DNA Hybridization on Walls of Electrokinetically

Controlled Microfluidic Channels

by

Lu Chen

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy

Department of Chemistry University of Toronto

© Copyright by Lu Chen 2010

ii

DNA Hybridization on Walls of Electrokinetically Controlled

Microfluidic Channels

Lu Chen

Doctor of Philosophy

Department of Chemistry University of Toronto

2010

Abstract

The use of microfluidic tools to develop two novel approaches to surface-based

oligonucleotide hybridization assays has been explored. In one of these approaches,

immobilized oligonucleotide probes on a glass surface of a microfluidic channel were able to

quantitatively hybridize with oligonucleotide targets that were electrokinetically injected into

the channel. Quantitative oligonucleotide analysis was achieved in seconds, with nM

detection limits and a dynamic range of 3 orders of magnitude. Hybridization was detected by

the use of fluorescently labeled target. The fluorescence intensity profile evolved as a

gradient that could be related to concentration, and was a function of many factors including

hybridization reaction rate, convective delivery speed, target concentration and target

diffusion coefficient. It was possible to acquire kinetic information from the static

fluorescence intensity profile to distinguish target concentration, and the length and base-pair

mismatches of target sequences. Numerical simulations were conducted for the system, and

fit well with the experimental data.

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In a second approach, a solid-phase nucleic acid assay was developed using

immobilized Quantum Dot (QD) bioprobes. Hybridization was used to immobilize QDs that

had been coated with oligonucleotides having two different sequences. The hybridization of

one oligonucleotide sequence conjugated to a QD (a linker sequence) with a complementary

sequence that was covalently attached to a glass substrate of a microfluidic channel was

shown to be an immobilization strategy that offered flexibility in assay design, with intrinsic

potential for quantitative replacement of the sensing chemistry by control of stringency. A

second oligonucleotide sequence conjugated to the immobilized QDs provided for the

selective detection of target nucleic acids. The microfluidic environment offered the ability to

manipulate flow conditions for control of stringency and increasing the speed of analytical

signal by introduction of convective delivery of target sequences to the immobilized QDs.

This work introduces a stable and adaptable immobilization strategy that facilitates solid-

phase QD-bioprobe assays in microfluidic platforms.

iv

Acknowledgments

I would like to thank my supervisor, Prof. Ulrich J. Krull, for giving me the

opportunity to pursue my study with him and the insightful guidance he has given me

throughout my graduate studies. I am especially grateful for his unwavering support and

encouragement from the beginning and especially in the end. Also, I want to thank Prof.

Aaron Wheeler and Prof Julie Audet for being in my advisory committee and giving helpful

comments over the years.

I also want to thank Prof. Claudiu Gradinaru and Mr. Amir Mazouchi at the Chemical

and Physical Science (CPS), University of Toronto at Mississauga, for collecting FCS data

for diffusion coefficient estimation. I would also like to thank Prof. David R. McMillen at the

CPS, University of Toronto at Mississauga, for giving me fluorescence microscope use time.

I would like to give special thanks to Dr. Henry Lee and Mr. Yimin Zhou at Emerging

Communications Technology Institute (ECTI), University of Toronto, for giving me clean

room training. My thanks also go to Dr. Sergei Musikhin for providing expertise in laser

alignment.

I would also like to thank my colleagues, Taufik Al-Sarraj, W. Russ Algar, Andrew

Chan, Yevgenia Kratvsova, Dr. Larry Liu, Melissa Massey, Omair Noor, Eleonora

Petryayeva, Anthony J. Tavares, and Lim Ying for the discussion about research and their

friendship. I would like to give special thanks to colleagues who I have worked closely;

specifically Dr. Yali Gao for her help with numerical simulations, W. Russ Algar and

Anthony J. Tavares for their help with work associated with QDs. I am grateful for the extra

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help and great ideas provided by former summer students: Didi Cheung, Omair Noor and

Solongo Wilson. I must thank the administrative staff, Carmen Bryson and Anna Liza

Villavelez, for offering great help when I need it the most.

I would like to thank my parents for their unconditional love and support over the

years. Lastly, I wish to thank my wife, Iris Zhou, and my Son, Ian Chen, for their invaluable

support. I shall forever be in debted to them.

vi

Overview of Author Contributions

Throughout the projects presented here, I was aided by various members of the

Chemical Sensors Group and other colleagues. Here I outline the contributions that each

person has made towards the work presented herein.

Chapter 4.2 outlines the simulations of surface hybridization kinetics and

electrokinetic transport using a finite element method. I was fortunate to work with Dr. Yali

Gao on this project. The geometry of the channel used for simulation was based on the

microfluidic chips I used in experiments described in Chapter 4.1. I defined the parameters

used for simulations, either from experimental results or literature values. Dr. Gao entered the

parameters and boundary conditions into the COMSOL software. Together we simulated the

processes using the software. I performed all the subsequent computational analyses, and

evaluated the fit of the models with the experimental results.

Chapter 4.3 outlines a regenerable solid-phase nucleic acid assay in an

electrokinetically driven microfluidic platform using immobilized QD-bioprobes. This work

was performed with W. Russ Algar and Anthony J. Tavares, graduate students in CSG group.

Together, Algar and I performed the QD surface modification and preparation of

oligonucleotide-QD conjugates. Tavares collected the spectral overlap and quantum yield

data, which served as background data for characterization of the materials. All other work

was performed by me.

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Table of Contents Acknowledgments.......................................................................................................................... iv

Overview of Author Contributions ................................................................................................ vi

Table of Contents.......................................................................................................................... vii

Symbols and Abbreviations ........................................................................................................... xi

List of Tables ............................................................................................................................... xvi

List of Figures ............................................................................................................................. xvii

List of Equations ......................................................................................................................... xxii

1 Introduction ................................................................................................................................ 1

1.1 Properties of nucleic acids .................................................................................................. 1

1.1.1 Energetic considerations of nucleic acid hybridization .......................................... 1

1.1.2 Denaturation of nucleic acid hybrids ...................................................................... 2

1.1.3 Effect of pH and ionic strength on stability of DNA hybridization........................ 3

1.1.4 Determination of DNA sequences .......................................................................... 4

1.1.5 Nucleic acid probes................................................................................................. 5

1.1.6 Nucleic acid hybridization assay techniques .......................................................... 6

1.1.7 Transducers for DNA detection .............................................................................. 8

1.2 Microfluidics for nucleic acid hybridization assay........................................................... 10

1.2.1 Introduction of microfluidics ................................................................................ 10

1.2.1.1 Electrophoresis ....................................................................................... 11

1.2.1.2 Electroosmotic flow (EOF) .................................................................... 12

1.2.1.3 Controlling of EOF and DNA transport ................................................. 14

1.2.2 Microfluidic system for DNA analysis ................................................................. 15

1.2.3 Electrokinetically driven microfluidic system...................................................... 17

1.2.3.1 Advantages of electrokinetically driven microfluidic system ................ 17

viii

1.2.3.2 Joule heating........................................................................................... 18

1.2.4 Microfluidic device fabrication............................................................................. 20

1.2.5 PDMS, plasma treatment and its aging effects ..................................................... 21

1.2.6 Kinetic model of nucleic acid surface hybridization and its relation to convective flow..................................................................................................... 22

1.2.6.1 Factors affecting surface hybridization kinetics and equilibrium .......... 22

1.2.6.2 Kinetic models of nucleic acid surface hybridization ............................ 24

1.2.6.3 Models coupled with convective flow.................................................... 27

1.3 Using Quantum Dots as donors in FRET for nucleic acid hybridization assay................ 28

1.3.1 Introduction........................................................................................................... 28

1.3.2 Quantum dots ........................................................................................................ 29

1.3.3 Surface modification of quantum dots for bioanalytical applications .................. 33

1.3.4 Quantum dots for FRET-based bioanalytical applications ................................... 36

1.3.5 Solid-phase assays using immobilized quantum dots as donors in FRET, and potential for use in microfluidic systems ....................................................... 38

2 Purpose of this thesis................................................................................................................ 40

3 Experimental ............................................................................................................................ 43

