6912 Langmuir 2003, 19, 6912-6921

Covalent Attachment of Poly(ethylene glycol) to Surfaces,

Critical for Reducing Bacterial Adhesion
Peter Kingshott,*,† Jiang Wei,† Dorthe Bagge-Ravn,‡ Nikolaj Gadegaard,† and
Lone Gram‡
Danish Polymer Centre, Risø National Laboratory, Building 124, P.O. Box 49,
DK-4000 Roskilde, Denmark, and Department of Seafood Research, Danish Institute for
Fisheries Research, Søltofts Plads, c/o Technical University of Denmark, Building 221,
DK-2800 Kgs. Lyngby, Denmark

Received January 8, 2003. In Final Form: May 28, 2003

The effects of different poly(ethylene glycol) (PEG) attachment strategies upon the adhesion of a Gram-
negative bacteria (Pseudomonas sp.) was tested. PEG was covalently immobilized, at the lower critical
Downloaded via UNIV NACIONAL AUTONOMA MEXICO on November 14, 2022 at 20:37:54 (UTC).

solution temperature of PEG, to a layer of branched poly(ethylenimine) (PEI). PEI was both physically
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adsorbed to a stainless-steel (SS) substrate and covalently immobilized to a carboxylated poly(ethylene

terephthalate) (PET-COOH) surface. On both substrates, the PEI and PEG grafting conditions were
optimized so that the levels of surface coverage after each step were maximized and were the same on both
substrates, as judged by X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry
(ToF-SIMS). Also, ToF-SIMS imaging showed that both substrates were chemically uniform after each
surface modification step. Thus, the two surfaces differ only in the mode of attachment of PEI to the
substrate. In bacterial adhesion experiments, the optimal SS-PEG surface was not capable of reducing
the number of adherent Pseudomonas sp. when compared to the controls. However, the PET-PEG surface
reduced the level of adhesion by between 2 and 4 orders of magnitude for up to 5 h. ToF-SIMS analysis
showed that both PEG surfaces adsorbed low but comparable levels of proteinaceous growth medium
components (tryptic soy broth), as indicated by the addition of unique amino acid fragment ions in the
spectra, most likely small peptides. Thus, bacterial adhesion was strongly dependent on the PEG
immobilization strategy and not on the extent of peptide/protein adsorption. However, for the best PEG
surfaces the residual bacterial adhesion is most likely from recognition of the small amount of adsorbed
peptides. This highlights the necessity for preventing the adsorption of small biological species that can
even penetrate PEG layers of high graft density, in the quest for the ultimate “nonfouling” surface.

Introduction components of a protein film,5 or specific binding to sugar

Bacterial colonization of surfaces has a major impact
in a broad range of technologically significant areas.
Examples where detrimental consequences arise in-
clude: (1) the medical device industry where implants
with adherent bacteria cause infections,1 (2) the food
processing industry where lack of hygiene can cause food
spoilage or pose a serious risk to public health,2 and (3)
industries where degradation of materials by adherent
bacteria is very costly,3 to name a few.
Pseudomonas sp. is one bacterium that is ubiquitous in
the environment that tenaciously and rapidly colonizes
surfaces. The colonies eventually develop into mature
biofilms and drastically increase the risk of severe
infections with improper hygiene.4 The initial attachment
of bacteria is speculated to be mediated by either the
specific recognition of elements on the surface, such as
* Corresponding author. Telephone ++ 45 4677 5480. Fax: ++
45 4677 4971. E-mail: peter.kingshott@risoe.dk.
† Risø National Laboratory.
‡ Danish Institute for Fisheries Research.
receptors.6 Also, nonspecific, physicochemical interactions
such as electrostatic or hydrophobic interactions between
the bacteria and the surface can play a role in adhesion.7
Once the bacteria are attached, the transcription of specific
genes is activated, resulting in the synthesis of ex-
opolysaccharides (EPSs; or slime) that encase the bacteria
and enable them to form a fully established biofilm.8
Biofilms are exceedingly resistant to antibiotics and
biocides as a result of the impenetrable slime protecting
the bacteria.9 Therefore, preventing biofilm formation
requires the development of surfaces that could ideally
fully resist the initial attachment of the bacteria, or at
least discourage them from “switching on” the genes that
enable them to produce EPSs and develop microcolonies
that eventually lead to the formation of a mature biofilm.4
To date no surface exists that can fully prevent bacteria
adhesion, and this remains a major challenge to the field
of biological surface science, defined as the interdiscipli-
nary area where properties and processes at interfaces
between synthetic materials and biological environments
are investigated and new materials are designed.10 From

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10.1021/la034032m CCC: $25.00 © 2003 American Chemical Society

