Browsing by Author "Huang, Yun-Tzu"

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Enhanced bioconjugation on sputtered palladium nano-thin-film electrode
(2019-03-04)
Chang, Chia-Yu
;
Chen, Wei
;
Su, Chien-Hao
;
Chang, Pang-Chia
;
Huang, Yun-Tzu
A highly reactive surface with an enhanced ability for chemical bonding relies on the presence of specifically coordinated atoms and step edges at the surface. In this study, an electrode with a unique Stranski-Krastanov-like thin film, with an epitaxial sputtering of a palladium (Pd) nanoparticle double layer on the polyethylene terephthalate substrate, was developed. On the surface of this flexible Pd-nano-thin-film (NTF) electrode with a (1 1 1) containing surface, DNA probes can be quickly immobilized in as short a period as 20 min, which is 24 times faster than that on the gold electrode. A DNA-based anticancer compound (ACC) sensing and screening process that would use the DNA functionalized Pd-NTF electrode as the biosensor was then proposed. Interestingly, the developed biosensor could detect DNA and ACCs, such as doxorubicin, tetra-O-methyl nordihydroguaiaretic acid, and Taxol via interactions with solutions containing 1 mu l ACCs within 11 min, and the sensitivity of the ACC solution is similar to 0.1 mu M (similar to 36 pg per-test), as detected by electrochemical impedance spectroscopy. Moreover, this highly reactive surface can be used in regular sensors and other interfaces, in scientific applications. (C) 2019 Author(s).
Functional Investigation of Transmembrane Helix 3 in H+-Translocating Pyrophosphatase
(2013-12-01)
Lee, Ching-Hung
;
Chen, Yen-Wei
;
Huang, Yun-Tzu
;
Pan, Yih-Jiuan
;
Lee, Chien-Hsien
H+-translocating pyrophosphatase (H+-PPase, EC 3.6.1.1) plays an important role in acidifying vacuoles by transporting protons across membranes at the expense of pyrophosphate (PPi) hydrolysis. Vigna radiata H+-PPase (VrH(+)-PPase) contains 16 transmembrane helices (TMs). The hydrophobicity of TM3 is relatively lower than that of most other TMs, and the amino acids in this TM are highly conserved in plants. Furthermore, TM5 and -6, which are the core TMs involving in H+-PPase functions, are near TM3. It is thus proposed that TM3 is associated with H+-PPase activity. To address this possibility, site-directed mutagenesis was applied in this investigation to determine the role of TM3 in VrH(+)-PPase. Upon alanine/serine substitution, T138 and S142, whose side chains face toward the center TMs, were found to be involved in efficient proton transport. G149/S153 and G160/A164 pairs at the crucial termini of the two GxxxG-like motifs are indispensable in maintaining enzymatic activities and conformational stability. Moreover, stability in the vicinity surrounding G149 is pivotal for efficient expression. S153, M161 and A164 are critical for the K+-mediated stimulation of H+-PPase. Taken together, our results demonstrate that TM3 plays essential roles in PPi hydrolysis, proton transport, expression, and K+ stimulation of H+-PPase.
Single Molecule Take-and-Place Technique for Positioning a Membrane Protein on a Lipid Bilayer
(2015-09-10)
Liu, Tseng-Huang
;
Huang, Yun-Tzu
;
Cheng, Hui-Wen
;
Chen, Yen-Wei
;
Lee, Ching-Hung
Manipulating a single protein molecule on liposomes or planar lipid bilayers is a useful technique for studying membrane-bound proteins, receptors, or ion transporters and for delicate applications including biosensor chips, drug screening, and clinical diagnoses. However, several key breakthroughs are required for success through difficult techniques such as single protein take-and-place with reasonable spatiotemporal control. In this report, a novel method was established for placing a single transmembrane protein onto a patterned lipid bilayer. A surface-functionalized 1.8 nm gold nanoparticle was first engineered at the tip of an atomic force microscope. A single transmembrane protein, H+-translocating pyrophosphatase with six-histidine residues tag, was then picked up using such a microscopy tip where its nanoparticle was modified by nickel-nitrilotriacetic acid. Two-step fluorescence bleaching observation and quantum dot blinking analysis subsequently verified successful manipulation of a single functional protein on a lipid membrane in a predetermined manner. Furthermore, the enzymatic activity from the single H+-translocating pyrophosphatase was determined, demonstrating that membrane proteins retain their functions on the lipid bilayer through a single-molecule take-and-place technique. This innovative technique overcomes current limitations and provides a single biomolecule nanomanipulation system for versatile studies of membrane-bound proteins.
Surface active flexible palladium nano-thin-film electrode development for biosensing
(2019-09-01)
Chang, Chia-Yu
;
Huang, Yun-Tzu
;
Chang, Pang-Chia
;
Su, Chien-Hao
;
Hsu, Kuo-Chen
Metal atoms on surface edges or with specific coordinates possess high surface potentials, and serve as core atoms for organic molecule bonding. However, these active metal atoms may lose their activity by self-diffusion on the surface. A polyethylene terephthalate (PET) substrate provided a stable rough surface in nanoscale for metal atoms absorption and formation of a stable and active nano-thin-film (NTF) surface. However, the NTF surface roughness decreased with increasing thickness of palladium (Pd) granules, indicating that the surface growth of the Pd film followed a Stranski-Krastanov-like growth model. Therefore, the proper thickness of Pd-NTF on PET substrate (Pd NTF-PET) was developed for electrochemical impedance spectroscopy (EIS). Protein was immobilized on the Pd NTF-PET electrodes within 15 min and its biosensing sensitivity was as low as 0.1 ng per-test in 1 mu l. We expect that the developed Pd NTF-PET electrode can be used for other biosensing applications as well.
The proximity between C-termini of dimeric vacuolar H(+)-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
(2009-08-01)
Liu, Tseng-Huang
;
Hsu, Shen-Hsing
;
Huang, Yun-Tzu
;
Lin, Shih-Ming
;
Huang, Tsu-Wei
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 PP(i) 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 6xHis 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.
The proximity between C-termini of dimeric vacuolar H+-pyrophosphatase determined using atomic force microscopy and a gold nanoparticle technique
(2009-08-01)
Liu, Tseng-Huang
;
Hsu, Shen-Hsing
;
Huang, Yun-Tzu
;
Lin, Shih-Ming
;
Huang, Tsu-Wei
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 6xHis 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.