Sy of Copolymer Stablized Silver Nps

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ORIGINAL CONTRIBUTION

Synthesis of copolymer-stabilized silver nanoparticles

for coating materials

Jukka Niskanen & Jun Shan & Heikki Tenhu &

Hua Jiang & Esko Kauppinen & Violeta Barranco &

Fernando Pic & Kirsi Yliniemi & Kysti Kontturi

Received: 7 October 2009 /Revised: 3 December 2009 /Accepted: 4 December 2009 /Published online: 7 January 2010# Springer-Verlag 2010

Abstract Silver ions being less toxic than silver nano-

particles, a more safe material can be obtained to be used asantimicrobial coating. This can be achieved by using thiol

chemistry and covalently attach the silver nanoparticles in

the coating. Our aim is to produce a coating having

antimicrobial properties of silver ions but with the silver

nanoparticles firmly attached in the coating. Here, we

present a way to produce silver nanoparticles that can be

used as a component in a coating or as such to produce an

antimicrobial coating. The silver nanoparticles presented

here are stabilized by a copolymer (poly(butyl acrylate

methyl methacrylate)) that is soft and has well-known

good film-producing properties. The reversible addition-

fragmentation chain transfer radical polymerization technique

used to prepare the polymers provides conveniently a thiol

group for effective binding of the silver nanoparticles to the

polymers and thus to the coating.

Keywords Nanoparticles . Block copolymers .

Antimicrobial coating

Introduction

Nanoparticles are used and investigated widely for medical

and biological applications. In medical research, it has been

shown that gold nanoparticles can interfere with growth

factor proteins that cause angiogenesis. This could be

utilized in cancer treatment since cancer cells release these

proteins for promoting angiogenesis in tumors [15].

Another application is to functionalize gold nanoparticles

with, e.g., antibodies for detection of hormones and be used

as sensors for example in pregnancy tests [6].

Silver nanoparticles, on the other hand, are mostly used

in antimicrobial applications since the antimicrobial effect

of silver ions is well known [79]. There are many

commercial products for wound treatment that contain

silver as an antimicrobial agent. Nanocrystalline silver in

wound dressings is used to treat ulcers, and silver

sulfadiazine is used as pastes or creams for treating burn

wounds [5].

Even though the antimicrobial effect of silver ions has

been known for long, there is still some discussion about

the actual mechanism of the toxicity of silver. It is widely

believed that the toxicity arises from silver ions interacting

with proteins in the cells [1012]. Feng et al. [10] have

shown that silver ions interact with thiol groups in proteins

and deactivate enzymes this way. They also found that the

condensed form of DNA loses its ability to replicate in the

presence of silver. Schreurs and Rosenberg [11] reported

that silver ions inhibit the respiratory chain of cells and that

the silver removes chlorine ions from the cytoplasm of cells

as AgCl.

J. Niskanen : J. Shan : H. Tenhu (*)

Laboratory of Polymer Chemistry, Department of Chemistry,

University of Helsinki,

P.O. Box 55, 00014 Helsinki, Finland

e-mail: [email protected]

H. Jiang : E. Kauppinen

NanoMaterials Group, Department of Applied Physics and Center

for New Materials, Helsinki University of Technology (TKK),P.O. Box 5100, 02150 Espoo, Finland

V. Barranco : F. Pic

Department of Ionic Solids, Materials Science Institute of Madrid

(ICMM), Spanish National Research Council (CSIC),

Cantoblanco,

Madrid, Spain

K. Yliniemi : K. Kontturi

Laboratory of Physical Chemistry and Electrochemistry,

Helsinki University of Technology,

P.O. Box 6100, 02015 Espoo, Finland

Colloid Polym Sci (2010) 288:543553

DOI 10.1007/s00396-009-2178-x


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The antimicrobial effect of silver nanoparticles is also well

documented [1315]. The efficacy is good against gram-

negative bacteria (e.g., Escherichia coli) and fungi, but for

gram-positive bacteria (e.g., Staphylococcus aureus), the

effect is not as prominent. It is believed that gram-positive

bacteria are not so sensitive to silver ions due to the thicker

peptidoglycan layer in the cell wall than in the gram-

negative bacteria [13].Recently, some studies of the toxicity of silver nano-

particles to mammalian cells have been conducted. As

could be expected, the silver nanoparticles interfere with

DNA in mammalian cells as well. However, Ahamed et al.

[16] have shown that the surface chemistry of silver

nanoparticles has an effect on their ability to cause DNA

damage in cells. They report that polysaccharide-coated

nanoparticles caused more severe damage to the genome

than hydrocarbon-coated ones. The difference in toxicity is

believed to arise from the aggregation and surface area of

the particles inside cells. Recent studies also suggest that

silver nanoparticles could be taken into mammalian cells byendocytosis [17, 18].

