Plasma deposited metal Schiff-base compounds as antimicrobials

Article abstract of DOI:10.1039/c1nj20091g

This paper details the synthesis, characterisation including crystal structure, and testing for antimicrobial efficacy of two compounds: a zinc centred bis(N-allylsalicylideneiminato)-zinc (ZSB) and its known copper analogue, bis(N-allylsalicylideneiminat

Full text of DOI:10.1039/c1nj20091g

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PAPER
Plasma deposited metal Schiff-base compounds as antimicrobialsw
Neil Poulter,a Matthew Donaldson,a Geraldine Mulley,ab Luis Duque,a
Nicholas Waterfield,b Alex G. Shard,c Steve Spencer,c A. Tobias A. Jenkinsa and
Andrew L. Johnson*a
Received (in Montpellier, France) 3rd February 2011, Accepted 28th March 2011
DOI: 10.1039/c1nj20091g
This paper details the synthesis, characterisation including crystal structure, and testing for
antimicrobial efficacy of two compounds: a zinc centred bis(N-allylsalicylideneiminato)-zinc (ZSB)
and its known copper analogue, bis(N-allylsalicylideneiminato)-copper (CSB). Differences in
antimicrobial efficacy of the two compounds were observed, suggesting possible mechanisms for
antimicrobial activity. The ZSB system was plasma deposited under various pulse conditions onto
non-woven fabric, and the antimicrobial efficacy of the resultant film measured for Staphylococcus
aureus and Pseudomonas aeruginosa. These results suggest the potential utility of this compound
as an effective antimicrobial thin film, and confirm the critical role the ligands play in effecting
antimicrobial activity.
infection and dermatitis, with consequences such as bed/
Introduction
pressure sores, and opportunistic infection by yeasts such as
Candida albicans.4
Bacterial infection, which at one time was thought to be a
battle won, returned in the 20th century to become a major
Subsequently, there is an unmet clinical and sub-clinical
health issue, causing increasing numbers of deaths in both
need for a cheap and effective method for imparting safe, low
clinical and community environments, with bacterial strains
level antimicrobial activity to objects such as catheters, wound
dressings and hospital protective clothing.5 There are a
such as Methicillin Resistant Staphylococcus aureus (MRSA)
receiving much media attention.1 The treatment of choice for
number of approaches for making surfaces antimicrobial,
bacterial infections since the 1940s has been antibiotics, but
ranging from impregnating polymers with chlorinated phenols
such as Triclosant, to the use of silver6 or quaternary
ammonium cations.7,8 Consumer concern over the safety of
with increasing evolved resistance, in part due to overuse, their
efficacy is starting to diminish.2 Most species of bacteria can
attach to biotic or abiotic surfaces and form biofilm communities
chlorinated organic compounds, particularly in the US has led
to a decline in its use.9 Silver is finding increasing usage, in
which are typically resistant to adverse environmental conditions
and often antimicrobial agents. Bacterial survival on everyday
everything from the inside of refrigerators, computer
keyboards to urinary catheters.10 It is often included either
objects such as hospital bedside curtains is believed to be a
part factor in the rise of hospital acquired infections in the
United Kingdom, for example.3 As well as well publicised
the polymer melt prior to injection moulding, or on post
in nanocrystalline form, or impregnated into zeolites as part of
mould/spin application as an additional thin polymer film.11
consequences of infection in clinical situations, enormous
numbers of people suffer from the effect of low level infection,
The difficulty with such films is that they add cost to clinical
which may not always be life threatening, but can seriously
objects, to a point where health care providers may not pay for
diminish quality of life, particularly in the case of elderly
their use. There are also concerns about the efficacy of such
or disabled people. Skin infections and dermatitis are two
films, and with increasing consumer concern about the
common conditions which are treatable, but often at some
safety/toxicity of nanoparticles, it is possible in the future that
use of silver nanoparticles may become restricted.12
cost. Skin covered in fabric can be particularly susceptible to
Zinc and copper compounds have previously been used as
a Department of Chemistry University of Bath, Bath, BA2 7AY,
United Kingdom. E-mail: A.L.Johnson@bath.ac.uk;
antimicrobial agents. For example zinc is incorporated into
