Browsing by Author "Wang, Tzu-Pin"

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Applying FDEM, ERT and GPR at a site with soil contamination: A case study
(2015-10-01)
Wang, Tzu-Pin
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Chen, Chien-Chih
;
Tong, Lun-Tao
;
Chang, Ping-Yu
;
Chen, Yi-Chieh
This study employed the combination of three methods, namely the Frequency Domain Electromagnetic (FDEM), Electrical Resistivity Tomography (ERT) and Ground Penetrating Radar (GPR) to evaluate a heavy-metal contaminated site for both pre- and post-remediation investigations. The main goals were to verify the position and the integrity of the underground storage tanks (UST), and to determine the effectiveness of remediation to ensure no contaminants remained at the site. In general, the GPR survey was effective at locating shallowly buried objects. However, due to the highly conductive nature of the heavy-metal laden sludge, the GPR signals were attenuated severely. Thus, the first attempt to use GPR in the pre-remediation investigation did not achieve the desired results and other methods were deployed. The existence of the UST and the sludge within were confirmed by ERT and the UST shape was mapped by FDEM. The principal remediation scheme was soil replacement by replacing the contaminated soil with clean silt Based on the distinctive property differences of the contaminated soil and the clean silt, the completion of the remediation was confirmed by the differences between pre-remediation and post-remediation in GPR, ERT and FDEM results. (C) 2015 Elsevier B.V. All rights reserved.
Azide-alkyne cycloaddition for universal post-synthetic modifications of nucleic acids and effective synthesis of bioactive nucleic acid conjugates
(2014)
Su, Yu-Chih
;
Lo, Yu-Lun
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Hwang, Chi-Ching
;
Wang, Li-Fang
;
Wu, Min Hui
The regioselective post-synthetic modifications of nucleic acids are essential to studies of these molecules for science and applications. Here we report a facile universal approach by harnessing versatile phosphoramidation reactions to regioselectively incorporate alkynyl/azido groups into post-synthetic nucleic acids primed with phosphate at the 5\' termini. With and without the presence of copper, the modified nucleic acids were subjected to azide-alkyne cycloaddition to afford various nucleic acid conjugates including a peptide-oligonucleotide conjugate (POC) with high yield. The POC was inoculated with human A549 cells and demonstrated excellent cell-penetrating ability despite cell deformation caused by a small amount of residual copper chelated to the POC. The combination of phosphoramidation and azide-alkyne cycloaddition reactions thus provides a universal regioselective strategy to post-synthetically modify nucleic acids. This study also explicated the toxicity of residual copper in synthesized bioconjugates destined for biological systems.
Development of a Gd(III)-Based Receptor-Induced Magnetization Enhancement (RIME) Contrast Agent for beta-Glucuronidase Activity Profiling
(2012-11-19)
Chen, Shih-Hsien
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Kuo, Yu-Ting
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Singh, Gyan
;
Cheng, Tian-Lu
;
Su, Yu-Zheng
beta-Glucuronidase is a key lysosomal enzyme and is often overexpressed in necrotic tumor masses. We report here the synthesis of a pro receptor induced magnetization enhancement (pro RIME) magnetic resonance imaging (MRI) contrast agent ([Gd(DOTA-FP beta Gu)]) for molecular imaging of beta-glucuronidase activity in tumor tissues. The contrast agent consists of two parts, a gadolinium complex and a beta-glucuronidase substrate (beta-D-glucopyranuronic acid). The binding association constant (K-A) of [Gd(DOTA-FP beta Gu)] is 7.42 X 10(2), which is significantly lower than that of a commercially available MS-325 (K-A = 3.0 x 10(4)) RIME contrast agent. The low K-A value of [Gd(DOTA-FP beta Gu)] is due to the pendant beta-D-glucopyranuronic acid moiety. Therefore, [Gd(DOTA-FP beta Gu)] can be used for detection of beta-glucuronidase through RIME modulation. The detail mechanism of enzymatic activation of [Gd(DOTA-FP beta Gu)] was elucidated by LC-MS. The kinetics of beta-glucuronidase catalyzed hydrolysis of [Eu(DOTA-FP beta Gu)] at pH 7.4 best fit the Miechalis-Menten kinetic mode with K-m = 1.38 mM, K-cat = 3.76 x 10(3), and k(cat)/K-m = 2.72 x 10(3) M-1 s(-1). The low K-m value indicates high affinity of beta-glucuronidase for [Gd(DOTA-FP beta Gu)] FP beta Gu) at physiological pH. Relaxometric studies revealed that T-1 relaxivity of [Gd(DOTA-FP beta Gu)] changes in response to the concentration of beta-glucuronidase. Consistent with the relaxometric studies, [Gd(DOTA-FP beta Gu)] showed significant change in MR image signal in the presence of beta-glucuronidase and HSA. In vitro and in vivo MR images demonstrated appreciable differences in signal enhancement in the cell lines and tumor xenografts in accordance to their expression levels of beta-glucuronidase. [GRAPHICS] .
