Stepwise functionalization of SiNx surfaces for covalent immobilization of antibodies

Stéphanie Dauphas a, Soraya Ababou-Girard b, Aurélie Girard c, France Le Bihan c,Tayeb Mohammed-Brahim c, Véronique Vié d, Anne Corlu e, Christiane Guguen-Guillouzo e,Olivier Lavastre a, Florence Geneste a,⁎a Université de Rennes 1, UMR-CNRS 6226, Laboratoire des Sciences Chimiques de Rennes, Equipe catalyse et organométalliques, Campus de Beaulieu, 35042 Rennes cedex, Franceb Université de Rennes 1, UMR-CNRS 6251, Institut de Physique de Rennes, Equipe Physique des surfaces et interfaces, Campus de Beaulieu, 35042 Rennes cedex, Francec Université de Rennes 1, UMR-CNRS 6164, Institut d'Electronique et Télécommunications de Rennes, Groupe de Microélectronique, Campus de Beaulieu, 35042 Rennes cedex, Franced Université de Rennes 1, UMR-CNRS 6251, Institut de Physique de Rennes, Equipe Biophysique, Campus Beaulieu, 35042 Rennes, Francee Université de Rennes 1, INSERM U522, IFR 140, Campus de Villejean, 35042 Rennes cedex, France

a b s t r a c ta r t i c l e i n f o

Article history:Received 19 November 2008Received in revised form 25 April 2009Accepted 4 May 2009Available online 18 May 2009

Keywords:SilaneSurface functionalizationSilicon nitrideTransferrinAtomic force microscopyX-ray photoelectron spectroscopy

A stepwise functionalization of silicon nitride surfaces is followed by X-ray photoelectron spectroscopy(XPS). The first step involves a silanization reaction leading to the formation of a silane filmwith a thicknessestimated by XPS of one or two molecular layers. A monoprotected homobifunctionalized linker is thenused to avoid the formation of bridge structures on the surface. The linker reacts quantitatively with theamino groups of the surface as outlined by the absence of residual unreacted CNH2/CNH3

+ groups in XPSanalyses. Deprotection of the ester groups of the immobilized linker and subsequent reaction with N-hydroxysuccinimid lead to N-hydroxysuccinimid activated surfaces able to react with biological species.These surfaces were then incubated with anti-transferrin antibodies. As seen by XPS and atomic forcemicroscopy analyses, the concentration and incubation conditions of antibodies are important to obtain acompact layer of antibodies on the surface. All chemical steps of the procedure are compatible withmicroelectronic process on silicon. Moreover, antibodies introduced under native conditions atphysiological pH, in the last step of the immobilization process, recognized specifically antigens, as shownby fluorescence competitive assay.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Analysis of protein markers is essential to establish accuratediagnosis for pathologies, especially in cardiovascular diseases andcancers. However, the high cost of these analyses makes difficult asystematic approach which would provide meaningful information toclinicians to help reaching accurate diagnosis. Biosensors based onmicroelectronic devices such as field effect transistors [1–7], giantmagnetoresistive sensors [8–10] or light-addressable potentiometricsensors [11–13] have received increased interest in the last 30 yearsfor protein detection as time-saving technology characterized by a lowcost due to miniaturization and mass production.

Silicon nitride films are key materials employed in the electronicindustry as etch stop and protecting layer from degradation (ionicspecies, moisture, oxygen). Their inert nature and their biocompat-ibility make them very useful supports for biosensing applications.The functionalization of silicon nitride is usually performed by an

oxidation step to form a SiOy film, followed by silanization with 3-aminopropyl triethoxysilane (APTS) [14], leading to the formation ofamino groups on the surface. Different methods for the subsequentimmobilization of biomolecules on these modified surfaces have beendeveloped, depending on the biological species to anchor [4,6–8,11,15–20]. One of the most frequently used is the homobifunctionalcross-linking with glutaraldehyde [8,11,15–18]. It allows the attach-ment of proteins by the abundant amino groups present in lysine toamine functionalized surfaces. However, this linker presents severaldisadvantages. It tends to polymerise at alkaline pH (above 7.5) andunder heating [21], and it leads to the formation of readilyhydrolysable imine bonds by reaction with amino groups [22].Moreover, as all homobifunctional linkers with long alkyl chains,glutaraldehyde can also form bridged structures with both ends fixedto the surface [14,23]. All these phenomenon can contribute to thenon-reproducibility and poor stability of bioelectronic sensors.

