Evidence for why tri(ethylene oxide) functionalized Si-C linked monolayers on Si(111) have inferior protein antifouling properties relative to the equivalent alkanethiol monolayers assembled on gold

Article abstract of DOI:10.1071/CH05121

High quality methoxy-terminated monolayers containing a tri(ethylene oxide) moiety were formed on Si(111)-H surfaces in thermal hydrosilylation reactions. X-ray photoelectron spectroscopy, contact angle, and X-ray reflectivity measurements suggested that the suboptimal protein anti-fouling properties of these Si-C linked monolayers were due to a reduced lateral packing density of the chains resulting in a disordered layer with insufficient internal and external hydrophilicity. CSIRO 2005.

Full text of DOI:10.1071/CH05121

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CSIRO PUBLISHING
Aust. J. Chem. 2005, 58, 660–663
www.publish.csiro.au/journals/ajc
Evidence for Why Tri(ethylene oxide) Functionalized Si–C Linked
Monolayers on Si(111) Have Inferior Protein Antifouling Properties
Relative to the Equivalent Alkanethiol Monolayers Assembled on Gold
Till Böcking,A,B Michael Gal, Katharina Gaus, and J. Justin Gooding
B
C
A,D
A
School of Chemistry, University of New South Wales, Sydney NSW 2052, Australia.
School of Physics, University of New South Wales, Sydney NSW 2052, Australia.
Centre for Vascular Research, School of Medical Sciences, University of New South Wales,
Sydney NSW 2052, Australia.
Corresponding author. Email: Justin.Gooding@unsw.edu.au
B
C
D
High quality methoxy-terminated monolayers containing a tri(ethylene oxide) moiety were formed on Si(111)–H
surfaces in thermal hydrosilylation reactions. X-ray photoelectron spectroscopy, contact angle, and X-ray reflectivity
measurements suggested that the suboptimal protein anti-fouling properties of these Si–C linked monolayers were
due to a reduced lateral packing density of the chains resulting in a disordered layer with insufficient internal and
external hydrophilicity.
Manuscript received: 16 May 2005.
Final version: 19 July 2005.
The formation of inert coatings designed to prevent bio-
Si
Si
H
fouling on surfaces is crucial for improving the selectivity
and sensitivity of devices for monitoring biomolecular inter-
actions such as biosensors and microarrays. Self-assembled
monolayers (SAMs) of alkanethiols on gold with hydroxyl
or methoxy terminated oligo(ethylene oxide) (EOn, n ≥ 3)
C11EO3Me in 1,3,5-triethylbenzene,
00ЊC
2
O
O
[
1,2]
moieties resist the non-specific adsorption of proteins.
O
O
The mechanism of protein resistance is thought to involve
[
3,4]
the binding of interfacial water by the EO moieties
Scheme 1. Surface modification of Si(111)–H by thermal hydrosilyl-
ation of C11EO3Me.
amongst other factors including the internal and termi-
nal hydrophilicity, and the chain packing which impacts
the lateral compression in the monolayer.[ Recently, the
5]
by hydrosilylation of 11-(2-(2-(2-methoxyethoxy)ethoxy)-
ethoxy)undec-1-ene (C11EO3Me) using a thermal method
with 1,3,5-triethylbenzene as the solvent for the reaction
(Scheme 1) is reported. The chemical composition and
structure of this monolayer were characterized by X-ray pho-
toelectron spectroscopy (XPS), water contact angles, and
X-ray reflectometry (XR). The purpose of this paper is to
analyze and discuss the XPS, contact angle, and XR data in
the context of previously observed differences in the protein
resisting properties of EO3Me terminated layers on silicon
and gold.
