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Developments in bio-nanotube science and technology

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Developments in bio-nanotube science and technology
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Enzymes ( jstor )
Molecules ( jstor )
Nanoparticles ( jstor )
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DEVELOPMENTS IN BIO-NANOTUBE SCIENCE AND TECHNOLOGY


By

DAVID TANNER MITCHELL

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


2002



























This dissertation is dedicated to my parents, Dr. James P. and Terri Tanner Mitchell.














ACKNOWLEDGMENTS

I would like to thank Dr. Charles Martin and the Martin group members I have had the

opportunity to work with over the course of my tenure. Dr. Martin never stopped

thinking of applications for my research and taught me much about effective scientific

communication. The Martin group members have been unanimously supportive and

terrific examples of ingenuity and perseverance. Kshama Jirage, Veronica Cepak, John

Hulteen, and Silvia di Vito Luebben showed great patience in training me in various

analytical techniques and instrumentation. Michelle Steen and Shawn Sapp gave

insightful advice on experiment design. In particular I would like to thank Charlie

Patrissi for his help in proofreading this dissertation.

Lacramioara Trofin and Naichao Lee spent many hours growing aluminum oxide for

my experiments, for which I am in their debt. Marc Wirtz and Erich Steinle taught me

the fundamentals of impedance spectroscopy and both were helpful in running

experiments on ion channel mimetic membranes. The enantioselective antibody

experiments would have been impossible without the aid of Sang Bok Lee who helped

both procure the antibody and analyze the extraction experiment results.

Scott Miller, Elizabeth Medeiros, and Shufang Yu discussed the experiments in this

dissertation with me at length and provided many helpful insights.

Professor Vaneica Young was very kind to run and help interpret the XPS

experiments. Drs. Bob Lee, John Chandler and Ameila Dempere gave me excellent

training in electron microscopy. Professor Dave Grainger was instrumental in my








decision where to attend graduate school and has been a continual source of sage

scientific and career advice. Drs. Donn Dennis and Tim Morey were very helpful in

discussing experimental results and planning future experiments. Drs. Hans Soderlund

and Tarja Nevanen kindly provided the enantioselective antibody ENAI lHis.

My life during graduate school would have been much less interesting without the

leavening influences ofLiisa Kauri, Cathy Zelenski, Dean Medeiros, and Nathalie Fosse.

Special thanks for keeping me climbing to my friends Craig Luebben, Lane Hale, Corey

Salzer, Andrea Koenig, Jason McDannold, Joie and Jann Contado, Mitzy Carlo, and

Shirley Blandon.

Finally, my family provided unflagging moral support throughout my graduate career.

For this support I thank Heather, John, Benjamin, and James Howell; Jonathan,

Rebekkah, Anthony, Rachel, Adam, and Tanner Mitchell; Heidi Mitchell, Adam and

Shannon Mitchell, and Jenni Mae, Matthew, and Ashley Mitchell. Above all, thanks to

my parents Jim and Terri Mitchell for instilling in me a love of learning.















TABLE OF CONTENTS
page

A CK N O W LED G M EN TS ................................................................................... iii

ABSTRACT .... ..... .................................... viii

CHAPTER

1 INTRODUCTION AND TEMPLATE SYNTHESIS........................................ 1

Introduction................... ................................. ......... .. ................. ....... 1
B ack g ro u n d ............................................................................. ... ................. ...... 3
Template Synthesis .......................... ........................................... .................3
Three-dim ensional templates ............................................ ................... 3
Two-dim ensional tem plates ............................. .... ......... ................. 5
One-dimensional templates................................... 5
A lum ina G row th ............................................ ....... ..................................... 11
P olishing ................................................................... ... ...... 11
Perfect pores ......................................................................... .............. 15
Separation of the alumina film ................ .............................. .......... ..... 16
Sol-G el C hem istry ................ ...................... ... 19
S ila n iz atio n ..................................................................... ................. 2 0
C chapter Sum m aries...................................... ................................ ......... ... 24

2 CHEMICAL MODIFICATION OF SILICA NANOTUBES.................................... 26

In tro d u ctio n ......................................................................................................... 2 6
B background .......................................................... ......... ........ ............ .. 26
D rug D delivery .................... .. .. .......... .... ................... 27
Types of nanoparticles for drug delivery ..................................................... 27
Advantages of using nanoparticles for drug delivery ..................................31
Engineered Sorbants and Extractants .....................................................32
E xperim mental ............................................ ............. ................. ............... .... 33
T em plate Synthesis ............................................................................................. 33
Silanization ...................... ...... ....... .................. ....... 34
Protein Modification ............................................................ .......... 39
Biotinylation .................................... ............................... ........ 39
C om posite N anotubes ............................................... ................ ................ 41
R esu lts and D discussion ............................................................................................. 42
D ifferential Inside/Outside M odification ............................. ........................... 42









Hydrophilic/lipophilic nanotubes ............................................ ............ 42
Quinine/Au nanoparticle nanotubes.................. .. ............ ..... 45
Au nanoparticle/fluorescent protein nanotubes .............................................47
N anotubes as Extractants .............................................. .............................. 49
Cls nanotubes for drug detoxification.............................. ............. 49
EDTA nanotubes for copper(II) extraction .................................... 56
Composite Silica-Polymer Nanotubes .................... .................... 57
Insulating polymers................................................ 58
P o ly (p y rro le)........................................................................ .................. 59
Self-A ssem bly .............. ............... ................................ .... ......... .......63
Biotin/avidin self-assem bly......................... ........................................ 64
D ithiol self-assem bly ............... .......................................................... 69
C conclusions .................. ................................ ......... .. ............... 70

3 PROTEIN MODIFICATION OF SILICA NANOTUBES ....................................... 72

In tro d u ctio n ......................................................................................................... 7 2
Background .................................. ................. ............ .............. ..... 72
S m art N ano particles ............................................................................................ 73
Biomolecule-Modified Particles............................................... 76
E x p erim mental ................................................................ ...... .... ........ .... .... 7 8
R esu lts and D iscu ssion ..................................................................................... 8 1
P rotein C oupling ............................... ................................ .... ....... .............. 82
Extractions Using Protein-Modified Nanotubes ................ .................. 85
A vidin/biotin ............................ .. ... ............... .... ............... 85
Immunoprotein extractions ............... .. ......................... ................ 87
Enantioselective extractions............................ .............. ................... 88
Directed Nanotube Binding............................ .. ................... ...................... 95
Enzyme Attachm ent and Assays ................................................................... 100
G lucose oxidase ................................... ......... .... ........... ........ ... 100
Cytocrome P450 enzymes for drug detoxification ................................... 102
Conclusions ............... ......... ....................................... ....... ............ 106

4 ION CHANNEL MEMETIC SENSORS............. ................. 108

Introduction ...................... .. ............................ 108
B ackg ro u nd .................. ....... .......... .. .... .. .... .... .. ................... .......... ...... 10 8
Experimental .................................... ................ 10
M aterials.................................................. ............ .. .... ... .. ................ 110
M em brane Preparation........................................................ .................. 110
AC Impedance Measurements......................................... 112
X-Ray Photoelectron Spectroscopy (XPS) ............................................ 112
Transport Experim ents.......................................... 113
Ion-Current Measurements.......................................... .................... 114
R results and D isscussion......................... ....................................................... 114
XPS Studies................................................................... ................ 114
AC Impedance Experiments with DBS Analyte ........................................ 115


vi









Transport Experim ents......................................................... ................. 118
M easurem ents of Ion Current....................................................................... 120
Effect of Alkyl Chain Length and Nature of the Surfactant Head Group ........... 121
D election of D rug M molecules .......................... ....................... .............. 123
The Effect of Pore Density and Pore Diameter on Analyte Detection................ 124
C o n clu sio n s..................................................... ................ 12 6

5 C O N C LU SIO N S ........................................................................ ................. 127

LIST OF REFERENCES ......................... ................ 130

BIOGRAPHICAL SKETCH ..................................... ............................................ 140














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

DEVELOPMENTS IN BIO-NANOTUBE SCIENCE AND TECHNOLOGY

By

David Tanner Mitchell

December 2002
Chair: Dr. Charles R. Martin
Department: Chemistry

Chapter 1 provides background information on the template synthesis of

nanomaterials. The template synthesis method is examined with special attention to the

use of membranes containing monodisperse cylindrical pores as templates. Several

examples of the utility of template-synthesized nanomaterials are given. The production

of one type of template membrane, nanopore alumina, is reviewed. Reviews of sol-gel

and silane chemistry are also provided.

In Chaper 2, a sol-gel template synthesis process is used to produce silica nanotubes

within the pores of alumina templates. The nanotubes can be modified using a variety of

chemistries, typically via a silanization process. Because the nanotubes are formed in a

template, the interior and exterior surface can be modified independently. Modified

nanotubes can be used for drug detoxification or as extractants for the removal of metal

ions. The nanotube surface can also be biotinylated, which causes binding to avidinated

surfaces. Composite microtubes of silica and various polymers are also prepared.








Additionally, Au nanowires are shown to assemble with colloidal Au particles using

dithiols as linkers.

Chapter 3 describes the attachment of proteins onto template-synthesized silica

nanotubes. The proteins are covalently linked via an aldehyde silane bridge that binds to

pendant primary amino moieties on the protein. Protein-modified nanotubes function as

highly specific extractants. Avidin-modified nanotubes extract biotin-coated Au

nanoparticles from solution with high extraction efficiency. Immunoprotein-modified

nanotubes extract the corresponding antibody from solution with high specificity.

Antibody-modified nanotubes extract one enantiomer from a racemic mix. Enzymes,

including drug detoxification enzymes, were also attached to the nanotubes and were

shown to retain their catalytic activity. Immunoproteins on the outside of nanotubes can

be used to direct nanotube binding, creating specific labeling agents.

Chapter 4 describes the production of synthetic microporous membranes that mimic

ligand-gated ion channels. The membranes are prepared from microporous alumina that

is silanized with a silane containing a hydrophobic C18 chain. The hydrophobic C18

chains exclude aqueous solutions from entering the pores. Ionic surfactant molecules act

as a chemical stimulus to allow water to flood the pores, which in turn permits molecular

flux across the membrane. The effect of concentration and hydrophobicity of the surfact

analyte species are examined, as is the effectiveness of several drug compounds as

analytes. The results and conclusions of this dissertation are summarized in Chapter 5.













CHAPTER 1
INTRODUCTION AND TEMPLATE SYNTHESIS

Introduction

Nanomaterials constitute an emerging subdiscipline at the intersection of the chemical

and materials sciences (1, 2). This field poses an important fundamental question-how do

the electronic, optical, and magnetic properties of a nanoscopic particle differ from those

of a bulk sample of the same material? This issue is of particular importance because all

properties of a material change as the particle size approaches molecular dimensions and

because it is often the unique properties of the nanomaterial that make it useful for a

particular application.

One example of this concerns the optical properties of gold. The yellow metallic

luster of bulk gold is well known. In contrast, solutions ofnanoparticles of gold are

colored from ruby red to purple, depending on the nanoparticle size. This is due to a

characteristic visible absorption band called the plasmon resonance absorption (3).

Extinction coefficients for these nanoparticles are extremely high, ranging from 108 to

1011 MTcm1', depending on particle size. The high extinction coeffients of gold

nanoparticles make them useful as labeling agents in optical microscopy or as indicators

for colorimetric tests, such as pregnancy tests (4).

There is no universally agreed-upon definition of what constitutes a nanomaterial. It

is generally considered that at least one dimension (d) must be less than or equal to 100

nm. Just larger than this are microscale materials, with dimensions in the range of 100

nm < d< -10 im. Materials in both these regimes will be discussed in this dissertation.








In addition to size, control of shape is of primary importance in nanomaterials science.

Because (at the nanometer scale) surface properties are dominant, particle shape often

determines material properties. Again using gold nanoparticles as an example, a large

shift in particle color is observed when gold nanoparticles are transformed from an

elliptical to a spherical shape (5).

There are two general methods for controlling the shape of a material. These are

usually termed top-down or bottom-up. An example of top-down shape control is the

production of a two-by-four. A round tree of dimensions larger than 2" x 4" is sawn

down to those dimensions. Bottom-up shape control is exemplified by the pouring of

concrete. Because the concrete is fluid before setting, it will take the shape of the

recepticle or form into which it is poured. After setting, the shape of the concrete is

locked and the form can be removed.

In recent years, our laboratory has pioneered a bottom-up method for the production of

nanomaterials. This method involves synthesizing the nanomaterials inside of a porous

template (and therefore is called template synthesis). This chapter provides background

information on the template synthesis of nanomaterials. The template synthesis method

is examined with special attention to the use of membranes containing monodisperse

cylindrical pores as templates. Several examples of the utility of template-synthesized

nanomaterials are given. The production of one type of template membrane, alumina, is

then reviewed. Reviews of sol-gel and silane chemistry that are extensively used in later

chapters are then provided.








Background

Template Synthesis

One of the easiest ways to control the shape and size of a material is to synthesize it

inside a form or template. This process is ubiquitous in the manufacturing world. Car

tires, concrete walls, and gelatin desserts are all examples of products produced by

casting the precursor material into a mold or form. The size and shape of the resulting

object is determined by the size and shape of the template. This remains true down to

nanoscopic dimensions. Several natural and synthetic materials have pores or voids in

the micro- or nanoscale and have been used as templates. Typically these templates are

classified by the dimensionality of their void spaces and therefore the dimensionality of

the resulting templated structures: three-dimensional, two-dimensional, or

one-dimensional.

Three-dimensional templates

Three-dimensional template materials include micelles, glasses, zeolytes, and block

copolymers. In the case of glasses or block copolymers, the pores are the result of

dissolution of a heterogenous phase. For example Vycor 7930 glass, manufactured by

Corning, is produced by annealing borosilicate glass to produce intercalating phases that

are correspondingly boron rich or silica rich. The boron-rich phase can then be dissolved

with acid to leave a three-dimensional network of pores. The average size of the pores

can be controlled by the length of the annealing time. Commercially available sizes

range from 4 to 20 nm. Indium has been templated into Vycor glass, resulting in an

electronically conducting composite material (6). Vycor has also been used to template

polymers such as poly(acrylic acid) (7). The composites were found to have restricted

polymer chain dynamics compared to bulk polymer (7).








Zeolites, also known as molecular sieves, are crystalline solids that contain highly

ordered pores. The framework of zeolites are usually made of oxides of Si and Al. The

pores may be empty or may contain water, ions, or molecules. As the moniker

'molecular sieves' would indicate, the pores are generally molecule sized, from

sub-Angstrom to a few nanometers in diameter. The pores are the result of the structure

of the highly crystalline oxides that make up the framework. As such they are almost

perfectly monodisperse. There are over a hundred types of zeolites, including natural and

synthetic varieties. Zeolites have been widely used as templates, including for such

substances as carbon (8, 9), polymers (10), semiconductors (11, 12) and metals (13). Due

to the sub-nanometer size of the template, the resulting materials can have extremely high

surface areas. The surface area of carbon templated in zeolites has been reported as high

as 2000 m2/g (8).

Pores in block copolymers are formed in a similar manner to those in glasses. The

block copolymer is made of two chemically distinct polymer types that phase separate.

One phase is then dissolved with an appropriate solvent. The size and shape of the

resulting pores are dependant on the molecular weight and concentrations of the polymer

types. In contrast to glasses, which have a random pore structure, the pores formed by

block copolymers can be highly ordered. This also is a function of the phase separation.

The three-dimensional network of pores in block copolymers has allowed them to be

used as templates for synthetic molecular sieves, allowing control over pore dimensions

(14, 15). They have also been used to template nanoparticles of magnetic oxides (16) and

nanowires of gold (17).








An interesting recent three-dimensional template is synthetic colloidal crystal or

synthetic opal. The crystals are made by physical close packing ofmicrospheres or

nanospheres, usually either latex or silica, from a solution. After or during packing, the

liquid is removed and a monomer solution is added. The monomer is then polymerized

and the original colloidal particles dissolved away (18). The distance between layers can

be controlled by the diameter of the colloids used. Materials templated in this way

include photopolymers (19) Ge (20) and titania (21). The resulting structures have been

shown to be photonic band gap materials (20, 21). This 'lost wax' replica of the crystal

can then be further used as a template in its own right. For example, the laser dye

rhodamine 6G was templated using a reverse opal and found to exhibit morphology

dependant resonances due to the confinement (19).

Two-dimensional templates

Two-dimensional templates are layered materials. Some examples include minerals,

Langmuir-Blodgett (L-B) films, and ionic polymer multilayers. Most of the layered

minerals used as templates, such as muscovite and montmorillonite, are oxides of Al and

Si, similar to zeolites. As with zeolites, the layers are the result of the molecular

structure. Polymer multilayers and L-B films, in contrast, are built up manually, one

layer at a time, usually by repeated dipping of a substrate into a solution containing or

supporting the layer. Examples of two-dimensional template synthesis include

organization of colloids on polyelectrolyte templates (22), and graphite synthesis in

montmorillonite clay (23).

One-dimensional templates

The primary concern of this dissertation is template materials of one dimension.

These are typically membranes with pores running through the thickness of the








membrane. The pores may be naturally occurring or may be the result of extensive

preparation and engineering.

Carbon and lipid nanotubes are naturally occurring one-dimensional templates.

Nanomaterials that have been deposited inside carbon nanotubes include metals (24, 25),

metal oxides (26), and semiconductors (27). Lipid nanotubes have been used to template

nanoscopic structures ofpolypyrrole (28).

Another natural one-dimensional template material is aluminum oxide or alumina

(A1203). When grown under appropriate conditions, cylindrical pores form normal to the

surface of the membrane (Figure 1-1). The pores result from the electrochemical

oxidation of aluminum. Pore size is dependant on the voltage used for oxidation and can

be varied from -10 nm to -350 nm. The pores are highly monodisperse. Pore densities

range from 109 to 1012 pores/cm2. Alumina membranes with pores of nominally 200 nm

diameter are available from Whatman International, Maidstone, England. Nominal pore

sizes of 100 nm and 20 nm are commercially available as well. For sizes other than 200

nm, however, the stated pore size reflects less than 1% of the pore length. The other

99+% of the pore is -200 nm in diameter. Many groups, including ours, have used

porous alumina as a template material. A sampling of the materials that have been

prepared by template synthesis includes metals (29), polymers (30), lithium ion battery

materials (31), thermoelectric materials (32), carbon (33), and semiconductors (34, 35).

A recent interesting application is the use of alumina to template nanometer scale

metallic barcodes (36). To form the bar codes, the researchers electrochemically

deposited layers of metal in the alumina membrane pores. By varying the metal type and

layer thickness, a unique set of nanowires could be composed each time. The nanowires













































Figure 1-1. Scanning electron micrographs of aluminum oxide. A) Side view of
homegrown 60 nm. B) Top view of commercial 200 nm (Whatman).






8






















Figure 1-2. Scanning electron micrograph of track-etched polycarbonate membrane
with 5 pm pores.








are then freed by dissolving the template. Nanobarcodes can be mixed in with another

substance as a type of tracer or marker or used in bioassays (36).

In contrast to the above one-dimensional template materials, polymeric and mica

membranes have pores created by irradiation with radioactive decay particles (Figure

1-2). The high-energy decay particles cause damage tracks as they pass through the

polymer or mica. These damage tracks are etched much faster than nondamaged

material. Etchants are usually either acid or base. The resulting porous membranes are

said to be 'track-etched'. Pores produced by this method tend to be highly monodisperse.

In mica the pores have a diamond-shaped cross-section while in polymers the

cross-section is circular. In both cases they are predominantly normal to the membrane

surface although there is some deviation in angle. The distribution of pores is random for

the same reason. Pore size is dependant on etching time and ranges from -10 nanometers

to tens of microns. Porosity depends on the pore density and size of the pores but is

typically from 15% to 0.01%.

Track-etched mica was one of the earliest materials used as a one-dimensional

template. Possin (37) deposited nanowires of several types of metals including Zn, Sn,

and In as early as 1970. Other researchers later modified the technique to produce

nanowires of gold as small as 8 nm in diameter (38).

Track-etched polymer membranes have been widely used as templates by the Martin

lab (30, 39, 40) and others (41). Metal nanostructures have been synthesized by

electrochemical reduction (42) or an electroless process (39). Solution casting (30), melt

casting, and polymerization from a monomer solution (43) have all been used to template

polymeric materials in track-etched membranes. Examples of the polymers templated








include many types of insulating polymers (30) and conducting polymers (44, 45).

Template-synthesized nanofibers of conducting polymers have been shown to be more

conductive than the bulk material (44, 45). Polymers and metals have been templated

sequentially to form concentric composites consisting of an outer metal shell, a

passivating layer of insulating polymer, and an inner wire of conducting polymer (46).

An early application of template-synthesized nanomaterials entails preparing

nanoscopic electrodes. Research in our lab has shown that an electroless plating method

can be used to prepare ensembles of gold nanodisk electrodes with a disk diameter as

small as 10 nm. Such nanoelectrode ensembles (NEEs) have potential applications in

electroanalytical chemistry because the signal-to-background ratio (S/B) observed at the

NEE can be orders of magnitude higher than at a conventional gold macroscopic

electrode disk. Thus the detection limit at the NEE that can be orders of magnitude lower

than that for conventional macroscopic electrodes (39).

The NEEs were obtained by electroless plating of gold within the pores of the

template membrane for sufficiently long times so that solid nanowires were produced. If

plating is done for shorter times, ensembles ofnanotubes that span the complete thickness

of the template membrane can be obtained. These nanotubes can have inside diameters

of molecular dimensions, (<1 nm) and can be used to separate molecules on the basis of

size (47). By applying a bias to the gold nanotubes, the membranes can be rendered

cation permselective, anion permselective, or nonpermselective (48). Furthermore, the

inside of the nanotubes can be modified using thiol chemistry to create chemically

selective filters (40, 49).








An additional type of engineered one-dimensional template is nanochannel glass (50,

51). This differs from the Vycor glass discussed earlier in that the pores are all parallel to

each other, normal to the membrane surface, and highly monodisperse. Nanochannel

glass is created by bundling fibers of glass together, heating to soften and bind the fibers,

then pulling the softened collection of fibers to decrease their cross-section. The process

is repeated until there are as many as 101 fibers/cm2. Each fiber consists of a sheath of

high-silica-content glass and a core of high-boron-content glass. The high-boron-content

glass is then etched away with acid to leave an equivalent density of pores. Nanochannel

glass has been used to template ferromagnetic wires that show enhanced magnetic

coercivity (50), and to pattern mesoscopic ring structures of Au (51).

Alumina Growth

As cited earlier, aluminum oxide or alumina can be produced with highly

monodisperse cylindrical pores (Figures 1-1 and 1-3). This is a result of the

electrochemical oxidation process in acid electrolytes. (If a basic electrolyte is used, a

nonporous barrier oxide is formed) (52). In the growth process, high-purity aluminum

metal (99.99%) is used as the anode. A stainless steel cathode is used as the other half of

the cell. Both cathode and anode are immersed in aqueous acid electrolye and voltage is

supplied by a variable power supply. The geometry of cathode and anode is important

for uniform alumina growth. Our system uses a cylindrical cathode that surrounds the

aluminum anode to assure homogenous current density on both sides of the aluminum

plate (Figure 1-4).

Polishing

Because the alumina film is converted directly from the aluminum metal, a rough

metal surface will produce a rough alumina film, albeit with the incorporation of pores.






















I \ I
A B
Scanning electron micrographs of homegrown aluminum oxide.
A) Aluminum metal. B) Barrier layer.


Figure 1-3.


'^M'
'416 Vol
ikfe Jeb.'Jl. jb -0^































Stainless I
Stainless Electrolyte
steel cathode
Electrochemical growth cell for anodic oxidation of aluminum.


Figure 1-4.








To produce an even and flat template, highly polished aluminum metal is needed. The

aluminum may be mechanically polished if the surface is severely scratched or pitted.

Mechanical polishing is done with a slurry of either silicon carbide or alumina particles.

Larger particles (>10 urn) are used to remove material quickly, and polishing is continued

with slurries of progressively smaller particles until the particles are submicron in size.

The final polishing stage is done electrochemically. This process is analogous to that

of alumina film formation but it is done in concentrated acid electrolyte solutions and at

high temperatures to favor immediate dissolution of the alumina. The electropolishing

solution used in our lab is 95% concentrated phosphoric acid, 5% concentrated sulfuric

acid with 20 g/L Cr03 added to prevent pitting. The aluminum is again the anode and a

lead plate is used as the cathode. The temperature of the electrolyte is kept around 70C

and between 10 and 15 V is applied for periods of roughly 5 minutes. If the aluminum

has been previously polished to a submicron surface roughness, two or three

electropolishing steps will generally result in a mirror finish.

The rate of the oxidation reaction is also important to final film characteristics. If this

becomes too high during the oxidation process, the resulting heat will cause a runaway

reaction that will preferentially etch the aluminum at the air/electrolyte interface. This

will eventually result in loss of electrical connection to the aluminum. To keep the

reaction rate low, the electrolyte is chilled to between 0 and 4 degrees C.

Polyprotic acids are used as the electrolyte (usually sulfuric acid, phosphoric acid, or

oxalic acid). The type of acid used is dependant on the size of the pores to be grown.

Smaller pore sizes require low applied voltages and therefore require more highly

conductive electrolytes (e.g., sulfuric acid) (53). Larger pores need larger voltages that








would cause a runaway reaction in a highly conductive electrolyte. Thus large-pore

formation uses lower conductivity electrolytes (such as oxalic acid). The concentration

of the electrolyte is also important. Higher concentrations increase the current density for

any given voltage. This increase in current density results in an increased rate of film

formation but also enhances the possibility of a runaway reaction.

The most important parameter in determining the final pore size for the alumina film

is the applied voltage. When proper electrolyte choice and temperature control constrain

the current density between 1 and 6 mA/cm2, the pore size is observed to be -1.4 times

the applied voltage (54).

Perfect pores

During the initial stages of film growth, the pores are somewhat random in shape and

packing, and there is some heterogeneity in size. This is a result of the pores nucleating

from random pits and valleys on the aluminum surface (55). As film growth continues,

the pores self organize into a hexagonal array and the pores become highly monodisperse

(56). A good review of the theory of this process can be found in the dissertation of G. L.

Homyak (54). To produce membranes with a "perfect" hexagonal packing and uniform

pore size, a two-step process was developed by Matsuda and coworkers (56) in 1995.

The first step in the process is growth of alumina film for sufficient time to allow

self-organization and homogenization of pore size. That film is then removed with acid.

This leaves an indentation or pit in the underlying aluminum corresponding to each pore.

The aluminum is then re-anodized at the same voltage and in the same electrolyte as the

original anodization. The pores nucleate in the pits and thus are already highly ordered

and monodisperse, and continue to grow as patterned (55, 56).








"Perfect" pores can also be formed by indenting the aluminum on a nanoscopic scale

preceding anodization. Matsuda and coworkers created a SiC stamp by electron beam

lithography and used this to pattern the aluminum surface. Pore formation again initiated

in the preformed indentations and gave rise to highly ordered, monodisperse pores (57).

Separation of the alumina film

There are two methods of removing the alumina film from the underlying aluminum

after growth is complete. The most conceptually simple process is to dissolve the

aluminum metal with HgCl2. This does not damage the alumina. This method works

best if thin foils of aluminum are being used to grow the alumina films. Thin films have

lower quantities of aluminum to dissolve, especially after anodizatoin. It should be noted

that the pores in alumina do not extend all the way through to the aluminum metal. Each

pore is separated from the metal by a thin barrier layer that is continuously forming and

dissolving as the film grows (Figure 1-3) (58). When the aluminum is dissolved, the

pores on the resulting alumina are closed on one end by this barrier layer. It can be

removed by acid or base etching, but as all of the alumina is exposed to the etchant this

may slightly widen the pores.

The other method of freeing the alumina from the aluminum involves a process called

voltage reduction (59). After the film reaches the desired thickness, the voltage is

reduced to about 70% of its original value. Because the pore size is determined by the

applied voltage, the pores at the barrier layer branch to smaller sizes. The current also

decreases but subsequently proportionally to the increasing voltage. The thickness of the

barrier layer, like the pore size, is proportional to anodization voltage. The voltage

reduction process is continued through many branching cycles until the barrier layer is

very thin and there are many small pores branching into it. Anodization is stopped, and








the alumina/aluminum composite is placed in an acid or base etchant solution. This

dissolves the alumina everywhere. However, the thin barrier layer and the small pores

are most quickly dissolved and the alumina is detached from the underlying aluminum

metal. The alumina film separates en masse if thick (above 20 mun) or flakes off in pieces

if thin. The resulting membrane has a branching pore structure that resembles a tree. A

silica replica templated from alumina demonstrating the structure of the barrier layer is

shown in Figure 1-5. The process used to make this replica is discussed in detail in

Chapter 2.

A membrane produced by voltage reduction has two distinct faces: the solution side

and the barrier side. Pores on the barrier side are always smaller than the solution side

due to the voltage reduction and pore branching. Voltage reduction can be done rapidly,

such that the branching constitutes only a fraction of the total pore length. For example,

in commercially available 20 nm alumina (Whatman), the pores branch from 200 nm to

20 nm over just 250 nm distance. Since each pore is 60 pm long, this represents only

0.42% of the overall pore length. If a membrane with uniform pores on both sides is

desired, the barrier side can be removed with acid, base, or reactive ion etching.

For several types of template synthesis, it is not necessary to separate the alumina film

from the underlying aluminum metal. The metal is often used to give mechanical support

to the membrane, as will be shown in Chapter 2. Although there is a barrier layer of

alumina preventing direct electrical contact, various materials have been electrodeposited

in alumina templates using AC deposition in which the attached aluminum metal acts as

the cathode. Examples include Ag (60)and CdS (61).






































Transmission electron micrograph of silica templated in branched pore
alumina.


Figure 1-5.








Sol-Gel Chemistry

Sol-gel technology originated in the 1970s as researchers attempted to find low

temperature routes to glass synthesis (62, 63). Glass is an amorphous material, however

it is generally made from crystalline oxide precursors. The high temperatures usually

needed to form glass (1300 to 2000* C) are a result of the need to destroy the crystallinity

of the precursors. A method using noncrystalline precursors was sought. As glass is

mostly SiO2, liquid alkoxysilanes were used. It was found that alkoxysilanes hydrolyze

readily in the presence of water to form silanols:

R'3Si-O-R + H20 -- R'3Si-O-H + R-OH (1-1)

The silanols can then undergo polymerization reactions with other silanols or with

other alkoxysilanes.

R'3Si-O-H + HO-SiR'3 -4 R'3Si-O- SiR'3 + H20 (1-2)

R'3Si-O-H + RO-SiR'3 -> R'3Si-O- SiR'3 + ROH (1-3)

In either case, for alkoxysilanes containing two or more alkoxy groups, the result is

formation of a three-dimensional siloxane network. At the start of polymerization, many

small particles of siloxane are formed. These are well dispersed in the liquid phase to

form a colloid. When the particles are well isolated from each other, the density of the

suspension resembles that of the solvent. At this stage the colloid is called a sol. As

polymerization continues, the particles increase in size and the liquid increases in

viscosity. When polymerization has progressed to the point where the particles develop a

three-dimensional network throughout the liquid, the colloid becomes very viscous and is

termed a gel (64).

Several parameters influence the rate of the hydrolysis and polymerization reactions.

These include temperature, pH, water concentration, and type of alkyl group (65). An








increase in temperature increases the rate of gelation. The reactions can be either acid or

base catalyzed; extremes of either high or low pH will speed the reaction, as will

increasing the amount of water (66). A larger, more sterically bulky alkyl group slows

the reaction rate (66).

Gels can be converted to silica through two routes. In the first method, the gel is

heated or placed under vacuum to remove the solvent phase, typically alcohol, as well as

water and other alcohols produced during polymerization. The resulting surface tension

collapses the open three-dimensional network of the silica phase of the colloid,

condensing it to a dense phase called a xerogel (65). If the solvent and water are

extracted instead through a critical-point drying procedure, the surface tension is

eliminated and the open porous nature of the gel is maintained (67). This type of

monolith is termed an aerogel. The maximum temperature for either process can be kept

under 100'C (66).

Since its inception, this process has been extended from silica to many types of metal

oxides (68). Because at the sol stage the colloid has liquid properties, it can be molded or

dip coated into nearly any size or shape, including nanomaterials. On dip coating, a gel is

formed at the interface of the material immersed in the sol. This gel layer is compacted

by drying to form a dense layer that replicates the surface topology of the immersed

template (65). Sol-gel chemistry has been used to fabricate template-synthesized

nanostructures of TiO2 for photocatalysis (34), V20s as a lithium ion battery material

(69), and WO3 (70).

Silanization

As mentioned above, the surface properties of a material become increasingly

important as the size decreases. It is therefore useful to have a method for controlling or








modifying the surface chemistry of materials. One relatively simple method for

modifying surface chemistry is the use of organosilanes. These are compounds

containing an Si-C bond. Because Si is in the same chemical group as carbon, these

compounds share much of their chemistry with organic compounds. Organosilanes form

stable covalent bonding with siliceous materials (e.g., silicates and aluminates) and many

metal oxides (71). The predominant types of organosilanes used for surface

modifications are chlorosilanes and alkoxysilanes. Both are usually deposited from

alcohol solutions. The attachment chemistry of both types is equivalent because when

chorosilanes are dissolved in alcohol, they react with the alcohol to form alkoxysilanes

and HCl (71).

R'3Si-Cl + ROH R'3Si-OR + HCI (1-4)

The extent of this reaction can be monitored by measuring the pH.

Alkoxysilane chemistry is analogous to the chemistry of sol-gel production. Silica

sol-gel precursors are organosilanes, generally either tetramethoxysilane (TMOS) or

tetraethoxysilane (TEOS). Due to its high vapor pressure and high reactivity,

tetrachlorosilane is used for vapor phase reactions to produce fumed silica. All of these

compounds have four reactive sites and thus can form three-dimensional networks. The

reactions are analogous to Equations 1-1 to 1-3, although in the chlorosilane case, the

chlorine is the leaving group.

If only a monolayer of surface modification is desired, an organosilane is used which

has only one reactive chloro- or alkoxy- substituent. Because there is only one reactive

site, these molecules can either dimerize or bind to the surface. Dimers cannot bind








further and are rinsed away. Binding to the surface takes place via surface hydroxyl

groups through the reaction

R'3Si-OH + R-O-SiR'3 -> Si-O-SiR'3 + ROH (1-5)

Often, a higher degree of surface modification is desired. To improve the density of

coverage, silanes with two or, more commonly, three reactive groups are used. These are

first allowed to oligimerize in a slightly aqueous alcohol solution (71). The presence of

water, typically around 5% vol/vol, initiates the formation of silanols (Equation 1-1)

increasing the reaction rate. The pH is adjusted to between 4.5 and 5.5 with acetic acid to

further facilitate the substitution reaction of the alkoxysilanes (71). The material to be

surface modified is then added to this solution and the oligimers bind through surface

hydroxyl sites (Figure 1-6). Surface modification by this route generally requires only a

few minutes of immersion. If a 2% solution of trialkyoxy- or trichlorosilane is used and

five minutes is allotted for oligimer formation, the resulting surface modification is

normally from 3-8 monolayers thick (72). After solution deposition, the silanized surface

must be cured. This increases cross-linking and removes residual solvent. It can be done

by heating to 120C for 20-30 min, or for longer times at lower temperatures. At room

temperature, 24 hours is generally sufficient for full curing.

