Senkei Umehara

Label-free biosensing with functionalized nanopipette probes

Nanopipette technology can uniquely identify biomolecules such as proteins based on differences in size, shape, and electrical charge. These differences are determined by the detection of changes in ionic current as the proteins interact with the nanopipette tip coated with probe molecules. Here we show that electrostatic, biotin-streptavidin, and antibody-antigen interactions on the nanopipette tip surface affect ionic current flowing through a 50-nm pore. Highly charged polymers interacting with the glass surface modulated the rectification property of the nanopipette electrode. Affinity-based binding between the probes tethered to the surface and their target proteins caused a change in the ionic current due to a partial blockade or an altered surface charge. These findings suggest that nanopipettes functionalized with appropriate molecular recognition elements can be used as nanosensors in biomedical and biological research.

On-chip single-cell microcultivation assay for monitoring environmental effects on isolated cells.

We have developed a on-chip single-cell microcultivation assay as a means of observing the adaptation process of single bacterial cells during nutrient concentration changes. This assay enables the direct observation of single cells captured in microchambers made on thin glass slides and having semipermeable membrane lids, in which cells were kept isolated with optical tweezers. After changing a medium of 0.2% (w/v) glucose concentration to make it nutrient-free 0.9% NaCl medium, the growth of all cells inserted into the medium stopped within 20 min, irrespective of their cell cycles. When a nutrient-rich medium was restored, the cells started to grow again, even after the medium had remained nutrient-free for 42 h. The results indicate that the cells growth and division are directly related to their nutrient condition. The growth curve also indicates that the cells keep their memory of what their growth and division had been before they stopped growing.

Measurement of incident angle dependence of swimming bacterium reflection using on-chip single-cell cultivation assay

We have developed an on-chip single-cell microcultivation assay as a means of continuously observing certain single swimming cells in order to trace their movement. The single cells were captured in microchambers fabricated on thin glass slides and having semipermeable membrane lids, in which cells can swim within the space for a long term without escaping. This assay enables the direct measurement of the reflection of certain cells against the microchamber walls depending on their incidence angles. Using this assay, the reflection was examined. We found that the ratio of reflection of cells to those of non-reverse was almost the same, though most of cells reflected when their incident angle was perpendicular to the wall.

Simultaneous Measurement of Growth and Movement of Cells Exploiting On-Chip Single-Cell Cultivation Assay

We have developed an on-chip single-cell microcultivation assay as a means of simultaneously observing the growth and movement of single bacterial cells during long-term cultivation. This assay enables the direct observation of single cells captured in microchambers fabricated on thin glass slides and having semipermeable membrane lids, in which the cells can swim within the space without escape for the long periods. Using this system, the relationship between the cell cycle and the tendency of movement was observed and it was found that the mean free path length did not change during the cell cycle, and that the growth and the swimming were not synchronized. The result indicates that the ability of movement of the cells was independent of the cell cycle.