Micro- and nanochamber array system for single enzyme assays

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  • Iijima, Kazuki
    Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University
  • Kaji, Noritada
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, Institute of Nano‑Life‑Systems, Institutes of Innovation for Future Society, Nagoya University
  • Tokeshi, Manabu
    Institute of Nano‑Life‑Systems, Institutes of Innovation for Future Society, Nagoya University Division of Applied Chemistry, Faculty of Engineering, Hokkaido University
  • Baba, Yoshinobu
    Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University Institute of Nano‑Life‑Systems, Institutes of Innovation for Future Society, Nagoya University Institute for Quantum Life Science, Quantum Life and Medical Science Directorate, National Institutes for Quantum Science and Technology School of Pharmacy, College of Pharmacy, Kaohsiung Medical University

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Arrays of small reaction containers, ranging from 624 femtoliters (10^<–15> L) to 270 attoliters (10^<–18> L), for capturing a single enzyme molecule and measuring the activity were developed along with a new reversible sealing system based on a pneumatic valve actuator made of polydimethylsiloxane (PDMS). The valve was actuated by PBS solution, effectively preventing evaporation of the solution from the micro- and nanochambers and allowing the assay to be performed over a long period of time. The hydrolysis rates of β-D-galactosidase (β-gal), k_<cat>, were decreased according to the decrease of the chamber size, and the overall tendency seems to be symmetrically related to the specific surface area of the chambers even under the prevented condition of non-specific adsorption. The spatial localization of the protons in the chambers, which might could affect the dissociation state of the proteins, was also investigated to explain the decrease in the hydrolysis rate. The developed chamber system developed here may be useful for artificially reproducing the confined intracellular environment and molecular crowding conditions.

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