Protein Micropatterning on Bifunctional Organic -InorganicSol-Gel Hybrid Materials

Woo-Soo Kim,†,‡ Min-Gon Kim,§ Jun-Hyeong Ahn,§ Byeong-Soo Bae,† andChan Beum Park*,†

Department of Materials Science and Engineering, Korea AdVanced Institute of Science and Technology,373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Republic of Korea, and Bio-Nano Technology

Research Center, Korea Research Institute of Bioscience and Biotechnology, 52 Oeun-dong, Yuseong-gu,Daejeon, 305-806, Republic of Korea

ReceiVed January 10, 2007. In Final Form: February 25, 2007

Active protein micropatterns and microarrays made by selective localization are popular candidates for medicaldiagnostics, such as biosensors, bioMEMS, and basic protein studies. In this paper, we present a simple fabricationprocess of thick (∼20µm) protein micropatterning using capillary force lithography with bifunctional sol-gel hybridmaterials. Because bifunctional sol-gel hybrid material can have both an amine function for linking with protein anda methacryl function for photocuring, proteins such as streptavidin can be immobilized directly on thick bifunctionalsol-gel hybrid micropatterns. Another advantage of the bifunctional sol-gel hybrid materials is the high selectivestability of the amine group on bifunctional sol-gel hybrid patterns. Because amine function is regularly containedin each siloxane oligomers, immobilizing sites for streptavidin are widely distributed on the surface of thick hybridmicropatterns. The micropatterning processes of active proteins using efficient bifunctional sol-gel hybrid materialswill be useful for the development of future bioengineered systems because they can save several processing stepsand reduce costs.

Introduction

The ability to control the selective immobilization of bio-materials on micro and nanoscale areas is important for thedevelopment of bioengineered systems, such as biosensor, lab-on-a-chip, or bio-MEMS structures.1 Selectively immobilizedmicroarray technology enables high-throughput drug screeningand rapid sensing analysis for medical diagnostics.2 Reliable,simple, and inexpensive patterning methods are essentiallyrequired for the commercialization of protein microarrayed chipsin the biomedical diagnostic field.3 The design and fabricationof biologically functional surfaces are significant steps in theproduction of such cheap microdevices.

Two existing processes for producing patterned surfacemodifications for bioapplications are optical photolithography4

and soft lithography.5 Photolithography is a well-establishedtechnique for the bulk manufacture of integrated circuits.However, the conventional solutions used to develop and stripthe photoresist are largely incompatible with biological molecules.

For example, proteins easily malfunction in the harsh environ-ments of photolithography. It is difficult to preserve their characterduring processes due to their complex and fragile structures. Asa result, the processes for protein microarray formation must begentle enough to preserve the protein functionality. Softlithography, including imprint lithography,6 microcontact print-ing,7 inkjet printing process,8 and capillary force lithography(CFL),9 are emerging lithographies for bioapplications. Inparticular, CFL uses capillary force as a tool for the patterningof polymeric materials and fulfills patterning without harshsolvents or chemicals. When a liquid wets a capillary tube andif the wetting results in the reduction of free energy, the wettingleads to a capillary rise in the liquid.10

Selectively immobilized substrate materials for proteins mayhave carboxylic acid11 or aldehyde-terminated12 chemistry toconnect with different kinds of proteins. Proteins can be attachedto aldehydes through free amines, forming imine bonds.13Becausephotoactive materials typically used for patterning do not containthese kinds of bioactive chemistry, there have been many studiesfor conferring these biofunctionalities to patterned surfaces.Molecular assembly patterning by lift-off (MAPL) was introducedrecently.14The MAPL technique converts a pre-structured resist

* Corresponding author. Phone:+82-42-869-3340. Fax:+82-42-869-3310. E-mail: parkcb@kaist.ac.kr.

† Korea Advanced Institute of Science and Technology.‡ Present Address: Department of Materials Science and Engineering,

Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge,MA.

