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Biomimicking Micropatterned Surfaces and Their Effect on Marine Biofouling

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DOI: 10.1021/la502006s · Source: PubMed

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Biomimicking Micropatterned Surfaces and Their Effect on Marine

Biofouling
Agata M. Brzozowska,† Fernando J. Parra-Velandia,‡ Robert Quintana,† Zhu Xiaoying,† Serina S. C. Lee,‡
Lim Chin-Sing,‡ Dominik Jańczewski,*,† Serena L.-M. Teo,*,‡ and Julius G. Vancso*,§,∥
†
Institute of Materials Research and Engineering, Agency for Science, Technology and Research, 3 Research Link, 117602 Singapore
‡
Tropical Marine Science Institute, National University of Singapore, 18 Kent Ridge Road, 119227 Singapore
§
Institute of Chemical and Engineering Sciences, 1 Pesek Road, 627833 Singapore
∥
MESA+ Institute for Nanotechnology, Materials Science and Technology of Polymers, University of Twente, 7500 AE Enschede,
The Netherlands
*
S Supporting Information

ABSTRACT: When synthetic materials are submerged in

marine environments, dissolved matter and marine organisms
attach to their surfaces by a process known as marine fouling.
This phenomenon may lead to diminished material perform-
ance with detrimental consequences. Bioinspired surface
patterning and chemical surface modifications present
promising approaches to the design of novel functional
surfaces that can prevent biofouling phenomena. In this
study, we report the synergistic effects of surface patterns,
inspired by the marine decapod crab Myomenippe hardwickii in
combination with chemical surface modifications toward
suppressing marine fouling. M. hardwickii is known to maintain a relatively clean carapace although the species occurs in
biofouling communities of tropical shallow subtidal coastal waters. Following the surface analysis of selected specimens, we
designed hierarchical surface microtopographies that replicate the critical features observed on the crustacean surface. The
micropatterned surfaces were modified with zwitterionic polymer brushes or with layer-by-layer deposited polyelectrolyte
multilayers to enhance their antifouling and/or fouling-release potential. Chemically modified and unmodified micropatterned
surfaces were subjected to extensive fouling tests, including laboratory assays against barnacle settlement and algae adhesion, and
field static immersion tests. The results show a statistically significant reduction in settlement on the micropatterned surfaces as
well as a synergistic effect when the microtopographies are combined with grafted polymer chains.

■ INTRODUCTION
The unwanted attachment and colonization of marine
organisms on submerged surfaces in seawater poses a scientific
challenge and has a severe technological impact. The
development of biofilms enhances the corrosion of the
submerged elements of manmade structures,1 it affects the
performance of heat exchangers,2 the fuel consumption and
performance of naval vessels due to increasing drag,3 and it
interferes with the performance of sensor equipment.4 The
process of attachment, often called biofouling, follows several
stages of ecosystem development, is species-dependent and not
yet fully understood.5 The lack of understanding hampers
technological efforts to provide materials that feature
antifouling surfaces in marine environments. Thus, until
recently, antifouling surfaces featured biocide-loaded coatings,
which bring about significant environmental consequences.6 As
the use of paints containing biocides has become increasingly
restricted owing to legal limitations, several nontoxic
alternatives have been considered.7 Among the various
strategies investigated for the suppression of biofouling, the
two most popular approaches have employed chemical8,9 and
physical patterning with microtopographic10 surface modifica-
tions.
Nature provides wonderful inspiration for achieving the
objective of preparing synthetic materials that do not foul.10
Research oriented toward bioinspired surface topographies
derived from sea-dwelling organisms is of particular interest.
Materials designed on the basis of the surface features observed
in many organisms, such as shark skin,11,12 muscles,13 pilot
whale skin,14 snail shells, butterfly or cicada wings, and plant
leaves,15,16 have been shown to reduce fouling to a significant
degree.17 However, the majority of the reported studies refer to
the antifouling performance of the patterns being directly
replicated from the natural surfaces. The observed antifouling
effects have been attributed to surface topography, wettability,
and secreted substances. Surface wettability and microtopog-
Received: May 23, 2014
Revised: July 9, 2014

© XXXX American Chemical Society A dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX

