protein adsorption kinetics under an applied
TRANSCRIPT
PROTEIN ADSORPTION KINETICS UNDER AN APPLIED ELECTRIC FIELD: AN OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY STUDY
by
MICHELLE A. BRUSATORI
DISSERTATION
Submitted to the Graduate School
of Wayne State University,
Detroit, Michigan
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
2001
MAJOR: CHEMICAL ENGINEERING Approved by: _______________________________ Advisor Date _______________________________ _______________________________ _______________________________
iii
Acknowledgements
I would like to acknowledge my advisor, Prof. Paul Van Tassel, for his
guidance and support and Dr. Joseph Smolinski for his assistance in the
development of experimental equipment.
iv
Table of Contents
Dedication ii
Acknowledgements iii
List of Tables viii
List of Figures ix
1. Introduction 1
1.1 Problem Description 1
1.2 Previous Work 2
1.3 Approach 4
2. Background 6
2.1 Basic Protein Chemistry 6
2.2 Protein Adsorption: Fundamental Principles 7
2.3 Protein Adsorption Models: Theoretical Analysis 7
2.3.1 Langmuir Approach 8
2.3.2 Simple Particle Model 9
2.3.3 Spreading Particle Model 10
2.3.4 Adsorption Model Curves 11
2.4 Adsorption Measurement Technique 13
2.4.1 Propagation of Light 13
2.42 Opitcal Waveguides 18
2.4.3 Optical Waveguide Lightmode Spectroscopy 20
2.4.4 Sensor Chips 24
2.5 Electric Field Systems 31
v
3. Experimental 36
3.1 Materials 36
3.1.1 Proteins 36
Cytochrome c 36
Albumin 37
Apo-Transferrin 37
3.1.2 Deionized Water 37
3.2 Equipment 38
3.2.1 Indium Tin Oxide Sensor Chip Specifications 38
3.2.2 Sensor Chip Preparation 40
3.2.3 Optical Biosensor 41
3.2.4 Flow Cell 43
3.3 Electric Field Set-Up 45
3.3.1 Electrical Circuit 46
3.4 Types of Experiments 47
3.5 Experimental Procedure 48
3.6 Electrode Potential 49
4. Results and Discussion 52
4.1 Effect of Electric Field on Instrument Readings 52
4.2 Protein Adsorption: Transport Modes 54
4.3 Protein Adsorption in an Applied Electric Field 56
4.3.1 Adsorption Curves 56
Albumin 56
vi
Cytochrome c 59
Apo-Transferrin 61
4.3.2 Transport-Limited Regime 63
Albumin 64
Cytochrome c 65
Apo-Transferrin 67
4.3.3 Linear Region of the Adsorption-Limited Regime 68
Albumin 69
Cytochrome c 70
Apo-Transferrin 71
4.3.4 Asymptotic Adsorption Rate 73
4.3.5 Current Versus Time During Adsorption 77
Albumin 78
Cytochrome c 80
Apo-Transferrin 83
4.3.6 Electrode Potentials 85
4.4 Discussion 89
5. Conclusion 96
Appendix A 99
A.1 Scaled Particle Theory 99
Appendix B 102
B.1 Sensor Chip Cleaning 102
B.2 Sensor Chip Soaking 104
vii
Appendix C 106
C.1 Fluid Flow 106
Appendix D 109
D.1 Adsorption Data 109
D.1.1 Albumin (Waveguide A) 109
D.1.2 Albumin (Waveguide B) 112
D.1.3 Cytochrome c (Waveguide C) 115
D.1.4 Cytochrome c (Waveguide D) 118
D.1.5 Apo-Transferrin (Waveguide E) 121
D.1.6 Apo-Transferrin (Waveguide F) 123
D.2 Current 125
D.2.1 Albumin (Waveguide A) 125
D.2.2 Albumin (Waveguide B) 128
D.2.3 Cytochrome c (Waveguide C) 130
D.2.4 Cytochrome c (Waveguide D) 133
D.2.5 Apo-Transferrin (Waveguide E) 135
D.2.6 Apo-Transferrin (Waveguide F) 137
D.3 Electrode Potential 139
D.3.1 Albumin (Waveguide G) 139
D.3.2 Cytochrome c (Waveguide H) 141
References 144
Abstract 147
Autobiographical Statement 149
viii
List of Tables
Table 3.1: ASI 2400 Sensor Chip Specifications 39
Table 3.2: ITO Coating Specifications 40
Table 4.1 Apparent Initial Adsorption Rate Constant, ka 72
Table 4.2 Asymptotic Rate Constant, kb 75
ix
List of Figures
Figure 2.1: Spreading Particle Model. A depiction of the … 10
Figure 2.2: Experimental data of 1 x 10 – 4 g/cm3 fibrinogen 12
versus the Langmuir and RSA models.
Figure 2.3: The three propagation vectors when n1<n2. 14
Figure 2.4: The transmitted wave propagates parallel to the 15
surface when φi = φc.
Figure 2.5: Lateral displacement of the totally internally reflected 16
beam because of the penetration of the evanescent …
Figure 2.6: Ei is normal to the plane of incidence. 17
Figure 2.7: Ei, is parallel to parallel to the plane of incidence. 17
Figure 2.8 Planar Dielectric Waveguide 19
Figure 2.9: Light incident on diffraction grating. 21
Figure 2.10 The sensor chip depicted as a three layer planar… 22
Figure 2.11: Sensor chip depicted as a four-layer planar dielectric 25
waveguide. (S) is a glass substrate …
Figure 2.12: Ions in solution accumulating at the surfaces of the 34
non-conductive electrode coating.
Figure 3.1: ITO coated sensor chip. 38
Figure 3.2: Main components of the scanner, IOS-1 … 42
Figure 3.3: Side and bottom view of the flow channel … 44
Figure 3.4: Flow cell sealed to an ITO coated sensor chip … 45
Figure 3.5: Flow cell and sensor chip depicted as a circuit … 47
x
Figure 3.6: The potential of the ITO or platinum electrode 51
is measured relative to a reference electrode.
Figure 4.1: Refractive index of a 5.0 x 10 –3 g/cm3 glucose 53
solution flowing through the channel at a rate …
Figure 4.2: Surface density of albumin adsorbed onto an ITO 54
coated sensor chip when a potential of …
Figure 4.3 Change in surface density with time versus surface 55
density for albumin …
Figure 4.4: Surface density of albumin adsorbed onto 57
waveguide A. Data is obtained (every 23.5 s) …
Figure 4.5: Surface density of albumin adsorbed onto 58
waveguide B. Data is obtained (every 2.9 s) …
Figure 4.6: Surface density of cytochrome c adsorbed onto 59
waveguide C. Data is obtained (every 23.5 s) ……
Figure 4.7: Surface density of cytochrome c adsorbed onto 60
waveguide D. Data is obtained (every 2.9 s) …
Figure 4.8: Surface density of apo-transferrin adsorbed onto 61
waveguide E. Data is obtained (every 23.5 s) …
Figure 4.9: Surface density of apo-transferrin adsorbed onto 62
waveguide F. Data is obtained (every 23.5 s) …
Figure 4.10 dΓ/dt as a function of time for albumin adsorbing onto 64
waveguide B at an applied potential of 1.0 V.
xi
Figure 4.11 Adsorption rate dΓ/dt, as a function of time for 65
albumin…
Figure 4.12 Adsorption rate dΓ/dt, as a function of time for 66
cytochrome c….
Figure 4.13 Adsorption rate dΓ/dt, as a function of time for 67
apo-transferrin….
Figure 4.14: Change in surface density of adsorbed protein 68
with time verses density. Apparent initial …
Figure 4.15: Change in surface density of adsorbed protein 69
with time verses density for albumin …
Figure 4.16: Change in surface density of adsorbed protein 70
with time versus density for cytochrome c …
Figure 4.17: Change in surface density of adsorbed protein 71
with time versus density for apo-transferrin …
Figure 4.18: The equilibrium constant, k, for albumin, 76
cytochrome c, and apo-transferrin ….
Figure 4.19: Surface density, as time goes to infinity, of albumin, 77
cytochrome c, and apo-transferrin ….
Figure 4.20: Density and current versus time for 1.0 x 10 – 4 g/cm3 78
albumin under a 2.0 V applied potential.
Figure 4.21: Current as a function of time during the adsorption 79
of albumin onto waveguides A and B.
xii
Figure 4.22: Density and current versus time for 1.0 x 10 – 4 g/cm3 81
cytochrome c under a 2.0 V applied potential.
Figure 4.23: Current as a function of time during the adsorption 82
of cytochrome c onto waveguides C and D.
Figure 4.24: Density and current versus time for 1.0 x 10 – 4 g/cm3 83
apo-transferrin under a 2.0 V applied potential
Figure 4.25: Current as a function of time during the adsorption 84
of apo-transferrin onto waveguides E and F.
Figure 4.26: Surface density of 1.0 x 10 – 4 g/cm3 albumin 86
adsorbed onto waveguide G …
Figure 4.27: Surface density of 1.0 x 10 – 4 g/cm3 cytochrome c 87
adsorbed onto waveguide H …
Figure B.1: Fibrinogen, 1.0 x 10 – 4 g/cm3, adsorbed onto a 103
Si0.25Ti0.75O2 film at a flow rate of …
Figure B.2: The effective refractive index, N(TE), measured 104
with time for deionized water flowing at a rate of …
Figure C.1: Experimental data of the refractive index of a 107
5.0 x 10 – 3 g/cm3 glucose solution flowing through
the channel at a rate of 1.33 x 10 – 3 cm3/s …
Figure C.2: Experimental data of the refractive index of a 108
5.0 x 10 –3 g/cm3 glucose solution flowing …
Figure D.1: Effective refractive indices of 1.0 x 10 – 4 g/cm3 109
human albumin adsorbing onto waveguide A…
xiii
Figure D.2: Effective refractive indices of 1.0 x 10 – 4 g/cm3 110
human albumin adsorbing onto waveguide A.
At t = 300 s, 0.5 volts is applied to the …
Figure D.3: Effective refractive indices of 1.0 x 10 – 4 g/cm3 110
human albumin adsorbing onto waveguide A.
At t = 360 s, 1.0 volts is applied to the…
Figure D.4: Effective refractive indices of 1.0 x 10 – 4 g/cm3 111
human albumin adsorbing onto waveguide A.
At t = 300 s, 1.5 volts is applied to the…
Figure D.5: Effective refractive indices of 1.0 x 10 – 4 g/cm3 111
human albumin adsorbing onto waveguide A.
At t = 300 s, 2.0 volts is applied to the …
Figure D.6: Effective refractive indices of 1.0 x 10 – 4 g/cm3 112
human albumin adsorbing onto waveguide B
when no voltage is applied to the electrodes…
Figure D.7: Effective refractive index of 1.0 x 10 – 4 g/cm3 113
human albumin adsorbing onto waveguide B
At t = 300 s, 0.5 volts is applied to the …
Figure D.8: Effective refractive index of 1.0 x 10 – 4 g/cm3 113
human albumin adsorbing onto waveguide B.
At t = 300 s, 1.0 volts is applied to the …
Figure D.9: Effective refractive index of 1.0 x 10 – 4 g/cm3 114
human albumin adsorbing onto waveguide B…
xiv
Figure D.10: Effective refractive index of 1.0 x 10 – 4 g/cm3 114
human albumin adsorbing onto waveguide B.
At t = 300 s, 2.0 volts is applied to the…
Figure D.11: Effective refractive indices of 1.0 x 10 – 4 g/cm3 115
cytochrome c adsorbing onto waveguide C
when no voltage is applied to the electrodes …
Figure D.12: Effective refractive indices of 1.0 x 10 – 4 g/cm3 116
cytochrome c adsorbing onto waveguide C.
At t = 300 s, 0.5 volts is applied to the…
Figure D.13: Effective refractive indices of 1.0 x 10 – 4 g/cm3 116
cytochrome c adsorbing onto waveguide C. At
t = 240 s, 1.0 volts is applied to the electrodes.
At t = 1740 s, the protein solution enters the …
Figure D.14: Effective refractive indices of 1.0 x 10 – 4 g/cm3 117
cytochrome c adsorbing onto waveguide C. At
t = 300 s, 1.5 volts is applied to the electrodes.
At t = 2100 s, the protein solution enters the …
Figure D.15: Effective refractive indices of 1.0 x 10 – 4 g/cm3 117
cytochrome c adsorbing onto waveguide C.
At t = 360 s, 2.0 volts is applied to the …
Figure D.16: Effective refractive indices of 1.0 x 10 – 4 g/cm3 118
cytochrome c adsorbing onto waveguide D
when no voltage is applied to the electrodes …
xv
Figure D.17: Effective refractive index of 1.0 x 10 – 4 g/cm3 119
cytochrome c adsorbing onto waveguide D.
At t = 300 s, 0.5 volts is applied to the …
Figure D.18: Effective refractive index of 1.0 x 10 – 4 g/cm3 119
cytochrome c adsorbing onto waveguide D.
At t = 300 s, 1.0 volts is applied to the …
Figure D.19: Effective refractive index of 1.0 x 10 – 4 g/cm3 120
cytochrome c adsorbing onto waveguide D.
At t = 300 s, 1.5 volts is applied to the …
Figure D.20: Effective refractive index of 1.0 x 10 – 4 g/cm3 120
cytochrome c adsorbing onto waveguide D. At
t = 300 s, 2.0 volts is applied to the electrodes.
At t = 8400 s, the protein solution enters the …
Figure D.21: Effective refractive indices of 1.0 x 10 – 4 g/cm3 121
apo-transferrin adsorbing onto waveguide E
when no voltage is applied to the electrodes.
At t= 600 s, the protein solution enters the …
Figure D.22: Effective refractive indices of 1.0 x 10 – 4 g/cm3 122
apo-transferrin adsorbing onto waveguide E.
At t = 300 s, 0.5 volts is applied to the …
Figure D.23: Effective refractive indices of 1.0 x 10 – 4 g/cm3 122
apo-transferrin adsorbing onto waveguide E.
At t = 300 s, 1.0 volts is applied to the …
xvi
Figure D.24: Effective refractive indices of 1.0 x 10 – 4 g/cm3 123
apo-transferrin adsorbing onto waveguide E.
At t = 300 s, 2.0 volts is applied to the …
Figure D.25: Effective refractive indices of 1.0 x 10 – 4 g/cm3 124
apo-transferrin adsorbing onto waveguide F
when no voltage is applied to the electrodes …
Figure D.26: Effective refractive indices of 1.0 x 10 – 4 g/cm3 124
apo-transferrin adsorbing onto waveguide F.
At t = 600 s, 0.5 volts is applied to the …
Figure D.27: Effective refractive indices of 1.0 x 10 – 4 g/cm3 125
apo-transferrin adsorbing onto waveguide F. At
t = 300 s, 1.0 volts is applied to the electrodes.
At t = 1800s, the protein solution enters the …
Figure D.28: Current versus time during the adsorption of 126
human albumin onto waveguide A. At t = 300 s,
0.5 volts is applied to the electrodes. At t = 1800 s,
the protein solution enters the flow cell …
Figure D.29: Current versus time during the adsorption of 126
human albumin onto waveguide A. At t = 360 s,
1.0 volts is applied to the electrodes …
Figure D.30: Current versus time during the adsorption of 127
human albumin onto waveguide A. At t = 300 s,
1.5 volts is applied to the electrodes …
xvii
Figure D.31: Current versus time during the adsorption of 127
human albumin onto waveguide A. At t = 300 s,
2.0 volts is applied to the electrodes …
Figure D.32: Current versus time during the adsorption of 128
human albumin onto waveguide B. At t = 300 s,
0.5 volts is applied to the electrodes …
Figure D.33: Current versus time during the adsorption of 129
human albumin onto waveguide B. At t = 300 s,
1.0 volts is applied to the electrodes …
Figure D.34: Current versus time during the adsorption of 129
human albumin onto waveguide B. At t = 300 s,
1.5 volts is applied to the electrodes. At t = 2280 s,
the protein solution enters the flow cell …
Figure D.35: Current versus time during the adsorption of 130
human albumin onto waveguide B. At t = 300 s,
2.0 volts is applied to the electrodes. At t = 2880 s,
the protein solution enters the flow cell …
Figure D.36: Current versus time during the adsorption of 131
cytochrome c onto waveguide C. At t = 300 s,
0.5 volts is applied to the electrodes …
Figure D.37: Current versus time during the adsorption of 131
cytochrome c onto waveguide C. At t = 240 s,
1.0 volts is applied to the electrodes …
xviii
Figure D.38: Current versus time during the adsorption of 132
cytochrome c onto waveguide C. At t = 300 s,
1.5 volts is applied to the electrodes …
Figure D.39: Current versus time during the adsorption of 132
cytochrome c onto waveguide C. At t = 360 s,
2.0 volts is applied to the electrodes …
Figure D.40: Current versus time during the adsorption of 133
cytochrome c onto waveguide D. At t = 300 s,
0.5 volts is applied to the electrodes …
Figure D.41: Current versus time during the adsorption of 134
cytochrome c onto waveguide D. At t = 300 s,
1.0 volts is applied to the electrodes. At t = 1800 s,
the protein solution enters the flow cell …
Figure D.42: Current versus time during the adsorption of 134
cytochrome c onto waveguide D. At t = 300 s,
1.5 volts is applied to the electrodes. At t = 1800 s,
the protein solution enters the flow cell …
Figure D.43: Current versus time during the adsorption of 135
cytochrome c onto waveguide D. At t = 300 s,
2.0 volts is applied to the electrodes …
Figure D.44: Current versus time during the adsorption of 136
apo-transferrin onto waveguide E. At t = 300 s,
0.5 volts is applied to the electrodes …
xix
Figure D.45: Current versus time during the adsorption of 136
apo-transferrin onto waveguide E. At t = 300 s,
1.0 volts is applied to the electrodes …
Figure D.46: Current versus time during the adsorption of 137
apo-transferrin onto waveguide E. At t = 300 s,
2.0 volts is applied to the electrodes …
Figure D.47: Current versus time during the adsorption of 138
apo-transferrin onto waveguide F. At t = 600 s,
0.5 volts is applied to the electrodes …
Figure D.48: Current versus time during the adsorption of 138
apo-transferrin onto waveguide F. At t = 300 s,
1.0 volts is applied to the electrodes …
Figure D.49: Effective refractive indices for 1.0 x 10 – 4 g/cm3 139
human albumin adsorbed onto waveguide G …
Figure D.50: Current as a function of time. At t = 300 s, 1.0 volts 140
is applied to the electrodes. At t= 2563 s, the …
Figure D.51: Potential of the ITO and platinum electrodes … 141
Figure D.52: Effective refractive indices for 1.0 x 10 – 4 g/cm3 142
cytochrome c adsorbing onto waveguide H…
Figure D.53: Current as a function of time. At t = 900 s, 1.0 volts 143
is applied to the electrodes. At t = 2845 s, the …
Figure D.54: Potential of the ITO and platinum electrodes 143
relative to a gold reference electrode. At …
1
1. Introduction
1.1 Problem Description
Proteins are biological macromolecules vital to cell structure and
function. The ability to incorporate proteins onto or within synthetic
materials offers the promise of new devices and processes of high potential
impact on the quality of human life. An important subclass of these
materials are those onto which a monolayer of protein molecules is
immobilized. Uses for such materials include supports for reusable
enzymes, thrombosis inhibiting biomaterials, bioelectric components, tissue
engineering substrates, and biosensing surfaces. The function of a surface-
immobilized protein monolayer depends critically on its structural properties;
these include lateral density, spatial homogeneity, relative molecular
orientation, and internal conformation. For example, surface-attached
enzymes (in catalysis) and matrix proteins (in cell attachment applications)
are oftentimes only effective if the proteins are oriented with their active sites
facing away from the surface and if the proteins retain (at least part of) their
native internal conformation. As a second example, layers of retinal
proteins, useful in photovoltaic devices, often function in a way that depends
critically on adsorbed layer uniformity.
