LAYER-BY-LAYER DEPOSITION OF GRAFTED PHEMA-g-PAA/PAH
POLYELECTROLYTE MULTILAYER FILMS AND LYSOZYME BINDING
By
Yiding Ma

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Chemistry
2011

ABSTRACT
LAYER-BY-LAYER DEPOSITION OF GRAFTED PHEMA-g-PAA/PAH
POLYELECTROLYTE MULTILAYER FILMS AND LYSOZYME BINDING
By
Yiding Ma
Improving the binding capacity of polyelectrolyte multilayers (PEMs) is important
for their application as a platform for protein capture and immobilization. In this work,
we aimed to create PEMs from comb-like poly(2-hydroxyethyl methacrylate)-graftpoly(acrylic acid) (PHEMA-g-PAA) and poly(allyl amine hydrochloride) (PAH), and
subsequently adsorb lysozyme throughout the film. At all deposition pH values, the
thickness of (PAH/PHEMA-g-PAA)n films grow faster and more exponential than
(PAH/PAA)n. In addition, (PAH/PAA)n and (PAH/PHEMA-g-PAA)n films show similar
trends in thickness as a function of adsorption pH. The presence of supporting electrolyte
in the deposition solutions, 0.5 NaCl, leads to thicker films by varying pH in the absence
of salt. Lysozyme binding capacities of (PAH/PHEMA-g-PAA)5 films, deposited at pH
values ≥5, are 2 to 5 fold higher than the values of corresponding (PAH/PAA)5 coatings,
and (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films deposited from solutions containing
0.5 M NaCl greatly enhances lysozyme adsorption, regardless of the deposition pH. The
relatively high lysozyme binding capacity of our new (PAH/PHEMA-g-PAA)n films
provides potential applications of these PEM systems for protein purification or
immobilization.

ACKNOWLEDGMENTS

Firstly, I would like to thank my advisor, Professor Gregory L. Baker, for being a great
mentor over the past three years. I really appreciate his kindness, patience and encouragement
throughout my graduate study at Michigan State University. His valuable guidance leads me to
think as a scientist, and I will benefit from his advice not only for my research in chemistry, but
also for my future career and life.
I also want to thank Professor Merlin L. Bruening for his great help on my research. He is
an excellent research advisor who thinks critically and has a special attitude for scientific
research. I would not have completed this project without his idea and patience. I also want to
express my gratitude to my committee member, Dr. Xuefei Huang, who has exceptional
knowledge in organic chemistry and gave me useful advice.
I would also like to express gratitude to past and present Baker’s group members for their
help and suggestions: Sampa, Qin, Gina, Hui, Heyi, Wen, Quanxuan, Greg, Zhe and Salinda. I
also enjoyed working with Bruening group members, especially Somnath, Fei, Weihan, Nishotha
and Jinlan for their directly involvement in my research project. Thanks to all the faculty and
staff in Department of Chemistry for their kindness to me.
Lastly, I express my special gratitude to my parents for all of their support and
understanding through the years. And I will never forget my friends at MSU and elsewhere.

iii

TABLE OF CONTENTS

List of Figures ..................................................................................................................vi
List of Schemes.................................................................................................................ix
Abbreviations................................................................................................................... x
Chapter 1. Introduction ....................................................................................................1
1.1. Organic thin films ...............................................................................................1
1.2. Layer-by-layer (LbL) deposition of polyelectrolyte multilayers (PEMs)...........2
1.2.1. Mechanism of polyelectrolyte adsorption..................................................2
1.2.2. Factors that affect LbL polyelectrolyte adsorption....................................4
1.3. Application of PEMs for binding proteins..........................................................7
1.4. Grafted copolymers prepared by atom transfer radical polymerization
(ATRP)...............................................................................................................10
1.5. Research motivation and objectives....................................................................12
Chapter 2. Layer-by-layer deposition of PHEMA-g-PAA/PAH bilayers on Au
substrates 14
2.1. Synthesis of PHEMA-g-PAA .............................................................................14
2.2. Formation of (PAH/PHEMA-g-PAA)n films .....................................................18
Chapter 3. Lysozyme Binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n
multilayers............................................................................................................27
3.1. Lysozyme binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n multilayers
deposited at various pH values ............................................................................27
3.2. Lysozyme binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n multilayers
deposited with a supporting electrolyte ...............................................................29
3.3. Comparison of lysozyme binding capacities of (PAH/PHEMA-g-PAA)n and
(PAH/PAA)n multilayers .....................................................................................31
Chapter 4. Experimental .................................................................................................34
4.1. Materials ...........................................................................................................34
4.2. Preparation of poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid)
(PHEMA-g-PAA) ..........................................................................................35
4.2.1. ATRP of HEMA ........................................................................................35
4.2.2. Preparation of macroinitiator, poly(2-(2-bromoisobutyryloxy)ethyl methacrylate)
(PBIEM).....................................................................................................35
4.2.3. Preparation of PHEMA-g-PtBA ................................................................36
4.2.4. Hydrolysis of PHEMA-g-PtBA to PHEMA-g-PAA .................................36
4.3. Preparation of polyelectrolyte multilayer (PEM) films ......................................36
4.4. Characterization of polymers and PEM films.......................................................37
iv

4.5. Lysozyme binding.................................................................................................38
References

....................................................................................................................40

v

LIST OF FIGURES

Figure 1.1.

Layer-by-layer (LbL) deposition of polyelectrolyte multilayers (PEMs).
..................................................................................................................2

Figure 1.2.

Schematic drawing showing the buildup of a polyelectrolyte “bilayer”
during linear growth and exponential growth..........................................4

Figure 1.3.

Growth behavior for PAA/PAA PEMs resulting from charge mismatch.
The PAA/PAH charge ratios for increasing pH, expressed as the linear
charge density, are (I to V) 4:7, 7:12, 7:6, 7:8, and 7:6. Regions II and IV
show exponential growth, while I, III, and V show linear growth.
..................................................................................................................6

Figure 1.4.

Schematic of protein adsorption onto/into PEMs. Apparent monolayers (a
and b), and apparent multilayers (c and d)...............................................9

Figure 1.5.

Three main strategies for preparing grafted copolymers. ........................10

Figure 1.6.

Cross-section of layer-by-layer deposition of (a) linear polymers and (b)
grafted polymers. .....................................................................................13
1

6

Figure 2.1.

