Download Imaging surface plasmon resonance Olof Andersson Link¨ oping Studies in Science and Technology

Linköping Studies in Science and Technology
Dissertation No. 1205
Imaging surface plasmon resonance
Olof Andersson
Department of Physics, Chemistry and Biology
Linköpings universitet, SE-581 83 Linköping, Sweden
Linköping 2008
During the course of the research underlying this thesis, Olof Andersson was
enrolled in Forum Scientium, a multidisciplinary doctoral programme at
Linköping University, Sweden.
c Olof Andersson 2008, unless otherwise noted.
Copyright All rights reserved.
Imaging surface plasmon resonance
ISBN 978-91-7393-820-4
ISSN 0345-7524
Printed in Sweden by LiU-Tryck, Linköping 2008
To my family
Abstract
The central theme of this thesis is the use of imaging Surface Plasmon Resonance (iSPR) as a tool in the characterization of surfaces with laterally varying
properties. Within the scope of this work, an instrument for iSPR analysis was
designed and built. SPR is a very sensitive technique for monitoring changes
in optical properties in the immediate vicinity of a sensor surface, which is very
useful in biosensing and surface science research. We have employed SPR in the
Kretschmann configuration, wherein surface plasmons are excited by means of an
evanescent field arising from total internal reflection from the backside of the sensor surface. In iSPR, the signal is the reflectivity of TM-polarized light which
is measured using an imaging detector, typically a CCD camera. Advantages of
this technique include extreme surface sensitivity and, because detection is done
from the backside, compatibility with complex samples. In addition, SPR is a
non-labeling technique, and in imaging mode, a lateral resolution in the µm range
can be attained.
The imaging SPR instrument could be operated in either wavelength interrogation mode or in intensity mode. In the former case, the objective is to find the
SPR wavelength, λSP R , which is the wavelength at which the reflected intensity is
at a minimum. In intensity mode, a snapshot of the intensity reflectance is taken
at a fixed wavelength and incidence angle.
In biosensor science, the use of an imaging technique offers a major advantage
by enabling parallelization and thereby increasing throughput. We have, for example, used iSPR in biochemical interaction analysis to monitor immobilization and
specific binding to protein and synthetic polypeptide micro arrays. The primary
interest has been the study of soft matter surfaces that possess properties interesting in the field of biomimetics or for applications in biosensing. Specifically, the
surfaces studied in this thesis include patterned self-assembled monolayers of thiolates on gold, a graft polymerized poly(ethylene glycol) (PEG) based hydrogel, a
dextran hydrogel, and a polyelectrolyte charge gradient. Our results show that the
PEG-based hydrogel is very well suited for use as a platform in protein immobilization in an array format, owing to the very low unspecific binding. In addition, well
defined microarray templates were designed by patterning of hydrophobic barriers
on dextran and monolayer surfaces. A polypeptide affinity microarray was further
designed and immobilized on such a patterned monolayer substrate, in order to
demonstrate the potential of analyte quantification with high sensitivity over a
large dynamic range.
v
Furthermore, iSPR was combined with electrochemistry to enable laterally
resolved studies of electrochemical surface reactions. Using this combination, the
electrochemical properties of surfaces patterned with self assembled monolayers
can be studied in parallel, with a spatial resolution in the µm regime. We have
also employed electrochemistry and iSPR for the investigation of potential and
current density gradients on bipolar electrodes.
vi
Populärvetenskaplig sammanfattning
Det genomgående temat i denna avhandling är användandet av en optisk metod,
avbildande ytplasmonresonans (iSPR), som ett verktyg för att karakterisera mönstrade molekylära skikt. SPR är en extremt känslig teknik som i huvudsak mäter
förändringar i de optiska egenskaperna, såsom brytningsindex, inom ett avstånd
mindre än 100-200 miljondels millimeter (nm) från en yta. Själva experimenten går
ut på att mäta förändringen hos intensiteten av ljus som reflekteras mot baksidan
av en tunn metallfilm, i allmänhet guld. Molekyler som binder till den tunna guldfilmen ger upphov till en förändring i ljusintensiteten vilket sedan kan relateras till
koncentrationen av molekylerna. Denna teknik används flitigt inom forskningen
för studier av interaktioner mellan biomolekyler eller för ytkaraktärisering. Vi har
utvecklat ett instrument för att mäta avbildande SPR, vilket i kombination med
tekniker för mönstring ger möjlighet att samtidigt utföra ett stort antal analyser,
något som är av intresse bland annat inom läkemedelsforskning eller diagnostik,
där ökad analyskapacitet är starkt efterfrågat.
Med hjälp av en metod baserad på gummistämplar med mönster i storleksordningen tusendels millimeter (µm) har vi skapat väldefinierade mikroskopiska
rutmönster på tunna (∼50 nm) guldytor, till vilka proteiner och korta polypeptider (syntetiskt framställda kedjor av cirka 10-20 aminosyror) sedan har länkats
fast. Med hjälp av vårt avbildande SPR-instrument har därefter interaktionerna
mellan dessa immobiliserade molekyler och andra målmolekyler kunnat studeras.
Även inom andra områden kan avbildande ytplasmonresonans vara intressant,
vi har bland annat studerat gradienter i elektrisk potential och ström i syfte att
utveckla ett nytt verktyg för mönstring och karaktärisering av molekylära skikt.
Ett antal olika typer av kemiska ytmodifikationer har använts, bl.a. så kallade
självorganiserande monoskikt som utgörs av ett enkelt molekyllager med en tjocklek av ∼2-5 nm. Med hjälp av SPR har även de proteinavstötande/attraherande
egenskaperna hos polymera skikt med en tjocklek i storleksordningen 5-100 nm
studerats. Dessa typer av ytor är intressanta då de kan användas som modellsystem för att studera adsorption till olika ytskikt eller som plattformar för immobilisering av proteiner i matrisformat, “protein micro-arrayer”, vilka kan bestå av
tusentals unika proteinfläckar. Då proteiner har mycket viktiga uppgifter som funktionsreglerare i kroppen förväntas micro-arrayer få stor betydelse inom forskning
som syftar till att kartlägga och förstå biomolekylära mekanismer. Tillgängligheten
till ytkemi som tillåter stabil immobilisering av proteiner med bibehållen biologisk
funktion är då av yttersta vikt.
vii
Acknowledgements
These past few years at IFM have brought a lot of joy, there has been much to learn,
many people to learn from and I have had some great co-workers, many of whom I
consider close friends. Much of the work in this thesis would not have been possible were
it not for the help of quite a few people. For this I would like to express my sincere
thankfulness and appreciation. In particular, I would like to thank...
Bo Liedberg, my main supervisor, who introduced me to surface plasmon resonance
and biosensing, initiated the project and provided this opportunity. Thank you for your
excellent advice in all matters, never-ending support, encouragement and confidence in
me.
Fredrik Björefors, co-supervisor, for excellent advice in the field of electrochemistry.
Your wise counseling and encouragement has been invaluable.
Thomas Ederth, co-supervisor, who helped me get started. Thank you for your
constructive criticism, and for sharing some excellent ideas.
Karin Enander, for enthusiastic support during the affinity array project and excellent
tutoring in the field of biochemistry and biointeraction analysis.
My closest collaborators, authors and co-authors of the papers, Christian Ulrich,
Tobias Ekblad, Andréas Larsson, Ye Zhou, and Feng-I Tai are all thankfully acknowledged. It has been a pleasure doing research with you.
Former and present co-workers in the Sensor Science and Molecular Physics Group,
for the interesting Thursday meetings and all on- and off-topic discussions: Goran K,
Mattias Ö, Annica M, Andreas C, Annika B, Rodrigo P, Kajsa U, Cecilia V, Emma E,
Robert S, Linnéa S, Jenny C, Luminita, Tomas R, Andréas L, Chun-Xia D, Erik M,
Maria A, Daniel K, Lan B, Patrik N, Timmy F, Daniel A, Tobias E, Christian U, Feng-I
T, Ramūnas V, Hung Hsun L, Alexander O, Sophia F, Ye Z, Magnus F, Gunnar B, and
anyone I might have forgotten.
Pia Blomstedt, Susanne Årnfeldt, and Anna-Maria Uhlin for making the administration run smoothly, organizing travels and for putting up with all the incomplete forms.
Bo Thuner, Agneta Askendal, and Jörgen Bengtsson, thank you for invaluable practical assistance in the lab. Rolf Rohback is acknowledged for his skills in the workshop.
Stefan Klintström, thank you for your optimism and engagement within the graduate
school Forum Scientium.
The people at Biacore, Uppsala were a great help early in the project.
ix
The diploma-workers, in order of appearance, Choy-Hsien Lin, Erik Martinsson, and
Henrik Nikkinen, are all acknowledged for their help in parts of the projects.
My room-mates at various stages, Mattias Andersson, Annica Myrskog, and Sara
Arvidsson, thank you for interesting discussions concerning any and all work-related
matters as well as other worldly issues.
The people in the lunch-room, all of the 800 hours coffee people, without whom the
days would never have started, and all friends who I forgot to mention. Tobias, Daniel,
Lars, Anders for all the good times and discussions since we started at TB together so
many years ago. Daniel, for when we climbed that mountain and those ahead.
My family, for unconditional support and for nurturing an oasis to which we visit to
seldom.
Elin, love, thank you for taking care of me, Ida and our home during the hectic times.
I could never have done this without your support. Ida, you will probably not remember
much of these overwhelming past three and a half months, but it was wonderful coming
home to you and mom and your fantastic smile, even though sometimes you were a bit
reluctant to go to sleep. Thank you for always making me happy.
Olof Andersson, Linköping 2008
x
Contents
Abstract . . . . . . . . . . . . . . . .
Populärvetenskaplig sammanfattning
Acknowledgements . . . . . . . . . .
List of figures . . . . . . . . . . . . .
List of publications . . . . . . . .
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1 General introduction
1.1 Imaging methods in biosensing . . . . . . . . . . . . . . . . . . . .
1.2 Biomolecules and biomolecular interactions . . . . . . . . . . . . .
1.3 On the significance of gradients . . . . . . . . . . . . . . . . . . . .
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2 Surface plasmon resonance
2.1 Theory . . . . . . . . . . . . . . . .
2.2 Stratified medium matrix model .
2.3 Optical SP excitation . . . . . . .
2.4 Imaging surface plasmon resonance
2.5 Imaging SPR and electrochemistry
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3 Soft matter surfaces
3.1 Self assembled monolayers . . . . . . . . . . . . . . . . . . . . . . .
3.2 Matrix surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3 Surface pattern techniques . . . . . . . . . . . . . . . . . . . . . . .
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4 Instrument design
4.1 Optical setup . . . . . . . . . . . . .
4.2 Fluidics and cell for electrochemistry
4.3 Interface . . . . . . . . . . . . . . . .
4.4 Data processing and analysis . . . .
4.5 Performance . . . . . . . . . . . . . .
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5 Future perspectives
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Bibliography
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List of Figures
1.1
1.2
1.3
1.4
Working principle of an array biosensor. . . . . . . . . .
Sensorgram example from an affinity array. . . . . . . .
Examples of microarray platforms . . . . . . . . . . . .
SPR image of protein adsorption to a polymer gradient.
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2.1
2.2
Schematic illustration of an SP wave. . . . . . . . . . . . . . . . . .
Schematic illustration of an SP wave, propagation length and probe
depth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Layer stack in the stratified medium model. . . . . . . . . . . . . .
Four layer model of an SPR biosensor. . . . . . . . . . . . . . . . .
Optical constants of glass and gold. . . . . . . . . . . . . . . . . . .
Seven layer model of an SPR biosensor. . . . . . . . . . . . . . . .
Otto- and Kretschmann SPR configurations. . . . . . . . . . . . . .
Reflectivity curves in the Otto- and Kretschmann- configurations. .
Reflectivity curves for different gold film thicknesses. . . . . . . . .
Propagation length and probe depth. . . . . . . . . . . . . . . . . .
Reflectivity as a function of angle of incidence and wavelength. . .
Contrast in imaging SPR . . . . . . . . . . . . . . . . . . . . . . .
Sensitivity in intensity modulation. . . . . . . . . . . . . . . . . . .
Intensity mode SPR. Example 1, Monolayers. . . . . . . . . . . . .
Intensity mode SPR. Example 2, Calmodulin sensor. . . . . . . . .
Sensitivity in spectral mode. . . . . . . . . . . . . . . . . . . . . . .
Imaging SPR in spectral mode. Example, affinity array. . . . . . .
Electrochemistry and imaging SPR . . . . . . . . . . . . . . . . . .
10
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
3.1
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Overview of the different soft matter surface modifications in Papers
I-VI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Schematic overview of the self-assembly of alkanethiols on gold. . .
Micro-contact printing. . . . . . . . . . . . . . . . . . . . . . . . . .
Electrochemical gradients, experimental setup. . . . . . . . . . . .
Piezodispensation. . . . . . . . . . . . . . . . . . . . . . . . . . . .
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4.2
4.3
4.4
4.5
4.6
Schematic overview and photograph of the optical configuration in
the imaging SPR instrument. . . . . . . . . . . . . . . . . . . . . .
Prism/Substrate configuration for iSPR. . . . . . . . . . . . . . . .
Flowcell and cuvette. . . . . . . . . . . . . . . . . . . . . . . . . . .
Software flowchart. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Data processing, raw and normalized spectra. . . . . . . . . . . . .
Imaging SPR spectroscopy - resolution . . . . . . . . . . . . . . . .
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5.1
Imaging SPR study of barnacle cyprid footprint deposition . . . .
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3.2
3.3
3.4
3.5
4.1
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List of publications
This thesis is based on the following papers, referred to in the text by their
Roman numerals (I-VI). The papers VII-XI are related to this work but not
included in the thesis.
Paper I
Protein microarrays on carboxymethylated dextran hydrogels: immobilization, characterization and application
Y.Zhou, O.Andersson, P.Lindberg, and B.Liedberg. Microchimica Acta, 2004,
147, 21-30.
Author’s contribution
Participated in planning and evaluation. Performed the imaging SPR and half of
the fluorescence experiments.
Paper II
Imaging SPR for detection of local electrochemical processes on patterned surfaces
O.Andersson, C.Ulrich, F.Björefors, and B.Liedberg. Sensors and Actuators B:
Chemical, In Press, doi:10.1016/j.snb.2008.05.042, 2008.
Author’s contribution
Responsible for planning, performing, and evaluating the experimental work. I
wrote the manuscript.
Paper III
Formation of molecular gradients on bipolar electrodes
C.Ulrich, O.Andersson, L.Nyholm, and F.Björefors. Angew. Chem. Int. Ed., 2008,
47 (16), 3034-3036.
Author’s contribution
Shared equally with C.U. in planning, performing, and evaluating the experimental
work. Responsible for the imaging SPR experiments and the thiol chemistry.
Contributed to the writing.
xv
Paper IV
A gradient hydrogel matrix for microarray and biosensor applications:
an imaging SPR study
O.Andersson, A.Larsson, T.Ekblad, B.Liedberg. In manuscript, 2008.
Author’s contribution
Responsible for planning, performing, and evaluating the experimental work. A.L.
and T.E. participated in planning and evaluation and prepared some of the samples. I wrote the manuscript.
Paper V
Lateral control of protein adsorption on charged polymer gradients
T.Ekblad, O.Andersson, F-I.Tai, T.Ederth and B.Liedberg. In manuscript, 2008.
Author’s contribution
Contributed significantly to the planning and evaluation of the experimental work.
Responsible for the imaging SPR experiments. Contributed to the writing of the
manuscript.
Paper VI
A multiple-ligand approach to extending the dynamic range of analyte
quantification in protein microarrays
O.Andersson, H.Nikkinen, and K.Enander. In manuscript, 2008.
Author’s contribution
Responsible for planning, performing, and evaluating most of the experimental
work. Co-supervised H.N. during his undergraduate diploma work, during which
he synthesized the polypeptides and determined the affinities. I did a major part
of the writing.
xvi
Additional publications, not included in thesis
Paper VII
Reversible Hydrophobic Barriers Introduced by Microcontact Printing:
Application to Protein Microarrays
Y.Zhou, O.Andersson, P.Lindberg, and B.Liedberg. Microchimica Acta, 2004,
146, 193-205.
Paper VIII
Microarray production on polymeric hydrogels using microcontact printing
B.Liedberg, Y.Zhou, O.Andersson, and P.Lindberg. Proc. of the ESF Workshop,
2004.
Paper IX
Photografted poly(ethylene glycol) matrix for affinity interaction studies
A.Larsson, T.Ekblad, O.Andersson, and B.Liedberg. Biomacromolecules, 2007, 8,
287-295.
Paper X
Evaluation of the potential and current density distributions at electrodes for bipolar patterning
C.Ulrich, O.Andersson, L.Nyholm, and F.Björefors. In manuscript, 2008.
Paper XI
DNA chips with conjugated polyelectrolytes in resonance energy transfer mode
J.Wigenius, K.Magnusson, P.Björk, O.Andersson and O.Inganäs. Submitted, 2008.
xvii
xviii
Chapter 1
General introduction
In January 2005, at the time of the American presidential inauguration campaign,
researchers at the United States Naval Research Laboratory (NRL) in Washington DC where at hard work, collecting, preparing and analyzing samples from
patients admitted at nearby military hospitals, exhibiting symptoms of flu. They
were working on the “Silent Guardian” project, which purpose was the demonstration of the potential for around the clock large scale bio-surveillance, based on
state of the art Deoxyribonucleic acid (DNA) microarray technology.1 The Silent
Guardian demonstration succeeded in this sense, and dozens of natural pathogens
were identified within 24 hours. Luckily, none of the biological agents commonly
associated with bioterrorism was found in the clinical samples, but control experiments showed that they would have, were they present. What made the work
of the scientists at NRL possible was the fast-paced progress within the recent
decades in the field of biological microarray technology.