Chlorosilanes can also be deposited from aprotic solvents, such as toluene and

tetrahydrofuran (71). If these solvents are anhydrous (and the substrate is free of bound

water), there will be no chance for alkoxysilanes to form. There can therefore be no

polymerization of the silane. The reaction must proceed by nucleophilic attack from

surface hydroxyl sites. As a result, only a monolayer can form (71). Surface

modification by this route requires longer times, from 12-24 hours. Higher temperatures








ROi-OR RO- OR
R0 jifOR + RO- O


OR


OR


OR


OR


9
+RO i-OR
OR


OR


..H
SiO2


RO-- i---- iO--i-OR

Trialkoxysilane oligier formation and attack ent to surface.
Trialkoxysilane oligimer formation and attachment to surface.


Figure 1-6.


RO- i-O--i-O--i-OR








or refluxing are also usually required, however the process does not need further curing

(71).

Chapter Summaries

In Chaper 2, a sol-gel template synthesis process is used to produce silica micro- and

nanotubes from alumina templates. The nanotubes can be modified with a variety of

chemistries, typically via a silanization process. Because the nanotubes are formed in a

template, the interior and exterior surface can be modified independently. For example,

the interior of the nanotubes can be modified with fluorophores while the exterior is

modified independently to make the nanotubes hydrophilic or hydrophobic. Modified

nanotubes can be used for drug detoxification or as extractants for the removal of metal

ions. The nanotube surface can also be biotinylated, which causes binding to avidinated

surfaces. Composite microtubes of silica and various polymers are also prepared.

Additionally, Au nanowires are shown to assemble with colloidal Au particles using

dithiols as linkers. The position of linkage can be controlled by using the template as a

mask.

Chapter 3 describes the attachment of proteins onto template synthesized micro- and

nanotubes. The proteins are covalently linked via an aldehyde silane bridge that binds to

pendant primary amino moieties. Because most proteins have pendant primary amines, a

wide range of proteins can be used to modify the nanotubes. Protein-modified nanotubes

function as highly specific extractants. Avidin-modified nanotubes extract biotin coated

Au nanoparticles from solution with high extraction efficiency. Immunoprotein-modified

nanotubes extract the corresponding antibody from solution with high specificity.

Antibody-modified nanotubes extract one enantiomer from a racemic mixture. Enzymes,

including drug detoxification enzymes, are also attached to the nanotubes and are shown








to retain their catalytic activity. Nanotubes modified in this way function as removable

bioreactors. Immunoproteins on the outside of nanotubes can be used to direct nanotube

binding, creating specific labeling agents.

Chapter 4 describes the production of synthetic microporous membranes that mimic

ligand-gated ion channels. The membranes are initially 'off, that is, they do not allow

molecules or ions to pass. On addition of analyte, the membranes switch to an 'on' state

and become permeable to molecules and ions. The membranes are manufactured from

microporous alumina that is silanized with a C18 silane. The hydrophobic C18 chains

prevent aqueous solutions from entering the pores. Ionic surfactant molecules act as a

chemical stimulus to turn the membranes 'on'. The hydrophobic portion of the surfactant

partitions into the C18 layer, while the ionic portion of the molecule becomes the

prevalent chemistry at the pore wall. Water can then flood the now ionic pores and

molecules and ions can cross. The effect of concentration and hydrophobicity of the

surfact analyte species are examined, as is the effectiveness of several drug compounds as

analytes. The results and conclusions of this dissertation are summarized in Chapter 5.













CHAPTER 2
CHEMICAL MODIFICATION OF SILICA NANOTUBES



Introduction

In this chapter, a sol-gel template synthesis process is used to produce silica micro-

and nanotubes within the pores of alumina templates. These silica nanotubes can be

modified via silanization with a variety of chemical functional groups. The nature of the

template synthesis process allows independent modification of the inner and outer

nanotube surfaces. Several examples of independent modification are given. Nanotubes

modified with C18 on the inside and unmodified on the outside are effective at sorbing

drugs and drug analogs from aqueous solution. Modifying the nanotubes with chelating

agents makes them useful as extractants of metal ions. Template synthesized structures

can also be used as building blocks for the assembly of larger structures. For example,

silica microtubes can be biotinylated, which causes binding to avidinated microspheres.

Additionally, Au nanowires are shown to assemble with colloidal Au particles using

dithiols as linkers. Composite microtubes of silica and various polymers are also

prepared.

Background

A recurrent theme in the recent literature involving nanoparticles concerns their use as

drug delivery agents (73-76). This process is the inverse of using nanoparticles as

extractants, which will be a primary subject of this chapter. Because of the similarities, a








review of the basic tenants of nanoparticle drug delivery is instructive. The use of

macroscopic particles as extractants is also reviewed.

Drug Delivery

There is increasing interest in using nanoparticles for drug delivery (73-76). Spherical

nanoparticles are almost always used because that shape is easy to make. The term

'nanoparticle' as used in the drug delivery literature encompasses everything from 5 nm

to 5 pm diameter. There are a few biologically important size parameters. Particles

below 200 nm in diameter are less susceptible to clearance by the reticulo-endothelial

system (RES), which increases their dwell time in the bloodstream and thus their efficacy

(77). Particles larger than 5 pm diameter will not be able to pass through capillaries and

will therefore not be well distributed by the circulatory system (77). Particles less than

100 nm in diameter are said to be 'ideal' in that they avoid RES uptake and facilitate

access to cells and tissues (77).

Types of nanoparticles for drug delivery

Most nanoparticles used for drug delivery are self-assembled from block copolymers

(27, 77, 78). Polymers are formed by linking together small monomer molecules. A

homopolymer is formed if only one type of monomer is used. When two different types

of monomers are joined in the same polymer chain, the result is called a copolymer (79).

Two types of monomer molecules can be made into a copolymer in many different

ways. If the two types of monomer are labeled A and B, the copolymer could be formed

by alternating A and B units:

-A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-








This type of copolymer is called an alternating copolymer. If the monomer units are

arranged in a random fashion, the copolymer is called a random copolymer. An example

would be as follows:

-A-A-B-A-B-B-B-A-A-B-A-B-B-B-A-A-B-A-B-A-A-

In a block copolymer, the monomers of each type are grouped together. A block

copolymer can be thought of as two homopolymers joined together (80):

-A-A-A-A-A-A-A-A-A-A-A-B-B-B-B-B-B-B-B-B-B-B-

Block copolymers used for drug delivery usually consist of either two or three

components (77). For a two-component system, one block is hydrophilic and the other

hydrophobic. In a three-block system, the scheme is

hydrophilic-hydrophobic-hydrophilic. These systems form micelles in a manner

analogous to that of surfacts. In aqueous systems, the hydrophobic regions cluster

together to form the interior or core of the micelle, while the hydrophilic regions create

an outside shell or corona.

Poly(ethylene oxide) (PEO), or the related polymer poly(ethylene glycol) (PEG), are

often used as the hydrophilic component (77). Poly(ethylene oxide) and PEG have nearly

unlimited water solubility, and therefore a high degree of hydration and large excluded

volume (81). These properties help to stabilize the micelles and prevent protein

adsorption (77). The type of hydrophobic polymer used varies widely. For a partial list

of biocompatible hydrophobic polymers used for micelle formation see Table 1 of

reference (77).

There are two typical routes to micelle formation, dissolving the block copolymer

directly in water or other aqueous solvent (e.g., buffer), and dialysis (77). The easiest








method is direct dissolution, in which the block copolymer is simply dissolved in the

aqueous system at a concentration high enough to cause spontaneous formation of

micelles. This concentration is known as the critical micelle concentration (CMC).

However, in many cases the block copolymers are not soluble in water. If they are not

soluble, the polymer is dissolved first in a water miscible organic solvent or

organic/aqueous mix, and then dialyzed against water or heated until the organic solvent

is removed. Again the micelles form spontaneously. For drug delivery applications, the

core of the micelle is hydrophobic and the exterior shell or corona is hydrophilic (77).

It is generally important that the working concentration of the block copolymer (that

is, the concentration in the blood after injection) be kept above the critical micelle

concentration (CMC) or the micelles may degrade (77). This can be avoided by cross-

linking either the core or the corona polymer. However, if the ratio of the hydrophobic

core material to the hydrophilic corona is very high, micelles can be kinetically stable

over long times even below their CMC (77). This is also true if the core material is well

below the temperature at which there is easy movement of the polymer chains, known as

the glass transition temperature (Tg), or if it is highly crystalline (77).

The size of the block copolymer micelle is influenced by the lengths of the core and

corona blocks (77). The size of the core generally determines the amount of drug the

micelle can carry. A typical core volume is only about 0.5% of the overall micelle

volume (77). The amount of drug a micelle can hold is also dependant on the

hydrophobicity of the drug, the drug size, and the physical state of the core (crystalline

vs. liquid-like) (77). These factors also influence how quickly the drug is released from








the micelle, as do other factors such as the density of the corona and biodegradation rates

of the polymers (77).

Non-micellar nanoparticles can be prepared by polymerization of monomer in the

presence of a stabilizing agent. Polymers preferred for this are usually hydrophobic and

biodegradable. Examples include poly(esters) (82), poly(alkylcyanoacrylates) and

analogs of poly(methylidenemalonate) (83). Stabilizers are usually surfactants or

carbohydrate polymers (e.g., dextrans). Nanoparticle size is often controlled by

templating the polymer inside the stabilizer micelle or in the oil phase of an oil-in-water

emulsion.

Dr. Karen Wooley and coworkers have modified block copolymer micelle formation

by cross-linking the corona (shell) after assembly. The nanoparticles can have liquid-like

or solid-like interiors, depending on the Tg of the hydrophobic polymer. For example,

poly(styrene) (Tg = 105"C), yields solid nanoparticles, while poly(isoprene) (Tg = -75C)

cores are liquid (78). The cores can also be comprised of a degradable material, which

can be subsequently removed to leave a nanocage structure (84). The outer hydrophilic

shells are usually poly(acrylic acid) with multiple functional sites to allow cross-linking

(78).

The other major synthetic route to nanoparticles for drug delivery is dendrimer

chemistry. Compared to polymer micelles, dendrimers are very highly monodisperse and

have precisely defined chemistries. Research in this area is typified by the work of

Frechet, who has synthesized amphiphilic dendrimers hydrophilicc PEG exterior,

hydrophobic phenolic core), and demonstrated their ability to incorporate and release

model drugs, notably phenol (85).








Micelles formed from biological macromolecules such as proteins, lipids, and nucleic

acids are called coacervates. Like the micelles mentioned above, these form

spontaneously in aqueous systems as a result of aggregation of hydrophobic regions of

the molecules. Like block copolymers, coacervates are often used as a delivery vehicle.

Because the macromolecules from which they are made are ionic, the coacervates are

also ionic in character. They typically have a densely charged ionomer core and a more

sparsely charged ionomer corona of the opposite sign (86). The inability of close ion

paring caused by the different charge densities causes the sparsely charged ionomer to

extend into solution, which stabilizes the particles (86). Because of their strongly ionic

nature, they are not often used as drug carries, as most drugs are hydrophobic. Their

niche is rather the transport of charged species which can be part of the coacervate, for

example DNA (87). One 'smart' example of this is discussed in Chapter 3.

Advantages of using nanoparticles for drug delivery

Paul et al. found that using nanoparticles to deliver drugs increased the drug efficacy

versus the free drug (88). They used pentamidine, an antileishmanial drug to treat

Leishmania-infected mice. (Leishmania are protozoan parasites, usually spread by sand

flea bites. The disease is most common in India and Bangladesh). The efficacy was

measured by the amount of drug needed to inhibit the disease. The micelles were 130 to

150 nm in diameter. When they used poly(D,L-lactide), which is biodegradable, as the

core polymer they observed a three fold increase in drug efficacy. When they used

poly(methacrylate) as the core, the efficacy increase was even greater- 6.5 times the free

drug, but this polymer is not biodegradable (88).

Nanoparticles can reduce drug toxicity. Perkins et al. made nanoparticles of 50 to 100

nm diameter using drug molecules as the core and PEG-conjugated lipids as the corona.








They found that administering Taxol (an anticancer drug) in this way to treat ovarian

cancer in mice resulted in far less toxicity than intraperitoneal (shot) or intravenous (IV)

administration of the free drug (89).

Encapsulation in nanoparticles can also protect fragile drugs from severe

gastrointestinal conditions and allow alternate means of treatment. Carino et al.

encapsulated insulin in nanoparticles of poly(lactide-co-glycolide) which were then taken

orally. The nanoparticles protect the insulin from enzymatic degradation in the intestinal

tract, and then cross the intestinal epithelium and release the insulin into the bloodstream

(90). The particles produced in this study were widely variable in size, from 100 nm to 5

um. Since only particles smaller than about 4 um can pass the intestinal epithelium, and

since the percentage of passage increases with decreasing size, most of the effective

insulin was probably encapsulated in the smaller particles.

Engineered Sorbants and Extractants

Macroscale engineered sorbants have proven useful in a diversity of applications

(91-93). For example, the addition of hygroscopic promoters such as CaCl2 and LiBr to

silica solutions improves water adsorption of the resulting material (91). Composites of

SiO2-CaCI2 and SiO2-LiBr prepared by a sol-gel process have been shown to more than

double the amount of water absorbed versus conventional zeolytes and silica gels (91).

Other examples of engineered bulk silica sorbant materials include solgel prepared

optical chemical sensors (93) and metal ion selective sorbants for drinking water

purification (92). There are limited examples ofnano-sized particles specifically

engineered as sorbants and extractants. Radioactive Cs can be removed from waste

materials using nanoporous silica silanized with Cs-specific binders (94). Polyurethane

nanoparticles have been used to remove a model pollutant from soil (95).








In this chapter, we build on these prior systems to synthesize silica based nanotube

systems that can be engineered to selectively extract a variety of species from solution.

Experimental

Commercial Anopore alumina membranes used as templates were obtained from

Whatman (Clifton, NJ). These were disks 47 mm in diameter with cylindrical pores of

nominally 200 nm diameter. Alumina filter membranes of nominally 20 nm pore

diameter were purchased from the same source. All other sizes of templates were grown

in house by anodic oxidation of high purity aluminum (see Chapter 1). Unless otherwise

noted, chemicals were reagent grade, obtained from commercial sources and were used as

received. Water was 18 MOhm pure from a Barnstead Epure model D4641 system.

UV-VIS spectra were obtained using a Hitachi U-3501 spectrophotometer.

Inductively-coupled plasma (ICP) analysis was done on a Varian Vista RL ICP-AES.

Scanning electron microscopy (SEM) was performed using a Phillips 505 scanning

electron microscope. SEM samples were made conductive by sputtering a thin layer of

Au (-15 nm), using a Hummer model 6 sputter coater. Transmission electron

microscopy (TEM) was performed on a JEOL model 200CX TEM.

Template Synthesis

A sol-gel template process was used to prepare the nanotubes (70). See Chapter 1 for

a review of the chemistry involved. First, a sol-gel silica precursor was prepared by

mixing absolute ethanol, tetraethylorthosilicate (TEOS, Figure 2-1A), and 1 M HCI

(50:5:1 vol/vol). This solution was allowed to hydrolyze for 30 minutes. Alumina

template membranes were then immersed into the sol-gel with sonication for 1 minute,

after which they were air dried for 10 minutes at room temperature and oven cured

overnight at 1500C. To produce plain silica nanotubes, the sol-gel treated membranes








were polished on both surfaces with an aqueous aluminum oxide slurry of-lum diameter

particles. This removed the surface layers of SiO2 that would otherwise bind the

nanotubes together. The membranes were then immersed overnight in a 25% (wt/wt)

solution ofH3P04 to dissolve the aluminum oxide template. The nanotubes were filtered

from the acid solution and repeatedly rinsed with pH 7.0 buffer and water, then dried. An

SEM micrograph of silica nanotubes templated in commercial 200 nm alumina can be

seen in Figure 2-2. In Figure 2-3, an alumina membrane produced in house with a pore

diameter of 30 nm was used as the template.

Silanization

Structures of all silanes used for surface modification are shown in Figure 2-1. Cls

functionalized surfaces were prepared by immersing either the SiO2-coated alumina

membrane or freed nanotubes in a 5% aqueous, 5% octadecyltrimethoxysilane (Aldrich,

Figure 2-1B) solution (vol/vol) in ethanol, pH adjusted to 5.0 with acetate (72). The

deposition solution was stirred for 20 minutes before addition of the membrane to allow

formation of siloxane oligimers. Silane deposition was usually done overnight to insure

maximum coverage of the C18. Following deposition, the membranes were rinsed with

ethanol and dried 20 minutes at 150C to cure the silane layer. The silicon dioxide

surface layers of the membrane were removed by mechanical polishing with a slurry of

-1 um alumina particles and the template alumina was dissolved with a 25% wt/wt

solution of phosphoric acid. The nanotubes were filtered and rinsed, as above.

Modifications with N-triethoxysilylpropylquinineurethan (Gelest, Morrisville, PA,

Figure 2-1C), N-triethoxysilylpropyldansylamide, (Gelest, Figure 2-1D),

2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (Shearwater, Huntsville, AL,

Figure 2-1E), or 3-mercaptopropyltrimethoxysilane (Sigma, Figure 2-1F), were







A) Tetraethylorthosilicate
OCH2CH3
H3CH2COi--OCH2CH3
6CH2CH3


C) N-triethoxysilylpropylquinineuretha

(C2H50)Si(CH2
(C2H50)3Si(CH2)3NH60 -N


B) N-octadecyltrimethoxysilane
OCH3
H3CO-i-(CH2)17CH3
OCH3


D) N-triethoxysilylpropyldansylamide
S02NH(CH2)3Si(OC2H5)3



N(CH3)2
E) 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane
OCH3
H3CO-iCH"(OCH2CH2)6-90CH3
6CH3

F) 3-mercaptopropyltrimethoxysilane
OCH3
H3CO- i-SH
6CH3


Structures of silanes used for modifications.


Figure 2-1.







G) N-(trimethoxysilylpropyl)ethylenediamine triacetic acid, trisodium salt
Na OOC COO Na
N-CH2CH2-N
Na OOC bH2
H2
(H3CO)3Si-C
H2

H) 3-trimethoxysilanealdehyde
H3CH2CO
H3CH2CO-i/^/C H
H3CH2CO 6


I) 3-aminopropyltriethoxysilane
H3CH2CO
H3CH2COi/\/NH2
H3CH2Cd
Figure 2-1. Continued
analogous. Each was deposited from an ethanolic solution containing 5% (wt./vol.) of
the silane. The deposition solution was also 5% in distilled water by volume and was
adjusted to pH 5.0 with sodium acetate. In all cases the silane solution was allowed to
polymerize 20 minutes before addition of the membrane. Silane deposition was
accomplished by immersing the SiO2 coated template into these solutions, followed by an
ethanol rinse and heat fixing at 140C for 20 minutes. Depositions were usually
performed overnight to maximize coverage. The modified nanotubes were released from
the template by dissolving with 25% H3PO4, filtered from the acid solution, and rinsed
repeatedly with water.









A L w-


Figure 2-2. Scanning electron micrographs of silica nanotubes templated in
commercial 200 nm diameter alumina membranes. A) Nanotubes held
together by a film of silica. B) Dispersed nanotubes.









A B


















Figure 2-3. Scanning electron micrographs of silica nanotubes templated in 30 nm
diameter alumina membranes produced in house. A) Nanotubes held
together by a film of silica. B) Dispersed nanotube.

EDTA-functionalized nanotubes were prepared in a similar manner but from an

aqueous silane solution. The EDTA silane, N-(trimethoxysilylpropyl)ethylenediamine

triacetic acid, trisodium salt (Figure 2-1G), is not soluble in alcohol and is supplied as a

50% solution in water (Gelest). This solution is added to water 10:90 vol/vol, which

makes the final silane concentration of the deposition solution ~5%. The pH is adjusted

to 5.0 with sodium acetate/acetic acid. Silica nanotubes produced by template synthesis

were added to this solution and stirred 3-4 hours. The nanotubes were then filtered by

vacuum filtration, rinsed with water and dried. Curing of the silane was accomplished by

heating in a 120' oven for 25 minutes.

Extraction experiments were generally performed using either 2.5 or 5.0 mL of

solution. Unless otherwise noted, the concentration of nanotubes added was ~1.0 mg

nanotubes per mL solution.








Protein Modification

Protein functionalized SiO2 nanotubes were prepared from the unmodified nanotubes

via an aldehyde terminated siloxane linker (96). (See Chapter 3 for a detailed discussion

of protein attachment). Unmodified nanotubes (as prepared above) were stirred in a 5%

aqueous, 10% aldehyde methoxysilane (PSX1050, United Chemical Technologies,

Figure 2-1H) in ethanol, pH adjusted to 5.0 with acetate. The aldehyde-modified

nanotubes were filtered, rinsed with ethanol, and cured 24 hours in an oxygen-free glove

box at room temperature. After drying, the nanotubes were incubated overnight at 4C

with a solution of protein in buffer (-1 mg protein/ml buffer). Aldehydes have been

shown to react with pendant primary amines on proteins to covalently link the proteins to

the substrate (97-99). Protein-modified nanotubes were filtered from the protein solution

and extensively rinsed with buffer before use. Unless otherwise noted, proteins were

obtained from Sigma in the highest purity possible and used as received.

Biotinylation

The nanotubes were biotinylated using a multistep modification process (see Figure

2-4). An amino silane was first deposited as a coupling agent. A 2% (wt./vol.) aqueous

solution of 3-aminopropyltriethoxysilane (United Chemical Technologies, Figure 2-11)

was stirred 20 minutes to allow oligimer formation. Silica nanotubes from commercial

templates were added to this solution and sonicated briefly to suspend. The nanotubes

were stirred in solution for a further 60 minutes, removed by filtration, and rinsed with

water. Heating to 120 for 20 minutes cured the aminosilane layer. The amino-modified

nanotubes were then added to 5 mL of 10 mM sulfosuccinimidyl

6-(biotinamido)hexanoate (Sigma) in N,N-dimethylformamide in water and again

sonicated to suspend. Sonication was continued for 1 hour. The nanotubes were







-OH H3CH2CO
-OH + H3CH2CO-i^/"\ NH2
-OH H3CH2Cd






0 Na O0 0 H
i/ NH2 + 03 N-
+ NO
0

NH
HN-



00 H 0


S
NH
HN-\
Figure 2-4. Schematic ofbiotinylation process.
removed from the reaction solution by vacuum filtration. They were rinsed with water
and then with pH 8.0 phosphate buffer.
Avidinated silica microspheres in buffer were purchased from Bangs Labs and used as
received. Two sizes were purchased, 5.0 gm and 0.5 lim diameter. Both sizes were
stored at 4'C until used.








Composite Nanotubes

Composite nanotubes of two types were prepared: polymer exterior with a silica

interior, or a silica exterior with a polymer interior. The ordering of the silica versus the

polymer layer was determined by order of deposition, with the outermost material being

the first deposited into the template. If the silica layer was to make up the outside of the

nanotube, it was deposited as detailed above. Inner layers of silica were deposited after

polymer deposition from the same sol-gel precursor solution. The cure, however, was

modified to 70C for 24 hours to prevent oxidation of previously deposited polymer.

Insulating polymers were deposited by dip coating the template membrane into a

polymer solution and allowing it to air dry. Poly(ethylene-vinyl acetate) (PEVA), was

dissolved in toluene to make a 0.5% (wt./vol.) solution. The PEVA was 40% by weight

vinyl acetate and had an average molecular weight of-100,000 Da. Poly(methyl

methacrylate) (PMMA) was prepared as a 4% (wt./vol.) solution in chloroform. The

molecular weight of this polymer was ~35,000 Da. Both polymers were supplied by

Scientific Polymer Products (Onterio, NY). Residual solvent was driven off by heating

the membrane to 70C for 15 minutes. The conducting polymer poly(pyrrole) was

deposited chemically from an aqueous 0.2 M monomer solution. The pyrrole monomer

(99%, TCI, Portland, OR) was twice distilled under argon and stored at -20C prior to

use. The template was immersed in 20 mL of the aqueous 0.2 M monomer solution and

an equivalent volume of a solution containing 0.5 M FeC3 and 0.5 Mp-toluenesulfonic

acid was added, which oxidatively polymerized the pyrrole monomer (100). This

reaction was done at 4' C. The alumina templates were removed with 25% phosphoric

acid and the nanotubes were collected by filtration.








Biotinylated Au nanoparticles of several sizes were supplied by Sigma. Other Au

nanoparticle colloid solutions were produced in house by reduction of hydrogen

tetracholoraurate (101, 102). To make the Au colloid, 300 mL of 0.5 mM HAuC14'3H20

was heated to boiling. 15 mL of boiling 1% (wt./wt.) sodium citrate dihydrate was

added. The combined solution is boiled a further 10 minutes, then allowed to cool to

room temperature. The resulting citrate-stabilized Au nanoparticles have an absorbance

maximum at 519 nm and average 15 nm in diameter, as measured by TEM.

Results and Discussion

Differential Inside/Outside Modification

Perhaps the most important attribute of the nanotubes is their distinct inner and outer

surfaces, which can be independently chemically and biochemically functionalized.

Furthermore, the template method provides a very simple way to differentially modify

the inner vs. outer surfaces. This concept is illustrated in Figure 2-5. While still

embedded within the pores of the template membrane, the inner surfaces are reacted with

one type of silane to attach this chemistry to the inner surfaces. The outer surfaces are

masked because they are in contact with the pore walls of the template membrane. The

amphoteric alumina template is then dissolved with acid or base to liberate the nanotubes.

These are collected by filtration, resuspended and reacted with the second silane. This

second silane attaches to the now accessible outer surfaces of the nanotubes. Several

experiments were conducted to prove this concept.

Hydrophilic/lipophilic nanotubes

As conclusive proof of independent inside/outside modification, we modified the

exterior nanotube surface chemistry in a way that would control nanotube partitioning in

an organic/aqueous two-phase system. One batch of nanotubes was labeled on their inner







A) Cross-section ofAl203 template membrane showing two pores.

Pore Pore


B) Sol-gel synthesis of SiO2 nanotubes.


J E 0

C) Attach first silane to inner tube surfaces.





D) Remove surface films on faces of membrane
and dissolve template membrane.




E) Attach second silane to outer surfaces.





Figure 2-5. Schematic of inside/outside modification of nanotubes.








surfaces with the fluorescent silane N-triethoxysilylpropylquinineurethan while still in

the alumina template. The outer surfaces of these nanotubes were protected by the

template and were therefore not silanized. After silanization, the template was dissolved

and the bare silica outer surface of the nanotubes was exposed. These nanotubes were

added to a vial containing 10 mL of water and 10 mL of the immiscible organic solvent

cyclohexane. The solvents were mixed by shaking the vial and then allowed to separate.

Because silica is naturally hydrophilic, when these nanotubes are added to the

cyclohexane/water two phase system, the pale blue fluorescence of the quinineurethan

(445 nm) is only seen from the lower aqueous phase (Figure 2-6A). The fluorescence at

the top of the cyclohexane phase is due to reflection of light from the meniscus. This

result confirms that exterior chemistry can be modified independent of interior chemistry.

If the initial quinineurathane silanazation were not confined to the inner nanotube

surface, the hydrophobic nature of the fluorophore would cause partitioning into the

cyclohexane phase.

A separate batch ofnanotubes was prepared with the fluorescent silane

N-(triethoxysilylpropyl)dansylamide attached to their inner surfaces. This fluorophore

emits in the green region (525 nm) when excited by UV light. The template membrane

was then dissolved and the outer surfaces were derivatized with the very hydrophobic

octadecyl silane (Cls). The nanotubes were added to an identical cyclohexane/water

system which was shaken and allowed to phase separate. The fluorescence image in

Figure 2-6B shows that because these nanotubes are hydrophobic on their outer surfaces,

they partition into the upper, hydrophobic cyclohexane phase. Hence, the pale green

fluorescence from the dansylamide is only seen from this phase.



























A B C
Figure 2-6. Fluorescently modified nanotubes in biphasic cyclohexane/water mix
(cyclohexane on top). A) Quinineurathane inside/bare silica outside
nanotubes partition into water phase. B) Dansylamine inside/C18 outside
nanotubes partition into cyclohexane phase. C) Mix of both types
independently phase separate.

When both sets of nanotubes are added to the solvent mixture in the same vial, the

tubes with the Ci8 outer surface chemistry partition into the cyclohexane and the tubes

with the silica outer surfaces go to the aqueous phase (Figure 2-6C).

Quinine/Au nanoparticle nanotubes

We first created nanotubes with a quinineurethane silanized interior and an

avidinated/biotin-Au nanoparticle exterior. Details of the modifications are given in the

experimental section. The interior silanization with quinineurethane was done while the

nanotubes were still in the alumina template. For this reason, the quinineurathane must

attach on the interior surface only. The outer nanotube surface is masked from

silanization by contact with the alumina template. In this experiment, 200 nm

commercial alumina templates were used to make the nanotubes. After quinine








silanization, the alumina template was dissolved in acid. This did not affect the attached

quinine, which showed both absorption and fluorescence following the acid treatment

(Figure 2-7).

0.12
oA
0.1
oB
0.08

.0 0.06

0.04

0.02

0 I I- I
250 300 350 400 450 500 550 600 650
Wavelength (nm)
Figure 2-7. UV-Vis spectra of modified silica nanotubes. A) Nanotubes modified
inside with quinineurathane and outside with avidin. B) The same
nanotubes after incubation with biotinylated Au nanoparticles.

Freed nanotubes were then silanized with an aldehyde silane (96). Because the

interior had already been functionalized, this attachment is posited to be preferential to

the nanotube exterior. Avidin was incubated with the aldehyde modified nanotubes

overnight. Avidin is a tetrameric 68 kDalton protein found in egg whites (103-105).

Each monomer unit is essentially identical and has a molecular weight of 17 kDaltons

(103). Incubation resulted in Shiffbase formation between the aldehyde and pendant

primary amino groups on the avidin, covalently binding it to the nanotube exterior (96).

Details of the protein attachment process are given in Chapter 3.

Avidin is well known to bind the small molecule biotin (104, 105). The binding

constant for the reaction is among the highest known for protein systems, -1 x 1015 NM"








(105). Because avidin is a tetramer, it can bind up to four molecules of biotin (103). To

be able to monitor the biotin binding to the nanotubes, 20 nm diameter biotinylated Au

nanoparticles (Sigma) were used. As mentioned in Chapter 1, nanoparticles of gold have

extremely high molar extinction coefficients due to their surface plasmon resonance

(106). The absorbtion maximum for the Au nanoparticles is 540 nm, well separated from

the 340 nm maximum absorbance of the quinineurathane.

Biotinylated Au nanoparticles were incubated with the nanotubes and bound to the

attached avidin. A UV-Vis spectrum of the resulting dual-functionalized nanotubes

shows two distinct absorption peaks, one at 340 nm due to the interior bound

quinineurathane and another at 540 nm caused by the exterior bound Au nanoparticles

(see Figure 2-7). The above results are encouraging, although not conclusive proof that

the Au nanoparticles are attached only to the exterior surface. To further prove this

concept, a control experiment was attempted in which both the inside and outside

surfaces of the nanotubes were modified with quinineurathane. This was done by

silanizing after template dissolution. It was then attempted to silanize these

quinineurathane-modified nanotubes with aldehyde silane and incubate them with

protein, but this proved impossible as the nanotubes would not suspend in aqueous

solutions. This result increases the probability that avidinization, and the subsequent

binding of biotinylated Au nanoparticles, occurs on the outer surface of the nanotubes.

Au nanoparticle/fluorescent protein nanotubes

Another example of independent inside/outside modification is the preparation of

nanotubes with Au nanoparticles on the inside and FITC-labeled antibodies on the outer

surface. As previously, we began by silanizing the nanotubes while still in the template.








In this case, the nanotubes were functionalized with a mercaptosilane. See experimental

section for details.


0.08 vA
oB
0.06


0.04


0.02


0 .I
400 500 600 700 800 900
Wavelength (nm)
Figure 2-8. UV-Vis spectra of modified silica nanotubes. A) Nanotubes modified
inside with mercaptosilane to which Au nanoparticles have been bound.
B) The same nanotubes after outside modification with FITC-labeled
protein.

Citrate stabilized Au nanoparticles (diameter = 15 nm) prepared in our lab were bound

by the mercapto group to the inside of the nanotubes while still in the template. The

template was then removed with acid. Acid dissolution did not remove the Au

nanoparticles, as evidenced by UV-Vis spectrophotometry (Figure 2-8). Note that the

absorption band for the mercapto-bound Au nanoparticles is much broader than that for

the avidin-bound biotinylated Au nanoparticles (Figure 2-8). This is a function of the

agglomeration of the mercapto bound nanoparticles that changes their surface plasmon.

This shift in Au nanoparticle plasmon resonance caused by agglomeration has been used

as a transducer for the detection of analyte species, for example by Mirkin and coworkers

(107).








As before, subsequent silanization with aldehyde silane is thought to occur primarily

on the silacious nanotube exterior, as the interior has already been modified. A protein

labeled with a fluorescent tag was then attached via the aldehyde-amino Shiffbase

reaction (96). The protein attached was immunoglobulin G (IgG), which had a

covalently-attached fluorescein isothiocyanate (FITC) group. The FITC absorbs in a

narrow region centered at 495 nm and thus serves as a label for protein attachment. The

dual-modified nanotubes again show two distinct absorbance peaks in the UV-Vis. The

broad 550-650 nm peak is due to the agglomerated Au nanoparticle absorbance (inside)

while the sharp 495 peak is a result of the FITC labeled protein (outside).

Nanotubes as Extractants

C1i nanotubes for drug detoxification

Differentially modified nanotubes have a practical application as drug detoxification

agents. Drug overdoses affect hundreds of thousands of people per year in the United

States alone (108). If the overdosed drugs are ingested, the patient's stomach can be

pumped and/or filled with a slurry of carbon black to sorb the remainder. However, if the

drugs are administered intraveinously or have already been absorbed into the

bloodstream, health professionals have little recourse.

Most drugs are soluble in aqueous solution but generally prefer a more lipophilic

environment due to the prevalence of hydrophobic moieties. For this reason, drugs tend

to deposit in fatty tissue in the body. It was posited that addition of lipophilic

(hydrophobic) particles to the blood would sorb the drug and therefore decrease toxicity.

Purely lipophilic particles would function as sorbants but may not disperse in aqueous

solution. Agglomeration of the particles in the bloodstream could lead to blockage,

compromising the health and even life of the patient. As demonstrated above, silica








nanotubes can be differentially modified to have two different surface chemistries, inside

and outside. Our goal was to create nanotubes with a lipophilic interior to sorb drugs and

a hydrophilic exterior to ensure suspension in aqueous solution.

Silica nanotubes embedded in an alumina template were silanized with Cis silane as

detailed in the experimental section. The template masked the outer nanotube surface

and consequently only the nanotube interior was modified. The template was dissolved

in acid and the nanotubes collected by filtration. The bare silica outer surface remains

hydrophilic, which causes the nanotubes to suspend in aqueous solution. Aqueous

suspendibility of nanotubes with bare silica outer surfaces is illustrated by Figure 2-6. In

this figure, nanotubes with bare silica exteriors and fluorescently silanized interiors are

shown to suspend in the water phase of a cyclohexane/water mixture. Nanotubes

modified on the exterior surface with C18 do not suspend in aqueous solution but rather

float on top.


