Browsing by Author "Hsu, Hsin-Yun"

Now showing 1 - 20 of 34

A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay
(2016-01)
Huang, Cheng-Yeh
;
Tsai, Po-Yen
;
Lee, I-Chin
;
Hsu, Hsin-Yun
;
Huang, Hong-Yuan
Here, we describe a technique to manipulate a low number of beads to achieve high washing efficiency with zero bead loss in the washing process of a digital microfluidic (DMF) immunoassay. Previously, two magnetic bead extraction methods were reported in the DMF platform: (1) single-side electrowetting method and (2) double-side electrowetting method. The first approach could provide high washing efficiency, but it required a large number of beads. The second approach could reduce the required number of beads, but it was inefficient where multiple washes were required. More importantly, bead loss during the washing process was unavoidable in both methods. Here, an improved double-side electrowetting method is proposed for bead extraction by utilizing a series of unequal electrodes. It is shown that, with proper electrode size ratio, only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. It allows using only about 25 magnetic beads in DMF immunoassay to increase the number of captured analytes on each bead effectively. In our human soluble tumor necrosis factor receptor I (sTNF-RI) model immunoassay, the experimental results show that, comparing to our previous results without using the proposed bead extraction technique, the immunoassay with low bead number significantly enhances the fluorescence signal to provide a better limit of detection (3.14 pg/ml) with smaller reagent volumes (200 nl) and shorter analysis time (<1 h). This improved bead extraction technique not only can be used in the DMF immunoassay but also has great potential to be used in any other bead-based DMF systems for different applications. (C) 2016 AIP Publishing LLC.
A Rapid SNAP-Tag Fluorogenic Probe Based on an Environment-Sensitive Fluorophore for No-Wash Live Cell Imaging
(2014-10-01)
Liu, Tao-Kai
;
Hsieh, Pei-Ying
;
Zhuang, Yu-De
;
Hsia, Chi-Yang
;
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.
AMPFLUID: Aggregation Magnified Post-Assay Fluorescence for Ultrasensitive Immunodetection on Digital Microfluidics
(2015-02-01)
Huang, Cheng-Yeh
;
Shih, Po-Huai
;
Tsai, Po-Yen
;
Lee, I-Chin
;
Hsu, Hsin-Yun
Several strategies are currently employed to enhance the detection limit of bead-based assays, but all these approaches improve the sensitivity by varying the assay procedures chemically or biologically. In previous digital microfluidic setups for bead-based immunoassay, the magnetic beads were suspended for detection. We investigated the effect of bead aggregation in such an immunoassay system and propose the aggregation magnified post-assay fluorescence for ultrasensitive immunodetection on digital microfluidics (AMPFLUID). The detection signal and sensitivity are further enhanced even at the post-assay stage without altering the original assay protocol on employing magnetically triggered post-assay aggregation of beads in a digital microfluidic setup followed by processing of the fluorescent signal. This method is shown to enhance the fluorescent signal with increased consistency and sensitivity after appropriate charge-coupled device (CCD) calibration. This method of on-chip detection allows the fulfilment of consumption of a volume at the nanoliter level and a limit of detection in the range picogram/mL. In our sTNF-RI model immunoassay, only 2.5 nL of sample is required; a detection limit 15 pg/mL is achieved. The decreased uncertainty of the measure is indicated by the error bars and coefficient of variation.
An NAD(P)H: quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles for tumor targeted drug delivery in vitro and in vivo
(2016)
Gayam, Srivardhan Reddy
;
Venkatesan, Parthiban
;
Sung, Yi-Ming
;
Sung, Shuo-Yuan
;
Hu, Shang-Hsiu
The synthesis and characterization of an NAD(P)H: quinone oxidoreductase 1 (NQO1) enzyme responsive nanocarrier based on mesoporous silica nanoparticles (MSNPs) for on-command delivery applications has been described in this paper. Gatekeeping of MSNPs is achieved by the integration of mechanically interlocked rotaxane nanovalves on the surface of MSNPs. The rotaxane nanovalve system is composed of a linear stalk anchoring on the surface of MSNPs, an alpha-cyclodextrin ring that encircles it and locks the payload "cargo" molecules in the mesopores, and a benzoquinone stopper incorporated at the end of the stalk. The gate opening and controlled release of the cargo are triggered by cleavage of the benzoquinone stopper using an endogenous NQO1 enzyme. In addition to having efficient drug loading and controlled release mechanisms, this smart biocompatible carrier system showed obvious uptake and consequent release of the drug in tumor cells, could selectively induce the tumor cell death and enhance the capability of inhibition of tumor growth in vivo. The controlled drug delivery system demonstrated its use as a potential theranostic material.