In the quest of understanding life, the role of the fundamental building blocks
of DNA and proteins are of great significance. While DNA molecules provide
the basic instructions, it is the proteins that are the laborers of the cells at the
1
2
General introduction
molecular level. Studying the DNA may give great insights, but it is not until one
appreciates the role of the proteins that a more complete picture can be drawn.
Proteins, however, are intrinsically more difficult to work with than DNA. This is
because while DNA polymers are rather stable and fairly homogeneous in charge
and structure, proteins can possess a wide variety of properties relating to their
backbone composition. Additionally, a vital part of the function of proteins lies
in their secondary and tertiary structures, which are sensitive to harsh treatment.
This is one of the reasons why DNA microarrays have been around for a longer
period of time than protein arrays.
Recent large scale efforts within the research community, such as the Human
Genome Project (HGP),2, 3 or the Swedish Human Protein Atlas (HPA) project,4
has been geared towards deciphering the code of life. The huge amount of information made available through these and other projects alike can be used to gain
a deeper understanding on the fundamentals of life’s processes at the molecular
level. However, interpretation and implementation of this newly gained knowledge into actual applications or devices puts forward new requirements on the
techniques available. In pharmaceutical research, for example, in the screening
for disease biomarkers, or in the selection process of new drug candidates, there
is a great demand for high throughput instrumentation capable of accurate analysis and parallelization. One such technique, that could be used as a transducer
element in screening studies of the kind performed at NRL, is imaging Surface
Plasmon Resonance (iSPR).
The recurring theme of the papers included in this thesis is the use of iSPR as a
tool in the study of biomolecular interactions, and of surfaces possessing properties
that are of interest in biomolecular analysis. In Papers I, IV, V and VI, iSPR
is used to study molecular binding and interactions with surfaces. Papers II and
III focus on the use of iSPR in combination with electrochemical techniques. The
iSPR instrument used throughout the papers (with the exception of Paper I) was
developed and built within the scope of this work.
The remainder of this chapter will be devoted to giving a general introduction
and motivation, while the goal of the subsequent chapters is to provide a theoretical
foundation for the included papers. It should be noted, although much of the
discussion is focused on biological interaction analysis, that iSPR is an equally
applicable technique in many other fields of chemical sensing.
1.1
Imaging methods in biosensing
Biosensors, which are a type of chemical sensors, have two main components. First,
there is a recognition part, wherein a chemical reaction or binding takes place which
ultimately gives rise to a signal. The second part is a transducer element at which
the sensor signal is transformed into an amount or equivalent measure. In an
1.1 Imaging methods in biosensing
3
Analyte solution
Recognition layer
Substrate / Transducer
Figure 1.1: Working principle of an array biosensor. The principal component is the
recognition layer which captures analyte molecules from solution. The substrate can
sometimes provide connectivity to or be a working part of the transducer.
array biosensor (Figure 1.1), the recognition part can be subdivided into smaller
groups each of which provides a separate signal. In this case, the transducer must
be capable of multiplexing, that is to provide means of separating the individual
signals from one another. In the type of iSPR instrument employed in this thesis,
this is accomplished by using an imaging detector. The advantage of an imaging
approach, is that it allows for increased throughput by enabling more and many
different analyses to be performed in parallel.
Many other types of imaging transducer technologies suitable for use in biosensors exist. These are for example based on optical, acoustical/mechanical or electronic read-out. For instance, Quartz Crystal Microbalance (QCM), which is based
on measuring very small changes in mass, is a technique suitable for miniaturization and large numbers of quartz crystals could be arranged in an array format
to facilitate parallelization. Ellipsometry, for example in Total Internal Reflection
(TIR) mode, is an optical technique closely related to surface plasmon resonance.5
Ellipsometry under SPR conditions offers a very high sensitivity at the expense of
a somewhat more intricate instrumental setup. The technique is also not limited
to thin metal film substrates. Techniques based on labeling, such as fluorescence
microscopy are traditionally strong within biosciences, much thanks to the intrinsic fluorescence of proteins, the large number of commercially available fluorescent
probes and the ease of labeling of e.g. DNA. Much more information than mere
quantification can be extracted from fluorescence measurements, for instance, fluorescence decay times can give information on the structure of biomacromolecules.6
The advantage of iSPR over the aforementioned techniques lies in the high surface
sensitivity, and that there is no need for labeling. For non-labeling techniques,
however, there is a high demand on functional surface chemistry, that provides
specificity towards desired analytes while preserving sensitivity. Therefore, a lot
of research within the biosensor field is devoted to the development of novel surface
coatings.
4
1.2
General introduction
Biomolecules and biomolecular interactions
The fundamental building blocks that form the various types of biomacromolecules
are in themselves remarkably simple. For example, only four different nucleotides
make up the DNA molecule, which is responsible for encoding all the genetic information in living organisms. Proteins, which are the laborers in cell biology,
are comprised of 20 different amino acids which combine to form a polypeptide
backbone, the primary structure of the protein. Much of the function of proteins,
however, lies in their elaborate secondary and tertiary structures. While the covalent peptide bonds in proteins are very stable, the three-dimensional structure
is very sensitive to the environmental conditions. A prerequisite for a successful
protein-based biosensor surface is therefore the availability of a friendly, nativelike environment. Other important biochemical building blocks are lipids, which
constitute membranes, and carbohydrates, which are vital in energy storage and
for many cellular processes. Because lipids and carbohydrates are abundant in the
native environment of proteins, molecules with similar structure and properties
are a natural template choice when designing protein friendly surfaces.
Affinity sensors
A large portion of biosensors are affinity-based, wherein the sensor signal originates
from a specific interaction between a ligand (L) immobilized on a surface, and the
analyte (A) molecule. For a monovalent interaction, the kinetics (association ka ,
and dissociation kd ) of formation of the LA complex is described by the following
differential equation (Eqn. 1.1):7
d[LA]
= ka [L][A] − kd [LA]
dt
(1.1)
At equilibrium, the rate of formation is zero and the equation reduces to:
[L][A]
kd
=
= KD
ka
[LA]
(1.2)
wherein KD is the dissociation constant. A low dissociation constant is a characteristic of high affinity sensors. If the binding process can be monitored over time,
the kinetic parameters can be extracted by fitting to the response curves during
the analyte injection and post-injection phases. Information on kinetics is important in many cases, for instance, it can provide thermodynamic information on
the specific interaction. Knowing the kinetics of an interaction is also important
in drug screening tests, where the affinity is a measure of the potentiality of a new
drug candidate.
In terms of quantifying the analyte concentration, affinity sensors are most
reliable when the concentration of the analyte is in the range (0.1-10)·KD .6 This
means that when the analyte concentration is unknown, as is most often the case,
1.2 Biomolecules and biomolecular interactions
Injection phase Post-injection phase
[A] = 50 nM
0.1
1.0 nM
0.05
13 nM
0
Approximate KD
ΔRTM/RTE
5
370 nM
0
5
10
Time / s
Figure 1.2: Injection and post-injection phases of an analyte binding to three ligands
immobilized in an array format monitored by imaging SPR. The ligands are in this case
three synthetic polypeptides with different affinities to a particular antibody fragment
(the analyte, A). (Data in this figure adapted from Paper VI).
systematic dilution of the sample is required to obtain a concentration within the
defined range. This calls for a series of experiments to be performed. A way of circumventing laborious sample preparation is to use an affinity array sensor wherein
separate elements of the array have different affinities toward the analyte. If the
affinity array spans a wide enough range, one can ensure that the analyte concentration is in the same order of magnitude as the KD of some of the array elements
and, ideally, quantification can be made in a single experiment.8 Figure 1.2 shows
an example of the SPR responses from three elements in an affinity array during
the injection and post-injection phases. In this model system, the dynamic range
of quantification was extended by using three ligands with affinities spanning two
orders of magnitude towards the analyte. A large dynamic range can be particularly desired in biomarker screening studies, where concentrations are known to
vary over three to four orders of magnitude in some cases.9 The concept of affinity
arrays is explored further in Paper VI of this thesis.
Protein microarray scaffolds
Since a few years back, protein chips comprising several thousand proteins from
the human proteome are commercially available.10 Commonly, these are based
on physisorption to e.g. nitrocellulose membranes, or covalent linkage by functional groups on glass surfaces directed towards amine- or carboxyl groups on the
proteins.11, 12 While these methods of immobilization are convenient and fairly
straightforward the ligands typically become randomly oriented in an uncontrollable fashion on the surface upon binding.13 It has been demonstrated that directed, i.e. tag-mediated or site-specific, immobilization methods which offer some
control of the orientation of the active site of the ligands are superior in terms
6
General introduction
λSPR / nm
A.
B.
740
680
620
0
0.5
y / mm
0
0.5
1.0
x / mm
1.5
Figure 1.3: Platforms for protein microarrays. A) SPR image of a poly(ethyleneglycol)
containing hydrogel matrix spot gradient for immobilization of proteins (Paper IV).
B) Microwetting image showing a patterned hydrophobic grid (125 µm wide brighter
regions) on a dextran hydrogel chip. (Adapted from Paper I).
of retained biomolecular activity.14 Such concerns are less important on threedimensional array supports, which provide a flexible, natural-like environment for
the protein ligands, and also offers immobilization capacity in excess to a monolayer. One such platform is the dextran based hydrogel developed for the Biacore
SPR-based biosensors.15 In highly condensed microarrays, one concern which
constrains the attainable immobilization density is the risk of cross-talk or contamination of neighboring spots. Ligands are commonly delivered to the surface
by dispensation, a process described more fully in section 3.3. Various solutions
to reduce cross-talk has been presented, for instance through the introduction
of microwells16 or by surface energy modification of regions on the substrate.17
Some of the work in this thesis has been directed towards development of scaffolds
for protein microarrays. In Paper I, we present a method of introducing hydrophobic barriers by micro stamping onto a dextran hydrogel chip (Figure 1.3B),
which facilitates dense microarrays to be created on three-dimensional surfaces.
A poly(ethylene glycol) based UV-patterned hydrogel matrix is demonstrated in
Paper IV, wherein three-dimensional spots for immobilization are separated by
a two-dimensional hydrophobic barrier region (Figure 1.3A). This polymer matrix particularly excels in terms of low levels of non-specific binding, and might
also act as a kind of ‘molecular sieve’, separating biomacromolecules with regard to
size. Further, a two-dimensional microarray platform based on biotin tag-mediated
binding is employed in Paper VI.
1.3
On the significance of gradients
Diffusion, the spontaneous intermixing of molecular species, is a fundamental process ubiquitous in everyday life. Due to diffusion, concentration gradients or spatial
molecular distributions will occur spontaneously at molecular system interfaces.
Such gradient patterns are believed to play a large role in morphogenesis∗ , first
∗ From
Greek: ”Beginning of the shape”
1.3 On the significance of gradients
PCEA
7
PAEMA
Increasing PCEA content
A.
O
O
O
n
B.
720
700
680
O
O
ΔλSPR / nm λSPR / nm
H 3N
O
n
2.0
1.5
50
0
0.5
y / mm
1.0
0.5
x / mm
0 0
Figure 1.4: Imaging SPR study of protein (lysozyme and pepsin) adsorption to an
amphoteric polymer surface. In A, an SPR image of the polymer gradient is shown. B
shows the shift in SPR wavelength upon introduction of proteins in solution. (The data
in this figure was adapted from Paper V).
suggested by Turing in an interesting work from 1952.18 At a cellular level, gradients of chemoattractants are important for cell motility,19 and in guidance of
axonal outgrowth.20 Synthetic gradients are very important tools for in vitro
studies of such phenomena.21
Surfaces with laterally varying properties can be used as means of increasing
throughput in materials research, as an alternative to the manufacture of large
numbers of discrete samples. Using gradients, a range of properties can be incorporated and studied in one single sample. An imaging method, such as iSPR, is
an excellent tool in the study of interactions at such surfaces.
Among the first type of surface gradients to be manufactured were wettability
gradients,22 based for instance on monolayers of alkanethiols on gold.23 There
are many other approaches to the creation of chemical surface gradients, some of
which are based on polymers.24 In this thesis, gradients were prepared using a
top-down approach, by modification of a uniform surface film, in Paper III and
using a bottom-up approach based on graft polymerization in Papers IV and V.
An example of an SPR image, showing the adsorption of proteins to an amphoteric
polymer surface with a laterally varying composition, is given in Figure 1.4. From
this data, it is straightforward to determine the surface composition which, in this
case, gives a minimum in protein adsorption.
Two comprehensive reviews on soft matter surface gradients were recently published.25, 26
8
General introduction
Chapter 2
Surface plasmon resonance
A little more than a century ago, Wood, at the Johns Hopkins University, observed what he referred to as anomalies in the reflection spectra from diffraction
gratings when varying the angle of incidence.27∗ These anomalies were attributed
to excitation of surface waves by Fano in 194128 and theoretically explained by
Ritchie in 1957.29 Otto, Kretschmann and Raether pioneered the modern work on
Surface Plasmon Resonance (SPR) in the end of the 60’s, devising an experimental
setup that allowed for excitation of plasmon waves on flat metal films.30, 31 The
Kretschmann type of setup is widely used in commercial SPR sensors of today.
Because the conditions under which surface plasmon resonance occurs is extremely
sensitive to the optical properties of the proximity, SPR can be used to probe for
changes in the refractive index or thickness of surface films. This was exploited
in the end of the 70’s by Pockrand and Swalen in thin film studies.32, 33 The high
sensitivity of SPR to changes in the optical parameters of thin films relates to the
existence of an evanescent field associated with the surface plasmon. In a biosensor
∗ In principle, these observations could be explained by the work of Fresnel in the first part
of the 19th century and modeled using the important theory of Maxwell from 1873.
9
10
Surface plasmon resonance
dielectric
z
E
z
x
y
−−−
+++
−−−
+++
−−−
+++
Ez
plasma
Figure 2.1: Schematic illustration of a TM-polarized SP wave at a semi-infinite metaldielectric interface. The exponential dependence of the z-component of the electric fields
is shown on the right. Adapted from Raether.37
context this was first explored in 1982 by Liedberg et al.,34, 35 who showed how
SPR can be used in the study of biomolecular interactions at surfaces. Principally, in a light-mediated SPR experiment, the objective is to find the conditions
under which coupling of the incident light to a surface plasmon mode occurs. A
comprehensive review on applications of surface plasmon resonance was recently
published.36
In this chapter, the theoretical basis of surface plasmon resonance is presented
and some different configurations and important characteristics are discussed.
2.1
Theory
A Surface Plasmon (SP) ∗ is a surface-bound electromagnetic wave that propagates along the interface of a metal and a dielectric.37 The origin of SP waves is
longitudinal charge density fluctuations in the free electron gas (plasma).29 Therefore, a prerequisite for SP excitation is that the metal can be described by the free
electron model† . A schematic representation of an SP wave at a semi-infinite
metal-dielectric interface is shown in Figure 2.1. The most frequently employed
metals are silver and gold. Even though the resonance width of thin silver films
is smaller than for gold, which theoretically leads to a higher sensitivity, the relative chemical inertness and the ease of functionalization of gold makes it the most
applicable metal in biosensor contexts. Gold can be considered free-electron like
only at optical wavelengths longer than about 500 nm because at higher energies,
electron excitations can occur and the light will be absorbed. Aside from the
book by Raether,37 several excellent reviews describing the theory behind surface
plasmons can be found in literature.38, 39 Most optics textbooks also treat surface
plasmons in detail.40–42 In the following subsections some fundamental theory is
presented and a theoretical model of an SPR sensor is given in section 2.2.
The interactions of electromagnetic waves with matter are described by Maxwell’s
∗ An
equivalent expression is ”Surface Plasma Polariton”
by the German physicist Arnold Sommerfeld
† Developed
2.1 Theory
11
equations.43 In SI units these are:
∂B
= 0,
∂t
∂D
∇×H−
= J,
∂t
∇ · D = ρ,
∇×E+
∇·B =
(2.1)
(2.2)
(2.3)
0.
(2.4)
wherein,
D = εε0 E,
(2.5)
B = µµ0 H,
(2.6)
J = σE.
(2.7)
in which ε denotes the permittivity∗ , µ the permeability and σ the conductivity
tensors. For isotropic materials these become scalar functions of frequency. When
there are no external charges, ρ = 0 and the current density J = 0. At optical
frequencies it is also generally assumed that the permeability µ = 1. Recall that
2
c2 = 1/µ0 ε0 , Eqn. 2.2 then becomes: ∇ × µ0 H − ∂E
∂t ε/c = 0.
Dispersion relation
In the most simple case, a single (TM-polarized) SP wave is bound at the interface
between two (isotropic) semi-infinite media as shown in Figure 2.1. The electric
and magnetic fields associated with the SP wave in this case are given by Eqn. 2.8
and Eqn. 2.9.
Ej
Hj
=
=
(Exj , 0, Ezj )ei(kx x−ωt)±(ikz z)
i(kx x−ωt)±(ikz z)
(0, Hyj , 0)e
(j = 1, 2)
(2.8)
(2.9)
Wherein j denotes the material (1 for the metal and 2 for the dielectric), ± indicates positive or negative z-axis and the positive lies in the dielectric. The
first exponential term accounts for propagation along the interface (in positive
x-direction), while the imaginary kz describes the exponential decay in the zdirection. Since the SP wave is TM-polarized there is no electric field component
in the y-direction (the vector set is Exj , Hyj , and Ezj ). These fields are continuous
at the boundary:
Ex1
= Ex2 ,
(2.10)
Hy1
= Hy2 ,
(2.11)
= ε2 Ez2 .