§ Korea Research Institute of Bioscience and Biotechnology.(1) (a) Macbeath, G.; Schreiber, S. L.Science2000, 289, 1760. (b) Frey, W.;

Meyer, D. E.; Chilkoti, A.AdV. Mater.2003, 15, 248. (c) Wilson, D. S.; Nock,S. Angew. Chem., Int. Ed.2003, 42, 494. (d) Zhu, H.; Snyder, M.Curr. Opin.Chem. Biol.2003, 7, 55.

(2) (a) Patel, N.; Padera, R.; Sanders, G. H. W.; Cannizzaro, S. M.; Davies,M. C.; Langer, R.; Roberts, C. J.; Tendler, S. J. B.; Williams, P. M.; Shakesheff,K. M. FASEB J.1998, 12, 1447. (b) Ostuni, E.; Kane, R.; Chen, C. S.; Ingber,D. E.; Whitesides, G. M.Langmuir2000, 16, 7811.

(3) (a) Lee, K. B.; Yoon, K. R.; Woo, S. I.; Choi, I. S.J. Pharm. Sci.2003,92, 933. (b) Lee, K. B.; Lim, J. H.; Mirkin, C. A.J. Am. Chem. Soc. 2003, 125,5588. (c) Lim, J. H.; Ginger, D. S.; Lee, K. B.; Heo, J.; Nam, J. M.; Mirkin, C.A. Angew. Chem., Int. Ed.2003, 42, 2309.

(4) Brack, H. P.; Padeste, C.; Slaski, M.; Alkan, S.; Solak, H. H.J. Am. Chem.Soc. 2004, 126, 1004.

(5) Lopez, G. P.; Biebuyck, H. A.; Harter, A. R.; Kumar, A.; Whitesides, G.M. J. Am. Chem. Soc. 1993, 115, 10774.

(6) (a) Hoff, J. D.; Cheng, L. J.; Meyhofer, E.; Guo, L. J.; Hunt, A. J.NanoLett.2004, 4, 853. (b) Kim, W. S.; Choi, D. G.; Bae, B. S.Nanotechnology2006,17, 3319.

(7) Shirahata, N.; Yonezawa, T.; Minura, Y.; Kobayashi, K.; oumoto, K.Langmuir2003, 19, 9107.

(8) Kimura, J.; Kawana, Y.; Kuriyana, T.Biosensors1988, 4, 41.(9) (a) Suh, K. Y.; Kim, Y. S.; Lee, H. H.AdV. Mater. 2001, 13, 1386. (b)

Suh, K. Y.; Seong, J.; Khademhossenini, A.; Laibinis, P. E.; Langer, R.Biomaterials2004, 25, 557.

(10) Kim, E.; Xia, Y.; Whitesides, G. M.Nature1995, 376, 581.(11) Kenseth, J. R.; Harnisch, J. A.; Jones, V. W.; Porter, M. D.Langmuir

2001, 17, 4105.(12) Wadu-Mesthrige, K.; Xu, S.; Amro, N. A.; Liu, G. Y.Langmuir1999,

15, 8580.(13) Christman, K. L.; Requa, M. V.; Enriquez-Rios, V. D.; Ward, S. C.;

Bradley, K. A.; Turner, K. L., Maynard, H. D.Langmuir2006, 22, 7444.(14) (a) Falconnet, D.; Koenig, D.; Assi, A.; Textor, M.AdV. Funct. Mater.

2004, 14, 749. (b) Falconnet, D.; Pasqui, D.; Park, S.; Eckert, R.; Schift, H.;Gobrecht, J.; Barbucci, R.; Textor, M.Nano Lett.2004, 4, 853.

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10.1021/la070074p CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 03/23/2007

film into a pattern of biointeractive chemistry in a non-interactivebackground using the spontaneous, monomolecular assembly ofbiofunctional moieties.14 An attractive feature of MAPL lies inits ability to vary and control the surface density of bioactivematerials in the biointeractive regions of the pattern. However,this process needs photoresist patterning and the self-assemblyof biofunctional materials onto the pre-patterned surface.Unfortunately, these several steps have drawbacks. The cost ishigh and they require long, serial processes in sample preparation.

