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Issue 20, 2018
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Digital enzyme assay using attoliter droplet array

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Abstract

Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed ‘nanocell’. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using β-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicability of digital bioassay for enzymes under non-optimum conditions or enzymes of low turnover rate. The sealing of the reactor with air would also expand the applicability, allowing the use of fluorescent dyes that leak into oil.

Graphical abstract: Digital enzyme assay using attoliter droplet array

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Publication details

The article was received on 22 Jun 2018, accepted on 18 Aug 2018 and first published on 17 Sep 2018


Article type: Paper
DOI: 10.1039/C8AN01152D
Citation: Analyst, 2018,143, 4923-4929
  • Open access: Creative Commons BY license
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    Digital enzyme assay using attoliter droplet array

    T. Ono, T. Ichiki and H. Noji, Analyst, 2018, 143, 4923
    DOI: 10.1039/C8AN01152D

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