S N





Figure 2-9. 7,8-benzoquinoline.

A model molecule was chosen to examine the efficacy of the nanotubes as sorbents.

The model needed to be of a moderate molecular weight, highly lipophilic, yet at least

somewhat soluble in water. It should also be easy to monitor, and stable over time. The

compound 7,8-benzoquinoline (BQ, Figure 2-9) satisfies all these conditions. It has a

molecular weight of 179 g/mol and is water soluble to about 2 x 10.4 M. Additionally the

tricylic structure of BQ results in strong absorbance in the UV. Concentrations down to








1.0 x 10"7 M can be easily monitored with UV spectroscopy using the absorbance peaks

at 233 or 266 nm. It is also stable under ambient conditions.

For the BQ extraction experiments, an initial concentration of 1.0 x 10-5 M BQ was

used. A UV spectrum of this concentration was taken to serve as a reference (see the

diamond top trace in Figure 2-10). To 2.5 mL of this solution was added 2.5 mg of

nanotubes modified with Cs1 on the interior surface. This amount of 1.0 mg

nanotubes/mL solution was arbitrarily set as a benchmark. A higher concentration of

nanotubes would obviously remove more drug.

o Control Solution (10^-5 M benzoquinoline)
0.3 1 A After Extraction with SiO2 Tubules
*.* ~ After Extraction with C-18 SiO2 Tubules
0.25
S 7 After 2nd Extraction with C-18 SiO2 Tubules
0.2

0.15
0.1


0.05

0
220 240 260 280 300
Wavelength (nm)
Figure 2-10. UV-Vis spectra of 7,8-benzoquinoline extracted with C18 modified silica
nanotubes.

The solution was briefly sonicated to suspend the nanotubes and then filtered through

a 20 nm alumina filter. The lipophilic BQ should partition into the lipophilic nanotube

interior and be removed from solution when the nanotubes are filtered. After filtration,

the concentration of the remaining BQ was again determined by UV-Vis spectroscopy.

The results are shown in the circle trace of Figure 2-10. Results are tabulated in Table

2-1.








Table 2-1. Absorbance of 7,8-Benzoquinoline Before and After Extractions.
1.0 x 10^-5 M Extraction with Extraction with 2nd Extraction
Starting Sol. Silica Nanotubes C18 Nanotubes C18 Nanotubes

Abs. @ 265 nm 0.1889 0.1708 0.0339 0.015
% Extracted 10% 82% 92%

It is evident that most of the BQ has been removed. The absorbance of BQ remaining

in solution was 0.0339, meaning that 82% of the BQ was extracted by the C18 modified

nanotubes. If the BQ solution is extracted twice, each time with a concentration of 1 mg

modified nanotubes per mL of solution, the amount of BQ removed increases to 92%.

The bottom trace of Figure 2-10 shows this result.

To insure that the lipophilic C18 was responsible for the extractions, a control

experiment was performed with unmodified silica nanotubes. Again 1.0 mg

nanotubes/mL solution was added, sonicated to disperse, and filtered. As shown in the

upper triangle trace of Figure 2-10, only a small amount of BQ was removed from the

solution by the unmodified nanotubes, -10%. It should be noted that for all the lipophilic

extraction experiment glass transfer pipettes, syringes, and vials were used. The

lipophilic nature of polymer (generally poly(ethylene)) storage and transfer devices

would otherwise cause sorbtion of the BQ and skew the results.

To more closely examine the effect of compound lipophilicity on relative extraction, a

series of increasingly lipophilic compounds was chosen. These included the moderately

hydrophilicp-cresol, and, in order of increasing lipophilicity, 2,4-dimethylphenol and

2,4,6-trimethylphenol. The increasing lipophilicity of these compounds is confirmed by

their increasing octanol/water partition coefficients. The log of the octanol/water

partition coefficient for p-cresol is 1.97 (109), for 2,4-dimethylphenol it is 2.35 (109), and

for 2,4,6-trimethylphenol it is 2.73 (110). Structures of all compounds are shown in








Figure 2-11. Solutions of all compounds at a concentration of 1.0 x 10-5 M were made in

pH 9.0 phosphate buffer. The buffer was used to insure protonation of the phenol.

Deprotonation of the phenol would alter the lipophilicity of the compounds. Extractions

with interior modified Csi nanotubes were analogous to those for 7,8-benzoqinoline.

Each extraction used 1 mg nanotubes/mL solution. The absorbances of the solutions

were monitored before and after extraction using UV-Vis spectroscopy. Results are

shown in Figure 2-12 and tabulated in Table 2-2.

CH3 CH3 CH3




H3 H3C CH3
A. OH B. H C. H
Figure 2-11. Structures of Lipophilicity Study Compounds A)p-cresol.
B) 2,4-dimethylphenol. C) 2,4,6-trimethylphenol.


Table 2-2. Absorbance Before and After Extraction.
Compound Wavelength Starting Abs. Abs. after extraction % Extracted

p-cresol 277 0.01559 0.01547 0.10%

2,4-dimethyl- 278 0.02131 0.01484 30%
phenol

2,4,6-trimethyl- 280 0.01267 0.00735 42%
phenol

It would be expected that the most hydrophilic compound would be extracted the least.

As shown in Figure 2-12A, this is the case. The concentration of p-cresol before and

after extraction is essentially identical. The more lipophilic 2,4-dimethylphenol is

extracted by the Cs1 modified nanotubes, as seen if Figure 2-12B. The concentration

after extraction is 7.0 x 106 M, thus 30% was extracted. Figure 2-12C shows that










1 x 10"-5 M p-cresol
After C-8 1()02 tubule extraction


0.016
0.014
0.012
0.01
0.008
0.006
0.004
0.002
0 2
265


270 275 280 285 290
Wavelength (nm)

S1 x 10^-5 M 2,4-dimethylphenol
SAtter extraction with C-l18 i-)2 tubules


0 ---------------------------------------
265 270 275 280 285 290 295
Wavelength (nm)
Figure 2-12. UV-Vis spectra showing increasing extraction for more lipophilic
compounds. A) p-cresol. B) 2,4-dimethylphenol.
C) 2,4,6-trimethylphenol.


0.025

0.02

0.015

0.01

0.005









C 1 x 10^-5 M 2,4,6-trimethylphenol
A After C-18/Si02 tubule extraction
0.014
0.012 .
0.01 on
S0.008 loss:" AA',
o A"A A
0.006 A "A
0.004 AA
AU
0.002 & .
0 .
265 270 275 280 285 290 295
Wavelength (nm)
Figure 2-12. Continued.

extraction efficiency increases for 2,4,6-trimethylphenol, the most lipophilic of the

compounds. Approximately 42% is sorbed by the Cls nanotubes.

The Cs1 modified nanotubes were next used to extract an actual drug. An aqueous

solution ofbupivacaine, a commonly used local anesthetic, was prepared at a

physiological dosage level of 5 x 10'6 M. The structure of bupivacaine is shown in

Figure 2-13. Due to the cationic nature of the bupivacaine, it is not as lipophilic as the

7,8-benzoquinoline and would not expect to be as readily extracted by the Cs1 nanotubes.



H3C H3C
C 3 HCI

N

CH3
Figure 2-13. Bupivacaine

Extraction with 1.0 mg Cs1 nanotubes per mL solution removed 65% of the

benzoquinoline, or 1.1 ug bupivacaine per mg of nanotubes. More bupivacaine could be








extracted from a more concentrated starting solution. The same concentration of

nanotubes removed 15 ug bupivacaine per mg nanotubes from a 1 x 104 M solution.

However, the efficiency of the extraction was decreased, with only 45% of the total

bupivacaine removed.

EDTA nanotubes for copper(H) extraction

As a second type of chemical extraction, the nanotubes were modified with an EDTA

analog (Figure 2-1G) which we refer to as EDTriA. The details of this modification are

discussed in the experimental section. Note that the free silica nanotubes were silanized,

which results in modification of both interior and exterior surfaces. The EDTriA, like

EDTA, is hydrophilic and the tubes suspend easily in aqueous systems, even if modified

on the exterior.

The EDTriA is pentadentate, with one acetic acid group of EDTA replaced by an

organosilane through which it is bound to the nanotubes. EDTA and related multidentate

compounds are known to strongly chelate many types of metal ions (111). It was

therefore hypothesized that the EDTriA-modified nanotubes would be useful in removing

metal ions from solution. Copper (II) was chosen as a representative ion for this

extraction system. The binding constant for Cu2+ to hexadentate EDTA is 6.3 x 1018

(112). Although the binding constant for the pentadentate analog will be somewhat

smaller, is should still be considerable.

Extraction experiments were similar to those involving C18 nanotubes. First the

concentration of a buffered solution of Cu2+ was measured using Inductively Coupled

Plasma-Atomic Emission Spectroscopy (ICP-AES). The initial concentration of Cu2'

was 2.0 ppm. EDTriA-modified nanotubes were then added to the solution and sonicated

to suspend. As before, 1 mg nanotubes was used per mL solution. The nanotubes were








filtered and the Cu2+ concentration of the filtrate solution remeasured by ICP-AES. As

shown in Figure 2-14 and Table 2-3, extraction removed 52% of the Cu2+. The control

experiment, using unmodified SiO2 nanotubes, removed only 5% of the Cu2+ (Figure

2-14 and Table 2-3).



Table 2-3. Cu(II) Extraction with EDTriA Nanotubes.
2.0 ppm Extraction with Extraction with
Starting Sol. Silica Nanotubes C18 Nanotubes

ICP-AES Area Counts 503 481 247
Percent Extracted 5% 52%

The results of the C8/benzoquinoline and EDTriA/Cu2+ experiments demonstrate that

our modified nanotubes can function effectively as extractants. The interior and exterior

surfaces can be differentially modified to tailor nanotube chemistry for a particular use,

(e.g., suspension in aqueous solution).

Composite Silica-Polymer Nanotubes

During template synthesis, most materials preferentially deposit on the template

surface. This minimizes surface energy (113), of particular importance for a high surface

energy template such as alumina (114). Because of this phenomenon, nanotubes are the

most common structure produced by template synthesis. Solid rods or wires can be

templated under certain conditions, for example if electroless metal deposition is

performed at a slow rate for long times (48). However, it is typical for the pores in the

template membrane to remain open after deposition, although with a smaller diameters.

This fact is useful in the differential silanization of silica nanotubes as it allows exclusive

alteration of the interior surface. It also allows a second material to be template

synthesized inside the first.









E 2.0 ppm Cu(II)
[ After extraction with silica nanotubes
600 1- After extraction with EDTA-silcia nanotubes

500

400

300

200

10


Figure 2-14. ICP-AES data showing Cu2+ extraction with EDTriA-modified nanotubes.

The Martin group has used this approach to manufacture concentric metal-insulating

polymer-conducting polymer composites (46). Using 3 pm diameter polyester track

etched membranes as templates, Au nanotubes were deposited by an electroless process.

This yields an Au microtube in each pore of the template membrane. Using the Au

nanotubes as working electrodes, poly(phenylene oxide) was next deposited

electrochemically from a solution of 2,6-dimethylphenol monomer. This polymer is

insulating and passivates the gold nanotubes. After gold and polymer depositions,

electronmicrographs showed that the pores of the membrane were still open. Finally, a

conducting wire of poly(pyrrole) was grown electrochemically to fill the center of the

pores (46).

Insulating polymers

These results show that composite structures can be synthesized using polymer track

etched templates. The same process should be amenable to templating with alumina

membranes. To explore this area, nanotubes of silica and polymer were prepared as








described in the experimental section. Two types of insulating polymers were used,

poly(ethylene-vinyl acetate) (PEVA), and poly(methyl methacrylate) (PMMA). Both

were deposited from solution by dip coating. Polymers could be deposited either before

or after silica nanotube formation. The silica was deposited from a sol-gel precursor, as

previously discussed. Commercial 200 nm alumina membranes were used as the

templates. Electronmicrographs show that the membrane pores are still open after

polymer and silica deposition (see Figure 2-15). The template can be removed by acid or

base treatment to free the composite structures (Figure 2-16).

Although it is difficult to see the silica or polymer layers from Figure 2-16, proof of

the composite nature is given by the nanotube solubilities. As with the C18 modified

nanotubes, the dispersion of polymer-silica composite nanotubes is controlled by the

chemistry of the outer surface. Composites with silica on the outer surface and polymer

inside suspend well in aqueous systems but do not disperse in organic solvents.

Conversely, composites with polymer exteriors do not disperse in water but suspend in

many organic solvents, such as ethanol or toluene. Care must be taken to not use solvents

that will dissolve the polymer. The polymer type can also affect dispersion. For

example, nanotubes with PMMA on the outside disperse readily in ethanol, while

nanotubes coated with the more hydrophobic PEVA do not.

Poly(pyrrole)

Conducting polymer-silica nanocomposites were also prepared. In this process the

silica was deposited first, followed by chemical polymerization of poly(pyrrole) (Ppy)

from an aqueous solution of the monomer. Details of the synthesis are available in the

experimental section. A homegrown alumina membrane with -60 nm pores was used as

the template. The resulting nanostructures are seen in Figure 2-17.









A


















B















Figure 2-15. Scanning electron micrographs of alumina templates after polymer and
silica depostion showing open pores. A) Poly(methyl methacrylate) and
silica. B) Poly(ethylene-vinyl acetate) and silica.






61


A


















B















Figure 2-16. Scanning electron micrographs of composite polymer outside/silica inside
nanotubes templated in 200 nm commerical alumina templates.
A) Poly(methyl methacrylate) and silica. B) Poly(ethylene-vinyl acetate)
and silica.









A B



















Figure 2-17. Scanning electron micrographs ofpoly(pyrrole) inside/silica outside
composite nanotubes templated in homegrown 60 nm dia. alumina
membranes. A) Nanotubes held together by a film of silica. B) Dispersed
nanotubes.

In Figure 2-17A the structures are held together by a surface layer of silica. Typically

this layer would be polished away to free the nanotubes, but here it was left intact and the

nanotubes protrude like bristles from a brush. After template dissolution, the nanotubes

are rinsed with water, then dried. The surface tension of the evaporating water draws

together collections of nanotubes to form the islands visible in Figure 2-17A. Figure

2-17B shows composite Ppy-silica nanotubes prepared in the same fashion but with the

surface silica layer removed. As the template is dissolved, these nanotubes are freed and

suspend in solution.

To prove that Ppy had been deposited into the silica nanotubes, an absorbance profile

of an aqueous dispersion of the composite nanotubes was taken over the visible region.

Poly(pyrrole) has a characteristic broad absorbance from 600 nm to -2 pm. Absorbance









0.02


8 0.015


0.01


0.005



300 400 500 600 700 800
Wavelength (nm)
Figure 2-18. UV-Vis spectra of poly(pyrrole)/silica composite nanotubes showing
poly(pyrrole) absorbance.

in this region is not seen for the bare silica nanotubes. The UV-Vis spectrum is seen in

Figure 2-18. The broad peak starting at -600 nm indicates that Ppy has been deposited.

This type of composite nanotube may have analytical applications, as the Ppy being

oxidized or reduced could generate an analytical signal.

Self-Assembly

The small size of micro- and nanoscale materials often makes it difficult to assemble

them into organized assemblies. Physical manipulation in this size regime is challenging,

even with highly specialized equipment. A better solution is relying on interacting

particle chemistries to self-assemble. The Whitesides group has been very active in this

area, using a variety of techniques to self-assemble structures from macro to sub-micro

scale (115-118). Natan and coworkers have used various chemical and biochemical

strategies to self-assemble colloidal Au particles onto surfaces (119, 120). Mirkin's

group used thiol linkers to bind colloidal Au particles to single stranded oligonucleotides.

These nanoparticles were subsequently self-assembled into a polymeric network by








addition of the complementary oligonucleotide (121, 122). Thiol linkers have also been

used to attach Au nanoparticles to carbon nanotubes (123).

Biotin/avidin self-assembly

To date, template synthesized micro- and nanostructures have not been self-assembled

in this way. To examine the possibility of self-assembling silica nanotubes, the

nanotubes were modified with biotin. As noted above, biotin binds very tightly to the

protein avidin. Incubation ofbiotinylated nanotubes with avidinated microspheres should

result in binding of the two to form an agglomerated structure.

Biotinylation of silica nanotubes is described in detail in the experimental section.

Briefly, the nanotubes were first silanized with a primary amino group that then bound a

succinimide ester containing the biotin. The biotin was at the end of a long linker arm to

ensure that steric hindrance would not compromise binding (124). Biotinylation occurred

on both the inside and outside surfaces of the nanotubes.

The biotin-modified nanotubes were incubated with commercially available avidinated

silica microspheres (Bangs Labs). Two sizes of microspheres were used, 5.0 gm and 0.5

pm diameter. Control experiments were performed with poly(ethylene glycol) (PEG)

silanized silica nanotubes. Poly(ethylene glycol) is known to prevent adsorbtion to

proteins. If bare nanotubes were used as a control, the silica would likely bind to the

avidin on the microspheres non-specifically.

When biotinylated nanotubes were incubated with the 5.0 pm avidinated silica

particles, they adhered to the particle surface (Figure 2-19). If PEG coated nanotubes

were used, no adhesion was seen (Figure 2-20). Similarly, 0.5 pm silica particles

agglomerated with the biotinylated nanotubes (Figure 2-21), but not with PEG-ylated

nanotubes (Figure 2-22).















































Figure 2-19. Scanning electron micrographs of biotinylated silica nanotubes on 5.0 pm
diameter avidinated silica spheres.














































Figure 2-20. Scanning electron micrographs ofpoly(ethylene glycol) modified silica
nanotubes and 5.0 pun diameter avidinated silica spheres.















































Figure 2-21.


Scanning electron micrographs of biotinylated silica nanotubes and 0.5
pm diameter avidinated silica spheres. A) Ball/stick clusters.
B) Agglomerated ball/stick mass.









A



















B
















Figure 2-22. Scanning electron micrographs ofpoly(ethylene glycol) coated silica
nanotubes and 0.5 gm diameter avidinated silica spheres. A) Isolated balls
and sticks. B) Agglomerated stick mass and isolated balls.








Dithiol self-assembly

While the self-assembled structures described thus far have dimensions in the

micrometer regime, there is no fundamental reason that this strategy cannot be applied to

much smaller nanowires and nanoparticles. An alternative self-assembly chemistry was

used to prove this point. This entailed the use of 1,9-nonanedithiol to assemble colloidal

Au particles (10 nm diameter) to the ends of 30 nm diameter template synthesized Au

nanowires (see experimental section for details).

A gold film was sputtered onto one face of a polycarbonate template membrane with

30 nm diameter pores. This film served as the working electrode for deposition of Au

nanowires within the pores of this membrane (125). The nanowire-containing membrane

was then immersed overnight into a 1 mM ethanolic solution of the dithiol. This resulted

in chemisorption of the dithiol onto the ends of the Au nanowires and also onto the

sputtered Au surface film. The Au film was removed by polishing with a laboratory

tissue. Because this polishing removed the surface film, the only remaining dithiolated

surface was the exposed ends of the Au nanowires. The membrane was then immersed

overnight into an aqueous colloid of Au nanoparticles, which resulted in binding of the

Au nanoparticles to the ends of the dithiolated nanowires. The membrane was then

rinsed with water to remove any unbound Au nanoparticles. The template membrane was

dissolved away by immersion in CHCl3 to yield a suspension (125) of the Au

nanowire/colloidal particle assemblies. A drop of this suspension was applied to a

transmission electron microscopy grid.

Figure 2-23 shows transmission electron micrographs of gold nanowire/colloidal

particle assemblies. Note that because the nanowires were embedded within the template

when they were exposed to the dithiol and colloidal nanoparticle suspension, the Au








particles are self-assembled to only the ends of the Au nanowires. About 10% of the

nanowires in such images were terminated with colloidal particles.


Figure 2-23. Transmission electron micrographs of template synthesized 30 nm
diameter Au nanowires to which 10 nm Au particles have been
self-assembled using 1,9-nonanedithiol.

Conclusions

Novel nanoparticulate silica-based extractants have been prepared with a variety of

chemistries. The initial step involves the template synthesis of silica nanotubes in a

porous alumina membrane. The nanotubes can then be modified with different chemical

moieties depending on the extraction desired, typically via a silanization process.

The inner and outer surface chemistries can be modified differentially. Mixed

nanotubes can be prepared in which the inside is silanized with a quinineurathane and the

outside coated with protein-bound Au nanoparticles. A further example is the

preparation of nanotubes with thiol-bound Au nanoparticles on the inside and

fluorescently labeled proteins on the outside. Outer surface chemistry can direct








nanotube phase segregation. Nanotubes with unmodified silica exteriors partition into

and suspend in aqueous solutions. In contrast, nanotubes modified with Cis on the outer

surface partition into and suspend in hydrophobic organic solvents.

Template synthesized nanotubes can be used as extractants. Using C18 modified

nanotubes, model and actual drugs can be extracted from aqueous solution. Extraction

efficiencies range from 45% to 85%, with higher efficiencies for lower initial drug

concentrations. Nanotubes modified with an EDTA analog extract copper ions from

water.

Composite polymer-silica nanotubes have been prepared. The polymer can be

compose either the external or internal surface. As with the differentially modified

nanotubes, surface chemistry controls dispersion properties.

Template synthesized nanotubes and nanowires can be used as building blocks for

self-assembly. Biotinylated silica nanotubes bind to avidinated silica microspheres.

Dithiol chemistry can be used to link Au nanowires with Au nanoparticles.













CHAPTER 3
PROTEIN MODIFICATION OF SILICA NANOTUBES

Introduction

This chapter describes the attachment of proteins onto template synthesized nanotubes.

The proteins are covalently linked via an aldehyde silane bridge that binds primary amino

groups. Because most proteins have pendant primary amines, a wide range of proteins

can be used to modify the nanotubes. The molecular recognition capabilities of certain

proteins can be retained after binding to make the protein-modified nanotubes

exceptionally specific extractants. Avidin-modified nanotubes extract biotin coated Au

nanoparticles from solution with high extraction efficiency. Immunoprotein-modified

nanotubes extract the corresponding antibody from solution with high specificity.

Nanotubes modified with an enantioselective antibody extract one enantiomer from a

racemic mix. Enzymes, including drug detoxification enzymes, are also attached to the

nanotubes and shown to retain their catalytic activity. Nanotubes modified in this way

function as bioreactors. Immunoproteins on the outside of nanotubes can be used to

direct nanotube binding, creating specific labeling agents.

Background

Nanoparticles have recently found use in a variety of biomedical applications. These

include enzyme encapsulation (126, 127), DNA transfection (128, 129), biosensors (36,

130-132), and, as detailed in Chapter 3, drug delivery (73-76). Linkage of the

biomolecule of interest to the nanoparticle is a critical step to increasing nanoparticle

utility. Several examples ofbiomolecule particle modification are reviewed, as well as








'smart' nanoparticles which have been engineered to deliver material to or bind to a

specific biological site.

Smart Nanoparticles

Nanoparticles can be engineered to deliver to specific organs or sites in the body. The

majority of this work again comes from drug delivery research. For example, drugs that

do not normally cross the blood/brain barrier (BBB) can be carried across in designed

nanoparticles. Schroeder et al. incorporated two analgesic peptides, dalargin and

kyotorphin, which are known to not cross the BBB, into nanoparticles (75). The

nanoparticles were made of poly(butylcyanoacrylate) cores and non-ionic surfactant

coronas, either of dextran 70,000 or polysorbate 85. A coating of another non-ionic

surfactant, polysorbate 80, was added prior to injection into mice to enhance the BBB

crossing rate. Nanoparticle size was about 200 nm for the dextran 70,000 stabilized

particles and about 300 nm for those stabilized with polysorbate 85. Tests found that

neither drug caused analgesia in the mice when administered free, nor did the non-drug

carrying nanoparticles. When the nanoparticles were loaded with either drug, analgesia

was induced, indicating that the drugs passed the BBB (75).

In another interesting nanoparticle modification, Levy et al. increased the arterial

uptake of drug carrying nanoparticles by changing the nanoparticle surface charge (74).

The need for arterial drug delivery stems from the high recurrence of artery obstruction

after surgery (30-50% of patients develop reoclusion within 3-6 months) (74). In this

study, a model drug, U-86, was used to test the efficacy of the delivery system. The

nanoparticles were composed of polylactic-polyglycolic acid copolymer and were

10040 nm in diameter. Several modifying agents, (including heparin, cyanoacrylate,

and ferritin) were coated on the surface of the nanoparticles; a complete list is presented








in Table 1 of reference (74). The most effective modifier was the cationic surfactant

didodecylammonium bromide (DMBA). The free U-86 concentration in the artery walls

was 7-10 times higher after treatment with nanoparticles modified with DMBA compared

to non-modified (but still drug containing) nanoparticles (74). The authors postulate that

this is due to the change in the surface charge of the particles. The unmodified particles

have an electrical surface charge, or zeta potential, of-28 mV while the DMBA modified

particles have a zeta potential of+22 mV. This change in charge may increase the

permeability of the arterial walls (74).

Bae and coworkers modified nanoparticles to specifically interact with cancerous liver

cells. They made their nanoparticles from block copolymers ofcurdlan (corona) and

sulfonylurea (core). Curdlan is a naturally occurring polysaccharide produced by

microbes, and is known to have anti-tumor activity. Although the mechanism for this

activity is not understood, the authors propose that the interaction between curdlan and

cancer cells may make nanoparticles of this substance effective anti-tumor drug delivery

agents. Three different ratios ofcurdlan to sulfonylurea were tested, but all of the

nanoparticles were about 200 nm in their swollen state and 30-50 nm when dried (73).

Because Bae and coworkers were specifically interested in treatment of liver cancers,

they further modified the surface of the nanoparticles with glactose moieties (73). Liver

cells have glactose receptors and preferentially uptake glactosylated proteins and

macromolecules. A fluorescent probe, rhodamine isothiocyanate (RITC), was also

attached to the nanoparticles to quantify their uptake. Results show that the glactosylated

nanoparticles are well taken up by the cancerous liver cells (73). Control experiments








with other cell types or with non-glactosylated nanoparticles show only 20% of the

uptake (73).

In another example of'smart' nanoparticles, Wooley et al. functionalized the surface

of shell cross-linked particles with a protein derived from HIV that facilitates particle

incorporation into cells (84). They first prepared this protein (known as the protein

transduction domain or PTD) by peptide synthesis on a solid support. Then, in a one step

process, they detached the PTD from the support, bound it to the nanoparticles, and

degraded the interior core of the particles. This yielded hollow nanocages coated with

PTD. The size of the nanoparticles was 55 20 nm (84). The PTD nanocages were then

labeled with the fluorophore fluorescein-5-thiosemicarbazide (FTSC), and incubated with

Chinese hamster ovary (CHO) cells and HeLa cells. (HeLa cells are cancer cells often

used for research that originated from a patient named Henrietta Lacks). The fluorescent

PTD-coated nanoparticles attached to and were incorporated by both the CHO and HeLa

cells (84). Similar nanoparticles without the PTD coating showed no evidence of binding

to either cell type. The nanoparticle labeled cells could be separated from unlabeled cells

via flow cytometry (84). Drugs have not yet been incorporated into the nanoparticles for

use in targeted drug delivery, but this is the goal.

Coacervate nanoparticles, which consist of self-assembled nanospheres of ionic

polymers, have also been engineered to be 'smart'. The composition of coacervates is

described in Chapter 2. August et al. synthesized DNA-gelatin coacervate nanoparticles

coated with the protein transferring and containing calcium and the drug chloroquine, all

of which are said to increase transfection (87). Transfection is the transfer of the

nanoparticle DNA into the cell nucleus. The extent of transfection is determined by








measuring which cells produce a protein coded by the nanoparticle DNA after incubation

with the nanoparticles. Gelatin and DNA were used as the ionic polymers to create these

coacervate nanoparticles (87). The resulting particles were about 400 nm in diameter.

The cells used for transfection were human tracheal epithelial cells. Transferrin, an iron-

binding protein present in blood plasma, was bound to the outside of the nanoparticles.

This was found to be necessary for transfection (87). Nanoparticles lacking transferring

showed no transfection ability. The researchers presume that this protein acts as a ligand

to a cell surface receptor, effectively docking the nanoparticle and allowing DNA transfer

into the cell. Calcium incorporated into the nanoparticles (in the form of CaPO4) was

also found to increase cell transfection about 10 fold versus nanoparticles without

calcium (87). This may be due to the calcium enhancing the permeability of the cell

membrane. The researchers also incorporated the drug chloroquine, postulating that it

would protect the DNA in the nanoparticles from degradation from DNAse enzymes

(87). There were, however no control experiments of nanoparticles without chloroquine

to prove or refute the supposed protection.

Biomolecule-Modified Particles

An interesting recent example of biological modification of nanoparticles concerns the

functionalization of carbon nanotubes. Dai and coworkers used a pyrene-based

succinimide ester to link proteins to the exterior surface of single-walled carbon

nanotubes (133). The pyrene structure irreversibly adsorbs to the nanotube via

n-stacking. The succinimide ester is then used to covalently bind to amine groups on the

protein. The group bound both ferritin and Au nanoparticle-labeled streptavidin, and

showed the presence of both using transmission electron microscopy (133).








The group of Dr. Weihong Tan at the University of Florida has also developed

bio/nano particles for labeling applications (134). Using a sol-gel microemulsion

process, silica nanoparticles were formed containing luminescent chromophores

immobilized in the silica matrix. This silica network protects the luminophores from

quenching by environmental oxygen (134). To make the luminescent nanoparticles into

labels, they were covalently coated with immunoproteins using a cyanogen bromide

coupling reaction. First, the hydroxylated silica surface was derivatized with cyanogen

bromide. Amino groups on the antibody then bound to the carbon of the cyanogen

bromide. The nanoscopic labels were used to identify leukemia cells, reportedly easily,

clearly, and with high efficiency (134).

Recently, nanoparticles of ZnS have been arranged into highly ordered crystals by

attaching a self-ordering virus (135). Belcher and coworkers first screened a collection of

viruses to find those that effectively bound to ZnS surfaces. A virus that bound

effectively was cloned and amplified. A solution of the amplified viruses was then

incubated with ZnS nanoparticles. The ZnS binding sites on the virus bound to the ZnS

nanoparticles such that each virus contained a nanoparticle at one end (135). Because the

virus is water soluble, this resulted in a viral/nanoparticle suspension. The

viral/nanoparticles formed ordered arrays in the suspension similar to liquid crystals due

to the van der Walls interactions of the long, rod-shaped viruses (135). Ordered packing

over several micrometers was observed when the viral/nanoparticle suspension was cast

onto smooth surfaces. The authors postulate that because the ZnS nanoparticles are

magnetic, the highly ordered arrays may find application in high-density information

storage (135).








Polymer particles have been modified with a variety of biomolecules and used

effectively as extractants (136, 137). Because the polymer particles in solution resemble

colloids of polyterpene produced by rubber plants, the polymer particles are termed

'latex' particles and this type of extraction process is termed 'affinity latex'. The

pioneers in this area are the Kawaguchi group at Keio University in Japan. Latex

polymer particles are usually on the order of 200 to 1000 nm, slightly larger than true

nanoparticles. Affinity latex particles have been modified with DNA to detect point

mutations in genes (136) and with proteins for identification of drug receptors (137).

Experimental

Silica nanotubes were prepared as described in Chapter 3. Protein functionalized SiO2

nanotubes were typically prepared from the unmodified nanotubes via an aldehyde

terminated siloxane linker (96). Unmodified nanotubes were stirred in a 5% aqueous,

10% aldehyde methoxysilane (PSX1050, United Chemical Technologies) in ethanol, pH

adjusted to 5.0 with acetate. The aldehyde-modified nanotubes were filtered, rinsed with

ethanol, and dried 24 hours in an oxygen free glove box. After drying, the nanotubes

were incubated with a solution of protein in buffer (-1 mg protein/ml buffer). Incubation

was at 4 *C overnight. Aldehydes have been shown to react with pendant primary amines

on proteins (lysine residues) to covalently link the proteins to the substrate (97-99). This

resulted in proteins being bound to both the interior and exterior of the nanotubes. The

protein-modified nanotubes were filtered from the protein solution and extensively rinsed

with buffer before use.

Nanotubes were modified with proteins on the inside only using a three step

gluteraldehyde process. While still in the alumina template membrane, the nanotubes

were silanized with an amino silane. The silica-coated alumina membrane was immersed








for two hours in an ethanol solution containing 5% by volume

3-aminopropyltriethoxysilane and 5% (vol./vol.) water. After rinsing with ethanol, the

silane was cured by heating two hours at 80C. The alumina template was then dissolved

with aqueous 25% phosphoric acid and the nanotubes were filtered and extensively

rinsed. The amino-modified interior surface was further modified with aldehyde by

suspending the nanotubes in a 150 mM pH 7.4 phosphate buffer containing 2.5%

(vol./vol.) gluteraldehyde for five hours. The nanotubes were again filtered, rinsed with

buffer, and suspended in protein solutions overnight at 4C for protein attachment.

Poly(ethylene glycol) (PEG) was attached to the nanotubes using a 5000 MW PEG

silane obtained from Shearwater Inc, Huntsville, AL. An ethanol solution containing 5%

water by volume was stirred and the PEG silane was added until it reached a

concentration of 5% by volume. The pH of the solution was adjusted to 5.1 with sodium

acetate/acetic acid. To this mixure, silica nanotubes were added and stirred for 30

minutes. The nanotubes were then filtered, rinsed with ethanol to remove excess silane,

and cured at 60C overnight.

Avidin, bovine serum albumin, biotinylated gold nanoparticles, and immunoproteins

other than ENAl lHis were purchased from Sigma and used as received. Unless

otherwise noted, all instruments used were as listed in Chapter 2.

The a (R,S) and d(S,R) enantiomers of

4-[3-(4-fluorophenyl)-2-hydroxy- 1-[1,2,4]triazol- 1-yl-propyl]-benzonitrile and the Fab

antibody fragment ENA1 lHis were kindly provided Dr. Hans Soderlund and coworkers

at VTT Biotechnology, Finland. All extraction experiments were from pH 8.5 sodium

phosphate buffer containing 5% (vol./vol.) dimethylsulfoxide. Separation and








concentration determination of the enantiomers was done using a Shimadzu VP system

High Performance Liquid Chromatograph (HPLC) under the following conditions: An

Ultron ES-OVM column was used at 30 C with a running buffer of 20 mM ammonium

phosphate adjusted to pH 5.0 containing 20% methanol by volume. The flow rate was

1.0 mL/min. Detection was at 230 nm and the detection limit was at a signal to noise

level of 5. The injection loop had a volume of 20 pL. Chiral HPLC data were obtained

by Dr. Sang Bok Lee.

Extraction experiments were conducted with either 2.5 or 5.0 mL of solution. Unless

otherwise stated, the concentration of nanotubes added was -1.0 mg nanotubes per mL

solution.

The activity of glucose oxidase was assayed according to a modified literature

procedure (138). First a 50 mM, pH 5.1 acetate buffer was prepared containing 0.17 mM

o-dianisidine and 1.72% (wt./vol.) 3-D-glucose. To 2.90 mL of this solution was added

0.10 mL of aqueous peroxidase solution at a concentration of 60 purpurogallin units/mL.

Glucose oxidase-modified nanotubes were added to this mixture and shaken to disperse

the nanotubes. Production of the oxidized o-dianisidine was monitored using visible

spectroscopy at 500 nm.