(2.12)
ε1 Ez1
∗ also
denoted the dielectric function, ε(ω).
12
Surface plasmon resonance
This implies that kx1 = kx2 = kx (where medium 1 is the metal and 2 the
dielectric). The Maxwell relations (Eqs. 2.1-2.4) applied on the waves in Eqn. 2.8
and Eqn. 2.9 lead to the following set of partial differential equations:
∂Exj
− ikx Ezj − iωµ0 Hyj
∂z
∂Hyj
iωεj
µ0
− 2 Exj
∂z
c
∂Hyj
iωεj
µ0
+ 2 Ezj
∂x
c
∂Ezj
+ ikx Exj
∂z
=
0,
(2.13)
=
0,
(2.14)
=
0,
(2.15)
=
0. (j = 1, 2)
(2.16)
Eqn. 2.14 together with the boundary conditions Eqs. 2.10-2.12 yield the following
dispersion relation:
kz1
kz2
+
=0
(2.17)
ε1
ε2
Combination of Eqs. 2.13-2.14, and Eqn. 2.16 gives the following relation for kz :
r
ω 2 εj
(2.18)
kzj =
− k2x
c2
By substitution of Eqn. 2.18 in Eqn. 2.17 the dispersion relation for surface plasmons at a semi-infinite metal-dielectric interface is reached:
r
ω
ε1 ε2
kx =
(2.19)
c ε1 + ε2
Propagation length and Probe depth
Some notable features of SP waves can be extracted from Eqn. 2.19. Firstly, since
the dielectric function of the metal is complex (ε̃1 (ω), while ε2 (ω) for the dielectric
00
is real), the SP wavevector will have an imaginary part, kx . Therefore the surface
plasmon is a damped wave and has a finite propagation length in the x-direction.
The energy of the SP is absorbed and dissipated as heat in the metal film, the
00
damping term is denoted Γi = kx where the subscript i indicates internal damping.
Furthermore, the absence of propagating waves in the metal or dielectric requires
ε2
> ε2 ∗ .
that kz in Eqn. 2.8 and Eqn. 2.9 be purely imaginary. This implies that εε11+ε
2
The wavevector of light incident through the dielectric medium is given by kin =
ω√
c ε2 and therefore kin will always be smaller than kx . Consequently, surface
plasmons can not be excited optically at a metal-dielectric interface by plane wave
light incident through the dielectric. One way to circumvent this problem is to
employ an Attenuated Total Reflection (ATR) configuration as will be described
in more detail in section 2.3 v.i. The above findings are summarized in Figure 2.2
∗ Here we have assumed that the real part of the dielectric function of the metal is much
0
00
larger than the imaginary part (| ε1 | ε1 ).
2.1 Theory
13
Ez
0
0
1
0
2
20
x / µm
21
22
1
z / µm
Figure 2.2: Schematic illustration of the electric field Ez (z, x) at the interface of semiinfinite metal and dielectric (water) layers (at λ = 633nm). The field decays exponentially
in the z-direction, the probe depth is in the sub-micron range for the dielectric and about
an order of magnitude smaller for the metal film. The wave is attenuated in the direction
of propagation (x) over a distance of some 10-30 microns. Note that the x-axis has been
cut off to illustrate the attenuation.
which schematically shows the exponentially decaying evanescent field and the
attenuation of the SP wave propagating in the x-direction.
The intensity, which is directly proportional to the energy density, of the SP
is given by the square of the electric field, for the fields in Eqn. 2.8 this yields
00
(wherein denotes the imaginary part):
00
Izj
Ix
∝ e−2kzj z , (j = 1, 2)
∝ e
00
−2kx x
.
(2.20)
(2.21)
Two important parameters of the SP wave are the propagation length (Lx ) and
the probe depth∗ (δzj ).39, 44 These are defined as the distances (along x or z
respectively) at which the intensity of the electric field has dropped to a value of
1/e and are hence given by:
Lx
=
δzj
=
1
2k00x
1
00 (j = 1, 2)
2kzj
(2.22)
(2.23)
The effect of a thin metal film
So far only the special case of a semi-infinite metal-dielectric system has been
treated. Typically, in optical excitation of surface plasmons one deals with an
asymmetric system, wherein the metal film is thin and supported on top of another,
dielectric material (typically glass). A metal thickness of 50 nm, which is typical,
∗ Distinguished from the penetration depth in that the latter applies to the electric field
strength rather than the intensity (different value by a factor of two).
14
Surface plasmon resonance
is of the same order of magnitude as the probe depth δz1 of the SP. Consequently,
the evanescent field will penetrate the film and probe the substrate leading to
an alteration in the resonant wavevector of the SP. The new wavevector can be
expressed as:37
kx = k∞
(2.24)
x + ∆kx
wherein k∞
x denotes the wavevector in the semi-infinite case. It is important to
note that ∆kx is a complex quantity which means that the propagation length will
00
be influenced. There is now an additional complex term, Γrad = ∆kx (radiative
damping term), caused by reradiation of the light associated with the SP.
In the next section a model that can be used to calculate the intensity reflectance of an asymmetric SPR system consisting of many thin film layers is
presented.
2.2
Stratified medium matrix model
The general ATR SPR coupler is based on total internal reflection within a glass
prism coated with a thin metal film. This type of SPR coupler can be optically
modeled as a stratified medium wherein the outermost prism and ambient (generally water or air) layers skirts the intermediate thin metal layer. For biosensor
applications, several additional thin film layers are incorporated in the model.
These can be for instance; molecular linker layer, immobilized biomolecular layer,
and analyte layer. In the following subsections, the Fresnel reflection formulas45
are used to calculate the intensity reflectance from a general N -layer stack. An
alternative approach would be to analytically solve Maxwell’s equations as was
done in the previous section, a tedious undertaking for a system of more than
one layer. The matrix formulation, on the other hand, can easily be employed to
solve for an arbitrary number of layers. Many descriptions of the matrix model
exist in literature, see for example the work by Hansen,46 Azzam and Bashara47
or Abelés.48
Assumptions
The basis of the stratified medium matrix model is a layer stack comprised of homogeneous layers connected through interfaces (Figure 2.3). In the general case,
the stack will be comprised of N + 1 (smooth and perfectly parallel) layers, with
varying thicknesses, dj , and complex refractive indices ñj = nj + ikj . Note that,
in this section, we will write the complex refractive index with a plus sign∗ ,49, 50
because of known problems in how for instance MATLAB will handle the complex
algebra when a minus sign is used.51 The first (incident) and last layers are considered semi-infinite and non-absorbing. The incident light can be parallel- (denoted
∗ See
the citations for a discussion about sign conventions.
2.2 Stratified medium matrix model
15
Incident light
Reflected light
E||
φ0
E┴
x
y
0th layer, (∞; n0)
1st layer, (d1; n1+ik1)
φ1
z
2nd layer, (d2; n2+ik2)
φ2
jth layer, (dj; nj+ikj)
φj
Nth layer, (∞; nN)
φN
Figure 2.3: Layer stack in the stratified medium model. The 0th and Nth layers are
semi-infinite and have real refractive indices. The incident wave is p- (TM, k) or s- (TE,
⊥) polarized. The planes show the interfaces, Iij .
p, T M or k) or perpendicular- (denoted s∗ , T E or ⊥) polarized. The Cartesian
coordinate system in Figure 2.3 defines the z-direction as parallel to the plane of
incidence with the positive direction into the layer stack. Electric fields will be superscripted + for positive and - for negative z-direction corresponding to refracted
and reflected waves respectively. For our purposes it is convenient (although not
necessary) to consider all layers isotropic, hence all fields are independent of x or
y. Just as before, it is assumed that the permeability µ = µ0 for all layers.
Derivation
Since there is no dependence on x or y, we have for the total electric field amplitude
at a certain distance along the z-axis:
+
−
Etot
z = Ez + Ez
(2.25)
Where the subscript indicates the z-dependence. Eqn. 2.25 holds for both TMpolarized and TE-polarized light respectively. For the relation between the electric
field vectors at two points, z1 and z2 we have:
∗ From
E+
z1
E−
z1
=
M11
M21
M12
M22
E+
z2
E−
z2
the German word senkrecht, meaning orthogonal.
=M
E+
z2
E−
z2
(2.26)
16
Surface plasmon resonance
in which M denotes the scattering matrix. When the points z1 and z2 are located
within the same layer the relation can be written as:
+ + Ez2
Ez1
(2.27)
=
L
j
−
E−
Ez1
z2
Wherein Lj is the layer matrix of the j:th layer. Conversely, when z1 and z2
are located at the interface of separate adjacent layers i and j we introduce the
interface matrix Iij ∗ . We then have:
+ + Ez1
Ez2
= Iij
(2.28)
−
E−
E
z1
z2
In the general case where z1 and z2 are within separate non-adjacent layers we can
write:
+ + Ez1
Ez2
(2.29)
= Ii(i+1) L(i+1) I(i+1)(i+2) . . . L(j−1) I(j−1)j
E−
E−
z1
z2
The scattering matrix in the case of N consecutive layers is hence given by:


N
−1
Y
M=
I(j−1)j Lj  I(N −1)N
(2.30)
j=1
For the electric fields at the 0:th and N :th layers we have:
+ " incoming #
E0
E0
=
lected
E−
Eref
0
0
(2.31)
E+
N
E−
N
=
Etransmitted
N
0
From the definition of the complex Fresnel reflection and transmission coefficients
we then find:
E−
r = E0+
0
(2.32)
τ=
E+
N
E+
0
By substitution of Eqn. 2.32 into Eqn. 2.26 the reflection and transmission coefficients of the layer stack can be expressed in terms of the scattering matrix elements:
r=
M21
M11
τ=
1
M11
(2.33)
∗L
is sometimes also called the propagation matrix and I the transmission matrix.
2.2 Stratified medium matrix model
17
We need now to determine the scattering matrix, M, for the layer stack. The
layer matrices, Lj , describe the phase shift undergone upon propagation through
a uniform film and have the form:
−iϕ
e j
0
(2.34)
Lj =
0
eiϕj
In which the film phase thickness, ϕj , is given by:
ϕj =
2πdj
Nj cos φj
λ
(2.35)
Where Nj is the complex refractive index. Turning then to the interface matrices,
we start by expanding the Eqn. 2.28. For two adjacent layers i and j we then have:
+
−
E+
i = I11 Ej + I12 Ej
(2.36)
E−
i
=
I21 E+
j
+
I22 E−
j
When the light is incident from layer i, we have E−
j = 0. By direct comparison
with Eqn. 2.32 we immediately find the first two coefficients:
I11 =
1
τij
I21 =
rij
τij
(2.37)
Consider next the case where E+
i = 0 (light is incident from layer j) we then have:
−
E−
i = τji Ej
(2.38)
−
E+
j = rji Ej
By combination of Eqn. 2.36 with Eqn. 2.38 it is possible to find expressions for the
remaining two coefficients:
rji
I12 = − τij
(2.39)
r r
I22 = τji − ijτijji
From the Fresnel reflection formulae we have the relations: rji = −rij , and τji =
2
1−rij
τij .
The complete interface matrix then has the form:
Iij =
1
τij
1
rij
rij
1
(2.40)
Since the general expression Eqn. 2.26 is valid for both p- and s-polarized light we
have so far omitted the polarization notation. The Fresnel reflection and transmis-
18
Surface plasmon resonance
φ0
0
Glass prism
1
2
3
Free electron metal film
Adsorbate layer
Ambient
Figure 2.4: Four layer model of an SPR biosensor. In the most simple case an SPR
sensor can be modeled as a four layer stratified medium where the constituents are a glass
prism, coated with a thin metal film, to which the organic sensing layer is adsorbed. The
semi-infinite ambient layer is typically aqueous buffer.
sion coefficients, Eqn.
2.41 are,
however, different for different states of polarization.
k
ñj cos φi −ñi cosφj
ñj cos φi +ñi cosφj
τij =
k
2ñi cos φi
ñj cos φi +ñi cosφj
⊥
rij
=
ñi cos φi −ñj cosφj
ñi cos φi +ñj cosφj
⊥
τij
=
2ñi cos φi
ñi cos φi +ñj cosφj
rij =
(2.41)
The intensity reflectance and transmittances of the layer stack are found as the
square modulus of the coefficients in Eqn. 2.33.
Implementation
As the number of layers increase, the matrix algebra required to find the reflection
coefficient of the stack quickly becomes tedious. However, the stratified medium
matrix model as described v.s. can easily be implemented in a computer program,
for instance MATLAB. A special case which is somewhat useful in SPR biosensing
concerns a stack consisting of two thin film layers on top of a glass prism and
in an aqueous ambient (Figure 2.4). Some algebra will give an expression for the
intensity reflection from such a layer stack:
r01 (1 + r12 r23 e2iϕ2 ) + e2iϕ1 (r12 + r23 e2iϕ2 ) 2
R = (2.42)
1 + r01 e2iϕ1 (r12 + r23 e2iϕ2 ) + r12 r23 e2iϕ2 In the above expression, which is valid for ñj = nj + ikj , the response upon introduction of an analyte can be modeled as a thickness increase or as an increase in
refractive index of the adsorbate layer. The former is most suitable for immobilization of biomacromolecules to a two-dimensional organic linker layer, whereas
the latter can be employed when a sensing matrix (for instance a hydrogel) is used.
By eliminating all terms with r23 in Eqn. 2.42, an expression for the more simple
19
1.8
6
1.5
5
1.2
4
0.9
3
0.6
2
0.3
1
0
500
550
600
650
700
750
800
0
850
k
n
2.2 Stratified medium matrix model
nAu
kAu
nBK7
nSF10
Wavelength / nm
Figure 2.5: Refractive indices of BK7 glass, SF10 glass, and complex refractive index
of gold. Gold data was measured using spectroscopic ellipsometry, while optical data for
glass was obtained from Schott.52
φ0
0
Glass prism
1
2
3
4
5
6
Adhesion layer
Free electron metal film
Biomolecular linker layer
Sensing layer
Response layer
Ambient
Figure 2.6: Complete seven layer model of an SPR biosensor.
three-layer case is reached. When the refractive indices and thicknesses of the
constituents of the layer stack are known, calculation of the reflectivity is straightforward. Figure 2.5 shows refractive indices for two different types of commonly
employed glasses and for gold.
A more general model of an SPR biosensor is given in Figure 2.6. In this
case, an additional adhesion layer between the prism and the metal film has been
included. Furthermore, the active part of the biosensor consists in this model
of a biomolecular linker layer, a sensing layer and a response layer. Typically,
the biomolecular linker layer serves as an attachment point for the sensing layer
to which the molecules making up the response layer can adhere. Although an
analytical expression for the reflectance of the layer stack in Figure 2.6 could be
derived, the intensity reflectance is best found using a computer program.
20
Surface plasmon resonance
Semi-infinite
metal layer
Ambient
Dielectric gap
Thin metal
film
Prism
Prism
Otto configuration
Kretschmann configuration
Figure 2.7: Principal outline of the Otto- and Kretschmann SPR prism couplers. In
the Otto setup, a thin dielectric gap (typically air or water) is introduced between the
two semi-infinite prism and metal layers. The SP propagates on the side of the metal
film facing the dielectric. In the Kretschmann configuration, the metal film is thin and
evaporated directly on top of the prism. In this case the surface plasmon is excited at
the metal-ambient interface.
2.3
Optical SP excitation
We saw in section 2.1 that for two semi-infinite dielectric - metal layers the momentum of incident plane wave light can never match the surface plasmon momentum.
In 1968, Otto presented the ATR configuration for SP excitation through an air
gap between a glass prism and a semi-infinite metal film.30 In the same year,
Kretschmann and Raether devised a setup in which the metal film was evaporated
directly on the prism.31 These two configurations are referred to as prism couplers
and utilize the evanescent field of light reflected at the prism boundary to excite
surface plasmons.
Prism coupler
In order to have resonance, the x-component of the incident light wavevector needs
to match the SP wavevector. This resonance condition can be expressed:
0
kSP = kphoton
=
x
ω√
ε0 sinφ0
c
(2.43)
in which ε0 is the dispersion of the prism and φ0 the internal angle of incidence. In
order to satisfy Eqn. 2.43 it is required that nprism sinφ0 > na sinφa (the subscript
a denotes ambient), and the resonance condition can therefore only be fulfilled
at values of φ0 greater than the critical angle of incidence, φc . In Figure 2.7 the
principal outlines for the experimental configurations (Otto and Kretschmann)
used to fulfill this condition are shown. In general, when for instance an equilateral
prism is employed, and when the angle of incidence deviates from the prism angle,
the incident light will be refracted at the prism boundaries upon entry and exit.
2.3 Optical SP excitation
21
RTM
1
0.5
0
in water
in air
40
60
AOI / deg.
Otto configuration
80
40
60
AOI / deg.
80
Kretschmann configuration
Figure 2.8: Calculated reflectivity curves for light incident in the Otto (left) and
Kretschmann (right) configurations. The wavelength λ = 633 nm and the dielectric
gap is 300 nm between a BK7 glass prism and a semi-infinite gold film. In the case of
Kretschmann, the gold film thickness is 50 nm. Curves are shown for air (∗) and water
(n = 1.33, ◦) as the dielectric.
Accordingly, the external angle of incidence will be different from the internal
angle within the prism. For clarity, all angles considered here are always the
internal angle of incidence and the refraction, and possible loss, of light at the
prism boundaries are disregarded.