Published on Web 07/23/2003
Grafting of PEG To Reduce Bacterial Adhesion
the literature, poly(ethylene glycol) (PEG) as a surface
modifying agent is clearly the most effective molecule at
reducing bioadhesion (protein adsorption, bacterial and
cell adhesion).11-16,54 The best reports have shown that
protein adsorption can be suppressed to a fraction of the
uncoated surface or even remain undetected when ana-
lyzed by the most sensitive analytical methods.12-15,17,18
However, the most promising reports investigating bacte-
rial adhesion to PEG surfaces have so far only been
marginally successful, by suppression of at best up to 1-2
orders of magnitude.19-23 Why is this the case? Theoretical
considerations predict that PEG can fully prevent
adhesion;24-26 however, the question remains whether
these predictions can ever be put into practice by experi-
ments: in particular, the generation of stable PEG layers
with a sufficiently high graft density and surface uni-
formity to provide the optimal steric repulsive barrier
against bioadhesion. Furthermore, investigations into the
specific structural properties and physicochemical forces
involved in the interaction of proteins and biological
material with PEG layers27,28 would greatly assist in the
design of the ultimate surface. Finally, the design of the
ultimate “nonfouling” surface is dependent on being able
to detect protein adsorption below the threshold where no
subsequent events can occur (such as bacterial adhesion).
This requires extremely sensitive surface analytical tools
capable of detecting fractions of a monolayer. In addition,
many studies have demonstrated that adsorption from
single solutions of model proteins can be prevented by
PEG. However, recent work has shown that low-molecular-
weight proteins and peptides can adsorb from complex
biological media, such as human tears, onto surfaces such
as contact lenses.29 Preventing the adsorption of low-
molecular-weight proteins (<5000), which may be able to
penetrate PEG layers and render the surface ineffective,
is, thus, highly desirable.
In this report, we attempt to address some of these
issues, such as how optimal PEG coverage, uniformity,
(11) Harris, J. M., Ed. Poly(ethylene glycol) ChemistrysBiotechnical
and Biomedical Applications; Plenum Press: New York, 1992.
(12) Sofia, S. J.; Premnath, V.; Merrill, E. W. Macromolecules 1998,
31, 5059-5070.
(13) Malmsten, M.; Emoto, K.; Van Alstine, J. J. Colloid Interface
Sci. 1998, 202, 507-517.
(14) Kingshott, P.; Thissen, H.; Griesser, H. J. Biomaterials 2002,
23, 2043-2056.
(15) Kingshott, P.; Griesser, H. J. Curr. Opin. Solid State Mater. Sci.
1999, 4 (4), 403-412.
(16) Huang, N.-P.; Michel, R.; Voros, J.; Textor, M.; Hofer, R.; Rossi,
A.; Elbert, D. L.; Hubbell, J. A.; Spencer, N. D. Langmuir 2001, 17,
489-498.
(17) Ostuni, E.; Chapman, R. G.; Liang, M. N.; Melulent, G.; Pier,
G.; Ingber, D. E.; Whitesides, G. M. Langmuir 2001, 17, 6336-6343.
(18) Chapman, R. G.; Ostuni, E. O.; Liang, M. N.; Meluleni, G.; Kim,
E.; Yan, L.; Pier, G.; Warren, H. S.; Whitesides, G. M. Langmuir 2001,
17, 1225-1233.
(19) Holmberg, K.; Bergstrom, K.; Brink, C.; Osterberg, E.; Tiberg,
F.; Harris, J. M. J. Adhes. Sci. Technol. 1993, 7, 503-517.
(20) Hendricks, S. K.; Kwok, C.; Shen, M.; Horbett, T. A.; Ratner, B.
D.; Bryers, J. D. J. Biomed. Mater. Res. 2000, 50, 160-170.
(21) Vacheethasanee, K.; Marchant, R. E. J. Biomed. Mater. Res.
2000, 50, 302-312.
(22) Cunliffe, D.; Smart, C. A.; Alexander, C.; Vulfson, E. N. Appl.
Environ. Microbiol. 1999, 65, 4995-5002.
(23) Park, K. D.; Kim, Y. S.; Han, D. K.; Kim, Y. H.; Lee, E. H. B.;
Suh, H.; Choi, K. S. Biomaterials 1998, 19, 851-859.
(24) Jeon, S. I.; Lee, J. H.; Andrade, J. D.; De Gennes, P. G. J. Colloid
Interface Sci. 1991, 142, 149-158.
(25) Jeon, S. I.; Andrade, J. D. J. Colloid Interface Sci. 1991, 142,
159-166.
(26) Szleifer, I. Physica A 1997, 244, 370-388.
(27) Sheth, S. R.; Leckband, D. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 8399-8404.
(28) Feldman, K.; Hahner, G.; Spencer, N. D.; Harder, P.; Grunze,
M. J. Am. Chem. Soc. 1999, 121, 10134-10141.
(29) Kingshott, P.; St. John, H. A. W.; Chatelier, R. C.; Griesser, H.
J. J. Biomed. Mater. Res. 2000, 49, 36-42.
Langmuir, Vol. 19, No. 17, 2003 6913
and surface stability influence bacterial adhesion. In
particular, we aim at preventing the colonization of a
Gram-negative bacterium (Pseudomonas sp.) relevant to
processing equipment in the fish industry.30,31 The linear
PEG [molecular weight (MW) 5000] in our study is
attached to both stainless-steel (SS) and poly(ethylene
terephthalate) (PET) substrates. The graft density of PEG
is optimized in a two-step process: by (1) attachment to
an intermediate branched poly(ethylenimine) (PEI; MW
25 000) linker layer that contains a very high level of free
reactive amino groups for the PEG immobilization and
(2) grafting at the lower critical solution temperature
(LCST) of PEG, which is necessary for maximal surface
coverage. The only difference between the two final PEG
surfaces is the mode of PEI attachment. In the case of SS,
it is physically adsorbed through strong electrostatic
interactions, and in the case of PET, it is covalently
immobilized after the generation of reactive groups on
PET by a wet-chemical method. The use of surface
characterization, including X-ray photoelectron spectros-
copy (XPS) and time-of-flight secondary ion mass spec-
trometry (ToF-SIMS), were used to optimize the surface
modification strategies and also investigate protein ad-
sorption from the bacterial growth medium.
Experimental Section
Materials. PET film (Mylar, 150-µm thickness) was purchased
from Trafoma A/S (Denmark). AISI 316 SS sheets (1-mm
thickness) were purchased from Avesta Polarit (Finland).
N-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride
(EDAC), N-hydroxysuccinimide (NHS), sodium cyanoborohydride
(NaCNBH3), trifluoroacetic anhydride (TFAA), and bromoacetic
acid were all purchased from the Sigma Chemical Company.
Pentafluorophenol (PFP) was purchased from Fluka, and PEI
(MW 25 000) was purchased from Aldrich. Linear methoxy-
terminated PEG (M-PEG-aldehyde) was purchase from Shear-
water Polymers, Inc. All chemicals were analytical grade or better.
Functionalization of PET. PET substrates were function-
alized by wet chemistry according to a published method32-34 to
initially generate hydroxyl groups, which are then converted
into carboxyl groups. Substrates (2 × 3 cm) were cleaned in
acetone (5 min), followed by methanol (5 min), and then dried
with a jet of CO2. Substrates were initially hydroxylated (PET-
OH) by exposure to 18.5 vol % formaldehyde in 1 M acetic acid
for 4 h at room temperature, followed by copious rinsing with
Milli-Q H2O (Figure 1B). The surface hydroxyl groups on the
PET-OH substrates were derivatized in a TFAA vapor according
to the method of Pan et al.35 (Figure 1C). In brief, substrates
were placed in the bottom of a small watch glass, which was
placed in a glass Petri dish. TFAA (0.5 mL) was pipetted into the
Petri dish, and the lid was sealed with paraffin wax. The reaction
was allowed to proceed for 1 h prior to XPS analysis. The hydroxyl
groups were carboxymethylated (PET-COOH) by overnight
exposure to 1 M bromoacetic acid in 2 M NaOH at room
temperature (Figure 1D), followed by copious rinsing with Milli-Q
H2O. PFP was used to specifically derivatize introduced carboxyl
functionality according to the method of Hyun et al.34 (Figure
1E). Both 0.1 M EDAC and 0.2 M PFP were dissolved in 10 mL
of ethanol (99%) and a PET-COOH substrate was added. The
reaction was allowed to proceed for 20 min, and the substrate
