含二茂铁的双嵌入型电化学核酸传感器杂交指示剂的设计合成及在基因传感检测中的应用

With the help of lable-free electrochemical nucleic acid based sensor, detection of target nucleic acids with specific sequence has been adapting in molecular diagnostics, biomedicine, forensic, food safety and environmental science. Due to the poor selectivity, poor water solubility, free-radical-induced nucleic acid probe damage and the unsuitable potential windows of traditional electrochemical nucleic acid hybridization indicators, their applications are limited in lable-free electrochemical nucleic acid based sensor. Kinds of novel indicators were synthesized to avoid the disadvantages. In our plan, a serious of electrochemical nucleic acid hybridization indicators will be designed and synthesized, including dumbbell-shaped, T-Shaped and comb-shaped indicators. Different number ferrocene groups are combined with bisintercalators in the indicator as electrical signal units. The length and structure of the spacers in the indicator are varied to adjust the binding selectivity between the indicator and single/duplex strand nucleic acid and to improve the water-solubility of the indicators. The binding selectivity, sensitivity and water solubility of the synthetic indicator are compared by UV-Vis titration, fluorescence titration, viscosity titration and cyclic voltammetry methods. Then the selected excellent indicators are used in lable-free electrochemical nucleic acid hybridization sensors which are adapted to detect short DNA species of Chronic Myelogenous Leukemia or miRNA 21 and so on. After the plan was finished, it is beneficial to our further study in rational design of new kinds of electrochemical hybridization indicators and also contributed to build electrochemical sensors which was used in detection of sequence specific nucleic acids.

非标记型电化学核酸传感器对特定序列核酸的测定在分子诊断、生物医学、法医学、食品安全和环境科学等方面有着极为广泛的应用。传统电化学杂交指示剂通常有选择性差，水溶性差，会氧化损伤核酸探针或电化学窗口不适合等缺点而使其在非标记型电化学核酸传感器中的使用受到限制。本项目拟设计合成哑铃型、T型和梳型结构以二茂铁为电化学信号单元的双嵌入型电化学核酸传感器杂交指示剂，通过改变嵌入基团间链长、链的结构调节其对单双链核酸的结合能力和选择性。在合成的杂交指示剂中，通过紫外滴定、荧光滴定、粘度滴定及循环伏安法等方法选出具有高结合能力和结合选择性、良好水溶性、灵敏的电化学活性的优秀核酸杂交指示剂。以其为基础构建非标记型电化学核酸传感器用于测定慢性髓细胞白细胞DNA、miRNA21等目标核酸片段。本项目完成后，将为合理设计双嵌入型电化学杂交指示剂提供经验和基础，并为电化学核酸传感器的构建和应用提供支持。

非标记型电化学核酸杂交指示剂是构建非标记型电化学核酸传感器的一个重要组成部分，现有的非标记型电化学核酸杂交指示剂在氧化还原窗口、水溶性、结合选择性及抗干扰性方面存在一种或多种缺点而限制了非标记型电化学核酸传感器在核酸检测中的优势。本项目针对这一问题，在前期工作中发现双嵌入型分子对双链核酸具有更好的结合能力和选择性的基础上，结合具有良好电化学活性及氧化还原电位窗口的二茂铁基团合成一系列新的非标记型电化学核酸杂交指示剂。通过考察所合成的电化学指示剂与核酸的结合能力、结合模式，在此基础上进一步比较单、双链核酸结合选择性及抗干扰能力，最终筛选出具有应用潜力的嵌入型电化学核酸传感器杂交指示剂。该类型电化学指示剂较常用的非标记型电化学指示剂亚甲蓝和道诺霉素有更大的电化学信号以及更好的单、双链核酸结合选择性和抗干扰能力。以其为信号单元构建非标记型核酸电化学传感器用于模拟肿瘤标志物样本的检测，在未使用任何信号放大策略时检测限就能够达到pmol水平，简单使用金纳米粒子修饰电极可提高检测限至fmol水平，该类型电化学核酸杂交指示剂已申请专利。在此基础上，还通过在非标记型核酸电化学传感器构建中引入环糊精改善含二茂铁的嵌入型电化学指示剂的溶解度，从而进一步提高传感器的检测限和灵敏度。该方法在改善采用此类指示剂构建的非标记型电化学核酸传感器检测限和灵敏度方面具有良好的普适性。此外，我们在合成相关杂交指示剂的过程中还发现了一些新的二茂铁衍生物的合成方法以及非天然氨基酸的合成方法，并申请了专利。基于上述指示剂构建的非标记型电化学核酸电化学核酸传感器较传统指示剂构建的传感器在检测限、选择性和抗干扰能力方面均有所提升，我们将在后续研究中，结合不同信号放大策略优化传感器参数，以便将其进一步推广使用。