基于无酶核酸信号放大的细胞内端粒酶活性高灵敏检测与成像研究

In this project, it is proposed to develop nanoparticle-mediated enzyme-free nucleic acid signal amplification strategy for highly sensitive detection and imaging of intracellular telomerase activity in situ. Through rational designing nucleic acid probes for telomerase-triggered release of target molecules and assisting of enzyme-free signal amplification methods such as catalyzed hairpin assembly, hybridization chain reaction, and DNAzyme, a series of new recognition and signal amplification strategies will be developed. In addition, by utilizing nanoparticles with biodegradation, low cytotoxicity, and high nucleic acid delivery efficiency as the carrier, a novel intracellular highly sensitive fluorescence sensing platform will be constructed, and new technology and new method will be developed for highly sensitive and highly selective detection and imaging of telomerase activity in living cells. Moreover, Real-time monitoring the changes of telomerase activity in different physiological and pathological processes will be realized, which is benefit for exploring the association between the changes of telomerase activity and the occurrence and development of cancer, further providing technical and methodological support for the early diagnosis and prognosis assessment of cancer. Therefore, this project has important scientific research significance and high value of practical application, and some innovative research results are also expected with the execution of current project.

本项目拟开展基于纳米粒子介导的无酶核酸信号放大新策略，实现细胞中端粒酶活性的高灵敏原位检测与成像。通过合理设计核酸探针进行端粒酶触发的靶标分子释放，借助催化发夹组装、杂交链式反应和DNAzyme循环等无酶核酸信号放大手段，发展识别与信号放大新策略，结合可生物降解、细胞毒性低、核酸运载效率高的功能化纳米粒子，构筑细胞内高灵敏荧光传感新平台，建立细胞内端粒酶活性高灵敏、高选择性分析检测新技术和新方法。实时监测不同生理病理学过程中端粒酶活性的变化，探索端粒酶活性变化与癌症发生、发展之间的关联，为癌症的早期有效诊断和预后评估等提供技术和方法支持，具有重要的科学研究意义和实际应用价值，有望获得一定特色的创新性研究成果。

发展高灵敏荧光传感新策略，实现细胞内端粒酶活性简单、快速、高灵敏、高特异性检测，为癌症的早期有效诊断和预后评估等提供技术和方法支持，具有十分重要的研究意义。本工作开展了高灵敏荧光传感新方法研究，基于催化发夹自组装和外切酶III切割反应，首次构建了免标记比率荧光传感策略，实现了癌细胞内端粒酶活性的高灵敏、高选择性检测；基于DNA纳米结构，酶辅助目标物循环、杂交链式反应等技术，发展了多种信号放大新策略，建立了若干肿瘤标志物高灵敏均相传感新方法，实现了癌细胞、癌细胞分泌的外泌体的高灵敏、高特异性检测；基于DNA纳米复合物探针，建立了均相传感策略，实现了细胞内和细胞外过氧化氢、环境污染物砷离子的简单、高效、灵敏检测。在国家自然科学基金资助下，在Analytical Chemistry、Chemical Communications、Biosensors & Bioelectronics等学术期刊上发表SCI收录论文8篇，影响因子均大于5.0；申请国家发明专利1件。指导在读硕士研究生2人。项目负责人获2020年度山东省自然科学二等奖（第四位）、2019年度山东省高等学校科学技术一等奖（第三位）各1项。