Transportation, release behavior, and stability of a green fluorescent protein (GFP, 3x4 nm) in self-assembled organic nanotubes with three different inner diameters (10, 20, and 80 nm) have been studied in terms of novel nanocontainers. Selective immobilization of a fluorescent acceptor dye on the inner surface enabled us to not only visualize the transportation of GFP in the nanochannels but to also detect release of the encapsulated GFP to the bulk solution in real time, based on fluorescence resonance energy transfer (FRET). Obtained diffusion constants and release rates of GFP markedly decreased as the inner diameter of the nanotubes was decreased. An endo-sensing procedure also clarified the dependence of the thermal and chemical stabilities of the GFP on the inner diameters. The GFP encapsulated in the 10 nm nanochannel showed strong resistance to heat and to a denaturant. On the other hand, the 20 nm nanochannel accelerated the denaturation of the encapsulated GFP compared with the rate of denaturation of the free GFP in bulk and the encapsulated GFP in the 80 nm nanochannels. The confinement effect based on rational fitting of the inner diameter to the size of GFP allowed us to store it stably and without denaturation under high temperatures and high denaturant concentrations.
Glow discharge plasma formed in solution under atmospheric pressure has been expected to provide reaction fields with characteristic physical and chemical properties owing to the frequent collisions and reactions of reactive particles inside and the rapid quenching of the products by the surrounding cold solutions. In particular, when an aqueous solution is utilized as the surrounding solution, the atmospheric-pressure in-solution glow (ASG) plasma contains hydrogen and hydroxyl radicals showing large activities for reduction and oxidation, respectively. In addition, because the ASG plasma is formed under atmospheric pressure, the collision frequencies between the particles contained in the plasma are higher than those in other plasmas ordinarily formed under low pressure. This feature should result in rapid energy redistribution among particles contained in the plasma. In the present study, time-resolved optical emission spectroscopy with nanosecond time resolution was applied for the diagnostics of the ASG plasma with chemical species selectivity. The time-resolved measurements revealed that the temporal evolutions of the temperatures of blackbody, hydrogen radical, and hydroxyl radical contained in the ASG plasma consist of two stages: initial rise within 0.15 µs (rising stage) and fluctuation around certain values for about 1 µs (fluctuating stage). In the time region corresponding to the rising stage, the electron number density is about ten times larger than the value temporally averaged during the plasma emission. The initial rise should result from frequent collisions between charged particles accelerated by the applied voltage and unexcited particles. In the fluctuating stage, the electron number density strongly correlates with the increase in the radical temperatures. It is concluded that the electron number density, rather than the electron temperature, is a key parameter determining the temperatures of reactive species in the ASG plasma.
The correlation between the fluorescent dynamics of excited anilinonaphthalene sulfonate (ANS) and the microviscosity of solvent molecules surrounding ANS is investigated by time-resolved fluorescence spectroscopy. ANS has been widely used to probe the local hydrophobicity due to the drastic change in its intensity. It is revealed that the fluorescence lifetime from the charge transfer (CT) state of ANS sensitively reflects the microviscosity. The higher sensitivity of 2,6-ANS than of 1,8-ANS demonstrates that the spatial freedom of the rotating phenylamino group in the photoexcited ANS is an important factor that determines the sensitivity. As an application, the measurements of the microviscosity of water in biologically important systems, such as hyaluronan, gellan gum, and gelatin aqueous solutions are also presented. The present results suggest that the fluorescence lifetime of ANS enables the estimation of the solvent microviscosity and provide a useful probe molecule for fluorescence lifetime imaging microscopy.
Stimulated Raman scattering (SRS) from water molecules in the air-water and water-silver interfacial regions is measured when laser-induced plasma are generated at these interfaces. In both cases, characteristic lower wavenumber shifts of the OH stretching vibrational modes of the interfacial water molecules are observed. The mechanisms of the characteristic lower wavenumber shifts are considered from two viewpoints. One is the electrostatic interactions between the interfacial water molecules and the excess electrons induced by the plasma generation at the interfacial area. The other is the coupling of the SRS with Brillouin scattering induced by a shock wave generation by abrupt volume expansion due to the phase transition from liquid to plasma. Differences between the obtained SRS spectra and vibrational spectra previously measured by a sum-frequency generation technique are also discussed.
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