三链DNA编码的纳米孔技术用于肿瘤标志物的同步分析

Nanopores-based sensing technology is leading a technological revolution due to its unique advantages of ultrafast, not requiring labeling/immobilization, and the ability of obtaining biomolecular information at single-molecule level. Presently, the nanopore sensing for nucleic acid analysis has been well developed. In comparison, the study on the detection of proteins by using nanopore is still facing with many challenges, such as signal interference, low throughput, and difficulty in multiplex analysis. Focusing on the clinical needs of combined detection of tumor markers in primary liver cancer, this project would bring about a new nanopores-sensing strategy for converting protein analysis to nucleic acid analysis. This strategy combines the molecular structure characteristic of the triple-helix DNA with the separation concept of probe recognizing-part and the signal reporting-part. On this basis, multiplexed sensing of tumor markers with nanopores will be achieved by designing a library of encoded triple-helix DNA with each member containing a unique length or secondary structure, which would produce different blockage currents. In order to eliminate various interfering signals, electrophoresis/dielectrophoresis technique will be used in this project. The execution of this project would not only provide powerful tool for exploring the relation between tumor markers and tumor occurrence, development, and prognosis, but also open up a new avenue for the development of a convenient, high throughput nanopore-based instruments.

纳米孔传感技术因其快速、无需标记、能从单分子水平获得生物分子相关信息等优点，正引领着一次新的技术变革。目前基于纳米孔的核酸分析技术已发展成熟，而利用纳米孔检测蛋白质的研究面临信号干扰大、通量低、难以多目标分析等挑战。本项目以原发性肝癌肿瘤标志物联合检测的临床需求为出发点，结合DNA三链结构可实现信号转换并使探针识别单元与信号报告单元分离的特性，提出将蛋白质信息直接获取转化为纳米孔核酸分析的解决思路。在此基础上，利用不同长度/二级结构的DNA报告单元通过纳米孔时产生阻断电流信号的差异进行信号编码，从而达到多种蛋白同时检测的目的。此外，通过电泳/介电泳技术消除干扰物带来的信号干扰。本项目的执行为探究生物活性物质变化与疾病成因提供强有力的工具，同时为开发便捷式、高通量的纳米孔分析仪器提供基础。

发展准确高效的生物信息获取技术，是当前分析化学研究的核心内容之一。本项目以疾病生物标志物联合检测的临床需求为出发点，结合DNA三链分子开关（TMS）可实现探针识别单元与信号报告单元分离的特性和纳米孔技术具有无需标记、能从单分子水平获得生物分子相关信息的优点，开发了长度和二级结构编码的三链分子开关纳米孔检测平台用于阿尔兹海默多重蛋白标志物的同时检测。TMS与待测蛋白质特异性结合后释放DNA信号报告探针，利用长度和二级结构迥异的 DNA 报告探针通过纳米孔产生差异性的阻断电流进行信号编码，从而实现对阿尔兹海默症三种蛋白类肿瘤标志物的完全区分。该策略实现了对AD靶蛋白的高灵敏检测，并适用于自然浓度迥异的多种靶标同时分析，为复杂疾病的联合诊断技术提供了新的选择。