Surface modification of parylene C and indium tin oxide for retinal and cortical prostheses

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SURFACE MODIFICATION OF PARYLENE C AND INDIUM TIN OXIDE FOR
RETINAL AND CORTICAL PROSTHESES

by

Paulin Nadi Wahjudi
________________________________________________________________________

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(CHEMISTRY)

May 2007

Copyright 2007 Paulin Nadi Wahjudi

ii
Dedication

This thesis is dedicated to my family: Papa, Mama, An, and Lus for all of the love,
support, optimism, and patience.

iii
Acknowledgements

As this thesis symbolizes the final chapter of my graduate study at University
of Southern California, I would like to take the opportunity to thank Dr. Mark E.
Thompson for accepting me as a member of his research group, and for the advice
and guidance during my study. I would also like to extend my thanks to all of the
committee members, Dr. Hanna Reisler, Dr. Robert Bau, Dr. William Weber, and
Dr. Katherine Shing.
In the years I have spent in this research group, I am grateful for the
guidance, suggestions and discussion, as well as the friendship that was extended to
me from current and former lab members, Dr. Laurent Griffe, Dr. Alexandre
Dokoutchev, Dr. Peter Djurovich, Dr. Bert Alleyne, Dr. Simona Garon, Dr. James
Ly, Dr. Arnold Tamayo, Dr. Xiaofan Ren, Dr. Jian Li, Dr. Biwu Ma, Tissa Sajoto,
Azad Hassan, Jin Oh, Marco Curreli, Eugene Polikarpov, Yun Tao, Chao Wu, Wei
Wei, Alex Alexander. Thank you for Judy, Michele and Heather for all the
administrative assistances over these years.
I would also like to extend my thank you to all of my research collaborators,
Dr. Mark S. Humayun, Dr. Theodore Berger, Dr. James Weiland, Dr. Yu-Chong Tai
Salam Salman, Adrian Rowley, M.D., Dr. Walid Soussou, Samuel Lee, Jason
Seabold, and Damien Rodger.

iv
My sincere thank you to Wyatt and Joan Brumfields, my “American family”,
for their support and friendship extended to me from the first day I arrived in the
United States, easing my transition to the American life and culture.
There are no words that can express how thankful I am for the love,
guidance, support and confidence of my parent Budijanto Wahjudi and Nancy
Tetanel, my older brother Anthony Nadi Wahjudi, and my younger brother Paulus
Nadi Wahjudi. I share this joyful end of a journey with my family as I would not be
who I am today without them.

“Ora et labora”

v
Table of Contents

Dedication ii

Acknowledgements iii

List of Tables vii

List of Figures viii

Abstract xiii

Chapter 1. Introduction 1
1.1.Introduction to Neural Prosthesis 1
1.2. Retinal Prosthesis 4
1.3. Cortical Prosthesis 8
1.4. Surface Modification for Retinal and Cortical Prosthesis 11
1.4.1. Surface Modification for Retinal Prosthesis 12
1.4.2. Surface Modification for Cortical Prosthesis 16
1.5. Chapter 1 References 21

Chapter 2. Parylene Surface Modification for Metal and Tissue Adhesion 25
2.1. Introduction 25
2.2. Surface Modification 29
2.2.1. Experimental Procedure 29
2.2.2. Results and Discussion on Surface Modification 35
2.3. Metal and Tissue Adhesion Toward Various Parylene Films 52
2.3.1. Experimental Procedure 52
2.3.2. Results and Discussion on Metal and Tissue Adhesion on
Parylene Film
53
2.3.2.1. Metal Adhesion 53
2.3.2.2. Tissue Adhesion 58
2.4. Summary 62
2.5. Chapter 2 References 64

Chapter 3. Surface Modification of Indium Tin Oxide (ITO) for Cortical
Prosthesis
68
3.1. Introduction 68
3.2. Surface Modification 71
3.2.1. Experimental Procedure 71
3.2.2. Results and Discussion on ITO Surface Modification 78
3.2.2.1. Selective Modification on ITO Surface 78

vi
3.2.2.2. Immobilization of IKVAV via Peptide Coupling 79
3.2.3.3. Peptide Immobilization via Dies-Alder Reaction 84
3.3. Neuron Cell Culture Results and Discussion 88
3.3.1. Neuron Cell Culture Results: Comparing mono-IKVAV
Layers vs. oligo-IKVAV Layers
88
3.3.1.1. Neuron Adhesion on mono-IKVAV Layer 88
3.3.1.2. Neuron Growth on mono-IKVAV Layer 90
3.3.2. Neuron Cell Culture Results: Varying mono-IKVAV
Surface Density on ITO
96
3.4. Investigating Strength of Neuron Adhesion on Surface Modified
ITO
98
3.5. Summary 103
3.6. Chapter 3 References 106

Bibliography 111

Appendix: Direct Formation of Hydrogen Peroxide from Hydrogen and
Oxygen by Utilizing Viologen-Palladium Embedded in Inert
Scaffold
119

vii
List of Tables

Table 1.1 Various cell surface receptor of laminin as reported by Kleinman
et. al
18

Table 1.2

Biologically active peptides of laminin and their functions 19

Table 2.1

Static water droplet contact angle measurement of various
parylene C film
39

Table 2.2

Peak intensity ratio of pNIPAM on PC surface varying the
ATRP condition
49

Table 3.1

Contact angle measurement data. Increase in contact angle value
was observed as the length of alkyl chain increases
79

Table 3.2

Flow rate settings and correlated shear stress applied to detach
neurons from ITO surface
102

viii
List of Figures

Figure 1.1 Illustration of cochlear implant courtesy of Mayo Clinic. 2

Figure 1.2 Cochlear prosthesis by Advance Bionics, Corp consist of the
implanted cochlear electrode array and receiver (1), the external
speech processor and transmitter (2), or the behind-the-ear style
processor and transmitter (3) courtesy of Mayo Clinic.
3

Figure 1.3 Vagus nerve stimulation by Cyberonics. The device provides
regular periodical electrical stimulation to the left vagus nerve.
Image courtesy of the Epilepsy Foundation.
3

Figure 1.4

Sagital illustration of human eye. 4

Figure 1.5

Illustration of retina cross-section showing the tissue layers. 5

Figure 1.6

Illustration of retinal prosthesis components and MEA location
for epiretinal and subretinal implant.
6

Figure 1.7

Utah penetrable electrode array for cortical prosthesis. 8

Figrue 1.8

Illustration of cortical prosthesis under development targeting
the hippocampus region, courtesy of http://www.neural-
prosthesis.com (accessed June 23
rd
, 2006).
9

Figure 1.9

Chemical structure of poly-D-lysine (PDL) and
polyethylenimine (PEI).
14

Figure 2.1

Known structures of various parylene films. 26

Figure 2.2 Illustration of chemical modification on PC film via Friedel-
Crafts acylation.
28

Figure 2.3

Schematic of PC modification, a: CH
3
COCl, AlCl
3
, b:
aminothiophenol, c: NaBH
4
. d: bromopropyl phthalimide, NaH,
e:N
2
H
4
, f: 3-maleimidobenzoic acid N-hydroxy succinimide
ester, g: CPC, AlCl
3
, h: NIPAM, CuCl, HMTETA.
31

Figure 2.4 FTIR spectra monitoring acylation of PC to form PC-CO. The
C=O bands were observed at 1710 cm
-1
and 530 cm
-1
.
35

ix
Figure 2.5 FTIR spectra monitoring conversion of C=O (bands observed at
1710 cm
-1
and 530 cm
-1
) to C=N (band observed at 1680 cm
-1
).
The conversion immobilized thiophenyl group indicated by the
presence of C-S stretching at 660 cm
-1
.
37

Figure 2.6 Ketone reduction of PC-CO by NaBH
4
resulting in formation of
secondary alcohol as observed in the FTIR spectra of PC-OH (-
OH bands observed at 3300 cm
-1
).
38

Figure 2.7 FTIR spectra showed conversion of alcohol on PC-OH to ether
as a pathway to immobilized phthalimide group. Phthalimide
characteristic bands at 1715 cm
-1
and 1680 cm
-1
were observed
on PC-phth.
40

Figure 2.8 FTIR spectra monitoring formation of primary amine (NH
2

stretching band at 3350 cm
-1
) due to cleavage of the
phthalimide group (characteristic bands at 1715 cm
-1
and 1680
cm
-1
) by hydrazine.
41

Figure 2.9 FTIR spectra showing immobilization of maleimide group from
PC-NH
2
. Characteristic maleimide stretching at 1680 cm
-1
and
1715 cm
-1
were observed along with C=O stretching at 530 cm
-1
.
43

Figure 2.10 Illustration of initiation and propagation steps in ATRP carried
out on solid surface.
44

Figure 2.11 FTIR spectra showing the two products of reaction of PC with
CPC in presence of AlCl
3
. PC-CPC 7a was obtained when
reaction was carried out neat without any solvent where the C=O
stretching was observed at 1715 cm
-1
. PC-CPC 7b with C=O
stretching at 1693 cm
-1
was observed when the concentration of
CPC was lowered by dilution in dichloromethane.
45

Figure 2.12 Structures of 2-phenylpropionaldehyde ( ν
C=O
= 1718 cm
-1
) and α-
chloropropionphenone ( ν
C=O
= 1695 cm
-1
).
46

Figure 2.13 Spectra of PC-CPC 7a before and after ATRP reaction. 47

Figure 2.14 FTIR spectra of PC-CPC 7b before and after ATRP of NIPAM.
Successful grafting of pNIPAM was concluded with the
appearance of bands associated with pNIPAM at 3400 cm
-1
,
1640 cm
-1
, 1550 cm
-1
, and 1248 cm
-1
.
48

x
Figure 2.15 FTIR spectra of PC-CPC7b and the film after immobilization of
laminin where the amide and other various functional groups of
the protein resulted in appearance of new broad bands in the
spectra of the film.
50

Figure 2.16 Vapor-deposited gold on PC film, (a) before and (b) after
Scotch® tape test. The diameter of each of the gold pads is 4
mm.
55

Figure 2.17 Vapor-deposited gold layer on PC film after Scotch® tape test at
50× magnification using optical microscope.
56

Figure 2.18 Vapor-deposited gold on thiol-modified PC film, (a) before and
(b) after Scotch® tape test. The diameter of each of the gold
pads is 4 mm.
57

Figure 2.19 Vapor-deposited gold layer on thiol-modified PC film after
Scotch® tape test at 50× magnification using optical microscope.
57

Figure 2.20 Adhesion of cortical tissue on PC-pNIPAM film at temperature
higher than pNIPAM LCST (left), and after addition of ice water
to lower the temperature condition (right).
61

Figure 3.1 Chemical structure of poly-D-lysine (PDL) and
polyethyleneimine (PEI), two common polymeric coatings for
promotion of cell adhesion.
69

Figure 3.2 Illustration of selective neuron adhesion promoted by CAM
immobilized on electrode surface.
70

Figure 3.3 Common illustration of immobilization of CAM onto
aminophase ITO surface via EDC coupling, implying formation
of single peptide layer on the surface.
79

Figure 3.4 Chemical structure of the five amino acid sequence of IKVAV, a
cell adhesion molecule that promote neuron adhesion and
growth. Functional groups in red indicate the primary amine of
the N-terminus and the carboxylic acid of the C-terminus.
Primary amine functional group in blue indicates the free amine
of lysine that could also participate in the peptide coupling
reaction.
80

xi
Figure 3.5 Illustration of immobilization of oligo-CAM onto aminophase
ITO surface via EDC coupling, where 1) oligo-CAM form in
solution prior to immobilization onto ITO surface, or 2) oligo-
CAM growth on the ITO surface.
81

Figure 3.6 MALDI-MS spectra of reaction mixture used in IKVAV
immobilization via peptide linkage onto aminophase ITO, where
presence of monomer and oligomers of IKVAV were observed.
82

Figure 3.7 Strategy for immoblilization of mono-IKVAV on aminophase
ITO surface by protecting all amine groups on IKVAV with t-
BOC groups. The protecting groups were removed once the
peptides were covalently bound to the ITO surface, providing the
primary amines groups on the N-terminus and lysine group.
83

Figure 3.8

Illustrated schematic for peptide immobilization on conducting
substrate.
85

Figure 3.9 Synthetic scheme of Cp*-IKVAV. 86

Figure 3.10 A representative of chronoamperometry spectra showing
decreased in the quantity of quinone groups on ITO surface that
are able to be electrochemically reduced post reaction with Cp*-
IKVAV.
87

Figure 3.11 Neuron attachment on various modified ITO surface after 6
hours of seeding. Presence of mono-IKVAV on the surface
promotes neuron adhesion with similar effect as PDL coated
surface.
89

Figure 3.12 Representative microscope images for each sample at 3 days of
culture.
91

Figure 3.13 Representative microscope images for each sample at 5 days of
culture.
92

Figure 3.14 Representative images for each sample of ITO after 7 days of
neuron culture, where neuron was seeded in the presence of
cellular debris.
94

Figure 3.15 Surface coverage percentage for each sample where neurons
were seeded along with cellular debris.
95

Figure 3.16 .Neuron cell culture on varied IKVAV surface density. 97

xii
Figure 3.17 Schematic and dimension of flow channel used for assessing
neurons adhesion on surface modified ITO.
100

Figure 3.18 Instrument setup for assessing neurons adhesion on ITO surface. 101

xiii
Abstract

In the world of prosthesis, the interaction between the material and the tissue
is as essential as the efficiency of the device itself. Development in medical devices
to improve health and healthcare would be immensely benefited by the ability to
tailor the interface of tissue and material according to the specific needs of each
device. In this thesis, works on surface modification of parylene C film and indium
tin oxide (ITO) for retinal and cortical prosthesis are presented. As the works
combine the multidisciplinary fields of chemistry, material science, and biology,
chapter one is targeted to provide general background of the retinal and prosthesis,
and the tissue-material interface that are required for the two prostheses. The second
chapter will discuss how selective chemical surface modifications of parylene C thin
film were carried out to introduce various functional groups and grafting of poly-N-
isopropylacrylamide onto the parylene surface via atom transfer radical
polymerization. The improvement of gold metal electrode and tissue adhesion onto
the modified thin film will also be discussed in this chapter. Works on ITO surface
will be describe in chapter three, including the selective surface modification of ITO,
methods to functionalized ITO surface with cell adhesion molecules (CAMs), and
the neuron cell culture results. Lastly, the appendix will discussed previous work on
direct production of hydrogen peroxide utilizing viologen-palladium embedded in
inert scaffold.

1
Chapter 1. Introduction

1.1 Introduction to Neural Prosthesis
Prosthesis as defined by Merriam-Webster’s Medical Dictionary is an
artificial device to replace or augment a missing or impaired part of the body.
Although the common known prosthetic are external extension of the body that are
removable, increasing number of researches are carried out in the area of implantable
prosthetic especially as active implantable medical devices. Neural prosthesis
defined by the National Institute of Neurological Disorders and Strokes (NINDS) are
prosthesis that restore or supplement the body’s nervous system by establishing links
between the nervous system with external environment
1
usually via functional
electrical stimulation. The most popular example of neural prosthesis is heart pace
maker which directly stimulate a muscle. Another type of implantable neural
prosthesis could be describes as neural stimulation device, in which the device
function is to modify electrical nerve activity. Some examples of neural stimulation
device already available in world market are cochlear implants and Vagus Nerve
Stimulation (VNS) for seizure control in epilepsy.

2

Figure 1.1. Illustration of cochlear implant courtesy of Mayo Clinic
2

In cochlear implant, sound captured by the microphone (1) is processed by
the processor unit (2) into electrical signals as illustrated on Figure 1.1. The signals
is transmitted to the implanted receiver (3) which relay the codes to the thin layer
containing multielectrode array at various location in the cochlea based on the pitch
of the audio received. The auditory nerve on the cochlear received the stimulation
from the electrodes and carries the signal into the brain where it interprets as a form
of hearing. Currently there are three manufacturers of Food Drug and
Administration (FDA) approved cochlear implant, Advanced Bionics Corp (Figure
1.2), Cochlear LTD, and Med-El Corp.

3

Figure 1.2. Cochlear prosthesis by Advance Bionics, Corp consist of the implanted
cochlear electrode array and receiver (1), the external speech processor and
transmitter (2), or the behind-the-ear style processor and transmitter (3) courtesy of
Mayo Clinic.
3

Figure 1.3.Vagus nerve stimulation by Cyberonics. The device provides regular
periodical electrical stimulation to the left vagus nerve. Image courtesy of the
Epilepsy Foundation.
4

Vagus nerve stimulation (VNS) is an alternative treatment to prevent seizure
occurrences for epilepsy patients who do not respond to medication. The system is
4
approved for partial onset seizure and about 32,000 people have received the VNS
system.
4
VNS deliver short bursts of electrical stimulation to the brain via the vagus
nerve, in which the frequency is programmed by physician depending on the needs
of the patient. Short pulse independent of the programmed stimulation can be
achieved by passing the provided magnet over the implanted device to stop and/or
shorten seizure. Similarly, the stimulation can be temporarily stop by placing the
magnet over the device.

1.2 Retinal Prosthesis
The retinal prosthesis targets blind patients due to illness such as Age
Macular Degeneration (AMD) and Retinitis Pigmentosa (RP).

Figure 1.4. Sagital illustration of human eye.
5

5
Loss of sight in AMD patients is due to degeneration or breakdown of macula, the
retina part that forms the “center” of the image. Some known treatments to minimize
progression include laser treatment to seal off blood vessels that grow beneath the
retina, repair of the macula’s weak spots or by removing worn-out tissue and
allowing new tissue growth, but there is no known cure.
6
There are approximately
70,000 new patients of AMD each year in United States.
7, 8

Figure 1.5. Illustration of retina cross-section showing the tissue layers.
9

RP is a collective generic name for genetic defects that resulted in photoreceptor
lost.
10, 11
Over 100 genetic defects have been linked to RP,
12
and the overall
incidence of RP is 1 every 4000 live births.
8
Unfortunately, there is no known
treatment for RP patients.
6
In both of these cases, the nerve optics of the retina is still functioning.
Retinal prosthesis is under development to “by-pass” the damage region of the retina
while still utilizing the presence nerve optics by supplying input signal directly onto
the nerve optic cells.
The multielectrode array (MEA) of the prosthesis will be placed in the
epiretinal surface, in contact with the inner limiting membrane (ILM) as illustrated in
Figure 1.5.

Figure 1.6. Illustration of retinal prosthesis components and MEA location for
epiretinal and subretinal implant.
13

Digitized image collected from a camera is sent to an external processing unit
which will process and further transmit the data to the epiretinal multielectrode array.
7
The signals on the multielectrode array can be sensed by the nerve cells (ganglion
cell, amacrine cells and/or bipolar cells) which will carry the stimulus to the visual
region of the brain utilizing the regular optical nerve pathway (Figure 1.6).
Placement of the multielectrode array on epiretinal surface is preferred due to
the possibilities of heat dissipation into the vitreous, thus minimizing cell and/or
tissue thermal damage from microelectrode activities.
13
It is also preferable than the
subretinal implant. Subretinal inplant will utilized the retina to hold the implant
implace
14
and stimulate the bipolar cells (Figure 1.6).
14,15
Disadvantages of
subretinal implant occur due to the limited subretinal space to place the electronics
and higher risk of thermal injury due to the close proximity of the electrode and the
neurons.
Another successful approach to restore vision was reported by William
Dobelle.
16
In his approach, the eye is by-passed altogether and signals were directly
transferred to the visual area of the brain cortex. Since the electrodes were placed on
the surface of the cortex, the risk greatly increases for implant insertion and
placement. To improve Dobelle’s approach, researches are carried out to investigate
application of penetrable MEA for improvement of specificity in targeting stimulus
into visual region of the brain.
17

The ideal retinal stimulating electrode would have the flexibility to conform
to the retina curvature. To achieve the flexibility requirement, polymeric thin films
are utilized as support material followed by deposition of patterned metal electrode
array onto the support. The support materials would also serve as electrical insulator
8
and water barrier. Other requirements for the polymer substrate are (1) good
adhesion between the substrates and the patterned metal electrodes and (2)
compatible surface that can provide prolonged attachment of the device onto the
ILM.

1.3 Cortical Prosthesis
In the area of brain implants, besides the work of Dobelle to restore vision,
many researches have been focused on translating brain signals into movement of
muscles or robotic limbs.
18
There are two basic approaches on cortical electrode
array. One method is to stimulate the brain by placing the array on the cortex surface
such as in Dobelle cortical prosthesis for vision and BrainGate chip. The second
method is utilizing penetrable electrode to achieve higher precision on stimulating
area. The most common penetrable multi electrode arrays for cortical prosthesis are
based on the Utah electrode array.

Figure 1.7. Utah penetrable electrode array for cortical prosthesis.
17

9
Another cortical prosthesis under development focused on the activities of
hippocampus, a cortical region of the brain that is known to be responsible in
formation on new long-term memories. The idea of this prosthesis is to develop a
microchip that can mimic collective neuron activities in hippocampus region and
capable for bi-directional signaling transmission, such that the microchip can be
applied as replacement of a damage region in the hippocampus.
19

Figure 1.8. Illustration of cortical prosthesis under development targeting the
hippocampus region, courtesy of http://www.neural-prosthesis.com (accessed June
23
rd
, 2006)

A penetrable MEA
20
will be placed on a specific region of hippocampus
where signal from nearby neurons is collected and transmitted to the processing unit.
The signal will then undergo mathematical algorithms such that the output signal
10
transmitted by the second penetrable MEA at a different location in hippocampus
mimics the input signal received previously. A proof of concept experiment was
conducted in which a flat MEA can mimic the signal from the hippocampus slice on
top of it.
21
One of the big obstacles on this project is the tissue reaction towards the
penetrable MEAs.
Similarly as with any other long-term and/or permanent implants, the biotic-
abiotic interface is an important issue to address. For this particular prosthesis where
bi-directional communication between the prosthesis’ MEA and neuron/tissue need
to be established, biotic-abiotic interface demand more consideration. The penetrable
MEA will have two basic types of material, a conductive electrode pads and an
insulator support. The biostability and biocompability requirement for the device will
be different between the conductive and the insulator part. The surface properties for
the two materials toward cells and/or tissue are not the same.
On the electrode pads, selective and/or specific neuron adhesion is required
such that 1) the electrode can receive neuron signals and vice versa, 2) surviving
neuron network can interact with the electrode, 3) prevent mixed signaling between
the neurons and the glia cells, and 4) prevent device encapsulation and/or tissue
inflammation.
On the insulating pads, the surface should be biostable and biocompatible
toward the surrounding tissue such that 1) interaction between the insulator and the
tissue can anchor the device on the designated location, 2) prevent tissue
encapsulation that could damage the device, and 3) prevent tissue inflammation.
11
1.4 Surface Modification for Retinal and Cortical Prosthesis
As the biomedical engineering field pushes forward in designing new
implantable devices to improve health and healthcare, biomaterials scientists always
face the dilemma of choosing the right materials to use and/or any further
modifications that need to be carried out to fulfill the properties required for the
implant. Scientists have the options of using metal, ceramic composites, and
polymers to provide stiffness or flexibility, conductivity or insulation, low friction
and lubrication or surface roughness, depending on the prosthesis need. Special
consideration must be taken on how the body would interact with the material and
whether this interaction can 1) caused health problem and 2) interfere with device
performance. The two most common approaches to achieve biocompatibility and/or
biostability are 1) prevention of tissue interaction by creating a non-fouling surface
or by application of bioinert polymer thin film coating, or 2) promotion of tissue-
interaction by creating a tissue and/or cells adhesive layers by coating of a charged
polymer, extracellular matrix protein, or any combinations of the above.
The Biomimetic Microelectronic System-Engineering Research Center
(BMES-ERC) at the University of Southern California is focusing on development of
neuromuscular, retinal, and cortical prostheses. The research projects on this thesis
are part of Interface 1 Thrust that supports the Retinal Testbed and Cortical Testbed,
to proved a seamless mechanical and electrical at the biotic-abiotic interface.
22

Focusing on the abiotic-biotic interface, the goal of the thrust is to provide a
seamless interaction between the device and the tissue. Various chemical surface
12
modifications will be employed to find a general procedure and/or approach that is
adaptable for various materials that could be used in the retinal prosthesis and/or
cortical prosthesis develop by the BMES-ERC.

