基于双级、多维编码及信号放大的高灵敏多元检测技术及其应用研究

Semi-conductive quantum dots (QDs)-encoded microbeads have shown great potential in multiplex assays of nucleic acids and proteins. However, this technology currently has several limitations in almost every step from encoding, decoding and bio-molecular detection. And more seriously, these limitations are linked to and affected by one another. Therefore, in this project, a systematic and comprehensive study on QDs-encoded microbeads technology will be carried out, which includes: combining droplet microfluidics with various materials to develop an accurate and controllable method for QDs encapsulation; using the size and shape of microparticles as additional elements for multi-dimensional encoding, so as to greatly improve the code quantity without increasing the QDs colors or intensity levels; integrating bio barcode with QDs-encoded microbeads for "tandem encoding & signal amplification", which leads to high sensitive multiplex assay; exploring novel decoding technology based on multispectral imaging, which will provide solutions for recognizing microbeads encoded with more QDs colors or even multi-dimensional information. Since all the aspects from beads synthesis, identification, to application have been considered as a whole, an important feature of this project is the integrity and innovativeness, which is expected to overcome the current drawbacks thoroughly, and promote the application of QDs-encoded microbeads in the fields of genomics and clinical diagnostics.

半导体量子点（QDs）荧光编码微珠在核酸及蛋白分子的多元检测中具有十分重要的价值。但当前该技术无论在微珠制备、解码识别还是实际样品检测等环节均存在一定不足，而且这些不足之处相互联系、相互影响。为此，本申报项目拟开展全面、系统的QDs荧光编码微珠技术研究：结合液滴微流控与多种优势材料发展一种高度精确、可控的微珠编码方法；引入微珠粒径与形状变化实现简单而有效的多维信息编码，从而在不增加QDs颜色和强度水平的条件下大幅度提高编码容量；结合生物条码技术，实现双级编码及信号放大，发展一种高灵敏的多元检测技术；探索多光谱成像微珠解码技术，为更多QDs颜色及多维信息编码微珠的识别提供解决方案。由于综合考虑了从编码微珠制备、解码识别到生物分子检测的所有环节，本项目的最大特色在于方案的完整性和创新性，可望较彻底地解决当前存在的诸多局限，促进QDs荧光编码微珠技术在基因组学与临床诊断等领域的研究与应用。

以半导体量子点（Quantum dots，QDs）荧光编码微珠为代表的悬液芯片在核酸、蛋白质等生物分子的多元检测中具有重要价值。然而，当前的QDs荧光编码微珠技术在编码（重复性、均匀性、编码容量）、解码（过多依赖于流式检测）、以及实际样品检测（光谱重叠，弱样品信号与强编码信号之间的矛盾）等环节均面临一定挑战。为此，本项目以液滴微流控技术为基础，并结合多种优势材料，如光固化材料聚乙二醇二丙烯酸酯、两亲性嵌段共聚物聚1,4-丁二炔-b-聚环氧乙烷和低温琼脂糖，发展了多种QDs荧光编码微珠，不但可以大大提高QDs的装载量、均匀性和重复性，同时还可以将微珠直径作为编码信息以实现三维编码（荧光颜色、强度、微珠直径），从而大幅度增加编码容量。此外，本项目还发展了多种分子检测信号放大方法，以解决弱样品信号与强编码信号之间的矛盾，从而提高多元检测的灵敏度。特别是发展了一种定位探针介导剪切技术（AMC）以及以此为基础的核酸等温指数扩增方法（AMC-SDA）。前者在核酸等温扩增、质粒构建以及目前研究最火热的基因编辑等领域均具有良好的应用潜力；后者不但具有高灵敏度、高特异性，而且还具有良好的通用性，可以对任一核酸序列进行扩增检测。考虑到AMC-SDA的启动不从引物的延伸开始，后续的研究中可以在引物上做各种修饰，以使其既不作为聚合酶的引物，也不作为反应模板，从而抑制在大多数等温指数扩增方法普遍存在的primer-dimer及non-priming synthesis等非特异性扩增作用。解决这一难题将使得AMC-SDA在疾病早期诊断等许多领域具有非常好的应用前景。