Prokaryotic cells differ from eukaryotic cells having a

prominent nucleus, complex DNA repair mechanisms, and

a cell cycle that controls the cell death and survival.

However, similar respiratory chains make both sensitive for

the interruption of respiratory chain caused by silver ions.

AshaRani et al. [17] report that hydrogen peroxide involved

in the respiratory chain can oxidize silver nanoparticles that

have entered the mitochondria and cause increased release

of silver ions, thus increasing the toxicity of silver

nanoparticles. From their findings, they conclude that silver

nanoparticles are cytotoxic, genotoxic, and antiproliferative.

Larese et al. [19] have studied the permeability of human

skin regarding silver nanoparticles. They found that silver

nanoparticles can easily penetrate in and through the skin

through pores and glands. In transmission electron micro-

scope (TEM) analysis of skin, silver nanoparticles were

observed to be distributed throughout the skin on all levels,

and damaging the skin naturally increased the penetration

considerably.

Considering these concerns, it is vital that the silver

nanoparticles used in antimicrobial coatings are firmly attached

to the coating. This way the release of silver nanoparticles to

the environment is limited and only silver ions are released.

This can be achieved by using thiol chemistry and covalently

attach the silver nanoparticles in the coating.

The synthesis of various nanoparticles is well reported

using many different approaches. In short, nanoparticles are

commonly produced in solutions by reducing a precursor

metal salt in the presence of stabilizing ligands (e.g.,

sodium citrate or alkane thiols) [2022]. The stabilizing

ligands make the dispersion of nanoparticles colloidally

stable by preventing the aggregation of the particles. A

well-known process is the BrustSchiffrin method that

produces highly monodisperse gold nanoparticles. It is a

two-phase system of toluene and water where a phase

transfer agent (tetraoctylammonium bromide) is used to

transfer the auric acid ions into the organic phase from

water. Dodecanethiol is dissolved in the organic phase, and

upon addition of aqueous NaBH4 solution, the auric acid

ions are reduced and form nanoparticles stabilized bydodecanethiol. Alkane thiols are very suitable for stabiliz-

ing nanoparticles since they possess a thiol group that

will firmly attach to the surface of the nanoparticles

[22]. Another two-phase system that produces highly

monodisperse silver nanoparticles utilizes oleylamine as a

stabilizing agent. In the process, silver nitrate is dispersed

in liquid paraffin together with oleylamine, and the particles

are formed upon heating [23]. The polyol synthesis is yet

another method of producing nanoparticles. Feldmann and

Jungk [24] have shown that diethylene glycol can produce

metal oxide nanoparticles by heating aqueous solutions of

metal salts and diethylene glycol. This procedure has laterbeen used to produce silver nanoparticles by several groups

[25, 26]. Wiley et al. [25] produced silver nanoparticles of

different morphologies, i.e., pyramids, cubes, and rods

using the polyol process and stabilizing the particles with

poly(vinyl pyrrolidone) showing that the morphology of the

silver nanoparticles can be adjusted by heating the particles

in the presence of oxygen and halogen ions. Recently,

Yliniemi et al. [27] prepared ultrathin coatings with silver

nanoparticles on steel and glass surfaces utilizing silanes

with amine functionalities to attach the nanoparticles to the

surface.

Polymers produced by reversible addition-fragmentation

chain transfer radical polymerization (RAFT) can easily be

used as stabilizing ligands for nanoparticles. The chain

transfer agent provides an easy access to a thiol group for

effective binding to the surface of the nanoparticles. Gold

nanoparticles can be easily produced in a batch reaction by

dissolving auric acid together with polymer produced by

RAFT and reducing them both simultaneously with, e.g.,

NaBH4 or LiB(C2H5)3H [28, 29].

In this report, we describe the synthesis of the polymer-

stabilized silver nanoparticles which might be used as a

component of a coating material or as such to produce a

coating. The dissolution of silver ions from this hybrid

material is shortly discussed.

Experimental

Background

The polymer of choice was a soft copolymer of butyl

acrylate (BuA) and methyl methacrylate (MMA) which was

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prepared by RAFT polymerization. Thus, we could easily

obtain polymers with low polydispersity and a thiol end

group by hydrolyzing the chain transfer agent fragment at

the chain end. The mechanical properties of this copolymer

can easily be adjusted by changing the ratio of BuA and

MMA in the polymer. The one with the ratio of 70% BuA

and 30% MMA has been shown to have good film-

forming properties [30, 31]. The copolymer is hydrophobic,and thus, there is a concern of silver ion released from the

films since water might not penetrate the films in such

extent that silver ions could be released. To overcome this

possible obstacle, a small block of poly(acrylic acid) (PAA)

was incorporated in the copolymers. da Silva Paula et al.