3.1 Materials ........................................................................................................................... 43

3.2 Instrumentation ................................................................................................................. 45

3.3 Procedures......................................................................................................................... 48

3.3.1 Preparation of glass substrates .............................................................................. 48

3.3.2 Functionalization of glass substrates with GOPS ................................................. 48

3.3.3 Surface immobilization of probe oligonucleotides ............................................... 49

3.3.4 Details of microfabrication and chip assembly process........................................ 51

3.3.5 EOF mobility measurements................................................................................. 56

3.3.6 DNA hybridization detection protocols ................................................................ 57

ix

3.3.7 Quantum dot surface modification and preparation of QDs conjugated with two different oligonucleotide sequences............................................................... 58

3.3.8 QD characterization, determining quantum yields and Förster distance .............. 61

3.3.9 Immobilization of QDs in channels ...................................................................... 63

3.3.10 Regeneration of immobilized QDs in channels .................................................... 64

4 Results and discussion.............................................................................................................. 65

4.1 Quantitative DNA hybridization assay in an electrokinetically controlled microfluidic chip............................................................................................................... 65

4.1.1 Introduction........................................................................................................... 65

4.1.2 Chip design ........................................................................................................... 67

4.1.3 Buffer solution selection ....................................................................................... 69

4.1.4 Electrokinetically controlled sample loading, delivery and washing ................... 73

4.1.5 Quantitative DNA hybridization in electrokinetically driven microfluidic chip........................................................................................................................ 77

4.1.6 Quantitative DNA analysis using DNA hybridization.......................................... 80

4.1.7 Hybridization signal profiles for distinguishing different target sequences ......... 83

4.1.8 Summary ............................................................................................................... 86

4.2 Simulations of surface hybridization kinetics and electrokinetic transport ...................... 87

4.2.1 Introduction........................................................................................................... 87

4.2.2 Mathematical model and numerical method......................................................... 88

4.2.3 Simulation results for quantitative extraction of DNA samples by surface hybridization in an electrokinetically driven microfluidic channel ...................... 94

4.2.4 Axial fluorescence intensity profile from hybridization for determination of target concentration .......................................................................................... 95

4.2.5 Comparison between simulation and experimental results................................. 100

4.2.6 Summary ............................................................................................................. 101

4.3 Use of QDs within microfluidic channels for solid-phase nucleic acid hybridization assay.......................................................................................................... 102

4.3.1 Introduction......................................................................................................... 102

x

4.3.2 Characterization of QDs and oligonucleotide-QD conjugates............................ 104

4.3.3 Confirmation of hybridization of oligonucleotide-QD conjugates in bulk solution................................................................................................................ 106

4.3.4 Immobilization and stability of QDs in microfluidic channels........................... 109

4.3.5 Nucleic acid hybridization assay using immobilized QDs ................................. 112

4.3.6 Removing and re-coating modified QDs within channels.................................. 116

4.3.7 Summary ............................................................................................................. 120

5 Future directions..................................................................................................................... 122

References................................................................................................................................... 124

xi

Symbols and Abbreviations A Adenine or acceptor

Across Cross sectional area

µe Electrophoretic mobility

µeo Electroosmotic mobility

Abs. Absorption

C Cytosine or concentration

COC Cycloolefin copolymer

D Donor or detection or diffusion coefficient

Da Damköhler number

DHLA Dihydrolipoic acid

DIPEA "N,N-diisopropylethylamine "

E Intensity of the electrical field or energy tranfer efficiency

E. coli Escherichia coli

EB Ethidium bromide

Em. Emission

EOF Electroosmotic flow

F Fluorescence intensity

FC Fully complementary

FCS Fluorescence correlation spectroscopy

xii

FISH Fluorescence in situ hybridization

FRET Fluorescence Resonance Energy Transfer

G Guanine

GOPS 3-glycidoxypropyltrimethoxysilane

h Characteristic length

H Concentration of DNA target-probe hybrids

IPA Isopropyl alcohol

J Spectral overlap integral

Keq Equilibrium constant

knr Rate of non-radiative decay

koff Dissociation rate constant

kon Association rate constant

kr Rate of radiative decay

L Linker

l Channel length

LIF Laser-induced fluorescence

LOD Limit-of-detection

MAA Mercaptoacetic acid

MPA Mercaptopropionic acid

MSA Mercaptosuccinic acid

xiii

n Refractive index

NA Numerical aperture

NC Non-complementary

P Concentration of probe

P0 Initial probe density

PC Polycarbonate

PCR Polymerase Chain Reaction

PDMS Polydimethylsiloxane

Pe Peclet number

PE Polyethylene

PEG Polyethylene glycol

PETG Polyethylenetetraphthalate glycol

PL Photoluminescence

PMMA Polymethyl methacrylate

PMT Photomultiplier tube

PP Polypropylene

PS Polystyrene

PVC Polyvinylchloride

PVP Polyvinylpyrrolidone

QCM Quartz crystal microbalance

xiv

QD Quantum dot

r Radius or donor-acceptor distance

R0 Förster distance

RNA Ribonucleic acid

RT-PCR Real-time PCR

SAW Surface acoustic wave

SHOM Sequencing by hybridization to oligonucleotide microchip

SMA Spinal Muscular Atrophy

SMN Survival motor neuron

SMN1 A sequence of survival motor neuron 1 gene

SNP Single Nucleotide Polymorphism

SPR Surface plasmon resonance

T Thymine or target

TBMP 1 × TB + 30 mM MgSO4 + 0.1 % (w/v) PVP

TCEP Tris(2-carboxyethyl)phosphine

Tm Melting temperature

TOP Trioctyl phosphine

TOPO Trioctyl phosphine oxide

Tris Tris(hydroxymethyl)aminomethane

u Sample flow velocity

xv

UidA A sequence for diagnostic of E. coli.

ve Electrophoretic velocity

veo Electroosmotic velocity

vnet Net velocity

W Length of surface that is coated with immobilized probes

x Distance

X Distance between the inlet and the initial point on the surface that is

coated with immobilized probes

xeq Equilibrium fraction

z Electrical charges on a species

ε Molar absorptivity

η Viscosity

κ2 Orientation factor

λ Wavelength of light or bulk conductivity

μTAS Micro total analysis systems

τ Time

Φ Quantum yield

xvi

List of Tables Table 3.1 Oligonucleotide Sequencesa used in the work of quantitative hybridization assay 44

Table 3.2 Voltage Program for sample loading and subsequent hybridizationa .....................58

Table 3.3 Oligonucleotide Sequencesa used for the experiments with QDs ...........................60

Table 4.1 Comparison of a static solid phase assay using modified QDs and the solid phase

assay done within microfluidic channels. ..............................................................................116

xvii

List of Figures Figure 1.1 Schematic picture of the electrical double layer which is composed of a compact

layer and a diffuse layer. (adopted from ref. [54])...................................................................13

Figure 1.2 (A) Electronic energy states of a semiconductor in the transition from discrete

molecules to nanosized crystals and bulk crystals. Blue denotes ground state electron

occupation. (B) Structure of a colloidal quantum dot. (C) Absorption (upper) and

fluorescence (lower) spectra of CdSe semiconductor. (Adopted and modified from ref. [103])

..................................................................................................................................................31

Figure 1.3 Modification with thioalkyl acid ligand to make QD soluble in aqueous solution:

a) mercaptoacetic acid (MAA); b) mercaptosuccinic acid (MSA); and c) dihydrolipoic acid

(DHLA). The ionization state of the ligand is determined by the pH of the solution. In this

diagram, only the acidic hydrogen atoms are shown. (adopted from ref. [107]).....................35

Figure 3.1 Schematic representation of the epi-fluorescence microscope setup. ....................47

Figure 3.2 Glass surface functionalization reaction. The silanol groups on the glass can react

with GOPS molecules. After the reaction, the surface was modified with GOPS which has an

epoxy group for further linkage to biomolecules.....................................................................49

Figure 3.3 Oligonucleotides surface immobilization reaction. Immobilization solution was

deposited onto the GOPS modified substrate and reacted with the epoxy group. Through a

ring opening reaction, the amine end group on the oligonucleotides formed a covalent bond

with the GOPS and immobilized on the substrate surface.......................................................51

