Browsing by Author "Liu, Cheng-Hsien"

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A highly efficient bead extraction technique with low bead number for digital microfluidic immunoassay
(2016-01)
Huang, Cheng-Yeh
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Tsai, Po-Yen
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Lee, I-Chin
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Hsu, Hsin-Yun
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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 microfluidic chip with a U-shaped microstructure array for multicellular spheroid formation, culturing and analysis
(2014-03-01)
Fu, Chien-Yu
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Tseng, Sheng-Yang
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Yang, Shih-Mo
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Hsu, Long
;
Liu, Cheng-Hsien
Multicellular spheroids (MCS), formed by self-assembly of single cells, are commonly used as a three-dimensional cell culture model to bridge the gap between in vitro monolayer culture and in vivo tissues. However, current methods for MCS generation and analysis still suffer drawbacks such as being labor-intensive and of poor controllability, and are not suitable for high-throughput applications. This study demonstrates a novel microfluidic chip to facilitate MCS formation, culturing and analysis. The chip contains an array of U-shaped microstructures fabricated by photopolymerizing the poly(ethylene glycol) diacrylate hydrogel through defining the ultraviolet light exposure pattern with a photomask. The geometry of the U-shaped microstructures allowed trapping cells into the pocket through the actions of fluid flow and the force of gravity. The hydrogel is non-adherent for cells, promoting the formation of MCS. Its permselective property also facilitates exchange of nutrients and waste for MCS, while providing protection of MCS from shearing stress during the medium perfusion. Heterotypic MCS can be formed easily by manipulating the cell trapping steps. Subsequent drug susceptibility analysis and long-term culture could also be achieved within the same chip. This MCS formation and culture platform can be used as a micro-scale bioreactor and applied in many cell biology and drug testing studies.
A novel optoelectrofulidic system for cells/particles manipulation and sorting
(2010)
Yang, Shih-Mo
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Yu, Tung-Ming
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Huang, Hang-Ping
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Hsu, Long
;
Liu, Cheng-Hsien
A novel optoelectrofulidic system integrated optical image concentration and alignment system, dielectrophoresis phenomenon, microfluidic and friendly real-time control interface is first reported in this article. A new application of photoconductive material oxotitanium phthalocyanine (TiOPc) for microparticle applying has been first described and demonstrated by our research group. Basis on the special character of the photoconductive material, a TiOPc-based optoelectronic tweezers (Ti-OET) is utilized for single and massive cells/particles manipulation. The objects wanted to be manipulated are defined with different behaviors (e.g., press, release, drag and move) using Flash (R) software when the cursor acts on them. It also reveals the application for biological application to form the cells trapping with three sorts of cells, HMEC-1, HepG2 and HEK293t. Another application of our optoelectrofulidic system is to fabricate a TiOPc-based flow cytometry chip which can be used for sorting the 15 mu m diameter particles with 105 mu m/s velocity. When the 10Vp. p. voltage and 45 kHz AC frequency apply on the top and button ITO electrode, the illuminated light pattern will become a spatially virtual switch inside the microchannel. The dielectrophoresis force between top ITO glass and button photoconductive layer controlled by the friendly interface will concentrate the cells/particles as a straight line and individually direct each one in different paths. In summary, we have established an optoelectronfulidic-based chip and spatially virtual switch system which are applied for cell pattern and particles sorting. In the future, this easy manipulation approach can place the full power of optoelectronfulidic chip into the biological operators' hands.
