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Sensors and Actuators B 129 (2008) 841–849

Preparation of micropatterned hydrogel substrate via surfacegraft polymerization combined with photolithography

for biosensor application

Woojin Lee, Dongkil Choi, Yeol Lee, Dae-Nyun Kim, Jinwon Park, Won-Gun Koh ∗Department of Chemical Engineering, Yonsei University, 134 Sinchon-Dong, Seodaemoon-Gu, Seoul 120-749, Republic of Korea

Received 31 May 2007; received in revised form 5 September 2007; accepted 24 September 2007Available online 6 October 2007

bstract

This paper presents a simple method to create protein micropattern on the poly(ethylene glycol) (PEG) hydrogels through the surface graftolymerization and photolithography. The modification of the protein-repellent PEG hydrogel surface was achieved by a two-step process usingmmobilization of benzophenone on the PEG hydrogel as surface initiator and subsequent surface-initiated polymerization of acrylic acid by UVrradiation. Surface modification of PEG hydrogels was demonstrated with FTIR/ATR spectroscopy and XPS by confirming the presence of carboxylroups in the poly(acrylic acid) (PAA). The photograft polymerization through the designed photomask produced well-defined, pH-responsiveAA micropatterns with diameters ranging from 50 to 300 �m on the PEG hydrogels. The size of PAA micropatterns was controlled by changinghe environmental pH, such that a 300 �m diameter and 17 �m thick PAA micropattern at pH 4 swelled to 480 �m diameter and 80 �m thick at pH

. Activation of the carboxyl groups in PAA allowed covalent immobilization of proteins only on the PAA micropatterns due to the nonadhesivityf PEG. Based on these results, biotin was micropatterned on the PEG hydrogels and binding of streptavidin was qualitatively and quantitativelynvestigated, demonstrating the possibility of micropatterned PEG hydrogels for various biosensor systems.

2007 Elsevier B.V. All rights reserved.

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eywords: Poly(ethylene glycol) hydrogels; Surface graft polymerization; Pho

. Introduction

Over the last decade, the synergetic applications of microfab-ication technology and biotechnology have allowed for proteinatterning, which is spatially controlled immobilization of pro-eins on solid surfaces with a micro- or nano-meter length scale.rotein patterning provides a powerful tool for studying the inter-ctions between proteins, antibodies, and other biomolecules,or application in many fields such as cell biology, biosensorechnology, biomedical devices, and tissue engineering [1–6]. Aumber of patterning technologies such as direct dispensing orpotting, photolithography, and soft lithography, have demon-

trated successful protein localization at a micro/nanometerength scale [5,7–12]. Combined with patterning techniques,roteins are immobilized onto various solid surfaces through

∗ Corresponding author. Tel.: +82 2 2123 5755; fax: +82 2 312 6401.E-mail address: wongun@yonsei.ac.kr (W.-G. Koh).

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925-4005/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2007.09.085

graphy; Benzophenone; Poly(acrylic acid) micropatterns

on-specific adsorption or covalent binding to the tetheredunctional groups on the surfaces. Generally, most protein pat-ernings are performed on hard materials such as glass, siliconnd gold [13–17]. The main problem associated with proteinatterning on a hard surface is that the conformational state ofany proteins is very labile and unable to withstand patterning

rocess conditions, eventually losing their native structures andunction [18,19]. One solution for these problems is to use aydrogel, which is three-dimensional polymeric structures thatwell in water or other biological fluids [20–24]. In contrast tohysical adsorption or protein tethering to a hard surface, theoft and hydrated environment of a swollen hydrogel can pro-ide proteins with near-physiological conditions that minimizeenaturation and help them to carry out their full biological func-ions [19,20,25]. Among the various hydrogels, poly(ethylene

lycol) (PEG)-based hydrogels have been widely used in biol-gy and medicine for biosensors, drug delivery devices, tissuengineering and cell transplantation for many years because ofheir high water content, hydrophilicity, and biocompatibility

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22,26–30]. PEG is a nondegradable, hydrophilic polymer thatas been employed as a biomaterial to obtain biocompatibilityecause of its remarkable nonadhesivity towards proteins andells [31]. Different molecular weight (MW) of PEGs could beasily converted into acrylates such as PEG diacrylate (PEG-A), and polymerization of the acrylated PEGs yields a highlyrosslinked hydrogel network. Physical properties of PEG-basedydrogels can be easily controlled by varying the MW of PEGnd the transparent nature of PEG hydrogels also makes themuitable for various detection schemes when they are used iniosensing applications [32–35]. PEG-based hydrogels could beabricated into various microstructures through the lithographicechniques [36,37], however, the dynamic swelling behavior,oft elastic nature, unavailable functional groups, and protein-epelling behavior of PEG hydrogels limit the micropatterningf proteins on their surfaces.