Langmuir
raphy, in addition to secreted substances, have been shown
either to enhance or to deter the larval settlement of many
sessile marine organisms, adding to the controversy.18 In this
context, some work has been carried out on the surface
microstructure of decapod crabs,19 some of which exhibit
fouling-free appearances. Such decapods have been extensively
studied only in relation to underwater robotics (mechanisms of
movement), sound generation (mechanical energy, acoustics in
snapping shrimp), and, more recently, as biomimetic models
for understanding biomineralization.20
Recently, we have uncovered an unprecedented complexity
of surface armory in a number of crustaceans. The armory is
organized in complex configurations, the functions of which
remain to be discovered. The low degree of fouling observed on
decapod shells has been attributed to burrowing, grooming, and
moulting behaviors as well as the secretion of bioactive
substances.21 Some studies suggest that surface topography
does not effectively suppress the accumulation of biomass22 at
solid−liquid interfaces. The most effective antifouling mecha-
nisms may include the physicochemical modification of the
(micropatterned) surfaces in the form of epidermal secretions14
or the adhesion of symbiotic organisms.23 The formation of
hairlike structures can also contribute by increasing the
separation distance between a given surface and the fouling
molecules, thereby weakening the interactions between the
surface and foulants. Although these mechanisms appear to be
actively used by some species, they do not account for the
relatively unfouled appearance of others.
In this report, we describe the effect of microtopographies
inspired by the shell of the crab M. hardwickii in combination
with chemical modifications of the surface. Because many
natural antifouling mechanisms usually involve the secretion of
chemical substances, e.g., enzymes,14 we hypothesized that an
antifouling surface should include some element of chemistry in
its design. We have chosen two independent chemical
techniques for the modification of micrometer-sized topological
features, namely, the use of surface-initiated polymerization to
form zwitterionic polymeric brushes (grafting-from techni-
que),24 and the layer-by-layer (LbL) deposition of thin
polymeric films.25 Both approaches allow a large amount of
control over the thickness of the surface coating, which is
particularly important when the modified surface chemistry is
combined with micrometer-sized surface topography.26
With respect to chemical surface modification, the antifouling
performance of polymer brushes is usually discussed in the
context of protein and bacteria adsorption.24,27 A brush forms a
kinetic barrier that the adsorbing molecule must overcome to
reach the surface. Recently, zwitterionic brushes received
attention for their potential application as antifouling surface
coatings.28 Due to their chemical composition, zwitterionic
brushes show prolonged stability, are charge-neutral, and form a
strong hydration layer,29 which has a significant effect on the
initial deposition of proteins.9,30 Another chemical modification
approach to the fabrication of highly hydrated and charge-
neutral thin polymeric films includes the LbL assembly of
polyelectrolytes. Due to their simplicity of fabrication, thickness
control, and the significant variety of possible chemical
composition,31 the LbL approach also enables the formation
of surfaces for antifouling applications. The assembly of LbL
films is driven mainly by electrostatic interactions between
oppositely charged polyelectrolytes25 but also may include
hydrogen bonding or hydrophobic interactions. The structure
of the film may be further strengthened by, for example, film
B dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
Article
cross-linking, which can provide stability in solutions of high
ionic strength.
Here, we report on the preparation of model surfaces,
fabricated by combining bioinspired patterns and surface
chemical treatments, and we assess their antifouling perform-
ance with particular emphasis on possible synergistic effects.
■ EXPERIMENTAL SECTION
Acquisition and Characterization of Myomenippe hardwickii
Specimens. Myomenippe hardwickii specimens were collected from
fouling communities located in Tuas, on the west coast of Singapore. A
fragment from the dorsal carapace was removed for examination using
scanning electron microscopy (SEM). The fragments were critically
dried at temperatures of up to 40 °C in a Balzers CPD 030, then were
platinum-coated in a JEOL JFC-1600 sputter coater, and finally were
examined with a JEOL JSM 6510LV SEM microscope. In total,
samples from 19 species were examined.
Fabrication of Silicon Molds. The silicone molds used for the
replication of the microtopography in PDMS were fabricated using a
two-mask design. Each mask design consists of 20 mm × 20 mm
hexagonal arrays of circular holes as well as smooth control areas. The
first array consists of 3-μm-diameter holes with a distance of 7.5 μm
between their centers. The second array consists of 100-μm-diameter
holes with a distance of 250 μm between their centers. The masks
were used to fabricate silicon molds with etched cylinders, featuring
depths of 20 μm (ø 100 μm) and 10 μm (ø 5 μm), respectively. The
molds were used to fabricate PDMS pattern replicates, as described in
the following section.
A silicon wafer (Prime silicon wafer 4 in., thickness 525 ± 25 μm,
single-side polished, MOS Group Pte Ltd) was vapor-coated with
hexamethyldisilazane (HMDS, Fluka Analytical 52619-250 mL) and
subsequently spin-coated with photoresist AZ4620 (AZ Electronic