formation of inert monolayers has been extended to sili-
con surfaces.[
6–8]
Silicon substrates have advantages over
gold, including smooth surfaces and compatibility with exist-
ing semiconductor nanofabrication technologies, and can be
modified with extremely robust Si–C linked monolayers in
thermal, catalyzed, or photochemical hydrosilylation reac-
Many functional terminal groups have
been incorporated on Si–C linked monolayers for immobi-
tions of alkenes.[
9,10]
lization of DNA,[
11–14]
saccharides,
[15]
and peptides,
[16]
and
chemicaltransformationsandcouplingchemistrieshavebeen
investigated using model compounds.[
17–19]
Photochemical
Monolayer formation was carried out in a solution of the
◦
hydrosilylation of EOn-functionalized alkenes terminated
alkene C11EO3Me in 1,3,5-triethylbenzene at 200 C. The
[6]
with hydroxyl groups (n = 3) or methoxy groups (n = 3, 6,
EOn moieties of the functionalized alkenes are expected
to trap traces of water. During the hydrosilylation reaction
this trapped water reacts with the hydride-terminated sili-
con surface, resulting in low-quality monolayers associated
with high levels of silicon dioxide. To reduce the amount of
water remaining in the alkene solution, the alkene and solvent
7,8]
7
, 9)[ on Si(111)–H yielded monolayers with anti-fouling
properties. In these studies monolayers with three EO units
were less efficient in reducing the non-specific adsorption of
proteins[ than the equivalent layers of EO3 functionalized
thiols on gold. Here the formation of monolayers formed
7]
©
CSIRO 2005
10.1071/CH05121
0004-9425/05/090660

Tri(ethylene oxide) Functionalized Si–C Linked Monolayers on Si(111) Have Inferior Protein Antifouling Properties
661
the C–C and C–O bonded carbons was 9.4:8–9.9:8, close
to the stoichiometric ratio of 10:8 for the surface-attached
C11EO3Me molecule.
(
a) Survey
Si 2s
Si 2p
Based on XPS data, the molecular surface coverage was
estimated to be 0.38–0.40 chains per silicon surface atom.[
This surface coverage is consistent with that reported for the
photochemically attached monolayer of C11EO3Me,[ and
points to a significantly reduced packing density compared
with the 0.5 chains per silicon surface atom proposed and
determined experimentally for unfunctionalized Si–C linked
20]
O 1s
C 1s
7]
O Auger
F 1s
[
20,21]
monolayers.
The reduced grafting density is expected
to result in a some disorder in the adsorbed monolayer. Some
of the oligoethoxy moieties may partially penetrate into the
1000
800
600
400
200
0
[
22]
underlying alkyl layer,
leading to mixing of the alkyl and
Binding energy (eV)
EO3Me chains. This picture of orientational disorder is sup-
ported by the XPS data. For an ordered bilayer structure in
which the EO3Me moieties form a separate layer on top of
the alkyl chain layer, the intensity of photoelectrons originat-
ing from the alkyl layer would be attenuated by the overlying
EO3Me layer. Taking this attenuation into account, the theo-
retical ratio of the peak area for C–C bonded carbons to that
of C–O bonded carbons in the carbon 1s narrow scan would
be approximately 1:1 (see Accessory Material). This attenu-
ation has been observed for highly ordered EOn terminated
(
b) O 1s
(c) C 1s
(d) Si 2p
C–C
C–O
105 102
537 533 529 291 288 285 282 105 102
99
Binding energy (eV) Binding energy (eV) Binding energy (eV)
[3]
alkanethiol SAMs on gold. For the Si–C linked monolay-
ers described here the attenuation is not evident and instead
a ratio close to the stoichiometric ether carbon-to-alkyl car-
bon ratio is observed, signifying disorder in these types of
monolayers.
Fig. 1. (a) XPS survey spectrum, and XPS narrow scans of the (b) O 1s,
(
c) C 1s, and (d) Si 2p regions of Si(111) derivatized with C11EO3Me.
The inset in (d) shows an enlargement of the Si 2p region where the peak
due to oxidized silicon is expected (102–105 eV).
The wettability of the surface was studied using water con-
tactangle measurements.The advancing andrecedingcontact
were stirred over sodium, redistilled under reduced pressure,
and stored over molecular sieves under an argon atmosphere.
