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ABSTRACT: We report a new two-axis active optical fiber manipulator for on-chip optical manipulation and detection in microfluidic environment. The system comprising of air chambers, fiber channels, controllable moving walls, and membrane structures were fabricated by using microelectromechanical systems technology. By adjusting air pressures to control the deflection of the pneumatic chambers placed orthogonal to and underneath the fiber channels, accurate alignment of a pair of approximately coaxial optical fibers, which was indicated by maximizing fiber-to-fiber optical-coupling measured in real time, has been achieved. A maximum displacement of a buried fiber as large as 13 mum at an applied pressure of 40 lb/in<sup>2</sup> for one air chamber has been demonstrated. It was sufficient to accurately align two approximately coaxial optical fibers to maximize the optical coupling efficiency. The maximum coupling efficiency for two single-mode optical fibers facing each other at a distance of 200 mum was measured to be 4.1%. The following features have been successfully demonstrated with this system: 1) stable optical trapping and stretching of a single red blood cell; 2) stable optical trapping of multiple microparticles; 3) optically driven controlled motion of single and multiple microparticles; and 4) integration of a counterpropagating dual-beam trap with single-beam optical tweezers. In addition to optical trapping and manipulation, the proposed device is promising for applications requiring coaxial input/output fibers for in-line optical analysis. Furthermore, it can be easily integrated with other microfluidic devices such as microcapillary electrophoresis channels or microflow cytometers for DNA, protein, and cell analysis.
Journal of Microelectromechanical Systems 07/2008; · 2.10 Impact Factor
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ABSTRACT: The formation of emulsification droplets is crucial for many industrial applications. This paper reports a new microfluidic chip capable of formation and collection of micro-droplets in liquids for emulsion applications. This microfluidic chip comprising microchannels, a micro-chopper and a micro-switch was fabricated by using micro-electro-mechanical-systems (MEMS) technology. The microfluidic chip can generate uniform droplets with tunable sizes by using combination of flow-focusing and liquid-chopping techniques. The droplet size can be actively fine-tuned by controlling either the relative sheath/sample flow velocity ratios or the chopping frequency. The generated droplets can be then sorted to a specific collection area utilizing an active pneumatic micro-switch formed with three micro-valves. Experimental data showed that the olive oil and sodium-alginate (Na-alginate) droplets with diameters ranging from 3 mum to 70 mum with a variation less than 14% is successfully generated and collected. The development of this microfluidic system can be promising for emulsion, drug delivery and nano-medicine applications.
Biomedical Microdevices 06/2008; 10(5):749-56. · 3.03 Impact Factor
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ABSTRACT: Hemagglutinin (HA) is the major immunogen on the envelope of avian influenza virus (AIV). Therefore we constructed two recombinant baculoviruses: Bac-HA, expressing histidine-tagged HA with the cytoplasmic domain (CTD) derived from HA, and Bac-HA64, expressing histidine-tagged HA with the CTD derived from baculovirus envelope protein gp64. After infection, HA with either CTD was expressed and anchored on the plasma membrane of Sf-9 cells, as revealed by confocal microscopy. Immunogold electron microscopy demonstrated that both Bac-HA and Bac-HA64 displayed HA on the viral surface. However, analyses of purified viruses revealed that significantly more HA was incorporated into Bac-HA64 than into Bac-HA. In comparison with Bac-HA, Bac-HA64 significantly improved the gene delivery and transgene expression in mammalian cells, as determined by quantitative real-time polymerase chain reaction and flow cytometry. Bac-HA64 elicited significantly higher hemagglutination inhibition titers in mouse models than Bac-HA and the negative controls. These data collectively confirmed that the gp64 CTD, in comparison with HA CTD, resulted in more efficient HA incorporation into baculovirus, more efficient transgene delivery and expression, and elevated immunogenicity. This is the first report demonstrating the potential of HA-pseudotyped baculovirus as an avian influenza vaccine and that the choice of CTD tremendously affects baculovirus properties and vaccine efficacy.