Cell patterning via diffraction-induced optoelectronic dielectrophoresis force on an organic photoconductive chip
(2013)
Yang, Shih-Mo
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Tseng, Sheng-Yang
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Chen, Hung-Po
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Hsu, Long
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Liu, Cheng-Hsien
A laser diffraction-induced dielectrophoresis (DEP) phenomenon for the patterning and manipulation of individual HepG2 cells and polystyrene beads via positive/negative DEP forces is reported in this paper. The optoelectronic substrate was fabricated using an organic photoconductive material, TiOPc, via a spin-coating process on an indium tin oxide glass surface. A piece of square aperture array grid grating was utilized to transform the collimating He-Ne laser beam into the multi-spot diffraction pattern which forms the virtual electrodes as the TiOPc-coating surface was illuminated by the multi-spot diffraction light pattern. HepG2 cells were trapped at the spot centers and polystyrene beads were trapped within the dim region of the illuminated image. The simulation results of light-induced electric field and a Fresnel diffraction image illustrated the distribution of trapped microparticles. The HepG2 morphology change, adhesion, and growth during a 5-day culture period demonstrated the cell viability through our manipulation. The power density inducing DEP phenomena, the characteristics of the thin TiOPc coating layer, the operating ac voltage/frequency, the sandwiched medium, the temperature rise due to the ac electric fields and the illuminating patterns are discussed in this paper. This concept of utilizing laser diffraction images to generate virtual electrodes on our TiOPc-based optoelectronic DEP chip extends the applications of optoelectronic dielectrophoretic manipulation.
Concentration of Magnetic Beads Utilizing Light-Induced Electro-Osmosis Flow
(2011-10-01)
Yang, Shih-Mo
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Harishchandra, Punde Tushar
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Yu, Tung-Ming
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Liu, Ming-Huei
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Hsu, Long
Magnetic beads are widely utilized for separating biomolecules, DNA and RNA. Traditionally, bulk magnet is utilized for manipulation these particles but when it comes to microscale bulk magnet is not the efficient method. Here, we utilize an organic photoconductive material, TiOPc, to generate light-induced electro-osmosis flow on chip. The fabrication process is convenient to be handled by the researchers and biologists without cleanroom IC fabrication facility. When specifically designed light pattern is projected onto the TiOPc substrate, the conductivity of the organic material layer within the illuminating region increases and the charges are locally assembled on its surface to form a virtual electrode. With an external ac voltage of 5 Vpp at 10 kHz, numerous magnetic beads are attracted from the nonilluminating region toward the center of light-pattern illuminating region. Driven by the moving light image, the grouped magnetic beads can be manipulated and merged in a desired way or direction. The light manipulation process provides a flexible and convenient approach for in vitro control of magnetic beads. We expect that this light-driven technology would display a multifunctional platform for manipulation of microparticles.
DIELECTROPHORETIC CONCENTRATOR FOR ENHANCED PERFORMANCE OF POLY-SILICON NANOWIRE FIELD EFFECT TRANSISTOR FOR BIOSENSING APPLICATION
(2012)
Puttaswamy, Srinivasu Valagerahally
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Su, Yi-, Jr.
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Sivashankar, Shilpa
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Yang, Shih-Mo
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Yang, Yuh-Shyong
Biological and medical fields have authenticated stupendous advancement in the development of biosensors and biochips. The development of highly sensitive biological sensors is need of the hour. This research work utilizes the dielectrophoretic concentrator chip with titanium (Ti) electrodes to shape the electric field, to pre-concentrate deoxyribo nucleic acid (DNA) within microfluidic channel onto nanowire region of poly-silicon nanowire field effect transistor (poly-Si NWFET). Preconcentration of biomolecules at the constriction site was demonstrated by focusing the DNA with negative dielectrophoresis (nDEP) manipulation technique. After integrating the DEP device with poly-Si NWFET, the I-V-G curve was plotted to ensure n-type behavior.
Digital Microfluidic Dynamic Culture of Mammalian Embryos on an Electrowetting on Dielectric (EWOD) Chip
(2015-05-01)
Huang, Hong-Yuan
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Shen, Hsien-Hua
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Tien, Chang-Hung
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Li, Chin-Jung
;
Fan, Shih-Kang
Current human fertilization in vitro (IVF) bypasses the female oviduct and manually inseminates, fertilizes and cultivates embryos in a static microdrop containing appropriate chemical compounds. A microfluidic microchannel system for IVF is considered to provide an improved in-vivo-mimicking environment to enhance the development in a culture system for an embryo before implantation. We demonstrate a novel digitalized microfluidic device powered with electrowetting on a dielectric (EWOD) to culture an embryo in vitro in a single droplet in a microfluidic environment to mimic the environment in vivo for development of the embryo and to culture the embryos with good development and live births. Our results show that the dynamic culture powered with EWOD can manipulate a single droplet containing one mouse embryo and culture to the blastocyst stage. The rate of embryo cleavage to a hatching blastocyst with a dynamic culture is significantly greater than that with a traditional static culture (p<0.05). The EWOD chip enhances the culture of mouse embryos in a dynamic environment. To test the reproductive outcome of the embryos collected from an EWOD chip as a culture system, we transferred embryos to pseudo-pregnant female mice and produced live births. These results demonstrate that an EWOD-based microfluidic device is capable of culturing mammalian embryos in a microfluidic biological manner, presaging future clinical application.
