Browsing by Author "Hsieh, Pei-Ying"

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A Rapid SNAP-Tag Fluorogenic Probe Based on an Environment-Sensitive Fluorophore for No-Wash Live Cell Imaging
(2014-10-01)
Liu, Tao-Kai
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Hsieh, Pei-Ying
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Zhuang, Yu-De
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Hsia, Chi-Yang
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Huang, Chi-Ling
One major limitation of labeling proteins with synthetic fluorophores is the high fluorescence background, which necessitates extensive washing steps to remove unreacted fluorophores. In this paper, we describe a novel fluorogenic probe based on an environment-sensitive fluorophore for labeling with SNAP-tag proteins. The probe exhibits dramatic fluorescence turn-on of 280-fold upon being labeled to SNAP-tag. The major advantages of our fluorogenic probe are the dramatic fluorescence turn-on, ease of synthesis, high selectivity, and rapid labeling with SNAP-tag. No-wash labeling of both intracellular and cell surface proteins was successfully achieved in living cells, and the localization of these proteins was specifically visualized.
Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells
(1970-01-01)
Awasthi, Kamlesh
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Chang, Feng-Lin
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Hsieh, Pei-Ying
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Hsu, Hsin-Yun
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Ohta, Nobuhiro
Monitoring fluorescence properties of endogenous fluorophores such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) in normal and cancerous cells provide substantial information noninvasively on biochemical and biophysical aspects of metabolic dysfunction of cancerous cells. Time-resolved spectral profiles and fluorescence lifetime images of NADH and FAD were obtained in human lung nonsmall carcinomas (H661 and A549) and normal lung cells (MRC-5). Both fluorophores show the fast and slowly decaying emission components upon pulsed excitation, and fluorescence spectra of NADH and FAD show blue- and red-shifts, respectively, during their decay. All identified lifetime components of NADH and FAD were found to be shorter in cancerous cells than in normal cells, no matter how they were measured under different extra-cellular conditions (cells suspended in cuvette and cells attached on glass substrate), indicating that the changes in metabolism likely altered the subcellular milieu and potentially also affected the interaction of NADH and FAD with enzymes to which these cofactors were bound. The intensity ratio of NADH and FAD of cancerous cells was also shown to be larger than that of normal cells.
Diatom-assisted biomicroreactor targeting the complete removal of perfluorinated compounds
(2020-02-15)
Albert, Karunya
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Hsieh, Pei-Ying
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Chen, Tsai-Hsuan
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Hou, Chia-Hung
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Hsu, Hsin-Yun
Persistent perfluorinated compounds (PFCs) have been recognized as a global environmental issue. Developing methods without leading to additional burden in nature will be essential for PFCs removal. Herein, we functionalized iron nanoparticles on living diatom (Dt) to efficiently enable the Fenton reaction and reactive oxygen species (ROS) production. Iron nanoparticles at the surface of living diatom act as promising catalytic agents to trigger (OH)-H-center dot radical generation from H2O2. Dt plays dual roles: i) as solid support for effective adsorption, and ii) it supplies oxygen and inherently produces ROS under stress conditions, which improves removal efficiency of PFCs. We also demonstrated its reusability by simple magnetic separation and 85% of decomposition efficiency could still be achieved. This newly developed diatom-assisted bioremediation strategy enables green and efficient PFC decomposition and shall be readily applicable to other persistent pollutants.
Differentiation between normal and cancer cells with autofluorescence lifetime microscopy and nanosecond pulsed electric field effects
(2019-01-01)
Ohta, Nobuhiro
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Awasthi, Kamlesh
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Hsu, Hsin-Yun
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Hsieh, Pei-Ying
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交大名義發表
Fluorescence lifetime images of the endogenous nicotinamide adenine dinucleotide (NADH), which is well known as autofluorescence chromophores, were obtained for rat normal fibroblast cells (WFB) and H-ras oncogene-transfected WFB cancer cells (W31) and for human normal lung fibroblast (MRC-5) and human lung large carcinoma (HCI-H661). In both cases, the average lifetime of the NADH autofluorescence was shorter in cancer cells than in normal cells, indicating that the difference in metabolism between healthy and cancer cells might alter the level of coenzymes such as NADH. It is also shown that application of nanosecond pulsed electric field (nsPEF) induces apoptosis in these cell, resulting in the morphological changes and lengthening the autofluorescence lifetime of NADH. Furthermore, we found that nsPEF more efficiently affected cancer cells than normal cells in cell viability, suggesting the possibility of drug-free cancer therapy by nsPEF.
