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Issue 15, 2015
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A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate

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Abstract

Digitalization of fluorogenic enzymatic assays through the use of femtoliter chamber array technology is an emerging approach to realizing highly quantitative bioassays with single-molecule sensitivity. However, only a few digital fluorogenic enzyme assays have been reported, and the variations of the digital enzyme assays are basically limited to fluorescein- and resorufin-based fluorogenic assays. This limitation hampers the realization of a multiplex digital enzyme assay such as a digital enzyme-linked immunosorbent assay (ELISA). In this study, after optimization of buffer conditions, we achieved a single-molecule digital enzyme alkaline phosphatase (ALP) assay with a cumarin-based fluorogenic substrate, 4-methylunbelliferyl phosphate (4-MUP). When ALP molecules were encapsulated in a 44-femtoliter chamber array at a low ratio of less than 1 molecule per chamber, each chamber showed a discrete fluorescence signal in an all-or-none manner, allowing the digital counting of the number of active enzyme molecules. The fraction of fluorescent chambers linearly decreased with the enzyme concentration, obeying the Poisson distribution as expected. We also demonstrated a dual-color digital enzyme assay with a ALP/4-MUP and β-galactosidase (β-gal)/resorufin-β-D-galactopyranoside combination. The activities of single ALP and β-gal molecules were clearly detected simultaneously. The method developed in this study will enable us to carry out a parallelized, multiplex digital ELISA.

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

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Publication details

The article was received on 14 Apr 2015, accepted on 31 May 2015 and first published on 01 Jun 2015


Article type: Paper
DOI: 10.1039/C5AN00714C
Citation: Analyst, 2015,140, 5065-5073
  • Open access: Creative Commons BY-NC license
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    A single-molecule digital enzyme assay using alkaline phosphatase with a cumarin-based fluorogenic substrate

    Y. Obayashi, R. Iino and H. Noji, Analyst, 2015, 140, 5065
    DOI: 10.1039/C5AN00714C

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