用于单细胞水平上检测干细胞内多种miRNA原位表达的多色纳米传感探针的研究

The recent studies show that the special expression of miRNA in stem cell is closely related to its behaviors of multipotent differentiation. The quantitative measurement of in-situ miRNA expression in stem cells is highly important for predication of differentiation direction of stem cells. The traditional methods for miRNA detection is labor-intensive and time-consuming which needs additional steps of cell lysis and miRNA extraction. Moreover, these traditional methods can not realize real-time miRNA detection in living stem cells. The emerging in-situ miRNA detection methods can realize real-time miRNA detection in living cells, but mainly focus on single type of miRNA detection in tumor cells. There are very few studies of in-situ miRNA detection in living stem cells. The purpose of this project is to develop a bio-functionalized gold nanoparticle (AuNP)-molecular beacon-graphene quantum dot (GQD) based fluorescence resonance energy transfer (FRET) nanoprobe for multiplexed simultaneous detection of miRNAs in single human Mesenchymal stem cell (hMSc). The combination of multi-color GQDs with good fluorescence property, nanoparticle based fluorescence resonance energy transfer (FRET) sensing mechanism with high sensitivity, and the chemically synthesized aptamer molecules as recognition elements for stem cells with high specificity and stability, provides a novel cellular nanoprobe for multiplexed in-situ detection of miRNA with high sensitivity and specificity. Successful delivery of the proposed milestones of this project can provide a new nano-biosensing platform to realize the multi-functionality including cell recognition, transfection and in-situ intracellular miRNA detection in single stem cells at the same time, which will have important applications for evaluation and prediction of stem cell differentiation status.

最近的研究表明，干细胞中miRNA 特异性的表达，与其各种定向分化行为密切相关,定量分析miRNA的原位表达对干细胞差异性分化的预测有着重大意义。传统的miRNA检测方法需要进行细胞裂解及提取等繁琐步骤，无法实现活体细胞内的原位检测。而目前miRNA原位检测技术集中在肿瘤细胞的应用，且主要是单类miRNA的检测。本项目拟合成多色石墨烯量子点，与具有高度特异性的核酸分子信标偶联进行荧光编码，建立纳米金粒子-分子信标-石墨烯量子点的FRET传感系统，并通过链接特异性配体分子实现对干细胞的靶向识别，利用表面功能化增强细胞转染效率及保护核酸分子信标免受干扰，在对传感特性进行研究分析的基础上，最终构建出功能化的纳米生物分子检查探针，实现对人类骨髓基质活体干细胞(hMSc)单细胞水平的识别、转染以及miRNA高灵敏度的原位同步检测的一体化，这将对干细胞多元分化预测的研究，提供一个全新的检测分析平台。

传统的细胞检测基于RT-PCR和ELISA等传统生化检测手段。无法实现对活体细胞内的原位检测且检测灵敏度和稳定性不佳。对活体细胞内的多种生物分子进行灵敏快速原位检测依然是目前的一个难题。本项目基于类石墨烯纳米材料的其高化学和光学稳定性以及独特的荧光特性等优点构建了纳米荧光检测探针，基于各种纳米荧光材料和光淬灭纳米材料，构建荧光能量转移探针。同时对各种纳米材料表面进行表面改性，利用自组装修饰技术，用化学交联剂对纳米材料进行功能化处理，嫁接生物识别分子用于特异性识别。对传感探针的检测灵敏度和特异性进行研究分析，阐明其传感响应机制及传感机理，构建出纳米生物分子检测平台。并利用微纳米加工技术制备了结合纳米检测探针的微纳阵列检测平台，可进行多个样品的平行检测。最终对传感探针的检测灵敏度和特异性进行研究分析，构建出用于活细胞原位检测的纳米生物分子检测平台。最后利用构建的生物纳米检测探针实现对活体细胞的多种生物分子高灵敏度的原位同步检测的一体化。本项目对活体细胞原位检测以及检测和诊疗一体化探针的研发具有重要的意义。