, metal nanoclusters etc.) in the field of ECL, showing excellent luminous efficiency. Therefore, nanomaterials have been widely used in the ECL analysis method. Based on this, this article mainly from the use of nano materials to improve the reaction efficiency and total development cost of reagent cost, good biocompatibility and nano materials luminescence material ECL excellent performance of the two aspects, the new ECL system development and construction of efficient, sensitive detection of ECL Biosensor Applied to protein and small biological molecules. The specific research work is divided into the following parts: 1. magnetic graphene gold nanoparticles functionalized based on high sensitive chemiluminescence immunosensor Research Co reaction reagent consumption, complex operation and increase the measurement error problem, directly on the bottom liquid detection reagent therefore, explore an effective method to co reaction reagent immobilized on the electrode interface in order to solve the above problems. Carbon nanomaterials are widely used as nano carriers for biosensors because of their large specific surface area, excellent electrical conductivity and good biocompatibility. In order to further increase the active sites on the surface of carbon nanomaterials, the surface functionalization of carbon nanomaterials is usually needed. First of all, nano gold functionalized magnetic graphene (Au@Fe_3O_4-rGO) composite materials were synthesized by one pot. The composite nanomaterials have larger specific surface area, more active sites and easier separation, which can significantly improve the immobilization of biomolecules. Then, the Au-N bond was used to modify the surface of the Au@Fe_3O_4-rGO composite surface with the bipyridine ruthenium (Ru (bpy) 32+) Co reaction reagent, arginine two peptide (bi-Arg), and then continued to assemble the anti purine / deacinase endonuclease 1 (APE-1) two resistance, and the enhanced two anti coupling probe was prepared. Based on the sandwich immunoreaction, a highly sensitive immunosensor was constructed for the quantitative analysis of APE-1. The sensor has a good linear response to the APE-1 in the concentration range of 1 FG m L-1~5.0 PG m L-1, and the detection limit is low to 0.3 FG m L-1. 2., based on nano gold functionalized C60 nanocomposites, a label free electrochemiluminescence aptamer sensor was developed. In ECL research, nanomaterials were usually used as nano carriers for immobilized co reagents, but the amount of immobilized co reagents was very limited. Therefore, in order to expand the type of CO reagents, the possibility of nanomaterials as co reagents is studied in this paper. C60 nanoparticles (nano-C60) were synthesized by solvent exchange. Then, bovine serum albumin (BSA) was modified onto nano-C60 surface by hydrophobic interaction, so that its surface was rich in amino and sulfhydryl groups. Then, nano gold was reduced to nano-C60 surface in situ, and gold nanoparticles functionalized C60 nanocomposite (Au@nano-C60) was obtained. The composite nanomaterial has many advantages, such as large surface area, multiple surface active sites and easy to film, etc. it is an ideal electrode modifier. It is worth noting that the Au@nano-C60 composite materials can effectively enhance the S_2O_8~ (2-) ECL signal -O_2 system, combined with the histidine co reaction reagent and gold nanoparticles to promote the role of constructing "on-off-on" mode ECL signal conversion label free aptamer sensor for kanamycin sensitive detection. The prepared body sensor has a good linear relationship with kanamycin in the range of 0.15 nmol L-1~170 mmol L-1, and the detection limit is as low as 45 pmol L-1. This work broadens the application space of nanomaterials in the field of ECL biosensing. 3. hemin/G- four split DNAzyme on self enhancement based on ruthenium (II) signal for reducing the ECL reaction of ECL sensor in the traditional body building three kinds of quenching compound and co reaction reagent on luminescence material ECL enhancement were all done by intermolecular electronic transfer. However, there is a large energy loss in this intermolecular electron transfer, thus limiting the efficiency of the co reagents to enhance the ECL. In view of this, this paper will glow bipyridine ruthenium derivatives and co reaction reagent of polyethyleneimine (PEI) by covalent cross-linking in organic semiconductor nano materials Xi four formic acid (PTCA) on the surface, prepared from ruthenium (II) complexes enhanced. The interaction between the self reacting ruthenium (II) complex and the luminescent materials is achieved by intramolecular electron transfer, which shortens the distance of electron transfer, thus greatly enhancing the luminous efficiency. The self reinforced ruthenium (II) compound droplets were coated on the electrode interface, and the co reacting reagents and luminescent substances were effectively immobilized. Using hemin/G- four partial deoxy ribozyme (hemin/G-quadruplex DNAzymes) to enhance the three ECL quenching effect of self reinforced ruthenium (II) complex, a signal reduced ECL aptamer sensor was set up for thrombin detection. The aptamer sensor is sensitive to the high sensitivity of thrombin in the range of 1 * 10-14 mol L-1~1.0 x 10-10 mol L-1
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：O657.3;TB383.1

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