HCR介导的核酸功能化碳点/氧化石墨烯荧光探针用于miRNA超灵敏检测及肿瘤细胞筛选

The kinds and contents of microRNA (miRNA) play an important role in early in situ diagnosis and screening of different tumor cells. Owing to intracellular miRNA in different tumor cells has very low content, variety, and easy digestion et al, the fluorescent probe techniques face challenges in the realization of high sensitivity, multiplex, and in situ detection of the miRNA. Herein we propose to develop DNA functionalized carbon dots (CDs)/ graphene oxide (GO) nanocomplex fluorescent probes with hybridization chain reaction (HCR) signal amplification technology for ultrasensitive detection of miRNAs in living cells. In this nanocomplex fluorescent probes, the environmentally friendly, high quantum yield CDs are used as fluorophores, GO is used as a quencher and carrier platform. The hairpin nucleic acids are modified onto surface of the CDs to form DNA-CDs, and then the DNA-CDs are adsorbed onto GO to assemble nucleic acid functionalized CDs/GO nanocomplex fluorescent probes, showing the quenched fluorescence of CDs by GO. At the presence of the target miRNA, the miRNA hybridize with DNA-CDs to form DNA/RNA double chain, leading to DNA hybridization with each other. The DNA-CDs release from the surface of GO, and the fluorescence of CDs is recovered with “ON” model to ultrasensitive detect of the miRNA. The nanocomplex fluorescent probes are expected to multiplex detect of miRNA with high sensitivity and specificity. Moreover, owing to good biocompatibility and cell penetrability of the nanocomplex fluorescent probes, we are also able to develop measurement of miRNA in living cells and screening of tumor cells. Therefore HCR mediated nucleic acid functionalized CDs/GO nanocomplex fluorescent probes will provide avenue for ultrasensitive early in situ diagnosis research of different tumor cells.

肿瘤细胞内miRNA种类和含量对不同肿瘤细胞的早期原位诊断及筛选等至关重要。由于细胞内miRNA具有含量低、种类多，易降解等特点，使用荧光探针技术在实现超灵敏性、多元、原位检测miRNA以及肿瘤细胞筛选方面面临挑战。本项目拟以DNA包覆的高量子产率碳点(CDs)作为荧光团，氧化石墨烯(GO)作为猝灭剂和载体，结合杂交链式反应（HCR）信号放大技术，组装成HCR介导的核酸功能化CDs/GO纳米荧光探针，通过miRNA诱导核酸功能化碳点之间发生HCR，控制碳点在GO表面的吸附或脱附，实现碳点荧光信号放大，以荧光“开”的方式实现超灵敏、多元检测miRNA。该纳米荧光探针具有低毒性和良好的细胞穿透性，可实现细胞内miRNA超灵敏原位检测及肿瘤细胞筛选。发展新型HCR介导的核酸功能化CDs/GO纳米荧光探针技术，对不同肿瘤超灵敏早期原位诊断具有重要理论意义。

肿瘤细胞内miRNA、离子、生物分子等种类和含量对不同肿瘤细胞的早期原位诊断及筛选等至关重要。由于细胞内miRNA、离子、以及生物分子具有含量低、种类多等特点，在实现超灵敏性、多元、原位检测miRNA、离子、生物分子以及肿瘤细胞筛选方面面临挑战。本项目开发了核酸功能化CDs/GO纳米荧光探针，实现了对miRNA-21的检测，并且实现了原位成像，为血癌的原位诊断和治疗提供基础研究。同时，构建了多功能纤维素碳点（CNC-CDs）并成功应用于环境和生物样品中检测。在纤维素原位合成碳点，通过荧光猝灭方式检测Hg2+；通过FRET机制，以卟啉（TSPP）为受体，构建了比率Fe3+荧光传感器。并应用于细胞内半定量的Fe3+成像。以柠檬酸、生物质材料等为碳源，制备了碳点，并应用于Fe3+、多巴胺等检测。该纳米荧光探针具有低毒性和良好的细胞穿透性，可实现细胞内miRNA、离子、生物分子超灵敏原位检测。对肿瘤超灵敏早期原位诊断具有重要理论意义。