Immobilizing protein monolayers with tailored structural properties
that could be independently optimized for a given application would be ideal.
In reality, we are far from this situation. Considering the diversity of systems
and applications, few established protein placement techniques exist.
2
A promising means for controlling the spatial homogeneity, mean
orientation, and growth rate of protein monolayers is through the application
of an external electric field. Due to a net charge and a permanent dipole,
most proteins align and migrate in an electric field.
Currently, little is known quantitatively of the effect of an electric field
on protein adsorption to a solid surface. One reason for this is the
experimental difficulty of simultaneously measuring adsorption and applying
the electric field. The purpose of this thesis is to develop a method for
following the time evolution of an adsorbed protein layer in the presence of
an electric field and to use this method to study the electric field dependence
of the adsorption kinetics of certain proteins.
1.2 Previous Work
Previous investigations of protein adsorption in the presence of an
external electric field have demonstrated an influence on adsorbed amounts,
orientation and antibody-antigen binding regulation by means of
electrochemical polarization [1, 2, 3, 4, 5].
Asanov, et al [1], studied the use of electrochemical polarization to
regulate antibody-antigen binding. Experiments were performed with biotin
covalently bound to an indium tin oxide electrode with strepavidin (or
polyclonal anti-biotin) subsequently adsorbed onto the biotinylated surface.
When no potential was applied to the ITO electrode (open circuit potential),
the irreversibly bound biotin-avidin (or antibody-antigen) complex
3
dissociated extremely slowly when rinsed with a pure buffer solution.
However, square wave polarization of the ITO electrode, -0.9 to +1.3 V for a
period of 5 s, during the rinse (time interval 2000-3000s) showed an
increase in the rate of dissociation and resulted in almost complete
regeneration of the biotinylated surface. Based on an earlier proposed
model, which assumed that with a variable double electric layer (DEL), a
protein molecule at the electrode surface would not have sufficient time to
adjust its structure and orientation to accommodate the new conditions and
thus rapidly desorb from the surface, it was concluded that a similar
mechanism could also describe the electrochemical stimulation of
dissociation of the biospecific complexes.
A study of the orientation of adsorbed cytochrome c as a function of
electrical potential of the adsorbing surface was presented by Fraaije, et al
[3]. Conclusions were that the adsorbed protein orientation on a tin oxide
electrode could only be affected when a potential was applied during the
adsorption process. No affect on orientation was observed when an
external potential was applied on previously adsorbed proteins.
Fievet, et al [4], studied the adsorption of a hydrophobic peptide onto
a carbon electrode. The adsorption was modeled by two consecutive
reactions occurring at the interface. The first reaction corresponded to the
irreversible adsorption of the peptide to the surface, and the second to a
change in conformation of the adsorbed molecules. Experimental conditions
were such that the peptide had an overall positive charge while the charge
4
of the surface was varied. It was determined that rate of adsorption and
coverage of molecules in an unaltered state (i.e. without a post-adsorption
change in conformation) and the coverage of molecules in an altered state
reached a maximum near the vicinity of a potential of zero charge, while the
rate of conformational change seemed to be independent of the charge of
the interface. It was suggested that because of the hydrophobic nature of
the peptide and carbon electrode (in addition to irreversible adsorption of the
peptide), the hydrophobic interactions were much stronger than the
coulombic interactions.
Bernabeu and Caprani [5] studied the adsorption of fibrinogen and
albumin onto the surface of a carbon electrode. Experimental conditions
were such that both proteins were negatively charged. It was found that the
density of protein adsorbed to the electrode increased with increasing
negative charge of the surface (i.e. the more negative the surface, the
greater the adsorption). To explain the favored adsorption of negative
proteins onto a negatively charged surface, it was proposed that cations
from the protein solvent adsorbed to the electrode surface creating a
positively charged layer with which the proteins could interact.
1.3 Approach
While previous research has established that an applied voltage can
have a significant impact on the adsorption process, it is difficult to draw
general conclusions from such studies. One reason for this is that the
5
kinetics of the adsorption process, from which much can be learned of the
underlying mechanisms, has not been systematically investigated. In this
work, it is proposed that a full kinetic analysis will allow one to determine the
affects of surface and protein charge and electrochemical properties of the
electrode surface on the adsorption process.
A method for measuring protein adsorption onto the surface of an
indium tin oxide (ITO) electrode based on Optical Waveguide Lightmode
Spectroscopy (OWLS) is developed. OWLS is a premier optical technique
that allows for the continuous measurement of adsorbed protein mass and
layer thickness, and shown by the results presented here, is capable of
yielding highly precise and accurate adsorption data over a range of applied
potentials. The proteins human albumin, cytochrome c, and apo-transferrin
are investigated in this work. These are chosen so that a range of size and
charge is considered.
6
2. Background
2.1 Basic Protein Chemistry
Proteins are biomolecules that are central to virtually every aspect of
cell structure and function [6]. Proteins can be thought of as medium
molecular weight flexible polymer chains. Chemically, proteins are linear
polymers of amino acids linked head to tail, from the carboxyl group to the
amino group, through covalent bonds.
Proteins can be assigned to one of three broad classes based on
their shape and solubility: fibrous, globular, and membrane. Fibrous
proteins are typically insoluble in water or dilute salt solutions, and tend to
have linear structures. Globular proteins, which are usually very soluble in
aqueous solutions, fold into compact units that are roughly spherical in
shape. Globular proteins tend to fold such that the hydrophobic amino acid
side chains are in the interior of the molecule while the hydrophilic side
chains are on the outside, exposed to the solvent. In contrast, membrane
proteins, which have their hydrophobic amino acid side chains oriented
outward, are characteristically insoluble in aqueous solutions.
The biological activity of proteins generally depends on their
conformation. The natural structure of proteins is dictated by (1) their amino
acid sequence, (2) their interaction with solvent molecules, and (3) the pH
and ionic composition of the solvent. Proteins tend to fold in such a way as
to form the most stable i.e. lowest free energy structure. Structural stability
primarily results from (1) the formation of large numbers of intramolecular
7
hydrogen bonds and (2) the reduction in surface area accessible to solvent
that occurs upon folding [6].
The ionic properties of proteins, determined primarily by their amino
acid side chains, are pH dependent. The pH value at which the sum of the
proteins positive and negative electrical charges is zero is the isoelectric
point, PI. At a pH value below the PI, the net charge of the protein is
positive. Charged residues are normally located on the surface of the
protein where they may interact with the solvent.
2.2 Protein Adsorption: Fundamental Principles
Most protein/surface combinations result in adsorption (i.e. sticking at
the interface). Physical adsorption at a liquid-solid interface is due to
favorable van der Waals, ionic and/or polar interactions. Most proteins
possess heterogeneous surfaces and may therefore exhibit more than one
mode of interaction with the adsorbing surface. The study of protein
adsorption onto solid surfaces is interesting theoretically and of practical
importance in areas such as (1) biocompatibility of materials, (2) separation
of biological solutions and (3) bioanalytical sensing.
2.3 Protein Adsorption: Theoretical Analysis
One would like to be able to predict the amount of protein adsorbed
to a surface as a function of time and certain protein and surface properties.
In flow experiments, protein molecules undergo convective diffusion toward
8
the surface. This is the rate limiting mechanism until a critical concentration
is established near the surface. However, in the absence of transport-
limitations, adsorption to the surface becomes the rate limiting process. In
this section, a few methods for predicting the adsorption rate under these
conditions are reviewed.
2.3.1 Langmuir Approach
One of the simplest and frequently used adsorption models is the
Langmuir approach. The key assumptions are: (1) adsorption onto the
surface cannot exceed a monolayer, (2) the adsorbing surface is composed
of discrete, identical, non-interacting sites and (3) the ability of a molecule to
adsorb to a given site on the surface is independent of the occupation of
neighboring sites [7].
The resulting kinetic equation is:
where ρmonolayer is the concentration of adsorbate corresponding to complete
monolayer coverage (µg/cm2), ρ is the amount of protein adsorbed onto the
surface (µg/cm2), c is the bulk concentration of adsorbing species at the
surface (µg/ml), and ka and kd are the adsorption and desorption rate
constants, respectively.
ρ−ρ
ρ−=∂ρ∂
dmonolayer
a k)1(ckt
(2.1)
9
2.3.2 Simple Particle Model
Since the Langmuir approach accounts only trivially for surface
blockage, a particle level approach in which the protein molecules are
modeled as geometric objects that are subject to surface exclusion (no
overlap) is favored.
The simplest particle level model is Random Sequential Adsorption
(RSA). In this approach, particles adsorb to a surface sequentially, at
randomly chosen positions, subject to no overlap with previously placed
particles. No desorption or surface diffusion occurs. The kinetic equation
becomes
where Φ is the (usually highly non-trivial) fractional surface blockage with the
property that Φ(0) = 1 and Φ(ρ∞) = 0. An interesting aspect of this model is
that a jammed state (saturation) is approached asymptotically with time. At
long times, the kinetics are described by an algebraic power law,
where ∞ρ is the saturation density and ν is a positive real number whose
value depends on the particle geometry [8 - 11]. Desorption may also be
incorporated into the simple particle approach. In this case, the approach to
saturation becomes exponential.
(2.2) )(ckt a ρΦ=
∂ρ∂
[ ] 1)t(t −νν
∞ν− ρ−ρ≈≈Φ (2.3)
10
2.3.3 Spreading Particle Model
An improvement to the Simple Model is the Spreading Particle Model
in which conformation/orientation changes of the surface adsorbed protein
are incorporated [12, 13].
As indicated in Figure 2.1, the Spreading Particle Model depicts
protein molecules as particles that adsorb sequentially and randomly onto
the surface without overlap. Once adsorbed, two competing events take
place, the molecule may desorb or may spread symmetrically and
instantaneously to a particle of larger diameter. Both of these occur at given
rates. Spreading can occur if space allows and represents a post-
adsorption transition in conformation or orientation. (Of course, adsorption
is also subject to size exclusion.)
The key assumptions of this model are: (1) proteins interact laterally through
a hard core potential, and (2) only a single altered state is possible.
1. Transport from bulk to surface
2. Adsorption onto surface
3. Surface induced conformational or orientation changes
4. Desorption
Figure 2.1: Spreading Particle Model. A depiction of the events occurring during protein adsorption. Solution state protein (α state).
Surface altered protein (β state).
11
The kinetic equations for this process are
where ρα is the density of protein in the unspread state, ρβ is the density of
protein in the surface altered state, Φα is the adsorption probability
(fractional surface available for adsorption), Ψαβ is the spreading probability
(the probability that an already adsorbed molecule has sufficient space to
spread), ks is the spreading rate, ka and kd are the adsorption and desorption
rates, and c is the bulk concentration at the surface.
Assuming that the proteins (or more generally, “particles”) on the
surface are at all times in an equilibrium distribution, and that their surface
projections are disk shaped, analytical expressions for the adsorption and
spreading probabilities may be derived via the Scaled Particle Theory [14,
15]. (See Appendix A for details.)
2.3.4 Adsorption Model Curves
Both Langmuir and Particle Models predict an initial linear increase in
adsorbed amounts with a slope proportional to the surface concentration.
The approach to saturation of the Langmuir Model is strictly exponential,
αβα Ψρ−ρ−Φ=∂ρ∂
ααα
sd kkckt a
(2.4)
αβαβ Ψρ=
∂ρ∂
skt
(2.5)
12
and thus very fast. In the Particle Model, this approach is much slower due
to the more realistic manner in which the surface is blockage is treated. (In
the case of purely irreversible adsorption, the approach is algebraic, i.e.
ν−∞ ≈ρ−ρ t)t()( .) These models may be coupled to transport models that
predict the bulk concentration near the surface as a function of time and flow
conditions [16].
Figure 2.2: Experimental data of 1.0 x 10 – 4 g/cm3 fibrinogen versus the
Langmuir and RSA models. Experimental data is obtained at 25°C and at a flow rate of 1.33 x 10 - 3 cm3/s. Experimental details are given in chapters 3 and 4.
Time (s)
0 1000 2000 3000 4000
Den
sity
(µg/
cm2 )
0.0
0.1
0.2
0.3
0.4
0.5
Experimental DataLangmuirRSA
13
2.4 Adsorption Measurement Technique
There are a number of techniques used to measure the amount of
protein adsorbed onto a surface. Some of these are based on optical
principals, for example, Optical Waveguide Lightmode Spectroscopy, Total
Internal Reflection Fluorescence, Scanning Angle Reflectometry, and
Ellipsometry. Non-optical methods also exist, such as Quartz Crystal
Microbalance, which is based on a weight measurement. Each of these
methods or techniques offers various advantages (and, of course,
disadvantages). Total Internal Reflection Fluorescence requires proteins
with either a natural or attached fluorescent label. Quartz Crystal
Microbalance requires careful accounting of viscous drag of the contacting
liquid. In contrast, Optical Waveguide Lightmode Spectroscopy suffers from
neither of the problems and has been shown to provide accurate and
precise kinetic adsorption data for several protein/surface systems [17 - 20].
In this work, Optical Waveguide Lightmode Spectroscopy is used to obtain
continuous measurements of surface adsorbed protein.
2.4.1 Propagation of Light
Light propagates through space in a wave-like nature and yet, during
the processes of absorption and emission, behaves in a particle-like fashion.
The wave nature of light can be represented by the classical
electromagnetic field equations of Maxwell. Consider light (in the form of a
plane wave) impinging on an optical material. The boundary conditions of
14
Maxwell’s equations can be satisfied assuming the existence of three waves
[32]: an incident wave, a reflected wave, and a transmitted wave, shown in
figure 2.3.
A well-known law of optics that is a direct consequence of Maxwell’s
equations is the Law of Reflection [32, 33]: the angle light is incident on an
optical material is equal to the angle it is reflected, ri φ=φ . For the
transmitted wave, the angle of refraction can be related to the angle of
incidence through Snell’s Law of Refraction [32, 33], ttii sinnsinn φ=φ ,
where ni is the refractive index of media (i) and nt is the refractive index of
media (t).
As seen from Snell’s Law, when ni<nt, the angle of the transmitted
wave, tφ , will be real and the refracted wave will propagate in media (t).
However, when ni>nt, as iφ becomes larger, the transmitted ray approaches
Figure 2.3: The three propagation vectors when n1<n2.
φr Reflected
φ t Transmitted
Incident φ i
nt = 1.52 (glass)
ni = 1 (air) Media (i)
Media (t)
15
tangency with the boundary (between media (i) and media (t)). As this
occurs, more and more of the incoming energy appear in the reflected
beam. When o90t =φ the transmitted wave will propagate parallel to the
boundary, as shown in figure 2.4. The value of iφ for which o90t =φ is
called the critical angle ( itc nnsin =φ ) [32, 33].
When φi > φc and ni > nt, the transmitted wave will travel in the x-direction,
that is, parallel the boundary, but with its amplitude decreasing exponentially
in the z-direction (into media (t)). The penetration depth of this evanescent
(surface) wave into media (t) becomes negligible at a distance of only a few
wavelengths [26, 32]. Even though the transmitted wave penetrates into
media (t), there is no energy flow across the boundary and all of the
incoming energy is reflected back into the incident media in the process
known as total internal reflection [26, 32]. Due to the penetration of the
Figure 2.4: The transmitted wave propagates parallel to the surface when ni > nt and φi = φc = arc sin (nt /ni).
φr Reflected
Transmitted
Incident φ i ni
nt
z
x
16
evanescent wave into the media of smaller refractive index there is a lateral
displacement of a totally internally reflected beam [26], as shown in figure
2.5. The reflected wave will undergo a phase change of ϕ with respect to
the incident wave. The phase change will be different for the electric and
magnetic components of the incident light. Total internal reflection is
exploited in many applications where it is desired to transmit light without
intensity loss. Optical techniques such as OWLS make use of this
phenomenon.
No matter what the polarization of the light wave, its electric and
magnetic fields can be resolved into components parallel and perpendicular
to the plane of incidence [32]. Considered here are plane waves. For the
incident wave, the electric field can be written as the vector sum
//iii EEE += ⊥ where ⊥iE is the perpendicular component (shown in figure
2.6), and //iE is the parallel component (shown in figure 2.7).
Figure 2.5: Lateral displacement, D, of the totally internally reflected beam caused by the penetration of the evanescent wave into the media of lower refractive index.
Incident ni
nt
Reflected
Penetration
D
17
These two components behave differently at the boundary between media 1
and media 2. The corresponding direction of the magnetic field, B , can be
found from the condition that BXE is in the direction of propagation, k (i.e.
k,B,E are mutually perpendicular) [33].
Figure 2.6: Ei is perpendicular to the plane of incidence. All of the electric fields are shown directed away from the viewer
Interface
Plane of incidence
Media1
Media 2
ki
Er
Ei
Et
Bi Br
Bt
kr
kt
Interface
Plane of incidence
Media1
Media 2
ki
Br
Bt
Bi
Ei Er
Et kt
kr
Figure 2.7: Ei is parallel to the plane of incidence. All of the magnetic fields are shown coming out of the page.
18
The interdependence of the amplitudes of the incident, reflected, and
transmitted waves is shown by Fresnel’s equations that evaluate the
amplitude reflection coefficient, ioro EE , and the amplitude transmission
coefficient, ioto EE [26, 32, 33]. The Fresnel equations obtained for the
electric field being perpendicular to the plane of incidence and that in which
it is parallel provides a means to determine the phase shift, TETM and ϕϕ ,
associated with total internal reflection.
2.4.2 Optical Waveguides
Of practical importance is the confinement and propagation of light
through optical waveguides. The key to high-speed telecommunications is
the transmission of visible or infrared light that has been modulated with a
signal, through small optical fibers. Optical Waveguide Lightmode
Spectroscopy, the technique used in this work to study protein adsorption,
utilizes a sensor chip that is also a dielectric waveguide. An import aspect
of such waveguides is that light can be transmitted over a long distance with
little loss of intensity.
Consider a simple planar waveguide consisting of a dielectric film of
thickness df in the z-direction and infinite in the other two directions. A
dielectric media, M, of refractive index nm surrounds the film, F, which has a
refractive index value of nf, where nf > nm. Figure 2.8 shows light confined
inside of the film as it propagates in the x-direction. Geometrically, any ray
19
that makes an angle φb with the z-axis that is greater than the critical angle
φc, evaluated at the F-M interfaces, is totally internally reflected where φc =
sin-1 (nm/nf).
When light propagates through the film, the reflected beam will
undergo a phase shift and when the accumulation of phase on the path from
point 1 to just beyond point 2 (i.e. two internal reflections) is an integer
multiple of 2π, a stable transverse field will result [26]
2 k df cosφb + 2ϕ F,M = 2mπ
where m is a non-negative integer, k df cosφb is the phase shift due to the
wave traversing the film where k = 2πnf / λ, and ϕ F,M is the phase shift
associated with total internal reflection at the F-M interface.
The phases associated with total internal reflection, according to Fresnel
formulas, are found to be
Film, nf
nm
nm
φb df
Point 1 Point 2
x
z
Figure 2.8: Planar dielectric waveguide.
(2.6)
20
where the subscripts TE and TM correspond to the electric field component
of the light being perpendicular and parallel to the plane of incidence,
respectively. For a given value of the propagation angle, φb, the phases for
the TM mode will differ from that of TE. Therefore, equation 2.6 will equal
2mπ at a different propagation angles for the TE mode than the TM mode.