was copiously rinsed three times with ethanol and stored in
ethanol prior to XPS analysis. PET and PET-OH samples were
run as controls.
(30) Vogel, B. F.; Huss, H. H.; Ojeniyi, B.; Ahrens, P.; Gram, L. Appl.
Environ. Microbiol. 2001, 67, 2586-2595.
(31) Bagge-Ravn, D.; Ng, Y.; Hjelm, M.; Christiansen, J. N.; Johansen,
C.; Gram, L. Int. J. Food Microbiol. 2002, in press.
(32) Massia, S. P.; Hubbell, J. A. J. Biomed. Mater. Res. 1991, 25,
223-242.
(33) Yang, Z.; Chilkoti, A. Adv. Mater. 2000, 12 (6), 413-417.
(34) Hyun, J.; Zhu, Y.; Liebmann-Vinson, A.; Beebe, T. P., Jr.; Chilkoti,
A. Langmuir 2001, 17 (20), 6358-6367.
(35) Pan, S.; Castner, D. G.; Ratner, B. D. Langmuir 1998, 14, 3545-
3550.
6914 Langmuir, Vol. 19, No. 17, 2003
Figure 1. Reaction schemes for the wet chemical modification
of PET. Included in the scheme are the reactions for the
derivatization of the surface functional groups.
The PET-COOH substrates were further functionalized with
PEI to generate an aminated surface (PET-COO-NH2) using
water-soluble carbodiimide chemistry (Figure 1F). Both 0.1 M
′′EDAC and 0.1 M NHS were dissolved in PEI solution (either
1 or 30 mg/mL in phosphate buffered saline (PBS; pH 7.4). PET-
COOH substrates were immediately added and shaken gently
for 2 h, followed by copious rinsing with Milli-Q H2O.
Functionalization of SS. The AISI 316 SS sheets were cut
to a size of 10 × 20 mm. The disks were cleaned by immersion
in a Piranha solution (3:1 solution of concentrated H2SO4 and
30% H2O2) for 10 min, followed by a thorough rinsing with Milli-Q
water. (CAUTION: Piranha solution is explosive and should
not be placed in contact with organic solutions.) PEI was dissolved
in water to a concentration of either 1 or 30 mg/mL. After cleaning
of the SS disks, they were immediately immersed in PEI solution
for 2 h at room temperature to minimize the adsorption of
contaminating atmospheric hydrocarbon and were then thor-
oughly rinsed three times with water and air-dried. The use of
a 30 mg/mL PEI concentration has been shown to produce an
optimal PEI layer.36
Grafting of PEG. M-PEG-aldehyde was grafted to the
PET-COO-NH2 and SS-NH2 substrates at the LCST of PEG
according to the method of Kingshott et al.14 K2SO4 (0.6 M) was
dissolved in PBS (pH 7.4) containing M-PEG-aldehyde (1 mg/
mL) and NaCNBH3 (3 mg/mL) and incubated at 60 °C overnight,
followed by copious rinsing with Milli-Q H2O (Figure 1G).
Surface Characterization. XPS analysis was performed on
all samples using a SSX-100 X-ray photoelectron spectrometer
(Surface Science Laboratories, U.S.A.), with a monochromatized
Al KR X-ray source (1487 eV) at a power of 150 W. The pressure
in the main chamber during the analysis was in the range of 10-8
mbar. Survey scans were recorded using a 1000-µm spot size and
a pass energy of 150 eV. High-resolution spectra of the individual
elements were recorded with a 600-µm spot size and a pass energy
of 50 eV. Atomic concentrations of each element were calculated
(36) Wei, J.; Bagge-Ravn, D.; Gram, L.; Kingshott, P. Colloid Surf.,
B, accepted for publication.
Kingshott et al.
by determining the relevant integral peak intensities using a
linear-type background. The systematic error is estimated to be
of the order of 5-10%. Curve-fitting was performed using the
Spectra Data Processor Version 2.3 software using a linear
background subtraction. All binding energies are referenced to
the hydrocarbon component (CHx) at 285.0 eV.
Surfaces were also characterized with a time-of-flight second-
ary ion mass spectrometer (Ion-Tof GmbH, Münster, Germany).
A pulsed 25-kV (1.7-mA current) 69Ga+ primary ion beam incident
at 45° to the surface normal was used for analysis. The pulse
width for spectral acquisition was 200 ns. The ion dose of primary
ion was maintained to ensure static conditions (less than 1012
ions/cm2) and avoid sample damage. A low-energy electron gun
was used for charge compensation on insulating samples. The
primary ion beam was rastered over the surface with an area of
100 × 100 µm. The resulting secondary ions were analyzed with
the ToF mass analyzer equipped with a reflectron. The mass
resolution (M/∆M) was typically >5000 in positive mode. Spectra
were calibrated using the known masses of hydrocarbon ions
(H+, C+, CH+, CH2+, CH3+, C3H2+, C4H6+, C5H9+, C6H5+, and
C7H7+). SIMS imaging was obtained by selecting masses of
interest and recording their intensities with respect to the position
of the primary ion. The analysis conditions were the same as
those used for spectral acquisition.
Atomic force microscopy (AFM) measurements were performed
in air on a Digital Instruments Dimension 3000 in tapping mode
using standard Si3N4 tips.
Bacterial Adhesion. A Gram-negative bacterium (Pseudomo-
nas sp.) was isolated from the processing equipment in a
processing plant producing cold-smoked salmon after cleaning
and disinfection.30,31 The adhesion of Pseudomonas sp. to the
different SS surfaces was tested according to the method of Bagge-
Ravn et al.37 The surfaces investigated were sterilized by
immersion in 97% ethanol for 10 min. The Pseudomonas sp. was
precultured in tryptic soy broth (TSB; Oxoid CM129) with
agitation at 25 °C. After 24 h, the bacteria were harvested (3000×
g for 10 min), resuspended, and diluted in PBS (0.8% NaCl, 0.02%
KCl, 0.144% Na2HPO4, and 0.0024% NaH2PO4). The assay for
studying bacterial adhesion is deliberately carried out in PBS so
the bacteria will not grow. Bacterial growth is avoided to create
a system where we can study the immediate interaction between
the bacterial cell and the surface. The bacterial density in PBS
at every sampling-time point is monitored by the total aerobic
plate counts in the beginning and in the end of each experiment,
and there is no killing of bacteria throughout the experiment.
The number of bacteria remains constant within the first 8 h;
thereafter, there is a slight increase in the numbers.37
Sterile disks and holders were conditioned in dilute (1:7) TSB
for 30 min with agitation; thereafter, the holders were transferred
to a new sterile beaker containing the investigated bacteria
suspended in PBS at approximately 106 cfu (colony-forming units)/
mL. Adhesion was allowed to take place on both sides of the
disks under slow stirring at room temperature. At different time
points, disks were sampled and the number of adhered micro-
organisms was quantified by indirect conductometry.37,38 The
disks were transferred to a test tube containing a TSB growth
medium. The growth of the adherent bacteria developed CO2,
which diffused to an inner tube containing sterile NaOH.
Electrodes measured the change of conductance in NaOH as
CO2 dissolved in the alkali. The detection time, that is, the time
point at which a significant change in conductance occurs, is
inversely related to the initial number of bacteria. By use of a
calibration curve constructed by a 10-fold dilution series of each
of the bacteria, the initial number of bacteria on the surfaces
was calculated. The conductometric detection system will detect
as low as one bacterial cell per vial. Two batches of PEG-coated
PET surfaces were tested.
Protein Adsorption. Surfaces that were preconditioned
with the TSB growth medium were also subjected to XPS and
ToF-SIMS analysis to elucidate whether any of the proteinaceous
components adsorbed to the PET-PEG or SS-PEG surfaces.
(37) Bagge-Ravn, D.; Hjelm, M.; Johansen, C.; Huber, I.; Gram, L.
Appl. Environ. Microbiol. 2001, 67, 2319-2325.
(38) Johansen, C.; Falholt, P.; Gram, L. Appl. Environ. Microbiol.
1997, 63, 3724-3728.
Grafting of PEG To Reduce Bacterial Adhesion Langmuir, Vol. 19, No. 17, 2003 6915

