表面等离子共振传感技术检测核酸新策略研究

发布时间：2018-05-12 00:21

本文选题：沙门氏菌 + MicroRNA　；参考：《重庆医科大学》2015年硕士论文

【摘要】：核酸作为一类最基本的生命物质,在遗传信息处理过程中具有至关重要的作用,包括遗传信息的存储、复制和传递等,因此成为生命科学中广泛研究的热点之一。作为一类生物标志物,核酸可能在一些疾病(例如癌症)的发生和发展过程中,提供了一些很有价值的信息。尤其是在一些疾病的早期,这些相关核酸生物标志物的浓度处于很低的水平。因此,建立快速、灵敏和特异的方法来检测这些低丰度的疾病相关核酸标志物,对于基因治疗、突变分析和临床诊断具有重要意义。表面等离子共振传感器是20世纪90年代提出的一种用于生物分子间相互作用分析(BIA)的亲和型生物传感技术。近年来,由于该技术具有免标记、实时监测、耗样量少和灵敏度高等特点,受到了相关领域研究人员的极大的关注,并广泛应用于生物标志物的分析。本研究以表面等离子共振生物传感器为检测平台,结合生物分析化学和分子生物学相关技术,提出了简便灵敏的核酸检测策略。本论文主要包括以下两个部分：1.亲和素适体放大的表面等离子共振传感器检测沙门氏菌invA基因基于表面等离子共振传感芯片,整合了信号放大元件亲和素适体和修饰的不对称PCR反应,建立了一种检测沙门氏菌的高灵敏方法。首先在传感芯片表面修饰巯基修饰的探针,然后加入靶物质和亲和素适体形成三明治杂交结构。当亲和素加入体系后,亲和素适体可与亲和素形成复合体,从而实现信号的放大。在最优的条件下,该方法检测invA基因的线性范围是50 pM到200 nM,最低检测限为20 pM。该策略成功地检测到低达60 CFU mL-1的沙门氏菌。所建立的方法灵敏度高、选择性强、稳定性好,这些优点使得该策略在食物、临床和环境样本中对沙门氏菌的筛查具有很好的应用前景。2.基于表面等离子共振传感器的新型DNA纳米技术检测microRNAMicroRNA (miRNA)在许多疾病发生和发展中具有十分重要的调节作用,已成为一类很有前景的疾病生物标志物。本研究基于表面等离子共振生物传感器平台,结合错配型的催化茎环自组装(CHA)和链霉亲和素适体,构建了一种放大策略,用于靶miRNA高灵敏特异的检测。在靶miRNA的存在下,可触发CHA放大反应,从而形成靶miRNA的循环使用并产生大量的CHA反应产物。另外,大量的CHA反应产物一端可与修饰在裸金芯片表面修饰成功的捕获探针杂交结合,另一端则可耦合链霉亲和素,完成检测信号的放大和输出。由于无酶的CHA放大反应和免标记SPR生物传感器的优良性能,在最优的实验条件时,该方法能够检测靶miRNA的线性范围为5 pM到100nM,最低检测限可达到1 pM,相关系数为0.9968,并且具有很好的特异性。综上所述,基于SPR的生物传感策略很有潜力发展成为一种用于miRNA检测的新型手段,以更好地用于医学研究和临床早期诊断。
[Abstract]:Nucleic acid, as the most basic life substance, plays an important role in the process of genetic information processing, including the storage, replication and transmission of genetic information, so it has become one of the hot spots in life science. As a kind of biomarker, nucleic acid may provide some valuable information in the occurrence and development of some diseases (such as cancer). Especially in the early stages of some diseases, the concentration of these related nucleic acid biomarkers is very low. Therefore, it is important for gene therapy, mutation analysis and clinical diagnosis to establish a rapid, sensitive and specific method for the detection of these low-abundance disease-related nucleic acid markers. Surface plasmon resonance sensor is a kind of affinity biosensor technology proposed in 1990s for biomolecular interaction analysis (BIA). In recent years, the technology has been paid great attention by researchers in related fields because of its characteristics such as no marking, real-time monitoring, small sample consumption and high sensitivity, and has been widely used in the analysis of biomarkers. In this study, a simple and sensitive nucleic acid detection strategy was proposed on the basis of surface plasmon resonance biosensor and related techniques of bioanalytical chemistry and molecular biology. This thesis mainly includes the following two parts: 1. Detection of Salmonella invA Gene based on Surface Plasmon Resonance Sensor Chip, the affinin-amplified aptamer and modified asymmetric PCR reaction are integrated. A highly sensitive method for the detection of salmonella was established. First, the thiol modified probe was modified on the surface of the sensor chip, and then the target material and the aptamer were added to form the sandwich hybrid structure. When affin is added into the system, the aptamer can form a complex with the aptamer, which can amplify the signal. Under the optimal conditions, the linear range of detection of invA gene by this method is from 50 pm to 200 nm, and the minimum detection limit is 20 pm. The strategy successfully detected salmonella as low as 60 CFU mL-1. The established method has the advantages of high sensitivity, high selectivity and good stability, which makes the strategy has a good application prospect in food, clinical and environmental samples for Salmonella screening. A novel DNA nanotechnology based on surface plasmon resonance (SSRR) has played an important role in regulating the occurrence and development of many diseases and has become a promising biomarker of disease. Based on the surface plasmon resonance biosensor platform and the mismatched catalytic stem ring self-assembly (cha) and streptavidin aptamer, an amplification strategy was constructed for the detection of target miRNA with high sensitivity and specificity. In the presence of target miRNA, the CHA amplification reaction can be triggered, resulting in the recycling of target miRNA and the production of a large number of CHA reaction products. In addition, a large number of CHA products can be hybridized with the successfully modified capture probe modified on the surface of bare gold chip, and the other end can be coupled with streptavidin to amplify and output the detection signal. Due to the excellent performance of non-enzymatic CHA amplification reaction and labeled free SPR biosensor, under the optimal experimental conditions, This method can detect target miRNA in the linear range from 5 pm to 100 nm, the minimum detection limit can reach 1 pm, the correlation coefficient is 0. 9968, and it has good specificity. In conclusion, the biosensor strategy based on SPR has the potential to develop into a new method for miRNA detection, which can be better used in medical research and early clinical diagnosis.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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