2.4.3 Optical Waveguide Lightmode Spectroscopy
Optical Waveguide Lightmode Spectroscopy (OWLS) [21 - 25] is a
technique, based on multiple total internal reflections, that is used to study
the adsorption of protein or other macromolecules onto the surface of a
sensor chip. The sensor chip is comprised of a glass substrate coated with
a thin, optically transparent, metal oxide film and a relief grating embossed
into the film’s surface. Polarized light from a He-Ne laser is directed onto
the sensor chip at the grating region. The sensor chip is rotated between ±
12.6° relative to the fixed laser beam. At a well-defined angle, α, light from
the laser is coupled into the film of the sensor chip by means of the grating.
(2.7)
φ−
−φ
−=ϕ
5.0
b22
f2f
2mb
22f
2
m
f)TM(M,F
sinnn
nsinnnn
arctan2
(2.8)
φ−
−φ−=ϕ
5.0
b22
f2f
2mb
22f
)TE(M,Fsinnn
nsinnarctan2
21
The incoupled light propagates through the film via multiple total internal
reflections. The intensity of light coupled out of the film is detected by
photodectors (one located at each end of the chip) and is recorded as a
function of the incident angle of the laser beam. The incident angles at
which light is maximally coupled into the film of the sensor chip are the basic
physical values determined by the biosensing system.
When light impinges on a diffraction grating (figure 2.9) it is scattered
and multiple diffracted beams b = 0, b = 1± … will arisen according to [33]
Λλ
=φ−φb
sinnsinn ifbf
where b is the order of the diffraction grating, λ is the wavelength of the
incident laser, and 1/Λ is the grating period.
For a diffracted beam to be coupled inside of the film of the sensor chip
(which is depicted in figure 2.10 as a three layer waveguide) and propagate,
the angle at which the light is diffracted must be greater than the critical
angles (evaluated at each interface), that is φb > φc (F,S) and φb > φc (F,C). If
(2.9)
φb φi 1st order (b = -1)
0 th order (b = 0)
1st order (b = +1)
Media of refractive index nf
Diffraction Grating
Figure 2.9: Light incident on a diffraction grating
22
cb φ<φ , total internal reflection will not occur. The sensor chips used in
OWLS are designed so that only one diffracted beam is coupled into the
waveguiding film. This is due to the grating period of the diffraction grating
and the refractive index of the waveguiding film.
From the diffraction equation (2.9), an effective refractive index, N, for
either the TE or TM mode of polarization is defined as [21, 22]
Λλ
±α=φ=±b
sinnsinnN airbf (2.10)
-x
Figure 2.10: The sensor chip is depicted as a three layer planar dielectric waveguide: (S) is a glass substrate, (F) is a thin film onto which a diffraction grating is embossed, and (C) is a media that is in contact with the film at the grating region.
+x
Laser
φ i
Grating
φb
nc C
nf
γs
α nair
S ns
F
23
Given that nair< ns< nf, according to Snell’s Law (nair sinα = ns sinγs = nf sinφi),
the term nf sinφi in equation 2.9 can be replaced by nair sinα where α is the
incident angle of the laser beam measured in air. Since the sensor chip is
rotated between ± 12.6° relative to the fixed laser beam, light can propagate
in either the ± x-directions. When propagation occurs in the +x direction,
Λλ+α= ++ bsinN and when the direction of propagation is negative,
Λλ−α= −− bsinN . The situation is fully symmetric with respect to the ±
directions such that the effective refractive indices of the modes are
identical, N+ = N-, as well as the incoupling angles, α+ = α_.
Although there is a range of angles, φb, that will result in the
propagation of light, due to multiple total internal reflections through the
waveguiding film of the sensor chip, only certain discrete values will satisfy
the phase condition (described in section 2.4.2). When light propagates
through the film, the reflected beam will undergo phase shifts and when the
accumulation of phase is equal to πm2 , maximum irradiance (intensity) will
be detected at the photodetectors. As described in section 2.4.2, the phase
for the TE mode differs from that of the TM mode and will equal πm2 for
different values of the propagation angle, φb. The propagation angles that
satisfy this condition are related to the incident angles of the laser beam, α,
with equation 2.10. Therefore, by scanning over an angular segment (α>0
or α<0) the transverse electric and transverse magnetic modes are
distinguished. A perpendicular incidence of the laser beam onto the sensor
24
chip results in a standing wave of light such that propagation through the
film occurs both in the +x and –x directions. The angular position halfway
between two peaks of light intensity (one peak resulting from light
propagating in the +x direction and the other in the –x direction) of the same
polarization is the angle of autocollimation. When light is coupled into the
waveguiding film, the angular position of the resonance peaks of light
intensity corresponding to the (TE ± ) and (TM ± ) modes along with the
angle of autocollimation is used to determine the incoupling angles for the
different modes. For reasons of symmetry α(TE+)=α(TE-) and
α(TM+)=α(TM-). [25]
When a protein solution is brought in contact with the film of the
sensor chip, as illustrated in Figure 2.11, the propagation angle changes
due to result the adsorption of molecules onto the film surface. The
propagation angle, φb, which is dependent on the optical properties of the
sensor chip (film and substrate) as well as on the surrounding media, is
related to the incoupling angle, α, by equation 2.10. Therefore, by
monitoring the incoupling angles, the amount of surface adsorbed protein
can be determined.
2.4.4 Sensor Chips
The theory of integrated optics for planar dielectric waveguides is
used to compute the refractive index and thickness, as a function of time, of
a protein layer deposited onto the film surface of the sensor chip. The
25
sensor chip and contacting protein, as depicted in Figure 2.11, is a four layer
planar waveguide where (S) is the glass substrate, (F) is the waveguiding
film, (A) is the protein adsorbed layer and (C) is the solution-state protein.
Sensor chip specifications are given in Table 3.1.
Due to total internal reflection at the film-substrate (F, S) and film
adlayer (F, A) interfaces, light is confined inside of the film as it propagates
in the x direction. Total internal reflection occurs at the film-substrate and
film-adlayer interfaces provided (1) the refractive index of the film is higher
than that of the substrate and adlayer and (2) the propagation angle, φb,
incident on the film at the F-A and F-S interfaces is greater than or equal to
Figure 2.11: Sensor chip depicted as a four-layer planar dielectric waveguide: (S) is a glass substrate, (F) is a thin metal oxide film with a diffraction grating embossed into its surface, (A) is an adsorbed layer of protein and (C) is the protein solution state.
C
F
A
S
z
- x + x
Grating
α+
φb
Laser
Detector Detector
26
the critical angles, φc, at each of the two interfaces [φb ≥ φc (F,A) and φb ≥ φc
(F,S)]. Since the penetration depth of the evanescent (surface) wave into the
less dense media (S and A) is of a few wavelengths, the cover media needs
to be considered when the adlayer thickness is less than (or of the same
order of magnitude as) the wavelength of light. In the following discussion it
is given that φb >sin-1 (nC/nF) and φb >sin-1 (nS/nF) = φc (F,S).
A stable traverse field and coherent propagation in the x-direction will
result (i.e. maximum intensity will be detected) when the propagation
condition is satisfied
where k z,F d F is the phase shift due to the wave traversing the film, ϕ F,S and
ϕ F,A,C are the phases associated with total internal reflection at the film-
substrate and film-adlayer interfaces, respectively.
When N < nF and N > nA (i.e. φf >sin -1(nA/nF) = φc (F, A)), the
mathematical expressions for these phases are:
m2dk2 C,A,FS,FFF,z π=ϕ+ϕ+ (2.11)
−−
−=ϕ
2/1
22F
2S
2p2
S
FS,F Nn
nNnn
arctan2(2.11 a)
−+
−−
−=ϕ
)dk2exp(ba
)dk2exp(ba
k
k
nn
arctan2AA,z
AA,z
F,z
A,zp2
A
FC,A,F
(2.11 b)
27
where
N is the effective refractive index of a guided mode of polarization (TE or
TM), nf and df are the refractive index and thickness of the film, nA and dA
are the refractive index and thickness of the protein adsorbed layer, ns is the
refractive index of the substrate, nc is the refractive index of the cover
media, and p is a number equal to zero or one. To obtain the expressions
for the transverse electric mode of polarization one sets p = 0. The
expressions for the transverse magnetic mode are obtained by setting p = 1.
In the above expression of ϕ F,A,C, it is assumed that the adlayer (protein
adsorbed layer) is a homogeneous monolayer. This assumption is
reasonable if the surface heterogeneity is on a length scale smaller than the
light.
When N < nF and N < nA the mathematical expressions for the
phases are:
2/122FF,z )Nn(
2k −
λπ
= p2C
C,zp2
A
A,z
n
k
n
ka +=
2/12A
2A,z )nN(
2k −
λπ
= p2C
C,z
p2A
A,z
n
k
n
kb −=
−−
−=ϕ
2/1
22F
2S
2p2
S
FS,F Nn
nNnn
arctan2 (2.11 c)
28
where
The sensor chips used with the biosensor support only the zeroth
modes of polarization, therefore m=0 in equation (2.11). The number of
modes supported by the waveguide can be approximated from the one-
dimensional phase-space estimate [26].
NOTE and NOTM are the number of transverse electric and transverse
magnetic modes supported by the waveguide, φb is the propagation angle,
φc is the critical angle at the film interface, fd is the film thickness, and k is
the propagation number. For example, if 3NN OTMOTE =≈ , the waveguide
( )oo 90
2f90
ff
k
k
zfOTMOTE
cc
max
min
sin1kd
coskd
2k
dNNθφ
φ−π
−=θ∂
π−
=π
∂≈≈ ∫∫ (2.12)
2/122FF,z )Nn(
2k −
λπ
=2/122
AA,z )Nn(2
k −λπ
=
−−
−=ϕ
2/1
22A
2C
2p2
c
AC,A Nn
nNnn
arctan2
ϕ+
=ϕ
2dktan
kk
nn
arctan2 C,AAA,z
F,z
A,zp2
A
FC,A,F
(2.11 d)
29
will support three TE modes and three TM modes (m=0,1,and 2). Using the
one-dimension phase estimate for the sensor chips used in OWLS
where bf sinnN φ= . Nmax is determined from the maximum value of φb,
which is 90°. The value of Nmin can be approximated using the largest value
of critical angle either at the film-substrate or film-adlayer interface (i.e. Nmin
= ni, where i = A or S). From the phase space estimate it is seen that the
sensor chips support only one TE and one TM mode (m = 0). Since only the
zeroth transverse electric and transverse magnetic modes of polarization
are supported by the waveguide, the values of φb are discrete.
When the effective refractive indices for both the transverse electric,
N(TE), and transverse magnetic, N(TM), modes of polarization are
continuously measured, the refractive index and thickness of the protein
layer can be determined with time by equation (2.11). By simultaneously
solving the two resulting expressions (one for the TE mode and another for
the TM mode), the values nA and dA are calculated provided that the values
of ns, nc, nf, and df are known. The refractive index of the glass substrate,
nS, is provided by the sensor chip manufacturer. The refractive index of the
solution state protein, nC, is determined by an abbey refractometer. The
refractive index, nF, and thickness, dF, of the film are measured with the
biosensor. The values of nF and dF are determined from baseline data, prior
( )
( )
5.0Nnd2
NN)(fnminN
nmaxN
22f
fOTMOTE
c
f
≈−λ
≈≈φ
30
to the onset of protein adsorption using the two expressions obtained by
equation (2.11), where nA is set equal to nC and dA is set equal to zero.
For given values of nA, and dA, the density of protein adsorbed onto
the surface of the film can be calculated by assuming a uniform layer of
constant density, of thickness dA, and of refractive index nA [21]:
where ρ is the surface density of protein adsorbed onto the film, and dn/dc,
which can be determined experimentally with a refractometer, is the change
in refractive index of a bulk solution with a change in concentration. For
many proteins, a linear dependence is observed with dn/dc=1.88 x 10 – 1
cm3/g over a large concentration range.
When the effective refractive index for only one of the two modes of
polarization is continuously measured, the density of adsorbed protein can
be determined with time. Assuming the values of nA and nC are constant
[21]
where ∆N is the change in the effective refractive indices resulting from
protein adsorption and
(2.14)
cn
NNd
)nn( ACA
∂∂
∆∂∂
−=ρ
( )
cn
dnn ACA
∂∂
−=ρ (2.13)
31
p
1)nN()nN(1)nN()nN(
)nn()nn(
dN
dN
2F
2C
2A
2C
2C
2F
2C
2A
FA
−+−+
−−
∂∂=
∂∂
( )
−
+
−
πλ+
−=
∂∂
∑=
−
−
C,SJ
2
J
2
F
2/12J
2F
22F
Fp
1nN
nN
nN2
dN
)Nn(dN
In equation 2.14 b, J = S or C corresponding the cover media and
substrate. To obtain the expressions for the transverse magnetic mode of
polarization one sets p equal to 1. Similarly, p is set equal to zero for the
transverse electric mode
Optical Waveguide Lightmode Spectroscopy provides a means to
measure the rate and amount of surface adsorbed protein. The rate at
which protein adsorbs to a surface is governed by diffusion and protein
surface interactions. In this work, the adsorption kinetics of protein in an
external electric field is studied to determine if the rate, saturation density
and adsorbed state can be influenced.
2.5 Electric Field Systems
A promising means for controlling the mean orientation and growth
rate of protein monolayers is through the application of an electric field. Due
(2.14 a)
(2.14 b)
32
to a net charge and a permanent dipole, most proteins align and migrate in
an electric field. An electric field will exert a force on any charge that is
present in the field. Positive charges will experience a force in the direction
of the field and negative charges in the opposite direction, where the force
on a unit of charge, q, is
Polar molecules will align or orient themselves in an electric field due to
torque resulting from forces acting on charges throughout the molecule.
Currently, little is known quantitatively of the effect of an electric field
on protein adsorption to a solid surface. One reason is the experimental
difficulty of simultaneously measuring adsorption and applying the electric
field. To investigate the influence of an electric field on protein adsorption
using OWLS, the limitations posed by the measurement technique must be
understood.
The sensor chips used in the biosensor provide a surface onto which
protein adsorbs. To examine the effect of an electric field on adsorption, it is
desired that the direction of the field be perpendicular to the adsorbing
surface. With this configuration, the electric field forces acting on the
molecule should oppose or act in the direction of diffusion (toward the
surface). An electric field between two oppositely charged parallel plates is
constant in magnitude and directed normal to the plates. The electric field
strength is then
EqFrr
= (2.15)
33
zd
VE plates∆
=
where ∆Vplates is the voltage difference between the plates and d is their
separation.
To create a perpendicular electric field, a thin conducting layer must
be placed on the waveguide. This allows the sensor chip to act as one of
the conducting plates in a parallel plate setup. So long as the conducting
layer is extremely thin and its conductivity relatively low the theory of
integrated optics for planar dielectric waveguides can be applied, as
demonstrated in Section 3.3.2 of this work, to calculate the amount of
surface adsorbed protein.
Proteins are usually dissolved in a buffer solution of relatively high
ionic strength. When an electrolytic solution is placed between the plates,
ions will in solution will experience a uniform electric field of magnitude E =
∆Vplates /d. At or above the electrode reduction/oxidation potential, ions in
solution (or water itself) will participate in electron exchange thus allowing
current to flow through the system. One such possible reaction is 2H+ (aq) +
2e- → H2 (g). The amount of gas produced is dependent upon the number
of reacting ions. If the amount of gas exceeds the solubility limit of the
solution, formation of a second phase occurs. The presence of gas bubbles
in the system interferes with instrument measurements and may interfere
with the adsorption process.
(2.16)
34
Decreasing the potential difference between the plates (i.e. current
flow) such that many of ions in solution cannot react with the electrodes can
impede gas formation. However, non-reacting ions will accumulate at the
electrode surfaces leading to a significant decrease in field strength.
Reducing current flow by means of a physical barrier can also slow
gas formation. Encasing the electrodes in a poorly conductive barrier, as
depicted in Figure 2.12, will inhibit electron exchange but lead to an
accumulation of non-reacting ions at the barrier surface and thus decrease
field strength.
Since current flow is necessary and gas may be evolved, it is
concluded that by increasing the resistance of the solution, thus decreasing
current flow, is the only viable means by which there will be appreciable
electric field strength without the formation of bubbles. So long as the
amount of gas produced is below the solubility limit of the solution, bubbles
Figure 2.12: Ions in solution accumulating at the surfaces of the poorly conductive electrode coating.
_ _ _ _ _ _ _ _ σ -
+ + + +
+ + + + + + + + σ +
_ _ _ _ d
+ Eo
Eind
Eind
Eind
35
will not form. Deionized water is used as the protein solvent for this work
since it has an extremely small number of charge carriers and thus has a
very high resistivity. Non-aqueous solvents can also be considered since
they do not undergo the equivalent of hydrolysis.
36
3. Experimental
3.1 Materials
3.1.1 Proteins
The proteins used in the electric field studies are horse heart
cytochrome c (type VI), human albumin (fraction V1), and human apo-
transferrin. All are purchased from Sigma Chemical Company, Missouri,
USA. Aqueous solutions of 1.0 x 10 -4 g/cm3 of each are prepared by
dissolving the protein in deionized water (pH of 5.5 – 6.0 and conductivity of
1.30 ± 0.05 µS at room temperature) for 30 minutes at 37 °C. Solutions not
used within 8 hours are discarded. Due to the high affinity of protein to
glass surfaces, Teflon vials are used to contain the protein solution before
and during experiments.
Cytochrome c
Cytochrome c is found in the mitochondria of all eukaryotic organisms
and is an essential component of the mitochondrial respiratory chain. It is a
hemoprotein that contains an iron-porphryn complex that functions as an
electron carrier. Cytochrome c, from horse heart, is a small globular protein
consisting of a single polypeptide chain of 104 residues. All cytochrome c
polypeptide chains have a cysteine residue at position 17 that serves to link
the heme prosthetic group to the protein. This protein has a molecular
weight of ≈ 12,370 and is soluble in water up to 2.0 x 10 -1 g/cm3. The
isoelectric point is approximately 10 and the redox potential is +0.251 volts
37
[27]. The conductivity of prepared aqueous solutions, determined
experimentally is 7.2 ± 0.4 µS at 25 °C.
Albumin
Serum albumin is a blood protein whose main biological function is to
regulate osmotic pressure of blood. Human albumin has 584 amino acid
residues. Albumin is water-soluble and has a molecular weight of ≈ 66,300
and an isoelectric point of 4.7. The solubility of albumin in water is 5.0 x 10 -
2 g/cm3 [27]. The conductivity of prepared aqueous solutions is
experimentally determined to be 3.8 ± 0.3 µS at 25 °C.
Apo-Transferrin
Human transferrin is a glycoprotein found in human serum. It is a
non-heme iron transport protein (that facilitates the transport of iron to cells).
The iron poor form, apo-transferrin, combines with an iron ion to become
halo-transferrin, the iron saturated form. Apo-transferrin is water soluble, up
to 2.0 x 10 –2 g/cm3, and has a molecular weight of ≈ 78,500 [27]. The
isoelectric point is 5.5 [28] and the conductivity of prepared aqueous
solutions, determined experimentally, is 3.9 ± 0.4 µS at 25 °C.
3.1.2 Deionized Water
Deionized water with a conductivity of 1.30 ± 0.05 µS and a pH of
5.5 - 6.0 at 25 °C is used as the protein solvent in this work.
