Specific binding of avidin to biotin immobilised on modified gold surfaces

Specific binding of avidin to biotin immobilised onmodified gold surfaces

Fourier transform infrared reflection absorptionspectroscopy analysis

Claire-Marie Pradier a,*, Mich�eele Salmain b, Liu Zheng a, G�eerard Jaouen b

a Laboratoire de Physico-Chimie des Surfaces, UMR CNRS 7045, Ecole Nationale Sup�eerieure de Chimie de Paris,

11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, Franceb Laboratoire de Chimie Organom�eetallique, UMR CNRS 7576, Ecole Nationale Sup�eerieure de Chimie de Paris,

11 rue Pierre et Marie Curie, 75231 Paris Cedex 05, France

Abstract

The immobilisation of biomolecules on gold surfaces has attracted a lot of attention due to the various ways of

transducing biorecognition events at such surfaces and of course to its broad field of applications. The current challenge

is two-fold: first, to produce a functionalised surface able to bind a given biomolecule in a complex environment, but

also resistant to nonspecific adsorption; second, to assess the protein–ligand interaction by a suitable physical method.

In our attempt to conceive a new type of biosensor, the biotin/avidin system was chosen as a model of biological

(ligand/receptor) interaction.

The first task was achieved by developing a two-step procedure, briefly consisting in the chemisorption of the short

diamine disulfide cystamine to a gold substrate followed by chemical coupling with the N-succinimidyl ester of biotin.

The presence of biotin molecules both specifically and unspecifically bound to the gold surface was assessed by Fourier

transform infrared reflection absorption spectroscopy (FT-IRRAS) and XPS. Undesired nonspecific binding of biotin

was minimised by increasing the chain length of the chemisorbed amine to which was coupled the N-succinimidyl ester

of biotin. Chemical characterisation of the adsorbed layer was performed after each step by FT-IRRAS.

For the second task, the protein avidin was labelled with an alkyne Co2(CO)6 probe, a transition metal carbonyl

entity that yields three characteristic mCBO signals far from the peptide region of absorption, before its interaction

with the modified surface. Molecular recognition was checked and quantified by FT-IRRAS. The occurrence of

nonspecific adsorption of avidin was measured by exposing the biotinylated substrates to a solution of labelled avidin

pre-saturated by biotin. Methods to reduce nonspecific binding of avidin were also shown. � 2002 Elsevier Science

B.V. All rights reserved.

Keywords: X-ray photoelectron spectroscopy; Infrared absorption spectroscopy; Self-assembly; Surface chemical reaction; Gold

1. Introduction

FT-IRRAS is a surface analysis technique,sensitive to a fraction of monolayer adsorbed on a

Surface Science 502–503 (2002) 193–202

www.elsevier.com/locate/susc

*Corresponding author. Tel.: +33-1-44-27-67-36; fax: +33-1-

46-34-07-53.

E-mail address: [email protected] (C.-M. Pradier).

0039-6028/02/$ - see front matter � 2002 Elsevier Science B.V. All rights reserved.

PII: S0039-6028 (01 )01932-X

reflective material. Much interest in this techniquehas been demonstrated for the investigation of theadsorption of simple molecules like CO or SO2 onmetals [1], but also more complex organic mole-cules, in particular biomolecules [2]. Measuring theIR signal after reflection on a surface at grazingincidence results in the enhancement of the signalfor dipole moments normal to the surface mak-ing the surface sensitive to the orientation of themolecular groups.Our long-term purpose is to implement an easy-

to-handle, Fourier transform infrared reflectionabsorption spectroscopy (FT-IRRAS)-based bio-sensor to enable quantification of analytes ofclinical or environmental interest by molecular re-cognition with specific antibodies immobilised at agold surface.The biotin/avidin system is a very good example

of biomolecular recognition with a very high af-finity constant (Ka � 1015 M�1) and a very highspecificity. It has often been used as a model forbiosensor development where one of the counter-parts, often the biotin molecule, is immobilisedon the transducer element. The key step is theimmobilisation of biotin molecules that shouldallow the protein avidin to bind to the transducerin an exclusively specific way. Indeed, nonspecificinteractions may (i) denature proteins, inducingfalse signals, and (ii) induce undesired irrevers-ible binding of these macromolecules. The con-struction of a monolayer of biotin molecules ontoa gold substrate generally proceeds via one of thetwo following pathways. The first one consists inthe direct chemisorption of a biotinylated thiol ordisulfide [3–6] by immersing clean gold substratesinto solutions of these compounds. The control ofthe density of biotin coverage is usually achievedby ‘‘dilution’’ of the biotinylated thiol with a long-chain thioether to prevent any further nonspecificinteraction of avidin [7,8]. The other pathwayconsists in the prior adsorption of a thiol with anadequate terminal function followed by its chemi-cal coupling with an appropriate biotin derivative[9–11].In an effort to overcome the risk of nonspecific

