Sustainable and bio-inspired chemistry for robust antibacterial activity of stainless steel

Article abstract of DOI:10.1039/c1jm11380a

We report on the original synthesis of a poly(methacrylamide) bearing (oxidized) 3,4-dihydroxyphenylalanine specially designed to (i) insure film growth by covalent coupling, (ii) covalently bind an antibacterial peptide and (iii) contribute to the film c

Full text of DOI:10.1039/c1jm11380a

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COMMUNICATION
Sustainable and bio-inspired chemistry for robust antibacterial activity of
stainless steel†
a
a
a
b
Emilie Faure, Philippe Lecomte, Sandrine Lenoir, Christelle Vreuls, Cecile Van De Weerdt,
b
ꢀ
c
b
a
d
ꢀ ^
Catherine Archambeau, Joseph Martial, Christine Jerome, Anne-Sophie Duwez
and Christophe Detrembleur
a
*
Received 1st April 2011, Accepted 18th April 2011
DOI: 10.1039/c1jm11380a
imine adsorbed onto SS,18 or the post-quaternisation of amino
groups grafted onto SS pre-functionalized by cold plasma treat-
ment19,20 or by silane coupling agents.21 However, these strategies are
multi-steps, sometimes complex processes and require the use of
aggressive chemicals and/or toxic molecules that make difficult the
implementation at the industrial scale.
We report on the original synthesis of a poly(methacrylamide)
bearing (oxidized) 3,4-dihydroxyphenylalanine specially designed to
(i) insure film growth by covalent coupling, (ii) covalently bind an
antibacterial peptide and (iii) contribute to the film cross-linking
that is essential for the durability of the properties.
Here we report on a very simple and robust strategy to impart
durable antibacterial properties to stainless steel by the Layer-by-
Layer (LbL) technique22 using a tailored multifunctional polymer. It
relies on the original synthesis of a homopolymer of methacrylamide
bearing (oxidized) 3,4-dihydroxyphenylalanine (mDOPA) specially
designed to (i) insure LbL film growth by covalent coupling, (ii)
covalently bind an AB peptide and (iii) contribute to the film cross-
linking that is essential for the durability of the properties. Further-
more the whole process occurs in aqueous solution without specific
surface treatments, providing a green approach. We therefore exploit
the redox property of DOPA moieties, an important component
responsible for the adhesion of mussels on various substrates,23–26
present in a tailored polymer (P(mDOPA)) to get cross-linked
multilayers and biocides grafting. This catechol derivative can be
switched from a water insoluble (reduced state) to a water-soluble
and highly reactive state towards primary amine (oxidized state). This
reactivity makes this polymer unique and versatile for adjusting
properties as demonstrated hereafter and for growing multilayers
faster than works previously described elsewhere.26,27
Stainless steel is widely used in daily life because of its resistance to
corrosion and chemicals, and its mechanical and aesthetic properties.
It can be found in many areas such as medicine and household
appliances, building and food industries.1 One main limitation of
stainless steel is its inability to stop bacteria proliferation. Therefore
for hygienic reasons, there is a strong need for developing robust
surface modifications to give antibacterial (AB) properties to stainless
steel. The literature is extremely rich in describing strategies for
coating substrates with inorganic (silver,2–4 copper,5.) and organic
(antibiotics,6,7 peptides,8 (co)polymers,9–12.) biocides. However,
most of the time, the AB activity is time limited by the diffusion of the
active species out of the coating. Stainless steel producers and users
are now expecting long-lasting coatings essential for the durability of
the functionality. It is therefore necessary to strongly bind the
biocides to the surface. Furthermore, considering environmental
issues, current development of new products has to prevent toxic
wastes. Green coating processes are thus urgently required.