38
3.2 Equipment
3.2.1 Indium Tin Oxide Sensor Chip Specifications
Traditionally, indium tin oxide has been used for transparent heating
elements of car windows, as antireflective coatings, and in early electro-
optic devices such as liquid crystal displays. More recently, indium tin oxide
thin films are being used as electrodes for integrated optical chemical and
biochemical sensors [29]. The major benefit of this application is to exert
electrochemical control over interactions taking place on waveguides. For
use as electrode overlayers for waveguides used in optical techniques such
OWLS and TIRF, the ITO thin film must have high transparency over the
wavelength range of operation and be of relatively low resistivity (≈ 1 X 10 – 4
Ωcm).
The ITO coated sensor chips used for electric field studies are
purchased from Microvacuum Ltd., Budapest, Hungary. A schematic of a
sensor chip is presented in Figure 3.1.
16 mm
48 mm
2 mm
0.55 mm
∼ 200 nm 10 nm
Substrate
Film
ITO Grating
Figure 3.1: ITO coated sensor chip
39
The sensor chips are ASI type-2400 (Artificial Sensing Instruments, Zurich),
coated with a thin ITO film. Specification for the ASI type 2400 sensor chip
and ITO film are given in Tables 3.1 and 3.2.
Table 3.1: ASI 2400 Sensor Chip Specifications.
ASI Type 2400 Sensor Chips
• Waveguide film Material Si(1-x)TixO2 x=0.25 ± 0.05 Refractive Index (25 °C) nf 1.77 ± 0.03 Thickness df 170 – 220 nm
• Substrate Material Glass (SiO2) Refractive Index (25 °C) ns 1.52578 Thickness ds 0.55 mm
• Diffraction Grating Relief Structure Surface of film Grating Periodicity 2400 lines/mm 0.4166 µm Diffraction Order 1
Grating Dimensions Depth 20 nm Length 2 mm Width 16 mm Grating Line Direction Parallel to width of sensor chip
• Sensor Chip Dimensions Length 48 mm Width 16 mm
40
Table 3.2: ITO Coating Specifications.
ITO Coating
Coating Location Surface of waveguide film Refractive Index (25 °C) nITO ∼ 1.78 Thickness dITO ∼ 10 nm Linear Resistance ∼ 2.08 x 104 Ω/m
3.2.2 Sensor Chip Preparation
Both new and used ITO coated sensor chips are cleaned using the
following procedure. The sensor chip is placed in an ultrasonic bath (of
frequency of 55 kHz), containing a cleaning solution, for 10 minutes and
then is extensively rinsed with deionized water. A cleaning solution at a
concentration of 1.0 x 10 - 2
g/cm3 is prepared by dissolving Terg-A-Zyme
from Alconox (a laboratory detergent with protease) in deionized water. The
effect of the cleaning procedure on the properties of the ITO coated sensor
chip has not yet been determined. However, an analysis of the ASI (Type
2400) sensor chip indicates that the cleaning procedure may affect film
thickness. The analysis of the cleaning procedure is presented in Appendix
B.
Sensor chips are soaked in deionized water (the protein solvent for
this work) for several hours prior to use. Due to the porosity of the
waveguiding film [22, 30], it is found experimentally (and confirmed by
41
literature) that effective refractive index measurements will vary until an
equilibrium condition is reached. Experimental data is presented in
Appendix B.
3.2.3 Optical Biosensor
An integrated optical biosensor, BIOS-1 (Artificial Sensing
Instruments, Zurich, Switzerland) is used to perform all OWLS experiments
[21-25, 31]. The biosensor uses sensor chips, which are comprised of a
glass substrate coated with a thin optically transparent metal oxide film. At
the center of the chip, a relief grating embossed onto the film surface acts to
couple laser light into the film through diffraction. The sample to be
investigated is brought in contact with the film at the grating region by
means of a flow through cuvette.
Measurements are performed and recorded by the biosensor’s
integrated optics scanner, IOS-1. The main components of the scanner are
a He-Ne laser, a mirror (M), the measuring head (MH), a turntable (T) in
which a lever arm (LA) is fixed, a stepper motor (SM), a micrometer screw
(MS) and an optical encoder (E). A schematic of the scanner is presented in
Figure 3.2.
The aluminum-measuring head (MH) of the biosensor’s integrated
optics scanner supports the sensor chip/flow cell apparatus. A photodiode
(D) and a digital potentiometer are located at each end of the measuring
42
head. The sensor chip is mounted into the measuring head such that the
two end faces of the chip, along its width, are aligned with the photodiodes.
Polarized light form a He-Ne laser is directed by a mirror (M) onto the sensor
chip. The measuring head, which is fixed to a turntable (T), is rotated
relative to the fixed beam so that the center of rotation (P1) goes through the
grating region of the sensor chip. A micrometer screw (MS), driven by the
Figure 3.2: Main components of the scanner, IOS-1: He-Ne laser, (M) mirror, (MH) measuring head, (T) turntable, (LA) lever arm, (SM) stepper motor, (MS) micrometer screw, and (E) optical encoder. P1 is the center of rotation, P2 is the engagement point, and XMS is the measured position of the engagement point from XMS=0.
X MS = 0 X MS
Laser
E SM
M
MS
D D
MH
LA
P2
T
P1
43
stepper motor (SM) actuates the lever arm (LA), which is attached to the
turntable (T). An optical encoder (E) measures the position of the stepper
motor. The micrometer screw will contact the lever arm at point (P2) and
from the given distance between P1 and P2, and the measured the position
(XMS) of P2 (from XMS=0), the angular position of the turntable is calculated.
The integrated optics scanner, IOS-1, scans an angular width of up to ± 12.6
degrees. During an angular scan, the photodiodes (D) measure the
intensity of light coupled out of the end faces of the sensor chip. A computer
records the angular peak position of light power as a function of the incident
angle of the laser beam onto the chip (i.e. the angular position of the
turntable).
The incident angle of the laser beam onto the sensor chip at which
light is maximally coupled into the waveguiding film are the basic physical
values determined by the instrument. As protein adsorbs onto the film
surface of the sensor chip, the angles change due to the formation of the
protein adlayer. A computer tracks the values of the incoupling angles with
time.
3.2.4 Flow Cell
The flow cell of the biosensor allows liquid to be brought in contact
with the film surface of a sensor chip at the grating region. The flow cell is
sealed to the surface of the sensor chip with a (n-buna) gasket to create a
flow cavity of volume 7.0 x 10 –2 cm3. Fluid is drawn into the cavity via a
44
peristaltic pump through a 7.62 x 10 -2 cm I.D. bore in the solid interior of the
flow cell and exits through an outlet bore of the same dimension. The flow
cavity is a rectangular channel of cross section (h x w) 5.5 x 10 -2 cm2. The
area (l x w) of the sensor chip wetted by the liquid is 7.0 x 10 -1 cm2. A
schematic of the flow cavity is presented in Figure 3.3. When a test solution
is drawn into the flow cavity through an inlet line of 21.0 cm in length (5.8 cm
inlet bore length plus a 15.2 cm Teflon tubing of 7.62 x 10 -2 cm ID) at a rate
of 1.33 x 10 –3 cm3/s, assuming axial flow, a Reynolds number of
approximately 0.5 is obtained indicating a laminar flow regime. An analysis
of the flow inside of the cavity (mixing effects) is presented in Appendix C.
Due to the significant dependence of refractive index on temperature,
the temperature of the cell is maintained at 25 ± 0.5 °C. Water from an
external bath is circulated inside of the Teflon flow cell body as shown in
Figure 3.3: Side and bottom view of the flow channel created by the flow cell and sensor chip.
O-ring
Flow cell
Sensor chip
Flow Channel
Inlet/outlet bore
w = 0.55 cm
l = 1.27 cm h = 0.10 cm
45
figure 3.4. The large thermal mass of the flow cell mediates temperature
fluctuations observed in the lab. Teflon was chosen for its chemical
resistance to most solvents as well as its thermal and electrical insulating
properties.
3.3 Electric Field Set-Up
The flow cell, shown in Figure 3.4, is constructed allowing for an
electric field to be directed perpendicular to the adsorbing surface. A disk
shaped platinum electrode is mounted flush with the upper surface of the
Figure 3.4: Flow cell sealed to an ITO coated sensor chip. A disk shaped platinum electrode is mounted with the upper surface of the flow cavity.
+-
Sensor chip
Inlet line
Circulating chamber
Electrode (ITO)
ITO film
Electrode (Pt)
Thermocouple
Outlet line
O-ring
Solid core
Stainless steel rod
Flow cavity
Pt.
Grating
46
flow cavity at a distance of 1.0 x 10 -1 cm above the surface of the sensor
chip. The ITO coating (protein adsorbing surface) of a sensor chip acts as
the second electrode in the parallel plate set-up. Electrical contact is made
with the ITO coating through the end of a small steel rod pressed against the
ITO film. Contact is made outside of the flow cavity at a distance of 1.5 cm
from the center of the sensor chip.
3.3.1 Electrical Circuit
The electrical circuit for the parallel plate set-up, which includes the
ITO film of the sensor chip, the platinum electrode, and the solution inside of
the flow cavity, can be thought of as resistors connected in series. Figure
3.5, depicts the solution inside of the flow cell as one resistor, Rsol, the ITO
film as another resistor, RITO, and the platinum and ITO interfaces as
resistors, Rint, each of which are in parallel with a capacitor. Current flow
through the system is monitored by measuring the voltage drop across an
external 1.0 x 10 5 Ω resistor. Applied voltage, ∆Vapp, across the ITO and
platinum electrodes is measured with a voltmeter meter after the external
resistor. The resistances of the wires, connectors, and platinum are
assumed to be negligible. Even though the voltage drop through the ITO
film is estimated to be negligible at less than 0.005 V (at the currents being
measured), the resistance is included. The electric field acting on ions in the
cell is:
47
dR
dV
E solsol Ι=
∆=
where Ι is the measured current and d is the distance between the two
electrodes.
3.4 Types of Experiments
Software accompanying the ASI Biosensor allows for three types of
tracking experiments to be performed, each having a minimum cycle time.
A tracking experiment in which the incoupling angles for a single mode
(TE+, TE-, TM+, TM-) are determined has a minimum cycle time of 2.9
seconds. The angle of autocollimation, from which the incoupling angles are
Figure 3.5: Flow cell and sensor chip depicted as a circuit. Current is measured across a 100 kΩ external resistor.
(3.1)
Volt- meter
V
Power supply
100 kΩ resistor
ITO film
Platinum
Stainless steel rod
Stainless steel rod
Solution inside flow cavity Rsol
RITO
Vapp
Rint
48
determined, is measured once at the beginning of the experiment. This is
not as accurate as measuring the angle at each scan since drift may occur
during an experiment. To calculate the surface density of protein adsorbed
onto the surface of the sensor chip, equation (2.14) may be used. However,
this expression requires a known value of the refractive index of the protein-
adsorbed layer. This type of experiment has the lowest cycle time and can
be useful when looking at trends involving short-time kinetics.
When performing a two mode or four mode tracking experiment, the
surface density of adsorbed protein can be determined with equation (2.13)
with calculated values of the refractive index and thickness of the protein-
adsorbed layer (from equation 2.11). A two mode (TE+ and TM+) or (TE-
and TM-) tracking experiment has a minimum cycle time of 13.7 seconds
with the angle of autocollimation being measured once, at the beginning of
the experiment. A four mode tracking experiment (TM ± and TE ± ) which
has a minimum cycle time of 23.5 seconds allows the angle of
autocollimation to be measured at each scan providing an even more
accurate measurement.
3.5 Experimental Procedure
The prepared sensor chip and flow cell is mounted into the
biosensing system. Pure solvent (DI water with no protein) is introduced
through the sensor chip/flow cell assembly at a rate of 1.33 x 10 –3 cm3/s.
The temperature of the external water bath is adjusted to maintain the body
49
of the flow cell at 25 ± 0.5 °C. Two types of tracking experiments are
performed. One set of data is obtained such that both effective refractive
indices, N(TE) and N(TM), are recorded at the minimum cycle time of 23.5 s.
A second set of data is obtained with N(TE) recorded at a minimum cycle
time of 2.9s. Once a stable baseline is achieved, a voltage is applied across
the electrodes. It is observed that when a potential is applied, the values of
N(TE) (and N(TM)) increase sharply. These values will either plateau,
reaching steady values within minutes, or gradually decrease reaching
steady values with an hour (the behavior depends on the applied potential).
Examples of this are given in section 4.1. After stable values are reached,
the protein solution is introduced into the system. Protein adsorption is
monitored for approximately 1 hour, after which deionized water is
introduced. If desorption is observed, it is monitored for approximately 15 -
20 minutes. Following completion of an experiment, the electrodes are
disconnected from the power supply and all surfaces, including the sensor
chip are cleaned.
Electric field studies are done at applied voltages of 0.0, 0.5, 1.0, 1.5
and 2.0 V. For each electric field experiment, the ITO film of the waveguide
acts as the anode, while the platinum electrode is the cathode.
3.6 Electrode Potential
In the above experiments, a potential is applied across the ITO and
platinum electrodes. To better understand the adsorption process, it is
50
desired to know the potential difference across the ITO/solution interface.
However, the potential difference across the interface cannot be measured
directly, but the potential of the ITO electrode relative to a reference
electrode can be measured with a high impedance voltmeter. Electrode
potentials are of interest since they are the governing parameter in
controlling electro-chemical reactions that can occur at the electrode
surface.
The potential of ITO electrode (and that of the platinum) is measured
with an electrometer (model 6514 from Keithley, Ohio, USA) relative to a
saturated gold reference electrode. Theoretically, there should be no
current flow through the reference electrode (current flow is restricted to the
ITO/platinum circuit). However, all potential detection systems are operated
by current. When current is passed through the reference electrode, an
error is induced in the measurement. To minimize this error, a high
impedance (resistance) voltmeter is used.
Potential measurements are done with a gold reference electrode
being placed in the solution reservoir, as shown in figure 3.6. Through the
inlet line leading into the flow cell, the reference electrode is in contact with
the solution near the working electrode (the electrode of interest) inside of
the flow cavity. Two gold reference electrodes, of the same surface area,
are utilized in these measurements. One electrode is placed in a solution
vial that contains deionized water (no protein) and the other is placed in a
reservoir that contains the protein solution. Each of the electrodes is
51
allowed to equalize in their respective solutions for approximately one half
hour before use. The experimental procedure outlined in section 3.5 is
implemented. When switching from water to the protein solution, the
electrometer is disconnected from the reference electrode contained in the
water vial and is reconnected to the reference electrode that is contained in
the protein solution reservoir.
Power supply
Figure 3.6: The potential of the ITO or platinum electrode measured relative to the reference with an electrometer. The external source establishes a current between the electrodes, and its effect on the potential difference of either of them relative to the reference electrode is observed. No current flows through the reference circuit.
Solution reservoir
Solution inside flow cavity
ITO film
EM
Gold reference electrode
Electro-meter Inlet line
Platinum
Reference circuit
Platinum/ITO circuit
52
4. Results and Discussion
4.1 Effect of Electric Field on Instrument Readings
Before examining protein adsorption, it is necessary to determine the
effect of an electric field on instrument readings. To accomplish this, the
refractive index of a glucose solution in both the presence and absence of
an applied field is measured. For each experiment, the flow cavity is initially
filled with deionized water of refractive index 1.33101 ± 1x10 -5 at 25 °C.
Glucose dissolved in deionized water at a concentration 5.0 x 10 -3 g/cm3
and refractive index 1.33173 ± 1x10 -5 at 25 °C (solution indexes
determined with an Abbey refractometer) is allowed to flow through the
channel at a rate of 1.33 x 10 –3 cm3/s. The refractive index of the solution,
at the surface of the sensor chip, versus time is shown in figure 4.1. The
steady state values of the refractive indices for the two experiments
(1.33173 ± 3 x 10 –5 when a potential of 0.0 V is applied and 1.33173 ± 5 x
10 –5 when 5.0 V is applied) are extremely close, indicating that the electric
field does not influence instrument readings. Additionally, the steady state
refractive index values measured at the surface of the sensor chip match
those determined by an Abbey Refractometer (1.33173 ± 1 x 10 –5).
The refractive indices for each of the experimental data sets are
determined with the instrument software. The software uses the mode
equations for planar dielectrics as described with equation (2.11). Since the
ITO film is extremely thin compared to the silicon-titanium dioxide layer, and
its conductivity is relatively low, the two layers are treated as one single film
53
layer in the calculation of film refractive index and thickness. Adsorption
measurements, using OWLS, rely on the incoupling and propagation of light
through the waveguiding film of the sensor chip as described in section
2.4.3. Since the penetration depth of the evanescent (or surface) wave into
the solution contacting the surface of the film is a few wavelengths, the
above verification applies to protein molecules as well as glucose.
Figure 4.1: Refractive index of a 5.0 x 10 –3 g/cm3 glucose solution
flowing through the channel at a rate of 1.33 x 10 – 3 cm3/s at 25 °C. The channel was initially filled with deionized water.
Time (s)
0 100 200 300 400 500 600 700
Ref
ract
ive
Inde
x
1.3310
1.3312
1.3314
1.3316
1.3318
1.3320
5.0 volts applied0.0 volts applied
54
4.2 Protein Adsorption: Transport Modes
In general, two distinct kinetic regimes (shown in figure 4.2) can
describe a protein adsorption curve, the transport-limited and adsorption-
limited regimes. In a flow experiment, protein molecules undergo convective
diffusion toward the surface. This is the rate limiting mechanism until a
critical concentration is established near the surface. As adsorption
proceeds, surface availability diminishes thus reducing the rate of
adsorption. When a significant fraction of the surface is covered, the rate of
protein attachment is equal to the rate of detachment and saturation is
reached.
Figure 4.2: Surface density of albumin adsorbed onto an ITO coated sensor chip when a potential of 0.0 V is applied.
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg/
cm2 )
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Adsorption limited region
Transport-limited regime
Approach to saturation
55
By recasting the data presented in figure 4.2 and plotting the
adsorption rate, ∂Γ/∂t, versus the adsorbed amount, Γ, of protein, the
different kinetic regimes are further distinguished. Figure 4.3 shows three
distinct regions: an initial transient transport-limited region, a linear region of
the adsorption-limited regime, and an asymptotic region of the adsorption-
limited regime.
Γ (µg/cm2)
0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35
dΓdt
(µg/
cm2 /s
)
-0.0002
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
Figure 4.3: Change in surface density with time versus surface density for albumin when a potential of 0.0 V is applied.
Transient transport- limited regime
Adsorption-limited regime (linear region)
Adsorption-limited regime (asymptotic region)
56
A transient region is observed during the very early stage of adsorption
because a stable concentration gradient has not yet been established
(Appendix C). Following sufficient adsorption to the surface, an adsorption-
limited regime is evident, where surface availability is the rate limiting
mechanism. In this regime the rate of adsorption, dΓ/dt, decreases linearly
with increasing surface density, Γ. This linear behavior is predicted by each
adsorption model discussed in section 2.3. When a significant fraction of
the surface covered, a non-linear approach to saturation is observed. In
section 4.3, experimental data for human albumin, cytochrome c, and apo-
transferrin are presented and an analysis of the affect of an applied potential
on the various regions of the adsorption curve is performed.
4.3 Protein Adsorption in an Applied Electric Field
Electric field studies are done at applied potentials of 0.0, 0.5, 1.0,
1.5, and 2.0 volts. For the case of an applied potential of 0.0 V, the two
electrodes are left as an open circuit. For each experiment, the ITO film of
the sensor chip acts as the anode and is the protein-adsorbing surface.
