Tsu-Wei Huang

Dynamics of hydrogen nanobubbles in KLH protein solution studied with in situ wet-TEM

Although the stability of the nanobubble remains a controversial issue that is subject to the classical predictions of high Laplace pressure, we demonstrate that a hydrogen nanobubble can be generated and stabilized in an aqueous solution of Keyhole limpet hemocyanin (KLH) protein via an electron radiolysis process. The hydrogen gas inside the nanobubble is in a “dense gas” phase that is characterized by a Knudsen number and number density of hydrogen molecules. The dynamics of nanobubbles are analyzed using time-series electron microscopy images. The growth of small nanobubbles will be affected by the largest neighboring nanobubble; however, a diffusive shielding effect for small nanobubbles is observed. Locally, anti-Ostwald ripening of nanobubbles can be observed; however, the global growth behavior among the nanobubbles is randomly correlated because the characteristic diffusion length of the hydrogen molecules is considerably greater than the average spacing among the nanobubbles.

The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique

Vacuolar H+‐translocating inorganic pyrophosphatase [vacuolar H+‐pyrophosphatase (V‐PPase); EC 3.6.1.1] is a homodimeric proton translocase; it plays a pivotal role in electrogenic translocation of protons from the cytosol to the vacuolar lumen, at the expense of PPi hydrolysis, for the storage of ions, sugars, and other metabolites. Dimerization of V‐PPase is necessary for full proton translocation function, although the structural details of V‐PPase within the vacuolar membrane remain uncertain. The C‐terminus presumably plays a crucial role in sustaining enzymatic and proton‐translocating reactions. We used atomic force microscopy to visualize V‐PPases embedded in an artificial lipid bilayer under physiological conditions. V‐PPases were randomly distributed in reconstituted lipid bilayers; approximately 43.3% of the V‐PPase protrusions faced the cytosol, and 56.7% faced the vacuolar lumen. The mean height and width of the cytosolic V‐PPase protrusions were 2.8 ± 0.3 nm and 26.3 ± 4.7 nm, whereas those of the luminal protrusions were 1.2 ± 0.1 nm and 21.7 ± 3.6 nm, respectively. Moreover, both C‐termini of dimeric subunits of V‐PPase are on the same side of the membrane, and they are close to each other, as visualized with antibody and gold nanoparticles against 6×His tags on C‐terminal ends of the enzyme. The distance between the V‐PPase C‐terminal ends was determined to be approximately 2.2 ± 1.4 nm. Thus, our study is the first to provide structural details of a membrane‐bound V‐PPase dimer, revealing its adjacent C‐termini.

Distance Variations between Active Sites of H+-Pyrophosphatase Determined by Fluorescence Resonance Energy Transfer

Homodimeric H+-pyrophosphatase (H+-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H+-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PPi), for H+ translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PPi binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H+-PPase are 49.3 ± 4.0 and 67.2 ± 5.7 Å, respectively. Furthermore, putative PPi binding motifs on individual subunits are found to be relatively far away from each other (70.8 ± 4.8 Å), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H+-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H+-PPase upon substrate binding.

Gradient Strain Chip for Stimulating Cellular Behaviors in Cell-laden Hydrogel

Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 - 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.

Gold-coated polystyrene bead array and the investigation of their plasmon coupling abilities

Many of SERS nanoparticles took advantage of the surface roughness for the significant improvement of their Raman sensing ability. Nevertheless, few papers analyzed the characteristics of surface roughness nanostructures that contribute to the SERS. Thus, this paper investigates the characteristics of the corrugated polystyrene bead (PSB) array etched by a series of oxygen plasma etching time for giving a criterion to fabricate appropriate SERS-active nanoparticles. Three factors were considered in this paper: (1) the effect of plasma coupling among neighboring particles, (2) the vertical surface roughness of nanocorrugations, and (3) the pitch size, the lateral surface roughness, of nanocorrugations. By the analysis of SEM and AFM images, those factors were quantifiable. The correlation coefficient between each factor and SERS Raman enhancement was also investigated to verify that the pitch size of nanocorrugations (ranging from ~6 nm to ~12 nm on the surface of PSBs) dominates the SERS enhancement. Therefore, the maximum improvement of Raman intensity that derives from surface roughness treatment is 12 times compared to smooth surface. Moreover, it has a high enhancement factor of ~106.