Enzymatic assay of the human drug detoxification enzyme cytochrome P450 3A4 was

based on a standard method (139). An aqueous potassium phosphate buffer (0.10 M) was

prepared at a pH of 7.4. To 930.4 pL of this buffer was added 50 pL of a solution

containing 20mg/mL glucose-6-phosphatate, 20mg/mL nicotinamide adenine

dinucleotide phosphate (NADP), and 13.3 mg/mL magnesium chloride hexahydrate. Ten

microliters of a 5 mM tribasic sodium citrate solution containing 40 units/mL of








glucose-6-phosphate dehydrogenase were also added. To this mixture was added 9.6 pL

of 20.8 mM testosterone in acetonitrile. The testosterone serves as the substrate for the

enzymatic reaction. The final solution volume was 1 mL and thus the final testosterone

concentration was 200 pM. Before addition of the enzyme, the solution was warmed to

35"C. To the prewarmed mix was added 5 mg ofP450 3A4 modified silica nanotubes.

Incubation at 35C was continued for 60 minutes. At 20 minutes, half the solution (0.50

mL) was removed and 250 pL of acetonitrile was added to denature and precipitate the

enzyme and to stop product formation. As a further impediment to the reaction, the

denatured mix was transferred to an ice bath immediately after addition of acetonitrile.

This procedure was repeated for the remaining half of the assay solution at 60 minutes.

Precipitated enzyme was removed by centrifuging at 14,000 rpm for 5 minutes. The

supernatant was then filtered through 20 nm diameter alumina filters (Whatman) to

remove the enzyme-modified nanotubes.

If the enzyme is active, the testosterone substrate will be converted to

6-P-hydroxytestosterone. Analysis of product formation was performed on a Shimadzu

VP HPLC system. The separation conditions were as follows: the running buffer was

60:40 methanol:water at a flow rate of 1 mL/min. Temperature was constant at 45'C.

The column used was a Supelcosil C18 that was 15 cm x 4.6 mm with an average particle

size of 5 pum. Absorbance was monitored at 254 nm. Under these conditions the

6-1-hydroxytestosterone eluted at 2.75 minutes and the testosterone at 5.3 minutes.

Results and Discussion

Over the course of millennia, Mother Nature has designed highly specific molecular

recognition agents. Examples include enzymes, transport proteins, and immunoproteins.

These agents are termed 'smart' due to their ability to recognize one particular molecule








in an array of molecular species. The capacity to couple these agents to our nanotubes

would give the nanotubes the same molecular recognition ability, and thus render them

'smart' as well. As noted by the examples, many of the most selective of these molecular

recognition agents are proteins. A method is therefore needed to couple proteins to the

silica nanotubes.

Protein Coupling

The primary difficulty in protein attachment is the preservation of protein structure.

The molecular recognition ability of proteins is based on three-dimensional structure and

this must be preserved for retention of selectivity. Protein attachment must therefore

occur without denaturation. If the protein is denatured during the binding process, the

resulting modified nanotubes would show little selectivity or molecular recognition.

A typical approach for protein modification is the gluteraldehyde route (97, 98)

(Figure 3-1). In this approach, the surface to which the protein is to be attached is first

modified with primary amino groups. For a glass or silica surface, this is usually

accomplished via silanization with an amino silane. The surface is then treated with a

solution containing gluteraldehyde, a five-carbon dialdehyde. The aldehyde moieties on

the gluteraldehyde can bind to primary amino groups to form a Schiffbase. In this

manner, the amino surface is converted to an aldehyde surface after gluteraldehyde

treatment. Proteins are then incubated with the aldehyde-modified surface. Primary

pendant amino groups in the protein, (e. g., lysine residues), can bond to the remaining

aldehyde functionality of the gluteraldehyde. Schiffbase formation for the

protein/gluteraldehyde linkage is identical to the reaction linking the gluteraldehyde to

the amino modified surface. The result of this procedure is that protein is covalently

linked to the surface.







-OH H3CH2CO
A OH + H3CH2CO Si /' NH2
-OH H3CH2CO



1

-0 0 0
B -Oi/^ NH2 + H H
-0








-0
-ON H HProtein





_0'i/"'N=C iH --N- Protein
Figure 3-1. Schematic of gluteraldehyde protein attachment process. A) Silanization
of silica surface with aminosilane. B) Attachment ofgluteraldehyde.
C) Attachment of protein.
There are several drawbacks to the gluteraldehyde attachment route. It is a three-step
process, involving first amino modification, then dialdehyde modification, and then
modification with the protein. The total amount of protein coverage will be the product
of the coverage for each step. A loss of coverage in any one step cannot be recovered in
subsequent steps. For example, if the amino modification of the surface is low, only a








low degree of protein coverage can be expected even if the subsequent steps in the

process have high yields.

-OH H3CH2CO H
A OH + H3CH2CO-ir V' H
-OH H3CH2CO







l0
B oH
T f + H2N Protein









-0
S-0 i ----N
~-O K\^-( :Protein

Figure 3-2. Schematic of aldehyde silane protein attachment process. A) Silanization
of silica surface with aldehyde silane. B) Attachment of protein.

A particular problem is the gluteraldehyde step. Because gluteraldehyde is a

dialdehyde, it can bond both aldehyde ends to amino groups during exposure to the

amino modified surface. If this cross-linking occurs, there can be no further bonding of

the protein.

In an attempt to simplify this process, an aldehyde silane route was chosen (Figure

3-2) (96). The surface is first derivitized with the aldehyde silane, followed by








incubation with protein and attachment. The three-step process is decreased to two steps,

obviating the need for amino derivitization of the surface. This process also eliminates

the possibility of cross-linked aldehyde groups and insures that the aldehyde moiety will

be available for binding of the protein (96). The exact procedure for aldehyde

silanization and protein attachment can be found in the experimental section.

Extractions Using Protein-Modified Nanotubes

Avidin/biotin

To determine if protein binding to the nanotubes occurred successfully and without

denaturation, we attempted to bind the protein avidin. Avidin and its binding to the small

molecule biotin are discussed in Chapter 2. To be able to monitor the binding of biotin

by the avidin, biotinylated 20 nm Au nanoparticles (Sigma) were chosen as the analyte.

Au nanoparticles have very strong absorbance in the visible region due to their surface

plasmon resonance (106), which allows straightforward monitoring of their concentration

using UV-Vis spectroscopy.

The idea was to modify the surfaces of the nanotube with avidin, then use the

modified nanotubes to extract the biotinylated Au nanoparticles from solution. It has

been seen in Chapter 3 that chemically modified nanotubes are effective extractants.

Here we wanted to use specific binding between proteins and ligands to enhance the

specificity of the extraction. If the extraction was successful, it would also indicate that

the protein is attached to the nanotubes and not significantly denatured by the attachment

process.

An extraction experiment was performed that was similar to the drug extraction

experiments in Chapter 2. The absorbance spectra of the Au nanoparticle solution was

measured prior to addition of the nanotubes to determine the initial Au nanoparticle









concentration. Avidin modified nanotubes were added to an aqueous dispersion of

biotinylated Au nanoparticles at a concentration of 1 mg modified nanotubes per mL

solution. The solution was briefly sonicated after addition of the nanotubes to suspend

them. Following sonication, the solution was filtered, removing the nanotubes and any

biotinylated Au nanoparticles bound to the avidinated surfaces. The absorbance of the

filtered solution was then measured again to determine the amount ofAu nanoparticles

removed (Figure 3-3).


o A
0.15
OB

0.1


q 0.05


0

450 500 550 600 650

Wavelength (nm)
Figure 3-3. UV-Vis spectra biotinylated Au nanoparticle solution. A) Starting
solution. B) After extraction with 1 mg/mL bovine serum albumin-
modified nanotubes. C) After extraction with 1 mg/mL avidin-modified
nanotubes.

As a control experiment, the SiO2 nanotubes were modified with bovine serum

albumin, which should have little specific affinity for the biotinylated Au nanoparticles,

and the extraction efficiency was determined in the same manner. In this case, significant

nonspecific adsorption of the Au nanoparticles was observed, with 34% removed from

solution (Figure 3-3). This is likely due to the fact that the gold nanoparticles are

stabilized with albumin, which may bind non-specifically to the albumin-coated silica








nanotubes. For the extraction with the avidinated SiO2 nanotubes, the extraction

efficiency was much higher, with 100% of the Au nanoparticles removed from solution

within our ability to measure (Figure 3-3).

These results show that the protein binding via the aldehyde silane is successful. The

protein is not significantly denatured in that the three-dimensional binding pocket for

avidin is still intact and functional.

Immunoprotein extractions

To mitigate the possibility of nonspecific adsorption, we next attempted to use a more

specific protein/protein interaction for our extraction. Immunoproteins have long been

known to exhibit high specificities, with little or no affinity for analytes other than those

against which they were raised (140, 141). For example, antihuman immunoglobulin

proteins bind strongly to human immunoglobulins, but not to immunoglobulins from

other species, (e.g., mouse or rat immunoglobulins). The most common type of

immunoglobulin is Immunoglobulin G (IgG) (140). This is a Y-shaped molecule with a

molecular weight of 146 kDaltons (142). It consists of two identical top fragments called

the Fab region, and one stem fragment, called the Fc region. The molecule can be

cleaved into its Fab and Fc fragments using enzymes (142). The overall dimensions of

the molecule are -10nm tall and 10 nm wide (140). The Fab fragments are 6.0 nm by 3.5

nm and the Fc fragments are 4.0 nm wide and 4.5 nm tall. The binding site for antigens

is on the end of each of the Fab fragments (142). The binding site area has dimensions of

3.0 nm by 1.0 nm by 0.6 nm (140).

Human IgG was attached via aldehyde silanes to one batch of SiO2 nanotubes (see

experimental). Rat IgG was attached in the same way to another batch of nanotubes to

serve as a control. The analyte for these experiments was a Fab fragment of antihuman








IgG antibody that had been labeled with a fluorescent fluorescein isothiocyanate (FITC)

tag (143) for absorbance monitoring. The structure of FITC is seen in Figure 3-4.

HO OO


OH





C
II
S
Figure 3-4. Structure of fluorescein isothiocyanate (FITC).

A solution of the FITC-labeled antihuman IgG was extracted with the modified

nanotubes in a manner analogous to the avidin/biotin extractions. The absorbance before

and after extraction was measured by UV-Vis spectroscopy. Extractions used I mg of

modified nanotubes per mL of solution. Results are seen in Figure 3-5. As expected, the

human IgG-modified nanotubes effectively extracted the FITC-labeled antihuman IgG.

Comparing the initial absorbance to the absorbance after extraction shows that 76% of

the FITC-labeled antihuman IgG has been removed from solution (Figure 3-5). In

contrast, Figure 3-5 also shows that there was no appreciable antihuman extraction using

the rat IgG-functionalized nanotubes. The concentration ofFITC-antihuman IgG is

indentical before and after extraction, as indicated by the overlapping absorbance spectra.

This result proves that protein modified nanotubes can retain the selectivity of the protein

used for modifications.

Enantioselective extractions

The most specific and therefore most difficult extraction is the selective removal of

one enantiomer from a racemic mixture. Enantiomeric extraction is contingent on having









0.16
oA
0.14
A B
0.12 -
0 O c

0 0.08 -

0.06

0.04

0.02
0 .
400 450 500 550

Wavelength (nm)
Figure 3-5. UV-Vis spectra FITC-labled antihuman IgG solution. A) Starting
solution. B) After extraction with 1 mg/mL rat IgG-modified nanotubes.
C) After extraction with 1 mg/mL human IgG-modified nanotubes.

an extractant species that shows selectivity for one of the enantiomers. The recombinant

antibody fragment ENA IHis, developed by Dr. Hans Soderlund and coworkers, is

known to be selective for one stereoisomer of the drug

4-[3-(4-fluorophenyl)-2-hydroxy-l-[1,2,4]triazol-1-yl-propyl]-benzonitrile (FTB) (144).

As there are two stereocenters for this molecule, there are four stereoisomers, (R,S),

(S,R), (R,R), and (S,S). The (R,R) and (S,S) isomers are diastereomers to the (R,S) and

(S,R) isomers and can be separated by differences in physical properties. Structure of the

a (R,S) and d (S,R) enantiomers are shown in Figure 3-6. FTB is used for postsurgical

treatment of breast cancer. It functions by inhibiting the conversion of testosterone to

estradiol (144).

To produce enantioselective antibody fragments, Soderlund and coworkers first

injected a mouse with FTB. They then screened antibodies produced by the mouse for

their ability to bind each enantiomer of FTB using an enzyme-linked immunosorbant








assay (ELISA) (144). Once an antibody that selectively bound the a enantiomer

selectively had been identified, the DNA which coded the production of the Fab fragment

of that antibody was spliced into an E. coli host. The E. coli then produced a monoclonal

version of the enantioselective Fab antibody fragments, which was termed ENA1 IHis

(144). These enantioselective fragments were then attached to chromatographic gel

material and placed in a chromatographic column. A column containing the

ENA1 1His-modified gel retained the a enantiomer but allowed the d enantiomer to pass

through (144).


A B













Figure 3-6. 3-D structure of the enantiomers of
4-[3-(4-fluorophenyl)-2-hydroxy-1-[1,2,4]triazol-1-yl-propyl]benzonitrile.
A) The a or (R,S) enantiomer. B) The d or (S,R) enantiomer.

ENAI IHis obtained from Dr. Soderlund was attached to silica nanotubes via an

aldehyde siloxane linker (see experimental). The antibody fragment modified-nanotubes

were used to extract racemic solutions of the FTB enantiomers. A concentration of 1 mg

nanotubes per mL solution was used for these initial extraction experiments. After

extraction and filtering to remove the nanotubes, the amounts of a and d enantiomer

remaining in solution were determined by chiral HPLC. Chromatography data were









obtained by Dr. Sang Bok Lee. Results show that the a enantiomer was indeed

selectively extracted from solution (Figures 3-7, 3-8, and 3-9).


Retention Time (min)
Chromatograms from enantioselective extractions. Absorbance was at
230 nm. A) Racemic 5.0 LM solution after extraction with 1.0 mg/mL
ENA IHis-modified nanotubes. B) Racemic 3.0 piM solution after
extraction with 1 mg/mL ENA 1His-modified nanotubes. C) Racemic 2.0
pM solution after extraction with 1 mg/mL ENA 11His-modified
nanotubes. D) Racemic 1.0 VM solution after extraction with 1 mg/mL
ENA1 His-modified nanotubes. E) Racemic 1.0 pM solution
(unextracted) for comparison. HPLC data were obtained by Dr. Sang Bok
Lee.


The extraction ratio of the a versus the d enantiomer is dependant on the initial

concentration of the racemic mixture. For a solution that was initially 1 gM in both

enantiomers, all of the a enantiomer was extracted by the nanotubes, within our ability to

measure (Figures 3-7 D and 3-8 B). Approximately half of the d enantiomer was

extracted. As the starting racemate concentration is increased to 2 pM, 3 pM, and 5 pM,


100
90
80
70
60
50
40
30
20


10
0


Figure 3-7.


~cl~l~rrylCYhSAFk~' *C~C~' '




Full Text

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DEVELOPMENTS IN BIO-NANOTUBE SCIENCE AND TECHNOLOGY By DA YID TANNER MITCHELL A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002

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This dissertation is dedicated to my parents Dr. James P. and Terri Tanner Mitchell

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ACKNOWLEDGMENTS I would like to thank Dr. Charles Martin and the Martin group members I have had the opportunity to work with over the course of my tenure Dr. Martin never stopped thinking of applications for my research and taught me much about effective scientific communication The Martin group members have been unanimously supportive and terrific examples of ingenuity and perseverance Kshama Jirage Veronica Cepak John Hulteen, and Silvia di Vito Luebben showed great patience in training me in various analytical techniques and instrumentation Michelle Steen and Shawn Sapp gave insightful advice on experiment design In particular I would like to thank Charlie Patrissi for his help in proofreading this dissertation Lacramioara Trofin and Naichao Lee spent many hours growing aluminum oxide for my experiments for which I am in their debt. Marc Wirtz and Erich Steinle taught me the fundamentals of impedance spectroscopy and both were helpful in running experiments on ion channel mimetic membranes The enantioselective antibody experiments would have been impossible without the aid of Sang Bok Lee who helped both procure the antibody and analyze the extraction experiment results Scott Miller Elizabeth Medeiros and Shufang Yu discussed the experiments in this dissertation with me at length and provided many helpful insights Professor Vaneica Young was very kind to run and help interpret the XPS experiments Drs. Bob Lee John Chandler and Ameila Dempere g a ve me excellent training in electron microscopy Professor Dave Grainger was instrumental in my lll

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decision where to attend graduate school and has been a continual source of sage scientific and career advice Drs Donn Dennis and Tim Morey were very helpful in discussing experimental results and planning future experiments. Drs Hans Soderlund and Tarja Nevanen kindly provided the enantioselective antibody ENAl lHis. My life during graduate school would have been much less interesting without the leavening influences ofLiisa Kauri Cathy Zelenski Dean Medeiros, and Nathalie Fosse Special thanks for keeping me climbing to my friends Craig Luebben Lane Hale Corey Salzer Andrea Koenig Jason McDannold Joie and Jann Contado Mitzy Carlo and Shirley Blandon Finally my family provided unflagging moral support throughout my graduate career. For this support I thank Heather John Benjamin and James Howell ; Jonathan Rebekkah Anthony Rachel Adam and Tanner Mitchell ; Heidi Mitchell Adam and Shannon Mitchell and Jenni Mae Matthew, and Ashley Mitchell Above all, thanks to my parents Jim and Terri Mitchell for instilling in me a love of learning IV

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TABLE OF CONTENTS ACKNOWLEDGMENTS ..... ...... . .......... .......... . . ...... . .......... ...... . .... ..... .... ...... ....... iii ABSTRACT .................. ..... ......... . ................ ............ ............. .... ........... ......... ..... . viii CHAPTER 1 INTRODUCTION AND TEMPLATE SYNTHESIS ......... .................. ...... ... ............ . 1 Introduction ....... ................ ... ........... ..... . ............ ..... ...... . ....... ..... . ........ ...... .......... 1 Background ................ ............ ............ ..... ........... ... ........................ ............ .......... .... 3 Template Synthesis .... . . ......... ...... ..... ........ . ... ....... ....... ........... .... ................ .... 3 Three-dimensional templates ... .... . . .... . ....... .... . .... ............. .... . ... . . .... .... 3 Two-dimensional templates ... .............. . ..... ............ . ..... ....... ... .... .... . ...... 5 One-dimensional templates . .... ............ . ........ ... .... .... ... ... .... ...... . .... . .... . 5 Alumina Growth . ...... ................ . ....... ...... ............... ....... .... . ..... .... . ... . ....... .... 11 Polishing .... ....... .... ............... ....... ... ... ...... . . ..... ................................. ........ 11 Perfect pores ..... ..................... . ............. ... .... . . ..... . ............. ........ .... . . .... 15 Separation of the alumina film . ..... ...... ...... ... . ..... ......... ............. ..... ....... 1 6 Sol-Gel Chemistry ............................ ......... ........... ........... ......... .... . ..... . ......... 19 Silanization . ........ .......... .............. .... ...... ... ... ....... . . ..... . ..... ........ .... . ... ... . ..... 20 Chapter Summaries ........ . ...... .................................... .... ..... ....................... . ... 24 2 CHEMICAL MODIFICATION OF SILICA NANOTUBES ........... ...... .... ............... 2 6 Introduction .......... ..... . ... ....... ... .... ... ...... ........................ . ... . .......... . ... ............... 2 6 Background ... ............... . ..... ..... . . . . ... . . . ... .... . ..... . . ... ... ......... .... ................. . .... 26 Dru g Delivery . ..... ............ . ....... . ......... ...... ... ... ........ ... . .... ... . ... . . . ..... .... 2 7 Types of nano particles for drug delivery ................. ............ ........ ... ......... ... 27 Advantages of using nanoparticles for drug delivery ..... ........................... . 3 1 Engineered Sorbants and Extractants .... .... ........ . .... .... .......... ...... . ................... 32 Experimental .... ................ ..... ....... ...... ..... ................. ..... ............. ........ ....... .... ...... 3 3 Template Synthesis ...... .... . ........ ......................... ..... ... ............. ... ............ . ..... 3 3 Silanization .......... ............. ............ ... . ............ ............. .......... ..... ..... .... ............ . 3 4 Protein Modification ......... ............... .................. ........ . ..... .... .... ....... .......... ... 3 9 Biotinylation ...... ......... ...... .... .... ......... . . ... ... . .... ... .... ..... .... . ...................... ... 39 Composite Nanotube s ... . .............. . .... .......... ..... ..... . ............ ............................ 41 Results and Discussion .............. .............................. ................... . ..... ...... ............. ..... 42 Diffe rential Insid e /Outsid e Modification ... ......... ..... ........ ... ..... ................. ..... 42 V

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Hydrophilic / lipophilic nanotubes . ............. ... ......... .... ......... ..... ... . . . . ..... 42 Quinine/ Au nanoparticle nanotubes .... ........... ....... . . ........ . . ... ... ............ ... 45 Au nanoparticle/fluorescent protein nanotubes .......................... .......... ....... 47 Nanotubes as Extractants ......................... ............ ....... .......... ....... ...... .... ........ 49 C1s nanotubes for drug detoxification ..................... ....... ............. ............... 49 EDTA nanotubes for copper(II) extraction .... . .... .... .... ............ . .... . .... ...... 56 Composite Silica-Polymer Nanotubes ......................... . ... . ............. .... ...... ........ 57 Insulating polymers ............ ............ ..... ... . ..... ....... ....... .......... . ..... .... ....... 58 Poly(pyrrole) . ............... ... .......... ... .... ... .... .......... ... . .... ........ . ........ .... .... ... 59 Self-Assembly .... .... . .... .... .... ... .... ........ ............. ...... .... ............ ......... . . ....... 63 Biotin/avidin self-assembly .... ....... .................. ................. ........ ... ............... 64 Dithiol self-assembly ......... .... ...... ............ ... ... .... .............. .............. ........ 69 Conclusions ........... . ..... .... .............. . . ..... ......... ....... ............... ....... . .... ... ... . . . . ... 70 3 PROTEIN MODIFICATION OF SILICA NANOTUBES ........................... ... .......... 72 Introduction ................... ...... ........ .......... ..... ......... ........ ... ............... ........ ....... ...... 72 Background .... . ........ ..... ....... .... ... ........ . ........ .................. ... .... ... . . . ... . ............ 72 Smart Nanoparticles ... ................... ...... .......... ................. ........... ..... ... . ....... ...... 73 Biomolecule-Modified Particles . ............. .... . . ............ . . .... ..... ........ . ............. 76 Experimental . ...... .............. .... .... . . ......... . . ..... ...... . . .............. .... . ... ... .... ...... ... ...... 78 Results and Discussion . . .... . ... ...... ... ......... .... ... ...... ... ..... .... . .......... .... . ... ........ 81 Protein Coupling .... ...... ........... ..... ...... ............. ...... . ... ... ......... .......... ............... 8 2 Extractions Using Protein-Modified Nanotubes ...... ....... .................................... 85 Avidin/biotin .... .......... ..... ........ ....... ... .......................... ... ....... ...... ............. 85 Immunoprotein extract i ons . . .............. .... ....... ........... ... ... ..... . ....... .... . 87 Enantioselective extractions ....... .... ....... . . ... ........ . .... . ....... ......... . ...... .... 88 Directed Nanotube Binding . ................ ................. ........ ..... .... ..... ....... ..... ...... . 95 Enzyme Attachment and Assays ..... ........ . ..................... .............. . . ......... . .... 100 Glucose oxidase ..... .............. .......... . ....... ...... ........... ..... ... ................... 100 Cytocrome P450 enzymes for drug detoxification .. .. ... ............. ..... .......... 102 Conclusions ...... . ... ....... .... .... . . .... ... .... ........ .......... ................... .... ... ......... .... ...... 106 4 ION CHANNEL MEMETIC SENSORS .......... ... ............................ .... .... . ........... 108 Introduction ........... ........... ....... . . .... ...... ... . .... ........ ...... ..... ........................ ... ...... 108 Background ..... . .... . ............... .......... ...... .... ..... . ... .... ....... .............. .............. .... . 108 Experimental . . . .... . ..... ..... ... ............ ....... . ... .... ........ ............ .... . ... ......... . ... ... 110 Materials ....... .... ..................... ...... ... ...... ........ . ... ........... ...... .... ... ..... ... .......... 110 Membrane Preparation ........ ...... ..... .... ...... ... ...... . .......... .... ...... ..... ....... ......... 110 AC Impedance M e asur e ments ............................ . . ....... .... ........ . . . ... ...... ...... 112 X-Ra y Photoelectron Spectroscopy (XPS) ............. .............. ...... ........... ...... .... 112 Transport Experiments .... ...... ......... .......... . ............... .... ............ . ..... ... ..... ..... 113 Ion-Current Measurements ..................... ...... ... . .................. .... ........... ......... 114 Results and Disscussion .. ............. .... ... ........ ... ..... . . ..... .... .... ........ ...... .... ............. 114 XPS Studies ......... ....... . . . ..... .... . .... . . . .... ... . . ... ....... ..... ... . ..... ... ...... ....... . 114 AC Impedance E x periments with DBS Analyte ....... ..................... ..... ............. 115 V l

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Transport Experiments ..... . .... ... ....... ........ . . ...... .... ....... ...... ......... ..... ............ 118 Measurements of Ion Current .. ...... ..... .... .... ....... ......... .... .............................. 120 Effect of Alkyl Chain Length and Nature of the Surfactant Head Group ........... 121 Detection of Drug Molecules ..... ....... .... ........ .................................................. 123 The Effect of Pore Density and Pore Diameter on Analyte Detection ............ .... 124 Conclusions ... ... ........... ..................... ..... ....... ... ... .... .......... .. ..... ...... ....... ....... ..... 126 5 CONCLUSIONS .... ... ....................................... ... ............. ........... ..... ......... . ..... . 127 LIST OF REFERENCES ................. .................. .... ..... . .... . .... ................. .............. . 130 BIOGRAPHICAL SKETCH ... ............... ... . ... ... ........ ..... ... . .......... ... ....... ............ ... 140 v u

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPMENTS IN BIO-NANOTUBE SCIENCE AND TECHNOLOGY By David Tanner Mitchell December 2002 Chair : Dr. Charles R. Martin Department : Chemistry Chapter 1 provides background information on the template synthesis of nanomaterials The template synthesis method is examined with special attention to the use of membranes containing monodisperse cylindrical pores as templates Several examples of the utility of template-synthesized nanomaterials are given The production of one type of template membrane, nanopore alumina is reviewed Reviews of sol-gel and silane chemistry are also provided In Chaper 2 a sol-gel template synthesis process is used to produce silica nanotubes within the pores of alumina templates The nanotubes can be modified using a v ariet y of chemistries typically via a silanization process Because the nanotubes are formed in a template, the interior and exterior surface can be modified independently Modified nanotubes can be used for drug detoxification or as extractants for the removal of metal ions The nanotube surface can also be biotinylated which causes binding to avidin at ed surfaces Composite microtubes of silica and various polymers are also prepared Vlll

PAGE 9

Additionally Au nanowires are shown to assemble with colloidal Au particles using dithiols as linkers Chapter 3 describes the attachment of proteins onto template-synthesized silica nanotubes The proteins are covalently linked via an aldehyde silane bridge that binds to pendant primary amino moieties on the protein Protein-modified nanotubes function as highly specific extractants Avidin-modified nanotubes extract biotin-coated Au nanoparticles from solution with high extraction efficiency Immunoprotein-modified nanotubes extract the corresponding antibody from solution with high specificity Antibody-modified nanotubes extract one enantiomer from a racemic mix Enzymes including drug detoxification enzymes were also attached to the nanotubes and were shown to retain their catalytic activity Immunoproteins on the outside of nanotubes can be used to direct nanotube binding creating specific labeling agents Chapter 4 describes the production of synthetic microporous membranes that mimic ligand-gated ion channels. The membranes are prepared from microporous alumina that is silanized with a silane containing a hydrophobic C1s chain The hydrophobic C1s chains exclude aqueous solutions from entering the pores. Ionic surfactant molecules act as a chemical stimulus to allow water to flood the pores which in turn permits molecular flux across the membrane The effect of concentration and hydrophobicity of the surf act analyte species are examined, as is the effectiveness of several drug compounds as analytes The results and conclusions of this dissertation are summarized in Chapter 5 IX

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CHAPTER 1 INTRODUCTION AND TEMPLATE SYNTHESIS Introduction Nanomaterials constitute an emerging subdiscipline at the intersection of the chemical and materials sciences (1, 2) This field poses an important fundamental question-how do the electronic optical and magnetic properties of a nanoscopic particle differ from those of a bulk sample of the same material? This issue is of particular importance because all properties of a material change as the particle size approaches molecular dimensions and because it is often the unique properties of the nanomaterial that make it useful for a particular application One example ofthis concerns the optical properties of gold The yellow metallic luster of bulk gold is well known In contrast solutions of nano particles of gold are colored from ruby red to purple depending on the nanoparticle size This is due to a characteristic visible absorption band called the plasmon resonance absorption (3) Extinction coefficients for these nanoparticles are extremely high ranging from 108 to 1011 M1 cm 1 depending on particle size The high extinction coeffients of gold nanoparticles make them useful as labeling agents in optical microscopy or as indicators for colorimetric tests such as pregnancy tests ( 4) There is no universally agreed-upon definition of what constitutes a nano material. It is generally considered that at least one dimension (d) must be less than or equal to 100 nm Just larger than this are micro scale materials with dimensions in the range of 100 nm < d < 10 m Materials in both these regimes will be discussed in this dissertation

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2 In addition to size, control of shape is of primary importance in nanomaterials science Because ( at the nanometer scale) surface properties are dominant, particle shape often determines material properties Again using gold nanoparticles as an example, a large shift in particle color is observed when gold nanoparticles are transformed from an elliptical to a spherical shape (5) There are two general methods for controlling the shape of a material. These are usually termed top-down or bottom-up An example of top-down shape control is the production of a two-by-four A round tree of dimensions larger than 2" x 4" is sawn down to those dimensions Bottom-up shape control is exemplified by the pouring of concrete. Because the concrete is fluid before setting it will take the shape of the recepticle or form into which it is poured After setting the shape of the concrete is locked and the form can be removed In recent years our laboratory has pioneered a bottom-up method for the production of nanomaterials This method involves synthesizing the nanomaterials inside of a porous template (and therefore is called template synthesis). This chapter provides background information on the template synthesis of nano materials. The template synthesis method is examined with special attention to the use of membranes containing monodisperse cylindrical pores as templates. Several examples of the utility of template-synthesized nanomaterials are given The production of one type of template membrane alumina is then reviewed Reviews of sol-gel and silane chemistry that are extensively used in later chapters are then provided

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3 Background Template Synthesis One of the easiest ways to control the shape and size of a material is to synthesize it inside a form or template This process is ubiquitous in the manufacturing world Car tires concrete walls and gelatin desserts are all examples of products produced by casting the precursor material into a mold or form The size and shape of the resulting object is determined by the size and shape of the template This remains true down to nanoscopic dimensions Several natural and synthetic materials have pores or voids in the micro-or nanoscale and have been used as templates Typically these templates are classified by the dimensionality of their void spaces and therefore the dimensionality of the resulting templated structures: three-dimensional two-dimensional, or one-dimensional. Three-dimensional templates Three-dimensional template materials include micelles, glasses zeolytes, and block copolymers In the case of glasses or block copolymers the pores are the result of dissolution of a heterogenous phase For example Vycor 7930 glass manufacured by Corning, is produced by annealing borosilicate glass to produce intercalating phases that are correspondingly boron rich or silica rich. The boron-rich phase can then be dissol v ed with acid to leave a three-dimensional network of pores The average size of the pores can be controlled by the length of the annealing time Commercially availab l e siz es range from 4 to 20 nm. Indium has been templated into Vycor glass, resulting in an electronically conducting composite material (6). Vycor has also been used to template polymers such as poly(acrylic acid) (7) The composites were found to have restricted polymer chain dynamics compared to bulk polymer (7)

PAGE 13

4 Zeolites also known as molecular sieves, are crystalline solids that contain highly ordered pores The framework of zeolites are usually made of oxides of Si and Al. The pores may be empty or may contain water, ions or molecules As the moniker 'molecular sieves' would indicate, the pores are generally molecule sized, from sub-Angstrom to a few nanometers in diameter. The pores are the result of the structure of the highly crystalline oxides that make up the framework. As such they are almost perfectly monodisperse There are over a hundred types of zeolites, including natural and synthetic varieties Zeolites have been widely used as templates, including for such substances as carbon (8 9), polymers (10), semiconductors (11 12) and metals (13) Due to the sub-nanometer size of the template, the resulting materials can have extremely high surface areas The suface area of carbon templated in zeolites has been reported as high as 2000 m2/g (8) Pores in block copolymers are formed in a similar manner to those in glasses The block copolymer is made of two chemically distinct polymer types that phase separate One phase is then dissolved with an appropriate solvent. The size and shape of the resulting pores are dependant on the molecular weight and concentrations of the polymer types In contrast to glasses which have a random pore structure, the pores formed by block copolymers can be highly ordered This also is a function of the phase separation The three-dimensional network of pores in block copolymers has allowed them to be used as templates for synthetic molecular sieves, allowing control over pore dimensions (14 15) They have also been used to template nanoparticles of magnetic oxides (16) and nanowires of gold (17)

PAGE 14

5 An interesting recent three-dimensional template is synthetic colloidal crystal or synthetic opal. The crystals are made by physical close packing of micro spheres or nanospheres usually either latex or silica from a solution After or during packing the liquid is removed and a monomer solution is added The monomer is then polymerized and the original colloidal particles dissolved away (18) The distance between layers can be controlled by the diameter of the colloids used Materials templated in this way include photopolymers (19) Ge (20) and titania (21 ) The resulting structures have been shown to be photonic band gap materials (20 21) This lost wax replica of the crystal can then be further used as a template in its own right. For example the laser dye rhodamine 6G was templated using a reverse opal and found to exhibi t morphology dependant resonances due to the confinement (19) Two-dimensional templates Two-dimensional templates are layered materials Some examples include minerals Langmuir-Blodgett (L-B) films and ionic polymer multilayers Most of the layered minerals used as templates such as muscovite and montmorillonite are oxides of Al and Si, similar to zeolites As with zeolites, the layers are the result of the molecular structure Polymer multilayers and L-B films in contrast are built up manuall y one layer at a time usually by reapeated dipping of a substrate into a solution containin g o r supporting the layer Examples of two-dimensional template synthesis include organization of colloids on polyelectrolyte templates (22), and graphite synthesis in montmorillonite clay (23) One-dimensional templates The primary concern of this dissertation is template materials of one dimension These are typically membranes with pores running through the thickness of the

PAGE 15

6 membrane The pores may be naturally occurring or may be the result of extensive preparation and engineering Carbon and lipid nanotubes are naturally occurring one-dimensional templates Nanomaterials that have been deposited inside carbon nanotubes include metals (24, 25), metal oxides (26), and semiconductors (27) Lipid nanotubes have been used to template nanoscopic structures of polypyrrole (28) Another natural one-dimensional template material is aluminum oxide or alumina (AhO3). When grown under appropriate conditions cylindrical pores form normal to the surface of the membrane (Figure 1-1). The pores result from the electrochemical oxidation of aluminum. Pore size is dependant on the voltage used for oxidation and can be varied from 10 nm to 3 50 nm. The pores are highly monodisperse Pore densities range from 109 to 1012 pores/cm2 Alumina membranes with pores of nominally 200 nm diameter are available from Whatman International, Maidstone, England Nominal pore sizes of 100 nm and 20 nm are commercially available as well For sizes other than 200 nm, however, the stated pore size reflects less than 1 % of the pore length The other 99+% of the pore is 200 nm in diameter. Many groups including ours have used porous alumina as a template material. A sampling of the materials that have been prepared by template synthesis includes metals (29) polymers (30), lithium ion battery materials (31), thermoelectric materials (32) carbon (33), and semiconductors (34 35). A recent interesting application is the use of alumina to template nanometer scale metallic barcodes (36) To form the bar codes the researchers electrochemically deposited layers of metal in the alumina membrane pores By varying the metal type and layer thickness a unique set of nanowires could be composed each time The nano wires

PAGE 16

7 A B Figure 1-1. Scanning electron micrographs of aluminum oxide. A) Side view of homegrown 60 nm. B) Top view of commercial 200 nm (Whatman).