As far as stainless steel (SS) is concerned, the direct covalent
grafting of organic biocides onto the surface is not trivial because of
the absence of appropriate functional groups allowing strong
anchoring. Only few examples can be found in the literature such as
the electrografting of acrylates13,14 post-modified to obtain AB
properties,15–17 the grafting of lysozymes or trypsin onto polyethylene
The developed strategy to build the multilayer (Scheme 1a) starts
with a first immersion of the substrate in an aqueous solution of P
(mDOPA)-co-P(DMAEMA+) (2 g Lꢀ1), a polycationic copolymer
bearing 15 mol% DOPA moieties (Scheme 1b; chemical structure (3))
at room temperature.4 This DOPA-functionalized polycation is
designed to strongly anchor to the surface by DOPA/metal interac-
tions.28 The next layers are then built by the successive dipping of the
surface into an aqueous solution of a poly(methacrylamide) bearing
oxidized DOPA moieties on each monomer unit (Scheme 1b,
formula (2)) (Pox(mDOPA)), and then in a polymer bearing primary
amines, polyallylamine (PAH). The appropriate combination of this
polymer with PAH in LbL multilayers, by adequately controlling
both the redox state of the P(mDOPA) polymer (Scheme 1b;
chemical structures (1) and (2)) and the pH of the solutions, has been
optimized to grow efficiently a durable LbL coating.
aCenter for Education and Research on Macromolecules (CERM),
University of Lieꢁge, Sart-Tilman, B6, B-4000 Lieꢁge, Belgium. E-mail:
christophe.detrembleur@ulg.ac.be
bCentre of Biomedical Integrative Genoproteomics, University of Lieꢁge,
B34 Sart-Tilman, 4000 Lieꢁge, Belgium
cArcelorMittal Lieꢁge Research, Bd de Colonster B57, 4000 Lieꢁge, Belgium
dNanochemistry and Molecular Systems (NANOCHEM), University of
Lieꢁge, Sart-Tilman, B6, B-4000 Lieꢁge, Belgium
† Electronic supplementary information (ESI) available: Monomer
P(mDOPA),
synthesis,
characterizations. See DOI: 10.1039/c1jm11380a
polymerization,
oxidation
of
and
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(pKa z 6) of nisin (Scheme S2, ESI†) with oxidized catechol35,36
cannot be excluded in these conditions.
As a first evidence of the multilayer film build-up, Quartz Crystal
Microbalance coupled with Dissipation (QCM-D) was used to follow
the film growth in real time on SS sensors by measuring the variation
of the resonant frequency (Df) vs. time. A decrease in Df indicates
polymer deposition.37 Fig. 1 shows that all partners are successfully
deposited according to the selected deposition sequence and redox/
pH conditions and remain on the substrate even after rinsing with
water.
According to this procedure, 15 bilayers of Pox(mDOPA)/PAH,
followed by 5 bilayers of Pox(mDOPA)/nisin, were built on
substrates provided by the steel industry (Arcelor-Mittal, SS 304 2B).
Surface coverage and film thickness were probed by Field Emission
Gun Scanning Electron Microscopy (FEG-SEM). A cross-section of
coated steel (Fig. S1, ESI†) clearly evidences that the film is homo-
geneously covering the substrate, even in the substrate defects, with
an average film thickness of about 50–60 nm. XPS analysis confirms
the presence of nisin with its detected sulfur atoms (Fig. S2, ESI†).
Antibacterial activity of the film has been assessed against
B. subtilis using the standard JIS Z method specially designed for
contact-killing coatings. Coated substrates were inoculated with
about 106–108 cells mLꢀ1 of bacteria for 24 hours and the number of
survival bacteria was evaluated by counting them after spreading on
agar plates. A strong AB activity was observed as demonstrated by
a very high log decrease of survival bacteria compared to bare SS
(Fig. 2, controls; according to this JIS Z 2801:2000(E) procedure, a 2
log decrease is considered as antibacterial). The permanent func-
tionality of the coating was then evaluated by immersing the coated
substrate in tap water for one night (the immersion test, Fig. 2) and by
cleaning it with a wet sponge (30 back and forth movements, the
mechanical test, Fig. 2). It was then tested again following the same
procedure and AB activity was maintained at the same level, sup-
porting the chemical grafting of nisin without denaturation. In order
to test further the efficiency of the coating activity, the amount of
bacteria was then considerably increased by one order of magnitude
(from 108 to 109 bacteria mLꢀ1). The AB activity was still present but
lowered, certainly due to the saturation of the surface by bacteria.
Different control samples were prepared to assess the role of nisin
grafting and of the developed strategy. First, films containing 20
bilayers of Pox(mDOPA) and PAH without nisin were tested and
Scheme 1 (a) LbL process for imparting long-lasting AB properties to
stainless steel and (b) chemical structures of (1) P(mDOPA), (2) Pox
(mDOPA) and (3) P(mDOPA)-co-P(DMAEMA+).
