The standard method of detaching adherent cells from a culture substrate involves the application of trypsin to digest the extracellular matrix. However, the trypsin treatment is time-consuming and sometimes causes damage to the cells when harvesting cells from a culture dish. We have developed a novel nanostructured scaffold composed of a lightresponsive gas-generating film (gas-generation nanoscaffold) that serves the dual functions of cell adhesion and cell detachment. Cell adhesion was based on the nanostructured surface topography with numerous holes of 230 nm diameter and 200 nm depth; the structure was created by soft lithography. Cell detachment was accomplished by the light irradiation of the photodecomposable polymer film to generate gas, which weakens the adhesive forces between the cells and the substrate. 11 µl of N 2 gas was generated in 90 s and the gas-generation rate was 0.1 µl/s. It was found that twice as many human bronchioalveolar carcinoma cells (NCL-H1650) adhered to the nanoscaffold than to the same scaffold without the nanostructure. The nanoscaffold exhibited no cytotoxicity according to cell viability and cytotoxicity assays. After culturing the cells on the gas-generation nanoscaffold, they were irradiated with light to generate gas, which causes the cells on the film to detach. We successfully demonstrated cell adhesion and cell detachment on the novel gas-generation nanoscaffold. This novel material is expected to be useful as an alternative cell culture substrate without the need for the standard trypsin treatment.

Bioluminescence resonance energy transfer (BRET) is a useful technique for visualizing cellular functions and responses to stimuli. To construct efficient biosensing protein probes using BRET, novel luminescent and red fluorescent protein pairs, which have separate peaks of luminescence and fluorescence and can cause energy transfer efficiently, were screened. The red fluorescent protein, mScarletI, used as an acceptor, induced a highly efficient BRET signal from green or blue luminescent proteins [Emerald Luc (ELuc) or NanoLuc (NLuc)]. Novel pairs of luminescent and red fluorescent protein (mScarletI) could be applied to the analysis of calcium ions (Ca 2+). The BRET-based biosensing protein pair of mScarletI and NLuc showed an increased intensity of the BRET signal, depending on the concentration of Ca 2+ (0-4 μM). Intracellular Ca 2+ influx was monitored in HEK293A cells stimulated with 50 mM KCl and 15 mM arginine using the BRET-based biosensing protein probe with the novel protein pair. This pair of proteins was particularly suited to cellular imaging in vitro and even in vivo. Therefore, it could be useful for various BRET-based analyses of cell and tissue samples.