核酸适体在蛋白质和小分子检测中的新方法研究

发布时间：2018-05-30 08:24

本文选题：G四股螺旋 + 核酸适体　；参考：《湖南大学》2010年硕士论文

【摘要】： 蛋白质如免疫球蛋白E及小分子如腺苷等生物分子在维系生物个体的生命活动中起着至关重要的作用。对这些生命物质进行快速而有效的监测对保持生个体正常的机能有着直接而现实的意义。生物传感器在众多分析方法中独树一帜,他不仅具有常规分析方法的优点,而且能模拟生命活动过程,能提供原位、实时分析。核酸适体是一段自核酸文库中随机筛选出的功能化单链短DNA或RNA序列,具有化学稳定,尺寸小,价格低廉,高亲和力,高特异性以及靶物质广泛等的特点,这使它在构建一系列目标分子检测体系中成为人们的首选。本文以核酸适体为目标识别探针,主要在以下几个方面开展工作： (1)基于G四股螺旋和信号增强的荧光寡核苷酸探针用于蛋白质的超灵敏检测【第2章】。本章中,我们报道了一种标记芘的富G核酸探针及其在IgE检测中的应用,为探究人类端粒的生物学功能提供了有益的参考。基于分子间G四股螺旋结构,结合S1核酸酶和目标识别核酸适体,我们设计了一种末端单芘标记的寡核酸探针并提出一种新的蛋白质检测方法。该方法不仅有效回避了报告基团特异性位点优化和芘单体荧光易被核酸碱基猝灭的问题,同时还提出了一种信号增强的响应机理,成功用于均相中IgE超灵敏检测。该传感器的线性范围为4.72×10-12～7.56×10-9M,回归系数为0.9941,检测下限9.45×10-14M。 (2)基于背景消除和信号增强的电化学核酸适体传感器用于小分子超灵敏检测[第3章]。本章中,我们以腺苷为目标模型提出了一种多功能的电化学核酸适体传感机理用于小分子的超灵敏检测。巯基修饰的核酸适体探针上标记二茂铁电活性分子,并将其固定在电极表面,在核酸适体序列的两端引入大肠杆菌核酸内切酶Ⅰ的酶切位点,这不仅能够扣除背景电流,同时形成一种信号增强的响应机理。没有目标物腺苷存在时,核酸适体折叠成发夹结构,在茎部形成一个酶切双链区,酶切后二茂铁脱离电极表面,没有二茂铁的响应电流信号产生。有腺苷存在时,腺苷和核酸适体结合诱导核酸适体发生构型转变,酶切双链区域消失,核酸内切酶就无法切断适体探针序列。此时二茂铁分子仍离电极表面很近,故能够观察到二茂铁的响应电流峰。这种传感检测体系,在腺苷浓度极低的情况下仍能检测到电流信号。检测下限达到10-13 M,线性响应范围为3.74×10m～3.74×10-8M。我们提出的这种新型电化学传感检测方法极大的拓展了各种基于核酸适体传感体系用于目标检测的分析范围。 (3)基于分子信标核酸适体荧光生物探针用于腺苷的高选择性检测【第4章】。本章中,基于核酸适体结合目标诱导构型转换和DNA杂交原理,以腺苷的核酸适体序列为主体设计分子信标,提出了一种信号减小的腺苷检测响应机理。当没有腺苷存在时,互补DNA序列和分子信标杂交,形成DNA双螺旋结构,分子信标打开,产生很强的荧光。有腺苷存在时,分子信标和腺苷结合后发生构型转换,再向其加入互补DNA序列,荧光响应信号变化很小。随着腺苷分子浓度的降低,体系的荧光响应相应增强,从而实现对腺苷小分子的特异性检测与精确定量。
[Abstract]:Proteins such as immunoglobulin E and small molecules, such as adenosine, play a vital role in maintaining individual life activities. Rapid and effective monitoring of these living substances has a direct and realistic significance for maintaining the normal functioning of the individual. Biological sensilla is unique in many analytical methods, He not only has the advantages of conventional analytical methods, but also simulates the process of life activity and provides in situ, real-time analysis. The aptamer is a functional single strand short DNA or RNA sequence randomly selected from the nucleic acid library. It has the characteristics of chemical stability, small size, low price, high affinity, high specificity and wide range of target materials. This makes it the first choice in building a series of target molecular detection systems. This paper uses aptamers as the target identification probe, which mainly works in the following aspects:
(1) the use of G four strand helix and signal enhanced fluorescent oligonucleotide probe for protein ultra sensitive detection [chapter second]. In this chapter, we reported a rich G nucleic acid probe for labeling pyrene and its application in IgE detection, which provided useful reference for exploring the biological function of human telomere. Based on intermolecular G four strands spiral structure In combination with S1 nuclease and target identification of aptamers, we designed a terminal single pyrene labeled oligonucleotide probe and proposed a new method for protein detection. This method not only effectively avoids the problem of the optimization of the specific site of the report group and the fluorescence of pyrene monomers easily to be quenched by the nucleic acid base, but also proposes a signal enhancement. The response mechanism is successfully used for IgE ultra sensitive detection in homogeneous phase. The linear range of the sensor is 4.72 x 10-12 to 7.56 x 10-9M, the regression coefficient is 0.9941, and the detection limit is 9.45 x 10-14M..
(2) electrochemical nucleic acid aptamers based on background elimination and signal enhancement are used for small molecular hypersensitive detection [Third]. In this chapter, we use adenosine as the target model to propose a multi-functional electrochemical aptamer sensing mechanism for the ultra sensitive detection of small molecules. A sulfhydryl modified nucleic acid aptamer labeled two metallocene electric probe. The active molecule is immobilized on the surface of the electrode, and the enzyme tangent site of Escherichia coli endonuclease I is introduced at both ends of the nucleotide sequence. This can not only deduct the background current, but also form a signal enhancement response mechanism. When the target adenosine exists, the aptamer is folded into a hairpin structure, and an enzyme is formed in the stem. After the enzyme was cut, two ferrocene was separated from the surface of the electrode, and there was no response current signal produced by the two ferrocene. When adenosine existed, the binding of adenosine and aptamer induced the transformation of the aptamer, the region of the double chain disappears, and the endonuclease could not cut off the sequence of the aptamer probe. At this time, the two ferrocene molecules were still close to the surface of the electrodes, so they could be found to be very close to the surface of the electrode. It is enough to observe the response current peak of two ferrocene. This sensing detection system can still detect the current signal when the concentration of adenosine is very low. The detection limit is 10-13 M, and the linear response range is 3.74 * 10m to 3.74 x 10-8M.. The new electrochemical sensing method proposed by us has greatly expanded a variety of aptamer based sensing. The system is used for the analysis of target detection.
(3) high selectivity detection of adenosine based on molecular beacon aptamer fluorescent biological probe (fourth chapter). In this chapter, a molecular beacon is designed based on aptamer binding target induced transformation and DNA hybridization, and a signal reduction adenosine detection response mechanism is proposed. When adenosine exists, the complementary DNA sequence and molecular beacon hybridize to form a DNA double helix structure, and the molecular beacon opens and produces a strong fluorescence. When the adenosine exists, the molecular beacon and adenosine are combined after the combination of the molecular beacon and the complementary DNA sequence, and the fluorescence response signal becomes small. With the decrease of the adenosine molecular concentration, the fluorescence of the system is fluorescence. In response to the corresponding enhancement, specific detection and accurate quantification of adenosine small molecules were achieved.
【学位授予单位】：湖南大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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