[32] have shown that when a copolymer of styrene and

acrylic acid was used to stabilize silver nanoparticles, the

composites showed antibacterial efficacy both against

E. coli and S. aureus. They also concluded that the acrylic

acid groups induce motility of silver ions from the composite

material.

One copolymer (poly(BuA-co-MMA), CP) and twodifferent block copolymers (PAA-block-poly(BuA-co-

MMA), B1 and B2) were made. The difference between

the block copolymers is in the location of the PAA block,

which is located either at the thiol end of the copolymer or

at the opposite one. This places the PAA block either near

the surface of the silver nanoparticles or on the outer corona

of the stabilizing polymer (Scheme 1). Using small blocks

of PAA, instead of making a random copolymer of all three

repeating units, preserves the good film-forming properties

of the poly(BuA-co-MMA) copolymer.

Considering a possible practical application, the nano-

particles in the coating should be fairly big and uniform in

size so that the lifetime for a coating could be estimated.

Silver nanoparticles of roughly the size of 3 to 4 nm were

obtained in our syntheses, which were considered to be too

small for the application in mind. Increasing the amount of

silver nitrate in the synthesis leads to bigger particles but at

the expense of the monodispersity of size and morphology.

However, there are reported methods to grow nanoparticles

in size without losing the monodispersity.

In a self-seeding process, Jiang et al. [33] grew silver

nanoplates from a solution of silver nitrate, ascorbic acid,

and citric acid by adding a trace amount of NaBH4 in the

system immediately after preparing the solution. A seed of

nanoparticles was produced fast by NaBH4 and then grown

during the slower coreduction of silver nitrate by ascorbic

and citric acid. Jana et al. [34] grew gold nanoparticles in a

slightly different way using seed-mediated growth. They

first produced the seed separately and added it to a

growth solution containing auric acid, where the seedsfinally grew upon addition of reductant. We chose the

latter method for growing our polymer-stabilized silver

nanoparticles.

Materials

The initiator azobisisobutyronitrile (AIBN; Fluka,

>98.0%) was recrystallized from methanol. The mono-

mers n-BuA (Fluka, >99.0%), tert-butyl acrylate (tBA;

Aldrich, 98%), and MMA (Imperial Chemical Industries

PLC) were distilled before use. Ascorbic acid (Merck,

99.7%), 2-butanone (Fluka, >99.0), 1,4-dioxane (Merck,>99.5%), ethanol (Altia, 99.5%), n-hexane (Lab-Scan,

95%), hydrochloric acid (J.T. Baker, 3638%), silver

nitrate (VWR international, Ph. Eur.), sodium borohydride

(Sigma-Aldrich, >98.5%), and tetrahydrofuran (THF; Lab-

Scan, 99.8%) were used as received. The chain transfer

agent, cyanopentanoic acid dithiobenzoate (CPA), was

synthesized by Shan et al. [29].

Synthesis of copolymers

The CP was prepared as follows: The reactants BuA

(0.191 mol, 27.3 ml, 3.11 M), MMA (0.180 mol, 19.2 ml,

2.93 M), CPA (0.964 mmol, 0.27 g, 0.016 M), and AIBN

(0.123 mmol, 20.2 mg, 0.002 M) were dissolved in 2-

butanone (14.9 ml), and oxygen was removed by bubbling

with nitrogen gas. The solution was lowered into an oil bath at

70C for 4 h. AIBN was used with a ratio of CPA/AIBN= 8/1.

The polymer was purified by precipitating it first in hexane

and then twice in THF/hexane (1:1) from THF. The purified

polymer was first air-dried and finally dried in vacuo.

The synthesis of the block copolymer B1 was started by

preparing a poly(BuA-co-MMA) block, in a way as

Scheme 1 Schematic

illustration of the three different

polymers used to stabilize

silver nanoparticles. Yellow

represents the copolymer poly

(BuA-co-MMA), blue PAA,

and red the thiol groups

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described above. BuA (0.40 mol, 57.7 ml, 2.00 M), MMA

(0.40 mol, 42.7 ml, 2.00 M), CPA (9.15 mmol, 2.6 g,

0.046 M), AIBN (1.15 mmol, 0.19 g, 0.006 M), and 2-

butanone (100 ml) were measured into a 250-ml flask. The

solution was bubbled with nitrogen for 1 h before lowering

it into a 70C oil bath for 24 h. The nitrogen flow was

stopped after 1.5 h of heating. The polymer was purified by

dissolving it in THF and precipitating it in hexane threetimes and dried in vacuo. A poly(tBA) block was then

added to this polymer; 27.0 g (0.003 mol, 0.12 M) polymer,

tBA (0.06 mol, 9.2 ml, 2.58 M), AIBN (0.34 mmol,

55.8 mg, 0.015 M), and 2-butanone (14.1 ml) were all

mixed in a 100-ml flask. The flask was sealed and oxygen

was removed as previously, and the solution was lowered

into an oil bath at 70C for 24 h. The polymer was purified

by precipitating it in hexane twice and then dried in vacuo.