Among the prism couplers, the Kretschmann configuration is prevalent much
thanks to its simplicity. The Otto configuration requires precise manufacture
and positioning techniques in order to achieve perfectly parallel prism and metal
surfaces with an exact and sub-micron wide gap in between. Direct evaporation
of the metal film on a prism surface is on the contrary quite straightforward, but
seldom used. Instead, an index matching oil or gel is used to optically couple
gold coated glass slides to the prism. This allows for exchange of the functional
gold film and reuse of the prism. In addition, because the light is incident from
below, a flow cell could be docked to the sensor surface, making the Kretschmann
configuration well suited for application within biosensing wherein samples are
sequentially injected over the surface.
The reflectivity in the Otto and Kretschmann configuration can be calculated
using the matrix model described in section 2.2. Figure 2.8 shows the reflectivity
of TM-polarized light, RT M , in the Otto and Kretschmann setups respectively.
For the Otto-configuration, the reflectivity curve for a 300 nm air gap displays
a pronounced minimum at an angle of incidence of around 42◦ (λ = 633 nm)
corresponding to fulfillment of the resonance condition. When the gap is filled
22
Surface plasmon resonance
RTM
1
0.5
0
60
10 nm
20 nm
30 nm
40 nm
50 nm
60 nm
70 nm
65
70
AOI / deg.
75
80
Figure 2.9: Calculated reflectivity curves for different gold film thicknesses in the
Kretschmann configuration. The substrate is BK7 glass and the ambient is water
(n = 1.33), λ = 633 nm. The optimal film thickness is close to 50 nm as indicated
by the thicker curve.
with water the minimum shifts to about 72◦ and the reflectivity becomes nearly
zero. In the Kretschmann setup the minimum shifts in a similar fashion. A
noteworthy observation is that the width of the resonance becomes larger at high
angles. At angles below the critical angle of incidence, φc , which is manifested
as a ‘kink’ in the reflectivity at about 41◦ and 61◦ respectively, the reflectivity is
still quite high, and the 50 nm gold film then acts as a mirror. The reflectivity at
resonance becomes zero when the radiative damping of the SP equals the internal
damping (Γrad = Γi ).37 This happens only for a very precisely defined metal film
thickness which is also a function of the wavelength. Figure 2.9 shows calculated
reflectivity curves for 633 nm light for different gold films in the Kretschmann
configuration. The optimal thickness is in this case close to 50 nm. This gold
film thickness is suitable for most parts of the red spectrum. We will refer to the
angle of incidence or the wavelength of the incident light that meets the resonance
condition and gives a minimum in reflected intensity as the SPR angle (φspr ) or
the SPR wavelength (λspr ) respectively.
Grating coupler
Corrugated surfaces can also be used to couple light into surface plasmon modes,
this configuration is referred to as a Grating Coupler (GC). GCs are attractive
as large-volume low-cost devices because they are easily manufactured using established plastic moulding technology. The dispersion relation of a GC device is
2.4 Imaging surface plasmon resonance
given by
23
Eqn. 2.4437
ω√
2π
εd sinφd ± u
(2.44)
c
Λ
wherein Λ is the grating constant, u is an integer and the subscript d denotes the
dielectric ambient. In this type of device, light is typically incident from above,
through the medium, which could be troublesome in biosensors with complex samples, i.e. blood. In addition, in wavelength spectroscopy mode, the sensitivity of
GC-SPR devices is lower than for ATR-based sensors.53 Several instances of GCSPR sensors has been presented. For example, the FlexchipTM instrument which
is part of the Biacore line-up (now GE Healthcare) utilize a grating coupler in
imaging SPR mode.54 Recently, an approach based on angular interrogation that
enables measurements in parallel along a two-dimensional array was presented.55
Grating coupled SPR sensors have also been used in combination with electrochemical measurements.56
kx =
2.4
Imaging surface plasmon resonance
If the optical detection unit of the SPR sensor is a Charge-Coupled Device (CCD)
chip or any other type of array detector and the substrate is evenly illuminated
with a large beam, the reflected light from the surface can be displayed as a twodimensional image, in which each pixel depicts a part of the illuminated surface
area. This type of sensor geometry is referred to as imaging surface plasmon
resonance∗ or surface plasmon microscopy and was first suggested by Knoll et al. in
1988.57, 58 Another way of achieving lateral resolution is to narrow the illuminating
beam and scan the substrate.59 iSPR sensors can be based on intensity modulation
in which case the wavelength and angle of incidence are fixed and the reflected
intensities measured as functions of in plane position and/or time. The observed
contrast in the SPR image is then obtained when different regions of the substrate
have different refractive indices so that the resonance condition varies over the
surface. Alternatively, angular or wavelength spectra can be acquired for each
depicted region of the substrate and the SPR angle or wavelength can be calculated
to render an SPR map of the sample.60–62
As for all optical microscopy techniques, the limit in resolution for iSPR is
governed by diffraction. This means that the highest attainable lateral resolution
is in the µm range, of the same order of magnitude as the wavelength of the light.
However, this only applies to the direction perpendicular to the plane of incidence.
In the direction of SP propagation, the resolution is limited by the plasmon propagation length (LSP , Eqn. 2.22). Since LSP is inversely proportional to the sum of
the radiative and internal damping (Γrad and Γi ) and these in turn depend on the
wavelength (from the dispersion relation), the propagation length will be different for different wavelengths. Figure 2.10 illustrates this wavelength dependence.
∗ Sometimes
the term “surface plasmon resonance imaging” (SPRI) is used
24
Surface plasmon resonance
LSP / μm
δz / nm
200
10
100
50
1
0.1
500
600
700
800
10
500
dielectric
metal
600
700
800
Figure 2.10: Calculated propagation length (LSP ) and probe depth (δz ) for 50 nm gold
on BK7 glass in aqueous buffer (n = 1.33) as a function of wavelength. The probe depth
(which was calculated for the semi-infinite case only) is shown for both the ambient (/)
and the gold film (◦). Note that the y-axes are logarithmic.
From the leftmost graph in Figure 2.10, it can be seen that the propagation length
in an aqueous ambient is about an order of magnitude larger than the diffraction
limit which means that LSP is the limiting factor in iSPR lateral resolution. It
should be noted that the calculated values for the propagation length are valid only
00
at resonance because we have used
q the assumption that Γrad = Γi = k∞ , which
εa
)], but this holds at resonance only. The
means that LSP = 1/[4 · =m( ωc εεmm+ε
a
probe depth (which in this case was calculated for a semi-infinite gold-dielectric
system) is shown in the rightmost graph of Figure 2.10.
Choice of wavelength and incidence angle
In intensity mode iSPR the choice of wavelength and incidence angle have a large
impact on the sensitivity and dynamic range. Figure 2.11 shows calculated reflectivity as a function of wavelength and incidence angle for Kretschmann based
SPR sensors of two different prism materials. The dark streaks in the graphs corresponds to fulfillment of the resonance condition. In order to obtain contrast in an
SPR image, it is necessary to select angles and wavelengths that lie on one of the
flanks of the resonance curve. As is seen in Figure 2.11, this can be attained with
a multitude of combinations of wavelength and incidence angle. In most cases,
one strive to obtain as low an angle of incidence as possible. This is because the
oblique angle in a simple iSPR configuration distorts the image, thereby leading to
a reduced lateral resolution. Since the SPR angle decreases with wavelength, it is
then recommendable to work in the far red of the visible spectra. Most CCD detectors, however, are designed to be less sensitive in the infrared posing a restraint
2.4 Imaging surface plasmon resonance
80
25
1
AOI / deg.
RTM
BK7
70
0
60
50
80
1
SF10
RTM
AOI / deg.
0.5
70
0.5
0
60
50
500
550
600
650
700
Wavelength / nm
750
800
850
Figure 2.11: Calculated reflectivity as a function of angle of incidence and wavelength.
The reflectivity for two different glass substrates (BK7 and SF10) is shown, the gold film
thickness is 50 nm and the ambient is water (n = 1.33) in both cases. The darkest areas
correspond to fulfillment of the resonance condition.
on the choice of wavelength∗ . The main reason for choosing shorter wavelengths
lie, however, in the desire to have as short a propagation length as possible. The
trade-off is then between propagation length and image distortion both of which
affect the lateral resolution negatively. In the Kretschmann geometry, in the red
and in aqueous ambient, propagation lengths lie in the 10 µm range (Figure 2.10),
this resolution is quite acceptable for most applications within biosensing.
In terms of sensitivity† or contrast in SPR imaging, many considerations need
to be taken into account. First of all, SPR is sensitive to both bulk refractive index
change and changes in refractive index or thickness of adsorbed thin films. This
∗ This
is primarily due to an (often removable) filter in front of the CCD of most digital
cameras and this problem can be circumvented through normalization.
† Here, we will only consider the theoretical instrumental sensitivity in terms of response to
changes in optical properties. In real biosensing systems an additional contributor to the real
sensitivity is the concentration dependent change in optical properties, ∂n
, or ∂d
.
∂c
∂c
26
Surface plasmon resonance
means that there are at least two separate sources that can give rise to contrast
in an SPR image; local differences in refractive index or adsorbed molecules. In
most biosensing applications, the change in bulk refractive index is equal over the
substrate and it is only molecular binding events that give rise to contrast. In this
case, a relevant model system is the growth in thickness of a thin film adsorbed on
the gold surface, or an increase in refractive index of an already present film. We
define sensitivity as the slope of the calibration curve, which for the three cases
described above renders:
Sn
=
Sd
=
Snf
=
∂∆R
,
∂na
∂∆R
,
∂df
∂∆R
.
∂nf
(2.45)
(2.46)
(2.47)
wherein, na denotes refractive index of the ambient (bulk), df and nf , thickness
and refractive index of a surface film. Figure 2.12 shows calculated reflectivity
curves for different thicknesses of a thin film with refractive index (n = 1.5).
These calculations were made at a wavelength of 750 nm. The SPR angle for the
gold film in the absence of an organic film is in this case about 66.45◦ . We see from
the difference curves in Figure 2.12B and C that the highest contrast is obtained at
a slightly lower angle than φSP R , in this specific case at about 66.2◦ (thick line in
the graphs). This is in agreement with theoretically derived findings by Yeatman
et al. who found that the highest sensitivity (defined in terms of the slope of
the SPR curve) is obtained
the
√ when Γrad = Γi /2 and at an angle offset from
63
resonance by ∆φ = −w/ 3, wherein w is the width of the resonance dip. The
slope (calculated as the numerical gradient of the curves in Figure 2.12C and shown
in D) shows that the maximum change in reflected intensity is about 12%/nm and
drops to about 6%/nm at an organic film thickness of 5 nm. This can give an
appreciation of the dynamic range of the iSPR sensor in intensity mode. From the
reflectivity maps in Figure 2.11 we see that the width of the resonance decrease
with wavelength, thereby increasing the slope of the flanks. Therefore, we expect
the sensitivity to increase with wavelength. On the other hand, there is a decrease
in dynamic range when the slope increases, making a relinquishment in sensitivity
beneficial for the dynamic range of the iSPR sensor.
Approximate analytical expressions for the sensitivity can be derived by applying the Cauchy-Lorentz distribution, which can accurately describe the reflectivity in the range close to SPR resonance. This is a commonly employed approach
in other work.63 Another approach is to take the derivative of the expression
Eqn. 2.42, or of the dispersion relation Eqn. 2.19.64 We will settle for a numerical
calculation based on the Fresnel formalism (section 2.2), the result of which is
presented in Figure 2.13. The dashed lines in the left graph of Figure 2.13 show
2.4 Imaging surface plasmon resonance
R
ΔR
A.
1
27
B.
1
TE
0.5
0.5
0
−0.5
0
64
66
68
AOI / deg.
ΔR
−1
64
70
66.4°
66.3°
66.2°
0.1
66.4°
66.3°
66.2°
66.1°
66.0°
0.01
0
5
Thickness / nm
10
70
D.
66.1°
66.0°
0.5
0
68
AOI / deg.
∂ΔR/∂d / nm-1
C.
1
66
0
5
Thickness / nm
10
Figure 2.12: Reflectivity curves for different thicknesses of an organic adlayer (n = 1.5)
in an aqueous ambient (n = 1.33). The wavelength, λ = 750 nm in all cases. A) Calculated reflectivity for different thicknesses (0, 5, and 10 nm) of the organic film. The SPR
angle, φSP R = 66.45◦ in the absence of an organic film. The reflectivity of TE-polarized
light is also included (dashed line). B) Change in reflectivity as a function of incidence
angle for growth of a 10 nm thick organic film, in 1 nm steps. The arrows indicate increasing thickness. C) Reflectivity change as a function of film thickness at different angles of
incidence. D) Numerical gradient of the curves in C. Note the logarithmic y-axis.
28
Surface plasmon resonance
Sensitivity /
mRIU-1 / nm-1
AOI / deg.
80
max(∂ΔR/∂na)
max(∂ΔR/∂d)
0.1
75
max(∂ΔR/∂n)
0.01
70
65
550
600
650
700
750
Wavelength / nm
800
0.001
550
Bulk
Hydrogel
Thin film
600
650
700
750
Wavelength / nm
800
Figure 2.13: Sensitivity for three different SPR response incentives; Change in refractive
index of the semi-infinite bulk (×), change in refractive index within a 20 nm thick sensing
layer, e.g. a hydrogel, (◦), and growth of a thin organic film, n = 1.5, (/). The left graph
shows the optimal angle, which is the angle that gives the highest sensitivity, as a function
of wavelength (solid lines) for the three different cases. The dashed lines represents
φspr . In the right graph the maximum sensitivity defined as the slope of the change in
reflectivity is plotted as a function of wavelength. Note that the scale is logarithmic.
The unit is either mRIU−1 or nm−1 depending on whether the monitored change is in
refractive index or thickness. It is assumed that the optimal angle of incidence is chosen.
2.4 Imaging surface plasmon resonance
HDT
Au
500
HDT
ΔRTM
Au
29
0
−500
4000
66.2°
Au
Au
HDT
Intensity (RTM)
65.9°
HDT
θc
3000
2000
1000
66.6°
66.9°
200 μm
60
62 64 66
AOI / deg.
68
Figure 2.14: SPR images taken at four different AOIs (marked by • on the angular
axis in the upper graph) showing circa 2.1 nm thick lines of micro contact printed 1mercaptohexadecanethiol (HDT) on gold. The image contrast shifts when the AOI pass
the SPR angle as indicated in the upper graph. The critical angle of reflection (Θc ) has
been indicated in the (unnormalized) SPR curves.
the SPR angle as a function of wavelength, these curves are similar to the maps
in Figure 2.11. The solid lines show the optimal choice of incidence angle in order
to maximize the sensitivity. In the right graph, the sensitivities of three different
incentives for shifting the resonance condition have been plotted; bulk refractive
index change, index change within an already present (non-dispersive) organic film,
and thickness increase of a thin film. In all three cases, the sensitivity increase
with wavelength.
An example on SPR imaging is given in Figure 2.14. In this case, a gold
substrate has been patterned with a self assembled monolayer of an alkanethiolate
using micro contact printing. In array biosensors, imaging SPR can be used in
intensity mode to study molecular binding events in real time. Figure 2.15 shows
an example of this, wherein a synthetic peptide with specificity to calmodulin
is covalently immobilized to a sensorchip, and the subsequent interaction with
calmodulin monitored. The shift in reflectivity is plotted as a function of time.
Imaging SPR in spectral mode
There is a fundamental limit to the dynamic range of intensity modulated SPR
sensors since shifts larger than the width of the resonance can not be detected. One
way of increasing the dynamic range is to acquire angular or wavelength reflectivity
spectra. With iSPR it is then possible to determine λspr or φspr with lateral
resolution. Angular spectral interrogation mode is a very common implementation
in commercial SPR sensors. However, in imaging mode, one concern with angular
30
Surface plasmon resonance
Patterned substrate
ΔRTM / RTE
0.4
CBD immobilization
Activation
Calmodulin binding
Dectivation
0.3
0.2
0.1
0
2000
2500
3000
3500
Time / s
4000
4500
Figure 2.15: Sensorgram showing the covalent immobilization of a calmodulin binding
peptide using reactive esters in a thin polymer film on gold. Binding (in the presence
of calcium ions) of calmodulin and the subsequent dissociation, when the Ca2+ ions are
removed, can be followed as shift in reflectivity. (The surface chemistry is more fully
described in Paper IV)
interrogation is the variation in image distortion which needs to be corrected for.
This can be accomplished, with some effort, by image post-processing or through
clever optical design. By fixing the incidence angle and scanning the wavelength,
correcting for distortion becomes much simpler. The sensitivity in wavelength
spectral mode can be defined as ∂λspr /∂n, or ∂λspr /∂d, depending on whether the
incentive is an increase in refractive index or in thickness, and can be calculated
in the same way as described v.s. Figure 2.16 (right graph) shows the calculated
sensitivity as a function of wavelength for two different cases, either a bulk index
change or addition of a thin film. As can be seen, the sensitivity increases with
wavelength. Hence, it is generally better from a sensitivity point of view to work
at longer wavelengths, particularly so for bulk index sensing. For addition of a
thin film, however, the wavelength dependence can also be seen but is in this case
much smaller. One consequence of this is that for sensor applications in which
one want to discriminate between an (often unspecific) change in bulk refractive
index to a change in film thickness (as is the case in many biosensor applications)
it might be beneficial to design sensors to work in the shorter wavelength regime.
The values in Figure 2.16 are valid in aqueous ambient. In air, the sensitivity is
generally higher and the wavelength dependence more pronounced.65
The left graph in Figure 2.16 shows calculated reflectivity curves in wavelength
interrogation mode. The drop in reflectivity at shorter wavelengths is caused
by electron excitation related absorption. At these energies, the gold film can no
longer be considered free electron like and the surface plasmon resonance condition
can not be fulfilled.