Fig. 1a shows the XP survey scan of a monolayer formed
by reaction of C11EO3Me with Si(111)–H under conditions
described above. As expected, the oxygen 1s and carbon 1s
signals from the organic monolayer were detectable at 533
and 285 eV respectively, and the silicon 2s and 2p signals
from the underlying substrate were present at 150 and 99 eV
respectively. A small fluorine 1s signal at 686 eV indicated
traces of fluoride ions remaining on the surface after etching.
The high quality of the surface was evident from the silicon 2p
narrow scan (Fig. 1d). Significant oxidation of the Si–H sur-
face during or after sample preparation (as observed for low
quality samples) would be detectable as a broad peak between
◦
◦
angles of the C EO Me modified surface (57 , 55 ) were
11
3
in good agreement with those reported for the same mono-
◦
◦
[7]
layer prepared using photochemical methods (59 , 56 ).
However, the advancing contact angle was considerably lower
than reported for the equivalent monolayer on gold and silver
◦
[3,5]
(63–65 ),
indicating greater exposure of the ether oxy-
gen atoms to the liquid due to disorder in the chains. In
contrast, the contact angles observed here were higher than
thosedeterminedforSi–Clinkedmonolayersterminatedwith
[
7]
EO Me (n = 6, 9) moieties, which indicated that in the
n
monolayers reported here the underlying alkyl chains were
insufficiently shielded by the EO Me chains. Finally, the low
3
hysteresis observed here compared to thiol SAMs on gold
◦
[5]
1
02 and 105 eV. Here the peak corresponding to oxidized sili-
con was barely detectable in the silicon 2p region.The oxygen
s narrow scan (Fig. 1b) contained a main peak at 533.3 eV
(∼10 ) is attributed to the extremely low surface rough-
ness of the Si(111) substrate (etching in 40% ammonium
fluoride solution yields atomically smooth surfaces[ ).
An indication of the packing density and structure of
the monolayers can be obtained using XR. XR has been
used previously for the characterization of Si–C linked
23]
1
mainly due to the oxygen atoms of the organic monolayer
and also a small shoulder at 531.9 eV, which was tentatively
∗
assigned to the presence of low levels of oxidized silicon.
[
19,21,24]
The C 1s envelope (Fig. 1c) was fitted with two peaks. The
peak at 285.0 eV was assigned to the C–C bonded carbons of
the alkyl chain and the peak at 287.0 eV to the C–O bonded
carbons of the triethylene oxide moiety. The area ratio of
monolayers.
Fig. 2 shows the reflectivity curve as
a function of momentum transfer (Q ) of Si(111) derivatized
z
◦
with dilute C EO Me in 1,3,5-triethylbenzene at 200 C.
11
3
Structural parameters such as the thickness, electron density,
∗
The detection of oxygen is more sensitive than that of silicon in XPS. Thus, oxide can be detected more readily in the oxygen 1s region than in the silicon
p region. The level of oxide detected here was considered to be insignificant. Even samples prepared from non-functionalized alkenes often reveal similar
2
levels of surface oxidation.

6
62
T. Böcking et al.
1
1
1
1
1
1
1
1
1
01
The ability of EOn and EOnMe terminated monolay-
ers to resist non-specific adsorption of proteins depends on
factors including the packing density of the chains in the
monolayer, which influences the conformation of the EOn
Layer
d (Å)
00
C11EO Me
16
3
0Ϫ1
[5]
units and their ability to coordinate water. The protein-
resistance of EO3Me terminated monolayers is reduced when
the packing density is too high, as observed for these types
0Ϫ2
[
26]
0Ϫ3
of SAMs on silver.
