Thin Solid Films 460(2004) 286–290

0040-6090/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.tsf.2004.01.082

Short Communication

Co-patterning chitosan and bovine serum albumin on an aldehyde-enriched glass substrate by microcontact printing

Jie Feng, Changyou Gao*, Bo Wang, Jiacong Shen

Department of Polymer Science and Engineering, No. 38, Zheda Road, Zhejiang University, Hangzhou 310027, PR China

Received 24 September 2003; received in revised form 16 January 2004; accepted 16 January 2004Available Online 25 March 2004

Abstract

Chitosan and bovine serum albumin were co-patterned onto a glass substrate by microcontact printing technique. The processuses a microfabricated polydimethylsiloxane stamp to transfer chitosan on an aldehyde functionalized glass substrate, followed byadding a drop of albumin solution to the patterned side and holding for 30 min. After being washed with phosphate-bufferedsaline and cleaned by ultrasonic, the co-patterns of chitosan and albumin, each with their own micropatterns, were formed on thesame surface. In this procedure, ultrasonic cleaning takes an important role to obtain clear patterns, whereas the printingyaddingsequence has less influence. Moreover, patterns printed could give higher contrast than those assembled from solution. These co-patterns could find applications in cell localization and cell growth guidance.� 2004 Elsevier B.V. All rights reserved.

Keywords: Microcontact printing; Glass; Surface structure; Biomaterials

1. Introduction

The ability to create micropatterns of biomoleculeson material surfaces is important for selective cellattachment and cell guidance, which in turn find appli-cations in cell-based biosensorw1–3x, tissue engineeringw4,5x and fundamental studies of cell biologyw6,7x.There are several ways to pattern proteins and otherbiomolecules on solid surfaces, such as photolithographyw8,9x, soft lithographyw10–12x and spottingw13x tech-niques. Among these methods, microcontact printing(mCP), one of the soft lithography techniques, has beenwidely applied recently.As a bridge of cells and their adhesion substrates,

biomacromolecules can be patterned onto substrates byadsorption on regions of different self-assembled mono-layers (SAMs). Whitesides and coworkersw14x usedmCP to fabricate patterns of alkanethiolates SAMs thathave different terminal groups on gold substrate, realiz-

*Corresponding author. Tel.:q86-571-87951108; fax:q86-571-87951948.

E-mail address: cygao@mail.hz.zj.cn(C. Gao).

ing selective adsorption of proteins. Mikos et al.w15xfabricated glass substrates with desired octadecyltrich-lorosilane(OTS) patterns usingmCP and realized selec-tive attachment and function holding of human retinalpigment epithelium cells. Biziosw16x prepared a co-patterned surface ofN w3-(trimethoxysilyl)propylxdi-1

ethylenetriamine and OTS on a same silicon substrateand realized cell selective attachment as well. Bioma-cromolecules can also be directly printed onto substratesby just using their solutions as ink, via physical adsorp-tion or covalently graftingw4,5,17x. Moreover, by suc-cessive stamping, different proteins can also be printedonto a same substrate, each with its own patternsw17,18x.Co-patterning two types of biomacromolecules that

have opposite effect on cell adhesion could realize moreaccurate control of cell attachment, especially when thisco-pattern is complementary. Successive printing couldnot achieve such co-patterns. They can only be preparedby other techniques, such as elastomeric membrane andlift-off technique w19x, combination ofmCP and layer-by-layer assemblyw20x. Here we introduce an alternative

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Fig. 1. Schematic illustration to show the process of co-patterningchitosan and BSA on the aldehyde-enriched substrate.

way to achieve such co-patterns of chitosan and bovineserum albumin(BSA) by a modifiedmCP process. Thebasic procedures include microcontact printing one kindof biomacromolecules onto an activated substrate, fol-lowed by incubation with another biomacromoleculesolution. Similar as method used in co-patterning SAMson gold w14x, the present process is simple and flexiblein co-patterning biomacromolecules on activated orcharged surfaces. Since chitosan is a cell-adhesive poly-saccharidew21x and BSA is a cell-resistant proteinw19x,their co-patterns could in turn find applications in celllocalization and cell growth guidance.

