Browsing by Author "Chen, Kuang-Po"

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Effective Construction of a High-Capacity Boronic Acid Layer on a Quartz Crystal Microbalance Chip for High-Density Antibody Immobilization
(2019-01-01)
Lin, Pei-Heng
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Huang, Sheng-Cih
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Chen, Kuang-Po
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Li, Bor-Ran
;
Li, Yaw-Kuen
Boronic acids (BAs) provide strong potential in orientation immobilization of antibody and the modification method is crucial for efficiency optimization. A highly effective method has been developed for rapid antibody immobilization on gold electrodes through the electrodeposition of a BA-containing linker in this study. Aniline-based BA forms a condense layer while antibody could automatically immobilize on the surface of the electrode. Compare to traditional self-assembled monolayer method, the electrodeposition process dramatically reduces the modification time from days to seconds. It also enhances the immobilized efficiency from 95 to 408 (ng/cm(2)) with a strong preference being exhibited for shorter aniline-based linkers.
Infrared Spectra of (Z)- and (E)-(C2H3C)-C-center dot(CH3)I Radicals Produced upon Photodissociation of (Z)- and (E)-(CH2I)HC=C(CH3)I in Solid para-Hydrogen
(2020-07-16)
Haupa, Karolina Anna
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Chen, Kuang-Po
;
Li, Yaw-Kuen
;
Lee, Yuan-Pern
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交大名義發表
Ozonolysis of isoprene to produce Criegee intermediates such as methyl vinyl ketone oxide (MVKO), C2H3C(CH3)OO, is an important process in atmospheric chemistry. MVKO was recently produced and identified in laboratories after photolysis of a gaseous mixture of 1,3-diiodobut-2-ene, (CH2I)HC=C(CH3)I, and O-2, but the mechanism of its formation remains unexplored. We synthesized pure (Z)- and (E)-1,3-diiodo-but-2-ene and measured their distinct IR spectra. Upon irradiation at 280 nm of (Z)-and (E)-1,3-diiodo-but-2-ene in solid p-H-2 at 3.3 K, the fission of the terminal C.I bond yields (Z)- and (E)-3-iodo-but-2-en-1-yl [(C2H3C)-C-center dot(CH3)I] radicals, respectively. These radicals were characterized with infrared absorption lines at 2962.4, 1423.8, 1265.3, 1120.9/1127.0, 921.4/922.3, and 792.5/791.7 cm(-1), and 16 additional weaker lines for (Z)-(C2H3C)-C-center dot(CH3)I and 1405.2, 1208.2, 1106.0/1103.9, 934.2/933.4, and 785.1/784.9 cm(-1) and five additional weaker ones for (E)-(C2H3C)-C-center dot(CH3)I. The assignments were derived according to behavior on secondary photolysis and comparison of the vibrational wavenumbers and the IR intensities of observed lines with those calculated with the B2PLYP-D3/aug-cc-pVTZ-pp method. These observations confirmed that only the terminal I atom, not the central one, was photodissociated at 280 nm and, in solid p-H-2, the excess energy after photodissociation induced no change in conformation. These new spectra of center dot C2H3C(CH3)I radicals can provide valuable information for the understanding of the mechanism of formation of Criegee intermediate MVKO from the source reaction of photolysis of (CH2I)HC=C(CH3)I in O-2 in the laboratory.
Linker and N-Terminal Domain Engineering of Pyrrolysyl-tRNA Synthetase for Substrate Range Shifting and Activity Enhancement
(2020-04-07)
Jiang, Han-Kai
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Lee, Man-Nee
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Tsou, Jo-Chu
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Chang, Kuan-Wen
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Tseng, Hsueh-Wei
The Methanosarcina mazei pyrrolysyl-tRNA synthetase (PylRS).tRNA(Pyl) pair can be used to incorporate non-canonical amino acids (ncAAs) into proteins at installed amber stop codons. Although engineering of the PylRS active site generates diverse binding pockets, the substrate ranges are found similar in charging lysine and phenylalanine analogs. To expand the diversity of the ncAA side chains that can be incorporated via the PylRS.tRNA(Pyl) pair, exploring remote interactions beyond the active site is an emerging approach in expanding the genetic code research. In this work, remote interactions between tRNA(Pyl), the tRNA binding domain of PylRS, and/or an introduced non-structured linker between the N- and C-terminus of PylRS were studied. The substrate range of the PylRS.tRNA(Pyl) pair was visualized by producing sfGFP-UAG gene products, which also indicated amber suppression efficiencies and substrate specificity. The unstructured loop linking the N-terminal and C-terminal domains (CTDs) of PylRS has been suggested to regulate the interaction between PylRS and tRNA(Pyl). In exploring the detailed role of the loop region, different lengths of the linker were inserted into the junction between the N-terminal and the C-terminal domains of PylRS to unearth the impact on remote effects. Our findings suggest that the insertion of a moderate-length linker tunes the interface between PylRS and tRNA(Pyl) and subsequently leads to improved suppression efficiencies. The suppression activity and the substrate specificity of PylRS were altered by introducing three mutations at or near the N-terminal domain of PylRS (N-PylRS). Using a N-PylRS.tRNA(Pyl) pair, three ncAA substrates, two S-benzyl cysteine and a histidine analog, were incorporated into the protein site specifically.