Figure 3.4 (a) Overview of the structure of the microfluidic chip: the assembly of PDMS

cover with microfluidic channel structure and substrate with immobilized probe sites. (b) H

shaped microfluidic channel structure for DNA hybridization: four reservoirs were labeled

with numbers that were used to identify points of applied electrical potential in Table 3.2. ..52

Figure 3.5 The process for making a negative PDMS cast. (a) transparent photomask with

microchannel structure was printed in local printing shop. (b) Su-8 5 negative photoresist was

spin-coated on a glass slide. (c) UV exposure when photomask was in close contact with the

xviii

Su-8 layer to create desired microchannel pattern. (d) creation of positive master with

crosslinked Su-8 photoresist. (e) liquid PDMS mixture casted onto the positive master and

cured. (f) cured negative PDMS cast after it was peeled from the positive master. ................54

Figure 4.1 A schematic of the H shaped chip which has one 30-mm-long center channel and

two 16-mm-long sidearms connecting four ports. All the channels are 250 µm wide and 9 µm

high. The chip layout is not to scale. .......................................................................................69

Figure 4.2 Comparison of different buffer solutions in terms of the conductivity and the

ability to support DNA hybridization (as determined by fluorescence intensity). Normalized

hybridization signal intensity acquired using standard microarray hybridization experiments

and normalized conductivity of the buffer solutions are compared. The results of 0.5 × SSC +

100 mM phosphate hybridization buffer was used as the standard for normalization. The error

bars represent the standard deviations of five replicates. ........................................................72

Figure 4.3 Typical current monitoring results of several cycles of buffer displacement. The

EOF mobility can be derived from the slopes indicated in the graph......................................75

Figure 4.4 Sample loading, and sample delivery and washing stages for the electrokinetically

driven microfluidic chip...........................................................................................................76

Figure 4.5 Electrokinetically controlled quantitative DNA hybridization in microfluidic

channel (1 µM targets are moving from left side to right side). Six fluorescence signal

intensity line scans were acquired at different times from the target loading, which were at

150, 180, 210, 240, 300 and 600 seconds respectively. No targets can be observed

downstream of region with immobilized probes (even after 600 s). .......................................79

Figure 4.6 Integrated fluorescence signal as a function of DNA target concentration (both in

log scale). The error bars represent the standard deviations of three replicates. .....................81

Figure 4.7 Normalized integrated fluorescence intensity as a function of the fractional

composition of complementary targets; oligonucleotide mixtures contained Cy5 labelled 19-

mer complementary target (SMN1-T19) and non-labelled 20-mer non-complementary strands

(NC) with total concentration of 1 μM. The error bars represent the standard deviations of

three replicates. ........................................................................................................................82

xix

Figure 4.8 Nomalized hybridization signal profile for targets with different length (19, 34 and

49 base pair target sequences)..................................................................................................84

Figure 4.9 Hybridization signal profile of fully complementary targets (SMN1-T19) and 3

base-pair mismatch sequences (SMN1-3BPM). The error bars represent the standard

deviations of at least two replicates. ........................................................................................86

Figure 4.10 Schematic geometry (2-D) of the microfluidic channel for DNA hybridization

used in the model (not to scale). DNA sample entered the microfluidic channel from the left

inlet and travelled through the channel with a plug-like profile. Probes were immobilized on

one channel wall in the centre of the construct. l and h are the length and the height of the

channel. X represents the distance between the inlet and the initial point on the surface where

immobilized probe molecules were present. W is the length of surface that is coated with

immobilized probes. In the simulation, the geometric parameters were: l = 1.7 mm, h = 9 µm,

X = 0.1 mm, and W = 1.0 mm. .................................................................................................92

Figure 4.11 Transient concentration distribution in cross-section showing the channel height

at position 1.5 mm, which is at the end of the portion of the channel surface that was coated

with immobilized probes..........................................................................................................96

Figure 4.12 The hybridization signal intensity profile along the channel at washing time of 45

s after delivering a 60 s plug of 100 nM oligonucleotide target. .............................................98

Figure 4.13 Hybridization signal intensity profiles of different concentrations of

oligonucleotide targets using a 30 s injection followed by 20 s of washing with buffer. Seven

profiles represented targets of 1, 10, 100, 200, 300, 500 nM and 1 µM concentration,

respectively. ...........................................................................................................................100

Figure 4.14 Comparison of hybridization signal intensity profile between experimental and

simulation results for the condition of 60 s injection of 100 nM oligonucleotide target in the

microfluidic channel. The error bars represent the standard deviations of three replicates. .102

Figure 4.15 The normalized absorption (Abs.) and emission (Em.) spectra of green emitting

QDs and Cy3 and the spectral overlap (shaded area) for the FRET pair...............................106

xx

Figure 4.16 Two different oligonucleotide sequences conjugated with each QD hybridize

with respective complementary targets in bulk solution. (a) QD conjugated with two different

oligonucleotides (LT and DP) and hybridization with complementary target sequences. The

hybridization was detected by FRET-sensitized Cy3 emission. (b) PL of DNA conjugated

QDs that hybridized with a stoichiometrically equivalent quantity of Cy3-LP2 in TB buffer:

(i) 0.1 µM QD―(1 × LT2, 1 × DP2), (ii) 0.1 µM QD―(2 × LT2, 2 × DP2), (iii) 0.1 µM

QD―(1 × LT2, 1 × DP2) hybridized with 0.1 µM Cy3-LP2, and (iv) 0.1 µM QD―(2 × LT2,

2 × DP2) hybridized with 0.2 µM Cy3-LP2. (c) PL of DNA conjugated QDs hybridized with

a stoichiometrically equivalent quantity of DT2 in TB buffer: (i) 0.1 µM QD―(1 × LT2, 1 ×

DP2), (ii) 0.1 µM QD―(2 × LT2, 2 × DP2), (iii) 0.1 µM QD―(1 × LT2, 1 × DP2)

hybridized with 0.1 µM DT2, and (iv) 0.1 µM QD―(2 × LT2, 2 × DP2) hybridized with 0.2

µM DT2. All PL spectra were background subtracted and normalized. ...............................108

Figure 4.17 Microfluidic chip showing pads of QDs. The inset picture depicts immobilization

of QDs in the channel by hybridization with complementary probes on the surface, while

leaving other oligonucleotides available for binding with a target sequence in solution. .....110

Figure 4.18 Images based on QD fluorescence intensities (all images were background

subtracted): (a) after QDs were injected into the channel. (b) after buffer washing at 125

V/cm. (c) after buffer washing at 250 V/cm. (d) after buffer washing at 375 V/cm. (e) after

buffer washing at 500 V/cm. (f) after buffer washing at 625 V/cm. (g) after buffer washing at

750 V/cm. (h) after re-injection of QDs, followed by buffer washing at 125 V/cm (as for (a)).

All steps were 10 minutes in duration....................................................................................113

Figure 4.19 Immobilization of modified QDs and detection of Cy3 labelled targets using

FRET-sensitized Cy3 emission. (a) Fluorescence image of immobilized QDs using

fluorescence emission from QDs (background subtracted). (b) FRET-sensitized Cy3 emission

using Cy3 channel (FRET background). (c) FRET-sensitized Cy3 emission after the addition

of 100 nM Cy3-NC. (d) FRET-sensitized Cy3 emission after the addition of 100 nM DT1. (e)

100 nM non-complementary and fully complementary sequences FRET-sensitized Cy3

emission signal profile along the channel (FRET signal profiles of (c) and (d) with

background subtracted). .........................................................................................................115

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Figure 4.20 Fluorescence images of cycles of QD immobilization, and demonstration of

nucleic acid assay. (a) image of immobilized QDs conjugates based on fluorescence emission

from the QDs (background subtracted). (b) subsequent image collected after washing using

TBMP buffer with 40% v/v formamide. (c) image after a second injection of QD conjugates

using the fluorescence emission from the QDs (background subtracted). (d) image using the