The poly(tBA) block was hydrolyzed into poly(acrylic

acid) with HCl as follows: All the resulting polymer was

dissolved in 100 ml of 1,4-dioxane with 24 ml of

concentrated HCl, and the solution was refluxed in an oilbath at 110C for 18 h. Next, the polymer was precipitated

in water (800 ml). The hydrolysis of the dithiobenzoate end

groups was done with NaBH4. Block copolymer (22.74 g,

0.0026 mol) was dissolved in THF (150 ml), and the

solution was turned basic by dropwise adding saturated

KOH/ethanol solution. NaBH4 (3.00 g, 0.079 mol) was

dissolved in water (10 ml) and added to the solution. The

color of the solution changed from red to pale brownish

after a few hours indicating the hydrolysis of the

dithiobenzoate. The polymer was precipitated first in

hexane and then in water from acetone. Water was made

acidic with HCl to ensure that PAA was in acid form.

NaNO3 was added to ensure complete precipitation.

The block copolymer B2 was synthesized by first

preparing a short block of poly(tBA), and the oligomer

was then used as a macro-initiator for polymerizing a

mixture of BuA and MMA. The poly(tBA) was prepared by

using 0.003 mol CPA (0.856 g, 0.060 M), 0.389 mmol

AIBN (63.9 mg, 0.008 M), 0.137 mol tBA (20.0 ml,

2.74 M), and 30.0 ml 2-butanone. The reaction time was

25 h and the yield of polymer was 10.16 g. Poly(BuA-

MMA) block was then prepared using 8.57 g poly(tBA)

(0.002 mol, 0.040 M), 14.5 ml BuA (0.101 mol, 2.00 M),

11.0 ml MMA (0.103 mol, 2.04 M), 29.0 mg AIBN

(0.177 mmol, 0.004 M), and 25 ml 2-butanone. Reaction

time was 24 h and yield of polymer was 21.54 g. The

t-butyl and dithiobenzoate groups were hydrolyzed in the

same manner as in the previous case. The oligomer poly

(tBA) was purified by precipitating from MeOH/H2O (1:1)

and dried in vacuo. The final block copolymer was purified

by precipitations from hexane and water. Care was taken to

remove chloride ions from the polymers, both B1 and B2,

with water so that they would not precipitate the silver

precursor (AgNO3) used later in the next step during the

silver nanoparticle synthesis.

The molar masses of the polymers were determined by

size exclusion chromatography (SEC) using THF as eluent.

For the block copolymers, this was done before the

hydrolysis of tBA to acrylic acid. The average amount of

acrylic acid groups in the polymer was calculated from the

difference in the molar masses before and after the blockcopolymerization.

NMR and FT-IR analysis of the polymers

CP:1

H nuclear magnetic resonance (NMR; poly(BuA-co-

MMA), 200 MHz, CDCl3, TMS, parts per million)3.91

and 3.87 (2H, B2), 3.56 and 3.51 (3H, M2), 1.84 and 1.80

(3H, M1), 1.74 (2H, C1, and B3), 1.51, 1.31, and 1.23 (2H,

B4), and 0.94, 0.89, 0.85, 0.82, and 0.76 (3H, B5)

(Scheme 2).

FT-IR (poly(BuA-co-MMA), solid, ATR) per centimeter

2,951 (m), 1,725 (s), 1,448 (m), 1,385 (m), 1,238 (s), 1,143(s), 1,064 (m), 986 (m), 842 (m), and 751 (m).

B1 : 1H NMR (PAA-block-poly(BuA-co-MMA),

200 MHz, THF, TMS, parts per million)4.02 and

3.99 (2H, B2), 3.63 (3H, M2), 2.44 (1H, A1), 1.95 and 1.86

(3H, M1), 1.59, 1.44, and 1.40 (2H, B4), and 1.15, 1.05,

1.00, 0.96, and 0.93 (3H, B5) (Scheme 4).

FT-IR (PAA-block-poly(BuA-co-MMA), solid, ATR)

per centimeter2,955 (m), 1,725 (s), 1,448 (m), 1,386

(m), 1,236 (s), 1,144 (s), 1,065 (m), 986 (m), 841 (m), 752

(m), and 667 (m).

B2 : 1H NMR (PtBA-block-poly(BuA-co-MMA),

200 MHz, CDCl3,TMS, parts per million)4.02 (2H,

B2), 3.65 and 3.60 (3H, M2), 2.25 (1H, A1), 1.92 and 1.84

(3H, M1), 1.61 (2H, C1, and B3), 1.45 (3H, M3), and 1.15,

1.05, 0.99, 0.95, 0.91, and 0.86 (3H, B5) (Scheme 3).

