【作者】 周慧；

【导师】 沈国励；

【作者基本信息】 湖南大学 ， 分析化学， 2009， 硕士

【摘要】 生物传感器具有选择性好、灵敏度高、分析速度快、成本低、能在复杂体系中进行在线连续监测等优点,在化学、生物医学、环境监测、食品、医药和军事等领域有重要的应用价值。核酸适体由于具有与目标结合的高特异性和强亲和力,以及生物活性稳定,易于合成和保存等优点而成为一种极富应用潜力的识别探针,为生物传感器的研究提供了一个新的平台。本论文针对目前构建检测蛋白质传感器界面的焦点和难点问题,结合本实验室的优势,利用目标蛋白质引起两种竞争机制的改变触发信号放大,发展了几种用于蛋白质检测的特异性好且灵敏度高的核酸适体传感器构建方法。主要工作如下:在第二章中,我们利用适体探针、互补序列和挂锁探针三者之间的竞争反应提出了一种基于核酸适体特异性识别目标物导致滚环扩增放大反应,用以电化学检测PDGF-BB的可再生适体识别系统。当新设计的适体序列与目标蛋白质发生特异性结合时,促发了由一互补DNA序列、线形挂锁探针、和引物探针引发的滚环扩增反应。本适体识别系统巧妙地把多个功能元件整合为一个信号检测系统:独特的电化学检测技术、引人注目的滚环扩增放大反应、电极表面可逆的DNA杂交以及理想的适体-目标物的识别体系。基于滚环扩增的适体识别系统不仅展现了优越的分析性能,而且克服了传统适体传感器的局限性(例如:对信号目标物与适体特异性结合力的依赖,或对拥有两个或更多识别位点的目标序列夹心分析的要求)。回收实验证实了本实验方案的可行性。据此,本适体识别体系在蛋白质或其它分析物的研究中展现了良好的应用前景。第三章,我们在第二章的基础上将连接-滚环信号放大与分子信标降低背景相结合发展了一种应用于蛋白质均相检测的新方法。连接-滚环信号放大同样由竞争反应触发。有目标蛋白存在时,适体探针与目标蛋白特异性结合形成适体探针/分析物复合物,使得适体探针发生末端链置换。原先与适体探针相结合的互补序列被释放,从而与挂锁探针杂交。在DNA连接酶的作用下,挂锁探针被进一步环化并在Phi 29 DNA聚合酶的作用下通过滚环放大反应进行扩增。其产物包含成千上万个可以和分子信标检测探针互补杂交的重复序列。在不含目标蛋白时,适体探针与互补序列杂交形成适体探针/互补序列的二元复合物,这样互补序列就不能与挂锁探针杂交从而不能进行连接-滚环放大反应。以PDGF-BB为模型分析物,可以检测到1.36amol的蛋白质分子。而且,该方法只需根据目标蛋白和其适体序列的不同设计不同的互补序列和挂锁探针就可以实现对其他蛋白质的检测。在第四章中,我们提出一种新的纳米金团聚机理,即发夹型核酸适体粘性末端匹配修饰诱导纳米金组装,从而降低胶体稳定性,加入较高浓度的盐引起团聚。当溶液中存在目标分子IgE时,适体稳定的发夹构型显著抑制纳米金组装。鉴于此,我们开发了一种以IgE为分析模型的基于纳米金稳定性增强的均相比色分析方法。与目标IgE结合后,核酸适体构象发生变化,空间位阻增大,修饰到纳米金表面使颗粒稳定性增强,呈分散状态,据此可用于IgE的分析测定。与现有IgE检测方法相比较,该方法灵敏度高,所需仪器设备简便,可通过目视观察颜色变化或紫外可见分光光度计监测吸光度的变化。这种发夹型核酸适体衍生纳米金的组装行为在生物传感技术中的成功应用有助于加深理解核酸构象变换对纳米金性能的影响,同时也为设计新颖的信号探针以及开发性能优良的比色检测技术提供新思路。更多还原

