Strong resistance of phosphorylcholine self-assembled monolayers to protein adsorption: Insights into nonfouling properties of zwitterionic materials

Article abstract of DOI:10.1021/ja054169u

In this work, we show the strong resistance of zwitterionic phosphorylcholine (PC) self-assembled monolayers (SAMs) to protein adsorption and examine key factors leading to their nonfouling behavior using both experimental and molecular simulation techniques. Zwitterions with a balanced charge and minimized dipole are excellent candidates as nonfouling materials due to their strong hydration capacity via electrostatic interactions.

Full text of DOI:10.1021/ja054169u

Published on Web 09/27/2005
Strong Resistance of Phosphorylcholine Self-Assembled
Monolayers to Protein Adsorption: Insights into Nonfouling
Properties of Zwitterionic Materials
Shengfu Chen, Jie Zheng, Lingyan Li, and Shaoyi Jiang*
Contribution from the Department of Chemical Engineering, UniVersity of Washington,
Seattle, Washington 98195
Received June 23, 2005; E-mail: sjiang@u.washington.edu
Abstract: In this work, we show the strong resistance of zwitterionic phosphorylcholine (PC) self-assembled
monolayers (SAMs) to protein adsorption and examine key factors leading to their nonfouling behavior
using both experimental and molecular simulation techniques. Zwitterions with a balanced charge and
minimized dipole are excellent candidates as nonfouling materials due to their strong hydration capacity
via electrostatic interactions.
Introduction
thin films with a high degree of structural control. Langmuir-
Blodgett deposition, spin-coating, and vesicle fusion are often
Poly(ethylene glycol) (PEG) is one of the best synthetic
nonfouling materials.1 However, it is now recognized that PEG
decomposes in the presence of oxygen and transition metal ions
found in most biochemically relevant solutions.1-3 It was also
shown that grafted PEG brushes exhibit protein resistance at
room temperature, but lose their protein repulsive properties
above 35 °C.4 It is of great interest to search for alternative
nonfouling materials other than PEG.1,3 It is generally believed
that water plays an important role in surface resistance to protein
adsorption.5-7 While hydrophilic and neutral PEG forms a
hydration layer via hydrogen bonds, zwitterions form a hydration
layer via electrostatic interactions. It is expected that zwitterions
are capable of binding a significant amount of water molecules
and therefore are potentially excellent candidates for super-low
fouling materials.
The lipid components that constitute the outside surface of a
cell membrane are mainly zwitterionic phospholipids and are
believed to be nonthrombogenic.8,9 2-Methacryloyloxyethacry-
late (MPC)-based polymers have been shown to significantly
reduce protein adsorption compared to relevant controls and
have been widely used for various applications.10-12 Langmuir-
Blodgett and self-assembly are two excellent methods to create
used to prepare planar supported lipid bilayers (PSLB).13
Polymerizable PC lipids were introduced to stabilize the fluid
PSLB.14 It was shown that the redox(poly) bis-SorbPC surface15
adsorbed only 6% of a monolayer (ML) of bovine serum
albumin (BSA) with respect to that on the arachidic acid
monolayer.
An alternative approach is to form PC-terminated SAMs on
gold as shown in the pioneering work by Whitesides’16 and
Cooper’s17 groups. However, fibrinogen (Fg) adsorption was
∼23% and ∼35% of a ML with respect to that on methyl-
terminated SAMs as measured in their own studies, respectively.
Recently, Chung et al.18 reported another study of PC SAMs.
Fibrinogen adsorption was 18% of a ML with respect to that
on methyl-terminated SAMs also measured in their experiments.
It is speculated15 that relatively high protein adsorption on PC
SAMs may be due in part to the packing arrangement of a PC
SAM, which is not likely to assemble with the same surface
area per PC moiety as that found in a fluidic PC lipid bilayer.