4.3.1 Adsorption Curves
Albumin
In figures 4.4 and 4.5, the adsorbed density versus time for human
albumin onto ITO coated waveguides A and B at applied potentials of 0.0,
0.5, 1.0, 1.5 and 2.0 volts is shown. At an applied potential of 0.0 V, the
57
adsorption curves plateau and saturation is reached within the time scale of
the experiment. At larger applied potentials, the adsorption curves no
longer reach saturation and the total amount of protein on each waveguide
is seen to increase with increasing applied voltage. It is also observed,
during the later stage of adsorption (t ≥ 1800 s), the slope of each
adsorption curve increases with increasing potential (i.e. the slopes rank
with applied voltage).
Albumin (Waveguide A)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
2.0 volts applied
0.5 volts applied1.0 volts applied
1.5 volts applied
0.0 volts applied
Figure 4.4: Surface density of albumin adsorbed onto waveguide A. Data is obtained (every 23.5 s) at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s.
58
Even though the trends mentioned above are similar for the adsorption of
albumin onto waveguides A and B, the absolute values of the surface
density differ. The variability between runs (less than 30%) is most likely
due to differences in the surface quality of each senor chip. From Appendix
B, it is observed that multiple runs on the same sensor chip yield far less
variability between runs.
Figure 4.5: Surface density of albumin adsorbed onto waveguide B. Data is obtained (every 2.9 s) at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s.
Albumin (Waveguide B)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg
/cm
2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
2.0 volts applied
1.5 volts applied
1.0 volts applied
0.5 volts applied
0.0 volts applied
59
Cytochrome c
In figures 4.6 and 4.7, the adsorbed density of cytochrome c onto the
ITO coated waveguides at applied potentials of 0.0, 0.5, 1.0, 1.5 and 2.0
volts is shown. At an applied potential of 0.0 V, the adsorption curves
plateau and saturation is reached with in the experimental time scale,
however, at larger applied potentials the adsorption curves no longer reach
saturation.
Figure 4.6: Surface density of cytochrome c adsorbed onto waveguide C. Data is obtained (every 23.5 s) at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s.
Cytochrome c (Waveguide C)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
2.0 volts applied
1.5 volts applied1.0 volts applied
0.5 volts applied
0.0 volts applied
60
On each waveguide, C and D, the total amount of adsorbed protein
increases with increasing applied potential with the exception of 0.5 volts.
On waveguide C, figure 4.6, the density of adsorbed protein is lower at an
applied potential of 0.5 volts than at 0.0 volts, while on waveguide D, figure
4.7, the density of adsorbed protein is higher at an applied potential of 0.5
volts than at 1.0 volt. Currently, no reason is known for the increase in
experimental error at the low applied potentials. During the later stage of
adsorption (t ≥1800 s), the slope of each adsorption curve generally
Figure 4.7: Surface density of cytochrome c adsorbed onto waveguide D. Data is obtained (every 2.9 s) at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s.
Cytochrome c (Waveguide D)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg
/cm
2 )
0.0
0.2
0.4
0.6
0.8
1.02.0 volts applied
1.5 volts applied
1.0 volts applied0.5 volts applied
0.0 volts applied
61
increases with increasing potential. As discussed previously, variations
between runs are expected when comparing data generated on two
separate sensor chips. However, at an applied potential of 0.5 V, the
variation between runs on waveguides C and D exceeds that what is
normally observed and appears to be specific for the case of cytochrome c.
Apo-Transferrin
In figure 4.8, the adsorbed density of apo-transferrin onto the ITO
coated waveguides at applied potentials of 0.0, 0.5, 1.0, and 2.0 volts is
shown.
Figure 4.8: Surface density of Apo-transferrin adsorbed onto Waveguide E. Data is obtained every 23.5 s.
Apo-Transferrin (Waveguide E)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
0.5 volts applied
1.0 volts applied
0.0 volts applied
2.0 volts applied
62
While, figure 4.9 shows adsorption at applied potentials of 0.0, 0.5, and 1.0
volts. At an applied potential of 0.0 V, the adsorption curves plateau and
saturation is reached within the time scale of the experiment. At larger
applied potential potentials, the adsorption curves no longer reach saturation
and the total amount of adsorbed protein (on each waveguide, E and F) is
seen to increase with increasing applied potential. Figures 4.8 and 4.9 also
show that during the later stage of adsorption (t ≥ 1800 s), the slope of each
curve increases with increasing potential (i.e. the slopes rank with applied
voltage). The variability between runs on waveguide E and F is less than
30%.
Figure 4.9: Surface density of Apo-transferrin adsorbed onto Waveguide F. Data is obtained every 23.5 s.
Apo-Transferrin (Waveguide F)
Time (s)
0 1000 2000 3000 4000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.0 volts applied
0.5 volts applied
0.0 volts applied
63
4.3.2 Transport-Limited Regime
In the absence of an applied electric field, in a flow experiment,
protein molecules undergo convective diffusion toward the surface. This is
the rate limiting mechanism until a critical concentration is established near
the surface. However, when adsorbing protein in the presence of an applied
electric field, forces resulting from the field act on the molecules. The
electric field forces acting on net negatively charged molecules are in the
vertical direction towards the surface, while those acting on a net positively
charged molecules are in the opposite direction. Any observed increase in
the rate of adsorption in this regime, for the case of negatively charged
proteins, indicates that the electric field forces are sufficient to enhance the
rate above that of concentration driven diffusion alone. Similarly, for
positively charged proteins, any observed decrease in rate would signify that
the electric field forces are sufficient to impede diffusion. When dissolved in
deionized water of pH of 5.5-6.0, albumin is net negatively charged,
cytochrome c is positively charged, and apo-transferrin is close to neutral.
To examine the affect of an applied potential on protein adsorption in
the transient transport-limited regime (as described in section 4.2) the data
presented in section 4.3 is recast. The adsorption rate, ∂Γ/∂t, as a function
of time, t, is plotted for values of t = 0 up to the function’s maximum (i.e. the
maximum value of ∂Γ/∂t). As seen from figure 4.10, the transient transport-
limited regime is described by an increase in the rate with time.
64
Albumin
In figure 4.11, the adsorption rate, ∂Γ/∂t, as a function of time for
albumin adsorbing onto waveguide B is shown. An increase in rate with
increasing applied potential is generally observed. This trend is seen for
adsorption on both waveguides A and B. From figure 4.11, the adsorption
curve obtained at an applied potential of 0.0 V show a difference in slope
when compared to those obtained at higher potentials. However, since the
adsorption data obtained for waveguide A is taken at 23.5 s intervals, there
are an insufficient number of data points to compare the slopes in this region
Figure 4.10: ∂Γ/∂t, as a function of time for albumin adsorbing onto waveguide B at an applied potential of 1.0 V.
Time (s)
0 50 100 150 200 250
dΓdt
(µg/
cm2 /s
)
0.000
0.001
0.002
0.003
0.004Maximum
Transient transport-limited region
Adsorption-limited regime
65
with those obtained for waveguide B (where data is taken at 2.9 s intervals).
Therefore, no inference regarding the slope can be made at this time.
Albumin (Waveguide B)
Time (s)
0 50 100 150 200
dΓ/d
t (µg
/cm
2 /s)
0.000
0.001
0.002
0.003
0.004
0.005
0.0062.0 volts applied1.5 volts applied 1.0 volts applied0.5 volts applied0.0 volts applied
Cytochrome c
In figure 4.12, the adsorption rate, ∂Γ/∂t, as a function time for
cytochrome c adsorbing onto waveguide D is shown. It is observed that the
initial adsorption kinetics is not greatly altered by an applied potential. This
Figure 4.11: Adsorption rate, ∂Γ/∂t, as a function time for albumin adsorbing onto waveguide B at applied potentials of 0.0, 0.5, 1.0, 1.5, and 2.0 V.
66
trend is seen for adsorption on both waveguides C and D. This result differs
from that of albumin, which shows an increase in the rate of adsorption with
increasing applied potential. From figure 4.12, the adsorption curves show
slight differences in slope. However, since the adsorption data obtained for
waveguide C is taken at 23.5 s intervals, there are an insufficient number of
data points to compare the slopes in this region with those obtained for
waveguide D (where data is taken at 2.9 s intervals). Therefore, no
inference regarding the slope can be made at this time.
Cytochrome c (Waveguide D)
Time (s)
0 20 40 60 80 100
dΓ/d
t (µg
/cm
2 /s)
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2.0 volts applied1.5 volts applied1.0 volts applied0.5 volts applied0.0 volts applied
Figure 4.12: Adsorption rate, ∂Γ/∂t, as a function time for cytochrome c adsorbing onto waveguide D at applied potentials of 0.0, 0.5, 1.0, 1.5, and 2.0 V.
67
Apo-Transferrin
In figure 4.13, the adsorption rate, ∂Γ/∂t, as a function time for
transferrin adsorbing onto waveguide E is shown. It is observed that the
initial adsorption kinetics is not greatly altered by an applied potential.
Since the adsorption data obtained for waveguides E and F are taken at
23.5 s intervals, there are an insufficient number of data points generate
slopes in this region. Therefore, no inference regarding the slope can be
made at this time.
Apo-Transferrin (Waveguide E)
Time (s)
0 50 100 150 200
dΓ/d
t (µg
/cm
2 /s)
0.000
0.001
0.002
0.003
0.004
0.005
2.0 volts applied1.0 volts applied0.5 volts applied0.0 volts applied
Figure 4.13: Adsorption rate, ∂Γ/∂t, as a function time for apo-transferrin adsorbing onto waveguide E at applied potentials of 0.0, 0.5, 1.0, and 2.0 V.
68
4.3.3 Linear Region of the Adsorption-Limited Regime
As discussed in section 4.3.2, during the very early stage of
adsorption, transport to the surface is the rate-limiting mechanism, which is
characterized by a continuous increase in the rate of adsorption. However,
when surface availability dominates, a decrease in rate is observed. From
plots of adsorption rate, ∂Γ/∂t, as a function of the adsorbed amount, Γ, of
protein, as presented in section 4.2, a linear decrease in the rate with
increasing surface coverage is evident.
Figure 4.14: Change in surface density of adsorbed protein with time versus
density. Apparent initial adsorption rate, ka (cm/s), is determined from the intercept of a line through the linear region of the adsorption-limited regime.
Γ (µg/cm2)
0.0 0.1 0.2 0.3 0.4 0.5 0.6
dΓ/d
t (µg
/cm
2 /s)
0.000
0.001
0.002
0.003
0.004
0.005kac
Transient transport – limited regime
Adsorption-limited regime (asymptotic region)
Adsorption-limited regime (linear region)
69
To examine the effect of an applied potential on adsorption in the
linear region of the adsorption-limited regime, an apparent initial adsorption
rate constant, ka, is determined. The apparent initial adsorption rate
constant is found by fitting a line, as predicted by Langmuir and other
models, to the linear region of the adsorption-limited regime (as shown in
figure 4.14) and extrapolating it to Γ = 0, where the intercept is kac and c is
the bulk concentration of protein [17].
Albumin
Figure 4.15: Change in surface density of adsorbed protein with time versus density for albumin onto waveguide B.
Albumin (Waveguide B)
Γ (µg/cm2)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
d Γ/d
t (µg
/cm
2 /s)
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2.0 volts applied1.5 volts applied1.0 volts applied0.5 volts applied0.0 volts applied
70
In figure 4.15, the adsorption rate, ∂Γ/∂t, as a function of the adsorbed
amount, Γ, of albumin onto waveguide B is shown. The apparent initial
adsorption rate constants, determined by the method previously described,
are seen to increase with increasing applied potential (the for the
experiments conducted on waveguides A and B are presented in table 4.1).
Cytochrome c
In figure 4.16, the adsorption rate, ∂Γ/∂t, as a function of the adsorbed
amount, Γ, of cytochrome c onto waveguide D is shown.
Figure 4.16: Change in surface density of adsorbed protein with time
versus density for cytochrome c onto waveguide D.
Cytochrome c (Waveguide D)
Γ (µg/cm2)
0.0 0.2 0.4 0.6 0.8 1.0
d Γ/d
t (µg
/cm
2 /s)
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2.0 volts applied1.5 volts applied1.0 volts applied0.5 volts applied
71
The apparent initial adsorption rate constants are not greatly altered by an
applied potential. This result differs from that of albumin, which shows an
increase in the rate constants with increasing applied potential. The values
of the apparent initial adsorption rate constants for the experiments
conducted on waveguides C and D are presented are table 4.1.
Apo-Transferrin
In figure 4.17, the adsorption rate, ∂Γ/∂t, as a function of the
adsorbed amount, Γ, of apo-transferrin on waveguide E is shown.
Figure 4.17: Change in surface density with time versus surface density for
apo-transferrin adsorbed onto waveguide E.
Apo-Transferrin (Waveguide E)
Γ (µg/cm2)
0.0 0.2 0.4 0.6 0.8 1.0
d Γ/d
t (µg
/cm
2 /s)
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
0.006
2.0 volts applied1.0 volts applied0.5 volts applied0.0 volts applied
72
The apparent initial adsorption rate constants are seen to increase (although
not as prominently as that of albumin) with increasing applied potential. The
values of the apparent initial rate constants for experiments conducted on
waveguides E and F are presented in table 4.1.
Table 4.1: Apparent Initial Adsorption Rate Constant, ka
Protein Applied Potential (V)
ka (cm / s)
Albumin (A) 0.0 1.1 x 10 -5 0.5 1.8 x 10 -5 1.0 3.2 x 10 -5 1.5 5.4 x 10 -5 2.0 1.1 x 10 -4 Albumin (B) 0.0 1.4 x 10 -5 0.5 3.0 x 10 -5 1.0 4.8 x 10 -5 1.5 3.4 x 10 -5 2.0 1.4 x 10 -4 Cytochrome c (C) 0.0 1.0 x 10 -4 0.5 4.1 x 10 -5 1.0 6.9 x 10 -5 1.5 9.2 x 10 -5 2.0 8.6 x 10 -5 Cytochrome c (D) 0.0 9.3 x 10 -5 0.5 8.8 x 10 -5 1.0 1.2 x 10 -4 1.5 8.8 x 10 -5 2.0 8.9 x 10 -5 Apo-Transferrin (E) 0.0 6.1 x 10 -5 0.5 9.3 x 10 -5 1.0 1.0 x 10 -4 2.0 8.1 x 10 -5 Apo-Transferrin (F) 0.0 2.4 x 10 -5 0.5 5.6 x 10 -5 1.0 7.5 x 10 -5
73
4.3.3 Asymptotic Adsorption Rate
To look at the affect of an applied potential on adsorption in the non-
linear asymptotic region of the adsorption–limited regime (as described in
section 4.2), the region of each adsorption curve (presented in section
4.3.1), is fit with the following two expressions for times, t ≥ 1800 s
where Γ(8) is the surface density of adsorbed protein when t? 8, kb is the
asymptotic rate constant and, ν is a time constant. When adsorbing protein
onto a solid surface it is observed experimentally and predicted theoretically,
that at long times, the surface density of protein asymptotically reaches a
steady state where the protein continues to adsorb and desorb. It has been
shown theoretically, by the Simple Particle Model, that an irreversible
approach to saturation is described by power law behavior (equation 4.2).
However, when desorption or surface diffusion is incorporated into the
model, the approach to saturation is described by the exponential function
(equation 4.1).
When examining the experimental data, surface density versus time
obtained for each of the three proteins for t ≥ 1800 s, it is observed that
equations 4.1 and 4.2 described the data equally well in terms of fit.
However, when applying equation 4.2 unrealistic large values of Γ(8) are
(4.1)
(4.2) ν−−∞Γ=Γ
ν−−∞Γ=Γ
tk)()t(
)texp(k)()t(
b
b
74
generated. By calculating the theoretical monolayer coverage, Γmonolayer =
mp / (d1d2), of a protein, (where mp is the mass of a single protein molecule
and d1 and d2 are the dimensions of the protein) and comparing this value to
those of obtained for Γ(8), several inference can be made. An analysis of
the adsorption curve (figure 4.5) for human albumin adsorbing onto
waveguige B at an applied potentials of 2.0 V will serve as an appropriate
example. The theoretical monolayer coverage for albumin is 0.2 µg/cm2,
where mp = 1.1 x 10 –13 µg, d1 = 1.5 x 10 –6 cm, and d2 = 3.8 x 10 –7 cm.
This value of Γmonolayer is in agreement with the saturation values seen with
the experimental results at an applied potential of 0.0 V. When fitting the
adsorption curve for albumin at an applied potential of 2.0 V, the power law
fit of the adsorption curve, for t ≥ 1800, gives a value of Γ(8) = 336.8 µg/cm2
where the exponential equation predicts Γ(8) = 1.7 µg/cm2. Even though
the results obtained with equations 4.1 and 4.2 both predict a surface
coverage that exceed the theoretical monolayer, it can be assumed that a
surface coverage exceeding that of a monolayer by four orders of magnitude
is unreasonable. Since the exponential equation gives realistic values of
Γ(8) that are in good agreement with the experimental data, it is concluded
that the approach to saturation is exponential in nature. For the adsorption
of albumin under applied potentials larger than 0.0 V, the values of Γ(8)
exceed that of the theoretical monolayer coverage. This is most likely due
to the formation of multiple layers, although at this time tighter packing of the
75
protein on the surface or changes in the protein’s orientation cannot be ruled
out as possible mechanisms. Table 4.2 lists the values of k, ν, and Γ(8)
obtained from the exponential fit of the adsorption data for each protein.
Table 4.2: Asymptotic Rate Constant, kb Γ(t) = Γ(8) - kbexp(-νt)
Protein Applied potential (V)
Γ(8) (µg/cm2)
kb (µg/cm2)
ν (1/s)
Alb. (A) 0.0 0.2573 0.0428 3.6 x 10 - 4 0.5 0.2632 0.0858 3.1 x 10 -4
1.0 0.6567 0.2871 2.3 x 10 -4
1.5 0.8414 0.4255 2.6 x 10 -4
2.0 1.7330 1.0850 2.7 x 10 -4
Alb. (B) 0.0 0.3414 0.661 1.3 x 10 -4
0.5 0.5128 0.2218 7.6 x 10 -5
1.0 1.0870 0.6947 5.7 x 10 -5
1.5 0.8095 0.4029 3.6 x 10 -4
2.0 1.7310 1.0400 3.8 x 10 -4
Cyt. (C) 0.0 0.4386 0.1230 1.1 x 10 -4
0.5 0.2467 0.0992 2.3 x 10 -4
1.0 0.4936 0.2437 2.7 x 10 -4
1.5 0.8761 0.5433 2.3 x 10 -4
2.0 1.5350 1.110 2.3 x 10 -4
Cyt. (D) 0.0 0.3584 0.1357 8.7 x 10 -4
0.5 0.5556 0.2876 3.1 x 10 -4
1.0 0.4853 0.2363 4.4 x 10 -4
1.5 0.9882 0.6345 2.5 x 10 -4
2.0 1.2430 0.8763 2.5 x 10 -4
Apo. (E) 0.0 0.3872 0.0900 4.1 x 10 -4
0.5 0.5331 0.2021 4.3 x 10 -4
1.0 0.7639 0.3822 3.4 x 10 -4
2.0 1.0820 0.6389 3.9 x 10 -4
Apo. (F) 0.0 0.2658 0.0612 4.4 x 10 -4
0.5 0.5514 0.2631 1.4 x 10 -4
1.0 0.6837 0.3219 2.4 x 10 -4
76
A plot of kb versus applied potential, presented in figure 4.18, shows
that within experimental error, the values of kb increases nearly linearly with
increasing applied potential. This result is unexpected since albumin,
cytochrome c and apo-transferrin differ considerably in their physical
properties as well as their biological function. This suggests, that during the
later stage of adsorption, the observed increase in density is independent of
the net charge of the protein.