FT-IR (PtBA-block-poly(BuA-co-MMA), solid, ATR)

per centimeter3,441 (w), 2,958 (m), 2,875 (m), 1,724

(s), 1,449 (m), 1,392 (m), 1,367 (s), 1,248 (s), 1,143 (s),

Scheme 2 Structure of the copolymer CP and designations for

protons



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1,066 (m), 988 (m), 963 (w), 909 (w), 844 (s), 811 (w), 752

(m), 689 (w), and 667 (w).1H NMR (PAA-block-poly(BuA-co-MMA), 200 MHz,

THF, TMS, parts per million)4.02 (2H, B2), 2.45 (1H,

A1), 1.93 and 1.86 (3H, M1), 1.61 (2H, C1 and B3), 1.43

(3H, M3), 1.29 (2H, B4), and 1.13, 0.99, 0.96, and 0.92(3H, B5) (Scheme 4).

FT-IR (PAA-block-poly(BuA-co-MMA), solid, ATR)

per centimeter2,953 (m), 2,162 (s), 1,979 (s), 1,725 (s),

1,448 (m), 1,386 (m), 1,236 (s), 1,144 (s), 1,064 (m), 987

(m), 841 (m), 803 (m), 753 (m), and 667 (m).

Batch synthesis of silver nanoparticles

The synthesized silver nanoparticles in a batch reaction was

done by reducing silver precursor (AgNO3) with sodium

borohydride in the presence of polymers serving as capping

agents. The molar ratio between the species was AgNO3/

polymer 10:1 and the concentration of AgNO3 was

0.002 M. A mixture of ethanol/THF (1:1) was used as a

solvent.

A typical synthesis was conducted as follows: The

polymer (0.01 mmol, 1.98 104

M) was dissolved in 50 ml

ethanol/THF (1:1). AgNO3 (0.1 mmol, 19.80104M) was

dissolved in 0.1 ml water and added to the solution followed

by the reductant (1.0 mmol, 0.0198 M) in 0.4 ml water. In

the experiments with the polymer B2, the amount of NaBH4was increased to 3 mmol, due to the higher amount of acrylic

acid in this polymer, to ensure the complete reduction of the

silver precursor.

The particles were purified by precipitating in hexane

and then dried. The dry nanoparticles were then washed

with water to remove excess of NaBH4.

Seed and feed growth of silver nanoparticles

Particles stabilized with B1 were grown with the seed

and feed method. Thus, first, a seed was prepared and

purified and then it was grown by repeated additions of

small amounts of the silver precursor followed by

reductant [34].

The seed was prepared by dissolving 86.8 mg polymer

B1 (0.010 mmol, 1.98104M) in 50 ml ethanol/THF;

16.6 mg AgNO3 (0.098 mmol, 19.37104M) dissolved

in 0.05 ml water was added, and the solution was left to

m ix f or 1 h. U po n a ddi ti on o f 37 .0 m g N aB H4

(0.978 mmol, 0.019 M) dissolved in 0.5 ml water, a cleardark brown solution was obtained. The UV/Vis absorption

spectrum was measured after 40 min. The solution was

centrifuged at 10,000 rpm for 15 min to remove the largest

particles and to obtain a seed as monodisperse as possible.

A tiny amount of precipitate was observed, and the

supernatant was carefully collected. The solvent was

evaporated with rotary evaporator. The particles were

rinsed with water, filtered, and dispersed in 50 ml

ethanol/THF.

Aqueous solutions (1 M) of AgNO3 and of the reductant

(ascorbic acid) were prepared for the growth of the

particles. The particles were grown by adding first 1 mlof the aqueous AgNO3 and then 1 ml of the aqueous

solution of ascorbic acid. This was repeated four times with

2-h intervals. On the last addition of reductant, the amount

was doubled to ensure complete reduction. The surface

plasmon resonance (SPR) peaks were measured 2 h after

each addition of AgNO3. This method is an adaption of the

method described by Jana et al. [34].

The particles (shown in the Fig. 3) were fractionated by

stepwise precipitation with hexane. In 50 ml of a particle

dispersion, hexane (54 ml) was added in small amounts

until the dispersion turned cloudy. The precipitate was left

to settle and the supernatant was collected. The supernatant

was yellow and was thus still containing nanoparticles. The

precipitate was dispersed in 50 ml ethanol/THF. The

particles still left in the yellow supernatant were precipitat-

ed by adding 2 ml hexane, and the precipitate was dispersed

in 20 ml ethanol/THF. The latter dispersion was used later

for dip-coating glass slides.

