Publications by authors named "Dong-Sik Shin"

38 Publications

Caffeoyl-Prolyl-Histidine Amide Inhibits Fyn and Alleviates Atopic Dermatitis-Like Phenotypes via Suppression of NF-κB Activation.

Int J Mol Sci 2020 Sep 28;21(19). Epub 2020 Sep 28.

Department of Life Science, Sogang University, Seoul 04107, Korea.

Caffeic acid (CA) is produced from a variety of plants and has diverse biological functions, including anti-inflammation activity. It has been recently demonstrated that caffeoyl-prolyl-histidine amide (CA-PH), which is CA conjugated with proline-histidine dipeptide, relieves atopic dermatitis (AD)-like phenotypes in mouse. In this study, we investigated the molecular mechanism underlying CA-PH-mediated alleviation of AD-like phenotypes using cell line and AD mouse models. We confirmed that CA-PH suppresses AD-like phenotypes, such as increased epidermal thickening, infiltration of mast cells, and dysregulated gene expression of cytokines. CA-PH suppressed up-regulation of cytokine expression through inhibition of nuclear translocation of NF-κB. Using a CA-PH affinity pull-down assay, we found that CA-PH binds to Fyn. In silico molecular docking and enzyme kinetic studies revealed that CA-PH binds to the ATP binding site and inhibits Fyn competitively with ATP. CA-PH further suppressed spleen tyrosine kinase (SYK)/inhibitor of nuclear factor kappa B kinase (IKK)/inhibitor of nuclear factor kappa B (IκB) signaling, which is required for nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. In addition, chronic application of CA-PH, in contrast with that of glucocorticoids, did not induce up-regulation of regulated in development and DNA damage response 1 (REDD1), reduction of mammalian target of rapamycin (mTOR) signaling, or skin atrophy. Thus, our study suggests that CA-PH treatment may help to reduce skin inflammation via down-regulation of NF-κB activation, and Fyn may be a new therapeutic target of inflammatory skin diseases, such as AD.
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http://dx.doi.org/10.3390/ijms21197160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7582254PMC
September 2020

Robust Magnetized Graphene Oxide Platform for In Situ Peptide Synthesis and FRET-Based Protease Detection.

Sensors (Basel) 2020 Sep 15;20(18). Epub 2020 Sep 15.

Department of Chemical and Biological Engineering, Sookmyung Women's University, Yongsan-gu, Seoul 04310, Korea.

Graphene oxide (GO)/peptide complexes as a promising disease biomarker analysis platform have been used to detect proteolytic activity by observing the turn-on signal of the quenched fluorescence upon the release of peptide fragments. However, the purification steps are often cumbersome during surface modification of nano-/micro-sized GO. In addition, it is still challenging to incorporate the specific peptides into GO with proper orientation using conventional immobilization methods based on pre-synthesized peptides. Here, we demonstrate a robust magnetic GO (MGO) fluorescence resonance energy transfer (FRET) platform based on in situ sequence-specific peptide synthesis of MGO. The magnetization of GO was achieved by co-precipitation of an iron precursor solution. Magnetic purification/isolation enabled efficient incorporation of amino-polyethylene glycol spacers and subsequent solid-phase peptide synthesis of MGO to ensure the oriented immobilization of the peptide, which was evaluated by mass spectrometry after photocleavage. The FRET peptide MGO responded to proteases such as trypsin, thrombin, and β-secretase in a concentration-dependent manner. Particularly, β-secretase, as an important Alzheimer's disease marker, was assayed down to 0.125 ng/mL. Overall, the MGO platform is applicable to the detection of other proteases by using various peptide substrates, with a potential to be used in an automated synthesis system operating in a high throughput configuration.
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http://dx.doi.org/10.3390/s20185275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570466PMC
September 2020

Microfluidic Generation of Amino-Functionalized Hydrogel Microbeads Capable of On-Bead Bioassay.

Micromachines (Basel) 2019 Aug 9;10(8). Epub 2019 Aug 9.

Department of Chemical and Biological Engineering, Sookmyung Women's University, 100 Cheongpa-ro 47-gil, Yongsan-gu, Seoul 04310, Korea.

Microfluidic generation of hydrogel microbeads is a highly efficient and reproducible approach to create various functional hydrogel beads. Here, we report a method to prepare crosslinked amino-functionalized polyethylene glycol (PEG) microbeads using a microfluidic channel. The microbeads generated from a microfluidic device were evaluated by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and confocal laser scanning microscopy, respectively. We found that the microbeads were monodisperse and the amino groups were localized on the shell region of the microbeads. A swelling test exhibited compatibility with various solvents. A cell binding assay was successfully performed with RGD peptide-coupled amino-functionalized hydrogel microbeads. This strategy will enable the large production of the various functional microbeads, which can be used for solid phase peptide synthesis and on-bead bioassays.
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http://dx.doi.org/10.3390/mi10080527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723060PMC
August 2019

Solid-Phase Synthesis of Peptide-Conjugated Perylene Diimide Bolaamphiphile and Its Application in Photodynamic Therapy.