Cy3 emission channel (FRET background). (e) FRET sensitized Cy3 emission image after

injection of 100 nM DT1 (fully complementary target FRET signal)...................................118

Figure 4.21 Idealized solution melting curves for three different DNA hybrids with melting

temperature of (i) 40 ˚C, (ii) 45 ˚C and (iii) 60 ˚C ................................................................119

Figure 4.22 The reproducibility of the immobilization of QD-oligonucleotide conjugates over

several cycles. (a) using QD―(2 × LT1, 2 × DP1). (b) using QD―(2 × LT2, 2 × DP2). ....120

xxii

List of Equations (1.1) Relationship between diffusion coefficient and time........................................................7

(1.2) Electrophoretic volocity expression ................................................................................11

(1.3) Factors determine electrophoretic mobility.....................................................................11

(1.4) Electroosmotic volocity expression ................................................................................14

(1.5) Net volocity expression...................................................................................................14

(1.6) DNA movement net velocity...........................................................................................15

(1.7) Langmuir isotherm for surface hybridization..................................................................26

(1.8) Damköhler number expression........................................................................................27

(1.9) Quantum yield described by radiative and non-radiative processes ...............................32

(1.10) FRET energy efficiency ................................................................................................36

(3.1) Electoomotic velocity......................................................................................................56

(3.2) Förster distance ......................................................................................62

(3.3) Quantum yield determined by relative method ...............................................................62

(3.4) Modified Quantum yield expression ...............................................................................62

(3.5) Spectral overlap ......................................................................................................62

(4.1) Surface hybridization reation ..........................................................................................90

(4.2) Mass balance on surface with hybridization reaction .....................................................90

(4.3) Modified mass balance on surface ..................................................................................90

(4.4) Covective delivery equation............................................................................................91

1

1

1 Introduction

1.1 Properties of nucleic acids

1.1.1 Energetic considerations of nucleic acid hybridization

The stability of the DNA double helix is very important for the fidelity of the DNA

replication. The proofreading ability of the enzymes involved in the DNA replication process

adds another layer of protection for the highly conservative process of DNA replication.

There are numerous forces that contribute to the stability of the double-stranded nucleic acid

structure. Among them, the most significant force for the stabilization of the double helix is

the hydrogen bonds between complementary Watson-Crick base pairs. Considering the DNA

hybridization process in aqueous solution, there is no significant gain or loss for number of

hydrogen bonds. However, DNA hybridization is a favorable process with an enthalpy

change of -9.8 kcal/mol per base pair. This is due to the fact that the hydrogen bonds formed

between complementary base pairs have a six-membered resonance stabilized ring structure

and inductively stabilized internal dipoles of DNA nucleotides [1]. London dispersion forces

and van der Waals forces are also important to the stability of the secondary structure of a

DNA double helix. The interactions of the π electrons of the aromatic purines and

pyrimidines, which is commonly referred to as “stacking interactions”, provide an average

stabilization energy of -8.1 kcal/mol per base pair dimer. An entropic gain is also realized

during the DNA hybridization process, which is the largest contributor to the overall negative

free energy change for the DNA hybridization process. This change in entropy is acquired

from the release of the loosely bound water molecules back to aqueous solution upon DNA

hybridization. The average number of water molecules associated with each base pair is

between 18 and 28. Upon DNA hybridization, an average of five tightly bound and many

2

2

more loosely bound water molecules, associated with the single-stranded DNA, are released

back into the bulk solution per base pair formation. There are also forces which are

unfavorable to DNA hybridization. The most significant of these is electrostatic repulsion

between the polyanionic phosphate backbones. The phosphate backbones of DNA are usually

negatively charged in solution when in biological conditions. Such electrostatic repulsion can

be reduced by the screening effect of ions in solution. So, the stability of the DNA double

helix depends on the ionic strength of the solution.

1.1.2 Denaturation of nucleic acid hybrids

The DNA double helix can also be destabilized by factors which can affect the

attractive and repulsive forces, including sequence composition, solution composition, ionic

strength, pH and temperature. The process of double-stranded DNA separating into two

single-stranded DNA is called DNA denaturation or DNA melting. If DNA denaturation is

due to temperature change, there is a temperature at which 50% of the population of double-

stranded DNA hybrids is separated into single-stranded DNA. This temperature is called

melting temperature, Tm. Melting temperature is a function of DNA length, sequence

composition, presence of mismatches and environment. Each extra base pair in a short

oligonucleotide (up to 14 base pair) will increase melting temperature of DNA by 2-4

degrees, depending on whether the interactions are A-T or G-C pairs [2]. This is because

breaking G-C pairs requires more energy since there are more hydrogen bonds between G-C

pair than for A-T pair. DNA melting temperature can be quantitatively predicted based on

interactions of nucleotides and knowledge of environmental conditions [3]. Single-stranded

DNA with mismatches has a lower thermodynamic stability in comparison to a fully

3

3

complementary sequence since there are less hydrogen bonds and stacking interactions to

break. Under appropriate conditions of temperature, such thermodynamic stability

differences can be used to discriminate a fully complementary sequence from a sequence

with a single mismatch, which is referred as single nucleotide polymorphism (SNP).

Chaotropic agents such as formamide and urea can promote denaturation of double-stranded

DNA since they can form inductively and resonance stabilized hydrogen bond structures

similar to that formed between complementary DNA base pairs. It is known that the

denaturing agent formamide lowers DNA melting temperatures linearly by 0.6 ˚C per volume

fraction percentage of formamide in the buffer with formamide volume fraction up to 40%

[4].

1.1.3 Effect of pH and ionic strength on stability of DNA hybridization

The electrostatic repulsion between the two negatively charged phosphate backbones

in DNA duplex can reduce the stability of the DNA double helix and even separate the DNA

into two single-stranded DNA. Ions in the solution can reduce this electrostatic repulsion

force by reducing the effective charges on the phosphate backbone through charge screening.

The higher the ionic strength of the solution, the smaller the electrostatic repulsion force

between two phosphate backbones. The stability of the DNA duplex (indicated by Tm)

increases with solution ionic strength. A linear relationship is observed between the

logarithm of salt concentration and Tm for salt concentration below 1 M.

The various ionizable states of the nucleobases can influence the structure of double-

stranded DNA. The degree of protonation of nucleobase influences hydrogen bond

4

4

formation, which is crucial for the stability of a DNA duplex. Ionization of nucleobases tends

to be constant in the pH range between 5 and 9, and DNA duplexes are stable at pH 5-9.

When solution pH is out of this range, the DNA duplex can become destabilized due to the

disruption of some inter-strand hydrogen bonding.

1.1.4 Determination of DNA sequences

Food and water contamination by pathogenic microorganisms continues to pose a

major threat with examples that include the tragedy in Walkerton, Ontario caused by drinking

water contamination with Escherichia coli (E. coli) bacteria, and the extensive recall of foods

by Maple Leaf Foods because of Listeria bacterial contamination [5, 6]. Both incidents

suggest the need for rapid and accurate detection and identification methods for pathogens.

Genetic testing is a potential universal method which one can perform for all organisms. The

invention of the polymerase chain reaction (PCR) technique makes it possible to acquire

enough DNA for testing from one or several copies of a piece of DNA or RNA [7-9]. PCR is

widely used for the detection of pathogens in foods. Real-time PCR (RT-PCR) has been

established as the choice for simultaneous amplification and quantitative determination of

nucleic acid targets.

Genetic testing has distinctive advantages over culturing methods for the detection of

pathogenic bacteria and offers the advantages of sensitive, specific, accurate and rapid

detection of target nucleic acids in a sample. Aside from pathogen detection, genetic tests can

also aid in the identification of whether an individual has a genetic disorder or is a carrier of a

genetic disorder, and can provide insight about predisposition to certain diseases. For both

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pathogens and genetic screening, the testing methods need to be rapid, simple, reliable,

sensitive and specific. Ideally, the methods will also allow for real time monitoring at a low

cost. This combination of attributes represents the holy grail of the genetic testing.

