Journal of Fluorescence

, Volume 18, Issue 5, pp 859–866 | Cite as

High Sensitivity, Quantitative Measurements of Polyphosphate Using a New DAPI-Based Approach

  • Roozbeh Aschar-Sobbi
  • Andrey Y. Abramov
  • Catherine Diao
  • Margaret E. Kargacin
  • Gary J. Kargacin
  • Robert J. French
  • Evgeny Pavlov
Original Paper


Polyphosphate (poly-P) is an important metabolite and signaling molecule in prokaryotes and eukaryotes. DAPI (4′,6-diamidino-2-phenylindole), a widely used fluorescent label for DNA, also interacts with polyphosphate. Binding of poly-P to DAPI, shifts its peak emission wavelength from 475 to 525 nm (excitation at 360 nm), allowing use of DAPI for detection of poly-P in vitro, and in live poly-P accumulating organisms. This approach, which relies on detection of a shift in fluorescence emission, allows use of DAPI only for qualitative detection of relatively high concentrations of poly-P, in the μg/ml range. Here, we report that long-wavelength excitation (≥400 nm) of the DAPI-poly-P complex provides a dramatic increase in the sensitivity of poly-P detection. Using excitation at 415 nm, fluorescence of the DAPI-poly-P complex can be detected at a higher wavelength (550 nm) for as little as 25 ng/ml of poly-P. Fluorescence emission from free DAPI and DAPI-DNA are minimal at this wavelength, making the DAPI-poly-P signal highly specific and essentially independent of the presence of DNA. In addition, we demonstrate the use of this protocol to measure the activity of poly-P hydrolyzing enzyme, polyphosphatase and demonstrate a similar signal from the mitochondrial region of cultured neurons.


Fluorescence DAPI Polyphosphate Inorganic phosphate Polyphosphatase 



We grateful to the late Dr. Arthur Kornberg, Department of Biochemistry at Stanford University, for providing us with purified scPPX1 enzyme. This work was supported by operating grants from Canadian Institutes of Health Research, and the Heart and Stroke Foundation of Alberta, NWT, and Nunavut.


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Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Roozbeh Aschar-Sobbi
    • 1
  • Andrey Y. Abramov
    • 3
  • Catherine Diao
    • 1
    • 2
  • Margaret E. Kargacin
    • 1
  • Gary J. Kargacin
    • 1
  • Robert J. French
    • 1
    • 2
  • Evgeny Pavlov
    • 1
    • 2
  1. 1.Department of Physiology and BiophysicsUniversity of CalgaryCalgaryCanada
  2. 2.Hotchkiss Brain InstituteCalgaryCanada
  3. 3.Department of Physiology and Mitochondrial Biology GroupUniversity College LondonLondonUK

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