Graphene Oxide Based Nanocarrier Combined with a pH-Sensitive Tracer: A Vehicle for Concurrent pH Sensing and pH-Responsive Oligonucleotide Delivery
(2015-06-03)
Hsieh, Chia-Jung
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Chen, Yu-Cheng
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Hsieh, Pei-Ying
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Liu, Shi-Rong
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Wu, Shu-Pao
We chemically tuned the oxidation status of graphene oxide (GO) and constructed a GO-based nanoplatform combined with a pH-sensitive fluorescence tracer that is designed for both pH sensing and pH-responsive drug delivery. A series of GOs oxidized to distinct degrees were examined to optimize the adsorption of the model drug, poly dT(30). We determined that highly oxidized GO was a superior drug-carrier candidate in vitro when compared to GOs oxidized to lesser degrees. In the cell experiment, the synthesized pH-sensitive rhodamine dye was first applied to monitor cellular pH; under acidic conditions, protonated rhodamine fluoresces at 588 mm (lambda(ex) = 561 nm). When the nanocarrier was introduced into cells, a rhodamine-triggered competition reaction occurred, and this led to the release of the oligonucleotides and the quenching of rhodamine fluorescence by GO. Our results indicate high drug loading (FAM-dT(30)/GO = 25/50 mu g/mL) and rapid cellular uptake (<0.5 h) of the nanocarrier which can potentially be used for targeted RNAi delivery to the acidic milieu of tumors.
Multifunctional silver nanocluster-hybrid oligonucleotide vehicle for cell imaging and microRNA-targeted gene silencing
(2017-04-01)
Chen, Hau-Yun
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Albert, Karunya
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Wen, Cheng-Che
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Hsieh, Pei-Ying
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Chen, Sih-Yu
Novel therapeutics is urgently needed to prevent cancer-related deaths. MicroRNAs that act as tumor suppressors have been recognized as a next-generation tumor therapy, and the restoration of tumor suppressive microRNAs using microRNA replacements or mimics may be a less toxic, more effective strategy due to fewer off-target effects. Here, we designed the novel multifunctional oligonucleotide nanocarrier complex composed of a tumor-targeting aptamer sequence specific to mucin 1. (MUC1), poly-cytosine region for fluorescent silver nanocluster (AgNC) synthesis, and complimentary sequence for microRNA miR-34a loading. MiR-34a was employed because of its therapeutic effect of inhibiting oncogene expression and inducing apoptosis in carcinomas. By monitoring the intrinsic fluorescence of AgNC, it was clearly shown that the constructed complex (MUC1-AgNCm-miR-34a) enters MCF-7 cells. To evaluate the efficacy of this nanocarrier for microRNA delivery, we investigated the gene and protein expression levels of downstream miR-34a targets (BCL-2, CDK6, and CCNDI) by quantitative PCR and western blotting, respectively, and the results indicated their effective inhibition by miR-34a. This novel multifunctional AgNC-based nanocarrier can aid in improving the efficacy of breast cancer theranostics. (C) 2017 Elsevier B.V. All rights reserved.
Plant Cell Wall-Penetrable, Redox-Responsive Silica Nanoprobe for the Imaging of Starvation-Induced Vesicle Trafficking
(2016-10-18)
Huang, Xin-Chun
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Inoue-Aono, Yuko
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Moriyasu, Yuji
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Hsieh, Pei-Ying
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Tu, Wei-Ming
Autophagy is a self-protection process against reactive oxygen species (ROS). The intracellular level of ROS increased when cells were cultured under nutrient starvation. Antioxidants such as glutathione and ascorbic acid play an important role in ROS removal. However, the cellular redox state In the autophagic pathway is still unclear. Herein, we developed a new redox-sensitive probe with a disulfide-linked silica scaffold to enable the sensing of the reduction environment in cell organelles. This redox-responsive silica nanoprobe (ReSiN) could penetrate the plant cell wall and release fluorescent molecules in; response to redox states. By applying the ReSiN to tobacco BY-2 cells and tracing the distribution of fluorescence, we found a higher reducing potential in. the central vacuole than in the autolysosomes. Upon cysteine protease inhibitor (E64-c) treatment in sucrose free medium, the disulfide-silica structures. of the ReSiNs were broken down in the vacuoles but Were not degraded and were accumulated in the autolysosomes. These results reveal the feasibility of our nanoprobe for monitoring the endocytic and Macroautophagic pathways. These pathways merge upstream of the-central vacuole, which is the final destination of both pathways. In addition, different redox potentials were observed in the autophagic pathway. Finally, the expression of the autophagy-related protein (Atg8) fused with green fluorescence protein confirmed that the ReSiN treatment itself did not induce the autophagic pathway under normal physiological conditions, indicating the versatility of this nanoprobe in studying stimuli triggered autophagy-related trafficking.