certain creams for the treatment of dermatitis. Furthermore,
Fax: +44 (0)1225 386231; Tel: +44 (0)1225 384467
b Department of Biology & Biochemistry, University of Bath, Bath,
BA2 7AY, United Kingdom
zinc has also been added to various consumer products to
extend their shelf lives, including toothpaste (zinc lactate and
zinc sulfate), where its low cost and low toxicity makes its use
attractive.13 Interestingly the ability of brass (a zinc/copper
alloy) to control bacterial growth has been used for hundreds
of years and recently, it has been suggested that copper and
c National Physical Laboratory, Teddington, Middlesex, TW11 0LW,
United Kingdom
w Electronic supplementary information (ESI) available. CCDC
reference numbers 771905 and 771906. For ESI and crystallographic
data in CIF or other electronic format see DOI: 10.1039/c1nj20091g
c
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brass fittings could be used on hospital door handles to reduce
cross-infection.14 Copper, like zinc, also has antimicrobial
effects that can inhibit bacterial growth surfaces.15,16 Despite
the wide use of these metals, the antimicrobial mode of action
of zinc and copper in bacteria is still not fully understood,
although Blencowe and Morby have recently reviewed of the
metabolism of Zn(II) by prokaryotes which may give some
understanding to antimicrobial modes of action.17
of characterisation techniques. In both cases elemental
analysis confirms the bulk product was within expected limits
for the desired complexes.
Single crystal X-ray diffraction studies reveal the ZSB
complex to be a dimer with bridging salicylaldiminato ligands
resulting in a distorted trigonal bipyramidal geometry around
each zinc centre (Fig. 1). The two zinc atoms in a discrete
molecule are symmetrically related by crystallographically
imposed Ci symmetry and connected by the O(2) atoms of
the bridging ligands. A result of this crystallographic arrangement
is that the imine substituents on the two bridging salicyl-
In addition to the direct antimicrobial activity of metals
such as silver, zinc and copper, various coordination
complexes can also exhibit antimicrobial activity.18–22
In this paper we discuss the synthesis of zinc and copper
Schiff-base complexes: bis(N-allylsalicylideneiminato)-zinc
(ZSB) and bis(N-allylsalicylideneiminato)-copper (CSB)
(Fig. 1 and 2 respectively) and the subsequent use of
these complexes in formation of antibacterial thin film
materials using plasma deposition. Plasma deposition is a
methodology that allows the low temperature deposition and
‘polymerisation’ of molecular compounds onto a wide range
of substrates and is discussed in detail by Yasuda.23 Film
analysis and measurement of antimicrobial activity for
both the Schiff-base complexes and their thin films is also
described.
aldiminato ligands posses
a cisoidal configuration as
do the imine substituents on the chelating salicylaldiminato
ligands.
Compared with the previously reported zinc Schiff-base
complexes the formation of the dinuclear motif in ZSB may
be caused by the smaller and more flexible imine substituent,22
and results in the oxygen atoms acting as m2 bridges The
chelating salicylaldiminato ligand on the zinc centre of this
dimeric species contains bond lengths similar to those
observed for related systems (ESIw)27 but as would be
expected, Zn–O bond distances for the bridging ligand are
slightly longer than the Zn–O bond in the non-bridging ligand.
The Oax–Zn–Oax angle is 171.31(9)1 and the equatorial angles
are not far from 1201 (122.65(11)1 [N(1)–Zn(1)–N(2)];
125.62(10)1 [O(2)–Zn(1)–N(2)] except for the equatorial angle
Results and discussion
[N(1)–Zn(1)–O(2)] which is 110. 49(16)1. The sum of the
P
Molecular structures
equatorial angles [
= 358.761] suggests there is very
little deviation of zinc out of the plane defined by the atoms
Zn(eq)
The allyl substituted Schiff-base, SB, was prepared by the
addition of allyl amine to salicylaldehyde in methanol using
standard literature techniques.24–26 Both zinc and copper
Schiff-base complexes where obtained by treating either zinc
acetate or copper acetate with two molar equivalents of the
ligand in the presence of triethylamine (Scheme 1).
N(1), N(2) and O(2).
The molecular structure of the CSB complex (shown in
Fig. 2) is revealed to be a monomeric complex with an
approximately planar configuration about the central copper
centre. The salicylaldiminato ligands are arranged with respect
to each other in a trans orientation such that one allyl
substituent lies above the [CuO2N2] plane and the other below.
