Poster Presentation 2014 International Biophysics Congress

Micro- and nanochamber array chips for a single enzyme activity assay and nucleus analysis (#511)

Noritada Kaji 1 , Ryo Koyama 1 , Takao Yasui 1 , Yoshinobu Baba 1 2
  1. Nagoya University, Nagoya, Japan
  2. Health Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan

Micro- and nanochamber array on a chip becomes a powerful new tool for bioanalysis since it could stochastically capture and measure biomolecules at a single molecule level. This experimental concept was introduced and elucidated by Prof. Noji at 2005 and applied for single molecule enzymology and measurements of ATP synthesis by F1-ATPase. Our fundamental interests are how reaction-space size affects an enzyme activity and how small space we could extrapolate enzyme kinetics in small space from old enzyme kinetic data in bulk. So we have measured a single β-galactosidase activity in various sizes of micro and nanochambers, from 5 μm to 500 nm cubic, and found that the activity gradually decreased according to its chamber size. Because the specific surface area increases according to the decrease of the chamber volume, non-specific adsorption might be a factor to suppress the activity. However, several experiments including surface coating and repetition of the enzyme capturing elucidated that non-specific adsorption is not a major factor affecting the activity.
Another application of micro and nanochamber array was for a single nucleus analysis. Nuclei were extracted from cells by gentle detergent treatments and captured into a PDMS or PMMA microchamber at a single nucleus level. After the confirmation of capturing a single nucleus into a microchamber, nucleotides and nuclear membrane stained nuclei, transportation of mRNA by molecular beacon were observed.