【作者】 储海虹；

【导师】 屠一锋；

【作者基本信息】 苏州大学 ， 分析化学， 2011， 博士

【摘要】 电化学发光或电致化学发光分析方法（Electrochemiluminescence,ECL）是指直接利用电化学反应形成激发态发光体而发光或通过电解产物之间、电解产物与体系中某组分之间进行化学反应产生光辐射而实现分析物测定的发光分析技术,是电化学与化学发光分析相结合的产物。与传统的化学发光分析法相比,ECL分析法不仅具有化学发光分析法的灵敏度高和线性范围宽等优点,而且在许多方面优于化学发光分析,包括如:第一,不稳定的化学试剂和中间体在电极表面定量生成,且迅速进行化学发光反应;第二,电化学反应可以通过改变所施加的电位加以控制,所以可以有选择地控制化学反应而不需要采取额外的分离手段;第三,电化学氧化能力是连续的,在同一化学条件下可以通过控制电位加以改变电化学氧化能力。本论文在全面总结和论述ECL分析的基本原理、常见ECL体系反应机理以及生物传感器的基本原理、分类、应用等方面的研究等基础上,进行了以下三方面的研究工作:一、鲁米诺电化学发光体系是基于电化学氧化鲁米诺生成自由基,所生成的自由基不稳定,再进一步被一些氧化剂氧化产生化学发光。虽然ECL分析法具有灵敏度高、线性范围宽和仪器设备简单等优点,但是在中性、弱碱性介质这样有利于生物分子保持活性的环境中,鲁米诺的ECL发光极弱,传统的鲁米诺ECL应用多在强碱性介质中进行。为保持生物分子的活性,考虑利用合理的措施来有效实现鲁米诺在中性、弱碱性介质环境中的ECL增敏,对其发展和应用都有很高的学术和实用价值。在已经建立的ECL分析体系的基础上,探讨了纳米材料如金溶胶（Au sol）和铂溶胶（Pt sol）、有机分子如氯霉素（Chloramphenicol,CAP）和半胱氨酸（Cysteine）、以及介质体系如微乳液（Microemulsion）和离子液体（Ion Liquid）在中性、弱碱性介质中对鲁米诺ECL的增敏作用,研究中发现ECL信号与增敏剂之间均存在可以被实际应用的确定的定量关系,并探讨了各体系的增敏机理。在此基础上,采用吸附、自组装等手段制备了相关的修饰电极,而且可以有效实现ECL物质鲁米诺的固定化,在成功提高检测灵敏度的前提下,实现微量、痕量生物分子（如维生素C、褪黑素、超氧化物歧化酶等）的检测。二、在中性或弱碱性介质中,对溶解氧、过氧化氢（H₂O₂）、辣根过氧化氢酶（HRP）/ H₂O₂、黄嘌呤氧化酶（XOD）/次黄嘌呤（Xanthine）、尿酸酶（Uricase）/尿酸（UA）以及谷丙转氨酶（ALT）等对鲁米诺电化学发光的增敏（或猝灭）行为进行了研究,进而对机理进行了探讨。研究结果表明,O₂、H₂O₂及其氧化还原过程中生成的具有更强氧化性的活性氧（Reactive oxygen species,ROSs）对鲁米诺的电化学发光具有显著的增敏效果,促进鲁米诺中间态自由基激发,导致ECL信号增强,在生物反应适合的酸度范围内,对鲁米诺ECL的增敏作用尤其显著,为研究生物ECL传感器奠定了良好的基础。由于辣根过氧化氢酶（HRP）催化H₂O₂的分解,故猝灭H₂O₂增敏的鲁米诺ECL。其他酶催化反应均可在电极表面产生活性氧物质,从而增敏鲁米诺的ECL,并可建立与相关底物浓度间的定量关系。研究中,结合循环伏安（Cyclic Voltammetry, CV）法,紫外-可见吸收光谱（Ultraviolet-Visible Absorption Spectrometry, UV-Vis）法等方法探讨了有关机理。在此基础上,采用能较好保持生物分子活性的固定化方法和材料,将生物活性物质修饰于电极表面,制备了响应性能优良的ECL生物传感器,稳定性好,灵敏度高。将制备的ECL生物传感器应用到一些生物分子（如超氧化物歧化酶、ALT、尿酸等）的检测中,均获得满意的结果。这些ECL生物传感器在保持生物分子活性的前提下,兼具了ECL的高灵敏度以及酶的高选择性,对于相关物质的检测的灵敏度高、检测限低、具有很强的实用性。三、DNA是生物体的基本遗传物质,是遗传信息的载体,它在生物的生长、发育和繁殖等生命活动中起着非常重要的作用。建立简单、敏感、特异和快速的病源、基因和药物检测方法,对疾病的预防、诊断和治疗具有重要的意义。论文研究了DNA在弱碱性介质中对鲁米诺ECL的猝灭作用,并用循环伏安法、紫外-可见吸收光谱法和荧光光谱法等讨论了其猝灭机理。采用金纳米粒子（Au NPs）和碳纳米管（CNTs）的复合纳米材料作为DNA的固定载体,应用鲁米诺的ECL作为对DNA响应的信号输出,制备了一种检测限低,灵敏度高、重现性好的DNA生物传感器。对应用ECL技术于不同互补状态的寡聚核苷酸杂交的表征进行了研究,结果表明ECL技术可以作为一种可靠的免标记表征杂交状态的手段。而药物与DNA相互作用的研究是认识某些疾病的致病机制和药物的治疗机制的基础,在阐明DNA结构和功能方面也具有重要意义。研究中探讨了利用ECL信号研究药物小分子如氯霉素（CAP）对DNA的损伤行为,并对其作用机理进行了探讨,为药物小分子损伤DNA的研究提供了一种可靠灵敏的检测模型。该研究对开发具有新颖特性的电化学发光探针和传感界面构造的新原理和新方法、以及设计和研制适用于检测蛋白质、基因或小分子药物的高灵敏度、高选择性、可重复使用的电化学发光DNA传感器具有很大的指导意义。更多还原

【Abstract】 Electrochemiluminescence (ECL) has received increasing attention in recent years, especially in the determination of organic or biochemical compounds and immune diagnostic field. As an analytical technique, ECL possesses some favorable advantages. First, it is repeatable due to the light-emitting process which is realized in ECL is in accurate controlling for synchronization and dimensional orientation with the electrochemical reaction on electrode surface. Second, the ECL detection is separated from the electrochemical excitation, it is beneficial for enhancement of signal/noise ratio therefore the sensitivity. Third, the ECL can be controlled by manipulating the applied potential and/or other electric parameters to realize selective detection. Forth, in ECL procedure, more than one of the reactive species is electrochemically produced in situ, therefore to avoid the problems of the use of some violent and instable chemicals. In addition, the ECL instrument is often simpler than many electrochemical or fluorescence instruments. Luminol, noticed as a typical strong chemiluminescence (CL) reagent, has also been widely researched for ECL application. However, to the