In this work, we present a simple fabrication process for athick, protein micropatterning using capillary force lithographywith bifunctional sol-gel hybrid materials. Because bifunctionalsol-gel hybrid material has both an amine function for linkingwith streptavidin and a methacryl function for photocuring, aprotein can be attached directly to the thick bifunctional sol-gelhybrid micropatterns. This thick, hybrid pattern has advantagesnot only with the organic flexibility of the polymeric surface butalso with the inorganic mechanical stability from the inorganicglassy nature of the hybrid material. Amino-functional materials

that attach biomolecules to surfaces are versatile because theyreact with many proteins and peptides under mild conditions.15

There is no need for any special chemical etching step of patterningbecause the capillary force lithography allows rapid prototypingfor high volume production of fully patterned substrates. Otheradvantages of the hybrid bioactive pattern are thermal stabilityover 300°C and easy pattern size control by changing the patternsize of the PDMS mold.

Experimental Section

Fabrication of a Bifunctional Sol-Gel Hybrid Material. Thebioactive hybrid material was newly designed and synthesized toinduce selective immobilization of proteins and to be easily UV-curable. Silicon alkoxide precursors for the bifunctional sol-gelhybrid material (biohybrimer) were (3-aminopropyl)trimethoxysi-lane(APTMS, Aldrich) for amino-functionality and 3-(trimethoxy-silyl) propyl methacrylate (MPTMS, Aldrich) as a polymerizable

(15) Lemieux, G. A.; Bertozzi, C. R.Trends Biotechnol.1998, 16, 506.

Figure 1. (a) Schematic diagram of major components and synthesized oligomer units of the biohybrimer. (b) Schematic diagram of thefabrication process for the biohybrimer micropatterns by capillary force lithography and SEM images of biohybrimer replica structures with∼20 µm line widths, respectively.

Letters Langmuir, Vol. 23, No. 9, 20074733

organo-alkoxy silane. APTMS and MPTMS were mixed at molarratios of 1:5, 1:3, 1:1, 3:1, and 5:1, respectively. These ratios werechosen to balance the two functionalities to make an optimalbiohybrimer. In addition, control of the quantities of APTMS andMPTMS could tune the photoactivity and immobilization site of thefabricated biohybrimer. APTMS and MPTMS were first hydrolyzedwith 0.75 equiv of 0.01 M hydrochloric acid (HCl) in an N2

atmosphere. After that, the hydrolyzed APTMS solution was addedto the pre-hydrolyzed MPTS solution and stirred for 1 h toadvancehydrolysis and condensation with propylene glycol methyl etheracetate (PGMEA, Aldrich, purity 99%) as an unreactive solvent.The mixed solution was reacted with additional water for 10 h tocomplete the hydrolysis and condensation reactions. The total amountof water contained 1.5 equiv of total alkoxides in the solution. Afterthe reaction to synthesize the biohybrimer solution, any residualproducts, such as alcohols, were removed at 50°C with an evaporator.The biohybrimer was optically transparent due to the highlycondensed methacrylic-oligosiloxane structure and highly amino-functionalizeddue to the largequantityofamino-groups.Theviscosityof the hybrimer could be controlled from 20 to 220 mPa.s by alteringthe content of PGMEA solvent and through thermal heating.16

Capillary Force Lithography of Bifunctional Biohybrimer. Inthe CFL presented here, the essential feature of imprinted lithography,i.e., molding a liquid biohybrimer, was combined with the majorprocess of microcontact printing, i.e., using a poly (dimethylsiloxane)(PDMS) elastomeric mold.17 As an advantage over microcontactprinting, a stringent thick micropattern for selective immobilizationof proteins can be acquired with the CFL method. We used PDMS(Sylgard 184, Dow Corning, Midland) as an elastomeric mold andfabricated a PDMS master that has a planar surface with protrudingmicropatterns by casting PDMS against a complimentary reliefstructure prepared by photolithography or electron beam lithography.Silicon wafer (100) was used as the substrate. After conformal contactof the PDMS mold to the bare silicon substrate, the biohybrimer wasinjected from the one side edge of the mold for capillary injectionalong pattern lines. For enhancing capillary force injection of thebiohybrimer into pattern lines, the PDMS mold, while in contactwith the substrate, was pressed in the vacuum chamber with vacuumpressure of 1000 mbar and a compression time of 60 s. Because thebiohybrimer has photocurable moiety, i.e., methacryl group contain-ing 1.0 wt % of photoinitiator (Irgacure369, Ciba Geigy), UV light(80 mJ/cm2, 365 nm wavelength) cross-linked biohybrimer oligomersonly within 20-30 s. Finally, the negative replica of the thickbiohybrimer micropattern was obtained after peeling off the PDMSmold.

