基于滚环DNA扩增和纳米材料的生物传感分析方法研究

发布时间：2018-09-19 19:19

【摘要】：随着分析科学的不断发展,生命科学领域中蛋白质、核酸、酶活性以及生物小分子的相关信息越来越多地需要借助于生物传感技术进行分析和检测取得。快速,准确的获取这些生命分子的信息对生物医学,临床诊断和治疗具有非常巨大的意义。近年来,新的分子生物技术不断涌现,并在科学研究和实际应用中得到不断的发展和完善。然而,随着医学诊断水平的不断提高和科学研究的持续深入,对生物分子检测方法的要求也越来越高。开发一些高灵敏度、高特异性、高准确性、低成本、简单快速的定性或定量的分析方法以更好的满足研究和实际应用的需要成为分析化学科研工作者面临的重大挑战。本论文针对上述问题,基于滚环DNA扩增技术和新型纳米材料发展了一系列高灵敏度、高特异性的生物分析方法用于酶活性、核酸、蛋白以及小分子检测,并进一步通过对实际样品的分析,初步验证了这些技术的可行性、可靠性以及准确性。具体内容如下:人鸟嘌呤糖基化酶(h OGG1)由于具有修复DNA氧化损伤的能力而在维持生命体基因完整度上发挥着重要作用。h OGG1的表达水平与很多包括癌症在内的疾病紧密相关。在第2章中,我们构建了一种新颖的荧光“light-up”型的生物传感器用于高灵敏检测h OGG1活性。该方法的建立基于目标物诱导形成5’端磷酸化探针和能够产生自催化DNA酶的滚环扩增技术。该方法对h OGG1的检测限可低至0.001 U/m L,如此低的灵敏度主要归因于基本没有增加的背景信号和我们构建的双信号放大策略。据我们所知,这是检测碱基修复酶的方法中最灵敏的检测方法之一。同时,该方法对可能影响目标物检测的干扰酶体现出很好的特异性,在实际样品的分析中有很大的应用潜力。单核苷酸多态性,是指基因组水平上单个核苷酸的变异,是最常见的遗传变异类型。如果突变发生在遗传基因的编码区,可能会引起所编码的蛋白质结构或功能的改变,从而引起基因疾病的发生。发展高灵敏,高特异性的检测方法用于单核苷酸多态性的检测对产前基因诊断具有重要意义。在第3章中,我们利用Ecoli DNA连接酶对单碱基错配的高特异性识别能力,结合上一章的滚环扩增技术和DNA酶循环放大技术构建了一种高灵敏和高特异性检测单核苷酸多态性的分析方法。挂锁探针只能与完全互补的目标DNA杂交后才能被Ecoli DNA连接酶连接成环,并引发级联信号放大反应。错配的DNA与挂锁探针不能发生完全杂交,Ecoli DNA连接酶能够识别不完全杂交的探针,因此,挂锁探针不能连接成环形探针,进而没有级联信号放大反应的发生,检测不到荧光信号。该分析方法对单核苷酸多态性具有很好的检测性能,对目标DNA检测的线性范围为0.01-1 n M,检测下限为2.6pm。此外,由于dna连接酶在连接位点对单碱基错配的识别能力,该方法在野生型和突变型以不同比例混合的样品中对突变型的dna具有很好的特异性。核酸内切酶iv(endoiv),作为一种dna修复酶,主要修复由脱碱基位点造成的dna损伤,因此,在维持基因完整度方面发挥着重要作用。在第4章中,我们证明了endoiv对单链dna(sdna)中脱嘌呤/脱嘧啶位点(ap)的裂解能力。我们发现,endoiv对ssdna中的ap位点有很好的裂解能力相比于对双链dna中ap位点的酶切活性。进一步地,我们利用endoiv裂解ssdna中ap位点性质构建了一种双信号放大体系用于高灵敏检测酶和蛋白的活性。双信号放大体系主要是将核酸外切酶iii(exoiii)辅助的信号放大方法和滚环扩增技术结合起来。通过这样的放大体系,我们构建的分析方法具有良好的分析性能,对endoiv的检测下限可达0.008u/ml,对链霉亲和素(sa)的检测下限可达2.5pm。此外,该方法对其他可能会有干扰的物质表现出良好的特异性。本章提供了一种新的可用于酶和蛋白检测的平台,并有潜力在生物分析、疾病诊断和药物发展方面找到更广泛的应用价值。乙酰胆碱酯酶,是动物中枢神经系统中很重要的一种关键酶,在阿兹海默疾病、炎症和神经毒性方面发挥着重要的功能。乙酰胆碱酯酶通过将乙酰胆碱水解成胆碱以维持神经递质的水平。而乙酰胆碱酯酶的抑制剂,能够使乙酰胆碱保持活跃状态,并在体内积累造成致命的后果。因此,测定乙酰胆碱酯酶活性和筛选其抑制剂具有重大意义。在第5章中,利用聚t-dna为模板合成的荧光铜纳米颗粒,我们构建了一种简捷、高灵敏、高特异性的荧光分析方法用于乙酰胆碱酯酶及其抑制剂的检测。该检测方法利用硫代乙酰胆碱做为乙酰胆碱酯酶的底物,在乙酰胆碱酯酶的水解作用下,硫代乙酰胆碱被水解成硫代胆碱,硫代胆碱的自由的巯基可以和铜纳米颗粒发生配位作用,进而猝灭铜纳米颗粒的荧光。该传感器具有较好的分析性能,对乙酰胆碱酯酶的检测范围为0-5mu/ml,检测下限达0.026mu/ml,并且对其他可能会干扰检测的蛋白具有很好的特异性。此外,我们还用该传感器进行了乙酰胆碱酯酶抑制剂的检测,有机磷杀虫剂对氧磷为被选作抑制剂的模型进行检测,检测机理主要是利用有机磷可以抑制乙酰胆碱酯酶对硫代乙酰胆碱的水解,进而没有硫代胆碱产物的生成,铜纳米颗粒的荧光不会被猝灭。通过铜纳米颗粒的荧光强度恢复信号实现对有机磷的检测。该传感器对对氧磷的检测ic50值约为84pg/ml。而且,我们还用该方法对实际样品中加入的对氧磷进行了检测,获得了满意的结果。本章的工作可能提供了一种在生物医学和临床应用方面具有潜力的可用于酶及其抑制剂的分析传感平台。目前,荧光纳米颗粒由于其在标记、传感器、成像和生物医学应用方面的前景受到了越来越多的关注。在第6章中,我们利用二氧化锰作为氧化剂用于合成荧光聚多巴胺纳米颗粒。二氧化锰存在时,可以快速将多巴胺氧化成多巴醌,紧接着多巴醌通过自发聚合反应形成荧光聚多巴胺纳米颗粒。进一步,利用荧光聚多巴胺纳米颗粒作为指示剂,构建了一种低成本、快速、灵敏、选择性好的传感器用于谷胱甘肽的检测。谷胱甘肽可以将二氧化锰氧化成锰离子,锰离子不能将多巴胺氧化成多巴醌,进而抑制了荧光聚多巴胺纳米颗粒的合成。荧光聚多巴胺纳米颗粒的荧光强度直接反应了谷胱甘肽的浓度。该传感器对谷胱甘肽的检测表现出很好的分析性能,具有高的灵敏度,理想的选择性。此外,该传感器在人全血的实际样品中对谷胱甘肽的检测有很好的响应,表明该分析方法在生物分析检测和临床诊断方面具有很大的应用潜能。
[Abstract]:With the development of analytical science, more and more information about protein, nucleic acid, enzyme activity and small biomolecules in the field of life sciences needs to be analyzed and detected by means of biosensor technology. In recent years, new molecular biotechnologies have emerged, and have been developed and improved in scientific research and practical applications. However, with the continuous improvement of medical diagnosis and scientific research, the