1.1.5 Nucleic acid probes

Nucleic acid probes are usually single-stranded sequences which can hybridize with a

single-stranded target sequence that is unique to certain species [10]. Targets for such probes

are many. Numerous research projects have focused on identification of unique sequences in

one or a few gene regions for species identification, which is now called DNA barcoding

[11]. The effort of establishing a universal DNA barcode library containing details about all

organisms is underway.

The nucleic acid probes have lengths ranging from 10 to 10,000 nucleotide bases. The

most common probe length is between 10 and 30 bases [10, 12]. A nucleic acid probe with a

certain minimum length is required in terms of statistical uniqueness to identify species using

hybridization. On the basis of probability, this length is determined to be 17 nucleotide bases

for identification of a unique sequence in the human genome, while 12 nucleotide bases are

required for E. coli. The use of the shorter nucleic acid probes offers advantages in terms of

speed and selectivity. Long nucleic acid probes require longer reaction times to equilibrate

with analyte because of reduced diffusion rates of a target and slower hybridization. For

example, nucleic acid probes with length of several hundred bases usually require

hybridization time of many hours, while short probes (10-15mer) could equilibrate in tens of

minutes [13]. Increased probe length provides for increased probability of forming a stable

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hybrid with a mismatched sequence, reducing the potential for selectivity. This is due to the

fact that the nucleic acid duplex stability increases with its length, and a mismatch

contributes less to the total energy as the hybrid becomes longer.

1.1.6 Nucleic acid hybridization assay techniques

Nucleic acid hybridization assays are based on the ability of single-stranded nucleic

acids to selectively hybridize with complementary strands. Although the basic underlying

principle of operation is the same, there are many formats in which the hybridization can

operate. Different formats include operation in the solid phase or liquid phase, use of labelled

and unlabelled reagents, application of separation steps and direct or indirect methods of

detection.

Microarray technology uses thousands of ordered DNA spots in an array format for

nucleic acid analysis. Each spot contains one particular probe sequence that is selective for

identification of a particular target. Hybridization is typically transduced by use of

fluorescence markers, and spatial registration allows identification of the specific target.

Microarray technology enables large-scale and parallel analysis of targets simultaneously

[14-16].

DNA microarrays usually require a long incubation time of several hours or even

days. The DNA microarray is typically expected to reach equilibrium before signal

acquisition takes place. This approach does not offer real-time signal acquisition and the

kinetic information which can be used for quantitative analysis is lost. Only recently has the

possibility of real-time DNA microarrays been explored [17]. However, new detection

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chemistries and analysis algorithms still need to be developed to make real-time microarrays

a viable solution for DNA analysis.

DNA microarrays are not well suited for small scale fast point-of-care applications

because of their complicated protocols and instrumentation, and diffusion limited reaction

kinetics [18]. The common DNA microarray technologies are slow since they are based on

passive DNA hybridization which relies solely on the diffusion of the target oligonucleotides

to the probes on a solid surface. Such passive DNA hybridization processes may take many

hours to complete based on typical diffusion coefficients of target oligonucleotides. For a

target oligonucleotide transported only by diffusion, the relation between the travel time, τ,

and the distance, x, is given by:

Dx2

2

=τ (1.1)

This equation describes the movement of a target molecule under diffusion, which is

independent of the concentration gradient. The concentration gradient only determines the

rate of mass transfer. For a typical 19 base pair oligonucleotide with diffusion coefficient of

9.5 × 10-7 cm2 s-1, it takes more than 1.5 hours to travel a distance of only 1 mm. The

dependence on diffusion of target oligonucleotides for hybridization necessitates the use of

relatively large amounts of sample and long hybridization time to achieve reproducible and

reliable hybridization signals. For this reason, a number of strategies for mixing and

alternative transport have been proposed to improve the speed and sensitivity of DNA

detection [19-24].

Moreover, probes of different sequence length and base composition intrinsically

have various melting temperatures [25]. From spot to spot the kinetics and selectivity of

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DNA hybridization will vary according to specific set of environmental conditions. It is

therefore extremely challenging to optimize hybridization conditions at all spots for

mismatch discrimination, and it is virtually impossible to engineer the chemistry of the

system so that one environmental condition concurrently optimizes selectivity at all spots.

1.1.7 Transducers for DNA detection

Transducers play a fundamental role in DNA biosensors and assays. Detection can be

categorized based on the transduction method. The most common transduction methods are

optical, electrochemical and mass based.

Electrochemical transducers have several advantages such as low cost, low power

requirements, simple instrumentation and are relatively easy to miniaturize [26-34]. Mass

based transducers including quartz crystal microbalance (QCM) and surface acoustic wave

(SAW) devices offer label-free methods and have the ability to carry out both steady state

and real time measurements [35-37]. However, they are prone to false positive results due to

non-specific adsorption. For more detailed discussion of transduction using electrochemical

and mass based methods, readers are referred to articles in references [33, 34, 38-43][40].

The most common optical methods are surface plasmon resonance (SPR) and

fluorescence. SPR experiments typically measure the change of the angle of the reflected

light as a function of change of optical mass at the interface. It is considered a label-free

detection method. The ability to carry out both steady state and real time measurements is

advantageous. However, SPR is prone to have false positive results due to non-specific

adsorption [44, 45]. In addition, the mass transport limitation and the heterogeneity of the

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surface probe spots are common issues that contribute to limiting the accuracy of surface

binding kinetics derived from SPR experiments for determining biomolecular affinity and

kinetic rate constants [46, 47].

Fluorescence based assays have many advantages including high sensitivity and

ability to multiplex using multiple fluorophores. Many DNA assay methods and detection

platforms use fluorescence intensity as the analytical signal, with examples being

fluorescence in situ hybridization (FISH), fiber optic-based biosensors and nucleic acid

microarrays. A variety of fluorescence detection schemes can be used for signal generation

using direct fluorescence measurements through optical fibers and waveguides or by using

evanescent wave methods [48]. For fluorescence detection, usually either nucleic acid probes

or targets are labelled directly with fluorophores (such as cyanine dyes used in this thesis)

and increased fluorescence intensity can be detected after hybridization events. Fluorescence

detection can also be achieved by using intercalating dye such as ethidium bromide (EB) and

picogreen. These intercalating dyes will experience a substantial increase in quantum yield

while intercalated into the nucleobase stacking region of DNA duplexes. The use of an

intercalating dye can avoid the requirement of a labeling step. However, this approach

introduces background signal into an experiment due to the presence of free dyes, and a weak

interaction the dyes with single-stranded DNA, resulting in reduction of performance in the

area of limit-of-detection (LOD). Many nucleic acid assay methods such as Taqman® and

molecular beacon techniques are also fluorescence transduction methods based on

fluorescence resonance energy transfer (FRET). A detailed introduction of FRET is in section

1.3.4. There are also many other fluorescence-based nucleic acid detection methods, and a

detailed review has been published [49]. Because of the great sensitivity offered by

fluorescence methods, they are the techniques used in this thesis. Cy5 dye, working as a

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direct label, was used in the study of DNA hybridization kinetics. Green quantum dots and

Cy3 dye were used as a FRET pair for the quantum dot study.

1.2 Microfluidics for nucleic acid hybridization assay

1.2.1 Introduction of microfluidics

Today, “Microfluidics is the science and technology of systems that process or

manipulate small (10-9 to 10-18 liters) amount of liquids, using channels with dimensions of

tens to hundreds of micrometers.” [50]. The study of microfluidic systems is also referred to

as lab-on-a-chip or micro total analysis systems (μTAS) which is a fast growing area in

modern analytical science. Microfluidic systems offer many important capabilities including:

portability; speed; high resolution; sensitivity; small quantities of samples and reagents; and

low fabrication cost. Because of these capabilities, areas such as genomic [51, 52], proteomic

[53], clinical and forensic analysis are all developing methods to take advantage of

microfluidic systems. The fluidic flow control can be achieved in various ways such as

pressure differential, electrical potential and centrifugal force. Fluidic flow control by

electroosmotic flow (EOF) is a widely used method. Although the performance of EOF is

often not satisfactory in a complex microfluidic system, its ease of use by just applying

different voltages without any pumps and valves makes EOF a rational choice for the work in

this thesis. It is noteworthy that the similarities of the dimension of the microfluidic channel

and capillaries used in electrophoresis suggest that many of the development in theory that

are applicable to capillaries can also be applied to microfluidic systems.