Artificial peptide-controlled protein release of Zn2+-triggered, self-assembled histidine-tagged protein microparticle
(2020-03-01)
Chen, Tzu-Yin
;
Cheng, Wan-Jung
;
Horng, Jia-Cherng
;
Hsu, Hsin-Yun
;
交大名義發表
Protein microparticles have received attention as drug delivery systems because of their high protein stability and prolonged release in vivo. However, most current preparation processes introduce chemical crosslinkers, which often lead to protein inactivation and limit drug efficacy in delivery systems. In this study, we employed the well-known hexahistidine (His)-tag recombinant protein technology and a metal-triggerable collagen-mimetic peptide to enhance the binding strength between the protein and metal ion and fine-tuned the protein drug release. His-tagged proteins self-assembled to form microparticles (similar to 2 mu m) upon zinc ion treatment and sustained protein drug release was achieved in physiological saline. The results also indicated that by adjusting the peptide concentration and N- and C-terminal hexahistidine-tags, protein release could be controlled. Moreover, no protein denaturation was observed. We developed a universal strategy for facile protein microparticle fabrication under mild conditions and we demonstrated its potential as a therapeutics formulation with tunable protein release.
Bacterial reprogramming of PBMCs impairs monocyte phagocytosis and modulates adaptive T cell responses
(2012-06-01)
Andre, Maya C.
;
Gille, Christian
;
Glemser, Philip
;
Woiterski, Jeanette
;
Hsu, Hsin-Yun
Septic diseases are characterized by an initial systemic, proinflammatory phase, followed by a period of anti-inflammation. In the context of the latter, monocytes have been described to display altered functions, including reduced TNF secretion and T cell-stimulating capacities in response to recall antigens. This hyporesponsiveness is supposed to be detrimental for coping with secondary infections. We here characterize bacterially reprogrammed PBMC-derived monocytes with special focus on their phagocytic activity. Hence, we have implemented a surrogate model of the early, postinflammatory period by exposing PBMCs to Escherichia coli on d0 and rechallenging them with bacteria on d2. This induced the emergence of a distinct monocytic phenotype with profound phagocytic impairments but a preserved ability for naive T cell stimulation. The compromising effects on phagocytosis required the presence of bacteria and were not mimicked by TLR4 ligation or exposure to isolated cytokines alone. Moreover, the impairments were specific for the engulfment of bacteria and were coupled to a selective down-regulation of Fc gamma R and SR expression. Intriguingly, this monocytic phenotype contributed to the stimulation of a T(H)17-polarized adaptive immune response in the context of secondary infection. Our findings extend the current knowledge of monocytic reprogramming and identify the phagocytic capacity of monocytes as a putative sepsis biomarker. J. Leukoc. Biol. 91: 977-989; 2012.
Bio-templated silica composites for next-generation biomedical applications
(2017-11-01)
Albert, Karunya
;
Huang, Xin-Chun
;
Hsu, Hsin-Yun
;
應用化學系
;
應用化學系分子科學碩博班
Silica-based materials have extensive biomedical applications owing to their unique physical, chemical, and biological properties. Recently, increasing studies have examined the mechanisms involved in biosilicification to develop novel, fine-tunable, eco-friendly materials and/or technologies. In this review, we focus on recent developments in bio-templated silica synthesis and relevant applications in drug delivery systems, tissue engineering, and biosensing.
Biothiol-triggered, self-disassembled silica nanobeads for intracellular drug delivery
(2015-09-01)
Huang, Xin-Chun
;
Wu, Li-Bang
;
Hsu, Jen-Fang
;
Shigeto, Shinsuke
;
Hsu, Hsin-Yun
Silica-based nanomaterials have demonstrated great potential in biomedical applications due to their chemical inertness. However, the degradability and endosomal trapping issues remain as rate-limiting barriers during their innovation. In this study, we provide a simple yet novel sol-gel approach to construct the redox-responsive silica nanobeads (ReSiNs), which could be rapidly disassembled upon redox gradient for intracellular drug delivery. The disulfide-linked scaffold of the nanobead was synthesized by employing the dithiobis-(succinimidyl propionate) to bridge (3-aminopropyI)-trimethoxysilane. Such silica matrix could be efficiently disrupted in response to intracellular glutathione, resulting in drug release and collapse of entire nanocarrier. Moreover, the ReSiNs exhibited insignificant cytotoxicity before and after the degradation. These results indicated the potential of using ReSiNs as a novel silica-based, biothiol-degradable nanoplatform for future drug delivery. (C) 2015 Acts Materialia Inc. Published by Elsevier Ltd. All rights reserved.