Consequently, the dipole orientation of the PC lipid bilayer and
associated water molecules may be altered in a PC SAM relative
to a PC lipid bilayer.15 However, it was observed that even
mixed N+(CH3)3 and SO3 zwitterionic SAMs adsorbed less
-
(1) Ostuni, E.; Chapman, R. G.; Holmlin, R. K.; Takayama, S.; Whitesides,
G. M. Langmuir 2001, 17, 5605.
than 1% of a ML of Fg from a 1 mg/mL solution in a study by
Holmlin et al.16
(2) Shen, M. C.; Martinson, L.; Wagner, M. S.; Castner, D. G.; Ratner, B. D.;
Horbett, T. A. J. Biomater. Sci., Polym. Ed. 2002, 13, 367.
(3) Luk, Y. Y.; Kato, M.; Mrksich, M. Langmuir 2000, 16, 9604.
(4) Leckband, D.; Sheth, S.; Halperin, A. J. Biomater. Sci., Polym. Ed. 1999,
10, 1125.
In this work, we synthesized PC thiols from a modified
synthetic route, characterized PC SAMs on Au(111) using X-ray
photoelectron spectroscopy (XPS) and atomic force microscopy
(5) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J.
Phys. Chem. B 1998, 102, 426.
(6) (a) Zheng, J.; Li, L.; Chen, S.; Jiang, S. Langmuir 2004, 20, 8931. (b)
Zheng, J.; Li, L.; Tsao, H.; Sheng, Y.; Chen, S.; Jiang, S. Biophys. J. 2005,
89, 158.
(13) Malmsten, M. J. Colloid Interface Sci. 1995, 4, 106.
(14) Hayward, J.; Chapman, D. Biomaterials 1984, 5, 135.
(15) Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O’Brien, D. F.; Saavedra,
S. S. Langmuir 2003, 19, 1766.
(7) Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. Y. J. Phys. Chem.
B 2005, 109, 2934.
(8) Lewis, A. L. Colloids Surf., B 2000, 18, 261.
(16) Holmlin, R. E.; Chen, X.; Chapman, R. G.; Takayama, S.; Whitesides, G.
M. Langmuir 2001, 17, 2841.
(9) Vermette, P.; Meagher, L. Colloids Surf., B 2003, 28, 153.
(10) Ishihara, K.; Fukumoto, K.; Iwasaki, Y.; Nakabayashi, N. Biomaterials 1999,
20, 1545.
(17) Tegoulia, V. A.; Rao, W.; Kalambur, A. T.; Rabolt, J. F.; Cooper, S. L.
Langmuir 2001, 17, 4396.
(18) Chung, Y. C.; Chiu, Y. H.; Wu, Y. W.; Tao, Y. T. Biomaterials 2005, 26,
2313.
(11) Durrani, A. A.; Hayward, J.; Chapman, D. Biomaterials 1986, 7, 121.
(12) Nakaya, T.; Li, Y. J. Prog. Polym. Sci. 1999, 143.
9
10.1021/ja054169u CCC: $30.25 © 2005 American Chemical Society
J. AM. CHEM. SOC. 2005, 127, 14473-14478
14473

A R T I C L E S
Chen et al.
Scheme 1. Synthesis route of 11-mercaptoundecylphosphorylcholine
silica using a mixed eluent (i.e., chloroform:ethanol:water, 4:8:4). The
fraction of 11-mercaptoundecylphosphorylcholine was concentrated in
vacuo by a rotary evaporator at room temperature. Anhydrous ethanol
was added to help remove water. The final product (5) is a white solid
powder (0.16 g, total yield 21%). H NMR (MeOD, 300 MHz): δ
1.27-1.48 14H, 1.55-1.75 4H, 2.51 2H, 3.24 9H, 3.65 2H, 3.88 2H,
(AFM), and evaluated the adsorption of Fg and BSA using a
surface plasmon resonance (SPR) sensor. In addition, we also
performed molecular simulation studies of the packing structure
and density of PC SAMs on Au(111) to provide molecular-
level information for the further interpretation of our experi-
mental results. The objectives of this work are to study the
nonfouling properties of PC SAMs and to provide insights into
the molecular-level nonfouling mechanism of zwitterionic
materials.
1
4.26 2H.