Effective and site-specific phosphoramidation reaction for universally labeling nucleic acids
(2014-03-15)
Su, Yu-Chih
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Chen, Hsing-Yin
;
Ko, Ni Chien
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Hwang, Chi-Ching
;
Wu, Min Hui
Here we report efficient and selective postsynthesis labeling strategies, based on an advanced phosphoramidation reaction, for nucleic acids of either synthetic or enzyme-catalyzed origin. The reactions provided phosphorimidazolide intermediates of DNA or RNA which, whether reacted in one pot (one-step) or purified (two-step), were directly or indirectly phosphoramidated with label molecules. The acquired fluorophore-labeled nucleic acids, prepared from the phosphoramidation reactions, demonstrated labeling efficacy by their F/N ratio values (number of fluorophores per molecule of nucleic acid) of 0.02-1.2 which are comparable or better than conventional postsynthesis fluorescent labeling methods for DNA and RNA. Yet, PCR and UV melting studies of the one-step phosphoramidation-prepared FITC-labeled DNA indicated that the reaction might facilitate nonspecific hybridization in nucleic acids. Intrinsic hybridization specificity of nucleic acids was, however, conserved in the two-step phosphoramidation reaction. The reaction of site-specific labeling nucleic acids at the 5'-end was supported by fluorescence quenching and UV melting studies of fluorophore-labeled DNA. The two-step phosphoramidation-based, effective, and site-specific labeling method has the potential to expedite critical research including visualization, quantification, structural determination, localization, and distribution of nucleic acids in vivo and in vitro. (C) 2013 Elsevier Inc. All rights reserved.
Imaging specificity of MR-optical imaging agents following the masking of surface charge by poly(ethylene glycol)
(2013-05-01)
Wu, Shou-Cheng
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Lin, Kun-Liang
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Wang, Tzu-Pin
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Tzou, Shey-Cherng
;
Singh, Gyan
The coupling of specific antibodies to imaging agents often improves imaging specificity. However, free amine groups designed for the coupling can cause nonspecific binding of the imaging agents. We report here development of a nanocarrier, MnMEIO-silane-NH2-mPEG nanoparticles (NPs), consisting of a manganese-doped iron oxide nanoparticle core (MnMEIO), a copolymer shell of silane and amine-functionalized poly(ethylene glycol) (silane-EA-mPEG). The key feature in MnMEIO-silane-NH2-mPEG is the flexible PEG, which masks the non-conjugated reactive amine groups (-NH2 <-> -NH3+) and reduces nonspecific binding of MnMEIO-silane-NH2-mPEG to cells. The amine groups on MnMEIO-silane-NH2-mPEG were conjugated with the fluorescent dye, Cy777 or antibodies [Erbitux (Erb)] to form a MR-optical imaging contrast agent (MnMEIO-silane-NH2-(Erb)-mPEG) for EGFR-expressing tumors. Confocal microscopic and flow cytometric analyses showed that MnMEIO-silane-NH2-(Erb)-mPEG displayed low nonspecific binding. Moreover, TEM images showed that MnMEIO-silane-NH2-(Erb)-mPEG were endocytosed by EGFR-expressing cells. In line with their EGFR expression levels, A431, PC-3, and Colo-205 tumors treated with MnMEIO-silane-NH2-(Erb)-mPEG NPs showed -97.1%, -49.7%, and -2.8% contrast enhancement, respectively, in in vitro T-2-weighted MR imaging. In vivo T-2-weighted MR imaging and optical images showed that MnMEIO-silane-NH2-(Erb)mPEG could specifically and effectively target to