Table 1. XPS Elemental Composition for the

Derivatization of the Modified PET Samples with either
TFAA or PFP
sample %C %O %F F/C
PET 70.8 29.2 0.0 0
PET (theory) 71.4 28.6 0.0 0
PET-OH 69.5 30.5 0.0 0
PET-TFAA 72.7 26.8 0.5 0.007
PET-OH-TFAA 67.7 26.2 6.0 0.09
PET-COOH 68.8 31.3 0.0 0
PET-PFP 68.6 29.5 1.8 0.03
PET-OH-PFP 68.9 30.1 1.0 0.01
PET-COOH-PFP 65.8 28.1 6.1 0.09

After rinsing with PBS, the surfaces were rinsed with water to
remove any buffer salts that could interfere with the analysis.

Results
Functionalization of PET. The two-step process to
functionalize PET with reactive carboxyl groups was
confirmed by derivatization with fluorinated probes that
could be detected by XPS, and the results are shown in
Table 1. Before derivatization, there is little difference
between the PET, PET-OH, and PET-COOH surfaces
with only small changes to the elemental composition after
either the hydroxylation or the carboxylation steps. This
is also shown in Table 2, which summarizes the data from
the curve-fitted high-resolution C(1s) spectra of the PET,
PET-OH, and PET-COOH surfaces. Each of the three
spectra contains three main components at 285.0 eV
(CdC/CsC/CsH), 286.5-286.7 eV (peak B; ether), and
288.9-289.1 eV (peak C, acid or ester), and there is little
change to the concentration of each species evident. Also
present are the π-π* shake-up satellite peaks. Therefore,
derivatization was necessary to provide evidence of the
presence of the functional groups. First, the hydroxyl
groups on the PET-OH substrates were derivatized in a
TFAA vapor, as depicted in Figure 1C. The increase in
the F/C ratio from 0.007 for the untreated PET to 0.09 for
PET-OH indicates the introduction of hydroxyl groups
on the PET surface. PET-COOH was derivatized with
PFP in ethanol, and the F/C ratio increased from 0.01 for
the PET-OH surface to 0.09 for the PET-COOH surface.
The presence of F on the PET and PET-OH surfaces after
each type of derivatization is indicative of either non-
specific binding of the probe or reaction with PET
endgroups.
For the PET-OH surface, the theoretical F/C ratio for
100% conversion is 0.23; therefore, a hydroxyl group is
introduced to only one in every two or three PET monomer
units. For the PET-COOH surface, the theoretical F/C
ratio is 0.26 for 100% conversion; therefore, one in three
PET monomer units gets functionalized with carboxyl
groups. The results are in good agreement with the
previous reports.33,34 It would appear that the derivati-
zation reactions confirm that the hydroxyl to carboxylation
step proceeds to almost 100% conversion; however, AFM
measurements (Figure 2) suggest that significant surface
erosion occurs and a significant number of carboxyl groups
are most likely generated from hydrolysis of PET. The
root-mean-square (RMS) roughness increases from 3.1
nm for the PET surface (Figure 3A) to 12 nm for the PET-
OH surface (Figure 3B) and 11.6 nm for the PET-COOH
surface (Figure 3C). Despite these findings, a significant
number of reactive groups are present on PET-COOH
for further functionalization.
Grafting of PEI to the PET and SS Surfaces. Both
the SS and the PET-COOH surfaces were modified
with a branched PEI linker layer to provide a high
concentration of reactive amine groups that would sub-
Figure 2. Positive-ion ToF-SIMS mass spectra for (A) the SS-
NH2 surface and (B) the PET-COO-NH2 surface. The con-
centration of PEI used for grafting was 30 mg/mL.
sequently produce a PEG surface of high graft density.
PEI is a highly branched polymer with about 25% primary
amine groups. The remaining amine groups are secondary
(50%) and tertiary (25%). Only a small fraction of the
groups react with the surface; thus, a high concentration
of free amino groups are available for PEG grafting. We
have recently shown that the level of PEI coverage on the
SS surface is optimal when adsorption takes place from
a concentrated solution (30 mg/mL).36 For PEI grafting to
the PET-COOH surface (Figure 1F), EDAC/NHS was
used for covalent immobilization, and a 30 mg/mL buffered
(pH 7.4) solution was used to provide a direct comparison
to the SS surface. For SS, surface modification occurs by
adsorption of positively charged PEI to the negatively
charged SS surface. The XPS elemental composition
results for both the SS-NH2 and PET-COO-NH2
surfaces are shown in Table 2. The successful grafting of
PEI to both surfaces is clearly shown from the substantial
increase in nitrogen content to >11%. On SS-NH2, the
N/C ratio increases to 0.282 compared to the PET-COO-
NH2 surface (0.166). However, some nitrogen is present
on the control SS surface (1.8%). Also detected on the SS
surface were metals (Cr, Fe, Mo, and Mn) that are present
in the native SS as metal oxides and hydroxides and S
introduced during the Piranha cleaning step.36 Despite
the optimal level of PEI achieved on both surfaces, from
XPS overlayer calculations the dry thickness for the PEI
layer is only 0.7 and 0.8 nm on the SS and PET-COOH
surfaces, respectively.39
(39) The thickness calculation (d) assumes an electron takeoff angle
of 55° and an inelastic mean free path (λ) for nitrogen photoelectrons
of 3 nm.40 I ) I∞ exp[-d/(λ cos θ)]. In the equation, I is the measured
intensity of the overlayer (in %) and I∞ is the intensity of an infinitely
thick PEI layer ()33%).
(40) Blomberg, E.; Claesson, P. M.; Fröberg, J. C. Biomaterials 1998,
19, 371-386.
6916 Langmuir, Vol. 19, No. 17, 2003 Kingshott et al.

Table 2. Summary of the XPS High-Resolution C(1s) Data Derived from Curve-Fitting
C1 C2 C3 C4a C5a
sample BE (eV) % BE (eV) % BE (eV) % BE (eV) BE (eV)
PET 285.0 53.5 286.6 22.7 289.0 23.8 291.1 292.8
PET-OH 285.0 53.2 286.5 28.8 289.0 18.1 290.9 292.2
PET-COOH 285.0 58.8 286.7 16.1 289.1 25.1 291.0 293.4
PET-COOH-PEI 285.0 59.9 286.5 24.4 288.9 15.7 291.3 292.4
a π-π* shake-up satellite peaks.

Figure 3. AFM images for the (A) PET, (B) PET-COOH, (C) PET-COO-NH2, and (D) PET-PEG surfaces. The concentration
of PEI used for grafting was 30 mg/mL.

