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Surface Science 601 (2007) 4563–4570

Construction of a novel multilayer system and its usefor oriented immobilization of immunoglobulin G

Gokhan Demirel a, Tuncer Caykara a,*, Barıs� Akaoglu b, Mehmet Cakmak b

a Department of Chemistry, Faculty of Art and Science, Gazi University, 06500 Besevler, Ankara, Turkeyb Department of Physics, Faculty of Art and Science, Gazi University, 06500 Besevler, Ankara, Turkey

Received 26 February 2007; accepted for publication 19 June 2007Available online 27 June 2007

Abstract

In this contribution, we have developed a novel multilayer system composed of 3-aminopropyltrimethoxysilane (APTS), biotin, strep-tavidin and biotinylated protein A on the Si(100) surface to immobilize of immunoglobulin G (IgG) molecule. Existence of the firstAPTS layer covalently attached on the silicon surface was revealed by X-ray photoelectron spectroscopy (XPS). Average thickness ofthe APTS overlayer was estimated by spectroscopic ellipsometry analysis as 6.3 nm. The surface topography of the APTS overlayerobserved by atomic force microscopy is found to be considerably different from the hydroxylated silicon surface and many additionalislands of various heights are observed over the substrate. The water contact angle of the APTS overlayer was determined as 38�, whereasthat of the hydroxylated silicon was obtained as 6�, indicating the difference in their surface hydrophobicility. Furthermore, multilayerstudies on the APTS overlayer were carried out by using biotin, streptavidin and fluorescein-labeled biotinylated protein-A molecules.The fluorescence images obtained by fluorescence microscopy showed the formation of the multilayer on the Si(10 0) surface. The resultsindicated that the protein A-terminated surfaces can be used to immobilize IgG molecules in a highly oriented manner and maintain IgGmolecular functional configuration on the multilayer system.� 2007 Elsevier B.V. All rights reserved.

Keywords: Of immunoglobulin G; Self-assembled monolayer; Surface characterization

1. Introduction

In the work of immunoassay protein chip, the process ofself-assembled monolayers (SAMs) and more recently, ofself-assembly of multilayer systems have attached a greatdeal of interest. Self-assembly of multilayers have beencomposed not only from simple organic molecules, suchas thiols, silanes, and carboxylic acids [1,2], but also frombiomolecules [3,4]. Multilayer systems of the latter sortare often based on the protein streptavidin. Streptavidinis particularly suitable for this purpose, as it possesses fourbinding sites for its ligand biotin – two on each side –allowing binding of streptavidin to biotinylated layers on

0039-6028/$ - see front matter � 2007 Elsevier B.V. All rights reserved.

doi:10.1016/j.susc.2007.06.034

* Corresponding author.E-mail address: caykara@gazi.edu.tr (T. Caykara).

both sides of the protein. In principle, therefore, it shouldbe relatively straightforward to build up a variety of multi-layer systems based on streptavidin layers. A versatile anduseful biotin-functionalized self-assembly of multilayer sys-tems on gold have been presented by Knoll and co-workers[5,6]. However, recently, there has been great interest in useof semiconductor materials, especially silicon, for directelectronic sensing of biomolecular binding at surface [7–10] and for their higher chemical and thermal stability[11,12]. In particular, organosilane overlayers formed onhydroxyl (–OH) bearing oxide surfaces though chemicalreaction of organosilane molecules (e.g. 3-aminopropyltri-methoxysilane, APTS) with –OH sites on the silicon sur-faces are promising for practical applications, becausethese overlayers are markedly stable mechanically andchemically due to the strong immobilization though silox-ane bonding. To the best of our knowledge, although a

4564 G. Demirel et al. / Surface Science 601 (2007) 4563–4570

great deal of research has been conducted on the aminosi-lane SAMs on silicon surface, a multilayer system com-posed of 3-aminopropyltriethoxysilane (APTS); biotin,streptavidin and biotinylated protein A covalently attachedon Si(1 00) surface has not been investigated.

