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Colloids and Surfaces B: Biointerfaces 87 (2011) 192– 197

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

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hort communication

urface self-assembly of N-fluorenyl-9-methoxycarbonyl diphenylalanine onilica wafer

un Liu, Xiao-Ding Xu, Jing-Xiao Chen, Han Cheng, Xian-Zheng Zhang ∗, Ren-Xi Zhuoey Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, PR China

r t i c l e i n f o

rticle history:eceived 15 February 2011eceived in revised form 19 April 2011

a b s t r a c t

N-Fluorenyl-9-methoxycarbonyl diphenylalanine (Fmoc-FF-OH) was chemically immobilized to the sur-face of silica wafer as the “seed”. When immersing this peptide attached silica wafer into the dipeptideaqueous solution, the occurrence of a pH triggered surface self-assembly resulted in the formation of pep-

ccepted 27 April 2011vailable online 6 May 2011

eywords:eptideodification

tide nanorods on the surface of silica wafer. This surface self-assembly exhibited a dependence on theconcentration of the dipeptide aqueous solution. It was proposed that the self-assembly of this dipeptideon the surface of silica wafer was similar to that in aqueous solution. In comparison with the conventionalphysical adsorption on the substrates, the chemically attached self-assembled nanorods exhibited muchimproved adsorption capacity on the substrate surface.

urface self-assembly

. Introduction

In recent years, surface modification has received increasingesearch interests, and extensive studies have been carried outndow the surfaces with specific chemical and physical proper-ies. To improve the surface biocompatibility and biofunctionalityf biomaterials, various molecules, such as oligo(ethylene glycol)1], DNA [2], proteins [3], carbohydrates [4], and antibodies [5] haveeen attached on the surfaces of biomaterials.

Among these modification molecules, peptides, due to theirnherent biocompatibility and biodegradability, have been widelytilized to attach on the surfaces of substrates for improving theirerformance in cell attachment and proliferation [6,7] or immobi-

ization of enzymes [8,9] and other biomolecules [10]. In this field,tudies have been carried out predominately in two aspects, i.e.hemical immobilization of peptide monolayer as mentioned abovend surface physical deposition of peptide nanofibres [11–14]. Theormer method is not perfect due to its molecule-sized defects15–19]. While the latter approach suffers from inherent instabil-ty because of the weak adsorption, and peptide molecules coulde easily rinsed away by aqueous solution.

On the other hand, peptides have also been exploited as multi-unctional molecules for the preparation of nanostructures, such

s one-dimensional (1D) nanofibres [20] and three-dimensional3D) constructions [21] through self-assembly. Up to now, differentpproaches have been developed to direct or drive the self-

∗ Corresponding author. Tel.: +86 27 68755993; fax: +86 27 68755993.E-mail address: xz-zhang@whu.edu.cn (X.-Z. Zhang).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.04.032

© 2011 Elsevier B.V. All rights reserved.

assembly of peptides, and a variety of peptides have been designedfor self-assembly triggered by an external stimuli including pH [22],enzymes [23], light [24] and temperature [25]. However, as far aswe know, the studies on the self-assembly of peptides on the sur-faces of materials are limited as compared with the self-assemblyin the solutions.

In this study, by using Fmoc-FF-OH chemically immobilizedon the surface of silica wafer as an initial “seed”, we investi-gated the effect of surfaces on the self-assembly of the dipeptide.When immersing the modified silica wafers into Fmoc-FF-OHaqueous solutions, nanorods formed via surface self-assembly ofthe dipeptides. Since the peptide self-assemblies were chemicallyimmobilized on the surface of silica wafers, the peptides couldnot be rinsed away by water. This study proposed an innovativestrategy for the surface modification of biomaterials.

2. Materials and methods

2.1. Materials

N-Fluorenyl-9-methoxycarbonyl protected phenylalanine(Fmoc-Phe-OH), 2-(1H-benzotriazol-1-yl)-1,1,1,1-tetramethyl-uroniumhexafluo-rophosphate (HBTU), 1-hydroxybenzotriazole(HOBt), triisopropylsilane (TIS), and 2-chlorotrityl chloride resin(100–200 mesh, loading: 1.35 mmol/g) were obtained from Shang-hai GL Biochem. Ltd. (China) and used directly. Trifluoroacetic

acid (TFA) was purchased from Shanghai Chemical Co. (China)and used as received. Diisopropylethylamine (DIEA) was fromShanghai GL Biochem. Ltd. (China) and used after distillation.N,N-Dimethylformamide (DMF), methanol and dichloromethane

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DCM) were obtained from Shanghai Chemical Co. (China) and usedfter distillation. Toluene was purchased from Shanghai Chemicalo. (China) and dried by distillation over sodium. (3-Aminopropyl)rimethoxysilane (APS) was purchased from ACROS (USA) and usedirectly. Silica wafers were purchased from Huihong Electricityo. Ltd. (China). All other reagents and solvents were of analyticalrades and used without further purification.