Materials, USA) and soft-baked at 100 °C for 6.5 min. The photoresist
layer was exposed to UV light for 5 s through a photolithography mask
(mask and bond aligner, SUSS Microtec, Germany), developed for 30
s in AZ Developer 400k (AZ Electronic Materials, USA) diluted with
deionized water (1:5), rinsed with deionized water, and blown dry
with N2 gas. The prepared wafer was subjected to reactive ion etching
(RIE) using an etching time of 20 min to form 10-μm-deep holes
measuring 5 μm in diameter. The residual photoresist was removed by
rinsing with acetone (Honeywell Specialist Chemicals Seelze GmbH,
10189037). Subsequently, the wafers were cleaned with hot piranha
solution (1 part 30% H2O2 (electronics grade, MGC Pure Chemicals
Singapore Pte. Ltd.) and 2 parts 95−97% H2SO4 (10189285 Puranal,
Honeywell Specialty Chemicals Seelze GmbH)) for 20 min, rinsed
with deionized water, and dried. Following the procedure described
above, we aligned the second array of holes (ø 100 μm) with the
already-etched array using a mask and bond aligner. The second array
of holes was subjected to RIE using an etching time of 20 min. The
residual photoresist was removed by rinsing with acetone. Sub-
sequently, the molds were cleaned with hot piranha solution for 20
min and were vapor-coated with 1H,1H,2H,2H-perfluorododecyltri-
chlorosilane (FDTS, Alfa Aesar, L16584) according to the following
procedure: a syringe filled with approximately 0.1 mL of FDTS in a 30
mL glass container was placed in a desiccator containing the molds.
The FDTS solution was vaporized by lowering the pressure in the
desiccator and was then left to react with the molds overnight. The
quality of the FDTS coating was monitored by means of static contact
angle measurements (119 ± 0.9°, VCA Optima). The quality of the
molds and the depth of the etched features were inspected with
scanning electron microscopy (Figure 4, JEOL SEM JSM5600) and
surface profilometry (Tencor P-10).
Fabrication of Patterned and Smooth PDMS Surfaces.
Poly(dimethylsiloxane) (PDMS, Sylgard 184 silicone elastomer kit,
Best Chemical Co(s) Pte Ltd) base was mixed with cross-linker in a
volume ratio of 10:1. The prepared mixture was poured over FDTS-
coated silicon molds and placed separately in disposable aluminum
dishes with the microstructured surfaces facing upward. Once the
molds were covered with PDMS, they were placed in a desiccator for
Langmuir
low-pressure degassing for 30−40 min. The degassed samples were
cured in the oven at 60 °C overnight. Following a similar procedure,
we prepared the smooth PDMS samples, casted against a flat silicon
wafer, for use as a control reference. After curing, the PDMS
microstructured samples were separated from the molds, cut into the
desired 20 × 20 mm2 pieces, and stored for further tests. The quality
of the PDMS microstructured samples was controlled with optical
microscopy (Olympus BX51 microscope equipped with an Olympus
TH4-200 light source and an Olympus DP70 digital camera) and SEM
(Figure 4).
Surface Modification. The micropatterned and smooth PDMS
samples, prepared as described in the previous sections, were further
modified by vapor-coating with FDTS, polymer brushes, or LbL-
deposited polyelectrolyte multilayers. Prior to modification, the
samples were cleaned by sonication in ethanol (Merck,
1.00983.2500 ethanol absolute for analysis EMSURE ACS, ISO,
Reag. Ph Eur) using an ultrasound bath for 5 min, followed by
sonication in acetone for 30 min. During this cleaning procedure, (i)
PDMS pillars that were stacked together during demolding were
separated without damage to the pattern and (ii) low-molecular-
weight polymer chains were removed by washing,32 lowering the risk
of polymer leaching during subsequent biological tests.
FDTS Coating. The cleaned PDMS substrates were exposed to 40
W oxygen plasma (Triple P microwave plasma process system,
Duratek, Taiwan) for 10 s to increase the number of hydroxyl groups
on their surfaces. Subsequently, the substrates were coated with FDTS
following the method described in the previous section.
Surface-Initiated Polymerization of the Polymer Brushes.
Copper(I) bromide (99.999%), 2,2′-bipyridyl (Bipy) (99%), and ([3-
(methacryloylamino)propyl]dimethyl(3-sulfopropyl) ammonium hy-
droxide inner salt (or sulfobetaine acrylamide, SBMAm) (96%) were
purchased from Sigma-Aldrich and used without further purification.
(p-Chloromethyl)phenyl-trichlorosilane (CMPS, 95%) was purchased
from Gelest, stored inside a nitrogen-filled glovebox, and used as
received. Deionized water (18 MΩ cm) and ultrapure nitrogen were
used throughout. Silicon wafers with a thickness of 0.56 mm, resistivity
of 10−20 Ω cm were purchased from Latech Scientific Supply Pte. Ltd
(Singapore) and cut into 2 × 2 cm2 pieces.
Both the smooth and patterned PDMS surfaces were exposed to
oxygen plasma for 10 s at 50 W. Subsequently, a self-assembled CMPS
ATRP-initiator layer was formed on the samples by vapor deposition,
followed by baking at 120 °C for 3 min. The organo-modified samples
were stored in a nitrogen-filled glovebox. SI-ATRP of SBMAm was
carried out as previously reported.33 In a typical procedure, 1.5 g of
SBMAm monomer (5.13 mmol) was dissolved in 10 mL of a 4:1
water/methanol mixture and purged with nitrogen for at least 30 min.
CuBr (14.7 mg, 0.1 mmol) and the ligand, Bipy (32.1 mg, 0.2 mmol),
were added to an oven-dried Schlenck flask, and oxygen was removed
by four vacuum−nitrogen refill cycles. The degassed solution
containing the monomer was transferred by cannula to the Schlenck
flask. The resulting mixture was stirred until homogenization and then
was transferred by cannula into a customized reactor containing the
organo-modified PDMS substrates, which were placed in the reactor
