Yung-Shin Sun

Fu Jen Catholic University, T’ai-pei, Taipei, Taiwan

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Publications (15)19.13 Total impact

  • Yung-Shin Sun, Xiangdong Zhu
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    ABSTRACT: Microarrays provide a platform for high-throughput characterization of biomolecular interactions. To increase the sensitivity and specificity of microarrays, surface blocking is required to minimize the nonspecific interactions between analytes and unprinted yet functionalized surfaces. To block amine- or epoxy-functionalized substrates, bovine serum albumin (BSA) is one of the most commonly used blocking reagents because it is cheap and easy to use. Based on standard protocols from microarray manufactories, a BSA concentration of 1% (10 mg/mL or 200 μM) and reaction time of at least 30 min are required to efficiently block epoxy-coated slides. In this paper, we used both fluorescent and label-free methods to characterize the BSA blocking efficiency on epoxy-functionalized substrates. The blocking efficiency of BSA was characterized using a fluorescent scanner and a label-free oblique-incidence reflectivity difference (OI-RD) microscope. We found that (1) a BSA concentration of 0.05% (0.5 mg/mL or 10 μM) could give a blocking efficiency of 98%, and (2) the BSA blocking step took only about 5 min to be complete. Also, from real-time and in situ measurements, we were able to calculate the conformational properties (thickness, mass density, and number density) of BSA molecules deposited on the epoxy surface. © 2015 Society for Laboratory Automation and Screening.
    Journal of the Association for Laboratory Automation 05/2015; DOI:10.1177/2211068215586977 · 1.50 Impact Factor
  • Yung-Shin Sun, X. D. Zhu
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    ABSTRACT: One key advantage of label-free biosensors involves the monitoring of binding between biomolecules. However, a number of experimental artifacts may lead to complicated real-time curves that do not fit well to a simple Langmuir model. As a result, the quality of the kinetic data must be improved to obtain accurate reaction rates. By carefully designing experiments, collecting the data, and processing the data, issues arising from signal drift, nonspecific binding, mass-transport effect in solution, mass-transport effect on surface, and curve fitting may be avoided or resolved. Using a label-free oblique-incidence reflectivity difference biosensor, key processes required to obtain reliable kinetic data and accurate reaction rates are described.
    Instrumentation Science & Technology 02/2015; 43(2). DOI:10.1080/10739149.2014.976830 · 0.80 Impact Factor
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    Yung-Shin Sun, Xiangdong Zhu
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    ABSTRACT: One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD) technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR). The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.
    Sensors 12/2014; 14(12):23307-20. DOI:10.3390/s141223307 · 2.05 Impact Factor
  • Yung-Shin Sun, Kit S. Lam, X. D. Zhu
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    ABSTRACT: Human serum is a mixture of various proteins which may interact with drugs. Therefore, it is of interest to investigate the binding kinetics of pharmaceuticals with their corresponding antibodies in serum. In this article, microarrays and a label-free biosensor were used to study these interactions. Microarrays provide a high-throughput platform for characterizing biomolecular interactions, and the label-free oblique-incidence reflectivity difference biosensor avoids the drawbacks of fluorescence-based methods. The experimental results show that the binding affinities between most of the drugs and their antibodies were reduced in human serum because the bulky proteins block the access to or reduce the stability of the reaction complexes. Therefore, one should be mindful when in vitro or in vivo testing the efficiency of potential drugs and their antibodies.
    Instrumentation Science & Technology 05/2014; 42(4). DOI:10.1080/10739149.2014.880149 · 0.80 Impact Factor
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    ABSTRACT: We demonstrate an effective method for generating libraries of encoded compounds for fabricating large compound microarrays on solid supports. This method is based on one-bead, one-compound synthesis and employs a novel trilayer bead-partition scheme that ensures sufficient quantity of synthesized compounds releasable from each bead for compound microarray fabrication in high-throughput protein–ligand discovery assays.[Supplementary materials are available for this article. Go to the publisher's online edition of Synthetic Communications® for the following free supplemental resource(s): Full experimental and spectral details.]