We have recently reported a method to covalently immobilizesensitive biomolecules as antibodies to carbon surfaces [14]. Thismethod involves the use of a monoprotected homobifunctional linker,avoiding the formation of bridge structures on the surface and leadingto a more compact layer of anchored antibodies than with traditionalhomobifunctional linker. This method is applied here to silicon nitride

Thin Solid Films 517 (2009) 6016–6022

⁎ Corresponding author. Tel.: +33 2 23 23 59 65; fax: +33 2 23 23 59 67.E-mail address: Florence.Geneste@univ-rennes1.fr (F. Geneste).

0040-6090/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.tsf.2009.05.014

Contents lists available at ScienceDirect

Thin Solid Films

j ourna l homepage: www.e lsev ie r.com/ locate / ts f

surfaces, leading to a robust and reproducible anchoring of antibodies.All chemical steps employed in themodification process are compatiblewith materials involved in the fabrication of microelectronic devices.

X-ray photoelectron spectroscopy (XPS) was used to monitor thestep of the modification process to obtain the maximum surfacecoverage. The stability and the compactness of the covalentimmobilization of antibodies were also checked by atomic forcemicroscopy (AFM). Finally, the specific recognition of antibodiesimmobilized according to our procedure toward antigens was clearlydemonstrated by fluorescence microscopy experiments. In this study,transferrin was chosen as antigen model because its level in blood istested to diagnostic several diseases such as hemochromatosis.

2. Experimental

Triethylamine, 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimidehydrochloride (EDC), 2,6-lutidine, anhydrous lithium iodide werepurchased from Acros and methyl adipoyl chloride, N-hydroxysuccini-mid (NHS), 3-aminopropyl-triethoxysilane (APTS) from Aldrich.

Goat anti-human transferrin antibodies, human holo-transferrin,human serum albumin (HSA) were purchased from Sigma anddelipidated fraction V bovine serum albumin (BSA) from Roche. TexasRed human apo-transferrin was fromMolecular Probes (Eugene, OR).

Phosphate buffered saline (PBS) was prepared at pH 7.4 from1.5 mM potassium phosphate monobasic, 8.6 mM sodium phosphatedibasic dodecahydrate, 137 mM sodium chloride, and 2.7 mMpotassium chloride in deionized water.

2.1. Preparation of silicon nitride surfaces

The silicon nitride films were deposited on n-type IC-grade siliconsubstrates, (100)-orientated. Silicon nitride films were deposited in a

LPCVD (Low Pressure Chemical Vapor deposition) furnace tube at600 °C and a pressure of 70 Pa, using gas flows of 50 sccm silane (SiH4)and 50 sccm ammonia (NH3). The film thickness was about 70 nm.The silicon nitride samples were cleaned in ultra pure grade acetoneand isopropyl alcohol.

Then, the films were oxidized in an oxygen plasma using anincident rf power of 30 W and a negative bias voltage, for 2 min, atroom temperature, under a pressure of 3000 Pa.

2.2. Functionalization of silicon nitride surfaces

Freshly oxidized silicon nitride surfaces were first heated at 100 °Cfor 2 h under vacuum (10 Pa) and exposed to the APTS atmospherecreated by 1 ml of APTS in the bottom of a 0.5-liter glass reactor underpressure (10 Pa) at room temperature. Some samples have been left ina degassed solution of APTS (55 mmol L−1) in toluene for 20 min. Allsamples were cured in an oven at 100 °C for 1 h. After heating, thesesurfaces were immersed in a degassed solution of toluene supple-mented with methyl adipoyl chloride (31 mmol L−1) and triethyla-mine (0.1 mol L−1) for 3 h at 40 °C. The surfaces were ultrasonicated 3times for 15 min. in toluene and dichloromethane before beingimmersed in 20 ml of 2,6-lutidine containing anhydrous lithiumiodide (240 mg, 1.8 mmol). The samples were heated at reflux for 16 hin the dark and ultrasonicated 3 times for 15 min. in distilled waterand acetone. Subsequent reaction with EDC 0.4 M and NHS 0.1 M inwater for 1 h at room temperature, followed by ultrasonication 3 timesfor 15 min in distilled water lead to the N-hydroxysuccinimidactivated surfaces.