H NMR spectra of (a) PHEMA and (b) PBIEM in CDCl3 with 10% d DMSO. .....................................................................................................15

Figure 2.2.

Gel-permeation chromatograms of (a) PBIEM and (b) PHEMA-g-PtBA
detected using a multi-angle light scattering detector..............................16

Figure 2.3.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n (triangles) and
(PAH/PAA)n films (squares) deposited at pH=7 on Au substrates modified
with a monolayer of MPA. The polyelectrolyte deposition solutions
contained no NaCl. Integer numbers of bilayers indicate films terminated
with PHEMA-g-PAA, and films with an extra half bilayer end in PAH.
..................................................................................................................19

Figure 2.4.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)5 (triangles) and
(PAH/PAA)5 (squares) films deposited from solutions with various pH
values and no supporting electrolyte. The numbers above the triangles
represent the ratio of the average thicknesses of the two types of films
deposited at the same pH. Data points are average of two trials and error
bars here and below represents the difference of two trials.....................21

vi

Figure 2.5.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)10 or (PAH/PAA)10
films deposited from polyelectrolyte solutions with various pH values and
no supporting electrolyte. Numbers in the figure represent the ratio of the
(PAH/PHEMA-g-PAA)10 and (PAH/PAA)10 thicknesses. ....................22

Figure 2.6.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n films deposited
from polyelectrolyte solutions with various pH values and no supporting
electrolyte. Integral bilayers indicate films terminated with PHEMA-gPAA, and films with an extra half bilayer end in PAH. ..........................23

Figure 2.7.

Thickness of (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers
deposited from polyelectrolyte solutions containing 0.5 M NaCl at various
pH values. The numbers above the triangles represent the ratio of the
average thicknesses of the two types of films deposited at the same pH.
..................................................................................................................24

Figure 2.8.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)5 multilayers
deposited from polyelectrolyte solutions at various pH values in the
presence and absence of 0.5 M NaCl.......................................................25

Figure 2.9.

Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n films deposited
from polyelectrolyte solutions containing 0.5 M NaCl at various pH values.
Integral bilayers indicate films terminated with PHEMA-g-PAA, and half
bilayer films end in PAH. Films with more than 5 bilayers layers are
relatively rough, so there is significant scatter in the ellipsometric results.
..................................................................................................................26

Figure 3.1.

Film thicknesses and the equivalent thickness of lysozyme sorbed in
(PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers deposited from
polyelectrolyte solutions with various pH values and no supporting
electrolyte. The numbers above the bars represent the ratios of the
lysozyme equivalent thickness to the film thickness. The equivalent
thickness is the thickness of spin-coated lysozyme that would give an
FTIR absorbance equivalent to that of the sorbed lysozyme...................29

Figure 3.2.

Film thicknesses and the equivalent thickness of lysozyme sorbed in
(PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers deposited from
polyelectrolyte solutions containing 0.5 M NaCl at various pH values. The
numbers above the bars represent the ratios of the lysozyme equivalent
thickness to the film thickness. The equivalent thickness is the thickness of
spin-coated lysozyme that would give an FTIR absorbance equivalent to
that of the sorbed lysozyme .....................................................................30
vii

Figure 3.3.

Lysozyme binding capacities of (PAH/PHEMA-g-PAA)n or (PAH/PAA)n
multilayers (n=1~5) deposited from polyelectrolyte solutions containing
0.5 M NaCl at pH=3. The numbers above the bars represent the ratios of
the lysozyme equivalent thickness to the film thickness. The equivalent
thickness is the thickness of spin-coated lysozyme that would give an
FTIR absorbance equivalent to that of the sorbed lysozyme.
..................................................................................................................32

Figure 3.4.

Lysozyme binding capacities of (a) (PAH/PHEMA-g-PAA)n and (b)
(PAH/PAA)n multilayers (n=1~5) deposited from polyelectrolyte
solutions at pH=3 both in the presence and absence of 0.5 M NaCl. The
numbers above the bars represent the ratios of the lysozyme equivalent
thickness to the film thickness. The equivalent thickness is the thickness of
spin-coated lysozyme that would give an FTIR absorbance equivalent to
that of the sorbed lysozyme .....................................................................33

viii

LIST OF SCHEMES

Scheme 1.1.

Kinetic scheme of ATRP .........................................................................11

Scheme 2.1.

Synthesis of poly(2-hydroxyethyl methacrylate)-g-poly(acrylic acid)
(PHEMA-g-PAA) ....................................................................................17

ix

LIST OF ABBREVIATIONS
A

Absorbance

ATRP

Atom transfer radical polymerization

bpy

2,2-Bipyridine

CRP

Controlled radical polymerization

DMSO

Dimethyl sulfoxide

EBiB

Ethyl 2-bromoisobutyrate

FTIR

Fourier transform infrared

GPC

Gel permeation chromatography

HEMA

2-Hydroxyethyl methacrylate

I

Ionic strength

kact

Activation rate constant

kdeact

Deactivation rate constant

kp

Polymerization rate constant

kt

Termination rate constant

LbL

Layer-by-layer

MALL

Multiangle light scattering detector

MEK

Methyl ethyl ketone

MHz

Million Hertz

Mn

Number average molecular weight

MPA

Mercaptopropionic acid

x

Mw

Weight average molecular weight

NMP

Nitroxide-mediated polymerization

NMR

Nuclear magnetic resonance

PAA

Poly(acrylic acid)

PAH

Poly(allylamine hydrochloride)

PBIEM

Poly(2-(2-bromoisobutyryloxy)ethyl methacrylate)

PDADMAC

Poly(diallyldimethylammonium chloride)

PEM

Polyelectrolyte multilayer

PHEMA

Poly(2-hydroxyethyl methacrylate)

PHEMA-g-PAA

Poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid)

PHEMA-g-PtBA

Poly(2-hydroxyethyl methacrylate)-graft-poly(tert-butyl acrylate)

PMDETA

N,N,N′,N′,N″-pentamethyldiethylenetriamine

ppm

Parts-per-million

PSS

Poly(styrene sulfonate)

PtBA

Poly(tert-butyl acrylate)

RAFT

Reversible addition-fragmentation chain transfer

SAM

Self-assembled monolayer

tBA

tert-Butyl acrylate

THF

Tetrahydrofuran

UV

Ultraviolet

xi

Chapter 1
Introduction

1.1. Organic thin films
Thin films of organic compounds on solid substrates have a variety of potential
applications as antibacterial or biocompatible coatings, substrates that capture or immobilize
proteins, and selective membrane skins for nanofiltration or gas separation.