1.4.1 Surface Modification for Retinal Prosthesis
As mentioned previously, an ideal material support for the MEA of epiretinal
implant is a thin polymer film that 1) allows metal patterning on the surface for
formation of the MEA pads, 2) have flexibility to conform to the retina curvature, 3)
can act as an electrical insulator, 4) have good water barrier property, 5) provide
good adhesion toward the metal pads, and 6) allow tissue attachment for device
anchorage.
In the development of retinal prosthesis, inert polymer films such as
polyimide and polydimethylsiloxane are used. Polyimide is a known thermally stable
insulator coating in the semiconductor industry that has low Young’s modulus. The
polymer is currently use as the support layer by Second Sight Medical Products for
fabrication of epiretinal MEA. The flexible polymer support has been plaque with
delamination from metal and water penetration. Polydimethylsiloxane (PDMS) has
been reported for a candidate for flexible support material, but the material may not
be as good water barrier as polyimide.
The material of interest for this research project is poly-p-xylylene. The
polymer (also refer as parylene) satisfy both the flexibility and water barrier
13
requirement for the MEA substrates. Parylene has been utilize in medical device
coating to provide bioinert conformal coating and is approved for long term and/or
permanent implant. The inertness of the material provides the long term stability and
lubrication properties when used for coating of limb prosthetic.
Since all of the materials above are bioinert thin films, long term stability
requirements for application in permanent retinal prosthesis would be achieved.
Unfortunately, the inertness of the material inhibits any interaction with the tissue,
preventing formation of stable adhesion between the implant and the surrounding
tissue. The problematic permanent placement of the thin film containing MEA pads
onto a specific location is currently solved by utilizing medical staples to
mechanically anchor the implant to the tissue. The disadvantage of the above
solution is that the surrounding tissue recognized both the thin film MEA pads and
the medical staples as foreign materials, in which the tissues will response by
encapsulating the implant and the medical staples with scar tissue, preventing the
device to function properly.
As mentioned previously, the most common approach to improve
biocompatibility of a material is either by preventing any tissue interactions, or by
promoting tissue interaction. Adapting from approaches used in cells cultures
procedures, promotion of tissue and/or cells adhesion can be achieve by formation of
adhesive layers on the surface of the implant, such that tricking the tissue to
recognized the implant not as a foreign material. Cell adhesion on abiotic materials
could occur due to charge interaction or cell adhesion receptor-ligand interaction. On
14
the first system, cell adhesion took place due to the interaction of the negatively
charge cells with the positively charge surface material. The most common approach
to create this type of adhesion is by decorating the surface with primary amine group.
One method to create an amine rich surface is via plasma treatment. The
approach could be utilized for surfaces that can withstand the harsh treatment, but
unsuitable for parylene thin film since the treatment will cause the polymeric film to
lose its mechanical properties.

HN
H
2
N
O
O
n
poly-D-lysine
H
N
N
H
2
N
x
y
polyethylenimine

Figure 1.9. Chemical structure of poly-D-lysine (PDL) and polyethylenimine (PEI).

Another method to introduce amine rich layer onto abiotic surface is by applying
polymeric coating. Charged polymers such as poly-D-lysine (PDL),
23, 24
and
polyethyleneimine (PEI)
24, 25
are commonly applied on various culture dishes to
provide cell adhesive surface. Although the charged polymeric coating provides a
strong and stable tissue adhesion, the coating is mainly anchored to the substrates
surface via hydrogen bond interaction. For parylene film, where the polymer
coatings ability to be anchored to the surface via hydrogen bonding was severely
15
limited, the polymeric coating can be easily removed. Thus, the method is ineffective
for promotion of stable tissue adhesion onto parylene film.
The second approach to promote tissue adhesion was via cell receptor-ligand
interaction, as found in the interaction between cells and the surrounding extra
cellular matrix proteins. Extra cellular matrix proteins are known to regulate various
biological processes of various cells. Coating of extra cellular matrix protein, such as
laminin, fibronectin, and collagen, on abiotic substrates is also a common approach
in cell culture practices to promote cells adhesion and growth. The approach utilizes
the binding of various surface cell receptors with the active sites of these proteins to
anchor the cells onto the coated substrates. Laminin coating is the most common
protein coating in neural cell cultures practices due to its role in regulating various
biological processes, including neural adhesion, growth and migration.
26-31
Laminin
itself is a glycoprotein that is composed of α, β, and γ polypeptide chains totaling of
over 900 kD. Laminin contains multiple active sites that can interact with various
cells and tissues receptors both with integrin receptors and non-integrin receptors.
26,
28, 29, 32
Similar with PDL and PEI coating, coating of laminin onto abiotic substrate
relies on hydrogen bonding between the various functional groups presence in the
proteins and the surface of the material.
Therefore, although there are available methods that can be apply to promote
tissue and cell adhesion onto abiotic surface, the main challenge in this project is to
find a method that would allow strong anchoring of the adhesive layers onto the
parylene film. Delamination of the polymeric coating and/or laminin coating can be
16
prevented by covalently immobilizing the adhesive layer onto parylene film surface.
With the inertness of parylene film, a method to carry out selective surface
modification of the film will need to be developed. Successful selective chemical
modification of parylene film will allow tailoring of the film surface properties
Thus, the two main goals for parylene surface modifications are 1) to improve the
metal adhesion onto parylene, and 2) to create an adhesive surface for
adhesion/anchorage of the parylene onto tissues such as the retina’s ILM.
Methodologies, experiments, results, and progress on this project will be discussed
on Chapter 2.

1.4.2 Surface Modification for Cortical Prosthesis
The requirement for a good biotic-abiotic interface for the cortical prosthesis
is slightly different compared to those of the retinal prosthesis. The biotic-abiotic
requirements are depended on whether the material is part of the electrode pad or the
insulator support and what the biotic target of the material is.
Concentrating on the biotic-abiotic interface of the MEA as the signal
receiver and/or transmitter, the material is required to have specific surface
properties. These properties are (1) allow and/or promote neuron cells adhesion, (2)
allow and/or promote formation of new synapses between the adhered neurons and
the surrounding intact neural network, (3) prevent mixed signal recording due to
17
non-selective cell attachment, and (4) prevent inflammatory and scar tissue
encapsulation.
As mentioned in the previous subsection, cell adhesion on to abiotic materials
could occur due to charge interaction with the polymeric coating on the surface, or
due to cell adhesion receptor-ligand interaction between the cells surface receptors
and the extra cellular matrix proteins. Assuming that covalent anchoring of the
adhesive layers could be achieved, the approach is a viable method for formation of
stable non-selective adhesion between the tissue and the abiotic surface.
Unfortunately, the adhesive coatings are not suitable for MEA in cortical prosthesis
application. The lack of cell specificity of PDL and laminin will allows any cells and
tissues in the surrounding implantable area to adhere onto the MEA surface. This
could resulted in mixed signal recording by the MEA.

18

Table 1.1. Various cell surface receptor of laminin as reported by Kleinman et. al
32

Adhesion of cells onto extra cellular proteins through receptor-ligand
interaction was found to be selective. That is, adhesion only occurred when the cell
surface receptor recognized a specific ligand that it can bind to in a particular active
site of the base membrane protein. and vice versa, the ligand in the active sites can
only bind to a specific type of cells surface receptors from particular type of cells.
20,
32
The cell surface receptors are transmembrane proteins that directly or indirectly
19
link the extra cellular matrix proteins to the cells cytoskeleton and act as a signaling
pathway between the cells and the extra cellular matrix. It is reported that neural
cells can bind to laminin both via integrins and non-integrins receptors (Table 1.1).

Table 1.2. Biologically active peptides of laminin and their functions.
33

20
Various studies have been conducted to identify specific location and the
amino acid sequences in the extra cellular matrix proteins that responsible for
specific cell functions such as adhesion, growth and proliferation (Table 1.2).
Sequences that were involved on the cell adhesion are known as cell adhesion
molecule (CAMs).
3, 9, 33-37
Of interest for this project is the amino acid sequence of
IKVAV, a peptide that interact with the non-integrin receptor protein of LBP-110 on
the neuron cells surface . The five amino acids have been reported to promote neuron
adhesion and growth onto various abiotic surfaces.
37-41
The cell specificity of the
CAM lend itself for utilization in surface modification for the cortical prosthesis.
One of possible electrode array material is indium tin oxide (ITO), a
transparent electrode that has been used in commercial MEA manufacturing.
Although ITO is not considered a toxic material, the surface properties did not
readily support neuron adhesion unless an adhesive layer such as laminin or PDL
coating was applied on top of the substrate.
42
The goal of this research project is to
selectively modified ITO surface to selectively promote neuron cells adhesion and
growth. Selectivity in neurons adhesion and growth could be achieved by creating
IKVAV layers on the surface of ITO. To avoid delamination of the IKVAV layer
from the ITO surface, a procedure to covalently bound IKVAV is needed, without
compromising the peptide ability in promoting neuron adhesion and growth.
Methodologies, experiments, results, and progress on this project will be discussed
on Chapter 3.

21
1.5 Chapter 1 References
1. National Institute of Neurological Disorders and Stroke :Neural Prosthesis
Program (NPP). http://www.ninds.nih.gov/funding/research/npp/ (July 17th, 2006),

2. Massia, S. P. H., Matthew M.; Ehteshami, Gholam R., In vitro Assessment of
Bioactive Coatings for Neural Implant Applications. Journal of Biomedical Material
Research 2004, 68A, 177-186.

3. Kam, L. S., W.; Turner, J.N.;Bizios, R., Selective Adhesion of Astrocytes to
Surfaces Modified with Immobilized Peptides. Biomaterials 2002, 23, 511-515.

4. Hasenbein, M. E. A., T.T.; Bizios, R., Micropatterned Surfaces Modified
with Select Peptides Promote Exclusive Interactions with Osteoblasts. Biomaterials
2002, 23, 3937-3942.

5. Choquet, D. F., Dan P.; Sheetz, Micahel P., Extracellular Matrix Rigidity
Causes Strengthening of Integrin-Cytoskeleton Linkages. Cell 1997, 88, 39-48.

6. Weisz, J. M.; O'Connell, S. R.; Bressler, N. M., Treatement Guidelines for
Age-Related Macular Degeneration Based upon Results from The Macular
Photocoagulation Study. Lippincott, Williams&Wilkins: Philadelphia, 2000; p 201-
211.

7. Curcio, C. A.; Medeiros, N. E.; Millican, C. L., Photoreceptor Loss in Age-
Related Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 1996, 37, (7), 1236-
1249.

8. Margalit, E.; Sadda, S. R., Retinal and Optical Nerve Diseases. Artif. Organs.
2003, 27, (11), 963-974.

9. Dee, K. C. A., Thomas T.; Bizios, R., Osteoblast Population Migration
Characteristics on Substrates Modified with Immobilized Adhesive Peptides.
Biomaterials 1999, 20, 221-227.

10. Berson, E. L., Rentinitis Pigmentosa : The Friedenwald Lecture. Invest.
Ophthalmol. Vis. Sci. 1993, 34, (5), 1659-1679.

11. Sharma, R. K.; B., E., Management of Hereditary Retinal Degenerations:
Present Status and Future Directions. Surv. Ophthalmol. 1999, 43, (5), 427-444.

12. Heckenlively, J. R.; Boughman, J.; Friedman, L., Diagnosis and
Classification of Rentinitis Pigmentosa. Lippincott: Philadelphia, 1998; p 21.

22
13. Weiland, J. D.; Liu, W.; Humayun, M. S., Retina Prosthesis. Annual Review
Biomedical Engineering 2005, 7, (361-401).

14. Volker, M.; Shinoda, K.; Sach, H.; Grneier, H.; Schwarz, T.; In Vitro
Assessment of Subretinally Implanted Microphotodiode Arrays in Cats by Optical
Coherencetomography and Fluoresecein Angiography. Arch. Ophthalmol 2004, 242,
(9), 792-799.

15. Zrenner, E.; Steet, A.; Weiss, S.; Aramant, R. B.; Guenther, E.; al., e., Can
Subretinal Microphotoiodides Successfully Replace Degenerated Photoreceptors? .
Vis. Res. 1999, 39, 2555-2567.

16. Dobelle, W. H.; Mladejovsky, M. G.; Girvin, J. P., Artificial Vision for The
Blind: Electrical Stimulation of Visual Cortex Offers Hope for a Functional
Prosthesis. Science 1974, 183, (4123), 440-444.

17. Kindlmann, G.; Normann, R. A.; Badi, A.; Bigler, J.; Keller, C.; Coffey, R.;
Jones, G. M.; Johnson, C. R., Imaging of Utah Electrode Array, Implanted in
Cochlear Nerve. In NIH Symposium on Biocomputation & Bioinformation Digital
Biology: The Emerging Paradigm, Bethesda, Maryland, 2003.

18. Serruya, M. D.; Hatsopoulos, N. G.; Paninski, L.; Fellows, M. R.; Donoghue,
J. P., Instant Neural Control of a Movement Signal. Nature 2002, 416, 141-142.

19. Berger, T. W.; Baudry, M.; Brinton, R. D.; Liaw, J.-S.; Mamarelis, V. Z.;
Park, A. Y.; Sheu, B. J.; Tanguay, A. R., Brain-Implantable Biomimietic Electronics
as the Next Era in Neural Prosthetics. Proceedings of the IEEE 2001, 89, (7), 993-
1012.

20. Beckerman, M., Chapter 10. Cell Adhesion and Motility. In Molecular and
Cellular Signaling, Springer: New York, 2005; pp 221-245.

21. Berger, T. W.; Ahuja, A.; Courellis, S. H.; Deadwyler, S. A.; Erinjipurath,
G.; Gerhardt, G. A.; Gholmieh, G.; Granacki, J. J.; Hampson, R.; Hsaio, M. C.;
Lacoss, J.; Marmarelis, V. Z.; Nasiatka, P.; Srinivasan, V.; Song, D.; Tanguay, A. R.;
Wills, J., Restoring Lost Cognitive Function. IEEE Engineering in Medicine and
Biology Magazine September/October, 2005, pp 30-44.

22. Humayun, M. S.; Loeb, G. E. Biomimetic Microelectronic Systems
Engineering Research Center; University of Southern California, California Institute
of Technology, University of California at Santa Cruz: 2005; p 47.

23
23. Jacobson, B. S.; Branton, D., Plasma Membrane: Rapid Isolation and
Exposure of The Cytoplasmic Surface by Use of Positively Charged Beads. Science
1977, 195, (4275), 302-304.

24. Ai, H. M., Hongdi; Ichinose, Izumi; Jones, Steven A.;Mills, David K.; Lvov,
Yuri M.; Qiao, Xiaoxi., Biocompatibility of Layer-by-Layer Self Assembled
Nanofilm on Silicone Rubber for Neurons. Journal of Neuroscience Methods 2003,
128, 1-8.

25. He, W.; Bellamkonda, R. V., Nanoscale Neuro-Integrative Coatings for
Neural Implants. Biomaterials 2005, 26, 2983–2990.

26. Bosman, F.; Stamenkovic, I., Functional Structure and Composition of The
Extracellular Matrix. J. Pathol 2003, 200, 423-428.

27. Li, J.; Zhang, Y.-P.; Kirsner, R. S., Angiogenesis in Wound Repair:
Angiogenic Growth Factors and The Extracellular Matrix. Microsc Res Tech 2003,
60, 107-114.

28. Mercurio AM, Laminin Receptors: Achieving Specificity Through
Cooperation. Trends Cell Biol 1995, 5, 419-423.

29. Smyth, J.; Walker, M., Surface Precoating in The 1980s: A First Taste of
Cell-Matrix Interactions. In Tissue Engineering of Vascular Prosthetic Grafts, Zilla,
P.; Greisler, H., Eds. R.G. Landes Company: Austin, 1999; pp 69-77.

30. Ratner, B., Perspective and Possibilities in Biomaterials Science. In
Bioimaterials Science: An Introduction to Materials in Medicine, Ratner, B.;
Hoffman, A.; Schoen, F.; Lemons, J., Eds. Academic Press: San Diego, 1996; pp
465-468.

31. Williams, D., Bioinertness: An Outdated Principle. In Tissue Engineering of
Vascular Prosthetic Grafts, Zilla, P.; Greisler, H., Eds. R.G. Landes Company:
Austin, 1999; pp 459-462.

32. Powell, S. K.; Kleinman, H. K., Neuronal Laminins and Their Cellular
Receptors. Int. J. Biochem. Cell Bio. 1997, 29, (3), 401-414.

33. Murtomaki-Repo, S. The Role of Laminin in Development, Regeneration,and
Injuries of The Nervous System. University of Helsinki, Helsinki, 2000.

34. Hersel, U. D., Claudia; Kessler, Horst., RGD Modified Polymers:
Biomaterials for Stimulated Cell Adhesion and Beyond. Biomaterials 2003, 24,
4385-4415.
24
35. Dee, K. C. A., Thomas T.; Bizios, R., Design and Function of Novel
Osteoblast-Adhesive Peptides for Chemical Modification of Biomaterials. Journal of
Biomedical Material Research 1998, 40, 371-377.

36. Ranieri, J. P. B., Ravi; Bekos, Evan J.; Gardella Jr., Joseph A.; Mathieu,
Hans J. ; Ruiz, Laurence; Aebischer, Patrick., Spatial Control of Neuronal Cell
Attachment and Differentiation on Covalently Patterned Laminin Oligopeptide
Substrates. Int. J. Devl. Neuroscience 1994, 12, (8), 725-735.

37. Nomizu, M. W., Benjamin S.;Weston, Christi A.;Kim, Woo Hoo; Kleinman,
Hynda K.; Yamada, Yoshihiko., Structure-Activity Study of a Laminin α1 Chain
Active Peptide Segment Ile-Lys-Val-Ala-Val (IKVAV). FEBS Letters 1995, 365,
227-231.

38. Santiago, L. Y. N., Richard W.; Rubin, J. Peter; Marra, Kacey G., Peptide-
Surface Modification of Poly(caprolactone) with Laminin-Derived Sequences for
Adipose-Derived Stem Cell Applications. Biomaterials 2006, 27, 2962-2969.

39. Silva, G. A. C., Catherine; Niece, Krista L.; Beniash, Elia; Harrington, Daniel
A.; Kessler, John A.;Stupp, Samuel I., Selective Differentiation of Neural Progenitor
Cells by High-Epitope Density Nanofibers. Science 2004, 303, 1352-1355.

40. Svedhem, S. D., D.; Ekeroth, J.; Kelly, J.; Hook, F.;Gold, J. , In situ Peptide-
Modified Supported Lipid Bilayers for Controlled Cell Attachment. Langmuir 2003,
19, 6730-6736.

41. Tong, Y. W.; Shoichet, M. S., Enhancing The Neuronal Interaction on
Fluoropolymer Surfaces with Mixed Peptides or Spacer Group Linkers. Biomaterials
2001, 22, 1029-1034.

42. Brinton, R. D.; Sousou, W.; Baudry, M.; Thompson, M. E.; Berger, T. W.,
The Biotic/Abiotic Interface: Achievements and Foreseeable Challenges. In Toward
Replacement Parts for the Brain, Berger, T. W. G., Dennis L., Ed. The MIT Press:
Cambridge, Massachusetts, 2005; pp 221-238.

25
Chapter 2. Parylene Surface Modification for Metal and Tissue Adhesion

2.1 Introduction
Parylene (poly-xylylene) was first discovered by Szwarc in 1947, as a
product of the pyrolysis of p-xylene.
1
The synthesis of parylene was improved by
Gorham twenty years later by substituting di-p-xylylene (para-cyclophane) for
xylene as starting material.
2
The cyclophane is quantitatively cleaved by vapor-
phase pyrolysis at 600°C, to form two α,α’-xylyl diradicals, which polymerize upon
condensation at room temperature, forming a hydrophobic conformal and pin-hole
free parylene film. Chemical structures of various parylene films are listed in Figure
2.1.
PC is known for its high resistivity,
3
and are also reported to be chemically
inert toward most common organic solvent and water up to 150°C.
4
The high
flexibility of parylene film (Young’s modulus = 4 GPa) and ability to form
conformal coating are of great interests for application of this material as a flexible
substrate for electrodes and electrode arrays
5
and in microelectromechanical system
(MEMS) applications.
6

26
Cl Cl
Cl
n
nn
Parylene N Parylene C Parylene D
Three most commons industrial parylene film:
Known parylene film structures:
n
CH
2
OH
n
CH
2
OCH
3
n
CH
2
COOCH
3
n
NH
2
n
COOCH
3
n
COOCH
3
COOCH
3
H
3
COOC
H
3
COOC
n
SO
3
CF
3
COCF
3
OCOF
CH
2
COOCF
3
x
y
x
y
x
y

Figure 2.1. Known structures of various parylene films.
7

Common applications for PC take advantage of its excellent mechanical
properties, barrier properties for water, and stability toward a wide range of organic
and inorganic materials. PC has been used as an electrical insulator in electric
27
motors and as the dielectric in capacitors.
8
PC has been used as a conformal moisture
barrier on circuit boards and semiconductor-based devices,
9, 10
as well as for the
conservation of paper and textile artifacts
11
and plant and animal specimens.
12
The
stability of parylene films toward organic solvents and biological fluids makes them
ideal for coating medical electronics, medical instruments, and prostheses,
preventing corrosion from moisture, biofluids or biogases.
4
Due to the high stability
of parylene to biological media, it is listed as a United States Pharmacopial (USP)
Class VI plastic, enabling its use in long term and/or permanent prostheses.
While parylene has many desirable characteristics, the fact that it is inert can
limit its usefulness in a range of applications. Many metals do not adhere strongly to
the bare parylene surface
13-16
and the inertness of the material might cause tissue
reactivity and encapsulation, affecting the use of the material in biomedical implants
both as a protective and as an interfacial material.
17

Chemical functionality has been incorporated into parylene films by adding
functional groups to the cyclophane, prior to chemical vapor deposition
7
The
modified parylene films were utilized in microengineering for cell patterning,
18

immobilization of biomolecules,
18
and initiation centers for polymer grafting.
19

While this is a useful approach to produce conformal coatings of functionalized PC
over the entire targeted object, deposition conditions must be adjusted and monitored
carefully for each of the dimers to ensure preservation of the functional groups
through the pyrolysis step. Moreover, the mechanical properties of the film may be
compromised, since the bulky side groups could prevent close packing of the
28
parylene oligomers compared to PN, PC, and PD. Parylene functionalization has
been achieved by plasma treatment,
20
treatment with activated water vapor,
21
and
photooxidation,
9, 22, 23
leading to improved biocompatibility,
24
cell adhesion,
25
and
stability of metallization layers toward hydrolysis.
21
All three of these methods lead
to incorporation of several different functional groups on the film surface making it
difficult to determine which of these functional groups is responsible for the
observed properties of the treated film. A chemical surface modification route for
PN film was reported via sulfonation and chloroamidomethylation for formation of
negatively- and positively-charged film surfaces.
26

As stated earlier, parylene film has properties that lend itself as an ideal
material for various biomedical devices. The range of parylene applications could be
expanded if the surface properties of the film can be tailored in such matter that 1)
the modified surface provides properties that are required for the application and 2)
the modification of the film complement and does not alter the desired properties of
the unmodified parylene film. This project is focused on selectively modifying the
parylene film by immobilization of various groups via Friedel-Crafts acylation.

Cl
Cl
Cl
Cl
R
O
AlCl
3
,
O
R Cl
n
n
n
n

Figure 2.2. Illustration of surface chemical modification on PC film via Friedel-
Crafts acylation
29
Chemical modification utilizing Friedel-Crafts acylation could provides 1)
selective control of functional groups to be immobilized to the surface; and 2)
possibilities to confined modification to film surface and preserved the properties of
the bulk of the film based on the reported study of acylation penetration depth on PN
film
26
. The obstacles of utilizing PC in biomedical devices were due to its poor
adhesion toward metal and tissues, thus modifications were geared towards
improving PC properties that addressed the above challenges.

2.2 Surface Modification
2.2.1 Experimental
Reagents. Anhydrous aluminum trichloride (AlCl
3
) was obtained from Fluka, acetyl
chloride (CH
3
COCl, 98.5+%) and gold shot (Au Premion®, 99.9999%) were
obtained from Alfa Aesar, 4-aminothiophenol (NH
2
C
6
H
4
SH, 96%) was obtained
from Acros. Sodium borohydride (NaBH
4
, 99%), N-(3-bromopropyl)-phthalimide
(C
11
H
10
NOBr, 98%), 3-maleimidobenzoic acid-N-hydroxy succinimide ester
(C
15
H
10
N
2
O
6,
97%), anhydrous hydrazine (N
2
H
4
, 98%), 2-chloropropionyl chloride
(CPC, C
3
H
4
OCl
2
) (97%), N-isopropylacrylamide (NIPAM, C
6
H
11
NO) (97%), 2,2’-
azobisisobutyronitrile (AIBN) (98%), copper(I)chloride (CuCl) (99.995+%) and
1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA,C
12
H
30
N
4
) (97%)were
purchased from Aldrich. Laminin from Engelbreth-Holm-Swarm murine sarcoma
basement membrane (laminin) was purchased from Sigma Chemical. Phosphate
buffer saline (PBS) (10×) was obtained from Gibco. Ethanol (100%) was purchased
30
from AAPER. Anhydrous N, N-dimethylformamide (DMF), hexane, and ether were
obtained from EMD. Dichloromethane (CH
2
Cl
2
) (100%) and tetrahydrofuran (THF)
(100%) were purchased from Mallinckordt All reagents were used without further
purification unless noted otherwise.

Instruments. Infrared spectroscopy was performed in both transmission and
attenuated total reflection (ATR) modes on a Perkin Elmer Fourier Transform
Infrared Spectrometer (Model Spectrum 200) equipped with MIR-acle single
reflection ATR with diamond crystal at 45° angle obtained from Pike Technologies,
Inc. Static water drop contact angle measurements were carried out using Tantec
Contact Angle Meter model CAM-F1 to asses changes in hydrophilicity of the
surface

Recrystallization of NIPAM. NIPAM (3.00 grams, 26.5 mmol) were dissolved in
50 mL of warm hexane. Once the NIPAM crystals were dissolved, the beaker was
placed in an ice bath. White solid precipitates were vacuum filtered and rinsed with
cool hexane three times and dried under air, to give a 92% yield.