All intermediate compounds and the final product were characterized
by a Bruker Esquire Ion Trap LC-Mass Spectrometer. All molecular
weight peaks match well with their theoretical values. 2 is closer to
100% pure from thin-layer chromatography (TLC). In the conversion
of 2 to 3, there were some unreacted hydroxyl groups although extra
2-chloro-1,3,2,-dioxaphospholane-2-oxide was added. However, these
unreacted hydroxyl groups do not affect the conversion of 3 to 4. 3
and 4 were obtained without further purification. Due to strong
electrostatic interactions between phosphorylcholine groups and silica,
the retention factor (Rf) value of 4 is only less than 0.2 even in the
eluent with high water content (the volume ratio of ethanol to water is
8:3). The Rf value of 5 is 0.35 when eluted by 8:3 of ethanol:water and
0.55 when eluted by 8:3:0.1 of ethanol:water:HCl in volume. In the
conversion of 3 to 4, an asymmetrical disulfide containing one PC group
and one phosphate [(CH2)11OPO2H(OCH2CH2OH)] group could be
produced. This negatively charged thiol could affect the quality of PC
SAMs and protein adsorption on PC SAMs. To check for the existence
of this side product in 5, a small amount of concentrated HCl was
added to the eluent to increase the difference in Rf value between PC
thiol and HS(CH2)11OPO2H(OCH2CH2OH) thiol.
Materials and Methods
Chemicals. 11-Mercaptoundecanol, 1-octanethiol, 1-hexadecanethiol,
iodine, triethylamine, trimethylamine, tetrahydrofuran (anhydrous,
99.9%), 1,4-dithio-D,L-threitol, and sodium bisulfite were purchased
from Sigma-Aldrich and used as received. 2-Chloro-1,3,2-dioxaphos-
pholane-2-oxide, and N,N-dimethanolamine were purchased from Acros
Organics. Human Fg and BSA were also purchased from Sigma-
Aldrich.
Synthesis of 11-Mercaptoundecylphosphorylcholine. The reaction
route for the synthesis of 11-mercaptoundecylphosphorylcholine (PC
thiol) is presented in Scheme 1. A methanol solution (30 mL) containing
11-mercapto-1-undecanol (2.05 g) (1) was titrated by 1 M iodine
methanol solution until the solution turned light yellow, and then the
reaction was quenched with sodium bisulfite. 11-Hydroxyundecyl
disulfide (2) was precipitated from the methanol solution at 0 °C and
recrystallized from ethanol with a yield of 1.85 g (90%). 2 (0.407 g, 1
mmol) and triethylamine (0.35 mL, 2.5 mmol) were dissolved in 15
mL of chloroform (CHCl3). This solution was slowly added into
2-chloro-1,3,2,-dioxaphospholane-2-oxide (0.21 mL, ∼2.4 mmol) an-
hydrous THF (40 mL) solution at -18 °C. The mixture was allowed
to warm to ambient temperature over 2 h. The reaction mixture was
then cooled to 0 °C and filtered. The filtrate was concentrated in vacuo
at room temperature without further purification, and the product (3)
is a colorless to light-yellow oil. 3 and trimethylamine (0.5 mL, 5.5
mol) in anhydrous methane chloride (35 mL) at -15 °C were sealed
in a bomb, which was heated to 55 °C and stirred for 24 h. The reaction
mixture was cooled and filtered to give a white solid powder (4, 0.4
g). The white solid powder (0.4 g) was dissolved in ethanol (3 mL),
and 1,4-dithio-D,L-threitol (DTT, 0.4 g) was added. The pH of the
solution was adjusted to 9 with concentrated NH3‚H2O. The solution
was stirred for half an hour and purified by flash chromatography on
SAM Preparation and Characterization by X-ray Photoelectron
Spectroscopy. Gold-coated chips were washed with ethanol and cleaned
in a UV cleaner for 15 min. They were dipped into either (acidic)
ethanolic or (basic) aqueous solution of 0.1 mM PC thiol overnight.
The chips covered with alkanethiolate SAMs and PC SAMs were
washed with ethanol and Millipore water, respectively, and then dried
with filtered, compressed air. The procedure was repeated three times.