    Synthetic Communications 04/2014; 44(7). DOI:10.1080/00397911.2013.840728 · 0.98 Impact Factor
  • Yung-Shin Sun, Juntao Luo, Kit S. Lam, X. D. Zhu
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    ABSTRACT: An oblique-incidence reflectivity difference (OI-RD) scanning microscope was developed for label-free detection of the formation and disintegration of micelles upon solid substrates. Micelles are made of polymers with hydrophilic heads in contact with the surrounding solvent and hydrophobic tails in the micelle center. This characteristic makes them very efficient drug carriers. Streptavidin molecules were first printed on glass slides for capturing biotinylated polymers. Micelles were formed when the concentration of polymers was higher than a critical value. The formation of micelles resulted in an increase in the oblique-incidence reflectivity difference signals. As the concentration of polymers decreased below the critical value, micelles were disintegrated, and a corresponding decrease in the oblique-incidence reflectivity difference signals was observed. This microscope was employed for the real-time monitoring of the formation and disintegration of two different micelles. The critical concentration above which micelles were formed was determined to be around 0.0006 mg/mL for micelles made of PEG5KCA8 polymers. The results suggest that this microscope would have practical application in testing the efficiency and durability of micellar drug carriers.
    Instrumentation Science & Technology 11/2013; 41(6). DOI:10.1080/10739149.2013.798808 · 0.80 Impact Factor
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    Kai-Yin Lo, Yun Zhu, Hsieh-Fu Tsai, Yung-Shin Sun
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    ABSTRACT: Reactive oxygen species (ROS) are known to be a key factor in the development of cancer, and many exogenous sources are supposed to be related to the formation of ROS. In this paper, a microfluidic chip was developed for studying the production of ROS in lung cancer cells under different chemical and physical stimuli. This chip has two unique features: (1) five relative concentrations of 0, 1/8, 1/2, 7/8, and 1 are achieved in the culture regions; (2) a shear stress gradient is produced inside each of the five culture areas. Lung cancer cells were seeded inside this biocompatible chip for investigating their response to different concentrations of H2O2, a chemical stimulus known to increase the production of ROS. Then the effect of shear stress, a physical stimulus, on lung cancer cells was examined, showing that the production of ROS was increased in response to a larger shear stress. Finally, two antioxidants, α-tocopherol and ferulic acid, were used to study their effects on reducing ROS. It was found that high-dose α-tocopherol was not able to effectively eliminate the ROS produced inside cells. This counter effect was not observed in cells cultured in a traditional chamber slide, where no shear stress was present. This result suggests that the current microfluidic chip provides an in vitro platform best mimicking the physiological condition where cells are under circulating conditions.
    Biomicrofluidics 11/2013; 7(6):64108. DOI:10.1063/1.4836675 · 3.77 Impact Factor
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    ABSTRACT: A combination of the microarray platform and oblique-incidence reflectivity difference (OI-RD) microscopy was used to study the dependence of kinetic constants of probe-target reactions on the surface density of immobilized targets. Streptavidin-biotin reactions with a very high binding affinity were employed as the study model. Oblique-incidence reflectivity difference microscopy, a label-free and surface-based detection technique, was developed for monitoring real-time binding curves between two interactive biomolecules, enabling the acquisition of kinetic constants such as on-rate, off-rate, and equilibrium dissociation constants. These kinetic constants are important in characterizing biomolecular interactions because in living cells all intercellular and intermolecular reactions are at dynamic rather than at stable equilibrium. The kinetic constant of streptavidin binding to surface-immobilized biotin-bovine serum albumin was demonstrated to be significantly affected by the density of surface bovine serum albumin conjugates, mainly due to mass-transport effects within targets.
    Instrumentation Science & Technology 09/2013; 41(5):535-544. DOI:10.1080/10739149.2013.775590 · 0.80 Impact Factor
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    ABSTRACT: Interactions of glycan-binding proteins (GBPs) with glycans are essential in cell adhesion, bacterial/viral infection, and cellular signaling pathways. Experimental characterization of these interactions based on glycan microarrays typically involves (1) labeling GBPs directly with fluorescent reagents before incubation with the microarrays, or (2) labeling GBPs with biotin before the incubation and detecting the captured GBPs after the incubation using fluorescently labeled streptavidin, or (3) detecting the captured GBPs after the incubation using fluorescently labeled antibodies raised against the GBPs. The fluorescent signal is mostly measured ex situ after excess fluorescent materials are washed off. In this study, by using a label-free optical scanner for glycan microarray detection, we measured binding curves of 7 plant lectins to 24 glycans: four β1-4-linked galactosides, three β1-3-linked galactosides, one β-linked galactoside, one α-linked N-acetylgalactosaminide, eight α2-3-linked sialosides, and seven α2-6-linked sialosides. From association and dissociation constants deduced by global-fitting the binding curves, we found that (1) labeling lectins directly with fluorescent agents change binding profiles of lectins, in some cases by orders of magnitude; (2) those lectin-glycan binding reactions characterized with large dissociation rates, though biologically relevant, are easily missed or deemed insignificant in ex situ fluorescence-based assays as most captured lectins are washed off before detection. This study highlights the importance of label-free real-time detection of protein-ligand interactions and the potential pitfall in interpreting fluorescence-based assays for characterization of protein-glycan interactions.