ACS Omega 2018 May;3(5):5896-5902

School of Chemical and Biological Engineering, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea.

Here, we describe a rapid and efficient synthetic method of peptide-conjugated perylene diimide (P-PDI) using solid-phase peptide synthesis (SPPS). Due to severe insolubility of perylene dianhydride (PDA) as a starting material of perylene diimide (PDI), PDA was initially conjugated with amino acids to obtain soluble PDI derivatives. Target peptides were synthesized on a 2-chlorotrityl chloride resin using the SPPS method and then conjugated with the amino acid-appended PDI. Various conditions such as loading levels, reaction times and solvents were optimized for introducing the peptides to both sides of the amino acid-appended PDI. The final P-PDI was obtained with a maximum yield of 80% in 12 h. Its singlet oxygen-derived phototoxicity on cells was confirmed, which could be applicable to photodynamic therapy.
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http://dx.doi.org/10.1021/acsomega.8b00040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6045400PMC
May 2018

Susceptibility of Diabetic Mice to Noise Trauma.

Biomed Res Int 2018 12;2018:7601232. Epub 2018 Feb 12.

Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Chungnam National University, Daejeon, Republic of Korea.

Diabetes can lead to many end-organ complications. However, the association between diabetes and hearing loss is not well understood. Here, we investigated the effect of noise exposure on diabetic mice compared with wild-type mice. Hearing threshold shifts, histopathologic changes in the cochlea, and inflammatory responses were evaluated over time. After noise exposure, more severe hearing threshold shifts, auditory hair cell loss, and synaptopathies were notable in diabetic mice compared with wild-type mice. Moreover, increased inflammatory responses and reactive oxygen species production were observed in the ears of diabetic mice. The results demonstrated that diabetic mice are more susceptible to noise trauma.
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http://dx.doi.org/10.1155/2018/7601232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5830016PMC
September 2018

Effects of dexamethasone on intracochlear inflammation and residual hearing after cochleostomy: A comparison of administration routes.

PLoS One 2018 30;13(3):e0195230. Epub 2018 Mar 30.

Department of Otolaryngology-Head and Neck Surgery, College of Medicine, Chungnam National University, Daejeon, Republic of Korea.

Preservation of residual hearing after cochlear implant is an important issue with regards to hearing performance. Various methods of steroid administration have been widely used in clinical practice to reduce inflammation and preserve residual hearing. Here we compare the effect of different routes of dexamethasone administration on intracochlear inflammation and residual hearing in guinea pig ears. Dexamethasone was delivered into the guinea pigs either through intracochlear, intratympanic or systemic route. The intracochlear concentration of dexamethasone, residual hearing, inflammatory cytokines and histopathologic changes were evaluated over time. A higher intracochlear dexamethasone concentration was observed after intracochlear administration than through the other routes. Residual hearing was better preserved with local dexamethasone administration as was supported by the reduced inflammatory cytokines, more hair cell survival and less severe intracochlear fibrosis and ossification concurrently seen in the local delivery group than in the systemic group. The results demonstrate that local dexamethasone delivery can reduce intracochlear inflammation and preserve residual hearing better than in systemically administered dexamethasone.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0195230PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5877881PMC
July 2018

Preparation of tri(ethylene glycol) grafted core-shell type polymer support for solid-phase peptide synthesis.

J Pept Sci 2018 Feb 13;24(2). Epub 2017 Dec 13.

School of Chemical and Biological Engineering, Seoul National University, Seoul, 08826, Republic of Korea.

A core-shell type polymer support for solid-phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on-bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core-shell type polymer supports (TEG SURE) for efficient solid-phase peptide synthesis and on-bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on-bead bioassays.
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http://dx.doi.org/10.1002/psc.3061DOI Listing
February 2018

Nanowire Aptasensors for Electrochemical Detection of Cell-Secreted Cytokines.

ACS Sens 2017 11 9;2(11):1644-1652. Epub 2017 Oct 9.

Department of Biomedical Engineering, University of California, Davis , Davis, California 95616, United States.

