单细胞水平的端粒酶活性可视化检测新方法的研究

Telomerase is closely related to the occurrence and development of cancer. Therefore, the detection of telomerase activity is crucial for the telomerase-related early diagnosis of cancer and anti-cancer drug screening. In view of the low sensitivity of visual detection methods reported, this project will focus on developing new methods for the visual detection of telomerase activity with ultra-high sensitivity. These methods are as follows. The multi-color visual detection of telomerase combining with the strand-displacement cycle reaction will be developed to amplify the detection signal. The liposome with high encapsulation efficiency will be selected as a carrier to trap large amounts of enzymes with high catalytic activity, in order to achieve double amplification of the visualization signal. An ultra-sensitive pH-responsive visualization method will be established through converting pH change to visualized signal using an indicator. With aggregation-induced emission luminogens with high quantum yield as a probe and coupling with the cyclic amplification strategy, the telomerase in cell lysate will be determined at single-cell level. And in situ imaging method will be further established to monitor the dynamic changes of intracellular telomerase. These highly sensitive detection methods will enable visualization of telomerase at single-cell level and provide a basis for telomerase-related cancer diagnosis and treatment assessment.

端粒酶与癌症的发生和发展密切相关，因此端粒酶活性的检测对与端粒酶相关的癌症早期诊断和抗癌药物筛选至关重要。针对目前报道的可视化检测方法灵敏度低的问题，本项目拟建立具有超高灵敏度的端粒酶活性可视化检测新方法。这些方法包括：将链置换循环反应用于端粒酶的多色可视化检测，放大检测信号；以高包封率的脂质体为载体封装高催化活性的酶，双重放大可视化信号；利用指示剂将pH值的变化转化为可视化信号，建立超灵敏的pH响应的可视化方法；以高量子产率的聚集诱导发光分子为探针，结合循环放大手段，对细胞裂解液内的端粒酶实现单细胞水平的检测，并进一步建立原位成像方法，监测细胞内端粒酶的动态变化。这些高灵敏度的检测方法将实现单细胞水平的端粒酶可视化检测，为以端粒酶为靶点的癌症诊断和治疗评价提供依据。

端粒酶与癌症的发生和发展密切相关，端粒酶活性的检测对于基于端粒酶的癌症早期诊断和预后具有重要意义。本项目针对可视化检测灵敏度较低这一问题，以端粒酶为研究对象，建立了多种高灵敏度、简单可靠的端粒酶活性可视化检测方法。基于催化发夹自组装信号放大和金纳米棒刻蚀等离子体比色，构建了灵敏的多色可视化端粒酶活性检测；基于封装酶的脂质体信号放大和金纳米双锥体刻蚀，发展了灵敏和分辨率更高的端粒酶活性等离子体比色分析方法，检出限低至1个HeLa细胞；基于脲酶介导水解和催化发夹自组装，开发了用于人端粒酶RNA检测的pH响应比色法；基于脲酶诱导金纳米双锥体金属化，在未引入信号放大策略的情况下，对端粒酶的检出限低至1个人乳腺癌细胞；设计了一种将滚环扩增和催化发夹组装在MnO2纳米片上形成级联扩增反应的纳米探针，实现了活细胞内端粒酶活性的高灵敏监测。此外，对高灵敏可视化检测方法进行了深入探究，包括基于纳米材料刻蚀或生长的比色可视化方法、光热可视化方法、酶介导的可视化方法，以及荧光增强检测方法等。这些研究工作丰富了肿瘤标志物的可视化检测策略，为肿瘤诊断和治疗评价提供了快速检测方法。