Dynamic manipulation and patterning of microparticles and cells by using TiOPc-based optoelectronic dielectrophoresis
(2010-06-15)
Yang, Shih-Mo
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Yu, Tung-Ming
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Huang, Hang-Ping
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Ku, Meng-Yen
;
Hsu, Long
We develop light-driven optoelectronic tweezers based on the organic photoconductive material titanium oxide phthalocyanine. These tweezers function based on negative dielectrophoresis (nDEP). The dynamic manipulation of a single microparticle and cell patterning are demonstrated by using this light-driven optoelectronic DEP chip. The adaptive light patterns that drive the optoelectronic DEP onchip are designed by using Flash software to approach appropriate dynamic manipulation. This is also the first reported demonstration, to the best of our knowledge, for successfully patterning such delicate cells from human hepatocellular liver carcinoma cell line HepG2 by using any optoelectronic tweezers. (C) 2010 Optical Society of America
Electrodeless dielectrophoretic concentrator for analyte pre-concentration on poly-silicon nanowire field effect transistor
(2013-03-01)
Puttaswamy, Srinivasu Valagerahally
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Lin, Chih-Heng
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Sivashankar, Shilpa
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Yang, Yuh-Shyong
;
Liu, Cheng-Hsien
The sensitivity of biosensors can be affected by mass transport limitations as a result of miniaturization. To enhance signal sensitivity, pre-concentration of deoxyribonucleic acid (DNA), in the vicinity of the sensing elements of n-type polycrystalline silicon nanowire field effect transistor (Poly-Si NWFET) is demonstrated using a microfluidic device based on insulator or gradient dielectrophoresis (iDEP) to overcome mass transport limitations. In insulator-based dielectrophoresis (iDEP), insulating microstructures produce non-uniform electric fields to drive dielectrophoresis (DEP) in microsystems. Fabrication of accurately controlled three-dimensional (3D) microstructure of poly (ethylene glycol) diacrylate (PEG-DA) with a narrow microchannel using maskless gray-scale lithography is described. The fabrication of three-dimensional structures at low cost with saving time is accomplished by the use of maskless exposure system. PEG-DA has been used due to its excellent biocompatibility and ease of fabrication. To enhance the stability of PEG-DA, we added another polymer pentaerythritol tetraacrylate (PETA). Electrical property of the Poly-Si NWFET is ensured by plotting I-V-G curve after the integration of iDEP micro device. (c) 2013 Elsevier B.V. All rights reserved.
EMBRYO LAB CHIP TAKING ADVANTAGE OF MICROFLUIDICS AND CELL CO-CULTURING
(2015)
Liu, Cheng-Hsien
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Chang, Kuo-Wei
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Chang, Pei-Yu
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Sung, Yi-Jung
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Huang, Hong-Yuan
This research serves as an in vitro platform for fertilization and embryonic development. Thin polydimethylsiloxane ( PDMS) membrane was etched to create an array of through-holes, and sandwiched between two PDMS channels to form an upper chamber and lower chamber. This device integrates the concentration gradient design and perfusion system for proposed embryo coculture with stromal cells.