Cytokines are small proteins secreted by immune cells in response to pathogens/infections; therefore, these proteins can be used in diagnosing infectious diseases. For example, release of a cytokine interferon (IFN)-γ from T-cells is used for blood-based diagnosis of tuberculosis (TB). Our lab has previously developed an atpamer-based electrochemical biosensor for rapid and sensitive detection of IFN-γ. In this study, we explored the use of silicon nanowires (NWs) as a way to create nanostructured electrodes with enhanced sensitivity for IFN-γ. Si NWs were covered with gold and were further functionalized with thiolated aptamers specific for IFN-γ. Aptamer molecules were designed to form a hairpin and in addition to terminal thiol groups contained redox reporter molecules methylene blue. Binding of analyte to aptamer-modified NWs (termed here nanowire aptasensors) inhibited electron transfer from redox reporters to the electrode and caused electrochemical redox signal to decrease. In a series of experiments we demonstrate that NW aptasensors responded 3× faster and were 2× more sensitive to IFN-γ compared to standard flat electrodes. Most significantly, NW aptasensors allowed detection of IFN-γ from as few as 150 T-cells/mL while ELISA did not pick up signal from the same number of cells. One of the challenges faced by ELISA-based TB diagnostics is poor performance in patients whose T-cell numbers are low, typically HIV patients. Therefore, NW aptasensors developed here may be used in the future for more sensitive monitoring of IFN-γ responses in patients coinfected with HIV/TB.
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http://dx.doi.org/10.1021/acssensors.7b00486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311073PMC
November 2017

Influence of limiting factors on biomass and lipid productivities of axenic Chlorella vulgaris in photobioreactor under chemostat cultivation.

Bioresour Technol 2016 Jul 22;211:367-73. Epub 2016 Mar 22.

Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong-gu, Daejeon 305-806, Republic of Korea; Green Chemistry and Environmental Biotechnology, University of Science and Technology (UST), Yuseong-gu, Daejeon 305-350, Republic of Korea. Electronic address:

The understanding of process parameters and limiting conditions on microalgal biomass and lipid productivities is scarce especially in chemostat cultivation. In this study, the factors limiting growth of axenic Chlorella vulgaris OW-01 in cylindrical photobioreactor under chemostat cultivation were overcome in two phases. Physiological and physicochemical analyses determined inorganic carbon, phosphorous and light intensity as major limiting factors. Their effect on system productivity was ascertained and optimized in the first phase resulting in maximum biomass and lipid productivities of 538 and 128 (mg/L/d), respectively. In the second phase, the effect of dilution rate was evaluated under optimized conditions. The biomass and lipid productivities in this phase reached to 1013 and 270 (mg/L/d), respectively at a dilution rate of 0.75d(-1), yielding >10-fold cumulative increase in productivities. The study demonstrates addressing resource limitations by constant monitoring and optimization of chemostat cultivation to achieve high biomass and lipid productivities in photobioreactors.
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http://dx.doi.org/10.1016/j.biortech.2016.03.109DOI Listing
July 2016

Sensing Conductive Hydrogels for Rapid Detection of Cytokines in Blood.

Adv Healthc Mater 2016 Mar 22;5(6):659-64, 627. Epub 2016 Jan 22.

Department of Biomedical Engineering, University of California, Davis, CA, 95616, USA.

Conducting polymer hydrogel is fabricated atop gold or ITO electrodes and is functionalized with monoclonal antibodies. Binding of interferon-γ molecules causes redox properties of conductive hydrogel to change in a concentration-dependent fashion without the need for washing or sample handling steps. This conductive hydrogel remains functional in a fouling media such as whole blood.
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http://dx.doi.org/10.1002/adhm.201500571DOI Listing
March 2016

Microfluidic compartments with sensing microbeads for dynamic monitoring of cytokine and exosome release from single cells.

Analyst 2016 Jan;141(2):679-88

Department of Biomedical Engineering, University of California, Davis, California 95616, USA.

Monitoring activity of single cells has high significance for basic science and diagnostic applications. Here we describe a reconfigurable microfluidic device for confining single cells along with antibody-modified sensing beads inside 20 picoliter (pL) microcompartments for monitoring cellular secretory activity. An array of ∼7000 microchambers fabricated in the roof of the reconfigurable microfluidic device could be raised or lowered by applying negative pressure. The floor of the device was micropatterned to contain cell attachment sites in registration with the microcompartments. Using this set-up, we demonstrated the detection of inflammatory cytokine IFN-γ and exosomes from single immune cells and cancer cells respectively. The detection scheme was similar in both cases: cells were first captured on the surface inside the microfluidic device, then sensing microbeads were introduced into the device so that, once the microcompartments were lowered, single cells and microbeads became confined together. The liquid bathing the beads and the cells inside the compartments also contained fluorescently-labeled secondary antibodies (Abs). The capture of cell-secreted molecules onto microbeads was followed by binding of secondary antibodies - this caused microbeads to become fluorescent. The fluorescence intensity of the microbeads changed over time, providing dynamics of single cell secretory activity. The microdevice described here may be particularly useful in the cases where panning upstream of sensing is required or to analyze secretory activity of anchorage-dependent cells.
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http://dx.doi.org/10.1039/c5an01648gDOI Listing
January 2016

Development of an aptasensor for electrochemical detection of exosomes.

