1
Polymer multilayers loaded with silver particles show
reduced bacterial growth.
Janet Ryu & Geetha Berera
Department of Materials Science and Engineering, Massachusetts Institute of
Technology, Cambridge, Massachusetts 02139
Received October 8, 2004. Final version October 27, 2004.
Silver (Ag) is a well known antibacterial agent1. This property provides surfaces
containing Ag the ability to kill bacteria. Here we show that a polymer multilayer
(PEM), loaded with Ag particles, has quantifyably less bacterial growth than a
non-Ag loaded PEM. The synthesized Ag particles, even at nanoscale sizes, are
affective in killing bacteria. In addition, our results show that increasing
concentrations of Ag particles lead to lower density of bacteria colonies on the
PEMs. The PEMs are fabricated with poly(acrylic acid) (PAA), polyacrylamide
(PAAm), and poly(allylamine hydrochloride) (PAH) at low pH so that Ag particle
synthesis is possible through a reduction reaction within the PEM1. Escherichia
coli bacteria is seeded and cultured on the surfaces of Ag particle loaded PEMs
composed of poly(acrylic acid) (PAA), polyacrylamide (PAAm), and
poly(allylamine hydrochloride) (PAH). The Ag particles were synthesized in
varying concentrations, which were then verified quantifyably through UV-visible
spectroscopy. Examinations of these PEMs and an Ag-free PEM, under an
inverted optical light microscope, demonstrated an inverse relation between
amount of Ag particles in the PEM and bacterial growth on its surface.
This experiment took advantage of nanotechnology by synthesizing Ag particles
at nanoscale sizes. Even at such small scale, the antibacterial property of silver was
observable. This experiment used PEMs, ultra-thin polyelectrolyte films, as the base for
Ag nanoparticle synthesis to occur. The PEMs were fabricated on glass slides with
hydrophilic polymers for strong interlayer bonding with hydrogen bonds (Fig. 1). The
order in which these different multilayers were created is shown in Table 1, along with
the duration which the slide was dipped. In addition, the polymer solutions and the
water used for rinsing between layers were specifically at a low pH of 3.0. This is
necessary because carboxylic acid is a weak acid that remains deprotenated only around
low levels of pH. The availability of deprotenated carboxyl groups in the PAA layers
ultimately determines how many silver ions (Ag+) can be dispersed throughout the
PEM 2. It is through this process of events, that the PEMs themselves essentially act as
nanoreactors for Ag particle synthesis to occur. The PEM is then heated for
crosslinking the PAA and PAAm layers with a peptide bond (Fig. 1). This process
creates cross links that allow the PEM to stabilize at a higher pH. More importantly,
there are many deprotenated carboxyl groups left, which are necessary for the Ag+
dispersion in the PEM.
Ag ions are initially dispersed throughout the PEM through the PAA layers.
When dipped in a solution containing silver ions, the ions have the ability to bond with
2
the carboxylic acid group, in place of the hydrogen2. This exchange is only possible if
the PEM is fabricated at a sufficiently low pH, which allows for the easy removal of the
hydrogen2. The Ag ions are subsequently reduced with a Borane Dimethylamine
(BDMA) solution. An exchange between Ag+ and a hydrogen, from the amine group,
in the BDMA reduces the Ag ion to a neutral Ag particle1,2. The carboxylic groups also
Figure 1 Polymers used to fabricate the PEMs. Ionic crosslinks are formed
between the PAA and PAAm layers.
Table 1 Dipping System for the Fabrication of Polyelectrolyte Multilayers
Solution
PAH
Water Rise (1)
Water Rise (2)
PAA
Water Rise (1)
Water Rise (2)
PAAm
Water Rise (1)
Water Rise (2)
PAA
Water Rise (1)
Water Rise (2)
PAAm
Water Rise (1)
Water Rise (2)
PAA
Water Rise (1)
Water Rinse (2)
pH
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
Dipping Time (min)
15
1
1
15
1
1
15
1
1
15
1
1
15
1
1
15
1
1
All solutions of 1.0 M and made with deionized water.
gain a hydrogen to replace the lost Ag+. This chain of events leads to metal Ag
particles which are uniformly dispersed throughout the PEM, between the carboxyl
groups of the poly(acrylic acid). Consequently, the pH at which the PEM substrate is
fabricated determines the size of the Ag particles dispersed throughout2.