For the development of an immunosensor, the immobi-lization of IgG on the solid substrate is very important.Several techniques for the immobilization of IgG wasreported and they can be divided into two main parts:physical immobilization and chemical immobilization.The physical immobilization technique has been little usedbecause of denaturation of the protein and poor repro-ducibility, in spite of its simplicity [13]. The chemicalimmobilization technique has been found to show goodreproducibility and coverage, because the protein is cova-lently immobilized on the substrate [14]. However, toimprove the sensitivity of the immunosensor, there is amajor problem that remains to be solved. The IgG mole-cule has a Y-shape, which is consisting of two Fab frag-ments used for binding specific antigens and Fc fragmentcontaining antibody-effector functions [15]. When IgGmolecules are chemically immobilized on an unmodifiedsurface with disordered orientation, their Fab fragmentsmay be hidden, which results in the antibody–antigen bind-ing being hindered. Moreover, a random orientation ofIgG can result in loss of biological activity [15]. Thus, whenIgG is immobilized on a surface with modified by proteinA, it should be done in a manner, which results in Fab frag-ments pointing away from the surface, in order to allow foreasy antigen–antibody binding, which is a very importantissue in the development of an immunosensor.

The objective of this research is to fabricate the biotinyl-ated protein-A terminated multiplayer system consisting ofAPTS, biotin and streptavidin covalently attached onSi(1 00) surface in order to eventually achieve highly ori-ented IgG layer and to use antibody-based immunosensorswith high efficiency, which is indispensable for the produc-tion of highly sensitive immunosensor or protein chip. Inour opinion, the immobilization of IgG by binding proteinis likely to be the most successful approach for obtaining awell-ordered orientation. These molecules were chosen forthe following reasons: (1) The APTS is one of the most use-ful molecules for both the preparation of amino-terminatedoverlayers on silicon surfaces because it consists of threemethoxy groups which provide chemisorptions to sub-strate, and steric hindrance prevents attachment of biotinto all the available amino groups. (2) Biotin (vitamin H)has an extremely high binding constant (Kdiss = 10�5 M)for tetravalent streptavidin. When streptavidin is boundto the overlayer, a biotinylated protein-A or a biotinylatedantibody (anti-hCG) are readily immobilized [16]. (3) Bio-tinylated protein A is used for IgG immobilization. Biotin-ylated protein A is a cell wall component of Staphylococcus

aureus that specifically bound to the Fc portion of IgGfrom many mammals [17], while leaving the Fab regionsavailable for antigen binding. It has been successfully usedto orient the antibodies in a variety of immunoassays, such

as magnetic beads and enzyme-linked immunosorbentassay (ELISA) [18,19]. The first APTS overlayer was char-acterized by XPS, ellipsometry, AFM and water contactangle measurements. The images of the fluorescein-labeledbiotinylated protein-A immobilized on the designed surfacewere determined by fluorescence microscopy. The orientedimmobilization of IgG on the biotinylated protein A-termi-nated surface was also investigated by using ellipsometrytechnique.

2. Experiments

2.1. Substrate preparation

The substrates used in these experiments were Si(1 00)wafers (n-type, obtained from Shin-etsu, Handoutai,Japan). The substrates were cut into 5 mm · 5 mm piecesfor further modification and cleaned with a mixture ofNH3 (25% in volume), H2O2 (30% in volume), and deion-ized water in the volume ratio of 1:1:5 at 70 �C for20 min and then thoroughly rinsed in deionized water, eth-anol by supersonic wave, respectively, and finally dried in astream of nitrogen. These substrates were also exposed inUV/ozone chamber (Irvine, CA: Model 42, Jelight Com-pany Inc. USA) for 15 min prior to modification to removehydrocarbon and produce a hydrophilic surface. For thissurface, the water contact angle was 6�. The lower contactangle is consistent with the presence of more hydroxylgroups on the surface.

2.2. Preparation of multilayer systems

For the preparation of amino-terminated surfaces, thehydroxyl-terminated substrates were allowed to react with2 mM 3-aminopropyltrimetoksisilane (APTS, Aldrich,USA) solution in absolute ethanol at 50 �C for 12 h. TheAPTS-bound surfaces prepared were then rinsed with eth-anol several times and dried in a stream of nitrogen. Inmost cases, the amino-terminated substrates were immedi-ately used in the following experiments in order to avoidcontamination, since impurities from ambient air arehighly attached to amino-terminated surfaces [20].