PC samples analyzed by XPS were put in a vacuum chamber within
30 min of preparation or stored in N2 before they were put in the
vacuum chamber. XPS measurements were conducted using a Surface
Science Instrument X-Probe spectrometer (Mountain View, CA)
equipped with a monochromatic Al KR source (KE ) 1486.6 eV), a
Hemispherical analyzer, and a multichannel detector. All XPS data were
9
14474 J. AM. CHEM. SOC. VOL. 127, NO. 41, 2005

Protein Resistance of PC Self-Assembled Monolayers
A R T I C L E S
Table 1. Elemental Compositions of PC SAMs on Au(111)
Determined from XPSa
acquired at a nominal photoelectron takeoff angle of 55°. SSI data
analysis software was used to calculate elemental compositions from
peak areas.
elements
theory
pH 10
acidic ethanol solutions
SAM Characterization by Ellipsometry. Ellipsometry was per-
formed using a spectroscopic ellipsometer (Sentech SE-850, GmbH).
Sample preparation is the same as in XPS experiments. Five separate
spots were measured at three different angles of incidence (50°, 60°,
and 70°) in the vis region. The same batch of gold-coated chips was
cleaned by UV-ozone cleaner for 20 min, washed with ethanol and
Millipore water, and dried with filtered air. These bare gold-coated
chips were used as reference. The thicknesses of films under study
were determined using the Cauchy layer model with an assumed
C
N
P
O
S
69.6
4.4
4.4
17.7
4.4
63.2
4.0
3.8
26.6
2.3
62.1
3.9
4.5
27.3
2.2
a The gold ratio is 23 ( 0.7% at pH 10 and 22.8 ( 0.9% from acidic
ethanol solution if gold is counted.
quality of PC SAMs and protein adsorption. Thus, by reducing
the disulfide to a thiol under mild conditions, pure PC thiol
can be obtained with simpler purification and higher yield (21%
after purified by flash chromatography).
16
refractive index of 1.45 and 1.50.19
Protein Adsorption by Surface Plasmon Resonance Sensor. A
custom-built highly sensitive SPR biosensor was used. Unlike many
SPR sensors based on angular measurements,20,21 this SPR biosensor
is based on wavelength interrogation. It was reported in our previous
studies that 5 ng/mL of staphylococcal enterotoxin B (SEB) was
detectable by a direct method and 0.5 ng/mL SEB with a sandwich
method using this sensor.22 The chips were coated with adhesion-
promoting chromium layer (thickness ≈ 2 nm) and surface plasmon
active gold layer (thickness ) 50 nm) by electron beam evaporation
in a vacuum. The chip modified with a sensing layer was attached to
the base of the prism, and optical contact was established using
refractive index matching fluid (Cargille). A dual-channel Teflon flow
cell containing two independent parallel flow channels with small
chambers was used to contain a liquid sample during experiments. A
peristaltic pump (Ismatec) was utilized to deliver liquid sample to the
two chambers of the flow cell. The flow rate of 0.05 mL/min was used
throughout experiments.
All protein adsorption measurements were performed in physiological
PBS solution (10 mM sodium phosphate, 138 mM NaCl, and 2.7 mM
KCl at pH 7.4). SPR was first stabilized with PBS solution for 20 min,
then the protein solution was flowed into the system at 1 mg/ml for 10
min, and the SPR was then flushed with PBS solution for 5 min.
Nanografting. A Nanoscope IV AFM (Veeco, CA) equipped with
E scanner was used for nanografting. A sample was mounted inside a
quartz fluid cell, which allowed the injection and removal of solution
from the flow cell for AFM lithography and imaging in a liquid
environment. Si3N4 cantilevers with a force constant of 0.32 N/m were
used. The AFM tip was used to remove C16 SAM chains within an
area of 100 nm × 80 nm in an aqueous solution (pH 10) containing
PC thiol (0.4 mM) under a minimal force of 5 nN. When C16 chains
were removed, PC thiols refilled the empty space. Both topographic
and frictional AFM images were acquired under 0.2 nN.
Preparation and Characterization of PC SAMs. The
quality of a PC SAM is critical to its nonfouling property and
will depend on its preparation conditions. As discussed above,
a trace amount of OPO2H(OCH2CH2OH)-terminated thiol from
impurity in the final product could alter the properties of PC
SAM from neutral to negatively charged, leading to protein
adsorption. In this work, we prepared PC SAMs from an acidic
ethanol solution and from an aqueous solution under the basic
condition at pH 10. An ethanol solution of PC thiol prepared
from PC thiol dried under vacuum is slightly acidic. It was found
that the N/P ratio of ∼1:1 was achieved for PC SAMs formed
from a basic assembly solution, while a ∼18% lower N/P ratio
was observed under an acidic assembly solution (Table 1). From
Table 1, under both basic and acidic conditions, bound sulfur
species and the relative film thickness based on gold elemental
percentages or C/Au ratios do not show obvious changes.
