Micro- and Nano-Chamber Array Chips for a Single Molecule Analysis

Abstract

This paper reports the design, fabrication, and application of micro- and nanoscale chamber array chips toward a single nucleus assay as a new experimental format for research in biology. Micro- and nanoscale chamber array on a chip becomes a powerful new tool for bioanalysis since it could stochastically capture and measure biomolecules at a single molecule level. 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. In the field of gene delivery systems, it is still unknown that virus vector showed much higher transportation efficiency of plasmid DNA from cytoplasm to nucleus in contrast to non-virus vectors. To improve gene transportation efficiency, which directly lead to increase transfection efficiency, gene transportation mechanism through nucleus membrane should be cleared. But unfortunately there are no experimental protocol to quantitatively evaluate the transportation efficiency through the nucleus membrane. So, in this study, 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.