inside a nitrogen-filled glovebox. The reactor was placed in an oil bath
at 40 °C, and the reaction was carried out for 24 h before removing the
reaction mixture from the reactor. The substrates were removed from
the reactor and rinsed several times with warm water and methanol;
next, the substrates were rinsed with abundant water and then were
dried under a stream of argon. An accessory for attenuated total
reflection (ATR) equipped with a single-refraction diamond crystal
mounted in a vacuum-purged Bruker Vertex 80v FTIR spectrometer
was utilized to verify the presence of polymer brushes on the PDMS
substrates. Static water contact angle (CA) measurements were
performed in a Ramé-Hart CA goniometer. The sessile drop method
was used with a 3 μL droplet, and at least four different drops were
measured for each surface. Because it is difficult to measure the
thickness of films on soft surfaces (PDMS), the thickness of the
polymer brushes was determined on silicon using an AFM
NanoWizard II instrument (JPK Instruments AG, Berlin, Germany)
equipped with a NanoWizard head and controller. Measurements in
C dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
Article
the dry state were performed in tapping mode. Quantitative imaging
mode was used to characterize hydrated brushes in liquid. The
measurements were performed in the SmallCell fluid cell with an O-
ring seal in the dry state as well as in DI water and in 0.7 M NaCl
solution. We used silicon probes with a gold reflex coating
(Budgetsensors, model Multi75GD). The nominal spring constant
of the cantilevers was 3.0 N/m. For quantitative imaging (QITM), the
deflection sensitivities were measured, followed by the determination
of the cantilever spring constants, showing values in the range of 2.07−
3.17 N/m for the Multi75GD probes. The thickness of the dry
polymer brush was determined to be approximately 10 nm. After
hydration in 0.7 M NaCl, the determined thickness was between 50
and 60 nm. Due to the similarities in surface chemistry, we assume that
brushes grafted from PDMS have similar characteristics to those
grafted from a silicon substrate.
Layer-by-Layer (LbL)-Deposited Polyelectrolyte Films. Poly-
(isobutylene-alt-maleic anhydride) (PIAMA, Mw 60 000), poly-
(ethylenimine) (PEI, Mw ≈ 25,000, branched), 3-aminopropyltrime-
thoxysilane, 4-(dimethylamino)pyridine (DMAP), and sodium hydrox-
ide were purchased from Sigma-Aldrich. N,N-Dimethylformamide
(DMF), dimethyl sulfoxide (DMSO), toluene, methanol, and ethanol
were purchased from Tedia. Dialysis membrane tubing (MWCO 12
000−14 000) was purchased from Fisher Scientific. Silicon wafers were
obtained from Latech Scientific Supply Pte Ltd. We used ultrapure
water (Millipore Milli-Q integral water purification system) to prepare
all aqueous solutions. The silicon wafers were treated with oxygen
plasma prior to surface modification. Poly(ethylenimine) was used as
received for the fabrication of the polycation layers, and the polyanion
was synthesized as reported previously.37
Cleaned patterned and smooth samples were exposed to 40 W
plasma for 10 s and subsequently were immersed in a 3-amino-
propyltrimethoxysilane solution in toluene (10 mM) for 5 h to impart
positively charged amino groups to the sample surfaces. The thus
prepared samples were immersed in the polyanion solution for 10 min,
followed by rinsing with deionized water for 2 min. Next, the samples
were immersed in PEI (polycation) solutions for 10 min, followed by
rinsing with deionized water for 2 min. This cycle was repeated until
the desired number of bilayers (5.5) was reached. Our previous reports
indicated that once the surface is fully covered, the LbL film shows an
antifouling effect which is not enhanced by an increased film
thickness;34 as such, the chosen number of bilayers was sufficient to
cover the substrate fully. The PDMS samples with the LbL-deposited
films were dried under vacuum with a nitrogen stream at room
temperature for 5 h. The cross-linking process was conducted by
holding the samples at 60 °C for 5 h. The quality of the samples was
controlled by monitoring with NMR (Bruker, 400 MHz), FTIR
(PerkinElmer), and XPS (VG ESCALAB 250i-XL spectrometer). The
thickness of the LbL films deposited on the silicon was measured by
removing stripes of the coating and measuring the step height between
the substrate and the top of the film using AFM. The height values
obtained were 61.2 ± 7.6 nm. Successful modification of the silica
surface was further confirmed with contact angle measurements. The
static contact angle was found to be 60°. Due to similar surface
chemistry, we assume that the LbL films deposited on PDMS have
similar characteristics to those deposited on the silicon substrate.
Settlement Tests. Barnacle Settlement Test. Amphibalanus
amphitrite barnacle larvae were spawned from adults collected from
the Kranji mangrove, Singapore. The nauplius larvae were fed a 1:1 v/v
algal mixture of Tetraselmis suecica and Chaetoceros muelleri (CSIRO
Microalgae Research Centre, Australia) at a density of approximately 5
×105 mL−1 and were reared at 27 °C in 27 ppt, 0.2 μm filtered
seawater. Nauplii metamorphosed into cyprids within 5 days, and the
cyprids were aged for 2 days at 4−6 °C prior to use in the settlement
assays.35
Prior to testing, all samples were immersed in sterile deionized
water under reduced pressure (approximately 320 mmHg). This step
was of particular importance for the patterned samples, for which
complete wetting can be achieved only if the air trapped between small
surface features is replaced by the wetting liquid. Next, 300 μL of
seawater containing approximately 10−20 cyprids was added to each
Langmuir Article