Although only a trace amount of phosphate-terminated thiol
occurs in our TLC results, it could preferentially absorb in acidic
conditions. PC SAMs prepared in a basic aqueous solution of
PC thiol will suppress the assembly of phosphate-terminated
thiol. The composition of PC SAMs formed under basic
conditions agrees well with the theoretical value except for
slightly lower elemental percentages of sulfur and higher
percentages of oxygen (Table 1). The slightly lower amount of
sulfur is due to the signal attenuation of bound sulfur species
on the gold surface by the top organic layer. The high-resolution
spectra of the S2p region from XPS shows that most of the sulfur
species are bound to the surface, with very low amounts of
unbound or oxidized sulfur species (Figure 1).23,24 XPS results
show that high-quality PC SAMs are formed. It can be seen
from Table 1 that the elemental composition of oxygen is higher
than the theoretical value, which was also observed previously
in other work.17,18 Since few oxidized sulfur species are
observed, oxidized sulfur species do not account for the large
increase in oxygen content. In the previous work on MPC
polymers, it was found that each MPC monomer contained two
hydrated water molecules.25 Some of these tightly associated
water molecules would remain even under high vacuum in XPS
experiments and contribute to the higher oxygen content. The
similar phenomenon of tightly associated water on cadmium
carboxylate layers was observed in earlier XPS experiments.26
Results and Discussion
Synthesis of PC Thiol. In this work, PC thiol was synthesized
following a modified scheme (Scheme 1) based on the methods
16
reported by Holmlin et al.
and Chung et al.18 with two
considerations. For synthesis of a thiol from a thioester in the
conventional method, such as the one by Holmlin et al.,16 the
thioester group is converted to the thiol group by hydrolysis,
which could also hydrolyze the phosphorylcholine group. When
starting with a disulfide, one can avoid the undesirable hydroly-
sis step, particularly after the formation of the PC group. For
synthesis of PC thiols starting from a disulfide by Chung et al.,
it is hard to purify the asymmetrical disulfide containing one
PC group and one phosphate group, which will later affect the
(19) Fabianowski, W.; Coyle, L. C.; Weber, B. A.; Granata, R. D.; Castner, D.
G.; Sadownik, A.; Regen, S. L. Langmuir 1989, 5, 35.
(20) Liedberg, B.; Lundstrom, I.; Stenberg, E. Sens. Actuators, B 1993, 11, 63.
(21) Melendez, J.; Carr, R.; Bartholomew, D. U.; Kukaskis, K.; Elkind, J.; Yee,
S. S.; Furlong, C.; Woodbury, R. Sens. Actuators, B 1996, 35-36, 212.
(22) Homola, J.; Dosta´lek, J.; Chen, S. F.; Rasooly, A.; Jiang, S. Y.; Yee, S. S.
Int. J. Food Microbiol 2002, 75, 61.
(23) Sun, F.; Grainger, D.; Castner, D.; Leachscampavia, D. Macromolecules
1994, 27, 3053.
(24) Castner, D.; Hinds, K.; Grainger, D. Langmuir 1996, 12, 5083.
(25) Umeda, T.; Nakaya, T.; Imoto, M. Makromol. Chem. 1982, 3, 457.
(26) Roberts, G. G. AdV. Phys. 1985, 34, 475.
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A R T I C L E S
Chen et al.
Figure 3. Fg adsorption on mixed C8 and PC SAMs from PBS buffer.
With the increase of C8 composition, Fg adsorption increases.
Figure 4. (a) Molecular configuration of PC SAMs from molecular
simulations and (b) antiparallel orientation of PC headgroups for dipole
minimization.