The actual size of the Ag particles can be estimate based on data from Wang et.
al3, which used PEMs made very similarly to ours. They found that PEMs made at pH
of 2.5 had average Ag particle diameter of 3.8 nm, and PEMs at a pH of 3.5 had average
size of 3.1 nm2. Taking the average of these two diameter values, we can estimate that
the average Ag particle diameter of PEMs made at pH 3.0 was 3.45 nm. The volume of
3
this particle (assuming it is a sphere) is 21.5 nm3. The diameter of an Ag atom is .252
nm, and its volume is .00838 nm3. Therefore, we can conclude that there were 2566
silver atoms per Ag particle in our PEMs.
Table 2 Normalized Absorption Intensity and vol% Ag as a Function of
Initial Ag-Acetate Concentration
Molarity
0.01
0.005
0.0001
0.00005
0.00001
0.000001
vol% Ag
5.11
5.26
1.95
2.51
0.651
0.233
The data from Wang et al.2 can be used again to estimate the silver concentration
as a volume percent (vol%). Their PEMs of pH 3.0 dipped in silver acetate solution of
5 mM concentration had 5.3 vol% Ag content3. Although their Ag ions were reduced
with H2 gas, as opposed to the BDMA solution the current study used, this does not
change the amount of Ag particles formed in the reduction reaction in the PEMs.
Therefore, we can assume that the absorbance measurement Wang found for PEMs with
5.3 vol% Ag, correlates with our absorbance measurements. This assumption allows us
to find a conversion factor for our own absorbance data based on the Wang data. To
find the vol% Ag in PEMs dipped in Ag solutions of different molarities, we first found
a proportionality constant to find absorption as a function of vol% Ag. We used Beer’s
Law which shows a direct relationship between absorption and concentration:
A = εbc
(1)
where A is the absorption intensity, ε is the proportionality constant, b is the pathlength,
and c is the concentration of the absorbing species. The absorption intensity is the value
from UV-visual spectroscopy of our PEMs. In our situation, the Ag particles are the
absorbing species, and our concentration is the vol% Ag. Because the pathlength is the
same for all of our different molarities, we can treat εb as the proportionality constant.
For .005 mM silver acetate, our relative absorption intensity was 0.113 and
above we had estimated our vol% as 5.3. Dividing 0.113 by 5.3, the proportionality
constant is found to be 0.0213. This proportionality constant is the conversion factor to
find the vol% of Ag in the PEMs dipped in silver acetate solution of other molarities:
vol% = A/εb
(2)
Using eq. 2 we find the vol% Ag for the different molarities, the values of which are
shown in Table 2. We can see the correlation between absorption and vol% Ag in Fig.
2. The amounts of Ag particles dispersed within the PEMs vary greatly depending on
the molarity of the initial silver acetate solution. There were 22 times as many silver
particles, based on vol% Ag, in the PEM made with a silver acetate solution of 0.01 M,
than in the PEM made with 1 x 10 6 M because of the higher initial concentration of Ag
ions dispersed throughout the PEM.
4
6
5
4
3
2
1
0
0.01
0.005
0.0001
0.00005
0.00001
0.000001
Silver Acetate Concentration (M)
Figure 2 Vol% Ag (▲) and normalized absorption intensity (■), both as a
function of silver acetate concentration. Raw absorption data normalized by
multiplying by 5.3 (vol% Ag of PEM dipped in 5 mM silver acetate) and dividing
by .113 (absorbance of PEM dipped in 5 mM silver acetate). 3
We found that the existence of Ag particles in the PEM gives PEM surfaces the
ability to kill bacteria. When comparing PEMs, we found quantifiably less bacterial
growth on PEMs with Ag particles than on the control samples that had none. Fig. 3
shows the images of the slides of PEMs taken from observing them under an inverted
optical light microscope. Pictures of the slides were taken at five different positions for
the Ag-free PEM and PEM dipped in the 1 x 10-6 M silver acetate solution. On average,
the Ag-free PEM had around 12 bacteria colonies in each photographic plate. Each
plate had an area of around 12.6 mm2, so there was approximate 1 colony per 1 mm2.