Fabrication of 3D Microfluidic Chip integrating Concentration gradient design and Perfusion system for Embryo coculture with Stromal cells
(2012-01-01)
Chang, Pei-Yu
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Chang, Kuo-Wei
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Lin, Ciou-Wen
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Shih, Po-Chen
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Liu, Cheng-Hsien
Fabrication of 3D chip that enables cell culture under continuous flow perfusion and repeated in situ observation by light microscopy is described. Thin polydimethylsiloxane PDMS) membrane was etched to create an array of through-holes, and sandwiched between two PDMS channels to form an upper chamber and lower chamber. Thus we fabricated a 3D microfluidic chip which integrates the concentration gradient design and perfusion system for proposed embryo coculture with stromal cells.
Fabrication of PEG Hydrogel Microstructures in Microfluidic System for Multicellular Spheroid Formation, Culturing and Analysis
(2011)
Fu, Chien-Yu
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Tseng, Sheng-Yang
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Yang, Shih-Mo
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Hsu, Long
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Liu, Cheng-Hsien
This study presents a novel microfluidic device for 3 dimension cell culturing and analysis. Through defining the exposure to ultra-violet light by a photomask, localized photopolymerization of poly (ethylene glycol) diacrylate hydrogels form biocompatible and permselective microstructures within a microfluidic device. The designed geometry of microstructures allowed cells to be trapped into structures and to form spheroids in situ. Subsequent analysis and long-term three-dimension cell culture can achieve within the same device. This technique may apply as a microscale bioreactor or a platform for spheroid-based drug screening and study.
Finger-powered agglutination lab chip with CMOS image sensing for rapid point-of-care diagnosis applications
(2020-01-21)
Lu, Chung Hsiang
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Shih, Ting-Sheng
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Shih, Po-Chen
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Pendharkar, Gaurav Prashant
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Liu, Cheng-En
Agglutination is an antigen-antibody reaction with visible expression of aggregation of the antigens and their corresponding antibodies. Applications extend to the identification of acute bacterial infection, hemagglutination, such as blood grouping, and diagnostic immunology. Our finger-powered agglutination lab chip with external CMOS image sensing was developed to support a platform for inexpensive, rapid point-of-care (POC) testing applications related to agglutination effects. In this paper, blood grouping (ABO and Rh grouping) was utilized to demonstrate the function of our finger-powered agglutination lab chip with CMOS image sensing. Blood antibodies were preloaded into the antibody reaction chamber in the lab chip. The blood sample was pushed through the antibody reaction chamber using finger-powered pressure actuation to initiate a hemagglutination reaction to identify the blood type at the on-chip detection area using our homemade CMOS image sensing mini-system. Finger-powered actuation without the need for external electrical pumping is excellent for low-cost POC applications, but the pumping liquid volume per finger push is hard to control. In our finger-powered agglutination lab chip with CMOS image sensing, we minimized the effects of different finger push depths and achieved robust performance for the test results with different push depths. The driving sample volume per finger push is about 0.79 mm(3). For different chips and different pushes, the driven sample volume per finger push was observed to vary in the range of 0.64 to 1.18 mm(3). The red blood cells were separated from the plasma on-chip after the whole blood sample was finger pumped and before the red blood cells reached the antibody chamber via an embedded plasma-separation membrane. Our homemade CMOS image mini-system robustly read and identified the agglutination results on our agglutination lab chip.
Identification of Protein Domains on Major Pilin MrkA That Affects the Mechanical Properties of Klebsiella pneumoniae Type 3 Fimbriae
(2012-05-15)
Chan, Chia-Han
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Chen, Feng-Jung
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Huang, Ying-Jung
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Chen, Shin-Yu
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Liu, Kuo-Liang
The Klebsiella pneumoniae type 3 fimbriae are mainly composed of MrkA pilins that assemble into a helixlike filament. This study determined the biomechanical properties of the fimbriae and analyzed 11 site-directed MrkA mutants to identify domains that are critical for the properties. Escherichia coli strains expressing type 3 fimbriae with an Ala substitution at either F34, V45, C87, G189, T196, or Y197 resulted in a significant reduction in biofilm formation. The E. coli strain expressing MrkAG189A remained capable of producing a normal number of fimbriae. Although F34A, V45A, T196A, and Y197A substitutions expressed on E. coli strains produced sparse quantities of fimbriae, no fimbriae were observed on the cells expressing MrkAC87A. Further investigations of the mechanical properties of the MrkAG189A fimbriae with optical tweezers revealed that, unlike the wild-type fimbriae, the uncoiling force for MrkAG189A fimbriae was not constant. The MrkAG189A fimbriae also exhibited a lower enthalpy in the differential scanning calorimetry analysis. Together, these findings indicate that the mutant fimbriae are less stable than the wildtype. This study has demonstrated that the C-terminal beta strands of MrkA are required for the assembly and structural stability of fimbriae.