For the preparation of the surfaces with biological activ-ities such as biotin (Sigma Co., USA) and streptavidin (Sig-ma Co., USA), the amino-terminated substrates were firstlyplaced into the biotin solution (5 mg biotin + 10 mL pH7.5 phosphate buffer + 1 mg 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDAC, Aldrich Co., USA,)), andwaited for 6 h at 4 �C. The substrates were then washedwith deionized water. After that, biotinylated substrateswere immersed into streptavidin solution (1 mg streptavi-din + 10 mL pH 7.5 phosphate buffer) at 4 �C for 12 h,and then washed with deionized water several times anddried in a stream of nitrogen.

To test of biotinylated protein A adsorption, thestreptavidin attached substrates were placed into fluores-cein-labeled biotinylated protein-A solution (1 mg fluores-

Fig. 1. Schematic representation of the designed self-assembled multilayersystem.

G. Demirel et al. / Surface Science 601 (2007) 4563–4570 4565

cein-labeled biotinylated protein-A (Sigma Co., USA) +10 mL pH 7.5 phosphate buffer)), and waited in a darkroom at 4 �C for 6 h. Surfaces were then washed withdeionized water several times and dried under a nitrogenstream in a dark room.

2.3. Characterization of APTS overlayer

Chemical composition of the surface was determined byXPS, using s Kratos ES300 spectrometer with Mg KaX-rays (1253.6 eV) under a vacuum of ca. 2 · 10�9 mbarat electron take-off angle 90� (with respect to the samplesurface), corresponding to analysis depth of about 10 nm.All binding energies were referenced to the C 1s hydrocar-bon peak at 285.0 eV. Ellipsometry measurements wereperformed in air at angles of incidence of 74�, 76� and78� with UVISEL variable angle spectroscopic phase mod-ulated ellipsometer (Jobin Yvon–Horiba) with a spectralrange from 1.5 to 4.7 eV in steps of 50 meV. The analyzerhead focuses the light beam originating from the 75 W Xe-non light source on the sample with a polarizing lens. Asthe light beam is reflected from the sample, it passesthrough a photoelastic modulator and a polarizer in themodulator head. The arms holding the analyzer and mod-ulator heads are mounted to a goniometer unit that allowssetting of angle of incidence (AOI) with steps of 0.01�. Theorientations of analyzer and modulator were set to 45 and0, respectively. In this configuration, the system measureIs = sin(2w)sin(D)and Ic = sin(2w)cos (D) where w and Dare conventional ellipsometric angles [21]. The surface mor-phology was investigated using an atomic force microscopy(AFM) (Omicron-VF STM-AFM system). All images werecollected under a vacuum of ca. 2 · 10�9 mbar using thetapping mode and a monolithic silicon tip. The drive fre-quency was 1000 ± 50 kHz, and the voltage was between0.3 and 1.0 V. Static water contact angles of the samplesurfaces were measured at 298 K in air using an automaticcontact angle goniometer combined with a flash cameraequipment (model DSA 100, Kruss, Germany) applyingthe sessile drop method. The volume of the drop usedwas always 1 lm. The contact angle is calculated by a com-puter program. All reported values are the average of atleast nine measurements taken at three different locationson each sample surface and have a maximum error of±1�. Fluorescence microscope measurements were per-formed with a reflected fluorescence system (Nikon EclipseE600) equipped with a mercury lamp.

2.4. Immobilization of IgG on biotinylated protein

A-terminated surface

The streptavidin-terminated surface was first treatedwith biotinylated protein A (Sigma Co., USA) modifica-tion by immersion in 0.1 mg/mL biotinylated protein Asolution until the saturation surface concentration of bio-tinylated protein A was obtained. The biotinylated proteinA-modified surface was then washed with deionized water

several times. Afterwards, biotinylated protein A-modifiedsurface was incubated in 1.0 mg/mL IgG solution for dif-ferent length of time. After rinsed with deionized waterand dried with nitrogen, the thickness of surface-boundIgG was determined by ellipsometry. The measurementswere carried out at an angle of incidence of 75�.