Carbon-Based Materials for Photo-Triggered Theranostic Applications
(2016-11)
Albert, Karunya
;
Hsu, Hsin-Yun
;
應用化學系
;
應用化學系分子科學碩博班
;
Department of Applied Chemistry
Carbon-based nanomaterials serve as a type of smart material for photo-triggered disease theranostics. The inherent physicochemical properties of these nanomaterials facilitate their use for less invasive treatments. This review summarizes the properties and applications of materials including fullerene, nanotubes, nanohorns, nanodots and nanographenes for photodynamic nanomedicine in cancer and antimicrobial therapies. Carbon nanomaterials themselves do not usually act as photodynamic therapy (PDT) agents owing to the high hydrophobicity, however, when the surface is passivated or functionalized, these materials become great vehicles for PDT. Moreover, conjugation of carbonaceous nanomaterials with the photosensitizer (PS) and relevant targeting ligands enhances properties such as selectivity, stability, and high quantum yield, making them readily available for versatile biomedical applications.
Characterization of endogenous fluorescence in nonsmall lung cancerous cells: A comparison with nonmalignant lung normal cells
(1970-01-01)
Awasthi, Kamlesh
;
Chang, Feng-Lin
;
Hsieh, Pei-Ying
;
Hsu, Hsin-Yun
;
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.
Chip-size wavelength detector based on a gradient grating period guided-mode resonance filter
(2016-01-01)
Lin, Hsin-An
;
Hsu, Hsin-Yun
;
Huang, Cheng-Sheng
;
機械工程學系
;
Department of Mechanical Engineering
In this study, we designed, fabricated and demonstrated a compact wavelength detection system based on a gradient grating period guided-mode resonance filter (GGP-GMRF) mounted on a linear charge-coupled device (CCD) camera. The GGP-GMRF was first fabricated through nanoreplica molding on a plastic substrate, followed by the deposition of a thin TiO2 film. The grating periods of the GGP-GMRF vary from 250 to 550 nm with a 2 nm increment in each period consisting of 100 cycles. The results show that a 6 mm long GGP-GMRF has a filtering range of 506 to 915 nm. Upon illumination, the GGP-GMRF reflects a particular wavelength of light resulting in the minimum transmission of that wavelength. Hence, the GGP-GMRF provides a spatially dependent minimum transmission depending on the wavelength of the incident light. The linear CCD underneath the GGP-GMRF measures the transmitted intensity, and the wavelength of the incident light can be correlated with the location of the minimum intensity. For the demonstrated GGP-GMRF and CCD system, a spectral resolution of 1 nm can be achieved.
Compact spectrometer system based on a gradient grating period guided-mode resonance filter
(2016-05-16)
Lin, Hsin-An
;
Hsu, Hsin-Yun
;
Chang, Chih-Wei
;
Huang, Cheng-Sheng
;
機械工程學系
We demonstrate a compact spectrometer system by using a gradient grating period guided-mode resonance filter-mounted on a linear photodetector array-that exhibits spatially dependent resonance characteristics; a specific incident wavelength is reflected such that the underlying array pixels measure minimum intensity. A precalibrated transmission efficiency matrix is used to determine each pixel's transmission efficiency for specific wavelengths. Unknown spectral information can be calculated from the measured intensity. Grating periods of 250-388 nm in 2-nm increments are used in each 100-cycle period. Device length is 2.23 mm. Spectral range of 506-700 nm is measurable with 1-nm resolution. (C) 2016 Optical Society of America
Compact Wavelength Detection System Based on a Gradient Grating Period Guided-Mode Resonance Filter
(2016)
Lin, Hsin-An
;
Hsu, Hsin-Yun
;
Huang, Cheng-Sheng
;
機械工程學系
;
Department of Mechanical Engineering
We designed a linear filter based on a gradient grating period guided-mode resonance. By incorporating this device with a photodetector, the system can measure wavelength ranging from 500 to 700 nm with 1 nm resolution.
Design and Fabrication of Compact Spectrometer based on Gradient Grating Period Guided-Mode Resonance Filter
(2017-01-01)
Lin, Hsin-An
;
Hsu, Hsin-Yun
;
Chang, Chih-Wei
;
Huang, Cheng-Sheng
;
機械工程學系
We designed and fabricated a compact spectrometer based on a gradient grating period guided-mode resonance filter (GGP-GMRF). The GGP-GMRF of 2.23 mm long consisted of gratings with periods varying from 250-388 nm in 2-nm increments. With such compact size, the device can measure spectrum ranging from 506 to 700 nm.
