基于核酸放大及共反应放大策略构建的电致化学发光生物传感器的研究

发布时间：2018-01-10 23:27

本文关键词：基于核酸放大及共反应放大策略构建的电致化学发光生物传感器的研究　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：电致化学发光(ECL)生物传感器是一类以生物分子如核酸分子、适体、抗体等为分子识别元件,结合ECL技术实现目标物检测的传感器。ECL生物传感器具有灵敏度高、选择性好、响应速度快、操作简便、成本低等优点,被广泛应用于疾病相关核酸分子、蛋白质、功能小分子等的检测。通常,向ECL体系中引入共反应试剂可显著增强ECL信号强度,从而提高ECL生物传感器的检测灵敏度。然而,一些共反应试剂不稳定且难以标记,从而增加了固载难度。同时,其与发光体相互作用时,电子传输距离长、能量损失大。鉴于此,本论文主要从提高发光体和共反应试剂之间的相互作用效率着手,通过合成自增强型ECL试剂和引入新型共反应促进剂放大策略,并结合核酸扩增技术以及多种纳米复合材料,构建了多个ECL生物传感器,实现了多种生物分子如micro RNAs、蛋白等的高灵敏检测。本论文主要的研究工作如下:1.基于目标物催化发夹组装和分子内/分子间共反应信号放大策略构建的ECL生物传感器研究目前,将电致化学发光技术和核酸扩增技术结合起来用于micro RNAs(mi RNAs)的检测日渐成熟。然而,大多数的核酸扩增技术都依赖酶的参与,因此会受到一些不利因素,如操作步骤繁琐、操作条件苛刻和测试成本较高等的制约。目标物催化发夹组装(T-CHA)技术能在无酶的条件下实现,其不仅具有灵敏度高、分析时间短、操作简单等特点,而且还能同时实现目标物的循环扩增。另外,在构建ECL生物传感器过程中,引入合适的共反应试剂,同时有效的提高共反应试剂和发光体的作用效率尤为重要。通过共价交联作用,将共反应试剂和发光体合成为一个分子,制得自增强型钌复合物发光试剂(PEI-Ru(II)),缩短了发光体Ru(II)与共反应试剂聚乙烯亚胺(PEI)之间电子传输距离,减少了Ru(II)与PEI作用中能量的损失,提高了发光体与共反应试剂的作用效率,进而获得更强且稳定的ECL信号。本研究首次将新型的共反应试剂氨基硫脲(TSC)引入PEI-Ru(II)自增强发光体系中,实现了分子内/分子间双重共反应放大,进一步结合无酶T-CHA构建了生物传感器来高灵敏的检测mi R-21,其检测范围为1.0×10-16 mol/L到1.0×10-11 mol/L,检测限低至30 amol/L。2.基于傒衍生物作为新型共反应促进剂信号放大构建的ECL生物传感器研究量子点(QDs)具有尺寸和发射波长可控、发光产率高和化学稳定性好等优点,其作为ECL试剂已被广泛用于生物标志物的分析中。值得注意的是,QDs的固载是固态ECL生物传感器构建过程中的关键步骤。石墨烯凭借优良的导电性和大的比表面积等优势,可作为QDs理想的固载基质。同时,过硫酸根(S2O82-)作为QDs最常用的共反应试剂使QDs产生高效稳定的ECL信号,但是由于其难以固载,使其应用受到了一定的限制。本研究在S2O82-/QDs体系中引入共反应促进剂解决了这一难题。其中,L-赖氨酸功能化的傒四甲酸的复合物(PTC-Lys)作为一种新型的傒衍生物,能够促进S2O82-产生更多的强氧化剂中间体SO4·-,从而增强QDs的ECL信号。基于PTC-Lys作为新型的共反应促进剂构建了高灵敏的适体传感器用于检测凝血酶(TB),检测范围为1.0×10-16 mol/L~1.0×10-8 mol/L,检测限为34 amol/L。3.基于金属有机骨架材料/量子点复合材料作为多功能信号探针构建的ECL生物传感器研究金属有机骨架材料(MOFs)一般是以金属离子为连接点,有机配体为支撑构筑的3D空间网络结构材料,具有比表面积大、孔尺寸和孔形状可调等优点,使其成为理想的固载材料。本研究为了固载更多的发光试剂Cd Te QDs,同时采用了“内腔填充”、“内表面修饰”以及“外表面修饰”方法制得异格金属有机骨架材料-3(IRMOF-3)内外表面均修饰有Cd Te QDs的复合材料Cd Te@IRMOF-3/Cd Te QDs。值得注意的是,IRMOF-3不仅可作为Cd Te QDs的固载基质,而且能促进S2O82-产生更多的强氧化剂中间体SO4·-发射更强的ECL信号,即起到了共反应促进剂的作用。因此,本文基于IRMOF-3和Cd Te QDs的复合材料作为多功能信号探针,构建了一个高灵敏的免疫传感器用以检测心肌肌钙蛋白I(c Tn I),并获得了较宽的响应范围(1.1 fg/m L~11 ng/m L)和低的检测限(0.46 fg/m L)。
[Abstract]:Electrochemiluminescence (ECL) biosensor is a kind of biological molecules such as nucleic acid molecules, such as antibodies, aptamers, molecular recognition element, sensor.ECL biosensor target detection with high sensitivity, good selectivity with ECL technology, fast response speed, simple operation, low cost, is widely used in disease related nucleic acid molecules, proteins, small molecules detection function. Usually, introducing common reagent can significantly enhance the signal intensity of ECL to ECL system, thus improving the detection sensitivity of ECL biosensor. However, some common reagents unstable and difficult to mark, thus increasing the difficulty of immobilization. At the same time, with the light body electronic interaction, long transmission distance, large energy loss. In view of this, this paper mainly from the body and improve the luminous efficiency of interaction between the co reaction reagent to the synthesis of self reinforced ECL test The introduction of a new agent and co reaction accelerator amplification strategy, combined with the nucleic acid amplification technology and a variety of nano composite materials, construction of a number of ECL biological sensors, to achieve a variety of biological molecules such as micro, RNAs, high sensitive protein detection. The main works of this thesis are as follows: 1. based on the target assembly and molecular / catalytic hairpin the intermolecular co response signal amplification of ECL biosensor construction strategy at present, electrochemiluminescence technology and nucleic acid amplification technology together for micro RNAs (MI RNAs) detection is becoming more and more mature. However, most of the nucleic acid amplification techniques rely on enzymes, so it will have some adverse factors, such as complicated steps harsh operating conditions, and the test cost is higher. The restricted object assembly (T-CHA) hairpin catalytic technology