Figure 1. High-resolution spectra of the S2p region from XPS for
11-mercaptoundecylphosphorylcholine SAMs prepared from a solution at
(a) acidic and (b) pH 10. The peaks were fit using two S2p doublets with
2:1 area ratio and splitting of 1.2 eV. The high-resolution sulfur data show
up to 89% sulfur species at 162 eV, indicating that the majority of sulfur
species is bound to the gold surface.
Thus, the PC headgroups are intrinsically nonfouling. It should
be emphasized that the N/P ratio is critical to the nonfouling
behavior of a zwitterionic surface for charge balance. Comparing
our XPS results in Table 1 with those published previously,17,18
it can be seen that the N/P ratio from our PC thiol SAM formed
at pH 10 is 1.05:1, which is very close to the theoretical value
of 1:1 and which has 1% of a ML of Fg adsorption. The N/P
ratio of our PC thiol SAM formed at acidic ethanol solution
was 0.87:1, while Fg adsorption on the PC SAM is 3.5% of a
ML. This may be the key reason our PC surface resists protein
adsorption. However, in the work by Tegoulia et al.17 and Chung
et al.,18 the N/P ratio is 0.59:1, and protein adsorption is
relatively high. It should be pointed out that different methods
were used to measure protein adsorption on PC SAMs, such as
angular measurement-based SPR sensors,16 wavelength inter-
rogation-based SPR sensor in this work, radio-label,17 and
QCM.18 Each method has its own assumption to convert its
response to the amount of adsorbed proteins. Thus, the absolute
amount of adsorbed protein is often hard to obtain. For fair
comparison, the percentage of protein (Fg or BSA) adsorption
presented in this work is normalized with respect to that on
methyl-terminated SAMs or hydrophobic surfaces as measured
with the same method. Furthermore, since protein adsorption
depends on the ionic strength and the pH value of a protein
solution, protein adsorption is compared in physiological PBS
buffer. Therefore, the N/P ratio from XPS could serve as an
indicator for the quality of the PC SAMs.
Figure 2. Adsorption of 1 mg/ml Fg and BSA in PBS buffer (0.13 M and
pH 7.4) on PC SAMs from SPR. The typical SPR wavelength shifts for Fg
and BSA adsorption on methyl-terminated SAMs are 16 nm7 and 7 nm,33
respectively; 1 nm wavelength shift in SPR is equivalent to 0.15 mg/m2
adsorbed proteins.
In addition, the contact angle of PC SAMs measured with
Millipore water under ambient condition is 17 ( 2°.
Protein Adsorption on PC SAMs. The adsorption of Fg and
BSA on PC-terminated SAMs prepared from the aqueous
solution of PC thiol at pH 10 was investigated by an SPR sensor.
It is shown in Figure 2 that the adsorbed amounts of Fg and
BSA on PC SAMs are 0.03 mg/m2 (or 1% of a ML) and 0.01
mg/m2 (or 0.7% of a ML) from PBS buffer containing 1 mg/
mL Fg and BSA, respectively. Protein adsorption is much lower
on our PC SAMs than on those PC SAMs reported previously
and is even lower than on the redox(poly) bis-SorbPC surface.15
Protein Adsorption on Mixed SAMs. Protein adsorption on
mixed PC and CH3 SAMs was also investigated. It was shown
in our previous work that OEG SAMs of moderate surface
density were more resistant to protein adsorption than the
compact OEG SAM due to a large number of hydration water
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Protein Resistance of PC Self-Assembled Monolayers
A R T I C L E S
Figure 5. In situ AFM lithography for determining the height of PC SAMs on Au(111). The background is HS(CH2)15CH3 (C16) SAMs and the dark 100
nm × 80 nm area in the topographic image (a) is back-filled with PC thiol. The corresponding white square in frictional image (b) shows higher friction on
PC SAMs than on C16 SAMs. The cross-sectional analysis (c) shows ∼5.6 Å height difference between C16 SAM and PC SAM.
molecules around the OEG chains and the more flexible
structure of the OEG chains.6,7 Results in Figure 3 show that
protein adsorption increases on mixed SAM surfaces coadsorbed
from an (acidic) ethanol solution of PC and HS(CH2)7CH3 (C8)
thiols. The amount of adsorbed proteins on mixed SAMs
coadsorbed from PC and C8 thiols even containing only 1%
C8 is 3 times higher than that on the PC SAM formed in an
(acidic) ethanol solution and 10 times higher than that on the
PC SAM formed in an aqueous solution at pH 10 (Figure 2).
