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Applied Surface Science 255 (2009) 8953–8959
Review
Characterization of self-assembled monolayers (SAMs) on silicon substratecomparative with polymer substrate for Escherichia coli O157:H7 detection§
Carmen Moldovan a,*, Carmen Mihailescu c, Dana Stan b, Lavinia Ruta c, Rodica Iosub a,1, Raluca Gavrila a,1,Munizer Purica a,1, Schiopu Vasilica a,1
a National Institute for R&D in Microtechnologies, IMT-Bucharest, 126A Erou Iancu Nicolae, 077190 Bucharest, Romaniab DDS Diagnostic, 1 Segovia Street, Bucharest, Romaniac University of Bucharest, 90-92 Sos Panduri, Bucharest, Romania
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.1. Reagents and material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.2. Pre-treatment of gold substrates and the preparation of mixed SAMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.3. Activated with EDC/sulfo-NHS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.4. Binding of anti-E. coli O157:H7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.5. Blocking by BSA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
2.6. Characterization of the monolayers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8954
3. Results and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8957
3.1. Scanning electron microscopy and AFM (atomic force microscopy) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8957
3.2. Cyclic voltammetry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8957
3.3. FTIR-ATR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8958
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8959
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8959
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8959
A R T I C L E I N F O
Article history:
Received 18 December 2008
Received in revised form 6 May 2009
Accepted 26 June 2009
Available online 3 July 2009
Keywords:
Mixed self-assembled monolayers
Escherichia coli O157:H7 gold
Immunosensor
FTIR-ATR
A B S T R A C T
This article presents the characterization of two substrates, silicon and polymer coated with gold, that
are functionalized by mixed self-assembled monolayers (SAMs) in order to efficiently immobilize the
anti-Escherichia coli O157:H7 polyclonal purified antibody.
A biosurface functionalized by SAMs (self-assembled monolayers) technique has been developed.
Immobilization of goat anti-E. coli O157:H7 antibody was performed by covalently bonding of thiolate
mixed self-assembled monolayers (SAMs) realized on two substrates: polymer coated with gold and silicon
coated with gold. The F(ab0)2 fragments of the antibodies have been used for eliminating nonspecific
bindings between the Fc portions of antibodies and the Fc receptor on cells. The properties of the
monolayers and the biofilm formatted with attached antibody molecules were analyzed at each step using
infrared spectroscopy (FTIR-ATR), atomic force microscopy (AFM), scanning electron microscopy (SEM)
and cyclic voltammetry (CV). In our study the gold-coated silicon substrates approach yielded the best
results.
These experimental results revealed the necessity to investigate each stage of the immobilization
process taking into account in the same time the factors that influence the chemistry of the surface and
the further interactions as well and also provide a solid basis for further studies aiming at elaborating
sensitive and specific immunosensor or a microarray for the detection of E. coli O157:H7.
� 2009 Elsevier B.V. All rights reserved.
§
Contents lists available at ScienceDirect
Applied Surface Science
journa l homepage: www.e lsev ier .com/ locate /apsusc
The work has been performed at IMT Bucharest, by all authors using IMT’s Nanobiolab and characterization facilities.
* Corresponding author. Tel.: +40 214908412; fax: +40 214906238.
E-mail addresses: [email protected] (C. Moldovan), [email protected] (C. Mihailescu), [email protected] (D. Stan), [email protected]
(L. Ruta), [email protected] (R. Iosub), [email protected] (R. Gavrila), [email protected] (M. Purica), [email protected] (S. Vasilica).1 Tel.: +40 214908412; fax: +40 214906238.
