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Issue 24, 2010
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A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

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Abstract

The enzyme assay in a femtoliter chamber array is a simple and efficient method for concentrating the reaction product; it greatly improves the detection sensitivity down to the single-molecule level. However, in previous methods, controlling the initiation and termination of the reaction in each chamber is difficult once enclosed. Furthermore, the recovery of the enzyme and product is also difficult. To overcome these drawbacks, we developed a femtoliter droplet array in which the individual droplets are fixed on the substrate and are directly accessible from outside. A hydrophilic-in-hydrophobic micropatterned surface was used for the preparation of the droplets. When the aqueous solution on the surface is exchanged with oil, the hydrophilic surface retains the aqueous solution, and more than 106 dome-shaped droplets that are usable for further assay can be prepared simultaneously. The curvature radius of the droplet obeys the Young–Laplace equation, and the volume can be precisely controlled by the micropipette, which applies pressure into the droplet. Changing the pressure makes the addition, collection, and exchange of the aqueous content for individual droplets possible. Using these advantages, we successfully measured the kinetic parameters of the single-molecule enzyme β-galactosidase and rotary motor protein F1-ATPase enclosed in a droplet.

Graphical abstract: A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

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Publication details

The article was received on 01 Jun 2010, accepted on 14 Sep 2010 and first published on 28 Oct 2010


Article type: Paper
DOI: 10.1039/C0LC00062K
Citation: Lab Chip, 2010,10, 3355-3362
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    A single-molecule enzymatic assay in a directly accessible femtoliter droplet array

    S. Sakakihara, S. Araki, R. Iino and H. Noji, Lab Chip, 2010, 10, 3355
    DOI: 10.1039/C0LC00062K

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