Collagen Structure Control and Electrohydrodynamic Flow in Non-Uniform Electric Fields

7/29/2019 Collagen Structure Control and Electrohydrodynamic Flow in Non-Uniform Electric Fields

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Collagen Structure Control and

Electrohydrodynamic Flow in Non-Uniform

Electric Fields

by

cAndrew R. Morrow

A Thesis submitted to the Department of Physics and Physical Oceanography inpartial fulfillment of the requirements for the degree of

Bachelor of Science with Honours

Department of Physics and Physical Oceanography

Memorial University of Newfoundland

December 2011

St. Johns Newfoundland


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Abstract

In these experiments, PMMA and silica microspheres, as well as collagen fibrils

and monomers are placed in non-uniform AC electric fields. The motivation behind

this research is the medical applications of collagen, and microfluidics mixing. The

non-uniform field is created by patterned electrodes in a non-uniform field cell. This

is to determine if any of the collagen experiences the dielectrophoretic force. Both

untagged and tagged collagen monomers are tried, and no dielectrophoresis is ob-

served, although a pattern is visible that does not disappear with increasing field.

PMMA and silica microspheres are tested to ensure that they do experience the ex-

pected DEP effect. During these latter experiments, an interesting flow is observed in

some cells containing PMMA spheres that cannot be explained using dielectrophore-

sis alone. This flow is believed to be a combination of dipole interaction between

polarized spheres, fluid flow caused by sphere rotation, and dielectrophoresis. This

only appears at high field and certain frequencies.

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Acknowledgements

For my family, who always believed that education was a top priority.

For Dr. Anand Yethiraj and Dr. Erika Merschrod, who are dedicated educators

and researchers who go above and beyond.

For Dr. Yethirajs lab group, who showed me how to run the equipment.

For the physics students at Memorial who were good friends.

Thank you.

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Table of Contents

Abstract ii

Acknowledgments iii

Table of Contents v

List of Tables vi

List of Figures viii

1 Introduction 1

1.1 Micro scale structure control . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Collagen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.3 Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.4 Electrohydrodynamic flow . . . . . . . . . . . . . . . . . . . . . . . . 4

1.5 Goals of Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.6 Motivation of Research . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2 Theory 6

2.1 Dielectrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

2.2 Electrohydrodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . 8

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List of Tables

3.1 Instrumentation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

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List of Figures

1.1 Collagen triple helix. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

3.1 Non-uniform field cell schematic, top and side views (a. microscope

slide b. top electrode c. bottom electrode d. top electrode wire e.bottom electrode wire f. PET spacers g. pattern in bottom electrode). 12

3.2 White light image , collagen fibrils. . . . . . . . . . . . . . . . . . . . 14

3.3 Polarized light image, collagen fibrils. . . . . . . . . . . . . . . . . . . 17

3.4 Light path through the microscope for polarized light microscopy. White

light microscopy is similar except polarizer # 2 is removed from the

light path. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

3.5 Absorption and emission curves for various materials. . . . . . . . . . 19

3.6 Light paths for regular fluorescence and scanning confocal microscopy.

For laser reflection microscopy the dichroic mirror is removed. . . . . 20

3.7 Fluorescent image, 488 nm laser with 515/30 nm filter, FITC labeled

Collagen in capillary tube. . . . . . . . . . . . . . . . . . . . . . . . . 21

3.8 Fluorescein isothiocyanate (FITC) molecule attached to collagen. . . 22

3.9 Striped electrode, laser reflection microscopy (488 nm laser, 450/35

filter). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

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4.1 0.8 micron PMMA in decalin and TCE, left is 0 V/m, center is 1.2

V/m, right is 3.4 V/m. . . . . . . . . . . . . . . . . . . . . . . . . 25

4.2 Collagen fibrils in a 0.42 V/m 1 kHz field, 10x air objective, left t=0s,

right t=2.0s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

4.3 6.4 mg/ml collagen in a capillary tube, 408 nm laser with 450/35 nm

filter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4.4 6.4 mg/ml fluorescent untagged collagen in field cell, left 0 V/m, right