1-3

Techniques for

preparation of organic thin films have been developed for a long time including spin coating,
Langmuir-Blodgett assembly and adsorption of self-assembled monolayers (SAMs).
However, these classical techniques have significant limitations such as the need for special
equipment, specific functional groups in film-forming molecules, and restrictions with regard
to substrate topology and chemical compositions. In the last 20 years, the growth of polymer
2,4-6

brushes

7-15

and layer-by-layer (LbL) deposition of complementary polymers

have

emerged as versatile tools for forming a wide range of stable, thin films on many types of
3

substrates including 3-dimensional structures such as porous membranes. The growth of
polymer brushes often produce films with high grafting densities and thicknesses, and
2

provides accurate control over chemical composition and architecture. However, this
process requires both initiator immobilization and polymerization under controlled conditions
with a high concentration of monomer. The next section discusses the main focus of this
thesis, layer-by-layer deposition, which also has assets and limitations.

1

1.2. Layer-by-layer (LbL) deposition of polyelectrolyte multilayers (PEMs)
14

Decher and co-workers

popularized LbL deposition in the 1990s. This technique

simply includes alternating immersion of a substrate into polycation and polyanion solutions
13

along with rinsing steps (Figure 1.1).

LbL adsorption can occur on a wide range of
16-19

substrates, including flat surfaces, nanoparticles

20-25

and membranes.

Furthermore, a

variety of charged molecules, including colloids and biomacromolecules, can serve as the
polyelectrolytes in LbL films. Unfortunately, LbL deposition has the inherent drawback of
multiple processing steps. Although new spray methods may overcome this challenge to
some extent,

26-31

this is a major challenge for large-scale, low-cost applications.

1. Polycation

2. Wash

1. Polycation

3. Polyanion
4. Wash

4. Wash

2. Wash

3. Polyanion
13

Figure 1.1 Layer-by-layer (LbL) deposition of polyelectrolyte multilayers (PEMs).

For

interpretation of the references to color in this and all other figures, the reader is referred
to the electronic version of this thesis.

1.2.1. Mechanism of polyelectrolyte adsorption
The driving force for adsorption can arise from electrostatic interactions that displace
2

32

counterions on the surface to increase entropy,

as well as non-electrostatic interactions,
33

such as van der Waals forces, charge-transfer halogen interactions,
hydrophobic effects.

35

hydrogen bonds

34

and

Two growth modes of PEMs are investigated: linear and exponential.

Some systems showed linear increases in film thickness with the number of layers deposited,
36-37

e.g. poly(styrene sulfonate)/ poly(allylamine hydrochloride) (PSS/PAH) films.

This

suggests that each polyelectrolyte layer interpenetrates only its neighboring layers, and the
roughness and surface potential remain the same after a full deposition cycle. On the other
hand, the film thickness of some polyelectrolyte systems increases exponentially with the
number of layers. Polyelectrolyte diffusion ‘in’ and ‘out’ of the film was found to be a key
feature in film growth. Hoda et al. suggested a theoretical model for exponential growth,
38

which features polyelectrolyte diffusion ‘in’ and ‘out’ of the film during deposition.

One of

the mobile polyelectrolytes, with low charge density and high swelling in water, diffuse into
the films to balance the chemical potential. Upon addition of the oppositely charged
polyelectrolyte, this mobile polyelectrolyte diffuses out of the entire film to form a
polyanion/polycation complex at the surface. These complexes of both types of
polyelectrolytes lead to the additional mass and thickness of the multilayer. The number of
more mobile polyelectrolyte which can complex with the oppositely charged polyelectrolytes
increases with film thickness, thus the increment of film thickness increases with the number
of layers, which leads to an exponential growth. It also appears that exponential growth
39

becomes dominant when NaCl concentrations increase

3

40

or when temperature is increased.

Figure 1.2 Schematic drawing showing the buildup of a polyelectrolyte “bilayer” during
38

linear growth and exponential growth.

1.2.2. Factors that affect LbL polyelectrolyte adsorption
The amount of adsorbed polyelectrolyte and surface structure of PEMs can be controlled
by a number of factors. Here we mainly discuss the effect of deposition pH and the
supporting electrolyte, which provide simple and effective adjustments of PEMs, especially
where one polyelectrolyte is weakly charged. In addition, other adsorption parameters, such
39,41

as supporting electrolyte concentration and composition,
42-45

polyelectrolyte,

adsorption time

41

and temperature,

molecular weight of the

40,46-47

also influence

polyelectrolyte adsorption.
The ionization of weak polyelectrolytes is strongly influenced by the pH value of the
48-50

solution, resulting in PEMs highly sensitive to the deposited pH.

Therefore, it is possible

to control the properties of multilayers with small changes in solution pH value, such as the
4

thickness of adsorbed polyelectrolyte, the bulk and surface composition, and charge density
on the surface. The mechanism of pH-dependent growth behavior for PAA/PAH multilayers
49

were studied by Shiratori

48

and Bieker,

and five distinct pH regimes (3~4.5, 4.5~6, 6~8,

8~10, 10~12) of PAA and PAH solutions were identified. The behavior is symmetric around
an intermediate pH in the neutral region (pH 6~8). Both polyions have a similar high charge
density and form rigid and thin layers with a linear growth law as they are strong
polyelectrolytes. Next to this regime (pH 4.5~6 and 8~10), one of the two polyelectrolytes
becomes less charged. Interpenetration of polymer chains is strongly enhanced due to lower
electrostatic interactions, resulting in exponential growth and thick layers. At an even higher
or lower deposited pH, the larger mismatch of charge density leads to extremely asymmetric
layers and a linear growth law.
The supporting electrolyte is another important factor on the growth of PEMs. It is
widely reported that additional salt in polyelectrolyte dipping solutions increases the
32,39,50

thickness and roughness for various polyanion/polycation systems.

Due to the

extrinsic charge compensation of polyelectrolyte and counterions, charges along the
polyelectrolyte chains are screened, resulting in coiled conformations and more exponential
growth.

