基于交联增强自组装策略构建高稳定性量子点荧光探针及其在肺癌miRNA标志物检测中的应用

Due to the great need for early diagnostics of lung cancer, this project is aimed to develop a novel high sensitive detection technology of miRNA biomarkers for lung cancer based on Quantum dots (QDs). In order to solve the QDs fluorescent probe’s instability problem which seriously constrains the improvement of sensitivity and liability of the detection system, we propose a ‘cross-linking enhanced self-assembly’ strategy for QD’s surface modification. This method utilizes a newly designed thiol-containing amphiphilic diblock polymer as the surface modifier. The coordinating bonds between thiol groups and metal ions on the QD’s surface are firstly used to assembly the polymer to form ordered surface modification layer. Then the excess thiol groups in the polymer shell will function as cross-linking agent via oxidation between thiol groups, which provides strong covalent bonding and significantly enhances the stability of the surface modification layer. This strategy can break through the limit of stability-luminescence contradiction derived from either traditional surface modification strategies and produces high performance QD fluorescent probes in controlled manner. Based on the high quality QDs, QD-DNA fluorescent probes can be prepared and used to construct a highly sensitive miRNA fluorescence detection platform under base-pair complementary principle combined with magnetic separation of nano-magnetic beads. QDs with different emission colors can be used to prepare fluorescence programmed bioprobes and to simultaneously detect multiple lung cancer miRNA biomarkers with high sensitivity and specificity. This project will provide a versatile surface modification strategy for both QDs and other nanomaterials, and can offer potentially useful detection technology for lung cancer diagnostics.

本项目面向肺癌早期诊断的重大需求，以实现基于量子点的肺癌微小核糖核酸（miRNA）标志物的高灵敏荧光检测为研究目标，针对当前量子点荧光探针稳定性不足而严重制约检测灵敏度与可靠性的问题，重点发展 “交联增强自组装”的量子点表面修饰策略，设计具有巯基链段的两亲嵌段聚合物作为表面修饰剂，利用巯基与金属离子间的配位键在量子点表面组装有序的聚合物修饰层，进而利用巯基之间的氧化交联增强表面修饰层的稳定性，突破传统策略中稳定性与荧光性能难以兼顾的局限，实现高性能量子点荧光探针的可控构建。以此为基础，制备量子点-DNA荧光探针，基于碱基互补配对原则，结合纳米磁珠的磁分离能力，发展高灵敏、高特异性的miRNA荧光检测技术。利用不同发射波长的量子点对核酸探针进行荧光编码，实现肺癌miRNA标志物的多元联合检测。本项目的实施将为量子点和其它纳米材料提供一种通用的表面修饰策略，并为肺癌的早期诊断提供技术支持。

肺癌是一种在全球范围内发病率与死亡率最高的恶性肿瘤。为提高肺癌的早期诊断水平，当前迫切需要发展具有高灵敏度、高特异性的肺癌标志物检测的新技术和新方法。作为新一代的荧光标记物，量子点（QDs）相对于有机染料具有很多优异的荧光性能。尽管当前基于量子点的荧光检测技术已经展现出良好的发展前景，但是量子点稳定性不足的问题严重制约着检测灵敏度与可靠性的提高。针对以上问题，本项目利用活性聚合可控制备了新型多齿巯基嵌段聚合物，利用巯基的配位能力、交联能力和多齿协同作用，显著提高了量子点的光稳定性、盐稳定性、pH稳定性等性能，为提高量子点的稳定性提供了新的表面修饰策略。基于相似的策略，通过沉淀聚合制备了含有多齿巯基的聚合物微球，利用巯基的配位作用和协同作用，制备了具有较高负载效率的量子点荧光微球。发展了厚壁核壳型量子点微波水相合成技术，制备的CdTe/CdS量子点的壳层厚度可调，量子点的荧光发射波长覆盖可见光到近红外的区域（500-900 nm），而且量子点的荧光量子效率可达85%，显著优于普通的水相量子点。利用量子点优异的光学性能和磁珠的高效磁分离能力，发展了量子点-磁珠荧光传感体系（QD-MB），利用不同发光波长的量子点实现两种肺癌标志物miRNA（miRNA-21和miRNA-200b）的高灵敏、高特异性的联合检测。在血清体系中QD-MB传感体系对miRNA-200b和miRNA-21的检测限分别为6.09 fM和3.90 fM，检测线性范围分别为10 fM-10 pM和10 fM-100 pM。与其它的miRNA荧光传感体系相比，QD-MB传感体系具有较高的检测灵敏度、较宽的线性范围和良好的特异性，并且操作简单，为肺癌的早期诊断提供了技术支持。在此基础上，通过结合双链特异性核酸酶（DSN）的选择性剪切能力，发展了基于DNS酶信号放大的QD-MB荧光传感体系，实现对肺癌标志物miRNA-21的高灵敏检测。