4.16 V/m, 408 nm laser with 450/35 nm filter. . . . . . . . . . . . . 28

4.5 Normalized intensity plots, 0 V/m, 1.04 V/m, 2.12 V/m, 3.04

V/m, and 4.16 V/m. . . . . . . . . . . . . . . . . . . . . . . . . . 29

4.6 FITC tagged collagen above the electrode, top from left: 0 V/m 0.4

V/m, bottom from left: 0.6 V/m 1.0 V/m. Lines faintly visible in

0 V/m and 0.4 V/m, gone in 0.6 V/m and 1.0 V/m. . . . . . . 30

4.7 Silica in water, left is t=0s, center is t=5s, right is blown up section

from center. Note the movement of the large object and the clustering

of particles along the electrode edge and center of the glass. . . . . . 31

4.8 Electrode overlap in field cell with patterned electrodes on the top and

bottom. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

4.9 Left is flow bands of different densities, right is two sequential frames

subtracted from each other to show flow direction. . . . . . . . . . . . 33

4.10 Wave at the band interface, 5.4 V/m. . . . . . . . . . . . . . . . . . 34

4.11 Perpendicular wave at 19.52 V/m. . . . . . . . . . . . . . . . . . . . 35

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Chapter 1

Introduction

1.1 Micro scale structure control

There is a lot of interest in being able to control the structure of matter on the micro

scale. Such control would be useful in many different applications. For example,

people have used these ideas to capture living cells in an electrode array,1 create

photonic crystals2 and assemble semiconductor nanowires,3 among other things.

One way to control matter on this scale is by using a non-uniform electric field

to create a dielectrophoretic force. This type of control mechanism has been explored

using colloidal suspensions of micro-particles such as silica and PMMA in various

solvents. Bartlett4 was able to show that these particles exhibited positive or negative

dielectrophoresis (movement to the high or low field region, depending on the dielectric

properties of the particles and the solvent), and could control the particles depending

on the pattern of the field employed. These experiments all deal with particles that

are around 1 m in size. The following experiments deal with both micro-particles

and collagen, which is a long molecule found in biological matter. Experiments have

been performed before using biological materials in electric fields, such as Pohl using

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an AC field to attract yeast cells,5 and to separate viable and nonviable yeast cells.6

While collagen itself has been examined in the presence of an electric field, 7 what

we are interested in is whether or not it displays the same sorts of behavior seen

in the colloid experiments. The strength of the dielectrophoretic force on a sphere

is proportional to its volume.8 This means that the dielectrophoretic force becomes

very weak for particles with small radii. A single collagen molecule is quite small,

however they can come together to form much larger fibrils that can be visible to the

naked eye. We are interested in how these single molecules and larger fibrils behave

under a dielectrophoretic force.

1.2 Collagen

Collagen is a component in many different parts of the body such as tendons, blood

vessels and the skin.9 There are 16 different types of collagen, however the type of

collagen used in these experiments is type I collagen, which is the most abundant

form of collagen in the body. The collagen molecule is about 1.5 nm wide by 300 nm

long, made up of three chains twisted together to form a right-handed helical shape,

as seen in Figure 1.1. In type I collagen, there are two different chains, two 1 chains

and one 2 chain. Each chain is made up of 1050 amino acids, which are the building

blocks of all proteins. The collagen monomers can aggregate together to form larger

units known as fibrils. These fibrils are 50-200 nm in diameter, and the monomers are

staggered by a distance of 67 nm. This structure is very strong, and these fibrils form

parts of the connective tissues of mammals. When heated, the three strands in the

collagen helix unwind, and form a gelatin-like substance. This gelatin is used in foods,

pharmaceuticals, cosmetics and other industrial uses, while regular collagen can be

used for things such as artificial heart valves, burn dressings, and blood vessels.10