2.4 Imaging surface plasmon resonance
31
Sensitivity /
mRIU-1 / nm-1
R
1
10
TE
∂λ/∂na
0.5
∂λ/∂d
0
500
600
700
Wavelength / nm
800
1
600
Bulk
Thin film
650
700
750
Wavelength / nm
800
Figure 2.16: Calculated reflectivity curves and sensitivity for SPR in wavelength interrogation mode. The left graph shows reflectivity curves at an angle of incidence of 72◦ ,
for a 50 nm thick gold film in aqueous ambient (n = 1.33), the arrow indicates the shift
upon increasing the bulk refractive index or for addition of a thin film. The reflectivity
of TE-polarized light is also shown (dotted curve). In the right graph, the wavelength
dependence of the sensitivity defined in terms of shift in λspr is shown. The curves show
both bulk refractive index sensitivity (×) and the thin film sensitivity (n = 1.5, /).
An example where imaging SPR in wavelength interrogation mode is employed
in detection of biointeraction events is given in Figure 2.17. The reflectivity curves
in Figure 2.17 shows how λSP R shifts towards longer wavelengths upon binding of
biomolecules. The z-value of each pixel in the surface maps corresponds to a λSP R
value calculated from measured reflectivity curves.
Sensitivity in affinity biosensors
In the previous section, the instrumental sensitivity was discussed in terms of ∂∆R
∂n
∂λ
. In affinity biosensors, the measured change in refractive index is caused by
and ∂n
a change in the concentration of an analyte present over the surface. The induced
refractive index shift is given by:
∆n =
∂n
∂c
∆Γ
d
(2.48)
in which ∂n
∂c is the refractive index increment, d is the thickness of the sensing
layer and ∆Γ is the change in surface concentration of the analyte molecules. For
−1 66
proteins, a typical value for ∂n
.
∂c is 0.18 ml g
32
Surface plasmon resonance
Biotin patterned
SAM
λSPR / nm
760
750
740
730
1
b
0.5
x / mm
Fab
1
a
c
1
0
1
0.5
x / mm
0.5
x / mm
0
a
1
0
1
0.5
x / mm
0.5
y / mm
0 0
b
c
RTM/RTE
750
λSPR / nm
Biotinylated
peptide
Neutravidin
740
730
0
0.5
x / mm
1
0
650
700
750
λ / nm
800 650
700
750
λ / nm
800
Figure 2.17: Interactions on a biosensor array chip monitored by imaging SPR. A
self-assembled monolayer was patterned with biotin using micro-contact printing (•).
The surface was then incubated with Neutravidin (×), a biotin-tagged peptide () and
exposed to a Fragment Antigen Binding (Fab) part of an antibody with specificity towards
the peptide (J). The white markers in the first panel indicate the regions of interest from
which the line profiles (a), and the SPR curves (b and c) where acquired. (Data for this
Figure part of the work in Paper VI).
2.5 Imaging SPR and electrochemistry
2.5
33
Imaging SPR and electrochemistry
Electrochemistry primarily deals with the study of chemical reactions that takes
place at the interface of an electrode and an electrolyte. Because of the high
sensitivity to optical changes in the immediate vicinity of metal surfaces, SPR is
a particularly suited technique for the study of electrochemical processes at the
electrode/electrolyte boundary. For this reason, SPR has been combined with
electrochemistry in a number of applications, for example in studies of monolayers of redox-proteins67 and for detection of enzymatic reactions.68, 69 Ertl and
coworkers70–72 employed imaging surface plasmon resonance in a novel study of
spatiotemporal pattern formation on electrodes. They also studied the formation of stationary concentration patterns on electrode surfaces,73 and suggested
potential future applications regarding tailoring of electrode patterns.
When electrochemistry is combined with imaging surface plasmon resonance,
the substrate is used as an SPR sensor surface and as a working electrode simultaneously. Electrochemical reactions taking place at the electrode interface can
then be studied with spatial resolution (Paper II). Figure 2.18A shows a section
view of a typical experimental setup wherein the sensor surface can be used either
as working or as a bipolar electrode. An open cuvette is tightly pressed towards
the sensor surface which is connected to the electroanalytical equipment∗ using
a contact pad. Reference and counter electrodes are then immersed in the electrolyte solution. Elaborate descriptions of typical experimental setups and details
of electroanalysis can be found in textbook literature.74, 75
In electrochemical SPR, several phenomena can affect the optical properties
and induce an SPR signal. First and foremost, when a redox reaction takes
place, the different forms (reduced/oxidized) can have different refractive indices,
in which case a refractive index change takes place over the electrode surface as
the reaction proceeds in either direction. Secondly, when a potential is applied
to the surface in an ionic solution, the composition of the Electric Double Layer
(EDL) consisting of a surface layer of ions and a diffuse layer of counter-ions is
changed at the interface. The thickness of the EDL is typically a few nanometers† ,
at least an order of magnitude less than the probe depth of SPR. Even so, the
presence of a charged double layer can give rise to significant SPR signals. In this
case, the SPR condition is influenced by the structuring of ions at the surface.
There is also an additional contribution, by the presence of an electric field at the
surface/electrolyte interface, which affects the optical properties of the gold film.
The latter effect is observed in particular when the surface charge is positive and
there is a deficiency in electron density.76 The presence of the EDL can influence
both the position and the width of the resonance curve, because both the real and
∗ For instance a potentiostat, wherein the potential of the working electrode (vs. a reference
electrode) can be controlled and the resulting current measured.
† Given by the Debye screening length.
34
Surface plasmon resonance
imaginary part of the dielectric constant will be altered by the interfacial electric
field.77 Furthermore, in particular at sustained large positive potentials a layer
of gold oxide with significantly different optical properties will form that might
change the SPR condition.78
The relation between redox species concentration and applied potential at an
interface is given by the Nernst equation (Eqn. 2.49)
c
RT
E = E 00 −
ln
(2.49)
zF
1−c
wherein E is the potential at the working electrode, E 00 the formal potential, z
the number of electrons transferred in the redox reaction and c is the fraction of
[Red]
). The SPR response to changes in
molecules in either redox form (c = [Red]+[Ox]
redox species concentration, ∂R/∂c, can be subdivided into two components, the
refractive index increment, ∂n/∂c and the sensitivity to changes in refractive index,
∂R/∂n. Figure 2.18D shows measured SPR signal (solid squares) over the working
electrode/sensor surface as a function of applied potential in a three electrode
setup. The solid line is the calculated response from the Nernst equation. The
calculation was performed using a value of ∂R/∂n from Figure 2.13 and ∂n/∂c from
literature.79 The Cyclic Voltammetry (CV) measurement shows how the current
varies with the potential. From the CV it can be seen that the shift in SPR
response coincides with the formal potential, which is the potential between the
two peaks in the CV. The dependence of the SPR response to applied potential in
electrolyte solution in the absence of redox species is shown by the filled triangles in
Figure 2.18D. When the surface charge is positive, attraction of negatively charged
ions and the change in optical properties of the gold leads to a positive shift in the
SPR signal. In the presence of redox species, however, the redox reaction induced
shift in bulk refractive index appears to be the dominating factor.
On bipolar electrodes, in which the surface functions as an anode and a cathode
simultaneously, a spatial gradient in the potential drop across the electrode/electrolyte
interface is setup. The potential drop in the gradient region can be visualized using imaging SPR (Figure 2.18C) (Papers II and III). Using the calibration curve
in Figure 2.18D, the SPR response of the line profiles in Figure 2.18C can then be
converted into surface potential.
2.5 Imaging SPR and electrochemistry
Reference electrode
Electrode 1
35
A.
Electrolyte
RTM/RTE
Gold surface
Incident light
Reflected light
KNO3
[Fe(CN)6]3-/4-
0.8
Electrode 2
Contact pad
B.
1
0.6
0.4
0.2
0
600
Prism
650
700
750
Wavelength / nm
C.
0.05
D.
[Fe(CN)6]3-/4-
10 mA
∆RTM/RTE
1 mA
0
5 mA (in KNO3)
0
KNO3
−1
−0.05
4
3.5
3
x / mm
2.5
2 -0.1
0
0.1
0.2
E/V
0.3
0.4
Figure 2.18: A). Schematic view of the electrolytic cell used in combined imaging surface
plasmon resonance and electric measurements. B). SPR curves in supporting electrolyte,
0.5 M KNO3 (solid), and in a mixture of 0.5 M KNO3 and 10+10 mM [Fe(CN)6 ]3−/4−
(dashed). The vertical lines mark the respective wavelengths at which the data in C and
D was acquired. C). SPR response when an electric current is passed in the electrolyte
between electrodes 1 and 2 and the sensor surface acts as a bipolar electrode. A gradient
in the potential drop between the sensor surface and solution causes oxidation to occur
on one side of the surface and reduction on the other. The slope of the gradient depends
on the current passed through the solution. D). SPR response at different potentials
when the sensor surface acts as a working electrode in a three electrode setup. In the
presence of redox species () and in supporting electrolyte only (J). The solid curve is
a calculated fit based on the Nernst equation (see text). Also shown is a CV (dashed).
The left axis in C and D applies to both figures, while the right axis is for the CV only.
(Part of this figure to appear in Paper X)
0.5
i / mA
1
5 mA
36
Surface plasmon resonance
Chapter 3
Soft matter surfaces
Cleverly designed surface chemistry is an important path to the introduction of
functionality, e.g. specificity and selectivity in biosensors. Soft matter surface coatings such as polymers, organic molecular monolayers, biological membranes and
biomacromolecules can for instance be used as linker layers and provide means of
molecular immobilization in biosensor applications. The wide variety of available
chemistries and the endless possibilities of synthesis of new compounds enables
tailoring of desired surface properties.
In this chapter, an introduction to the various surface chemistries that were
used in the papers included in this thesis (Figure 3.1) is given.
3.1
Self assembled monolayers
Self Assembled Monolayers (SAMs) has become a very important tool for functional surface modification in biosensors. Although studies of molecular film for37
38
Soft matter surfaces
Paper I
Paper II
Paper III
O
HS
O
OH
8
HS
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
Paper V
S
Paper VI
10
Paper IV
S
O
6
SH
HS
O
HO
O
H3N
O
O
O
O
HS
O
O
O
n
N
H
O
HS
n
O
10
O
O
O
O
n
N
H
O
OH
11
O
HO
Figure 3.1: Overview of the different soft matter surface modifications employed in
studies within this thesis (not to scale). The substrates are in all cases glass slides coated
with a thin layer of gold. The different surface chemistries are: I) Hydrophobic barriers of
an ionic surfactant patterned onto a dextran hydrogel. II) Micro contact printed patterns
of thiolates on gold. III) Composition gradient of thiolates on gold. IV) Ethyleneglycolcontaining polymer array with variable thickness. V) Amphoteric polymer composition
gradient. VI) Mixed thiolate monolayer on gold.
3.1 Self assembled monolayers
39
mation at interfaces and on metal surfaces had been of interest early on∗ , it was
in the early 80’s when Nuzzo and Allara reported on the adsorption of organic
disulfides to gold surfaces80 that the path for applications of SAMs within the
field of biosensors was cleared. Early on it was found that monolayers of ωsubstituted alkanethiolates on gold were of special interest, due to the high degree
of ordering,81, 82 potential for tailoring of surface properties,83, 84 and ease of further functionalization of SAMs of such. A fairly recent review by Whitesides and
coworkers covers the details of formation and gives several examples on applications of SAMs.85
Monolayers of alkanethiolates form spontaneously upon immersion of a gold
substrate in a thiol solution with a concentration in the micro- to millimolar regime.
The initial adsorption step is generally very rapid, but the layer structure ill defined
and the alkyl chains in a largely gauche conformation. Over time, however, a
well organized crystalline like (all-trans) structure is adopted, as illustrated in
√
√
Figure 3.2. On the Au(111) crystal plane, an overlayer with a ( 3 × 3)R30◦
configuration is formed.86 The crystalline structure of the alkyl chains is stabilized
by intermolecular van der waals forces, the energy of which is about 1 kcal/mol
for each CH2 -group. Since the distance between the adsorption sites of sulfuric
atoms is about 5 Å, which is slightly more than the van der waal radii of the CH2
groups, a chain tilt with respect to the surface normal, α = 30◦ , is induced. The
strength of the Au-S bond is in the order of 40 kcal/mol, which is of the same
magnitude as covalent interactions. All the same, the thiolate layer is of a very
complex dynamic structure and reorganization and lateral diffusion seems to be
frequently occurring. A large number of defects are embedded in SAMs on gold,
these include crystal defects in the gold substrate such as terraces, islands, grain
boundaries and impurities, but also defects in the monolayer itself due to phase
transitions or vacancies in the overlayer structure.
SAMs of organosulfur compounds more complex than plain alkyl-derivatives
are frequently used in real applications. Unfortunately, the incorporation of bulky
tail-groups, e.g. carboxyls, amines, amino-acid derivatives etc., usually has a negative impact on the stability and structural organization of the monolayers. Therefore, the compounds providing the end-function are generally mixed with spacer
thiols to form heterogeneous SAMs that can still be highly ordered. Care needs
to be taken when optimizing concentrations and incubation times, particularly for
mixed monolayers, as these parameters will influence the composition and organization of the monolayer. In some cases introduction of hydrogen bond donors can
be used to improve the stability, an example is the inclusion of secondary amides
in the thiol compound, which facilitates lateral hydrogen bonding.87 When the
intended end-function of the SAM is chemical binding of biomacromolecules, the
dilution of functional groups on the surface can serve a secondary purpose by reducing steric hindrance in the interaction between species much larger than the
∗ Langmuir
pioneered this work in 1920 in studies of amphiphilic molecular layers.
40
Soft matter surfaces
α=30°
Tail group
Alkyl spacer
Head group
Overlayer (√3×√3)R30°
∼1 kcal/mol CH2
Au(111)
∼40 kcal/mol
Within minutes
Over several hours
Assembly from solution
Figure 3.2:
Schematic overview of the self assembly of alkanethiols (1mercaptohexadecanoic acid) from solution on gold (111). Assembly is a spontaneous
process that proceeds in several steps. Adsorption occurs within minutes in dilute solu√
√
tions while it takes longer for the highly organized crystalline ( 3 × 3)R30◦ layer to
form. The strength of the Au-S bond is in the order of magnitude of 40 kcal/mol and the
alkyl chains are stabilized by lateral interactions of about 1 kcal/mol per methyl-group.
Also shown is the approximate tilt-angle of the alkyl chains, α = 30◦ .
tail-group.
The presence of a self assembled monolayer on the surface of an electrode
can be detected electrochemically, since densely packed, defect deficient SAMs of
organic molecules can block electron transfer from solution to the substrate surface.88 Depending on the quality and characteristics of the monolayer the blocking
properties will be different. Therefore, electrochemical methods, for example CV,
is a very useful analytical technique when studying the properties of SAMs. Electrochemical analysis can be combined with imaging surface plasmon resonance, to
enable laterally resolved measurements (Paper II).
Ethylene glycol containing thiolates
Of special interest in biosensor applications are SAMs containing oligo(ethylene
glycol) (OEG) chains∗ . This is because OEG SAMs have been shown to suppress unspecific adsorption of proteins.89, 90 The protein repellent properties of
ethylene glycol derivatives can be attributed in part to their hydrophilic character. In addition, chains of more than a few EG-units can adopt a helical like
conformation in SAMs, the occurrence of which has also been correlated with
protein-resistance.91, 92
Within this thesis, SAMs of OEG containing thiolates have been used as a bioinert anchoring layer for an ethylene glycol based hydrogel (Paper IV and V),
∗ With
the general structure: . . . − (O−(CH2 )2 )n − . . .
3.2 Matrix surfaces
41
for specific protein immobilization (Paper III), and as means of immobilization
of synthetic polypeptides in an array format for biosensor purposes (Paper VI).
3.2
Matrix surfaces
In covalent immobilization of biomacromolecules, it is in many cases important to
ensure that the native state of the molecules is preserved. Immobilization on rigid
structures, such as dense alkanethiolate monolayers, can sometimes cause proteins
to loose their secondary structure or impede biointeractions sterically. Therefore,
in biosensors for interaction analysis, controlled immobilization to more flexible
three-dimensional structures is mostly preferred. In SPR sensors specifically, since
the evanescent field extends a few hundred nanometers into the ambient, by using a
three-dimensional matrix the sensor signal can be enhanced. For example, matrices
based on dextran polymer have been very successfully employed in the Biacore
line-up of SPR sensors.15, 93 A very important characteristic of matrix surfaces for
immobilization is a low degree of non-specific binding. One way of accomplishing
this is to employ poly(ethylene glycol) (PEG) based polymers.94 PEG matrices
have for instance been used in the suppression of biological fouling.95 In Paper IV
of this thesis, a PEG based matrix is employed as a template for immobilization
in biointeraction studies.
3.3
Surface pattern techniques
Micro contact printing
Micro Contact Printing (µCP) is a soft lithographic technique developed to fulfil
the need for a cheap and uncomplicated way of producing patterns of biomolecules
on rough and non-planar surfaces. In soft lithography an elastomer is used either
as a stamp, as is done in µCP, to print a molecular pattern, or as a mould,
which is the case in micro moulding in capillaries, replica moulding and micro
transfer moulding, to create three-dimensional structures of polymers or patterns
of biomolecules.96, 97 With soft lithography, a wide variety of materials can be
patterned on flat as well as rough surfaces. Micro contact printing is most suitable
for patterning two-dimensional structures; for example protein layers98 or SAMs
of alkane thiolates.99, 100
In micro contact printing, an elastomeric stamp is first submerged or covered
with a solution (ink) of the molecule to be printed. The inked stamp is then
brought into conformal contact with the surface to be patterned (Figure 3.3). The
ink concentration, ink transfer time and the surface contact time are important
parameters that affect the quality of the pattern produced. These parameters need
to be optimized for different types of inking solutions.
