电致化学发光生化新体系及其分析策略研究

发布时间：2018-06-11 22:27

本文选题：电致化学发光 + 自增强　；参考：《西南大学》2017年博士论文

【摘要】：电致化学发光(Electrochemiluminescence,ECL)以电化学为基础,通过电极表面电化学反应导致的化学发光现象进行特定物质的定量分析检测,其同时结合了电化学的高可控性和化学发光法的超灵敏性,极大地提高了分析检测方法的性能,并赋予了其独特的优势,诸如灵敏度高、线性范围宽、操作简单、可控性强、分析快速简便等。近年来,伴随着纳米技术及生物技术的快速发展,电致化学发光技术更是取得了蓬勃的发展进步,作为一种高效、灵敏的分析检测技术,在生命分析、环境监测、食品安全等领域备受关注。目前,电致化学发光材料正朝着高效、绿色、多功能的方向发展;而电致化学发光分析技术亦正朝着高灵敏、高通量、普适等方向发展。然而,针对一些特殊研究体系,现有电致化学发光体系尚存在灵敏度不足、检测过程复杂繁琐、适用性不强等方面的缺陷。基于此,本论文主要从电致化学发光检测体系的灵敏度、高通量、适用性等方面出发,通过高效自增强电致化学发光技术、原位酶促反应增强、核酸扩增放大及高效电致化学发光材料等方法提高电致化学发光技术的分析检测灵敏度;通过多元分析、竞争反应等策略提高电致化学发光技术的适用性。在此基础上,实现了多种疾病标志物及细胞功能检测研究。本论文的研究工作主要分为以下几个部分:1.基于自增强复合纳米材料的超灵敏细胞传感器构建及其在药物筛选中的应用研究抗癌药物研究具有极其重要的科学意义及社会价值,尤其是高效、低廉、特异、甚至个体化的抗癌药物。基于细胞凋亡分析的药物筛选评价系统亟需更为灵敏、准确、有效、便捷的分析检测技术。灵敏度高、反应可控性好、便捷低廉的电致化学发光技术具有极大的潜在应用价值,然而电致化学发光材料及共反应剂等的生物毒性、分析检测方法灵敏度等方面尚需进一步改善。本研究提出了一种自增强电致化学发光纳米材料的制备方案。通过羧基化联吡啶钌与氨基侧链修饰的甲氧基硅烷偶联,制得功能化的二氧化硅纳米材料前体,其中联吡啶钌作为电致化学发光中心,氨基侧链作为共反应试剂。在此基础上,通过碱性条件下水解作用,成功制得自增强电致化学发光纳米材料,并成功将其应用于基于细胞凋亡分析的抗肿瘤药效评价系统的构建,实现了高效、快速、便捷的抗肿瘤药效评价。实验研究同时采用乳腺癌肿瘤细胞(MDA-MB-231)和抗肿瘤药物(紫杉醇)作为模型研究了所构建的抗肿瘤药效评价系统的适用性,取得了较好的分析效果。该工作提出了自增强电致化学发光纳米材料的设计构建方式,并对其自增强机理进行了相应的研究;同时,实现了电致化学发光分析技术在亟需的药物筛选评价系统中的应用,进一步拓展了电致化学发光分析技术的研究应用,为进一步的电致化学发光技术及药物筛选评价等提供参考借鉴。2.基于多元分析的多组份电致化学发光分析策略研究电致化学发光分析技术由于其低背景、高灵敏度、宽线性范围、低耗用等突出优点而倍受关注。然而,截至到目前为止,绝大多数的电致化学发光分析仅仅能在同一界面上实现单一目标物的定量分析。而临床检测、环境分析、食品安全等方面迫切需求一种更为高效、灵敏的多组份电致化学发光分析检测技术。本研究设计了一种基于多元分析的多组份电致化学发光分析策略,结合以杂交连锁反应-滚环扩增反应为基础的级联放大策略,构建一种高灵敏的多组份电致化学发光分析系统。采用心肌损伤类标志物,N末端B型利钠肽原和肌钙蛋白I为检测模型,分别以鲁米诺和联吡啶钌为信号探针,研究讨论了该方法的分析检测性能。其中,通过多元分析有效地解决了不同电致化学发光信号探针间相互影响的关键问题;采用HCR-RCA为基础的级联放大策略有效地提高了检测灵敏度,实现了同一界面多组份的高灵敏分析检测,为进一步构建高通量的电致化学发光检测系统奠定了良好的基础。3.基于竞争法的电致化学发光免疫传感技术用于单一界面多目标物比率分析的研究现今,采用抗肿瘤药物来消灭肿瘤细胞仍作为抗击肿瘤的一种重要手段而备受关注。然而,由于长时间使用药物等易导致肿瘤细胞耐药而出现疗效差,甚至无疗效的情况。若能在肿瘤细胞耐药早期,通过调整治疗方案,则可以有效地避免或减少细胞耐药,然而尚缺乏高效、高灵敏、准确的早期细胞耐药侦检系统。电致化学发光分析作为一种高效、灵敏的潜在可用分析技术而备受关注。通常细胞耐药性可以通过P糖蛋白表达情况来确定,而对于P糖蛋白表达分析而言,不仅仅需要对P糖蛋白的高灵敏定量分析,同时需要辅助以细胞计数或者辅助以另外一种恒定表达蛋白的高灵敏定量分析作为对照,通过计算P糖蛋白与恒定表达蛋白的浓度比值来实现P糖蛋白的表达分析。虽然采用如前所述的基于多元分析的多组份电致化学发光分析策略可以实现了同一界面上多种目标物的同时检测。但是,多元分析仍比较耗时,亟需更为便捷有效的检测方式实现细胞耐药分析。值得注意的是,通过蛋白质表达来评估细胞耐药程度,仅仅需要耐药相关蛋白与恒定表达蛋白的浓度比值即可,而不需要两者的准确浓度。本研究通过基于免疫识别的目标物转换结合滚环扩增信号放大策略和电致化学发光传感器表面修饰核酸的竞争反应构建了一种用于比率分析的高灵敏电致化学发光分析策略,以P糖蛋白和甘油醛-3-磷酸脱氢酶为检测模型评估肿瘤细胞耐药程度。首先,磁珠表面的免疫识别反应,以夹心法检测模式,通过固定于磁珠表面生物素化抗体分别特异性识别P糖蛋白和甘油醛-3-磷酸脱氢酶,并进一步结合核酸标记二抗,实现目标物转化;为了提高分析检测灵敏度,采用滚环扩增放大策略,以标记于抗体上的核酸为引发链,延伸得到以滚环为模板的重复序列,并经DSN酶剪切实现1㑳N目标物扩增放大;剪切获得的重复序列部分一致,能够与电极表面的捕获序列形成竞争反应,并进一步结合电致化学发光材料,联吡啶钌标记信号探针,通过电致化学发光信号指示竞争反应结果,进而实现P糖蛋白和甘油醛-3-磷酸脱氢酶的比率检测,实现对细胞耐药程度的评估。该比率分析策略亦可推广于电化学、荧光、化学发光法等对于多种目标物,诸如蛋白、核酸等的高效、便捷、高灵敏的比率分析测定,同时提供了一种有效策略用于生化分析、临床诊断,尤其是细胞功能测定等。4.基于时间调控的检测范围可调式电致化学发光技术研究近年来,高灵敏电致化学发光分析技术更是得到了长足的发展进步。然而,截至目前为止,绝大多数的电致化学发光分析技术由于修饰界面单一,仅仅能够实现固定灵敏度及固定检测范围内的目标物分析。由于同一检测目标物在不同样本中的浓度差异悬殊,通常电致化学发光分析技术难于直接应用于样本分析检测,而繁琐的分离富集/稀释等操作成为定量分析的隐性必需内容之一,这无疑增加了分析检测工作量,并在一定程度上降低了分析检测的准确性。以Pb2+离子的分析检测作为模型,本研究通过在电极表面的核酸特异性杂交,层层组装核酸网状结构,并于网状结构内部设计DNA酶,且于网状结构的双链内镶嵌电致化学发光材料构建针对Pb2+离子的可调式电致化学发光传感器。由Pb2+离子介导的DNA酶循环剪切所组装的核酸网状结构,同时释放电致化学发光材料,导致电致化学发光信号降低,以此实现对Pb2+离子的定量分析。而剪切效率不仅与Pb2+离子浓度相关,同时与反应时间相关,由此通过控制反应时间以调节检测线性范围、检测灵敏度等。实验研究所构建的可调式电致化学发光策略亦可推广适用于其他诸如蛋白、核酸等的灵敏可调式检测,为环境安全及临床检验提供一种新型的分析策略。5.基于铱配合物纳米材料及原位酶放大的电致化学发光传感器研究三苯基吡啶铱配合物作为一种高量子转化效率、高电子转移效率的优良电致化学发光材料,有望以此为基础构建更高检测灵敏度的电致化学发光技术。然而其水溶性差、难于标记等缺陷限制了其在电致化学发光传感技术领域的广泛应用。本研究采用三苯基吡啶铱配合物掺杂二氧化硅纳米材料实现了电致化学发光材料的高效固载,并极大程度地改善了其水溶性。实验研究以心衰标志物,N末端B型利钠肽原作为研究模型,探究了基于铱配合物纳米材料及原位酶放大的电致化学发光传感器的性能。电致化学发光传感器采用Nafion分散三苯基吡啶铱配合物掺杂二氧化硅纳米材料包覆于电极表面,并进一步偶联固定抗N末端B型利钠肽原抗体来构建。基于此,所制备的电致化学发光传感器具有较强的电致化学发光信号。当检测样本中含有N末端B型利钠肽原时,其能够通过特异性免疫识别结合于电极表面,并进一步结合葡萄糖氧化酶标记的二抗。通过葡萄糖氧化酶催化葡萄糖产生过氧化氢对电致化学发光信号的猝灭作用实现目标物N末端B型利钠肽原的定量分析检测。实验研究所采用的电致化学发光分析策略亦可适用于以其他目标物,诸如核酸、细胞等物质的高灵敏分析检测应用。