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1.2.1.1 Electrophoresis

Charged species such as DNA and quantum dots will move through aqueous solution

under the influence of an electrical field. This motion caused by electrical field is called

electrophoresis. Depending on the charges of the species, positively charged species will

move following the direction of the electrical field while negatively charged species will

move countering the direction of the electrical field. During electrophoresis, charged species

experience two forces: one is the electrostatic columbic force that arises from the applied

electrical field. The other is the frictional force created between charged species and the

surrounding aqueous solution that hinders movement. The two forces have same magnitude

but opposite direction in steady state, and the electrophoretic movement of charged species

will maintain at a constant velocity.

The velocity, ve, of the charged species is proportional to the magnitude of electrical

field E and electrophoretic mobility, µe:

ee Ev μ= (1.2)

The electrophoretic mobility µe is a function of charges on the species, z, viscosity of

the aqueous solution, η, and the radius of the sphere of hydration, r. The relation between

these parameters is shown in equation 1.3:

rz

e πημ

6= (1.3)

The electrophoretic mobility µe is proportional to charge density, z/r (charge/size), of

the species. Species with different charge density will have different electrophoretic mobility

and can be separated using electrophoresis, while species with similar charge density will

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have similar electrophoretic mobility and can not be separated using electrophoresis in

aqueous solution. DNA falls into the latter situation since DNA with different numbers of

bases have similar charge density. DNA with different lengths can not be separated

efficiently using only electrophoresis. Separation can be achieved using gels (such as

polyacrylamide gels) or sieving media which separates DNA with different lengths based on

size. Although, we did not separate different oligonucleotides in our experiments, the ability

of the gel electrophoresis which can separate DNA with different lengths based on their size

and mass to charge ratio offers us the potential opportunity to process complex real samples

before the step of analytical detection.

1.2.1.2 Electroosmotic flow (EOF)

The concept of electroosmotic flow (EOF) originally became important from its role

in capillary electrophoresis. In a capillary made by bare fused silica, the functional groups on

the inner wall of the capillary are silanol (SiOH) groups. In a buffer solution of neutral pH,

these silanol groups are deprotonated to exist as SiO-. The inner wall of the capillary is

therefore negatively charged. Cations in the buffer solution will move to the surface to form

an electrical double layer (as shown in Figure 1.1). The electrical double layer is composed

of a compact layer which tightly binds with the deprontonated silanol groups and a diffuse

layer which is loosely bound and mobile. Upon applying voltage across the length of the

capillary column, cations in the loosely bound diffuse layer move towards the cathode. Such

movement concurrently drags the bulk buffer solution in the capillary. This flow of solution

caused by the combination of surface charge, electrical double layer and electrical potential is

called electroosmotic flow (EOF).

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Figure 1.1 Schematic picture of the electrical double layer which is composed of a compact

layer and a diffuse layer. (adopted from ref. [54])

The channels in a microfluidic system have similar dimensions to those of a capillary,

and EOF can occur in microfluidic channels even if they are not cylindric as would be the

case in a capillary. The channels in the microfluidic system are usually fabricated using

techniques that originated in building microelectronic circuits. The solution displacement

using EOF in rectangular microchannels has also been simulated using mathematical models

[55]. EOF can even be observed in a microfluidic system completely fabricated from

polydimethylsiloxane (PDMS). This may seem anomalous because PDMS is a polymer with

hydrophobic properties. The functional groups on the PDMS surface are hydrophobic methyl

groups instead of ionizable groups. However, it has been suggested that ions in the buffer

solution may adsorb onto PDMS to form a charged surface which can support EOF [56].

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A unique feature of EOF is its flat velocity profile in capillary which is different from

the parabolic velocity profile of hydrodynamic flow. The flat velocity profile of EOF flow

provides the advantage of superior separation resolution in comparison to hydrodynamic flow

since it virtually has no band broadening caused by the velocity profile.

The EOF velocity veo is proportional to the magnitude of electrical field E and EOF

mobility µeo:

eoeo Ev μ= (1.4)

EOF mobility is also a function of buffer solution pH as this affects the surface charge

density.

1.2.1.3 Controlling of EOF and DNA transport

The EOF mobility is related to the surface charge density and the choice of buffer

solution. Control of the surface properties of the capillary/microfluidic channel by surface

modification is therefore an important element of design of microfluidic systems. Surface

modifications includes covalent surface derivatization [57-59], and surface dynamic coating

[60, 61] by including additives in running buffer solution.

In a capillary/microfluidic channel, the net velocity vnet of a charged species is the

vector sum of its electrophoretic velocity ve and the EOF velocity veo both of which are

proportional to the mobility, and the magnitude of the electrical field:

Evvv eoeeoenet )(→→→→→

+=+= μμ (1.5)

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Since DNA is negatively charged, it will be driven towards the anode by the

electrophoretic force when electrical potential is applied. The EOF in a glass-PDMS hybrid

microfluidic channel moves to the cathode when using a neutral pH buffer, which is opposite

to the DNA movement caused by the electrophoretic force. Therefore, the DNA transport

velocity can be represented by:

Ev eoenetDNA )( μμ −=− (1.6)

This suggests that the DNA transport velocity in a microfluidic channel can be controlled by

EOF mobility which can be changed using different surface modification methods.

1.2.2 Microfluidic system for DNA analysis

Aside from the shortcomings described earlier, DNA microarray has other

disadvantages including: (1) large amount of sample material is required for the analysis,

since large detection area need to be fully covered by the sample solution. (2) The linkage

chemistry of the probe to the substrate can undergo solution-dependant cleavage over the

extended incubation time, lowering the reproducibility and sensitivity of the assay. By using

microfluidic systems for DNA analysis, there are a number of advantages such as, reduction

in reagent costs (sample consumption reduced from more than hundreds of μL to several μL),

reduction in the hybridization assay time, and automation for the whole analysis. Many

researchers are using microfluidic systems to improve the speed and sensitivity of DNA

analysis.

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Microfluidic systems offer opportunity to detect DNA with high speed. Several

techniques have been used to enhance mass transport of targets and to reduce diffusion

distances to decrease the hybridization time. Continuous flow of targets by hydrodynamic

pumping within microfluidic channels has been used to improve DNA hybridization [19-22].

Flow of the target solution over the probe surface provides enhanced mass transport of targets

to surface-immobilized probes. In this case, the target transport does not solely depend on

diffusion but also on convection, which leads to reduction of hybridization time.

Introduction of DNA samples with high velocity can induce extensional strain on

targets, which will reduce hybridization time and increase hybridization efficiency [23, 24]. It

has been suggested that long DNA targets tend to change into a super coil form in solution,

which reduces hybridization efficiency due to the inaccessibility of the target sequence by

probes. However, extensional strain created by flow can help to uncoil long DNA targets,

which leads to improved hybridization efficiency. By using this method, a nearly nine-fold

increase of hybridization signal was acquired for a 1.4 kbp single-stranded DNA target.

Active mixing of samples can also be used to reduce hybridization time. The small

dimensions of the microfluidic channel set conditions for small Reynolds number and

resulting laminar flow. This means that the only mixing is by molecular diffusion. The

hybridization speed can be improved by active mixing because it can replenish the target

molecules in the proximity of probes which is depleted by hybridization. Yuen et al. used

fluidic circulation and mixing to improve hybridization reaction efficiency on DNA arrays

[62]. Vijayendran et al. evaluated a three-dimensional micromixer for the purpose of a

surface-based biosensor [63]. Liu et al. observed enhanced signals and fast nucleic acid

hybridization using chaotic mixing [64].

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The rapid detection of DNA target sequences by hybridization has also been

demonstrated by using microfluidic systems with other liquid controlling methods such as

centrifugal pumping [65] and electrokinetic pumping [66]. The detection of single nucleotide

polymorphisms (SNPs) was also demonstrated by using electrokinetically controlled

microfluidic system [67].