The molecular structure of CSB has previously been described
by Bhatia et al.27 and was collected as part of this study for
completeness and is presented here for comparison with the
related zinc complex, ZSB.
Subsequent isolation of the metal complexes by extraction
into dichloromethane and recrystallisation from hot toluene
allowed the complexes ZSB and CSB to be isolated in high
1
yields (74–78%) as crystalline materials. While H NMR and
13C NMR data were obtainable for the zinc complex, the
paramagnetic nature of the copper complex limited the choice
Scheme 1 Simplified scheme showing synthesis of the Schiff-base ligand and subsequent complexation with zinc or copper to yield bis-
(N-allylsalicylideneiminato)-zinc (ZSB) and bis(N-allylsalicylideneiminato)-copper (CSB).
c
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Fig. 1 The molecular structure of bis(N-allylsalicylideneiminato)-zinc
(ZSB).
Fig. 2 The molecular structure of bis(N-allylsalicylideneiminato)-
copper (CSB).
Tables containing relevant bond lengths and bond angles
for CSB and experimental details relating to the single-crystal
X-ray crystallographic studies are provided in the ESI.w
Plasma deposited film analysis
The mass of the deposited films was estimated by depositing
compounds onto quartz crystal microbalance chips and
measuring the frequency shift, and estimating the mass change
using the Sauerbrey equation.28 Film thickness was measured
using a TLC Tencor P-10 alpha step profiler. Data relating to
film thickness and density following a 30 min, 1/40 pulse
plasma deposition are shown in Table 1.
Atomic absorbance analysis was run on 1 cm2 squares of
treated non-woven polypropylene following deposition of
ZSB and CSB under optimised conditions. Plasma film treated
samples were placed in 4 ml of filtered Milli-Q water for 24 h.
A zinc concentration at 20 ppb was measured and copper was
10 ppb. A control of a non-coated Petri dish was measured
with no zinc or copper was detected. The values obtained from
this analysis were close to the accuracy limit of the equipment.
A survey scan was measured (ESIw) and a narrow scan used
to look for the presence of zinc at 1030 eV. No zinc was
detected in the film, suggesting it is either buried, or at a
concentration below the limit of detection of the XPS instrument.
A linear background was applied to the C1s region and mixed
Gaussian–Lorentzian peaks with 30% Lorentzian character
were used to fit the envelope (Fig. 3).
The widths of the peaks were fixed to be equal, with the
exception of the putative shake-up satellite appearing about
6 eV higher than the hydrocarbon (lowest binding energy)
peak. The positions of the individual peaks were fixed to the
following values and the spectra were charge-compensated by
shifting the spectra so that the hydrocarbon peak appeared at
Fig. 3 (A) C1s spectrum of pp-ZSB following 30 min deposition with
a 1/40 duty cycle; (B) N1s spectrum of pp-ZSB.
Table 1 Film thickness, mass and density properties of ZSB and CSB
films deposited under 1/40 pulse plasma conditions after 30 min
deposition
285.0 eV.29 There is some ambiguity in the peak assignments,
since imine, nitrile and amide environments may exist in
plasma films due to rearrangement and damage of the
monomer in the plasma environment.30 The overlap between
the peaks is also very strong, so the values should be taken as
pp-ZSB
pp-CSB
Mass (mg cmꢀ2
Thickness (nm)
)
14.6
32 ꢁ 2
6.4
30 ꢁ 4
Density (g cmꢀ3
)
4.55
2.14
c
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Table 2 Fitted peak positions from C1s and N1s XPS spectra of 1/40 pp-ZSB film together with approximate assignments, contribution to the
total peak area for each element and comparison with expected values for the ZSB monomer
Energy/eV
Assignment
Measured
Expected for Schiff-base monomer
285.0
286.0
286.6
287.8
289.0
B291
399.5
401.8
Hydrocarbon
C–N
C–O, CQN
CQO
67% of carbon
12% of carbon
14% of carbon
4.5% of carbon
0.5% of carbon
1% of carbon
70% without shake up
10% without shake up
20% without shake up
None
None
B7% of total intensity, based on XPS of polystyrene.