[Abstract]:Electrochemiluminescence (Electrochemiluminescence, ECL) is based on electrochemistry and quantificationally analyses specific substances by electrochemiluminescence caused by electrochemical reaction on the surface of the electrode. It combines the high controllability of electrochemistry and the hyper sensitivity of chemiluminescence, which greatly improves the performance of the analytical method. It has given its unique advantages, such as high sensitivity, wide linear range, simple operation, strong controllability, and rapid and simple analysis. In recent years, with the rapid development of nanotechnology and biotechnology, electrochemiluminescence technology has made vigorous progress, as a highly efficient, sensitive analysis and detection technology, in life analysis, ring At present, electrochemiluminescence materials are developing in the direction of high efficiency, green and multifunction, and electrochemiluminescence analysis technology is developing towards high sensitivity, high throughput and universality. However, the electrochemiluminescence system is still sensitive to some special research systems. This paper is based on the sensitivity, high throughput and applicability of the electrochemiluminescence detection system, through high efficiency autofilt electrochemiluminescence technology, in situ enzymatic reaction enhancement, nucleic acid amplification amplification and high efficiency electrochemiluminescence materials and other aspects. The method improves the sensitivity of electrochemiluminescence technology and improves the applicability of electrochemiluminescence (electrochemiluminescence) by multivariate analysis and competitive reaction. On this basis, the study of various diseases markers and cell function detection is realized. The research work of this paper is divided into the following parts: 1. based on the self enhancement composite nano The construction of super sensitive cell sensor and its application in drug screening are of great scientific significance and social value, especially high efficiency, low, specific, and even individualized anticancer drugs. The drug screening evaluation system based on apoptosis analysis needs more sensitive, accurate, effective and convenient. The electrochemiluminescence technology with high sensitivity, good reaction controllability and convenient and inexpensive electrochemiluminescence has great potential application value. However, the biological toxicity of electrochemiluminescence materials and co reactants and the sensitivity of analytical methods need to be further improved. A kind of self enhanced electrochemiluminescence is proposed in this study. The preparation of nanomaterials is prepared by coupling of carboxyl bipyridine ruthenium with amino side chain modified methoxy silane to prepare functionalized silica nanomaterial precursors, in which Ru is an electrochemiluminescence center and an amino side chain is used as a co reaction reagent. Enhanced electrochemiluminescence nanomaterials have been successfully applied to the construction of an anti-tumor efficacy evaluation system based on apoptosis analysis, which has achieved efficient, rapid