Key Laboratory of Eco-chemical Engineering, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China.
Anal Chem. 2010 Apr 1;82(7):2850-5. doi: 10.1021/ac902818w.
On the basis of the extension reaction of a telomerase substrate (TS) primer in the presence of the telomerase, the inherent signal-transduction mechanism of the hairpin fluorescence probe, and the strand-displacement property of polymerase, an amplified fluorescence detection of telomerase activity in the cancer cells was described in this manuscript. A hairpin fluorescence probe was used as not only the fluorescence signal carrier but also a template of the telomere elongation reaction. In the presence of the telomerase, the stems of the hairpin probes were opened and the telomerase activity could be determined with the fluorescence enhancement. The telomerase activity in the HeLa extracts equivalent to 40-1000 cells was detected by this method, with the multiple rounds of isothermal strand replication, which led to strand displacement, and constituted consecutive signal amplification for the novel detection paradigm that allowed measurement of telomerase activity in crude cell extracts equivalent to 4 cultured HeLa cells. Using magnetic beads as both the separation tool and the immobilization matrix of the aptamer of Ramos cells (CRL-1596, B-cell, human Burkitt's lymphoma), the detection of the amount of the Ramos cell with the low concentration of 100 cells mL(-1) confirmed the reliability and practicality of the protocol, which reveal a good prospect of this platform for analysis.
基于端粒酶底物（TS）引物在端粒酶存在下的延伸反应、发夹荧光探针的固有信号转导机制以及聚合酶的链置换特性，本文描述了一种用于检测癌细胞中端粒酶活性的放大荧光检测方法。发夹荧光探针不仅可用作荧光信号载体，还可用作端粒延伸反应的模板。在端粒酶存在下，发夹探针的茎部被打开，通过荧光增强可以测定端粒酶的活性。该方法可检测相当于 40-1000 个细胞的 HeLa 提取物中的端粒酶活性，通过多次等温链复制，导致链置换，并构成连续的信号放大，为新型检测范式提供了测量粗细胞提取物中端粒酶活性的可能性，相当于 4 个培养的 HeLa 细胞。通过将磁珠用作 Ramos 细胞（CRL-1596，B 细胞，人Burkitt 淋巴瘤）的适体的分离工具和固定基质，可检测到浓度低至 100 个细胞 mL(-1)的 Ramos 细胞的量，这证实了该方案的可靠性和实用性，该平台为分析提供了广阔的前景。