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Figure 1.1: Collagen Triple Helix.11

1.3 Dielectrophoresis

Dielectrophoresis is the movement of uncharged dielectric material in the presence of a

non-uniform electric field.12 A dielectric material is something that can become polar-

ized in the presence of an electric field, which can be AC or DC. This phenomenon was

discovered in the 1950s by Dr. Herbert Pohl,13 who performed experiments mentioned

earlier.5 In the presence of a non-uniform electric field, dielectric particles suspended

in some medium will migrate to either the high or low field regions, depending on

the permittivity of the particle and the medium. If the permittivity of the particle is

higher than the medium, the particle will be attracted to the region of high field, and

to the region of low field if the permittivity is lower. This force affects a wide range

of particle sizes, from as small as 20 nm to 10 m and more.14

The dielectrophoreticforce is the main control mechanism for collagen and microparticle experiments.


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1.4 Electrohydrodynamic flow

Electrohydrodynamics is the movement of particles in a fluid, and the flow of the

fluid itself due to electric forces.15 Some experimentation has already been done in

this field, such as using high voltage low frequency AC fields to change the shape of

a region of barium titanate particles suspended in Castor oil,16 and causing 1.5 m

particles to circulate in a KCl solution using a wide range of AC electric fields.17

1.5 Goals of Research

The end goal of this research is to understand how biological molecules, specifically

collagen is affected by the dielectrophoretic force. To begin, we will experiment with

the same spheres used in the experiments done by Bartlett and others.4 Then, we

will try smaller spheres and collagen in the same types of non-uniform electric fields.

Although collagen has a natural fluorescence, we had to tag the collagen using a

fluorescent marker as the natural fluorescence was difficult to observe. Also, we will

try to find the cause of the electrohydrodynamic flow observed in some of our field

cells.

1.6 Motivation of Research

One important reason to study collagen is that it has a wide range of medical appli-

cations, such as artificial blood vessels and heart valves, tablets for protein delivery,

and in the treatment of various burns and wounds.18

The Collagen would have tobe modified in some fashion to be used in all these different applications, and an

understanding of how to control it on a micro-scale would be quite useful. A use

for the hydrodynamic flow we observe is the mixing of microfluids, which are fluids


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as the particles move through the medium. The forces that causes the motion is called

the diffusional force, and the strength of this force is given roughly by the equation

Fdiff =kbT

2r (2.2)

where kb is the Boltzmann constant, T is the temperature of the system, and r is

the radius of the particle. The kbT comes from the thermal energy of the system, while

the 2r is the diameter of the particle, which is the shortest distance that has meaning

in the context of diffusion. This is difficult to apply to the collagen monomers, which

are 1.5 nm in diameter and 300 nm long. Two possibilities exist, that the monomers

stay stretched out, and the force acts on them like a long chain of 1.5 nm spheres, or

that they coil up into a loose ball that is on the order of tens of nanometers. The size

of this ball is estimated using the hydrodynamic radius, which for a collagen monomer

has been estimated to be on the order ofRh = 24.7 nm.22 It is most likely that the

monomers coil up due to the collisions they are experiencing.

2.2 Electrohydrodynamics

Electrohydrodynamics encompasses all forms of fluid motion caused by electric forces.15

The dielectrophoretic force, which is not an electrohydrodynamic force, is what is nor-

mally used to control microspheres. Certain microparticle movement patterns that

were observed in one type of cell could not be explained simply using dielectrophoresis,

but rather a combination of dielectrophoresis and electrohydrodynamic forces. These

flows are discussed in Chapter 4.