Methods 2016 Mar 21;97:88-93. Epub 2015 Oct 21.

Department of Biomedical Engineering, University of California, Davis, CA, United States. Electronic address:

Exosomes are small (50-100 nm in diameter) vesicles secreted from various mammalian cells. Exosomes have been correlated with tumor antigens and anti-tumor immune responses and may represent cancer biomarkers. Herein, we report on the development of an aptamer-based electrochemical biosensor for quantitative detection of exosomes. Aptamers specific to exosome transmembrane protein CD63 were immobilized onto gold electrode surfaces and incorporated into a microfluidic system. Probing strands pre-labeled with redox moieties were hybridized onto aptamer molecules anchored on the electrode surface. In the presence of exosomes these beacons released probing strands with redox reporters causing electrochemical signal to decrease. These biosensors could be used to detect as few as 1×10(6) particles/mL of exosomes, which represents 100-fold decrease in the limit of detection compared to commercial immunoassays relying on anti-CD63 antibodies. Given the importance of exosome-mediated signal transmission among cells, our study may represent an important step towards development of a simple biosensor that detects exosomes without washing or labeling steps in complex media.
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http://dx.doi.org/10.1016/j.ymeth.2015.10.012DOI Listing
March 2016

A microsystem integrating photodegradable hydrogel microstructures and reconfigurable microfluidics for single-cell analysis and retrieval.

Lab Chip 2015 Feb;15(3):637-41

Department of Biomedical Engineering, University of California, Davis, USA.

We developed a micropatterned photodegradable hydrogel array integrated with reconfigurable microfluidics to enable cell secretion analysis and cell retrieval at the single-cell level. The activity of protease molecules secreted from single cells was monitored using FRET peptides entrapped inside microfabricated compartments. Antibody-modified gel islands tethering cells to the surface could be degraded by UV exposure to release specific single cells of interest.
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http://dx.doi.org/10.1039/c4lc00884gDOI Listing
February 2015

Photodegradable hydrogels for capture, detection, and release of live cells.

Angew Chem Int Ed Engl 2014 Jul 16;53(31):8221-4. Epub 2014 Jun 16.

Department of Biomedical Engineering, University of California Davis, One Shields Ave, Davis, CA 95616 (USA).

Cells may be captured and released using a photodegradable hydrogel (photogel) functionalized with antibodies. Photogel substrates were used to first isolate human CD4 or CD8 T-cells from a heterogeneous cell suspension and then to release desired cells or groups of cells by UV-induced photodegradation. Flow cytometry analysis of the retrieved cells revealed approximately 95% purity of CD4 and CD8 T-cells, suggesting that this substrate had excellent specificity. To demonstrate the possibility of sorting cells according to their function, photogel substrates that were functionalized with anti-CD4 and anti-TNF-α antibodies were prepared. Single cells captured and stimulated on such substrates were identified by the fluorescence "halo" after immunofluorescent staining and could be retrieved by site-specific exposure to UV light through a microscope objective. Overall, it was demonstrated that functional photodegradable hydrogels enable the capture, analysis, and sorting of live cells.
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http://dx.doi.org/10.1002/anie.201404323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380505PMC
July 2014

Development of micropatterned cell-sensing surfaces.

Methods Cell Biol 2014 ;121:75-90

Department of Biomedical Engineering, University of California, Davis, California, USA.

Microfabricated surfaces have been widely utilized for defining adhesion of single cells or groups of cells of various kinds. Beyond simple control of cell attachment, it is often important to monitor the molecules released by cells. Co-immobilizing miniature sensors alongside cells enables more sensitive detection of secreted factors and may allow for such detection to happen within the context of local microenvironment. Methods for interfacing cells and sensors are central to the notion of local in situ detection of cell function. This chapter describes the use of hydrogel photolithography for integrating cells and sensing elements on culture surfaces. Two types of micropatterned sensing surfaces are described: (1) arrays of microwells for single cell capture that contain antibodies against secreted proteins and (2) entrapment of enzymes inside hydrogel microstructures for local detection of cell metabolism. In both cases, poly(ethylene glycol) hydrogel lithography was employed to control cell attachment, in the second approach hydrogel structures also carried enzymes and functioned as sensors. The development of robust cell/sensor interfaces has implications for diagnostics, tissue engineering, and drug screening.
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http://dx.doi.org/10.1016/B978-0-12-800281-0.00006-3DOI Listing
November 2014

Micropatterned sensing hydrogels integrated with reconfigurable microfluidics for detecting protease release from cells.