This paper describes the simple method to create proteinicropattern on PEG hydrogels. Surfaces of the PEG hydro-

els were modified with a functional group by photoinducedurface graft polymerization to covalently bind proteins. Thehotolithographic method was used to direct graft polymeriza-ion at selective regions of PEG hydrogel with micrometer scale,roducing functionalized micropatterns on the PEG hydrogel.fter protein immobilization on the PEG hydrogels was demon-

trated, the feasibility of micropatterned PEG hydrogels for theiosensor application was investigated.

. Experimental

.1. Materials

PEG with a MW of 3400 g/mol, acryloyl chloride, triethy-amine, hexane, tetrahydrofuran (THF), ethyl alcohol, acryliccid, benzophenone, benzyl alcohol, sodium periodate, 1-vinyl--pyrrolidinone, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-ide (EDC), bovine serum albumin conjugated with fluorescein

socyanate (FITC-BSA), N-hydroxysuccinimide (NHS) and,2-dimethoxy-2-phenylacetophenone (DMPA) were purchasedrom Sigma–Aldrich (St. Louis, MO, USA). Biotinyl-3,6,9-ioxaoctanediamine (biotin–NH2) and FITC-labeled strepta-idin (FTIC-SA) were obtained from Pierce (Rockford, IL,SA) and BiosourceTM (Camarillo, CA, USA), respectively.hosphate buffered saline (PBS, 0.1 M, pH 7.4) was preparedith 1.1 mM potassium phosphate monobasic, 3 mM sodiumhosphate dibasic heptahydrate, and 0.15 M NaCl in deionizedater (ρ = 18 M�) (Milli-Q Ultrapure, Millipore).PEG was converted to PEG diacrylate (DA) according

o published protocol [38] and acrylation of PEG was con-rmed by attenuated total reflectance/Fourier transform infraredATR/FTIR) spectroscopy (Thermo Nicolet Corp., Madison,

I, USA).

.2. Preparation of hydrogel substrate

PEG hydrogel substrates were prepared by UV-initiated free-adical polymerization. Purified PEG-DA was dissolved in PBSo form 50% (w/v) PEG solution. A 300 mg/mL solution of

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MPA in 1-vinyl-2-pyrrolidinone was added and mixed to theEG solution at 5 �L/mL PEG-DA solution to initiate pho-

opolymerization. This precursor solution was cast into a Teflonold (1 cm diameter, 500 �m thick) and exposed to a 75 W

enon ultraviolet (UV) light source (Oriel Instruments, Moun-ain View, CA, USA) for 5 min after being covered with glasslates. Upon UV exposure, the precursor solution underwentree-radical induced gelation and became insoluble in commonEG solvents such as water. The resultant PEG hydrogel wasashed extensively for a minimum of 3 days in deionized water

o remove any unreacted components and to reach equilibriumwelling in deionized water.

.3. Surface graft polymerization

The surface modification of PEG hydrogels was achievedy two-step process based on photoinduced graft polymeriza-ion of acrylic acid, which was developed by Ma et al. [39].n the first step, surfaces of the hydrogels were dabbed drynd 100 �L of a 10 wt% benzophenone solution in ethanol wasrop-cast on the hydrogel and spread evenly over the surface.he solvent was then allowed to evaporate in a fume hood

o ensure deposition of the benzophenone on the hydrogel.V irradiation was performed using 365 nm, 300 mW/cm2 UV

ight (EXFO Omnicure UV spot lamp, Mississauga, Ontario,anada). After irradiation for a selected number of passes, the