These surfaces were allowed to react with anti-transferrin anti-body following two different conditions. For the procedure 1: 100 μgmL−1 in pH 7.4 PBS at 4 °C overnight, and for the procedure 2: 2 g L−1

in pH 7.4 PBS at room temperature for 3 h. The modified surfaces werewashed either 6 times for 15 min in pH 7.4 PBS before use or indeionized water for XPS analyses.

2.3. Surface analyses

XPS analyses were performed under a base pressure of 7×10−8 Pausing a VSW Scientific Instruments HA100 spectrometer with amultichannel detection. The analyser was operatedwith constant passenergy of 22 eV. X-ray source used Mg Kα excitation radiation at1283.6 eV. The total energy resolution was about 1.2 eV. Due to theinsulating character of silicon nitride which shifts the XP spectra tohigher binding energies, all surfaces were referenced to the C1s maincomponent set at 285.0 eV. Simulation of the experimental peaks wascarried out using a Gaussian–Lorenzian product function after Shirleybackground removal. The sampling depth is 3λ, where λ is theelectrons mean-free-path [24]. It is 4 nm and 2 nm in an organic layerand in silicon, respectively.

Topographic images were acquired with an AFM Pico-plus(Molecular Imaging, Phoenix, US) in constant force mode usingsilicon nitride tips with an estimated radius of 10 nm on integral

Scheme 1. Schematic representation of the process of covalently immobilizingantibodies on the silicon nitride surface.

Table 1XPS elemental surface analysis of silicon nitride after the sequential modification steps.

Steps/XPS (at.%±0.8%) C1s O1s N1s Si2p

Oxidation 7 50 6 37APTS 15 45 7 33APTS/linker 16 46 6 32APTS/linker/antibody procedure 1a 58 20 12 10APTS/linker/antibody procedure 2b 65 18 10 7

a 100 mg L−1, 0 °C, 1 night.b 2 g L−1, r.t., 3 h.

6017S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022

cantilevers. The spring constant was 0.06 N m−1. The pixel size was512×512. The surfaces were imaged in PBS with a scan rate of 1 Hz.Reproducibility is based on 3 different samples and at least 3 places onthe sample have been observed.

Preparation of samples for fluorescent analyses were done asfollowed. Silicon nitride surfaces modified with anti-transferrinantibodies (see functionalization of silicon nitride surfaces) weresaturated by a solution of BSA (10 g L−1) in PBS for 1 h at roomtemperature to avoid non-specific binding of antigens. Then, theywere placed in a BSA solution containing 5 μg mL−1 of Texas Red apo-transferrin and supplemented or not with HSA (500 μg mL−1) orunlabeled human apo-transferrin (500 μg mL−1). After three washesin PBS, fluorescence intensity was detected using an Olympus BXmicroscope. The wavelengths were set at 560±20 nm (excitation)and 630±37.5 nm (emission). A 20×0.7 objective and acquisitiontimes of 8 s were used. Quantification of fluorescence intensity wasperformed from at least 4 fields of each sample by imaging analysisusing SimplePCI software (Compix Inc).

3. Results and discussion

3.1. Surface functionalization

Plasma anodization [25] of silicon nitride leads to the formation of aSiOy film over the SiNx surface. This highly oxygenated silicon oxynitride

layer was chemically reacted with APTS in vapour phase, followed byacylation reaction with methyl adipoyl chloride (Scheme 1).

The elemental surface composition deduced from XPS measure-ments after the various steps of modification is given in Table 1.