In fact, our simulation results show that pure PC SAMs have
packing densities similar to those of PC lipids and the charge
interaction of PC groups is the dominant factor for the packing
density. Thus, it is not surprising that pure PC SAMs are more
resistant to protein adsorption than mixed PC SAMs. Further-
more, AFM images of mixed-SAM surfaces show strong phase
segregation. This is also a part of the reason for higher protein
adsorption even with a small amount of C8 thiol in the
coadsorption solution. This result also indicates that the purity
of PC thiols is important to the formation of protein-resistant
PC SAMs.
Molecular Packing Structure of PC SAMs. To provide
molecular-level information regarding the packing structures and
properties of PC SAMs, we performed molecular simulation
studies of PC SAMs on Au(111). All simulations were
performed using the BIO_SURFACE6 program developed in
our group, capable of simulating biomolecular interactions with
various material surfaces. Molecular mechanics was performed
first in continuous medium followed by molecular dynamics
simulations at 300 K in both implicit and explicit solvents. The
all-atom CHARMM27 force field was used. To obtain the
lowest-energy structure of PC SAMs on Au(111), we need to
consider two important parameters in our molecular simula-
tions: (a) the lattice structure of PC SAMs ranging from three
to eight gold atoms per unit cell and (b) the packing pattern of
a pair of PC chains, which could have the parallel, antiparallel,
or random orientation of their dipoles (the vector pointing from
P to N). A simulation box containing 36 PC chains was used in
this work, and all possible lattice structures and packing patterns
were considered. Our simulation results show that the lowest-
energy configuration of PC SAMs has a (x7×x7)R60° lattice
structure with a thickness of 14.4 Å. This corresponds to a chain-
chain spacing of ∼7.63 Å or an area of 50.42 Å2 per chain,
which is very close to that of membrane lipids obtained from
X-ray deflection experiments.27 Unlike lipids for which each
PC headgroup has two alkyl chains, each of the PC headgroups
in our PC SAMs has only one alkyl chain. In our AFM studies,
it was found that PC SAMs were more easily penetrated by an
AFM tip, indicating that PC SAMs have less dense alkyl chains
than alkanethiolate SAMs or lipid layers. Our simulation results
also show the angle between the vector pointing from P to N
and the normal gold surface is about 80°, indicating that the
orientation of PC headgroups lies nearly parallel to the gold
surface as shown in Figure 4a. This was also observed
previously for lipids.28 Furthermore, our simulation results show
that PC headgroups prefer to have an antiparallel orientation
with respect to each other as shown in Figure 4b. This was
also observed for zwitterionic peptide amphiphiles.29 With the
antiparallel configuration, the electrostatic energy and net dipole
moment of PC SAMs are minimized. Large dipole accumulation
on the surfaces will lead to protein adsorption. Detailed
simulation results will be published elsewhere.
(27) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta
1981, 650, 21.
(28) Sheng, Q.; Schulten, K.; Pidgeon, C. J. Phys. Chem. B 1995, 99, 11018.
(29) Tsonchev, S.; Troisi, A.; Schatz, G. C.; Ratner, M. A. J. Phys. Chem. B
2004, 108, 15278.
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A R T I C L E S
Chen et al.
seen that the spot having lower friction is higher in the
topographic image in Figure 6a. As can be seen from Figure 5,
C16 has lower friction. Thus, C16 chains are higher than PC
chains in the mixed SAMs.
The thickness of the PC SAMs was also measured by
ellipsometry to be 9.5 ( 0.2 or 8.9 ( 0.2 Å if a refractive index
of 1.4516 or 1.5019 is used. It should be pointed out that 1.45
and 1.50 are obtained for C16 thiol and phosphorylcholine lipid,
respectively. They are also used to describe the compact
structure of C16 SAMs and lipid bilayers. However, PC SAMs
have loose structures with (x7×x7)R60° (∼50% less dens than
CH3 and OH SAMs). Thus, it is expected that the thickness of
PC SAMs obtained from ellipsometric measurements is sig-
nificantly lower than their actual height if the refractive index
of 1.45 or 1.50 is used. Therefore, AFM results confirm those
from simulations that the orientation of PC headgroups lies
nearly parallel to the gold surface. Further ellipsometric
measurements suggest that the packing density of PC SAMs
should be lower than that of CH3 SAMs as also predicted in
molecular simulations.