general knowledge, the researches of CL and ECL of luminol have been limited in strong alkaline solution algate, and its ECL behavior in neutral medium has almost been ignored. With regard to the bioactive molecules, the neutral medium would be more suitable for keeping their original properties. For example, it is affirmed that neutral medium is of the most favorable condition for noctilucent systems because the organisms such as amino acid, protein in disadvantageous conditions would alter their qualities to induce wrong exhibitions in reactions. During the discussions between the colleagues around the world, most researchers declared that the ECL of luminol in neutral medium was too weak to be practically applied. That is to say, as a preferential opinion that the luminol-based ECL detection for biomolecules was suffered from the usual requirement of high pH condition, it is difficult to use it directly for the detection of important biological compounds at physiological pH.Due to the exceeding ECL sensitivity, expedience and inexpensiveness, the authors paid strong interest on luminol to attempt its feasibility for determination of biomolecules contained in bio-fluids with trace amount. The ECL detection of luminol in neutral medium is realized if there was proper intensifier presented. In the ECL system of luminol, the ECL intensity of luminol was enhanced obviously in the presence of nanosized Au and Pt particles which were synthesized by reduction methods and characterized by transmission electron microscope (TEM) method, and the sensitization was attributed to nanoparticle’s character. Organic molecules including chloramphenicol (CAP) and Cysteine also could enhance the ECL of luminol. Microemulsion and ionic liquid which have become hot topics in many fields for having several unique physical and chemical properties are enhancers for ECL of luminol, too. Experimental results showed that significant increases in ECL intensity of the luminol were observed when adding above-mentioned materials. The sensitization efficiency was always related to the content of the enhancers, which could be used to improve the sensitivity of determined method based on the ECL of luminol. The paper lucubrated the mechanisms of all these enhancers enhanced ECL of luminol in neutral and the weak alkaline media by other techniques. Based on the sensitization, some biomolecules were determined. For example, Vitamin C and melatonin could quench the ECL signals. The ECL luminous intensity linearly decreases with the concentration of quenchers, and the samples have been determined with satisfactory results. It should be a sensitive and reliable method for the determination of such biomolecules.As reported in this paper, the sensitization greatly improved the ECL performance of luminol in neutral and the weak alkaline media, part of which were related with the reactive oxygen species (ROSs) which could enhance the ECL intensity of luminol. The results indicated that whether the enhancers were dissolved in solutions or immobilized on the surface of conventional electrodes, stronger ECL intensity of luminol could be obtained. Enhanced ECL by ROSs helped to provide ground work for the detection of biomolecules for which would further enhance or quench the ECL signals. The powerful sensitization from ROSs related with organics and organisms were examined under selected conditions which were suitable for biochemical analysis. And the