EGFR-expressing tumors in nude mice; the relative contrast enhancements were 7.94 (at 2 h) and 7.59 (at 24 h) fold higher in A431 tumors as compared to the EGFR-negative Colo-205 tumors. On the contrary, MnMEIO-silane-NH2-(Erb) NPs showed only 1.44 (at 2 h) and 1.52 (at 24 h) fold higher in EGFR-positive tumors as compared to the EGFR-negative tumors. Finally, antibodies can be readily changed to allow imaging of other tumors bearing different antigens. These data indicate that masking surface charges on contrast agents is a useful strategy to improve imaging efficacy. (c) 2013 Elsevier Ltd. All rights reserved.
In Vitro Selection and Characterization of a Novel Zn(II)-Dependent Phosphorothiolate Thiolesterase Ribozyme
(2012-01-10)
Wang, Tzu-Pin
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Su, Yu-Chih
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Chen, Yi
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Liou, Yi-Ming
;
Lin, Kun-Liang
Here we present the in vitro selection of a novel ribozyme specific for Zn(2+)-dependent catalysis on hydrolysis of a phosphorothiolate thiolester bond. The ribozyme, called the TW17 ribozyme, was evolved and selected from an artificial RNA pool covalently linked to a biotin-containing substrate through the phosphorothiolate thiolester bond. The secondary structure for the evolved ribozyme consisted of three major helices and three loops. Biochemical and chemical studies of ribozyme-catalyzed reaction products provided evidence that the ribozyme specifically catalyzes hydrolysis of the phosphorothiolate thiolester linkage. A successful ribozyme construct with active catalysis in trans further supported the determined ribozyme structure and indicated the potential of the ribozyme for multiple-substrate turnover. The ribozyme also requires Zn(2+) and Mg(2+) for maximal catalysis. The TW17 ribozyme, in the presence of Zn(2+) and Mg(2+), conferred a rate enhancement of at least S orders of magnitude when compared to the estimated rate of the uncatalyzed reaction. The ribozyme completely lost catalytic activity in the absence of Zn(2+), like Zn(2+)-dependent protein hydrolases. The discovery and characterization of the TW17 ribozyme suggest additional roles for Zn(2+) in ribozyme catalysts.
Successful preparation and characterization of biotechnological grade agarose from indigenous Gelidium amansii of Taiwan
(2012-03-01)
Wang, Tzu-Pin
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Chang, Li-Lin
;
Chang, Sheng-Nan
;
Wang, Eng-Chi
;
Hwang, Long-Chih
This paper reports the first successful preparation of biotechnological grade agarose from Gelidium amansii found in Taiwan. The scale-efficiency preparation was achieved by shortening EDTA treatment time through dispersing G. amansii agar in water in the presence of heat and EDTA, removing agaropectin impurity with a heat-compatible and strong-anion exchange resin, and precipitating agarose with a cost-effective isopropanol method. The yield of agarose from prepared G. amansii agar was 11.3%. The acquired agarose has a gel strength of 853 g cm(-2), a sulfate content of 0.14%, a pyruvate content of 1.03%, a degree of electroendosmosis of 0.16 and very limited binding affinity to DNA. The excellent properties of agarose from G. amansii of Taiwan confirm its potential diverse biotechnological applications. This innovative agarose preparation method with the significantly improved scale-efficiency can be modified for large-scale preparation of agarose for use in biotechnological industry and biochemical research. (C) 2011 Elsevier Ltd. All rights reserved.