100% from imine
None
O–CQO
p to p* shake up
Imine
Ammonium
92.5% of nitrogen
7.5% of nitrogen
indications only. Schiff-base monomer expected values are
also shown in Table 2.
against both bacteria above 600 mmol dmꢀ3. For this reason,
attention focussed on the plasma deposition of the zinc
compound. The MIC of zinc acetate is approximately twice
that of ZSB against S. aureus, which suggests that for this
organism the Zn2+ is the antimicrobial moiety since ZSB is a
dimer containing two zinc centres per molecule. However, the
data for P. aeruginosa suggest that for this organism, the zinc
ions cannot be the primary antimicrobial agent, since the MIC
of zinc acetate (5.1 mmol dmꢀ3) is around 8 times larger than
that of ZSB (615 mmol dmꢀ3). The Schiff-base ligand has a low
antimicrobial activity against both S. aureus and P. aeruginosa
(approx. 2–3 mmol dmꢀ3).
The N1s spectrum (Fig. 3) is dominated by a peak at
B399.5 eV, which is midway between the typical positions
for amine (B399 eV) and amide (B400 eV) indicating a
possible mixture of these two common environments. It may
also arise from imine (which is present in the monomer) or
nitrile groups. The shoulder on the high binding energy side of
the main peak (fitted with a peak at 401.8 eV) is typical of an
ammonium environment.
FTIR of monomer and polymers
FTIR analysis of ZSB monomer and pp-ZSB plasma depos-
ited film showed the expected transition from well defined
sharp absorption peaks in the monomer to broader absorption
peaks on the plasma deposited film. The spectra are shown in
the ESI,w but of particular note are the retention of the C–H
stretch at 2900 cmꢀ1, but the appearance of a clear hydrogen
bonding O–H stretch at 3400 cmꢀ1, not present in the monomer.
This may have arisen from water absorbing into the more
hydrophilic film or OH groups from oxidation of the ZSB
during plasma deposition. The absorbance at 1620 cmꢀ1 in the
ZSB monomer and the broad absorbance around 1660 cmꢀ1
in the pp-ZSB is due to the CQN in the imine, showing
evidence of retention of this group in the pp-ZSB.30
Bioluminescence assay
To determine whether the antimicrobial activity of ZSB is due
to an accumulation of toxic concentrations of intracellular
Zn2+, a Zn2+-responsive E. coli strain was used to monitor
the presence of intracellular Zn2+ arising from dissolved ZSB
monomer. The change in intensity of bioluminescence
produced by this bacterial strain following titration of ZSB,
zinc acetate and the Schiff-base ligand is shown in Fig. 4,
with luminescence intensity normalised to the background
luminescence of bacteria not exposed to zinc.
The MIC for the E. coli was also measured for these
molecules, and is shown in Table 4.
Fig. 4 shows that bioluminescence intensity increased in
response to increased concentration of zinc acetate from
1 mmol dmꢀ3 to 1250 mmol dmꢀ3, this gave a maximum rate
Antimicrobial activity of monomeric ZSB/CSB and
pp-ZSB/pp-CSB
The antimicrobial activity of the ZSB and CSB compounds
against Staphylococcus aureus MSSA 476 and Pseudomonas
aeruginosa PAO1 was quantified using standard Minimum
Inhibitory Concentration (MIC) assays. The results are sum-
marised in Table 3 below, with MIC curves provided in the
ESI.w
of bioluminescence that was sustained up to at least 3 mmol dmꢀ3
,
which is 50% higher than the MIC for E. coli (Table 3)
showing that zinc acetate is only inhibitory to growth up to
3 mmol dmꢀ3 rather than toxic bactericidal. ZSB also stimulated
an increase in bioluminescence between 1 mmol dmꢀ3 and
400 mmol dmꢀ3 at concentrations below the MIC, suggesting
that if the whole molecule is entering the cell, it must be
dissociating to the ligand and Zn2+ in the cytoplasm. This
reporter system specifically responds to the concentration of
The CSB complex prevented growth of S. aureus above
515 mmol dmꢀ3
,
but was much less effective against
P. aeruginosa (43.5 mmol dmꢀ3). In contrast, ZSB is effective
Table 3 Minimum Inhibitory Concentrations of ZSB, CSB, Schiff-base ligand, zinc acetate and copper acetate
S. aureus MSSA
P. aeruginosa PA01
MIC100 (mmol dmꢀ3
MIC100 (mmol dmꢀ3
)
MIC100 (mg mlꢀ1
)
)
MIC100 (mg mlꢀ1
)
Complex
ZSB
CSB
Schiff-base ligand
Zinc acetate
Copper acetate
360
515
2109
700
4200
160
198
337
154
500
615
280
1300
483
1120
430
3500
3020
5100
3500
c
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Fig. 4 Plot of normalised luminescence of E. coli Zn2+-responsive
biosensor in response to intracellular zinc ions, as a function of ZSB,
zinc acetate and Schiff-base ligand concentration.