51

5

Figure 1.3 Growth behaviors for PAA/PAA PEMs resulting from charge mismatch. The
PAA/PAH charge ratios for increasing pH, expressed as the linear charge density, are (I to V)
4:7, 7:12, 7:6, 7:8, and 7:6. Regions II and IV show exponential growth, while I, III, and V
show linear growth.

48

(Reprinted from Macromolecules 2010, 43, 5052. Copyright 2010

American Chemical Society.)

6

The supporting electrolyte concentration shows a dramatic effect on the thickness of
PEMs. The increase in thickness d for different polyanion–polycation combinations is
B

proportional to I (I: ionic strength), and an exponent B as the salt varies between 0.5 and
51

1.

McAloney et al. examined the morphology of multilayer PDADMAC/PSS films
-4

deposited from solutions with salt concentrations ranging from 10 to 1.0 M.

39

Significant

differences were observed between films formed under low salt concentrations and those
produced under high concentrations. (PDADMAC/PSS)10 films deposited from solutions
with less than 0.3 M added NaCl were flat and featureless, and the thickness increased
linearly with the number of adsorbed bilayers, a consequence of the extended rod
configuration of the polyelectrolyte. In contrast, PDADMAC/PSS films deposited from an
ionic strength of 0.3 M or higher had a vermiculate morphology, and the growth rate and
roughness increased. However, very high salt concentrations were reported to dissolve the
52

film.

1.3. Application of PEMs for binding proteins
One attractive application of PEM assemblies is embedding bioactive proteins into thin
films, and utilizing their unique functions in opto-electrical devices, sensors, drug delivery,
9

cell seeding and growth, tissue engineering, and implantable materials. The simple growth
of PEMs on nanostructured substrates is a powerful tool to prepare biological devices.
Adsorption of proteins onto PEMs is attributed to electrostatic forces and
non-electrostatic interactions, such as hydrogen bonds, hydrophobic interactions, and
53-55

hydrophilic repulsion.

The effects of PEMs surface charge on the adsorption behavior of

different types of proteins, including serum albumin, fibrinogen, and lysozyme, have been
7

53-58

studied.

In these cases, proteins strongly interacted with the PEMs films regardless of

the sign of the charges for both the multilayers and the proteins. For like-charged multilayer
and proteins, one monolayer of protein adhered on the film because of hydrogen-bonding and
hydrophobic interactions. However, the interaction of oppositely charged protein and
multilayer is dominated by electrostatic force, thus forming dramatically thicker protein
layers which extend up to several times the dimension of the single protein.

8

Figure 1.4 Schematic of protein adsorption onto/into PEMs. Apparent monolayers (a and b),
55

and apparent multilayers (c and d).

(Reprinted with permission from Biomacromolecules

2004, 5, 1089. Copyright 2004 American Chemical Society.)

This thesis focuses on developing new PEMs as a platform for protein capture and
immobilization. In particular, at physiological ionic strength, PEMs containing poly(acrylic
acid) (PAA) exhibit minimal nonspecific adsorption of proteins, and activation of –COOH
groups provides a means for covalent immobilization of antibodies and other
55,59-61

proteins.

Moreover, these films also adsorb highly positively charged proteins such as

lysozyme. However, improving binding capacity is still important in applications of these
films for protein purification or immobilization. We hypothesized that LbL deposition of
cylindrical, comb-like PAA might lead to thicker films with much greater binding capacities
than corresponding multilayer films containing linear PAA.

9

1.4. Grafted copolymers prepared by atom transfer radical polymerization (ATRP)
Grafted copolymers can be prepared via various strategies: homopolymerization of
62-66

macromonomers (‘grafting through’),
67-68

(‘grafting to’),

and growth of side chains by polymerization from a macroinitiator

69-71

(‘grafting from’).

attachment of side chains to the backbone

Here we applied a ‘grafting from’ strategy via atom transfer radical

polymerization (ATRP), which allows the control of length and molecular weight distribution
of both the backbone and the side chains.

Figure 1.5 Three main strategies for preparing grafted copolymers.

72

Since ATRP was first reported in 1995,

this method was widely developed to prepare
73

various materials. A general mechanism is shown in Scheme 1.1.

Radicals are generated

through a reversible redox process catalyzed by a transition metal complex which undergoes
10

a one electron oxidation with abstraction of a halogen atom from a dormant species. This
process reduces the concentration of active radicals and termination, providing polymers with
a narrow molecular weight distribution. The diminished termination rate makes ATRP
attractive for synthesis of copolymers with controlled compositions and topologies.

Scheme 1.1 Kinetic scheme of ATRP

Other controlled radical polymerization (CRP) methods, such as nitroxide-mediated
polymerization (NMP) and reversible addition-fragmentation chain transfer polymerization
(RAFT) have also attracted significant interest for preparing copolymers with complex
74

architectures, and these methods have certain advantages and limitations.

ATRP initiators,

transition metal catalysts, and ligands are commercial products, and various monomers have
been successfully polymerized using ATRP, including styrenes, (meth)acrylates,
(meth)acrylamides, dienes, acrylonitrile, and other monomers which contain substituents that
73

stabilize the propagating radicals.

However, ATRP of acidic monomers is a challenging

problem and usually requires protection or neutralization, and in industrial applications, the
catalyst must be removed from the final product. For NMP, polymerization of both
methacrylates and less reactive monomers is very challenging, and RAFT is incompatible
with basic monomers and those with primary amino groups.

11

1.5. Research motivation and objectives
In this work, we applied poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid)
(PHEMA-g-PAA) as polyanions to form PEMs (Scheme 2). PHEMA provides a relatively
hydrophilic backbone modified by densely grafted PAA. LbL deposition of this cylindrical
polyanion will involve the same interaction as adsorption of linear PAA, but the cylindrical
shape may lead to thicker films. Thicker surface layers, in particular, might lead to additional
protein binding. Additionally, cylindrical polyelectrolyte layers might form a looser
multilayer to improve the penetration of proteins into the bulk of the film.
In addition to examining differences between films containing PHEMA-g-PAA and PAA,
we also aimed to investigate the effect of deposition pH on protein-binding properties of the
films. Deposition of PAA films at pH 3 and below leads to free –COOH groups in the film,
and subsequent immersion of these films into pH 7 buffer should deprotonate these groups to
increase film swelling and create adsorption sites. In fact, as this work shows, the deposition
pH has a much stronger effect on protein binding to PAA-containing films than the structure
of the PAA, i.e. linear versus graft copolymer.

