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Assaying for Inorganic Polyphosphate in Bacteria
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High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach.

Roozbeh Aschar-Sobbi1, Andrey Y Abramov, Catherine Diao

  • 1Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1, Canada.

Journal of Fluorescence
|January 23, 2008
PubMed
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This study enhances polyphosphate (poly-P) detection using DAPI by employing long-wavelength excitation. This novel method significantly increases sensitivity and specificity for poly-P, enabling new biological applications.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Polyphosphate (poly-P) is a crucial metabolite and signaling molecule in various organisms.
  • DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye commonly used for DNA staining.
  • DAPI's interaction with poly-P causes a detectable shift in its fluorescence emission spectrum.

Purpose of the Study:

  • To develop a more sensitive and specific method for detecting polyphosphate.
  • To investigate the utility of long-wavelength excitation for DAPI-poly-P complex detection.
  • To explore applications in enzyme activity measurement and cellular imaging.

Main Methods:

  • Utilizing long-wavelength excitation (≥ 400 nm, specifically 415 nm) of the DAPI-poly-P complex.
  • Measuring fluorescence emission at 550 nm to detect poly-P.

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  • Assessing the specificity of the method by evaluating DAPI-DNA and free DAPI fluorescence.
  • Applying the method to measure polyphosphatase enzyme activity and image neuronal mitochondria.

    • Main Results:

    • Long-wavelength excitation dramatically increases the sensitivity of poly-P detection, down to 25 ng/ml.
    • Detection at 550 nm with 415 nm excitation provides a highly specific signal, independent of DNA presence.
    • The enhanced method successfully measured polyphosphatase activity.
    • The DAPI-poly-P signal was observed in the mitochondrial region of cultured neurons.

    Conclusions:

    • Long-wavelength excitation of DAPI offers a significantly more sensitive and specific method for poly-P detection compared to traditional approaches.
    • This improved DAPI-based assay has broad applications in biochemistry, cell biology, and diagnostics.
    • The method allows for qualitative and quantitative analysis of poly-P in vitro and in vivo, including enzyme activity and cellular localization.