0169-4332/$ – see front matter � 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.apsusc.2009.06.113
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–89598954
1. Introduction
Escherichia coli O157:H7 is a representative food-borne patho-gen that produces large amounts of a potent toxin in the lining ofthe intestine and can induce the disorder that may lead to death,particularly in children. The necessary dose of infective has beenestimated to be as low as 10 cells [1]. The development of newmethod for the detection of this kind of serotype remains achallenge to the scientists in various fields [2]. Also many researchfields involve the study of interaction of biomolecules with solidsurfaces in particular for new biosensor elaboration [3]. Actually,these sensors can be considered to be the improved versions of theconventional method (ELISA), particularly in respect of decreasedassay time. Most of these sensors are based on standard sandwichimmunoassay, which involves the formation of sandwich immune-complex consisting of immobilization to capture antibodies in asubstrate [3]. These antibodies will capture bacteria and finally fordetection it will use an antibody labeled detection.
Immobilization of antibodies via self-assembled monolayers
(SAMs) or mixed SAMs offers one of the simplest ways to provide areproducible, ultrathin and well-ordered layer suitable for furthermodification with antibodies. Mixed SAMs are generally con-stituted of one thiolate with a functional headgroup (like acarboxylic acid) at a low mole fraction and another ‘‘diluting’’thiolate at a high mole fraction. The SAMs fabricated using a thiolmixture reduced the steric hindrance caused by the carboxyl-terminated groups of SAMs [4]. Based on these researches, it wasthought that one of the thiols, the smaller, was acting as spacer toreduce steric hindrance and the other was the ligand thatimmobilizes the antibody [5]. Characterization of surfaces coveredwith mixed SAMs is still the subject of numerous studies [5–7].
Antibody immobilization is vital in successful development ofimmunosensor, and the present immobilization methods arebased on four stages: (1) pre-treatment of gold substrates and thepreparation of mixed SAMs; (2) activated with EDC/sulfo-NHS; (3)binding of anti-E. coli O157:H7; and (4) blocking by BSA. Assubstrates for coating with gold have been used to distinguishmaterials: a polymer substrate and a silicon substrate.
The formation of SAMs and the sequential binding of anti-E. coli
O157:H7 were assessed and characterized by FTIR-ATR. Theconductive properties of the Au substrate allow exploitingelectrochemical properties of alkylthiolate assemblies on thetwo surfaces.
Biofilm formatted with SAMs was morphologically character-ized using scanning electron microscopy (SEM) and AFM (atomicforce microscopy).
Those are preliminary experiments in order to obtain a goodsurface to immobilize an important pathogen bacterium: E. coli
O157:H7 with wide range applications in the construction of animmunosensor or fabricating of a microarray.
2. Experimental
2.1. Reagents and material
Unlabeled goat affinity purified antibodies to E. coli O157:H7(used as a capture antibody) and fluorescein labeled goat anti-E.
coli O157:H7 (also used as a capture antibody in microarrayexperiment) were purchased from Kirkegaard & Perry Labora-tories (Gaithersburg, MD). They are F(ab0)2 fragments to immu-noglobulins that were used to remove nonspecific bindingbetween the Fc portions of antibodies and the Fc receptor oncells. 11-Mercaptoundecanoic acid (11-MUA), 3-mercaptopropa-nol (3-MPOH), sulfo-N-hydroxysuccinimide (NHS), and 1-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydro-chloride(EDC) were purchased from Pierce; Bovine serum albumin
(BSA) and phosphate buffered saline, Tween (PBST) werepurchased from Sigma–Aldrich (Germany).
Silicon substrates (Si p-type, 10–20 V cm) and poly(methylmethacrylate) polymer substrates (PMMA) (2 cm � 2 cm) werecoated with 25 nm chromium and 300 nm gold deposited byevaporation.
2.2. Pre-treatment of gold substrates and the preparation of mixed
SAMs
Evaporated gold (300 nm thickness) was deposited on silicon 40
wafers cut in 2 cm � 2 cm slices, using a chromium base layer(25 nm thickness) as substrate. Prior to immobilization of antibodyon the gold-coated silicon, the surface was thoroughly cleanedsequentially in methanol, acetone and isopropanol-2 for 15 mineach. This was followed by quick 2 min dip in piranha solution(H2SO4 70%:H2O2 30% = 3:1, v/v). Gold-coated silicon surface wasthen rinsed with deionized water for about 15 min. Finally it waswashed with ethanol and dried at 70 8C for 30 min. Polymer(PMMA), coated with gold, was cleaned with methanol for 15 minand rinsed with water and ethanol and finally was dried withnitrogen flow. For each characterization was kept a substrate forreference.