12

Figure 1.6 Cross-section of layer-by-layer deposition of (a) linear polymers and (b) grafted
polymers.

13

Chapter 2
Layer-by-layer deposition of PHEMA-g-PAA/PAH bilayers on Au
substrates

2.1 Synthesis of PHEMA-g-PAA
Synthesis of the grafted copolymer, PHEMA-g-PAA, proceeds in four steps (Scheme 2.1).
Copper-catalyzed ATRP of HEMA gives linear PHEMA and subsequent esterification of
PHEMA with 2-bromoisobutyryl bromide yields the macroinitiator,
1

poly(2-(2-bromoisobutyryloxy)ethyl methacrylate) (PBIEM). H NMR spectra indicate
essentially 100% esterification (Figure 2.1). Based on GPC data (Mn = 119 000, Mw/Mn =
1.03, see Figure 2.2), PBIEM has an average degree of polymerization of 430.

14

O

Cl

a

c
O

b

OH

d

d

a

c

n

b

5.0

4.5

4.0

3.5

3.0

2.5
f1 (ppm)

2.0

1.5

1.0

0.5

0.0

0.5

0.0
0.0

(a)

e

c
a

b

d
e
d

5.0

4.5

b

c

4.0

3.5

3.0

2.5
f1 (ppm)

2.0

1.5

a

1.0

(b)
1

6

Figure 2.1 H NMR spectra of (a) PHEMA and (b) PBIEM in CDCl3 with 10% d -DMSO

15

Figure 2.2 Gel-permeation chromatograms of (a) PBIEM and (b) PHEMA-g-PtBA detected
using a multi-angle light scattering detector.

16

O

CuCl/bpy

OH

O

Cl

OH

O

MEK/isopropanol
(2:1)

Br

Br

O

Cl

O

O

O

Pyridine
3 h, 0o C
12 h, r.t.

n

16 h, r.t.

Br

O
O

n

O
Acetone
23 h, 60 o C

O
CuBr/PMDETA/CuBr 2

Cl

HO

O
O
n

O

O
O

Scheme 2.1 Synthesis
(PHEMA-g-PAA)

m

Br

8M HCl
Dioxane
2h
reflux

of

Cl

O

O
O

O

O

n

poly(2-hydroxyethyl

m

O

Br

methacrylate)-g-poly(acrylic

acid)

Grafting PtBA from PBIEM also occurs via ATRP. GPC of the grafted copolymer shows
6

a very high average molecular weight with a relatively narrow distribution (Mn = 7.99×10 ,
Mw/Mn = 1.44, see Figure 2.2), indicating successful grafting of PtBA with reasonable
control over the polymerization. The GPC data correspond to an average degree of
polymerization of 140 for the PtBA side chains if we assume 100% initiation efficiency from
71

PBIEM, which is consistent with reports of similar reactions.

Thus the Mn for each PtBA

grafted chain is 61,500. Finally, deprotection of the tert-butyl groups of PHEMA-g-PtBA
proceeds in refluxing dioxane with 8 M hydrochloric acid for 2h. The disappearance of the
1

H NMR signal from the methyl protons of the tBA groups (1.35 ppm) confirms deprotection

to PHEMA-g-PAA.

17

2.2 Formation of (PAH/PHEMA-g-PAA)n films
PHEMA-g-PAA chains can serve as extremely large, somewhat 3-dimensional
polyanions in novel PEMs. Chemically, PHEMA-g-PAA should behave like linear PAA due
to the high degree of polymerization of the PAA side chains. Literature reports show only a
slightly higher pKa value for star-shaped PAA than linear PAA.

75-76

However, the length of

the PAA side chains in PHEMA-g-PAA is 1/3 of the PHEMA backbone, so the grafted
copolymers should be somewhat cylindrical. Steric constraints due to interactions with
neighboring side chains may lead to extended chains and more rapid film growth compared to
77-79

PEMs with linear polymers or even star-like polymers

80-84

and dendrimers.

The high

molecular weight of the grafted polymer might also increase the thickness of PEMs, although
26,44,85

typically, molecular weight has a relatively small effect on polyelectrolyte adsorption.

Figure 2.3 shows the ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n and
control (PAH/PAA)n films as a function of the number of bilayers (n) adsorbed from pH 7
solutions. After adsorption of the first two priming bilayers, the (PAH/PHEMA-g-PAA)n
films grow much faster than (PAH/PAA)n. At pH 7, both the ionized PAA side chains in
PHEMA-g-PAA and linear PAA will extend due to electrostatic repulsion. In the case of
linear PAA, however, this likely leads to adsorption of thin films with chains extended
parallel to the surface. In contrast, with PHEMA-g-PAA, adsorption might occur with either
the backbone or the side chains parallel to the surface (or some intermediate orientation), but
all orientations lead to thicker films at full surface coverage. Nevertheless, the highest
increase in thickness on deposition of an additional PAH/PHEMA-g-PAA bilayer is <10 nm.
Given the average degree of side-chain polymerization of 140, which corresponds to a fully

18

extended side-chain length of 35 nm (0.25 nm per repeat unit), bilayer thicknesses of 10 nm
are well within reason. (Films with more than 10 bilayers were visibly rough, so we could not
determine their ellipsometric thickness.)

Figure

2.3

Ellipsometric

thicknesses

of

(PAH/PHEMA-g-PAA)n

(triangles)

and

(PAH/PAA)n films (squares) deposited at pH=7 on Au substrates modified with a monolayer
of MPA. The polyelectrolyte deposition solutions contained no NaCl. Integer numbers of
bilayers indicate films terminated with PHEMA-g-PAA, and films with an extra half bilayer
end in PAH.

Figure 2.3 suggests that the thickness of (PAH/PHEMA-g-PAA)n films increases
exponentially with the number of bilayers, even though (PAH/PAA)n films show reasonably
48

linear growth film both in this and other studies.

Previous research suggests that diffusion
38,86

of polymer chains throughout the film leads to exponential growth,

and low
44

polyelectrolyte molecular weights favor exponential increases in thickness.

However, the

molecular weight of PHEMA-g-PAA (4500 kDa, calculated from the molecular weight of
PHEMA-g-PtBA assuming complete hydrolysis) is much higher than that of linear PAA (90
19

kDa), Perhaps the extended side chains of PHEMA-g-PAA allow diffusion of PAH
throughout the film.
Adsorption pH dramatically affects the thickness of (PAH/PAA)n films
have

a

similar

effect

on

(PAH/PHEMA-g-PAA)n

coatings.