3. Results and discussion

The work we report here focuses on the preparation of anovel self-assembled multilayer system consisting of APTS,biotin, streptavidin and biotinylated protein-A moleculeson Si(1 00) and its utilization for immobilization of IgGmolecules. Our self-assembled multilayer system on theSi(10 0) surface is fundamentally distinct from those ongold and silver because on silicon the functionalizationwith APTS occurs by formation of covalent bonds fromsubstrate to three methoxysilane groups of the molecules.This difference is significant because conventional SAMson gold and silver can optimize alkyl chain packing by lat-eral diffusion, whereas the molecules covalently bonded tosilicon surface prevent any lateral movement of the mole-cules. Immobilization of a protein onto a transducer sur-face causes the binding of the specific target molecule ofinterest. In this respect, the self-assembled multilayer sys-tem prepared in this study has been composed not onlyfrom simple organic component (e.g. APTS) but also frombiological components such as biotin, streptavidin and bio-tinylated protein A (Fig. 1). In this study, we discussed the

4566 G. Demirel et al. / Surface Science 601 (2007) 4563–4570

structural and surface properties of a novel amino-termi-nated-SAM system prepared in detail. In addition, it wasalso investigated that the feasibility of thebiotinylated pro-tein A-terminated surface to immobilize IgG molecules asan alternative to direct immobilization by adsorption foran immunoassay protein chip with ellipsometry equipment.

3.1. XPS characterization

Fig. 2 depicts the XPS spectra of APTS overlayer as wellas the hydroxylated silicon surface recorded at an electrontake-off angle of 90�. The XPS spectrum of the hydroxyl-ated silicon consists of a single but broad O 1s peak at532.2 eV, an intense Si 2p peak at 99 eV and a peak witha lower intensity at 103.5 eV. The 103.5 eV peak is due topresence of silicon oxides of the SiO2 and Si–OH types.However, in the corresponding spectra of the APTS over-layer the hydrocarbon component in the C 1s and the ami-no component in the N1s regions can be recognized. The C1s region can be deconvoluted into two chemical compo-nents with binding energies at 284.9 and 286.5 eV, whichare assigned to –C–H, and –C–N groups, respectively[22,23]. In addition, the N 1s region can also be deconvo-luted into two chemical components with binding energiesat 399.6 and 400.9 eV, which are assigned to –N–H and–NH2 groups, respectively.

3.2. Ellipsometry measurements

Ellipsometry measures the change of polarization of apolarized light upon specular reflection from a surface[21]. The refractive index, absorption coefficient and thick-ness of the thin film are generally determined by fitting aparameterized model to the measured data. In the analysisof the hydroxylated surface and the APTS overlayerformed on it, a three-phase model consisting of silicon sub-strate/overlayer/air is assumed. The designed overlayersare assumed to be transparent; a generally reasonableapproximation for organic layers with C-chains [24]. Sincethe thickness and refractive index are highly correlated for

Fig. 2. Part of the XPS spectra of: (a) hyd

very thin films (<10 nm), refractive index of the overlayercan be reasonably assumed and then thickness of the over-layer can be determined [25,26]. We applied this approachin our analysis and the dielectric function of SiO2 is as-sumed to represent the dielectric function of the overlayers[27]. The values given in [28] are used for the refractive in-dex of silicon substrate. The model parameters are fitted byminimizing the following function:

v2 ¼ 1

2N �M � 1

XN

j¼1

I exps;j � Icalc

s;j

rIs;j

!2

þIexp

c;j � Icalcc;j

rIc ;j

!224

35;

where Iexps;j ,I exp

c;j and Icalcs;j , Icalc

c;j are experimental and calcu-lated quantities at energy j, respectively while N is the totalnumber of data points and M is the number of fittedparameters. rIs;j (�0.01) and rIc;j (�0.01) are experimentalerrors associated with the experimental values of Is and Ic.Effective dielectric functions obtained at three differentAOIs are given in Fig. 3. The agreement between the exper-imental and fitting results is quite good (v < 1). The effec-tive dielectric function (hei = he1i�ihe2i) can be envisagedas an average dielectric response of the silicon substrateand the overlayer. Its difference from the actual e dependson how two-phase model represents the sample [21]. Thehe2i peak values at energies of about 3.4 eV (E1) and4.3 eV (E2) are attributed to interband transitions of siliconand they are sensitive to surface properties. Althoughhe2ivalues at E1 for each designed surface do not much differ,the he2i values at E2 decreases for all overlayers. Thisbehavior is very similar to the behavior of silicon coveredwith SiO2 thin film [21]. The W and D values as a functionof AOI for APTS overlayer are given in Fig. 4a. Thesubstantial and sharp change in D occurs at AOIs of about70–80� whereas W goes through a minimum at this angleinterval. Since most information from experimental datais obtained at this range of AOI, measurements arepreferred to be performed in this range of AOI. Thethicknesses of the overlayers are given in Fig. 4b. The aver-age thickness of APTS layer is determined as 6.8 nm. In

roxylated silicon and (b) APTS–SAMs.

