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Microfabricated arrays of femtoliter chambers allow single molecule enzymology

Nature Biotechnology volume 23, pages 361–365 (2005)Cite this article

Abstract

Precise understanding of biological functions requires tools comparable in size to the basic components of life1,2,3,4. Single molecule studies have revealed molecular behaviors usually hidden in the ensemble- and time-averaging of bulk experiments5,6. Although most such approaches rely on sophisticated optical strategies to limit the detection volume7,8, another attractive approach is to perform the assay inside very small containers9,10,11,12,13,14,15,16. We have developed a silicone device presenting a large array of micrometer-sized cavities. We used it to tightly enclose volumes of solution, as low as femtoliters, over long periods of time. The microchip insures that the chambers are uniform and precisely positioned. We demonstrated the feasibility of our approach by measuring the activity of single molecules of β-galactosidase and horseradish peroxidase. The approach should be of interest for many ultrasensitive bioassays at the single-molecule level.

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Figure 1: Microfabrication of the PDMS chambers.
Figure 2: Water enclosure in the microchambers, and evaluation of the sealing.
Figure 3: Detection of the activity of single β-galactosidase molecules.

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Acknowledgements

This project was performed in the framework of the Laboratory for Integrated Micro Mechatronic Systems (LIMMS-CNRS/IIS), and financially supported by the Japan Society for the Promotion of Science (H.N., S.T. and H.F.), Bio-Oriented Technology Research Advancement Institution (H.N. and S.T.) and Mitsubishi Foundation (H.N. and S.T.). We thank all members of Noji, Takeuchi and Fujita laboratories for help and advice, and I. Baba and T. Kitamori for critical reading and R. Yasuda for programming of image analysis software. Y.R. and G.T. are Research Fellows of the Japan Society for the Promotion of Science.

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Authors and Affiliations

  1. Laboratory for Integrated Micro Mechatronic Systems (LIMMS-CNRS/IIS), University of Tokyo, 4-6-1 Komaba, Meguroku, Tokyo, 153-8505, Japan

    Yannick Rondelez & Guillaume Tresset

  2. Institute of Industrial Science (IIS), University of Tokyo, 4-6-1 Komaba, Meguroku, Tokyo, 153-8505, Japan

    Kazuhito V Tabata, Hideyuki Arata, Hiroyuki Fujita, Shoji Takeuchi & Hiroyuki Noji

  3. Research on Advanced Medical Technology, Japan Association for the Advancement of Medical Equipment (JAAME), 3-42-6, Hongo, Bunkyou-ku, Tokyo, Japan

    Kazuhito V Tabata

Authors
  1. Yannick Rondelez
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  2. Guillaume Tresset
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  3. Kazuhito V Tabata
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  4. Hideyuki Arata
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  5. Hiroyuki Fujita
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  6. Shoji Takeuchi
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  7. Hiroyuki Noji
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Corresponding author

Correspondence to Hiroyuki Noji.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

The activity of other single enzymes can be detected inside the microchambers. (PDF 501 kb)

Supplementary Fig. 2

In the assay we used, β-Gal catalyzes the hydrolysis of nonfluorescent FDG to highly fluorescent fluorescein. (PDF 282 kb)

Supplementary Video (MOV 697 kb)

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Rondelez, Y., Tresset, G., Tabata, K. et al. Microfabricated arrays of femtoliter chambers allow single molecule enzymology. Nat Biotechnol 23, 361–365 (2005). https://doi.org/10.1038/nbt1072

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