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A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate.

Yusuke Obayashi1, Ryota Iino, Hiroyuki Noji

  • 1Department of Applied Chemistry, The University of Tokyo, Japan. hnoji@appchem.t.u-tokyo.ac.jp.

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|June 24, 2015
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Summary

This study introduces a new digital enzyme assay for alkaline phosphatase (ALP) using a coumarin-based substrate. This advancement enables single-molecule detection and multiplexing capabilities for digital enzyme-linked immunosorbent assays (ELISAs).

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Molecular Biology

Background:

  • Digitalization of fluorogenic enzymatic assays offers high quantification and single-molecule sensitivity.
  • Current digital enzyme assays are limited to fluorescein and resorufin, hindering multiplexing applications like digital ELISA.
  • Development of novel digital enzyme assays is crucial for expanding multiplexing capabilities.

Purpose of the Study:

  • To develop a single-molecule digital enzyme assay for alkaline phosphatase (ALP) using a coumarin-based substrate.
  • To demonstrate the feasibility of a dual-color digital enzyme assay for simultaneous detection of multiple enzymes.
  • To enable parallelized and multiplexed digital ELISA applications.

Main Methods:

  • Optimization of buffer conditions for enzymatic assays.
  • Utilizing femtoliter chamber array technology for single-molecule encapsulation.
  • Employing 4-methylumbelliferyl phosphate (4-MUP) as a fluorogenic substrate for ALP.
  • Developing a dual-enzyme system with ALP and beta-galactosidase (β-gal) using distinct substrates.

Main Results:

  • Achieved a single-molecule digital enzyme assay for ALP with high sensitivity.
  • Demonstrated discrete, all-or-none fluorescence signals in femtoliter chambers, enabling digital counting of active enzyme molecules.
  • Successfully performed a dual-color digital enzyme assay, simultaneously detecting ALP and β-gal activities at the single-molecule level.
  • Confirmed linear decrease in fluorescent chambers with enzyme concentration, following Poisson distribution.

Conclusions:

  • The developed method allows for precise quantification of enzyme activity at the single-molecule level.
  • This approach overcomes limitations of existing digital enzyme assays, paving the way for multiplexed digital ELISAs.
  • The dual-color assay demonstrates the potential for simultaneous detection of multiple analytes in a single reaction.