基于toehold链置换辅助目标循环放大的新型荧光生物传感器的研究

发布时间：2018-04-17 07:16

本文选题：荧光生物传感器 + 无酶信号放大　；参考：《西南大学》2017年硕士论文

【摘要】：荧光生物传感器是一种将特定分子识别事件与荧光转换过程相结合的装置。由于它们具有设备简单、选择性好和响应快速等的优点,自从首次构建,便被期望在疾病诊断、环境监测和食品安全中发挥重要的分析作用。然而,在实际分析检测中目标物的浓度通常处于相对较低的水平,这就需要研究人员设计信号放大策略来提高传感器的灵敏度。考虑到酶辅助循环放大反应容易受到温度、酸碱度等环境因素的影响,因此本研究主要致力于开发简单、方便的非酶信号放大方法用于检测不同的目标分子,并获得了一系列有效的结果。我们将所进行的研究工作的具体内容总结如下:1.MicroRNA激活的分子机器用于非酶目标循环放大检测癌细胞中的microRNA失调表达的microRNA可以作为不同癌症早期诊断的有用的生物标志物。基于新颖的目标激活的非酶目标循环放大DNA机器,本文提出了一种简单高灵敏的荧光方法用于检测前列腺癌细胞中的microRNA。目标物miR-141与荧光猝灭的DNA复合探针的末端区域进行toehold链置换反应(TSDR),导致了FAM标记的信号探针从复合探针中释放,并且产生了另一个toehold区域用于随后与燃料链杂交。一旦与该toehold区域结合,燃料链便通过第二个toehold链置换反应置换了杂交在复合探针上miR-141并且激活了该DNA机器,使得miR-141进行目标物循环并导致大量的释放信号探针用于放大检测miR-141,其检测限达到80fM。该方法也展示了高的选择性,同时可以用于监测癌细胞中的miR-141。此外,我们构建的方法避免了使用蛋白酶进行目标物循环放大,因此在未来的生物分析领域可以提供重要的应用价值。2.目标响应适配体机器用以免标记的和灵敏的非酶循环放大检测ATPATP的浓度异常与许多疾病相关,开发简单和灵敏的方法用于鉴定和检测ATP在临床诊断中至关重要。在这项工作中,基于一种新颖的ATP响应分子适配体机器,我们开发了一种非酶信号放大方法,这种方法可以敏感的检测人类血清中的ATP。目标ATP与三链DNA复合探针中的ATP适配体结合使得该适配体的构型改变,并导致toehold区域的暴露。燃料DNA随后杂交到该区域上,通过toehold介导的链置换反应释放了G-四链体活性序列和目标ATP。这样,ATP分子可以循环再利用使得释放许多G-四链体活性序列,这些被释放的活性序列与N-甲基卟啉二丙酸IX(NMM)结合产生显著放大的荧光信号,用于灵敏的检测低至25nM的ATP。此外,这种方法对于其他对照分子也展示出高选择性,并且可以用于检测稀释的血清样品中的ATP。另外,我们的方法避免使用酶和纳米材料进行信号放大,也未标记信号读出探针。因此,经过精心的改进设计,这里提出的传感平台具有很大的潜力用于简单的和敏感的检测其他小分子。3.金属toehold激活催化发夹组装形成的三向DNAzyme连接体用于放大荧光检测汞离子由于汞离子对环境和人体不可逆的毒性影响,开发一种可靠的并具有高选择性和灵敏的汞离子检测方法具有重要意义。借助于通过金属toehold触发催化发夹组装(catalytic hairpin assembly,CHA)形成的三向DNAzyme连接体的高效的信号放大能力,我们构建了一种可用于测定不同水环境样品中汞离子的高灵敏和高选择性的方法。存在目标汞离子可导致形成T-Hg~(2+)-T碱基错配金属toehold结构,这样便触发了含有分裂DNAzyme的三个发夹经过催化组装以形成依赖于镁离子进行底物链剪切的DNAzyme连接体。接着,镁离子循环切割荧光猝灭的底物链结构产生了显著增强的荧光信号用于灵敏的检测汞离子,达到了4.5pM低的水平。由于在金属toehold区域应用了T-Hg~(2+)-T桥状结构化学,我们开发的传感器对其他的竞争离子展示了高的选择性,而且可用于检测参杂在环境水样中的汞离子。考虑到核酸引发序列和组装序列的核酸本性,所开发的方法具有很大的潜质来设计新的无酶信号放大方法用以检测不同的DNA和RNA目标。
[Abstract]:Fluorescence biosensor is a device specific molecular recognition events and fluorescence conversion process combination. Because it has the advantages of simple equipment, good selectivity and fast response advantages, for the first time since the building was expected to play a role in disease diagnosis, analysis of important environmental monitoring and food safety. However, in the actual concentration analysis target detection is usually at a relatively low level, which requires researchers to design the signal amplification strategy to improve the sensitivity of the sensor. Considering the enzyme assisted circulation amplification reaction due to temperature effect, acid alkalinity and other environmental factors, this study focused on developing a simple, non enzymatic signal amplification method for convenience detection of different target molecules, and obtained a series of effective results. The main content of our research work will be carried out by the activation of 1.MicroRNA are summarized as follows: The molecular machine used for microRNA non enzymatic target detection cycle amplification in cancer cells microRNA expression imbalance can be used for the early diagnosis of cancer useful biomarkers. Non enzymatic target cycle novel target activation amplification of the DNA based machine, this paper presents a simple and high sensitive fluorescence method for toehold strand displacement reaction of terminal the regional DNA composite probe for the detection of prostate cancer cells microRNA. target miR-141 and fluorescence quenching (TSDR), the resulting signal FAM labeled probe was released from the composite probe, and produced another toehold region for subsequent and fuel chain hybridization. When combined with the toehold region, by the second fuel chain toehold strand displacement reaction of hybrid composite probe replacement in miR-141 and