Silicon Quantum Dot Nanoparticles with Antifouling Coatings for Immunostaining on Live Cancer Cells
(2016-06-08)
Tu, Chang-Ching
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Chen, Kuang-Po
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Yang, Tsu-An
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Chou, Min-Yuan
;
Lin, Lih Y.
Fluorescent silicon quantum dots (SiQDs) have shown a great potential as antiphotobleaching, nontoxic and biodegradable labels for various in vitro and in vivo applications. However, fabricating SiQDs with high water-solubility and high photoluminescence quantum yield (PLQY) remains a challenge. Furthermore, for targeted imaging, their surface chemistry has to be capable of conjugating to antibodies, as well as sufficiently antifouling. Herein, antibody-conjugated SiQD nanoparticles (SiQD-NPs) with antifouling coatings composed of bovine serum albumin (BSA) and polyethylene glycol (PEG) are demonstrated for immunostaining on live cancer cells. The monodisperse SiQD-NPs of diameter about 130 nm are synthesized by a novel top-down method, including electrochemical etching, photochemical hydrosilylation, high energy ball milling, and "selective-etching" in HNO3 and HF. Subsequently, the BSA and PEG are covalently grafted on to the SiQD-NP surface through presynthesized chemical linkers, resulting in a stable, hydrophilic, and antifouling organic capping layer with isothiocyanates as the terminal functional groups for facile conjugation to the antibodies. The in vitro cell viability assay reveals that the BSA-coated SiQD-NPs had exceptional biocompatibility, with minimal cytotoxicity at concentration up to 1600 mu g mL(-1). Under 365 nm excitation, the SiQD-NP colloid emits bright reddish photoluminescence with PLQY = 45-55% in organic solvent and 5-10% in aqueous buffer. Finally, through confocal fluorescent imaging and flow cytometry analysis, the anti-HER2 conjugated SiQD-NPs show obvious specific binding to the HER2-overexpressing SKOV3 cells and negligible nonspecific binding to the HER2-nonexpressing CHO cells. Under similar experimental conditions, the immunofluorescence results obtained with the SiQD-NPs are comparable to those using conventional fluorescein isothiocyanate (FITC).
Time-Gated Imaging on Live Cancer Cells Using Silicon Quantum Dot Nanoparticles with Long-Lived Fluorescence
(2017-06-01)
Tu, Chang-Ching
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Awasthi, Kamlesh
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Chen, Kuang-Po
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Lin, Chih-Hsiang
;
Hamada, Morihiko
In this work we demonstrate time-gated confocal fluorescence imaging on live cancer cells immunostained by antibody-conjugated silicon quantum dot nano particles (SiQD-NPs) and organic dyes, for simultaneous detection of two biological targets and removal of background autofluorescence. With almost all radiative recombinations occurring through oxide-related defect states located on the SiQD surface, the SiQD-NPs have very long photoluminescence lifetimes of about 25 its, in contrast to the nanosecond range lifetimes of other commonly used biological fluorophores. This drastic lifetime difference enables a time-gated imaging method here, in which the time-resolved photon distribution of each pixel of a fluorescence image is measured by using a time-correlated single-photon counting technique. Then, by integrating the photon histogram of each pixel over respective time windows, the long-lived component of the fluorescence image comprising only the fluorescence emitted from the SiQD-NPs is separated from all other short-lived signals resulting from the organic dyes and the cell endogenous luminescence. For instance, the membrane and nucleus of a single cancer cell or two types of cancer cells, immunostained with the SiQD-NPs and the organic dyes, respectively, can be clearly distinguished from each other by time-gating, which otherwise cannot be accomplished by conventional multiplexing due to spectral overlap in the wavelength domain.