High sensitivity, quantitative measurements of polyphosphate using a new DAPI-based approach

J Fluoresc. 2008 Sep;18(5):859-66. doi: 10.1007/s10895-008-0315-4. Epub 2008 Jan 22.


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 microg/ml range. Here, we report that long-wavelength excitation (> or = 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescent Dyes / chemistry
  • Fluorescent Dyes / metabolism*
  • Fluorometry
  • Indoles / metabolism*
  • Kinetics
  • Polyphosphates / chemistry*
  • Sensitivity and Specificity
  • Spectrometry, Fluorescence


  • Fluorescent Dyes
  • Indoles
  • Polyphosphates
  • DAPI