applications for biodetecting target related to superoxide dismutase (SOD), horseradish peroxidase (HRP), xanthine oxidase (XOD), uric acid (UA) and alanine aminotransferase (ALT) became true based on the progress. Different enzyme biosensors were fabricated according to enzymatic reactions. The ECL-based biosensor showed excellent character for some certain measurement. It can detect the lower concentration of samples due to the highly sensitivity of ECL and highly selectivity of enzyme. So, the studies on sensitization ECL of luminol not only extended the region of ECL research into the physiological category, but also established a basis for ECL research of biomaterials. The technique may provide new means in a variety of fields such as clinical diagnostics, immunological analysis and environmental monitoring due to its simplicity and high efficiency.Rapid detection, quantification and sequencing of deoxyribonucleic acid (DNA) are important tasks in the fields of biology, drug discovery, medical diagnostics, agriculture, as well as environmental science. In the thesis, we have reported the quenching effect of DNA on ECL of luminol and the further development of a DNA sensing device. The typical design of a DNA sensor involves the appropriate immobilization of the DNA molecules or introduction of an effective indicator or so called labeled probe. In this field, the nano-material modified electrodes are inherently ideal because of the enormous specific surface, which is highly susceptible to heterogeneous redox with surrounding environments. With the pre-functionalization by composite of carbon nano-tubes (CNTs) and Au nanoparticles (Au NPs), the sensor provides a novel and valuable label-free approach for DNA sensing. The results indicated that the ECL response was correlative with the presence of dsDNA. Based on the researches, the possible quenching mechanism of dsDNA for ECL of luminol has also been discussed. It might be resulted from the interaction of luminol with dsDNA, and the elimination of ROSs by dsDNA.Based on the ability of CAP to enhance the ECL signals, the above-mentioned DNA-biosensor was used for the study of interaction of DNA and CAP in a neutral aqueous solution in the thesis. The interaction of DNA and CAP had been examined in detail using ECL method, Ultraviolet-Visible Absorption Spectrum, fluorescence spectroscopy, cyclic voltammogram technique and the mechanism was proposed based on the experiments data. The method which was presented here could be act as a model leading to applications in DNA damage caused by other chemicals in clinical practice, medicine and basic research.The thesis reports an ECL sensing technique for label-free characterization of oligonucleotide complementation. Followed the pre-function for glassy carbon electrode by composite of Au NPs and CNTs, the sensor was constructed by immobilizing the probing oligonucleotide with ECL of luminol as sensing signal. Its availability for characterization of oligonucleotide hybridization and further potentiality for DNA detection were reported herein. The variational regression slope is distinguishable for complementation status of oligonucleotide hybridization. It was found that high degree of complementation caused efficient quenching for ECL of luminol. Furthermore, it was revealed that the response could be attributed to the interaction between luminol and oligo-strands, and also related to the reducing of ROSs.更多还原