binding of avidin, we first tested a two-step pro-cedure consisting in the covalent coupling of bio-tin to a cystamine monolayer [12]. Some results

will recalled here for sake of clarity. As some di-rect interaction of biotin with the surface wasstill observed, we then increased the length ofthe attachment amine-terminated layer. These so-prepared biotinylated layers were exposed to anavidin solution and FT-IRRAS detection of bind-ing was greatly facilitated by prior labelling of avi-din with an alkyne dicobalt hexacarbonyl probe.The nature of the avidin/biotin interaction wasalso tentatively optimised by pre-saturation withthe protein bovine serum albumin or by includinga blocking step with mercaptoethanol after cysta-mine functionalisation. The metal surface wascharacterised at each step by FT-IRRAS analysis.

2. Experimental procedures

Reagents. Cystamine dihydrochloride [CA](Aldrich), mercaptothanol [ME] (Merck) glutar-aldehyde [GA] (grade II, 25% aqueous solution,Aldrich), 2,20-(ethylenedioxy) bis(ethylenediamine)[DADOO] (Aldrich), biotin (Sigma), O-(N-succinimidyl)-N,N,N,N0,N0-tetramethyluroniumtetrafluoroborate [TSTU] (Sigma) and N,N-diiso-propylethylamine [DIPEA] (Aldrich) were used aspurchased. Double-distilled grade water, absoluteethanol and DMF were deoxygenated under argonbefore use. HEPES buffer (10 mM HEPES, pH7.4) was prepared from double distilled gradewater. Neutravidin (Pierce chemicals) and bovineserum albumin [BSA] (grade V, Sigma) labelled byalkyne dicobalt hexacarbonyl probes [NAV-Co,BSA-Co] were prepared according to a previouslypublished procedure [13].

Substrate preparation. Solid gold substrates(Goodfellow, 12:5� 12:5� 2 mm3) were polishedon one side with Si–C papers, followed by dia-mond paste (grain 5, 2, and 0.5 lm). The polishedgold substrates were then cleaned by soaking andsonicating in hexane, absolute ethanol and waterfor 15 min each time, followed by drying underclean air.

2.1. Sample preparation

Preparation of amine-terminated monolayers.Cystamine monolayers were obtained by immers-

194 C.-M. Pradier et al. / Surface Science 502–503 (2002) 193–202

ing gold substrates in a 0.1 M solution of CA indeionised water under argon at room temperaturefor 3 h. To prepare the long-chain amine-termi-nated monolayer, the CA monolayer was treatedwith a 0.1 M solution of GA in ethanol for one dayfollowed by a 0.01 M solution of DADOO inethanol. After exposure to GA and DADOO, thefilms were washed repeatedly with ethanol anddried under a stream of air. A supplementary stepconsisting in exposing the CA substrates to a so-lution of 0.1 M mercaptoethanol in ethanol 3 hwas also performed.

Biotinylation of amine-terminated monolayers.The carboxylic acid function of biotin was acti-vated by treatment of a 0.01 M solution of biotinin 10 ml of DMF with 1.2 molar equivalent ofTSTU and DIPEA 2 h at room temperature toform the N-succinimidyl ester of biotin (NHS-biotin) in situ [14]. The amine-terminated goldsubstrates were then immersed in the solution ofNHS-biotin and incubated over night under argonat room temperature, followed by rinsing withDMF and ethanol and finally drying under cleanair.

Binding of NAV-Co to biotinylated surfaces.Biotinylated substrates were immersed into a 2 lMsolution of NAV-Co in HEPES buffer (10 mM, pH7.4) for 3 h, followed by rinsing with buffer andwater, and finally drying under clean air.