31
Cl
O
Cl
N
SH
Cl
HO
n
n
n
Cl
n
Cl
n
Cl
O
n
O
N
O
O
H
2
N
2
Cl
O
n
HN
O
O
O
Cl
n
O
Cl
Cl
n
O
7b
8
HN
O
x
1
3
4
5
6
Cl
n
O
7a
a
b
c
d
e
f
g
h

Figure 2.3. Schematic of PC modification, a: CH
3
COCl, AlCl
3
, b: aminothiophenol,
c: NaBH
4
. d: bromopropyl phthalimide, NaH, e:N
2
H
4
, f: 3-maleimidobenzoic acid
N-hydroxy succinimide ester, g: CPC, AlCl
3
, h: NIPAM, CuCl, HMTETA

32
General Procedures. Unless noted otherwise, reactions were carried out by
immersion of PC film samples into the mixtures of the dissolved reactants. After
each chemical reaction, the PC film samples were isolated, rinsed with each of the
solvents used in that particular step, rinsed copiously with distilled water and lastly
rinsed with acetone and let dry at room temperature under a N
2
stream.

PC-CO(1). PC samples (each approximately 2 × 2 cm in size) were reacted with 0.1
grams of anhydrous AlCl
3
and 2 mL of acetyl chloride for 2 hours in which the
apparatus was immersed in a water bath to maintain the reaction temperature at
45°C. The PC-CO films were worked-up as described in the general procedure
section.

PC-SH (2). Functionalization of PC with thiol group was achieved by reacting PC-
CO film samples with 0.1 M of aminothiophenol in anhydrous DMF for 1 day. The
film samples were isolated and subjected to the general work-up procedure described
above.

PC-OH (3). Reduction of the acyl group on PC-CO to form PC-OH was carried out
by reacting PC-CO with 0.5 M NaBH
4
in ethanol for 16 hours at room temperature
in a closed vial. The film samples were carefully taken out of the container and
cleaned based on the general procedure described above.

33
PC-phth (4). Attachment of phthalimide group on PC surface was achieved by
reacting PC-OH with 0.16 M of NaH in anhydrous DMF, followed by 0.15 M of N-
(3-bromopropyl) phthalimide under N
2
atmosphere at room temperature for 16 hours.
The PC-phth film was isolated and subjected to the general work-up procedure
described above.

PC-NH
2
(5). PC-phth can easily be converted into PC-NH
2
by cleavage of the
phthalimide group. PC-phth was immersed in anhydrous hydrazine for 16 hours at
room temperature. The general work-up procedure was applied to the isolated film
after the reaction.

PC-ml (6). Immobilization of maleimide groups to form PC-ml was achieved by
reacting PC-NH
2
with 0.010 g of 3-maleimidobenzoic acid N-hydroxy succinimide
ester in anhydrous DMF at room temperature for 16 hours. The PC-ml film was
isolated and subjected to the general work-up procedure described above.

PC-CPC (7a and 7b). PC samples (each approximately 15 × 15 mm in size) were
reacted with 0.1 grams (0.75 mmol) of anhydrous AlCl
3
, 1mL (10 mmol) of CPC,
and 4 mL of dichloromethane for 16 hours at room temperature. The PC-CPC
samples were then rinsed with dichloromethane, dried with N
2
stream, and checked
for reaction completion by FTIR.

34
PC-pNIPAM (8). PC-CPC samples were placed in a round-bottom flask and
bubbled under N
2
(g), as were each of the reactants: 9 mg (0.09 mmol) CuCl, 1.0 g (9
mmol) NIPAM, 20 mL of ethanol. After 30 minutes of deoxygenating, 0.02 mL
(0.07 mmol) of the ligand, HMTETA, was added to the flask containing CuCl. 5 mL
of ethanol were then added to both flasks containing NIPAM and CuCl/HMTETA.
Following the addition of ethanol, both the solution were added to the PC-CPC
sample and allowed to react for 16 hours at room temperature. The samples were
then sonicated in DI H
2
O for 40 minutes and checked for reaction completion via
FTIR. The same procedures were also repeated for reaction times of 72 hours, as
well as at an elevated temperature of 65°C.

Immobilization of Laminin. 0.01% laminin (v/v) in 1x PBS was prepared from the
stock solution of laminin based on the accompanying procedure provided by the
supplier. The diluted laminin solution was reacted with PC-CPC in DMF to achieve
final concentration of 0.002%. The reaction was carried out for 16 hours at 4°C to
prevent decomposition of laminin. The sample was immersed in 1x PBS for at least
16 hours at 4°C to remove any unbound laminin and excess DMF and immediately
used for tissue adhesion experiments.

35
2.2.2 Results and Discussion on Surface Modification
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC
PC-CO

Figure 2.4. FTIR spectra monitoring acylation of PC to form PC-CO. The C=O
bands were observed at 1710 cm
-1
and 530 cm
-1

Considering the application range of functionalized PC films, it would be
desirable to have a reliable, general method of surface treatment, in which selective
functional groups can be immobilized onto the surface of the film.
Friedel-Crafts acylation of PN film has been reported previously.
26
Adapting
from the reported procedure, acylation of PC film was carried out by reacting acid
chloride, such as acetyl chloride and CPC, with the PC film in the presence of AlCl
3

36
anhydrous. The concentration of AlCl
3
was lowered in comparison to the reported
procedure and acylation was carried out at milder temperature with longer reaction
time to prevent brittleness of the PC film. Formation of the acetophenone group on
PC was observed in FTIR spectra by the appearance of the C=O stretching at 1715
cm
-1
(Figure 2.4).
The reaction of an amine with a carbonyl group leads to elimination of water
and the formation of an imine (C=N). Treatment of the acetophenone groups on the
PC-CO surface with aminothiophenol leads to quantitative conversion of the acyl
groups to imines.

37
3000 1500 1000 500
%T
wavenumber
PC-CO
PC-SH

Figure 2.5. FTIR spectra monitoring conversion of C=O (bands observed at 1710
cm
-1
and 530 cm
-1
) to C=N (band observed at 1680 cm
-1
). The conversion
immobilized thiophenyl group indicated by the presence of C-S stretching at 660 cm
-
1
.

Monitoring the reaction with FTIR (Figure 2.5), the disappearance of C=O
stretching band at 1715 cm
-1
and appearance of 1680 cm
-1
band assigned to C=N
stretching was observed. In the fingerprint region of the FTIR spectra, a C-S
stretching band at 660 cm
-1
was also observed and confirmed the conversion of the
acetophenone on the PC film to form PC-SH.
38
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-CO
PC-OH
Figure 2.6. Ketone reduction of PC-CO by NaBH
4
resulting in formation of
secondary alcohol as observed in the FTIR spectra of PC-OH (-OH bands observed
at 3300 cm
-1
)

As the goal of the project is to be able to selectively modify a specific region
of PC film surface to have a range of chemical functionalities, the PC-CO surface is
a starting point for further chemical modification. Reduction of the acetophenone
groups on PC-CO to benzyl alcohols was successfully achieved with the use of
sodium borohydride in ethanol. The conversion process was monitored with FTIR
spectroscopy, where a clear indication of 100% conversion can be observed by
disappearance of C=O stretching band at 1715 cm
-1
and appearance of a band at
39
3300 cm
-1
were consistent with the formation of –OH groups (Figure 2.6).
Unfortunately, observation at the other characteristic infrared absorptions for the
alcohol group at the range of 680-620 cm
-1
was not clearly identified due to
overlapping of the -OH bands with the IR stretching of the PC itself. The contact
angle measurement of PC-OH showed that the PC surface become significantly
more hydrophilic (contact angle dropped to 80° from 98° for PC-CO), as expected
for -OH groups at the surface.

Sample Functional group Contact angle
measurement
PC 118°
PC-CPC C(O)CH(Cl)CH3 78°
PC-CO C=O 98°
PC-OH -OH 80°
PC-phthalimide C-O-C(CH
2
)
3
-phthalimide 112°
PC-NH
2
-NH
2
78°

Table 2.1. Static water droplet contact angle measurement of various parylene C
films.
40
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-OH
PC-phth
Figure 2.7. FTIR spectra showed conversion of alcohol on PC-OH to ether as a
pathway to immobilized phthalimide group. Phthalimide characteristic bands at 1715
cm
-1
and 1680 cm
-1
were observed on PC-phth.

Conversion of the secondary alcohol into an ether group was carried out as a
method to anchor propyl-phthalimide onto the surface of PC. Reaction of sodium
hydride and the alcohol group on the surface of PC-OH produced the corresponding
sodium alkoxide, facilitating the nucleophilic attack at the N-bromopropyl
phthalimide to form the ether linkage between the film and the propyl phthalimide
(Figure 2.3). FTIR spectra of PC-phth clearly showed the characteristic phthalimide
bands at 1715 cm
-1
and 1680 cm
-1
(Figure 2.7). Again, the ether band at 1150-1070
41
cm
-1
was not observed due to the overlap with the PC modes. The surface of the
film PC-phth was expected to be more hydrophobic than PC-OH and was confirmed
with the contact angle measurement.
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-phth
PC-NH
2
Figure 2.8. FTIR spectra monitoring formation of primary amine (NH
2
stretching
band at 3350 cm
-1
) due to cleavage of the phthalimide group (characteristic bands at
1715 cm
-1
and 1680 cm
-1
) by hydrazine.

Anchoring of phthalimide groups provides an approach to form an amine on
a PC surface. The deprotection of phthalimide to give a primary amine was carried
out by immersing the film in a hydrazine solution at room temperature. The
disappearance of the characteristic phthalimide bands was observed as 100%
42
conversion was achieved. A weak band was observed at 3350 cm
-1
(Figure 2.8) and
assigned to the primary amine stretching. Contact angle measurements showed
changes of the film surface properties in which a decrease from 112° to 78° was
observed as the surface became more hydrophilic due to the presence of amine
groups on the surface of PC-NH
2
.
The maleimide group is known to react with the thiol groups of cystein and is
used as a biomolecule linker. A maleimide surface is expected to bind proteins
efficiently, making it accessible to covalently anchor a wide range of proteins to the
parylene surface. Immobilization of this group was achieved by formation of an
amide bond from the benzyl amine of PC-NH
2
surface with the phenylcarbonyl of
the maleimide molecule. The reaction was monitored with FTIR, where the presence
of maleimide group was detected on the PC-ml film with the appearance of the
amide bands at 1680 cm
-1
and a much weaker band at 1715 cm
-1
(Figure 2.9).
43
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-NH
2
PC-ml

Figure 2.9. FTIR spectra showing immobilization of maleimide group from PC-
NH
2
. Characteristic maleimide stretching at 1680 cm
-1
and 1715 cm
-1
were observed
along with C=O stretching at 530 cm
-1
.

As succesfull functionalization of PC film was achieved, trials on grafting poly-N-
isopropylacrylamide (pNIPAM) onto PC film were carried out. The polymer’s lower
critical solution temperature (LCST) at 31°C dictates the interaction of the polymer
and proteins. Below the LCST, pNIPAM chains are extended, providing a
hydrophilic surface that is non-adhering for proteins. While at higher than LCST
point, the chains collapsed and form a hydrophobic surface that strongly adhered to
proteins.
27-30
Grafting pNIPAM on solid surface can be carried out via plasma
44
processing and atom transfer radical polymerization (ATRP). For this project,
plasma treatment would not be suitable since the procedure could harshly react with
the thin film polymer, inducing chain scission that resulted in increase of brittleness
of the PC film. Prior to applying ATRP approach to graft pNIPAM onto PC film,
immobilization of the initiator on PC surface needs to be accomplished.

R
1
X
M
t
n
/Ligand
+
R
1
X M
t
1+n
/Ligand +
+ M
R
1
P
n
M
t
n
/Ligand +
X M
t
1+n
/Ligand
+
R
1
P
n
X
k
act k
deact
k
p
k
dormant

Figure 2.10. Illustration of initiation and propagation steps in ATRP carried out on
solid surface.

As in any radical polymerization, there are 3 stages in ATRP; 1) initiation, 2)
chain propagation, and 3) termination. The uniqueness of ATRP in comparison with
thermal radical polymerization was on the first two stages of the polymerization
(Figure 2.10). In the initiation step, the transition metal catalyst (M
t
n
/L), where M
t
n
is
the transition metal in the lower oxidation state n complexed with appropriate
ligand(s) L, reacts reversibly with the initiator that is immobilized on the PC surface
and generates an oxidized transition metal halide complex (X-M
t
n+1
/L) and a radical
(R*) on the PC surface. This radical propagates with addition of (M), and is rapidly
45
deactivated by reaction with the oxidized transition metal halide complex to reform
the lower oxidation state transition metal catalyst and an oligomeric X-terminated
chain (P
1
-X) as the dormant species. The sequence is repeated as the polymer chain
propagates.
25, 30-33

3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC
PC-CPC7b
PC-CPC7a
Figure 2.11. FTIR spectra showing the two products of reaction of PC with CPC in
presence of AlCl
3
. PC-CPC 7a was obtained when reaction was carried out neat
without any solvent where the C=O stretching was observed at 1715 cm
-1
. PC-CPC
7b with C=O stretching at 1693 cm
-1
was observed when the concentration of CPC
was lowered by dilution in dichloromethane.

ATRP generally utilizes secondary or tertiary alkyl halides which provides
stable radical upon activation with Cu(I) catalyst. To immobilize ATRP initiator to
46
PC film surface, we utilized Friedel-Crafts acylation reaction between the PC film
and CPC in presence of AlCl
3
. In the first acylation attempt, reaction was carried out
in neat CPC with similar conditions to those optimized for acylation of acetyl
chloride. The resulted PC film showed appearance of C=O stretching at 1715 cm
-1
in
the FTIR spectra, (Figure 2.11 PC-CPC 7a) suggesting that substitution on the
aromatic ring resulted in formation of aldehyde functional group similar in chemical
structure to 2-phenylpropionaldehyde (Figure 2.12, 7a*).
34, 35
Thus, it is
hypothesized that reaction of PC with neat CPC gave an alkylated PC film (as
illustrated in Figure 2.3 for 7a) When the acylation was carried out at lower
concentration of CPC in dichloromethane while keeping the AlCl
3
concentration the
same, the C=O stretching of the acylated PC film shift to 1693 cm
-1
.(Figure 2.11,
PC-CPC7b). This C=O stretching value is in agreement to the reported value for α-
chloropropionphenone (Figure 2.12, 7b*),
36
thus suggesting that aromatic
substitution on PC film was due to acylation of CPC. (Figure 2.3 , 7b).

O
2-phenylpropionaldehyde
7a* 7b*
O
Cl
α-chloropropionphenone

Figure 2.12. Structures of 2-phenylpropionaldehyde ( ν
C=O
= 1718 cm
-1
)
34
and α-
chloropropionphenone ( ν
C=O
= 1695 cm
-1
).
36

47

The two films (7a and 7b) were tested for pNIPAM grafting via ATRP. CuCl
was used as the system catalyst with HMTETA as the complexing ligand. The
reaction was carried out in N
2
atmosphere for 16 hours to minimize presence of
oxygen that could act as radical scavenger and quench the radical polymerization
reaction.

3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-CPC 7a
PC-CPC 7a post ATRP

Figure 2.13. FTIR spectra of PC-CPC 7a before and after ATRP reaction.

48

Post ATRP reaction, FTIR spectra of 7a showed loss of the C=O stretching at
1715 cm
-1
, and no appearance of pNIPAM band (Figure 2.13). We expected that
pNIPAM would not be grafted onto the parylene film surface since there are no
available alkyl halides that could be activated by the copper catalyst to start the
initiator step in ATRP. No further investigation was carried out to resolve the loss of
C=O functional groups from the film surface.
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-pNIPAM
PC-CPC 7b

Figure 2.14. FTIR spectra of PC-CPC 7b before and after ATRP of NIPAM.
Successful grafting of pNIPAM was concluded with the appearance of bands
associated with pNIPAM at 3400 cm
-1
, 1640 cm
-1
, 1550 cm
-1
, and 1248 cm
-1
.

49

Subjecting ATRP condition to 7b did resulted in appearance of new bands at
1640 cm
-1
, 1550 cm
-1
, and 1248 cm
-1
on the film’s FTIR spectra. These bands
correspond to the reported FTIR bands produced by thermal radical polymerization
of pNIPAM with AIBN initiator. No decreased on the intensity of these peaks was
detected post 30-minute sonication in DI H
2
O, which would remove any acrylamide
from the PC film which is due to physisorption. This concluded that the pNIPAM
bands observed on the PC film of (8) was due to the covalently bound pNIPAM as a
result of succesfull grafting via ATRP.

Reaction 16 hrs, r.t 72 hrs, r.t 16 hrs, 65ºC
Peak ratio
(1655 cm
-1
/ 1050 cm
-1
)

1.00

1.04

3.60

Table 2.2. Peak intensity ratio of pNIPAM on PC surface varying the ATRP
condition.

Extending the reaction time to 72 hours did not appear to increase the yield of
pNIPAM on the surface of the film, since the intensity ratio of peaks corresponding
to pNIPAM and PC did not change significantly (Table 2.3). Repeating the reaction
at 65ºC for 16 hours resulted in an increase of the pNIPAM on the surface as
observed from the increase of the peak intensity ratio. Figure 2.14 shows the FTIR
spectra of PC-CPC 7b, and PC-pNIPAM grafted at 65 ºC. Unfortunately no
conclusion can be drawn on whether the increase of pNIPAM on the surface was due
50
to an increase in the number of pNIPAM chains grafted on the surface, increase of
the pNIPAM chain length, or both.
Although pNIPAM was successfully grafted onto the film surface via the
living polymerization approach, we were unable to observe any increases in the peak
intensity ratio for samples that were subjected to additional cycles of ATRP.
3500 3000 1500 1000 500
%T
wavenumber (cm
-1
)
PC-CPC 7b
PC-laminin
Figure 2.15. FTIR spectra of PC-CPC7b and the film after immobilization of
laminin where the amide and other various functional groups of the protein resulted
in appearance of new broad bands in the spectra of the film.

The reactivity of the Cl substituent in 7b was utilized for immobilization of
proteins such as laminin as observed in the FTIR spectra of the film post reaction
51
with laminin (Figure 2.15). Laminin is a 900 kD trimeric glycoproteins present in
extracellular matrix and the major constituents of basement membranes. The
basement membrane is a dense matrix layer found beneath many types of cell sheets,
particularly the epithelium and endothelium (such as skin, or the linings of blood
vessels and the respiratory and digestive tracts).
37
The laminin in basement
membranes is actually produced by a wide variety of cells, and this protein appears
to be involved in an equally wide variety of activities, including cell adhesion,
migration and differentiation, embryonic development, and angiogenesis (blood
vessel formation) during wound healing.
37-39

Laminin is of interest in the biomaterials field because of the possibility that
coating a device with the protein may improve the host-device interface. Since cells
are typically in contact with a basement membrane or some other form of
extracellular matrix, a laminin coating on a biomaterial might create a more "natural"
environment, and thus encourage more "natural" or "normal" interactions between
cells and the material (as opposed to chronic inflammation, foreign body reactions,
etc.). For example, in the case of a tissue-engineered construct with a laminin
coating, a formerly non-cell adherent material could become attractive for cell
adhesion, migration and proper differentiation, thus demonstrating an improved
healing response when implanted within a host.
40-42

Stable coating of laminin via formation of covalent bonds to the PC film
allows another approach for improving tissue adhesion. This method is advantageous
for application where permanent adhesion is required, especially where cell
52
proliferation and/or cellular network formation need to occur on top of the parylene
film.

2. 3 Metal and Tissue Adhesion Toward Various Parylene C Films
As method for surface modification of PC film was successfully developed,
analysis need to be carry out to test whether the modifications could improve the
film properties toward metal and tissue adhesion.

2.3.1 Experimental Procedure
Gold deposition. Thin films of PC and PC-SH were loaded into a high vacuum
chamber (1×10
-6
Torr ) for thermal vapor-deposition of gold thin-films. The
deposition was carried out through a metal shadow mask that defines several circular
areas (approximately 4 mm diameter per circle). The gold was deposited at a rate of
2 Å/s to achieve 554 Å thick films. The thickness of the deposited gold layers was
measured with ellipsometry. The substrate holder containing the samples was cooled
with liquid nitrogen during gold deposition to prevent damage on the surface of the
PC films.

Scotch
®
tape test for adhesion. Untreated PC and PC-SH used for gold deposition
were secured to a microscope slide using vacuum tape. Scotch
®
tape was uniformly
placed on top of the deposited gold with slight pressure applied evenly on the tape to
53
ensure good adhesion between the tape and the deposited gold. A pull test was
applied by slowly pulling the Scotch
®
tape in one continuous motion.

Tissue adhesion assessment. Tissue adhesion toward parylene C film was tested for
PC, PC-CPC 7b, PC-pNIPAM prepared at room temperature and at 65ºC, and PC-
laminin. Cortical tissue of E-18 Sprague-Dawley rats were isolated and placed on
top of various PC films. Approximately 2 mL of 1× PBS solution was added into
each sample to prevent the tissue form drying during 4 hours incubation at 37°C and
5% CO
2
. After the incubation period, the film samples are removed from the Petri
dish and the tissue adhesion were tested by lifting and shaking the film samples. The
samples were also immersed in distilled water to eliminate possibilities of tissue
adhesion onto the film due to surface tension.
To test the temperature dependent bio-fouling properties of PC-pNIPAM, ice water
was added to the petri dish containing the tissue and PC-pNIPAM. The system was
allowed to cool down for 2 minutes before the tissue adhesion properties of the film
was tested as describe above.

2.3.2. Results and Discussion on Metal and Tissue Adhesion on Parylene C Films
2.3.2.1. Metal Adhesion
It has been reported that parylene often demonstrates poor adhesion toward
vapor-deposited metal electrodes.

Several methods have been used to increase
adhesion, including application of A-174 adhesion promoter,
4
plasma treatment
13

54
and the use of glow-discharge polymerized methane as a primer.
14-16
Another
common method of surface modification of parylene involves photooxidation,
however this leads to increased brittleness of the film. Thiol groups have been
known to form strong interactions with various metal electrode materials, thus
selective functionalization of PC surfaces with thiol groups is a possible approach to
enhance the metallic adhesion on the film without damaging the film surface.
Although gold is not the electrode of choice for usage as part of long term
and/or permanent prosthesis due to its toxicity, vapor depositing gold can be carried
out in relative easiness with the current instrument. Moreover, the adhesion of
platinum, the current material for prosthesis electrode, toward thiol group is
comparable of that of gold.

55

Figure 2.16. Vapor-deposited gold on PC film, (a) before and (b) after Scotch® tape
test. The diameter of each of the gold pads is 4 mm.

A test of gold adhesion toward PC and PC-SH films was conducted by
applying a Scotch
®
tape test. Observations were made by visually comparing
thermally evaporated gold pads on the two films, before and after the pull test. As
seen in Figure 2.16b and Figure 2.17, a pull test using Scotch® tape removed some
of the gold layer from the PC film.

56

Figure 2.17. Vapor-deposited gold layer on PC film after Scotch® tape test at 50×
magnification using optical microscope

Visual observation of the pull test carried out on PC-SH film did not show
any delamination of the gold layers from PC-SH (Figure 2.18). Observation using an
optical microscope at 50× magnification (Figure 2.19) showed no sign of
delamination of the gold layer at finer scales either.
57

Figure 2.18. Vapor-deposited gold on thiol-modified PC film, (a) before and (b)
after Scotch® tape test. The diameter of each of the gold pads is 4 mm.