Fig. 3. Experimental and calculated (solid lines) effective dielectricfunctions of designed surfaces on silicon substrates at angles of incidenceof: (a) 74�, (b) 76� and (c) 78�.

Fig. 4. (a) Variation of W and D as a function of angle of incidence fordesigned APTS overlayer. (b) Variation of thicknesses for: (i) Si–OH and(ii) APTS overlayers obtained at angle of incidences of 74�, 76� and 78�.

G. Demirel et al. / Surface Science 601 (2007) 4563–4570 4567

addition, the hydroxylated surface layer on silicon sub-strate is similarly modeled and its thickness is found as3.3 nm. This interfacial layer is probably a mixture ofSiO2 and Si–OH which is also evident in XPS results. Itshould be noted that surface roughness was not includedin the model; therefore these thicknesses need to be consid-ered as an effective thickness, not absolute thickness. If it isreminded that APTS overlayers are grown on hydroxylatedsilicon surfaces as they are immersed corresponding solu-tions as quick as possible, their surface oxidation can be ex-pected to be less than bare hydroxylated surface. Thethickness of the APTS overlayer of 6.8 nm is higher than

its theoretical molecular length of 1.2 nm obtained byab initio calculations [29]. Since APTS thickness dependson the solution concentration and dipping time [30], andconsequently, APTS may be formed on hydroxylated sur-face as a multilayer; the high value of APTS thickness ob-tained in this work suggests multilayer formation.

3.3. Surface topography by AFM

Figs. 5a and 6a exhibit the AFM images of the surfacetopographies of the hydroxylated Si(1 00), and APTS over-layers, respectively. The hydroxylated Si(1 00) surface ap-pears small pits with homogeneous distribution becauseof forming by the wet chemical etching. However, the sur-face topographies of the APTS overlayers are considerablydifferent from the hydroxylated silicon surface and theycontain some aggregates or clusters which lower the surfacehomogeneity due to the presence of covalently boundAPTS molecules at the surface. The covalent binding ofAPTS molecules to the hydroxylated silicon surface andshort-range van der Waals forces between the molecules

Fig. 5. AFM images of hydroxylated silicon: (a) height, (b) phase and (c)cross section.

Fig. 6. AFM images of APTS-SAM: (a) height, (b) phase and (c) crosssection.

4568 G. Demirel et al. / Surface Science 601 (2007) 4563–4570

lead to the formation of some ordered clusters with ran-dom distributions. All similar observations were confirmedby phase images.

The nano-scale measurements of the hydroxylatedSi(1 00) and APTS overlayers are shown in Figs. 5b and6b. The morphology of APTS overlayers in the picturesis completely different from the hydroxylated Si(1 00) sub-strate and indicates nonuniform coverage to the hydoxy-lated silicon surface. The APTS overlayers showed well-defined light domains surrounded by shallowly etched darkregions. The light regions are most likely comprised ofAPTS-rich phases, while darker regions can be attributedto the uncovered surface containing larger amount of –Si–OH groups. These images strongly suggest that theshort-range van der Waals interactions between the APTSmolecules allowed for the formation of domains with ran-dom distributions on the surface. These differences are fur-ther highlighted in Fig. 6b. Heterogeneous mechanicalproperties were also found for the phase imagines and weconcluded that traces of domain structures, usually ob-

served for local multilayer structures which form annonuniform distributions of covalently bound APTS mole-cules on the Si(1 00) surface. On the other hand, these non-uniform characters may become more significant becauseof providing an evidence for covalently bound APTS mol-ecules on the Si(1 00) surface.

Furthermore, cross-sections (Figs. 5c and 6c) of thehydroxylated Si(100), and APTS overlayers clearly indi-cated the root-mean-square roughness (RMS) variations(their RMS values are 0.323 and 2.672 nm, respectively)these are consistent with a thin covalently bound APTSmultilayer formation. In addition, as mentioned before,variation in the ellipsometric thickness over a 1 lm · 1 lmsurface area for the APTS overlayers is 3.5 nm, indicatingthat is microscopically roughness due to the presence ofAPTS multilayers on the silicon surfaces.