Anal Chem 2013 Dec 26;85(24):11893-901. Epub 2013 Nov 26.

Department of Biomedical Engineering, University of California , Davis, California 95616, United States.

Matrix metalloproteinases (MMPs) play a central role in the breakdown of the extracellular matrix and are typically upregulated in cancer cells. The goal of the present study is to develop microwells suitable for capture of cells and detection of cell-secreted proteases. Hydrogel microwells comprised of poly(ethylene glycol) (PEG) were photopatterned on glass and modified with ligands to promote cell adhesion. To sense protease release, peptides cleavable by MMP9 were designed to contain a donor/acceptor FRET pair (FITC and DABCYL). These sensing molecules were incorporated into the walls of the hydrogel wells to enable a detection scheme where cells captured within the wells secreted protease molecules which diffused into the gel, cleaved the peptide, and caused a fluorescence signal to come on. By challenging sensing hydrogel microstructures to known concentrations of recombinant MMP9, the limit of detection was determined to be 0.625 nM with a linear range extending to 40 nM. To enhance sensitivity and to limit cross-talk between adjacent sensing sites, microwell arrays containing small groups (∼20 cells/well) of lymphoma cells were integrated into reconfigurable PDMS microfluidic devices. Using this combination of sensing hydrogel microwells and reconfigurable microfluidics, detection of MMP9 release from as few as 11 cells was demonstrated. Smart hydrogel microstructures capable of sequestering small groups of cells and sensing cell function have multiple applications ranging from diagnostics to cell/tissue engineering. Further development of this technology will include single-cell analysis and function-based cell sorting capabilities.
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http://dx.doi.org/10.1021/ac402660zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370905PMC
December 2013

Micropatterned photodegradable hydrogels for the sorting of microbeads and cells.

Angew Chem Int Ed Engl 2013 Aug 19;52(35):9224-8. Epub 2013 Jul 19.

Department of Biomedical Engineering, University of California Davis, One Shields Ave, Davis, CA 95616, USA.

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http://dx.doi.org/10.1002/anie.201303965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4370904PMC
August 2013

Multilayered heparin hydrogel microwells for cultivation of primary hepatocytes.

Adv Healthc Mater 2014 Jan 5;3(1):126-32. Epub 2013 Jul 5.

Department of Biomedical Engineering, University of California, Davis, CA 95616, USA.

The biomaterial scaffolds for regenerative medicine need to be rationally designed to achieve the desired cell fate and function. This paper describes the development of hydrogel microstructures for cultivation of primary hepatocytes. Four different micropatterned surfaces are tested: 1) poly(ethyelene glycol) (PEG) microwells patterned on glass, 2) heparin hydrogel microwells patterned on glass, 3) PEG microwells patterned on heparin hydrogel-coated substrates, and 4) heparin hydrogel microwells patterned on heparin hydrogel-coated substrates. The latter surfaces are constructed by a combination of micromolding and microcontact printing techniques to create microwells with both walls and floor composed of heparin hydrogel. Individual microwell dimensions are 200 μm diameter and 20 μm in height. In all cases, the floor of the microwells is modified with collagen I to promote cell adhesion. Cultivation of hepatocytes followed by analysis of hepatic markers (urea production, albumin synthesis, and E-cadherin expression) reveals that the all-heparin gel microwells are most conducive to hepatic phenotype maintenance. For example, ELISA analysis shows 2.3 to 13.1 times higher levels of albumin production in all-heparin gel wells compared with other micropatterned surfaces. Importantly, hepatic phenotype expression can be further enhanced by culturing fibroblasts on the heparin gel walls of the microwells. In the future, multicomponent all-heparin gel microstructures may be employed in designing hepatic niche for liver-specific differentiation of stem cells.
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http://dx.doi.org/10.1002/adhm.201300054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4354952PMC
January 2014

Characterizing the effects of heparin gel stiffness on function of primary hepatocytes.

Tissue Eng Part A 2013 Dec 16;19(23-24):2655-63. Epub 2013 Aug 16.