In dielectrophoresis, polarization of the dielectric spheres is the cause of the

motion. However, with the flows observed, there are several forces working together

to cause it. The other forces occur due to the fact the decalin/TCE (trichloroethylene)


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Chapter 3

Experimental Procedures

In order to determine the behavior of the collagen monomers in an electric field, we

must construct a cell that can apply a non-uniform electric field across a volume

of solution. This chapter will detail the construction of such a cell, as well as the

techniques used to image the solution, such as polarized light microscopy, and confocal

microscopy.

3.1 Cell Construction

A schematic of the non-uniform field cell can be seen in Figure 3.1. The base of the

cell is constructed from a microscope slide and a patterned electrode. The Fisher

glass microscope slide is washed with distilled water and canned air (Dust-Off). A

smaller glass plate covered with indium tin oxide (ITO) made by Structure Probe Inc.

is attached to the slide using Norland Optical Adhesive (NOA-68) epoxy, and left to

cure under a 100 watt Spectroline SB100P UV lamp for 20 minutes. This small ITO

glass plate is the bottom electrode. To create the pattern on the bottom electrode,

0.5 ml of Shipley S-1813 photoresist is put onto the ITO and the plate is spun for

30 seconds at 4000 rpm. The slide and bottom electrode is then heated at 100 oC

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NA = nSin() (3.1)

where n is the index of refraction for the medium around the objective (1.0 for

air, 1.33 for water, around 1.5 for some oils),28 and is the widest angle from vertical

that light can enter the objective.29

The basic rule is that a higher the value of numerical aperture means that the

objective can resolve smaller objects.30 The limit of resolution of the objective is given

by

d = 2NA (3.2)

where is the wavelength of the light used to illuminate the sample and d is

spacing of a particular detail in the sample we want to image. It is resolved when

the numerical aperture of the objective is large enough to capture the first order

diffraction pattern caused by the detail at a light wavelength . This is known as the

Abbe limit.

This brings us to the difference between oil and air immersion objectives. The

numerical aperture of an air objective is limited to around 0.95. This is due to the

internal reflection of high angle light rays at the interface between the cover slip glass

over the sample, and the air between the glass and the objective. This is due to the

difference in the refractive index in air and glass, which are 1 and 1.5 respectively.

The maximum angle can be found using Snells law

max = arcsin(n2

n1) (3.3)

where n1 is the refractive index of the material the light is passing through, and

n2 is the refractive index of what the light is entering. With air and glass, this angle is


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around 39 degrees. However, if we put a small volume of oil between the objective and

the cover slip, with a refractive index of around 1.5, this maximum angle increases to

around 67 degrees, and the effective numerical aperture of the objective goes up to

around 1.4. Therefore, if we want to image something in very high resolution, we can

use an oil immersion objective with a high numerical aperture.29,30

3.4 Polarized Light Microscopy

Polarized light microscopy is a technique that is used with the standard white light

microscope setup. The Nikon Eclipse 80i microscope is equipped with two polarizers,

one that is fixed below the sample stage, and one above that is removable. To examine

a sample using polarized light, the sample is first brought into focus using white light.

The top polarizer (the analyzer) is inserted and the bottom polarizer is rotated until

it is 90 degrees to the analyzer, blocking all unrotated light. If there is the right type

of structure present in the sample, it will rotate the light so it can pass through the

polarizers.

For this rotation to occur, the material must be birefringent.28 Birefringence, or

double refraction means that when a single ray of light passes through an anisotropic

material, it is split into two different rays of light with new polarizations that allow

them to pass through the polarizers. In a material that is anisotropic along a single

axis, one of the rays will be diffracted in a normal manner and the other will not.

This type of microscopy has been used to examine the collagen fibrils in cartilage

and bone.31 The sample can then be imaged using Qcapture software and the Qicam

digital camera, which is not currently attached to the z translation stage, so it is

limited to two dimensional imaging. This type of microscopy was only used for the

large collagen fibrils.