ToF-SIMS analysis was also performed on both the PEI-
modified surfaces, and the positive-ion spectra (m/z 0-100)
are shown in Figure 2. For the SS-NH2 surface (Figure
2A), three types of ions were observed: N-containing ions,
hydrocarbon ions, and metal ions. The most abundant
peaks are N-containing ions, which mainly arise from the
fragments of repeat unit: Rn ( H (m/z 42 and 44) and Rn
( CH2 ( H (m/z 30, 55, 56, 57, 68, and 70). The hydrocarbon
peaks (m/z 27, 39, 41, 43, 55, 57, and 69) arise from the
fragments, which are due to the C-N cleavage in the
backbone, loss of N, and most likely hydrocarbon con-
tamination. The most intense peaks in the spectrum are
the Cr and Fe ions, which come from the substrate SS.
The high intensity of these ions is most likely due to their
higher ionization probability and greater emission depth
of small atomic species. The latter phenomenon has been
observed for Si+ ion emission through polyelectrolyte
multilayers.41 For the PET-COO-NH2 surface (Figure
2B) the most intense peaks generated are also those that
arise from the fragmentation of the PEI backbone. The
only striking difference between the two surfaces is that
the most intense ion is the C3H8N+ ion (m/z 58), which has
a very low abundance in the spectrum for SS-NH2. We
speculate that a high concentration of protonated amine
groups exist in the PEI layer, thereby increasing the
ionization efficiency of the PEI endgroup. The covalent
immobilization of PEI with EDAC/NHS to the PET-
COOH surface occurs at pH 7.4, and the isoelectric point
of PEI is >10. On the other hand, the pH of the PEI solution
during physical adsorption to the SS surface was 11.2,
where very little protonation of PEI is expected.42
(41) Delcorte, A.; Bertrand, P.; Jonas, A.; Wischerhoff, E.; Mayer, B.;
Laschewsky, A. Surf. Sci. 1996, 366, 149.
Grafting of PEG To Reduce Bacterial Adhesion Langmuir, Vol. 19, No. 17, 2003 6917

Table 3. XPS Elemental Compositions (Atomic % and Ratios) for PEI- and PEG-Modified SS and PET Surfaces
sample %C %O %N N/C O/C % Cr % Fe %S other
PET-COOH 68.8 31.3 0 0 0.455
PET-COO-NH2 66.9 22.0 11.1 0.166 0.329
PET-PEG 68.8 28.9 2.4 0.035 0.420
PET-PEG + TSB 67.6 29.5 2.9 0.043 0.436
SS (Piranha cleaned) 24.8 55.2 1.8 0.072 2.225 8.9 2.5 2.7 4.1
SS-NH2 43.2 37.1 12.1 0.282 0.858 4.5 2.8
SS-PEG 60.8 36.7 1.4 0.023 0.606 0.9
SS-PEG + TSB 60.9 34.4 2.9 0.048 0.565 1.8

Figure 4. Surface analysis of the SS-PEG and PET-PEG surfaces: (A) high-resolution C(1s) spectrum of the PET-PEG surface;
(B) corresponding positive-ion ToF-SIMS spectrum of the PET-PEG surface; (C) high-resolution C(1s) spectrum of the SS-PEG
surface; and (D) corresponding positive-ion ToF-SIMS spectrum of the SS-PEG surface. The concentration of PEI used for grafting
was 30 mg/mL.

Grafting of PEG to the SS-NH2 and PET-COO-
NH2 Surfaces. Linear PEG chains (methoxy-terminted
PEG-aldehyde, MW 5000) were chemically grafted
directly onto the SS-NH2 and PET-COO-NH2 surfaces
by reductive amination in an attempt to generate a surface
that was resistant to protein adsorption and bacterial
adhesion. The grafting conditions were chosen to optimize
the graft density. This was performed at the LCST (60 °C
in 0.6 M K2SO4) of PEG.13,14 Under these conditions, PEG
in the solution starts to phase separate as a result of the
disruption of the hydration shell around the individual
polymer chains. Subsequently, when an individual mol-
ecule first couples to the surface it exists in a collapsed
state and does not repel the next molecular chain to reach
(42) The pH of the PEI solution at a concentration of 30.0 mg/mL was
measured to be 11.2. It has been shown that the number of charged
amine groups decreases with increasing pH of the PEI solution (Smits,
R. G.; Koper, G. J. M.; Mandel, M. J. Phys. Chem. 1993, 97, 5754-
5751), and as a result, very few charges exist in the PEI backbone.
However, we expect that the principle interfacial driving force for PEI
self-assembly on SS is still electrostatic in nature because it has also
been shown that only a small number of charged groups are needed to
neutralize the opposite changes on the planar substrate (Claessen, P.
M.; Paulson, O. E. H.; Blomberg, E.; Burns, N. L. Colloid Surf., A 1997,
123-124, 341-353).
the surface away from neighboring reactive sites. because
this process is reversible, upon rehydration a PEG layer
is regenerated with the maximum possible graft density
necessary for optimal steric repulsion properties.24,25 The
final degree of PEG coverage is, thus, controlled by the
initial density of reactive groups on the surface.
Both XPS and ToF-SIMS was used to evaluate the
success of the PEG grafting to the SS-NH2 and PET-
COO-NH2 surfaces. From the XPS elemental composition
data in Table 3 and high-resolution C(1s) spectra in Figure
4, there is a clear indication that a high level of PEG
grafting is achieved on both surfaces. That is, for the PET-
PEG surface the O/C ratio increases from 0.329 to 0.420,
and the N/C ratio decreases from 0.166 to 0.035. For the
SS-PEG surface, the O/C ratio decreases from 0.858 to
0.606, and the N/C ratio decreases from 0.282 to 0.023.
The O/C ratios never reach the theoretical value of 0.5 for
a pure PEG layer because an individual PEG chain of
MW 5000 (radius of gyration: Rg ) 3 nm)43 is smaller
than the sampling depth of XPS (6 nm). In the case of
PET-PEG, the O/C is less than 0.5 as a result of a lower
(43) Malmsten, M.; Van Alstine, L. M. J. Colloid Interface Sci. 1996,
68, 3751.
6918 Langmuir, Vol. 19, No. 17, 2003 Kingshott et al.