Applied Potential (V)
0.0 0.5 1.0 1.5 2.0 2.5
k b ( µ
g/cm
2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Albumin Cytochrome cApo-Transferrin
Figure 4.14: The asymptotic rate constant, kb, for albumin, cytochrome
c, and apo-transferrin calculated for t ≥ 1800 s versus applied potential.
77
A plot of surface density, as time goes to infinity, versus applied
potential is presented in figure 4.19. The results show an increase in Γ(8)
with increasing applied potential.
4.3.5 Current versus Time During Adsorption
During the adsorption process, current through the circuit (described
in section 3.3.1) is monitored with respect to time. There is an observed
decrease in current with time when DI water (no protein) is present in the
flow cell as shown in figure 4.20. This phenomenon is observed for each
experiment at each of the applied potentials. This is most likely due to the
Figure 4.19: Surface density, as time goes to infinity, of albumin, cytochrome c, and apo-transferrin, versus applied potential.
Applied Potential (V)
0.0 0.5 1.0 1.5 2.0 2.5
Γh(µ
g/cm
2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
AlbuminCytochrome cApo-Transferrin
78
passivation of the ITO electrode. At the onset of adsorption, current may
increase or decrease depending upon the potential and the protein of study.
However, during the adsorption process, current continuously decreases for
each protein at each of the applied potentials.
Albumin
Figure 4.20 shows the surface density and current versus time for
albumin adsorbing at an applied voltage of 2.0 Volts. Prior to the onset of
adsorption, when DI water is in the flow cell, current decreases with time.
Albumin (Waveguide A)
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Cur
rent
(A)
0
1e-6
2e-6
3e-6
4e-6
5e-6DI Water Only Protein Solution
Figure 4.20: Density and current versus time for 1 x 10-4 g/cm3 albumin under a 2.0 V applied potential
79
At the onset of adsorption, a rapid decrease in current is observed followed
by a gradual decrease. The above effect is also observed at an applied
potential of 1.5 volts. However at applied potentials of 1.0 and 0.5 volts,
there is an increase in current at the onset of adsorption, after which current
continually decreases.
Current, as a function of time, during the adsorption of human
albumin (figures 4.4 and 4.5) at each of the applied potentials is presented
in figure 4.21. On waveguide A, at an applied potential of 2.0 volts, current
rapidly decreases (40%) during the first 600 s of adsorption after which the
decrease is slight (11%).
Figure 4.21: Current as a function of time during the adsorption process of albumin onto waveguides A and B.
Albumin
Time (s)
0 1000 2000 3000 4000
Cur
rent
(A)
0.0
5.0e-7
1.0e-6
1.5e-6
2.0e-6
2.5e-6
2.0 volts applied ( A, B)
1.0 volts applied ( A, B)
1.5 volts applied ( A, B)
0.5 volts applied ( A, B)
80
At an applied potential of 1.5 volts current changes little with time (10%
decreases from the onset of adsorption). However, at applied potentials of
1.0 and 0.5 volts, there is a slight increase in current at the onset of
adsorption (14% during the first 58s and 7% during the first 164s,
respectively), after which current continuously decreases (28% and 19%,
respectively). Original data is presented in Appendix D.
A similar trend is observed for experiments performed on waveguide
B. At an applied potential of 2.0 volts, current decreases rapidly (22%)
during the first 800 s of adsorption after which the decrease in current is
slight (8%). At an applied potential of 1.5 volts the current decreases from
the onset of adsorption is 18%. Again, when 1.0 and 0.5 volts is applied,
current increases (13% during the first 127s and 7% during the first 92s,
respectively) during the initial stage of adsorption, after which it continuously
decreases (49% and 33%, respectively).
Cytochrome c
Figure 4.22 shows surface density and current versus time for
cytochrome c adsorbing at an applied potential of 2.0 volts. Prior to the
onset of adsorption, when water is in the flow cell, current decreases with
time. When the protein solution is injected into the flow cell, current
increases at the onset of adsorption, then continually decreases throughout
the remainder of the experiment. This effect is observed at each of the
applied potentials.
81
Figure 4.23 shows current as a function of time, during the adsorption
of cytochrome c (figures 4.6 and 4.7) at each of the applied potentials. For
waveguide C, at an applied potential of 2.0 volts, current increases 20%
during the first 212 s of adsorption and then decreases, changing only 4%.
At applied potentials of 1.5, 1.0 and 0.5 volts, the increase in current from
the onset of adsorption is 0.3% during the first 212s, 52% during the first 70s
and 7% during the first 70s. After which current continuously decreases
(9%, 56%, and 23%, respectively).
Cytochrome c (Waveguide C)
Time (s)
0 2000 4000 6000 8000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4C
urre
nt (A
)
0
1e-6
2e-6
3e-6
4e-6DI Water Only Protein Solution
Figure 4.22: Density and current versus time for 1 x 10-4 g/cm3 cytochrome c under a 2.0 V applied potential
82
Similarly, for waveguide D, at an applied potential of 2.0 volts, current
increases 28% during the first 513 s of adsorption then decreases showing a
2% change from 513 s to the end run. At applied potentials of 1.5, 1.0 and
0.5 volts, there is a 17%, 14%, and 6% increase from the onset of
adsorption during the first 264s, 107s, and 118s, respectively. After which
current continuously decreases (13%, 35%, and 29%, respectively).
Original data is presented in Appendix D.
Figure 4.23: Current as a function of time during the adsorption process of cytochrome c onto waveguides C and D.
Cytochrome c
Time (s)
0 1000 2000 3000 4000
Cur
rent
(A)
0.0
5.0e-7
1.0e-6
1.5e-6
2.0e-6
2.5e-6
2.0 volts applied ( C, D)
1.0 volts applied ( C, D)
1.5 volts applied ( C, D)
0.5 volts applied ( C, D)
83
Apo-Transferrin
Figure 4.24 shows the surface density and current versus time for
apo-transferrin adsorbing at an applied potential of 2.0 Volts. Prior to the
onset of adsorption, when water is in the flow cell, current decreases with
time. When the protein solution is injected into the flow cell, current
increases at the onset of adsorption, then continually decreases throughout
the remainder of the experiment. This is observed at each of the applied
potentials.
Apo-Transferrin (Waveguide E)
Time (s)
0 1000 2000 3000 4000 5000 6000
Sur
face
Den
sity
(µg
/cm
2 )
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Cur
rent
(A)
0.0
5.0e-7
1.0e-6
1.5e-6
2.0e-6
2.5e-6DI Water Only Protein Solution
Figure 4.24: Density and current versus time for 1 x 10-4 g/cm3 apo-transferrin under a 2.0 V applied potential
84
Figure 4.25 shows current as a function of time for the adsorption of
apo-transferrin (figures 4.8 and 4.9) at each of the applied potentials. For
waveguide E, at an applied potential of 2.0 volts, current increases 10%
during the first 142 s of adsorption and then decreases, changing 11%. At
applied potentials of 1.0, and 0.5 volts, current increases from the onset of
adsorption (12% during the first 47s, and 3% during the first 94s,
respectively), after which current continuously decreases (35% and 29%,
respectively).
Figure 4.25: Current as a function of time during the adsorption process of apo-transferrin onto waveguides E and F.
Apo-Transferrin
Time (s)
0 1000 2000 3000 4000
Cur
rent
(A)
0
5e-7
1e-6
2e-6
2e-6
1.0 volts applied ( E, F)
2.0 volts applied ( E)
0.5 volts applied ( E, F)
85
For waveguide F, at applied potentials of 1.0, and 0.5 volts, current
increases 11% during the first 117s, and 5% during the first 95s of
adsorption. After which there is a continuous decrease in current, 31%, and
27%, respectively. Original data is presented in Appendix D.
As seen in figures 4.20, 4.22, and 4.24, at the onset of adsorption
current increases with time. This is followed by a steady decrease in current
flow. This phenomenon is observed for each protein at each of the applied
potentials with the exception of human albumin. At the higher applied
potentials (1.5 and 2.0 volts), a decrease in current flow is observed at the
onset of adsorption. This is of note since albumin is the only protein tested
that has a net charge opposite of that of the adsorbing surface and which an
applied potential has an impact on the initial adsorption rate.
4.3.6 Electrode Potentials
In the above experiments, a potential is applied across the ITO (the
protein adsorbing surface) and platinum electrodes. To better understand
the adsorption process, it is desired to know the potential difference across
the ITO/solution interface. However, the potential difference across the
interface cannot be measured directly, but the potential of the ITO electrode
relative to a reference electrode can be measured with a high impedance
voltmeter. Electrode potentials are of interest since they are the governing
parameter in controlling electro-chemical reactions that can occur at the
electrode surface.
86
At an applied potential 1.0V, the surface density of adsorbed protein
and the potential of the ITO electrode (with respect to a saturated gold
reference electrode) is recorded as a function of time. The potential of the
platinum electrode is given in Appendix D. For each experiment, prior to the
onset of adsorption, when deionized water (no protein) is in the flow cell, the
potential of the ITO electrode increases with time, while current decreases.
This result may be due to the passivation of the ITO electrode.
Albumin (Waveguide G)
Time (s)
0 500 1000 1500 2000 2500 3000 3500
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.1
0.2
0.3
0.4
Pot
entia
l of I
TO
(V
)
0.58
0.60
0.62
0.64
0.66
0.68
0.70
DensityPotential
Figure 4.26: Surface density of 1.0 x 10 –4 g/cm3 albumin adsorbed onto waveguide G. Data is obtained at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s. The potential of the ITO electrode is measured with respect to a gold reference electrode.
87
During the adsorption of human albumin, figure 4.26, a rapid
decrease in the ITO potential is observed during the first 500 s of
adsorption, after which the potential slowly decreases with time. Whether
the rapid decrease in potential is a real effect or a consequence of the
measurement is undetermined at this time. More experimental data is
needed to verify these findings. Current behavior is similar to that described
previously for an applied potential of 1.0 volts.
Figure 4.27: Surface density of 1.0 x 10 –4 g/cm3 cytochrome c adsorbed onto waveguide H. Data is obtained at 25 °C, at a flow rate of 1.33 x 10 –3 cm3/s. The potential of the ITO electrode is measured with respect to a gold reference electrode.
Cytochrome c (Waveguide H)
Time (s)
0 500 1000 1500 2000 2500 3000 3500 4000
Sur
face
Den
sity
(µg/
cm2 )
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Pot
entia
l of I
TO
(V
)
0.56
0.58
0.60
0.62
0.64
0.66
0.68
DensityPotential
88
During the adsorption of cytochrome c, figure 4.27, a rapid decrease
in potential is observed during the fist 500 s of adsorption, after which
potential is seen to increase with time. The rapid decrease during the early
stage of adsorption is also observed for the case of albumin. To verify this
affect is real and not a consequence of measurement, additional
experimental are required. Again, current behavior is similar to that
described previously. Current data is presented in Appendix D.
The method presented for measuring electrode potential is in the
preliminary stage of development, however, some generalization about the
experimental results can be made. During the adsorption of albumin and
cytochrome c at an applied potential of 1.0 V current, in general, decreases
with time. Figure 4.26 shows, during the adsorption of albumin, the
electrode potential, for the most part, also decreases with time. A decrease
in current with decreasing electrode potential is expected. However, figure
4.27 shows, a general increase in electrode potential during the adsorption
of cytochrome c. Since current is decreasing with time, this indicates that
some species may be poisoning the electrode.
The next phase of this ongoing research is to further refine the
system. By monitoring and maintaining the electrode potential it may be
possible to determine the reactions occurring at the electrode.
89
4.4 Discussion
In general, two kinetic regimes can describe a protein adsorption
curve, the transport-limited and adsorption-limited regimes. During the initial
stage of adsorption, when no electric field is applied, diffusion is the rate-
limiting step at short times. Following sufficient adsorption to the surface, an
adsorption-limited regime is evident. The adsorption-limited regime can be
divided into two regions, one in which a linear decrease in the adsorption
rate, dΓ/dt, with increasing surface density, Γ, is observed and a second
region in which a non-linear approach to saturation is seen. This discussion
is divided into three sections, which describes the affect of an applied
electric field on protein adsorption in the transport-limited regime and the
two regions of the adsorption-limited regime.
When adsorbing protein in the presence of an electric field, forces
resulting from the field act on the molecules. The magnitude of this force is
dependent on the field strength and the total charge of the molecule.
Experiments are conducted such that the electric field forces acting on a net
negatively charged protein molecules are in the vertical direction toward the
surface, while those acting on a net positively charged molecules are in the
opposite direction. Even though a protein molecule has a net charge
(positive, negative, or neutral), there are both negative and positively
charged groups on the molecule. Because of this, protein molecules will
also align or orient in an electric field due to torque (resulting from forces
acting on the charges through out the molecule).
90
Transient Transport-Limited Regime
The affect of an applied potential on protein adsorption in the
transport-limited regime, where transport to the surface is the rate limiting
mechanism, is analyzed in section 4.3.2. From this analysis, it is found that
the adsorption rates, ∂Γ/∂t, of albumin increases with increasing applied
potential, while the adsorptions rates of cytochrome c and apo-transferrin
are unaffected by the presence of an applied potential in this regime.
When dissolved in deionized water (pH of 5.5 – 6.0), albumin is net
negatively charged. The electric field forces acting on this protein are in the
vertical direction toward the surface. Since an increase adsorption rate is
observed in the transport-limited regime, it is concluded that the magnitude
of the electric field forces acting on this protein are sufficient to enhance the
rate of protein adsorption. Since in this regime the adsorption process is
transport limited, one can infer that electric field induced migration is acting
to increase the effective transport rate for albumin.
Cytochrome c is net positively charged at a pH of 5.5 – 6.0. The
electric field forces acting on this protein should be in the vertical direction
away from the surface, in a direction opposite that of concentration driven
diffusion. The results obtained for cytochrome c show the adsorption rates
are essentially unaffected by an applied electric field. Therefore, it is
concluded that the electric field forces acting on this protein are not sufficient
to impede concentration driven diffusion.
91
At a pH of 5.5 – 6.0, apo-transferrin is close to neutral (or slightly
negatively charged). The results presented in section 4.3.2, show no
noticeable affect on rates of adsorption of this protein. For the case of
neutral molecules it is expected that an applied electric field will have little or
no affect on the rate of adsorption in the transient transport-limited regime,
which is consistent with the experimental findings.
In the presence of an applied electric field, the behavior of albumin
and apo-transferrin in this transport-limited regime are, as one would expect.
However, the behavior of cytochrome c is somewhat surprising. To analyze
this region further, the ratio of the electric field forces to the diffusive forces
(in the absence of an applied electric field), for each of the three proteins at
each of the applied potentials, needs to be determined. To calculate the
electric field induced force acting on a protein molecule, on needs determine
to the strength of the applied field and the apparent charge of the protein
molecule in solution. While the electric field strength can easily be
estimated from current measurements and knowledge of the solution
conductivity, the effective charge of the protein needs to be evaluated
experimentally with electrophoretic measurements. To estimate the
magnitude of the diffusive forces acting on the protein in this initial regime,
one must know the diffusivity of the proteins as well as the concentration
gradients that exist within the flow cell. Theoretical models for the diffusion
of proteins in the initial phases of the adsorption are present by Van Tassel
et. al. [17]. A comparison of these forces may yield some explanation as to
92
why cytochrome c is not affected by the presence of an electric field in this
initial transport limited regime.
Linear Region of the Adsorption-Limited Regime
When surface availability is the rate-limiting mechanism, it is
observed experimentally and predicted theoretically, by Langmuir and other
models, that a linear decrease in the adsorption rate, ∂Γ/∂t, with increasing
adsorbed amounts of protein, Γ, occur. The adsorption rates in this region
are consistent with the protein interacting with the surface (i.e. through the
formation of attractive bonds between the protein and the surface. These
bonds may be physical or chemical in nature). The affect of an applied
potential on protein adsorption in the linear region of the adsorption-limited
regime, is analyzed in section 4.3.3. From this analysis, it is found that the
apparent initial adsorption rate constants, ka, obtained for albumin increase
with increasing applied potential, those obtained for apo-transferrin are only
a slight enhanced, and for cytochrome c, the apparent initial adsorption rate
constants are unaffected. Since surface adsorption is the rate-limiting step
in this region, as described by Langmuir kinetics, one can conclude that the
enhancement in initial rates observed with albumin are not due to electric
field induced migration, as was the case in the transport-limited regime. Of
the three proteins tested, albumin is the only negatively charged protein.
Since the adsorbing surface is positively charged, electrostatic attraction
93
appears to be a likely mechanism. However, surface reactions must also be
considered.
Appling a potential to an electrode affects a number of physical and
chemical properties of the electrode surface, as well as the chemistry of the
solution at the electrode interface. Among these affects are changes in the
hydrophobcity of the electrode surface, localized pH gradients that may
develop near the electrode surface, and the possibility of protein/surface
electron exchange, any of which could possibly have an impact on the
adsorption process. To determine whether the results obtained for albumin
are simply some form of electrostatic attraction or a specific electrochemical
reaction, testing of additional proteins that also have a net negative charge
may provide insight to the nature of the enhancement in initial rate.
Asymptotic Region of the Adsorption-Limited Regime
During the later stage of adsorption, when no potential is applied,
plots of surface density versus time, for each of the three proteins tested,
show an approach to saturation within the time scale of the experiments.
However, when adsorbing protein in the presence of an applied electric field,
the adsorption curves no longer plateau, and a continuous increase in
adsorbed amounts is observed. This is seen for each of the three proteins
at each applied potential (0.5 – 2.0 volts). To look at the affect of an applied
potential on adsorption in the non-linear asymptotic region adsorption-limited
regime, the region of each adsorption curve (presented in section 4.3.1) for t
94
≥ 1800s is analyzed. It has been shown theoretically, by the Simple Particle
Model, that a reversible approach to saturation is exponential and since the
experimental data fit the exponential equation (section 4.3.4, equation 4.1),
it is concluded that the adsorption process in this region is reversible in
nature. From the exponential fit, an asymptotic rate constant, kb, is
obtained. It is observed that the rate constant increases nearly linearly with
increasing applied potential. This behavior is seen with each of the three
proteins tested, despite their difference in chemistry and net charge. Thus
the increase in kb with increasing applied potential is not an affect of electric
field induced migration.
The increase in surface density resulting from an applied potential
may be due one of the following:
1. Tighter packing of the adsorbed protein.
2. A surface reaction with elements common to each of the three
proteins. It is the amino acid side chains that give rise to the unique
properties of proteins. Since it is determined that the later stage of
adsorption is unaffected by protein type (i.e. the differences arising
between each), a reaction could be occurring with elements common
to each protein such as the carboxyl or amino groups.
3. An orientation that favors a higher packing density of adsorbed
protein.
Even though the exact mechanism or factors that account for the observed
effects has not yet been identified, it is evident that an applied potential has
95
a profound affect on adsorption that is highly reproducible over the range of
proteins examined. The ability to deposit multiple and/or thicker protein
layers without higher concentrations, or long adsorption times may have
significant uses in a variety of industrial applications. It seems clear that
both surface density and possibly orientation are influenced by an applied
potential and/or the resulting electric field. The ability to control and alter
these properties would be of great use in creation of biomaterial coatings
and sensing surfaces. Based on the results obtained in this research, this
may be possible with the use of an applied electric field during adsorption.