Figure 2.19. Vapor-deposited gold layer on thiol-modified PC film after Scotch®
tape test at 50× magnification using optical microscope

58
2.3.2.2. Tissue Adhesion
Poor tissue adhesion on parylene film has been reported along with formation
of scar tissue that encapsulated the film. For application in retinal prosthesis,
parylene film surface need to be able to adhere to the inner limiting membrane of
retina, providing a stable anchoring for the MEA pads in order to effectively relay
signals to the optic nerve. As discussed earlier, succesfull selective chemical
modification of thin film parylene can be achieved via Friedel-Crafts acylation.
Grafting of pNIPAM, a temperature-dependent tissue-fouling polymer, was also
successfully carried out via ATRP method. The tissue adhesion property of these
modified parylene film were tested toward cortical and retinal tissue.
The cortical tissue samples were extracted from 18 days old embryo of
Sprague-Dawley rat that were also utilized for neuron culture studies discussed in
chapter 3. Adhesion tests were carried out for PC, PC-CPC, PC-pNIPAM grafted for
16 hours via ATRP at 65 ºC, and PC-laminin. Each film was placed onto a 3 mm
culture dish where a whole E18 Sprague-Dawley rat cortical tissue was placed
carefully on the center of the film. After the placement of the tissue on top of the
film, the samples were incubated for 4 hours at 37ºC and 5% CO
2
in presence of 1
mL of phosphate buffer saline. The adhesion strength was crudely tested by lifting
the film from the culture dish and testing the tissue adhesion strength onto the film
against its own gravitational force.
In the first culture dish, PC film was tested for tissue adhesion. The film was
carefully lifted from the dish without disturbing the cortical tissue. Upon lifting, the
59
cortical tissue seemed to weakly adhere onto the PC film. Suspecting that the
adhesion was due to surface tension, the samples were returned to the culture dish
where additional 2 mL of phosphate buffer saline was added. Immediately after
addition of liquid, the adhesion of cortical tissue on PC film was loss. The results is
expected since the cortical tissue does not have any available anchoring methods to
form a stable adhesion on to the bioinert PC film sample.
In the second culture dish, PC-CPC 7b was tested for adhesion towards
cortical tissue. Similarly the film was carefully lifted from the dish without
disturbing the cortical tissue on top of the film. The interest to test PC-CPC 7b film
adhesion properties towards tissue arose due to succesfull laminin immobilization
onto the surface of this film. During the experiment, the cortical tissue appeared to
adhered strongly to the thin film. To ensure that the adhesion was not due to surface
tension as observed earlier on PC film, the sample was place back into the culture
dish where additional phosphate buffer saline was added until complete immersion
of the sample was achieved. The film was again lifted from the culture dish, in which
the cortical tissue was still strongly adhered to the film. The result is expected since
the cortical tissue surface contains multiple amine groups that could react with the
chlorine of 2-chloropropionyl group. This is a similar mechanism as that utilized in
the immobilization of laminin in PC-laminin where the amine groups can easily react
to form covalent bonds to the film surface with the formation of HCl as the
byproduct. Therefore, although the film showed strong tissue adhesion, the approach
60
might not be suitable for biomedical devices application where trace amount of HCl
could not be tolerated.
On the third sample, PC-pNIPAM was tested for cortical tissue adhesion. For
this particular sample, tissue adhesion was tested at two temperature condition to
asses the temperature dependent tissue-fouling properties of the pNIPAM that are
grafted on the parylene C film. Immediately upon removal of the sample from the 37
ºC incubator, the sample was tested for tissue adhesion. Again, the wet sample was
slowly lifted from the culture dish without disturbing the cortical tissue where the
tissue appeared to be strongly adhered to the film surface. The sample was returned
to the culture dish where ice water was added to completely submerge the samples in
effort to lower the temperature of the samples. Once the environment cooled down to
less than the LCST of pNIPAM (31 ºC), the film was again slowly lifted from the
culture dish where it was observed that the cortical tissue easily detached from the
parylene C film. The results are in agreement with pNIPAM tissue-fouling
properties, where at temperature higher than its LCST, pNIPAM chains collapsed to
form a hydrophobic surface that are adhesive towards proteins and loss the adhesive
properties once the chains got extended at temperature below the LCST point.

61

Figure 2.20. Adhesion of cortical tissue on PC-pNIPAM film at temperature higher
than pNIPAM LCST (left), and after addition of ice water to lower the temperature
condition (right)

Strong tissue adhesion was also observed on PC-laminin, the last sample
tested for cortical tissue adhesion. As in the previous testing, the film was slowly
lifted from the culture dish to test the tissue adhesion against its own gravitational
force in which the cortical tissue showed a strong adhesion onto PC-laminin surface.
The sample was also submerged into phosphate buffer saline to ensure that the
adhesion of tissue was not due to surface tension. The film was lifted from the
culture dish once more and the cortical tissue was still adhered strongly to the film.
These observations were expected as laminin is known to strongly adhere to cortical
tissue and has been widely used to improve tissue and/or cells adhesion.
PC-pNIPAM was also tested for adhesion onto inner limiting membrane of
retina. The in-vitro experiments were carried out by collaborators at University of
Southern California Doheney Eye Institute on porcine retina tissue. After removal of
the vitreous liquid, the PC-pNIPAM film was placed on top of the retina’s inner
limiting membrane. Similarly, the experiments were carried out at 37 ºC where
62
strong tissue adhesions were observed. The temperature was slowly lowered by
addition of ice water and loss of film-tissue adhesions were observed at lower
temperature.

2.4 Summary

Parylene C (PC) is a biocompatible and biostable organic thin film polymer.
It is an ideal candidate to be used as insulator coating of biomedical devices due to
the possibilities to form conformal coating and flexible thin film, the inertness of the
film towards various chemical and biological fluids up to 150ºC, its water and
moisture barrier capabilities.
Improvement on metal and tissue adhesion on parylene C film is of great
interest for possible incorporation of this material in various long term and/or
permanent prosthesis such as retinal and cortical prostheses. The objective of this
project is to utilize the positive characteristics of PC film as a coating agent by
performing specific surface modification reactions to increase its ability to adhere to
both cellular tissue and metallic electrodes. In this chapter, surface modification of
parylene C film is discussed where various functional groups can be introduce on to
the surface via Friedel-Crafts acylation in which acyl groups are added to the arene
groups at the surface of the parylene film by electrophilic addition. The presence of
the various functional groups attached to the parylene surface was confirmed by
Fourier-Transform infra red spectroscopy. Static water drop contact angle
measurements were also used to demonstrate the changes in hydrophilicity of the PC
63
film surface. Metallic adhesion was achieved by placing a thiol functionalized group
onto the acylated surface of PC film, which was tested via vapor deposition of gold
thin-films and a Scotch® tape test. Cellular adhesion was also achieved by grafting
poly-N-isopropylacrylamide (pNIPAM) onto the acylated surface of PC film via
atom transfer radical polymerization (ATRP) or by covalently immobilizing laminin
on to the film. The tissue adhesion properties were tested by incubating E18
Sprague-Dawley rat cortical tissue on top of the films for 4 hours at 37ºC, 5% CO
2
.
PC-CPC 7b, PC-pNIPAM, and PC-laminin showed very strong adhesion properties
toward the cortical tissue and can withstand movements and gravitational force. The
temperature dependent protein-fouling property of pNIPAM was also observed on
the film where the pNIPAM was grafted onto.
Further assessment of the tissue adhesion strength is currently carried out by
collaborators at the Doheney Eye Institute. To test the stability of the adhesion
toward movement, in vivo test will be carried out by placing the film onto rabbit’s
iris.

64
2.5. Chapter 2 References

1. Szwarc, M. J., New Monomers of The Quinoid Type and Their Polymers.
Journal of Polymer Science 1951, 6, 319-329.

2. Gorham, W. F., A New General Synthetic Method for The Preparation of
Linear Poly-p-xylylenes. Journal of Polymer Science, Part A 1966, 1-4, 3027-3039.

3. Greiner, A., Poly(1,4-xylylene)s: Polymer Films by Chemical Vapour
Deposition. Trend in Polymer Science 1997, 5, 12.

4. Stark, N., Literature Review:Biological Safety of Parylene. Medical Plastic
and Biomaterials 1999.

5. Weiland, J. D.; Liu, W.; Humayun, M. S., Retina Prosthesis. Annual Review
Biomedical Engineering 2005, 7, (361-401).

6. Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster,
J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.;
Mastrangelo, C. H.; Burke, D. T., An Integrated Nanoliter DNA
Analysis Device. Science 1998, 282, (484-487).

7. Lahann, J.; Langer, R., Novel Poly(p-xylylenes): Thin Films with Tailored
Chemical and Optical Properties. Macromolecules 2002, 35, 4380-4386.

8. Beach, W.; Lee, C.; Bassett, D., Encyclopedia of Polymer Science and
Engineering 1985, 17, 990.

9. Fortin, J. B.; Lu, T. M., Ultraviolet Radiation Induced Degradation of Poly-
para-xylylene (Parylene) Thin Films. Thin Solid Films 2001, 397, 223-228.

10. Lee, B. Q.; Maurer, R. H.; Nhan, E.; Lew, A., Int. J. Microcircuits Electron.
Packag. 1999, 22, 104.

11. Carter, H. A., The Chemistry of Paper Preservation Part 3. The Strengthening
of Paper. Journal of Chemical Education 1996, 73, (12), 1160-1162.

12. Grattan, D. W., Parylene at the Canadian Conservation Institute. Canadian
Chemical News 1989, 41, (9), 25-26.

13. Gadre, K. S.; Alford, T. L., Thermal Stability and Adhesion Improvement of
Ag Deposited on Pa-n by Oxygen Plasma Treatment. J. Vac. Sci. Technol. B 2000,
18, (6), 2814-2819.
65
14. Sadhir, R. K.; James, W. J.; Yasuda, H. K.; Sharma, A. K.; Nichols, M. F.;
Hahn, A. W., The Adhesion of Glow-Discharge Polymers, Silastic and Parylene to
Implantable Platinum Electrodes: Results of Tensile Pull Test After Exposure to
Isotonic Sodium Chloride. Biomaterials 1981, 2, (4), 239-243.

15. Yamagishi, F. G., Investigation of Plasma Polymerized Films as Primers for
Parylene C Coatings on Neural Prosthesis Materials. Thin Solid Films 1991, 202, 39-
50.

16. Nichols, M. F.; Hahn, A. W.; James, W. J.; Sharma, A. K.; Yasuda, H. K.,
Cyclic Voltammetry for The Study of Polymer Film Adhesion to Platinum
Electrodes. Biomaterials 1981, 2, (3), 161-165.

17. Hahn, A. W.; York, D. H.; Nichols, M. F.; Amromin, G. C.; Yasuda, H. K.,
Biocompatibility of Glow-Discharge Polymerized Films and Vacuum-Deposited
Parylene. Journal of Applied Polymer Science:Applied Polymer Symposium 1984,
38, 55-64.

18. Lahann, J.; Balcells, M.; Rodon, T.; Lee, J.; Choi, I. S.; Jensen, K. F.; Langer,
R., Reactive Polymer Coatings: A Platform for Patterning Proteins and Mammalian
Cells onto a Broad Range of Materials. Langmuir 2002, 18, 3632-3638.

19. Lahann, J.; Klee, D.; Pluester, W.; Hoecker, H., Bioactive Immobilization of
r-hirudin on CVD-coated Metallic Implant Devices. Biomaterials 2001, 22, 817-826.

20. Pruden, K. G.; Beaudoin, S. In Ammonia Plasma Treatment of Parylene-C,
Annual Meeting Archive - American Institute of Chemical Engineers,, Indianapolis,
IN, United States,, 2002; American Institute of Chemical Engineers,: Indianapolis,
IN, United States,, 2002; pp 857-863.

21. Martini, D.; Shepherd, K.; Sutcliffe, R.; Kelber, J.; Edwards, H.; Martin, R.
S., Modification of Parylene AF-4 Surfaces Using Activated Water Vapor. Applied
Surface Science 1999, 141, 89-100.

22. Bera, M.; Rivaton, A.; Gandon, C.; Gardette, J. L., Photooxidation of
Poly(para-xylylene). European Polymer Journal 2000, 36, 1753-1764.

23. Pruden, K. G.; Sinclair, K.; Beaudoin, S., Characterization of Parylene-N and
Parylene-C Photooxidation. Journal of Polymer Science: Part A: Polymer Chemistry
2003, 41, 1486-1496.

24. Lahann, J.; Klee, D.; Thelen, H.; Bienert, H.; Vorwerk, D.; Ho¨cker, H.,
Improvement of Haemocompatibility of Metallic Stents by Polymer Coating.
Journal of Material Science: Materials in medicine 1999, 10, (7), 443-448.
66
25. Savariar, E. N.; Thayumanavan, S., Controlled Polymerization of N-
Isopropylacrylamide with an Activated Methyacrylic Ester. Journal of Polymer
Science: Part A: Polymer Chemistry 2004, 42, 6340-6345.

26. Herrera-Alonso, M.; McCarthy, T. J., Chemical Surface Modification of
Poly(p-xylylene) Thin Films. Langmuir 2004, 20, 9184.

27. Alarcon, C. d. l. H.; Tamer Farhan, V. L. O.; Huck, W. T. S.; Alexander, C.,
Bioadhesion at Micro-Patterned Stimuli-Responsive Polymer Brushes. Journal of
Materials Chemistry 2005, 15, 2089-2094.

28. Kizhakkedathu, J. N.; Norris-Jones, R.; Brooks, D. E., Synthesis of Well-
Defined Environmentally Responsive Polymer Brushes by Aqueous ATRP.
Macromolecules 2004, 37, 734-743.

29. Xia, Y.; Yin, X.; Burke, N. A. D.; Stover, H. D. H., Thermal Response of
Narrow-Disperse Poly(N-Isopropylacrylamide) Prepared by Atom Transfer Radical
Polymerization. Macromolecules 2005, 38, 5937-5943.

30. Kizhakkedathu, J. N.; Kumar, K. R.; Goodman, D.; Brooks, D., Synthesis
and Characterization of Well-Defined Hydrophilic Block Copolymer Brushes by
Aqueous ATRP. Polymer 2004, 45, 7471-7489.

31. Matyjaszewski, K.; Xia, J., Atom Transfer Radical Polymerization. Chem.
Review 2001, 101, (2921-2990).

32. Wang, J.-S.; Matyjaszewski, K., “Living”/Controlled Radial Polymerization.
Transition-Metal-Catalyzed Atom Transfer Radical Polymerization in the Presence
of a Conventional Radical Initiator. Macromolecules 1995, 28, 7572-7573.

33. Bontempo, D.; Tirelli, N.; Masci, G.; Crescenzi, V.; Hubbell, J. A., Thick
Coating and Functionalization of Organic Surfaces via ATRP in Water. Macromol.
Rapid Commun. 2002, 23, 417-422.

34. Robinson, M. W. C.; Pillinger, K. S.; Graham, A. E., Highly Efficient
Meinwald Rearrangement Reactions of Epoxides Catalyzed by Copper
Tetrafluoroborate. Tetrahedron Letters 2006, 47, (5919-5921).

35. Meyers, A. I.; Walkup, R. D., Diastereofacial Selectivity via Aldol Reactions
Using Ethyl Dithioacetate and Ethyl Dithiopropionate Enolates. Tetrahedron 1985,
41, (22), 5089-5106.

67
36. Bergmark, W. R.; Barnes, C.; Clark, J.; Paparian, S.; Marynowski, S.,
Photoenolization with α-Chloro Substituents. Journal of Organic Chemistry 1985,
50, 5612-5615.

37. Bosman, F.; Stamenkovic, I., Functional Structure and Composition of The
Extracellular Matrix. J. Pathol 2003, 200, 423-428.

38. Mercurio AM, Laminin Receptors: Achieving Specificity Through
Cooperation. Trends Cell Biol 1995, 5, 419-423.

39. Li, J.; Zhang, Y.-P.; Kirsner, R. S., Angiogenesis in Wound Repair:
Angiogenic Growth Factors and The Extracellular Matrix. Microsc Res Tech 2003,
60, 107-114.

40. Pan, Y. V.; Wesley, R. A.; Luginbuhl, R.; Denton, D. D.; Ratner, B. D.,
Plasma Polymerized N-Isopropylacrylamide: Synthesis and Characterization of a
Smart Thermally Responsive Coating. Biomacromolecules 2001, 2, (32-36).

41. Smyth, J.; Walker, M., Surface Precoating in The 1980s: A First Taste of
Cell-Matrix Interactions. In Tissue Engineering of Vascular Prosthetic Grafts, Zilla,
P.; Greisler, H., Eds. R.G. Landes Company: Austin, 1999; pp 69-77.

42. Williams, D., Bioinertness: An Outdated Principle. In Tissue Engineering of
Vascular Prosthetic Grafts, Zilla, P.; Greisler, H., Eds. R.G. Landes Company:
Austin, 1999; pp 459-462.

68
Chapter 3. Surface Modification for Cortical Prosthesis

3.1 Introduction
The cortical prosthesis under development focused on the activities of
hippocampus. This region of the brain is responsible in the formation of new long-
term memories. In this prosthesis, a penetrable multi-electrode array (MEA) will be
utilized to record and transmit neuron activities in the surrounding areas of the
probe.
1, 2
MEA are microfabricated electrodes that can simultaneously monitor the
extracellular activity at multiple sites of a tissue sample. The MEA were designed
not only to record signals but also re-transmit the signal to the targeted tissue areas.
The bi-directional signaling between the MEA and the surrounding tissue leads to
the possibilities to apply the prosthesis as a replacement of a damage region in the
hippocampus.
3
Since the MEA will act as a bridge between one neural-network to
the other, the biotic-abiotic interface becomes a very important issue to be
addressed.
3

A close contact between the MEA and the targeted tissue is desired. Neurons
adhering directly on or in close proximity to the electrode pads could improve the
recording and transmission of electrical stimulation between the MEA and the
neurons. Improvement of signal recording and transmission not only depended on
the distance between the tissue and the MEA. The prosthetic device will utilize
algorithm developed to mimic the neurons signal transmission pathway. Therefore,
69
improvement of biotic-abiotic interface should also focus on selective spatial neuron
adhesion onto the electrode pads.
HN
O
O
n
poly-D-lysine
(PDL)
H
N
N
NH
2
x
y
polyethyleneimine
(PEI)
NH
2

Figure 3.1. Chemical structure of poly-D-lysine (PDL) and polyethyleneimine (PEI),
two common polymeric coatings for promotion of cell adhesion.

The most common methods for increasing/promoting cell attachment is by
coating the target substrates with polymers such as poly-D-lysine (PDL)
4-6
and
polyethyleneimine (PEI)
6, 7
which chemical structures are illustrated in Figure 3.1.
The highly positive charge polymer coating provides a strong anchoring site for the
cell through electrostatic interaction with the negatively charge cell surface.
Unfortunately since there is neglible charge difference between various cells, these
polymer coatings can not provide selective cell adhesion property.
Another common approach is by coating the target substrates proteins such as
laminin and fibronectin.
8-16
Various cell receptors on the cell surfaces can interact
with these large proteins and act as anchoring sites for the cells to adhere. While the
70
protein coating could provide more cell selectivity compare to the polymer coating,
the stability and degradation of the protein could be an issue.
The interaction of cell receptor and extracellular matrix was found to be
related to the recognition of the cell receptor towards amino acid sequence located on
the binding site of the extracellular matrix. Interaction of peptides containing the
specific amino acid sequence, which will be referred as cell adhesion molecules
(CAMs), were found to be selective towards various cell receptors.
5, 17-28
Therefore,
covalently attaching CAM onto substrate surface could provide the cell adhesion
specificity required in this project. These peptides are less prone to degradation and
could be covalently anchored to the target substrates in comparison to the
extracellular matrix proteins.

Figure 3.2. Illustration of selective neuron adhesion promoted by CAM immobilized
on electrode surface.

Neuron cell
Astroglia cell
Tissue
Anchored neuron
CAM
Electrode
71
In this chapter, selective surface modification of indium tin oxide (ITO) for
promotion of neuron adhesion via immobilization of neuron CAM will be discussed.
ITO is a transparent electrode commonly utilized in fabrication of MEA. First,
selective surface modification on ITO surface via phosphonic acid anchoring group
was tested. Investigations were carried out to study the effect of single CAM versus
oligo CAM bound covalently on the electrode surface. Preliminary tests study the
correlation between CAMs surface density and cell adhesion will also be discussed
as well as the effort to assess cell adhesion strength with microfluidic flow-system.

3.2 Surface Modification
3.2.1 Experimental Procedure
Reagents. 3-aminopropyl phosphonic acid (C
3
H
9
PO
3
N), 1-ethyl-3-(3-
dimethylaminopropyl)-carbodiimide (EDC, C
8
H
17
N
3
), α-cyano-4-hydroxycinnamic
acid (CHCA,C
10
H
7
NO
3
) and triethylamine were obtained from Aldrich. Peptide
with 5-amino acid sequence of isoleucine-lysine-valine-alanin-valine (IKVAV) was
obtained from Genemed Synthesis, Inc at 98% purity. The amine protected of
IKVAV was obtained from GenScript at 95% purity. Hoescht 33342 (C
28
H
31
N
5
O,
97%) was obtained from Fluka. Poly-D-lysine was obtained from Sigma. 4-
ethylmorpholine was obtained from Lancaster. Phosphate Buffer Saline (PBS), was
obtained from Gibco. Anhydrous dimethylformamide (DMF) was obtained from
EMD. Acetone was obtained from JT Baker. Trichloroethylene, acetic acid glacial,
sulfuric acid, and nitric acid were obtained from Mallinckrodt. Ethanol (200 proof)
72
were obtained from AAPER. All reagents are used without further purification
unless noted otherwise.

Sample substrates. Indium tin oxide (ITO) coated glass sheet obtained from
Universal Display Corporation were used as sample substrates in this study.

Surface cleaning procedure. Glass coated ITO approximately 2 ×2 cm
2
in size were
cleaned by sonication in detergent water for 10 minutes followed by copious rinsing
with acetone. The pieces were then immersed into boiling solvents of
trichloroethylene, acetone and ethanol in these following orders for 3 minutes each.
The samples were dried with N
2(g)
stream and underwent UV-Ozone treatment for 10
minutes.

Aminophase substrate. Aminophase ITO samples were prepared by submersion of
cleaned ITO coated glass samples into 0.1 M aqueous solution of 3-aminopropyl
phosphonic acid for 48 hours at room temperature in a closed container. The ITO
samples were rinsed with DI H
2
O and dried with N
2(g)
stream prior to peptide
immobilization.

Peptide immobilization via formation of amide bond. A mixture of peptide (0.1
mM), EDC (0.1 M) and 4-ethylmorpholine with 25:25:1 volume ratio was prepared
with anhydrous DMF as solvent. Into this mixture, amino phase samples were
73
immersed and let to react with the peptide for 24 hours in a closed container at room
temperature. Unreacted primary amine could be passivated by immersion in 0.1 M of
acetic anhydride for 2 hours, followed by copious rinsing of the samples in sterile
water to remove the adsorbed peptides from the sample surfaces. For samples treated
with amine-protected IKVAV, deprotection of the tBOC groups was carried out by
immersion of the appropriate samples into 0.1 mM acetic acid in 70% ethanol, for
15-30 minutes.

Synthesis of hydroquinone-propylphosphonic acid. Hydroquinone propyl
phosphonic acid was synthesized following reported procedure by Curreli et. al.
29

Synthesis of n-propylphthalimide pentamethylcylcopentadiene (Cp*-
phthalimide). Under N
2
atmosphere, bromopropyl phthalimide (1.398 grams) was
dissolved in 40 mL of anhydrous THF. Slight excess of sodium
pentamethylcyclopentadiene (10 mL, 0.5 M) was added dropwise into the mixture
under constant stirring at room temperature. Following addition, the mixture was
refluxed for 16 hours under N2 Reaction was quenched by addition of methanol
,followed by solvent removal under vacuum. The salt byproduct was separated via
ether-water extraction. Unreacted pentamethylcyclopentadiene must be removed
from the organic layer via column chromatography with hexane as eluent, prior to
elution of product with hexane/dichloromethane (1:2 ratio). After removal of the
solvent in vacuum, the yellowish powder of the product was collected at 65% yield
74
(the reaction has not been optimized).
1
H: 7.82ppm(2H, m), 7.73ppm(2H,m),
3.54ppm(2H, t), 1.73ppm(6H,s), 1.64ppm(6H, s), 1.43ppm(2H, m), 0.979ppm(1H,
m), 0.860ppm(1H, s).
13
C: 139.5ppm (2), 133.49ppm(2), 133.78ppm(2), 123.27
ppm(2), 123.01ppm(2), 55.25ppm(1), 38.21ppm(1), 32.1ppm(1), 22.47ppm(1),
21.86ppm(1), 13.99ppm(2), 10.85ppm(2). M/z = 323 (theoretical:323.3)

Synthesis of aminopropyl pentamethylcyclopentadiene (Cp*-NH
2
). Cp*-
phthalimide (0.280 grams) was reacted with slight excess of anhydrous hydrazine
(0.3 mL) in dry dichloromethane at room temperature for 24 hours under N2
atmosphere. The phthalazine-1,4-dione byproduct was removed via filtration, while
excess hydrazine and solvent were removed under vacuum. A yellowish clear oil of
aminopropyl pentamethylcyclopentadiene was obtained at 90.8% yield.
1
H:
3.54ppm (2H, broad), 2.67ppm(2H,t), 1.77ppm(6H, s), 1.68ppm(6H, s),
1.454ppm(2H, m), 1.273ppm(2H,m), 0.887ppm(3H,s).
13
C: 140.06 (2), 134.00 (2),
55.47(1), 52.26(1), 33.14 (1), 29.33(1), 22.04(1), 11.02(2), 9.67(2). M/z = 193
(theoretical:193.33)

Synthesis of peptide-propyl pentamethylcyclopentadiene (Cp*-IKVAV).
Aminopropyl pentamethylcyclopentadiene (0.024 mmoles) was reacted with amine
protected IKVAV (0.0180 grams, 0.0246mmoles) in dry DCM in presence of excess
dicyclohexyl carbodiimide (0.032 mmoles) at room temperature for 24 hours under
N2. The reaction was quenched by addition of methanol (5 mL). After solvent
75
removal by vacuum, byproduct and excess peptide was removed by extraction with
water in dichloromethane. Peptide-propylpentamethyl cyclopentadiene was collected
from the organic layer and dried under vacuum to give a 43.75% yield.
1
H : 1.73-
1.52ppm (26H, broad), 1.44-1.41ppm(20H), 1.265ppm(10H), 1.178-0.987ppm(8H),
0.920ppm (15H,m), 0.069ppm (2H, s), M/z= 876 (theoretical: 876.18).

Immobilized peptide to surface via Dies-Alder. Hydroquinone groups on ITO
surface was electrochemically oxidized to the quinone states in pH 7.4 buffer with Pt
wire as the counter electrode and Ag/AgCl as the reference electrode. The density of
the quinone groups was determined via chronoamperometry measurement. The ITO
samples were then rinsed with DI H2O and reacted with 0.01 mM of peptide-propyl
pentamethylcyclopentadiene via Dies-Alder reaction in dry DMF for 24 hours. The
density of unreacted quinone was again measured via chronoamperometry post Dies-
Alder reaction.