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Figure 3.7: Fluorescent image, 488 nm laser with 515/30 nm filter, FITC labeledCollagen in capillary tube.

isothiocyanate isomer 1). The FITC arrives in a powder form, so it must be mixed

with distilled water. 2.4 mg of FITC powder was mixed with 150 l of distilled water,

giving a dye solution of about 17 mg/ml. Then 5 l of dye solution was mixed with

100 l of Nutragen 6.4 mg/ml bovine collagen in a Millipore Ultra-free 0.5 micropore

tube with a Biomax high flux polyethersulfone membrane. This solution was left to

react overnight in the fridge. The solution was then centrifuged at 400 x g for ten

minutes, and then again for 20 minutes at 15000 x g as none of the solution had been

passed through after the first centrifuging. The purpose of this step is to remove the

excess dye from the collagen solution. After the second centrifuging, 25 l of collagen

solution was recovered from the top of the micropore tube, which was then mixed

with 75 l of 0.01 M HCl to return the collagen solution to the original 6.4 mg/ml.

The collagen will now fluoresce at 520 nm when excited by a 488 nm laser.35,36


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Figure 3.8: Fluorescein isothiocyanate (FITC) molecule attached to collagen.

3.7 Experiments

There were essentially three different rounds of experiments performed in this work.

All of the experiments were performed in the same way as described, by applying a

non-uniform field to some system by way of a patterned field cell, however the different

systems can be classed into three groups. The field cell was not altered until the third

group of experiments, however time was short at that point, so further experiments

with different geometries could be attempted for future work. The basic geometry of

the cell was a striped electrode on the bottom of the cell, Figure 3.9, with a plain

electrode on top. This type of cell will give alternating regions of high and low field

across the cell.

The first group of experiments was performed on 0.8 micron PMMA (poly(methyl

methacrylate)) particles suspended in a mixture of 60/40 decalin/TCE(trichloroethylene).

This mixture was the same one used in experiments mentioned earlier by Bartlett and

others.4

These experiments were done to ensure that everything was being done prop-

erly and everything was working.

The next group of experiments dealt with the collagen fibrils and monomers. We

had four different kinds of samples containing collagen. There was one where the


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Figure 3.9: Striped electrode, laser reflection microscopy (488 nm laser, 450/35 filter).

collagen had aggregated together to form fibrils, two untagged solutions of collagen

monomers at 3 mg/ml and 6.4 mg/ml in 0.01M HCl, and one solution of 6.4 mg/ml

collagen tagged with FITC. At the beginning, we tried the different imaging methods

mentioned above to try and find which one gave the best results. These mixtures

were then experimented on with the field cells. Finally the tagged collagen was tried

in a field cell.

In the third group, silica microspheres with a diameter of 100 nm in 0.01 M HCl

were examined, as it is on the same length scale as the collagen monomers. We also

tried a different electrode geometry, with a striped electrode on the top and bottom,

with 1.0 micron PMMA particles in the 60/40 decalin/TCE mix.


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Chapter 4

Results and discussion

4.1 0.8 micron PMMA

The samples in this group of experiments are 0.8 m PMMA in a 60/40 mix of decalin

and trichloroethylene (TCE). These experiments were mostly done to make sure that

the cell construction was correct by replicating earlier experiments done by Bartlett

and others.4 We also wanted to observe if the particles formed any kind of three

dimensional structure when they were in an equilibrium state. The decalin/TCE

mixture is useful as the ITO electrodes do not burn as readily at higher fields unlike

water.

As can be seen in Figure 4.1, the particles experience positive dielectrophoresis,

and are in a very well defined band at high voltage. There were z stacks taken at

all voltages, however we were unable to determine whether or not they were forming

three dimensional structure.

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Figure 4.1: 0.8 micron PMMA in decalin and TCE, left is 0 V/m, center is 1.2V/m, right is 3.4 V/m.

