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.
Highlights
When sensitivity of analytical methods is approaching singlemolecule levels, biomolecules and their reactions exhibit their intrinsic nature of discreteness
In dual-color enzyme assay, 50 μM FDP and 200 μM resorufin β-D-galactopyranoside (RGP) were used as substrates for 300 pM alkaline phosphatase (ALP) and 300 pM β-gal, respectively, and 2.5 mM 4-MU was used for position identification
We first conducted digital enzyme assay of β-gal with a fluorogenic substrate, fluorescein di-β-D-galactopyranoside (FDG), that is hydrolyzed into galactose and fluorescein to give a fluorescence signal
Summary
When sensitivity of analytical methods is approaching singlemolecule levels, biomolecules and their reactions exhibit their intrinsic nature of discreteness. Analyst protocol for digital ELISA, target non-enzyme molecules are recognized by enzyme-conjugated antibodies and individually encapsulated into micron-sized reactors with fluorogenic substrates for the conjugated enzyme. The reaction volume of nanocell reactors (nano-reactors) is reduced to the attoliter level and concentration of reaction products increased more rapidly, so that digital counting of enzymes became more accessible even under non-optimum conditions for enzymes.
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