48-50

and may

(PAH/PAA)5

and

(PAH/PHEMA-g-PAA)5 films show similar trends in thickness as a function of adsorption
pH (Figure 2.4). Comparable trends with greater overall thickness occur for PEMs with 10
bilayers (Figure 2.5). In the case of (PAH/PAA)n films, decreased ionization of PAA at pH
values <7 and of PAH at pH values >7 lead to increases in film thickness as shown
49

previously.

Less ionization presumably leads to polymer chains with more loops and tails,

and hence, greater thickness. Additionally, lower charge densities on polymer chains may
require deposition of more polymers to compensate the charge on the surface. The latter
explanation might better explain why the thickness of (PAH/PHEMA-g-PAA)5 films
increases at low deposition pH because the grafted copolymer should be most extended and
occupy the greatest volume at high degrees of ionization. At all deposition pH values,
(PAH/PHEMA-g-PAA)5 films are about twice as thick as (PAH/PAA)5 films (Figure 2.4).

20

Figure

2.4

Ellipsometric

thicknesses

of

(PAH/PHEMA-g-PAA)5

(triangles)

and

(PAH/PAA)5 (squares) films deposited from solutions with various pH values and no
supporting electrolyte. The numbers above the triangles represent the ratio of the average
thicknesses of the two types of films deposited at the same pH. Data points are average of
two trials and error bars here and below represents the difference of two trials.

21

Figure 2.5 Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)10 or (PAH/PAA)10 films
deposited from polyelectrolyte solutions with various pH values and no supporting electrolyte.
Numbers in the figure represent the ratio of the (PAH/PHEMA-g-PAA)10 and (PAH/PAA)10
thicknesses.

22

Figure 2.6 Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n films deposited from
polyelectrolyte solutions with various pH values and no supporting electrolyte. Integral
bilayers indicate films terminated with PHEMA-g-PAA, and films with an extra half bilayer
end in PAH.

A number of studies show that adding salt to adsorption solutions increases the thickness
32,39,50

and roughness of PEMs.

The excess electrolyte screens the charges along the
51

polyelectrolyte chains to give more coiled chain conformations and thicker films.

Adding

0.5 M NaCl to PHEMA-g-PAA, PAA, and PAH deposition solutions dramatically changes
trends in the thicknesses of (PAH/PHEMA-g-PAA)n and (PAH/PAA)n films as a function of
pH (Figure 2.7). The thickness of these films increases essentially monotonically with
solution pH, and the ratios of the thicknesses of (PAH/PHEMA-g-PAA)n and (PAH/PAA)n
films deposited under the same conditions range from 0.9 to 1.6. At a deposition pH of 3, the
supporting electrolyte has little effect on film thickness (compare Figures 2.6 and 2.7, see
23

Figure 2.8), but at all other adsorption pH values, film thicknesses increase by a factor
between 2 and 9 when using 0.5 M NaCl in the deposition solution. The thickness increase in
0.5 M NaCl is especially large for (PAH/PAA)n films. Compensation of the polymer charge
by ions in the electrolyte might result in a much more coiled conformation of linear
polyelectrolytes, but have a less significant transformation on grafted PHEMA-g-PAA.
Overall, the presence of salt leads to thicker films in the absence of salt. However,
rinsing of the films with 0.5 M NaCl rather than deionized water still leads to nonuniform
films (Figure 2.9).

Figure 2.7 Thickness of (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers deposited
from polyelectrolyte solutions containing 0.5 M NaCl at various pH values. The numbers
above the triangles represent the ratio of the average thicknesses of the two types of films
deposited at the same pH.

24

Figure 2.8 Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)5 multilayers deposited from
polyelectrolyte solutions at various pH values in the presence and absence of 0.5 M NaCl.

25

Figure 2.9 Ellipsometric thicknesses of (PAH/PHEMA-g-PAA)n films deposited from
polyelectrolyte solutions containing 0.5 M NaCl at various pH values. Integral bilayers
indicate films terminated with PHEMA-g-PAA, and half bilayer films end in PAH. Films
with more than 5 bilayers layers are relatively rough, so there is significant scatter in the
ellipsometric results.

26

Chapter 3
Lysozyme Binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n
multilayers

Protein adsorption on PEMs is a simple method for creating functional thin films, both
20-25

on flat surface and in membranes.

This study examines sorption of lysozyme, which is
87

positively charged in pH 7.4 phosphate buffer (the isoelectric point of lysozyme is 11.4 ), in
(PAH/PHEMA-g-PAA)n and (PAH/PAA)n multilayers with the polyanion as the outermost
layer. Initially, we thought that protein would adsorb primarily on the surface, and that the
large size of PHEMA-g-PAA molecules would enhance protein binding. However, in some
cases, sorption of proteins can occur throughout a PEM film to yield the equivalent of many
55

monolayers of protein.

3.1 Lysozyme binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n multilayers
deposited at various pH values
Initially, we examined lysozyme sorption from phosphate buffer (pH 7.4) into
(PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films deposited from polyelectrolyte solutions
with different pH values and no added supporting electrolyte. With the exception of films
deposited at pH 3, all (PAH/PAA)5 films sorb the equivalent of <5 nm of lysozyme (Figure
3.1). Thus, these films essentially adsorb a monolayer or less of lysozyme (14 600 Da,
3×3×4.5 nm)

88

on the surface. Strong ion-pairing in films deposited at pH values ≥5 likely
48-50

prevents lysozyme from entering the film.

(PAH/PHEMA-g-PAA)5 films deposited at

27

pH values ≥5 show 2-5-fold higher lysozyme sorption than corresponding (PAH/PAA)5
coatings. This could be due to more binding in the thicker layer at surface or a somewhat
looser film.
Both (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 deposited at pH 3 contain free COOH
groups that deprotonate in the pH 7.4 buffer. These new carboxylate groups should provide
lysozyme binding sites and increase film swelling to enhance diffusion of lysozyme into the
film. Both (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 deposited at pH 3 bind an amount of
lysozyme equivalent to the initial film thickness. These results are consistent with a surface
plasmon resonance study that showed a 2-fold increase in lysozyme adsorption to PAH/PAA
films deposited at pH 2.0 compared to films in which PAH was deposited at pH 7.5 and PAA
59

was deposited at pH 3.5.