2. Experimental details

2.1. Silanization and glutaraldehyde treatment of glassslides

Glass slides were cleaned by immersion in a solutionof H SO :H O (70:30 vyv) for 1 h, and then washed2 4 2 2

extensively in running distilled water. 3-Aminopropyl-trimethoxysilane(3-APS, Aldrich) was dissolved in anethanol:water(95:5 vyv) solution with a concentrationof 1.5 vol.%. The pH value was adjusted to 2.0 withconcentrated hydrochloric acid. The cleaned slides(1.5=1.5 cm) were dipped into 3-APS solution for 22

h and then were rinsed with ethanol and water for 3cycles. After dried by nitrogen and baked at 1108C for1 h, these amino functionalized glass slides were furtherimmersed into a 1% glutaraldehyde solution for 30 minto convert the free amino groups into aldehyde groups(glass-CHO).

2.2. Microfabrication of co-patterned substrates

2.2.1. Stamp fabricationPoly(dimethylsiloxane) (PDMS, Sylgard 184, Dow

Corning) elastomer stamps were cast from mastersw22xcontaining arrays of microwells or posts, which wereoriginally fabricated by photolithography using maskswith patterns measuring 50y50 mm in diameteryspace.Two types of stamps with posts or microwells werefabricated, which are designated the positive and thenegative stamps, respectively.

2.2.2. Co-patterning biomacromoleculesIn order to increase the surface hydrophilicity, PDMS

stamp was immersed in deionized water for at least 24h. Before patterning, stamp was taken out and dried bynitrogen flow, then coated with 2 mgyml of rhodamineisothiocyanate labeled chitosan(Rd-chitosan. Chitosanwith medium molecular weight was purchased fromAldrich)yacetic acid solution(pH 5.2) for 20 min. Afterdrying by nitrogen, the inked stamp was immediatelypressed onto the above modified glass-CHO slides witha force of 100 gycm for 20 min. After the stamp was2

removed, a drop of fluorescein isothiocyanate-labeledBSA (FITC-BSA, Aldrich) solution(2 mgyml) in phos-phate-buffered saline(PBS) was added onto the pat-terned side. Thirty minutes later, the co-patternedsubstrate was rinsed with PBS and cleaned by ultrasonic,in acid solution for 8 min. All procedures were con-ducted at room temperature. The process is schematical-ly presented in Fig. 1.For a comparison, the reverse printingyadding process

was also performed by printing BSA with a negativestamp, followed by addition of chitosan solution, andfinally cleaned by ultrasonic.

2.3. Characterization

The water contact angles of glass surface before andafter silanization were measured by DSA10-MK2 Con-tact Angle Measuring System from Kruss. The topo-¨graphic features of stamps were imaged with atomicforce microscopy(AFM, SPI3800 N, Seiko InstrumentsInc.) in dynamic mode under ambient conditions. Theco-patterned biomacromolecules on the glass substrateswere visualized using a Bio-Rad Radiance 2100 confocallaser scanning microscope(CLSM). FITC-BSA and Rd-chitosan were excited with lasers of 488 nm and 543nm, respectively. Independent images of chitosan andBSA patterns were obtained by a sequential scanningmode, and then merged into a co-pattern image. A M850fluorescence spectrum analyzer was used to measure

288 J. Feng et al. / Thin Solid Films 460 (2004) 286–290

Fig. 2. (a) Confocal image of chitosanyBSA co-patterns fabricated by first printing chitosan, then adding BSA solution, and finally ultrasoniccleaning. The separate areas are chitosan and the continuous regions are BSA.(b) The line profiles of the fluorescence intensity of the chito-sanyBSA co-patterns in(a). Line 1 and line 2 represent chitosan and BSA, respectively.

Fig. 3. Confocal image of BSAychitosan co-patterns fabricated by thereverse printingyadding process, e.g. first printing BSA with a nega-tive stamp, followed by addition of chitosan solution, and finallycleaned by ultrasonic. The separate areas are chitosan and the contin-uous regions are BSA.

fluorescence spectra of rd-chitosan and FITC-BSA print-ed or adsorbed on aldehyde-functionalized quartz.

3. Results and discussion

The silanization of glass slides was evaluated bywater contact angle measurements. Cleaned glass slidehad a static contact angle of 3.5"1.48, while increasedto 598 after silanized by 3-APS. These range of anglesare all representative of a short(C ) chain length silanes3

w23x, demonstrating 3-APS had been covalently coupledonto the slide surface.AFM measurements revealed that the final topograph-

ic dimensions of the two types of stamps are;50y50mm in diameteryspace and;4.5 mm in heightydepth.The diameter of the microwells in the negative stamp issomewhat larger than that of posts in the positive stamp.Using the positive stamp, the complementary co-patternsof chitosan and BSA on the same glass-CHO slide werefabricated by first printing chitosan, followed by additionof BSA solution, and finally cleaned by ultrasonic(Fig.2a). The confocal image shows that not only the co-patterns but also the borders between each kind ofpatterns are clear. The average signal-to-noise ratio(SyN, i.e. pattern intensityybackground intensity) of 6–8(Fig. 2b) demonstrates the successful localization ofthese two biomacromolecules. By more careful analysis,one can find that theSyN of chitosan patterns(approx.8) is better than that of BSA patterns(approx. 6).Fig. 3 shows the co-patterns of BSA and chitosan