Table 4 E. coli MIC (mmol dmꢀ3) for ZSB, zinc acetate and the
Schiff-base ligand
ZSB
Zinc acetate
Schiff-base ligand
400
1950
1600
free intracellular Zn2+ and we speculate that it is unlikely to
bind to the ZSB complex, due to the small size of the Zn2+
binding pocket. However, above 400 mmol dmꢀ3, at the MIC
of ZSB in this strain, luminescence rapidly decreases to below
the background intensity, which is likely due to toxicity of the
ZSB. These results suggest that the bactericidal properties of
ZSB are not solely due to the effect of free Zn2+ ions and that
the ZSB complex is having an additional antimicrobial
effect—at least in E. coli. We speculate that one possible
mechanism is transmetalation, with the ZSB possibly exchanging
zinc ions for a key trace metal in the cell.
Fig. 5 (a) Live–dead ratio for P. aeruginosa on pp-ZSB films.
(b) Live–dead ratio for S. aureus on pp-ZSB films.
(1/40). This is probably due to relatively greater retention of
the Schiff-base ligand in the film under these low power
conditions. It is known that using low power and/or pulsing
of input energy facilitates retention of monomer functional
groups in the plasma deposited film.23 The results in Fig. 5
correlate with the MIC measurements of the ZSB against
P. aeruginosa and S. aureus shown in Table 4.
Plasma deposition
Time course microbial assays for pp-ZSB on fabric against
Optimisation of plasma deposition parameters for maximum
antimicrobial efficacy
S. aureus and P. aeruginosa
With the plasma deposition conditions for ZSB optimised,
ZSB was plasma deposited under 1/40 pulse conditions onto
1 cm2 of non-woven polypropylene for 30 min.z This is a fabric
used in many products including diapers and wound dressings
and is frequently in direct skin contact. Following ZSB
deposition, the fabric was treated with a 30 s continuous wave
C2F6 plasma to render the surface more hydrophobic. All
fabric squares (and controls) were measured in triplicate. The
non-woven fabric was inoculated with 200 ml of a suspension
of either the P. aeruginosa or S. aureus in LB broth at a
concentration of 105 cfu mlꢀ1 (vide supra). Every two hours,
for a maximum of 16 h, fabric squares were removed and the
numbers of viable bacteria on the fabric quantified by the
colony counting method described above. Results are shown
In order to determine the optimum plasma deposition
conditions in terms of pulse sequence, a series of coatings
were made onto polystyrene Petri dishes (with no fluorination).
The relative efficacy of these coatings to kill bacteria was
assessed by growing bacteria on coated samples for 4 h and
then assessing bacterial viability using a live–dead fluorescent
assay (Invitrogen). This assay stains live bacterial cells green,
and dead ones red. The ratio of live to dead bacteria removed
from the treated samples could therefore be determined by
measuring the fluorescence emission ratios at 535 and 630 nm.
The live–dead ratio does not correlate quantitatively with
classical colony counting assays (Fig. 6), instead its gives a
qualitative, relative measure of antimicrobial efficacy: the
lower the ratio the more effective the antimicrobial surface.