oxygen content in the substrate, and for the SS-PEG

surface, the higher O/C ratio arises from residual metal
oxides that are not fully attenuated to the PEG overlayer.
Finally, the large attenuation of the nitrogen signal from
the underlying PEI layer also indicates that the surface
has a high degree of PEG grafting. The high-resolution
C(1s) XPS spectra of the PET-PEG (Figure 4A) and SS-
PEG (Figure 4C) surfaces also show that the surface
consists mainly of PEG. Both spectra are dominated by
the ether component (CsO, 286.5 eV), which contributes
up to 86.5 and 84.1% for the total carbon species on the
PET-PEG and SS-PEG surfaces, respectively. The C(1s)
spectra for both surfaces show small levels of neutral
carbon (CsH/CsC, 285.0 eV) and amide functionality
(CdOsN, 288.2 eV). The latter component arises from
the amide bond formation between PEG and PEI.
Figure 4B,D shows the positive-ion ToF-SIMS spectra
(0-100 m/z) for the PET-PEG (B) and SS-PEG (D)
surfaces. The most intense ions generated from both
surfaces come from the fragmentation of PEG, producing
the characteristic fingerprint, as shown for ions of Rn (
H+ (m/z 43, 45, 87, and 89) and Rn ( CH2 ( H+ (m/z 31,
59, 71, and 73) structure. The most intense fragment ion
is C2H5O+ (m/z 45), which is expected from PEG analysis
by SIMS. The characteristic ions of PEI were still detected
with very low intensities, suggesting that the PEI layer
was fully covered by a substantial PEG layer. The only
major difference between the two surfaces is the presence
of Cr+ (m/z 51.9) and Fe+ (m/z 55.9) ions that are still
detected even though the overlayer is approaching the
thickness (6 nm) of the XPS sampling depth. However,
they are of lower intensity than the SS-PEI surface
(Figure 2A). It has been estimated from XPS and X-ray
reflectivity that the SIMS emission depth of Si+ ions is
greater than 10 nm, when polyelectrolyte multilayers
uniformly cover a Si substrate.41 A decrease in emission
depth with increasing ion size was also observed. There-
fore, we speculate that the same phenomenon occurs with
Fe+ and Cr+ ion emission through the PEI and PEG layers.
Finally, to evaluate the homogeneity of the PEI and
PEG layers, ToF-SIMS imaging was performed. Under
the analysis conditions chosen (3-pA current, 600-ps pulse
width), the lateral resolution of the ToF-SIMS images is
6-10 µm. The data are presented in Figure 5 (100 × 100
µm images). Parts A and B of Figure 5 show the image of
the sum of several of the major nitrogen fragment ions Figure 5. ToF-SIMS chemical images for the (A) PET-COO-
and the total ion image for the PET-COOH-HN2 surface, NH2, the sum of the nitrogen fragments from the PEI layer; (B)
respectively. After PEG grafting, the image generated from PET-COO-NH2, the total ion image; (C) PET-PEG, the sum
the sum of several of oxygen fragment ions (Figure 5C) of the major oxygen fragments from the PEG layer; (D) PET-
and the total ion image (Figure 5D) are shown. Similarly, PEG, the total ion image; (E) SS-NH2, the sum of the nitrogen
the images for the SS-NH2 and SS-PEG surfaces are fragments from the PEI layer; (F) SS-NH2, the total ion image;
shown in Figures 5E-G. All images on both substrates (G) SS-PEG, the sum of the major oxygen fragments from the
PEG layer; and (H) SS-PEG, the total ion image. The
demonstrate that both steps in the modification on the concentration of PEI used for grafting was 30 mg/mL.
two substrates are chemically uniform (on the micrometer
scale). For the PET-PEG surface, the homogeneous (cleaned in the Piranha solution), and SS-NH2(1) were
nature of the surface is also confirmed by AFM measure- used as comparison surfaces. Our previous results have
ments, as shown in Figure 3D, where the RMS value is demonstrated that there is a significant difference in the
significantly reduced compared to those of the other steps adsorbed amount of PEI on SS when a 1 mg/mL solution
in the surface modification procedure (Figure 3A-C). From concentration is used compared to 30 mg/mL. Conse-
the AFM pictures of the SS-PEG surface (data not shown), quently, the resulting grafted PEG surface is lower in
the RMS values were of a magnitude similar to those surface coverage and was demonstrated to be less effective
achieved on PET-PEG, which is well below the reported at reducing protein adsorption.36 The number of bacteria
values where topography has an influence on bacterial adhering was not different on the four surfaces, and within
adhesion.44 2 h of exposure to the bacterial suspension, the final
Bacterial Adhesion to the Modified Surfaces. The saturation level was reached. Similarly, the SS-NH2(30)-
initial bacterial adhesion experiments were performed on PEG surface was also exposed to a suspension of
the SS-NH2(1)-PEG surface, and untreated SS, SS-clean Pseudomonas sp., and again similar numbers adhered on
this surface as compared to those on the SS surface.36 The
(44) Verran, J.; Boyd, R. D. Biofouling 2001, 17, 59-71. concentration of PEI used for surface modification was
Grafting of PEG To Reduce Bacterial Adhesion
Figure 6. Bacterial adhesion to the modified PET and SS
surfaces. (A) Adhesion of Pseudomonas sp. to PET-PEI(1)-
PEG, PET-OH, PET-PEI, and untreated SS; number of cfu/
mL ) 2.0 × 107. (B) Adhesion of Pseudomonas sp. to PET-
control, PET-PEI(30)-PEG, and untreated SS; number of cfu/
mL ) 2.7 × 106. (C) Adhesion of Pseudomonas sp. to PET-
control, PET-PEI(30)-PEG, and untreated SS; number of cfu/
mL ) 1.6 × 107.
30 mg/mL, and the surface analysis is described above.
Therefore, the optimal surface layer of PEG on SS was
incapable of preventing bacterial adhesion even though
there is a very high graft density, and a uniform layer of
PEG is presented to the bacteria.
Figure 6A-C shows the results for the adhesion of
Pseudomonas sp. to the modified PET surfaces used in
the study. In contrast to that of the modified SS substrate,
the level of adherent bacteria was markedly reduced by
Langmuir, Vol. 19, No. 17, 2003 6919
grafting PEG to the PET surface. In Figure 6A, results
are shown for adhesion to the PET-NH2(1)-PEG surface,
with covalent grafting of the PEI from a 1 mg/mL solution.
The data are presented as log cfu/cm2 versus time. The
initial level of Pseudomonas sp. adhesion to the PET-
NH2(1)-PEG surface after 1 h was approximately log 3
cfu/cm2, or 1000 bacteria/cm2, and this value increased to
log 4 cfu/cm2, or 10 000/cm2, after 5 h. In contrast, a level
of log 5 to log 6 cfu/cm2 adhered to the untreated SS and
PET-NH2(1) controls. Both the PET-OH and PET-NH2-
(1)-PEG surfaces were not significantly different after 5
h, and this could be due to variability in the experiment.
The surface analysis results for this particular surface
(data not shown) suggest that a very uniform but thinner
layer of PEG is present on the surface, compared to the
PET-NH2(30)-PEG surface (30 mg/mL PEI used). In this
case, less PEI was coupled to the surface, and, therefore,
fewer reactive groups were available for achieving the
higher level of PEG grafting.
In Figure 6B,C, the results are presented for the
Pseudomonas sp. adhesion to the PET-NH2(30)-PEG
surface in two separate experiments, where the PEI was
grafted from a 30 mg/mL solution and a very high level
of PEG grafting was achieved, comparable to the SS-
NH2(30)-PEG, as shown from XPS and ToF-SIMS mea-
surements. Adhesion was compared to that of the PET
and SS controls. In the first experiment (Figure 6B), the
level of adhesion varied from log 4 to log 5 cfu, or 104-105
bacteria/cm2, for the entire time course of the experiment
(5 h), compared to log 8 (108 bacteria/cm2) for the controls.
