用于G-四链体DNA识别的量子点/大环分子荧光探针研究

Discovery of the G-quadruplex DNA structure, as well as the revelation of it's close relationship with cancer conducted by modern molecular biotechnology have provided new opportunities for the development and screening of anti-cancer molecules. Small molecule, conjugated polymer, and quantum dot based fluorescent probes can be utilized to screen G-quadruplex DNA-targeted anti-cancer molecules. Owing to the polymorphism of the G-quadruplex structure, however, there are two challenges in the G-quadruplex DNA detection with fluorescent probes: the first is the specific interaction between G-quadruplex DNA and probes; and the second is the sensitivity of the detection. Small-molecule compounds have advantages of the highly specific recognition as well as the wide availability of molecular structure, whilst quantum dots have excellent fluorescent properties and the ease of surface functionalization. In the past, we developed a series of fluorescent probes based on quantum dots hybridized with small molecules. The hybrid fluorescent nano-probes have been successfully utilized in the detection of trace ions including Zn2+, Ba2+ and Cd2+ in water and living cells. Herein, we propose to develop novel hybrid quantum dot/macrocyclic molecules probes for selective and sensitive assay of G-quadruplex DNA detection. In such hybrid fluorescent probes, quantum dot is the fluorescent signal resource, while the macrocyclic molecule acts as the surface ligand. By the careful design of molecular structure, the macrocyclic molecule could be used to specifically interact with G-quadruplex DNA. The novel hybrid fluorescent nano-probes are expected to detect G-quadruplex DNA with high sensitivity and specificity. Moreover, in combination with the quantum dot-coding technique, we are also able to develop the methods of multiplex detection of G-quadruplex DNA with fluorescent nano-probes. Therefore, the hybrid quantum dot/macrocyclic molecule fluorescent probes are of significance in developing low-cost, rapid, in-situ, real-time, multiplexed, and visual screening techniques of targeted drugs.

荧光探针技术广泛应用于以G-四链体DNA为靶点的抗癌药物分子筛选。由于G-四链体DNA具有多态性，现有荧光探针技术在同时实现对G-四链体DNA的特异性、高灵敏性及多元检测等方面面临挑战。在成功实现杂化型纳米荧光探针对于活细胞中痕量离子检测的基础上，申请者在本课题中提出发展新型的量子点/大环分子杂化型荧光探针用于G-四链体DNA的识别研究。在杂化型荧光探针中，高荧光量子产率的量子点是荧光信号源；对G-四链体DNA特异性识别的大环分子如卟啉（酞菁）衍生物作为量子点的配体。杂化型纳米荧光探针可预期不仅同时实现对G-四链体DNA的高灵敏性、高特异性检测，同时结合量子点编码技术可发展出对G-四链体DNA实现多元检测的原理和方法。新型量子点/大环分子杂化型荧光探针对于发展低成本、快速、原位、实时、多元及可视化的靶向药物筛选方法具有重要的理论意义和应用价值。

荧光探针/传感器可广泛应用于以G4-DNA为靶点的抗癌药物筛选、药物输送，以及肿瘤细胞诊断。本课题基于量子点（QD）和大环/稠环分子发展出了多种超结构的荧光传感器用于G4-DNA的低成本、快速、原位、实时、多元及可视化检测。（1）基于双色量子点 “core-satellite”结构，利用稠环分子（DI）为外层QD配体，构建了比率荧光探传感器用于G4-DNA的快速、可视化检测。检测原理为靶标存在条件下配体置换反应调控QD表面电荷转移过程。内层QD荧光恒定，外层QD（不同颜色）荧光“关-开”，可实现G4-DNA的可视化检测。响应时间为10分钟，达到的检测限为7 nM。（2）基于量子点荧光编码微球（Qbead），结合锁式滚环扩增（RCA）技术和非标记的大环分子（ZnPc）荧光报告技术，构建了荧光传感器阵列用于G4-DNA的多元检测。检测策略为靶标G4-DNA触发Qbead表面的RCA反应，实现ZnPc荧光信号高效放大。利用量子点编码荧光为G4-DNA识别信号，ZnPc荧光为报告信号，通过流式细胞术实现了三种G4-DNA序列（telo、bcl2、k-ras）的同时检测，检测限达到2.2 nM。（3）基于生物素化的纤维素纸，利用生物素-链霉亲和素特异性作用组装Qbead，构建了纸基荧光传感器微阵列，用于G4-DNA低成本筛选和检测。检测原理为利用大环分子（ZnPc）、靶标序列、QD标记的报告序列、Qbead上捕获序列之间的竞争反应。基于比率荧光技术，该纸基荧光传感器微阵列实现了三种G4-DNA序列的 “信号灯”式多元检测；同时实现了G4-DNA定量，检测限达到10 nM。（4）基于生物分子（PEG和TAT）功能化的无毒InP/ZnS量子点，超声辅助组装大环分子酞菁锌（ZnPc），构建了超结构的新型纳米荧光探针。量子点荧光用于纳米示踪，ZnPc荧光用于G4-DNA特异性识别。通过共聚焦荧光成像，实现了人类肝癌（HepG2）细胞染色体上G4-DNA的原位检测。基于该探针高分辨荧光成像，实时监测HepG2细胞不同分裂周期，可实现肿瘤细胞诊断。这些新型、低成本、可视化荧光探针/传感器的发展对于基础生物学研究和临床医学诊断具有重要的理论意义和应用价值。