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Total internal reflection fluorescence microscopy or TIRF is an advanced microscopic technique used to visualize fluorophores in samples close to a solid surface with a higher refractive index, such as a glass coverslip. TIRF only allows fluorophores in proximity to the solid surface to be excited. When light from a medium with a lower refractive index (such as air) hits the glass coverslip at a critical angle, the light undergoes total internal reflection stead of passing through the glass.
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Fluorescence Imaging of Intracellular Telomerase Activity Using Enzyme-Free Signal Amplification.

Min Hong1,2, Lidan Xu2, Qingwang Xue2

  • 1College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University , Jinan, 250014, People's Republic of China.

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|February 15, 2017
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Summary

A new assay uses gold nanoflare probes and mimic-hybridization chain reaction for sensitive detection of intracellular telomerase activity in living cells. This method distinguishes cancer cells and monitors drug responses.

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Area of Science:

  • Biomedical Engineering
  • Molecular Biology
  • Nanotechnology

Background:

  • Telomerase is a key biomarker for cancer detection and progression.
  • Existing methods for telomerase detection often lack sensitivity or require cell lysis.

Purpose of the Study:

  • To develop a novel, enzyme-free assay for sensitive, in situ fluorescence imaging of intracellular telomerase activity.
  • To enable real-time monitoring of telomerase activity in living cells and its response to inhibitors.

Main Methods:

  • A gold nanoflare probe (AuNP)-based system was designed, incorporating nucleic acid sequences for telomerase primer extension.
  • The probe was coupled with a graphene oxide (GO)-anchored mimic-hybridization chain reaction (mimic-HCR) for signal amplification.
  • The assay utilizes the release of a fluorescent reporter (FAM) upon telomerase-mediated primer extension and subsequent mimic-HCR amplification.

Main Results:

  • The developed assay achieved highly sensitive, in situ fluorescence detection of intracellular telomerase activity.
  • The system successfully distinguished between normal and cancer cells based on telomerase levels.
  • The assay demonstrated the ability to monitor changes in telomerase activity upon treatment with a telomerase inhibitor.

Conclusions:

  • This enzyme-free, nanoflare probe-triggered mimic-HCR assay offers a sensitive and effective platform for intracellular telomerase activity detection.
  • The method holds potential for cancer diagnostics and therapeutic monitoring in living cells.