One concern with this method is that the ITO layer could be changing
with time, as indicated by the measurements of current. At each applied
potential, when deionized water in the flow cell (no protein), there is an
observed decrease in current with time, indicating passivation of the ITO
electrode. At the onset of adsorption, for each of the three proteins tested,
there is an observed current increase at applied potentials of 0.5 and 1.0
volts followed by a continuous decrease. While at applied potentials of 1.5
and 2.0 volts, current may increase, and then gradually decrease
(cytochrome c and apo-transferrin) or it may show a continuous downward
trend (albumin). By measuring and maintaining the ITO electrode potential,
it may be possible to determine what reactions are occurring. Future work in
this area seems promising.
96
5. Conclusion
An important accomplishment here is the modification of an OWLS
biosensor for the continuous measurement of protein adsorption under an
applied electric field. Using this modified system, it is shown that an applied
potential significantly affects the adsorption process. It is found that in the
transient transport-limited regime, an applied potential has a significant
influence on the initial rate of adsorption for albumin, while cytochrome c
and apo-transferrin are unaffected in this region. This implies that, in the
transport-limited regime, the electric field forces acting on albumin are
sufficient to increase the rate of protein transport. In the linear region of the
adsorption-limited regime, it is found that the apparent initial adsorption rate
constants, ka, obtained for albumin increase with increasing applied
potential, those obtained for apo-transferrin are only slightly enhanced, and
for cytochrome c, the apparent initial adsorption rate constants are
unaffected. Given that adsorption in this region is governed by surface
availability, as described Langmuir kinetics, the observed increase in the
initial rate constants seen with albumin must be due to some type of reaction
and not to an increase in transport rate. During the later stage of
adsorption, the density of each of the three proteins tested is considerably
enhanced by the presence of an applied electric field. When a potential of
0.0 V is applied, saturation is reached within the experimental time scale.
However, in the presence larger applied voltages, a continuous increase in
adsorbed amounts of protein is observed. The approach to saturation (for
97
data obtained with and without an applied potential) is found to be
exponential indicating that the adsorption process in this region is not strictly
irreversible in nature. It is observed for each of the three proteins tested that
the asymptotic rate constants, kb, increase similarly with increasing applied
potential. This indicates that the increase in surface density with increasing
applied potential is independent of the protein’s net charge. Kinetic data
such as these are useful for designing electric field methods of controlled
protein-surface placement.
5.1 Future Directions
• Since deionized water is the protein solvent for this work, a pH
gradient near the ITO surface will form. OH- ions will migrate
toward the ITO electrode and may cause the pH near the surface
to be much greater than 5.5-6.0. This would result in albumin,
cytochrome c, and apo-transferrin to be more negatively charged.
Repeating the adsorption experiments presented here with the
proteins being dissolved in a buffer solution (if possible) will
minimize the formation of a pH gradient near the electrode
surface. If the effects described above are due to the presence of
a pH gradient, then testing a buffer solution should have a
significant impact on the experimental findings. However, it is
noted that the use of a buffer solution may reduce electric field
strength, as discussed in section 2.5.
98
• Measure electrode potential during adsorption. Testing at a
constant electrode potential, rather than a constant applied
voltage, may provide insight into protein/surface interactions. In
addition, electrode potentials may prove to be a less system
dependent parameter for the scaling the observed effects than
applied voltage.
• The physical evaluation of surface morphology by techniques
such as AFM may be used to determine the influence of an
applied potential on the final state of the protein-adsorbed layer.
Questions regarding protein packing, orientation, and the
formation of multiple adsorbed layers on the electrode surface
may be answered.
• Examine protein adsorption under cathodic polarization of the
electrode surface rather than anodic. Since prolonged cathodic
polarization changes the optical properties of the ITO electrode,
making it unsuitable for OWLS experiments, other electrodes
needs to be investigated.
99
Appendix A
A.1 Scaled Particle Theory
Scaled Particle Theory is based on an approximate expression for the
work of adding a single solute hard sphere particle of radius R to a system
of hard sphere particles. For the case of protein adsorption, the system of
hard sphere particles is the surface adsorbed proteins. A protein molecule,
from the bulk, will be able to adsorb (be inserted into the system of hard
sphere particles) to the surface if space is made available.
Scaled Particle Theory relates the reversible work required to create
a cavity of radius R that is free from any part of any particle, W(R), to the
probability of finding such a cavity in the equilibrated system Po(R)
where β is the reciprocal of Boltzmann constant times the absolute
temperature. For a 2-D binary mixture of circular particles of radii Rα and Rβ
and densities ρα and ρβ, the value of P0 is known exactly for R=0 and can be
approximated as a power series in R for R>0
[ ]
0RRPR)0(W)0(W
0R)RR()RR(1ln)R(W
2
22
>πβ+′β+β=
≤ρ+π−ρ+π−−=β ββαα
(A.2)
)R(Pln)R(W o−=β (A.1)
100
where P is the pressure of the 2-D binary disk mixture. (Note that a cavity of
negative radius may be thought of as a point that may be approached by a
particle center up to a threshold distance that is less than the particle
radius.)
To obtain W to second order in R, P must be determined. This is
done by noting that the excess chemical potential of species α and β are just
the reversible work required to create cavities of size Rα and Rβ,
respectively: µαex=W(Rα) and µβ
ex =W(Rβ). By differentiating each of these
quantities with respect to ρα, employing the following form of the Gibbs-
Duhem equation,
α
ββ
α
αα
α ∂ρ∂βµ
ρ+∂ρ
∂βµρ=
∂ρ∂β exexexP
solving for the derivative of the excess pressure, and integrating with
respect to ρα, one obtains
[ ]222 RR1
)RR(P
ββαα
βααββα
ρπ−ρπ−
ρρ−π−ρ+ρ=β
The expression obtained for βP is then inserted into equation (A.2) for R>0
to obtain
(A.4)
(A.4)
101
The adsorption probability of the spreading particle model is defined as the
probability of finding a cavity of radius Rα, where Φα = Po(Rα) = exp (-
W(Rα)). The spreading probability is determined as the conditional
probability of finding a cavity of radius Rβ given that a particle of Rα exists at
its center, where Ψαβ = Po(Rβ)/ Po(Rα).
[ ] [ ]
[ ][ ]222
22
2222
RR1
)RR(R
RR1
RRR2RR1ln)R(W
ββαα
βααββα
ββαα
ββααββαα
ρπ−ρπ−
ρρ−π+ρ+ρπ+
ρπ−ρπ−
ρ+ρπ+ρπ−ρπ−−=β (A.5)
102
Appendix B
B.1 Sensor Chip Cleaning
The cleaning procedure for the ITO coated sensor chips is derived
from that of the ASI (type 2400) chips. New ASI (type 2400) sensor chips
are soaked in 0.1 N HCL for 10 minutes then rinsed in deionized water. This
procedure is done only once to remove any residue that may have resulted
from the packing material (i.e. the sensor chips are packaged in a Styrofoam
casing when shipped). After this procedure, the sensor chip is placed in an
ultrasonic bath, containing cleaning solution, at a frequency of 55k Hz for 20
minutes and then rinsed extensively with deionized water. A cleaning
solution at a concentration of 1.0 x 10 - 2
g/cc is prepared by dissolving Terg-
A-Zyme in deionized water.
After a protein adsorption experiment, the ASI type-2400 sensor chip
is cleaned in the ultrasonic bath containing a solution of Terg-A-zyme at the
above mention concentration for 20 minutes, and then rinsed extensively
with deionized water. To determine the effect of the cleaning procedure on
the ASI (type 2400) sensor chip, several experiments are performed. A
sensor chip is placed into the biosensor where the refractive index and
thickness of the film are measured. A 1.0X10 – 4 g/cm3 solution of human
fibrinogen is allowed to flow over the surface of the sensor chip for 30
minutes at a flow rate of 1.33 x 10 – 3 cm3/s and at a temperature of 25 °C.
The chip is then removed from the biosensor and cleaned. After cleaning is
complete, the sensor chip is soaked in the protein solvent (a buffer solution
103
without protein) over night. Three separate experiments are conducted on
the same chip. After each test, a 0.4% decrease in film thickness is
observed. When comparing the adsorption curves, N(TM) versus time,
shown in figure B.1, it is observed that the overall shape of each curve as
well as the total amount of adsorbed protein is not affected by the decrease
in film thickness. While no correlation between experimental error and
number of runs has been identified, it is assumed that after a sufficient
decrease in film thickness the sensor chip will become unusable. This
number of runs has not yet been determined.
Multiple Runs on Same Waveguide
Time (s)
0 1000 2000 3000 4000
∆ N(T
M+)
0.0000
0.0002
0.0004
0.0006
0.0008
0.0010
0.0012
0.0014
0.0016
0.0018
Run1Run 2Run 3
Figure B.1: Fibrinogen, 1 X 10 –4 g/cm3 adsorbed onto a Si0.25Ti0.75O2 film at a flow rate of 1.33 x 10 –3 cm3/s at 25 °C. The cleaning procedure in the text is applied between each run.
104
Due to these results, a similar cleaning procedure for the ITO coated
sensor chips (Section 3.2.2) is implemented.
B.2 Sensor Chip Soaking
By experimental observation it is determined that ASI type-2400
sensor chips need to be soaked for several hours prior to use. A sensor
chip that has not been soaked is placed into the biosensor. Deionized water
is allowed to flow over the surface of the sensor chip at a rate of 1.33 x 10 – 3
cm3/s and at a temperature of 25 °C.
Time (s)
0 2000 4000 6000 8000 10000 12000
N(T
E)
1.57860
1.57862
1.57864
1.57866
1.57868
1.57870
1.57872
Figure B.2: The effective refractive index, N(TE), measured with time
for deionized water flowing at a rate of 1.33 x 10 – 3 cm3/s and at a temperature of 25 °C.
105
Experiments are performed by measuring the effective refractive indices
N(TM) or N(TE) with time. As shown in figure B.2, it takes several hours for
the effective refractive indices to reach steady state values. Jeremy
Ramsden, of the University of Basle, has also observed this phenomenon.
He attributes this effect to the waveguiding film being porous [22, 30].
Because of these findings, the ITO coated sensor chips are soaked in
deionized water (the protein solvent for this work) for several hours prior to
use.
106
Appendix C
C.1 Fluid Flow
The flow cell of the biosensor allows liquid to be brought in contact
with the film surface of a sensor chip. The flow cell is sealed to the surface
of the sensor chip to create a flow cavity, as described in Section 3.2.4.
Transport limitations are observed inside the cavity when one fluid is
switched with another, as illustrated by the following experiment. At the
beginning of the experiment, the flow channel is filled with deionized water
of refractive index 1.331012 ± 1x10-5 at 25 C°. Glucose dissolved in
deionized water at a concentration 5.0 x 10 – 3 g/cm3 and refractive index
1.33173 ± 1x10-5 at 25 C° (solution indexes measured at 632.8 nm with an
Abbey refractometer, modified by Leica Microsystems, IL, USA) is then
allowed to enter the channel at a rate of 1.33 x 10 – 3 cm3/s. The refractive
index of the solution, at the surface of the sensor chip, is measured with
time.
As seen in Figure C.1, it takes approximately 200 s for the glucose
solution, at the surface of the chip, to reach a maximum refractive index
value. At times t<200 s, the concentration of glucose at the surface of the
chip is transient. At times t > = 200 s, the concentration is that of the bulk.
This observation is attributed to transient diffusion and/or to incomplete
mixing.
Experimental data is compared to refractive index values calculated
for an ideally mixed system where ( )( )Vtexp1CC o ν−−= .
107
C is the concentration of glucose as a function of time, Co is the bulk
concentration,ν is the volumetric flow rate, and V is the volume occupied by
the glucose solution inside of the flow channel. There is a linear relationship
between concentration (0 to 20 x 10 – 2 g/cm3) and the corresponding
measured refractive index values (determined with an Abbey refractometer).
From a plot of refractive index, RI, versus glucose concentration (g/cm3) it is
found that ( ) 1000/1.9357RI1.7030C −= .
Time (s)
0 200 400 600 800 1000
Ref
ract
ive
Inde
x
1.3310
1.3312
1.3314
1.3316
1.3318
ExperimentalIdeal Mixing
Figure C.1: Experimental data of the refractive index of a 5.0 x 10 - 3 g/cm3 glucose solution flowing through the channel at a rate of 1.33 x 10 - 3 cm3/s at 25 °C versus an ideally mixed system.
108
This procedure is repeated for the case of no applied voltage using
different inlet line lengths: 17.3, 22.3, and 25.3 cm. The ratio of the
residence time of the sample inside of the flow channel (52.6 s) to the
residence times of sample in the tubing leading into the flow channel are
1:1.13, 1:1.45, 1:1.65 for the respective lengths of the inlet line. Figure C.2,
shows that changing the length of the inlet line shows no significant effect on
the time it takes for the glucose solution to reach a maximum refractive
index values (i.e. that of the bulk).
Time (s)
0 100 200 300 400 500
Ref
ract
ive
Inde
x
1.3310
1.3312
1.3314
1.3316
1.3318
17.3 cm22.3 cm25.3 cm
Figure C.2: Experimental data of the refractive index of a 5.0 x 10 - 3 g/cm3
glucose solution flowing through the channel at a rate of 1.33 x 10 - 3 cm3/s at 25 °C for various inlet line lengths.
109
Appendix D
D.1 Adsorption Data
D.1.1 Human Albumin (Waveguide A)
Figures D.1 through D.5 show the effective refractive indices, N(TE)
and N(TM), as a function of time for the adsorption of 1.0 x 10-4 g/cm3
human albumin onto an ITO coated sensor chip. Data is obtained every
23.5 s at 25°C and at a flow rate of 1.33 x 10-3 cm3/s.
Figure D.1: Effective refractive indices of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide A when no voltage is applied to the electrodes. At t= 300 s, the protein solution enters the flow cell. At t=3900 s, a DI water rinse is initiated.
N(TE)N(TM)
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5710
1.5712
1.5714
1.5716
1.5718
1.5720
N(T
M)
1.5446
1.5448
1.5450
1.5452
1.5454
1.5456
N (TE) N (TM)
110
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5714
1.5716
1.5718
1.5720
1.5722
1.5724
1.5726
N(T
M)
1.5448
1.5450
1.5452
1.5454
1.5456
1.5458
1.5460
N(TE)N(TM)
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5710
1.5715
1.5720
1.5725
1.5730
1.5735
N(T
M)
1.5445
1.5450
1.5455
1.5460
1.5465
1.5470
N(TE) N(TM)
Figure D.2: Effective refractive indices of 1 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide A. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5520 s, a DI water rinse is initiated.
Figure D.3: Effective refractive indices of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide A. At t=360 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
111
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5735
1.5740
1.5745
1.5750
1.5755
1.5760
1.5765
N(T
M)
1.5460
1.5465
1.5470
1.5475
1.5480
1.5485
1.5490
N(TE) N(TM)
Time (s)
0 2000 4000 6000 8000
N(T
E)
1.570
1.571
1.572
1.573
1.574
1.575
1.576
N(T
M)
1.544
1.545
1.546
1.547
1.548
1.549
1.550
N(TE) N(TM)
Figure D.4: Effective refractive indices of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide A. At t=300 s, 1.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.5: Effective refractive indices of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide A. At t=300 s, 2.0 volts is applied to the electrodes. At t=2400 s, the protein solution enters the flow cell. At t=6000 s, a DI water rinse is initiated.
112
D.1.2 Human Albumin (Waveguide B)
Figures D.6 through D.10 show the effective refractive indices, N(TE),
as a function of time for the adsorption of 1.00 x 10-4 g/cm3 human albumin
onto an ITO coated sensor chip. Data is every obtained 2.9 s (except for
figure D.6 for which data is obtained every 23.5 s) at 25°C and at a flow rate
of 1.33 x 10-3 cm3/s.
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5690
1.5692
1.5694
1.5696
1.5698
1.5700
1.5702
N(T
M)
1.5438
1.5440
1.5442
1.5444
1.5446
1.5448
1.5450
N(TE)N(TM)
Figure D.6: Effective refractive indices of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide B when no voltage is applied to the electrodes. At t= 600 s, the protein solution enters the flow cell. At t=4200 s, a DI water rinse is initiated.
113
Time (s)
0 1000 2000 3000 4000 5000
N(T
E)
1.5696
1.5698
1.5700
1.5702
1.5704
1.5706
1.5708
1.5710
1.5712
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5685
1.5690
1.5695
1.5700
1.5705
1.5710
Figure D.8: Effective refractive index of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide B. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.7: Effective refractive index of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide B. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell.
114
Time (s)
0 2000 4000 6000 8000
N(T
E)
1.5695
1.5700
1.5705
1.5710
1.5715
1.5720
1.5725
Time (s)
0 2000 4000 6000 8000
N(T
E)
1.571
1.572
1.573
1.574
1.575
1.576
Figure D.9: Effective refractive index of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide B. At t=300 s, 1.5 volts is applied to the electrodes. At t=2280 s, the protein solution enters the flow cell. At t=5880 s, a DI water rinse is initiated.
Figure D.10: Effective refractive index of 1.0 x 10 – 4 g/cm3 human albumin adsorbing onto waveguide B. At t=300 s, 2.0 volts is applied to the electrodes. At t=2880 s, the protein solution enters the flow cell. At t=6480 s, a DI water rinse is initiated.
115
D.1.3 Cytochrome c (Waveguide C)
Figures D.11 through D.15 show the effective refractive indices,
N(TE) and N(TM), as a function of time for the adsorption of 1.0 x 10-4 g/cm3
cytochromce c onto an ITO coated sensor chip. Data is obtained every 23.5
s at 25°C and at a flow rate of 1.33 x 10-3 cm3/s.
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5690
1.5692
1.5694
1.5696
1.5698
1.5700
1.5702
1.5704
N(T
M)
1.5434
1.5436
1.5438
1.5440
1.5442
1.5444
1.5446
1.5448
N(TE)N(TM)
Figure D.11: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide C when no voltage is applied to the electrodes. At t= 600 s, the protein solution enters the flow cell. At t=4200 s, a DI water rinse is initiated.
116
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5702
1.5704
1.5706
1.5708
1.5710
1.5712
N(T
M)
1.5444
1.5446
1.5448
1.5450
1.5452
1.5454
N(TE)N(TM)
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5692
1.5696
1.5700
1.5704
1.5708
1.5712
N(T
M)
1.5436
1.5440
1.5444
1.5448
1.5452
1.5456
N(TE) N(TM)
Figure D.12: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide C. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.13: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide C. At t=240 s, 1.0 volts is applied to the electrodes. At t=1740 s, the protein solution enters the flow cell. At t=5340 s, a DI water rinse is initiated.
117
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5700
1.5705
1.5710
1.5715
1.5720
1.5725
1.5730
N(T
M)
1.5445
1.5450
1.5455
1.5460
1.5465
1.5470
1.5475
N(TE) N(TM)
Time (s)
0 2000 4000 6000 8000 10000
N(T
E)
1.569
1.570
1.571
1.572
1.573
N(T
M)
1.543
1.544
1.545
1.546
1.547
N(TE) N(TM)
Figure D.14: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide C. At t=300 s, 1.5 volts is applied to the electrodes. At t=2100 s, the protein solution enters the flow cell. At t=5700 s, a DI water rinse is initiated.
Figure D.15: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide C. At t=360 s, 2.0 volts is applied to the electrodes. At t=3900 s, the protein solution enters the flow cell. At t=7500 s, a DI water rinse is initiated.
118
D.1.4 Cytochrome c (Waveguide D)
Figures D.16 through D.20 show the effective refractive indices,
N(TE), as a function of time for the adsorption of 1.0 x 10-4 g/cm3
cytochrome c onto an ITO coated sensor chip. Data is obtained every 2.9 s
(except for figure D.16 for which data is obtained every 23.5 s) at 25°C and
at a flow rate of 1.33 x 10-3 cm3/s.