2.2. Surface characterisation

FT-IRRAS spectra were recorded on a NICO-LET Magna 550 spectrometer equipped withmirrors optimised for reflection at grazing inci-dence. A wide band HgCdTe detector, cooled withliquid nitrogen, was used to collect the data at4 cm�1 resolution. The FTIR beam was focusedonto the sample at an incidence angle of 87�. Theentire experimental setup was purged to eliminatewater and CO2 absorption contributions. A refer-ence spectrum was taken on a clean gold sample.For all spectra, 600 scans were collected.XPS analyses were performed on a VG MKII

spectrometer using AlKa radiation (hm ¼ 1486:6eV) at 10�10 mbar. After a survey spectrum re-corded at a pass energy equal to 50 eV, the S 2p,N 1s, C 1s and Au 4f region spectra were recorded

at 20 eV pass energy using a 90� takeoff angle. TheAu 4f7=2 peak at 84.0 eV was used as a reference forthe binding energies. The C 1s spectra were curvefitted using the VG software and applying a sym-metrical Gaussian/Lorentzian (50/50) function.

3. Results

3.1. Binding of biotin to a cystamine layer

Scheme 1 displays the reactions involved in theimmobilisation of biotin to the gold surface. Thesamples were rinsed and dried following the abovedescribed procedure. The C 1s, N 1s, S 2p high-resolution XPS spectra measured after sequentialexposure to CA and NHS-biotin are displayed inFig. 1. The C 1s peak, centred at 285.1 eV, wasassigned to the CH2 groups of CA. Dissymmetryobserved at the high energy side has been previ-ously ascribed to some ethanol molecules trappedat the surface after rinsing [15]. The intensity ofthe C 1s peak markedly increased after exposure toNHS-biotin and a new contribution at high bind-ing energy (288.6 eV) was assigned to the C@Obonds of the urea and peptide groups. A weak andbroad N1s signal (400.3 eV, FWHM ¼ 3 eV) wasalso observed after CA treatment as expected;it increased by a factor of 2 upon binding of NHS-biotin. Conversely, the broad S 2p peak at 162.4eV (characteristic of S–Au) slightly decreasedupon treatment by NHS-biotin as a result ofthe screening of the S 2p photoelectrons by theadsorbed organic layer. No peak in the Cl 2p re-gion could be detected indicating that chloride,the counterion of the CA salt, was washed awayduring the rinsing procedure [15]. The averagethickness of the CA layer was evaluated fromthe attenuation of the Au 4f7=2 peak. Assuming acontinuous layer and a mean free path of Auphotoelectrons through the cystamine layer of 42�AA [16], a value of 1.98 �AA was calculated.Functionalisation of gold substrates was con-

firmed by FT-IRRAS analysis of the metal sur-faces. Fig. 2 shows the spectra taken on the goldsurfaces after interaction with cystamine andNHS-biotin solutions, respectively. Very weak IRsignals were observed after treatment by CA. The

C.-M. Pradier et al. / Surface Science 502–503 (2002) 193–202 195

major one, located at 1575 cm�1, was ascribed tothe deformation vibration, dNH of the CA NH2

functions. The C–H stretching modes of the CH2

groups could not be distinguished from the base-line (data not shown), not unsurprisingly for sucha short alkyl chain [17]. After exposure of the CA-treated gold substrate to NHS-biotin, the IRRASspectrum displayed a broad band at �1620–1660cm�1 and three weaker bands at 1440, 1540 and1760 cm�1. The feature centred at 1660 cm�1 wasreadily assigned to the amide I band of a peptidegroup and provided evidence that chemical bind-ing of biotin to the terminal amine function of

CA occurred [18,19]. This band probably includeda contribution of the C@O stretch of the cyclicurea of biotin. In previous IR studies of chemi-sorbed alkylthiolates used to bind a second mole-cule via amide linkage, the C@O and C–Nstretching vibrations were shown to be IR-activedue to the preferential orientation of these bondsnormal to the surface [20]. Finally, the weak signalat 1760 cm�1, assigned to the carboxylic acidstretch of biotin suggested another mode of bind-ing of biotin molecules to the gold surface leavingsome free carboxylic functions. This unwantedbinding mode might be confirmed by the presence

Scheme 1. Preparation of NHS-biotin and immobilisation of biotin of a cystamine layer on gold.

Fig. 1. XPS analysis of the CA layer before (a) and after (b) exposure to NHS-biotin.


of a weak band at 1440 cm�1, assigned to the sym-metric C–O stretch of a carboxylate group.In conclusion, biotin molecules can be immo-

bilised on a gold surface by covalent coupling ofNHS-biotin to a monolayer of CA, although thisprocedure provides some extent of nonspecificbinding.