requirements for biomolecular detection methods have become increasingly high. Qualitative or quantitative methods with low cost, simplicity and rapidity to better meet the needs of research and practical applications have become a major challenge for analytical chemistry researchers. The method was applied to enzyme activity, nucleic acid, protein and small molecule detection, and the feasibility, reliability and accuracy of these techniques were preliminarily verified by the analysis of actual samples. In Chapter 2, we constructed a novel fluorescent "light-up" biosensor for highly sensitive detection of the activity of H OGG1. The method was based on the target-induced formation of 5'-terminal phosphorylation probes and the ability to produce self-catalysis. The detection limit of this method for H OGG1 can be as low as 0.001 U/m L. This low sensitivity is mainly attributed to the lack of increased background signal and the double signal amplification strategy we constructed. Single nucleotide polymorphism (SNP) is the most common type of genetic variation at the genome level. If the mutation occurs in the coding region of the genetic gene, it may cause the encoded egg. The development of highly sensitive, highly specific detection methods for single nucleotide polymorphisms is of great significance for prenatal genetic diagnosis. In Chapter 3, we utilize the highly specific recognition ability of Ecoli DNA ligase for single base mismatches in conjunction with the rolling ring in the previous chapter. A highly sensitive and specific method for detecting single nucleotide polymorphisms has been developed by amplification and DNA enzyme cycling amplification techniques. The padlocked probes can only be hybridized with fully complementary target DNA before they can be linked into rings by Ecoli DNA ligase and trigger cascade signal amplification. Mismatched DNA and the padlocked probes can not be completely heterozygous. The cross and Ecoli DNA ligase can identify incomplete hybridization probes, so the padlocked probes can not be connected into circular probes, and then no cascade signal amplification reaction occurs, so no fluorescence signal can be detected. The lower limit is 2.6 pm. In addition, because of the ability of DNA ligase to recognize single base mismatch at the junction site, this method has good specificity for mutant DNA in wild and mutant samples mixed in different proportions. In Chapter 4, we demonstrated the ability of endoiv to cleave AP sites in single stranded DNA (sdna). We found that endoiv has a better cleavage ability to AP sites in ssDNA than to AP sites in double stranded dna. A double-signal amplification system was constructed to detect the activity of enzymes and proteins with high sensitivity. The double-signal amplification system was mainly composed of exoiii-assisted signal amplification and ring-rolling amplification techniques. The detection limit of endoiv is 0.008u/ml and streptavidin (sa) is 2.5pm. In addition, the method has good specificity for other substances that may interfere with the detection of endoiv. This chapter provides a new platform for the