The PEM with .233 vol% Ag (made with an initial silver acetate concentration of 1 x
10-6 M) had around the same number of colonies; however, they ranged in size from
diameters of 0.3-0.4 mm. A large amount of the surface of the PEM with no Ag was
covered with bacteria growth (Fig. 3a), while there was visibly less bacteria growth on
the surface of PEM dipped in 1.0 x 10-6 M (Fig. 3b). A drastic reduction in bacterial
growth was seen in the PEMs dipped in 5 x 10-5 M, 1 x 10-5 M, and 1x 10-2 M. All three
of these showed one bacteria colony each per photographic plate, or approximately
0.079 colonies/mm2. The rest of the colonies were below the resolution of the inverted
optical light microscope to be observed. Fig. 3c and Fig. 3d shows the surface of the
PEM dipped in 1 x 10-5 M and 1.0 x 10-2 M, respectively, both of which has very little
bacteria growth.
The evidence suggests that there is a minimum concentration of silver acetate
necessary to observe the antibacterial property of silver. The minimum concentration
used was 1 x 10-6 M which had a noticeable amount of bacteria killed when compared
5
a
b
c
d
Figure 3 Images of PEMs with a, no Ag particles; b, dipped in 1.0 x 10-6 M; c,
dipped in 1.0 x 10-5 M; d, dipped in 1.0 x 10-2 M. Unit scale is 1.0 mm. The
scale bar in each images is 1.0 mm.
to the PEM with no Ag. However, there was still much bacteria growth at the lowest
concentration of silver acetate. This implies that at a concentration of 1 x 10-6 M silver
acetate, the silver was not effectively antibacterial.
The Ag nanoparticles were effective in killing bacteria. Even at nanoscale
diameters, silver’s antibacterial property was observed. These particles were dispersed
throughout the PEMs including the surfaces which we observed. The silver particles
inhibit the growth of these bacteria colonies. This is particularly visible in PEMs
dipped in higher concentrations of silver acetate, which had higher vol% Ag. As the
bacteria colony grows, it eventually comes in contact with the silver particles in the
PEM. In addition, the evidence suggests that the more silver particles there are
throughout the PEM, the more bacteria that are killed. With increasing concentrations
of silver acetate, there was higher vol% Ag (Table 1), and a corresponding decrease in
bacteria growth. The Ag particles were also dispersed throughout the entire PEM, and
not just the top surface, inhibiting bacteria growth from all directions.
Silver particles were successfully synthesized using polymers as nanoreactors.
In addition, the antibacterial property of silver was found to be effective at nanoscale
sizes. These two conclusions together create opportunities for many different
applications. Different initial concentrations of silver acetate and using different
substrates for Ag synthesis may be interesting steps to take next. Synthesizing Ag
particles at nanoscale diameters on different substrates will also create many new
applications for silver as an antibacterial agent.
6
Methods
PEM fabrication. The polymer thin films were pre-fabricated by dipping glass slides
using polymer solutions of PAH, PAA, and PAAm1. All of them had 0.01 M in H20
with a pH of 3.0. The slides were also dipped in deionized water to rinse between
layers of polymers. The pH of water was adjusted to 3.0 as well. The dipping order and
duration is shown in Table 2. After the dipping process was completed, the PEM was
heated at 130oC for 2 hours in an air furnace to create the crosslinks1.
Ag nanoparticle synthesis. The PEM slides were dipped in silver acetate solutions of
varying concentrations which were pre-made with deionized water1. They were in
molar concentrations of 1.0 x 10-6 M, 5.0 x 10-5 M, 1.0 x 10-5 M, 1.0 x 10-4 M, 5.0 x 10-3
M, and 1.0 x 10-2 M. The silver ions from the silver acetate solution bonded with the
carboxylic group of PAA. After being in silver acetate solution for ten minutes, the
slides were rinsed with deionized for one minute with agitation. The slides were then
dipped for one minute in borane dimethylamine solution of 0.05 M, also made with
deionized water. This was for the reduction of the silver ions to neutral particles. The
PEM slides were rinsed again in water for one minute with agitation. Subsequently, the
slides were dried with N2 gas.
Seeding of E. coli on Ag-loaded PEMs and controls. E. coli bacteria solution of 105
bacteria/mL was made. 20 mL of this bacteria solution was put onto each other PEM
slides, along with several control slides1. The control slides were both, plain glass
slides and Ag-free PEM slides. The slides were put into Petri dishes on sterile 1%
agarose gel and incubated overnight at 37oC1.
1. Berg, Michael C. Laboratory Experiment 1: Nanoreactors and Bacteria. 1-7 (2004).
2. Wang, T. C., Rubner, M. F. & Cohen, R. E. Polyelectrolyte Multilayer Nanoreactors
for Preparing Silver Nanoparticle Composites: Controlling Metal Concentration and
Nanoparticle Size. Langmuir. 18, 3370-3375 (2002).