It is evident from the results shown in Fig. 5 that the
optimum plasma conditions were pulsed: 1 ms on/40 ms off
z Non-woven polypropylene obtained from the topsheet of Bootst
brand disposable diapers.
c
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Fig. 6 (A) Time course measurements for P. aeruginosa growth/inhibition on treated and non-treated fabrics measured over 16 h. (B) Time course
measurements for S. aureus growth/inhibition on treated and non-treated fabrics measured over 16 h.
below in Fig. 6a and b. Further details regarding the
procedure for testing coated fabrics can be found in the ESI
from a previous communication by Poulter et al.w31
refluxed for 2 h before removal of methanol under vacuum,
resulting in a yellow oil, Schiff-base ligand ((E)-2-((allylimino)-
methyl)phenol). The oil was re-dissolved in dichloromethane and
dried using anhydrous MgSO4, which was removed via gravity
filtration. Removal of dichloromethane under vacuum yielded a
product of sufficient purity that no further purification was
needed. Yield 75%, 18.11 g. Anal. Calc. for C10H11ON:
C, 74.49; H, 6.89; N, 8.69. Found: C, 73.2; H, 6.71; N, 8.57.
The results in Fig. 6A and 6B show that the pp-ZSB coating
effectively suppresses growth of both S. aureus and
P. aeruginosa over the 16 h time period of the study.
P. aeruginosa starts to grow after 12 h, but still at a much
lower rate than on the control. One hypothesis to explain this
could be the ability of P. aeruginosa to secrete extracellular
polysaccharides (alginate) which might reduce the bacterial
exposure to the antimicrobial agent. The S. aureus results
show virtually no growth of bacteria over the whole 16 h time
period of the experiment.
1H NMR (CDCl3, 250 Hz) d/ppm: 4.2 (2H, d, CH2), 5.2 (2H, m,
ꢀ
ꢀ
QCH2), 6.1 (1H, m, CH), 7.1-7.8 (4H, m, CHAr), 9.9 (1H, s,
ꢀ
ꢀ
NQCH), 11.1 (1H, s, OH).13C NMR (CDCl3, 300 MHz) d/ppm:
61.7, 116.9, 117.4, 119.0, 119.3, 131.8, 132.7, 135.2,161.6, 166.1
ꢀ
Bis(N-allylsalicylideneiminato)-zinc (ZSB). The Schiff-base
ligand (E)-2-((allylimino)methyl)phenol, (100 mmol, 16.12 g)
was dissolved in 40 ml of methanol, triethylamine (100 mmol,
13.94 ml) and zinc acetate (50 mmol, 9.17 g) and the reaction
refluxed for two hours. The solution was allowed to cool to
room temperature, and the solvent was removed under
vacuum. The resulting yellow oil was re-dissolved in dichloro-
methane (50 ml) and washed with water (2 ꢂ 20 ml). The
organic layer was separated and dried using anhydrous
MgSO4. The solvent was removed under reduced pressure.
The resulting oil was stored at ꢀ18 1C and allowed to crystallise.
The resulting yellow powder was isolated using suction
filtration and washed with ice cold hexane and filtered to give
the complex ZSB. The product was recrystallised in the
minimum volume of hot toluene to give a yellow crystalline
solid. Yield 78%, 15.03 g, M.p. 112–114 1C. Anal. Calc. for
C20H20O2N2Zn: C, 62.26; H, 5.24; N, 7.26. Found: C, 60.9; H,
Conclusions
Attempting to understand the structure–function relationship
for these Schiff-base coordinated antimicrobials is a non-
trivial problem. The experiments reported here show that
two antimicrobials, with identical ligands but differing metal
centres appear to have quite different antimicrobial activities
from one another and for two species of bacteria, Pseudomonas
aeruginosa and Staphylococcus aureus. Our initial hypothesis was
that the free metal ions would constitute the antimicrobial
component of these novel complexes. However, the detailed
study of MIC, the intracellular Zn-reporter system and effect
of plasma duty cycle (and hence degree of ligand retention) all
suggest a more complex synergy between the metal and ligand.
However, whatever the precise mechanism of action, these
compounds suggest a utility as a new approach to making
surfaces antimicrobial. Moreover, plasma deposition offers a
potentially useful approach for industrial scale-up and for coating
non-planar surfaces, particularly fabrics.
1
5.17; N, 7.19. H NMR (CDCl3, 250 Hz) d/ppm: 4.1 (2H, d,
CH2), 5.0 (2H, m,QCH2, HB), 5.8 (1H, m, CH), 6.5–7.2 (4H,
ꢀ
ꢀ
ꢀ
m, CHAr), 8.3 (1H, s, NQCH). 13C NMR (CDCl3, 300 MHz)
ꢀ
ꢀ
d/ppm: 61.9, 113.5, 117.0, 118.6, 122.1, 132.2, 134.0, 134.7,
169.6, 170.4.