Figure 1. M. hardwickii: a model decapod for the design of hierarchical surface topographies for antifouling and/or fouling release applications. (a) A
specimen of M. hardwickii (the scale bar corresponds to 10 mm). (b) SEM image of the carapace of M. hardwickii (the scale bar corresponds to 500
μm). (c) SEM image of the carapace of M. hardwickii (the scale bar corresponds to 10 μm). (d) SEM image of the spines on the carapace of M.
hardwickii (the scale bar corresponds to 5 μm).

Figure 2. Distribution of the feature sizes of the carapace of M. hardwickii. The data were obtained from 2D SEM images of the surfaces of at least
five different specimens of M. hardwickii. Each histogram was constructed using at least 100 measured values. The solid lines identify the nonlinear
regression (Gaussian). The height of the bin between 4 and 5 μm may be attributed to new, growing spines.

sample. Each sample was placed in its own Petri dish and covered to plastic boxes. The humidity inside the boxes was maintained at a
minimize droplet evaporation. The Petri dishes were placed in black constant level by covering the insides and the openings with

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Langmuir
Figure 3. Schematic representation of the pattern development process: (a) Surface analysis using SEM (scale bar = 100 μm), (b) pattern design, (c)
fabrication of silicon molds for pattern replication in a polymeric material (scale bar top = 20 μm, scale bar bottom = 10 μm), (d) pattern replica in
PDMS (scale bar = 50 μm), and (e) modification of the surface chemistry by the application of polymer brushes or LbL-deposited films (surface
features and modifications not drawn to scale). We show a schematic representation of the chemical (zwitterionic) structure of the applied coatings.
thoroughly wet towels. The distribution of Petri dishes inside the
boxes was randomized. After 48 h, the samples were removed, and the
number of settled cyprids on each sample was counted.
Amphora Adhesion Assay. Amphora cof feaeformis (UTEX number
B2080) was maintained in an F/2 medium36 in tissue culture flasks at
24 °C under a 12 h light/12 h dark regime for at least 1 week prior to
use. The algae were gently removed from the culture flasks with a cell
scraper, and clumps were broken up by continuous pipetting and
filtering through a 35 μm Nitex mesh. The cell count was determined
with a hemocytometer, and a suspension of 10 000 cells/mL was made
up in 30 ppt, 0.22 μm filtered seawater (FSW). PDMS controls,
treated PDMS samples, and glass coverslips were randomly placed into
wells in a six-well plate. Then, 5 mL of an algal cell suspension was
added to each well. The experiment was allowed to incubate for 24 h
in a 12 h light/12 h dark cycle at 24 °C. At the end of the incubation
period, all samples were dip-rinsed in 30 ppt, 0.22 μm FSW to remove
any unattached cells. This rinsing step was repeated three times. The
samples were then drip-dried, and 10 random fields of view were
scored at 20× magnification (0.916 mm2) for each sample under a
fluorescence microscope.
Static Field Immersion Test. Four replicate samples for smooth
and patterned polymers were threaded with a nylon thread and
suspended in a frame at 0.5 m sea depth. The frame was covered with a
3
/8 in. mesh netting to reduce grazing. The samples were inspected
every week, and the number of settling organisms was enumerated.
The samples were photographed, and 10% of the area around the
edges was disregarded during counting.
Data Analysis. The effect of surface modification on the number of
organisms that settled in the laboratory assays was analyzed using the
two-way ANOVA test (α = 0.05). Patterned and smooth surfaces
coated with LbL and brush films were compared using FDTS surfaces
as a reference. For all data sets considered, the requirement of
normality and homoscedasticity of variance was met. Field tests were
evaluated using the repeated measurers mixed-effect model. All tests
were performed using the R (Development Core Team, 2010)
software package.
■ RESULTS AND DISCUSSION
Species Choice and Microtopography Analysis. We
selected patterns that mimic the surface topology of decapod
crustaceans, which are naturally occurring foul-free species in
E dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
Article
heavy-fouling environments. These ubiquitous organisms,
consisting of over 15 000 species, occur in almost all marine
habitats. Our choice for a model species was made on the basis
of (i) their occurrence in shallow water tropical habitats with
heavy fouling conditions, (ii) the generally low level or absence
of fouling on their carapaces, and (iii) the lack of burrowing
behavior or occurrence in a habitat where burrowing is unlikely.
Additionally, practical considerations, such as the availability of
fresh samples and the potential for cost-effective replication of
the microtopographical features, were also taken into account.
The research was focused on the decapod crab Myomenippe
hardwickii, which is commonly found living in rocky shores, in
shallow water, hiding under rocks and boulders or in crevices.
This species is also very abundant in subtidal fouling
communities.
The carapace surface of M. hardwickii (Figure 1) displays
low-aspect-ratio calcareous features (tubercles) in two size
classes as well as very few hairs. To the naked eye, the carapace
appears clean and smooth, with setae found only in bundles at
the edges of the carapace and legs. Examination with scanning
electron microscopy (SEM) showed that the carapace surface is
covered with almost flat circular tubercles (5 μm high and 25−
170 μm in diameter). Between the tubercles, clusters (4−8) of
small spines (0.6−1.2 μm in diameter, 3−13 μm long) are
present (Figure 1).
In Figure 2, we present detailed results of an analysis of the
surface features. The data were obtained from two-dimensional
(2D) SEM images of the surfaces of at least five different
specimens of M. hardwickii. Each histogram was constructed
using at least 100 measured values. The estimations of the
lengths of the small spines (Figure 2f) may contain an element
of error, as these spines often grow at different angles with
respect to the surface and may not always be correctly