esidual, unreacted benzophenone was extracted by soaking andashing the hydrogels in ethanol and drying the membrane at

oom temperature in air until constant weight. For the secondtep, a monomer solution composed of acrylic acid (50 wt%n water), benzyl alcohol (0.5 wt%) and 0.5 mM of sodiumeriodate was coated on the benzophenone-immobilized hydro-el surface, which was then exposed to the same UV source.fter UV exposure, the hydrogel substrates were rinsed witheionized water and immersed in ethanol to remove unreactedcrylic acid. Hydrogel substrates were further rinsed with waternd incubated in PBS buffer overnight to remove poly(acryliccid) (PAA) which was not covalently attached to the hydro-el. Characterization of the modified surfaces was performedsing ATR/FTIR spectroscopy and X-ray photoelectron spec-roscopy (XPS) (Kratos Analyrical Inc., ChestnutRidge, NY,SA).

.4. Micropatterning of hydrogel surface

Surface micropatterning was performed using photolithogra-hy as shown in Scheme 1a. As previously described, a monomerolution was coated on the benzophenone-immobilized hydro-el substrates and exposed to UV light through a photomask toirect surface modification at the selected areas. The photomaskor photolithography contained circular element arrays, whichere prepared using AUTO CAD and printed onto transparen-

ies using a standard laser jet printer (LaserWriter 16/600 PS,

pple Inc., Cupertino, CA, USA). After UV exposure, the pho-

omask was removed and the hydrogel substrate was rinsed usinghe procedure described above. The morphology of micropat-erns on the hydrogels was investigated with optical microscopy

W. Lee et al. / Sensors and Actuat

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3.1. Preparation of PEG hydrogel substrates

The formation of PEG hydrogels is based on the UV-initiatedfree-radical cross-linking of acrylate end groups on PEG-DA,

cheme 1. Schematic diagram of (a) micropatterning of PAA on the PEG hydro-el surface and (b) photoinduced graft polymerization using benzophenone.

nd alpha-step surface profiler (KLA-Tencor Co., San Jose, CA,SA).

.5. Immobilization of albumin on the hydrogel substrates

As a model protein, albumin was covalently immobilizedn the PEG hydrogel surface via a EDC/NHS-mediated reac-ion. Specifically, PAA-grafted hydrogels were immersed into

solution containing 2 mM EDC and 5 mM NHS for 3 h toonvert the carboxyl groups in PAA into reactive intermediateshich are susceptible to attack by amine groups from proteins.he activated hydrogel substrates were then washed with PBSnd immediately reacted with FITC-BSA solutions for 3 h at◦C, which resulted in the covalent attachment of the albu-in to the hydrogel surface by amide formation. Immobilization

f albumin onto PAA-grafted hydrogel was characterized withTR/FTIR spectroscopy and XPS. Fluorescence microscopyas used to confirm the attachment of albumin to the micropat-

erned hydrogels.

.6. Study of the streptavidin–biotin system

Micropatterned hydrogel substrates were used to studyolecular recognition between biotin and streptavidin. A 10 mM

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olution of biotin–NH2 in PBS was directly coupled to the PAAicropattern via the EDC/NHS-mediated reaction described

bove. After patterning of biotin–NH2 on the hydrogel, theydrogel substrates were incubated with different concentrationf FITC-SA solution for 10 min. The binding of streptavidino micropatterned biotin was visualized and quantified usinguorescence microscopy.

.7. Image acquisition and analysis

A Zeiss Axiovert 200 microscope equipped with an integratedolor CCD camera (Carl Zeiss Inc., Thornwood, NY, USA) wassed to obtain optical and fluorescence images. Image analysesere performed using commercially available image analysis

oftware (KS 300, Carl Zeiss Inc.).

. Results and discussion

ig. 1. FTIR spectra of (a) unmodified PEG hydrogel and (b) PAA-grafted PEGydrogel.