Sample preparation prior to neuron culture. The samples were sterilized by
immersion into 70% ethanol for 1 hour, followed by copious rinsing with 1x PBS to
reconditioned the samples to cell culture medium environment.

Neuron cell culture. Pregnant Sprague-Dawley rats were sacrificed by CO
2

inhalation after 18 days gestation. Following decapitation, the embryonic brains
were removed from the skull and placed in cold calcium and magnesium-free Hank’s
76
salt solution (Sigma) buffered with HEPES (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid) pH 7.3. Hippocampi were then dissected under a
stereo microscope and kept in cold HSS. Meningeal membranes were removed
during three HSS rinses, and then the hippocampi were first dissociated by a 5 min
incubation of 0.02% trypsin at 37 °C followed by mechanical dissociation by
repeated pipetting through a series of fire-polished Pasteur pipettes with decreasing
tip diameters. After centrifugation for removal of membranes and cellular debris,
dissociated cells were then passed through a 40 µm cell strainer to remove large
clumps. The cells were mixed in Neurobasal medium (NBM, InVitrogen, Carlsbad,
USA) supplemented with 0.5 mM glutamine, B-27 supplement (InVitrogen),
Penicillin-Streptavidin, and 25 µM glutamate. Cells were then plated at a density of
~2,000 cells/mm
2
per culture well each containing a substrate sample. Cultures were
kept in a humidified incubator at 37 °C in 5% CO
2
and medium was replaced
biweekly with NBM without additional glutamate. Phase contrast photomicrographs
were taken before feedings on day 3 and day 5 of culture period. One set of sample
were taken out after 6 hours of culture, in which the medium was replaced with
Hoescht 33342 (0.001 mg/mL in PBS) followed by incubation for 15 minutes at
culture condition. The stained sample set was rinse three times with PBS and used
for analysis of neuron adhesion on various substrates.

Ethics Section. The procedures performed on live animals will be the sacrifice of the
pregnant rats with CO2 and the decapitation of the fetal rats. The animals will not be
77
subjected to any discomfort, pain or distress. All of the experiments are conducted
on dissociated cells. The University of Southern California is a leader in the ethical
and humane use of animals for research. USC has been accredited under the
American Association for Accreditation for Laboratory Animal Care since 1966. An
institutional Animal Care and Use Committee review all applications to ensure
ethical and humane treatment of animals. This body follows the NIH Guide for the
Care and Use of Laboratory Animals prepared by the Institute of Animal Resources,
National Research Council, all applicable government regulations, and USC policies
governing the use and care of laboratory animals. All animals will be housed in
facilities maintained by the USC Vivaria.

Live neuron cell staining. Hoescht 33342 staining of the cell nuclei were
performed after 6 hours of seeding. The samples were immersed into the staining
solution (1 µg/mL Hoescht in 1x PBS) for 15 minutes at 37°C in the dark, followed
by three successive rinsing with 1x PBS.

Instrument. Ultra-Violet Ozone Cleaning Systems (UVOCS Inc.) was used in
cleaning sample surfaces prior to modification. Static water drop contact angle
measurements were carried out using Tantec Contact Angle Meter model CAM-F1
to assess changes in hydrophilicity of the surface. Optical microscope images were
taken from Nikon Eclipse ME600 equipped with fluorescence system and Olympus
Magnafire digital camera model S99800 and inverted microscope (DMIRB, Leica,
78
Germany) equipped with digital camera (SPOT RT, Diagnostic Instrument, MI,
USA). Acquisition of time-lapse image recording was carried out with ImagePro
(Media Cybernatics). Surface coverage was measured utilizing intensity threshold
function in Sigmascan Pro (SPSS, Inc.)

3.2.2 Results and Discussion on ITO Surface Modification
3.2.2.1 Selective Modification on ITO Surface
Formation of self-assembled monolayer of phosphonic acid onto ITO surface
has been reported previously.
29, 30
The selectivity of phosphonic acid toward ITO
incomparison with glass was tested by reacting the two substrates with alkyl
phosphonic acids with varied alkyl chain length. A sessile water drop contact angle
measurement was utilized to assess the phosphonic acid anchor groups toward ITO
surface compared to glass surface. Increasing the alkyl chain length of the alkyl
phosphonic acid should create a more hydrophobic monolayer on the ITO surface.
As expected, the hydrophobicity of ITO surface increased as the length of alkyl
chain increase due to succesfull formation of monolayer of alkyl phosphonic acid on
the surface.
The specificity of phosphonic acid group to ITO surface versus glass surface
was also tested. Based on contact angle measurements, hydrophobicity of glass
surface after treatment with alkyl phosphonic acid did not show any changes, an
indication that no anchoring of phosphonic acid onto glass surface occurred (Table
79
3.1). This result allows possibility in specific substrates modification by careful
selection of anchoring group.
Sample

Anchor group

Number of carbons
in alkyl chain
Contact angle

ITO n/a n/a 30
PO
3
2-
1 20
2 52
6 90
8 92
Glass n/a n/a 52
PO
3
2-
8 54

Table 3.1. Contact angle measurement data. Increase in contact angle value was
observed as the length of alkyl chain increases.

3.2.2.2 Immobilization of IKVAV via Peptide Coupling

P
NH
2
O
O
O
P
NH
2
O
O
O
P
NH
O
O
O
P
NH
O
O
O
CAM
O
CAM
O
PO
3
H
2
NH
2

Figure 3.3. Common illustration of immobilization of CAM onto aminophase ITO
surface via EDC coupling, implying formation of single peptide layer on the surface

ITO
Glass
80
The most common method for peptide immobilization utilizes formation of
amide bond between the peptide and targeted substrate.
17, 19, 22, 27, 31
For ITO samples,
formation of aminophase surface was carried out utilizing 3-aminopropyl phosphonic
acid where the phosphonic acid group will act as the anchoring group. The amide
bond linkage between the primary amine of the aminophase substrate and the
carboxylic acid of the peptide C-terminus was facilitated in presence of carbodiimide
(Figure 3.3). Reaction must be carefully carried out in a minimum moisture
condition to prevent quenching of carbodiimide prior to coupling of the peptide and
the aminophase substrates.

H
2
N
CH C
CH HN
O
CH
3
H
2
C
H
3
C
CH
C
CH
2
NH
O
H
2
C
CH
2
CH
2
H
2
N
CH C
CH HN
O
CH
3
H
3
CCH
C
CH
3
NH
OCH C
CH
OH
O
CH
3
H
3
C

Figure 3.4. Chemical structure of the five amino acid sequence of IKVAV, a cell
adhesion molecule that promote neuron adhesion and growth. Functional groups in
red indicate the primary amine of the N-terminus and the carboxylic acid of the C-
terminus. Primary amine functional group in blue indicates the free amine of lysine
that could also participate in the peptide coupling reaction.

81
In reported cell culture studies regarding cell adhesion and proliferation with
CAMs immobilized in presence of carbodiimide coupling agent, it is always implied
that adhesion and proliferation phenomena were the result of immobilization of
single CAM per amine group on the surface as illustrated by Figure 3.4. In this
project, the peptide of interest is the five amino acid sequence of IKVAV (Figure
3.4). This synthetic peptide is derived from laminin and has been reported to promote
neurons adhesion and growth.
5, 21, 23, 26, 32
To promote neuron adhesion and growth
on ITO surface, IKVAV was immobilized onto aminophase ITO in presence of
peptide coupling agent EDC. .

P
NH
2
O
O
O
P
NH
O
O
O
CAM
O
P
NH
O
O
O
CAM
O
CAM
CAM
CAM CAM
n
n
P
NH
O
O
O
CAM
O
CAM
n
P
NH
2
O
O
O
CAM CAM CAM
n
1)
2)

Figure 3.5. Illustration of immobilization of oligo-CAM onto aminophase ITO
surface via EDC coupling, where 1) oligo-CAM form in solution prior to
immobilization onto ITO surface, or 2) oligo-CAM growth on the ITO surface.

ITO
Glass
82
Carbodiimide coupling agent such as EDC and/or DCC
(dicyclohexanecarbodiimide), did not differentiate carboxylic acid of the peptide C-
terminus or one from the amino acid side group, nor does it differentiates primary
amine on the surface, of the N-terminus, or one from amino acid side groups.
Therefore, immobilization of oligo-CAM instead of a single CAM layer could occur.
The oligo-CAM could be formed 1) prior to coupling with aminophase surface due
to higher reaction rate in solution, or 2) post coupling of the first CAM onto the
aminophase surface in which primary amine on the immobilized peptide can further
react with the carboxylic acid group of another peptide (Figure 3.5).
500 505 510 515 520 525 530 535 540
513 (heptamer)
520 (dimer)
514 (mixture of tetramer,pentamer, hexamer)
529 (monomer)
Intensity
m/z

Figure 3.6. MALDI-MS spectra of reaction mixture used in IKVAV immobilization
via peptide linkage onto aminophase ITO, where presence of monomer and
oligomers of IKVAV were observed
83
Confirmation on the formation of oligo-IKVAV was obtained via analysis of
the peptide reaction mixture past immobilization via peptide bond reaction using
matrix assisted laser desorption ionization mass spectroscopy (MALDI-MS) with
CHCA as the sample matrix. Since each repeating unit of IKVAV on the
oligopeptide will increase the total mass of the molecule along with an increase of +1
in the molecule total charge, the analyzed mass per charge (m/z) of the oligo-peptide
will decrease as the number of peptide repeating unit increases. Presence of a
mixture consisting of monomer and oligomer of IKVAV in the solution was
observed in the MALDI-MS spectra (Figure 3.6).

I KV A V
NH
2 NH
2
NH
I
K
V
A
V
COOH
O
HN
I
K
V
A
V
O
NH
I
K
V
A
V
NH
2
H
2
N
O
HN
I
K
V
A
V
H
2
N
NH
2
O

Figure 3.7. Strategy for immoblilization of mono-IKVAV on aminophase ITO
surface by protecting all amine groups on IKVAV with t-BOC groups. The
protecting groups were removed once the peptides were covalently bound to the ITO
surface, providing the primary amines groups on the N-terminus and lysine group.

It has been reported that mutation on the amino acid sequence will deactivate
the IKVAV affect on neuron adhesion and proliferation. Specifically, mutation on
t-BOC group
84
the lysine position was crucial towards cell adhesion.
21
Thus, formation of oligo-
IKVAV due to peptide coupling between the C-terminus and the lysine pendant
group would alter the IKVAV structure and potentially deactivate the CAMs
function in promoting neuron adhesion. Investigation to assess whether presence of
oligo-IKVAV on the surface would affect cell adhesion and growth in comparison
with a mono-IKVAV was carried out. To create a mono-IKVAV layer and prevent
formation of oligo-IKVAV, all primary amine groups of IKVAV were protected
with tert-butoxycarbonyl (tBOC) group. When peptide coupling reaction was carried
out, formation of peptide bond can only occurred between the C-terminus of the
peptide and the amine group on the aminophase ITO surface, Therefore, no
oligomerization of the peptide could take place in the solution mixture or on the ITO
surface during the peptide coupling reaction. The protecting groups were cleaved off
prior to sterilization to re-form the original primary amine groups. The neuron
culture results will be discussed in subchapter 3.3.1.

3.2.2.3 Peptide Immobilization via Dies-Alder Reaction
Another disadvantage of immobilization via carbodiimide coupling agent in
is the control of peptide surface density. A method to modified gold surface with
hydroquinone monolayer which could be electrochemically oxidized to its quinone
states on gold has been reported.
33-35
The reversible redox states was utilized as
electrochemical tuning process for cell adhesion by Dies-Alder reaction between the
85
quinone groups and cyclopentadiene-RGDS (Cp-RGDS) for areas where fibroblast
cells adhesion are desired.
34, 35

A succesfull adaptation of the electrochemical tuning on ITO surface with
hydroquinone groups has been reported by Curelli et al,
29
by substituting the thiol
anchor group with phosphonic acid. This approach was implemented to investigate
the effect of IKVAV surface density towards neuron adhesion and proliferation. To
prevent dimerization of the cyclopentadiene-peptide during storage,
pentamethylcyclopentadiene, a bulkier derivative, was used (Figure 3.8).

PO
3
2-
HO
OH
P
HO
OH
O
O
P
O
O
O
O
P
O
O
O
Peptide
Cp*-peptide
H
2
O, r.t, 16 hours
O
O
O
O
2e
-
-2e
-

Figure 3.8. Illustrated schematic for peptide immobilization on conducting substrate.
86
Synthesis of pentamethyl cyclopentadiene (Cp*) coupled to the C-terminus of
IKVAV was achieved in 3 steps synthesis as illustrated in Figure 3.9.
H
2
N
Na
+
Br
N
O
O
reflux
THF, N
2
, 3 days
N
O
O
NH
2
-NH
2
CH
2
Cl
2
, r.t, 24 hrs
DCC
CH
2
Cl
2
, N
2
, r.t. 24 hrs
N
H
HN
O
N
H
O
NH
NH
O
NH
O
H
N
O
O
O
O
O
+
Cp*-phthalimide
Cp*-NH
2
Cp*-IKVAV

Figure 3.9. Synthetic scheme of Cp*-IKVAV

It is also demonstrated by Curreli et al, that the density of hydroquinone groups on
the surface can be measured via chronoamperometry.
29
Since the amount of charge
involve in the reaction is dependent to the surface density of the quinone groups on
the surface, it is possible to utilize this approach to measure the peptide surface
density by calculating the difference between the density of quinone groups prior and
post reaction with Cp*-IKVAV.
Chronoamperometric measurements were carried out on a fixed area of ITO
as the working electrode, Ag/AgCl reference electrode, and a Pt wire as a counter
electrode in a pH 7.4 buffer solution. To obtain a fixed surface area, for each sample,
cell culture chambers (Grace Bio-Labs) was placed on top of the modified ITO
87
surface. The chambers will seal the electrolyte such that a fixed 0.4 cm
2
circular area
of ITO surface will be measured during chronoamperometry experiment. The initial
potential was set to -300 mV and held to this value for 6 seconds to ensure complete
reduction of any quinone groups on the ITO surface. The potential was then raised to
400mV to oxidize the hydroquinone groups to the quinone state.
Chronoamperometric measurement provided the total charge involved in the
oxidation process as the integration of the current versus time. Complete oxidation of
the hydroquinone on the ITO surface was achieved after 16 seconds, where the
measured amount of charge involved in the process reached a constant value as seen
in Figure 3.10

0 6 8 1012 1416
0
2
4
6
8
10
12
14
16
Q (μC)
time (s)
prior to reaction with Cp*-IKVAV
post reaction with Cp*-IKVAV

Figure 3.10. A representative of chronoamperometry spectra showing decreased in
the quantity of quinone groups on ITO surface that are able to be electrochemically
reduced post reaction with Cp*-IKVAV.

88
The surface density of quinone on ITO surface was calculated according to
the formula :
F
Q
nA
2
=
where n is the number of mol of quinone on the surface, A is the measured area of
the working electrode (0.4 cm
2
), Q is the amount of charge involve in the
measurement, and F is the faraday constant.

3.3 Neuron Cell Culture Results and Discussion
3.3.1 Neuron Culture Results: Comparing mono-IKVAV Layer vs. oligo-IKVAV
Layer
3.3.1.1 Neuron Adhesion on mono IKVAV Layer Surface.
It is stated earlier that during coupling of IKVAV onto the aminophase ITO
facilitated by carbodiimide coupling agent, oligo-IKVAV was formed in the solution
mixture, a good indication that the ITO surface was decorated with oligo-IKVAV
instead of a layer of single IKVAV (mono-IKVAV layer). To investigate whether
immobilization of mono-peptide versus oligo-peptide have an affect on cell adhesion
and proliferation, ITO surface were modified with amine protected IKVAV. Since all
the amine groups on the peptide were protected with tBOC group, the primary amine
of aminophase ITO was the only available amine for formation of amide bond with
the C-terminus of IKVAV. Therefore, formation of oligo-IKVAV in the presence of
excess carbodiimide would not occur and the ITO surface will only be decorated
89
with mono-IKVAV layer. Prior to sterilization of the sample, deprotection of the
tBOC groups were carried out to return the primary amine of the N-terminus and of
lysine side group of the IKVAV peptide. These samples will be referred as ITO-
mIKVAV.
Dissociated neuron cell culture were carried out on ITO-mIKVAV for 6
hours, along with ITO coated with PDL to serve as the positive control, and cleaned
ITO samples as the negative control. Aminophase ITO samples were also tested for
cell culture to observed any cell adhesion and/or proliferation that could be due to
the presence of the primary amine on the surface.

ITO ITO-NH2 ITO-mIKVAV ITO-PDL
0
500
1000
1500
# Neuron cells attached/mm
2

Figure 3.11. Neuron attachment on various modified ITO surface after 6 hours of
seeding. Presence of mono-IKVAV on the surface promotes neuron adhesion with
similar effect as PDL coated surface.

90
Cell adhesion processes was completed 6 hours after culture was started. At
this point, quantitative cell adhesion analysis was assessed by image analysis. To
improve identification of the transparent neuron cell body under the microscope, the
cells were stained with Hoescht 33342 for 15 minutes at 37°C followed by three
times rinsing of the samples with PBS to remove any unattached neuron cells,
cellular debris, and excess Hoescht 33342 from the ITO surface. As presented on
Figure 3.11, the presence of mono-IKVAV layer on ITO surface promotes neuron
adhesion almost as efficient as PDL coating, the most common polymeric coating for
promotion of cell adhesion.
It should be noted that neuron cell adhesion process should be independent to
the states of the cells, i.e., both viable and damage cells would have similar affinity
towards adhesion to the surface. Thus, staining procedure with Hoescht 33342 will
stained both viable and damage cells.

3.3.1.2 Neuron Growth on mono-IKVAV Layer Surface.
One concern regarding the mono-layer IKVAV capabilities in supporting
neurons growth and formation of cellular network was due to the possible need of
longer linker between the peptide and the surface. Therefore, culture experiments
were carried out to investigate whether the mono-IKVAV layer on the surface could
support growth of the attached viable neurons.
Similar observations were obtained from the multiple neuron cell cultures
conducted on samples with mono-layer IKVAV surface. On each of the samples,
91
formation of axons and dendridites can be easily identified after 3 days of culture
(Figure 3.12). In comparison, attached neuron cells on non-treated surface did not
shows any growth. This is as expected for neuron cells, in which non-anchored
neurons could not sustain growth.
Growth of axons and dendridites were observed only on some samples with
oligo-IKVAV layer, The inconsistent neuron growth on these samples could be
attributed to the possible deactivation of the CAMs via formation of branching oligo-
IKVAV. We postulate that the growth and proliferation on these samples could occur
because the IKVAV at the end-chain of the oligomers could still retain the correct
conformation, recognizable by the neurons cell receptor.

Blank mIKVAV oIKVAV

0.25 mm

0.25 mm

0.25 mm

Figure 3.12. Representative microscope images for each sample at 3 days of culture.

92
Blank mIKVAV oIKVAV

0.25 mm

0.25 mm

0.25 mm

Figure 3.13. Representative microscope images for each sample at 5 days of culture

Unexpectedly, as the cell culture experiments continued on to day 5, the
quality of the neurons growth greatly diminished (Figure 3.13). Image analysis of the
mono-IKVAV samples showed a decrease in the cellular network formation, loss of
axons and dendrites when compared to the observation obtained on the 3
rd
day of
culture. Also observed on these samples is the increased on the aggregations of cell
bodies, and indication of neuron cell deaths. For samples with oligo-IKVAV layer,
not only that the neurons growth was diminished, the quantity of viable cells was
dramatically reduced. Past the fifth day of culture, there are virtually no viable cells
on both the mono-IKVAV samples and the oligo-IKVAV samples.
There are several scenarios that could be postulated to explain why the
adhered neurons cells to undergo apoptosis where lack of control over the peptide
surface density plays an important role. It is possible that the CAMs surface density
93
that the cell receptor can recognize and interact is very limited. Since the amount of
anchoring site was limited, the resulting adhesion of the cell to the surface will not
be optimal. Previous studies on cell adhesion promoted by immobilization of CAMS
usually were carried out utilizing cells from established cell lines instead of a
primary cell culture. Moreover, the peptide treated area is very restricted such that
the cells are “forced” to adhere in close contact with each other. The adhered cells
will create their own matrix to sustain adhesion and growth. When the cells are
adhering in close proximity with each other, the concentration of the matrix will
increase, thus cell growth and cellular network formation were sustained by the
matrix instead of due to the presence of CAM on the surface.
If the assumption is that the peptide surface density is sufficient, it is still
possible that although the layer of peptide did not completely cover the surface of the
substrates. Thus, there are “patches” of area where cell bodies, dendrites nor axons
could anchor themselves on to. In this case, cleavage of axons and/or dendrites from
the cell bodies could occur and the event will accelerate the cell death process. Most
studies involving IKVAV utilize longer peptide containing the IKVAV sequence
instead of just the 5 amino acid sequence. Thus, as the cells are adhered due to the
presence of IKVAV sequence, the rest of the amino acid in the peptide could cover
as much of the substrates surface with material that can interact with the surface of
the cells. This postulate is based on observations obtained when the culture
procedure was slightly modified.

94

Figure 3.14. Representative images for each sample of ITO after 7 days of neuron
culture, where neuron was seeded in the presence of cellular debris.

Cell culture procedure was modified in which the centrifugation step for
removal of membranes and cellular debris was eliminated. Thus, during cell seeding,
both neurons and cellular debris were introduced onto each sample. Under this
procedure, cell culture was able to be carried out past 5 days without increase of cell
death (Figure 3.14).
ITO
ITO-NH
2
ITO-oIKVAV ITO_mIKVAV
100 µm
95
ITO ITO-NH2 ITO-mIKVAV ITO-oIKVAV
0
5
10
15
20
25
30
35
% Surface Coverage

Figure 3.15. Surface coverage percentage for each samples where neurons were
seeded along with cellular debris.

After 7 days of culture, the samples were analyzed for surface coverage
percentage of the substrates with the neural network. As presented on Figure 3.15,
the percent surface coverage of the neural network on mono-IKVAV samples and on
oligo-IKVAV samples are very similar.
In the presence of extra cellular proteins, significant neurons adhesion and
proliferation was also observed on untreated ITO surface. This observation suggested
that the presence of extra cellular debris on the surface of the ITO can act as a matrix
that is able to not only promote neurons adhesion but also support the formation of
neural network. It is also indicating that efficiency of IKVAV to promote specific
96
neuron adhesion would be masked by the presence of a matrix that would promote
non-specific adhesion of proteins.

3.3.2. Neuron Cell Culture Results: Varying mono-IKVAV Surface Density on
ITO
Discussed earlier (section 3.2.2.3), a method to quantified IKVAV surface
density was developed based on the redox properties of hydroquinone on the
conducting material surface. For ITO surface, the hydroquinone was immobilized
with phosphonic acid acting as the anchoring group. The peptide was immobilized
onto the surface as a product of Dies-Alder reaction between the Cp*-IKVAV with
quinone, the electrochemically oxidized state of hydroquinone (Figure 3.8). Several
samples were prepared with IKVAV surface density varied from 10
12 -
10
13
molecules/cm
2
. These values are in the range of theoretical IKVAV nanofibers
reported by Silva et.al. in which the peptide promote adhesion and differentiation of
neural progenitor cells.
26
The samples were tested for neuron adhesion and
proliferation in one week period of neuron cell culture, where PDL coated ITO was
used as positive control.
97
B
D E
C
A
B
D E
C
A

Sample Peptide / cm
2

A 0
B 1.57×10
13

C 2.68×10
13

D 10.28×10
13

E PDL coating

Figure 3.16. .Neuron cell culture on varied IKVAV surface density.

After 7 days of culture, the cell adhesion on all ITO samples with IKVAV on
the surface is very similar. Unfortunately, there are no formations of dendrites
and/or axons observed on any of the samples (Figure 3.16). It is apparent that the
presence of unreacted hydroquinone/quinone groups on the ITO surface are toxic for
100 µm
98
neuron cells,
36, 37
where the groups could interfere the cells signaling and/or
processes due to their redox active property. Attempts to passivate the unreacted
hydroquinone groups were carried out by reacting the groups with Cp*-NH
2
, to be
followed with further cell culture investigation.