3.2. Binding of biotin to a long-chain, amine-terminated attachment layer

To decrease this observed nonspecific binding, asecond procedure consisting in increasing the

chain length of the amine-terminated attachmentlayer was adapted from the literature [21]. The CAmonolayer was first exposed to a solution of GAas cross-linking agent to yield an aldehyde-termi-nated layer then to a solution of the hydrophilicdiamine 2,20-(ethylenedioxy) bis(ethylenediamine)DADOO to provide a long-chain amine-termi-nated monolayer (Scheme 2). The IRRAS spectrarecorded after each of these steps are shown in Fig.3. The spectrum of the CA–GA layer displayed aband at 1730 cm�1, assigned to the C@O stretch ofthe aldehyde group, as expected.Upon reaction with DADOO, the IRRAS spec-

trum displayed intense features at 1660 andFig. 2. FT-IRRAS analysis of the CA layer on gold before (a)

and after (b) exposure to NHS-biotin.

Scheme 2. Preparation of a long-chain, amine-terminated layer and immobilisation of biotin.

Fig. 3. FT-IRRAS analysis of the CA layer after sequential

exposure to GA (a), DADOO (b) and NHS-biotin (c).


1560 cm�1, whereas the aldehyde C@O stretchcompletely disappeared, indicating that reactionbetween the aldehyde groupof theGA layerwith theDADOO amine function has gone to completion.The 1660 cm�1 band can be attributed to the C@Nimine group, and the 1560 cm�1 band to the dN–H

vibration of the amine function of DADOO. In-teresting is to note that, on spectrum a, Fig. 3, GAon the surface, the mC@N imine band, if present at�1650 cm�1 is very weak. Conversely, the sametype of bond yields an intense signal on the spec-trum of the CA–GA–DADOO modified surface.These features are a strong indication of a prefer-ential orientation of the molecular groups at thesurface: adsorption of cystamine in a gauche con-formation [22] and, successive bindings of GA andof DADOO according to the geometry representedin Scheme 2. Finally, chemical binding of biotin tothe CA–GA–DADOO layer was evidenced by anoticeable increase of the 1660 cm�1 band and ashift of the other band to 1540 cm�1, as a result ofthe formation of the peptide bond as seen above.As we have previously observed by XPS that

the layer of amine-terminated molecules formedfrom exposure to cystamine had a poor coverage,we thought that the subsequent treatment of thegold substrate with another thiol might optimisethe properties of the first organic layer regard-ing further functionalisation steps. Moreover, hy-droxyl terminated thiolates have been shown tosuccessfully inhibit protein binding at gold surfaces[23–25]. We thus submitted a gold substrate to se-quential exposure to CA, ME, GA, DADOO andNHS-biotin. Completion of each functionalisa-tion step was assessed by FT-IRRAS (Fig. 4). Thesame spectral changes as above were observed. Itis interesting to note that the density of DADOOmolecules immobilised at the gold substrate wasmore than twice that obtained when the ME stepwas omitted.Using the last two procedures, no IR band at

�1760 cm�1 was detected, indicating that nononspecific binding of biotin occurred.

3.3. Recognition of avidin

The next step is now the molecular recognitionof biotin molecules immobilised on the gold sur-

face by the protein avidin in solution. Biotin-functionalised gold substrates were exposed to asolution of the protein avidin that was previouslylabelled with an alkyne Co2(CO)6 probe (NAV-Co). Fig. 5c shows the IRRAS spectrum of theCA–biotin substrate after this treatment. Bindingof NAV-Co was assessed by the increase of the1660 and 1540 cm�1 bands and the presence of twoof the three expected mCBO bands at 2054 and 2020cm�1, characteristic of the alkyne Co2(CO)6 probe.Similarly, the same change in the IRRAS spectrumwas observed after exposure of the CA–GA–DA-DOO–biotin (Fig. 6d) and CA–ME–GA–DA-DOO–biotin (Fig. 7d) substrates to the solution of

Fig. 4. FT-IRRAS analysis of the organic layer obtained by

sequential exposure to CA, ME; GA (a), DADOO (b) and

NHS-biotin (c).

Fig. 5. FT-IRRAS analysis of the CA–biotin layer before (b)

and after exposure to a 2 lM solution of NAV-Co (c) or a 2 lMsolution of NAV-Co pre-incubated with biotin (100 lM) (d).