With deposition at pH 3 the (PAH/PHEMA-g-PAA)5 shows 50%

more lysozyme binding than (PAH/PAA)5, but this is likely due only to an increase in film
thickness.

28

Figure 3.1 Film thicknesses and the equivalent thickness of lysozyme sorbed in
(PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers deposited from polyelectrolyte
solutions with various pH values and no supporting electrolyte. The numbers above the bars
represent the ratios of the lysozyme equivalent thickness to the film thickness. The equivalent
thickness is the thickness of spin-coated lysozyme that would give an FTIR absorbance
equivalent to that of the sorbed lysozyme.

3.2 Lysozyme binding on (PAH/PHEMA-g-PAA)n and (PAH/PAA)n multilayers
deposited with a supporting electrolyte
Formation of (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films from solutions containing
0.5 M NaCl greatly enhances lysozyme sorption, regardless of the pH used for film
deposition (compare Figure 3.1 and 3.2). The (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5
films deposited at pH 3 in 0.5 M NaCl show some of the highest lysozyme binding capacities,
despite these films have the lowest thicknesses prior to lysozyme adsorption. Multilayer films
deposited at pH 7 and pH 9 also bind remarkably large amounts of protein, suggesting that

29

these films either contain a larger number of intrinsic ion-exchange sites, or the lysozyme
readily disrupts polyanion-polycation ion pairs to create adsorption sites.

Figure 3.2 Film thicknesses and the equivalent thickness of lysozyme sorbed in
(PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 multilayers deposited from polyelectrolyte
solutions containing 0.5 M NaCl at various pH values. The numbers above the bars represent
the ratios of the lysozyme equivalent thickness to the film thickness. The equivalent thickness
is the thickness of spin-coated lysozyme that would give an FTIR absorbance equivalent to
that of the sorbed lysozyme.

The relatively low sorption capacity of films prepared at pH 5 with 0.5 M NaCl may
indicate that these films have the strongest intrinsic ion-pairing between cations and anions.
Interestingly, only the film deposited at pH 5 shows significantly more binding to
(PAH/PHEMA-g-PAA)5 than (PAH/PAA)5. At this deposition pH, the graft copolymer may
have a somewhat looser structure and more binding at the film surface.

30

3.3 Comparison of lysozyme binding capacities of (PAH/PHEMA-g-PAA)n and
(PAH/PAA)n multilayers
Since PEMs deposited at pH 3 in 0.5 M NaCl clearly exhibit the highest ratio of bound
lysozyme to film thickness (Figure 3.2), we examined binding to these coatings as a function
of the number of layers in the film. For both (PAH/PHEMA-g-PAA)n and (PAH/PAA)n, the
amount of adsorbed lysozyme increases with film thickness, but the ratio of lysozyme
binding to film thickness decreases with the number of layers (Figure 3.3). This suggests that
the accessibility of the film interior decreases somewhat with the addition of more layers. For
the same number of layers, all (PAH/PHEMA-g-PAA)n films bind more lysozyme than
(PAH/PAA)n. However, the ratios of lysozyme binding to film thickness are similar for these
two polyelectrolyte systems. This result implies that the internal structure of
(PAH/PHEMA-g-PAA)n multilayers is similar to the structure of (PAH/PAA)n.

31

Figure 3.3 Lysozyme binding capacities of (PAH/PHEMA-g-PAA)n or (PAH/PAA)n
multilayers (n=1~5) deposited from polyelectrolyte solutions containing 0.5 M NaCl at pH=3.
The numbers above the bars represent the ratios of the lysozyme equivalent thickness to the
film thickness. The equivalent thickness is the thickness of spin-coated lysozyme that would
give an FTIR absorbance equivalent to that of the sorbed lysozyme.

32

(a)

(b)
Figure 3.4 Lysozyme binding capacities of (a) (PAH/PHEMA-g-PAA)n and (b)
(PAH/PAA)n multilayers (n=1~5) deposited from polyelectrolyte solutions at pH=3 both in
the presence and absence of 0.5 M NaCl. The numbers above the bars represent the ratios of
the lysozyme equivalent thickness to the film thickness. The equivalent thickness is the
thickness of spin-coated lysozyme that would give an FTIR absorbance equivalent to that of
the sorbed lysozyme.
33

Chapter 4
Experimental

4.1 Materials
Unless otherwise noted, all chemicals were obtained from Aldrich and used as received
without further purification. 2,2-Bipyridine (bpy, 99%) was recrystallized from hexanes and
sublimed prior to use. Triethylamine was distilled from calcium hydride under a nitrogen
atmosphere and stored under nitrogen. 2-Hydroxyethyl methacrylate (HEMA) (98%) and
tert-butyl acrylate (tBA) (98%), were passed through a column of activated basic alumina to
remove inhibitors (length

╳ 

diameter: ca. 10 cm

╳ 

3 cm). Methyl ethyl ketone (MEK) and

isopropanol were stored with 3 Å molecular sieves.
Poly(allyl amine hydrochloride) (PAH, molecular weight 120,000 ~ 200,000 Da) was
purchased from Alfa Aesar, and poly(acrylic acid) (PAA, molecular weight 90 000 Da, 25
wt% solution in water) was obtained from Polysciences. Aqueous solutions of 0.02 M PAH,
0.01 M PAA and 0.005 M PHEMA-g-PAA were prepared in deionized water (18.2 MΩcm,
Milli-Q), and pH values were adjusted with 0.1 M NaOH or HCl (polymer concentrations are
given with respect to the repeating unit). Au-coated silicon wafers were prepared by
electron-beam evaporation of 200 nm of Au on 20 nm of Cr on Si (100) wafers. Silicon (SiO2
surface) and Au-coated wafers were cleaned in a UV/O3 chamber for 15 min prior to use.

34

4.2 Preparation of poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid)
(PHEMA-g-PAA)
4.2.1

ATRP of HEMA

Poly(2-hydroxyethyl methacrylate) (PHEMA) was prepared by ATRP using a modified
89

literature procedure.

