研究目的
To develop a fluorogenic probe for the determination of telomerase activity in cell lysates using chimeric DNA-templated silver nanoclusters, aiming for a direct, amplification-free, and cost-effective method compared to existing assays like TRAP.
研究成果
The developed Ct9TS-AgNCs probe enables simple, rapid, and sensitive detection of telomerase activity in cancer cell extracts without amplification, showing improvements over TRAP in time, sensitivity, and cost. The quenching is attributed to parallel G-quadruplex formation in elongated products, with validated performance across multiple cell lines.
研究不足
The assay's applicability in more complex matrices such as tissue samples requires further investigation before clinical use. The quenching mechanism's full understanding, especially the role of buffer components, is not complete.
1：Experimental Design and Method Selection:
The study designed two routes (A and B) for AgNCs formation relative to telomerase elongation to investigate the quenching mechanism. It utilized fluorescence spectroscopy for detection and CD spectroscopy for structural analysis.
2：Sample Selection and Data Sources:
Cancer cell lines (MCF7, HT29, RPMI 2650) were used to extract telomerase. Synthetic DNA templates (Ct9TSRn) mimicked elongated products.
3：List of Experimental Equipment and Materials:
Instruments included a Hitachi F-7000 spectrofluorometer, Epoch Microplate Reader, FEI Tecnai G2-F20 HR-TEM, Jasco J-815 spectropolarimeter, Beckman Coulter centrifuge, Biorad thermal cycler, and ChemiDoc MP Imaging system. Materials included DNA oligonucleotides, silver nitrate, sodium borohydride, and various buffers.
4：Experimental Procedures and Operational Workflow:
AgNCs were synthesized via one-pot method with DNA templates. Telomerase extracts were prepared using CHAPS lysis. Fluorescence measurements were taken at optimized wavelengths. Proof-of-concept used synthetic DNA. TRAP assay was performed for validation.
5：Data Analysis Methods:
Fluorescence intensities were analyzed to calculate quenching ratios. CD spectra were interpreted for G-quadruplex identification. Statistical analysis included CV% and LOD calculations.
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