A C1s peak was observed in the spectra of oxidized silicon nitridesurfaces. It is probably due to pollution by oil vapor of the vacuumsystem because of the quite high base pressure used during the siliconnitride deposition by LPCVD. The efficiency of the plasma anodizationis clearly outlined by the O1s content (49.7%) and the immobilizationprocess clearly results in the decrease of the Si2p content and theincrease of the C1s content. However, quantitative comparison of eachstep of the modification process is difficult here, since the carbon andnitrogen contents can change from a silicon nitride sample to another.We obtained more information from the XPS deconvolution analysesof the modified surfaces after the silanization step and the introduc-tion of the linker (Fig. 1, Table 2).

Following the reaction with APTS, the XPS spectrum for the N1scurve shows the main component at 397.5 eV attributed to nitrogen inSiNx film, and two peaks for the N1s curve at binding energies of 399.7and 401.8 eV, previously assigned to the free and protonated aminesrespectively [26,27]. Since precautions were taken to avoid reactionwith CO2 and H2O present in the air, the peak corresponding to theformation of ammonium bicarbonate was not observed. In the samemanner, C1s spectrum was resolved into two components at 285 and286.6 eV. The main peak at 285 eV is assigned to CH2 groups of APTS(Fig. 1A) and to adventitious carbon already present on silicon nitride

Fig. 1. C1s (top) and N1s (bottom) photoemission spectra of oxidized (A), APTS (B) and APTS/linker (C) silicon nitride surfaces.

6018 S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022

surfaces, whereas the peak at 286.6 eV corresponds to the carbonlinked to free and protonated amino groups. We notice that the peakcorresponding to the bicarbonate group previously observed at288.8 eV [26] is missing from these spectra since the surface exposureto air contamination has been minimized. The XPS C1s spectra ofsilicon nitride after addition of the linker clearly showed emergence ofthe (CfO)O, (CfO)N and OCH3 peaks at 291.1, 288.6 and 287.1 eVrespectively. As expected, the content of each peak is about the same(6±1% of total carbon). The N1s spectrum exhibited a peak at400.3 eV corresponding to the CON groups [28]. The disappearance ofthe peak at 401.6 eV previously attributed to NH3

+ confirmed that theamino groups of APTS reacted with the linker. When the reactionwiththe linker was not complete, an increase of the peak at 400 eVattributed to NH2 was observed with the apparition of a peak at402.5 eV (NH3

+). In the C1s spectra, the peak at 287.1 eV (CNH2 andCNH3

+) increased, compared to the peaks at 291.1 and 288.6 eV. Allthese XPS data are in agreement with a total reaction between theamino groups of APTS and the COCl groups of the linker. This result isinteresting because it proves that in our case the amino groups areaccessible, whereas it has been reported that, on SiO2 surfaces, someamino groups can form hydrogen bonds with the OH groups of thesurface during the APTS deposition, making them inaccessible to anyadditional surface chemistry [27].

3.2. Layers thickness measurement

In order to estimate the thickness of the different layers grafted onthe silicon nitride modified surface, we consider the attenuatedintensity of silicon in SiNx according to the relation

I = I0exp −d = λ cos θð Þ

where I0 is the measured signal for SiNx electrons issued from theuncovered surface, I the signal measured of these electrons fromsilicon nitride covered by an organic layer of thickness d, and λ is theattenuation length of SiNx electrons in the organic layer [24]. For

electrons escaping through organic layers the value of λ falls withinthe range of 20–30 Å [29]. We estimated a layer thickness rangingfrom 8 to 12 Å for APTS and 15 to 22 Å for APTS/linker, from the signalattenuation of the SiNx component of Si2p. The obtained value wasconsistent with 1 or 2 layers of APTS since the thickness of an APTSmonolayer has been reported to be 7 Å [27]. Since chain length of thelinker was estimated to be around 10 Å (energy-minimizedMM2 levelby Chem 3D Ultra), the APTS/linker surface coverage corresponds to 1or 2 layers of APTS and 1 layer of linker. It has been previously reportedthat gas phase silanization of silicon surfaces at elevated temperatureleads to smooth, stable and reproducible silane films of monolayercharacter [30–32]. In our study, APTS deposition was performed atroom temperature for 5 min. Higher temperatures or deposition timesled to thicker APTS films. Moreover, experiments performed withAPTS in anhydrous toluene for 20 min gave a less homogeneous layerof APTS/linker with an average thickness of about 100 Å. These resultsare consistent with literature since it has been reported that non-uniform layer with thickness of 10 nm is obtained with incubationtime of 20 min in APTS solution [33].