Characterization of Biohybrimer. The synthesized transparentbiohybrimer was passed through a 0.45µm filter to remove impuritiesand bubbles. Then, the synthesis result of the structural evolutionsin the biohybrimer was examined through Fourier transform-infrared(FT-IR) spectroscopy (JASCO, FT-IR 460plus) and direct29Si NMRspectroscopy. Direct29Si NMR spectra were recorded using a BrukerFT 500 MHz instrument from a sample consisting of 30% volumeof the biohybrimer resin in chloroform-d. Chromium (III) acetyl-acetonate was added at a concentration of 30 mg/L as a relaxationagent for silicon. Pulse delays were 30 s, the sample temperaturewas 300K, and TMS was used as a reference.

Immobilization of Streptavidin onto the Biohybrimer Mi-cropattern. To confirm the biohybrimer pattern on silicon wafer,streptavidin-biotin affinity binding was applied to the formation ofthe biohybrimer pattern. The amine-functionalized, biohybrimerpatterned surface was washed and ultrasonicated with ethanol. Next,the biohybrimer pattern was reacted with 1 mg/mL NHS-biotin (EZ-Link, Pierce, USA) in a pH 7.4 phosphate-buffered saline (PBS)buffer to form the biotin surface. NHS-biotin was dissolved indimethyl sulfoxide to achieve a 10 mg/mL concentration and dilutedto a 1 mg/mL concentration with a pH 7.4 PBS buffer. Then, the

surface was blocked with 1 mg/mL BSA in pH 7.4 PBST. To forma biotin surface, 10µg/mL Cy5-labeled streptavidin (Sigma-Aldrich,Inc., USA) in pH 7.4 PBS was reacted on the biohybrimer patternfor 30 min. The fluorescence image was obtained with a GenePixprofessional 4200A fluorescence image scanner (Axon Instrumentsm,Union, CA) and photospectrometer (fluorescence microscope, Nikon,Japan, and CCD camera, Andor technology, USA). PBS containing137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 2 mM KH2PO4

was adjusted to pH 7.4 with HCl and filtered (0.22µm Milliporefilters). A PBST buffer was prepared with 0.05% Tween 20 in a pH7.4 PBS buffer.

Results and Discussion

A series of biohybrimer was prepared by variation of the relativecontent of two silanes. The ratios of the two were chosen bybalancing two functionalities for the optimal biohybrimer, i.e.,a relatively high portion of amino-group can bind with moreprotein on the surface and a relatively high portion of methacrylicgroup can cross-link for a much denser structural pattern. Increaseof the content of APTMS in the mixture may give considerablerise in the amounts of immobilized protein, but it was difficultto synthesize a biohybrimer with a high molecular portion ofAPTMS at over 60 mol % in the mixture because of the easygelation character of APTMS. From 60 to 100 mol % of APTMSin the mixture, biohybrimer became a gel within 24 h aftersynthesis. But the biohybrimer containing less than 60 mol %APTMS showed a stable solution condition. After all, the molarratio 1:1 of APTMS and MPTMS was chosen for the compro-mising ratio of biohybrimer patterning. Figure 1a schematicallyillustrates major components of the biohybrimer and the

(16) Kim, W. S.; Kim, K. S.; Kim, Y. C.; Bae, B. S.Thin Solid Fims2005,476, 181.