and convenient evaluation of antitumor efficacy. The experimental study was built on the use of breast cancer tumor cells (MDA-MB-231) and antitumor drugs (paclitaxel) as models. The application of the anti-tumor efficacy evaluation system has achieved good results. The design and construction of self enhanced electrochemiluminescence nanomaterials were put forward, and the self enhancement mechanism was studied. At the same time, the application of electrochemiluminescence analysis technology in the urgent drug screening evaluation system was realized. One step extended the research and application of electrochemiluminescence analysis technology to provide reference for further electrochemiluminescence technology and drug screening and evaluation..2. based on multicomponent electrochemiluminescence analysis based on multivariate analysis to study electrochemiluminescence analysis technology, because of its low background, high sensitivity, wide linear range and low consumption. So far, most of the electrochemiluminescence analysis can only achieve a single target quantitative analysis at the same interface, and a more efficient and sensitive multi component electrochemiluminescence detection technology is urgently needed in clinical detection, environmental analysis, food safety and so on. In this study, a multicomponent electrochemiluminescence analysis strategy based on multivariate analysis was designed, and a highly sensitive multicomponent electrochemiluminescence analysis system was constructed with a cascade amplification strategy based on the hybridization chain reaction rolling ring amplification reaction. The N terminal B type natriuretic peptide and the troponin I were used. The analysis and detection performance of the method is discussed with Lumino and bipyridine ruthenium as the signal probe, and the key problem of mutual influence between different electrochemiluminescence signal probes is effectively solved by multivariate analysis. The detection sensitivity is effectively improved by using the HCR-RCA based cascade amplification strategy. The high sensitivity analysis and detection of multiple components at the same interface have laid a good foundation for the further construction of high throughput electrochemiluminescence detection system..3. based on competition based electrochemiluminescence immunosensing technology is applied to the analysis of the ratio of multiple targets in a single interface. Nowadays, the use of antitumor drugs to eliminate tumor cells is still done by using antitumor drugs. However, it is not effective and sensitive, however, to avoid or reduce the drug resistance in the early stage of cancer cell resistance. An accurate early cell resistance detection system. Electrochemiluminescence analysis has attracted much attention as a highly efficient, sensitive and potentially useful analytical technique. Generally, cell resistance can be determined by the expression of P glycoprotein. For the analysis of P glycoprotein expression, it is not only required to be highly sensitive and quantitative analysis of P glycoprotein, but also need to be analyzed. The analysis of the expression of P glycoprotein by calculating the ratio of the concentration of P glycoprotein to the constant expressed protein was aided by a cell count or assisted by a high sensitivity quantitative analysis of a constant expressed