Integration of organic opto-electrowetting and poly(ethylene) glycol diacrylate (PEGDA) microfluidics for droplets manipulation
(2013-04-01)
Yu, Tung-Ming
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Yang, Shih-Mo
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Fu, Chien-Yu
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Liu, Ming-Huei
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Hsu, Long
This paper reports a fabrication technology which integrates organic opto-electrowetting (OEW) with PEGDA-based microfluidics between stacked ITO glass chips for droplets generation and manipulation. Organic OEW can be realized by using titanium oxide phthalocyanine (TiOPc) as a photoconductive layer. Optical images can be projected on an organic OEW area to induce the local virtual electrodes which reduce the surface energy for actuating droplets. Low-molecular-weight (LMW) PEGDA (poly(ethylene) glycol diacrylate) material is used to form stable and biocompatible microchannels between the stacked ITO glass chips by UV photopolymerization process. PEGDA-based microfluidics provides a simpler way to enhance droplets applications in organic OEW. We demonstrate (1) the droplet formation in PEGDA microstructure, (2) the motion of droplet-in-air and droplet-in-oil operated by TiOPc OEW in this platform. This technology requires only spin-coating and UV exposure and is more cost-effective than previous methods. (C) 2011 Elsevier B.V. All rights reserved.
Light-driven manipulation of picobubbles on a titanium oxide phthalocyanine-based optoelectronic chip
(2011-04-11)
Yang, Shih-Mo
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Yu, Tung-Ming
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Huang, Hang-Ping
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Ku, Meng-Yen
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Tseng, Sheng-Yang
Microbubbles have a variety of applications in science and biological technology. Here, we demonstrate the manipulation of the picoliter gas bubble (picobubble) based on the optoelectronic-mechanism. The organic photoconductive material, titanium oxide phthalocyanine (TiOPc), was developed to make the light-sensitive substrate of this optoelectronic chip. The virtual electrodes are formed by projecting the dynamic light pattern onto TiOPc layer for generating the desired nonuniform electric field. The picobubble suspended in silicone oil can be manipulated with the velocity of 40-50 mu m/s. The driving force up to 160 pico-Newtons could be generated for manipulating a gas bubble of 300 picoliters. (C) 2011 American Institute of Physics. [doi:10.1063/1.3580760]
Liver-cell patterning Lab Chip: mimicking the morphology of liver lobule tissue
(2013)
Ho, Chen-Ta
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Lin, Ruei-Zeng
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Chen, Rong-Jhe
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Chin, Chung-Kuang
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Gong, Song-En
A lobule-mimetic cell-patterning technique for on-chip reconstruction of centimetre-scale liver tissue of heterogeneous hepatic and endothelial cells via an enhanced field-induced dielectrophoresis (DEP) trap is demonstrated and reported. By mimicking the basic morphology of liver tissue, the classic hepatic lobule, the lobule-mimetic-stellate-electrodes array was designed for cell patterning. Through DEP manipulation, well-defined and enhanced spatial electric field gradients were created for in-parallel manipulation of massive individual cells. With this liver-cell patterning labchip design, the original randomly distributed hepatic and endothelial cells inside the microfluidic chamber can be manipulated separately and aligned into the desired pattern that mimicks the morphology of liver lobule tissue. Experimental results showed that both hepatic and endothelial cells were orderly guided, snared, and aligned along the field-induced orientation to form the lobule-mimetic pattern. About 95% cell viability of hepatic and endothelial cells was also observed after cell-patterning demonstration via a fluorescent assay technique. The liver function of CYP450-1A1 enzyme activity showed an 80% enhancement for our engineered liver tissue (HepG2+HUVECs) compared to the non-patterned pure HepG2 for two-day culturing.