4.2 Collagen

4.2.1 Collagen fibrils

The first sample tested in this group of experiments contained fibrils, which form

when a solution of collagen monomers is unrefrigerated for a period of time, and the

pH of the solution exceeds 4.0.37 Some of these fibrils are visible to the naked eye,

and are easily observed with white light and polarized light microscopy, as can be

seen in the figures in Chapter 3. This sample was sealed in a standard field cell, and

a field of 0.42 V/m at 1 kHz was applied across the cell. There was no movement

observed in this cell, as can be seen in Figure 4.2.

The clumps of fibrils observed are on the same length scale as the thickness of the

cell, so it is possible that they are stuck in place and cannot move. This was a short

test conducted at only one voltage as we were more interested in how the smaller

monomers would behave. Also, there were too many unknown variables about this

sample, such as fibril concentration, and storage conditions such as temperature. This

sample was mostly used to test the various types of microscopy at our disposal.


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Figure 4.2: Collagen fibrils in a 0.42 V/m 1 kHz field, 10x air objective, left t=0s,right t=2.0s.

4.2.2 Untagged Collagen Monomers

Two different concentrations of untagged collagen monomers were used, one at 3

mg/ml, and one at 6.4 mg/ml. Before using these solutions in the field cells, they

were imaged in capillary tubes to determine the correct type of microscopy. For this

type of collagen, the best results were found with a 408 nm laser combined with a

450/35 nm filter. The 3 mg/ml collagen did not fluoresce enough, so all electric field

tests were conducted with the 6.4 mg/ml samples.

When these samples were put into a field cell, there was a visible pattern in the

sample at zero field. The collagen appears to be concentrated over areas over where

the ITO on the electrodes was stripped away. The field was turned on at 1 kHz, and

images were recorded at 1.08 V/m, 2.12 V/m, 3.04 V/m and 4.16 V/m. The

images in Figure 4.4 have had the contrast adjusted in the same way so as the pattern

observed is more easily visible, however the analysis was done on the original pictures.

It appears that the pattern does not change between the different voltages, how-

ever to be sure, the images were analyzed using ImageJ,38 an image manipulation


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Figure 4.4: 6.4 mg/ml fluorescent untagged collagen in field cell, left 0 V/m, right4.16 V/m, 408 nm laser with 450/35 nm filter.

4.2.3 FITC tagged collagen

As the natural fluorescence of Collagen was quite weak, we labeled a sample with

FITC. Unfortunately, the fluorescence of the tagged collagen was not much stronger

than the natural fluorescence. It was tested in much the same way as the natural

collagen, except the images were recorded about halfway through the cell, as opposed

to right above the electrode. There was also series of images recorded along the z-axis

for each field. The fields used on this cell were at 1 kHz, and at 0.1 V/m, 0.2 V/m,

0.4 V/m, 0.6 V/m, and 1.0 V/m.

As can be seen in Figure 4.6, the collagen seems to be concentrating over theITO strips, instead of the glass as with the previous collagen at zero field. This can

be seen clearly in the 0.4 V/m picture with the narrow, bright strips, which are the

regions of highest concentration of collagen. This could be FITC dye in the solution


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Figure 4.6: FITC tagged collagen above the electrode, top from left: 0 V/m 0.4V/m, bottom from left: 0.6 V/m 1.0 V/m. Lines faintly visible in 0 V/m and0.4 V/m, gone in 0.6 V/m and 1.0 V/m.

4.3 Silica and offset field cells

In the collagen experiments, we did not see any structure formation or movement as

we did with the 0.8 micron particles. Due to the a3 term in the dielectrophoretic force

equation, the strength of force drops drops off rapidly for particles below 1 micron,

and the collagen monomers are quite small, around 1.5 nm thick and 300 nm long,9

with a hydrodynamic radius of around 24.7 nm.22 To see if there was any sort of effect

on objects that small, we did an experiment on 100 nm silica particles in water. The

field was slowly increased from 0 V/m to 0.6 V/m.