Here it is suggested that anti-fouling
properties are also reduced when the packing density falls
below a critical value. The packing density of C EO Me
0Ϫ4
0Ϫ5
11
3
−
2
SAMs on silicon with an estimated 3.0–3.1 molecule nm
based on XPS data) was considerably lower than that of
the chemically equivalent but highly inert SAMs on gold
(
0Ϫ6
−
2 [5]
(
3.61 molecule nm ). This arrangement is likely to result
0Ϫ7
in a collapse of the layer to the extent that partial mixing
of alkyl and EO3Me moieties occurs. As a consequence of
the mixing of the monolayer components, the hydrophilic-
ity of the interior and exterior of the SAM polyether region
is reduced so to compromise the anti-fouling properties. A
similar observation of protein adsorption to poorly packed
EO3Me terminated monolayers formed by electrochemical
reduction of the corresponding aryl diazonium salt on glassy
0
.0
0.1
0.2
0.3
0.4
0.5
Q (ÅϪ1)
z
Fig. 2. XR curve of Si(111) derivatized with a monolayer formed by
hydrosilylation of C11EO3Me. The solid line represents a model fit to
the reflectivity data.
[
27]
andinterfacialroughnessweredeterminedbyrefiningastruc-
tural model for the reflectivity data. A monolayer model
was sufficient to achieve a satisfactory fit. The thickness of
the layer was approximately 16 Å, which was around 3 Å
shorter than the estimated minimum thickness of 19 Å for
an ordered C11EO3Me monolayer assuming fully extended
carbon electrodes was reported.
In conclusion, it was demonstrated that high-quality (low
oxide) SAMs of C11EO3Me could be formed on Si(111)–
H by a thermal method. The monolayers exhibited a lower
grafting density than observed for unfunctionalized Si–C
linked monolayers, which may have lead to partial collapse
of the layer with possible partial mixing of hydrophilic and
hydrophobic moieties. This orientational disorder was sup-
ported by XPS and XR data and may be responsible for
the reduced hydrophilicity of the interface, and thus the
suboptimal anti-fouling properties of these monolayers. Fur-
ther experiments investigating the structure of these types
of monolayers on silicon are required to corroborate the
molecular picture suggested by the preliminary experiments
presented here.
◦
alkyl chains with a tilt angle of 40–45 from surface nor-
mal, and a helical or extended conformation of the EO3Me
chains.There was ambiguity in determining the electron den-
sity of the layer because it was found to vary between 0.35
and 0.42 e Å for fits of similar quality. The range was
between the electron densities of methyl-terminated Si–C
−
3
−
3 [21,24]
linked alkyl monolayers (∼0.31 e Å
)
and the value
reported for self-assembled monolayers of low molecular
weight EOn-terminated silanes on silicon dioxide surfaces
0.44 e Å ). Surprisingly, a bilayer model (assuming sep-
−
3 [25]
(
arate electron densities for the alkyl and EO3Me regions of
the monolayer) did not result in a significantly improved fit
to the XR data. The interfacial roughness at the substrate–
monolayer and the monolayer–air interfaces for the mono-
layer in Fig. 2 was 2–3 Å, as observed previously for these
Si–C linked layers.[
The considerably smaller than expected thickness
observed for the Si–C linked C11EO3Me monolayer further
supported the existence of a high degree of disorder leading
to partial collapse. Further, the inability to obtain an improved
fit using a bilayer model for the C11EO3Me SAM suggested
that the alkyl and EO3Me moieties were not present as distinct
separate layers.
Experimental
1
,3,5-triethylbenzene (97%) and octadecene (95%) were purchased
from Fluka, redistilled from sodium under vacuum, and stored over
molecular sieves under argon. Semiconductor grade chemicals were
used for cleaning (30% H2O2, 98% H2SO4) and etching (40% NH4F
solution) pieces of silicon wafer.
Si(111) wafer pieces (p-type, 1–10 ꢀ cm) were cleaned in Piranha
solution (concentrated H2SO4–30% H2O2, 3:1, v/v) at 90 C for
19,24]
◦
2
0–30 min followed by rinsing with excess Milli-Q water. Hydrogen-
terminated Si(111) surfaces were prepared by etching in deoxygenated
40% solution of NH4F for 15–20 min. Monolayers were formed using
thermal hydrosilylation reactions at 200 C in a deoxygenated 0.2 M
solutionofalkenein1,3,5-triethylbenzenefor7 h.Thesamplewasrinsed
several times with dichloromethane and ethyl acetate and blown dry
under a stream of argon.