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5702
1.5704
1.5706
1.5708
1.5710
1.5712
1.5714
N(T
M)
1.5440
1.5442
1.5444
1.5446
1.5448
1.5450
1.5452
N(TE)N(TM)
Figure D.16: Effective refractive indices of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide D when no voltage is applied to the electrodes. At t= 600 s, the protein solution enters the flow cell. At t=4200 s, a DI water rinse is initiated.
119
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5720
1.5722
1.5724
1.5726
1.5728
1.5730
1.5732
1.5734
1.5736
1.5738
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5710
1.5712
1.5714
1.5716
1.5718
1.5720
1.5722
1.5724
1.5726
1.5728
1.5730
1.5732
Figure D.17: Effective refractive index of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide D. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.18: Effective refractive index of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide D. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
120
Time (s)
0 2000 4000 6000 8000
N(T
E)
1.5710
1.5715
1.5720
1.5725
1.5730
1.5735
1.5740
Time (s)
0 2000 4000 6000 8000 10000 12000 14000
N(T
E)
1.5705
1.5710
1.5715
1.5720
1.5725
1.5730
1.5735
Figure D.19: Effective refractive index of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide D. At t=300 s, 1.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.20: Effective refractive index of 1.0 x 10 – 4 g/cm3 cytochrome c adsorbing onto waveguide D. At t=300 s, 2.0 volts is applied to the electrodes. At t=8400 s, the protein solution enters the flow cell. At t=12000 s, a DI water rinse is initiated.
121
D.1.5 Apo-Transferrrin (Waveguide E)
Figures D.21 through D.24 show the effective refractive indices,
N(TE) and N(TM), as a function of time for the adsorption of 1.0 x 10- 4 g/cm3
apo-transferrin onto an ITO coated sensor chip. Data is obtained every 23.5
s at 25°C and at a flow rate of 1.33 x 10-3 cm3/s.
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5696
1.5698
1.5700
1.5702
1.5704
1.5706
1.5708
N(T
M)
1.5434
1.5436
1.5438
1.5440
1.5442
1.5444
1.5446
N(TE)N(TM)
Figure D.21: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide E when no voltage is applied to the electrodes. At t= 600 s, the protein solution enters the flow cell. At t=4200 s, a DI water rinse is initiated.
122
Time (s)
0 2000 4000 6000 8000
N(T
E)
1.5700
1.5704
1.5708
1.5712
1.5716
1.5720
1.5724
N(T
M)
1.5436
1.5440
1.5444
1.5448
1.5452
1.5456
1.5460
N(TE)N(TM)
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5755
1.5760
1.5765
1.5770
1.5775
1.5780
1.5785
N(T
M)
1.5465
1.5470
1.5475
1.5480
1.5485
1.5490
1.5495
N(TE) N(TM)
Figure D.22: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide E. At t=300 s, 0.5 volts is applied to the electrodes. At t=2400 s, the protein solution enters the flow cell. At t=6000 s, a DI water rinse is initiated.
Figure D.23: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide E. At t=300 s, 1.0 volts is applied to the electrodes. At t=1860 s, the protein solution enters the flow cell. At t=5460 s, a DI water rinse is initiated.
123
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5690
1.5695
1.5700
1.5705
1.5710
1.5715
1.5720
1.5725
1.5730
N(T
M)
1.5430
1.5435
1.5440
1.5445
1.5450
1.5455
1.5460
1.5465
1.5470
N(TE) N(TM)
D.1.6 Apo-Transferrin (Waveguide F)
Figures D.25 through D.27 show the effective refractive indices,
N(TE) and N(TM), as a function of time for the adsorption of 1.00 x 10-4
g/cm3 apo-transferrin onto an ITO coated sensor chip. Data is obtained
every 23.5 s at 25°C and at a flow rate of 1.33 x 10-3 cm3/s.
Figure D.24: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide E. At t=300 s, 2.0 volts is applied to the electrodes. At t=2100 s, the protein solution enters the flow cell. At t=5700 s, a DI water rinse is initiated.
124
Time (s)
0 1000 2000 3000 4000 5000 6000
N(T
E)
1.5720
1.5722
1.5724
1.5726
1.5728
1.5730
N(T
M)
1.5454
1.5456
1.5458
1.5460
1.5462
1.5464
N(TE)N(TM)
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5732
1.5736
1.5740
1.5744
1.5748
1.5752
N(T
M)
1.5460
1.5464
1.5468
1.5472
1.5476
1.5480
N(TE)N(TM)
Figure D.26: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide F. At t=600 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.25: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide F. No voltage is applied to the electrodes. At t= 600 s, the protein solution enters the flow cell. At t=4200 s, a DI water rinse is initiated.
125
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5710
1.5715
1.5720
1.5725
1.5730
1.5735
1.5740
N(T
M)
1.5445
1.5450
1.5455
1.5460
1.5465
1.5470
1.5475
N(TE) N(TM)
D.2 Current
D.2.1 Human Albumin (Waveguide A)
Figures D.28 through D.31 show current as a function of time during
the adsorption of 1.0 x 10 – 4 g/cm3 human albumin onto an ITO coated
sensor chip.
Figure D.27: Effective refractive indices of 1.0 x 10 – 4 g/cm3 apo-transferrin adsorbing onto waveguide F. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
126
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
1.0e-8
2.0e-8
3.0e-8
4.0e-8
5.0e-8
6.0e-8
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A)
4.0e-8
6.0e-8
8.0e-8
1.0e-7
1.2e-7
1.4e-7
1.6e-7
Figure D.28: Current versus time during the adsorption of human albumin onto waveguide A. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5520 s, a DI water rinse is initiated.
Figure D.29: Current versus time during the adsorption of human albumin onto waveguide A. At t=360 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
127
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
2.5e-7
3.0e-7
3.5e-7
4.0e-7
4.5e-7
5.0e-7
Time (s)
0 2000 4000 6000 8000
Cur
rent
(A
)
1.0e-6
1.5e-6
2.0e-6
2.5e-6
3.0e-6
3.5e-6
4.0e-6
4.5e-6
5.0e-6
Figure D.30: Current versus time during the adsorption of human albumin onto waveguide A. At t=300 s, 1.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.31: Current versus time during the adsorption of human albumin onto waveguide A. At t=300 s, 2.0 volts is applied to the electrodes. At t=2400 s, the protein solution enters the flow cell. At t=6000 s, a DI water rinse is initiated.
128
D.2.2 Human Albumin (Waveguide B)
Figures D.32 through D.35 show current as a function of time during
the adsorption of 1.0 x 10 - 4 g/cm3 human albumin onto an ITO coated
sensor chip.
Time (s)
0 1000 2000 3000 4000 5000 6000
Cur
rent
(A)
0.0
2.0e-8
4.0e-8
6.0e-8
8.0e-8
1.0e-7
Figure D.32: Current versus time during the adsorption of human albumin onto waveguide B. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell.
129
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
0.0
1.0e-7
2.0e-7
3.0e-7
4.0e-7
Time (s)
0 2000 4000 6000 8000
Cur
rent
(A)
2.0e-7
3.0e-7
4.0e-7
5.0e-7
6.0e-7
7.0e-7
8.0e-7
Figure D.33: Current versus time during the adsorption of human albumin onto waveguide B. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.34: Current versus time during the adsorption of human albumin onto waveguide B. At t=300 s, 1.5 volts is applied to the electrodes. At t=2280 s, the protein solution enters the flow cell. At t=5880 s, a DI water rinse is initiated.
130
Time (s)
0 2000 4000 6000 8000
Cur
rent
(A
)
8.0e-7
1.2e-6
1.6e-6
2.0e-6
2.4e-6
2.8e-6
3.2e-6
D.2.3 Cytochrome c (Waveguide C)
Figures D.36 through D.39 show current as a function of time during
the adsorption of 1.0 x 10 - 4 g/cm3 cytochromce c onto an ITO coated
sensor chip.
Figure D.35: Current versus time during the adsorption of human albumin onto waveguide B. At t=300 s, 2.0 volts is applied to the electrodes. At t=2880 s, the protein solution enters the flow cell. At t=6480 s, a DI water rinse is initiated.
131
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
2.0e-8
4.0e-8
6.0e-8
8.0e-8
1.0e-7
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A)
0.0
1.0e-7
2.0e-7
3.0e-7
4.0e-7
5.0e-7
6.0e-7
Figure D.37: Current versus time during the adsorption of cytochrome c onto waveguide C. At t=240 s, 1.0 volts is applied to the electrodes. At t=1740 s, the protein solution enters the flow cell. At t=5340 s, a DI water rinse is initiated.
Figure D.36: Current versus time during the adsorption of cytochrome c onto waveguide C. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
132
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A)
3.0e-7
3.5e-7
4.0e-7
4.5e-7
5.0e-7
5.5e-7
6.0e-7
Time (s)
0 2000 4000 6000 8000 10000
Cur
rent
(A
)
1.0e-6
1.5e-6
2.0e-6
2.5e-6
3.0e-6
3.5e-6
4.0e-6
Figure D.38: Current versus time during the adsorption of cytochrome c onto waveguide C. At t=300 s, 1.5 volts is applied to the electrodes. At t=2100 s, the protein solution enters the flow cell. At t=5700 s, a DI water rinse is initiated.
Figure D.39: Current versus time during the adsorption of cytochrome c onto waveguide C. At t=360 s, 2.0 volts is applied to the electrodes. At t=3900 s, the protein solution enters the flow cell. At t=7500 s, a DI water rinse is initiated.
133
D.2.4 Cytochrome c (Waveguide D)
Figures D.40 through D.43 show current as a function of time during
the adsorption of 1.0 x 10 - 4 g/cm3 cytochrome c onto an ITO coated sensor
chip.
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A)
2.0e-8
3.0e-8
4.0e-8
5.0e-8
6.0e-8
7.0e-8
8.0e-8
Figure D.40: Current versus time during the adsorption of cytochrome c
onto waveguide D. At t=300 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
134
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
0.0
5.0e-8
1.0e-7
1.5e-7
2.0e-7
2.5e-7
3.0e-7
Time (s)
0 2000 4000 6000 8000
Cur
rent
(A
)
2.0e-7
2.5e-7
3.0e-7
3.5e-7
4.0e-7
4.5e-7
5.0e-7
Figure D.41: Current versus time during the adsorption of cytochrome c onto waveguide D. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.42: Current versus time during the adsorption of cytochrome c onto waveguide D. At t=300 s, 1.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
135
Time (s)
0 2000 4000 6000 8000 10000 12000 14000
Cur
rent
(A
)
8.0e-7
1.0e-6
1.2e-6
1.4e-6
1.6e-6
1.8e-6
2.0e-6
D.2.5 Apo-Transferrrin (Waveguide E)
Figures D.44 through D.46 show current, as a function of time during
the adsorption of 1.0 x 10 - 4 g/cm3 apo-transferrin onto an ITO coated
sensor chip.
Figure D.43: Current versus time during the adsorption of cytochrome c onto waveguide D. At t=300 s, 2.0 volts is applied to the electrodes. At t=8400 s, the protein solution enters the flow cell. At t=12000 s, a DI water rinse is initiated.
136
Time (s)
0 2000 4000 6000 8000
Cur
rent
(A
)
0.0
4.0e-8
8.0e-8
1.2e-7
1.6e-7
2.0e-7
2.4e-7
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
0.0
5.0e-8
1.0e-7
1.5e-7
2.0e-7
2.5e-7
3.0e-7
3.5e-7
Figure D.44: Current versus time during the adsorption of apo-transferrin onto waveguide E. At t=300 s, 0.5 volts is applied to the electrodes. At t=2400 s, the protein solution enters the flow cell. At t=6000 s, a DI water rinse is initiated.
Figure D.45: Current versus time during the adsorption of apo-transferrin onto waveguide E. At t=300 s, 1.0 volts is applied to the electrodes. At t=1860 s, the protein solution enters the flow cell. At t=5460 s, a DI water rinse is initiated.
137
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
8.0e-7
1.0e-6
1.2e-6
1.4e-6
1.6e-6
1.8e-6
2.0e-6
2.2e-6
D.2.6 Apo-Transferrin (Waveguide F)
Figures D.47 and D.48 show current as a function of time during the
adsorption of 1.0 x 10 - 4 g/cm3 apo-transferrin onto an ITO coated sensor
chip.
Figure D.46: Current versus time during the adsorption of apo-transferrin onto waveguide E. At t=300 s, 2.0 volts is applied to the electrodes. At t=2100 s, the protein solution enters the flow cell. At t=5700 s, a DI water rinse is initiated.
138
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
0.0
2.0e-8
4.0e-8
6.0e-8
8.0e-8
1.0e-7
1.2e-7
1.4e-7
1.6e-7
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A)
4.0e-8
6.0e-8
8.0e-8
1.0e-7
1.2e-7
1.4e-7
1.6e-7
Figure D.48: Current versus time during the adsorption of apo-transferrin onto waveguide F. At t=300 s, 1.0 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
Figure D.47: Current versus time during the adsorption of apo-transferrin onto waveguide F. At t=600 s, 0.5 volts is applied to the electrodes. At t=1800 s, the protein solution enters the flow cell. At t=5400 s, a DI water rinse is initiated.
139
D.3 Electrode Potential
D.3.1 Albumin (Waveguide G)
Figure D.49 shows the effective refractive indices, N(TE) and N(TM),
as a function of time for the adsorption of 1.0 x 10 - 4 g/cm3 human albumin
onto an ITO coated sensor chip. Data is obtained every 23.5 s at 25°C and
at a flow rate of 1.33 x 10 - 3 cm3/s.
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5836
1.5838
1.5840
1.5842
1.5844
1.5846
1.5848
1.5850
N(T
M)
1.5554
1.5556
1.5558
1.5560
1.5562
1.5564
1.5566
1.5568
N(TE)N(TM)
Figure D.49: Effective refractive indices for 1.0 x 10 – 4 g/cm3 human albumin adsorbed onto waveguide G. At t=300 s, 1.0 volts is applied to the electrodes. At t=2563 s, the protein solution enters the flow cell.
140
Figure D.50 shows current as a function of time when a potential of
1.0 volt is applied across the electrodes. While figure D.51 shows the
potentials of the ITO and platinum electrodes relative to a gold reference
electrode.
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
1.2e-8
1.4e-8
1.6e-8
1.8e-8
2.0e-8
2.2e-8
2.4e-8
2.6e-8
2.8e-8
3.0e-8
3.2e-8
3.4e-8
Figure D.50: Current as a function of time. At t=300 s, 1.0 volts is applied to the electrodes. At t=2563 s, the protein solution enters the flow cell.
141
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Pot
entia
l of I
TO
(V
)
0.45
0.50
0.55
0.60
0.65
0.70
Pot
entia
l of P
t. (V
)
-0.55
-0.50
-0.45
-0.40
-0.35
-0.30
ITOPlatinum
D.3.2 Cytochrome c (Waveguide H)
Figure D.52 shows the effective refractive indices, N(TE) and N(TM),
as a function of time for the adsorption of 1.0 x 10 - 4 g/cm3 cytochrome c
onto an ITO coated sensor chip. Data is obtained every 23.5 s at 25°C and
at a flow rate of 1.33 x 10-3 cm3/s.
Figure D.51: Potential of the ITO and platinum electrodes relative to a gold reference electrode. At t=300 s, 1.0 volts is applied to the electrodes. At t=2563 s, the protein solution enters the flow cell.
142
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
N(T
E)
1.5750
1.5755
1.5760
1.5765
1.5770
1.5775
1.5780
N(T
M)
1.5475
1.5480
1.5485
1.5490
1.5495
1.5500
1.5505
N(TE)N(TM)
Figure D.53 shows current as a function of time when a potential of
1.0 volt is applied across the electrodes. While figure D.54 shows the
potentials of the ITO and platinum electrodes relative to a gold reference
electrode.
Figure D.52: Effective refractive indices of 1.0 x 10 – 4 g/cm3
cytochrome c adsorbing onto waveguide G. At t=900 s, 1.0 volts is applied to the electrodes. At t=2845 s, the protein solution enters the flow cell.
143
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Pot
entia
l of I
TO
(V)
0.40
0.45
0.50
0.55
0.60
0.65
0.70
Pot
entia
l of P
t. (V
)
-0.54
-0.52
-0.50
-0.48
-0.46
-0.44
-0.42
-0.40
-0.38
-0.36
ITOPlatinum
Figure D.53: Current as a function of time. At t=900 s, 1.0 volts is applied to the electrodes. At t=2845 s, the protein solution enters the flow cell.
Figure D.54: Potential of the ITO and platinum electrodes relative to a gold reference electrode. At t=900 s, 1.0 volts is applied to the electrodes. At t=2845 s, the protein solution enters the flow cell.
Time (s)
0 1000 2000 3000 4000 5000 6000 7000
Cur
rent
(A
)
1.6e-8
1.8e-8
2.0e-8
2.2e-8
2.4e-8
2.6e-8
2.8e-8
3.0e-8
3.2e-8
3.4e-8
144
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Abstract
PROTEIN ADSORPTION KINETICS UNDER AN APPLIED ELECTRIC FIELD: AN OPTICAL WAVEGUIDE LIGHTMODE SPECTROSCOPY STUDY
by
MICHELLE A. BRUSATORI
December 2001
Advisor: Dr. Paul Van Tassel Major: Chemical Engineering Degree: Doctor of Philosophy
The controlled placement of protein molecules onto surfaces represents
a crucial step toward many new biotechnological devices and processes. An
applied electric field offers a promising means of controlling the rate of
adsorption to the surface and the structure of the adsorbed layer. A method for
monitoring the time evolution of an adsorbed protein layer in the presence of
an electric field is presented. In this work, Optical Waveguide Lightmode
Spectroscopy (OWLS) is used to measure the mass and layer thickness of
protein adsorbing onto an indium tin oxide (ITO) electrode. Over a range of
applied potentials, a kinetic analysis of human albumin, cytochrome c, and
apo-transferrin is performed to determine the affects of surface and protein
charge and electrochemical properties of the electrode surface on the
adsorption process. It is found that in the transport-limited regime an applied
potential has a significant influence on the initial rate of adsorption of albumin,
148
while cytochrome c and apo-transferrin are unaffected in this region. During
the later stage of adsorption the density of each of the three proteins tested is
considerably enhanced by the presence of an applied electric field. This
enhancement is found to be independent of the net charge of the protein.
149
Autobiographical Statement Name: Michelle A. Brusatori
Date of Birth: June 13, 1965, Detroit, MI.
Education: ♦ Ph.D. Chemical Engineering, Dec. 2001 Minor in Physics Wayne State University, Detroit, MI. ♦ MS Chemical Engineering, May 1998 Wayne State University, Detroit, MI. ♦ BS Chemical Engineering, May 1996 Wayne State University, Detroit, MI. Experience: ♦ Graduate Research Assistant, May 1998 – Dec. 2001 Wayne State University, Detroit, MI.
♦ Graduate Teaching Assistant, Sept. 1996 – May 1998 Wayne State University, Detroit, MI.
♦ Undergraduate Research, May 1995 – Sept. 1996 Wayne State University, Detroit, MI.
♦ Production Manager
July 1984 – Dec. 1989 Alden design Inc., Sterling Heights, MI. Areas of Interest: ♦ Protein Adsorption Kinetics and Surface Reactions
♦ Optics ♦ Electromagnetic Theory
Awards ♦ College of Engineering Excellence in Teaching Award Wayne State University, 1997