42
Soft matter surfaces
Ink solution
I) Incubation
Transferred ink
II) Dry stamp and
contact substrate
III) Remove stamp
Figure 3.3: Overview of the micro contact printing process. A PDMS stamp is incubated with the desired ink, dried and brought into conformal contact with the substrate.
The ink is transferred in the contact areas only.
Poly(dimethylsiloxane) (PDMS) is the elastomer most commonly used in micro
contact printing. Besides being elastic, it is optically transparent, highly biocompatible and durable, which makes it useful in many applications. Stamps are
generally cast off a master mould fabricated using traditional photolithographic
techniques. Many positive properties of PDMS are related to the low surface energy and chemical inertness, which assures that most molecules does not adhere
irreversibly to the stamp. The elasticity and softness of the PDMS is the cause of
some of the negative properties of the stamp. During curing, the stamp shrinks
by about 1% and especially non polar organic solvents tend to swell the cured
stamp. Transfer of residual PDMS to the substrate can also sometimes occur.101
Sagging or pairing of the structures on the stamp can take place if the aspect
ratio of the structures is too high or too low. When the molecule to be printed is
polar or dissolved in water, it is often necessary to modify the stamp and render
the PDMS surface more hydrophilic, this can be accomplished by a brief exposure
of the stamp to an oxygen plasma.102, 103 Gradient patterns has been made with
PDMS stamps by means of varying the thickness of the elastomer.104
In the papers included in this thesis, µCP has been used to create hydrophobic barriers for the confinement of tiny droplets of protein solution (Paper I), as
means of achieving multi-component SAMs (Paper II) and in bio-functionalization
of thiolate monolayers (Paper VI).
Electrochemical patterning
Although the S-Au interaction of adsorbed thiols on gold surfaces is very strong,
it is not entirely irreversible. Even at room temperature, immersed in a solution
with thiols, there appears to be a continuously ongoing replacement of thiols on
the surface with those present in solution. Most alkane thiols can be reductively
desorbed from gold surfaces at a potential of circa -1.0 V vs. Ag/AgCl.105 This
process is fully reversible, as the thiols will reattach upon restoration of the surface
3.3 Surface pattern techniques
43
potential. The exact potential required to remove the thiols from the gold surface
is dependent on the choice of electrolyte as well as the length of the alkyl chain
and the properties of any present tail-groups on the thiols. Therefore, different
thiols will leave the surface at different potentials. This can in fact be used as
means of patterning through selective desorption of thiols, as proposed by Tender
et al.106, 107 and Wolfbeis et al.108 They demonstrated how individual elements
of an array of gold microelectrodes could be selectively functionalized with different thiols. In their method the surface needed to be patterned and divided into
smaller elements that could be individually contacted. Bohn and coworkers, on
the other hand, have devised a means of electrochemical patterning that allows for
spatial patterns to be set up on electrodes,109–112 whereby for instance the thiol
monolayer composition can be varied laterally. Their method is based on passing
a current through a thin, thereby resistive, gold film, causing a potential gradient
to be set up over the surface. This potential gradient could then be used to desorb alkanethiols from a previously formed monolayer. The empty regions of the
surface was then backfilled with another thiol from solution, ultimately leading to
a gradient mixture of two different thiols.
Within the scope of this thesis, a novel method for electrochemical patterning of conducting surfaces was explored (Paper III). In this approach, an electric
field is induced in an electrolyte by passing a current between two feeder electrodes
(Figure 3.4). When the electric field is large enough, reductions will be induced
on one side of the surface and oxidations on the other as the surface becomes a
bipolar electrode. The potential difference between a point along the surface and
the solution will vary laterally and a potential gradient is thus induced. Various types of molecular gradients can be made with this method, since the redox
reactions can be of virtually any type. For example, electrodeposition of metals,
electropolymerization, or reductive desorption of thiols can all be facilely governed
on a variety of substrates using the setup in Figure 3.4.
Ink-jet printing
Dosage of very small droplets of liquid can be readily accomplished using conventional ink-jet printing technology that has been available in for instance consumer
desktop printers for years.113 Ink-jet printing is based on the application of a pressure pulse to a confined volume of liquid that allows a small part of the bulk liquid
to be ejected through an orifice. In a piezodispenser, a piezoelectric material is
used to generate the pressure pulse. Generally, the piezoelectric material is coated
around a glass capillary so that when a voltage is applied the walls of the capillary
will deform, resulting in the ejection of a liquid droplet from the nozzle. Ink-jet
printing can be used to print proteins,114, 115 thiol SAMs,116, 117 DNA,118, 119 and
other biomolecules for immobilization on surfaces.
Under optimal conditions, a single nearly spherical droplet following a straight
44
Soft matter surfaces
Anode
Cathode
Figure 3.4: Sketch of the experimental setup used in the electrochemical gradient
method. A variable voltage is used to control the total current between two electrodes.
When a conducting substrate is introduced, the current can take an alternate pathway
(i2 ), via redox reactions at both ends of the substrate. At the cathodic end, the reaction
can be for example the reductive desorption of thiols.
trajectory will be dispensed. Upon impact with the substrate, the droplet momentum, which is originally directed towards the surface, will cause a radial flow
parallel to the surface. This results in rapid spreading of the droplet, which forms
a liquid disk. If the kinetic energy of the droplet is much larger than the surface
energy, splashing will occur, and a number of smaller droplets will be formed.
However, if the surface energy is high enough, the liquid disk will retract to form a
sessile droplet with the shape of a spherical cap.120 The contact area of the sessile
droplet is governed by the difference in surface energy of the substrate and the
liquid. On hydrophilic, high energy surfaces, aqueous drops tend to spread over a
larger area. This can be disadvantageous in micro array applications since it limits
the maximum spot density and the risk of cross contamination is increased. One
way to circumvent this is the introduction of a hydrophobic grid, used to divide
the hydrophilic substrate into smaller elements.
In molecular arrays involving chemical binding or biomolecular interactions,
sufficient time needs to be given for the present species to interact. However,
under normal conditions, nano-liter sized water droplets evaporate very quickly.
Another related problem is the accumulation of solute at the rim∗ as the droplet
evaporates (Figure 3.5).121 The reason for this is that initially during evaporation, the contact radius of the spherical cap is pinned, leading to a net radial flow
within the droplet. Unless immobilization of the solute to the substrate is rapid,
some type of evaporation control is often necessary. This can be accomplished by
increasing the vapor pressure of the environment through humidity control or by
lowering the vapor pressure of the droplet through temperature control. Alternatively, the viscosity of the solvent can be increased by addition of i.e. glycerol
or sucrose.11, 13 Additives that act as hydrogen-bond donors can also significantly
enhance the stability of piezodispensed proteins.122, 123 In some instances, evap∗ The
coffee stain phenomenon.
3.3 Surface pattern techniques
45
Evaporation
Uncontrolled
Nozzle
Radial flow
I) Piezodispensation
Controlled
II) Droplet evaporation
Figure 3.5: Solute delivery using piezodispensation. Too rapid evaporation of the
solvent causes accumulation of solute molecules at the droplet boundaries, something
which can be prevented by control of the rate of evaporation.
oration might be beneficial, because the solute concentration increases when the
solvent evaporates.
46
Soft matter surfaces
Chapter 4
Instrument design
The instrumental platform for iSPR used herein was designed and built during
the course of this work. Our design was based on the Kretschmann configuration,
wherein surface plasmons are optically excited by means of an evanescent field.
The instrument was built on an optical bench using standard mounts and fashioned
mechanical components. The basic system, shown schematically in Figure 4.1, consists of the optical components, a light source with appropriate filters, a polarizer,
a prism, and a CCD detector, and a rotational translation stage for control of the
incidence angle. The system is controlled via an appropriate data acquisition software built within the NI LabVIEW environment. Moreover, some different flow
cells and sample compartments were designed to allow for measurements in a confined liquid volume or under controlled flow. While the specific instrument built
here was intended for laboratory use, the configuration is generic and adaptable
for field use.
In this chapter, a brief overview of the components of the imaging SPR instrument is presented along with some justification and discussion of the general
features.
47
48
Instrument design
Light source and
monochromator
Collimator
Polarizer
Mirrors
Prism
CCD detector
φin
Lens
Figure 4.1: Schematic overview and photograph of the optical configuration in the
imaging SPR instrument.
4.1
Optical setup
Prism
A 25 mm equilateral glass prism (BK7) with a refractive index of ∼1.51 was mainly
used in this work. The prism material was chosen primarily to match the supply
of gold covered glass substrates. Substrate gold surfaces are optically coupled to
the prism using an index matching oil, as illustrated in Figure 4.2A. When the
external incidence angle deviate from the prism angle (60◦ ), refraction of the beam
takes place at the prism boundaries as illustrated in Figure 4.2B. Because of the
oblique angle the illuminated/imaged area on the sample is larger than the width
of the incident beam and the image becomes compressed in the axis parallel to the
plane of incidence. The use of a high refractive index prism (for instance SF-10,
n ≈1.72) would lead to less distortion since the SPR angle becomes lower, and
hence closer to the prism angle, with the dispersion of the prism. Because image
distortion is a function of incidence angle, the image resolution will depend on
the chosen angle of incidence and will vary during an angular SPR scan. While
distortion leads to slightly lower resolution as well as a decrease in illuminating
intensity, it can easily be corrected for as described v.i.
Light source
Different types of light sources can be employed in SPR sensing. In the present
setup, two different broadband sources were put to use. Firstly, a Xenon discharge lamp (Oriel Inc.) used in conjunction with a set of interference filters can
be used for fixed wavelength iSPR. Additionally, a Xenon lamp connected to a
monochromator (Acton Research SpectraPro 300i) is used for iSPR in spectral
4.1 Optical setup
49
A.
B.
Index matching fluid
Refraction at prism boundaries
φinternal
φexternal
Prism
Figure 4.2: Prism/Substrate configuration for iSPR. A) An index matching oil is used
to optically couple the glass slide to the equilateral prism. B) The internal angle of
incidence is different from the external due to refraction at the prism boundaries.
interrogation mode. Of common use in laboratory setups is the HeNe laser with
a wavelength of 632 nm. In an imaging setup, a laser light source can be used
with a beam-expander to increase the diameter of the light beam. In the present
setup, light from the monochromator was collimated using a single condenser. The
divergence of the collimated beam is a trade-off with the beam diameter, larger
beam diameters inevitably leads to greater divergence.
Spectral resolution in a diffraction grating based monochromator partially depend on the groove distance which should be on the same order of magnitude
as the reciprocal of the wavelength. The grating efficiency, the relative intensity
of transmitted light, is generally optimized (blazed) for a particular wavelength
range. Because the efficiency also depends on the polarization, care must be taken
during SPR measurements when the TE-polarized beam is used for referencing.
In the present setup, for the most part, a 300 g/mm grating blazed at 500 nm was
used. While the efficiency at 600-800 nm is only about 50% for this particular
grating, there is very little difference between the TE- and TM-polarized beams
which justifies the via media choice of grating.
Detector
A monochromatic 12 bit 1 MPixel CCD camera (Retiga EXi, QImaging) was used
to acquire SPR images. In intensity mode SPR, an important parameter is the
bit depth of the detector. When a 12 bit A/D converter is used, in theory, the
minimal resolvable shift in reflectivity is around 0.025%. In practice, however, the
intensity resolution is governed by noise which is nearly an order of magnitude
larger and also depend on the gain setting of the camera. Since both the incident
light intensity and the detector sensitivity varies with wavelength, the intensity
resolution will also be wavelength dependent.
50
Instrument design
The lateral resolution∗ of the images in the direction perpendicular to the plane
of incidence depends solely on the magnifying optics, while in the direction parallel
to the plane of incidence the resolution is dependent on the angle of incidence.
In the perpendicular direction, the resolution is 2.2 µm/pixel, and the parallel
resolution at an internal angle of incidence of 72.5◦ is around 8 µm/pixel. The
parallel resolution could be well improved by tilting the CCD detector such that
the detector plane becomes largely parallel with the substrate surface. In this
manner, the depth of focus could also be increased. The specific camera used here
is, however, not suited for this type of modification. Alternatively, the use of a tiltshift imaging optics of the type commonly employed in architectural photography
or a similar optical configuration could be used to the same effect. In any case, the
image resolution can easily be calculated at any angle of incidence and distortion
correction is straightforward to implement.
4.2
Fluidics and cell for electrochemistry
There are virtually no limitations to the design of flow systems or cuvettes for the
imaging SPR system, as long as the substrate forms one side or the base of the
liquid cell. However, some important considerations need to be taken into account
when designing a flow system for Biomolecular Interaction Analysis (BIA) in an
array format.124 Typically, it is desired to keep the total volume of the liquid
handling system minimal, in order to reduce sample consumption. Additionally,
in array applications, one prerequisite is that the analyte can be reproducibly
delivered in equal concentration and roughly simultaneously over the entire array.125 This is best achieved by keeping the physical dimensions of the sample
compartment low. The flow system and cuvette predominately used in this work
are presented schematically in Figure 4.3. The volume of the sample compartment
in the liquid flow cell is circa three microliters. Therefore, with a flow speed of 30
µL per minute the liquid is fully replaced in around six seconds which is sufficient
for most array biosensor applications. In addition, a different cuvette for electrochemistry can be seen in Figure 2.18A. The electrochemical cuvette also includes
a contact for the surface as well as room for the immersion of counter and reference electrodes. A modified version of this cuvette was used in the electrochemical
bipolar patterning in Paper III.
4.3
Interface
While the user interface is of minor importance in an experimental setup for laboratory use, the physical interface between the computer and the devices that
∗ Note that we differentiate between resolution, which is the minimum resolvable response or
the limit of detection, and lateral resolution, which is the resolution in the SPR images.
4.4 Data processing and analysis
A.
51
B.
Reference
electrode
PMMA cell
PTFE cuvette
outlet
Counter
electrode
Contact pad
o-ring
o-ring
inlet
Figure 4.3: A) Cuvette for electrochemistry. B) Flow cell for sample delivery during
e.g. biological interaction analysis. To scale.
comprise the instrument might be very important for the reliability of a measurement. Figure 4.4 shows a flowchart of the instrument in operation, and illustrates
some of the tasks that the control and acquisition software must handle. The key
issue in an instrument composed of several distinct devices is timing. For instance,
an image supplied by the detector is synchronized with time in kinetic measurement mode, to the angle of incidence during an angular scan or to the current
wavelength in spectral mode. The software was written in LabVIEW, which by
default operates on an ‘execute on availability’ basis, meaning that subfunctions
within a program will run when all the input data has been provided. Because the
images from the detector are by far the largest blocks of data transferred during a
measurement, the image acquisition is the limiting step in execution. It is important that the software can handle temporary interrupts in the flow of data from
the detector, monochromator or the motion controller without losing synchronization between the devices. In addition, a properly designed control software should
be able to run reliably even if the computer is under load from other programs
running in parallel. All of these issues were addressed during the software design
process.
4.4
Data processing and analysis
Data processing is a very important step in any sensor system. In particular,
in indirect measurement methods, data analysis is a crucial step affecting both
instrumental resolution and sensitivity.
The two modes of operation which are employed in the iSPR sensor described
52
Instrument design
Manual control
On / Off
Motorized control
Storage buffer
Lightsource
Synchronized loop
Polarizer
Stored settings
Computer
Filter wheel
Set λ
Monochromator
Motion
controller
1. Scan λ
Detector
Image λ-stack
Frame
grabber
Image
2. Kinetic
mode
Image sensorgram
Figure 4.4: Simplified flowchart over the software controlled operation of the imaging
SPR instrument. The most critical steps are the synchronized loops. The frame grabber
is synchronized to the monochromator in order to acquire correct spectra. In an image
sensorgram, the time of acquire for each image is also stored.
4.5 Performance
53
in this work require slightly different data processing. In intensity or kinetic mode,
the signal is the reflected intensity as a function of time. The sample frequency
can be up to 10 Hz which is significantly faster than any expected signal response
caused by biomolecular interactions. Therefore, a convenient way of reducing noise
is to take the average over a certain time interval, in the present case this was
mostly done over 1 s. Analogously, CCD detector noise can be reduced to some
extent by lowering gain and increasing exposure time or through pixel binning.
An even more convenient route to noise reduction is to increase the size of the
Region of Interest (ROI), averaging the signal over a larger detector area. It has
also been shown recently, that incorporating multiple areas for referencing leads
to lower noise levels in intensity mode iSPR.126
In spectral mode, the resulting data is an image stack, consisting of a large
number of images acquired at different frequencies of the incident light, typically
at a sampling frequency of 1 nm−1 . These spectra are always normalized using
TE-polarized light as reference. Before normalization, because the TE- and TMpolarized spectra can not be acquired in parallel, the data needs to be interpolated to be equidistant. The appearance of raw and normalized spectra and the
data processing step is shown in Figure 4.5. There exists numerous methods of
determining the SPR wavelength from a reflectivity spectrum, each of which may
have different qualities.127, 128 Herein, a weighted centroid algorithm (Eqn. 4.1)
was used.129, 130
P
2
i λi · (L − Ri )
λSP R = P
(4.1)
2
i (L − Ri )
An important parameter in the weighted centroid algorithm is the threshold value,
L, which defines a reflectivity, R, at which all values above are neglected. Because
of the asymmetric shape of the SPR reflectivity curve, the weighted centroid algorithm introduces a slight deviation in the SPR wavelength from the true value.