3.4. Water contact angle measurements

The contact angle of water is sensitive to the polarity ofthe surface and it indicates the surface hydrophilicity.

Fig. 7. Florescence images (·100) of: (A) hydroxylated silicon and (B) APTS-Biotin-streptavidin multilayer systems. These systems were immersed influorescein-labeled protein-A solution and waited in a dark room for 6 h at 4 �C.

G. Demirel et al. / Surface Science 601 (2007) 4563–4570 4569

While the water contact angles of APTS attached surface is41� that of the hydroxylated silicon is 6� which reveals thedifferences in their surface hydrophilicity characteristics.These data are in good agreement with the contact anglesmeasured for water on amino-terminated layers reportedin the literature, which were in the range of 23–68� [31–33].

Fig. 8. Kinetics of the coupling of IgG to the protein A-modified surface.

3.5. Fluorescein-labeled protein A immobilization

As mentioned before, the APTS-Biotin-streptavidinmultilayer system was firstly designed and then exposedto the fluorescein- labeled biotinylated protein A. Fig. 7shows the fluorescence images of the hydroxylated siliconsurface (Fig. 7A) and the fluorescein-labeled biotinylatedprotein A molecules covalently immobilized on the de-signed multilayer system (Fig. 7B). As expected, while noany fluorescence signals were detected (molecules arenon-covalently adsorbed, probably remained on the sur-face even after several washing steps) on the hydroxylatedsilicon surface, the fluorescence signals with random distri-butions were observed on the designed surface. As a result,this fluorescence microscope image of the streptavidin ter-minated surface confirmed the benefits of streptavidin,which were used to capture biotinylated protein A.

3.6. Oriented immobilization of IgG

The protein-A terminated surfaces are commonly usedfor the oriented immobilization of the IgG molecules,which is a very important issue in the development of animmunosensor. It is well known that the protein-A mole-cules are interacted with the Fc fragment of the IgG mole-cules, leaving free Fab fragments which will be availablefor further interaction of these correctly oriented IgG mol-ecules with their complementary molecules [34].

The time dependence of the thickness of the IgG layer isgiven in Fig. 8. Reported thicknesses are mean values ofthree measurements and given with their standard devia-tions. The thickness of the IgG rapidly increased at thebeginning and then exhibited a slow variation with time,presumably in association with the formation of a multi-layer or the occurrence of structural rearrangement of the

immobilized IgG. IgG has a molecular weight of156 kDa, and is a Y-shaped molecule with a height ofapproximately 12 nm, and a thickness of 4 nm [35,36].Thus, when IgG is immobilized with the larger cross-sec-tion of IgG parallel to the surface, the thickness of a mono-layer of IgG is 4 nm, which is possible minimal thickness ofa monomolecular layer formed by IgG. Therefore, our elli-psometric analysis indicates that the IgG layer on the pro-tein-A terminated surface is formed almost as amonomolecular layer in approximately 80 min (Fig. 8).After 80 min, the constancy of the thickness of the IgGlayer shows that IgG is immobilized with the constant-ori-ented configuration, in which the Fc domain of IgG isbound to protein A, and the Fab domain faces away fromthe substrate as presented in Fig. 1. As a result, Fig. 1 isalso confirmed by the fact that the thickness of the IgGlayer remained constant.

4. Conclusion

In this study, a novel multilayer system consisting ofAPTS, biotin, streptavidin and biotinylated protein Awas successfully prepared on the Si(1 00) surface. The firstAPTS layer covalently attached on the silicon surface was

4570 G. Demirel et al. / Surface Science 601 (2007) 4563–4570

characterized by XPS, spectroscopic ellipsometry, AFMand contact angle measurements. All the experimental re-sults confirmed the presence of the first APTS layer thatis covalently attached on the silicon surface. In addition,the fluorescence images indicated the formation of the mul-tilayer system on the Si(100) surface. The results in thisstudy also show that a multilayer system with protein Ahas the potential to be applied in a sensitive immunoassayprotein chip.

Acknowledgements

The authors thank Professor S�efik Suzer for the X-rayphotoelectron spectroscopy measurements and helpfulcomments. The authors also acknowledge the financialsupport of DPT (Turkish State Planning Association), Pro-ject Nos: 2003K 120470-31 and 2001 K120 590.

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