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8 Figure 1-2. Scanning electron micrograph of track-etched polycarbonate membrane with 5 m pores.

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9 are then freed by dissolving the template Nanobarcodes can be mixed in with another substance as a type of tracer or marker or used in bioassays (36) In contrast to the above one-dimensional template materials polymeric and mica membranes have pores created by irradiation with radioactive decay particles (Figure 1-2) The high-energy decay particles cause damage tracks as they pass through the polymer or mica These damage tracks are etched much faster than nondamaged material. Etchants are usually either acid or base. The resulting porous membranes are said to be 'track-etched Pores produced by this method tend to be highly monodisperse In mica the pores have a diamond-shaped cross-section while in polymers the cross-section is circular In both cases they are predominantly normal to the membrane surface although there is some deviation in angle The distribution of pores is random for the same reason Pore size is dependant on etching time and ranges from -10 nanometers to tens of microns Porosity depends on the pore density and size of the pores but is typically from 15% to 0 01% Track-etched mica was one of the earliest materials used as a one-dimensional template. Possin (3 7) deposited nanowires of several types of metals including Zn, Sn and In as early as 1970 Other researchers later modified the technique to produce nanowires of gold as small as 8 nm in diameter (38) Track-etched polymer membranes have been widely used as templates by the Mart i n lab (30 39 40) and others ( 41 ) Metal nanostructures have been synthesized by electrochemical reduction ( 42) or an electroless process (39) Solution casting (30) melt casting and polymerization from a monomer solution ( 43) have all been used to template polymeric materials in track-etched membranes Examples of the pol y mers templated

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10 include many types of insulating polymers (30) and conducting polymers (44 45). Template-synthesized nanofibers of conducting polymers have been shown to be more conductive than the bulk material (44, 45) Polymers and metals have been templated sequentially to form concentric composites consisting of an outer metal shell a passivating layer of insulating polymer, and an inner wire of conducting polymer (46) An early application of template-synthesized nanomaterials entails preparing nanoscopic electrodes Research in our lab has shown that an electroless plating method can be used to prepare ensembles of gold nanodisk electrodes with a disk diameter as small as 10 nm Such nanoelectrode ensembles (NEEs) have potential applications in electroanalytical chemistry because the signal-to-background ratio (SIB) observed at the NEE can be orders of magnitude higher than at a conventional gold macroscopic electrode disk. Thus the detection limit at the NEE that can be orders of magnitude lower than that for conventional macroscopic electrodes (39) The NEEs were obtained by electro less plating of gold within the pores of the template membrane for sufficiently long times so that solid nanowires were produced If plating is done for shorter times ensembles of nanotubes that span the complete thickness of the template membrane can be obtained. These nanotubes can have inside diameters of molecular dimensions (<1 nm) and can be used to separate molecules on the basis of size ( 47). By applying a bias to the gold nanotubes the membranes can be rendered cation permselective anion permselective or nonpermselective ( 48) Furthermore, the inside of the nanotubes can be modified using thiol chemistry to create chemically selective filters ( 40, 49)

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11 An additional type of engineered one-dimensional template is nanochannel glass (50 51 ) This differs from the Vycor glass discussed earlier in that the pores are all parallel to each other normal to the membrane surface and highly monodisperse Nanochannel glass is created by bundling fibers of glass together heating to soften and bind the fibers then pulling the softened collection of fibers to decrease their cross-section The process is repeated until there are as many as 1011 fibers/cm2 Each fiber consists of a sheath of high-silica-content glass and a core of high-boron-content glass The high-boron content glass is then etched away with acid to leave an equivalent density of pores Nanochannel glass has been used to template ferromagnetic wires that show enhanced magnetic coercivity (50) and to pattern mesoscopic ring structures of Au (51) Alumina Growth As cited earlier aluminum oxide or alumina can be produced with highly monodisperse cylindrical pores (Figures 1-1 and 1-3) This is a result of the electrochemical oxidation process in acid electrolytes (If a basic electrolyte is used a nonporous barrier oxide is formed) (52) In the growth process high-purity aluminum metal (99. 99%) is used as the anode A stainless steel cathode is used as the other half of the cell Both cathode and anode are immersed in aqueous acid electrolye and voltage is supplied by a variable power supply The geometry of cathode and anode is important for uniform alumina growth Our system uses a cylindrical cathode that surrounds the aluminum anode to assure homogenous current density on both sides of the aluminum plate (Figure 1-4 ) Polishing Because the alumina film is converted directly from the aluminum metal a rough metal surface will produce a rough alumina film, albeit with the incorporation of pores

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12 A B Figure 1-3 Scanning electron micrographs of homegrown aluminum oxide. A) Aluminum metal. B) Barrier layer.

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+ Variable Power Supply Stainless steel cathode 13 -Aluminum anode Electrolyte Figure 1-4. Electrochemical growth cell for anodic oxidation of aluminum

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14 To produce an even and flat template, highly polished aluminum metal is needed The aluminum may be mechanically polished if the surface is severly scratched or pitted Mechanical polishing is done with a slurry of either silicon carbide or alumina particles Larger particles ( > 10 m) are used to remove material quickly and polishing is continued with slurries of progressively smaller particles until the particles are submicron in size The final polishing stage is done electrochemically This process is analogous to that of alumina film formation but it is done in concentrated acid electrolyte solutions and at high temperatures to favor immediate dissolution of the alumina The electropolishing solution used in our lab is 95% concentrated phosphoric acid 5% concentrated sulfuric acid with 20 g/L CrO3 added to prevent pitting The aluminum is again the anode and a lead plate is used as the cathode The temperature of the electrolyte is kept around 70 C and between 10 and 15 V is applied for periods of roughly 5 minutes If the aluminum has been previously polished to a submicron surface roughness two or three electropolishing steps will generally result in a mirror finish The rate of the oxidation reaction is also important to final film characteristics If this becomes too high during the oxidation process, the resulting heat will cause a runawa y reaction that will preferentiall y etch the alum i num at the air / electrolyte interfa c e This will eventually result in loss of electrical connection to the aluminum To keep the reaction rate low, the electrolyte is chilled to between 0 and 4 deg rees C Polyprotic acids are used as the electrolyte (usually sulfuric acid phosphoric acid or oxalic acid) The type of acid used is dependant on the size of the pores to be grown. Smaller pore sizes require low applied volta g es and therefore require more highl y conducti v e electrolytes (e. g., s ulfuric a c id) (53) Larger pores need lar ger v olta g es that

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15 would cause a runaway reaction in a highly conductive electrolyte Thus large-pore formation uses lower conductivity electrolytes (such as oxalic acid). The concentration of the electrolyte is also important. Higher concentrations increase the current density for any given voltage This increase in current density results in an increased rate of film formation but also enhances the possibility of a runaway reaction. The most important parameter in determining the final pore size for the alumina film is the applied voltage When proper electrolyte choice and temperature control constrain the current density between 1 and 6 mA/cm2 the pore size is observed to be 1 .4 times the applied voltage (54) Perfect pores During the initial stages of film growth, the pores are somewhat random in shape a n d packing, and there is some heterogeneity in size. This is a result of the pores nucleating from random pits and valleys on the aluminum surface (55) As film growth continues the pores self organize into a hexagonal array and the pores become highly monodisperse (56) A good review of the theory of this process can be found in the dissertation of G L. Homyak (54). To produce membranes with a "perfect hexagonal packing and uniform pore size a two-step process was developed by Matsuda and coworkers (56) in 1995 The first step in the process is growth of alumina film for sufficient time to allow self-organization and homogenization of pore size That film is then removed with acid This leaves an indentation or pit in the underlying aluminum corresponding to each pore The aluminum is then re-anodized at the same voltage and in the same electrolyte as the original anodization The pores nucleate in the pits and thus are already highly ordered and monodisperse and continue to grow as patterned (55 56)

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16 Perfect pores can also be formed by indenting the aluminum on a nanoscopic scale preceding anodization Matsuda and coworkers created a SiC stamp by electron beam lithography and used this to pattern the aluminum surface Pore formation again initiated i n the preformed indentations and gave rise to highly ordered monodisperse pores (57) Separation of the alumina film There are two methods of removing the alumina film from the underlying aluminum after growth is complete. The most conceptually simple process is to dissolve the aluminum metal with HgCh. This does not damage the alumina This method works best if thin foils of aluminum are being used to grow the alumina fil ms Thin films have lower quantities of aluminum to dissolve especially after anodizatoin It should be noted that the pores in alumina do not extend all the way through to the aluminum meta l Each pore is separated from the metal by a thin barrier layer that is continuously forming and dissolving as the film grows (Figure 1-3) (58) When the aluminum is dissolved the pores on the resulting alumina are closed on one end by this barrier layer It can be removed by acid or base etching but as all of the alumina is exposed to the etchant this may slightly widen the pores. The other method of freeing the alumina from the aluminum involves a process called voltage reduction (59) After the film reaches the desired thickness, the voltage is reduced to about 70% of its original value Because the pore size is determined by the applied voltage the pores at the barrier layer branch to smaller sizes The current also decreases but subsequently proportionally to the increasing voltage The thickness of the barrier layer like the pore size is proportional to anodi z ation volta g e The volta g e reduction process is continued through many branching cycles until the barrier layer is very thin and there are many small pores branching into it. Anodization is stopped and

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17 the alumina/aluminum composite is placed in an acid or base etchant solution This dissolves the alumina everywhere However the thin barrier layer and the small pores are most quickly dissolved and the alumina is detached from the underlying aluminum metal. The alumina film separates en masse if thick ( above 20 m) or flakes off in pieces if thin The resulting membrane has a branching pore structure that resembles a tree A silica replica temp lated from alumina demonstrating the structure of the barrier layer is shown in Figure 1-5 The process used to make this replica is di cussed in detail in Chapter 2 A membrane produced by voltage reduction has two distinct faces : the solution side and the barrier side Pores on the barrier side are always smaller than the solution side due to the voltage reduction and pore branching. Voltage reduction can be done rapidly such that the branching constitutes only a fraction of the total pore length For example in commercially available 20 nm alumina (Whatman) the pores branch from 200 nm to 20 nm over just 250 nm distance Since each pore is 60 rn long this represents only 0 .4 2% of the overall pore length If a membrane with uniform pores on both sides is desired the barrier side can be removed with acid base or reactive ion etching For several types of template synthesis it is not necessary to separate the alumina film from the underlying aluminum metal. The metal is often used to give mechanical support to the membrane as will be shown in Chapter 2 Although there is a barrier layer of alumina preventing direct electrical contact various materials have been electrodeposited in alumina templates using AC deposition in which the attached aluminum metal acts as the cathode Examples include Ag (60)and CdS (61)

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18 Figure 1-5. Transmission electron micrograph of silica templated in branched pore alumina.

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19 Sol-Gel Chemistry Sol-gel technology originated in the 1970s as researchers attemped to find low temperature routes to glass synthes i s (62 63) Glass is an amorphous material, however it is generally made from crystalline oxide precursors The high temperatures usually needed to form glass (1300 to 2000 C) are a result of the need to destroy the crystallinit y of the precursors A method using noncrystalline precursors was sought. As glass is mostly SiO 2 liquid alkoxysilanes were used It was found that alkoxysilanes hydrolyze readily in the presence of water to form silanols : (1-1) The silanols can then undergo polymerization reactions with other silanols or with other alkoxysilanes R'3Si-O-H + HO-SiR'3 R'3Si-OSiR'3 + H2O R'3Si-O-H + ROSiR'3 R 3 Si-OSiR 3 + ROH (1-2) (1-3) In either case for alkoxysilanes containing two or more alkoxy groups the result is formation of a three-dimensional siloxane network. At the start of polymerization man y small particles of siloxane are formed These are well dispersed in the liquid phase to form a colloid When the particles are well isolated from each other the density of the suspension resembles that of the solvent. At this stage the colloid is called a sol. As polymerization continues the particles increase in size and the liquid increases in viscosity When polymerization has pro g ressed to the point where the particles develop a three-dimensional network throughout the liquid the colloid becomes very viscous and i s termed a gel (64) Several parameters influence the rate of the hydrolys i s and polymerization reactions These include temperature pH, water concentration and type of alkyl g roup (65) An

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20 increase in temperature increases the rate of gelation The reactions can be either acid or base catalyzed ; extremes of either high or low pH will speed the reaction, as will increasing the amount of water (66) A larger more sterically bulky alkyl group slows the reaction rate (66) Gels can be converted to silica through two routes In the first method the gel is heated or placed under vacuum to remove the solvent phase typically alcohol as well as water and other alcohols produced during polymerization The resulting surface tension collapses the open three-dimensional network of the silica phase of the colloid condensing it to a dense phase called a xerogel (65) If the solvent and water are extracted instead through a critical-point drying procedure the surface tension is eliminated and the open porous nature of the gel is maintained (67) This type of monolith is termed an aerogel. The maximum temperature for either process can be kept under 1oo c (66) Since its inception this process has been extended from silica to many types of metal oxides (68) Because at the sol stage the colloid has liquid properties it can be molded or dip coated into nearly any size or shape including nanomaterials On dip coating a gel is formed at the interface of the material immersed in the sol. This gel layer is compacted by drying to form a dense layer that replicates the surface topology of the immersed template (65) Sol-gel chemistry has been used to fabricate template-synthesized nanostructures of Ti02 for photocatalysis (34), V 2 05 as a lithium ion battery material (69), and W03 (70) Silanization As mentioned above the surface properties of a material become increasingly important as the size decreases It is therefore useful to ha v e a method for controllin g or

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21 modifying the surface chemistry of materials One relatively simple method for modifying surface chemistry is the use of organosilanes These are compounds containing an Si-C bond Because Si is in the same chemical group as carbon these compounds share much of their chemistry with organic compounds Organosilanes form stable covalent bonding with siliceous materials (e.g ., silicates and aluminates) and many metal oxides (71 ) The predominant types of organosilanes used for surface modifications are chlorosilanes and alkoxysilanes Both are usually deposited from alcohol solutions The attachment chemistry of both types is equivalent because when chorosilanes are dissolved in alcohol, they react with the alcohol to form alkoxysilanes and HCl (71) R 3 Si-Cl + ROH R 3 Si-OR + HCl (1-4) The extent of this reaction can be monitored by measuring the pH Alkoxysilane chemistry is analogous to the chemistry of sol-gel production Silica sol-gel precursors are organosilanes generally either tetramethoxysilane (TMOS) or tetraethoxysilane (TEOS) Due to its high vapor pressure and high reactivity tetrachlorosilane is used for vapor phase reactions to produce fumed silica All of these compounds have four reactive sites and thus can form three-dimensional networks The reactions are analogous to E quations 1-1 to 1-3, al t hough in the chlorosilane case, the chlorine is the leaving group If only a monolayer of surface modification is desired an organosilane is used which has only one reactive chloro-or alkoxysubstituent. Because there is only one reacti ve site these molecules can either dimerize or bind to the surface Dimers cannot bind

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22 further and are rinsed away Binding to the surface takes place via surface hydroxyl groups through the reaction R 3 Si-OH + R-O-SiR 3 "'7 Si-O-SiR' 3 + ROH (1-5) Often a higher degree of surface modification is desired To improve the density of coverage silanes with two or, more commonly three reactive groups are used These are first allowed to oligimerize in a slightly aqueous alcohol solution (71 ) The presence of water, typically around 5% vol/vol initiates the formation of silanols (Equation 1-1) increasing the reaction rate The pH is adjusted to between 4 5 and 5 5 with acetic acid to further facilitate the substitution reaction of the alkoxysilanes (71 ) The material to be surface modified is then added to this solution and the oligimers bind through surface hydroxyl sites (Figure 1-6) Surface modification by this route generally requires only a few minutes of immersion If a 2% solution of trialkyoxy-or trichlorosilane is used and five minutes is allotted for oligimer formation the resulting surface modification is normally from 3-8 monolayers thick (72) After solution deposition the silanized surface must be cured This increases cross-linking and removes residual solvent. It can be done by heating to 120 C for 20-30 min or for longer times at lower temperatures At room temperature 24 hours is generally sufficient for full curing Chlorosilanes can also be deposited from aprotic solvents such as toluene and tetrahydrofuran (71) If these solvents are anhydrous (and the substrate is free o f bound water), there will be no chance for alkoxysilanes to form There can therefore be no polymerization of the silane The reaction must proceed by nucleophilic attack from surface hydroxyl sites As a result only a monola y er can form (71 ) Surface modification b y this rout e requires lon ger times from 1 2 -24 hours H i g her temperatures

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23 r, r, r, RO-Sr-OR Ro-Si-OR Ro-Si-oR I + I + I OR OR OR + r, r, r, RO-~i--0-~i-~~i-OR OR OR OR + r, r, r, RO-~i-o-~i-o-~i-oR H 0 Si02 Figure 1-6. Trialkoxysilane oligimer formation and attachment to surface.

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24 or refluxing are also usually required, however the process does not need further curing (71) Chapter Summaries In Chaper 2 a sol-gel template synthesis process is used to produce silica microand nanotubes from alumina templates. The nanotubes can be modified with a variety of chemistries, typically via a silanization process. Because the nanotubes are formed in a template the interior and exterior surface can be modified independently For example the interior of the nanotubes can be modified with fluorophores while the exterior is modified independently to make the nanotubes hydrophilic or hydrophobic Modified nanotubes can be used for drug detoxification or as extractants for the removal of metal ions The nanotube surface can also be biotinylated, which causes binding to avidinated surfaces. Composite microtubes of silica and various polymers are also prepared. Additionally Au nanowires are shown to assemble with colloidal Au particles using dithiols as linkers The position of linkage can be controlled by using the template as a mask. Chapter 3 describes the attachment of proteins onto template synthesized microand nanotubes The proteins are covalently linked via an aldehyde silane bridge that binds to pendant primary amino moieties Because most proteins have pendant primary amines a wide range of proteins can be used to modify the nanotubes Protein-modified nano tubes function as highly specific extractants. A vidin-modified nanotubes extract biotin coated Au nanoparticles from solution with high extraction efficiency Imrnunoprotein-modified nanotubes extract the corresponding antibody from solution with high specificity Antibody-modified nanotubes extract one enantiomer from a racemic mixture Enzymes, including drug detoxification enzymes are also attached to the nanotubes and are shown

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25 to retain their catalytic activity Nanotubes modified in this way function as removable bioreactors Imrnunoproteins on the outside of nanotubes can be used to direct nanotube binding creating specific labeling agents. Chapter 4 describes the production of synthetic microporous membranes that mimic ligand-gated ion channels The membranes are initially off' that is they do not allo w molecules or ions to pass On addition of analyte, the membranes switch to an 'on' state and become permeable to molecules and ions The membranes are manufactured from microporous alumina that is silanized with a C1s silane The hydrophobic C1s chains prevent aqueous solutions from entering the pores Ionic surfactant molecules act as a chemical stimulus to turn the membranes 'on' The hydrophobic portion of the surfactant partitions into the C 1 s layer while the ionic portion of the molecule becomes the prevalent chemistry at the pore wall Water can then flood the now ionic pores and molecules and ions can cross. The effect of concentration and hydrophobicity of the surfact analyte species are examined as is the effectiveness of several drug compounds as analytes The results and conclusions of this dissertation are summarized in Chapter 5

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CHAPTER2 CHEMICAL MODIFICATION OF SILICA NANOTUBES Introduction In this chapter, a sol-gel template synthesis process is used to produce silica microand nanotubes within the pores of alumina templates These silica nanotubes can be modified via silanization with a variety of chemical functional groups The nature of the template synthesis process allows independent modification of the inner and outer nanotube surfaces Several examples of independent modification are given Nanotubes modified with C1s on the inside and unmodified on the outside are effective at sorbing drugs and drug analogs from aqueous solution Modifying the nanotubes with chelating agents makes them useful as extractants of metal ions Template synthesized structures can also be used as building blocks for the assembly oflarger structures For example silica microtubes can be biotinylated, which causes binding to avidinated microspheres Additionally Au nanowires are shown to assemble with colloidal Au particles using dithiols as linkers Composite microtubes of silica and various polymers are also prepared Background A recurrent theme in the recent literature involving nanoparticles concerns their use as drug delivery agents (7376) This process is the inverse of using nanoparticles as extractants which will be a primary subject of this chapter Because of the similarities a 26

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27 review of the basic tenants of nanoparticle drug delivery is instructive. The use of macroscopic particles as extractants is also reviewed Drug Delivery There is increasing interest in using nanoparticles for drug delivery (73-76) Spherical nanoparticles are almost always used because that shape is easy to make The term nanoparticle as used in the drug delivery literature encompasses everything from 5 nm to 5 m diameter There are a few biologically important size parameters Particles below 200 nm in diameter are less susceptible to clearance by the reticulo-endothelial system (RES) which increases their dwell time in the bloodstream and thus their efficacy (77) Particles larger than 5 m diameter will not be able to pass through capillaries and will therefore not be well distributed by the circulatory system (77) Particles less than 100 nm in diameter are said to be 'ideal in that they avoid RES uptake and facilitate access to cells and tissues (77). Types of nanoparticles for drug delivery Most nanoparticles used for drug delivery are self-assembled from block copolymers (27 77 78) Polymers are formed by linking together small monomer molecules A homopolymer is formed if only one type of monomer is used When two different types of monomers are joined in the same polymer chain the result is called a copolymer (79) Two types of monomer molecules can be made into a copolymer in many different ways If the two types of monomer are labeled A and B the copolymer could be formed by alternating A and B units : -A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-B-A-

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28 This type of copolymer is called an alternating copolymer. If the monomer units are arranged in a random fashon the copolymer is called a random copolymer An example would be as follows : -A-A-B-A-B-B-B-A-A-B-A-B-B-B-A-A-B-A-B-A-AIn a block copolymer, the monomers of each type are grouped together. A block copolymer can be thought of as two homopolymers joined together (80): -A-A-A-A-A A-A-A-A-A-A-B-B-B-B-B-B-B-B-B-B-BBlock copolymers used for drug delivery usually consist of either two or three components (77) For a two-component system, one block is hydrophilic and the other hydrophobic In a three-block system, the scheme is hydrophilic-hydrophobic-hydrophilic These systems form micelles in a manner analogous to that of surfacts. In aqueous systems the hydrophobic regions cluster together to form the interior or core of the micelle, while the hydrophilic regions create an outside shell or corona Poly(ethylene oxide) (PEO), or the related polymer poly(ethylene glycol) (PEG) are often used as the hydrophilic component (77) Poly(ethylene oxide) and PEG have nearly unlimited water solubility, and therefore a high degree of hydration and large excluded volume (81 ) These properties help to stabilize the micelles and prevent protein adsorption (77). The type of hydrophobic polymer used varies widely For a partial list of biocompatible hydrophobic polymers used for micelle formation see Table 1 of reference (77) There are two typical routes to micelle formation dissolving the block copolymer directly in water or other aqueous solvent (e.g ., buffer) and dialysis (77) The easiest

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29 method is direct dissolution, in which the block copolymer is simply dissolved in the aqueous system at a concentration high enough to cause spontaneous formation of micelles. This concentration is known as the critical micelle concentration (CMC) However in many cases the block copolymers are not soluble in water If they are not soluble the polymer is dissolved first in a water miscible organic solvent or organic/aqueous mix and then dialyzed against water or heated until the organic solvent is removed Again the micelles form spontaneously. For drug delivery applications, the core of the micelle is hydrophobic and the exterior shell or corona is hydrophilic (77) It is generally important that the working concentration of the block copolymer (that is, the concentration in the blood after injection) be kept above the critical micelle concentration (CMC) or the micelles may degrade (77) This can be avoided by cross linking either the core or the corona polymer However, if the ratio of the hydrophobic core material to the hydrophilic corona is very high micelles can be kinetically stable over long times even below their CMC (77). This is also true if the core material is well below the temperature at which there is easy movement of the polymer chains known as the glass transition temperature (T g ) or if it is highly crystalline (77) The size of the block copolymer micelle is influenced by the lengths of the core and corona blocks (77) The size of the core generally determines the amount of dru g the micelle can carry A typical core volume is only about 0 .5% of the overall micelle volume (77) The amount of drug a micelle can hold is also dependant on the hydrophobicity of the drug the drug size and the physical state of the core ( crystalline vs. liquid-like) (77) These factors also influence how quickly the drug is released from

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30 the micelle as do other factors such as the density of the corona and biodegradation rates of the polymers (77) Non-micellar nanoparticles can be prepared by polymerization of monomer in the presence of a stabilizing agent. Polymers preferred for this are usually hydrophobic and biodegradable Examples include poly(esters) (82), poly(alkylcyanoacrylates) and analogs of poly( methylidenemalonate) (83) Stabilizers are usually surfactants or carbohydrate polymers (e g dextrans) Nanoparticle size is often controlled by templating the polymer inside the stabilizer micelle or in the oil phase of an oil-in-water emulsion Dr. Karen Wooley and coworkers have modified block copolymer micelle formation by cross-linking the corona (shell) after assembly. The nanoparticles can have liquid-like or solid-like interiors depending on the T g of the hydrophobic polymer For example poly(styrene) (T g = 105C) yields solid nanoparticles while poly(isoprene) (T g = -75 C) cores are liquid (78) The cores can also be comprised of a degradable material, which can be subsequently removed to leave a nanocage structure (84) The outer hydrophil i c shells are usually poly(acrylic acid) with multiple functional sites to allow cross-linking (78) The other major synthetic route to nanoparticles for drug delivery is dendrimer chemistry Compared to polymer micelles, dendrimers are very highly monodisperse and have precisely defined chemistries Research in this area is typified by the work of Frechet who has synthesized amphiphilic dendrimers (hydrophilic PEG exterior hydrophobic phenolic core) and demonstrated their ability to incorporate and release model drugs notably phenol (85)

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31 Micelles formed from biological macromolecules such as proteins, lipids, and nucleic acids are called coacervates Like the micelles mentioned above, these form spontaineously in aqueous systems as a result of aggregation of hydrophobic regions of the molecules Like block copolymers, coacervates are often used as a delivery vehicle Because the macromolecules from which they are made are ionic the coacervates are also ionic in character They typically have a densely charged ionomer core and a more sparsely charged ionomer corona of the opposite sign (86) The inability of close ion paring caused by the different charge densities causes the sparsely charged ionomer to extend into solution, which stabilizes the particles (86) Because of their strongly ionic nature, they are not often used as drug carries, as most drugs are hydrophobic Their niche is rather the transport of charged species which can be part of the coacervate, for example DNA (87) One 'smart' example of this is discussed in Chapter 3. Advantages of using nano particles for drug delivery Paul et al. found that using nanoparticles to deliver drugs increased the drug efficacy versus the free drug (88) They used pentamidine an antileishmanial drug to treat Leishmania-infected mice. (Leishmania are protozoan parasites, usually spread by sand flea bites The disease is most common in India and Bangladesh) The efficacy was measured by the amount of drug needed to inhibit the disease The micelles were 130 to 150 nm in diameter. When they used poly(D,L-lactide ), which is biodegradable, as the core polymer they observed a three fold increase in drug efficacy When they used poly(methacrylate) as the core, the efficacy increase was even greater6 5 times the free drug but this polymer is not biodegradable (88) Nanoparticles can reduce drug toxicity Perkins et al. made nanoparticles of 50 to 100 nm diameter using drug molecules as the core and PEG-conjugated lipids as the corona

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32 They found that administering Taxol (an anticancer drug) in this way to treat ovarian cancer in mice resulted in far less toxicity than intraperitoneal (shot) or intravenous (IV) administration of the free drug (89) Encapsulation in nanoparticles can also protect fragile drugs from severe gastrointestinal conditions and allow alternate means of treatment. Carino et al. encapsulated insulin in nanoparticles of poly(lactide-co-glycolide) which were then taken orally The nanoparticles protect the insulin from enzymatic degradation in the intestinal tract and then cross the intestinal epithelium and release the insulin into the bloodstream (90) The particles produced in this study were widely variable in size from 100 nm to 5 um Since only particles smaller than about 4 um can pass the intestinal epithelium and since the percentage of passage increases with decreasing size most of the effective insulin was probably encapsulated in the smaller particles Engineered Sorbants and Extractants Macroscale engineered sorbants have proven useful in a diversity of applications (91-93) For example, the addition of hygroscopic promoters such as CaCh and LiBr to silica solutions improves water adsorption of the resulting material (91). Composites of Si02-CaCh and Si02-LiBr prepared by a sol-gel process have been shown to more than double the amount of water absorbed versus conventional zeolytes and silica gels (91) Other examples of engineered bulk silica sorbant materials include solgel prepared optical chemical sensors (93) and metal ion selective sorbants for drinking water purification (92) There are limited examples of nano-sized particles specifically engineered as sorbants and extractants Radioactive Cs can be removed from waste materials using nanoporous silica silanized with Cs-specific binders (94) Polyurethane nanoparticles have been used to remove a model pollutant from soil (95)

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33 In this chapter we build on these prior systems to synthesize silica based nanotube systems that can be engineered to selectively extract a variety of species from solution Experimental Commercial Anopore alumina membranes used as templates were obtained from Whatman (Clifton NJ). These were disks 47 mm in diameter with cylindrical pores of nominally 200 nm diameter Alumina filter membranes of nominally 20 nm pore diameter were purchased from the same source All other sizes of templates were grown in house by anodic oxidation of high purity aluminum (see Chapter 1) Unless otherwise noted chemicals were reagent grade, obtained from commercial sources and were used as received. Water was 18 MOhm pure from a Barnstead Epure model D4641 system. UV-VIS spectra were obtained using a Hitachi U-3501 spectrophotometer Inductively-coupled plasma (ICP) analysis was done on a Varian Vista RL ICP-AES Scanning electron microscopy (SEM) was performed using a Phillips 505 scanning electron microscope SEM samples were made conductive by sputtering a thin layer of Au ( -15 nm) using a Hummer model 6 sputter coater. Transmission electron microscopy (TEM) was performed on a JEOL model 200CX TEM. Template Synthesis A sol-gel template process was used to prepare the nanotubes (70) See Chapter 1 for a review of the chemistry involved First, a sol-gel silica precursor was prepared by mixing absolute ethanol tetraethylorthosilicate (TEOS Figure 2-1 A) and 1 M HCl (50 : 5 : 1 vol/vol) This solution was allowed to hydrolyze for 30 minutes Alumina template membranes were then immersed into the sol-gel w i th sonication for 1 minute after which they were air dried for 10 minutes at room temperature and oven cured overnight at 150 C To produce plain silica nanotubes the solg el treated membranes

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34 were polished on both surfaces with an aqueous aluminum oxide slurry of 1 um diameter particles This removed the surface layers of Si02 that would otherwise bind the nanotubes together. The membranes were then immersed overnight in a 25% (wt/wt) solution of H3P04 to dissolve the aluminum oxide template The nanotubes were filtered from the acid solution and repeatedly rinsed with pH 7 0 buffer and water, then dried An SEM micrograph of silica nanotubes templated in commercial 200 nm alumina can be seen in Figure 2-2 In Figure 2-3, an alumina membrane produced in house with a pore diameter of 30 nm was used as the template. Silanization Structures of all silanes used for surface modification are shown in Figure 2-1. C 1 8 functionalized surfaces were prepared by immersing either the Si02 -coated alumina membrane or freed nanotubes in a 5% aqueous 5% octadecyltrimethoxysilane (Aldrich Figure 2-lB) solution (vol/vol) in ethanol pH adjusted to 5.0 with acetate (72) T he deposition solution was stirred for 20 minutes before addition of the membrane to allow formation of siloxane oligimers Silane deposition was usually done overnight to insure maximum coverage of the C18 Following deposition the membranes were rinsed with ethanol and dried 20 minutes at 150C to cure the silane layer The silicon dioxide surface layers of the membrane were removed by mechanical polishin g with a slurry of 1 um alumina particles and the template alumina was dissolved with a 25% wt/wt solution of phosphoric acid The nanotubes were filtered and rinsed, as above Modifications with N-triethoxysilylpropylquinineurethan (Gelest Morrisville PA, Figure 2-1 C) N-triethoxysilylpropyldansylamide (Gelest Figure 2-1D), 2-[ methoxy(polyethyleneoxy)propyl]trimethoxysilane (Shearwater Huntsville, AL Figure 2-IE), or 3-mercaptopropyltrimethoxysilane (Sigma Figure 2-IF), were

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35 A) Tetraethylorthosilicate B) N-octadecyltrimethoxysilane C) N-triethoxysilylpropylquinineurethan 0 II (C2HsOhSi(CH2hNHCO H3CO I# D) N-triethoxysilylpropyldansylamide S02 NH(CH2bSi(OC2Hsh E) 2-[ methoxy(polyethyleneoxy )propyl]trimethoxysilane F) 3-mercaptopropyltrimethoxysilane 9CH3 H3C0i~SH OCH3 Figure 2-1 Structures of silanes used for modifications

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36 G) N-(trimethoxysilylpropyl)ethylenediamine triacetic acid, trisodium salt H) 3-trimethoxysilanealdehyde I) 3-aminopropyltriethoxysilane H3CH2 CO H3CH2CO-~i/'yANH2 H3CH2 CO Figure 2-1 Continued analogous Each was deposited from an ethanolic solution containing 5% (wt./vol.) of the silane The deposition solution was also 5% in distilled water by volume and was adjusted to pH 5 0 with sodium acetate. In all cases the silane solution was allowed to polymerize 20 minutes before addition of the membrane Silane deposition was accomplished by immersing the SiO 2 coated template into these solutions followed b y an ethanol rinse and heat fixing at 140C for 20 minutes. Depositions were usually performed overnight to maximize coverage The modified nanotubes were released from the template by dissolving with 25% H 3PO4 filtered from the acid solut ion, and r i nsed repeatedly with water.

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37 A B Figure 2-2. Scanning electron micrographs of silica nanotubes templated in commercial 200 nm diameter alumina membranes. A) Nanotubes held together by a film of silica B) Dispersed nanotubes.