(17) Park, J. P.; Lee, K. B.; Lee, S. J.; Park, T. J.; Kim, M. G.; Chung, B. H.;Lee, Z. W.; Choi, I. S.; Lee, S. Y.Biotechnol. Bioeng.2005, 92, 160.

Figure 2. (a) Direct29Si NMR spectrum of biohybrimer condensedwith APTMS and MPTMS. (b) FT-IR spectra of the structuralevolutions of the biohybrimer before and after UV curing.

4734 Langmuir, Vol. 23, No. 9, 2007 Letters

synthesized different sized oligomers. These oligomer units canbe cross-linked with a sufficient UV dosage during CFLpatterning.

A schematic of the CFL for micropatterning of the biohybrimeris shown in Figure 1b. In our experiment, a PDMS mold wasplaced directly onto the bare silicon substrate and the biohybrimerresin was injected into the one side edge of the PDMS mold,forming a conformal contact with the surface. By applying vacuumpressure, the biohybrimer, in contact with the protruding PDMSfeatures, receded spontaneously and moved into the void spaceof the PDMS mold by means of capillary action. Figure 1b showsSEM images of the negative replica of the PDMS mold, whichhas 40µm size line widths. The replicated pattern shape showsthe volume shrinkage due to evaporation of the PGMEA solvent.No residual biohybrimer remained on the substrate surfacebetween pattern lines, and the substrate was exposed as depictedin Figure 1b.

The formation of biohybrimer oligomers was further studiedby 29Si NMR and FT-IR.29Si NMR was used to confirm thedegreeof condensationand the formulatedstructureof thesiloxanebond in biohybrimer reacted with APTMS and MPTMS in a 1:1ratio. Figure 2a shows29Si NMR spectra of the biohybrimer’ssiloxane structures. The notations of Si atoms in NMR

spectroscopy are as follows: Tn represents Si from APTMS andMPTS, respectively. The superscript,n, denotes the number ofsiloxane bonds of the Si atom. The chemical shifts of Si in29SiNMR spectroscopy according to its bond states are shown inTable 1.18 As shown in Figure 2a, there were no T0 peaks, whichsuggests that the hydrolysis and condensation reactions of twodifferent precursor molecules proceeded almost to completionand that the biohybrimer had been successfully synthesized. T2

and T3 peaks show a much higher degree of condensation thanthe T1 peaks. The T2 peak is the main peak, and T3 peaks are

(18) Soppera, O.; Croutxe-barghorn, C.J. Polym. Sci., Part A: Polym. Chem.2003, 41, 716.

Figure 3. (a) A red fluorescent micrograph of site-specific streptavidin patterning that was selectively adsorbed on the 20µm lines of thebiohybrimer surface. The image was obtained by the capillary force lithography of the biohybrimer followed by immobilization of Cy5-streptavidin. The graph shows the estimated fluorescence intensities. (b) A green fluorescent micrograph of site-specific streptavidin patterningon the array of 5µm lines of the biohybrimer surface and the estimated fluorescent intensities.

Table 1. Chemical Shifts of Silicon in the Liquid 29Si NMRSpectroscopy According to Its Bond Statesa

speciesTn chemical shifts

T0 -40 ppmT1 -48 to-52 ppmT2 -55 to-61 ppmT3 -65 to-71 ppm

aTn)RSi(OMe)3-n-m(OH)m(OSi)n,whereR)-CH2CH2CH2O(CO)C(CH3)CH2,-CH2CH2CH2NH2.

Letters Langmuir, Vol. 23, No. 9, 20074735

relatively small, which indicates the three-dimensional siloxanenetworks occur in a comparably smaller quantity. Moreover, theinorganic condensation degree in the biohybrimer could becalculated by integrating the individual signals from29Si NMRspectra of the biohybrimers, according to the different chemicalshift of the hydrolysis and condensation species of the silanecomponents (eq 1):19

where T is the Si-species with three hydrolyzable silanecompounds and n in Tn is the number of siloxane bonds on theSi-atom. According to the calculation, the degree of condensationof the biohybrimer was about 64%. This condensation valuemay be higher if the poly condensation reaction time waslengthened.