Oxidized DOPA moieties of Pox(mDOPA) are necessary for the
covalent grafting of PAH through amine/quinone reaction and/or
Schiff base formation at room temperature, and consequently for the
interlayer cross-linking (Scheme 1a and S1, ESI†).28–30 Preparation
conditions are crucial for the success of the film build-up. First,
P(mDOPA) is oxidized in aqueous media under basic conditions for
12 h to form the hydrosoluble Pox(mDOPA) (see ESI†) which is then
deposited at a concentration of 2 g Lꢀ1 on the first P(mDOPA)-co-P
(DMAEMA+) layer by immersing the surface for 2 min. Importantly,
the next solution of PAH (2 g Lꢀ1) is deposited at pH ¼ 11 in order to
obtain the polymer in the deprotonated state. The reaction between
primary amines and the quinone groups of Pox(mDOPA) is therefore
made possible (Scheme S1, ESI†). In acidic media, amine groups are
protonated and do not react with Pox(mDOPA), such that cross-
linking and growth of the film cannot occur. All synthetic steps are
performed in mild conditions and in aqueous media (see ESI†), which
make the building-block synthesis pathways relevant for the devel-
opment of an environment-safe process.
Finally, in order to impart AB activity to these coatings, PAH is
replaced by nisin in the 5 last layers (Scheme 1a). Nisin, a low
molecular weight AB peptide (3500 g molꢀ1), was selected because of
(i) its high activity against a broad range of Gram-positive bacteria,31
(ii) its wide use as food preservative, (iii) its lack of toxicity for
humans32 and (iv) its stability when involved in coatings.33 For this
purpose, nisin was dissolved in a phosphate buffer solution at
pH ¼ 7.4 to favor the peptide grafting through its N-terminal NH2
group (pKa ¼ 6.8–8) and not through the amino-groups of the three
internal lysines (pKa ¼ 11) (Scheme S2, ESI†) that are mainly
responsible for the AB activity of the peptide.34 It is important to
mention that the peptide grafting by the reaction of imidazole groups
Fig.
1 Frequency change upon deposition of P(mDOPA)-co-P
(DMAEMA+), Pox(mDOPA), PAH and nisin measured by QCM-D as
a function of time at 25 ꢁC, where the overtone number is 7.
7902 | J. Mater. Chem., 2011, 21, 7901–7904
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those made from self-polymerized dopamine,27 our strategy is faster
(ꢂ1 h vs. 24 h26) and does not require the use of strong oxidizing
agents. Moreover, our cross-linking process occurs at room temper-
ature in water and does not require the use of any toxic reagent. This
is in sharp contrast to conventional LbL PAA/PAH films that can
only be cross-linked using some toxic activators (such as dicyclo-
hexylcarbodiimide, DCC) and/or under thermal treatment that may
denaturate the peptide. Our versatile approach might be applied to
other biomolecules and surfaces, broadening considerably its general
scope.
The research was partly supported by BELSPO (IUAP VI/27), the
Walloon Region (PPP program BIOCOAT) and the National Fund
for Scientific research (F.R.S.–FNRS). We thank all the BIOCOAT
team for its contribution, in particular C. De Bona, G. Garitte,
Fig. 2 Antibacterial assessments against B. subtilis using the JIS Z
method, values are bacteria log survivals compared with bare stainless
steel; all durability experiments were done in triplicate; purple colour
corresponds to an initial bacteria log of 6.2, red colour to 7.7 and the
green one to 8.9 (controls ¼ AB coating on SS before mechanical and
immersion tests).
ꢀ
F. Farina, and G. Zocchi. We thank the Centre d’Ingenierie des
ꢀ
Proteines (ULg) for B. subtilis 168 strain.
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amount of bacteria one order of magnitude lower than for the test
carried out on the cross-linked system (((Pox(mDOPA)/PAH)15/
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between the layers by electrostatic interactions (PAA is negatively
charged and nisin is positively charged) is not sufficient for preserving
the durability of the functionality. Cross-linking of the film and
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3,4-dihydroxyphenylalanine groups. All the processing steps,
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