visualized with SEM. It was noted that the estimation of the
diameter of the larger features contains measurement error due
to the poorly resolved boundaries of these features as viewed in
the SEM images.
Langmuir
Figure 4. (a) SEM image of the silicon mold. The scale bar corresponds to 20 μm. (b) PDMS replica of the designed pattern, prepared using the
mold. The scale bar corresponds to 50 μm.
Previous studies on model surfaces37 have shown that 2 μm
features may deter the settlement of algal spores, whereas
features on the order of 20 μm appear to be effective against
barnacle larvae. As shown in ref 38, microtopographies of
similar size (2 μm width × 4−16 μm length) reduce and delay
the formation of biofilms of Staphylococcus aureus without the
need for prophylactic antibacterial agents. The M. hardwickii
model exhibited hierarchical features across both of these size
classes, suggesting the possible involvement of such structures
in the prevention of biomass accumulation. Hierarchical
microtopographies were also examined by Efimenko et al.39
as a series of folds and creases, but the effect of the discussed
surface features appears to be associated with improved fouling-
release properties rather than the reduced accumulation of
organic matter.10 We speculate that high-aspect-ratio features
on carapaces may have potential antifouling properties, while
the purpose of larger low-aspect-ratio features may be to reduce
mechanical damage to the small spines.
Fabrication of Antifouling Surfaces. On the basis of an
examination of the crab carapace, a simplified hierarchical
surface pattern, mimicking the topography of M. hardwickii, was
designed. Taking into account the reported dimensions of
surface microtopographies that effectively suppress fouling as
well as the limitations in the fabrication process, we simplified
the pattern by representing each bundle of high-aspect-ratio
carapace features with a single feature on the designed surface
(Figure 3). The designed pattern displays two regular,
hexagonal arrays of features on significantly different length
scales. The first array consists of cylindrical structures, with
each being an approximation of a single cluster of spines on the
M. hardwickii carapace, measuring 5 μm in diameter and having
an aspect ratio (height/width) of 2 and a center-to-center
distance of 7.5 μm. The second array consists of cylindrical
structures, with each being an approximation of a single
tubercle on the carapace of M. hardwickii, measuring 100 μm in
diameter, having an aspect ratio of 0.2 and a distance between
their centers of 250 μm.
The designed pattern was used to fabricate silicone molds by
applying lithographic and etching techniques. (The process is
shown schematically in the Supporting Information.) During
the process, two arrays of surface features (ø 100 μm and ø 5
μm) were etched independently in consecutive steps using two
different masks. A cross section of the resulting mold is shown
in Figure 4. The fabricated silicon molds were used for multiple
replications of the pattern in PDMS using a common casting
procedure.
For applications, the mechanical properties of the surface are
as important as the designed topography.40 It has been shown
that these properties influence the choice of the settlement
F dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
Article
location during surface exploration by fouling species. In this
study, we have chosen PDMS since it is relatively easy to
replicate patterned surfaces in this material. PDSM is also a
good candidate for a nontoxic fouling-release coating41 mainly
due to its low modulus and low surface energy.
To investigate the combined antifouling influence of surface
chemistry and topological patterning, the replicated surfaces
were further modified with zwitterionic polymer brushes and
thin films composed of LbL polyelectrolyte multilayers. Both
coatings consist of highly hydrated polymeric structures of well-
controlled thickness, ionic compensated bulk structure. The
chemical composition of both polymer coatings is shown
schematically in Figure 3 while details of the chemical reactions
leading to them are given in the Supporting Information. The
zwitterionic polymer brush that we used consists of
sulfobetaine; these brushes are effective against nonspecific
protein absorption.42 Moreover, they exhibit good resistance
against biofouling in both biomedical and marine environments
when used on hard surfaces such as glass or silicone.43 Surface-
initiated polymerization was used to modify the PDMS
substrates with polymeric brushes. In the LbL approach, thin
films composed of alternately charged polyelectrolytes were
deposited and cross-linked, i.e., were chemically anchored to
the top of the PDSM pattern. We used a recently published
novel polyanion34 that can be easily cross-linked under mild
conditions to form covalent bonds, combined with commer-
cially available PEI.
In all experiments, flat and patterned PDMS samples coated
with vapor-deposited monolayers of chemically inert and
hydrophobic perfluorododecyltrichlorosilane (FDTS) coatings
were used as a reference to ensure chemically homogeneous
PDMS surfaces. Because the settlement of algae and barnacles
(cyprids) on glass is well understood, cleaned glass slides were
used as an additional reference. If settlement on the glass slides
was lower than expected, then the results obtained on the
PDMS samples were discarded, and the tests were repeated.
Settlement Assays. Smooth and patterned samples coated
with FDTS, LbL-deposited films, and polymer brushes were
tested against two common foulers: diatoms (Amphora
cof feaeformis) and cyprids (Amphibalanus amphitrite). These
foulers have different dimensions, surface properties, and
settlement behaviors. In parallel experiments, the samples
were also exposed to marine environments during field testing,
bringing the samples into contact with a mixed population of
marine foulers.
Amphora cof feaeformis is one of the most commonly reported
species recovered from antifouling and fouling release coat-
ings;44 thus, this species was selected to evaluate the antifouling
performance of the patterns developed in this study. The
Langmuir Article