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A 38 B Scanning electron micrographs of silica nanotubes templated in 30 nm diameter alumina membranes produced in house. A) Nanotubes held together by a film of silica. B) Dispersed nanotube. EDTA-functionalized nanotubes were prepared in a similar manner but from an aqueous silane solution. The EDTA silane, N-(trimethoxysilylpropyl)ethylenediamine triacetic acid, trisodium salt (Figure 2-lG), is not soluble in alcohol and is supplied as a 50% solution in water (Gelest). This solution is added to water 10:90 vol/vol, which makes the final silane concentration of the deposition solution -5%. The pH is adjusted to 5.0 with sodium acetate/acetic acid. Silica nanotubes produced by template synthesis were added to this solution and stirred 3-4 hours. The nanotubes were then filtered by vacuum filtration, rinsed with water and dried. Curing of the silane was accomplished by heating in a 120 oven for 25 minutes. Extraction experiments were generally performed using either 2.5 or 5.0 mL of solution. Unless otherwise noted, the concentration of nanotubes added was 1.0 mg nanotubes per mL solution.

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39 Protein Modification Protein functionalized SiO2 nanotubes were prepared from the unmodified nanotubes via an aldehyde terminated siloxane linker (96) (See Chapter 3 for a detailed discussion of protein attachment) Unmodified nanotubes (as prepared above) were stirred in a 5% aqueous, 10% aldehyde methoxysilane (PSX1050, United Chemical Technologies, Figure 2-lH) in ethanol, pH adjusted to 5 0 with acetate The aldehyde-modified nanotubes were filtered, rinsed with ethanol, and cured 24 hours in an oxygen-free glo v e box at room temperature After drying the nanotubes were incubated overnight at 4 C with a solution of protein in buffer ( 1 mg protein/ml buffer) Aldehydes have been shown to react with pendant primary amines on proteins to covalently link the proteins to the substrate (97-99) Protein-modified nanotubes were filtered from the protein solut ion and extensively rinsed with buffer before use Unless otherwise noted, proteins were obtained from Sigma in the highest purity possible and used as received Biotinylation The nanotubes were biotinylated using a multistep modification process (see Figure 2-4) An amino silane was first deposited as a coupling agent. A 2% (wt./vol.) aqueous solution of 3-aminopropyltriethoxysilane (United Chemical Technologies Figure 2-11 ) was stirred 20 minutes to allow oligimer formation Silica nanotubes from commercial templates were added to this solution and sonicated briefly to suspend The nanotubes were stirred in solution for a further 60 minutes removed by filtration and rinsed with water Heating to 120 for 20 minutes cured the aminosilane layer The amino-modified nanotubes were then added to 5 mL of 10 mM sulfosuccinimidyl 6-(biotinamido )hexanoate (Sigma) in N N-dimethylformamide in water and again sonicated to suspend Sonication was continued for 1 hour The nanotubes were

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+ 40 Na O O H 03S0 N + N-oVV'v 0 NH HN--\i Figure 2-4 Schematic of biotinylation process 0 removed from the reaction solution by vacuum filtration They were rinsed with water and then with pH 8 0 phosphate buffer Avidinated silica microspheres in buffer were purchased from Bangs Labs and used as received Two sizes were purchased, 5 0 m and 0 5 m diameter Both sizes were stored at 4 C until used

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41 Composite Nanotubes Composite nanotubes of two types were prepared : polymer exterior with a silica interior, or a silica exterior with a polymer interior The ordering of the silica versus the polymer layer was determined by order of deposition with the outermost material being the first deposited into the template. If the silica layer was to make up the outside of the nanotube, it was deposited as detailed above Inner layers of silica were deposited after polymer deposition from the same sol-gel precursor solution. The cure, however, was modified to 70C for 24 hours to prevent oxidation of previously deposited polymer Insulating polymers were deposited by dip coating the template membrane into a polymer solution and allowing it to air dry Poly(ethylene-vinyl acetate) (PEVA) was dissolved in toluene to make a 0.5% (wt./vol.) solution The PEVA was 40% by weight vinyl acetate and had an average molecular weight of100,000 Da. Poly(methyl methacrylate) (PMMA) was prepared as a 4% (wt./ vol.) solution in chloroform The molecular weight of this polymer was -35,000 Da. Both polymers were supplied by Scientific Polymer Products (Onterio NY). Residual solvent was driven off by heating the membrane to 70C for 15 minutes. The conducting polymer poly(pyrrole) was deposited chemically from an aqueous 0 2 M monomer solution The pyrrole monomer (99% TCI, Portland OR) was twice distilled under argon and stored at -20 C prior to use The template was immersed in 20 mL of the aqueous 0 2 M monomer solut ion and an equivalent volume of a solution containing O 5 M F eCb and O 5 M p-toluenesulfonic acid was added which oxidatively polymerized the pyrrole monomer (100) This reaction was done at 4 C The alumina templates were removed with 25% phosphoric acid and the nanotubes were collected by filtration

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42 Biotinylated Au nanoparticles of several sizes were supplied by Sigma Other Au nanoparticle colloid solutions were produced in house by reduction of hydrogen tetracholoraurate (101 102) To make the Au colloid, 300 mL of0. 5 mM HAuCk3H20 was heated to boiling 15 mL of boiling 1 % (wt./wt ) sodium citrate dihydrate was added The combined solution is boiled a further 10 minutes then allowed to cool to room temperature The resulting citrate-stabilized Au nanoparticles have an absorbance maximum at 519 nm and average 15 nm in diameter as measured by TEM. Results and Discussion Differential Inside/Outside Modification Perhaps the most important attribute of the nanotubes is their distinct inner and outer surfaces which can be independantly chemically and biochemically functionalized Furthermore the template method provides a very simple way to differentially modify the inner vs outer surfaces This concept is illustrated in Figure 2-5 While still embedded within the pores of the template membrane the inner surfaces are reacted with one type of silane to attach this chemistry to the inner surfaces The outer surfaces are masked because they are in contact with the pore walls of the template membrane The amphoteric alumina template is then dissolved with acid or base to liberate the na n otubes These are collected by filtration resuspended and reacted with the second silane This second silane attaches to the now accessable outer surfaces of the nanotubes Several experiments were conducted to prove this concept. Hydrophilic/lipophilic nanotubes As conclusive proof of independent inside/outside modification we modified the exterior nanotube surface chemistry in a way that would control nanotube partitioning in an organic / aqueous two-phase system One batch of nanotubes was labeled on their inner

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43 A) Cross section of Al203 template membrane showing two pores. L Pore Pore B) Sol-gel synthes i s of Si 02 nanotu bes. C) Attach first silane to inner tu b e surfaces. II I I I ' $ ,.. ;; =. -I I I I D) Remove surface films on faces of membrane and dissolve template mem b rane. E) Attach second silane to outer surfaces. Figure 2-5. Schematic of inside / outside modification of nanotubes.

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44 surfaces with the fluorescent silane N-triethoxysilylpropylquinineurethan while still in the alumina template The outer surfaces of these nanotubes were protected by the template and were therefore not silanized After silanization, the template was dissolved and the bare silica outer surface of the nano tubes was exposed These nanotubes were added to a vial containing 10 mL of water and 10 mL of the immiscible organic solvent cyclohexane The solvents were mixed by shaking the vial and then allowed to separate Because silica is naturally hydrophilic, when these nanotubes are added to the cyclohexane/water two phase system, the pale blue fluorescence of the quinineurethan ( 445 nm) is only seen from the lower aqueous phase (Figure 2-6A) The fluorescence at the top of the cyclohexane phase is due to reflection of light from the meniscus This result confirms that exterior chemistry can be modified independent of interior chemistry If the initial quinineurathane silanazation were not confined to the inner nanotube surface, the hydrophobic nature of the fluorophore would cause partitioning into the cyclohexane phase A separate batch of nanotubes was prepared with the fluorescent silane N-(triethoxysilylpropyl)dansylamide attached to their inner surfaces. This fluorophore emits in the green region (525 nm) when excited by UV light. The template membrane was then dissolved and the outer surfaces were derivatized with the very hydrophobic octadecyl silane (C1s). The nanotubes were added to an identical cyclohexane/water system which was shaken and allowed to phase separate The fluorescence image in Figure 2-6B shows that because these nanotubes are hydrophobic on their outer surfaces they partition into the upper, hydrophobic cyclohexane phase. Hence, the pale green fluorescence from the dansylamide is only seen from this phase

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45 A B C Figure 2-6 Fluorescently modified nanotubes in biphasic cyclohexane/water mix ( cyclohexane on top). A) Quinineurathane inside/bare silica outside nanotubes partition into water phase. B) Dansylamine inside/Cl8 outside nanotubes partition into cyclohexane phase. C) Mix of both types independantly phase separate. When both sets of nanotubes are added to the solvent mixture in the same vial the tubes with the C,s outer surface chemistry partition into the cyclohexane and the tubes with the silica outer surfaces go to the aqueous phase (Figure 2-6C) Quinine/ Au nanoparticle nanotubes We first created nanotubes with a quinineurethane silanized interior and an avidinated/biotin-Au nanoparticle exterior. Details of the modifications are given in the experimental section. The interior silanization with quinineurethane was done while the nanotubes were still in the alumina template. For this reason, the quinineurathane must attach on the interior surface only The outer nanotube surface is masked from silanization by contact with the alumina template. In this experiment 200 nm commercial alumina templates were used to make the nanotubes. After quinine

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46 silanization, the alumina template was dissolved in acid. This did not affect the attached quinine, which showed both absorption and fluorescence following the acid treatment (Figure 27) 0.12 0. 1 0.08 Q) 0.06 0
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47 (105) Because avidin is a tetramer, it can bind up to four molecules of biotin (103) To be able to monitor the biotin binding to the nanotubes, 20 nm diameter biotinylated Au nanoparticles (Sigma) were used. As mentioned in Chapter 1, nanoparticles of gold have extremely high molar extinction coefficients due to their surface plasmon resonance (106) The absorbtion maximum for the Au nanoparticles is 540 nm, well separated from the 340 nm maximum absorbance of the quinineurathane Biotinylated Au nanoparticles were incubated with the nanotubes and bound to the attached avidin A UVVis spectrum of the resulting dual-functionalized nanotubes shows two distinct absorption peaks one at 340 nm due to the interior bound quinineurathane and another at 540 nm caused by the exterior bound Au nanoparticles (see Figure 2-7) The above results are encouraging, although not conclusive proof that the Au nanoparticles are attached only to the exterior surface To further prove this concept a control experiment was attempted in which both the inside and outside surfaces of the nanotubes were modified with quinineurathane This was done by silanizing after template dissolution It was then attempted to silanize these quinineurathane-modified nanotubes with aldehyde silane and incubate them with protein but this proved impossible as the nanotubes would not suspend in aqueous solutions This result increases the probability that avidinization and the subsequent binding of biotinylated Au nano particles occurs on the outer surface of the nanotubes Au nanoparticle/fluorescent protein nanotubes Another example of independent inside/outside modification is the preparation of nanotubes with Au nanoparticles on the inside and FITC-labeled antibodies on the outer surface As previously we began by silanizing the nanotubes while still in the template

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48 In this case the nanotubes were functionalized with a mercaptosilane. See experimental section for details V (.) C: ro .D '"' 0
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49 As before, subsequent silanization with aldehyde silane is thought to occur primarily on the silacious nanotube exterior, as the interior has already been modified. A protein labeled with a fluorescent tag was then attached via the aldehyde-amino Shiffbase reaction (96) The protein attached was immunoglobulin G (IgG), which had a covalently-attached fluorescein isothiocyanate (FITC) group The FITC absorbs in a narrow region centered at 495 nm and thus serves as a label for protein attachment. The dual-modified nanotubes again show two distinct absorbance peaks in the UV-Vis The broad 550-650 nm peak is due to the agglomerated Au nanoparticle absorbance (inside) while the sharp 495 peak is a result of the FITC labeled protein (outside) Nanotubes as Extractants C1s nanotubes for drug detoxification Differentially modified nanotubes have a practical application as drug detoxification agents Drug overdoses affect hundreds of thousands of people per year in the United States alone (108) If the overdosed drugs are ingested, the patient's stomach can be pumped and/or filled with a slurry of carbon black to sorb the remainder. However, if the drugs are administered intraveinously or have already been absorbed into the bloodstream health professionals have little recourse. Most drugs are soluble in aqueous solution but generally prefer a more lipophilic environment due to the prevalence of hydrophobic moieties For this reason, drugs tend to deposit in fatty tissue in the body It was posited that addition oflipophilic (hydrophobic) particles to the blood would sorb the drug and therefore decrease toxicity Purely lipophilic particles would function as sorbants but may not disperse in aqueous solution. Agglomeration of the particles in the bloodstream could lead to blockage, compromising the health and even life of the patient. As demonstrated above, silica

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50 nanotubes can be differentially modified to have two different surface chemistries inside and outside Our goal was to create nanotubes with a lipophilic interior to sorb drugs and a hydrophilic exterior to ensure suspension in aqueous solution Silica nanotubes embedded in an alumina template were silanized with C1g silane as detailed in the experimental section The template masked the outer nanotube surface and consequently only the nanotube interior was modified The template was dissolved in acid and the nanotubes collected by filtration The bare silica outer surface remains hydrophilic which causes the nanotubes to suspend in aqueous solution Aqueous suspendibility of nanotubes with bare silica outer surfaces is illustrated by Figure 2-6 In this figure nanotubes with bare silica exteriors and fluorescently silanized interiors are shown to suspend in the water phase of a cyclohexane/water mixture Nanotubes modified on the exterior surface with C1 8 do not suspend in aqueous solution but rather float on top. Figure 2-9 7 8-benzoquinoline A model molecule was chosen to examine the efficacy of the nanotubes as sorbents The model needed to be of a moderate molecular weight highl y lipophilic yet at least somewhat soluble in water It should also be easy to monitor and stable over time. The compound 7 8-benzoquinoline (BQ Fig ure 2 -9) satisfies all these cond i tions It has a molecular weight of 179 g/mol and is water soluble to about 2 x 10-4 M Additionall y the tricylic structure ofBQ results in strong absorbance in the UV. Concentrations do w n t o

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51 1.0 x 10-7 M can be easily monitored with UV spectroscopy using the absorbance peaks at 233 or 266 nm. It is also stable under ambient conditions. For the BQ extraction experiments, an initial concentration of 1 0 x 10-5 M BQ was used A UV spectrum of this concentration was taken to serve as a reference (see the diamond top trace in Figure 2-10) To 2 5 mL of this solution was added 2 5 mg of nanotubes modified with C1s on the interior surface This amount of 1.0 mg nanotubes/mL solution was arbitrarily set as a benchmark A higher concentration of nanotubes would obviously remove more drug. Q) 0 3 0 25 0.2 0.15 ,: 0.1 0 05 0 o Control Solution (10 "-5 M benzoquinoline) After Extraction with SiO2 Tubules 0 After Extraction with C-18 SiO2 Tubules 'v After 2nd Extraction with C-18 SiO2 Tubules 220 240 260 280 300 Wavelength (nm) Figure 2-10 UV-Vis spectra of 7, 8-benzoquinoline extracted with C 1 s modified silica nanotubes The solution was briefly sonicated to suspend the nanotubes and then filtered through a 20 nm alumina filter. The lipophilic BQ should partition into the lipophilic nanotube interior and be removed from solution when the nanotubes are filtered After filtration the concentration of the remaining BQ was again determined by UV-Vis spectroscopy The results are shown in the circle trace of Figure 2-10 Results are tabulated in Table 2-1.

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52 Table 2-1 Absorbance of 7,8-Benzoquinoline Before and After Extractions 1.0 x 10"-5 M Extraction with Extraction with 2nd Extraction Starting Sol. Silica Nanotubes C18 Nanotubes C18 Nanotubes Abs @ 265 nm 0 1889 % Extracted 0.1708 10% 0.0339 82% 0.015 92% It is evident that most of the BQ has been removed The absorbance ofBQ remaining in solution was 0 0339, meaning that 82% of the BQ was extracted by the C18 modified nanotubes. If the BQ solution is extracted twice, each time with a concentration of 1 mg modified nanotubes per mL of solution, the amount of BQ removed increases to 92% The bottom trace of Figure 2-10 shows this result. To insure that the lipophilic C1& was responsible for the extractions, a control experiment was performed with unmodified silica nanotubes Again 1 0 mg nanotubes/mL solution was added, sonicated to disperse, and filtered As shown in the upper triangle trace of Figure 2-10, only a small amount ofBQ was removed from the solution by the unmodified nanotubes, -10%. It should be noted that for all the lipophilic extraction experiment glass transfer pipettes, syringes and vials were used The lipophilic nature of polymer (generally poly( ethylene)) storage and transfer devices would otherwise cause sorbtion of the BQ and skew the results To more closely examine the effect of compound lipophilicity on relative extraction, a series of increasingly lipophilic compounds was chosen These included the moderately hydrophilic p-cresol and, in order of increasing lipophilicity 2 4-dimethylphenol and 2 4,6-trimethylphenol. The increasing lipophilicity of these compounds is confirmed by their increasing octanol/water partition coefficients The log of the octanol/water partition coefficient for p-cresol is 1.97 (109) for 2,4-dimethylphenol it is 2 35 (109) and for 2,4 6-trimethylphenol it is 2 73 (110) Structures of all compounds are shown in

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53 Figure 2-11 Solutions of all compounds at a concentration of 1. 0 x 10-5 M were made in pH 9 0 phosphate buffer The buffer was used to insure protonation of the phenol. Deprotonation of the phenol would alter the lipophilicity of the compounds Extractions with interior modified C1s nanotubes were analogous to those for 7 8-benzoqinoline Each extraction used 1 mg nanotubes/mL solution The absorbances of the solutions were monitored before and after extraction using UV-Vis spectroscopy Results are shown in Figure 2-12 and tabulated in Table 2-2 A. OH B. O H OH Figure 2-11 Structures of Lipophilicity Study Compounds A) p-cresol. B) 2 4-dimethylphenol. C) 2,4 6-trimethylphenol. Table 2-2 Absorbance Before and After Extraction Compound Wavelength Starting Abs Abs after extraction % Extracted p-cresol 277 0 01559 0 01547 0 10% 2,4-dimethyl278 0 02131 0 01484 30% phenol 2,4 6-trimethyl280 0.01267 0.00735 42% phenol It would be expected that the most hydrophilic compound would be extracted the least. As shown in Figure 2-l 2A, this is the case The concentration of p-cresol before and after extraction is essentially identical. The more lipophilic 2 4-dimethylphenol is extracted by the C 18 modified nanotubes as seen if Figure 2l 2B The concentration after extraction is 7 0 x 10-6 M thus 30% was extracted Figure 2-12C shows that

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A Q) (,) !a .&> .... B (I) 0 Vl ..D < 0 016 0 014 0 012 0.01 0.008 0 006 0.004 0 002 0 265 0 025 0.02 0 015 0 .01 0 005 0 54 D 1 x 10 -5 M p-cresol I:, After CI 8 /Si02 tubule extraction 270 275 280 285 290 Wavelength ( run ) 0 1 x 10 "-5 M 2 4-dimethylphenol 1:, After extraction with CI 8 /Si02 tubules . .... ..... _.. .. AAA . .... ... .... ~--~ - A 295 265 270 275 280 285 290 295 Wavelength (run) Figure 2-12 UV-Vis spectra showing increasing extraction for more lipophilic compounds. A) p-cresol. B) 2 4-dimethylphenol. C) 2 4 6-trimethylphenol.

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C Q) (.) C: ro ..0 1-.. 0 Vl ..0
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56 extracted from a more concentrated starting solution. The same concentration of nanotubes removed 15 ug bupivacaine per mg nanotubes from a 1 x 10-4 M solution. However, the efficiency of the extraction was decreased, with only 45% of the total bupivacaine removed EDTA nanotubes for copper(II) extraction As a second type of chemical extraction, the nanotubes were modified with an EDT A analog (Figure 2-1 G) which we refer to as EDTriA. The details of this modification are discussed in the experimental section Note that the free silica nanotubes were silanized which results in modification of both interior and exterior surfaces The EDTriA, like EDT A, is hydrophilic and the tubes suspend easily in aqueous systems even if modified on the exterior The EDTriA is pentadentate, with one acetic acid group of EDT A replaced by an organosilane through which it is bound to the nanotubes EDT A and related multidentate compounds are known to strongly chelate many types of metal ions (111) It was therefore hypothesized that the EDTriA-modified nanotubes would be useful in removing metal ions from solution Copper (II) was chosen as a representative ion for this extraction system The binding constant for Cu2+ to hexadentate EDT A is 6 3 x 1018 (112) Although the binding constant for the pentadentate analog will be somewhat smaller is should still be considerable Extraction experiments were similar to those involving C1s nanotubes First the concentration of a buffered solution of Cu2+ was measured using Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) The initial concentration of Cu2+ was 2 0 ppm EDTriA-modified nanotubes were then added to the solution and sonicated to suspend As before 1 mg nanotubes was used per mL solution The nanotubes were

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57 filtered and the Cu2+ concentration of the filtrate solution remeasured by ICP-AES As shown in Figure 2-14 and Table 2-3 extraction removed 52% of the Cu2+ The control experiment using unmodified SiO2 nanotubes removed only 5% of the Cu2+ (Figure 2-14 and Table 2-3) Table 2-3 Cu(II) Extraction with EDTriA Nanotubes. 2.0 ppm Extraction with Extraction with Starting Sol. Silica Nanotubes C18 Nanotubes ICP-AES Area Counts Percent Extracted 503 481 5% 247 52% The results of the C1g/benzoquinoline and EDTriA/Cu2+ experiments demonstrate that our modified nanotubes can function effectively as extractants The interior and exter ior surfaces can be differentially modified to tailor nanotube chemistry for a particular use (e g suspension in aqueous solution) Composite Silica-Polymer Nanotubes During template synthesis most materials preferentially deposit on the template surface This minimizes surface energy (113) of particular importance for a high surface energy template such as alumina (114) Because of this phenomenon nanotubes are the most common structure produced by template synthesis Solid rods or wires can be templated under certain condit i ons, for example if electroless metal deposition is performed at a slow rate for long times ( 48) However it is typical for the pores in the template membrane to remain open after deposition although with a smaller diameters This fact is useful in the differential silanization of silica nanotubes as it allows exclusive alteration of the interior surface It also allows a second material to be templ ate synthesized inside the first.

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58 D 2.0 ppm Cu(II) After extraction with silica nanotubes 600 D After extraction with EDTA-silcia nanotubes 500 r--400 N M s:= 0 300 .... Vl Vl .... 8 200 -8 ::s u 100 0 Figure 2-14. ICP-AES data showing Cu2+ extraction with EDTriA-modified nanotubes. The Martin group has used this approach to manufacture concentric metal-insulating polymer-conducting polymer composites ( 46). Using 3 m diameter polyester track etched membranes as templates, Au nanotubes were deposited by an electroless process. This yields an Au microtube in each pore of the template membrane. Using the Au nanotubes as working electrodes, poly(phenylene oxide) was next deposited electrochemically from a solution of 2,6-dimethylphenol monomer. This polymer is insulating and passivates the gold nanotubes. After gold and polymer depositions, electronmicrographs showed that the pores of the membrane were still open. Finally, a conducting wire of poly(pyrrole) was grown electrochemically to fill the center of the pores (46). Insulating polymers These results show that composite structures can be synthesized using polymer track etched templates. The same process should be amenable to templating with alumina membranes. To explore this area, nanotubes of silica and polymer were prepared as

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59 described in the experimental section Two types of insulating polymers were used, poly(ethylene-vinyl acetate) (PEVA), and poly(methyl methacrylate) (PMMA). Both were deposited from solution by dip coating Polymers could be deposited either before or after silica nanotube formation The silica was deposited from a sol-gel precursor as previously discussed. Commercial 200 nm alumina membranes were used as the templates Electronmicrographs show that the membrane pores are still open after polymer and silica deposition (see Figure 2-15). The template can be removed by acid or base treatment to free the composite structures (Figure 2-16) Although it is difficult to see the silica or polymer layers from Figure 2-16, proof of the composite nature is given by the nanotube solubilities As with the C1s modified nanotubes, the dispersion of polymer-silica composite nanotubes is controlled by the chemistry of the outer surface Composites with silica on the outer surface and polymer inside suspend well in aqueous systems but do not disperse in organic solvents. Conversely composites with polymer exteriors do not disperse in water but suspend in many organic solvents, such as ethanol or toluene Care must be taken to not use solvents that will dissolve the polymer The polymer type can also affect dispersion For example, nanotubes with PMMA on the outside disperse readily in ethanol, while nanotubes coated with the more hydrophobic PEV A do not. Poly(pyrrole) Conducting polymer-silica nanocomposites were also prepared In this process the silica was deposited first, followed by chemical polymerization of poly(pyrrole) (Ppy) from an aqueous solution of the monomer. Details of the synthesis are available in the experimental section A homegrown alumina membrane with -60 nm pores was used as the template The resulting nano structures are seen in Figure 2-17.

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60 A B Figure 2-15. Scanning electron micro graphs of alumina templates after polymer and silica depostion showing open pores. A) Poly(methyl methacrylate) and silica. B) Poly(ethylene-vinyl acetate) and silica.

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61 A B ~'!r.S'.::::!!!m-1!11 Figure 2-16. Scanning electron micrographs of composite polymer outside / silica inside nanotubes templated in 200 run commerical alumina templates. A) Poly(methyl methacrylate) and silica. B) Poly(ethylene-vinyl acetate) and silica.

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A Figure 2-17 62 B Scanning electron micrographs of poly(pyrrole) inside / silica outside composite nanotubes templated in homegrown 60 nm dia. alumina membranes. A) Nanotubes held together by a film of silica. B) Dispersed nanotubes. In Figure 2-17 A the structures are held together by a surface layer of silica. Typically this layer would be polished away to free the nanotubes but here it was left intact and the nanotubes protrude like bristles from a brush. After template dissolution, the nanotubes are rinsed with water, then dried. The surface tension of the evaporating water draws together collections ofnanotubes to form the islands visible in Figure 2-17A. Figure 2-178 shows composite Ppy-silica nanotubes prepared in the same fashion but with the surface silica layer removed. As the template is dissolved, these nanotubes are freed and suspend in solution. To prove that Ppy had been deposited into the silica nanotubes an absorbance profile of an aqueous dispersion of the composite nanotubes was taken over the visible region. Poly(pyrrole) has a characteristic broad absorbance from 600 nm to 2 m Absorbance

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(1) (..) .D I-< 0 en .D 0.02 0 015 0.01 0 005 0 300 63 400 500 600 700 Wavelength (mn) Figure 2-18 UV-Vis spectra ofpoly(pyrrole) / silica composite nanotubes showing poly(pyrrole) absorbance 800 in this region is not seen for the bare silica nanotubes The UV-Vis spectrum is seen in Figure 2-18 The broad peak starting at -600 nm indicates that Ppy has been deposited This type of composite nanotube may have analytical applications, as the Ppy being oxidized or reduced could generate an analytical signal. Self-Assembly The small size of microand nanoscale materials often makes it difficult to assemble them into organized assemblies Physical manipulation in this size regime is challenging even with highly specialized equipment. A better solution is relying on interacting particle chemistries to self-assemble The Whitesides group has been very active in this area, using a variety of techniques to self-assemble structures from macro to sub-micro scale (115-118) Natan and coworkers have used various chemical and biochemical strategies to self-assemble colloidal Au particles onto surfaces (119 120) Mirkin's group used thiol linkers to bind colloidal Au particles to single stranded oligonucleotides These nanoparticles were subsequently self-assembled into a polymeric network by

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64 addition of the complementary oligonucleotide (121 122). Thiol linkers have also been used to attach Au nanoparticles to carbon nanotubes (123) Biotin/avidin self-assembly To date, template synthesized microand nanostructures have not been self-assemb l ed in this way. To examine the possibility of self-assembling silica nanotubes, the nanotubes were modified with biotin As noted above, biotin binds very tightly to the protein avidin Incubation ofbiotinylated nanotubes with avidinated microspheres should result in binding of the two to form an agglomerated structure Biotinylation of silica nanotubes is described in detail in the experimental section Briefly, the nanotubes were first silanized with a primary amino group that then bound a succinimide ester containing the biotin The biotin was at the end of a long linker arm to ensure that steric hindrance would not compromise binding (124) B1otinylation occurred on both the inside and outside surfaces of the nanotubes The biotin-modified nanotubes were incubated with commercially available avidinated silica microspheres (Bangs Labs) Two sizes of microspheres were used 5.0 m and 0.5 m diameter Control experiments were performed with poly( ethylene glycol) (PEG) silanized silica nanotubes Poly( ethylene glycol) is known to prevent adsorbtion to proteins If bare nanotubes were used as a control the silica would likely bind to the avidin on the microspheres non-specifically When biotinylated nanotubes were incubated with the 5 0 m avidinated silica particles, they adhered to the particle surface (Figure 2-19). If PEG coated nanotubes were used no adhesion was seen (Figure 2-20) Similarly 0.5 m silica particles agglomerated with the biotinylated nanotubes (Figure 2-21 ) but not with PEG-y lated nanotubes (Figure 2-22)

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65 Figure 2-19. Scanning electron micrographs ofbiotinylated silica nanotubes on 5.0 m diameter avidinated silica spheres

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66 Figure 2-20. Scanning electron micrographs of poly( ethylene gl y col) modified silica nanotubes and 5.0 m diameter avidinated silica spheres.

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67 A B Figure 2-21. Scanning electron micrographs of biotinylated silica nanotubes and 0.5 m diameter avidinated silica spheres. A) Ball/stick clusters. B) Agglomerated ball/stick mass.

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68 A B Figure 2-22. Scanning electron micrographs of poly( ethylene glycol) coated silica nanotubes and 0.5 m diameter avidinated silica spheres. A) Isolated balls and sticks. B) Agglomerated stick mass and isolated balls.

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69 Dithiol self-assembly While the self-assembled structures described thus far have dimensions i n the micrometer regime, there is no fundamental reason that this strategy cannot be applied to much smaller nanowires and nanoparticles An alternative self-assembly chemistry was used to prove this point. This entailed the use of 1,9-nonanedithiol to assemble colloidal Au particles (10 nm diameter) to the ends of 30 nm diameter template synthesized Au nanowires (see experimental section for details). A gold film was sputtered onto one face of a polycarbonate template membrane with 30 nm diameter pores This film served as the working electrode for deposition of Au nanowires within the pores of this membrane (125) The nanowire-containing membrane was then immersed overnight into a 1 mM ethanolic solution of the dithiol. This resulted in chemisorption of the dithiol onto the ends of the Au nanowires and also onto the sputtered Au surface film The Au film was removed by polishing with a laboratory tissue. Because this polishing removed the surface film the only remaining dithiolated surface was the exposed ends of the Au nanowires The membrane was then immersed overnight into an aqueous colloid of Au nanoparticles which resulted in binding of the Au nanoparticles to the ends of the dithiolated nanowires The membrane was then rinsed with water to remove any unbound Au nanoparticles The template membrane was dissolved away by immersion in CHCIJ to yield a suspension (125) of the Au nanowire/colloidal particle assemblies A drop of this suspension was applied to a transmission electron microscopy grid Figure 2-23 shows transmission electron micrographs of gold nanowire / colloidal particle assemblies Note that because the nanowires were embedded within the template when they were exposed to the dithiol and colloidal nanoparticle suspension, the Au

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70 particles are self-assembled to only the ends of the Au nanowires. About 10% of the nanowires in such images were terminated with colloidal particles. -Figure 2-23. m '21\ II Ill 11111 21 -1 m ~AMOROOS -129054 188 ilU X288k 28n1 Transmission electron micrographs of template synthesized 30 nm diameter Au nanowires to which 10 nm Au particles have been self-assembled using 1,9-nonanedithiol. Conclusions Novel nanoparticulate silica-based extractants have been prepared with a variety of chemistries. The initial step involves the template synthesis of silica nanotubes in a porous alumina membrane. The nanotubes can then be modified with different chemical moieties depending on the extraction desired, typically via a silanization process. The inner and outer surface chemistries can be modified differentially. Mixed nanotubes can be prepared in which the inside is silanized with a quinineurathane and the outside coated with protein-bound Au nanoparticles. A further example is the preparation of nanotubes with thiol-bound Au nanoparticles on the inside and fluorescently labeled proteins on the outside. Outer surface chemistry can direct

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71 nanotube phase segregation Nanotubes with unmodified silica exteriors partition into and suspend in aqueous solutions. In contrast, nanotubes modified with C1s on the outer surface partition into and suspend in hydrophobic organic solvents Template synthesized nanotubes can be used as extractants Using C 1 s modified nanotubes, model and actual drugs can be extracted from aqueous solution Extraction efficiencies range from 45% to 85%, with higher efficiencies for lower initial drug concentrations Nanotubes modified with an EDTA analog extract copper ions from water. Composite polymer-silica nanotubes have been prepared The polymer can be compose either the external or internal surface As with the differentially modified nanotubes surface chemistry controls dispersion properties Template synthesized nanotubes and nanowires can be used as building blocks for self-assembly Biotinylated silica nanotubes bind to avidinated silica microspheres Dithiol chemistry can be used to link Au nanowires with Au nanoparticles

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CHAPTER3 PROTEIN MODIFICATION OF SILICA NANOTUBES Introduction This chapter describes the attachment of proteins onto template synthesized nanotubes The proteins are covalently linked via an aldehyde silane bridge that binds primary amino groups Because most proteins have pendant primary amines, a wide range of proteins can be used to modify the nanotubes The molecular recognition capabilities of certain proteins can be retained after binding to make the protein-modified nanotubes exceptionally specific extractants. Avidin-modified nanotubes extract biotin coated Au nanoparticles from solution with high extraction efficiency Immunoprotein-modified nanotubes extract the corresponding antibody from solution with high specificity Nanotubes modified with an enantioselective antibody extract one enantiomer from a racemic mix. Enzymes, including drug detoxification enzymes, are also attached to the nanotubes and shown to retain their catalytic activity Nanotubes modified in this way function as bioreactors Immunoproteins on the outside of nanotubes can be used to direct nanotube binding creating specific labeling agents Background Nanoparticles have recently found use in a variety of biomedical applicat i ons These include enzyme encapsulation (126, 127), DNA transfection (128 129) biosensors (36 130 132), and as detailed in Chapter 3, drug delivery (73-76) Linkage of the biomolecule of interest to the nanoparticle is a critical step to increasing nano particle utility Several examples of biomolecule particle modification are reviewed as well as 72

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73 'smart' nanoparticles which have been engineered to deliver material to or bind to a specific biological site. Smart N anoparticles Nanoparticles can be engineered to deliver to specific organs or sites in the body The majority of this work again comes from drug delivery research. For example, drugs that do not normally cross the blood/brain barrier (BBB) can be carried across in designed nanoparticles Schroeder et al. incorporated two analgesic peptides dalargin and kyotorphin which are known to not cross the BBB into nanoparticles (75) The nanoparticles were made of poly(butylcyanoacrylate) cores and non-ionic surfactant coronas either of dextran 70,000 or polysorbate 85. A coating of another non-ionic surfactant polysorbate 80, was added prior to injection into mice to enhance the BBB crossing rate Nanoparticle size was about 200 nm for the dextran 70,000 stabilized particles and about 300 nm for those stabilized with polysorbate 85 Tests found that neither drug caused analgesia in the mice when administered free, nor did the non-drug carrying nanoparticles When the nanoparticles were loaded with either drug, analgesia was induced indicating that the drugs passed the BBB (75) In another interesting nanoparticle modification Levy et al. increased the arterial uptake of drug carrying nanoparticles by changing the nanoparticle surface charge (74) The need for arterial drug delivery stems from the high recurrence of artery obstruction after surgery (30-50% of patients develop reoclusion within 3-6 months) (74) In this study a model drug, U-86, was used to test the efficacy of the delivery system. The nanoparticles were composed of polylactic-polyglycolic acid copolymer and were 100 nm in diameter Several modifying agents, (including heparin cyanoacrylate and ferritin) were coated on the surface of the nano particles ; a complete list is presented