This represents up to 4 orders of magnitude reduction in
bacterial adhesion. In the second experiment (Figure 6C),
the PEG coating also reduced the number of adhering
Pseudomonas sp. very significantly (up to 2 orders of
magnitude). Surface analysis revealed that chemically the
two batches were identical; hence, the difference in
adhesion could be associated with a difference in the
peptide composition of the TSB used to precondition the
surfaces. More peptide adsorption would allow for higher
numbers of adherent bacteria.
Protein Adsorption to the PEG Surfaces. Both XPS
and ToF-SIMS analysis were performed on SS-PEG and
PET-PEG (with 30 mg/mL PEI as the linker layer) after
exposure to the TSB growth medium. The adsorption
experiments were identical to that for the preconditioning
of the surfaces prior to the bacterial adhesion experiments,
that is, for 30 min in 1:7 TSB solution followed by PBS
rinsing. The elemental compositions for the SS-PEG and
PET-PEG after TSB adsorption are shown in Table 3.
For both surfaces, there is a slight increase in the N/C
ratio after exposure to the TSB, which may be associated
with the adsorption of peptides. However, the greater error
when measuring low surface concentrations with XPS
places uncertainty in this assumption. In addition, the
level of protein adsorption may be below that detectable
by XPS when nitrogen is present, as previously demon-
strated.14,45
To determine whether protein adsorption actually
occurs to these “low-fouling surfaces”, ToF-SIMS analysis
was performed before and after exposure of the SS-PEI-
(30)-PEG and PET-PEI(30)-PEG surfaces to the TSB
media. ToF-SIMS has previously been shown to be capable
of detecting very low levels of protein adsorption to PEG
surfaces when XPS measurements showed that no protein
adsorbed.45 This is due to the inherently higher surface
sensitivity and specificity of ToF-SIMS. In the SIMS
(45) Kingshott, P.; McArthur, S.; Thissen, H.; Castner, D. G.; Griesser,
H. J. Biomaterials 2002, 23 (24), 4775-4785.
6920 Langmuir, Vol. 19, No. 17, 2003
Figure 7. ToF-SIMS data for the PET-PEI(30)-PEG and
SS-PEI(30)-PEG surfaces after adsorption experiments with
the TSB growth medium. The sum of the intensities of the
amino acid fragment ions, C2H5S+ (m/z 61), C3H4NO+ (m/z 70),
C3H8NO+ (m/z 74), and C4H6NO+ (m/z 84), is normalized against
the sum of the two characteristic PEG fragment ions, C2H3O+
(m/z 43) and C2H5O+ (m/z 45). For the control samples, the
amino acid fragment ions were not detected; hence, the data
are not included in the figure.
ionization of proteins, characteristic immonium ions and
their fragment ions are generated from the amino acids
that constitute the backbone of the proteins.46,47 The
ToF-SIMS data for the two surfaces before and after TSB
adsorption are shown in Figure 7. The data show the sum
of the intensity of selected nitrogen-containing ions of
amino acids normalized against two PEG fragment ions.
That is, the sum of the intensity of the amino acid fragment
ions, C2H5S+ (m/z 61), C3H4NO+ (m/z 70), C3H8NO+ (m/z
74), and C4H6NO+ (m/z 84), is normalized against the sum
of the PEG fragment ions, C2H3O+ (m/z 43) and C2H5O+
(m/z 45). These nitrogen fragments were chosen because
they appeared in the spectrum acquired from a bulk film
of TSB deposited on a silicon wafer,48 and they are absent
in the spectrum for PEI. They originate from the frag-
mentation of methionine (C2H5S+), asparagine and proline
(C3H4NO+), and threonine (C3H8NO+).47 The increase in
intensity compared to that of the controls indicates the
adsorption of some peptide or protein from the TSB
solution, confirming speculations from XPS measure-
ments. The similarity in the intensities for both PEG
surfaces suggests that the level of adsorption is quite
comparable, and, hence, the profound difference in bacte-
rial adhesion to the two surfaces cannot be correlated with
differences in peptide or protein adsorption.
Discussion
An optimal PEG (MW 5000) surface in terms of surface
coverage has been created on two different substrates (SS
and PET) in a two-step process. The common features of
the attachment were (1) the use of a PEI linker layer to
provide reactive amino groups for the PEG grafting and
(2) grafting of PEG at the LCST. The only difference in
the procedure was that the PEI layer was physically
adsorbed to the SS surface but covalently attached to the
PET substrate. This combination of immobilization meth-
odology offers the best opportunity of creating a steric
repulsive barrier against bacterial adhesion using linear
(46) Mantus, D. S.; Ratner, B. D.; Carlson, B. A.; Moulder, J. F. Anal.
Chem. 1993, 65, 1431-1438.
(47) Wagner, M. S.; Castner, D. G. Langmuir 2001, 17, 4649-4660.
(48) Wei, J.; Bagge-Ravn, D.; Gram, L.; Kingshott, P. Manuscript in
preparation.
Kingshott et al.
PEG chains of MW 5000. Characterization of both surfaces
by high-sensitivity chemical surface analysis (XPS and
ToF-SIMS) after PEG grafting reveals that they are
identical in terms of both surface uniformity and surface
coverage, consisting of a very high graft density in both
cases. For both surfaces, a thickness of 4-4.5 nm for the
PEG layer is estimated from the attenuation of the N
signal using XPS overlayer calculations, which is slightly
more than the Rg (3 nm) of the PEG chains (MW 5000).
This suggests that the attached PEG chains exist in a
brush conformation, which is a necessary requirement
for good nonfouling behavior.24,25
Both the SS-PEG and PET-PEG surfaces were tested
for the adhesion of Pseudomonas sp. that was isolated
from a fish processing plant. However, contrary to the
expectations from surface analysis the adhesion results
revealed that the two substrates differed markedly in their
ability to prevent colonization. The SS-PEG surface was
incapable of having any effect in the level of Pseudomonas
sp. up to 5 h compared to controls, whereas the PET-
PEG surface showed up to a fourfold or 10 000 times
reduction in bacterial adhesion. ToF-SIMS analysis
showed that proteinaceous species adsorbed from the TSB
solution in comparable levels. The results clearly dem-
onstrate the importance of the covalent attachment of
surface modifying agents if bacteria adhesion is to be
prevented.
The bacterial adhesion results raise the question of why
one surface (the SS-PEG) cannot stop bacteria attach-
ment whereas another is extremely effective, despite the
surface chemistries of the final PEG layers being almost
identical. We thought that the sterilization procedure
(rinsing in 97% ethanol) prior to adhesion experiments
might have a profound effect on the stability of the two
different PEG layers. However, because the surface
composition remained unchanged after sterilization we
ruled this out as a possible explanation. Also, at the quite
harsh conditions of the PEG grafting step (high salt
concentration and temperature) no PEI delamination
occurs. The profound difference in bacterial adhesion
between the SS-PEG and PET-PEG surfaces, in the
absence of a conditioning film effect, clearly points to
additional mechanisms that may be used by bacteria to
colonize the surfaces. The tenacity that bacteria show in
adhering to surfaces appears significantly greater than
that shown by mammalian cells. For example, it has been
shown49 that the attachment and growth of cornea
epithelial cells, which synthesize their own extracellular
matrix proteins to help them attach to surfaces, can be
prevented by PEG (MW 5000) layers grafted to plasma
polymer layers at the LCST, even in the presence of serum
proteins. This raises the question about possible explana-