detection of enzymes and proteins, and has the potential for biological analysis, disease diagnosis and drugs. Acetylcholinesterase, a key enzyme in the animal central nervous system, plays an important role in Alzheimer's disease, inflammation, and neurotoxicity. Acetylcholinesterase maintains neurotransmitter levels by hydrolyzing acetylcholine to choline. Inhibitors of enzymes can keep acetylcholine active and accumulate in vivo with fatal consequences. Therefore, it is of great significance to determine the activity of acetylcholinesterase and screen its inhibitors. The method uses thioacetylcholine as the substrate of acetylcholinesterase. Under the hydrolysis of acetylcholinesterase, thioacetylcholine is hydrolyzed to thiocholine. The free thiol group of thioacetylcholine can coordinate with copper nanoparticles, and then the thioacetylcholine can coordinate with copper nanoparticles. The sensor has good analytical performance. The detection range of acetylcholinesterase is 0-5mu/ml, the detection limit is 0.026mu/ml, and the specificity of other proteins that may interfere with the detection is very good. In addition, the sensor has been used to detect acetylcholinesterase inhibitors. Phosphorus insecticides were used to detect the model of phosphorus oxide as a selective inhibitor. The detection mechanism was mainly that organic phosphorus could inhibit the hydrolysis of acetylcholinesterase to thioacetylcholine, and the fluorescence of copper nanoparticles could not be quenched without the formation of thiocholine products. Detection of organophosphorus. The IC50 value of the sensor for paraoxon is about 84pg/ml. Furthermore, the method has been applied to the determination of paraoxon in real samples with satisfactory results. The work in this chapter may provide a potential component for enzymes and their inhibitors in biomedical and clinical applications. At present, fluorescent nanoparticles have attracted more and more attention due to their potential applications in labeling, sensor, imaging and biomedical fields. In Chapter 6, we use manganese dioxide as an oxidant to synthesize fluorescent polydopamine nanoparticles. In the presence of manganese dioxide, dopamine can be rapidly oxidized to dopamine. Baquinone is then spontaneously polymerized to form fluorescent polydopamine nanoparticles. Further, a low-cost, rapid, sensitive and selective biosensor for the detection of glutathione is constructed using fluorescent polydopamine nanoparticles as indicators. Glutathione oxidizes manganese dioxide to manganese ions, and manganese is separated. The fluorescence intensity of fluorescent polydopamine nanoparticles directly reflects the concentration of glutathione. The sensor has good analytical performance for the detection of glutathione with high sensitivity and ideal selectivity. Sensors have a good response to the detection of glutathione in real human blood samples, indicating that this method has great potential in bioanalysis and clinical diagnosis.
【学位授予单位】：湖南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TP212.3;TB383.1

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