.2. Methods

.2.1. Synthesis of Fmoc-FF-OHFmoc-FF-OH was prepared manually on the 2-chlorotrityl chlo-

ide resin employing a standard Fmoc chemistry. The resin was firstmmersed in DMF for 2 h. Then Fmoc-Phe-OH was fixed on the resin

ith a DMF solution of the mixture of Fmoc-Phe-OH (4 equiv. rela-ive to resin loading) and DIEA (6 equiv. relative to Fmoc-Phe-OH).fter stirring at room temperature for 3 h, 20% piperidine/DMF (v/v)olution was added to remove the Fmoc protected groups. There-fter, a DMF solution of the mixture of Fmoc-Phe-OH (4 equiv.),BTU (4 equiv.), HOBt (4 equiv.), and DIEA (6 equiv.) was added and

tirred for 2 h at room temperature. The cleavage of the expectedeptide was performed using a mixture of TFA, deionized water,nd TIS in the volume ratio of 95:2.5:2.5 by stirring for 2 h at roomemperature. The cleavage mixture was collected, concentrated byotary evaporation and dropped in cold ether to precipitate theroduct. ESI-MS: 533.2, [M−H]−

.2.2. Modification of silica wafersSilica wafers (2 cm × 0.8 cm × 0.1 cm) were cleaned with deion-

zed water, ethanol and dichloromethane in succession underltrasonication for 30 min. After that, the wafers were dried in atream of nitrogen, kept in piranha solution (concentrate H2SO4nd H2O2 7:3 in volume) at 110 ◦C for 1 h, and then washed severalimes with deionized water. After the rinsing, the wafers were driednder nitrogen atmosphere. To introduce primary amine groups tohe surface, all the dried silica wafers were immersed into a tolueneolution containing 1% (v/v) APS at 60 ◦C for 15 min [26], and theninsed with dried toluene for five times and dried under nitro-en atmosphere. Thereafter, the amino-functionalized wafers wereodified with Fmoc-FF-OH by immersing them in a DMF solution

ontaining 0.4 mmol of Fmoc-FF-OH, 0.8 mmol HOBt and 0.8 mmolIC for 24 h, followed by washing with DMF, ethanol, deionizedater and ethanol, respectively and drying with nitrogen.

.2.3. Self-assembly of Fmoc-FF-OH on the surface of silica wafersThe aqueous solutions of Fmoc-FF-OH with different concen-

rations (0.8, 1.3 and 2.0 mg/mL) were prepared by dissolving theequired amount of Fmoc-FF-OH in the aqueous solutions at aH of ∼10.5. The Fmoc-FF-OH modified silica wafers were then

mmersed into these solutions. After that, the pH of the solutionsere adjusted to a value of 2 to trigger the self-assembly of the

moc-FF-OH on the surface of the modified silica wafers. After 1 h,he silica wafers were taken out, rinsed with the aqueous solutionf hydrochloric acid at a pH of 4 or 7 for several times and finallyried under nitrogen atmosphere.

.2.4. MS measurementThe molecular weight of Fmoc-FF-OH was analyzed by elec-

rospray ionization mass spectrometry (ESI-MS, LCQ Advantage,inigan, USA).

.2.5. CA measurements

Water contact angles (CAs) of the silica wafers were measured

y using a Dataphysics OCA20 (German) and the sessile dropethod was employed. A droplet of deionized water (2 �L) which

ormed at the end of the needle was dropped onto the surface of

iointerfaces 87 (2011) 192– 197 193

the silica wafers, and then contact angles were measured within20 s.

2.2.6. AFM measurementsAtomic force microscopy (AFM, SPM-9500J3, SHIMADZU, Japan)

was used to observe the surface morphology of the silica wafersusing a tapping mode.

2.2.7. XPS measurementsX-ray photoelectron spectroscopy (XPS) was carried out on

X-650 (HITACHI, Japan) using a monochromated beam from a mag-nesium source run at FRR analyzer mode.