All substrates were immersed in 10 mM mixed solutionsconsisting of 10:1 molar ratio of 3-MPOH to 11-MUA preparedin absolute ethanol. The prepared gold substrates were immersedin this solution for 24 h. Then the gold films were dried with argonand close packed in aluminium bag until utilization. For eachcharacterization was kept a substrate of mixed SAMs.
2.3. Activated with EDC/sulfo-NHS
The surfaces were immersed in a solution of sulfo-NHS (20 mM)and EDC (100 mM) in ultrapure water for 1 h to convert theterminal carboxylic group to an active sulfo-NHS ester.
2.4. Binding of anti-E. coli O157:H7
After rinsing with water and drying, 30 mL of 10 mg/mL anti-E.
coli O157:H7 antibodies were spread over the entire surface ofpolymer and silicon coated with gold and functionalized withSAMs techniques. Then the substrates are stored at 4 8C overnight(18 h). The excess antibodies were removed by rinsing with PBST.
2.5. Blocking by BSA
The antibody-modified substrates were treated with 1% BSA–PBS for 1 h to block the un-reacted and nonspecific sites. Afterrinsing with PBS and water the substrates were dried and used forcharacterization methods.
2.6. Characterization of the monolayers
Scanning electron microscopy was performed using electronmicroscopes SUPRATM 25.
AFM (atomic force microscopy) experiments were performedusing AFM equipment in contact working mode.
Infrared spectra of samples were recorded on a Tensor 27 Brukerspectrometer via the attenuated total internal reflection (ATR)method with the resolution of 4 cm�1 and 64 scans, in thewavelength ranging from 4000 to 400 cm�1.
Electrochemistry measurement was carried out with a VOLTALAB10 instrument. A silver/silver chloride electrode was used as areference, platinum gauze as the counter electrode, and the SAM-covered gold attached to a stainless steel strip as the workingelectrode reference electrode. For the intensity/V measurement,
Fig. 1. Scanning electron microscopy (SEM) images: (a) silicon with gold reference, (b) mixed SAMs in silicon substrate coated with gold, (c) activated with EDC/sulfo-NHS, (d)
image of binding the anti-E. coli O157:7 at Mag.42.42 KX, (e) image of binding the anti-E. coli O157:7 at Mag.7.60 KX, (f) image of binding the anti-E. coli O157:7 at Mag.62.53
KX (200 nm) and (g) image of the F(ab0)2 fragments immobilized on surface at Mag.143 66 KX (20 nm).
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–8959 8955
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–89598956
Fig. 3. Cyclic voltammetry of the silicon substrate coated with gold (scan rate
25 mV/s): (a) SAMs after the formation of the mixed layer (green curve), (b) after
activation with EDC/sulfo-NHS (blue curve) and (c) after immobilization of the
antibody (anti-E. coli O157:H7). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
Fig. 4. Cyclic voltammetry of the silicon substrate coated with gold (scan rate
25 mV/s): (a) after blocking the surface with BSA/PBS and (b) after immobilization
of the antibody fragments.
Fig. 5. Cyclic voltammetry of the polymer substrate coated with gold (scan rate
25 mV/s): (a) polymer substrate gold reference and (b) binding with the antibody.
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–8959 8957
the potential was stepped from �800 mV and then returning to600 mV with a rate of 25 mV/s.
PBS (pH 7.4) was chosen as an electrolyte solution because allexperiments were done in this physiologically relevant mediumfor the bacteria. All the voltammograms were recorded inobscurity.