1 Department of Biomedical Engineering, University of California , Davis, California.

In the liver, hepatocytes are exposed to a large array of stimuli that shape hepatic phenotype. This in vivo microenvironment is lost when hepatocytes are cultured in standard cell cultureware, making it challenging to maintain hepatocyte function in vitro. Our article focused on one of the least studied inducers of the hepatic phenotype-the mechanical properties of the underlying substrate. Gel layers comprised of thiolated heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA) were formed on glass substrates via a radical mediated thiol-ene coupling reaction. The substrate stiffness varied from 10 to 110 kPa by changing the concentration of the precursor solution. ELISA analysis revealed that after 5 days, hepatocytes cultured on a softer heparin gel were synthesizing five times higher levels of albumin compared to those on a stiffer heparin gel. Immunofluorescent staining for hepatic markers, albumin and E-cadherin, confirmed that softer gels promoted better maintenance of the hepatic phenotype. Our findings point to the importance of substrate mechanical properties on hepatocyte function.
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http://dx.doi.org/10.1089/ten.TEA.2012.0681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3856597PMC
December 2013

Micropatterned surfaces functionalized with electroactive peptides for detecting protease release from cells.

Anal Chem 2013 Jan 14;85(1):220-7. Epub 2012 Dec 14.

Department of Biomedical Engineering, University of California, Davis, 95616, United States.

Matrix metalloproteinases (MMPs) regulate composition of the extracellular matrix and play a critical role in cancer, fibrosis, and wound healing. This article describes a novel peptide-based electrochemical biosensor for detecting activity of cell-secreted protease MMP9. In this sensing strategy, a peptide specific to MMP9 was modified with a redox label (methylene blue (MB)) and immobilized on microfabricated 300 μm diameter Au electrodes. Challenging the electrodes with known concentrations of MMP9 resulted in the cleavage of the MB containing peptide fragment and caused a decrease in electrical signal measured by square wave voltammetry (SWV). The limit of detection for MMP9 was determined to be 60 pM with a linear range extending to 50 nM. In preparation to detect cell-secreted MMP9, glass surfaces with Au electrode arrays were further micropatterned with poly(ethylene glycol) (PEG) gel to define annular cell adhesive regions next to electrodes and render the remainder of the surface nonfouling. The surfaces were further modified with CD14 antibody to promote attachment of monocytes. The peptide-modified electrode arrays were integrated into PDMS microfluidic devices and incubated with U-937 cells, transformed monocytes known to produce MMPs. These studies revealed a 3-fold higher electrochemical signal from ∼400 activated monocytes after 10 min activation compared to nonactivated monocytes. Whereas this article focuses on MMP9 detection, the general strategy of employing redox-labeled peptides on electrodes should be broadly applicable for detection of other proteases and should have clinical as well as basic science applications.
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http://dx.doi.org/10.1021/ac302547pDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677045PMC
January 2013

Aptamer-containing surfaces for selective capture of CD4 expressing cells.

Langmuir 2012 Aug 14;28(34):12544-9. Epub 2012 Aug 14.

Department of Biomedical Engineering, University of California, Davis, California 95616, United States.

Aptamers have recently emerged as an excellent alternative to antibodies because of their inherent stability and ease of modification. In this paper, we describe the development of an aptamer-based surface for capture of cells expressing CD4 antigen. The glass or silicon surfaces were modified with amine-terminated silanes and then modified with thiolated RNA aptamer against CD4. Modification of the surface was first characterized by ellipsometry to demonstrate assembly of biointerface components and to show specific capture of recombinant CD4 protein. Subsequently, surfaces were challenged with model lymphocytes (cell lines) that were either positive or negative for CD4 antigen. Our experiments show that aptamer-functionalized surfaces have similar capture efficiency to substrates containing anti-CD4 antibody. To mimick capture of specific T-cells from a complex cell mixture, aptamer-modified surfaces were exposed to binary mixtures containing Molt-3 cells (CD4+) spiked into Daudi B cells (CD4-). 94% purity of CD4 cells was observed on aptamer-containing surfaces from an initial fraction of 15% of CD4. Given the importance of CD4 cell enumeration in HIV/AIDS diagnosis and monitoring, aptamer-based devices may offer an opportunity for novel cell detection strategies and may yield more robust and less expensive blood analysis devices in the future.
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http://dx.doi.org/10.1021/la2050338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3538135PMC
August 2012

Attachment of hydrogel microstructures and proteins to glass via thiol-terminated silanes.

Colloids Surf B Biointerfaces 2012 Oct 27;98:1-6. Epub 2012 Apr 27.

Department of Biomedical Engineering, University of California, Davis, CA 95616, United States.