There are also some significant challenges associated with the microfluidic systems

for DNA analysis such as provision of effective macro-to-micro interfaces, minimization of

non-specific analyte/wall interactions due to the high surface-to-volume ratio of the

microfluidics, development of low-cost manufacturing methods for microfluidic chips, and

developing materials that concurrently are compatible with biological samples and suitable

for the selected means of transduction.

1.2.3 Electrokinetically driven microfluidic system

1.2.3.1 Advantages of electrokinetically driven microfluidic system

Electrokinetically driven flow is a widely used method that does not require any

pumps and valves. Fluid control can be accomplished by changing applied voltages. The

EOF in a network of channels has been modeled by the electric current flowing in a network

of resistors using Kirchoff’s rules. The proper voltages for fluidic control can be easily

obtained by simple calculation.

The electrokinetically controlled microfluidic system can effectively deliver samples

to the reaction region. Because of its flat velocity profile, the electrokinetically driven flow is

more efficient at delivering targets to probes on the surface compared to the pressure-driven

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flow which has a parabolic velocity profile. It was shown that reaction equilibrium time

could be reduced by 20% using electrokinetically driven flow in comparison to pressure-

driven flow with the same bulk flow rate [68]. Also, it was suggested by Erickson et al. that

non-specific adsorption of DNA targets in the electrokinetically driven microfluidic channel

is minimal due to high surface shearing force [66]. This is a unique benefit since non-specific

analyte/wall interactions due to the high surface-to-volume ratio of the microfluidics are quite

common. With little or no non-specific surface adsorption, the detection limit can be greatly

increased. Moreover, Joule heating phenomena can also be used to control the stringency of

the hybridization condition to attain mismatch discrimination [67].

Electrokinetically driven flow is also well suited for point-of-care and field

applications since control requires only a single voltage supply. Pumps and valves for

conventional fluid handling techniques are not required. The potential usage of a small sized

power supply for fluidic flow control is a great fit for portable applications. Since our

microfluidic system is quite simple, the fluidic flow control using EOF is a rational choice.

1.2.3.2 Joule heating

While electrokinetic pumping can greatly simplify the fluidic flow control in

microfluidic systems, a significant drawback is Joule heating. Joule heating a unique

phenomenon associated with electrokinetic pumping and is caused by electrical current

through the buffer solution. A direct effect of Joule heating is the increase of the temperature

within the microfluidic channel. Since the temperature increase can destabilize the nucleic

acid duplex, and can lead to band broadening for separation caused by increasing the

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diffusion rate, it is very important to control Joule heating in terms of achieving stable DNA

hybridization and reproducible and effective separation in electrokinetically driven

microfluidic systems [69, 70]. Microfluidic systems must have the ability to rapidly transport

heat to the surroundings to maintain uniform and controlled buffer temperature. In fact, it is

the ability to dissipate this heat that limits the strength of the applied electrical field and thus

the maximum flow speed.

The extent of Joule heating is proportional to the applied electrical field and

conductivity of the buffer solution. The conductivity is also a function of temperature. When

Joule heating increases the temperature it will increase the buffer conductivity, which in turn

increases the current and results in more Joule heating.

Joule heating can be controlled by either using buffer with low conductivity or

improving heat dissipation by the microfluidic system. It was suggested by Erickson that

polymeric materials used for microfluidic system fabrication had lower thermal

conductivities than traditionally used glass and silicon. Experimental results suggested that

heat transfer for a PDMS/glass hybrid microfluidic system was more effective than for a

microfluidic system made purely from PDMS. Under high electrical field conditions, they

observed a 5-fold temperature increase in the PDMS/PDMS microfluidic system compared to

a PDMS/glass system [71]. Although the microfluidic system used in the thesis is made of

the PDMS/glass, a buffer with lower conductivity is still preferred to maintain uniform and

controlled buffer temperature within the microfluidic channel and maximizes flow speed.

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1.2.4 Microfluidic device fabrication

In the early 1990s, microfluidic devices were initially fabricated from silicon and

glass [72]. These substrates were used based on well established photolithography and

etching techniques that had been refined for the fabrication processes used in the

microelectronics industry. Glass has a number of properties that make it the ideal substrate

for microfluidic devices, such as stable and well known surface chemistry and optical

transparency. However, it is expensive and sealing of individual devices can be challenging

Since the late 1990s, polymeric materials have become widely used for fabricating

microfluidic devices. The primary attractiveness of polymers is that they typically require

simpler and significantly less expensive manufacturing techniques. Polymeric devices can be

manufactured in large numbers with lower cost by injection molding, casting, or hot

embossing using a high-resolution mold. These devices can be made cheaply enough to be

disposable, which can avoid cross contamination. Polymeric materials are also amenable to

surface modification and the wide variety of chemical and physical properties allows the

matching of specific polymers to particular applications [73]. There are many polymers that

have been used for microfluidic device fabrication including polyethylene (PE),

polypropylene (PP), polymethyl methacrylate (PMMA), polystyrene (PS), polycarbonate

(PC), polyethylenetetraphthalate glycol (PETG), polyvinylchloride (PVC), cycloolefin

copolymer (COC) and polydimethylsiloxane (PDMS).

Although many polymeric devices can be manufactured in large numbers with lower

cost, they are not cost effective in term of making a few prototype microfluidic devices. The

introduction of the soft lithography method by Duffy et al. solved this problem [74]. By using

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an elastomeric polymer, PDMS, it is possible to carry out a complete cycle of design,

fabrication and testing of microfluidic systems rapidly.

1.2.5 PDMS, plasma treatment and its aging effects

Soft lithography is a rapid and flexible method to fabricate microfluidic devices [75].

PDMS is one of the most widely used polymers for fabricating microfluidic devices using the

soft lithography method. PDMS is composed of an inorganic siloxane backbone that supports

organic methyl groups, which combine to offer unique properties including being relatively

chemically inert, non-flammable, non-toxic, and optically transparent.

PDMS is not an ideal polymer for an electrokinetically driven microfluidic system

due to its hydrophobic surface covered by methyl groups. However, using reactive plasma

treatment, PDMS surface can easily be made hydrophilic by the formation of surface silanol

groups [76, 77]. This hydrophilic PDMS can better support electrokinetic pumping and

simplifies aqueous solution filling within the microfluidic channel. A hydrophilic surface can

also help form an irreversible seal with other materials such as glass and silicon [74]. This

hydrophilic PDMS surface is not very stable when in contact with air and can gradually

regain the original hydrophobic property within an hour [78]. However, the hydrophilicity of

the surface can be maintained if the PDMS was placed in aqueous solution immediately after

plasma treatment, which is probably due to the silanol groups tending to stay in a more

hydrophilic environment [59, 79, 80]. Because of PDMS hydrophobic recovery, bringing two

bonding surfaces into contact immediately after plasma treatment is required to make an

irreversible seal. The surface properties of PDMS can also be changed by further chemical

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modification [57-59]. In addition, dynamic surface coating is an option for controlling

surface properties [60].

1.2.6 Kinetic model of nucleic acid surface hybridization and its

relation to convective flow

1.2.6.1 Factors affecting surface hybridization kinetics and equilibrium

In solid-phase analysis, hybridization happens between targets in solution phase and

probes immobilized on a solid support. It is also called heterogeneous hybridization since the

probes and targets are in different phases. Before modeling the nucleic acid surface

hybridization kinetics, it is important to consider each of the major factors that contribute.

The most obvious factor that affects surface hybridization is the concentration of the

reactants. As for most chemical reactions, rates depend on the concentration of the reactants

in solid-phase assays. So, the surface concentration of the probes and the concentration of the

target in the bulk solution are important factors for surface hybridization. Non-specific

surface adsorption of the targets also has an effect on surface hybridization. In fact,

adsorption precedes hybridization in solid-phase assays. The process involves a 2D surface

diffusion followed by hybridization and is particularly significant for surfaces supporting low

densities of immobilized probes [81]. However, when the surface probe density is high, then

hybridization through this mechanism is less likely to happen.

