DNA纳米技术调控的新型芯片体系开发及其microRNA检测研究

Sensitivity and specificity are still the two major bottlenecks that restrict the application of the microarray. The development of DNA nanotechnology brings new ideas and possibilities for the biodetection application of microarrays. In the project, DNA tetrahedral nanostructures were employed as capture probes. Through precisely nanoscale regulation of the recognition interface and the self-assembly process, recognition efficiency and specificity can be greatly improved. Signal amplification strategy based on hybridization chain reaction can trigger signaling cascades as long as there is a target, which would greatly improve the sensitivity, and thus the overall performance of the microarray system can be improved. We aim to develop a newly microarray detection system based on DNA technology with high specificity and sensitivity. Some advanced techniques, such as Quartz Crystal and Atomic Force Microscopy are employed to investigate the physicochemical mechanisms, thermodynamics and kinetics. Then, based on the DNA nanotechnology and nanoscale regulation, we design specific systems for detection of microRNAs with several inputs and realize multi target detection, then evaluate the performance of the microarray platform in complex environment.

灵敏度及特异性低仍是目前制约芯片技术应用的两大瓶颈，DNA纳米技术的发展给生物芯片检测带来了新思路和新可能。本项目从根本界面问题出发，以DNA四面体纳米结构作为探针，设计不同尺寸的纳米结构，对生物分子识别界面以及生物分子自组装进行纳米尺度调控，提高识别效率和特异性；以链式杂交反应（Hybridization Chain Reaction，HCR）为基础的体系为信号输出及放大策略，只要有目标物存在就可以引发信号产生级联放大，提高检测灵敏度，进而提升芯片的整体性能，最终设计一种高特异性、高灵敏度的新型芯片检测体系。利用各种先进技术（包括石英晶体微天平、原子力显微镜等），对整个过程的物理化学机制，热力学以及动力学进行测定和表征。在纳米尺度调控的基础上，设计多种针对microRNA的检测体系，实现多靶标联合检测，并在复杂体系中进行验证。

生物芯片（微阵列），因其杰出的平行性和高通量筛选的优点而广泛应用于生物分子检测领域。然而，多数应用于传统DNA芯片的荧光检测方法仍受限于特异性和灵敏度等指标，不能应用于低丰度的microRNA的检测。DNA纳米技术的高度可设计性及编程性给基于芯片的miRNA检测带来新的思路。本项目通过引入基于DNA四面体设计的探针以及基于链式杂交反应（Hybridization Chain Reacrtion, HCR）的信号放大策略，成功开发出了一种高特异性、高灵敏度的通用核酸检测芯片平台。我们首先设计并合成了3种不同大小四面体探针（DNA tetrahedral structured probe，DSTP）：17bp、26bp和37bp，其大小分别为5.8nm，8.8nm和12.6nm，随后系统考察各个大小探针的动力学检测性能，我们发现不同的探针可以实现不同的动力学检测范围及检测限。其中大小为8.8nm的四面体探针拥有最好的检测性能，其检测限可以低至10aM，优于目前为止所开发的各种检测方法的性能。此外，我们的芯片检测平台能够精确区分待测DNA的原始序列及其单碱基错配序列。最终，我们将优化后的芯片检测技术应用于模拟体系中的miRNA检测并成功实现了三种不同miRNA的同时检测。因此，此项芯片检测技术平台拥有良好的临床诊断应用潜能。