【Abstract】 Biosensors have valuable applications in chemistry, biomedicine, environmental protection, food industry, medicine and military affairs because of its good selectivity, high sensitivity, fast response, low cost and continuous on-line detection in complex system. Aptamers have become highly promising recognition probes due to the high specificity, high binding affinity, the stable bioactivity, and the easy systhesis and storage. Aptamers have provided an interesting alternative to biochemical analysis and biosensors. In this paper, in order to solve the focus and difficult problems in building interfaces of sensors for current detection of proteins, we combined with the advantages of our laboratory and made use of the competition reaction alone target proteins complement DNA and aptamer ,which can trigger signal amplification(eg RCA). We developed several high specifity and sensitivity biosensors construction methods for the detection of proteins and small molecules. Main works are as follows:In chapter two, a reusable aptameric recognition system was described for the electrochemical detection of protein PDGF-BB based on the target binding-induced rolling circle amplification (RCA). A complementary DNA (CDNA), linear padlock probe and primer probe were utilized to introduce a RCA process into the aptamer-target binding event while a newly designed aptamer probe was used to recognize the target protein. Towards this goal, this adapted aptamer probe was designed via lengthening an original aptamer sequence by the complement to CDNA and extending the outer helix of three-way junction. The aptameric sensing system facilitates the integration of multiple functional elements into a signaling scheme: the unique electrochemical technique, attractive RCA process, reversible DNA hybridization on an electrode surface and desirable aptameric target recognition. This RCA-based electrochemical recognition system not only exhibits the excellent performance,but also overcomes the limitations associated with conventional aptameric biosensors (e.g. dependence of signaling target binding on specific aptamer sequence and requirement of sandwich assays for two or more binding sites per target molecule). The recovery test demonstrated the feasibility of the designed sensing scheme for target protein assay. Given attractive characteristics, this aptameric recognition platform is expected to be a candidate for the detection of proteins and other ligands of interest in both fundamental and applied research. Based on chapter two, we explore a new method for protein detection in chapter three, which combined signal amplification of the L-RCA system and background elimination of molecular beacon. The strategy is depended on the competition reaction between target protein, aptamer probe and a complementary single-strand DNA (the CDNA) perfectly matched with the aptamer probe. The presences of target protein in the assay system made the aptamer probe bind specifically with the target protein to form aptamer probe/analyte complex and triggered the end of aptamer strand displace with itself, that made the CDNA released from aptamer and allowed the CDNA to hybridize with the padlock probe. With the assistance of DNA ligase, the padlock probe could be circularised and subsequently be amplified by RCA reaction with Phi 29 DNA polymerase. The RCA products contained thousands of repeated sequences which could hybridize with the molecular beacon (the detection probe). By contraries, In the absence of target protein, the aptamer probe hybridized with the CDNA to form aptamer probe/CDNA duplex, which hindered the binding of CDNA and the padlock probe and resulted in the failure of L-RCA reaction. Using platelet-derived growth factor BB (PDGF-BB) as the model analyte, as low as 1.36 amol of protein molecules could be detected. The proposed strategy is universal since the sequences of aptamer probe, CDNA, and the padlock probe could be easily designed to adapt for the aptamer-based detection of other proteins without other strict conditions.In chapter four, a new working mechanism, hairpin sticky-end pairing-induced GNP assembly, is introduced on the basis of the unique instability of aptamer-modified nanoparticles. The salt-induced aggregation of oligonucleotide-modified GNPs can readily occur while addition of target molecules favors the formation of hairpin structure and inhibits substantially the nanoparticle assembly. Along this line, we developed a proof-of-concept colorimetric homogeneous assay using immunoglobulin E (IgE) as analyte model via transforming commonly designed“light-down”colorimetric biosensor into“light-up”one. From the points of view of both conformational transition of aptamer and steric bulk, oligonucleotide-GNPs display an additional stability upon binding to target molecules. The assay showed an excellent sensitivity with both the naked eye and absorbance measurements. Compared with almost all existing IgE sensing strategies, the proposed colorimetric system possesses a substantially improved analytical performance. Success in this biosensing protocol indicates that investigating the assembly behavior of hairpin aptamer-modified GNPs would offer new insight into the dependence of GNP property on the structure switching and open a new way to design signaling probes and develop colorimetric assay schemes.更多还原