Scheme 3 Structure of the block copolymers B1 and B2 before the

hydrolysis and designations for protons

Scheme 4 Structure of the block copolymers B1 and B2 after the

hydrolysis and designations for protons



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Coating of glass slides

Quartz glass slides were dipped in a toluene solution of

silver nanoparticles prepared by the seed and feed

method. The dipping was done once with a dip coater

using a lowering and lifting speed of 3 mm/min and an

immersion time of 5 min.

Ion release tests

For the ion release test, a new designed experimental setup

including in situ pH, temperature, and light control and its

optimization for the determination of low silver ion

concentrations was performed. The silver ion release

through potential measurements of silver containing sam-

ples immersed in distilled water with an ion-selective

electrode was carried out. Inductively coupled plasma

atomic emission spectroscopy results were also obtained

for comparison purposes.

Besides the measurement of the studied silver-containingfilms, pure silver plates used as silver model surfaces

(99.999% Ag) were also studied. After 1 day of immersion

in distilled water, the obtained ion release from the silver

model surface was 1.19105M/cm2.

The measurements on the coated glass slides, with silver

nanoparticles stabilized by polymer B1, showed that the

silver concentration released in distilled water after 1 day of

immersion was 8.5 108M/cm2. The cumulative silver

released per square centimeter of sample increases with

time, reaching a value of 1.6107M/cm2 after 6 days of

immersion.

For comparison, Zhao and Stevens [12] have shown that

for a bacterial population of 1045 E. coli bacteria, the

growth inhibitory concentration of silver ions was 9.45

18.90M, whereas Schreurs and Rosenberg [11] showed

that a concentration of 2M silver ions caused inhibition of

phosphate exchange between growth media and E. coli

bacteria indicating damages to the cells.

Instrumentation and characterization

High-resolution TEM (HRTEM) micrographs were acquired

with a Philips CM200-FEG microscope equipped with a

Gatan slowscan CCD camera at VTT Technical Research

Centre of Finland. TEM micrographs were also obtained

with a Hitachi S4800 FE-SEM using a TEM probe and Inca

X-sight software (Oxford instruments).

Microstructural characterization of the surface morphol-

ogy of selected specimens was carried out by scanning

electron microscopy (SEM), using JEOL-6500F equipped

with a field emission gun and coupled with an energy

dispersive X-ray system for chemical analysis at CENIM,

Madrid. The samples were examined at different accelera-

tion voltages, depending of the nature of the studied

surfaces.

Topographical characterization of the surface morphology

of selected specimens was carried out by atomic force

microscopy (AFM) working in a tapping mode. A Dulcinea

AFM microscope (Nanotec Electronica S.L) at NANOTEC

ELECTRONICA, Spain, was used. Data acquisition was

performed using a Nanotec Dulcinea controller unit, poweredby the WSxM software.

The NMR instrument used was a 200-MHz Varian

Gemini 2000 NMR spectrometer. Sample concentrations

were between 20 and 40 mg/ml, and the deuterated solvents

chloroform, THF, or 1,4-dioxane were from Euriso-top.

Tetramethylsilane was the reference in all measurements.

SEC was used to determine the molar masses of the

polymers. Poly(methyl methacrylate) standards from PSS

Polymer Standards Service GmbH were used for calibration.

Eluents were either THF or dimethylformamide/LiBr

(1 mg/ml). The apparatus included the following instruments:

Biotech model 2003 degasser, Waters 515 HPLC pump,Waters 717plus auto sampler, Viscotek 270 dual detector,

Waters 2487 dual absorbance detector, Waters 2410

refractive index detector, and the software OmnisecTM from

Viscotek. Styragel HR 1, 2, and 4 columns and a flow rate of

0.8 ml/min were used in the measurements.

A Shimadzu UV-160 1PC UV/Vis spectrometer was

used to measure the absorption spectra of the silver

nanoparticles. Calorimetric analyses of the polymers were

made with a Mettler Toledo DSC822e which had a

Ts0801R0 sample robot, and the results were analyzed

with Star E-evaluation program. Heating rate was 10C/min;

the reported Tgs are from the second heating scan.

A KSV DC dip coater was used to dip coat the glass

slides. The IR spectra were measured with a Perkin-Elmer

Spectrum One spectrometer.