Figure 5. Settlement of Amphora cof feaeformis (a) and Amphibalanus amphitrite (b) on smooth and patterned PDMS samples modified with FDTS,
LbL films, and sulfobetaine brushes. The data are shown as the total number of settled organisms (a) and the fraction of settled organisms (b) per
square millimeter. The notation for the statistical significance was adapted as follows: * = 0.05 ≤ p < 0.10, ** = 0.01 ≤ p < 0.05, *** = p < 0.01.

Figure 6. Settlement of marine foulers on the smooth and patterned PDMS samples during the static field immersion test (a). The bar chart gives
the average number of individuals present on each sample surface (area of approximately 325 mm2) and the standard error. Examples of the fouling
condition on smooth and patterned PDMS samples, coated with FDTS, after 4 and 6 weeks of immersion (b), respectively. The size of each panel
shown b is 2 × 2 cm2. The notation of the statistical significance was adapted as follows: * = 0.05 ≤ p < 0.10, ** = 0.01 ≤ p < 0.05, *** = p < 0.01.

experimental results of the laboratory settlement assays are
shown in Figure 5a.
We observed that the settlement of Amphora on the
patterned PDMS surfaces was lower than that on the smooth
PDMS surfaces. However, the effect seemed to vary with
surface coating. The results of the statistical analysis indicate
that the surface microtopography and sulfobetaine brushes
significantly affect cellular adhesion (p = 0.002 and 0.036,
respectively, see footnote and ref 45). However, no significant
difference was identified in the number of Amphora cells settled
on the patterned surfaces when the samples coated with
sulfobetaine brushes and LbL films were compared (p = 0.108).
Fewer organisms settled on the polymer-coated surfaces than
on the FDTS-coated micropatterned surfaces. Combining the
microtopography with polymer brushes resulted in a reduction
of approximately 37% in terms of cell adhesion, whereas
combining the microtopography with the LbL film resulted in
an approximately 14% reduction of cell adhesion in comparison
to that of smooth surfaces coated with brushes and an LbL film,
respectively. Hence, the use of microtopography enhances the
antifouling performance of the brushes to a greater extent than
for the LbL films. On smooth surfaces, significantly less
G dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
settlement was observed on the surfaces coated with LbL films
than on the surfaces coated with FDTS and sulfobetaine
brushes.
The settlement of Amphibalanus amphitrite cyprids con-
stitutes another commonly applied test. This test is based on
the resilience that these organisms exhibit in fouling
communities in coastal environments, ship hulls, and man-
made marine structures as well as their wide geographical
distribution.46 Thus, this test was used to evaluate the
antifouling potential of the developed surfaces. The exper-
imental results are shown in Figure 5b.
The results indicate that fewer cyprids settled on the
patterned surfaces coated with LbL films and sulfobetaine
brushes than on smooth surfaces with corresponding coatings.
As in the case of Amphora, we observed the highest and most
significant (p = 0.072) reduction in cyprids settlement on
patterned surfaces coated with sulfobetaine brushes. In the case
of FDTS-coated reference surfaces, settlement on patterned
and smooth surface was comparable. Also any chemical
modification of a flat surface did not result in a significant
enhancement of antifouling according to ANOVA tests (p =
0.17). The effect of surface modification on settlement
Langmuir
Figure 7. SEM images of Amphora settled on a sulfobetaine brush. The scale bars are (a) 50, (b) 5, and (c) 2 μm.
suppression is however clearly visible on the surfaces covered
with zwitterionic brushes, where a synergistic effect of the
pattern and the brush can be observed.
The observed effect of sulfobetaine brushes on flat surfaces is
surprisingly weak compared to the data available in the
literature for zwitterionic-coated hard materials such as silicone
and glass.47 To the best of our knowledge sulfobetaine brushes
on flat PDMS for antifouling research were reported only once
and not for marine applications.48 As such there is no
comparative data for barnacles and Amphora algae available
to cross verify our findings. We speculate that two factors can
be responsible for the lower performance of flat PDMS surfaces
when compared to that of glass or silicon. First, the modulus of
the substrate may play a role when animal assays are performed
since PDMS is softer than typical materials in use. Second, the
surface reorganization of PDMS may affect the brush structural
conformation and thus its functionality. Nevertheless, one has
to bear in mind that exactly the same type of grafted polymer
was applied to patterned and smooth PDMS substrates and the
presence and stability of the graft was thoroughly supported by
the experimental evidence. Presented data allow us to draw
conclusions using direct sample comparison clearly showing the
importance of surface chemistry when applied in combination
with a patterned structure.
To evaluate further the antifouling properties of the
patterned surfaces, samples coated with FDTS were exposed
to a marine environment in a static field test. The experimental
results are shown in Figure 6.
The organisms settling on the PDMS consisted mainly of
calcareous tubeworms. Detailed information about other
groups of foulers present can be found in the Supporting
Information. We observed that during the immersion period
more organisms settled on the smooth surface than on the
patterned surfaces. The number of spirorbid tubeworms settling
on the patterned surface was consistently smaller than that
observed on the smooth PDMS, and the individuals present
were smaller than those observed on the smooth surfaces.
There was a decrease in the number of organisms at 6 weeks,
which was likely a result of the detachment of larger individuals
due to the inherent fouling-release properties of the PDMS
material. Barnacle settlement on the control (smooth) surface
was first observed after 4 weeks. No barnacles were recorded on
the patterned surfaces throughout the 7 week immersion
period. This observation appears to contradict the results from
the cyprid settlement test, for which no difference between the
smooth and patterned surfaces was recorded. However, it
should be noted that the hydrodynamic conditions between the
static laboratory versus field tests are different, and it has been
demonstrated that the antifouling properties of microtopog-
raphies may be associated with hydrodynamic forces.49 Finally,
we note that there was no observable difference in the extent of
H dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
Article
slimes and soft fouling on the respective surfaces. The field tests
were carried out on topologically patterned specimens without
polymer coatings. At this point, surface modification technol-
ogy using hydrophilic brushes does not allow field test exposure
for sufficient duration without compromising the quality of the
coating.39