Typically, it is the shift in λSP R that is of interest rather than the absolute value,
in which case subtraction of two images is performed. In that case, the deviation
is canceled and of no importance. Even in absolute measurements, the deviation
can be neglected most of the time, particularly if the threshold is cleverly chosen
to reduce the assymetry.127
4.5
Performance
The performance, in terms of resolution and sensitivity of the iSPR instrument in
any given measurement is highly dependent on the experimental circumstances.
While the instrumental sensitivity can be maximized, in accordance with
Figure 2.13, by proper choice of a combination of the wavelength and the angle of
incidence, the resolution will depend also on other experimental specific parame
ters, such as ∂n
∂c , unspecific binding, temperature drift, fluctuations in the light
source and camera noise.
54
Instrument design
Image stack
λSPR map
Determine λSPR of each pixel
Counts / au
4000
B.
TE
Weighted
centroid
λSPR
TM
3000
0.8
0.6
2000
Threshold
1000
0
650
1
0.4
RTM/RTE
A.
0.2
700
750
Wavelength / nm
800
650
700
750
Wavelength / nm
800
0
Figure 4.5: Data processing to determine the SPR wavelength from a stack of images
acquired during a spectral scan. A) Raw spectra. The TE-polarized spectrum is used
for normalization. B) Normalized spectrum ( ). All values below the threshold (×) are
used as input in a weighted centroid algorithm to determine λSP R . Each pixel in the
image stack is processed individually.
4.5 Performance
55
ΔRTM/RTE
0.25
0.2
0.15
680
700
Wavelength / nm
720
Figure 4.6: Normalized SPR curves from 200 µm× 200 µm regions of OEG SAM ()
and protein (◦) on a gold surface, acquired during eight consecutive wavelength scans
during continuous flow of room temperate aqueous buffer.
The limit of detection was determined for the experimental conditions in Paper VI. Herein, the angle of incidence was chosen in order to yield a reasonable
image resolution with limited distortion, and the wavelength was set to achieve
the highest sensitivity. By averaging the reflected intensity over ∼2200 pixels for
∼20 samples acquired during 5 seconds, the limit of quantification was assessed
to be in the order of 3 × 10−5 RIU. This value is expected in intensity modulated
SPR131 and comparable to the resolutions of other similar, imaging SPR configurations, reported in literature,132–134 but about two orders of magnitude inferior
to the non-imaging Biacore instruments. Figure 4.6 shows normalized SPR curves
acquired during eight consecutive wavelength scans over an OEG SAM surface patterned with spots of neutravidin (surface pattern procedure more fully described
in Paper VI). The standard deviation of the mean SPR wavelength, determined
using Eqn. 4.1, when treating the curves from the SAM and the protein separately
is σ ≈ 0.08 nm. However, by taking the mean of the difference between λSP R
of a protein spot and a SAM spot for each of the scans, a standard deviation of
σ ≈ 0.02 nm is obtained. Under the experimental conditions used in Figure 4.6
(∂λ/∂n ≈ 3 nm mRIU−1 ), this corresponds to a resolution of 2 × 10−5 RIU.
56
Instrument design
Chapter 5
Future perspectives
Since the launch of the first Biacore instrument in 1990, SPR has proven a tremendously useful tool in the field of biological interaction analysis. The search query
“surface plasmon resonance” currently∗ renders more than 11 000 hits in ISI Web
of Knowledge, out of which 1200 were published in 2008, and the rate of appearance of new papers is anticipated to increase steadily.
On the one hand, iSPR is very useful as a transducer in label free detection of
analytes, in which the tendency is towards the development of more sensitive and
higher-throughput instrumentation. One might envision future SPR appliances
as being low-cost, robust, portable, sensitive, and fast devices. However, because
SPR is a label-free method, specificity relies on the affinity of the sensing surface
towards the analyte, and might be limited by the background response. Even
if single molecular binding events could be resolved, one must be able to ensure
that it is the analyte that is being detected. Therefore, the development of novel
interfaces for affinity sensors will continue to be important, even more so when the
∗ August
17th , 2008
57
58
Future perspectives
instrumental resolution is improved.
There are many potential applications of imaging surface plasmon resonance
related to the field of biomimetics. For example, the behavior of eukaryotic cells,
which are in the size range 10-100 µm, are studied with a variety of techniques.
In particular, cell/substrate contact investigations are often performed using fluorescent labeling and total internal reflection microscopy. In an interesting work,
Giebel et al. have used imaging SPR in ex vivo studies of the movement and
adhesion of living cells on aluminium substrates.135 Using imaging SPR, it is further possible to quantify the cell-substrate distance in real time.136 The variety
of available tools for tailoring of surface properties of SPR substrates should also
enable many interesting studies of cell behavioral patterns.
The interaction of higher organisms with soft surfaces can also be studied with
imaging SPR. For example, barnacles, which can be a great nuisance in marine
biofouling, undergo a larval stage during which they adhere very strongly to surfaces.137, 138 Figure 5.1 shows a sequence of SPR images acquired while barnacle
larvae are ‘walking’ over the sensor surface, in this case an amine-terminated selfassembled monolayer, leaving footprints of a protein adhesive. Using this technique, the response of the larvae to different surface coatings can be investigated
by monitoring their behavior in real time, and the potential of anti-fouling surface
coatings can be assessed. The grafted polymer matrices described herein are particularly interesting model surfaces in such future studies. In addition, the ‘bipolar
patterning’ approach of gradient manufacturing presented in Paper III offers an
alternate, versatile route to the development of new functional materials.
There are some modifications to the imaging SPR instrument employed here
that could increase the resolution. For example, increasing the power of the light
source, or the efficiency of the monochromator would lower the noise, signal drift
could be tackled by temperature control, and the use of a trapezoidal prism would
lower the distortion. Further improvements in sensitivity could be reached if the
phase shift of the reflected light was also evaluated, as is done in ellipsometry.
Future alterations to the experimental setup are likely to include one or several of
these improvements.
59
1
0.4
0.3
1
1
1
0.2
0.1
2
2
2
ΔRTM/RTE
0.5
0
3
Figure 5.1: Use of imaging SPR to study deposition of footprints from cyprid larvae.
As the cyprid walks over the SPR surface, its antenulles (black arrows in photo) leaves
behind a temporary protein adhesive, a ’footprint’. The white arrows in the sequential
SPR images highlight the footprint deposition. The photograph shows a cyprid larva of
the barnacle Balanus amphitrite. Work in progress. (The inset photo is published by permission from
c
Inter-Research Science Center. Inter-Research
2007, Mar.Ecol.Prog.Ser. Vol. 340: 1-8, 2007.
60
Future perspectives
Bibliography
[1] F.S. Ligler, J.M. Schnur, A.W. Kusterbeck, C.R. Taitt, L.C. Shriver-Lake, B Lin, and D.A.
Stenger. 2006 nrl review - the silent guardian demonstration. Technical report, The Naval
Research Laboratory, Center for Bio/Molecular Science and Engineering, 2007.
[2] ES Lander et al. Initial sequencing and analysis of the human genome.
409(6822):860–921, Feb 15 2001.
Nature,
[3] JC Venter et al. The sequence of the human genome. Science, 291(5507):1304+, Feb 16
2001.
[4] S. Hober and M. Uhlen. Human protein atlas and the use of microarray technologies.
Current opinion in biotechnology, 19(1):30–35, Feb 2008.
[5] M Poksinski. Total Internal Reflection Ellipsometry. PhD thesis, Linkoping University,
2005.
[6] J.R. Lakowicz. Principles of Fluorescence Spectroscopy. Springer Science+Business Media,
3rd edition, 2006.
[7] A. Fersht. Structure and Mechanism in Protein Science. W.H. Freeman and Company,
1999.
[8] K. Enander. Folded Polypeptide Scaffolds for Biosensor and Biochip Applications - Design,
Synthesis, Functionalization and Characterization. PhD thesis, Linkoping University, 2003.
[9] Mark B. Pepys and Gideon M. Hirschfield. C-reactive protein: a critical update. The
journal of clinical investigation, 111(12):1805–1812, 2003.
[10] C Sheridan. Protein chip companies turn to biomarkers. Nature biotechnology, 23(1):3–4,
Jan 2005.
[11] G MacBeath and SL Schreiber. Printing proteins as microarrays for high-throughput
function determination. Science, 289(5485):1760–1763, Sep 8 2000.
[12] M Uttamchandani, DP Walsh, SQ Yao, and YT Chang. Small molecule microarrays: recent
advances and applications. Current opinion in chemical biology, 9(1):4–13, Feb 2005.
[13] H Zhu and M Snyder. Protein chip technology. Current opinion in chemical biology,
7(1):55–63, Feb 2003.
[14] H Zhu, M Bilgin, R Bangham, D Hall, A Casamayor, P Bertone, N Lan, R Jansen,
S Bidlingmaier, T Houfek, T Mitchell, P Miller, RA Dean, M Gerstein, and M Snyder.
Global analysis of protein activities using proteome chips. Science, 293(5537):2101–2105,
Sep 14 2001.
[15] S Lofas and B Johnsson. A novel hydrogel matrix on gold surfaces in surface-plasmon
resonance sensors for fast and efficient covalent immobilization of ligands. Journal of the
chemical society-chemical communications, (21):1526–1528, Nov 1 1990.
[16] H Zhu, JF Klemic, S Chang, P Bertone, A Casamayor, KG Klemic, D Smith, M Gerstein,
MA Reed, and M Snyder. Analysis of yeast protein kinases using protein chips. Nature
genetics, 26(3):283–289, Nov 2000.
[17] YS Sohn, J Kai, and CH Ahn. Protein array patterning on cyclic olefin copolymer (COC)
for disposable protein chip. Sensor letters, 2(3-4):171–174, Sep-Dec 2004.
61
62
Bibliography
[18] A.M. Turing. The chemical basis of morphogenesis. Philosophical Transactions of the
Royal Society of London. Series B, Biological Sciences, 237(641):37–72, 1952.
[19] Daniel S. Rhoads and Jun-Lin Guan. Analysis of directional cell migration on defined
FN gradients: Role of intracellular signaling molecules. Experimental cell research,
313(18):3859–3867, Nov 1 2007.
[20] GL Ming, ST Wong, J Henley, XB Yuan, HJ Song, NC Spitzer, and MM Poo. Adaptation
in the chemotactic guidance of nerve growth cones. Nature, 417(6887):411–418, May 23
2002.
[21] Moon Suk Kim, Gilson Khang, and Hal Bang Lee. Gradient polymer surfaces for biomedical
applications. Progress in polymer science, 33(1):138–164, Jan 2008.
[22] MK Chaudhury and GM Whitesides.
256(5063):1539–1541, Jun 12 1992.
How to make water run uphill.
Science,
[23] B Liedberg and P Tengvall. Molecular gradients of omega-substituted alkanethiols on gold
- preparation and characterization. Langmuir, 11(10):3821–3827, Oct 1995.
[24] T Wu, K Efimenko, and J Genzer. Combinatorial study of the mushroom-to-brush
crossover in surface anchored polyacrylamide. Journal of the american chemical society,
124(32):9394–9395, Aug 14 2002.
[25] Jan Genzer and Rajendra R. Bhat.
24(6):2294–2317, Mar 18 2008.
Surface-bound soft matter gradients.
[26] Sara Morgenthaler, Christian Zink, and Nicholas D. Spencer.
morphological gradients. Soft matter, 4(3):419–434, 2008.
Langmuir,
Surface-chemical and -
[27] R.W. Wood. On a remarkable case of uneven distribution of light in a diffraction grating
spectrum. Proc. Phys. Soc. Lond., 18:269, 1902.
[28] U. Fano. The theory of anomalous diffraction gratings and of quasi-stationary waves on
metallic surfaces (sommerfeld’s waves). J. Opt. Soc. Am., 31:213–222, 1941.
[29] R.H. Ritchie. Plasma losses by fast electrons in thin films. Physical Review, 106(5):874–
881, 1957.
[30] A. Otto. Excitation of nonradiative surface plasma waves in silver by the method of
frustrated total reflection. Z. Physik, 216:398–410, 1968.
[31] E. Kretschmann and H. Raether. Radiative decay of non radiative surface plasmons excited
by light. Z. Naturforsch, 23a:2135–2136, 1968.
[32] I Pockrand, J.D. Swalen, J.G. Gordon II, and M.R. Philpott. Surface plasmon spectroscopy
of organic monolayer assemblies. Surface Science, 74:237–244, 1977.
[33] J.D. Swalen, J.G. Gordon, M.R. Philpott, A. Brillante, I. Pockrand, and R. Santo. Plasmon
polariton dispersion by direct optical observation. Am. J. Phys., 48(8):669–672, 1980.
[34] Claes Nylander, Bo Liedberg, and T. Lind. Gas detection by means of surface plasmon
resonance. Sensors and Actuators, 3:79, 1982/83.
[35] Bo Liedberg, Claes Nylander, and Ingemar Lundström. Surface plasmon resonance for gas
detection and biosensing. Sensors and Actuators, 4:299, 1983.
[36] Jiri Homola. Surface plasmon resonance sensors for detection of chemical and biological
species. Chemical reviews, 108(2):462–493, Feb 2008.
[37] H. Raether. Surface Plasmons on Smooth and Rough Surfaces and on Gratings. SpringerVerlag, 1988.
[38] W. Knoll. Interfaces and thin films as seen by bound electromagnetic waves. Annu. Rev.
Phys. Chem., 49:569–638, 1998.
Bibliography
63
[39] J. Homola. Surface Plasmon Resonance Based Sensors, volume 4 of Springer Series on
Chemical Sensors and Biosensors. Springer-Verlag, Berlin Heidelberg, 2006.
[40] O.S. Heavens. Optical Properties of Thin Solid Films. Dover Publications Inc., corrected
reprint of 1st edition, 1955.
[41] A.D. Boardman, editor. Electromagnetic Surface Modes. John Wiley and Sons Ltd.,
Belfast, Northern Ireland, 1st edition, 1982.
[42] P. Yeh. Optical Waves in Layered Media. John Wiley and Sons Inc., USA, 1st edition,
1988.
[43] J.D. Jackson. Classical Electrodynamics. John Wiley and Sons Inc., 3rd edition, 1999.
[44] K. Johansen, H. Arwin, I. Lundström, and B. Liedberg. Imaging surface plasmon resonance sensor based on multiple wavelengths: Sensitivity considerations. Rev. of Sci. Instr.,
71(9):3530–3538, 2000.
[45] M. Born and E. Wolf. Principles of Optics. University Press, 7th (expanded) edition, 2006.
[46] W.N. Hansen. Electric fields produced by the propagation of plane coherent electromagnetic radiation in a stratified medium. J. Opt. Soc. Am., 58:380–390, 1968.
[47] R.M.A Azzam and N.M. Bashara. Ellipsometry and polarized light. North-Holland Physics
Publisher, Amsterdam, Netherlands, 1986.
[48] Florin Abelés. Researches sur la propagation des ondes électromagnétiques sinusoı̂dales
dans les milieux stratifiés. application aux coches minces. Annales de physique, 5:706–782,
1950.
[49] R.H. Muller. Definitions and conventions in ellipsometry. Surface Science, 16:14–33, 1969.
[50] R.T. Holm. Handbook of Optical Constants of Solids II, chapter 2, pages 21–55. Academic
Press Limited, 1250 Sixth Av, San Diego, CA 92101, 1991.
[51] E. Spiller. Phase conventions in thin film optics and ellipsometry.
23(18):3036–3037, 1984.
Applied Optics,
[52] Schott North America Inc. Optical Glass - Datasheets. 400 York Avenue, Duryea, PA
18642, USA, 2007.
[53] J Homola, I Koudela, and SS Yee. Surface plasmon resonance sensors based on diffraction
gratings and prism couplers: sensitivity comparison. Sensors and actuators b-chemical,
54(1-2):16–24, Jan 25 1999.
[54] Biacore website. http://www.biacore.com, June 2008.
[55] Jakub Dostalek and Jini Homola. Surface plasmon resonance sensor based on an array of
diffraction gratings for highly parallelized observation of biomolecular interactions. Sensors
and actuators b-chemical, 129(1):303–310, Jan 29 2008.
[56] Nan Zhang, Rudiger Schweiss, Yun Zong, and Wolfgang Knoll. Electrochemical surface plasmon spectroscopy - Recent developments and applications. Electrochimica acta,
52(8):2869–2875, Feb 10 2007.
[57] B Rothenhäusler and W. Knoll. Surface-plasmon microscopy. Nature, 332:615–617, 1988.
[58] Werner Hickel, Benno Rothenhäusler, and Wolfgang Knoll. Surface plasmon microscopic
characterization of external surfaces. J. Appl. Phys., 66(10):4832–4836, 1989.
[59] E Yeatman and EA Ash. Surface-plasmon microscopy. Electronics letters, 23(20):1091–
1092, Sep 24 1987.
[60] JS Yuk, HS Kim, JW Jung, SH Jung, SJ Lee, WJ Kim, JA Han, YM Kim, and KS Ha.
Analysis of protein interactions on protein arrays by a novel spectral surface plasmon
resonance imaging. Biosensors & bioelectronics, 21(8):1521–1528, Feb 15 2006.
64
Bibliography
[61] Jong Seol Yuk, Duk-Geun Hong, Hyo-Il Jung, and Kwon-Soo Ha. Application of spectral
SPR imaging for the surface analysis of C-reactive protein binding. Sensors and actuators
b-chemical, 119(2):673–675, Dec 7 2006.