As we can see from Figure 4.7, the 100 nm diameter silica particles do experience

some force, so the collagen monomers are not too small to experience dielectrophoresis,

as the hydrodynamic radius of the monomer is on the same length scale as the silica


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Figure 4.7: Silica in water, left is t=0s, center is t=5s, right is blown up section fromcenter. Note the movement of the large object and the clustering of particles alongthe electrode edge and center of the glass.

microspheres. The left image shows the particles when there is no field turned on,

and the particles are fairly evenly distributed across the cell. In the center image, the

field has been turned on, and the particles have moved away from the ITO strips to

the center of the glass area, as well as migrated to the high field region at the edge

of the ITO strips. The right image is a magnified image of the area enclosed in the

black square in the center image. Unfortunately, the cell failed at around 6 seconds

into the experiment, so the silica particles could not finish their migration. This is

common with water based solutions, as the ITO strips tend to react more in water.

If the cell had not failed, the majority of the silica would be in the glass area, away

from the high field region. To try and modify the non-uniform field, we constructed

cells that had patterned electrodes on the top and bottom, as opposed to simply on

the bottom in previous cells. The original idea was to have the electrode strips line

up exactly, however the strips ended up staggered at half widths, as can be seen in

Figure 4.8.

In this type of field cell, using 1 m PMMA particles in a 60/40 mix of decalin

and TCE, we observed the electrohydrodynamic flow discussed in the Chapter 2. We


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on the right hand side is moving down and to the right.

Figure 4.9: Left is flow bands of different densities, right is two sequential framessubtracted from each other to show flow direction.

The second important factor is the frequency dependence of the flow. By mea-

suring the resistance of the cell, and using this value to determine the conductivity

of the cell, the characteristic time was determined by using Equation 2.3, and from

this the critical frequency was determined to be around 50 Hz. It should be noted

that the conductivity was determined after the experiment, using an identical cell, as

we had not determined the cause of the flow at the time of experimentation. In the

second cell, a 2.52 V/m field was applied at frequencies of 50 Hz, 100 Hz, 200 Hz,

and 300 Hz. The 2.52 V/m field was chosen as it was high enough to cause flow, but

not so high as to cause disruptive waves, which will be discussed later. At 50 Hz, we

do observe flow, but the mass of particles oscillates rapidly in a manner perpendicular

to the flow. We make an association between this oscillation and the low frequency

of the AC field. At 100 Hz, we see flow at a rate that is similar to what is seen at 50

Hz, but without the oscillation. At 200 Hz there is still a small amount of flow, at a


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rate that is much lower than what is observed at 50Hz and 100Hz. At 300 Hz, there is

no flow whatsoever, and the images resemble regular positive dielectrophoresis. This

suggests that the electrohydrodynamic effects drop off very quickly once we are past

the critical frequency.

The third factor to discuss is what happens at the low and high ends of the

voltage range while we are at a frequency when flow occurs. The voltage must reach

a threshold value before flow will start. Below this value, the cell appears to undergo

regular dielectrophoresis. For this particular cell, this value is around 2 V/m, so we

see that it takes a large field to cause the flow. Around 5.4 V/m, we see a wave

action that runs perpendicular to the interface between the two flow bands. These

waves move very slowly, at a frequency of about 2 Hz, and the frequency does not

increase with increasing field. The wavelength of the wave is about 20 m, and this

also does not increase with increasing field. This can be seen in Figure 4.10.

Figure 4.10: Wave at the band interface, 5.4 V/m.

The amplitude of the wave grows as the voltage is increased, up to the point


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35

where it propagates across the entire field of view of the microscope. Interestingly,

the two domains remain intact, even with a massive field. Figure 4.11 shows the wave

at 19.52 V/m. Notably, the cell was able to survive even this very high field, in

contrast to the silica water system discussed earlier that burned out at a relatively

low voltage of around 0.8 V/m.

