双链DNA标记探针型电化学传感器的研究

Electrochemical DNA sensors have been widespread concern in the research field of life science. Traditional electronic DNA sensors have faced some challenges such as accurately located the electrochemical beacon and economical reageneration. This proposal will fabricate a novel electrochemical DNA sensor which is based on the competitive hybridization to detect the target DNA oligonucleotides. This sensor has inovations as follows: 1) To accurately locate the electrochemical beacon, the probe oligonucleotides employ a Watosn-Crick helix befor and after detect the target by resonable design the sequece of the probe. 2) The probe is shorten by dispart the recognition and signal-transformation functions to each of the single DNA oligonucleotide probe. 3) After detect the target, there are ssDNA probe oligonucleotides lefted on the electrode surface. The simple hybridization of the ssDNA assembled onto the electrodes with an unlabled-ssDNA could regenerate the DNA sensor. Despite the advantages of high selectivity and sensitivity, this study will make improvements to the electrochemical DNA detection such as high signal-stability and economically reageneration. This novel DNA sensor will probably be widely applied in the field of genetic engineering.

DNA电化学传感器因具备选择性好、灵敏度高等优点在生命科学研究领域受到越来越广泛的关注。针对DNA电化学传感器普遍存在的标记物难于准确定位、不易再生等关键科学问题，本项目拟构建基于双链标记探针的新型DNA电化学传感器，该传感器利用竞争杂交反应实现靶标DNA序列的测定，具体包括以下研究：1)通过对传感器探针序列的合理设计，确保在检测靶标前后探针自身均具有双螺旋结构，提高探针二级结构的稳定性。2)将传感器的探针识别功能和信号转换功能拆分到两条DNA链上，从而有效地缩短探针链的长度，使得标记在探针末端的电活性标记物被准确定位。3)在检测机理中引入竞争杂交反应，使得传感器在测定靶标后电极表面仅剩余单链探针，易于传感器再生。通过上述研究，构建电化学信号稳定、再生操作简单、再生成本低廉的新型DNA电化学传感器。基于以上创新性的工作，该传感器的应用研究有望在基因工程相关领域中得到长足的发展。

我们针对DNA寡核苷酸与其对应靶标（小分子、蛋白质、DNA互补链等）之间的强相互作用构建了基于双链DNA的电化学传感器。我们在电极表面固定了部分互补的双链DNA作为探针，通过竞争杂交实现针对靶标的识别。我们构建了一种基于计时库仑技术的DNA核酸适体电化学传感器，该传感器采用六氨合钌阳离子（RuHex）作为电化学响应元件，利用RuHex-DNA-电极体系构建传感器。采用该传感器可以实现大鼠脑透析液中ATP分子的定量测定，并且传感器易于再生。此外，我们利用DNA与其部分错配的互补链杂交形成的双链DNA体系，构建了AND和OR两种DNA逻辑门，此部分的实验数据仍在整理过程中。