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74 in Table I of reference (74) The most effective modifier was the cationic surfactant didodecylammonium bromide (DMBA) The free U-86 concentration in the artery walls was 7-10 times higher after treatment with nanoparticles modified with DMBA compared to non-modified (but still drug containing) nanoparticles (74). The authors postulate that this is due to the change in the surface charge of the particles The unmodified particles have an electrical surface charge, or zeta potential, of -28 m V while the DMBA modified particles have a zeta potential of +22 m V. This change in charge may increase the permeability of the arterial walls (74) Bae and coworkers modified nanoparticles to specifically interact with cancerous liver cells They made their nanoparticles from block copolymers of curdlan ( corona) and sulfonylurea (core) Curdlan is a naturally occurring polysaccharide produced by microbes and is known to have anti-tumor activity Although the mechanism for this activity is not understood, the authors propose that the interaction between curdlan and cancer cells may make nanoparticles of this substance effective anti-tumor drug delivery agents Three different ratios of curdlan to sulfonylurea were tested, but all of the nanoparticles were about 200 nm in their swollen state and 30-50 nm when dried (73) Because Bae and coworkers were specifically interested in treatment ofliver cancers, they further modified the surface of the nanoparticles with glactose moieties (73) Liver cells have glactose receptors and preferentially uptake glactosylated proteins and macromolecules A fluorescent probe rhodamine isothiocyanate (RITC), was also attached to the nanoparticles to quantify their uptake. Results show that the glactosylated nanoparticles are well taken up by the cancerous liver cells (73) Control experiments

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75 with other cell types or with non-glactosylated nanoparticles show only 20% of the uptake (73) In another example of 'smart' nanoparticles, Wooley et al. functionalized the surface of shell cross-linked particles with a protein derived from HIV that facilitates particle incorporation into cells (84) They first prepared this protein (known as the protein transduction domain or PTD) by peptide synthesis on a solid support Then in a one step process, they detached the PTD from the support, bound it to the nanoparticles and degraded the interior core of the particles This yielded hollow nanocages coated with PTD. The size of the nanoparticles was 55 20 nm (84) The PTD nanocages were then labeled with the fluorophore fluorescein-5-thiosemicarbazide (FTSC), and incubated with Chinese hamster ovary (CHO) cells and HeLa cells (HeLa cells are cancer cells often used for research that originated from a patient named Henrietta Lacks). The fluorescent PTO-coated nanoparticles attached to and were incorporated by both the CHO and HeLa cells (84) Similar nanoparticles without the PTD coating showed no evidence of binding to either cell type The nanoparticle labeled cells could be separated from unlabeled cells via flow cytometry (84) Drugs have not yet been incorporated into the nanoparticles for use in targeted drug delivery but this is the goal. Coacervate nanoparticles which consist of self-assembled nanospheres of ionic polymers have also been engineered to be smart' The composition of coacervates is described in Chapter 2 August et al. synthesized DNA-gelatin coacervate nanoparticles coated with the protein transferrin and containing calcium and the drug chloroquine all of which are said to increase transfection (87) Transfection is the transfer of the nanoparticle DNA into the cell nucleus The extent of transfection is determined by

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76 measuring which cells produce a protein coded by the nanoparticle DNA after incubation with the nanoparticles Gelatin and DNA were used as the ionic polymers to create these coacervate nanoparticles (87). The resulting particles were about 400 nm in diameter The cells used for transfection were human tracheal epithelial cells. Transferrin, an iron binding protein present in blood plasma, was bound to the outside of the nanoparticles This was found to be necessary for transfection (87). Nanoparticles lacking transferrin showed no transfection ability The researchers presume that this protein acts as a ligand to a cell surface receptor effectively docking the nanoparticle and allowing DNA transfer into the cell Calcium incorporated into the nanoparticles (in the form of CaP04 ) was also found to increase cell transfection about 10 fold versus nanoparticles without calcium (87) This may be due to the calcium enhancing the permeability of the cell membrane The researchers also incorporated the drug chloroquine, postulating that it would protect the DNA in the nanoparticles from degradation from DNAse enzymes (87) There were however no control experiments of nanoparticles without chloroquine to prove or refute the supposed protection Biomolecule-Modified Particles An interesting recent example of biological modification of nano particles concerns the functionalization of carbon nanotubes Dai and coworkers used a pyrene-based succinimide ester to link proteins to the exterior surface of single-walled carbon nanotubes (133) The pyrene structure irreversibly adsorbs to the nanotube via 1t-stacking The succinimide ester is then used to covalently bind to amine groups on the protein The group bound both ferritin and Au nanoparticle-labeled streptavidin and showed the presence of both using transmission electron microscopy (133)

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77 The group of Dr. Weihong Tan at the University of Florida has also developed bio/nano particles for labeling applications (134) Using a sol-gel microemulsion process silica nanoparticles were formed containing luminescent chromophores immobilized in the silica matrix This silica network protects the luminophores from quenching by environmental oxygen (134) To make the luminescent nanoparticles into labels they were covalently coated with immunoproteins using a cyanogen bromide coupling reaction First the hydroxylated silica surface was derivatized with cyanogen bromide Amino groups on the antibody then bound to the carbon of the cyanogen bromide The nanoscopic labels were used to identify leukemia cells reportedly easily clearly and with high efficiency (134) Recently nanoparticles of ZnS have been arranged into highly ordered crystals by attaching a self-ordering virus (135) Belcher and coworkers first screened a collection of viruses to find those that effectively bound to ZnS surfaces A virus that bound effectively was cloned and amplified A solution of the amplified viruses was then incubated with ZnS nanoparticles The ZnS binding sites on the virus bound to the ZnS nanoparticles such that each virus contained a nanoparticle at one end (135) Because the virus is water soluble this resulted i n a viral/nanoparticle suspension The viral/nanoparticles formed ordered arrays in the suspension similar to liqu i d crystals due to the van der Walls interactions of the long rod-shaped viruses (135) Ordered packing over several micrometers was observed when the viral/nanoparticle suspension was cast onto smooth surfaces The authors postulate that because the ZnS nanoparticles are ma g netic the highly ordered arrays may find application in high-density information storage (135)

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78 Polymer particles have been modified with a variety of biomolecules and used effectively as extractants (136, 137) Because the polymer particles in solution resemble colloids of polyterpene produced by rubber plants, the polymer partic.les are termed 'latex' particles and this type of extraction process is termed affinity latex' The pioneers in this area are the Kawaguchi group at Keio University in Japan Latex polymer particles are usually on the order of 200 to 1000 run, slightly larger than true nanoparticles Affinity latex particles have been modified with DNA to detect point mutations in genes ( 136) and with proteins for identification of drug receptors ( 13 7) Experimental Silica nanotubes were prepared as described in Chapter 3 Protein functionalized Si02 nanotubes were typically prepared from the urunodified nanotubes via an aldehyde terminated siloxane linker (96) Urunodified nanotubes were stirred in a 5% aqueous 10% aldehyde methoxysilane (PSX1050, United Chemical Technologies) in ethanol, pH adjusted to 5 0 with acetate The aldehyde-modified nanotubes were filtered, rinsed with ethanol, and dried 24 hours in an oxygen free glove box. After drying, the nanotubes were incubated with a solution of protein in buffer (-1 mg protein/ml buffer) Incubation was at 4 c overnight. Aldehydes have been shown to react with pendant primary amines on proteins (lysine residues) to covalently link the proteins to the substrate (97-99) This resulted in proteins being bound to both the interior and exterior of the nanotubes The protein-modified nanotubes were filtered from the protein solution and extensively rinsed with buffer before use. Nanotubes were modified with proteins on the inside only using a three step gluteraldehyde process While still in the alumina template membrane, the nanotubes were silanized with an amino silane The silica-coated alumina membrane was immersed

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79 for two hours in an ethanol solution containing 5% by volume 3-aminopropyltriethoxysilane and 5% (vol./vol.) water. After rinsing with ethanol, the silane was cured by heating two hours at 80C The alumina template was then dissolved with aqueous 25% phosphoric acid and the nanotubes were filtered and extensively rinsed The amino-modified interior surface was further modified with aldehyde by suspending the nanotubes in a 150 mM pH 7.4 phosphate buffer containing 2 .5% (vol./vol.) gluteraldehyde for five hours The nanotubes were again filtered rinsed with buffer, and suspended in protein solutions overnight at 4 C for protein attachment. Poly( ethylene glycol) (PEG) was attached to the nanotubes using a 5000 MW PEG silane obtained from Shearwater Inc, Huntsville, AL An ethanol solution containing 5% water by volume was stirred and the PEG silane was added until it reached a concentration of 5% by volume The pH of the solution was adjusted to 5. I with sodium acetate/acetic acid To this mixure silica nanotubes were added and stirred for 30 minutes The nanotubes were then filtered, rinsed with ethanol to remove excess silane and cured at 60C overnight. A vidin, bovine serum albumin, biotinylated gold nanoparticles, and immunoproteins other than ENAI lHis were purchased from Sigma and used as received Unless otherwise noted all instruments used were as listed in Chapter 2 The a (R,S) and d (S,R) enantiomers of 4-[3-( 4-fluorophenyl)-2-hydroxy-l-[ l 2,4]triazoll-yl-propyl]-benzonitrile and the Fab antibody fragment ENAI IHis were kindly provided Dr. Hans Soderlund and coworkers at VTT Biotechnology Finland All extraction experiments were from pH 8 5 sodium phosphate buffer containing 5% (vol./vol.) dimethylsulfoxide Separation and

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80 concentration determination of the enantiomers was done using a Shimadzu VP system High Performance Liquid Chromatograph (HPLC) under the following conditions : An Ultron ES-OVM column was used at 30 C with a running buffer of20 mM ammonium phosphate adjusted to pH 5 0 containing 20% methanol by volume The flow rate was 1.0 mL/min Detection was at 230 nm and the detection limit was at a signal to noise level of 5 The injection loop had a volume of 20 L. Chiral HPLC data were obtained by Dr. Sang Bok Lee Extraction experiments were conducted with either 2 5 or 5 0 mL of solution Unless otherwise stated the concentration of nanotubes added was -1. 0 mg nanotubes per mL solution The act i vity of glucose oxidase was assayed according to a modified literature procedure (138) First a 50 mM, pH 5 1 acetate buffer was prepared containing 0 17 mM o-dianisidine and 1.72% (wt./vol.) P-D-glucose To 2 90 mL of this solution was added 0 10 mL of aqueous peroxidase solution at a concentration of 60 purpurogallin units/mL Glucose oxidase-modified nanotubes were added to this mixture and shaken to disperse the nanotubes Production of the oxidized o-dianisidine was monitored using visible spectroscopy at 500 nm Enzymatic assay of the human drug detoxification enzyme cytochrome P450 3A4 was based on a standard method (I 39) An aqueous potass i um phosphate buffer (0 10 M) was prepared at a pH of 7.4 To 93 0 .4 L of this buffer was added 50 L of a solution containing 20mg/mL glucose-6-phosphatate 20mg/mL nicotinamide adenine dinucleotide phosphate (NADP), and 13.3 mg/mL magnesium chloride hexahydrate Ten microliters of a 5 mM tribasic sodium citrate solution containing 40 units / mL of

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81 glucose-6-phosphate dehydrogenase were also added To this mixture was added 9 6 L of 20.8 rnM testosterone in acetonitrile The testosterone serves as the substrate for the enzymatic reaction The final solution volume was 1 mL and thus the final testosterone concentration was 200 M. Before addition of the enzyme the solution was warmed to 35C To the prewarmed mix was added 5 mg of P450 3A4 modified silica nanotubes Incubation at 35 C was continued for 60 minutes At 20 minutes half the solut ion (0 50 mL) was removed and 250 L of acetonitrile was added to denature and precipitate the enzyme and to stop product formation As a further impediment to the reaction the denatured mix was transferred to an ice bath immediately after addition of acetonitrile This procedure was repeated for the remaining ha l f of the assay solution at 60 minutes Precipitated enzyme was removed by centrifuging at 14 ,000 rpm for 5 minutes The supernatant was then filtered through 20 nm diameter alumina filters (Whatman) to remove the enzyme-modified nanotubes If the enzyme is active the testosterone substrate will be converted to 6-P-hydroxytestosterone Analysis of product formation was performed on a Shimadzu VP HPLC system The separation conditions were as follows : the running buffer was 60:40 methanol : water at a flow rate of 1 mL/min Temperature was constant at 45C The column used was a Supelcosil C1s that was 15 cm x 4 6 mm with an average particle size of 5 m Absorbance was monitored at 254 nm Under these conditions the 6-P-hydroxytestosterone eluted at 2 75 minutes and the testosterone at 5 3 minutes Results and Discussion Over the course of millennia Mother Nature has designed highly specific molecular recognition agents Examples include enzymes transport proteins and immunoprote i ns These a g ents are termed smart due to their ability to reco g nize one particular molecule

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82 in an array of molecular species The capacity to couple these agents to our nanotubes would give the nanotubes the same molecular recognition ability and thus render them smart' as well. As noted by the examples, many of the most selective of these molecular recognition agents are proteins A method is therefore needed to couple proteins to the silica nanotubes Protein Coupling The primary difficulty in protein attachment is the preservation of protein structure The molecular recognition ability of proteins is based on three-dimensional structure and this must be preserved for retention of selectivity Protein attachment must therefore occur without denaturation If the protein is denatured during the binding process the resulting modified nanotubes would show little selectivity or molecular recognition A typical approach for protein modification is the gluteraldehyde route (97, 98) (Figure 3-1) In this approach the surface to which the protein is to be attached is first modified with primary amino groups For a glass or silica surface this is usually accomplished via silanization with an amino silane The surface is then treated with a solution containing gluteraldehyde a five-carbon dialdehyde The aldehyde moieties on the gluteraldehyde can bind to primary amino groups to form a Schiff base In this manner the amino surface is converted to an aldehyde surface after gluteraldehyde treatment. Proteins are then incubated with the aldehyde modified surface Primary pendant amino groups in the protein (e g lysine residues), can bond to the remaining aldehyde functionality of the gluteraldehyde Schiff base formation for the protein/gluteraldehyde linkage is identical to the reaction linking the gluteraldehyde to the amino modified surface The result of this procedure is that protein is covalently linked to the surface

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A B C + 83 H3CH2CO H3CH2CO-~i~NH2 H3CH2CO ! 0 0 H~H ~}i~N-Ci'.iVHG'=NProtein Protein Figure 3-1. Schematic of gluteraldehyde protein attachment process A) Silanization of silica surface with arninosilane B) Attachment of gluteraldehyde C) Attachment of protein There are several drawbacks to the gluteraldehyde attachment route It is a three-step process involving first amino modification, then dialdehyde modification, and then modification with the protein The total amount of protein coverage will be the product of the coverage for each step A loss of coverage in any one step cannot be recovered in subsequent steps For example if the amino modification of the surface is low only a

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84 l o w degree of protein coverage can be expected even if the subsequent steps in the process have high yields A B ~! + i to}i~H + ~}i~ H2N Protein Protein Figure 3-2. Schematic of aldehyde silane protein attachment process A) Silanization of silica surface with aldehyde silane B) Attachment of protein. A particular problem is the gluteraldehyde step Because gluteraldehyde is a dialdehyde it can bond both aldehyde ends to amino groups during exposure to the amino modified surface. If this cross-linking occurs there can be no further bonding of the protein In an attempt to simplify this process, an aldehyde si lane route was chosen (Figure 3 -2) (96) The surface is first derivitized with the aldehyde silane followed by

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85 incubation with protein and attachment. The three-step process is decreased to two steps, obviating the need for amino derivitization of the surface This process also eliminates the possibility of cross-linked aldehyde groups and insures that the aldehyde moiety will be available for binding of the protein (96) The exact procedure for aldehyde silanization and protein attachment can be found in the experimental section Extractions Using Protein-Modified Nanotubes A vidin/biotin To determine if protein binding to the nanotubes occurred successfully and without denaturation we attemped to bind the protein avidin Avidin and its binding to the small molecule biotin are discussed in Chapter 2 To be able to monitor the binding of biotin by the avidin biotinylated 20 nm Au nanoparticles (Sigma) were chosen as the analyte Au nanoparticles have very strong absorbance in the visible region due to their surface plasmon resonance (106) which allows straightforward monitoring of their concentration using UV-Vis spectroscopy The idea was to modify the surfaces of the nanotube with avidin then use the modified nanotubes to extract the biotinylated Au nanoparticles from solution It has been seen in Chapter 3 that chemically modified nanotubes are effective extractants Here we wanted to use specific binding between proteins and ligands to enhance the specificity of the extraction If the extraction was successful, it would also indicate that the protein is attached to the nanotubes and not significantly denatured by the attachment process. An extraction experiment was performed that was similar to the drug extraction e x periments in Chapter 2 The absorbance spectra of the Au nanoparticle solution was measured prior to addition of the nanotubes to determine the initial Au nanoparticle

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86 concentration A vidin modified nanotubes were added to an aqueous dispersion of biotinylated Au nanoparticles at a concentration of 1 mg modified nanotubes per mL solution The solution was briefly sonicated after addition of the nanotubes to suspend them Following sonication, the solution was filtered, removing the nanotubes and any biotinylated Au nanoparticles bound to the avidinated surfaces The absorbance of the filtered solution was then measured again to determine the amount of Au nanoparticles removed (Figure 3-3) 0 .15 0.1 g c,:i -e 0 .05 0 Figure 3-3 450 500 550 600 Wavelength (run) UV-Vis spectra biotinylated Au nanoparticle solution A) Starting solution B) After extraction with 1 mg/mL bovine serum albumin modified nanotubes. C) After extraction with 1 mg/mL avidin-modified nanotubes 650 As a control experiment, the Si02 nanotubes were modified with bovine serum albumin, which should have little specific affinity for the biotinylated Au nanoparticles, and the extraction efficiency was determined in the same manner In this case, significant nonspecific adsorption of the Au nanoparticles was observed, with 34% removed from solution (Figure 3-3) This is likely due to the fact that the gold nanoparticles are stabilized with albumin, which may bind non-specifically to the albumin-coated silica

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87 nanotubes. For the extraction with the avidinated Si02 nanotubes, the extraction efficiency was much higher with 100% of the Au nanoparticles removed from solution within our ability to measure (Figure 3-3) These results show that the protein binding via the aldehyde silane is successful. The protein is not significantly denatured in that the three-dimensional binding pocket for avidin is still intact and functional. Immunoprotein extractions To mitigate the possibility of nonspecific adsorption we next attempted to use a more specific protein/protein interaction for our extraction Immunoproteins have long been known to exhibit high specificities with little or no affinity for analytes other than those against which they were raised (140, 141) For example, antihuman immunoglobulin proteins bind strongly to human immunoglobulins but not to immunoglobulins from other species, (e g mouse or rat immunoglobulins) The most common type of immunoglobulin is Immunoglobulin G (IgG) (140) This is a Y-shaped molecule with a molecular weight of 146 k.Daltons (142) It consists of two identical top fragments called the Fab region, and one stem fragment called the Fe region The molecule can be cleaved into its Fab and Fe fragments using enzymes (142) The overall dimensions of the molecule are lOnm tall and 10 nm wide (140) The Fab fragments are 6 0 nm by 3 5 nm and the Fe fragments are 4 0 nm wide and 4 5 nm tall The binding site for antigens is on the end of each of the Fab fragments (142) The binding site area has dimensions of 3 0 nm by 1.0 nm by 0 6 nm (140) Human IgG was attached via aldehyde silanes to one batch of Si02 nanotubes (see experimental) Rat IgG was attached in the same way to another batch of nanotubes to serve as a control. The analyte for these experiments was a Fab fragment of antihuman

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88 lgG antibody that had been labeled with a fluorescent fluorescein isothiocyanate (FITC) tag (143) for absorbance monitoring The structure ofFITC is seen in Figure 3-4 HO N II C II s Figure 3-4 Structure of fluorescein isothiocyanate (FITC) A solution of the FITC-labeled antihuman IgG was extracted with the modified nanotubes in a manner analogous to the avidin/biotin extractions The absorbance before and after extraction was measured by UV-Vis spectroscopy Extractions used 1 mg of modified nanotubes per mL of solution Results are seen in Figure 3-5 As expected the human IgG-modified nanotubes effectively extracted the FITC-labeled antihuman IgG Comparing the initial absorbance to the absorbance after extraction shows that 76% of the FITC-labeled antihuman lgG has been removed from solution (Figure 3-5) In contrast Figure 3-5 also shows that there was no appreciable antihuman extraction using the rat IgG-functionalized nanotubes The concentration ofFITC-antihuman IgG is indentical before and after extraction, as indicated by the overlapping absorbance spectra This result proves that protein modified nanotubes can retain the selectivity of the protein used for modifications Enantioselective extractions The most specific and therefore most difficult extraction is the selective removal of one enantiomer from a racemic mixture Enantiomeric extraction is contingent on having

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89 0 16 0 14 0 .12 Q) () 0 1 a -e 0 0 08 "' < 0 06 0 04 0 02 0 4 00 450 500 550 Wavelength (run) Figure 3-5 UV-Vis spectra FITC-labled antihuman IgG solution A) Starting solution B) After extraction with 1 mg/mL rat lgG-modified nanotubes C) After extraction with 1 mg/mL human IgG-modified nanotubes an extractant species that shows selectivity for one of the enantiomers The recombinant antibody fragment ENAl lHis developed by Dr. Hans Soderlund and coworkers is known to be selective for one stereoisomer of the drug 4-[3-( 4-fluorophenyl)-2-hydroxy-l-[l ,2,4]triazol-l-yl-propyl]-benzonitrile (FTB) (144) As there are two stereocenters for this molecule there are four stereoisomers (R,S) (S R), (R,R) and (S,S) The (R,R) and (S,S) isomers are diastereomers to the (R,S) and (S R) isomers and can be separated by differences in physical properties Structure of the a (R,S) and d (S R) enantiomers are shown in Figure 3-6 FTB is used for postsurgical treatment of breast cancer. It functions by inhibiting the conversion of testosterone to estradiol ( 144) To produce enantioselective antibody fragments, Soderlund and coworkers first injected a mouse with FTB They then screened antibodies produced by the mouse for their ability to bind each enantiomer ofFTB using an enzyme-linked immunosorbant

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90 assay (ELISA) (144). Once an antibody that selectively bound the a enantiomer selectively had been identified, the DNA which coded the production of the Fab fragment of that antibody was spliced into an E. coli host. The E. coli then produced a monoclonal version of the enantioselective Fab antibody fragments, which was termed ENAI I His (144). These enantioselective fragments were then attached to chromatographic gel material and placed in a chromatographic column. A column containing the ENAl !His-modified gel retained the a enantiomer but allowed the d enantiomer to pass through (144). A B Figure 3-6. 3-D structure of the enantiomers of 4-[3-( 4-tluorophenyl)-2-hydroxy-l-[l 2,4 ]triazol-1-yl-propyl]benzonitrile. A) The a or (RS) enantiomer. B) The d or (S R) enantiomer ENAl I His obtained from Dr. Soderlund was attached to silica nanotubes via an aldehyde siloxane linker (see experimental). The antibody fragment modified-nanotubes were used to extract racemic solutions of the FTB enantiomers. A concentration of I mg nanotubes per mL solution was used for these initial extraction experiments. After extraction and filtering to remove the nanotubes the amounts of a and d enantiomer remaining in solution were determined by chiral HPLC. Chromatography data were

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91 obtained by Dr. Sang Bok Lee. Results show that the a enantiomer was indeed selectively extracted from solution (Figures 3-7 3-8 and 3-9) 100 90 80 70 60 Q,I 50 c.J = ,.Q 40 I. Q "' ,.Q 30 < 20 10 0 6 Figure 3-7 7 8 9 10 Retention Time (min) 11 A B C D E 12 Chromatograms from enantioselective extractions Absorbance was at 230 nm A) Racemic 5 0 M solution after extraction with 1 0 mg/mL ENA11His-modified nanotubes B) Racemic 3 0 M solution after extraction with 1 mg/mL ENA11His-modified nanotubes C) Racemic 2 0 M solution after extraction with 1 mg/mL ENA11His-modified nanotubes D) Racemic 1 0 M solution after extraction with I mg/mL ENAl lHis-modified nanotubes E) Racemic 1 0 M solution (unextracted) for comparison HPLC dat a were obtained by Dr. Sang Bok Lee The extraction ratio of the a versus the d enantiomer is dependant on the initial concentration of the racemic mixture For a solution that was initially 1 Min both enantiomers all of the a enantiomer was extracted by the nanotubes within our ability to measure (Figures 3-7 D and 3-8 B). Approximately half of the d enantiomer was extracted As the starting racemate concentration is increased to 2 M 3 M and 5 M

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92 the extraction ratio declines (Figure 37) However, at every concentration more a is extracted than d (Figure 3-7) 35 30 25 (,J 20 C c,s -e 0 "' 15 .Q < 10 5 0 6 Figure 3-8 A B C 7 8 9 10 11 12 Retention Time (min) Chromatograms from enantioselective extractions Absorbance was at 230 nm. A) Racemic 1.0 M solution after extraction with 1.0 mg/mL bovine serum albumin-modified nanotubes B) Racemic 1. 0 M solution after extraction with 1 mg/mL ENAl lHis-modified nanotubes C) Racemic 1 0 M solution (unextracted) for comparison HPLC data were obtained by Dr. Sang Bok Lee Figure 3-8 B shows the same data for extraction of a 1 M racemic starting solution as seen in Figure 3-7 D with the y-axis expanded to show detail. As noted by the signal to noise ratio in this graph we are very close to the detection limit for the HPLC system. Also shown in Figure 3-8 are the results of a control experiment extracting the 1 M racemic FTB solution with nanotubes modified with bovine serum albumin (Figure 3-8 A) Bovine serum albumin (BSA) should show no selectivity for one enantiomer over

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93 another, and Figure 3-8 A shows that equivalent amounts of both a and d enantiomers were removed by BSA-modified nanotubes 200 150 50 6 Fig ure 3-9 7 8 a enantiomer (antigen) / 9 Retention Time (min) d enantiomer 10 11 A B C D 12 Chromatograms from enantioselective extractions Absorbance was at 230 nm. A) Racemic starting solution 20 Min both a and d enantiomers B) Same solution after extraction with 18 mg/mL ENAl !His-modified nanotubes C) Racemic 10 M starting solution after extraction with 18 mg/mL ENAl !His-modified nanotubes D) Racemic 5 M starting solution after extraction with 18 mg/mL ENAl !His-modified nanotubes HPLC data were obtained by Dr. Sang Bok Lee. To better visualize and quantify the experiment both the concentration of the racemic mixture and the concentration of antibody-modified nanotubes used for extraction were increased A 20 M racemic mixture of both enantiomers was extracted with 18 mg antibody modified nanotubes / mL solution The results for this experiment are shown in Figure 3-9 After extraction 75% of the a enantiomer is removed without removal of any of the d enantiomer within our ability to measure Thus the a enantiomer can be cleanly separated from the d using antibody-modified nanotubes and with a high yield If a

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94 lower starting concentration of the racemic mixture of IO M is used and again extracted with 18 mg modified nanotubes/ mL solution, all of the a enantiomer can be removed within the ability to measure In this case, a small amount of the d enantiomer is also removed, about 15 % Eighty five percent of the d enantiomer remains in solution which means the d enantiomer can be cleanly separated from the a again in high yield (Figure 3-9). An even lower starting concentration of the racemic mixture, 5 M, also had 100% of the a enantiomer extracted using 18 mg/mL ENAI !His-modified nanotubes However at this low starting concentration, a larger percentage of d enantiomer was also extracted 25% A set of control nanotubes was taken through the same fabrication procedure but without attachment of the aldehyde silane coupling agent. At the 20 M level, these nanotubes did not extract measurable quantities of either enantiomer The above experiments were done with the Fab fragment immobilized to both the inner and outer surfaces of the nanotubes A chemistry was also developed to attach the protein to only the inner surfaces While still within the pores of the template membrane the inner surfaces of the nanotubes were treated with aminopropyltrimethoxysilane The outer surfaces of the nanotubes were protected from silanization by contact with the template membrane. The alumina template was then dissolved and the amino sites on the inner surfaces were coupled to free amino groups on the Fab fragment using the well-known glutaraldehyde coupling reaction (97 98)(Figure 31 ) When 18 mg of these interior-only Fab -modified nanotubes were incubated with I mL of a IO M racemic mixture of the drug 80% of the RS (and none of the SR) enantiomer was e x tracted This corresponds to 0.44 nmol RS enantiomer per mg tubes As expected, almost double that

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95 amount, 0 80 nmol/mg, was extracted by the nanotubes with Fab on both their inner and outer surfaces. For possible in vivo applications, biocompatibility is an important issue It is well known that materials can be made biocompatible, as evidenced by decreased protein adsorption by attaching poly(ethylene glycol) (PEG) chains to their surfaces (145 146) We have used a PEG silane (MW -5000, Shearwater, Huntsville, AL), to improve the biocompatibility of the silica nanotubes. When 5 mg of unmodified silica nanotubes were incubated with 1 mL of a buffer solution containing 1 mg IgG immunoprotiens, 52% of the protein was removed from solution via adsorption to the nanotubes, as measured by the decrease in solution UVVis absorbtion In contrast, when an identical amount of PEG-modified nanotubes was incubated with this IgG solution less than 3% of the protein was removed from the solution Directed Nanotube Binding The above experiments show that protein modified smart' nanotubes function as highly selective extractants It was demonstrated in Chapter 3 that the surface chemistry of the nanotubes could control the phase into which the nanotubes partition The logical next step was to attempt to use the specificity of the proteins on the nano tube surface to direct their binding As an initial proof of concept experiment we first modified glass coverslip slides with different immunoproteins Two slides were modified with rabbit IgG protein via the aldehyde silane method, and two with bovine IgG. Two batches of silica nanotubes were then prepared both from a 200 nm alumina template One batch was modified with antirabbit IgG protein and the other with antibovine IgG. The slides were then incubated in buffered solutions of the nanotubes One of the rabbit IgG coated slides was incubated with antirabbit IgG nanotubes the other with antibovine I g G

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96 A B Figure 3-10 Scanning electron micrographs oflgG coated slides and nanotubes. A) Antirabbit lgG coated nanotubes bound to a rabbit IgG coated glass slide after rinsing. B) Rabbit IgG coated glass slide incubated with antibovine IgG nanotubes after rinsing

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97 A B Figure 3-11. Scanning electron micrographs of lgG coated slides and nanotubes A) Antibovine lgG coated nanotubes bound to a bovine IgG coated glass slide after rinsing. B) Bovine lgG coated glass slide incubated with antirabbit lgG nanotubes after rinsing.

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98 A B Figure 3-12. Fluorescence microscope images of immunoprotein coated nanotubes labeled on the inside with fluorescent silanes. A) Antirabbit IgG/quinineurathane (blue) and antibovine IgG/dansylamide (green) nanotubes on rabbit IgG coated glass slide before rinse. B) After rinse, only antirabbit lgG/quinineurathane (blue) nanotubes remain.