3. Results and discussion
3.1. Scanning electron microscopy and AFM (atomic force
microscopy)
Immobilization of the F(ab0)2 fragments of goat affinity anti-body in functionalized silicon surface was morphologicallycharacterized with SEM. The successive steps of biointerfaceassembling in terms of changes in surface morphology are shownin Fig. 1. The images of scanning electron microscopy (SEM) atdifferent magnitudes revealed the existence of the fragment F(ab0)2
on the surface of the silicon substrate coated with gold as shown inFig. 1(d)–(g).
For the AFM experiments it has been checked the presence of anarea on clean gold surface that allowed studying the functionaliza-tion by the formation of SAMs. The scan size for the images was2 mm � 2 mm. Comparative AFM images for the polymer/goldversus silicon/gold are observed in Fig. 2(a) and (b), respectively.Fig. 2(c) and (d) indicates the existence of defects dispersed all overfor the polymer surface. It has been clearly observed the differencein homogeneity between the two surfaces and is recorded inFig. 2(e). Only the AFM images for SAMs monolayer was formattedin silicon substrates.
3.2. Cyclic voltammetry
The structural integrity of the absorbed monolayers may becharacterized using CV (cyclic voltammetry) method. The steps ofchemical procedure were: (a) first an orientated mixed monolayeris formatted via the Au–thiolate bond with the carboxylic group
Fig. 2. AFM images: (a) 3D topographic images of the gold surface on silicon substrate
substrate with gold reference, (c) polymer coated with gold reference three dimensional A
(e) topographic images of the gold surface on silicon substrate after immersion in thio
exposed at interface; (b) the second step is the activation of thecarboxylic group and a sulfo-NHS ester; (c) binding the anti-E. coli
O157:H7 by the primary amines of the antibody; and (d) blockingthe surface with BSA/PBS. Each step in the above procedure wasfollowed by an electrochemical characterization. Cyclic voltam-mograms of the functionalized silicon substrate are determined inFig. 3(a) and (b).
The formation of the SAMs on Au resulted a highly insulatingsurface and blocked almost all the faradic current as shown inFig. 3(a). After activated with EDC/sulfo-NHS, the layer became lessinsulating, and an increased current response is observed inFig. 3(b), which is probably due to the terminal carboxylic groupsof 11-MUA which in this electrolytic solution at pH 7.4 becomenegatively charged and cause the electrostatic repulsion andattraction to the surface. Replacement of the active NHS ester byantibody reduced the penetration of the redox pair and decreasedthe current response (Fig. 3(c)). Insulating was further improved
with gold reference, (b) 2D topographic images of the polymer surface on silicon
FM image, (d) polymer coated with gold reference two dimensional AFM image and
ls (1 mm).
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–89598958
after blocking the surface as is shown in Fig. 4. In Fig. 5 isrepresented the cyclic voltammograms curve obtained for polymersubstrate coated with gold. The current is much lower than thecurrent obtained from the silicon substrate but insulatingproperties are increased with the deposed monolayers.
Fig. 6. FTIR-ATR (carbon bands region, 3000–2700 cm�1) spectra of (a) the gold surfaces
SAMs and (b) the silicon substrate coated with gold and SAMs. FTIR-ATR (carboxyl an
immersion in mixture of thiols and binding with the antibody and (d) the silicon substrate
ATR (C–S region, 500–750 cm�1) spectra of (e) the polymer substrate coated with gold a
functionalization.
3.3. FTIR-ATR
The gold surfaces were characterized by FTIR-ATR providingchemical and structural information about thin organic filmsdeposited on gold surfaces. For each step of assembling FTIR-ATR
after all stages of chemical modification on polymer substrate coated with gold and
d amide region, 1300–1800 cm�1) spectra of (c) the gold polymer surfaces after
coated with gold, immersion in mixture thiols and binding with the antibody. FTIR-
nd SAMs functionalization and (f) the silicon substrate coated with gold and SAMs
C. Moldovan et al. / Applied Surface Science 255 (2009) 8953–8959 8959
spectrum was recorded and results will be presented anddiscussed step by step for both types of the substrates, siliconand polymer.Step 1: Co-adsorption of two thiols, MUA and 3-MPOHto form mixed SAMs is recorded in Fig. 6, for polymer substrate (a)and for silicon substrate (b) coated with gold.