formed by a reverse process. In this process BSA wasfirstly printed using a negative stamp, followed byaddition of chitosan solution, and finally cleaned byultrasonic in PBS. Similar as the results obtained above,the co-patterns of chitosan and BSA are clear as well.Interestingly, theSyN of the two patterns also reversedalong with the reverse process, i.e. theSyN of the

chitosan patterns is poorer than that of BSA patterns.Though the fluorophores and the labeling density mightinfluence on the fluorescence intensity, the inversion ofthe SyN demonstrates that the decisive factor is theprinting technique other than the difference of thefluorophores. Hence, one can deduct that the influenceof different fluorophores is very limited and can beneglected when compared with the printing technique.To further confirm this deduction, Rd-chitosan and

FITC-BSA were either printed with a flat stamp ordirectly adsorbed onto the aldehyde functionalized

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Fig. 4. Fluorescence spectra of Rd-chitosan and FITC-BSA printed oradsorbed on the aldehyde-functionalized quartz slides. The excitingband is 488 nm and 543 nm, respectively.

Fig. 5. Confocal image of chitosanyBSA co-patterns fabricated by firstprinting chitosan, then adding BSA solution, but without ultrasoniccleaning. No clear patterns could be created.

quartz slides for 20 min. After being rinsed with corre-sponding solvent and cleaned by ultrasonic for 8 min,their fluorescence spectra were recorded(Fig. 4). Fig. 4shows that all the fluorescence intensity of the printedslides is stronger than that of physically adsorbed,regardless of the fluorophores. The reason could be thata larger amount of the biomacromolecules are attachedonto the slide surface because of the pressing procedure,which can overcome the repulsion of the same speciesin a larger extent than physical adsorption. The pressingcan also result in multilayer accumulation of the bio-macromolecules, which is hardly achieved by the pureadsorption that generally yields a monolayer.

It is worth noting that the cleaning procedure takesan important role in obtaining clear co-patterns of thebiomacromolecules. Before ultrasonic cleaning, no clearpatterns of BSA or chitosan could be created, as illus-trated in Fig. 5. Here chitosan was firstly printed,followed by addition of BSA solution without cleaning.Similar results have also been found in co-patterningchitosan and BSA on aldehyde-enriched polycaprolac-tone surfaces.It is well known that chitosan and BSA have strong

interaction and can be assembled together by layer-by-layer deposition techniquew24x. In this experiment,when the BSA solution was added onto the chitosanpre-patterned glass-CHO surface, BSA adhered not onlyon the remaining bare substrate but also on the chitosanpatterns. No clear patterns of BSA could be found atthis stage(Fig. 5). After being cleaned by ultrasonic,however, the outmost layer of the chitosan patternsdesquamated from the substrate together with the BSAadhered on it, thus yielding the clear co-patterns. Fromthese results one can also deduct that the pre-patternedchitosan or BSA should be rather dense other than amonolayer. This can be understood from the nature ofthe microcontact printing and the biomacromolecules.In principle, only the closest layer of the biomacromo-lecules can be covalently attached by reaction betweenthe aldehyde on the substrate and the amino groups inthe biomacromolecules. Meanwhile, the physical adsorp-tion of extra biomacromolecules is unavoidable, leadingto the formation of a dense but removable outer layeron the covalently attached biomacromolecules. As aresult, co-patterning of the two different biomacromo-lecules could be realized easily by the present method.

4. Conclusion

Co-patterns of chitosan and BSA on glass substratewere successfully fabricated with high contrast and clearborders by a modifiedmCP technique. The averagesignal-to-noise ratio(SyN) in the co-patterns showedthat the printed patterns give higher contrast than thoseassembled from solution. Ultrasonic cleaning takes animportant role to obtain clear co-patterns, whereas theprintingyadding sequence of the biomacromolecules hasless influence. This modified technique may be extendedto co-pattern other biomacromolecules as well.

Acknowledgments

The authors would like to thank Prof. Bai Yang forproviding the photolithographic mask for preparing thePDMS stamp. Financial support by the Natural ScienceFoundation of China(Grant No. 50173024) and theMajor State Basic Research Project of China(Grant No.G1999054305) are greatly acknowledged.

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