activate the DNA machine, the miR-141 target circulation and cause a lot of Release signal probe for detection of miR-141 amplification, the detection limit is 80fM.. This method also shows high selectivity, and can be used for monitoring miR-141. in cancer cells in addition, we construct the method to avoid the use of protease of target cycle amplification, so the analysis in the field of biology in the future. It can provide important value of.2. target response. The machine with the concentration of labeled ligands to avoid sensitive and non enzymatic cycling amplification detection of ATPATP abnormalities associated with many diseases, the development of simple and sensitive method for the detection and identification of ATP is important in clinical diagnosis. In this work, a novel ATP molecular response aptamer based machine, we developed a non enzyme signal amplification method, this method can make the detection of ATP. ATP in human serum and three chain DNA composite probe sensitive in ATP aptamer binding Change the aptamer configuration, and lead exposed toehold area. Then to the hybrid fuel DNA region, through the chain replacement reaction mediated release of toehold G- four chain activity sequence and target ATP., ATP molecules can be recycled makes the release of many G- four chain activity sequences, which were released the activity sequence and N- methyl porphyrin two propionic acid IX (NMM) combined with fluorescence signal was significantly enlarged, for sensitive detection of low to 25nM ATP. in addition, this method is for other control molecules also exhibited high selectivity, and can be used for the detection of serum samples diluted in ATP. in addition, our method can avoid signal amplification the use of enzymes and nano materials, but also unlabeled probe signal. Therefore, the improved design meticulously, sensing platform proposed here has great potential for simple and sensitive detection of other small Molecular.3. metal toehold catalytic activation of hairpin assembly forming three to DNAzyme connector for amplifying fluorescence detection of mercury ions due to mercury ion toxicity to the environment and human irreversible effects on the development of a reliable and highly selective and sensitive method for detection of mercury ion has important significance. With the help of toehold triggered by metal catalytic hairpin assembly (catalytic hairpin assembly, CHA) formed three to DNAzyme connector efficient signal amplification ability, we construct a can be used for the determination of mercury ions in different water samples in high sensitive and high selective methods. There are mercury ions can lead to the formation of T-Hg~ (2+) -T mismatch metal toehold this will trigger structure, three hairpin containing DNAzyme split through catalytic assembly to form a dependence on the substrate chain of magnesium ions DNAzyme shear connector. Then, magnesium ion The substrate chain structure of circle cutting fluorescence quenching produced significantly enhanced fluorescence signal for detection of mercury ion sensitive, reached 4.5pM low level. Due to the metal toehold region T-Hg~ application (2+) the chemical structure of -T bridge shape, we developed the sensor exhibits high selectivity to other competing ions, and can be used for mixed detection of mercury ion in environmental water samples. Considering the nature of nucleic acid nucleic acid triggered sequence and assemble sequences, the developed method has great potential to design a new non enzymatic signal amplification method for detection of different DNA and RNA target.

【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

【相似文献】