Diatom-assisted biomicroreactor targeting the complete removal of perfluorinated compounds
(2020-02-15)
Albert, Karunya
;
Hsieh, Pei-Ying
;
Chen, Tsai-Hsuan
;
Hou, Chia-Hung
;
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.
Differential in situ sensing of extra- and intracellular glutathione by a novel redox-responsive silica matrix-Au nanoprobe
(2016-01-01)
Luo, Yun-Ling
;
Huang, Xin-Chun
;
Tu, Wei-Ming
;
Hsu, Hsin-Yun
;
應用化學系
We synthesized a biothiol-sensitive nanoprobe by assembling gold nanoparticles with a novel redox-responsive silica (ReSi) matrix using dithiobis (succinimidyl propionate) and (3-aminopropyl) trimethoxysilane. Thin layer disulfide bonded networks of the ReSi could differentially respond to extra and intracellular glutathione in cancer cells within 30 min; furthermore, targeted cellular uptake could be monitored ill situ by fluorescence recovery. Sigmoidal dose response pattern of the nanoprobes presented in this study were attributed to the buried disulfide linked 3D nanostructure of the ReSi nanoshell, optimized at an appropriate thickness, enabling not only buffering of small redox disturbances in the extracellular milieu but also the satisfied sensitivity for rapid redox sensing. Such a ReSi-functionalized gold nanoparticle-based nanoconjugate possesses the potential to serve as an effective intracellular drug carrier for future cancer theranostics. (C) 2015 Elsevier B.V. All rights reserved.
Differentiation between normal and cancer cells with autofluorescence lifetime microscopy and nanosecond pulsed electric field effects
(2019-01-01)
Ohta, Nobuhiro
;
Awasthi, Kamlesh
;
Hsu, Hsin-Yun
;
Hsieh, Pei-Ying
;
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
;
Chen, Yu-Cheng
;
Hsieh, Pei-Ying
;
Liu, Shi-Rong
;
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.
In situ patterning and controlling living cells by utilizing femtosecond laser
(2016-09)
Okano, Kazunori
;
Hsu, Hsin-Yun
;
Li, Yaw-Kuen
;
Masuhara, Hiroshi
;
應用化學系
Photo-induced processes have high potential in in situ patterning and controlling living cells, whose developments are introduced and recent progresses by utilizing femtosecond laser are described. Photochemical and photothermal surface modification performed by conventional light and nanosecond laser irradiation is summarized and their applicability is considered. Femtosecond laser ablation has superior features due to its photomechanical mechanism, which is confirmed by ultrafast spectroscopy and imaging of a model film under laser ablation. Femtosecond laser ablation of physiological solutions generates shockwave and cavitation bubbles, which is employed for patterning and manipulating living cells. Femtosecond laser ablation fabricating cytophobic and cytophilic domains enable us to form living cell patterns and to study cell migration and cell-cell interaction. Finally summary and perspective are presented. (C) 2016 Elsevier B.V. All rights reserved.
In vitro investigation of methylene blue-bearing, electrostatically assembled aptamer-silica nanocomposites as potential photodynamic therapeutics
(2015-11-01)
Ding, Tian-Shyng
;
Huang, Xin-Chun
;
Luo, Yun-Ling
;
Hsu, Hsin-Yun
;
應用化學系分子科學碩博班
Photodynamic therapy, that is, excitation of a photosensitizer with light to generate reactive oxygen species such as singlet oxygen, has emerged as a noninvasive technique for cancer theranostics. However, the clinical use of many photosensitizers is impeded by their hydrophobicity, the nonspecific damage they cause to normal tissues, and their susceptibility to environmental degradation. In this study, we developed a simple electrostatic adsorption strategy to fabricate aptamer silica nanocomposites by sequentially functionalizing nanocomposites with the cell surface-associated mucin 1 aptamer for tumor targeting and a hydrophilic photosensitizer, methylene blue, for photodynamic therapy applications. We investigated the relationship between the biophysical properties and cellular uptake of such nanocomposites to improve their formulation. Effective generation of singlet oxygen was achieved with a low photosensitizer dosage (0.5 mu M) and a short, low-power irradiation (1 min, 10 mW/cm(2)). With the current strategy, the efficiency of photodynamic therapy was determined by the cellular uptake of nanocomposites and the targeting molecules used. (C) 2015 Elsevier B.V. All rights reserved.