can be realized in non enzymatic conditions, it not only has high sensitivity, divided Analysis of short time, simple operation and other characteristics, but also realize the target of cyclic amplification. In addition, in the process of constructing ECL biosensor, introducing common reagent suitable, and effectively improve the co reaction reagent and luminous efficiency of the body is particularly important. Through covalent crosslinking, CO reaction reagent and luminescence the body into a molecule, prepared from ruthenium complexes enhanced luminescence reagent (PEI-Ru (II)), Ru phosphor (II) and shorten the reaction reagent of polyethyleneimine (PEI) between the electronic transmission distance, reduced Ru (II) and PEI in energy loss, improve the luminous body with the total reaction efficiency of reagents, and ECL signals were enhanced and stable. This is the first time of the co reaction reagent Aminothiourea model (TSC) into PEI-Ru (II) from the enhanced chemiluminescence system, the intramolecular / intermolecular co amplification reaction, into a No enzyme T-CHA to construct biosensor to the sensitive detection of MI combined with R-21, the detection range of 1 * 10-16 to 1 * mol/L 10-11 mol/L, the detection limit is low to 30 amol/L.2. based on Xi derivatives as novel co reaction accelerator signal amplification to construct ECL biosensor based on quantum dots (QDs) with the size and emission wavelength the advantages of light controlled, high yield and good chemical stability, as the ECL reagent has been widely used for biomarker analysis. It is worth noting that QDs supported is the key step in the process of solid ECL biosensors. Graphene with excellent electrical conductivity and large specific surface area and other advantages, solid matrix can be used as the ideal QDs. At the same time, persulfate (S2O82-) as the most commonly used QDs co reaction reagent QDs ECL signal is stable and efficient, but because of its difficult immobilization, the application by a certain Limited. This study introduces co reaction accelerator to solve this problem in the S2O82-/QDs system. Among them, compound L- lysine functionalized Xi four formic acid (PTC-Lys) as a new type of Xi derivatives can promote S2O82- produced more strong oxidizing agent intermediate SO4 - ECL, thereby enhancing the signal QDs PTC-Lys. As a new co reaction accelerator built high sensitive aptasensor for thrombin detection based on (TB), the detection range of 1 * 10-16 mol/L~1.0 * 10-8 mol/L, the detection limit is 34 amol/L.3. based on metal organic frameworks / quantum dots composite materials as ECL biosensor based on metal organic framework of multi functional signal probe the construction of the (MOFs) are generally based on metal ions as the connection point, to support the construction of organic ligand 3D space network structure material, has the advantages of large specific surface area, pore size and pore shape adjustable, Make it a solid material ideal. In this study, in order to Cd Te QDs reagent supported more at the same time, the "cavity filling", "modified" and "surface modification" prepared different lattice metal organic frameworks (-3 IRMOF-3) both internal and external surface composite modified Cd Te@IRMOF-3/Cd Te QDs. Cd Te QDs, it is worth noting that IRMOF-3 not only can be used as immobilization matrix Cd Te QDs, but also can promote S2O82- to produce more strong oxidant agent intermediate SO4 - emission stronger ECL signals, which play the role of CO reaction accelerator. Therefore, the composite materials of IRMOF-3 and Cd Te as QDs multifunctional signal probe based on the construction of a highly sensitive immunosensor for the detection of cardiac troponin I (C Tn I), and received a wide range of response (1.1 fg/m L~11 ng/m L) and low detection limit (0.46 fg/m L).

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：O657.3;TP212.3

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