protein. Although the multi component electrochemiluminescence analysis strategy, as described earlier, could be implemented, the strategy of multicomponent Electrochemiluminescence analysis, as described earlier, could be realized. At the same time, multiple targets are detected at the same interface. However, the multivariate analysis is still more time-consuming and needs more convenient and effective detection methods to realize cell resistance analysis. It is worth noting that the degree of cell resistance is evaluated by protein expression, only the concentration ratio of drug resistance related protein and constant expression protein is needed, but it is not necessary. In this study, a highly sensitive electrochemiluminescence analysis strategy for ratio analysis was constructed by using immune recognition based target conversion combined with rolling ring amplification signal amplification strategy and the competitive reaction of electrochemiluminescence sensor surface modified nucleic acid. The P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase was used as a method. The detection model evaluated the degree of tumor cell resistance. First, the immuno recognition reaction on the surface of the magnetic beads was detected by the sandwich method, and the P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase was identified by the biotinylated antibodies fixed on the surface of the magnetic beads, and the target transformation was further combined with the nucleic acid marker two, in order to improve the analysis and detection. Sensitivity, using the rolling ring amplification strategy, the nucleic acid marked on the antibody as the trigger chain, the repeat sequence of the roll ring as the template, and the amplification and amplification of the 1? N target by the DSN enzyme shear; the repeated sequence parts of the shear are consistent, which can form a competitive reaction with the capture sequence on the electrode surface, and further combine the electromotive force. Chemiluminescent material, bipyridine ruthenium labeled signal probe, indicated the ratio of P glycoprotein and glyceraldehyde -3- phosphate dehydrogenase by electrochemiluminescence signal, and realized the evaluation of the degree of cell resistance. The ratio analysis strategy can also be extended to various targets, such as electrochemistry, fluorescence, chemiluminescence, etc. High efficiency, convenient and highly sensitive ratio analysis, such as protein, nucleic acid, and so on, provides an effective strategy for biochemical analysis, clinical diagnosis, especially cell function determination,.4. based on time regulated detection range of tunable electrochemiluminescence technology, high sensitivity electrochemiluminescence analysis technology is more important. However, so far, most of the electrochemiluminescence analysis techniques have only been able to realize the target analysis in fixed sensitivity and fixed detection range due to the single modification interface. The electrochemiluminescence is usually caused by the difference in the concentration of the same target in different samples. Analysis technology is difficult to be applied directly to sample analysis and detection, and the complicated separation and enrichment / dilution operation becomes one of the recessive content of quantitative analysis, which undoubtedly increases the workload of analysis and