【作者】 吴超；

【导师】 吴朝阳；

【作者基本信息】 湖南大学 ， 分析化学， 2013， 硕士

【摘要】 小分子及蛋白质在人的生命活动中是不可或缺的重要物质。因此，对于小分子及蛋白质的检测也是一项重要的内容。而由于小分子物质分子量小、体积小等性质的限制，相对于大分子物质的检测来说实现小分子的检测更为困难。液晶生物传感器是Abbott研宄组将物相介于液态和晶态之间性能独特（即具有液态的流动性也具有晶态的光学各向异性）的液晶材料应用于生物传感检测技术中提出的一种新型生物传感器。由于其具有灵敏度高、易于操作等特点受到越来越多的分析研宄者的关注。本文运用核酸适配体与小分子特异性识别作用，构建具有灵敏度高、特异性强、操作简单、成本低等特点的新型液晶生物传感方法，实现小分子的快速检测。具体内容如下：(1)发展了一种基于裂开型适配体的新型液晶生物传感系统，以“适配体-靶分子-适配体”的方式相互特异性结合形成稳定的夹心结构来检测三磷酸腺苷(ATP)o将含部分ATP识别片段的寡核苷酸单链探针作为捕获探针固定在经三乙氧基丁醛基硅烷/N,N-二甲基-N-十八烷基-3-氨丙基三甲基硅烷（TEA/DMOAP)混合组装膜修饰的玻片表面。当ATP存在时，分别含有ATP识别片段的两部分适配体寡核苷酸单链与ATP相互特异性结合形成“适配体-ATP-适配体”三元复合物，结合前后空间几何结构的变化促使液晶分子取向排列变化，从而导致光学信号发生变化，来实现ATP的检测。本实验对实验条件、选择性、以及传感器的检测性能进行了考察。实验结果表明：该方法的灵敏度较高，检测ATP的浓度可低至10nM,并且具有良好选择性。(2)构建了一种基于胸腺嘧啶-汞离子-胸腺嘧啶（T-Hg2+-T)特异性配位作用的液晶生物传感器，实现了Hg2+的高灵敏检测。新设计探针分别为富含胸腺嘧啶(T)的发卡型探针、富含T的单链探针及末端带有活泼氨基的单链探针。将末端带有活泼氨基的单链探针作为捕获探针固定在TEA/DM0AP混合组装膜修饰的玻片基底表面，当待测液中有Hg2+存在时，由于T-Hg2+-T特异性配位作用，能够有效诱导富含T的发卡型探针与富含T的单链探针发生碱基错配形成稳定的刚性双链结构，并促使发卡探针打开，所释放出其余碱基与固定在基底表面的捕获探针碱基互配结合至玻片基底表面。结合前后空间构象及几何尺寸发生改变，导致液晶生物传感器在偏光显微镜下观察到的光学信号图像从均一黑色变为颜色鲜明的织构图像。从而实现对Hg2+的高灵敏检测。检测Hg2+浓度可低至0.1nM。(3)提出了一种基于构象转换的裂开型核酸适配体与ATP特异性结合的液晶生物传感方法检测ATP。设计具有发卡型结构和末端带有活泼氨基的两条分别含有部分ATP核苷酸适配体序列的核苷酸探针链。将末端带有活泼氨基的单链探针作为捕获探针固定在经TEA/DMOAP诱导液晶分子垂直排列的混合组装膜修饰的玻片表面。当存在ATP时，捕获探针、发卡型探针与ATP相互结合形成三元复合物而促使发卡打开并固定在玻片基底表面。构象发生变化，引发光学信号改变。从而实现对ATP的检测。该方法能够有效减少非特异性吸附对实验的影响，具有较高的灵敏度和选择性。更多还原

【Abstract】 Small molecules and proteins in the life activity of human are indispensablematerials, their detections are also of great importance. However, the small moleculesusually have small molecular weight and size. Due to those limitations, small moleculedetection is more difficult than protein detection.Liquid crystal (LC) is a kind of special material which can exhibit the mobility ofliquids and the anisotropy of solid crystals. Based on the special properties of the LC,Abbott and co-workers proposed a new concept named LC biosensor by using LCs assensing elements in the detection of biomolecules. The LC biosensor is simple, robust,and inexpensive, and it is easy to miniaturization and array.Aptamers can specifically recognize small moleculars，and it usually accompanyby a notable structural change aptamers. Such a structural change is able to induce ahomeotropic-to-tiled transition of the LC molecules surrounding them, and furtherproduce the optical signal change of optical images under the crossed polarized light.In the thesis, we developed several small molecular LC biosensors based on theprinciple. The detailed methods are as follows:(1)We presented a novel LC biosensor for the detection of adenosine triphosphate(ATP) based on the two split aptamer chips specifically recognize and binding withATP. The TEA/DMOAP mixed self-assembled monolayer (SAM) which can induceLC molecules homeotropic alignment modified on the glass surface and the5’-amino-modified oligonucleotide probe which contain a part of ATP aptameroligonucleotide immobilized on the glass surface via covalent bind. In the presence ofATP, those two split aptamer chips will be special hybridization with each other andthe ternary complex is formed. The aptamer structure change which can disrupt theorientational arrangement of LCs, and case the optical signal image change. We alsoinvestigated the experimental condition, specificity, and detection performance. Theresult showed that the proposed method can detect ATP concentration as low as10nMand it is a sensitive and simple sensor to realize the detection of ATP.(2)We constructed an ultrasensitive Hg2+LC sensor based on the specificalrecognization of Hg2+by two DNA thymine bases (T-Hg2+-T). There were threeessential elements: a thymine-rich (T-rich) hairpin oligonucleotide probe, a T-richsingle-stranded oligonucleotide probe, and a5’-amino-modified oligonucleotide probe. The5’-amino-modified oligonucleotide probe, as capture probe, was first immobilizedon the glass surface modified with the TEA/DMOAP mixed self-assembled monolayer(SAM). In the presence of Hg2+，the T-rich single-stranded oligonucleotide and theT-rich hairpin oligonucleotide probe probe then hybridized via the special T-Hg2+-Tinteraction, and maked the hairpin loop open. The latter further hybridized with thecapture probe immobilized on the SAM. The change of molecular space conformationand size before and after hybridization led to the optical signal change of LC sensor.The proposed method can detect Hg2+concentration as low as0.1nM, exhibiting highsensitivity.(3) We proposed a kind of ATP liquid crystal biosensor based on conformationtransition principle. A Hairpin oligonucleotide probe and A single-strandedoligonucleotide probe were designed for the recognization of ATP. The single-strandedoligonucleotide probe was first immobilized on the glass slide modified theTEA/DMOAP mixed SAM. The hairpin oligonucleotide probe and the single-strandedoligonucleotide probe possess different parts of the recognition sequences of ATPaptamer which can specifically recognize of ATP. In the presence of ATP, these twoprobes can combined together with ATP and formed a ternary complex, leading thehairpin probe to be opened and immobilized on the SAM. The introduction of the ATPternary complex after hybridization can cause the optical signal change from black tobirefringent texture. This LC sensing method has good sensitivity and selectivity sincethe hairpin oligonucleotide probe conformation transition.更多还原