Fig. 1 Thermograms of the polymers: 1 CP (poly(BuA-co-MMA))

Tg=36C, 2 poly(BuA-co-MMA) block used to prepare the block

copolymer B1 Tg=12C, 3 B1 (HS-poly(AA-block-BuA-co-MMA))

Tg=36C, and 4 B2 (HS-poly(BuA-co-MMA-block-AA)) Tg=39C



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Results and discussion

Polymers

The polymers used in this work are all amorphous, which

was considered as a requirement in order to obtain good

and homogenous films. Differential scanning calorimetry

analysis of the polymers shows only one glass transition for

the polymers, even though it could be expected that in the

block copolymers, the different blocks would show distinct

glass transitions. Thermograms 1 and 2 in Fig. 1 represent

random copolymers of butyl acrylate and methyl methac-

rylate. Curve 1 is of copolymer CP and curve 2 is from the

poly(BuA-co-MMA) which was used to prepare the block

copolymer B1. The difference in glass transition tempera-

ture between these polymers arises from different monomer

ratios (see Table 1) as an increase in MMA is known to

increase the Tg. The addition of acrylic acid units into either

end of the random copolymer (block copolymers B1 and

B2) increases the Tg presumably because of hydrogen

bonding which restricts the motional freedom of the

polymers.

Because the PAA blocks are very short, no separate glass

transition typical to polyacrylic acid is observed. Next, we

show that all the polymers can be used to stabilize silver

nanoparticles, with a special emphasis on the particles

prepared by using polymer B1. In this case, the hydrophilic

PAA unit is next to the metal surface and is expected to

facilitate the dissolution of silver ions [32]. Studies on the

antibacterial properties of the various particles are under-

way and will be reported separately.

Particles produced in batch reactions

Particles grafted with the three different polymers were

characterized by HRTEM. The particles prepared with the

polymer CP as a stabilizing agent had an average size of

17 nm (standard deviation 6 nm).

Particles stabilized with polymer B1 were considerably

smaller having a mean diameter of 4 nm (standard deviation

2 nm). The introduction of a hydrophilic PAA block to the

polymer and the lower molar mass of the polymer,

compared with CP, clearly influenced the size of the

particles. The particles produced with polymer CP are

heterogeneous, while the particles produced with B1 are

more monodisperse in their morphology, judging from the

TEM micrographs (Figs. 2, 3, and 4).

Reverse location of the PAA block in polymer B2 gave

particles with bimodal size distribution with an average

diameter of 6 nm (standard deviation 14 nm). While using

polymer B2, the reduction of the precursor into metallic

particles was slow, despite increasing the amount of the

Table 1 Polymer characteristics: denotations, molar masses (Mn), and polydispersity indices from SEC, the approximate number of acrylic acid

units, and the BuA/MMA monomer ratio in the polymers

Polymer Denotation Mn (g/mol) DPI n (AA) Tg (C) BuA/MMA

HS-poly(BuA-co-MMA) CP 13,500 1.24 36 1:1.44

HS-poly(AA-block-BuA-co-MMA) B1 8,600 1.88 9 36 1:1.26

HS-poly(BuA-co-MMA-block-AA) B2 21,200 1.45 44 39 1:1.31

Fig. 2 TEM micrographs of silver nanoparticles stabilized with

polymer CP (poly(BuA-MMA))


polymer B1 (HS-poly(AA-block-BuA-co-MMA))



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reductant. The particles stabilized with polymer B2 even-tually precipitated from the solution (ethanol/THF, 1:1)

after a few hours. This is due to the ionization of PAA in

the outer corona of the stabilizing polymer, caused by the

reductant. This clearly influenced the particle size, resulting

in a bimodal size distribution. Since in polymer B2 the

amount of PAA is roughly four times higher than in B1 and

when it is ionized, the solubility of the polymer in organic

solvent is too low compared to B1, which does not

precipitate upon ionization. Also, the location of the PAA

block being on the outer corona in particles stabilized with

polymer B2 masks somewhat the hydrophobic parts of the

polymer, thus decreasing further the solubility of theparticles in organic solvent. A summary of the sizes and

the optical properties of the particles are presented in

Table 2.

Seed and feed grown particles

Particles stabilized with polymer B1 were regarded as best

for the application in mind, i.e., using the particles as a

component in a commercial coating. The B1 polymer wasstill soluble in organic media after the reduction of silver

nitrate, and the nanoparticles were small and more uniform

than the particles produced with the other two polymers.

Also, the PAA block is located in the inner regions of the

polymer corona, thus creating a hydrophilic environment

for the silver nanoparticles that favors the dissolution of

silver ions from the coating [32]. However, the particles are

too small and not monodisperse enough that they could be

used as such in the coating; thus, they were grown using the

seed and feed method [34].

The final grown particles had a bimodal size distribution.