3.4. Investigating Strength of Neurons Adhesion onto Surface Modified ITO
Observation of neural network formation on cell cultures studies are
accumulation of various biological processes of the neurons that is started with the
cells adhesion onto the substrate support. Stable anchorage of neurons onto the
substrate support allows the neurons to carry out subsequent processes such as
growth, migration, and formation of neural network. These processes are depended
and regulated by the interaction of the cells surface receptors to the matrix on the
surface where the cells adhered. Theferore, achieving stable neuron cells anchorage
to the surface is a crucial start in formation cellular network on a solid surface. .
Although various studies have been carried out to promote cells attachment and
cellular network formation, assessment method for the adhesion strength between the
cells and the substrates was very limited. Observation of cell spreading and
migration studies are often used as indicator of adhesion strength which are
misleading since the two processes are the products of various processes and are not
solely depend on adhesion strength alone.
The most common method in assessment of cell adhesion strength was
through centrifugation.
15, 38-41
The culture samples were subjected to known
99
rotational speed. Measurement of shear force required to detach cells from the
substrates will be equivalent to the centrifugal force acting on the sample.
15, 38-41

Experiments were carried out by placing the substrate into a substrate holder on a
spinning disc which an adjustable rotational speed. The method was widely used due
to the relative easiness in experimental preparation and execution, as well as the
rapid analysis that could be carried out. The drawback of this method are lack of
adjustability in applying constant force per run and constrained in the magnitude of
force that could be applied due to equipment safety requirement.
Another method for direct measurement of cell adhesion that has been
explored was the used of atomic force microscopy (AFM) tips to mechanically
remove a cell from its focal adhesion site.
42
The method allows real-time imaging
and/or analysis with sensitive control of force applied to detach a cell from the
sample surface. Unfortunately, measurement can only be carried out for one cell at a
time. It is also need to be considered that measurement must be carried out in a short
period cautiously as not to damage the AFM tip during analysis of viable cell
adhesion.
An adaptation of microfluidic channel flow-system for assessing neuron
adhesion was utilized, in which the method allow for real-time imaging while
exerting controlled force to multiple cells. Similar setup has been reported for
assessing the adhesion of fibroblast cell lines WTNR6 on fibronectin coated glass.
43

100

Figure 3.17. Schematic and dimension of flow channel used for assessing neurons
adhesion on surface modified ITO.

A poly-dimethoxy silane (PDMS) flow channel was made from Sylgard
186® (Dow Corning) and a Teflon mold to achieve the desired specification (Figure
3.17). Real-time imaging was recorded every 10 seconds per frame for a total of 75
frames. An ultra-low adjustable peristaltic pump was used to control the flow rate in
which the flow rate was slowly increased to 6
th
pre-set values every 10
th
frame
starting on the 5
th
frame. The flow rate was measured manually by measuring
average time of deliverance for 10 mL of DI H
2
O in the exact setup as the one used
in the actual experiment. Correlation of flow rate to applied shear stress was
calculated according to the formula:
⎟
⎠
⎞
⎜
⎝
⎛
=
w h
Q
2
12 μ
τ
Out In
w
h
l
w = 0.5 mm
h = 1 mm
l = 1 mm
101
where τ is the shear stress exerted by the flow of liquid, Q is the flow rate of the
liquid, μ is the fluid viscosity, h is the height of the channel, and w is the width of the
channel.
43

Preliminary studies on assessing strength of neuron adhesion on ITO surface
with the flow systems were carried out on neurons cells after 6 hours of culture. To
improve imaging of the neurons, cells are stained with Hoescht 33342 as described
in the previous experimental section (subchapter 3.2.1).

Figure 3.18. Instrument setup for assessing neurons adhesion on ITO surface.

It was postulated before that cell adhesion to IKVAV layer on ITO surface
was not strong enough to support neurons growth and formation of neural network
due to the low IKVAV surface density that the neuron receptor could recognized. It
was also postulated that when neuron cell culture was carried out in presence of
cellular debris, a better adhesion was obtained, which could be due to additional
interaction between the cells and the cellular debris that settled on top of the surface
during culture. In attempts to test the hypothesis above, two sets of experiments were
102
carried out. For the first set of experiments, neurons were seeded onto ITO samples
based on the regular culture procedure describe in the experimental section
(subchapter 3.2). In the second set of experiments, neurons were seeded along with
cellular debris onto the ITO.

Setting # Flow rate (mL/s) Shear stress (dyne)
0 0 0
1
st
0.41 97.70
2
nd
0.46 109.40
3
rd
0.50 119.97
4
th
0.57 135.67
5
th
0.67 161.78
6
th
0.76 182.84
Table 3.2 Flow rate settings and correlated shear stress applied to detach neurons
from ITO surface.

In first set of experiment, neurons cultured on both mono-IKVAV layer and
oligo-IKVAV layer unexpectedly were instantaneously removed at the lowest flow-
rate setting, exerting 97.70 dyne. For the second set of experiment, neurons cells
adhered stronger on both mono-IKVAV layer and oligo-IKVAV layer. No adhered
neurons were detached from the surface at the highest flow-rate setting, in which
182.84 dyne was exerted.
103
As the preliminary studies showed, adhesion on neurons on ITO modified
surface was significantly enhanced by the presence of cellular debris on the surface.
The above results follow the hypothesis proposed in subchapter 3.3.1.2, that the
cellular debris on the surface can act as a friendly, non-selective adhesive matrix
which supports stronger adhesion and resulting in survival of neurons in cell culture
for more than 5 days.
The studies above provide a proof of concept that the strength of neuron
adhesion on IKVAV modified ITO can be asses with the flow-system setup. In order
to determine the adhesion strength of neurons onto the modified ITO surface, further
experiments must be carried out at much slower flow rate. Due to the limitation of
the peristaltic pump and the setup, it is not possible to lower the flow rate by
decreasing the diameter of the tubing. One possible modification was to create a new
Teflon mold such that a new PDMS flow-system stamp can be created with larger
flow channel width while keeping the height and the shape of the channel constant.

3.5. Summary
Selective surface modification on ITO, a transparent electrode commonly
utilized in fabrication of MEA, can be carried out utilizing phosphonic acid as
anchoring group. The approach allows immobilization of various functional groups
that could alter the surface properties of ITO, including formation of aminophase
ITO. To promote neuron cells adhesion and growth on the surface, IKVAV (a neuron
specific CAM) was covalently bound via formation of peptide linkage. The amide
104
bond formation was facilitated by carbodiimide, a known peptide coupling agent in a
similar procedure that was used in various reports regarding CAMs immobilization
on surface.
During the coupling of the peptide onto the aminophase surface, formation of
oligo-peptide in solution was observed based on MALDI analysis. In the contrary,
published literature reports implied that adhesion and proliferation phenomena were
the result of immobilization of single CAM per amine group on the surface. Cell
culture experiments on ITO surface with mono-IKVAV layer showed promotion of
cell adhesion after 6 hours of culture with similar efficiency with PDL coating. After
3 days of culture, growth of neurons on mono-IKVAV samples is considerable better
than that on oligo-IKVAV samples, an indication that the oligo-IKVAV formed
might include a deactivated branching oligo-IKVAV. Deterioration of neurons
health was observed on the fifth day of culture on both systems. The lack of neurons
growth after the fifth day of culture was attributed to possible low IKVAV surface
density and low cell adhesion strength.
Another approach for immobilization of IKVAV onto ITO could be carried
out via Dies-Alder reaction between Cp*-IKVAV with quinone groups on ITO
surface. The surface density of the electrochemically active quinone groups can be
measured via chronoamperometry. The difference between surface density of
quinone prior and post Dies-Alder reaction with Cp*-IKVAV will correspond to the
surface density of IKVAV on ITO surface. Unfortunately although the IKVAV on
the surface could increase the quantity of neurons adhering onto the surface, the
105
toxicity of unreacted of hydroquinone/quinone groups on the surface inhibits neurons
growth.
In effort to assess the strength of neuron adhesion on IKVAV decorated ITO
surface, a flow-system was utilized. It is hypothesized that presence of cellular
matrix during seeding of neuron cells could improve the adhesion of the neuron cells
onto the substrate. Experimental observations are in agreement with this hypothesis
where neuron cells adhered on IKVAV surfaces free of cellular debris, can be easily
detach at 97.70 dyne flow shear stress. For IKVAV samples with cellular debris
present on the surface of the substrate, exerting 182.84 dyne of shear stress did not
remove any of the attached cells.

106
3.6. Chapter 3 References
1. Berger, T. W.; Ahuja, A.; Courellis, S. H.; Deadwyler, S. A.; Erinjippurath,
G.; Gerhardt, G. A.; Gholmieh, G.; Granacki, J. J.; Hampson, R.; Hsaio, M. C.;
Lacoss, J.; Marmarelis, V. Z.; Nasiatka, P.; Srinivasan, V.; Song, D.; Tanguay, A. R.;
Wills, J., Restoring Lost Cognitive Function. IEEE Engineering in Medicine and
Biology Magazine September/October, 2005, pp 30-44.

2. Berger, T. W.; Baudry, M.; Brinton, R. D.; Liaw, J.-S.; Mamarelis, V. Z.;
Park, A. Y.; Sheu, B. J.; Tanguay, A. R., Brain-Implantable Biomimietic Electronics
as the Next Era in Neural Prosthetics. Proceedings of the IEEE 2001, 89, (7), 993-
1012.

3. Brinton, R. D.; Sousou, W.; Baudry, M.; Thompson, M. E.; Berger, T. W.,
The Biotic/Abiotic Interface: Achievements and Foreseeable Challenges. In Toward
Replacement Parts for the Brain, Berger, T. W. G., Dennis L., Ed. The MIT Press:
Cambridge, Massachusetts, 2005; pp 221-238.

4. Jacobson, B. S.; Branton, D., Plasma Membrane: Rapid Isolation and
Exposure of the Cytoplasmic Surface by Use of Positively Charged Beads. Science
1977, 195, (4275), 302-304.

5. Tong, Y. W.; Shoichet, M. S., Enhancing The Neuronal Interaction on
Fluoropolymer Surfaces with Mixed Peptides or Spacer Group Linkers. Biomaterials
2001, 22, 1029-1034.

6. Ai, H. M., Hongdi; Ichinose, Izumi; Jones, Steven A.;Mills, David K.; Lvov,
Yuri M.; Qiao, Xiaoxi., Biocompatibility of Layer-by-Layer Self Assembled
Nanofilm on Silicone Rubber for Neurons. Journal of Neuroscience Methods 2003,
128, 1-8.

7. He, W. B., Ravi V., Nanoscale Neuro-Integrative Coatings for Neural
Implants. Biomaterials 2005, 26.

8. Bosman, F.; Stamenkovic, I., Functional Structure and Composition of The
Extracellular Matrix. J. Pathol 2003, 200, 423-428.

9. Mercurio AM, Laminin Receptors: Achieving Specificity Through
Cooperation. Trends Cell Biol 1995, 5, 419-423.

10. Li, J.; Zhang, Y.-P.; Kirsner, R. S., Angiogenesis in Wound Repair:
Angiogenic Growth Factors and The Extracellular Matrix. Microsc Res Tech 2003,
60, 107-114.

107
11. Ratner, B., Perspective and Possibilities in Biomaterials Science. In
Bioimaterials Science: An Introduction to Materials in Medicine, Ratner, B.;
Hoffman, A.; Schoen, F.; Lemons, J., Eds. Academic Press: San Diego, 1996; pp
465-468.

12. Smyth, J.; Walker, M., Surface Precoating in The 1980s: A First Taste of
Cell-Matrix Interactions. In Tissue engineering of vascular prosthetic grafts, Zilla,
P.; Greisler, H., Eds. R.G. Landes Company: Austin, 1999; pp 69-77.

13. Williams, D., Bioinertness: An Outdated Principle. In Tissue Engineering of
Vascular Prosthetic Grafts, Zilla, P.; Greisler, H., Eds. R.G. Landes Company:
Austin, 1999; pp 459-462.

14. Choquet, D. F., Dan P.; Sheetz, Micahel P., Extracellular Matrix Rigidity
Causes Strengthening of Integrin-Cytoskeleton Linkages. Cell 1997, 88, 39-48.

15. Gallant, N. D. C., Jeffery R.;Frazier, A. Bruno; Collard, David M.; Garcia,
Andres J., Micropatterned Surfaces to Engineer Focal Adhesions for Analysis of Cell
adhesion Strengthening. Langmuir 2002, 18, 5579-5584.

16. Webster, T. J. E., Celaletdin; Doremus, Robert H.; Siegel, Richard W.;
Bizios, R., Specific Proteins Mediate Enchanced Osteoblast Adhesion on Nanophase
Ceramics. Journal of Biomedical Material Research 2000, 51, 475-483.

17. Dee, K. C. A., Thomas T.; Bizios, R., Design and Function of Novel
Osteoblast-Adhesive Peptides for Chemical Modification of Biomaterials. Journal of
Biomedical Material Research 1998, 40, 371-377.

18. Hersel, U. D., Claudia; Kessler, Horst., RGD Modified Polymers:
Biomaterials for Stimulated Cell Adhesion and Beyond. Biomaterials 2003, 24,
4385-4415.

19. Kam, L. S., W.; Turner, J.N.;Bizios, R., Selective Adhesion of Astrocytes to
Surfaces Modified with Immobilized Peptides. Biomaterials 2002, 23, 511-515.

20. Lin, X.; Takahashi, K.; Liu, Y.; Zamora, P. O., Enhancement of Cell
Attachment and Tissue Integration by a IKVAV Containing Multi-Domain Peptide.
Biochmica et Biophyscia Acta 2006, 1760, 1403-1410.

21. Nomizu, M. W., Benjamin S.;Weston, Christi A.;Kim, Woo Hoo; Kleinman,
Hynda K.; Yamada, Yoshihiko., Structure-Activity Study of a Laminin α1 Chain
Active Peptide Segment Ile-Lys-Val-Ala-Val (IKVAV). FEBS Letters 1995, 365,
227-231.
108
22. Saneinejad, S.; Shoichet, M. S., Patterned Glass Surfaces Direct Cell
Adhesion and Process Outgrowth of Primary Neurons of The Central Nervous
System. J. Biomed. Mater. Res 1998, 42, 13-19.

23. Svedhem, S. D., D.; Ekeroth, J.; Kelly, J.; Hook, F.;Gold, J. , In situ Peptide-
Modified Supported Lipid Bilayers for Controlled Cell Attachment. Langmuir 2003,
19, 6730-6736.

24. Yamada, M. K., Yuichi; Kasai, Shingo; Kato, Kozue; Mochizuki, Mayumi;
Nishi, Norio; Watanabe, Nobuhisa; Kleinman, Hynda K.; Yamada, Yoshihiko;
Nomizu, Motoyoshi, Ile-Lys-Val-Ala-Val (IKVAV)-Containing Laminin α1 chain
Peptides Form Amyloid-like Fibrils. FEBS Letters 2002, 530, 48-52.

25. Sorribas, H. B., Dieter; Leder, Lukas; Sonderegger, Peter; Tiefenauer, Louis.,
Adhesion Proteins for a Tight Neuron-Electrode Contact. Journal of Neuroscience
Methods 2001, 104, 133-141.

26. Silva, G. A. C., Catherine; Niece, Krista L.; Beniash, Elia; Harrington, Daniel
A.; Kessler, John A.;Stupp, Samuel I., Selective Differentiation of Neural Progenitor
Cells by High-Epitope Density Nanofibers. Science 2004, 303, 1352-1355.

27. Santiago, L. Y. N., Richard W.; Rubin, J. Peter; Marra, Kacey G., Peptide-
Surface Modification of Poly(caprolactone) with Laminin-Derived Sequences for
Adipose-Derived Stem Cell Applications. Biomaterials 2006, 27, 2962-2969.

28. Ranieri, J. P. B., Ravi; Bekos, Evan J.; Gardella Jr., Joseph A.; Mathieu,
Hans J. ; Ruiz, Laurence; Aebischer, Patrick., Spatial Control of Neuronal Cell
Attachment and Differentiation on Covalently Patterned Laminin Oligopeptide
Substrates. Int. J. Devl. Neuroscience 1994, 12, (8), 725-735.

29. Curreli, M. L., Chao; Sun, Yingua; Lei, Bo; Gundersen, Martin A.;
Thompson, Mark E., Zhou, Chongwu, Selective Functionalization of In
2
O
3

Nanowire Mat Devices for Biosensing Applications. Journal of American Chemical
Society 2005, 127, 6922-6923.

30. Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S., Systems for Orthogonal Self-
Assembly of Electroactive Monolayers on Au and ITO: An Approach to Molecular
Electronics. J. Am. Chem. Soc. 1995, 117, 6927-6933.

31. Dee, K. C. A., Thomas T.; Bizios, R., Osteoblast Population Migration
Characteristics on Substrates Modified with Immobilized Adhesive Peptides.
Biomaterials 1999, 20, 221-227.

109
32. Heller, D. A. G., Veronika; Kelleher, Keith J.; Lee, Tai-Chou; Mahbubani,
Sunil; Sigworth, Laura A.; Lee, T. Randall; Rea, Michael A., Patterned Networks of
Mouse Hippocampal Neurons on Peptide-Coated Gold Surfaces. Biomaterials 2005,
26, 883-889.

33. Hong, H.-G. P., Wonchoul., Electrochemical Characteristics of
Hydroquinone-Terminated Self-Assembled monolayers on Gold. Langmuir 2001,
17, 2485-2492.

34. Yousaf, M. N. H., Benjamin T.; Mrksich, Milan, Turning on Cell Migration
with Electroactive Substrates. Angew. Chem. Int. Ed. 2001, 40, (6), 1093-1096.

35. Yousaf, M. N. H., Benjamin T.; Mrksich, Milan, Using Electroactive
Substrates to Pattern The Attachment of Two Different Cell Populations. PNAS
2001, 98, (11), 5992-5996.

36. Tomiyama, T. K., Hideshi; Kataoka, Ken-ichiro; Asano, Satoshi; Endo,
Noriaki, Rifampicin Inhibits The Toxicity of Pre-aggregated Amyloid Peptides by
Binding to Peptide Fibrils and Preventing Amyloid-Cell Interaction. Biochem. J
1997, 322, 859-865.

37. Asanuma, M. M., Ikuko; Diaz-Corrales, Fransisco J.; Ogawa, Norio, Quinone
Formation as Dopaminergic Neuron-Specific Oxidative Stress in The Pathogenesis
of Sporadic Parkinson's Disease and Neurotoxin-induced Parkinsonism. Acta Med.
Okayama 2004, 58, (5), 221-233.

38. Lotz, M. M. B., Carol A.; Erickson, Harold P.; McClay, David R., Cell
Adhesion to Fibronectin and Tenascin:Quantitative Measurements of Initial Binding
and Subsequent Strengthening Response The Journal of Cell Biology 1989, 109,
1795-1805.

39. McClay, D. R.; Wessel, G. M.; Marchase., R. B., Intercellular Recognition:
Quantitation of Initial Binding Events. Proc. Natl. Acad. Sci. USA 1981, 78, :4975-
4979.

40. Garcı´a, A. J., Huber, F., Boettiger, D., Force Required to Break α5 β1
Integrin-Fibronectin Bonds in Intact Adherent Cells is Sensitive to Integrin
Activation State. J.Biol. Chem. 1998, 273, 10988–10993.

41. Garcı´a, A. J.; Ducheyne, P.; Boettiger, D., Quantification of Cell Adhesion
Using a Spinning Disk Device and Application to Surface-Reactive Materials.
Biomaterials 1997, 18, 1091–1098.

110
42. Simon, A. D., Marie-Christine, Strategies and Results of Atomic Force
Microscopy in The Study of Cellular Adhesion. Micron 2006, 37, 1-13.

43. Lu, H. K., Lily Y.; Wang, Wechung M.; Lauffenburger, Douglas A.; Griffith,
Linda G.; Jensen, Klavs F., Microfluidic Shear Devices for Quantitative Analysis of
Cell Adhesion. Analytical Chemistry 2004, 76, 5257-5264.

111
Bibliography

Ai, H. M., Hongdi; Ichinose, Izumi; Jones, Steven A.;Mills, David K.; Lvov, Yuri
M.; Qiao, Xiaoxi., Biocompatibility of Layer-by-Layer Self Assembled Nanofilm on
Silicone Rubber for Neurons. Journal of Neuroscience Methods 2003, 128, 1-8.

Asanuma, M. M., Ikuko; Diaz-Corrales, Fransisco J.; Ogawa, Norio, Quinone
Formation as Dopaminergic Neuron-Specific Oxidative Stress in The Pathogenesis
of Sporadic Parkinson's Disease and Neurotoxin-Induced Parkinsonism. Acta Med.
Okayama 2004, 58, (5), 221-233.

Beach, W.; Lee, C.; Bassett, D., Encyclopedia of Polymer Science and Engineering
1985, 17, 990.

Beckerman, M., Chapter 10. Cell Adhesion and Motility. In Molecular and Cellular
Signaling, Springer: New York, 2005; pp 221-245.

Bera, M.; Rivaton, A.; Gandon, C.; Gardette, J. L., Photooxidation of Poly(para-
xylylene). European Polymer Journal 2000, 36, 1753-1764.

Berger, T. W.; Ahuja, A.; Courellis, S. H.; Deadwyler, S. A.; Erinjippurath, G.;
Gerhardt, G. A.; Gholmieh, G.; Granacki, J. J.; Hampson, R.; Hsaio, M. C.; Lacoss,
J.; Marmarelis, V. Z.; Nasiatka, P.; Srinivasan, V.; Song, D.; Tanguay, A. R.; Wills,
J., Restoring Lost Cognitive Function. IEEE Engineering in Medicine and Biology
Magazine September/October, 2005, pp 30-44.

Berger, T. W.; Baudry, M.; Brinton, R. D.; Liaw, J.-S.; Mamarelis, V. Z.; Park, A.
Y.; Sheu, B. J.; Tanguay, A. R., Brain-Implantable Biomimietic Electronics as the
Next Era in Neural Prosthetics. Proceedings of the IEEE 2001, 89, (7), 993-1012.

Berson, E. L., Rentinitis Pigmentosa : The Friedenwald Lecture. Invest. Ophthalmol.
Vis. Sci. 1993, 34, (5), 1659-1679.

Bosman, F.; Stamenkovic, I., Functional Structure and Composition of The
Extracellular Matrix. J. Pathol 2003, 200, 423-428.

Brinton, R. D.; Sousou, W.; Baudry, M.; Thompson, M. E.; Berger, T. W., The
Biotic/Abiotic Interface: Achievements and Foreseeable Challenges. In Toward
Replacement Parts for the Brain, Berger, T. W. G., Dennis L., Ed. The MIT Press:
Cambridge, Massachusetts, 2005; pp 221-238.

112
Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.;
Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo,
C. H.; Burke, D. T., An Integrated Nanoliter DNA Analysis Device. Science 1998,
282, (484-487).

Carter, H. A., The Chemistry of Paper Preservation Part 3. The Strengthening of
Paper. Journal of Chemical Education 1996, 73, (12), 1160-1162.

Choquet, D. F., Dan P.; Sheetz, Micahel P., Extracellular Matrix Rigidity Causes
Strengthening of Integrin-Cytoskeleton Linkages. Cell 1997, 88, 39-48.

Curcio, C. A.; Medeiros, N. E.; Millican, C. L., Photoreceptor Loss in Age-Related
Macular Degeneration. Invest. Ophthalmol. Vis. Sci. 1996, 37, (7), 1236-1249.

Curreli, M. L., Chao; Sun, Yingua; Lei, Bo; Gundersen, Martin A.; Thompson, Mark
E., Zhou, Chongwu, Selective Functionalization of In
2
O
3
Nanowire Mat Devices for
Biosensing Applications. Journal of American Chemical Society 2005, 127, 6922-
6923.

Dee, K. C. A., Thomas T.; Bizios, R., Design and Function of Novel Osteoblast-
Adhesive Peptides for Chemical Modification of Biomaterials. Journal of
Biomedical Material Research 1998, 40, 371-377.

Dee, K. C. A., Thomas T.; Bizios, R., Osteoblast Population Migration
Characteristics on Substrates Modified with Immobilized Adhesive Peptides.
Biomaterials 1999, 20, 221-227.

Dobelle, W. H.; Mladejovsky, M. G.; Girvin, J. P., Artificial Vision for the Blind:
Electrical Stimulation of Visual Cortex Offers Hope for a Functional Prosthesis.
Science 1974, 183, (4123), 440-444.

Epilepy Foundation : Vagus Nerve Stimulation Therapy.
http://www.epilepsyfoundation.org/answerplace/Medical/treatment/vns/treatvns.cfm
(June 23rd, 2006),

Fortin, J. B.; Lu, T. M., Ultraviolet Radiation Induced Degradation of Poly-para-
xylylene (Parylene) Thin Films. Thin Solid Films 2001, 397, 223-228.

Gadre, K. S.; Alford, T. L., Thermal Stability and Adhsion Improvement of Ag
Deposited on Pa-n by Oxygen Plasma Treatment. J. Vac. Sci. Technol. B 2000, 18,
(6), 2814-2819.

113
Gallant, N. D. C., Jeffery R.;Frazier, A. Bruno; Collard, David M.; Garcia, Andres J.,
Micropatterned Surfaces to Engineer Focal Adhesions for Analysis of Cell adhesion
Strengthening. Langmuir 2002, 18, 5579-5584.

Garcı´a, A. J., Huber, F., Boettiger, D., Force Required to Break α5β1 Integrin-
Fibronectin Bonds in Intact Adherent Cells Is Sensitive to Integrin Activation State.
J.Biol. Chem. 1998, 273, 10988–10993

Garcı´a, A. J.; Ducheyne, P.; Boettiger, D., Quantification of Cell Adhesion Using A
Spinning Disk Device and Application To Surface-Reactive Materials. Biomaterials
1997, 18, 1091–1098.

Gardner, T. J.; Frisbie, C. D.; Wrighton, M. S., Systems for Orthogonal Self-
Assembly of Electroactive Monolayers on Au and ITO: An Approach to Molecular
Electronics. J. Am. Chem. Soc. 1995, 117, 6927-6933.

Gorham, W. F., A New General Synthetic Method For The Preparation of Linear
Poly-p-xylylenes. Journal of Polymer Science, Part A 1966, 1-4, 3027-3039.