Experimental
Bis(N-allylsalicylideneiminato)-copper (CSB). The analogous
complex containing copper, CSB was prepared in an identical
manner to ZSB with copper acetate (50 mmol, 9.10 g) used in
place of zinc acetate. The product afforded a dark green powder,
which was recrystallised from a minimum volume of hot toluene
Monomer synthesis
Allyl substituted Schiff-base (SB). Salicylaldehyde (150 mmol,
15.99 ml) was dissolved in 40 ml of methanol and allylamine
(150 mmol, 10.92 ml) added slowly.32 The mixture was then
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to afford a dark green crystalline solid. Yield 74%, 14.17 g, M.p
120 1C. Anal. Calc. for C20H20O2N2Cu: C, 62.56; H, 5.26; N,
7.30. Found: C, 62.5; H, 5.27; N, 7.29.
Colony counting assay based on the Japanese Industry Standard
JIS 1902. ZSB and CSB plasma treated (50 W/30 min/1–40 ms)
50 mm polystyrene Petri dishes and 30 ꢂ 30 mm treated non-
woven polypropylene fabric squares were prepared in triplicate.
Control samples were sterilised by exposure to UV/O3 for 10 min
prior to inoculation with bacterial broth culture. 4 ml of a 103
CFU mlꢀ1 bacterial culture was applied to the surface of Petri
dishes, subsequently sealed with parafilm and 200 mL of a 105
CFU mlꢀ1 bacterial broth culture was applied to each fabric
square, placed in Petri dishes and sealed with parafilm before
incubation at 37 1C for time periods between 2 and 18 h. After this
time the plasma treated samples were removed and added to 20 ml
of sterile physiological saline (0.9% w/v NaCl). These were
vortexed five times in five second bursts to physically remove
bacteria attached to the surfaces. To assess the number of viable
bacteria in these samples 100 ml aliquots of serial dilutions were
spread on LB agar plates, incubated at 37 1C for 18 h and the
number of colony forming units recorded.
Experimental details relating to the single-crystal X-ray
crystallographic studies are provided in the ESI and summarised
in Table S1.w
Plasma deposition
A reactor comprising of a 30 cm long, 10 cm diameter Pyrex
tube and stainless steel discs at each end, both connected to
ground, attached to an Edwards RV5 pump (used to create a
vacuum of ca. 10ꢀ2 mbar) was used for these experiments
based on the design described by Bullett et al.33 Oxygen for
reactor cleaning was fed in via a gas control valve and
monomer vapour introduced via a Young’s tap. A 13.56 MHz
RF coaxial system supplied power and was connected to the
reactor via a 2 mm diameter coil of copper wire, earthed
through the electrodes. A manual matching unit was used to
adjust input impedance, ensuring minimal reflected power
(40.5%) and an oscilloscope allowed pulsing of the input
power. A range of power and duty cycles were used in the
experiments, and are summarised in Table 5.
Bacterial bioluminescence assay. A Zn2+ biosensor strain of
E. coli was used to determine whether the antimicrobial
activity of ZSB is due to a toxic accumulation of intracellular
Zn2+. This strain carries two plasmids: one encodes the
zinc-responsive regulatory protein ZntR (pSLzntR) and the
other encodes the luciferase genes (lux) which are located
downstream of the ZntR-Zn2+-responsive ZntA promoter
(pDNPzntAlux). This strain becomes increasingly bioluminescent
as the intracellular Zn2+ concentration increases. The
construction and validation of this genetically modified strain
of E. coli has previously been reported by Ivask et al.34
The zinc-responsive E. coli strain was grown overnight at
30 1C, with shaking at 200 rpm, in M9 minimal medium
(per dm3: 33.9 g Na2HPO4, 15 g KH2PO4, 2.5 g NaCl,
5 g NH4Cl, 2 mM MgSO4, 0.5 mmol dmꢀ3 CaCl2) supplemented
with 1 mg Lꢀ1 thiamine, 0.2% (w/v) glucose, 0.2% (w/v) acid
hydrolysate of casein, 100 mg mlꢀ1 ampicillin and 10 mg mlꢀ1
tetracycline. 500 mL of this culture was sub-cultured in fresh
M9 media (with supplements as before) for 4 h to exponential
phase (OD600 = 0.4–0.6) before dilution to OD600 0.1. 100 mL
aliquots of culture were inoculated into a 96-well plate (Flat
clear bottom, black, Greiner) containing dilutions of Zn
acetate or ZSB (100 mL per well). Plates were incubated for
2 h at 30 1C (without shaking) and bioluminescence measured
using a Fluostar Omega plate reader (BMG Labtech).