The TEM micrograph (Fig. 5) shows particles of two verydifferent sizes having the diameters of 76 nm (standard

deviation 13 nm) and 12 nm (standard deviation 2 nm).


polymer B2 (HS-poly(BuA-co-MMA-block-AA))

Table 2 Characteristics of the silver nanoparticles

Polymer CPa B1a B2a SFsmallb

SFlargeb

Average size (nm) 17 4 6 12 76

Standard deviation (nm) 6 2 14 2 13

Abs. max (nm) 415 402 405 402 407

The half width of the absorption line is also given

Abs. max maximum wavelength of light absorptionaBatch reactionsbSeed and feed particles after the fractionation

Fig. 5 TEM micrograph of seed and feed grown particles before

fractionation, stabilized with polymer B1 (HS-poly(AA-block-BuA-

co-MMA))

Fig. 6 TEM micrograph of seed and feed grown particles after

fractionation, stabilized with polymer B1 (HS-poly(AA-block-BuA-

co-MMA)) at higher magnification



9/11

Having such a big difference in size, the particles could be

easily fractionated to a fraction containing only the smaller

particles (Fig. 6) to be used for coating glass slides. Theparticles obtained in this way were the most monodisperse

obtained in this work, both in size and morphology, making

them more optimal for the coating applications than any of

the other obtained particles.

UV/Vis spectroscopy

The SPR peak of the silver nanoparticles was monitored

during the growth of the particles. This is a convenient way

to observe if the particles start to aggregate and form

clusters in the dispersion, during the seed and feed

process. Moores and Goettmann [35] have shown that the

SPR shifts to longer wavelength with decreasing the

interparticle distance, and upon aggregation, the SPR peak

gets wider and shifts to wavelengths of about 700 nm.

Upon the growth of the particles (seed and feed), the

SPR peak changes a bit getting bimodal during the process

(Fig. 7). This is due to the aggregation and increasing

polydispersity during the process, as can also be seen from

the TEM image (Fig. 5); the particles obtained have a

bimodal size distribution.

Coating glass slides with silver nanoparticles

Glass slides were coated with the fractionated particles

from the seed and feed reaction. The coated slides were

characterized by microscopy and UV/Vis spectroscopy.

From the SEM image (Fig. 8), it can be seen how the

particles are distributed on the surface, and some aggrega-

25.15 nm

0.00 nm

Fig. 10 AFM topography image obtained in tapping mode from a

surface area of 2 m2 m of the silver nanoparticle-coated quartz

surface. The nanoparticles are from the seed and feed synthesis

stabilized with polymer B1 (HS-poly(AA-block-BuA-co-MMA))

Fig. 8 FESEM micrograph of a surface coated with nanoparticles

from the seed and feed synthesis stabilized with polymer B1 (HS-poly

(AA-block-BuA-co-MMA))

Fig. 9 Absorption spectra of silver nanoparticles in solution (solid

line) and on a quartz substrate (dashed line). These nanoparticles were

obtained from the seed and feed process using polymer B1 (HS-poly

(AA-block-BuA-co-MMA))

Fig. 7 SPR peak evolution during the growth of the particles during

the seed and feed reaction. Dotted line curve is the absorption from the

seed, dashed line after the first, dashdotdashed line after the 2nd,

solid line after the third, and dashdotdotdashed line after the fourth

growth step



10/11

tion can be observed. Same observations can be seen in

from the UV/Vis spectrum of the coated glass slides

(Fig. 9). The SPR peaks shifts to longer wavelengths than

observed from the particles in solution. The shift is

explained by both the change in the dielectric constant

and the close proximity of neighboring particles [35, 36].

Further investigations of SEM and AFM (Fig. 10)

images show that the silver nanoparticles on the surfacehad a mean size of 194 nm and covering 154% of the

surface area. Even though the particles are close to each

other on the surface, the polymer stabilization still keeps a

vast majority of them as individual particles. The AFM

image presented as Fig. 10 also gives an estimate of the

thickness of the coating.

Conclusions

The production of a hybrid material to be used as a

component in an antimicrobial coating, containing silvernanoparticles, is reported in this paper. The material

consists of a block copolymer of acrylic acid and butyl

acrylatemethyl methacrylate copolymer (polymer B1),

stabilized silver nanoparticles. This polymer produced the

most monodisperse silver nanoparticles of the three

polymers used in this work and was therefore chosen for

the final product. The nanoparticles produced with this

polymer were quite small (4 nm), so they were grown in

order to get larger (12 nm) fairly monodisperse nano-

particles in the final product. The acrylic acid block is

located in the polymer so that it forms a hydrophilic layer

on the silver nanoparticles and thus promotes dissolution of

silver ions from the nanoparticles. This way, the individual

nanoparticles are well attached to the model coating with

thiol-silver bonds but still capable of antimicrobial activity

through dissolved silver ions. During 6 days of investiga-

tion, the coating released 0.16M/cm2 silver ions into

deionized water.

Acknowledgments This was a MNT ERA-NET project funded by

the Finnish Funding Agency for Technology and Innovation, TEKES,

Finland. Dr. Pablo Aras and Dr. Adriana Gil from NANOTEC

ELECTRONICA are thanked for the AFM characterization.

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Sy of Copolymer Stablized Silver Nps