Molecular Therapy 06/2007; 15(5):989-96. · 6.87 Impact Factor
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ABSTRACT: Optical detection technique is critical for biomedical applications. The technique for optical fiber coupling is crucial for many optical detection applications. This study reports a new active two-axis micro coupler for buried optical fibers, which are commonly used for on-chip optical detection in microfluidic systems. The active micro coupler comprising air chambers, optical fiber channels, and controllable moving wall and membrane structures was fabricated by using micro electro-mechanical-systems technology. By adjusting air pressures to control the deflection of the pneumatic chambers placed orthogonal to and underneath optical fiber channels, precise alignment of buried head-on optical fibers can be achieved, which is crucial for on-line optical detection. Experimental results showed that the maximum deformation can be as high as 13 mum at an applied pressure of 40 psi for one air chamber, which is sufficient for precise coupling of two-axis optical fibers. The proposed device is promising for applications of head-on input/output fibers for the on-line optical analysis. It can be easily integrated with micro capillary electrophoresis or micro flow cytometer chips for DNA, protein and cell studies.
Micro Electro Mechanical Systems, 2007. MEMS. IEEE 20th International Conference on; 02/2007
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ABSTRACT: It has been shown that severe acute respiratory syndrome-associated coronavirus (SARS-CoV) 3a and 7a proteins, but not membrane (M) protein, induce apoptosis in mammalian cells. Upon expression of SARS-CoV M protein using the baculovirus/insect cell expression system, however, we found that the expressed M protein triggered accelerated apoptosis in insect cells, as characterized by rapid cell death, elevated cytotoxicity, cell shrinkage, nuclear condensation and DNA fragmentation. Conversely, the M protein expressed in mammalian cells did not induce apoptosis. This is the first report describing the induction of apoptosis by SARS-CoV M protein in animal cells and possible implications are discussed.
FEBS Letters 08/2006; 580(16):3829-34. · 3.54 Impact Factor
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ABSTRACT: To study the baculovirus/mammalian cell system for efficient expression of functional large hepatitis delta antigen (L-HDAg).
A recombinant baculovirus expressing histidine-tagged L-HDAg (L-HDAgH) was constructed to transduce baby hamster kidney (BHK) cells by a simplified transduction protocol.
The recombinant baculovirus transduced BHK cells with efficiencies higher than 90% as determined by flow cytometry. The expression level was significantly higher than that obtained by plasmid transfection and was further enhanced 3-fold to around 19 pg/cell by the addition of 10 mmol/L sodium butyrate. Importantly, the expressed L-HDAgH was localized to the cell nucleus and correctly isoprenylated as determined by immunofluorescence labeling and confocal microscopy. Moreover, L-HDAgH interacted with hepatitis B surface antigen to form virus-like particles.
The fusion with histidine tags as well as overexpression of L-HDAgH in the baculovirus-transduced BHK cells does not impair the biological functions. Taken together, the baculovirus/mammalian cell system offers an attractive alternative for high level expression of L-HDAgH or other proteins that require extensive post-translational modifications.
World Journal of Gastroenterology 04/2006; 12(10):1551-7. · 2.47 Impact Factor
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ABSTRACT: Enterovirus 71 (EV71) has been implicated as the etiological agent responsible for the recent outbreaks of hand, foot and mouth disease associated with severe neurological diseases in the Asia-Pacific region.
The assembly process was hypothesized to occur via an orchestrated proteolytic processing of the P1 precursor by the viral protease 3CD. To test this hypothesis, we constructed 3 recombinant baculoviruses: Bac-P1 expressing P1; Bac-3CD expressing 3CD; and Bac-P1-3CD co-expressing P1 and 3CD.
Both single infection by Bac-P1-3CD and co-infection by Bac-P1 and Bac-3CD resulted in correct cleavage of P1 to yield individual proteins VP0, VP1 and VP3, while the former approach yielded higher VLP production. In the cells, the structural proteins self-assembled into clusters of virus-like particles (VLP) resembling the authentic EV71 particle aggregates. After ultracentrifugation purification, the dispersed VLPs were indistinguishable from the authentic virus in size, appearance, composition and surface epitopes, as determined by SDS-PAGE, Western blot, transmission electron microscopy and immunogold labeling.
Our data, for the first time, suggest that in insect cells EV71 structural proteins adopt a processing and assembly pathway similar to poliovirus assembly. The preservation of particle morphology and composition suggest that the VLP may be a valuable vaccine candidate to prevent EV71 epidemics.