We estimated a surface concentration of the APTS chain of3.7×1014 cm−2 from the nitrogen signal at 399.5 and 401.6 eV specificto the APTS chain (the N1s XPS intensity relevant to APTS wasnormalized to the C1s intensity, corresponding to a graphene sheet[34]). This surface covering corresponds to about one chain of APTS fortwo OH groups present on the surface. This estimation seems to be inagreement with a monolayer of APTS.

3.3. Anchoring antibodies

The deprotection of the ester group has been previously done bysaponification with KOH [14]. However, hydroxide anions are notcompatible with materials usually used in microelectronics, such asaluminium and silicon. For this reason, the ester group wasdeprotected by LiI [35]. For SN2 dealkylation of methyl ester, LiI hasbeen proven to be very efficient (yields N90%) [36,37]. Subsequentreaction with EDC/NHS (1-(3-dimethyl-aminopropyl)-3-ethylcarbo-diimide hydrochloride/N-hydroxysuccinimid) led to N-hydroxysucci-nimid groups on the surface (Scheme 1). Anti-human transferrinantibodies were then covalently immobilized by reaction with the N-hydroxysuccinimid-activated surfaces under native conditions atphysiological pH to minimize their denaturation. All the steps of thegrafting procedure did not cause damage on aluminium, silicon,silicon nitride and silicon oxide surfaces.

XPS analysis of surfaces with immobilized antibodies according toprocedures 1 (100mg L−1 antibody concentration, O °C,1 night) and 2(2 g L−1 antibody concentration, r.t., 3 h) is given in Fig. 2.

After the addition of antibodies, the carbon amount increased,whereas the silicon amount decreased for procedures 1 and 2(Table 1). A significant difference was however observed betweenprocedures 1 and 2. After deconvolution, the C1s spectra exhibitedthree peaks at 285, 286.2 and 288.3 eV for both procedures. The twolast peaks are mainly due to C–N and (CfO)N groups highly present inthe protein. Two peaks were observed for the N1s curve at bindingenergies of 397.9 (397.3 eV for procedure 2) and 400.1 eV. Comparedto the APTS/linker surfaces, the peak at higher binding energyincreased markedly because of the peptide backbone of the antibody.However, the attenuation of the signal corresponding to the siliconnitride surface (peak at 400.1 eV) was not the same for bothprocedures: the peak nearly disappeared in procedure 2 whereas itis still present in procedure 1. This result shows that the surfacecovering is better for procedure 2 than for procedure 1, underlying theimportance of concentration and incubation conditions of antibodies.

These results were corroborated by atomic force microscopy(AFM). We first examined NHS-modified silicon nitride surfaces.Contact mode AFM image obtained in phosphate buffered saline (PBS)

Table 2Parameters obtained from the XPS measurements of the functionalized surfaces.

Nature of the felt Binding energy/eV FWHM/eVa

% Peak arearatiob

Plasma C1s 285.0} oil vapor 1.7 91287.3 1.8 9

N1s 397.9 SiNx 1.6 100APTS C1s 285.0 CH2+oil vapor 1.8 83

286.6 CH2NH2, CH2NH3+ 1.8 17

N1s 397.5 SiNx 1.6 72399.7 NH2 1.8 15401.8 NH3

+ 1.9 13APTS/linker C1s 285.0 CH2+oil vapor 2.2 81

287.1 OCH3 1.9 8288.6 (CfO)N 1.8 6291.1 (CfO)O 1.7 5

N1s 397.2 SiNx 1.5 87398.7 non-attributed 1.8 8400.3 CON 1.5 5

APTS/linker/antibodyprocedure 1a

C1s 285.0 1.7 60286.4} peptide

backbone1.7 26

288.2 1.6 14N1s 397.3 SiNx 1.6 18

400.1 mostly CON 1.8 82APTS/linker/antibodyprocedure 2b

C1s 285.0 1.7 64286.2} peptide

backbone1.7 21

288.3 1.8 15N1s 397.9 SiNx 1.8 3

400.1 mostly CON 1.8 97

a Full-Width Half-Maximum of peaks.b % C1s/C1s total or N1s/N1s total.