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esulting in the formation of polyacrylate networks highlyross-linked with PEG. Resultant three-dimensional, insolu-le PEG hydrogels were flexible and completely transparenthen swollen in deionized water (water content of hydrogel was3.4 ± 5.5%). Photopolymerization of PEG-DA was monitoredith a Bruker RFS100 FT–Raman spectrometer equipped with

liquid N2 cooled Ge-detector and a Nd-Yag laser providing anxcitation wavelength of 1064 nm (Bruker Optics Inc., Biller-ca, MA, USA) by measuring the decrease in the terminal C Ctretch of the PEG-DA macromer at 1635 cm−1 before and after

ig. 2. C 1s XPS spectra of (a) unmodified PEG hydrogel, (b) pure PAA, andc) PAA-grafted PEG hydrogel.

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V exposure. After the polymerization and rinsing process, theignal for the C C bonds was not detected, confirming that thereas no active groups in the PEG hydrogels.

.2. Photoinduced surface graft polymerization

Surfaces of the PEG hydrogels were modified by sequen-ial photoinduced graft polymerization using acrylic acid as a

onomer and benzophenone as a photoinitiator. In this method,he grafting method was divided into two steps as shown incheme 1b. First, the benzophenone was covalently immobi-

ized onto the hydrogel substrates after benzophenone moleculesbsorbed a photon and then abstracts a hydrogen atom fromubstrate, yielding a surface initiator. Second, polymerizationf acrylic acid occurred from the hydrogel surface with surfacenitiator by UV irradiation, which resulted in the grafting of PAAolymer chain to the PEG hydrogel substrates.

Formation of PAA layers on the PEG hydrogel substrates

as demonstrated by ATR/FTIR spectroscopy and XPS. Fig. 1

hows FTIR spectra of unmodified PEG hydrogel and PAA-rafted PEG hydrogel. Although the two spectra have a commonbsorption band at around 1730 cm−1 which is attributed to car-

ig. 3. Optical images of PEG hydrogel surfaces micropatterned with the 100nd 300 �m diameter circles of PAA.

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onyl bonds (C O), spectrum of PAA-grafted PEG hydrogelshows higher intensity of this band than that of unmodified PEGydrogel (MW 3400). The concentration of carbonyl groupsn the PEG hydrogel is relatively low, as they exist in acrylateroups at the termini of the large MW of PEG. On the otherand, for PAA, carbonyl bonds in the carboxyl groups appearn every acrylic acid monomer unit (MW 72). Therefore, PAA-rafted PEG hydrogels possess more carbonyl group than PEGydrogel, resulting in an increase of peak intensity at around730 cm−1 as shown in Fig. 1. Formation of PAA layers onhe PEG hydrogel substrates was further investigated with XPS.ig. 2 shows the C 1s XPS spectra obtained from pure PAA,nmodified PEG hydrogel and PAA-grafted PEG hydrogels. Forhe unmodified PEG hydrogel, the intensity of C O peak was

uch higher than C C peak due to the ether group in the mainhain of PEG. Compared with the unmodified PEG hydrogel,he C 1s spectra of PAA-grafted PEG hydrogel had an inten-ity increase of C C and O C O peak because PAA have more

C and O C O bonds than PEG. Based on these results, itas demonstrated that PAA was successfully grafted to the PEGydrogel substrate by surface-initiated photopolymerization. Ithould be noted that even though the thickness of PAA layersas micrometer scale, PAA-coated PEG hydrogel sample pos-

essed the peak at 286.5 eV which was originated from the PEG.his result indicated that packing density of PAA grafted ontoEG hydrogel was not high enough to perfectly cover the whole

rea of PEG hydrogel surface. As a control, same experimentas carried out without a benzophenone-immobilizing step. No

vidence of surface modification was detected, corroborating theole of benzophenone as a surface initiator.

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ig. 4. Effects of pH on the size of PAA micropattern. Optical image (left) and surfa

ors B 129 (2008) 841–849 845

.3. Micropatterning of PEG hydrogel substrates

To examine whether surface graft polymerization can pro-ide micro-order precision, a micropatterning process waserformed using a photomask with an array of circular ele-ents with diameters of 100 and 300 �m. Fig. 3 shows the

ptical images of the PEG hydrogel substrate micropatternedith PAA. The combination of photolithography and surface-

nitiated graft polymerization successfully created the clearlyefined microstructures that corresponded to the pattern of thehotomask. Only the UV-exposed region determined by the pho-omask, was susceptible to photoinduced graft polymerization,esulting in no residual polymer on the substrate after wash-ng. PAA micropatterns were firmly anchored to the hydrogelubstrates and did not delaminate during washing or prolongedxposure to an aqueous environment.