Greiner, A., Poly(1,4-xylylene)s: Polymer Films By Chemical Vapour Deposition.
Trend in Polymer Science 1997, 5, 12.

Grattan, D. W., Parylene at The Canadian Conservation Institute. Canadian
Chemical News 1989, 41, (9), 25-26.

Hahn, A. W.; York, D. H.; Nichols, M. F.; Amromin, G. C.; Yasuda, H. K.,
Biocompatibility of Glow-Discharge Polymerized Films and Vacuum-Deposited
Parylene. Journal of Applied Polymer Science:Applied Polymer Symposium 1984,
38, 55-64.

He, W.; Bellamkonda, R. V., Nanoscale Neuro-Integrative Coatings For Neural
Implants. Biomaterials 2005, 26, 2983–2990.

Heckenlively, J. R.; Boughman, J.; Friedman, L., Diagnosis and Classification of
Rentinitis Pigmentosa. Lippincott: Philadelphia, 1998; p 21.

Herrera-Alonso, M.; McCarthy, T. J., Chemical Surface Modification of Poly(p-
xylylene) Thin Films. Langmuir 2004, 20, 9184.

Hersel, U. D., Claudia; Kessler, Horst., RGD Modified Polymers: Biomaterials for
Stimulated Cell Adhesion and Beyond. Biomaterials 2003, 24, 4385-4415.

Hong, H.-G. P., Wonchoul., Electrochemical Characteristics of Hydroquinone-
Terminated Self-Assembled monolayers on Gold. Langmuir 2001, 17, 2485-2492.
114
Humayun, M. S.; Loeb, G. E. Biomimetic Microelectronic Systems Engineering
Research Center; University of Southern California, California Institue of
Technology, University of California at Santa Cruz: 2005; p 47.

Jacobson, B. S.; Branton, D., Plasma Membrane: Rapid Isolation and Exposure Of
The Cytoplasmic Surface By Use Of Positively Charged Beads. Science 1977, 195,
(4275), 302-304.

Kam, L. S., W.; Turner, J.N.;Bizios, R., Selective Adhesion of Astrocytes to
Surfaces Modified with Immobilized Peptides. Biomaterials 2002, 23, 511-515.

Kindlmann, G.; Normann, R. A.; Badi, A.; Bigler, J.; Keller, C.; Coffey, R.; Jones,
G. M.; Johnson, C. R., Imaging of Utah Electrode Array, Implanted in Cochlear
Nerve. In NIH Symposium on Biocomputation & Bioinformation Digital Biology:
The Emerging Paradigm, Bethesda, Maryland, 2003.

Lahann, J.; Balcells, M.; Rodon, T.; Lee, J.; Choi, I. S.; Jensen, K. F.; Langer, R.,
Reactive Polymer Coatings: A Platform for Patterning Proteins and Mammalian
Cells onto a Broad Range of Materials. Langmuir 2002, 18, 3632-3638.

Lahann, J.; Klee, D.; Thelen, H.; Bienert, H.; Vorwerk, D.; Ho¨cker, H.,
Improvement of Haemocompatibility of Metallic Stents By Polymer Coating.
Journal of Material Science: Materials in medicine 1999, 10, (7), 443-448.

Lahann, J.; Klee, D.; Pluester, W.; Hoecker, H., Bioactive Immobilization of R-
Hirudin on CVD-coated Metallic Implant Devices. Biomaterials 2001, 22, 817-826.

Lahann, J.; Langer, R., Novel Poly(p-xylylenes): Thin Films with Tailored Chemical
and Optical Properties. Macromolecules 2002, 35, 4380-4386.

Lee, B. Q.; Maurer, R. H.; Nhan, E.; Lew, A., Int. J. Microcircuits Electron. Packag.
1999, 22, 104.

Li, J.; Zhang, Y.-P.; Kirsner, R. S., Angiogenesis in wound repair: Angiogenic
Growth Factors and The Extracellular Matrix. Microsc Res Tech 2003, 60, 107-114.

Lin, X.; Takahashi, K.; Liu, Y.; Zamora, P. O., Enhancement of Cell Attachment and
Tissue Integration by a IKVAV Containing Multi-Domain Peptide. Biochmica et
Biophyscia Acta 2006, 1760, 1403-1410.

Lotz, M. M. B., Carol A.; Erickson, Harold P.; McClay, David R., Cell Adhesion to
Fibronectin and Tenascin:Quantitative Measurements of Initial Binding and
Subsequent Strengthening Response The Journal of Cell Biology 1989, 109, 1795-
1805.
115
Lu, H. K., Lily Y.; Wang, Wechung M.; Lauffenburger, Douglas A.; Griffith, Linda
G.; Jensen, Klavs F., Microfluidic Shear Devices for Quantitative Analysis of Cell
Adhesion. Analytical Chemistry 2004, 76, 5257-5264.

Margalit, E.; Sadda, S. R., Retinal and Optical Nerve Diseases. Artif. Organs. 2003,
27, (11), 963-974.

Martini, D.; Shepherd, K.; Sutcliffe, R.; Kelber, J.; Edwards, H.; Martin, R. S.,
Modification of Parylene AF-4 Surfaces Using Activated Water Vapor. Applied
Surface Science 1999, 141, 89-100.

Mayo Clinic : About Cochlear Implant. http://www.mayoclinic.org/cochlear-
implants/about.html (June 23rd, 2006),

McClay, D. R.; Wessel, G. M.; Marchase., R. B., Intercellular Recognition:
Quantitation of Initial Binding Events. Proc. Natl. Acad. Sci. USA 1981, 78, :4975-
4979Mercurio AM, Laminin Receptors: Achieving Specificity Through Cooperation.
Trends Cell Biol 1995, 5, 419-423.

Mercurio AM, Laminin Receptors: Achieving Specificity Through Cooperation.
Trends Cell Biol 1995, 5, 419-423.

National Institute of Neurological Disorders and Stroke:Neural Prosthesis Program
(NPP). http://www.ninds.nih.gov/funding/research/npp/ (July17th, 2006),

Nichols, M. F.; Hahn, A. W.; James, W. J.; Sharma, A. K.; Yasuda, H. K., Cyclic
Voltammetry for The Study of Polymer Film Adhesion to Platinum Electrodes.
Biomaterials 1981, 2, (3), 161-165.

Nomizu, M. W., Benjamin S.;Weston, Christi A.;Kim, Woo Hoo; Kleinman, Hynda
K.; Yamada, Yoshihiko., Structure-Activity Study of a Laminin α1 Chain Active
Peptide Segment Ile-Lys-Val-Ala-Val (IKVAV). FEBS Letters 1995, 365, 227-231.

Ogawa, M., Photoprocesses in Mesoporous Silicas Prepared by a Supramolecular
Templating Approach. Journal of Photochemistry and Photobiology C:
Photochemistry Reviews 2002, 3, 129-146.

Pruden, K. G.; Beaudoin, S. In Ammonia Plasma Treatment of Parylene-C, Annual
Meeting Archive - American Institute of Chemical Engineers,, Indianapolis, IN,
United States,, 2002; American Institute of Chemical Engineers,: Indianapolis, IN,
United States,, 2002; pp 857-863.

116
Pruden, K. G.; Sinclair, K.; Beaudoin, S., Characterization of Parylene-N and
Parylene-C Photooxidation. Journal of Polymer Science: Part A: Polymer Chemistry
2003, 41, 1486-1496.

Ranieri, J. P. B., Ravi; Bekos, Evan J.; Gardella Jr., Joseph A.; Mathieu, Hans J. ;
Ruiz, Laurence; Aebischer, Patrick., Spatial Control of Neuronal Cell Attachment
and Differentiation on Covalently Patterned Laminin Oligopeptide Substrates. Int. J.
Devl. Neuroscience 1994, 12, (8), 725-735.

Ratner, B., Perspective and Possibilities in Biomaterials Science. In Bioimaterials
Science: An Introduction to Materials in Medicine, Ratner, B.; Hoffman, A.; Schoen,
F.; Lemons, J., Eds. Academic Press: San Diego, 1996; pp 465-468.

Sadhir, R. K.; James, W. J.; Yasuda, H. K.; Sharma, A. K.; Nichols, M. F.; Hahn, A.
W., The Adhesion of Glow-Discharge Polymers, Silastic and Parylene to
Implantable Platinum Electrodes: Results of Tensile Pull Test After Exposure to
Isotonic Sodium Chloride. Biomaterials 1981, 2, (4), 239-243.

Saneinejad, S.; Shoichet, M. S., Patterned Glass Surfaces Direct Cell Adhesion and
Process Outgrowth of Primary Neurons of The Central Nervous System. J. Biomed.
Mater. Res 1998, 42, 13-19.

Santiago, L. Y. N., Richard W.; Rubin, J. Peter; Marra, Kacey G., Peptide-Surface
Modification of Poly(caprolactone) with Laminin-Derived Sequences for Adipose-
Derived Stem Cell Applications. Biomaterials 2006, 27, 2962-2969.

Serruya, M. D.; Hatsopoulos, N. G.; Paninski, L.; Fellows, M. R.; Donoghue, J. P.,
Instant Neural Control of a Movement Signal. Nature 2002, 416, 141-142.

Sharma, R. K.; B., E., Management of Hereditary Retinal Degenerations: Present
Status and Future Directions. Surv. Ophthalmol. 1999, 43, (5), 427-444.

Silva, G. A. C., Catherine; Niece, Krista L.; Beniash, Elia; Harrington, Daniel A.;
Kessler, John A.;Stupp, Samuel I., Selective Differentiation of Neural Progenitor
Cells by High-Epitope Density Nanofibers. Science 2004, 303, 1352-1355.

Simon, A. D., Marie-Christine, Strategies and Results of Atomic Force Microscopy
in The Study of Cellular Adhesion. Micron 2006, 37, 1-13.

Sorribas, H. B., Dieter; Leder, Lukas; Sonderegger, Peter; Tiefenauer, Louis.,
Adhesion Proteins For A Tight Neuron-Electrode Contact. Journal of Neuroscience
Methods 2001, 104, 133-141.

117
Stark, N., Literature Review:Biological Safety of Parylene. Medical Plastic and
Biomaterials 1999.

Smyth, J.; Walker, M., Surface precoating in the 1980s: A First Taste of Cell-Matrix
Interactions. In Tissue engineering of vascular prosthetic grafts, Zilla, P.; Greisler,
H., Eds. R.G. Landes Company: Austin, 1999; pp 69-77.

Svedhem, S. D., D.; Ekeroth, J.; Kelly, J.; Hook, F.;Gold, J. , In situ Peptide-
Modified Supported Lipid Bilayers for Controlled Cell Attachment. Langmuir 2003,
19, 6730-6736.

Szwarc, M. J., New Monomers of The Quinoid Type and Their Polymers. Journal of
Polymer Science 1951, 6, 319-329.

Tomiyama, T. K., Hideshi; Kataoka, Ken-ichiro; Asano, Satoshi; Endo, Noriaki,
Rifampicin Inhibits The Toxicity of Pre-Aggregated Amyloid Peptides By Binding
to Peptide Fibrils and Preventing Amyloid-Cell Interaction. Biochem. J 1997, 322,
859-865.

Tong, Y. W.; Shoichet, M. S., Enhancing The Neuronal Interaction on
Fluoropolymer Surfaces with Mixed Peptides or Spacer Group Linkers. Biomaterials
2001, 22, 1029-1034.

Volker, M.; Shinoda, K.; Sach, H.; Grneier, H.; Schwarz, T.; al, e., In Vitro
Assessment of Subretinally Implanted Microphotodiode Arrays in Cats by Optical
Coherencetomography and Fluoresecein Angiography. Arch. Ophthalmol 2004, 242,
(9), 792-799.

Webster, T. J. E., Celaletdin; Doremus, Robert H.; Siegel, Richard W.; Bizios, R.,
Specific Proteins Mediate Enchanced Osteoblast Adhesion on Nanophase Ceramics.
Journal of Biomedical Material Research 2000, 51, 475-483.

Webvision : Gross Anatomy of the Eye.
http://webvision.med.utah.edu/anatomy.html (July 17th, 2006),

Webvision: Simple Anatomy of the Retina
http://webvision.med.utah.edu/imageswv/schem.jpeg (July 17th, 2006),

Weiland, J. D.; Liu, W.; Humayun, M. S., Retina Prosthesis. Annual Review
Biomedical Engineering 2005, 7, (361-401).
Weisz, J. M.; O'Connell, S. R.; Bressler, N. M., Treatement Guidelines for Age-
Related Macular Degeneration Based Upon Results From The Macular
Photocoagulation Study. Lippincott, Williams&Wilkins: Philadelphia, 2000; p 201-
211.
118
Williams, D., Bioinertness: An Outdated Principle. In Tissue Engineering of
Vascular Prosthetic Grafts, Zilla, P.; Greisler, H., Eds. R.G. Landes Company:
Austin, 1999; pp 459-462.

Yamada, M. K., Yuichi; Kasai, Shingo; Kato, Kozue; Mochizuki, Mayumi; Nishi,
Norio; Watanabe, Nobuhisa; Kleinman, Hynda K.; Yamada, Yoshihiko; Nomizu,
Motoyoshi, Ile-Lys-Val-Ala-Val (IKVAV)-Containing Laminin α1 Chain Peptides
Form Amyloid-Like Fibrils. FEBS Letters 2002, 530, 48-52.

Yamagishi, F. G., Investigation of Plasma Polymerized Films as Primers for
Parylene C Coatings on Neural Prosthesis Materials. Thin Solid Films 1991, 202, 39-
50.

Yousaf, M. N. H., Benjamin T.; Mrksich, Milan, Turning on Cell Migration with
Electroactive Substrates. Angew. Chem. Int. Ed. 2001, 40, (6), 1093-1096.

Yousaf, M. N. H., Benjamin T.; Mrksich, Milan, Using Electroactive Substrates to
Pattern The Attachment of Two Different Cell Populations. PNAS 2001, 98, (11),
5992-5996.

Yu, Q.; Deffeyes, J.; Yasuda, H., Engineering The Surface and Interface of Parylene
C Coatings by Low-Temperature Plasmas. Progress in Organic Coating 2001, 41,
(4), 247-253.

Zrenner, E.; Steet, A.; Weiss, S.; Aramant, R. B.; Guenther, E., Can Subretinal
Microphotoiodides Successfully Replace Degenerated Photoreceptors? . Vis. Res.
1999, 39, 2555-2567.
119

Appendix:
Direct Formation of Hydrogen Peroxide from Hydrogen and Oxygen by
Utilizing Viologen-Palladium Embedded in Inert Scaffolds

A.1. Introduction
Hydrogen peroxide is used industrially as cleaning agent due to its oxidative
strength (Table A.1).
Oxidant Oxidation potential (V)
Fluorine 3.0
Hydroxyl radical 2.8
Ozone 2.1
Hydrogen peroxide 1.8
Potassium permanganate 1.7
Chlorine dioxide 1.5
Chlorine 1.4

Table A.1. Oxdiation potential of various oxidants.
1

Since the by-product of hydrogen peroxide oxidation are water and oxygen gas,
hydrogen peroxide is a environmentally friendly replacement for various chemical
and bleach based cleaners; especially with the efforts to pursue a “greener” industrial
processing. The largest usage of hydrogen peroxide come from the pulp and paper
120

bleaching processes in which the industries are converting their methods from the
chlorine based bleaching applications.
1, 2
Other than pulp and paper bleaching,
textile industry also employed hydrogen peroxide to bleach variety of material.
Another advantage of hydrogen peroxide application for the textile industry besides
the environmental aspect is that hydrogen peroxide has no effect on many modern
dyes.
Another industrial application of hydrogen peroxide is in the wastewater
treatment processing. Hydrogen peroxide is used for odor control, corrosion control,
organic, inorganic, and metal oxidation from wastewater, and increases the oxygen
content in water and lowers the Biological Oxygen Demand (BOD) and the
Chemical Oxygen Demand (COD).
1-3

Besides usage in bleaching and environmental applications, hydrogen
peroxide is also utilized in chemical plants for production of epoxides such as
propylene oxide, per-acid compounds by reaction with carboxylic acid. Another
large consumption of hydrogen peroxide come from the chemical manufacturing of
sodium perborate and sodium percarbonate that are of interest product of the soap
and detergent industry to replace the hard chlorine-base cleaning agent.
2

121

Figure A.1. Schematic of Reidl-Pfleiderer manufacturing process of H
2
O
2
based on
autooxidation of alkyl-anthrahydroquinone.
3

Various commercial manufacturing process of hydrogen peroxide were
reported, ranging from electrolytic method,
4, 5
autoxidation of alcohol,
6, 7
and 2-
alkylanthraquinone. Currently, the majority manufacturing process of hydrogen
peroxide is known as the Reidl-Pfleiderer process which involve autooxidation of 2-
ethyl-9,10-dihydroxyanthracene to 2-ethylanthraquinone (Figure A.1).
3
This process
is a two-batch process, where in the first reaction vessel, the antraquinone is reacted
with hydrogen gas to form the anthrahydroquinone. The formation of the
anthrahydroquinone occurs in the presence of palladium or platinum to act as the
hydrogenation catalyst. The batch is then transferred to the second reaction vessel
H
2
,

Pd -s upported

ca ta l y s t,

Pd-black
O
2
R
R
OH
OH
O
O
H
2
O
2
as

byproduct

A lk yl -a n th rahydroquinone
i n

s o lv en t/working

solution
Alkyl-anthraquinone

in

solvent
122

were the anthrahydroquinone will be oxidized by oxygen to re-form the
anthraquinone while producing hydrogen peroxide as the reaction by-product.
Because the process is carried out in two separate vessels, the danger on
mixing the two gasses is eliminated. The autoxidation process is a viable process
only for large scale manufacturing plant due to the expensive cost of production
which include extraction and replacement of the hydrogenated (also referred as
“contaminated”) anthraquinone, extraction of the hydrogen peroxide from the
working solution, and the cost of the hydrogenation catalyst. Purchasing and
delivery expenses for high concentration of hydrogen peroxide are other reasons for
increase interest for finding a method for smaller scale on-site hydrogen peroxide
production.
1-3

Direct formation of hydrogen peroxide from H
2(g)
and O
2(g)
could minimize
the cost of the hydrogen peroxide separation and recycling of the alkyl-
anthraquinone, and a possible approach for on-site production of hydrogen peroxide.
The process is hindered by the danger of mixing the two gases, thus researches have
been carried out in effort to develop a safe procedure for this approach in hydrogen
peroxide production. The common theme on direct formation method utilized
supported metal catalyst in an acidic aqueous media.
8-13
Various supports were
reported ranging from carbon, silica and alumina.
13-18

A catalyst for direct production of hydrogen peroxide has been developed by
this research group utilizing an organic species known as viologen,
19-22
which has
high reaction kinetic for reaction with oxygen gas in presence of hydroxyl radical.
23

123

The approach is to incorporate both the metal and the viologen into an inert support
to create a heterogeneous catalyst such that both hydrogen and oxygen will be
activated in one reactor vessel.

Figure A.2. Proposed mechanism of H
2
O
2
direct production in presence of viologen

Microporous layered zirconium or hafnium phosphonate with viologen
groups acting as pillar bridges (MPOPV, M=Zr or Hf) containing metal such as Pd or
Pt have been successfully developed and tested for hydrogen peroxide direct
production (Figure A.3).
20, 24

124

PO
4
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
P
P
P
P
PO
4
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
P
P
P
P
X
-
X
-
X
-
n H
2
O n H
2
On H
2
O
Pd
PO
4
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
P
P
P
P
PO
4
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
P
P
P
P
X
-
X
-
X
-
n H
2
O n H
2
On H
2
O
Pd
PO
4
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
N+
N+
P
N+
N+
P
F
F
F
F
P
P
P
P
PO
4
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
Zr
Zr
PO
4
Zr
Zr
P
P
F
F
F
F
P
P
P
P
X
-
X
-
X
-
n H
2
O n H
2
On H
2
O
Pd Pd

Figure A.3. Illustrated structure of MPOPV (M=Zr or Hf) with Pd filling the pores
between the viologen pillars.

The best performance was achieved by phosphonate viologen containing palladium
synthesized via hydrothermal method as higher metal dispersions and incorporation
in between the layers were obtained. Unfortunately, the yield of catalyst per reaction
was limited to the size of the hydrothermal reaction vessel.
In this appendix, possibility of large scale MPOPV synthesis via reflux
method and hydrothermal method were investigated. As continuation of the project,
investigation on incorporation of viologen-palladium into other inert support such as
125

microporous silica was carried out. Synthetic approaches and catalytic activity
towards H
2
O
2
direct production will be discussed briefly.

A.2. Experimental Procedure
Reagents. Hafnium dichloride (HfOCl
2
.8H
2
O,98+%) was obtained from Alfa Aesar.
4,4’-dipyridyl (C
10
N
2
H
12
), potassium tetrachloropalladate (K
2
PdCl
4
), tetraethyl ortho
silicate (TEOS, C
4
H
20
O
4
,95%), and cetyltrimethyl ammonium bromide (CTABr,
C
19
H4
2
BrN) were purchased from Aldrich. Phosphoric acid (H
3
PO
4
, 85%) was
purchased from Mallinkcrodt. Iodopropyl methoxysilane (C
6
H
15
SiO
3
I
2
) and
bromopropyl methoxysiliane (C
6
H
15
SiO
3
Br
2
) were obtained from Gelest, Inc.
Hydrofluoric acid (HF, 48%) was purchased from EM. All reagents were used
without further purification.

Instruments. UV-Vis measurement was performed on AVIV model 14DS UV-Vis-
IR spectrophotometer. Concentration of hydrogen and oxygen during H
2
O
2
direct
production were controlled by mass flowmeter / controller LB-366 equipped with
electronic controller model CM2 by Porter Instrument Company, Inc. Step-scanned
X-ray powder diffraction (XRD) data were collected on the finely ground sample
using Rigaku, Rotaflex RU02a with Cu K α radiation with a 2 Θ scan from 4º to 80 º.
Scanning electron microscope (SEM) images were obtained using Cambridge 360
Scanning Electron Microscope equipped with Link Energy Dispersive Spectroscopy
(EDS).
126

PV.Cl
2
. Diethyl-bromopropyl phosphonate (25 g) and 4,4’-dipyridyl (7.35 g) were
combined in 125 mL of distilled (DI) water and heated to reflux for 3 days.
Concentrated HCl (125 mL) was added and the reflux continued for 16 hours. The
solution was concentrated to approximately 120 mL, and chilled in an ice bath.
Isopropanol (550 mL) was added dropwise while stirring. The resulting mixture was
stirred in an ice bath for one hour followed by filtration and copious rinsing with
cold isopropanol. The product was air dried to give 95.4% yield.
1
H NMR (ppm in
D2O) : 9.1 (d), 8.5 (d), 4.2 (m), 2.0 (m)

Reflux synthesis of HfPOPV. Reflux was carried out in Teflon boiling flask and
condenser. Two mixtures were prepared in plastic beakers. Reactant mixture A
contain 1.865 g of HfOCl
2
.8H
2
O, 3.967 mL 50% HF (10x stoichiometri) and 85 mL
DI H
2
O. In the second beaker, reactant mixture B was prepared containing 2.536 g of
PV.Cl
2
and 0.389 mL 85% H
3
PO
4
, in 42.5 mL DI H
2
O. An equimolar aqueous
solution of K
2
PdCl
4
(1.865 g, 42.5 mL) was slowly added to mixture B under
ultrasonication to obtain fine precipitates. This mixture was then added slowly to A
in the boiling flask, also under ultrasonication condition and let refluxing for 7 days
under N
2
. The precipitates were collected and rinsed copiously with DI water and
acetone before let to air dried to give 3.476 grams.