6019S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022

(10 μm×10 μm) in a top view representation and accompanying lineprofiles is given in Fig. 3A.

The surface is flat and homogeneous showing topographicalvariations of 10–20 nm. Disperse nodules of approximately 200 nmin diameter were observed. Fig. 3B and C (10 μm×10 μm) shows asimilar set of data, image and profile, after immobilization ofantibodies according to procedure 1 and procedure 2, respectively.The roughness changes appreciably giving rise to topographicalvariations of 100 to 150 nm. The appearance of the surface issignificantly different with the presence of globular structures ofaround 100 nm in diameter scattered on the surface. These structureswere not attributed to aggregated or crystallized salts of PBS bufferbecause such salts exhibit on XPS measurements a peak at 263 eV dueto a Na KLL Auger transition. This peak was never observed in the XPSspectra of the surfaces modified according to our procedure (Fig. 2).We attributed the presence of nodules in the AFM images to area ofhigher concentration of antibodies, as observed by fluorescencemicroscopy using labeled-antibodies. The heterogeneous repartitionof antibodies on the surface is probably due to proteins adsorbed oraggregated on the uniform film formed by the covalently immobilizedantibodies. However, the difference of morphologies is clearlyobserved after immobilization of antibodies, compared to the NHS-modified silicon nitride surfaces, and confirms the success of thegrafting procedure. Compared to Fig. 3B, Fig. 3C exhibited a morecompact structure with less black area and a denser repartition of thewhite spots corresponding to aggregated proteins. This result is inaccordance with XPS analyses, showing that the surface coating isbetter with procedure 2 than with procedure 1. Moreover, samplesultrasonicated in PBS to desorb non-covalently anchored proteins [14]exhibited a morphology with globular structures similar to theprevious one (Fig. 3D). The more heterogeneous distribution on thez-axis is probably due to a partial degradation of the 3D-structure ofthe antibodies during ultrasonication. Thus, imaging of the modified

surface by AFM shows that the grafting procedure gives rise to ahomogeneous and stable layer of antibodies on the silicon nitridesurface.

3.4. Antigens recognition

To complete the validation of the grafting procedure, we checkedthat antibodies immobilized according to the present processspecifically recognized antigens. For this purpose, we used afluorescence microscopy approach. First, the non-specific bindingsites were saturated by a solution of delipidated fraction V bovineserum albumin (BSA). Then, a positive reference sample (A) wasprepared by incubation of apo-transferrin labeled with Texas Red asfluorescent dye in BSA solution with the coated surface. The obtainedfluorescent images were quantitated as mean grey per pixels (Fig. 4).

To assess the specificity of the antibody–antigen reaction, weperformed antigen competition experiments using unlabeled apo-transferrin and human serum albumin (Scheme 2).

Antibodies were combined at a concentration ratio of 1:100 ofTexas Red labeled apo-transferrin. As expected, the fluorescentintensity did not decrease when the competition experiments wereperformed with human serum albumin (B), whereas it decreasedsignificantly with unlabeled apo-transferrin (C). These results clearlyevidence that the antigen–antibody specific recognition takes placewhen antibodies are covalently anchored to silicon nitride surfacesaccording to our immobilization procedure.

4. Conclusion

SiNx surfaces were functionalized by a stepwise process allowingthe covalent immobilization of antibodies. The first step involved asilanization process leading to the formation of a silane filmcorresponding to 1 or 2 layers of APTS. Then, a monoprotected

Fig. 2. C1s (top) and N1s (bottom) photoemission spectra of silicon nitride surfaces with anchored anti-transferrin antibodies according to procedures 1 and 2.