The effect of surface topology on the settlement of foulers
has long been discussed in the literature. It has been suggested
that the effective antifouling pattern designs should consist of
dissimilar features, inducing a stress gradient within the lateral
plane of the surface membrane upon contact.50 Moreover, the
number of possible contact points should be minimized.51 The
distance between the patterned features should prevent the
penetration of depressed regions by fouling organisms,52 and in
the case of settlement on neighboring features, the organism
should not be able to rest on the bottom of the depressed
region.50 At the same time, the interfeature distance and feature
dimensions should prevent the stabilization of organisms on a
single element. It was reported that algae (Ulva) spores tend to
settle in areas where at least three points of contact with the
substrate are possible.53 A close examination of the patterned
surfaces in this study, after exposure to Amphora, clearly shows
that such spore positioning also takes place. Moreover, we
observed that the settling spore “pulls” the pattern features
(pillars) together, making the surface area available for
settlement larger (Figure 7). Using polymers of lower elasticity
than PDMS to replicate the designed microtopography (i.e.,
polyurethane or poly(methyl methacrylate)) may restrict such
surface adaptation. Increasing the surface area available for
settlement also may be possible due to the secretion of
exopolymers, which organisms use to (partially) fill the gaps
between surface features during settlement.54,55 Another crucial
issue is the relationship between the size of the settling
organism and the critical feature size of the patterns. For algae
spores, the effective feature sizes were reported to correspond
to the spore size.56 The case for cyprids is more complex, as it is
not clear which dimension is the most significant during the
settlement process, namely, the size of the oval attachment disc
or the size of its features (villi, sensory setae).57 Andersson et
al.58 have shown that artificial microtopographies with feature
sizes of between 50 and 100 μm reduced the settlement of
barnacle larvae. Schumacher et al.11 showed a strong correlation
between the aspect ratio of surface features and the settlement
of Ulva spores and A. amphitrite cyprids: as the aspect ratio
increased, the suppression of settlement became more effective.
The best results (approximately 85 and 90% reduction for Ulva
and A. amphitrite, respectively) were reported for a topo-
graphical aspect ratio value of 2. The simple surface structure
described in this work accounts for these findings: the design
consists of arrays of two feature length scales, including 100 and
Langmuir
5 μm with aspect ratio values of 2 and 0.2 for the small and
large features, respectively.
In this study, we focused on charge-compensated and highly
hydrated surfaces, as these characteristics have been reported to
suppress protein adsorption very effectively.9
It has also been reported that many bacteria, diatoms,59 and
barnacles60 tend to settle on hydrophobic surfaces. Here, we
demonstrate a consistent significant reduction in the attach-
ment of Amphora and barnacle cyprids on patterned surfaces
with zwitterionic brushes, indicating the synergistic enhance-
ment of the antifouling performance, which cannot be
explained by any observed effect obtained for the pattern or
brush alone. The same brushes were much less efficient on
planar surfaces of the elastomeric substrates used here
(PDMS), in comparison to situations in which they were
applied to hard substrates, as reported in the literature.16 This
observation underlines the importance of the substrate modulus
as a factor when determining the fouling performance.
Compared to the zwitterionic brushes, the LbL films were
less effective. Our research provides criteria for the rational
design of technologically relevant, even more complex
integrated antifouling surfaces.
■
CONCLUSIONS AND OUTLOOK
We have presented a design of bioinspired hierarchical surface
topography showing non-species-specific antifouling and
fouling release potentials. We have also proposed an efficient
method for further chemical modification of such surfaces with
thin polymeric films. The application of these does not affect
the existing surface topography but provides a highly hydrated
surface layer, further increasing its antifouling potential.
Moreover, we explored a synergistic effect between the
developed surface topography and the applied polymer brush
and provided arguments for more complex surface designs. The
pattern effect was greatly enhanced by the presence of specific
chemical modification; in our case, zwitterionic polymeric
brushes were particularly effective.
Antifouling properties of the proposed material were also
demonstrated in a real marine environment. The difference
between the results of the laboratory tests and the raft test
suggest that that the effect of pattern on settlement can vary
with hydrodynamic conditions and indicate the fouling-release
potential of the designed coating. The design presented was
investigated using small demonstrators and due to limitations in
fabrication can be applied only to small objects such as sensors.
Independent research efforts are currently directed toward
scaling-up issues using hot roll embossing technology, allowing
us to utilize technology for larger surfaces.
The discussed results indicate that the surface features of M.
hardwickii may be associated with antifouling. However, as we
have tested only a simplified topography, studies of more
complex surface topographies, closely resembling that of the
crab surface, are necessary to provide a definitive answer to this
question. This research is ongoing.
■
ASSOCIATED CONTENT
*
S Supporting Information
FTIR, schemes of chemical modifications, scheme of the
microfabrication procedure, and detailed contributions of
various fouling organisms detected during the raft test. This
material is available free of charge via the Internet at http://
pubs.acs.org.
I dx.doi.org/10.1021/la502006s | Langmuir XXXX, XXX, XXX−XXX
■
Article
AUTHOR INFORMATION
Corresponding Authors
*Tel: +65 6874 5443. Fax: +31 53 489 3823. E-mail: g.j.
vancso@utwente.nl.
*Tel: +31 53 489 2974. Fax: +65 6872 0785. E-mail:
janczewskid@imre.a-star.edu.sg.
*Tel: +65 6774 9887. Fax: +65 6776 1455. E-mail: tmsteolm@
nus.edu.sg.
Author Contributions
A.M.B. and F.J.P.-V. have contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
We gratefully acknowledge Mr. Vincent Lim Shien Fuh, Mr.
Chen Yi Fan, and Mr. Eric Tang Xiaosong from the SERC
Nano Fabrication, Processing and Characterisation (SnFPC)
Facility at the Institute of Materials Research and Engineering
(IMRE) for their help with photomask writing and silicon wafer
oxidation and discussions. We are grateful to the Agency for
Science, Technology and Research (A*STAR) for providing
financial support under the Innovative Marine Antifouling
Solutions (IMAS) Program.
■
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