Fig. 6(b) shows two typical bands appeared in the n(C–H)region, at 2855 and 2926 to the symmetric and asymmetricstretching vibrations of the chain CH2. The position of the CH2
asymmetric stretching vibration band at 2923 cm�1 is typical of adensely packed, quasi-crystalline arrangement of the alkyl chainsthiolates [8,9]. This band appeared in the spectrum of SAMsrealized in silicon substrate at 2926 cm�1 as it is shown in Fig. 6(b).This may be related to the difference of alkyl chain lengthsbetween MUA and MPOH, which induces some disorders withinthe thin film of SAMs. In case of polymer substrate, in Fig. 6(a),these bands appeared in a higher wave number (2960 cm�1) thanmixed SAMs realized in silicon substrate coated with gold indicatesa more disorder layer.
The bands at 1300–1800 cm�1, attributed to the symmetric andasymmetric COO� and C55O stretch vibrations, prove the presenceof adsorbed MUA for both layers as it is shown in Fig. 6(c) for thepolymer surface and Fig. 6(d) for the silicon surface.
The formation of a covalent gold–sulfur bond has been provenby the presence of the large band in the range 500–750 cm�1,which is assigned to stretch the mode of C–S. This band same toappear in both layers realized in polymer and silicon substrate as itis shown in Fig. 6(e) and (f), respectively.
Step 2: SAM-coated gold on both substrates were first treatedwith a mixture of sulfo-NHS and EDC in water to formintermediately surface NHS-ester. The surfaces were immersedin this solution for an hour at room temperature. In this step, inspectrum of silicon substrate was observed a band attributed to theC55O stretch of remaining NHS ester functions at 1743 cm�1 asshown in Fig. 6(d). Treatment with blocking solution of BSA/PBSinduced a disappearance of the residual ester bands and it shouldincrease of the amide ones (spectra not shown).
Step 3: The antibody binding step, is expected to be immobilizedin a covalent manner on activated MUA acid terminal group. Thebands assigned to the amides II and I are in range waves at 1550–1660 cm�1. These bands appeared more clearly in the spectrum ofsilicon substrate coated with gold (Fig. 6(d)).
4. Conclusions
A comparative study in two different substrates and a mixedself-assembled monolayer of MUA and thiolate were built on a
gold surface. A robust biosurface based on functionalized bySAMs (self-assembled monolayers) technique has been devel-oped. The high insulating proprieties of the thiol monolayerhave been characterized with cyclic voltammetry. Increasednumbers of runs demonstrate the stability of formatted SAMs.Topological AFM measurements have showed the formation of adense and homogeneous SAMs at the gold surface in case ofsilicon substrate comparative with polymer substrate. SEManalysis showed that the immobilization of the polyclonalantibody anti-E. coli O157:H7 did occur. The formation of SAMs,in both the substrates coated with gold, was characterized byFTIR-ATR. The analysis of the spectra recorded for siliconsubstrate showed the typical bands appeared in the carbonregion, at 2855 and 2926 cm�1 to the symmetric and asym-metric stretching vibration of the chain CH2. The position of theCH2 asymmetric stretching vibration band at the 2923 cm�1 istypical of quasi-crystalline arrangement of the alkyl chainsthiolates [8,9]. The analysis of the spectrum recorded forpolymer substrate showed a band in a higher wave number(2960 cm�1). In terms of morphology, AFM images showed auniform gold film formed on both substrates, with low porosityand relatively homogeneous. But the chemical treatmentrealized for surface functionalization disturbs the gold film incase of polymer substrate.
These results provide a solid basis for further studies aiming atelaborating sensitive and specific immunosensor or a microarrayfor the detection of E. coli O157:H7.
Acknowledgements
This work was supported by INTEGRAMplus European project(FP6, IST, 027540) and IMUNOSENSE national project (PNII, No. 51-083/2007).
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