Figure 2b shows the infrared spectra of the biohybrimer samplesbefore and after UV curing. It was observed that uncured andcured biohybrimer had no absorption at 3342-3420 cm-1, aband that corresponds to OH stretching of SiOH, CH3OH, andH2O. This also means that the almost hydrolyzed species werepoly condensed well to biohybrimer oligomers. The spectra ofeach film show signals at 1580 cm-1 that correspond to N-Hscissoring vibration and signals at 1486 cm-1 that shouldcorrespond to the symmetric NH+

3 deformation mode.20 Ac-cording to literature,21CdC stretching vibrational mode resultingfrom the methacrylic groups appears at 1640 cm-1. In the samplespectra before curing, this peak was observed, but there was noCdC stretching vibrational mode in that of the sample after UVcuring. This shows the complete cross-linking reaction betweenthe oligomers of biohybrimer during UV curing has been fulfilledfor highly compacted organic-inorganic hybrid materials.

In this work, streptavidin was used as a model protein. Forthe immobilization of streptavidin onto a biohybrimer micro-pattern by CFL, the NHS-biotin was first bonded with the amino-

function of biohybrimer pattern. Later, red fluorescent streptavidinwas reacted on the NHS-biotin connected biohybrimer pattern.In this manner, a well-defined pattern of 20µm stripe repeatswas fabricated on the thick biohybrimer on the silicon substrate.As shown in Figure 3a,b, the selective binding of the Cy5-labeledstreptavidin to two different types of biohybrimer patterns couldbe visualized as a red or a green fluorescent micrograph. Theestimated fluorescence intensities for each case are also illustratedin the Figure 3. To examine whether the binding of Cy5-labeledstreptavidinwasspecific to theNHS-biotinconnectedbiohybrimerpattern, a bare silicon substrate was prepared and used in thesame manner for generating the streptavidin protein micropatterns.According to our observation, no fluorescence was observed,which suggests that streptavidin was exactly bonded only ontothe biohybrimer patterns. In order to confirm whether streptavidinretained its binding ability after several days, the fluorescenceintensities of bound streptavidin were investigated after 15 days.Most streptavidin (∼98%) remained bound compared to the firstmeasured intensity of fluorescence, which means that the selectiveimmobilization of streptavidin onto the stable biohybrimer patterncould be maintained for 15 days or more.

Summary and ConclusionWe report here a simple protein micropatterning process with

bifunctional sol-gel hybrid materials by capillary force lithog-raphy. This process did not require any chemical etching step,high-temperature step, or solvent evaporation step for patterningprotein. By introducing thermally and chemically stable bifunc-tional sol-gel hybrid materials as the patterned media, spatiallywell-defined, thick images of selective protein adsorption wereobserved in a wide area. Because of the amino-function of well-condensed bifunctional hybrid material, this methodology canbe easily extended to the patterning of other peptides and proteins.In addition, sub-micron scale protein patterning may be possiblewith a sub-micron scale mold. As demonstrated by the stableimmobilization of streptavidin in a micropattern, the concept ofpatterning bifunctional sol-gel hybrid material appears to bevery flexible for establishing platforms for biomolecular im-mobilization.

Acknowledgment. This work was supported by the BrainKorea 21 project (BK21) and the Sol-Gel Innovation Project(SOLIP) of the Ministry of Commerce, Industry and Energy ofKorea.

LA070074P

(19) Sepeur, S.; Kunze, N.; Werner, B.; Schmidt, H.Thin Solid Films1999,351, 216.

(20) Shmizu, I.; Okabayashi, H.; Taga, K.; Nishio, E.Vib. Spectrosc.1997,14, 113.

(21) Kim, W. S.; Houbertz, R.; Lee, T. H.; Bae, B. S.J. Polym. Sci., Part B:Polym. Phys.2004, 42, 1979.

degree of condensation (%))0 × T0 + 1 × T1 + 2 × T2 + 3 × T3

3 × ∑n)0

3

Tn

(1)

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