detection, and reduces the accuracy of analysis and detection to a certain extent. The analysis of Pb2+ ions is used as a model. This study is carried out through the electrode table. The nucleic acid specific hybridization, the reticular formation of nucleic acid reticulation, and the design of the DNA enzyme in the reticular structure, and the construction of an adjustable electrochemiluminescence sensor for Pb2+ ions in the reticulate double chain of electrochemiluminescence materials. The nucleic acid reticular structure assembled by the Pb2+ ion mediated DNA enzyme cyclic shear was released and released at the same time. The discharge induced chemiluminescence material leads to the reduction of electrochemiluminescence signal to realize the quantitative analysis of Pb2+ ions. The shear efficiency is not only related to the concentration of Pb2+ ions, but also related to the reaction time, thereby regulating the linear range and detecting sensitivity by controlling the reaction time. The chemiluminescence strategy can also be applied to other sensitive and adjustable tests, such as proteins, nucleic acids, etc., and provides a new analytical strategy for environmental safety and clinical testing.5. based on iridium complex nanomaterials and in situ enzyme amplified electrochemiluminescence sensors for the study of the three phenyl pyridine iridium complex as a kind of high quantum conversion efficiency. The excellent electrochemiluminescence materials with high electron transfer efficiency and high electron transfer efficiency are expected to build higher detection sensitivity electrochemiluminescence (electrochemiluminescence) technology on this basis. However, their poor solubility in water, difficult to label and other defects restrict its wide application in the field of electrochemiluminescence sensing technology. This study uses three phenyl pyridine iridium complexes to doping two oxygen. Silicon nanomaterials have realized the high efficiency of electrochemiluminescent materials and greatly improved their water solubility. The performance of electrochemiluminescence sensors based on iridium complex nanomaterials and in situ enzyme amplified electrochemiluminescence sensors was investigated by using the markers of heart failure and the B natriuretic peptide at the end of N. The inductor was coated on the surface of the electrode with Nafion dispersed three phenyl pyridine iridium complex doped silica nanomaterials and further coupled with the immobilized anti N terminal B natriuretic peptide antibody. Based on this, the electrochemiluminescence sensor has a strong electrochemiluminescence signal. When the detection sample contains the N terminal B type natrium At the time of peptide, it can be combined with the surface of the electrode by specific immune recognition, and further combined with the two resistance of glucose oxidase labeling. The quantitative analysis and detection of the B type natriuretic peptide in the N terminal of the target by glucose oxidase catalyzing the quenching of glucose producing hydrogen peroxide to the electrochemiluminescence signal. The electrochemiluminescence analysis strategy can also be applied to high sensitive analytical applications of other targets, such as nucleic acids, cells and other substances.
【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

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