Micropatterning strategies often call for attachment of non-fouling biomaterials and immobilization of proteins in order to create biosensing surfaces or to control cell-surface interactions. Our laboratory has made frequent use of hydrogel photolithography - a micropatterning process for immobilizing poly(ethylene glycol) (PEG) hydrogel microstructures on glass surfaces. In the present study we explored the use of thiolsilane as a coupling layer for both covalent anchoring of hydrogel microstructures and covalent immobilization of proteins on glass. These new surfaces were compared to acryl-silane functionalized glass slides that allowed covalent attachment of gels but only physical adsorption of proteins as well as surfaces containing a mixture of both functional groups. We observed comparable attachment and retention of hydrogel microstructures on acryl and thiol-terminated silanes. Ellipsometry studies revealed presence of significantly higher level of proteins on thiol-functionalized glass. Overall, our studies demonstrate that thiol-silane functionalized glass surfaces may be used to create complex micropatterned surfaces comprised of covalently attached hydrogels and proteins. This simple and effective surface modification strategy will be broadly applicable in cellular engineering and biosensing studies employing hydrogel micropatterns.
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http://dx.doi.org/10.1016/j.colsurfb.2012.03.025DOI Listing
October 2012

Determination of protease subsite preference on SPOT peptide array by fluorescence quenching-based assay.

J Pept Sci 2012 Jun 30;18(6):394-9. Epub 2012 Apr 30.

School of Chemical and Biological Engineering, Seoul National University, Seoul 151-747, Korea.

A peptide SPOT array was synthesized on a glass chip and used to determine protease subsite preference. To synthesize a peptide array for positional scanning, the ratio of the isokinetic concentration was determined for every Fmoc-amino acid except Cys. Based on this ratio, a peptide array consisting of Dabcyl-X-X-P(2)-Arg-X-X-X-Lys(FITC) (X: equimolar mixture of 19 amino acids, P(2): one of 19 amino acids) was synthesized on a chitosan-grafted glass chip. Subsequently, the peptide substrates on the array were hydrolyzed by thrombin to screen for subsite specificity using a fluorescence quenching-based assay. The P(2) subsite specificity of thrombin was screened by the fluorescence images obtained after hydrolysis. Pro at the P(2) subsite showed the highest specificity for thrombin based on both the fluorescence quenching-based assay and the solution phase assay. From these results, we confirmed that our mixture-based peptide SPOT array format on the chitosan-grafted glass chips could be used to determine protease subsite preference.
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http://dx.doi.org/10.1002/psc.2409DOI Listing
June 2012

Photolabile micropatterned surfaces for cell capture and release.

Chem Commun (Camb) 2011 Nov 5;47(43):11942-4. Epub 2011 Oct 5.

Department of Biomedical Engineering, University of California, Davis, 451 East Health Sciences Dr #2619, Davis, CA 95616, USA.

A method for capture and release of cells was developed using a photolabile linker and antibody-attached glass surface with a poly(ethylene glycol) (PEG)-pattern.
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http://dx.doi.org/10.1039/c1cc15046dDOI Listing
November 2011

Development of high-throughput phosphorylation profiling method for identification of Ser/Thr kinase specificity.

J Pept Sci 2011 May 22;17(5):392-7. Epub 2010 Nov 22.

School of Chemical and Biological Engineering, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Korea.

Identification of substrate specificity of kinases is crucial to understand the roles of the kinases in cellular signal transduction pathways. Here, we present an approach applicable for the discovery of substrate specificity of Ser/Thr kinases. The method, which is named as the 'high-throughput phosphorylation profiling (HTPP)' method was developed on the basis of a fully randomized one-bead one-compound (OBOC) combinatorial ladder type peptide library and MALDI-TOF MS. The OBOC ladder peptide library was constructed by the 'split and pool' method on a HiCore resin. The peptide library sequence was Ac-Ala-X-X-X-Ser-X-X-Ala-BEBE-PLL resin. The substrate specificity of murine PKA (cAMP-dependent protein kinase A) and yeast Yak1 kinase was identified using this method. On the basis of the result, we identified Ifh1, which is a co-activator for the transcription of ribosomal protein genes, as a novel substrate of Yak1 kinase. The putative Yak1-dependent phosphorylation site of Ifh1 was verified by in vitro kinase assay.
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http://dx.doi.org/10.1002/psc.1312DOI Listing
May 2011

Antibody-free peptide substrate screening of serine/threonine kinase (protein kinase A) with a biotinylated detection probe.

Anal Biochem 2011 Jun 3;413(1):30-5. Epub 2011 Mar 3.

School of Chemical and Biological Engineering, Seoul National University, San 56-1, Shilim-dong, Kwnak-gu, Seoul 151-744, South Korea.