NAV-Co. However, the mCBO bands were approx-imately three and four times more intense in thetwo last cases, respectively (Table 1).At this point, the question is whether avidin

was exclusively bound to the gold substrate byspecific interaction with the immobilised biotinmolecules or not. Two different experiments wereperformed to check this. The first experiment con-sisted in exposing a gold substrate sequentiallyfunctionalised by CA, GA and DADOO to thesolution of NAV-Co. Only very weak mCBO bands

were observed on the resulting FT-IRRAS spec-trum (Table 1), indicating that the presence ofbiotin molecules at the surface was essential foravidin binding. In the second experiment, a solu-tion of NAV-Co was incubated with a large excessof biotin to saturate the four biotin binding sitesprior to exposure to the biotinylated gold sub-strates. In the case of the CA–biotin layer, theIRRAS spectrum of the gold substrate was com-pletely superimposable (in the mCBO bands region)to that recorded after exposure to NAV-Co alone(Fig. 5d, Table 1), indicating that binding of NAV-Co to CA–biotin layer was almost totally non-specific. Conversely, binding of NAV-Co saturatedby biotin to the CA–GA–DADOO–biotin layerwas almost fully inhibited as shown in Fig. 6e, witha percentage of nonspecific binding of 9% calcu-lated from the ratio of mCBO bands intensities(Table 1). Finally, exposure of the CA–ME–GA–DADOO–biotin substrate to biotin-saturatedNAV-Co resulted in a very low, almost undetect-able binding of avidin (Fig. 7e, Table 1), indicatingthat nonspecific binding of avidin was fully in-hibited in this manner.Therefore, sequential functionalisation of gold

substrates by CA, GA, DADOO and biotin pro-vided a monolayer of biotin molecules presenting agood capacity to bind avidin in an almost fullyspecific fashion. Completion of the CA layer withME increased the reactivity of DADOO towardsGA, which resulted in a higher density of biotinmolecules at the metal surface and in turns in-duced a larger and more specific binding of avidinto this surface.To improve the quality of binding of avidin to

the CA–biotin surface, we experimented with thefollowing procedure. Exposure of the CA–biotinsubstrate to a solution of the unrelated proteinBSA labelled with the same transition metal car-bonyl probe resulted in the presence of weak mCBObands at 2054 and 2020 cm�1 (Table 1), indicatingthat binding of this protein also occurred, al-though to a lower extent than with NAV-Co,probably as a result of incomplete surface cover-age by biotin molecules. This however opened theway to the blocking of the nonspecific proteinbinding sites with BSA. This was achieved by se-quential incubation of theCA–biotin gold substrate

Fig. 6. FT-IRRAS analysis of the CA–GA–DADOO–biotin

layer before (c) and after exposure to a 2 lM solution of NAV-

Co (d) or a 2 lM solution of NAV-Co pre-incubated with

biotin (100 lM) (e).

Fig. 7. FT-IRRAS analysis of the CA–ME–GA–DADOO–

biotin layer before (c) and after exposure to a 2 lM solution of

NAV-Co (d) or a 2 lM solution of NAV-Co pre-incubated with

biotin (100 lM) (e).


with BSA (in its unlabelled form), followed byNAV-Co. In these conditions, binding of avidinstill occurred (Table 1) and this must be in a spe-cific fashion since all nonspecific sites were blockedby BSA.

4. Discussion

The characterisation of the metal surface at thesuccessive steps of its functionalisation enabledus to estimate the nature of the interactions be-tween the organic molecules and gold and also tocompare the efficiency of the different modes ofimmobilisation of biotin regarding molecular rec-ognition of avidin.In agreement with the well-known affinity of

sulphur for gold, adsorption of cystamine wasreadily observed though not leading to a completecoverage of the surface [26]. Based on severalworks which demonstrate that the order of theSAMs on gold is improved by increasing thelength of the thiolate alkyl chains [27], we believethat the cystamine layer is disordered, some of themolecules adopting a trans, others a gauche con-figuration. These surface defects may explain thevery short average thickness of the layer that wecalculated. We indeed think that the adsorbedlayer is heterogeneous, leaving parts of the surfaceuncovered that can react with biotin or even withavidin.On the cystamine-biotin layer, binding of avidin

or biotin-saturated avidin was shown to lead to thesame amount of adsorbed material. This was de-

duced from the mCBO band intensities, and thehigher amide I signal, observed for surface-boundsaturated avidin is questionable. This may be be-cause though probably less avidin molecules in-teracted with the surface when pre-saturated withbiotin, each of them already included four biotinmolecules in their binding sites. Conversely, avidincan only bind to one or two immobilised biotinligands for steric reasons.Our results suggest that adsorption of mercapto-