3. Results and discussion

3.1. Characterizations of the silica wafers

In this study, Fmoc-FF-OH was selected to investigate the sur-face self-assembly of peptides on silica wafers since it has beenreported to be able to form supermolecular self-assembled nanos-tructures under physiological conditions [27]. As illustrated inScheme 1, before the self-assembly, the silica wafer was firstlyamino-terminated and then covalently linked with Fmoc-FF-OH.XPS was carried out to examine the chemical properties of the sur-face of the silica wafers. As shown in Fig. 1, the nitrogen atom ratioincreased after silylation (Fig. 1b) in comparison with the blanksilica wafer (Fig. 1a), indicating that primary amine groups weresuccessfully immobilized onto the surface of silica wafer after sily-lation using APS [28,29]. In addition, because of the presence ofpropyl amine groups on the surface of the silica wafer, a decreasein the oxygen atom ratio could be observed in Fig. 1b, also demon-strating the success of silylation. Furthermore, the result of XPS alsoshowed that the oxygen and nitrogen atom ratios both increasedafter the modification by Fmoc-FF-OH (Fig. 1c) when comparedwith the amino-terminated silica wafer because of the existence ofoxygen and nitrogen atoms in Fmoc-FF-OH. The XPS result provideda direct proof for the successful modification of the silica wafer byAPS and Fmoc-FF-OH.

The surface morphology of the silica wafers before and aftermodification was observed by AFM. As presented in Fig. 2, theamino-terminated wafer (Fig. 2b) was rougher than that of theblank silica wafer (Fig. 2a), and the average roughness of sil-ica wafers prior and after silylation was 0.421 and 1.173 nm,respectively. AFM characterization also showed. It was found thatFmoc-FF-OH modified silica wafer (Fig. 3c) was smoother than thatof the amino-terminated wafer, and the average roughness of silicawafers prior and after the modification of Fmoc-FF-OH was 1.173and 0.667 nm, respectively.

In the current study, contact angles (CAs) of the silica wafersprior and after the modification were measured to study thehydrophilicity change of the surfaces, which also offered evidencefor the successful surface modification of silica wafers. As shownin Fig. 3, due to the propyl group of APS, the CA of the amino-terminated wafer (Fig. 3b) was bigger than that of the blank silicawafer with hydrophilic hydroxyl groups (Fig. 3a). Moreover, afterlinked with Fmoc-FF-OH, the surface of modified silica wafer exhib-ited a further increased CA (Fig. 3c) because of the hydrophobicterminal. CA measurements further confirmed the attachment ofFmoc-FF-OH on the wafer surface.

3.2. Self-assembly of Fmoc-FF-OH on Fmoc-FF-OH modifiedsurfaces

According to our previous studies, Fmoc-FF-OHs could self-assemble through �–� interactions of the fluorenyl groups andphenyl rings in the phenylalanine side chains to form nanorods

194 Y. Liu et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 192– 197

modifi

wIcaonsam

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Scheme 1. The surface

ith an antiparallel �-sheet structure in aqueous solutions [21].n the current study, however, because of the restriction of theovalently linked Fmoc-FF-OH “seeds” on the surface, the self-ssemblies could not grow into long nanofibres. After self-assemblyf Fmoc-FF-OH molecules on the Fmoc-FF-OH modified surfaces,

anorods ranged from about 10 to 30 nm appeared on the silicaurfaces. Three different Fmoc-FF-OH concentrations of 0.8, 1.3nd 2.0 mg/mL were selected to carry out the self-assembly on theodified silica surfaces. It was found that concentration played an

ig. 1. XPS (N 1s 403 eV, O 1s 536 eV, C 1s 289 and Si 2s 153, 2p 107) spectra of blank silic N:O:C:Si = 2.8:21.6:61.4:14.2 (b) and silica wafer modified with Fmoc-FF-OH, atom % N:

cation of silica wafers.

important role in the process of self-assembly and determined theproperty and morphology of the self-assembled surfaces.

As shown in Fig. 3, water CAs of the self-assembled sur-faces were much smaller than that of the Fmoc-FF-OHmodified silica wafer, and CA decreased with increasing

Fmoc-FF-OH concentration for the self-assembly. Since thenanorods formed by self-assembly or nanofibres physicallyadsorbed on the surfaces were both hydrophilic, with the increas-ing concentration, there were a growing number of nanorods

a wafer, atom % N:O:C:Si = 0:36.6:40.5:23 (a), amino-terminated silica wafer, atomO:C:Si = 4.8:29.1:48.8:17.3 (c).