tions. It is known from the literature that Pseudomonas
sp. produce biosurfactants, which are used to solubilize
hydrocarbon material by micellization and increase
biodegradation rates.50 We speculate that such mecha-
nisms could be used by bacteria to desorb material and
assist in their colonization of surfaces. In such a case, the
SS-PEG system may not be stable enough in a bacterial
environment compared to the covalent PET-PEG system.
From the literature, it has been recently demonstrated
that densely packed, physically adsorbed PEG-containing
(49) Thissen, H.; Hayes, J. P.; Kingshott, P.; Johnson, G.; Harvey,
E. C.; Griesser, H. J. Smart Mater. Struct. 2002, 11, 792-799.
(50) Al-Tahhan, R. A.; Sandrin, T. R.; Bodour, A. A.; Maier, R. M.
Appl. Environ. Microbiol. 2002, 66 (8), 3262-3268.
Grafting of PEG To Reduce Bacterial Adhesion
layers such as those based on Pluronics51 or hydrophobic
surfactants21 can reduce the attachment of certain bacteria
up to 1 order of magnitude. Maybe these surfaces would
be more effective if covalently attached. However, from a
speculative point of view there could be a number of other
possible interrelated mechanisms that may explain our
findings, including: (1) On the PET-PEG surface, apart
from protein adsorption, quite possibly a small population
of the PEI-PEG molecules are not covalently attached.
These could be desorbed by bacteria and attachment using
the mechanisms described above. (2) PEG may be inca-
pable of repelling the EPS produced by the bacteria. Also,
it is known that both Gram-positive52 bacteria and Gram-
negative53 bacteria release natural virulent factors such
as lipopolysaccharides and potent hydrolytic enzymes,
which may also adsorb or even destroy the PEG layer
over time. These hypotheses regarding the coating stability
and bacterial attachment mechanisms need further
elucidation. In addition, further investigations into the
biochemical pathways used by bacteria to attach to
surfaces in the absence of a conditioning film are clearly
needed. Also, preventing the adsorption of conditioning
film components is in itself not a trivial exercise and needs
to be confirmed using very surface-sensitive methods such
as ToF-SIMS.36,45
Our results are extremely promising, but some bacteria
(1000/cm2) still adsorb to the best PET-PEG despite the
high graft density. From ToF-SIMS analysis, peptide or
protein adsorption is detected from the TSB solution in
comparable levels on both the SS-PEG and PET-PEG
surfaces. We have analyzed TSB by matrix-assisted laser
desorption/ionization mass spectrometry (data not shown)
and found that it consists of a number of low-molecular-
weight proteins (ca. <3000).48 These are most likely small
enough to prevent being repelled by the high hydrated
chains of the PEG and penetrate the layer where they
subsequently get recognized by the bacteria. This mo-
lecular-weight dependence is clearly seen from our previ-
ous work in which we investigated the adsorption of
β-lactoglobulin to the SS-PEG surface. Both XPS and
ToF-SIMS were not capable of detecting any β-lactoglo-
bulin adsorption, which has a molecular weight of 18 300.36
Therefore, it would appear that both the covalent attach-
ment of surface layers and preventing adsorption of low-
molecular-weight species are necessary if fully preventing
bacterial adhesion is to be achieved.
The role of differences at the molecular level in terms
of conformation, hydration, and residual charge between
the two PEG surfaces cannot be fully ruled out as a
contributing factor toward the large differences in bacterial
adhesion. We do not observe an influence of these factors
at the protein level, as indicated by the same degree of
adsorption on both surfaces; however, the surface of
bacteria most likely contains a large number of charged
(51) March, L. H.; Coke, M.; Dettmar, P. W.; Ewen, R. J.; Havler, M.;
Nevell, T. G.; Smart, J. D.; Smith, J. R.; Timmins, B.; Tsibouklis, J.;
Alexander, C. J. Biomed. Mater. Res. 2002, 61, 641-652.
(52) Ziebandt, A.-K.; Weber, H.; Rudolph, J.; Schmid, R.; Hoper, D.;
Engelmann, S.; Hesker, M. Proteomics 2001, 1, 480-493.
(53) Kadurugamuwa, J. L.; Beveridge, T. J. J. Antimicrob. Chemother.
1997, 40, 615-621.
(54) Kenausis, G. L.; Voros, J.; Elbert, D. L.; Huang, N.; Hofer, R.;
Ruiz-Taylor, L.; Textor, M.; Hubbell, J. A.; Spencer, N. D. J. Phys. Chem.
B 2000, 104, 3298-3309.
Langmuir, Vol. 19, No. 17, 2003 6921
extracellular polysaccharides that could be more influ-
enced by very subtle surface properties than proteins in
solution. Such information could be provided by techniques
such as surface force apparatus, AFM, and streaming
potential measurements in hydrated environments.
How do our coatings compare to those in the literature?
First of all, it is rather difficult to compare results for
bacterial adhesion between laboratories because of the
plethora of different test methods (flow, rotating disk or
static), quantification techniques, and different bacterial
strains. For example, it has been demonstrated that
surfaces tested in a modified Robbins device, used to
investigate bacterial adhesion under flowing conditions,
result in less adherence and longer times to reach steady
state when compared to those in the batch reactor system
used in the present study.37 Although a large volume of
literature exists reporting the use of PEG to modify
surfaces for preventing bioadhesion, we believe that the
PET-PEG surface demonstrated here compares very well
to the best reports on preventing bacterial adhesion.
Recent reports demonstrated that the use of PEI on a Au
self-assembled monolayer with di(ethylene glycol) (EG)
attached as the outer layer could reduce the adhesion of
Staphyloccus epidermidis, Staphyloccus aureas, and Es-
cherichia coli by up to 100 times when compared to bare
Au.18 This system was more effective than the Au-C11-
EG3OH surface, the most effective PEG-like model surface
at preventing protein adsorption.17 In another study20
where a hydrophilic PEG-like surface was created by the
plasma deposition of triethylene glycol dimethyl ether
(triglyme), adhesion of Pseudomonas aeruginosa was
suppressed by 2 orders of magnitude. Interestingly, when
the triglyme surface was manufactured to release the
antibiotic Ciprofloxacin, adhesion was reduced by an
additional order of magnitude.
Conclusions
We have demonstrated that a PEG layer, with an
optimal graft density, can reduce the adhesion of a Gram-
negative Pseudomonas sp. significantly when compared
to controls. This is at least 2 orders of magnitude better
than previous reports in the literature. We show that such
reductions are only possible if the PEG layer is covalently
bonded to the substrate, and we speculate that this is
necessary to overcome the possible deleterious biodeg-
radation mechanisms that opportunistic bacteria use to
colonize surfaces. We also highlight the necessity for using
surface-sensitive analysis methods such as XPS, AFM,
and ToF-SIMS to optimize surface modification protocols
because subtle differences in the surface chemistry and
structure can have profound effects on biological responses
to surfaces. Finally, XPS and ToF-SIMS show that low-
molecular-weight proteins and peptides from the bacterial
growth medium (TSB) are not fully prevented from
adsorbing to the best surfaces, and this may account for
the very low level of bacterial adhesion observed.
Acknowledgment. Funding for the HYDEKO Centre
Contract is from the Danish Ministry of Industry and
Trade. We thank Dr. K. Norrman (Risø) for his help with
the ToF-SIMS analysis and Dr Afshin Gianbari-Siahkali
and Dr. Niels Bent Larsen for helpful discussions.
LA034032M