A nicked molecular beacon (MB)-functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments: a shorter telomerase primer (TSP) sequence as a part of the 5'-end stem and a longer sequence to form a loop with one thiol-labeled 3'-end stem. The MB can be opened by substitutional hybridization of the telomerase-triggered stem elongation product, which leads to separation of the Cy5 at the 5'-end nick from the gold nanoparticle (AuNP) as the nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP. Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the fluorescence of Cy5. The probe provides a one-step incubation technique for quantification and monitoring of the telomerase activity in living cells. The practicality of the proposed approach for distinguishing tumor cells from normal cells and monitoring the decrease of telomerase activity during treatment with antitumor drugs demonstrates its potential in clinical diagnostic and therapeutic monitoring.

A smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3' end, which was just complementary with the loop of MB assembled on probe surface. Thus, the MB was opened upon hybridization to switch the fluorescent state from "off" to "on". The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of the cytoplasmic telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2 × 10(-11), 2.4 × 10(-11), and 8.6 × 10(-13) IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells. The proposed method could be employed for dynamic monitoring of the cytoplasmic telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs.

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