Fig. 3. Average line profiles and AFM images in PBS of NHS-modified silicon nitride surfaces (A), antibodies modified surfaces according to procedure1 (B) and procedure 2 (C), andafter ultrasonication in PBS for procedure 1 (D). The white lines on AFM images indicate where the line profiles were taken from.

6020 S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022

6021S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022

homobifunctional linker was added to assure a good control of thesurface modification, leading to a stable and compact layer ofbiomolecules, as seen in AFM. XPS analyses also showed that thechemical introduction of the linker was quantitative because all aminogroups of the APTS-modified surface reacted with the linker, and thathigher concentration of antibodies lead to a better surface covering.Antibodies introduced in the last step of the grafting procedure, undernative conditions at physiological pH, specifically recognized antigens,as shown by fluorescentmicroscopy experiments. We only studied therandom fixation of antibodies (IgG) directed against transferrin on thesurface, even a specific fixation by the Fc fragment of the antibodycould be obtained by anchoring protein A or G on the functionalized

surface before the addition of antibodies. Moreover, the compatibilityof each step of the modification procedure with materials usuallyemployed in microelectronics allows a direct application of themethod in electronic biosensors. The low thickness of the organic film,well-adapted to miniaturized systems and the compact layer ofimmobilized antibodies for enhancing the sensor response should alsomake easier the application of this well-controlled immobilizationprocess to electronic devices.

Acknowledgments

This work was supported by the Agence Nationale de la Recherche(Project DEPIST), the Institut National de la Santé et de la RechercheMédicale and the Centre National de la Recherche Scientifique. Wethank Dr. Remy Le Guevel from the Cell biochips platform, OuestGenopole.

References

[1] T. Mohammed-Brahim, O. Bonnaud, C. Simon, N. Coulon, A. Sabouni, K. Kandoussi,PCT Int. Appl. WO 2006114535, 2006.

[2] H. Mahfoz-Kotb, A.C. Salauen, F. Bendriaa, F. Le Bihan, T. Mohammed-Brahim, J.R.Morante, Sens. Actuators, B 118 (2006) 243.

[3] F. Bendriaa, F. Le Bihan, A.C. Salauen, T. Mohammed-Brahim, O. Bonnaud, J. Non-Cryst. Solids 352 (2006) 1246.

[4] M. Thust, M.J. Schoning, P. Schroth, U. Malkoc, C.I. Dicker, A. Steffen, P. Kordos, H.Luth, J. Mol. Catal. B: Enzym. 7 (1999) 77.

[5] S. Caras, J. Janata, Anal. Chem. 52 (1980) 1935.[6] L. Bandiera, G. Cellere, S. Cagnin, A. De Toni, E. Zanoni, G. Lanfranchi, L. Lorenzelli,

Biosens. Bioelectron. 22 (2007) 2108.[7] T. Uno, H. Tabata, T. Kawai, Anal. Chem. 79 (2007) 52.[8] J.P. Diao, D.C. Ren, J.R. Engstrom, K.H. Lee, Anal. Biochem. 343 (2005) 322.[9] D.L. Graham, H.A. Ferreira, P.P. Freitas, Trends Biotechnol. 22 (2004) 455.[10] J.C. Rife, M.M. Miller, P.E. Sheehan, C.R. Tamanaha, M. Tondra, L.J. Whitman, Sens.

Actuators, A 107 (2003) 209.[11] I.G. Mourzina, T. Yoshinobu, Y.E. Ermolenko, Y.G. Vlasov, M.J. Schoning, H. Iwasaki,

Microchim. Acta 144 (2004) 41.[12] G.X. Xu, X.S. Ye, L.F. Qin, Y. Xu, Y. Li, R. Li, P. Wang, Biosens. Bioelectron. 20 (2005)

1757.[13] A. Seki, S. Ikeda, I. Kubo, I. Karube, Anal. Chim. Acta 373 (1998) 9.[14] S. Dauphas, A. Corlu, C. Guguen-Guillouzo, S. Ababou-Girard, O. Lavastre, F.