Fluorination. The pp-ZSB and pp-CSB films were both very
hydrophilic on removal from the reactor. X-Ray Photo-
electron Spectroscopy analysis suggested a high degree of
oxidation of the film, possibly post-treatment. In order to
improve the antimicrobial efficacy of fabrics coated in ZSB or
CSB, the films were exposed to a brief (30 s) plasma of
hexafluoroethane (C2F6) immediately following plasma
deposition of the organometallics. This made the coated
fabrics extremely hydrophobic (water droplets formed spheres
and ran off) and improved antimicrobial performance
significantly on pp-ZSB, less so on pp-CSB samples. Contact
angles (sessile): control and oxygen treated non-woven =
ca. 01 (water was absorbed to fabric), pp-C2F6 treated non-
woven = av. 1261 ꢁ 0.801.
Microbiological analysis
MIC determination of monomeric zinc Schiff-base compounds,
zinc acetate and Schiff-base ligand. Dilution series (50ꢂ final
concentration) of ZSB, CSB and Schiff-base ligand were made in
microbiology grade DMSO (Sigma, 499.99% purity) due to the
insoluble nature of these complexes in water. A dilution series of
zinc acetate (50ꢂ final concentration) was made in sterile H2O
due to poor solubility in DMSO. 200 mL aliquots of these
solutions were added to a final volume of 10 ml Luria-Bertani
(LB) broth (DMSO was added separately to the LB/ zinc acetate
solutions at a final concentration of 2% v/v). LB/monomer
solutions were inoculated with exponential phase bacterial LB
broth culture (approx. 105 CFU) and incubated with shaking
(200 rpm) at 37 1C for 18 h, after which time the optical density
at 600 nm was measured.
X-Ray photoelectron spectroscopy (XPS)
X-Ray photoelectron spectroscopy (XPS) analysis was carried
out using a Kratos Axis Ultra instrument under ultra high
vacuum. A monochromated Al (1486.6 eV) source was
operated at 15 kV and 5 mA emission and photoelectrons
from the top few nanometres of the surface detected in
the normal emission direction. Spectra in the range 1400 to
ꢀ10 eV binding energy and a step size of 1 eV, using a pass
Table 5 Plasma deposition parameters for the optimised deposition of the two metal containing monomers
Monomer temperature Flow rate/sscm3 minꢀ1 Base pressure/mbar Film deposition rate Deposition time
Compound Duty cycles (peak power)
ZSB
CSB
CW, 40/40, 10/40, 1/40 (50 W) 135 1C
CW, 40/40, 10/40, 1/40 (50 W) 135 1C
0.53
0.26
0.020
0.022
1 nm minꢀ1
1 nm minꢀ1
30 min
30 min
c
This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011
New J. Chem., 2011, 35, 1477–1484 1483

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energy of 160 eV and 1 scan of 200 ms dwell per step, were
acquired from areas of approximately 700 ꢂ 300 micrometres
on each sample. The peak areas were measured and the
manufacturer’s calibration and sensitivity factors used to
determine the concentration of the elements present.
Narrow-scan spectra were acquired for C 1s and N 1s with a
step size of 0.1 eV and 20 eV pass energy using 500 ms dwell
per step, 1 scan in all cases apart from Zn where 2 scans were
used. For all spectra on both samples the charge neutraliser
was used, this has the effect of shifting the peak positions by
approximately 3.7 eV towards lower binding energies. The
scale was then corrected in Casa XPS software by referencing
to the C 1s hydrocarbon peak at 285 eV.
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All authors would like to thank the European Commission’s
7th Framework programme for funding via the consortium
EMBEK1-FP7 project # 211436. The authors would also like
to thank Dr A. Ivask for providing the reporter plasmids
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This journal is The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2011