Figure 6. Phase-segregated C16- and PC-mixed SAM for determining the
height of PC SAMs on Au (111) by AFM. The features (e.g., the one marked
by an arrow) having lighter color in the topographic image (a) are darker
in the corresponding frictional image (b).
The Thickness of PC SAMs. The thickness of PC SAMs
would be 21.2 Å (equivalent to that of C18 SAMs) if the PC
chains are all-trans and tile 30° from the normal gold surface
as do CH3- and OH-terminated alkanethiols on gold surfaces.5,30
Our molecular simulation results show that the angle between
the vector pointing from P to N and the normal gold surface is
about 80°, and the thickness of PC SAMs is 14.4 Å. In this
work, two AFM experiments were used to estimate the thickness
of PC SAMs and to verify the simulation results. One is the in
situ nanografting of PC thiols on HS(CH2)15CH3 (C16) SAMs
using AFM,31 in which the AFM tip scans over C16 SAMs to
remove C16 chains within a certain area, and PC thiols in the
solution then fill the empty space. The AFM tip scans again
with low force to obtain an image. It is found in the nanografting
experiment that C16 SAMs are higher than PC SAMs by ∼5.6
Å (Figure 5). Thus, the thickness of PC SAMs is 13.7 Å with
the thickness of C16 equal to 19.3 Å.5,30 To exclude the
possibility of gold layer plastic deformation by extremely high
forces in AFM,32 a minimal force (∼5 nN) was applied to
remove the C16 thiol. Since PC SAMs in the nanografting area
were formed in a short time before being imaged, these
(immature) SAMs could be lower in height than those (mature)
PC SAMs formed overnight. In the second method, mixed
SAMs were formed in two steps. First, cleaned Au(111) chips
were immersed in a solution of 1 mM C16 thiol for 10 min,
rinsed thoroughly, and immersed in a solution of 0.1 mM PC
thiol overnight. The topographic image of mixed PC and C16
SAMs in Figure 6a shows phase segregation with 2-4 Å height
difference. From the frictional image in Figure 6b, it can be
Conclusions
It is demonstrated in this work that zwitterionic PC SAMs
are highly resistant to protein adsorption. Our experimental
results show that PC SAMs have very low protein adsorption
when the N/P ratio is close to 1:1 and the charges are balanced.
The N/P ratio from XPS could be a good indicator of charge
balance. Due to the strong dipole of zwitterions, appropriate
packing is needed to minimize their net dipole. Our simulation
results show that PC head groups have similar packing densities
to membrane lipids and prefer to have an antiparallel orientation
for dipole minimization. Although zwitterions have a strong
hydration layer via electrostatic interactions and should highly
resist protein adsorption, balanced charge and minimized dipole
are two key factors for their nonfouling behavior.
Acknowledgment. We thank Prof. Buddy Ratner and Dr.
Esmaeel Naeemi for helpful discussion. This work is supported
by the Office of Naval Research (N000140410409). The XPS
experiments were performed at the National ESCA and Surface
Analysis Center for Biomedical Problems (NESAC/BIO) sup-
ported by NIBIB Grant EB02027. Ellipsometric measurements
were performed by Petr Tobiska in Jiri Homola’s group at the
Institute of Radio Engineering and Electronics, Academy of
Sciences of the Czech Republic.
(30) Bain, C. D.; Whitesides, G. M. J. Phys. Chem. B 1989, 93, 1670.
(31) Liu, G. Y.; Xu, S.; Qian, Y. L. ACC. Chem. Res. 2000, 33, 457.
(32) Salmeron, M.; Folch, A.; Neubauer, G.; Tomitori, M.; Ogletree, D. F.;
Kolbe, W. Langmuir 1992, 8, 2832.
(33) Li, L. Y.; Chen, S. F.; Jiang, S. Langmuir 2003, 19, 2974.
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