Measurement of the adhesive force between a single Klebsiella pneumoniae type 3 fimbria and collagen IV using optical tweezers
(2006-11-10)
Chang, Bo-Jui
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Huang, Ying-Jung
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Chan, Chia-Han
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Hsu, Long
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Peng, Hwei-Ling
Type 3 fimbriae are important adhesive filaments that assist Klebsiella pneumoniae to establish an infection. Different MrkD adhesin variants on the fimbriae are known to display distinct adherence capability for the bacteria to bind extracellular matrix proteins, although the difference has not been determined physically. For this reason, the adhesive force between type 3 fimbriae and collagen IV were measured using optical tweezers. The measured force data displayed a periodic histogram thus Fourier analysis was applied to group it to extract the adhesive force of a single molecular pair. Specifically, we showed that grouping should begin with an offset at the first half of the period. Finally, we first present the adhesive force between each mrkD(V2)-, mrkD(V3)-, and mrkD(V4)-expressed fimbriae and collagen IV is 2.03, 3.79, and 2.87 pN, respectively, This result can be referred to further research on mrkD allelic effect on bacteria infection. (c) 2006 Elsevier Inc. All rights reserved.
ORGANIC PHOTOCONDUCTIVE DIELECTROPHORESIS BY USING TITANIUM OXIDE PHTHALOCYANINE FOR MICRO-PARTICLES MANUPULATION
(2010)
Yu, Tung-Ming
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Yang, Shi-Mo
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Huang, Hang-Ping
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Ku, Meng-Yen
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Tseng, Sheng-Yang
This paper reports our updating development for the organic photoconductive dielectrophoresis (OPDEP) by using titanium oxide phthalocyanine (TiOPc) material for the manipulation of micro-objects. The advantages of organic photoconductive material include non-toxicity, low cost, easy coating fabrication, flexibility, panchromaticity and high photosensitivity [1]. TiOPc has the high photoconductivity and the optical absorptions in the near infrared region (550 - 850 nm) to be a candidate of the light-sensitive charge generation layer. OPDEP chip is easily mass-produced by using a spin-coating process. Here, we demonstrate two kinds of the manipulations of micro-particles on our OPDEP chip. Firstly, the trapping and movement of single micro-particle is achieved by the ring shaped virtual electrode. Secondly, the steel rim shaped virtual electrode is designed to rotate micro-particles and characterize the OPDEP induced force.
Simultaneous detection of two growth factors from human single-embryo culture medium by a bead-based digital microfluidic chip
(2020-02-15)
Lee, Meng-Shiue
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Hsu, Wensyang
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Huang, Hong-Yuan
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Tseng, Hsueh-Yang
;
Lee, Chia-Tung
The measurement of growth factors released in a culture medium is considered to be an attractive non-invasive approach, apart from the embryo morphology, to identify the condition of an embryo development after fertilization in vitro (IVF), but the available embryo culture medium in the current method is only a few microlitres. This small sample volume, also of small concentration, makes difficult the application of a conventional detection method, such as an enzyme-linked immunosorbent assay (ELISA). A reliable detection of the growth factor from each embryo culture medium of such a small concentration hence remains a challenge. Here for the first time we report the results of measurement of not just one, but two, growth factors, human IL-1 beta and human TNF-alpha, from an individual droplet of embryo culture medium with a bead-based digital microfluidic chip. The required sample volume for a single measurement is only 520 nL; the total duration of the on-chip process is less than 40 min. Using the culture media of human embryos with normal morphologic features, we found that the concentrations of TNF-alpha change little from day 3 to day 5-6, but the concentrations of IL-1 beta for some embryos might double from day 3 to day 5-6. For other embryos even with similar normal morphologic features, some growth factors, such as IL-1 beta, might exhibit different expressions during the culture period. Those growth factors could serve to distinguish the development conditions of each embryo, not merely from an observation of embryo morphology.