[62] JM Brockman, BP Nelson, and RM Corn. Surface plasmon resonance imaging measurements of ultrathin organic films. Annual review of physical chemistry, 51:41–63, 2000.
[63] EM Yeatman. Resolution and sensitivity in surface plasmon microscopy and sensing.
Biosensors & bioelectronics, 11(6-7):635–649, 1996.
[64] J. Homola. On the sensitivity of surface plasmon resonance sensors with spectral interrogation. Sensors and Actuators B, 41:207–211, 1997.
[65] S-H. Jung J-A. Han Y-M. Kim K-S. Ha J.S. Yuk, J-W. Jung. Sensitivity of ex situ and in
situ spectral surface plasmon resonance sensors in the analysis of protein arrays. Biosensors
and Bioelectronics, 20:2189–2196, 2005.
[66] E Stenberg, B Persson, H Roos, and C Urbaniczky. Quantitative-determination of surface concentration of protein with surface-plasmon resonance using radiolabeled proteins.
Journal of colloid and interface science, 143(2):513–526, May 1991.
[67] DD Schlereth. Characterization of protein monolayers by surface plasmon resonance combined with cyclic voltammetry ‘in situ’. Journal of electroanalytical chemistry, 464(2):198–
207, MAR 29 1999.
[68] Y Iwasaki, T Horiuchi, and O Niwa. Detection of electrochemical enzymatic reactions by
surface plasmon resonance measurement. Analytical chemistry, 73(7):1595–1598, Apr 1
2001.
[69] Y Iwasaki, T Tobita, K Kurihara, T Horiuchi, K Suzuki, and O Niwa. Imaging of electrochemical enzyme sensor on gold electrode using surface plasmon resonance. Biosensors &
bioelectronics, 17(9):783–788, Sep 2002.
[70] G Ertl. Pattern formation at electrode surfaces. Electrochimica acta, 43(19-20):2743–2750,
1998.
[71] G Flatgen, K Krischer, and G Ertl. Spatio-temporal pattern formation during the reduction
of peroxodisulfate in the bistable and oscillatory regime: A surface plasmon microscopy
study. Journal of electroanalytical chemistry, 409(1-2):183–194, Jun 7 1996.
[72] G Flatgen, K Krischer, B Pettinger, K Doblhofer, H Junkes, and G Ertl. 2-dimensional
imaging of potential waves in electrochemical systems by surface-plasmon microscopy. Science, 269(5224):668–671, Aug 4 1995.
[73] YJ Li, J Oslonovitch, N Mazouz, F Plenge, K Krischer, and G Ertl. Turing-type patterns
on electrode surfaces. Science, 291(5512):2395–2398, Mar 23 2001.
[74] A.J. Bard and L.R. Faulkner. Electrochemical Methods: Fundamentals and Applications.
John Wiley & Sons, Inc., 2:nd edition, 2001.
[75] Carl H. Hamann, Andrew Hamnett, and Wolf Vielstich. Electrochemistry. Wiley-VCH,
1998.
[76] J.G. Gordon II and S. Ernst. Surface plasmons as a probe of the electrochemical interface.
Surface Science, 101:499–506, 1980.
[77] A. Tadjeddine. Influence of the electrical polarization on the surface plasmon dispersion
at metal-electrolyte interfaces. Electrochimica Acta, 34:29–33, 1989.
[78] V. Liubimov, A Kolomenskii, A Mershin, D.V. Nanopoulos, and H.A. Scheussler. Effect of
varying electric potential on surface-plasmon resonance sensing. Applied Optics, 43:3426–
3432, 2004.
[79] H.J. Kragt, C.P. Smith, and H.S. White. Refractive index mapping of concentration profiles. Journal of Electroanalytical Chemistry, 278:403–407, 1990.
Bibliography
65
[80] Ralph G. Nuzzo and David L. Allara. Adsorption of bifunctional organic disulfides on gold
surfaces. Journal of the American Chemical Society, 105(13):4481–4483, 1983.
[81] MD Porter, TB Bright, DL Allara, and CED Chidsey. Spontaneously organized molecular assemblies .4. structural characterization of normal-alkyl thiol monolayers on gold by
optical ellipsometry, infrared-spectroscopy, and electrochemistry. Journal of the american
chemical society, 109(12):3559–3568, Jun 10 1987.
[82] CD Bain, EB Troughton, YT Tao, J Evall, GM Whitesides, and RG Nuzzo. Formation
of monolayer films by the spontaneous assembly of organic thiols from solution onto gold.
Journal of the american chemical society, 111(1):321–335, Jan 4 1989.
[83] CD Bain and GM Whitesides. Formation of 2-component surfaces by the spontaneous assembly of monolayers on gold from solutions containing mixtures of organic thiols. Journal
of the american chemical society, 110(19):6560–6561, Sep 14 1988.
[84] CD Bain, J Evall, and GM Whitesides. Formation of monolayers by the coadsorption of
thiols on gold - variation in the head group, tail group, and solvent. Journal of the american
chemical society, 111(18):7155–7164, Aug 30 1989.
[85] JC Love, LA Estroff, JK Kriebel, RG Nuzzo, and GM Whitesides. Self-assembled monolayers of thiolates on metals as a form of nanotechnology. Chemical reviews, 105(4):1103–1169,
Apr 2005.
[86] A Ulman. Formation and structure of self-assembled monolayers.
96(4):1533–1554, Jun 1996.
Chemical reviews,
[87] R Valiokas, M Ostblom, S Svedhem, SCT Svensson, and B Liedberg. Thermal stability
of self-assembled monolayers: Influence of lateral hydrogen bonding. Journal of physical
chemistry b, 106(40):10401–10409, Oct 10 2002.
[88] H.O. Finklea. Electrochemistry of organized monolayers of thiols and related molecules on
electrodes, volume 19 of Electroanalytical Chemistry: a series of advances, pages 109–335.
Marcel Dekker, New York, 1996.
[89] C Palegrosdemange, ES Simon, Kl Prime, and GM Whitesides. Formation of selfassembled monolayers by chemisorption of derivatives of oligo(ethylene glycol) of structure
hs(ch2)11(och2ch2)meta-oh on gold. Journal of the american chemical society, 113(1):12–
20, Jan 2 1991.
[90] KL Prime and GM Whitesides. Self-assembled organic monolayers - model systems for
studying adsorption of proteins at surfaces. Science, 252(5009):1164–1167, May 24 1991.
[91] K. Feldman, G. Hahner, N.D. Spencer, P. Harder, and M. Grunze. Probing resistance
to protein adsorption of oligo(ethylene glycol)-terminated self-assembled monolayers by
scanning force microscopy. Journal of the American Chemical Society, 121(43):10134–
10141, 1999.
[92] P. Harder, M. Grunze, R. Dahint, G.M. Whitesides, and P.E. Laibinis. Molecular conformation in oligo(ethylene glycol)-terminated self-assembled monolayers on gold and silver
surfaces determines their ability to resist protein adsorption. Journal of Physical Chemistry
B, 102(2):426–436, 1998.
[93] S Lofas, M Malmqvist, I Ronnberg, E Stenberg, B Liedberg, and I Lundstrom. Bioanalysis
with surface-plasmon resonance. Sensors and actuators b-chemical, 5(1-4):79–84, Aug-Dec
1991.
[94] Andreas Larsson, Tobias Ekblad, Olof Andersson, and Bo Liedberg. Photografted
poly(ethylene glycol) matrix for affinity interaction studies. Biomacromolecules, 8(1):287–
295, Jan 2007.
66
Bibliography
[95] Bo (contact) Liedberg, Tobias Ekblad, Gunnar Bergström, Thomas Ederth, Sheelagh Conlan, Robert Mutton, Anthony Clare, Su Wang, Yunli Liu, Qi Zhao, Fraddry D’Souza, Glen
Donnelly, Peter Willemsen, Michala Pettitt, Maureen Callow, and J. Callow. Poly(ethylene
glycol)-containing hydrogel surfaces for antifouling applications in marine and freshwater
environments. Submitted, 2008.
[96] YN Xia and GM Whitesides. Soft lithography. Angewandte chemie-international edition,
37(5):551–575, MAR 16 1998.
[97] RS Kane, S Takayama, E Ostuni, DE Ingber, and GM Whitesides. Patterning proteins
and cells using soft lithography. Biomaterials, 20(23-24):2363–2376, Dec 1999.
[98] A Bernard, E Delamarche, H Schmid, B Michel, HR Bosshard, and H Biebuyck. Printing
patterns of proteins. Langmuir, 14(9):2225–2229, Apr 28 1998.
[99] E Delamarche, H Schmid, A Bietsch, NB Larsen, H Rothuizen, B Michel, and H Biebuyck.
Transport mechanisms of alkanethiols during microcontact printing on gold. Journal of
physical chemistry b, 102(18):3324–3334, Apr 30 1998.
[100] M Mrksich and GM Whitesides. Patterning self-assembled monolayers using microcontact
printing - a new technology for biosensors. Trends in biotechnology, 13(6):228–235, Jun
1995.
[101] K Glasmastar, J Gold, AS Andersson, DS Sutherland, and B Kasemo. Silicone transfer
during microcontact printing. Langmuir, 19(13):5475–5483, Jun 24 2003.
[102] J. Lahiri, E. Ostuni, and G.M. Whitesides. Patterning ligands on reactive sams by microcontact printing. Langmuir, 15(6):2055–2060, 1999.
[103] E Delamarche, M Geissler, A Bernard, H Wolf, B Michel, J Hilborn, and C Donzel. Hydrophilic poly (dimethylsloxane) stamps for microcontact printing. Advanced materials,
13(15):1164+, Aug 3 2001.
[104] T Kraus, R Stutz, TE Balmer, H Schmid, L Malaquin, ND Spencer, and H Wolf. Printing
chemical gradients. Langmuir, 21(17):7796–7804, Aug 16 2005.
[105] CA Widrig, C Chung, and MD Porter. The electrochemical desorption of n-alkanethiol
monolayers from polycrystalline au and ag electrodes. Journal of electroanalytical chemistry, 310(1-2):335–359, Jul 25 1991.
[106] LM Tender, RL Worley, HY Fan, and GP Lopez. Electrochemical patterning of selfassembled monolayers onto microscopic arrays of gold electrodes fabricated by laser ablation. Langmuir, 12(23):5515–5518, Nov 13 1996.
[107] LM Tender, KA Opperman, PD Hampton, and GP Lopez. Fabrication of microscopic
biosensor arrays without microscopic alignment. Advanced materials, 10(1):73+, Jan 2
1998.
[108] M Riepl, VM Mirsky, and OS Wolfbeis. Electrical control of alkanethiols self-assembly on
a gold surface as an approach for preparation of microelectrode arrays. Mikrochimica acta,
131(1-2):29–34, 1999.
[109] KM Balss, BD Coleman, CH Lansford, RT Haasch, and PW Bohn. Active spatiotemporal
control of electrochemical reactions by coupling to in-plane potential gradients. Journal of
physical chemistry b, 105(37):8970–8978, Sep 20 2001.
[110] ST Plummer and PW Bohn. Spatial dispersion in electrochemically generated surface
composition gradients visualized with covalently bound fluorescent nanospheres. Langmuir,
18(10):4142–4149, May 14 2002.
[111] ST Plummer, Q Wang, PW Bohn, R Stockton, and MA Schwartz. Electrochemically
derived gradients of the extracellular matrix protein fibronectin on gold. Langmuir,
19(18):7528–7536, Sep 2 2003.
Bibliography
67
[112] RH Terrill, KM Balss, YM Zhang, and PW Bohn. Dynamic monolayer gradients: Active
spatiotemporal control of alkanethiol coatings on thin gold films. Journal of the american
chemical society, 122(5):988–989, Feb 9 2000.
[113] M.J. Madou. Fundamentals of microfabrication - The science of miniaturization. CRC
Press, second edition, 2002.
[114] L Pardo, C.W. Jr. Wilson, and T. Boland. Characterization of patterned self-assembled
monolayers and protein arrays generated by the ink-jet method. Langmuir, 19:1462–1466,
2003.
[115] M Mosbach, H Zimmermann, T Laurell, J Nilsson, E Csoregi, and W Schuhmann.
Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures. Biosensors & bioelectronics, 16(9-12, Sp. Iss.
SI):827–837, Dec 2001.
[116] G Klenkar, R Valiokas, I Lundstrom, A Tinazli, R Tampe, J Piehler, and B Liedberg. Piezo
dispensed microarray of multivalent chelating thiols for dissecting complex protein-protein
interactions. Analytical chemistry, 78(11):3643–3650, Jun 1 2006.
[117] Ramunas Valiokas, Goran Klenkar, Ali Tinazli, Robert Tampe, Bo Liedberg, and Jacob
Piehler. Differential protein assembly on micropatterned surfaces with tailored molecular
and surface multivalency. Chembiochem, 7(9):1325–1329, Sep 2006.
[118] T Okamoto, T Suzuki, and N Yamamoto. Microarray fabrication with covalent attachment
of DNA using Bubble Jet technology. Nature biotechnology, 18(4):438–441, Apr 2000.
[119] A Bietsch, M Hegner, HP Lang, and C Gerber. Inkjet deposition of alkanethiolate monolayers and DNA oligonucleotides on gold: Evaluation of spot uniformity by wet etching.
Langmuir, 20(12):5119–5122, Jun 8 2004.
[120] XG Zhang and OA Basaran. Dynamic surface tension effects in impact of a drop with a
solid surface. Journal of colloid and interface science, 187(1):166–178, Mar 1 1997.
[121] R.D. Deegan, O Bakajin, T.F. Dupont, G. Huber, S.R. Nagel, and T.A. Witten. Capillary
flow as the cause of ring stains from dried liquid drops. Nature, 389:827–829, 1997.
[122] S.D. Risio and N Yan. Piezoelectric ink-jet printing of horseradish peroxidase. effect on
ink viscosity modifiers on activity. Macromolecular Rapid Communications, 28:1934–1940,
2007.
[123] N Xia, J.S. Shumaker-Parry, M.H. Zareie, C.T. Campbell, and D.G. Castner. A streptavidin linker layer that functions after drying. Langmuir, 20:3710–3716, 2004.
[124] LD Ward and DJ Winzor. Relative merits of optical biosensors based on flow-cell and
cuvette designs. Analytical biochemistry, 285(2):179–193, OCT 15 2000.
[125] Rebecca L. Rich, Michelle J. Cannon, Jerry Jenkins, Prabhakar Pandian, Shankar Sundaram, Rachelle Magyar, Jennifer Brockman, Jeremy Lambert, and David G. Myszka.
Extracting kinetic rate constants from surface plasmon resonance array systems. Analytical biochemistry, 373(1):112–120, Feb 1 2008.
[126] Daniel Boecker, Alexander Zybin, Kay Niemax, Christian Grunwald, and Vladimir M.
Mirsky. Noise reduction by multiple referencing in surface plasmon resonance imaging.
Review of scientific instruments, 79(2, Part 1), Feb 2008.
[127] K. Johansen. Surface Plasmon Resonance - Sensitivity and Resolution. PhD thesis, Linkopings Universitet, 2000.
[128] P Tobiska and J Homola. Advanced data processing for SPR biosensors. Sensors and
actuators b-chemical, 107(1):162–169, May 27 2005.
[129] K Johansen, I Lundstrom, and B Liedberg. Sensitivity deviation: instrumental linearity
errors that influence concentration analyses and kinetic evaluation of biomolecular interactions. Biosensors & bioelectronics, 15(9-10):503–509, Nov 2000.
68
Bibliography
[130] K Johansen, R Stalberg, I Lundstrom, and B Liedberg. Surface plasmon resonance:
instrumental resolution using photo diode arrays. Measurement science & technology,
11(11):1630–1638, Nov 2000.
[131] J Homola, SS Yee, and G Gauglitz. Surface plasmon resonance sensors: review. Sensors
and actuators b-chemical, 54(1-2):3–15, Jan 25 1999.
[132] BP Nelson, AG Frutos, JM Brockman, and RM Corn. Near-infrared surface plasmon
resonance measurements of ultrathin films. 1. Angle shift and SPR imaging experiments.
Analytical chemistry, 71(18):3928–3934, Sep 15 1999.
[133] JS Shumaker-Parry and CT Campbell. Quantitative methods for spatially resolved adsorption/desorption measurements in real time by surface plasmon resonance microscopy.
Analytical chemistry, 76(4):907–917, Feb 15 2004.
[134] E Fu, T Chinowsky, J Foley, J Weinstein, and P Yager.
Characterization of a
wavelength-tunable surface plasmon resonance microscope. Review of scientific instruments, 75(7):2300–2304, Jul 2004.
[135] KF Giebel, C Bechinger, S Herminghaus, M Riedel, P Leiderer, U Weiland, and M Bastmeyer. Imaging of cell/substrate contacts of living cells with surface plasmon resonance
microscopy. Biophysical journal, 76(1, Part 1):509–516, Jan 1999.
[136] T Zhang, H Morgan, ASG Curtis, and M Riehle. Measuring particle-substrate distance
with surface plasmon resonance microscopy. Journal of optics a-pure and applied optics,
3(5):333–337, Sep 2001.
[137] Catherine Dreanno, Richard R. Kirby, and Anthony S. Clare. Involvement of the barnacle
settlement-inducing protein complex (SIPC) in species recognition at settlement. Journal
of experimental marine biology and ecology, 351(1-2):276–282, Nov 23 2007.
[138] Catherine Dreanno, Richard R. Kirby, and Anthony S. Clare. Smelly feet are not always a
bad thing: the relationship between cyprid footprint protein and the barnacle settlement
pheromone. Biology letters, 2(3):423–425, Sep 22 2006.