Since PAA achieved different levels of swelling with the pHy the ionization of carboxyl group above its pKa (4.7), a changen the pH was expected to alter the morphology of PAA micropat-erns. To investigate the pH-response on the PEG hydrogel, PAA

icropatterns were fabricated using a photomask with 300 �miameter circles. After the micropatterned PEG hydrogels werelaced in pH 4 and 7 buffers, morphological change was inves-igated with optical microscopy and a surface profiler, as shownn Fig. 4. At pH 4, the diameter of PAA micropatterns did nothange, having the same size of patterns on the photomask

ecause no ionization occurred below the pKa of PAA. However,t pH 7, the size of the PAA micropatterns increased both lat-rally and vertically because PAA became charged and swelledbove the pKa value. From pH 4 to 7, diameter and height of

ce profile (right) of micropatterned PEG hydrogel (a) in pH 4 and (b) in pH 7.

8 ctuators B 129 (2008) 841–849

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AA micropattern increased from 300 to 480 �m and from 17o 80 �m, respectively. The PAA became protonated and con-racted at a pH lower than 4.7, allowing for the swollen PAA

icropattern to reversibly return to the initial size by decreasinghe pH to 4.

.4. Albumin immobilization onto the micropatternedubstrates

PAA possesses carboxyl groups that could serve as sitesor immobilization of various proteins. Using the EDC/NHS-ediated reaction, carboxyl groups in PAA are converted to-hydroxysuccinimide ester, which can react with amine groups

n proteins to form a stable amide linkage. Before the micropat-erning of albumin on the PEG hydrogel, BSA was incubatedith the PAA-modified PEG hydrogel to confirm the ability ofrafted PAA to react and form the covalent bond with albu-in. Immobilization of albumin on the PEG hydrogels was

emonstrated with ATR/FTIR and XPS. Comparing with Fig. 1,he FTIR spectra in Fig. 5a indicated new peaks at 1653 and540 cm−1, which were characteristic of the C O stretchingnd the N H bending of the amide bond. As shown in Fig. 5b,

Fig. 6. Fluorescence image of the micropatterned PEG hydrogel after FITC-BSA was attached to the activated PAA area.

Fig. 5. Protein immobilization on the PAA-grafted PEG hydrogel. (a) FTIR and (b) XPS spectra of protein-immobilizing PEG hydrogel.

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lbumin immobilization was further investigated with XPS inhich a new N 1s signal not present for the PAA-modified PEGydrogel was observed for the albumin-immobilized hydrogel,

hich is indicative of peptide linkage on the hydrogel surface.TIR and XPS results suggest that albumin was successfully

ncorporated to the PEG hydrogel surface through surface mod-fication.

ig. 7. Biotin–streptavidin assay on the micropatterned PEG hydrogel. (a) Flu-rescence image of FITC-SA bound to micropatterned biotin. (b) Fluorescencentensity profile across the sample shown in (a). (c) Relationship between con-entration of FITC-SA and the fluorescence intensity.

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Because of nonadhesivity of PEG hydrogel towards proteins,icropatterning of the PEG hydrogel created a clear contrast

etween the adhesion promoting PAA micropattern and adhe-ion resisting PEG hydrogel surface. Feasibility of immobilizinglbumin on the micropatterned PAA was investigated by incubat-ng FITC-BSA with the micropatterned PEG hydrogel substrateo visualize the localization and patterning of the albumin. Theesult is shown in Fig. 6 where fluorescent and dark regionsorrespond to the PAA micropatterns with immobilized BSAnd unmodified PEG region, respectively. As indicated by theseesults, albumin was immobilized only onto the PAA micropat-ern with PEG hydrogel serving as effective barrier to albumindsorption, demonstrating the spatial control of protein immo-ilization was possible on the PEG hydrogel substrates with aicrometer scale.Finally, biotin–streptavidin assay was performed to test

he potential application of the micropatterned PEG hydro-el as a biosensor. Biotin–streptavidin system was chosenecause it provides a universal platform for patterning otheriomolecules [40,41]. To confirm the molecular recognitionediated, specific binding between biotin and streptavidin,