Quinone-Modified Mn-Doped ZnS Quantum Dots for Room-Temperature Phosphorescence Sensing of Human Cancer Cells That Overexpress NQO1
(2015-11-25)
Sung, Yi-Ming
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Gayam, Srivardhan Reddy
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Hsieh, Pei-Ying
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Hsu, Hsin-Yun
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Diau, Eric Wei-Guang
Early detection of cancer cells in a rapid and sensitive approach is one of the great challenges in modern clinical cancer care. This study has demonstrated the first example of a rapid, selective, and sensitive phosphorescence probe based on phosphorescence energy transfer (PET) for cancer-associated human NAD(P)H:quinone oxidoreductase isozyme 1 (NQO1). An efficient room-temperature phosphorescence NQO1 probe was constructed by using Mn-doped ZnS quantum dots (Mn:ZnS QDs) as donors and trimethylquinone propionic acids as acceptors. Phosphorescence quenching of Mn:ZnS QDs from the Mn:ZnS QDs to a covalently bonded quinone was achieved through PET. Phosphorescence of Mn:ZnS QDs was turned on by the rapid reduction-initiated removal of the quinone quencher by NQO1. This probe shows low cellular toxicity and can rapidly distinguish between NQO1-expressing and -nonexpressing cancer cell lines through phosphorescence imaging.
Real-time CRP detection from whole blood using micropost-embedded microfluidic chip incorporated with label-free biosensor
(2018-01-21)
Tsai, Meng-Zhe
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Hsiung, Chan-Te
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Chen, Yang
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Huang, Cheng-Sheng
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Hsu, Hsin-Yun
We demonstrate the detection of C-creative protein (CRP) from whole blood samples without sample pretreatment by using a lab-on-a-chip system consisting of a microfluidic chip and a label-free biosensor. The microfluidic chip includes an array of microposts for filtering blood cells and allows only plasma to flow through and reach the guided-mode resonance (GMR) biosensor for real-time monitoring. The developed GMR sensor can achieve a bulk sensitivity of 186 nm RIU-1, which supports a limit of detection of 3.2 ng mL(-1) for recombinant CRP spiked in human serum. The results are comparable with those obtained using enzyme-linked immunosorbent assay. In addition, we demonstrate the efficacy of filtration of blood cells using microposts and simultaneous measurement of CRP concentration using a GMR sensor by using whole blood and plasma samples.
When cells divide: Label-free multimodal spectral imaging for exploratory molecular investigation of living cells during cytokinesis
(2015-12-03)
Hsu, Jen-Fang
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Hsieh, Pei-Ying
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Hsu, Hsin-Yun
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Shigeto, Shinsuke
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應用化學系
In vivo, molecular-level investigation of cytokinesis, the climax of the cell cycle, not only deepens our understanding of how life continues, but it will also open up new possibilities of diagnosis/prognosis of cancer cells. Although fluorescence-based methods have been widely employed to address this challenge, they require a fluorophore to be designed for a specific known biomolecule and introduced into the cell. Here, we present a label-free spectral imaging approach based on multivariate curve resolution analysis of Raman hyperspectral data that enables exploratory untargeted studies of mammalian cell cytokinesis. We derived intrinsic vibrational spectra and intracellular distributions of major biomolecular components (lipids and proteins) in dividing and nondividing human colon cancer cells. In addition, we discovered an unusual autofluorescent lipid component that appears predominantly in the vicinity of the cleavage furrow during cytokinesis. This autofluorescence signal could be utilized as an endogenous probe for monitoring and visualizing cytokinesis in vivo.