◦
Preliminary studies to determine the ability of the Si–C
linked C11EO3Me SAM prepared using thermal conditions to
resist the non-specific adsorption of protein were carried out
using fluorescein-labelled bovine serum albumin (F-BSA). It
was found that the surface had partial anti-fouling properties,
reducing the adsorption of F-BSA by 70–80% compared with
adsorption on Si–C linked octadecyl monolayers.
XPS spectra were obtained by means of an EscaLab 220-IXL spec-
trometer with a monochromated AlKα source (1486.6 eV), hemispheri-
cal analyzer, and multichannel detector. The spectra were accumulated
◦
2
at a take-off angle of 90 with a 0.79 mm spot size at a pressure of less
−
8
than 10 mbar.
Samples for X-ray reflectometry were packaged under argon in
screw-cap glass vials for transport and storage. X-ray reflectivity
curves were acquired at the Brisbane Surface Analysis Facility using

Tri(ethylene oxide) Functionalized Si–C Linked Monolayers on Si(111) Have Inferior Protein Antifouling Properties
663
a Bruker D8 Advance diffractometer in reflectometer mode. Parratt32
software[ was used to fit model parameters to measured sets of XR
data with linear background correction as described previously.[19]
Protein adsorption was studied by immersing the sample into a solu-
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−
1
tion of fluorescein-labelled BSA (1 mg mL ) in phosphate-buffered
saline (pH 7.4) for 1 h, followed by rinsing with Milli-Q H2O for 1 min
and drying under argon. The samples were mounted with a glycerol
based mounting medium (Vectashield, Vector Labs) and covered with
coverslips. Fluorescence was measured using a Fujifilm FLA 5000 flu-
orescence scanner. The fluorescence signal determined after adsorption
of F-BSA onto an octadecyl monolayer on Si was taken as monolayer
coverage with protein.The background fluorescence was determined on
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1-(2-(2-(2-Methoxyethoxy)ethoxy)ethoxy)undec-1-ene (C11EO3Me)
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[
1
1-bromoundecene (9.33 g, 95%, 38 mmol). The reaction mixture was
allowed to warm to room temperature and refluxed over night. After fil-
tration by suction, the filtrate was concentrated under reduced pressure.
The crude product was taken up in light petroleum and the organic phase
was washed with brine, dried over Na2SO4, and concentrated under
reduced pressure.The crude product was purified by column chromatog-
raphyonsilicagel(lightpetroleum:ethylacetate1:1, v/v)toyieldthetitle
compound as a colourless oil (8.76 g, 73%). For monolayer preparation
the compound was then dried and further purified by vacuum distilla-
[
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◦
tion from sodium (0.01–0.02 mmHg/176 C). The distilled product was
[
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stored over molecular sieves under an Ar atmosphere. δH (300 MHz,
CDCl3) 5.67 (m, 1 H, CH2=CH), 4.81 (m, 2 H, CH2=CH), 3.49–3.46
1
1513. doi:10.1021/JA992032W
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LA010484S
(
m, 12 H, (OCH2CH2)3), 3.32 (t, 2 H, (CH2)7CH2CH2O), 3.24 (s, 3 H,
OCH3), 1.91 (q, 2 H, =CHCH2), 1.44 (quint, 2 H, (CH2)7CH2CH2O),
1
.16 (bsr, 12 H, =CHCH2(CH2)6).
[
[
[
[
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Accessory Materials
1H NMR spectrum of C11EO3Me and calculation of attenua-
tion factors for XPS analysis are available from the authors or,
until September 2010, the Australian Journal of Chemistry.
Acknowledgments
[
This work was supported by theAustralian Research Council
and the Australian Institute for Nuclear Science and Engi-
neering. The authors thank Dr Jeremy Ruggles for help with
X-ray reflectometry.
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