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Indicator displacement assays inside live cells.

Amir Norouzy1, Zahra Azizi, Werner M Nau

  • 1Department of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen (Germany).

Angewandte Chemie (International Ed. in English)
|November 29, 2014
PubMed
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Synthetic receptors detect analytes entering live cells. The p-sulfonatocalix[4]arene (CX4) and lucigenin (LCG) complex enables fluorescence detection of choline, acetylcholine, and protamine uptake by V79 and CHO cells.

Area of Science:

  • Biomedical Engineering
  • Chemical Biology
  • Analytical Chemistry

Background:

  • Host-guest complexation is crucial for molecular recognition.
  • Fluorescent dyes can report on molecular interactions.
  • Cellular uptake of analytes is challenging to monitor in real-time.

Purpose of the Study:

  • To develop a novel chemosensing ensemble for detecting bioorganic analyte uptake in live cells.
  • To utilize indicator displacement assays with synthetic receptors for cellular analysis.
  • To establish the functionality of p-sulfonatocalix[4]arene (CX4) and lucigenin (LCG) for live-cell sensing.

Main Methods:

  • Formation of a stable host-guest complex between p-sulfonatocalix[4]arene (CX4) and lucigenin (LCG), resulting in fluorescence quenching.
Keywords:
bioanalytical chemistryfluorescencehost-guest complexesmacrocyclessupramolecular chemistry

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  • Incubation of live V79 and CHO cells with the CX4/LCG ensemble to observe spontaneous cellular uptake.
  • Addition of analytes (choline, acetylcholine, protamine) to monitor fluorescence recovery due to analyte-induced displacement of LCG from CX4.

    • Main Results:

    • The CX4/LCG complex was readily taken up by live V79 and CHO cells.
    • Displacement of LCG from CX4 by high-affinity analytes (choline, acetylcholine, protamine) led to a fluorescence 'switch-on' response.
    • Demonstrated the ability to detect the cellular uptake of specific bioorganic molecules using this system.

    Conclusions:

    • The CX4/LCG chemosensing ensemble is effectively internalized by cells.
    • Indicator displacement assays with synthetic receptors are viable for detecting intracellular analyte uptake.
    • This approach offers a promising method for real-time monitoring of bioorganic analyte interactions within live cells.