Y. Liu et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 192– 197 195

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Fig. 2. Three-dimensional AFM images (1 �m × 1 �m) of blank silica wafer (a)

ormed or nanofibres physically adsorbed on the Fmoc-FF-OHodified surfaces, which made the surface more hydrophilic. As a

esult, water CAs got decreased.AFM measurements revealed the surface morphology of the

ilica wafers after surface self-assembly. At the concentration of.8 mg/mL (Fig. 4a), uniformly distributed nanorods were formedn the Fmoc-FF-OH modified surface, while only a few nanorods

ormed on the blank and amino-terminated silica wafer under theame conditions (Fig. 5a and d). With the increasing concentration,he self-assembled nanorods occupied the Fmoc-FF-OH modifiedurface entirely at the concentration of 1.3 mg/mL (Fig. 4b). In

ig. 3. Water contact angles of blank silica wafer (a), amino-terminated silica wafer (b),

.8 (d), 1.3 (e), 2.0 mg/mL (f) aqueous solutions of Fmoc-FF-OH and then rinsed with wate

o-terminated silica wafer (b) and silica wafer modified with Fmoc-FF-OH (c).

addition, a small amount of nanofibres were physically adsorbedon the layer of nanorods, which could not be removed by rins-ing. While for the surfaces of blank and amino-terminated silicawafer (Fig. 5b and e) after self-assembly, only several nanorodscould be observed. When the concentration further increased to2.0 mg/mL, more physically adsorbed nanofibres could be foundon the Fmoc-FF-OH modified surface (Fig. 4c). Several nanorods

could also be seen on the blank silica wafer (Fig. 5c) and manynanorods formed on amino-terminated silica wafer because of theincreased hydrogen bonding between amino groups and dipep-tides with the increasing concentration (Fig. 5f). In our study, it

silica wafer modified with Fmoc-FF-OH (c) and silica wafers after self-assembly inr of pH ∼ 4.

196 Y. Liu et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 192– 197

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ig. 4. Three-dimensional AFM images (4 �m × 4 �m) of Fmoc-FF-OH modified siliF-OH and then rinsed with aqueous solutions of hydrochloric acid at pH ∼ 4 and aqueous solutions of hydrochloric acid at pH ∼ 7 (d).

as found that the physically adsorbed nanofibres were difficult toe rinsed away. Here, the rinsing aqueous solutions of hydrochlo-ic acid at two different pHs (pH ∼ 4 and pH ∼ 7) were used toemove the physically adsorbed nanofibres after self-assembly.

ig. 5. Three-dimensional AFM images (4 �m × 4 �m) of blank silica wafers after self-asseerminated silica wafers after self-assembly in 0.8 (d), 1.3 (e), 2.0 mg/mL (f) aqueous solut pH ∼ 4.

ers after self-assembly in 0.8 (a), 1.3 (b), 2.0 mg/mL (c) aqueous solutions of Fmoc-lf-assembly in a 0.8 mg/mL aqueous solution of Fmoc-FF-OH and then rinsed with

Obviously, there were very few nanorods left on the self-assembledsurfaces when rinsed by aqueous solution of hydrochloric acid atpH ∼ 7 (Fig. 4d) because most of the self-assembled nanostructuresgot disassembled at pH ∼ 7. On the contrary, the self-assembled

mbly in 0.8 (a), 1.3 (b), 2.0 mg/mL (c) aqueous solutions of Fmoc-FF-OH and amino-tions of Fmoc-FF-OH, and then rinsed with aqueous solutions of hydrochloric acid

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anorods remained integrated after rinsing by aqueous solutionf hydrochloric acid at pH ∼ 4 (Fig. 4a–c). Thus, rinsing solution ofH ∼ 4 cannot destroy the self-assemblies.

. Conclusions

Surface self-assembly of Fmoc-FF-OH on the surface of silicaafers was studied and different morphologies were obtained

hrough adjusting the concentration of Fmoc-FF-OH aqueous solu-ions. With the increasing concentration, there was a growingensity of the self-assembled nanorods formed on the surfaces. Thistudy provides a new methodology of surface modification, whichas great potential in biomedical applications such as cell culturend biosensors.

cknowledgements

This work was financially supported by the Ministry of Sciencend Technology of China (2011CB606202), Trans(New)-Centuryraining Programme Foundation for the Talents from the Ministryf Education of China and Natural Science Foundation of Hubeirovince, China (2009CDA024).

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