ethanol, an alcohol-terminated thiol, within thecystamine layer ensured a strictly covalent bind-ing of biotin. Moreover the density of DADOOand successively of biotin molecules was increased.A possible explanation of the increased reactivityof the gluraraldehyde layer would be that thetwo terminal aldehyde functions of glutaraldehydemay, on a disordered cystamine layer, react withtwo neighbouring amines hence creating inactivemolecular bridges. Adding mercaptoethanol with-in the adsorbed cystamine molecules is likely tomaintain a sufficient distance between the CAmolecules to prevent ‘‘ring closure’’ to occur.The reason for the nonspecific interaction of

avidin to the CA–GA–DADOO–biotin layer alsodeserves some discussion. This direct interactionmay take place either on some DADOO groups,noncovered with biotin, or directly on the goldsurface if the CA layer is incomplete leaving bareareas from the initial functionalisation step. Thefirst hypothesis would involve for instance elec-trostatic interactions between the slightly nega-tively charged avidin protein (pI ¼ 6, pH ¼ 7:4)and the protonated amine groups of DADOO. We

Table 1

FT-IRRAS analysis of functionalised gold substrates exposed to a 2 lM solution of NAV-Co or BSA-Co or to a 2 lM solution of

NAV-Co containing 100 lM biotin in HEPES buffer pH 7.4

Sample Treatment Intensity of the mCBO bands(area between 2075 and 2000 cm�1)

% nonspecific binding

CA–BIO NAV-Co 0.0084 100%

NAV-Coþ biotin 0.0084

CA–BIO BSA-Co 0.0018 –

BSAþNAV-Co 0.0058

CA–GA–DADOO–BIO NAV-Co 0.0237 9%


CA–ME–GA–DADOO–BIO NAV-Co 0.0312 3%


CA–GA–DADOO NAV-Co 0.0032 –


hardly believe that such interactions would surviverepeated rinses in water. Moreover, the interactionbetween avidin and a DADOO layer on gold wastested and revealed almost no binding. The secondhypothesis involves an incomplete coverage of thesurface from the initial step of functionalisationsince we saw, from the evolution of the IR bands,that the reactions between, on one hand, CA andGA, and on the other hand, GA and DADOO hadgone to completion. The presence of ‘‘holes’’ in theCA layer, probably due to a disordered structureof the short-length cystamine layer [15,28] is con-firmed by the beneficial effect of mercaptoethanolwhich blocks the empty surface sites. We there-fore rather believe that the nonspecific binding ofavidin originates from an unsaturated and dis-ordered layer of CA on the gold surface ratherthan by an incomplete reaction of biotin with theDADOO layer.Aside from the consideration of coverage and

reaction efficiency, our results demonstrate thenecessity of maintaining a certain distance betweenthe metal surface and the bioactive ligand for op-timal protein binding [29]. They also demonstratethe sensitivity of the IRRAS technique for de-tecting covalent binding at a reflective materialand also for the detection of biorecognition eventsthanks to the labelling of biomolecules with metalcarbonyl probes with preservation of their bioac-tivity.

5. Conclusion

We have reported on different strategies for theimmobilisation of biotin onto a gold surface andcompared the quality of the further binding ofavidin. Labelling of avidin with a metal carbonylprobe enabled us to qualitatively and quantita-tively detect binding events.On a short-length thiolate layer, the crowding of

biotin groups sterically hindered recognition ofavidin. The inclusion of sequential cross-linkers tocreate a long-chain attachment layer probably in-creased the density of bound biotin moleculeswhile removing them further away from the metalsurface and appeared to greatly favour the recog-nition of avidin by facilitating the insertion of

biotin groups into the binding pockets. Our find-ings support previously published studies thatshowed that too densely packed biotin layers arenot optimal for the further binding of avidin [30].Moreover, we showed that both factors, i.e. thedensity and the chain length of the attachmentlayer built prior to the immobilisation of biotin,are determining for the step of protein immobili-sation.FT-IRRAS, combined with transition metal

carbonyl probes labelling, provides a sensitive,quantitative tool to optimise the attachment offunctionalised thiols and of biotin at a gold surfaceas well as to monitor the molecular recognition ofavidin by immobilised biotin. It opens a new waytowards the building of reliable, easy to handle,biosensors.

Acknowledgements

The Centre National de la Recherche Scientifi-que is gratefully acknowledged for its financialsupport.

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Specific binding of avidin to biotin immobilised on modified gold surfaces

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