Figure 4.11: Perpendicular wave at 19.52 V/m.

The beginnings of this wave can be seen at lower voltages on the bottom of the

cell. This was not included as it is not obvious in a single frame. This wave is most

likely caused by the same combination of dielectrophoresis and spinning particles that

cause the flow observed, however it is not immediately obvious how the mechanism

works.


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Chapter 5

Conclusions

The goal of this research was to determine the effects of a non-uniform AC electric

field on Bovine collagen monomers. To do this, we used field cells constructed from

patterned ITO glass plates, along with various types of microscopy, most notably

laser confocal microscopy. In attempts to construct a variation on our regular cell,

interesting electrohydrodynamic flow was observed.

5.1 Collagen

To begin with, regular collagen was experimented with to determine if it would display

a similar reaction to a non-uniform electric field that was seen with PMMA particles in

earlier experiments, namely a dielectrophoretic flow to high or low field regions. This

collagen was placed in a series of 1 kHz alternating fields ranging from 1.04 V/m to

4.16 V/m. A pattern was present in the images from zero field, but it was determined

that this pattern was most likely caused by higher collagen concentration over glass

areas as opposed to ITO areas. For better image quality, the same collagen was tagged

with a fluorescent marker, FITC. This turned out to give a weaker signal than the

untagged collagen, most likely due to not all the monomers being attached to FITC

36


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37

molecules. This tagged collagen was examined in identical field cells, with similar 1

kHz alternating fields, at voltages between 0.4 V/m and 1.0 V/m. There was a

similar pattern observed with the tagged collagen, with two different characteristics.

The first was that it was strongest over the ITO strips, as opposed to the glass

areas of the previous untagged collagen, and at fields above 0.6 V/m the pattern

disappeared. This does not show dielectrophoretic effects, as the signal remained

uniform throughout the cell. These results show that collagen does not experience

any serious dielectrophoretic effect at these field strengths.

5.2 Electrohydrodynamic flow

During experimentation with different kinds of electrode geometry in our cells, an elec-

trohydrodynamic flow was observed. This flow circulated in bands that corresponded

to the high and low field regions of the cell, and had several defining characteristics.

Firstly, it required a high field to start the flow, around 2 V/m in our particular cell.

Secondly, there were waves created at higher fields that flowed perpendicular to the

interface between the flow bands. These waves had a very low frequency of only 1.5

or 2 Hz, that does not seem to increase with a stronger field. At extremely high field,

of around 19.52 V/m, the amplitude of the wave increased to fill the entire field of

view of the microscope, but the bands remain. The third characteristic of the flow

is the small frequency band that it the flow occurs in. The characteristic frequency

of this cell was determined to be around 50 Hz, and at 200Hz the flow was severely

reduced. This flow is thought to be caused by a combination of forces, dielectrophore-

sis and fluid flow due to rotating particles. A major contributing factor is that the

decalin/TCE medium is a leaky dielectric. This type of flow may have applications

in the mixing of microfluids.


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5.3 Future Work

For the experiments concerning collagen, there are a few things that could be tried

for future experiments. It would be interesting to attempt different cell geometries to

see if any sort of motion could be seen. Also, as the fields tried were relatively low

compared to the PMMA experiments, for future work it would be useful to be able

to apply a much larger field due to the small hydrodynamic radius of the collagen.

The ITO electrodes do not support high fields in water, as they tend to burn quickly,

however there are other materials such as conductive carbon nanotubes39 that could

work with the water solution. Another thing to try is a different fluorescent tag for

the collagen, to see if the opposite pattern will still appear in the field cells.

The flow observed in the second type of cell is interesting, however as it came

about late in the experiments, a proper study was unable to be done of it. A more

thorough study would be done over a number of cells, with many different frequencies

of applied field, so as to explore the frequency dependence of the flow.


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Collagen Structure Control and Electrohydrodynamic Flow in Non-Uniform Electric Fields

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