Geneste, New J. Chem. 32 (2008) 1228.[15] T. Sakata, M. Kamahori, Y. Miyahara, Mater. Sci. Eng., C C24 (2004) 827.[16] S.H. Lee, C.S. Lee, D.S. Shin, B.G. Kim, Y.S. Lee, Y.K. Kim, Sens. Actuators, B B99

(2004) 623.[17] R. Stine, C.L. Cole, K.M. Ainslie, S.P. Mulvaney, L.J. Whitman, Langmuir 23 (2007)

4400.[18] A. Tlili, M.A. Jarboui, A. Abdelghani, D.M. Fathallah, M.A. Maaref, Mater. Sci. Eng., C

C25 (2005) 490.[19] P. Wu, P. Hogrebe, D.W. Grainger, Biosens. Bioelectron. 21 (2006) 1252.[20] M. Manning, G. Redmond, Langmuir 21 (2005) 395.[21] J. Blass, C. Verriest, A. Leau, J. Chromatogr. 107 (1975) 383.[22] J. Donoso, F. Munoz, A. Garcia Del Vado, G. Echevarria, F. Garcia Blanco, Biochem. J.

238 (1986) 137.[23] B. Xia, S.J. Xiao, D.J. Guo, J. Wang, J. Chao, H.B. Liu, J. Pei, Y.Q. Chen, Y.C. Tang, J.N. Liu,

J. Mater. Chem. 16 (2006) 570.[24] D. Briggs, M.P. Seah, Practical Surface Analysis (Pt. 1) (1990).[25] G.P. Kennedy, O. Buiu, S. Taylor, J. Appl. Phys. 85 (1999) 3319.[26] I. George, P. Viel, C. Bureau, J. Suski, G. Lecayon, Surf. Interface Anal. 24 (1996) 774.[27] E.T. Vandenberg, L. Bertilsson, B. Liedberg, K. Uvdal, R. Erlandsson, H. Elwing, I.

Lundstroem, J. Colloid Interface Sci. 147 (1991) 103.[28] B. Thierry, F.M. Winnik, Y. Merhi, H.J. Griesser, M. Tabrizian, Langmuir 24 (2008)

11834.[29] C.J. Powell, A. Jablonski, NIST Electron Inelastic Mean Free Path Database—Version

1.1, National Institute of Standards and Technology, Gaithersburg, 2000.[30] U. Joensson, G. Olofsson, M. Malmqvist, I. Roennberg, Thin Solid Films 124 (1985)

117.[31] N. Crampton, W.A. Bonass, J. Kirkham, N.H. Thomson, Langmuir 21 (2005) 7884.[32] Y. Lyubchenko, L. Shlyakhtenko, R. Harrington, P. Oden, S. Lindsay, Proc. Natl. Acad.

Sci. U. S. A. 90 (1993) 2137.[33] Y. Han, D. Mayer, A. Offenhaeusser, S. Ingebrandt, Thin Solid Films 510 (2006) 175.[34] S. Ababou-Girard, H. Sabbah, B. Fabre, K. Zellama, F. Solal, C. Godet, J. Phys. Chem. C

111 (2007) 3099.[35] E. Haslam, Tetrahedron 36 (1980) 2409.[36] C. Mayato, R.L. Dorta, J.T. Vazquez, Tetrahedron Lett. 49 (2008) 1396.[37] B.M. O'Leary, T. Szabo, N. Svenstrup, C.A. Schalley, A. Luetzen, M. Schaefer, J. Rebek

Jr., J. Am. Chem. Soc. 123 (2001) 11519.

Fig. 4. Fluorescent analysis of the antibodies modified surfaces in the presence offluorescent transferrin antigens alone, or in competition with human albumin and non-labeled antigens. F is the fluorescence intensity obtained in the presence of fluorescenttransferrin antigens and F0 is the signal in absence of antigen.

Scheme 2. Antigen competition experiments to assess the specific antigen–antibodyrecognition (Alb: albumin, Tran: transferrin).

6022 S. Dauphas et al. / Thin Solid Films 517 (2009) 6016–6022