    Molecular BioSystems 12/2011; 7(12):3343-52. DOI:10.1039/c1mb05332a · 3.18 Impact Factor
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    ABSTRACT: Conventional fluorescence microscopy is routinely used to detect cell surface markers through fluorophore-conjugated antibodies. However, fluorophore-conjugation of antibodies alters binding properties such as strength and specificity of the antibody in often uncharacterized ways. Here we present a method using an oblique-incidence reflectivity difference (OI-RD) microscope for label-free, real-time detection of cell surface markers, and apply it to analysis of stage-specific embryonic antigen 1 (SSEA1) on stem cells. Mouse stem cells express SSEA1 on their surfaces, and the level of SSEA1 decreases when the cells start to differentiate. In this study, we immobilized mouse stem cells and non-stem cells (control) on a glass surface as a microarray and reacted the cell microarray with unlabeled SSEA1 antibodies. By monitoring the reaction with an OI-RD microscope in real time, we confirmed that the SSEA1 antibodies bind only to the surface of the stem cells and not to the surface of non-stem cells. From the binding curves, we determined the equilibrium dissociation constant (Kd) of the antibody with the SSEA1 markers on the stem cell surface. Thus, the OI-RD microscope can be used to detect binding affinities between cell surface markers and unlabeled antibodies bound to the cells; this information could be useful for determination of stem cell stages.
    BioTechniques 06/2011; 50(6):381-8. DOI:10.2144/000113670 · 2.75 Impact Factor
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    ABSTRACT: Using a label-free high-throughput optical scanning microscope we detected endpoints and binding kinetics of vascular endothelial growth factor (VEGF) protein with microarrays of small molecule compounds from the NCI Developmental Therapeutics Program.
    Frontiers in Optics; 10/2009
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    ABSTRACT: Using a combination of an oblique-incidence reflectivity difference (OI-RD) scanning microscope and a customized 8-chamber sample cartridge, we detect 300 surface-immobilized molecular targets reacting with up to 8 different analytes simultaneously on a single slide.
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    ABSTRACT: We investigated surface chemistry platforms for immobilizing two types of synthetic, small-molecule compound libraries, each with over 6000 compounds, and for screening these compounds for potential proteins ligands by a label-free optical scanning microscope.
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    ABSTRACT: We describe a recently developed oblique-incidence reflectivity difference (OI-RD) microscope, a form of polarization-modulated imaging ellipsometer, for label-free-high-throughput detection of biomolecular reactions on DNA and protein microarrays. We present examples of application of this technique to end-point and real-time investigations of DNA-DNA hybridization, antibody-antigen capture, and protein-small-molecule binding reactions. Compared to a conventional imaging ellipsometer based on the polarizer-compensator-sample-analyzer scheme and under the off-null condition, a polarization-modulated OI-RD microscope is inherently more sensitive by at least 1 order of magnitude to thickness changes on a solid surface. Compared with imaging surface plasmon resonance microscopes based on reflectance change on falling or rising slopes of the surface plasmon resonance, the OI-RD microscope (1) has a comparable sensitivity, (2) is applicable to conventional microscope glass slides, and (3) easily covers a field of view as large as the entire surface of a 1 in. x 3 in. (2.54 cm x 7.62 cm) microscope slide.
    Applied Optics 05/2007; 46(10):1890-5. DOI:10.1364/AO.46.001890 · 1.69 Impact Factor
  • Yung-Shin Sun, James P. Landry, Xiangdong Zhu
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    ABSTRACT: One of the enabling platforms in proteomic research is parallel (high-throughput) detection of multiple biomolecular interactions on a microarray. To keep conformational and in turn functional integrity of protein molecules, label-free detection is desirable. We have developed an oblique-incidence optical reflectivity difference (OI-RD) technique for label-free measurements of protein reactions with molecular targets in microarray format immobilized on functionalized glass surface. As an ellipsometric technique, OI-RD measures changes in thickness and/or optical dielectric response instead of fluorescence. By incorporating total internal refection geometry and a multi-element photodiode array detector, we demonstrate how such the OI-RD technique can be efficiently used to measure multiple protein reactions in real time with surface-immobilized molecules or molecular groups on a glass substrate.