Being different from anti-phosphotyrosine antibodies, anti-phosphoserine- or anti-phosphothreonine-specific antibodies with high affinity for the detection of serine/threonine kinase substrates are not readily available. Therefore, chemical modification methods were developed for the detection of phosphoserine or threonine in the screening of protein kinase substrates based on β-elimination and Michael addition. We have developed a biotin-based detection probe for identification of the phosphorylated serine or threonine residue. A biotin derivative induced a color reaction using alkaline phosphate-conjugated streptavidin that amplified the signal. It was effective for the detection and separation of the target peptide on the resin. The detection probe was successfully used in identifying PKA substrates from peptide libraries on resin beads. The peptide library was prepared as a ladder-type, such that the active peptides on the colored resin beads were readily sequenced with the truncated peptide fragments by MALDI-TOF/MS analysis after releasing the peptides from the resin bead through photolysis.
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http://dx.doi.org/10.1016/j.ab.2011.02.005DOI Listing
June 2011

Automated maskless photolithography system for peptide microarray synthesis on a chip.

J Comb Chem 2010 Jul;12(4):463-71

School of Chemical and Biological Engineering and School of Electrical Engineering and Computer Science, Seoul National University, Seoul 151-744, Korea.

Maskless photolithographic peptide synthesis was performed on a glass chip using an automated peptide array synthesizer system. The peptide array synthesizer was built in a closed box, which contained optical and fluidic systems. The conditions for peptide synthesis were fully controlled by a computer program. For the peptide synthesis on a glass chip, 20 NVOC-protected amino acids were synthesized. The coupling efficiencies of two model peptide sequences were examined on ACA/APTS and PEG/CHI/GPTS chips. PEG/CHI/GPTS chip gave higher average stepwise yields of GIYWHHY (94%) and YIYGSFK (98%) than those of ACA/APTS chip. To quantify peptide-protein binding affinity, HPQ- or HPM-containing pentapeptides were synthesized on a PEG/CHI/GPTS chip and the binding event of Cy3 labeled-streptavidin was quantified. The peptide sequence of IQHPQ showed highest binding affinity with Cy3 labeled-streptavidin. The results demonstrated that the photolithographic peptide array synthesis method efficiently quantified the binding activities of protein-peptide interactions and it can be used for additional biological assay applications.
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http://dx.doi.org/10.1021/cc100009gDOI Listing
July 2010

Substrate screening of protein kinases: detection methods and combinatorial peptide libraries.

Biopolymers 2010 ;94(6):753-62

School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-744 Korea.

The study of protein kinases has become a matter of great importance in the development of new drugs for the treatment of diseases, including cancer and inflammation. Substrate screening is the first step in the fundamental investigation of protein kinases and the development of inhibitors for use in drug discovery. Towards this goal, various studies have been reported regarding the development of phospho-peptide detection methods and the screening of phosphorylated peptide sites by protein kinases. This review introduces the detection methods for phosphorylation events using the reagents with (γ(32)P)ATP, ligand-linked ATP, phospho-peptide-specific antibodies and metal chelating compounds. Chemical modification methods using β-elimination for the detection of phospho-Ser/Thr peptides are introduced as well. In addition, the implementations of combinatorial peptide libraries for screening peptide substrates of protein kinases are discussed. The phage display approach has been suggested as an alternative method of using synthetic peptides for screening the substrate specificities of protein kinase. However, a solid phase assay using a peptide library-bound polymer resin or a peptide-arrayed glass chip is preferred for high throughput screening (HTS).
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http://dx.doi.org/10.1002/bip.21506DOI Listing
March 2011

Two-Step Enzymatic Modification of Solid-Supported Bergenin in Aqueous and Organic Media.

Tetrahedron Lett 2010 Feb;51(8):1220

Department of Chemical Engineering, University of California, Berkeley, Berkeley, California 94720.

The natural flavonoid bergenin was directly immobilized onto carboxylic acid functionalized controlled pore glass (carboxy-CPG) at 95% yield. Immobilized bergenin was brominated via chloroperoxidase in aqueous solution and then transesterified with vinyl butyrate in diisopropyl ether by subtilisin carslberg (SC) extracted into the organic solvent via ion pairing. Enzymatic cleavage of 7-bromo-4-butyrylbergenin from carboxy-CPG (9.6% final yield) was accomplished using lipase B (LipB) in an aqueous/organic mixture (90/10 v/v of water/acetonitrile), demonstrating the feasibility of solid phase biocatalysis of a natural product in aqueous and non-aqueous media.
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http://dx.doi.org/10.1016/j.tetlet.2009.12.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2822990PMC
February 2010

High-throughput screening of glycan-binding proteins using miniature pig kidney N-glycan-immobilized beads.

Chem Biol 2008 Mar;15(3):215-23

Interdisciplinary Program for Biochemical Engineering and Biotechnology, Seoul National University, Seoul 151-742, Korea.

Glycan recognition leading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Without any derivatization steps, the purified pig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.
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http://dx.doi.org/10.1016/j.chembiol.2008.02.009DOI Listing
March 2008