Conformation of the probes on the surface is also related to surface hybridization

processes. Oligonucleotide probes on the surface with secondary structures such as hairpins

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have an energy barrier to cross before they can hybridize with targets in bulk solution. This

has an effect on the overall process of surface hybridization. The conformation of the probes

on a surface can be affected by electrical fields. This is particularly important since the

electrical potential was used to control fluidic flow and movement of target DNA molecules

in our microfluidic system. Since DNA oligonucleotide is a non-spherical particle, its

orientation parallel to the external electrical field is often attributed to the interactions

between the external electrical field and the induced dipole of DNA. Also, a DNA molecule

stretches in an external electrical field [82]. When DNA molecules are immobilized on the

surface, they are align in parallel and stretch in the direction of the external electrical field

[83]. Since the conformation of the probes have an effect on surface hybridization, it is likely

that such alignment and stretching will have an impact on the surface hybridization. Such

alignment and stretching is expected to increase the surface hybridization rate by extending

and unfolding probe strands similar to the extensional strain created by flow which can help

to uncoil long DNA targets and lead to improved hybridization efficiency [23, 24]. At the

same time, the external electrical field could also have an effect on the stability of DNA

hybrids. It was estimated that an external force up to 150 pN is required to melt DNA hybrids

of λ DNA molecule [84]. Smaller forces of tens of pN are required to melt shorter DNA

hybrids. The force exerted on a DNA by external electrical field was estimated as well. With

applied field strength of 1000 V/cm, the force is around 0.2 pN which appears to be at least

one to two orders of magnitude smaller than the force required to melt DNA hybrids [85]. So

the influence of the external force induced by the electrical field on DNA hybrids is at best

marginal.

The target diffusion coefficient and convective delivery in the solution also play a

role in the surface hybridization process. The DNA diffusion coefficient is a function of its

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length, where longer DNA strands have smaller diffusion coefficients. Convective delivery

can enhance the rate of surface hybridization, and the enhancement depends on the relative

rate of target diffusion and the surface hybridization reaction. If the target diffusion rate is

much smaller than the surface hybridization reaction rate, then enhancement by convective

delivery can be great.

Sequences composition, solution composition, ionic strength, pH of solution, and

temperature are also related to surface hybridization, as discussed in 1.1.2. To accurately

model DNA surface hybridization, all of these factors need to be considered. Simplification

is achieved by considering only the most significant factors while neglecting others.

1.2.6.2 Kinetic models of nucleic acid surface hybridization

Several attempts have been made to develop comprehensive models of nucleic acid

hybridization kinetics between immobilized probes on a surface and free targets in bulk

solution. An early model was published by Chan et al. in 1995 [86], which was based on the

receptor-ligand model developed by Axelrod et al [87]. The model developed by Chan et al.

proposed two mechanisms for surface hybridization: 1) direct hybridization between targets

in the bulk solution with probes immobilized on surface; 2) non-specific adsorption of the

targets on surface followed by two-dimensional diffusion towards the probes on surface and

subsequent hybridization. They are referred to as direct and indirect hybridization,

respectively. Several assumptions were made by Chan et al. to build the model including: a)

fixed number of probe molecules immobilized on surface that are equally spaced and

exposed to the same chemical environment; b) each immobilized probe reacts irreversibly

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25

with only one target molecule in solution (no DNA hybrids dissociate); c) surface coverage

of non-specifically adsorbed target molecules is well below a monolayer so that the lateral

interactions between adsorbed molecules can be neglected; d) the number of available probes

is constant throughout the hybridization process and is independent to the hybridization

reaction rate. The assumption of constant probe numbers is only valid initially. As the

hybridization reaction progresses, the number of available probes inevitably changes. So the

model can only predict the initial rate of hybridization. Also, the authors believed that for all

hybridization reactions, hybridization was the rate limiting reaction. Actually, many surface

hybridization reactions are target diffusion limited due to depletion which makes the

hybridization enhancement by convective target delivery a mechanism to improve surface

hybridization rates.

A comprehensive model developed by Erickson et al. considers both direct and

indirect hybridization mechanisms [81]. The authors suggested that the indirect hybridization

mechanism is an important part of the overall surface hybridization. Instead of providing only

the initial rate of hybridization, this model can be applied to provide quantitative dynamic

predictions of surface hybridization. The model uses a numerical simulation method (finite

element method) to quantitatively calculate the evolution of the surface hybridization

process. However, many parameters required for the model are difficult to measure

quantitatively and may change over the course of the surface hybridization reaction,

including such details as the two-dimensional surface diffusion coefficients, and the

adsorption and desorption rate constants.

Surface hybridization models for competitive multiple hybridization reactions were

published by Zhang et al. [88] and Bishop et al. [89]. Diffusion effects of targets were not

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considered in the model by Zhang et al. They considered the situation where one form of

target hybridized with two types of differently immobilized probes on the surface. The two

forward hybridization reactions were considered the same while the dissociation constants of

the hybrids were different. Bishop et al. used a similar model to simulate surface

hybridization reactions between one type of immobilized probe and two types of different

targets (matched and mismatched targets) [89]. Thus two types of targets were in competition

for hybridization with the same probes. Diffusion effects were considered in this model, but

only the direct hybridization mechanism was considered. They observed that the

hybridization process had two stages: in an early stage, both targets were bound with the

same probes; in a later stage, the matched targets gradually displaced the mismatched targets

from the surface due to thermodynamic stability considerations. Such predictions were

confirmed experimentally by the authors [90].

Alternative approaches have been used for building surface hybridization models.

Vainrub et al. modeled electrostatic charging effects of DNA surface hybridization by

treating DNA targets as ion-penetrable charged spheres interacting with a charged surface

immersed in electrolyte. This corresponds to the hybridization system characterized by a low

surface density of immobilized probes. The authors concluded that the surface electrostatic

interactions (even at zero surface charge or potential) drastically affected binding parameters

[91-94]. The Langmuir isotherm has also been applied as the basis for a surface hybridization

model. The appropriate Langmuir isotherm for surface hybridization is:

1eq

eqeq

xCK

x=

− (1.7)

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where xeq is the equilibrium fraction of hybridized probes, C is the initial concentration of the

target and Keq is the equilibrium constant for the hybridization reaction at the surface which is

independent of xeq. This form applies to a small spot limit, where the hybridization at the

surface does not affect C in the bulk solution. Halperin et al. investigated the role of

electrostatic interactions and competitive surface hybridization using modified Langmuir

isotherms for a polyelectrolyte brush layer with finite thickness [13, 95-97]. Wong et al.

modeled surface hybridization at high DNA densities accounting for the changing

electrostatic interactions within the surface layer which alters rapidly as hybridization

proceeds. The application of positive voltages allowed highly enhanced hybridization and

accelerated kinetics at very high DNA probe density [98]. These models focus on

hybridization equilibrium and seldom consider dynamic target transport effects and the

changing density of hybrids as the hybridization process continues over time. Static models

can not be applied to provide dynamic predictions of surface hybridization.

1.2.6.3 Models coupled with convective flow

Diffusion of DNA targets to a surface coated with immobilized probes has been

recognized as a rate limiting step in the DNA surface hybridization. Such mass transport

limitations arise when the surface hybridization reaction is faster than the rate of delivery of

DNA targets to the surface due to diffusion. The relative rates of surface hybridization

reaction and target diffusion can be represented by Damköhler number Da,

0

/onk PDa

D h= (1.8)

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where kon is the hybridization association constant, P0 is the initial probe density on the

surface, D is the diffusion coefficient of DNA target and h is the characteristic length.

Several attempts have been made to couple surface hybridization models with convective

flow for target delivery. The model based on both direct and indirect hybridization developed

by Erickson et al. has been coupled to convective flow. The coupled model was used to

model the dynamics of hybridization on a biochip surface that had a temperature gradient. It

is shown how the dynamic transport of DNA targets is likely to affect the rate and location of

hybridization [81]. Bishop et al. also coupled their competitive surface hybridization model

with convective flow. When the rate of surface hybridization was much higher than diffusion

transport, convective flow enhanced mass transport and caused higher hybridization rates.

The convective enhancements for a multi-component sample are controlled by target

concentrations, association rate constants, and the dissociation constant of the lower affinity

species through competitive displacement [99][90]. Models of other surface reactions have