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99 nanotubes Corresponding incubations were prepared with the bovine IgG coated slides It is important to note that these incubations were carried out separately; a glass slide was exposed to either antirabbit IgG nanotubes or antibovine IgG nanotubes, but never both After incubation, the slides were rinsed gently with buffer and water and allowed to dry The dried slides were then sputtered with gold and examined in an SEM. As shown in Figures 3-10 and 3-11 the binding of the nanotubes was as predicted Antirabbit modified nanotubes bound in abundance to the rabbit IgG glass slides but did not bind appreciably to the bovine IgG slides Conversely antibovine nanotubes bound well to the bovine IgG coated slides but only slightly to the slides modified with rabbit IgG Both types of nanotubes were incubated with unmodified bare glass slides as a control. In each case there was some nonspecific binding, although much less binding than for the specific protein modified slides To prove that one type of nanotube could be selectively bound from a mixture we performed a similar experiment. Two batches of nanotubes were created the first of which was silanized with a dansylamide silane This fluorophore fluoresces green with UV excitation The other batch of nanotubes was silanized using a quinineurathane silane which fluoresces blue under UV light. Both batches were modified with the fluorophores while still in the alumina template and therefore on the interior surface only. These two silanes can be easily distinguished in a fluorescence microscope by varying a filter that controls the passing wavelength. The dansylamide silanated nanotubes were futher modified on the exterior with antibovine IgG. Antirabbit IgG was used to modify the outside of the quinineurathane nanotubes Both nanotubes were then incubated together with a rabbit IgG modified

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100 coverslip slide Before the slide is rinsed, both types of nano tube are clearly identifiable in the fluorescent microscope image (Figure 3-12) After rinsing however, only the blue antirabbit nanotubes remain The unbound green antibovine nanotubes have all rinsed away (Figure 3-12) Enzyme Attachment and Assays Glucose oxidase The above experiments show that proteins can be attached to the nanotubes and that after attachment the proteins are still functional and active These proteins exhibit high binding specificities but stop short of catalyzing reactions Enzymes, however, are by definition catalytic proteins Enzyme-modified nanotubes may function as bioreactors if the enzyme can be attached without denaturation Glucose oxidase, a robust and well-studied enzyme ( 14 7) was chosen as a model system The attachment of the enzyme to the nanotubes was carried out via an aldehyde silane bridge, as described in the experimental section. Assaying the protein for activity after attachment was necessary to determine if the protein structure survives the attachment process intact. The activity of the attached glucose oxidase was determined using a modified literature assay (138) Details are given in the experimental section and in Figure 3-13 The assay uses hydrogen peroxide, a byproduct of the oxidation of glucose by glucose oxidase, to oxidize o-dianisidine When reduced o-dianisidine does not absorb in the visible region. After oxidation, it shows a strong absorbance peak at 500 nm Thus monitoring that wavelength with visible spectroscopy should show an increase in absorbance if the glucose oxidase is active This increase should be linear over time as long as there are ample amounts of substrate and cofactors to continue the reaction

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A) B ) H202 + o-dianisidine (reduced) 101 GOD .. POD .. D-glucono-1 ,5-lactone + H202 o-dianisidine ( oxidized) Figure 3-13. Schematic of glucose oxidase activity assay A) Glucose is converted to glucono-1 5-lactone and hydrogen peroxide in the presence of glucose oxidase B) Hydrogen peroxide produced in first reaction oxidizes o dianisidine in the presence of peroxidase Figure 3-14 shows this to be the case For the first 100 seconds of this graph the absorbance of the assay solution was monitored to show that no active enzyme was present. At -100 seconds, the glucose oxidase modified nanotubes were added to the solution and the absorbance at 500 nm began increasing immediately The increase in absorbance is linear as long as the nanotubes are left in the solution By measur i ng the rate of product formation the activity of the glucose oxidase on the nanotubes can be determined This activity was found to be 0 6 units per mg nanotubes One unit of glucose oxidase activity is the amount required to oxidize one mo! of glucose per minute Figure 3-14 also proves that the glucose oxidase is strongly bound to the nanotubes and does not leach off i n appreciable amounts This is shown by filtering the solution through 20 nm diameter alumina to remove the enzyme-modified nanotubes Any leached or unbound glucose oxidase would be passed through the filter and would continue to catalyze oxidation of o-dianisidine resulting in increasing absorbance. In Figure 3-14 the reaction mixture was filtered at 350 seconds The absorbance after filtration drops to near zero because the oxidized o-dianisidine product forms micelles

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102 0 .65 0. 55 0.45 C) a 0.35 -e 0 "' 0.25 .0 < A 0.15 0.05 \ -0 05 0 100 200 300 400 500 600 Time (sec) Figure 3-14. Activity assay of glucose oxidase-modified nanotubes. Absorbance was at 500 nm. A) Enzyme-modified nanotubes added to assay solution. B) Nanotubes filtered from solution that are removed from solution by filtering. Absorbance at 500 nm was then monitored a further 200 seconds. No increase is seen during this period verifying that the enzyme is strongly bound to the nanotubes via the aldehyde silane route. Cytocr ome P45 0 enzymes for drug detoxification The above experiments with glucose oxidase demonstrate our ability to attach enzymes to the nanotubes and create enzyme bioreactors. To increase the utility of such bioreactors attachment of an enzyme with drug detoxification properties was next attempted The most prominent group of enzymes used by the body to break down foreign chemicals or drugs is the cytochrome P450 family (148). These enzymes get their name from the fact that they absorb strongly at 450 nm under certain conditions The P in the name stands for pigment. Of particular interest is the isozyme CYP3A4, which is the most abundantly expressed protein in human liver (148). This enzyme is unique in its

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103 abilty to use a variety of different molecules as substrates In fact CYP3A4 is known to degrade 50% of all xenobiotics, as foreign chemicals or drugs are collectively known (149) This represents tens of thousands of individual compounds including such well-known drugs as cocaine and acteomenophen Such a broadband enzyme would have obvious utility for detoxification 0 07 0 A 0 06 0 05 6. B 0 04 Q) (.) a ... 0 03 0 Cl) 0 02 < 0 .01 0 400 440 480 520 Wav elength (nm) Figure 3-15 Spectra of free (unbound) P450 in solution A) Oxidized B) Reduced There are two major methods of determining ifCYP3A4 is functional and active The first concerns the eponymous 450 nm absorbance (150) All P450 enzymes contain a heme which absorbs at 420 nm when oxidized the normal state of the enzyme if exposed to air When the enzyme is reduced and bound to carbon monoxide ( CO ) this absorbance shifts to 450 nm (Figure 3-15) Note that not all of the 420 nm peak shifts to 450 nm There is still a significant fraction of enzyme absorb i ng at 420 nm and this fraction is thought to be non functional or denatured For the functional P450 this shift of absorbance with reduction and CO binding is completely reversible On exposu r e to

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104 oxygen the absorbance peak at 450 run decays until eliminated and the peak at 420 run increases to its original intensity. CYP3A4 was attached to 200 run diameter silica nanotubes using the aldehyde silane chemistry described above. The enzyme-modified nanotubes were then suspended in buffer and examined using spectroscopy in the visible region (Figure 3-16). As with the native enzyme solution, the modified nanotubes show a absorption peak of -420 nm when oxidized (under ambient conditions). When the enzyme on the nanotubes is reduced using sodium dithionite and CO is bubbled through the solut ion, part of the peak shifts to 450 nm. Again not all of the enzyme is reduced; there is a non-functional percentage which remains absorptive at 420 run. The shift is full y reversible with reo x idation of the enzyme. 0.24 6 0 A 6 0 .22 Cl) 0 2 u a .D .... 0 rfl 0.18 .D --< 0 .16 0 .14 380 400 4 2 0 440 460 480 500 5 2 0 Wavelength (nm) Figure 3-16. Spectra of P450-modified nanotubes. A) Oxidized. B) Reduced. T he second method of determining the activity of CYP3A4 is an enzymatic assay (139). Testosterone is used as a substrate and in the presence of a ctive enzyme is converted to 6-P hydroxytestosterone. This is a complex reaction requirin g a variety of

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NADPH NADP+ )( 105 Reductase (ox) ) / Reductase (red) .._\ I I Cytochrome b5 I 0 P450 (red) P450 (ox) Testosterone (Substrate) ) OH 6-P-hydroxystosterone (Product) Figure 3-17 Schematic of P450 assay reaction and electron transport pathway

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1 000 0 9000 A 8000 Cl) 7000 u C: 6000 "' .rJ ... 0 5000 Vl .rJ <: 4000 3000 2000 1000 0 0 50 100 106 A 150 200 Elution time (sec) <> O After 20 min to A After 60 min A Q
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107 appropriate antigen from solution Immunoprotein-modified nanotubes can be used to separate enantiomers Further, immunoprotein-coated nanotubes bind selectively on surfaces coated with the corresponding antigen Finally enzymes including drug detoxification enzymes, can be attached to the nanotubes to make removable bioreactors

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CHAPTER4 ION CHANNEL MEMETIC SENSORS Introduction This chapter describes synthetic micropore and nanotube membranes that mimic the function of a ligand-gated ion channel; i e these membranes can be switched from an "off" state (no or low ion-current through the membrane) to an "on" state (higher ion-current) in response to the presence of a chemical stimulus. Ion-channel mimics based on both microporous alumina and Au-nanotube membranes are investigated The off state is obtained by making the membranes hydrophobic by chemisorbing either a C,s silane (alumina membrane) or a C,s thiol (Au nanotube membrane) Water and electrolyte are forbidden from entering these very hydrophobic pores/nanotubes The transition to the on state is induced by the partitioning of a hydrophobic ionic species (e.g., a drug or a surfactant) into the membrane. The membrane switches to the on state because at a sufficiently high concentration of this ionic analyte species the pores / nanotubes flood with water and electrolyte. Background As mentioned in Chapter 1, the Martin group has been exploring the transport and electrochemical properties ofnanotube membranes (40 47, 48 151-158) prepared by the template method (159-161), a general approach for preparing nanomaterials One possible application for these nanotube membranes is in electroanalytical chemistry where the membrane is used to sense analyte species (152) 108

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109 In prior work done in the Martin group, membranes containing gold nanotubes with inside diameters that approached molecular dimensions (1 nm to 4 nm) were used (152) The Au nanotube membrane was placed between two salt solutions and a constant transmembrane potential was applied The resulting transmembrane current, associated with migration of ions through the nanotubes, was measured When an analyte molecule with a diameter comparable to the inside diameter of the nano tubes was added to one salt solution, this molecule partitioned into the nanotubes and partially occluded the pathway for ion transport This resulted in a decrease in the transmembrane ion current, and the magnitude of the drop in cqrrent was found to be proportional to the concentration of the analyte (152) This principle of decreasing current caused by blockage of pores is analogous to that used in a commercial cell counting device made by the Coulter company and known as a Coulter counter. In both the above experiment and the cell counting device, a baseline transmembrane ion current was established, and the analyte molecule, in essence, turned off this current. It occurred that there might be an advantage in doing the opposite ; i.e., starting with an ideally zero current situation and having the analyte molecule switch on the ion current. That is, it may be possible to make a synthetic membrane that mimics the function of a ligand-gated ion channel. An example is the acetylcholine-gated ion channel (162), which is closed ("off' state) in the absence of acetylcholine but opens (and supports an ion current "on" state) when acetylcholine binds to the channel. This concept of ion-channel mimetic sensing originally proposed by Umezawa's group (163) has been of considerable interest in analytical chemistry ( 164-166) There is also considerable

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110 interest in using naturally-occurring and genetically-engineered protein channels as sensors (see (167) and references therein). Experimental Materials Tris (2,2 '-bipyridyl)ruthenium(II) (Ru(bpy)/+ ) dichloride, I, 5-napthalene disulfonic acid tetrahydrate, octadecyltrimethoxysilane, 1-dodecanesulfonic acid, 1-hexadecanesulfonic acid, 1-octanesulfonic acid, 4-octylbenzensulfonic acid, 1-propanesulfonic acid, and ethanesulfonic acid were obtained from Aldrich and used as received Tetradecylammonium bromide was obtained from ICN Biomedicals Hexadecyltrimethylammonium chloride, decyltrimethylammonium chloride, dodecyltrimethylammonium chloride, and dodecylbenzenesulfonic acid were obtained from Acros Chemicals. Octadecyl thiol was obtained from Aldrich The alumina membranes were Anopore (Whatman Inc Clifton, New Jersey) that had nominally 200 nm-diameter pores and were 60 m thick Microporous track-etched polycarbonate membranes (Osmonics) were also investigated ; these membranes have 30 nm-diameter pores and a pore density of 6x 108 cm2 The hydrochlorides of three cationic drug molecules arniodarone, bupivacaine, and amytriptyline (Sigma)were investigated as part of this work Amiodarone is an antiarrhythmic and antianginal bupivacaine is a local anesthetic, and amytriptyline is a tricyclic antidepressant. Their chemical structures will be discussed below. Membrane Preparation A solution that was 5% (v/v) in octadecyltrimethoxysilane was prepared in ethanol; to this solution was added acetate buffer (0 1 M, pH= 5 1) to make the solution 5% (v/v) in this buffer. The resulting solution was stirred for 5 minutes and the alumina membrane

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111 was then immersed. After 2 hours, the membrane was removed from the solution rinsed with ethanol and cured at 150 C in air for 20 min. Following silanization, the membrane was mounted into the assembly device shown in Figure 4-1 After assembling the various components, the assembly was heated for three minutes at 150 C in an oven to melt the Parafilm which acted as a glue to hold the various pieces together. This assembly exposes 0.079 cm2 of membrane area to the contacting electrolyte solutions. B B Figure 4-1. Membrane assembly. A) Glass slide. B) Parafilm. C) Membrane We also conducted a limited number of experiments with gold nanotube membranes prepared by electroless deposition of Au within the pores of the microporous track-etched polycarbonate membrane ( 40). These membranes have been discussed in detail in our previous publications (40, 47 48, 151-154, 158). The electroless plating procedure has been previously described ( 40); membranes containing Au nanotubes with inside diameters of 2 nm were used. The Au nanotubes were rendered hydrophobic by chemisorbing octadecylthiol to the nanotube walls ( 40).

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112 AC Impedance Measurements The membrane assembly was mounted between the halves of a U-tube permeation cell, and both half-cells were filled with -20 mL of 0.1 M KCI. A Ag/AgCl working electrode was immersed into one half-cell solution, and a Pt counter electrode and a Ag/ AgCl reference electrode were placed in the other half-cell ( 168-170) Impedance data were obtained using a Solartron 1287 electrochemical interface module (Solartron Analytical, Hampshire England) connected to a Solartron 1255B frequency response analyzer and a personal computer The magnitude of the sinusoidal potential perturbation applied across the membrane was mV ; impedance data were obtained over a frequency range of 106 Hz to 0 1 Hz. Zplot and Zview software packages (Scribner Associates, Inc Southern Pines North Carolina) were used to control the impedance experiments and analyze the impedance data. X-Ray Photoelectron Spectroscopy (XPS) The surface analysis technique of XPS was used to show that the prototypical analyte dodecylbenzene sulfonate (DBS) is present on the C1s-modified alumina surface after exposure of the membrane to DBS solution All XPS data was collected and analyzed by Dr. Vaneica Young. The XPS cross-section for S from the DBS proved too weak to obtain unambiguous evidence; furthermore 0, C and Na+ (the counterion for the DBS) are ubiquitous and therefore not useful as probes to prove that DBS is present on the surface For this reason we used a surface ion exchange reaction to replace Na+ with Cs + as the counterion for the surface-bound DBS. We then used XPS to look for the presence of Cs + on the C18-modified surface that had been treated with DBS using an i dentical surface that was exposed to the Cs + solution but not to DBS as the control.

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113 A section of the C1s membrane was immersed for 5 minutes into an aqueous 2 0 mM solution of Na+ -DBS Following exposure to surfactant, the membrane was rinsed repeatedly with deionized water and then immersed for 5 minutes in a 100 mM aqueous solution of CsNO3. After rinsing again with deionized water the sample was air-dried XPS studies were performed on a Kratos XSAM 800 spectrometer using Al Ka excitation (200 W) The samples were mounted onto a stainless steel sample stub by means of a 7 mm diameter carbon sheet disk (SPI Supplies), so as to completel y cover both the carbon disk and the sample stub The samples were inserted in t o the sample analyzer chamber via a quick insertion probe and spectral acquisition using the fixed anal y zer transm i ssion mode commenced after the pressure decreased to 5x109 torr. An incident angle of 75 relative to the sample surface was employed Transport Experiments The membrane was mounted between the two halves of a U-tube permeation cell and 0 1 M KCl was added to each half-cell. The feed half-cell was also 50 Min either Ru(bpy)/+ or naphthalene disulfonate (NDs2), the permeate ions These permeate ions were chosen because transport from the feed solution through the membrane and into t he permeate solution can be easily monitored by measuring the UV absorbance of the permeate solution (Both permeate ions were detected at 286 nm ) Transport occurred b y diffusion of the permeate ion down the concentrat ion gradient across the me m brane ; bo th the feed and permeate half-cells were vigorously stirred during the permeation experiments. The experimental protocol used was as follows : An aliquot of the analyte surfactant (for these experiments DBS) was added to both the feed and permeate half-cells and permeation was allowed to occur for 24 hours After this time th e permeate halfcell wa s

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114 sampled and the UV absorbance was used to determine the moles of the permeate i on transported The permeate solution was then returned to the permeate half-cell and a second increment of DBS was added Permeation was again allowed to occur for 24 hours and the amount of permeate ion transported was again determined This process was repeated for various DBS concentrations over the range from I o 8 to I 04 M Ion-Current Measurements The membrane was mounted between the two halves of a U-tube permeation cell and 0.1 M KCl was added to each half-cell A Pt wire electrode was inserted into each half-cell and the Solartron potentiostat was used to apply a constant transmembrane voltage of I 5 V and measure the resulting transmembrane current. The current was monitored for 30 min and then the half-cell solutions were spiked with DBS to a total concentration of 109 M The current was again measured for 30 minutes and the half-cells were spiked again with DBS This process was repeated for various DBS concentrations over the range from 10-9 to 103 5 M Results and Disscussion XPS Studies Cesium ions (Cs + ) were used as a probe to prove that the prototypical analyte DBS is present on the C1s -modified alumina surface after exposure of the membrane t o DBS solution Figure 4-2 shows XPS data for a C1s-modified membrane that had been exposed to DBS, rinsed exposed to Cs+, and then rinsed extensively again The Cs 3d peaks at 724 and 738 eV are clearly evident (171) This may be contrasted to the control surface -a C1s-modified alumina membrane that was exposed to the Cs + solution but not to DBS where no Cs signal is seen (Figure 4-2) These data show that exposure of the

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115 membrane to DBS results in partiti o ning of this a nalyte species onto the C1s-modified surface All XPS data were provided by Dr. Vaneica Young 30,000 With DBS ,-.... (/) -i::: = 2 0 ,000 0 c.) _, .0 (/) i::: 0 1 0 ,000 -i::: Contro l no DBS 7 50 74 0 730 720 Binding energy (eV) Fig u re 4-2. XPS data for a C1s-modifie d alumina-membrane surface that was exposed to a DBS solution and then to a Cs+ solution and for an identical surface that was exposed to Cs+ but not to DBS Data for this figure were provided by Dr. Vaneica Young AC I mp eda n ce Experime n ts wi th DBS Ana l yte AC Impedance measurements proved to be a useful way to demonstrate the analyte-induced switching of the membrane between the off and on states The upper-most curve in Figure 4 3 is the Nyquist plot for a C1s-modified alumina membrane with 0 1 M KCl solutions and no DBS, on either side of the membrane As per prior investigations of ion-channel and ion-channel-mimetic membranes (172, 173) the impedance data were interpreted in terms of the equivalent circuit shown in the inset of Figure 4-3 where Rs is the solution res i stance, Rm is the membrane resistance and C is the membrane capacitance The dashed curve is the best fit to the experimental data, from which the Rm (Figure 4-4) and C values were obtained Also shown in Figure 4-3

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116 are impedance data after spiking the half-cell electrolyte solutions t o the indicated concentrations with the analyte DBS. -1. 5e8 .--------------,-------------. C R s -----1.0e8 C: .......... Cl) (.) "Cl Cl) 0... 8 .... -5.0e7 s ] 0 ia---fl.J--->eJ....-------'--------'----_j__--_j__---->....,;...._---'-----'--'--_J 0 5.0e7 1.0e8 1.5e8 2.0e8 Real Impedance (.0) Figure 4-3. Nyquist plots for a C 1 s modified alumina membrane upon exposure to increasing concentrations of DBS in 0 1 M KC!. The points are the experimental data The lines are calculated data obtained using the equivalent circuit shown in the inset. Concentrations of DBS were as follows : DO nM, o 1.0 nM, \] 5 0 nM, /Y 10 nM. In the absence of DBS, the membrane resistance is very large > 180 M.O as opposed t o 5 n for the alumina membrane before modification with the C1s s i lane The transport experiments described below show that this is because the very h y drophobi c C1s-modified pores are not wetted by water This is supported by contact angle measurements on the membrane surface, where a water contact an gle of 130(8) was obtained for the C1s-treated alumina membrane as opposed to 8( 1)0 for the untreated membrane It should be noted that the contact angle for the C1s-treated alumina is

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117 somewhat higher than literature values for alkane-derivatized surfaces, which are typically around 110 (174, 175) This is l ikely due to the increased roughness of the microporous alumina surface 7 8 6 ...... f 6 "'d f . Q) ..... : .................... t 0 0.. Cl) .---, @ -a 5 . b -. 4 + N ,.., -.. '--' >-, bO 0.. 0 4 . . f" ,.D --2 Cl) Q) ........ ~ 0 3 8 :J.. 0 -8 -7 -6 -5 4 log ([DBS] M) Figure 4 4 Plots of log membrane resistance (left y-axis) and mmoles Ru(bpy)2 + transported across the mem b rane ( r ight y-axis) vs. log[DBS] for a C18 -modified alumina membrane The error bars represent the standard deviation of three separate experiments While over the concentration range 10-9 to 10-7 M there is some drop in membrane resistance with increasing DBS concentration, Rn remains very large (>20 M.O) However, over the DBS concentration range between 10-6 and 10-5 M there is a precipitous four-order-of-magnitude, drop in Rm (Figure 4-4). This drop signals the analyte-induced switching of the membrane from the off to the on states The concentration of DBS that induces this off to on transition is well below the critical micelle concentration for DBS (1. 1 mM) (176) The capacitance data also show the

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118 effect of this off/on transition At concentrations of DBS below the transition, the membrane capacitance is extremely low (e.g 0.46 nF per cm2 of membrane surface area at a DBS concentration of 10-6 M) but jumps by two orders of magnitude when the membrane is switched to the on state (22 nF per cm2 at [ DBS] = 105 M). Transport Experiments The protocol used entailed spiking the electrolyte solutions as per the impedance experiments allowing permeation to occur for 24 hours and then measuring the quanti ty of permeate ion transported to the permeate solution. The data obtained for Ru(bpy) / + transport are shown in Figure 4-4 At DBS concentrations below 106 M there is no detectable Ru(bpy)/+ in the permeate solution It is important to emphasize t hat each permeation data point in Figure 4-4 corresponds to an additional 2 4 hours o f per m eation time Hence, by the time the DBS concentration was increased to 9x107 M, the to t al permeation time was 5 days The inabil i ty to detect Ru (bpy)/+ in the permeate solution after five days of permeation shows that over the DBS concentrat i on range 0 to 1 0-6 M, the pores in the C1s membrane are not wetted by water making the rate ofRu (bpy)/+ transport immeasurably small. These data again show that at DBS concentrations belo w 1 o 6 M the membrane is in the off state At DBS concentrations above 10-6 M, Ru ( bpy) / + t ra nsport is switc h ed o n and flux increases with concentration of DBS for concentrations abo v e this value T h e impedance and transport data tell a consistent story about the effect of DBS on the C18-derivatized membrane (Figure 4-4) At low DBS concentrations ( <10"6 M) where the membrane resistance is in the 107 ohm range Ru(bpy) / + is not tra n sported. The sudden drop in Rm at DBS concentrations above 1 o6 M is seen in the transport expe r ime nt s as a abrupt switching on of Ru(bpy) 32+ transport across the membr a ne

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119 In order to understand the nature of this abrupt switch to the on state, we compared Ru(bpy)/+ and NDs2 fluxes across bare (no C1s) alumina membranes with fluxes across C1s-modified membranes that had been exposed to 104 M DBS The Ru(bpy)/+ fluxes for the bare alumina membrane and for the C1s-modified membrane that had been exposed to 104 M DBS were found to be identical (data not shown) In the case of the bare membrane, the pores are flooded with water and transport occurs by diffusion through these water filled pores. The equivalence of the flux for the C1s-modified membrane that had been exposed to 104 M DBS clearly shows that the pores in this membrane are also flooded with water. The same results were obtained for the flux of the anionic permeate ion NDs2 An anion was studied to insure that transport of the cationic Ru(bpy)32+ was not being facilitated in some way by the anionic surfactant incorporated within the pores Again, these data show that the transition from the off state to the on state occurs because at DBS concentrations above 10-6 M sufficient DBS has partitioned into the membrane that the pores spontaneously flood with water and electrolyte There is, however, one issue that remains unexplained The Ru(bpy)/+ and NDs2 flux data discussed above clearly show that exposure of the C1s-alurnina membrane to 104 M DBS fully wets the pores making them equivalent, in terms of this transport experiment, to the pores in the bare alumina membrane. This equivalence is not, however, observed in the membrane resistance experiment. That is the resistance of the C1s-alurnina membrane after exposure to 104 M DBS does not become equivalent to that of the bare alumina membrane. In fact the C 1 s-membrane resistance after exposure 104 M DBS remains two orders above the bare alumina resistance One possible explanation

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120 for this observation is that the resistance measurement for the bare alumina membrane contains a component associated with ionic conductivity along the pore wall, and that when the membrane is treated with the C1s silane, this component is lost. Measurements of Ion Current While the transport experiments show that the analyte DBS can switch on ion ( e g., Ru(bpy)/+ and NDS2") transport across the membrane a direct measure of the ion current was also desired To do this a constant transmembrane potential of 1 5 V was applied and the resulting transmembrane ion current was measured Figure 4-5 shows the measured ion current vs time data At the indicated times the electrolyte solutions were spiked to the indicated concentrations with DBS The ion-current data show the same general trend as both the impedance and transport data at concentrations below 1 o6 M the ion-current is at a very low baseline value and at concentrations above -10-6 M the ion current abruptly switches on. In addition the 10-5 5 M datum shows that the transit i on from the low-current to the high-current state occurs very abruptly Both the impedance and ion-current data show that when t he membrane is in the off state some small baseline current does flow across the membrane It is important to note that the resistance value for the off state obtained by the impedance and ion-current measurements are essentially iden t ical. As shown in Figure 4-4 the impedance measurement yields a value of-107 n. The ion-current in the off state i s 1.5x10 7 A which for a 1 5 V transmembrane potential yields a membrane resistance of 1 0 7 n The issue left to resolve however is what is supporting this baseline ion current when the membrane is in the off state? At this point there is no definitive answer other than to suggest that this current results from some surface conduction process that occurs along the pore walls when the pores are devoid of water In the absence of DBS this surface

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121 c o nducti o n process may involve residual surface hydroxyl sites The impedance data (Figure 4 4) indicate that in the presence of DBS, the surfactant itself i s involved in the conduction process 3 eC l 2e l,-. ,_ 8. 1 e .._, i u 0 Blank 0 1 M KCI I ~ / 1 o-s. s M 10-8 M 1 Q-6. S M / I., I I I u I I I 1 Q-9 M 10-7 M 10-6 M I I I I I c -1e O 1 000 200 0 3000 4000 Time (seconds) Figure 4-5 Ion current through a C18-modified alumina membrane v s time The contacting so l ution phases were spiked with the indica t ed concentrations of DBS at the indicated times The electrolyte was 0 1 M KCI. A constant transmembrane potentia l of 1 5 V was applied Effect o f Alkyl Chain Lengt h an d Na tu re o f the Surfactant Head G r oup The above data show that partitioning of the analyte DBS into the membrane is responsible for the transition to the on state It seems likely that this partitioning process is driven by the h y drophobic effect. To prove this point C1 8-modified alumina membranes were exposed to 0 1 wt% solutions of various alkyl sulfona t e alkyl benzen e sulfonate, and trimethyl alkyl ammonium surfactants (0 1 wt% corresponds t o 8 1 mM

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122 9--------------------------, 8 7 6 5 4 3 2 1 Figure 4-6 a ......... . . . ........ ................ : : : : ~ : :: : : : : : : : \ . . . \ .. 2 4 6 8 10 12 14 Number of Carbons Atoms in Alkyl Chain Plots of log membrane resistance for C18-modified alumina membranes as a function of the carbon chain length of the surfactant exposed to the membrane Prior to measurement, the membranes were soaked in 0 1 wt% surfactant solutions CJ Benzenesulfonate, Sulfonate, and Trimethylammonium surfactant families Data for this graph was provided by Dr. Erich Steinle for the smallest surfactant and 2 8 mM for the largest.) The membranes were then rinsed and the impedances determined in 0 1 M KCl that was devoid of surfactant. These experiments were done in conjuction with Dr. Erich Steinle, who collected the impedance data Figure 4-6 shows plots of log membrane resistance vs. number of carbon atoms in the alkyl side chain for all of the surfactants investigated These data show that in order for a surfactant to switch the membrane to the on state the alkyl chain must be above some minimum length For the alkyl benzene sulfonates, a 12-carbon chain is required

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123 For the alkyl sulfonates and alkyl ammoniums, the alkyl group must be at least 14 carbons long (Figure 4-6) These data clearly show that the hydrophobic effect is responsible for driving the surfactant into the C1s-modified membrane The difference between the alkyl benzene sulfonates and the other two classes of surfactants reflects the added contribution of the benzene group to the hydrophobicity With the added benzene group a 12-carbon alkyl chain can switch the membrane to the on state; without the benzene group a 14-carbon chain is needed (Figure 4-6) The data in Figure 4-6 also show that for the long-chain surfactants the adsorption of the surfactant to the membrane is essentially irreversible i f the membrane is exposed to aqueous solution However we have found that the surfactant can be removed from the membrane by rinsing with ethanol. After the ethanol rinse the membrane resistance returns to values equivalent to the off state obtained pr i o r to exposure to surfactant. Detection of Drug Molecules To further explore the role of the hydrophobic effect in driving the analyte species into the C 1 s-derivatized alumina membrane we investigated the effect of hydrophobic cationic drug molecules on the membrane resistance The molecules and thei r molecular wei g hts are amiodarone (645 g mor1 ) amitriptyline (278 g mor1 ) and bupi v acaine (288 g mor1 ) (Figure 4-7) Because its molecular weight is more than doub l e those of th e other drugs and because it contains very hydrophobic iodo substituents amiodarone is b y far the most hydrophobic of these molecules If the hydrophobic effect is respons i ble for driving molecules into the C1s -derivatized membr a ne then t he tran sition from the off t o the on state would occur at lowest concentrations for arniodarone and this i s what is observed e x perimentally (Figure 4-7) There is only a 3% d i fference in t he mole c ular

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124 w e ight s o f amitriptyline and bup iva c ain e ; however, b upivacaine presents two additional oppo rtunities for hydrogen b o nding with water the lo n e pairs on the carbonyl group and the lone p air of the non-prot o nate d nitrogen For this reason, bup i vacaine is much more hydrophi l ic, a n d it would be expected to b e the mostly poorly detected of the three drugs ; Figure 4 7 shows that this is also o b serve d experimentally "'}? 9 H O I if" # 8 CH3 B u ivacaine 7 I C 6 I ' d I I 5 I oO I ..3 I 4 I I 0 I 3 "" ~ ~CH:i I 6t9 I # I -+(CH:i I I I i 2 Amioda rone I ..,:; ,..CH3 1 l Amitrip ty lin eGH3 8 -7' 6 5 -4 -3 2 -1 L o g ([Drug] (M)) Figure 4-7 Plots of log membrane resistance vs log[Drug] for the indicated drugs and a C1s-modified alumina membrane The Effect o f Pore D e n sity a n d Por e D iameter o n Analyte D etection Au nanotube membranes that were ren d ered hydrophobic by chemisorbing octadecyl thiol ( 4 0 ) were used to explore these iss u es This work was done primarily by Dr. Erich Steinle and is included here as supplementary proof of the mechanism of pore opening These membranes contained 2 nm inside-diameter Au nanot u bes and had a total porosity of only 2x10 3 %. This may be contrasted to the alumina membranes which

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125 contain pores that are 2 orders of magnitude larger in diameter and are -30% porous. In a study analogous to Figure 4-4, the resistance of the thiol-modified Au nanotube membranes was examined with increasing concentration of DBS. An abrupt transition from a very high-resistance (109 Q) off state to a 4-order-of-magnitude lower-resistance on state is observed over essentially the same concentration range as is observed for the C1s-derivatized alumina membranes. These results show that for membranes containing either C18-modified alumina micropores or C1s-modified Au nanotubes, the concentration of DBS that induces the transition form the off to the on state does not depend on porosity or pore/tube diameter. It is important to point out that changing the porosity or pore/nanotube diameter in reality changes only the surface area of C1s groups available for the adsorption of analyte i.e., the low-porosity Au nanotube membranes have a lower surface area available for analyte adsorption than the alumina membranes. This situation can be modeled by assuming that the analyte is adsorbed onto the C18-modified surfaces via a Langmuir isotherm and that a certain critical fraction (0c) of the surface must be covered with analyte in order to flood the pores with electrolyte. The critical fraction is related to the equilibrium constant for the adsorption reaction(~) and the activity of DBS in the solution phase (a08s) via (177) 0c = (~aoss) I (1 + ~aoss) (2-1) Equation 2-1 shows that 0c depends only on the value of the equilibrium constant (the same for both membranes, since both contain C 1 s alkyl groups) and the activity of surfactant in solution. Because surface area does not enter into the equation the concentration of DBS required to switch the membrane to the on state should be

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126 independent of membrane porosity and pore diameter and this is what is observed experimentally Conclusions We have shown that synthetic rnicropore and nanotube membranes can mimic the function ofligand-gated ion channels ; i e they can be switched from an off state to an on state in response to the presence of a chemical stimulus The off state was obtained by making the membranes hydrophobic, and the on state was obtained by introducing ions and electrolyte into the membrane Ions were introduced by partitioning a hydrophobic ionic species (e g a drug or a surfactant) into the membrane The concentration of surfactant required to switch a C18-derivatized membrane to the on state does not depend on membrane pore diameter or porosity It does however depend on the hydrophob icity of the analyte with more hydrophobic analytes yielding the on state at lower concentrations In this regard these sensors are qualitatively very similar to ion-selective electrodes developed for hydrophobic cations that were based on a hydrophob i c anion exchange reagent (178 179) In both cases the sensor could be useful in an environment where only one hydrophobic ionic analyte is present ; i.e., all other ions in the analyte solution being hydrophilic

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CHAPTER 5 CONCLUSIONS A common theme of this dissertation has been the use of aluminum oxide membranes Chapter 1 introduces these membranes as well as other types of membranes used for template synthesis It also outlines how alumina membranes are prepared from aluminum metal, and how to control important membrane parameters such as pore size and membrane thickness Silanization and sol-gel chemistry are also explained in Chapter 1 as these are used frequently in subsequent chapters Chapters 2 and 3 involve the use of alumina membranes as templates for the manufacture of silica nanotubes. The size and shape of the nanotubes are directly dependant on the size and shape of the pores in the alumina membranes Because the pores of such membranes can be highly monodisperse in size, the resulting nanotubes show an analogously monodispersity Experiments in Chapter 2 show how the surfaces of the silica nano tubes can be chemically modified Both the interior and exterior surface chemistries can be controlled and these chemistries can be independent. For example the i nside suface can be hydrophobic and the outside hydrophilic Nanotubes modified in this way are shown to be effective extractants for drug molecules Nanotubes modified with a chelating agen t are shown to extract metal ions It is also shown that the nanotube surface could be biotinylated which resulted in binding to avidinated microspheres A similar experiment using much smaller particles shows that dithiols can be used to link Au nanoparticles to 127

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128 template-synthesized Au nanowires Differential inner and outer surface chemistry of nanotubes is also achieved by making composite polymer/silica nanotubes Chapter 3 examined protein attachment to the nanotubes A convenient and practical method involving an aldehyde silane linker is most commonly used Proteins attached by this method are shown to be functional and active, retaining the protein s nat i ve selectivity or catalytic properties Several protein-modified nanotubes are used as highl y specific extractants Examples include avidinated nanotubes for removing biotinylated particles and immunoprotein-coated nanotubes that selectively extract their respec t ive antigens from solution Experiments show that immunoprotein-coated nanotubes can selectively extract one enantiomer from a racemic drug mixture The protein coating o f the nanotube was also shown to control where the nanotube binds Because of this nanotubes modified on the inside with a fluorophore and on the outside with a protein function as specific labeling agents Enzymes attached to the nanotubes are shown to retain their activity and to function as removable bioreactors In Chapter 4 the alumina membrane served as the starting material for production o f a gated membrane The gate on the membrane is initially closed, and no molecules or ions can pass through When the membrane is exposed to analyte, the pores of the membrane open and molecules and ions can pass from one side of the membrane to the other The gate is formed by hydrophobic alkyl chains, each eighteen carbons in length which are deposited on the alumina using silane chemistry The hydrophobic nature of the alkyl groups prevents water from entering the pores Because the pores are not filled with water water-borne molecules and ions have no means of transport through the membrane Experiments show that when surfactant analyte is added the pores flood wit h

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129 water. This is caused by the hydrophobic regions of the surfactant interacting with the hydrophobic C1s chains on the pore wall. The ionic surfactant headgroup lines the pore walls, and this permits water to flood the pore Water soluble species can now be transported across the membrane. Analyte species which cause the membrane pores to open include cationic and anionic surfactants, and drug molecules The concentration of the analyte needed to cause pore opening is shown to be largely dependant on the analyte hydrophobicity with more hydrophobic surfactants causing pore opening at lower concentrations

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BIOGRAPHICAL SKETCH David Mitchell was born in Ojai California, the first of e i ght children in the fami l y of Jim and Terri Mitchell. As a baby he moved with h i s paren t s t o Gainesville Florida where he lived until age eight. He and his family then transferred to Logan Utah where he graduated from high school. David developed an interes t in science at an early age starting a Chemistry club in middle school. This interest continued through college at Utah State University, where he majored in Chemistry with an emphasis in Biology H i s studies continued after graduation first at Colorado State Universit y in Fort Collins then at the University of Florida in Gainesville, where he moved with his professor In addition to science David enjoys outdoor activities especially rock climbing rafting and hiking 140

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a thesis for the degree of Doctor of Philo ~ ef__ ~--Charles R. Martin Chairman Professor of Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality as a thesis for the degree of Doctor of Philosophy. V aneica Young Associate Profess I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality, as a thesis for the degree of Doctor of Philosophy. r aduate Research Professor of Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a thesis for the degree of Doctor of Philosophy.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate in scope and quality as a thesis for the degree of Doctor of Philosophy. Associate Professor of Electrical and Computer Engineering This thesis was submitted to the Graduate Faculty of the Department of Chemistry in the College of Liberal Arts and Sciences and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 2002 Dean, Graduate School

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