127

Large scale hydrothermal synthesis of HfPOPV. Teflon-lined bombs (PARR 4748
Acid Digestion Bomb) with maximum volumes of 125 mL were used for
hydrothermal synthesis. Reactant mixture A was prepared by dissolving 2.802 g of
HfOCl
2
.8H
2
O along with 1.095 mL of 50% HF (5 times the stoichiometri amount) in
51 mL of water. Reactant mixture A was prepared directly in the bomb. Reactant
mixture B contained 1.518g of PV.Cl
2
and 0.234 mL 85% H
3
PO
4
, along with 25.5
mL DI water. An equal volume of aqueous solution of K
2
PdCl
4
(0.855g in 25.5 mL
DI water) was slowly added to mixture B under ultrasonication to obtain fine
precipitates. This mixture was the added slowly to A in the bomb, also under
ultrasonication condition. The bomb was sealed and placed in an oven kept at 125ºC
for a period of 5 days. Product was collected by filtration followed by copious
rinsing with DI water and acetone before dried at room temperature to give 2.30
grams (60.65% yield)

SiV.Br
2
. 4,4’-dipyridyl (1. 406g) and bromopropylmethoxysilane (5.61 mL) were
refluxed in dry CH
3
CN (125 mL) under N
2
for 4 days. The mixture was
concentrated to approximately half of its original volume, followed by precipitation
of the product by addition of ethyl ether to give the product (45% yield).
1
H NMR
(ppm, CD
3
OD): 9.17 (4H,d), 8.59 (4H, d), 4.63 (4H, t), 3.47 (18H, s), 2.07 (4H, m),
and 0.63 (4H, m)

128

Hydrothermal synthesis of silica-viologen (SV). In a polyethylene bottle, CTABr
(0.437 g) was dissolved in 10 mL DI H
2
O. TEOS (2.086 g) was slowly dissolved in
CTABr under stirring condition, followed by addition of NH
4
OH (4.53 mL).
Aqueous solution of SiV.Br
2
(0.0643g, 10 mL) was slowly introduced to the CTABr-
TEOS mixture under sonication and let stirring for additional 10 minutes. The
mixture was let to react inside the closed polyethylene bottle for 5 days at 85 ºC. The
solid were collected and was with copious amount of DI H
2
O. CTABr were removed
at 200ºC under high vacuum (10
-2
torr) for 2 hours to give the silica-viologen
scaffold (0.650 g). Pd loading was achieved by ion-exchanged of the product with of
K
2
PdCl
4
(0.2146 g per gram of silica-viologen scaffold) for 60 minutes

Hydrothermal synthesis of silica-viologen/PdCl
4
(SVPd). Aqueous SiV.Br
2

(0.0643 g, 5 mL) was ion-exchanged with equal volume of K
2
PdCl
4
(0.0326 g) under
rapid stirring for 20 minutes. In a polyethylene bottle, CTABr (0.437 g) was
dissolved in 10 mL DI H
2
O. TEOS (2.086 g) was slowly dissolved in CTABr under
stirring condition, followed by addition of NH
4
OH (4.53 mL). The ion-exchanged
SiV. Mixture was slowly introduced to the CTABr-TEOS mixture under sonication
and let stirring for additional 10 minutes. The mixture was let to react inside the
closed polyethylene bottle for 5 days at 85 ºC. The solids (0.739 g) were collected
and washed with copious amount of DI H
2
O. CTABr were removed at 200ºC under
high vacuum (10
-2
torr) for 2 hours.

129

Catalyst pretreatment. Pre-treatment of the catalyst prior to any hydrogen peroxide
production experiment was needed since the catalyst might contain oxidized metal
(PdO) during storage for an extended period.
24
Pre-reduction of the catalyst was
carried out by bubbling a stream of pure H
2(g)
into the mixture of catalyst in DI
water at room temperature 45-50 ºC for 3 hours.

Line A, O
2
Line B, H
2
Flow control
valves
Flow control
valves
Bubblers
Reactors
V1
A
V2
A
V3
A
V4
A
V1
B
V2
B
V3
B V4
B
B1 B2
B3
B4
R1
R2
R3
R4

Figure A.4. Schematic of experimental setup for H
2
O
2
direct production.

Production of H
2
O
2
. Reactions were carried out at 0ºC. Water jacketed centrifuge
tube was used as reactor, charged with a slurry of catalyst in 30 mL methanol,
consisting of 25 mg of HfPOPV catalyst or 0.1 gram of Silica-Viologen catalyst, 51
µL of H
2
SO
4
conc. The flow of oxygen and hydrogen were controlled by flowmeters
130

to 200 cc/min and 20 cc/min respectively. The 10:1 ratio is in the safe range of
mixing of the two gases. Hydrogen gas was introduced to the system via gas
dispersion tube to achieve better mixing. To minimize medium evaporation due to
the high flow rate of oxygen, the gas was passed through a bubbler filled with
methanol prior to entering the reactor.

Measurement of H
2
O
2
concentration. The measurement of hydrogen peroxide
concentration was carried out colorimetrically. An aliquoat of the reaction medium
was removed and diluted appropriately. Reaction of hydrogen peroxide in the sample
with a commercial CHEMETRICS indicator (a mixture of ferrous ion and
ammonium thiocyanate) resulted in formation of a red solution with an absorbance
maximum at 475 nm. The intensity of this peak can then be correlated with the
concentration of the H
2
O
2
in the reactor by Beer’s Law.

131

A.3. Results and Discussion
A.3.1. Hafnium Phosphonate-Viologen for Direct Formation of Hydrogen
Peroxide
0 5 10 15 20 25 30
Counts (a.u)
2 Θ
reflux synthesis

Figure A.5. XRD spectra of HfPOPV. synthesized via reflux synthesis

Based on the XRD spectra collected for the two catalysts, reflux synthesis
(Figure A.5.) appeared to form the unfavored structure of a dense layer hafnium
phosphonate with smaller pores size HfPV (Figure A.6a), instead of the more porous
structure of HfPOPV (Figure A.6b).

132

a b

Figure A.6. Illustration of two layered phosphonate viologen reported by Poojary, et.
al a) HfPV, and b) HfPOPV. The viologen is represented by the positiviely charged
groups and the solid blocks illustrate the phosphonate layers.
25

This result is in agreement with previous XRD spectra reported by Poojary,
et. al (Figure A.7c).
25
Although the product did incorporate viologen and palladium
metal, no catalytic activity was observed. This could be due to 1) less intake of
palladium metal into the smaller pore sizes, 2) the difficulty of the oxygen gas to
come close to the viologen radical, thus preventing production of hydrogen peroxide.

133

Figure A.7. Reported XRD spectra of possible porous phosphonate layer containing
viologen. a) pure MPV, b) mixture of MPV and MPOPV, c) pure MPOPV.
26

0 5 10 15 20 25 30
Counts (a.u)
2 Θ
hydrothermal synthesis

Figure A.8. XRD spectra of HfPOPV. synthesized via large scale hydrothermal
synthesis.

134

Hydrothermal synthesis is reported to exclusively form the more porous
hafnium phosphonate.
21, 22, 24
When the scale of the reaction was increase 3 folds,
the XRD spectra shown a mix structure of HfPV and HfPOPV (Figure A.8) in
referenced to the reported spectra (Figure A.7b). The reaction product was tested for
hydrogen peroxide direct formation in 0-4ºC methanol with 10:1 ratio of O
2
and H
2
,
and was found to be an active catalyst. The hydrogen peroxide production in the
presence of this mixed hafnium phosphonate reached 190 mM after 3.5 hours, which
are comparable to the reported catalytic performance of pure HfPOPV (Figure
A.9).
24

0 50 100 150 200 250
0
50
100
150
200
Concentration (mM)
Time (minutes)

Figure A.9. Catalytic direct formation of hydrogen peroxide in presence of HfPOPV

It is apparent that large scale synthesis of the phosphonate viologen catalyst
must be carried out via hydrothermal synthesis method. Experimental trials to
synthesizing the catalyst via reflux produced a structurally denser layered
135

phosphonate with embedded viologen pillars. This layered phosphonate-viologen
(HfPV) was found to be catalytically inactive for H
2
O
2
production.
Attempts to scale-up the hydrothermal synthetic method resulted in product
with mixed phosphonate layer structures based on XRD analysis. It is not known
whether the product is a mixture of two separate type of phosphonate layers HfPV
and HfPOPV, or one product with mixed pillared structure. The mix phosphonate
layer is able to catalytically produced hydrogen peroxide directly from H
2
and O
2
in
a similar activity as the pure HfPOPV catalyst reported earlier.

A.3.2. Silica- Viologen for Catalytic Direct Formation of H
2
O
2

Formation of periodic porous silica with various pore sizes and shapes
utilized various hydrocarbon surfactants as site directing agent have been a topic of
various researches.
27-33
The hydrocarbon surfactants act as site directing agent as a
result of the interaction between the surfactant and the inorganic species. The soluble
anionic inorganic species displace the surfactant anions and induce a reorganization
of the organic surfactant molecules to form micelles structures surrounded by the
inorganic species.
27, 32, 33
At higher temperature, polymerization of the inorganic
species occurred producing periodic structures which shapes are based on the
templates created from the reorganization of the surfactant and the molecular
inorganic species at lower temperature. Removal of the surfactants from the product
produced periodic mesoporous structures. The ability to incorporate organic
molecules, including viologen, onto the walls of the periodic structures has also been
136

reported.
30, 31
Removal of the hydrocarbon surfactant will forms periodic mesoporous
silica with viologen groups covalently bound to the wall of the pores. The structures
could be utilized for direct production of H
2
O
2
with a similar mechanism as HfPOPV
by incorporating palladium into the pores of the silica, in close distant with the
viologen groups.
Two methods were tested to form the silica viologen catalyst. In the first
method, SiV.Br
2
(Figure A.10) were embedded onto the silica pores via
hydrothermal synthesis according to the reported procedure.
30, 34, 35

.
N N
Si
O
O
O
Si
O
O
O
2Br

Figure A.10. Chemical structure of SiV.Br2

The surfactant was removed at 200ºC at 10
-2
torr for 2 hours and complete removal
awas confirmed by disappearance of the CTABr peaks in the FTIR spectra at 1470
cm
-1
(Figure A.11) to give a yellowish powder product.
137

1800 1700 1600 1500 1400 1300
Absorbance (a.u)
Wavelenght number (cm
-1
)
hydrothermal synthesis
after vacuum dried
after ion-exchanged

Figure A.11. FTIR spectra of SV. The characteristic viologen and the site directing
agent CTABr bands were observed at 1650 cm
-1
and at 1470 cm
-1
respectively.

The quantity of viologen that were bound covalently to the silica structure
was determined from elemental mass analysis and indicated 0.66 mmoles of
viologen per gram of product. This value translates to 82.7 % incorporation of the
viologen into the silica.
The reported pore size of the silica is 3.8 nm,
30
which allow the possibility
for Pd loading into the pores and be spatially close with the viologen groups.
Palladium loading was achieved via ion-exchange of PdCl
4
2-
with the viologen
counter ions. Palladium aggregates were then reduced to form Pd
(0)
by H
2(g)
prior to
catalytic H
2
O
2
formation test. Catalyst activity of the product (SV) was tested with
the same experimental setup as the one used for HfPOPV.
138

The H
2
O
2
formation in presence of the ion-exchanged silica-viologen was monitored
for 9 hours, where H
2
O
2
production at about 4.5 mM was observed (Figure A.12).
0 50 100 150 200 250 300 350 400
0
1
2
3
4
5
Catalyst: 0.1 g SV
Solvent : MeOH (30 mL) + H
2
SO
4
(51 μL)
Flowrate: H
2
= 20 cc/min; O
2
= 200 cc/min
Concentration (mM)
Time (minutes)

Figure A.12. Direct H
2
O
2
production with SV as catalyst at 4ºC in methanol.
0 100 200 300 400 500 600
0
5
10
15
20
Concentration (mM)
Time (minutes)
Catalyst: 0.10 g SVPd
Solvent : MeOH (30 mL) + conc. H
2
SO
4
(51 μL)
Flow rate : O
2
= 200 cc/min
H
2
= 20 cc/min

Figure A. 13. Direct formation of H
2
O
2
in presence of SVPd at 4ºC in methanol.
139

In the second method, SiV.Br
2
was ion-exchanged with K
2
PdCl
4
prior to
formation of silica structure to form orange powder product (SVPd). Similarly, the
catalyst was reduced prior to catalytic H
2
O
2
production experiment. The product was
tested for catalytic H
2
O
2
production with the same setup as the one used for HfPOPV
and SV catalyst, where formation of H
2
O
2
was observed. Ion-exchange of PdCl
4
2-

with SiV. Br
2
prior to formation of the silica scaffold appears to increase hydrogen
peroxide production (Figure A.13).

Figure A.14. SEM image of SVPd showing two different particles presence in the
product. EDS measurements found palladium only exist in the larger particles.

Although the catalytic activity appeared higher than the first approach, it is
still significantly lower than HfPOPV. Examination using scanning electron
microscopy (SEM) equipped with energy dispersive spectrometer (EDS) showed the
product consists of a mixture of two different particle aggregrates. The larger
140

particles were determined by EDS to mainly contain palladium elements with no
silica presence (Figure A.14). Examination on SEM images of SV also provided a
similar observation (Figure A.15).

Figure A.15. SEM image of SV. Similar to SVPd, two different clusters were
presence in the product where EDS measurements found palladium only exist in the
larger particles.

The increase in H
2
O
2
production of SVPd in comparison with SV could be due to
higher Pd loading into the silica viologen structure as the reduced catalyst of SVPd
appear darker in color than that of SV.
141

Figure A.16. TEM image analysis of HfPOPV catalyst where no large cluster of Pd
were observed
19

Comparing the silica-viologen system with the HfPOPV system, significant
difference was observed on the size of the Pd cluster. In HfPOPV, no large Pd
structures were observed (Figure A.16), allowing the metal to be in close distant to
viologen group,
19, 24
SEM analysis of SV and SVPd showed that in presence of Pd
metal, the silica-viologen did not form a periodic mesoporous structure and appear to
encapsulated the Pd cluster. Further investigation should be targeted on the study of
the silica-viologen structure and on finding a procedure that can improve the Pd
loading into the silica-viologen scaffold, such that high metal loading can be
achieved while preserving porosity to allow access for oxygen and hydrogen to react
with the two active component of the catalyst. Usage of finer Pd particle produce by
142

citrate reduction or sonochemical method
36
could be an interesting viable approach
to incorporate Pd particles into the silica scaffold.

A.4. Summary
Direct production of H
2
O
2
have been a topic of various researches in attempt
to resolve the safety challenges in reacting H
2(g)
and O
2(g)
and provide a viable
system that can be applied for on-site production. Previous work in the group
proposed a system where H
2
O
2
direct production was assisted by layered
phosphonate viologen. The advantage of the catalyst lies on the possibility to activate
both gases for H
2
O
2
formation, Pd aggregates to activate H
2(g)
and viologen for O
2(g)
.
Synthesis of the active catalyst in large scale via reflux method and hydrothermal
method were explored. Both methods produced layered phosphonate layer
containing Pd clusters. Reflux method produced HfPV, a denser layered phosphonate
containing viologen pillars, which was found to be inactive toward H
2
O
2
production.
The product of hydrothermal synthesis was analyzed with XRD and showed to
contain a mixture of HfPV and HfPOPV. The mixture showed the same level of
catalytic activity as the reported value for pure HfPOPV.
Preliminary investigation on adaptation of the above catalyst into a slilica
scaffold was carried out. Embedding viologen into the scaffold was achieved by
modifying the end group of PV.Cl
2
with methoxy-silane group as in SiV. Reaction of
SiV and TEOS according to the published method was carried out and determined to
incorporate 82.7% of the SiV reagents into the silica scaffold. Attempt for Pd
143

incorporation post formation of the silica-viologen scaffold was performed and
catalytic direct formation of H
2
O
2
was observed. The low yield of H
2
O
2
production
suggested that the synthesis did not include oxygen activation by the viologen groups
embedded inside the silica scaffold. Higher production of H
2
O
2
was achieved by
introducing viologen-palladium complex prior to formation of the silica scaffold.
Upon examination of SEM images of the two catalysts, it appears that the increase of
H
2
O
2
production in presence of SVPd was due mainly to the increase in Pd loading
into the scaffold. Thus in both catalytic systems, viologen groups embedded into the
silica scaffold were not involve in the production of hydrogen peroxide.
Improvement of the catalyst can be achieved if oxygen gas could react with
the activated hydrogen to produce H
2
O
2
with the assistance of violgen groups. To
increase viologen participation, the size of Pd clusters need to be decreased
significantly such that access to viologen groups are not blocked. Another possibility
is to investigate the usage of other site-directing agent in the synthesis of silica-
viologen scaffold to increase the pore sizes of the scaffold.

144

A.5. Appendix References
1. Introduction to Hydrogen Peroxide-Application Overview.
http://www.h2o2.com/intro/overview.html

2. Local and In-situ Generation of Hydrogen Peroxide: a Position Paper.
http://www.iac.org.uk/pages/pospaper3.html (September 18, 2003),

3. Hess, W. T., Hydrogen Peroxide. In Encyclopedia of chemical Technology,
4th Edition, Kirk, R.; Othmer, D., Eds. Wiley Interscience: New York, 1995; Vol.
13, pp 961-995.

4. Berzins, T.; Gosser, L. W. Electrochemical catalysis of H
2
O
2
. U.S. Pat.
5,112,702, May 12, 1992, 1992.

5. McIntrye, J. A.; Phillips, R. F. Method for Electrolytic Production of
Alkaline Peroxide Solutions. U. S. Pat. 4,431,494, 1984.

6. Albal, R. S.; R.N.Cochran Production of Hydrogen Peroxide. U. S. Pat
4,994,625, 1991.

7. Cochran, R. N.; Candela, L. M. Recovery of Hydrogen Peroxide U. S. Pat.
4,897,085, 1990.

8. Maraschino, M. J. Process for Producing Hydrogen Peroxide. U. S. Pat.
5,169,618, 1992.

9. Kanada, T.; K.Nagai; Nawata, T. Process for Making Hydrogen Peroxide. U.
S. Pat. 5,104,635, 1990.

10. Michaelson, R. C. Preparation of Hydrogen Peroxide from Its Elements. U. S.
Pat. 4,347,232, 1982.

11. Mosley, F.; Dryer, P. N. Method of Manufacturing Hydrogen Peroxide. U.S.
Pat. 4,336,240, 1982.

12. Dryer, P. N.; Mosley, F. Synthesis of Hydrogen Peroxide. U.S. Pat.
4,128,627, 1978.

13. Izumi, Y.; Miyazaki, H.; Kawahara, S.-i. Process for Preparing Hydrogen
Peroxide. U.S. Pat. 4,009,252, 1977.

14. Chuang, K. T.; Zhou, B. Production of Hydrogen Peroxide. U.S. Pat.
5,338,531, 1994.
145

15. Gosser, L. W.; Paoli, M. A. Method for Catalytic Production of Hydrogen
Peroxide. U.S. Pat. 5,135,731, 1992.

16. Huckins, H. A. Method for Producing Hydrogen Peroxide. U.S. Pat.
5,641,467, 1997.

17. Augustine I. Dalton, J.; Greskovich, E. J.; Skinner, R. W. Process for
Production of Hydrogen Peroxide. U.S. Pat. 4,389,390, 1983.

18. Zhou, B.; Lee, L.-K. Catalyst and Process for Direct Catalysist Production of
Hydrogen Peroxide (H
2
O
2
). U.S. Pat. 6,168,775, January 2, 2001, 2001.

19. Krishnan, V. V.; Dokoutchaev, A. G.; Thompson, M. E., Direct Production of
Hydrogen Peroxide with Palladium Supported on Phosphate Viologen Phosphonate
Catalysts. Journal of Catalysis 2000, 196, 366-374.

20. Vermeulen, L. A.; Thompson, M. E., Synthesis and Photochemical Properties
of Porous Zirconium Viologen Phosphonate Compounds. Chem. Mater. 1994, 6, 77-
81.

21. Thompson;, M. E.; Krishnan, V. V.; Dokoutchaev, A. G.; Abdel-Razzaq, F.;
Rice, S. C. Method for Catalytic Production of Hydrogen Peroxide and Catalyst
Therefor. U.S. Pat. 5,976,486, 1999.

22. Thompson, M. E.; Krishnan, V. V.; Dokoutchaev, A. G. Method of Making a
Bulk Catalyst. U. S. Pat. 6,143,688, 2000.

23. Farrington, J. A.; Ebertb, M.; Landb, E. J.; Fletcher, K., Bipyridylium
Quaternary Salts and Related Compounds. V. Pulse Radiolysis Studies of The
Reaction of Paraquat Radical with Oxygen. Implications for The Mode of Action of
Bipyridyl Herbicides Biochimica et Biophysica Acta (BBA) - Bioenergetics 1973,
314, (3), 372-281.

24. Dokoutchaev, A. G.; Krishnan, V. V.; Thompson, M. E.; Balsubramanian,
M., Platinum and Palladium Incorporation into Phosphate/Viologen-Phosphonate of
Zirconium and Hafnium: Synthesis and Characterization. Journal of Molecular
Structure 1998, 469, 191-205.

25. Poojary, D. M.; Vermeulen, L. A.; Vicenzi, E.; Clearfield, A.; Thompson, M.
E., Structure of a Novel Layered Zirconium Diphsphonate Compound:
Zr2(O3PCH2CH2-viologen-CH2CH2PO3)F6.2H2O. Chemistry of Materials 1994,
6, (1845-1849).

146

26. Byrd, H.; Clearfield, A.; Poojary, D.; Reis, K. P.; Thompson, M. E., Crystal
Structure of a Porous Zirconium Phosphate/Phosphonate Compound and
Photocatalytic Hydrogen Production from Related Materials. Chem. Mater. 1996, 8,
2239-2246.

27. Zhao, D.; Huo, Q.; Feng, J.; Chmelka, B. F.; Stucky, G. D., Nonionic
Triblock and Star Diblock Copolymer and Oligomeric Surfactant Synthesis of
Highly Prdered, Hydrothermally Stable, Mesoporous Silica Structures. J. Am. Chem.
Soc. 1998, 120, 6024-6036.

28. Ogawa, M., Photoprocesses in Mesoporous Silicas Prepared by a
Supramolecular Templating Approach. Journal of Photochemistry and Photobiology
C: Photochemistry Reviews 2002, 3, 129-146.

29. Che, S.; Lim, S.; Kaneda, M.; Yoshitake, H.; Terasaki, O.; Tatsumi, T., The
Effect of the Counteranion on the Formation of Mesoporous Materials under the
Acidic Synthesis Process. J. Am. Chem. Soc. 2002, 124, 13962-13963.

30. Alvaro, M.; Ferrer, B.; Fornes, V.; Garcia, H., A Periodic Mesoporous
Organosilica Containing Electron Acceptor Viologen Units. Chem. Commun 2001,
2546-2547.

31. Alvaro, M.; Ferrer, B.; Garcia, H.; Rey, F., Photochemical Modification of
The Surface Area and Tortuosity of a Trans-1,2-bis(4-pyridyl)ethylene Periodic
Mesoporous MCM Organosilica. Chem. Commun 2002, 2012-2013.

32. Yang, Q.; Kapoor, M. P.; Inagaki, S., Sulfuric Acid-Functionalized
Mesoporous Benzene-Silica with a Molecular-Scale Periodicity in the Walls. J. Am.
Chem. Soc. 2002, 124, 9694-9695.

33. Huo, Q.; Margolese, D. I.; Cisela, U.; Demuth, D. G.; Feng, P.; Gier, T. E.;
Sieger, P.; Firouzi, A.; Chmelka, B. F.; Schuth, F.; Stucky, G. D., Organization of
Organic Molecules with Inorganic Molecular Species into Nanocomposite Biphase
Arrays. Chem. Mater. 1994, 6, 1176-1191.

34. Bookbinder, D. C.; Lewis, N. S.; Wrighton, M. S., Heterogeneous One-
Electron REduction of Metal Containing Biological Molecules Using Molecular
Hydrogen as the Reductant: Synthesis and Use of a Surface-Confined Viologen
Redox Mediator That Equilibrates with Hydrogen. J. Am. Chem. Soc. 1981, 103,
7656-7659.

35. Dominey, R. N.; Lewis, N. S.; Bruce, J. A.; Bookbinder, D. C.; Wrighton, M.
S., Improvement of Photoelectrochemical Hydrogen Generation by Surface
147

Modification of p-Type Silicon Semiconductor Photocathodes. J. Am. Chem. Soc.
1982, 104, 467-482.

36. Dokoutchaev, A. G.; James, J. T.; Koene, S. C.; Pathak, S.; Prakash, G. K. S.;
Thompson, M. E., Colloidal Metal Deposition onto Functionalized Polystyrene
Microsphere. Chem. Mater. 1999, 11, 2389-2399.

Asset Metadata

Creator Wahjudi, Paulin Nadi (author)

Core Title Surface modification of parylene C and indium tin oxide for retinal and cortical prostheses

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Chemistry

Publication Date 05/31/2010

Defense Date 03/19/2007

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag cortical prosthesis,indium tin oxide,OAI-PMH Harvest,Parylene C,retinal prosthesis,surface modification

Language English

Advisor Thompson, Mark E. (committee chair), Bau, Robert (committee member), Reisler, Hannah (committee member), Shing, Katherine S. (committee member), Weber, William P. (committee member)

Creator Email wahjudi@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m362

Unique identifier UC1232034

Identifier etd-Wahjudi-20070410 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-327639 (legacy record id),usctheses-m362 (legacy record id)

Legacy Identifier etd-Wahjudi-20070410.pdf

Dmrecord 327639

Document Type Dissertation

Rights Wahjudi, Paulin Nadi

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email uscdl@usc.edu

Abstract (if available)

Abstract In the world of prosthesis, the interaction between the material and the tissue is as essential as the efficiency of the device itself. Development in medical devices to improve health and health care would be immensely benefited by the ability to tailor the interface of tissue and material according to the specific needs of each device. In this thesis, works on surface modification of parylene C film and indium tin oxide (ITO) for retinal and cortical prosthesis are presented. As the works combine the multidisciplinary fields of chemistry, material science, and biology, chapter one is targeted to provide general background of the retinal and prosthesis, and the tissue-material interface that are required for the two prostheses. The second chapter will discuss how selective chemical surface modifications of parylene C thin film were carried out to introduce various functional groups and grafting of poly-N-isopropylacrylamide onto the parylene surface via atom transfer radical polymerization. The improvement of gold metal electrode and tissue adhesion onto the modified thin film will also be discussed in this chapter. Works on ITO surface will be describe in chapter three, including the selective surface modification of ITO, methods to functionalized ITO surface with cell adhesion molecules (CAMs), and the neuron cell culture results. Lastly, the appendix will discussed previous work on direct production of hydrogen peroxide utilizing viologen-palladium embedded in inert scaffold.