World Journal of Gastroenterology 03/2006; 12(6):921-7. · 2.47 Impact Factor
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ABSTRACT: Baculovirus has emerged as a promising vector for in vivo or ex vivo gene therapy. To date, the infectious titer and multiplicity of infection (MOI) based on the ability of baculovirus to infect insect cells are commonly adopted to indicate the virus dosage. However, the infectious titer and MOI do not reliably represent the baculovirus transducing ability, making the comparison of baculovirus-mediated gene transfer difficult. To determine the baculovirus transducing ability more rapidly and reliably, we developed a protocol to evaluate the transducing titers of baculovirus stocks. The virus was diluted twofold serially and used to transduce HeLa cells. The resultant transduction efficiencies were measured by flow cytometry for the calculation of transducing titers. Compared to the infectious titer, the determination of transducing titer is more reproducible as the standard deviations among measurements are smaller. Also, the transducing titers can be obtained in 24 h, which is significantly faster as opposed to 4-7 days to obtain the infectious titer. More importantly, we demonstrated that baculoviruses with higher transducing titers could transduce cells at higher efficiency and yield stronger and longer transgene expression, confirming that the transducing titer was representative of the baculovirus transducing ability. This finding is particularly significant for ex vivo gene delivery whereby unconcentrated viruses are used for transduction and long-term transgene expression is desired. In this regard, our titration protocol provides a simple, fast, and reliable measure to evaluate the quality of virus stocks during virus production and purification, and is helpful to predict the performance of vector supernatants and ensure reproducible gene delivery experiments.
Biotechnology and Bioengineering 03/2006; 93(3):564-71. · 3.95 Impact Factor
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ABSTRACT: We have recently demonstrated the assembly of hepatitis delta virus-like particles (HDV VLP) by co-transducing hepatoma cells using two recombinant baculoviruses, one encoding hepatitis B surface antigen (HBsAg), and one encoding large delta antigen (L-HDAg). In this study, we further demonstrated the assembly and secretion of VLP in other mammalian cells. The assembly efficiency varied depending on cell lines, the baculovirus constructs and the relative dosage of both recombinant viruses. The co-transduction of BHK cells led to the formation of VLPs resembling authentic virions in size and appearance. The production process was transferred to a novel oscillating packed bed bioreactor, BelloCell, in which the transduction efficiency was up to approximately 90% for a high cell density of 1.5 x 10(7) cells/cm(3) bed and a total yield of 427 microg based on HBsAg in the VLP (harvested from 940 ml medium) was obtained. The particle yield corresponded to an average volumetric yield of 454 ngml(-1) and a specific yield of 285 microg/10(9) cells, and is significantly superior to that can be obtained by the commonly employed transfection method. The combination of baculovirus transduction and BelloCell reactor, thus, may represent a simple and efficient approach for the production of HDV VLP and viral vectors.
Journal of Biotechnology 09/2005; 118(2):135-47. · 3.05 Impact Factor
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ABSTRACT: Histidine-tagged N (rNH) and E (rEH) proteins of Severe Acute Respiratory Syndrome (SARS)-coronovirus were expressed in the baculovirus/insect cell system and purified by immobilized metal affinity chromatography. rNH and rEH proteins differed markedly with respect to expression levels, cell death kinetics and subcellular localizations that led to different extraction and purification schemes. The features of both proteins are compared and the potential applications of purified rNH and rEH are discussed.
Biotechnology Letters 08/2005; 27(13):883-91. · 1.68 Impact Factor
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ABSTRACT: This study reports a new microfluidic chip capable of delivering and pre-positioning cells in a predefined trapping zone, and followed by manipulation of buried optical fibers for on-chip, dual-beam, optical trapping and stretching. In this microfluidic system, microchannels, micropumps, microvalves, dielectrophoretic (DEP) electrodes and active fiber manipulators were fabricated and integrated using micro-electro-mechanical-systems technology to perform several crucial functions including transportation, pre-positioning and manipulation of cells. Experimental results showed that by integrating three micropumps connected in series, the cell samples were automatically delivered into the flow focusing area and then transported to the trapping zone. A single cell can be confined by microvalves and then elevated towards the optical trapping zone by a negative-DEP force operated at a low voltage (20 Vp–p) and at a specific frequency (900 kHz). The active fiber manipulators can be used for optical trapping, manipulation, and stretching. A red blood cell was successfully trapped and stretched by a dual-beam, optical trap using the proposed microfluidic system. The developed system is promising for further applications that require trapping, manipulation and biomechanical analysis of a single cell or particle.
Sensors and Actuators B: Chemical.