Ethyl 2-bromoisobutyrate (EBiB) (97 mg, 0.50 mmol) bpy (156 mg,

1.00 mmol), MEK (2 mL) isopropanol (1 mL) and HEMA (6.44 g, 6.0 mL, 50 mmol) were
added to a 25 mL Schlenk flask, and degassed by three freeze-pump-thaw cycles. CuCl (50
mg, 0.50 mmol) was added under a flow of N2, and the mixture was stirred at room
temperature for 16 h. The polymer solution was diluted in acetone/isopropanol (volume ratio
2:1) and passed through a basic alumina column. Evaporating the solvent gave the purified
1

6

polymer. H NMR (CDCl3, 10% d -DMSO): δ = 3.87 (-CH2-OCO, s, 2H), 3.59 (-CH2-OH, s,
2H), 2.05-1.60 (-CH2-C, br, 2H), 0.89 (-CH3, s, 1H), 0.73 (-CH3, s, 2H) ppm.
4.2.2

Preparation of macroinitiator, poly(2-(2-bromoisobutyryloxy)ethyl

methacrylate) (PBIEM)

71

During 60 min, α-bromoisobutyryl bromide (7.4 g, 32 mmol) was added dropwise to a
0 °C solution of PHEMA (2.0 g, 15 mmol of OH groups) in anhydrous pyridine (30 mL). The
mixture was stirred for 3 h at 0 °C and then for 12 h at room temperature. The insoluble
pyridinium salt was removed by filtration, and the solvent was removed by rotary evaporation.
The crude polymer was dissolved in 10 mL THF and purified by precipitation into 250 mL
1

6

methanol. Yield: 31 %. H NMR (CDCl3, 10% d -DMSO): δ = 4.17 (-CH2-OCO, s, 2H),
4.00 (-CH2-OCO, s, 2H), 1.77 (-C(Br)(CH3)2, s, 6H), 1.65 (-CH2-C, s, 2H), 0.87 (-CH3, s,
1H), 0.72 (-CH3, s, 2H) ppm.
35

4.2.3

71

Preparation of PHEMA-g-PtBA

In a 25 mL Schlenk flask, PBIEM (0.23 g, 0.82 mmol 2-bromoisobutyryloxy groups),
CuBr2 (9 mg, 41 mmol), N,N,N',N',N"-pentamethyldiethylenetriamine (PMDETA) (150 g,
86 mmol), tBA (10.5 g, 12 mL, 82 mmol) and acetone (3 mL) were combined and degassed
by three freeze-pump-thaw cycles. CuBr (0.118 g (0.82 mmol) was added under a flow of N2,
and the mixture was stirred at 60 °C for 23 h. The resulting polymer was dissolved in acetone
and purified by passage through a basic alumina column (to remove catalyst) and
1

precipitation in water. H NMR (CDCl3): δ = 2.36-2.07 (-CH2-C, br, 2H), 1.90-1.68
(-CH-COOtBu, br, 1H), 1.47-1.20 (-OC(CH3)3, br, 9H) ppm.
4.2.4

90

Hydrolysis of PHEMA-g-PtBA to PHEMA-g-PAA

In a 10 mL round bottom flask equipped with a condenser, a solution of PHEMA-g-PtBA
(3.0 g, 23 mmol tert-butyl ester groups), dioxane (45 mL) and concentrated HCl (15 mL, 0.48
mol), was heated to reflux. After about 2 h, the solution was cooled, and the excess reagents
1

were removed by evaporation under vacuum. H NMR (D2O, pH>10 adjusted by NaOD):
δ = 2.37-2.03 (-CH-COOH, br, 1H), 1.90-1.35 (-CH2-C, br, 2H) ppm.
4.3 Preparation of polyelectrolyte multilayer (PEM) films
Au-coated Si substrates (24 mm × 11 mm) were immersed in 5 mM 3-mercaptopropionic
acid (MPA) in ethanol for 2 h, rinsed with ethanol and dried with N2 to form a monolayer of
MPA and create a negatively charged surface at neutral pH. Then the substrates were
immersed in 0.02 M aqueous PAH, adjusted to the desired pH, for 5 min and subsequently
rinsed with deionized water and blown dry with N2. Substrates were then immersed in a
polyanion-containing solution (0.01 M PAA or PHEMA-g-PAA adjusted to the desired pH

36

value) for 5 min followed by the same rinsing and drying procedures. In some cases, the PAH
and polyanion solutions also contained 0.5 M NaCl. The process was repeated to form
multilayer films.
4.4 Characterization of polymers and PEM films
1

H and

13

C NMR analyses were carried out at room temperature on a Varian

UnityPlus-500 spectrometer at 500 and 300 MHz, respectively, with the chemical shifts
reported in ppm and referenced to signals from residual protons in the solvent.
Ellipsometric measurements were performed with a rotating analyzer ellipsometer (model
M-44, J. A. Woollam) using WVASE32 software. The angle of incidence was 75 °, and the
film refractive index was assumed to be 1.500 for thickness calculations. Reflectance Fourier
Transform Infrared (FTIR) spectroscopy was performed using a Nicolet 6700 FTIR
spectrometer containing a PIKE grazing angle (80 °) attachment. Spectra were typically
collected with 128 scans using a UV/ozone-cleaned, Au-coated wafer as a background.
Polymer molecular weights were determined by gel permeation chromatography with
multi angle light scattering detector (GPC-MALLS) at 35 °C using two PLgel 10µ mixed-B
6

columns in series (manufacturer-stated linear molecular weight range of 500-10╳10 g/mol).
The eluting solvent was THF at a flow rate of 1 mL/min. An Optilab rEX (Wyatt Technology
Co.) refractive index detector and a DAWN EOS 18-angle light scattering detector (Wyatt
Technology Co.) with a laser wavelength of 684 nm were used to calculate absolute
molecular weights.

37

4.5 Lysozyme binding
To immobilize lysozyme, substrates coated with PEM films were immersed in 1.0 mg/mL
lysozyme in 20 mM phosphate buffer (pH 7.4) for 16 h at room temperature. Subsequently,
these substrates were rinsed with 10 mL washing buffer (20 mM phosphate buffer containing
0.1% Tween-20 surfactant) and 10 mL water for 1 min each and dried with N2. The amount
of lysozyme binding was determined by reflectance FTIR spectroscopy and express as the
20

equivalent thickness of spin-coated lysozyme that would give the same absorbance.

The
-1

equivalent thickness d is calculated from the difference of absorbance (ΔA) at 1680 cm

(amide band I of lysozyme) before and after binding lysozyme. d (nm) =ΔA / 0.0017. Some
of these thicknesses were confirmed using ellipsometry.

38

References

39

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