iotin–NH2 was covalently immobilized to the activated PAAicropatterns and the hydrogel substrates were incubated withFITC-SA. Fluorescent image demonstrated the streptavidin

pecifically bound to the biotin-immobilized PAA micropat-erns (Fig. 7a). Furthermore, Fig. 7b shows that the fluorescencentensity of biotin–streptavidin micropatterns was nearly identi-al at each binding site, with minimal background fluorescenceetween micropatterns. These results indicate that the densityf binding sites at each micropatterned location was simi-ar and that non-specific adsorption was insignificant in theEG region. As shown in Fig. 7c, the fluorescence intensityf biotin–streptavidin micropatterns was linearly dependent onhe concentration of streptavidin, suggesting the potential oficropatterned PEG hydrogels for various bioassays.

. Conclusion

This paper described a simple method for the patterned,ovalent modification of PEG hydrogel surfaces to create pro-ein micropatterns. A protein-repellent PEG hydrogel surfaceas modified by grafting PAA via photoinduced surface-

nitiated polymerization using acrylic acid as a monomer andenzophenone as a surface initiator. Surface-initiated graftolymerization via the photolithographic method producedell-defined, pH-responsive PAA micropatterns on the PEGydrogels, which did not delaminate during washing or pro-onged exposure to an aqueous environment. The carboxylroups in PAA were activated using EDC and NHS to cova-ently immobilize the albumin. Albumin was immobilized on thectivated PAA micropattern, while the unpatterned PEG regionffectively prevented non-specific adsorption. For the futurepplication of micropatterned PEG hydrogels as a biosensor,

he biotin–streptavidin system was investigated and the molec-lar recognition mediated, specific binding between biotin andtreptavidin was successfully assayed. In addition to the biosen-or application, it is expected that techniques developed in this

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tudy can be also used for tissue engineering, considering themportance of hydrogel as scaffold for soft tissue engineeringpplication.

cknowledgements

This work was supported by the Korea Research Founda-ion Grant funded by the Korean Government (MOEHRD)KRF-2006-311-D00455) and grants from Seoul Research andusiness Development Program (10816).

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iographies

oojin Lee was born in 1980 in Seosan, Korea. He received his BS degree in004 from Kyunghee University. He is now a MS candidate in Department ofhemical Engineering in Yonsei University. His research area is biosensor and

issue engineering.

ongkil Choi was born in 1980 in Seoul, Korea. He received his BS degreen 2006 in Kyunghee University. He is now a MS candidate in Department ofhemical Engineering in Yonsei University. His research interests are Lab on ahip device for the detection of enzymatic reaction.

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eol Lee was born in 1977 in Jeonju, Korea. He received his BS degree fromonsei University in Chemical Engineering in 2007. He is now a MS candidate

n Department of Chemical Engineering, Yonsei University. His research areasre surface modification and bead-based biosensor.

ae-Nyun Kim was born in 1981 in Seoul, Korea. After receiving his BS degreen Chemical Engineering from Yonsei University at 2007, he is currently a MSandidate majoring in Chemical Engineering in Yonsei University. The area

f his research includes protein patterning, microfluidic devices, and hydrogelanofiber.

inwon Park received his BS degree in 1983, MS degree in 1985, and PhDegree in 1990 in Yonsei University, Korea. Furthermore, he received another

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octoral degree from Tokyo Institute of Technology, Japan in 1994. He vis-ted University of California, Riverside as a visiting professor in 2000 for oneear. His research interests are recovery of CO2 from fossil fuel, land filled gaseparation, waste cellulose utilization.

on-Gun Koh was born in Seoul, Korea in 1974. He received his BS degree in997, MS degree in 1999 from Yonsei University, Korea and PhD degree withrofessor Michael V. Pishko from Pennsylvania State University in 2004. From

004 to 2005 he was a postdoctoral research fellow at Stanford University forurtis W. Frank whose research interest was in artificial cornea. He is now anssistant professor at the Department of Chemical Engineering, Yonsei Univer-ity. His research interests include biosensor, drug delivery, bioMEMS, tissuengineering, and microfluidic device.