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Linköping Studies in Science and TechnologyLicentiate Thesis No. 1573
Biosensor surface chemistry for oriented protein immobilization
and biochip patterning
Emma Ericsson
Division of Molecular PhysicsDepartment of Physics, Chemistry and Biology
Linköping University, Sweden
Linköping 2013
This is a Swedish Licentiate Thesis. The Licentiate Degree comprises 120 ECTS credits of postgraduate studies. During the course of the research underlying this thesis, Emma Ericsson was enrolled in Forum Scientium, a multidisciplinary graduate school at Linköping University, Sweden. Copyright © 2013 Emma Ericsson unless otherwise noted ISBN: 978-91-7519-698-5 ISSN: 0280-7971 Linköping Studies in Science and Technology Licentiate Thesis No. 1573 LIU-TEK-LIC-2013:7 Electronic publication: www.ep.liu.se Printed by LiU-Tryck, Linköping, Sweden, 2013
To Daniel and Ludvig
Acknowledgements
I have had the pleasure of getting to know many inspiring, intelligent and interesting
people during my years as a graduate student. I would like to sincerely thank them for
sharing their knowledge and for making all the work worthwhile, after all.
Thank you, Karin Enander, for being my advisor. I appreciate your clarity and
honesty and your support has been invaluable. Bo Liedberg, my previous advisor, for
initiating the research projects and introducing me to the subject and the research division
of Molecular Physics. Lan Bui for your energy and hard work during the startup of our
twinning project. Tomas Rakickas and Ramūnas Valiokas for a very nice collaboration
and for showing me around in Vilnius.
Thank you, my present and former co-workers, including (but absolutely not limited
to) Olof Andersson, Daniel Kanmert, Robert Selegård, Annica Myrskog, Tobias Ekblad,
Daniel Aili, Linnéa Selegård and Maria Ahrén, for your help, suggestions and many nice
chats. Kristina Buchholt for your friendship and support during the hard times. Stefan
Klintström for your commitment to Forum Scientium and for always having time to listen.
Agneta Askendal for being a nice co-worker with lots of knowledge and lots of stories. Bo
Thunér for helping me with practical issues in the lab and for the entertaining discussions
at lunch time. Thanks to all other senior researchers, PhD students, technical staff and
administrators for help and good advice.
Thank you, my parents, brothers, in-laws and friends, for standing by me during all
these years and for trying to understand what all this work was really about, but especially
for taking my mind off it. Daniel, my dear husband, for your love, understanding and
support. Ludvig, my wonderful son, for your love, curiosity and joy. I have learned so
much during the work with this thesis, but thank you both for reminding me every day that
there are more important things in life.
Linköping, January 2013
Emma
Abstract
This licentiate thesis is focused on two methods for protein immobilization to biosensor
surfaces for future applications in protein microarray formats. The common denominator
is a surface chemistry based on a gold substrate with a self-assembled monolayer (SAM)
of functionalized alkanethiolates. Both methods involve photochemistry, in the first case
for direct immobilization of proteins to the surface, in the other for grafting a hydrogel,
which is then used for protein immobilization.
Paper I describes the development and characterization of Chelation Assisted
Photoimmobilization (CAP), a three-component surface chemistry that allows for covalent
attachment and controlled orientation of the immobilized recognition molecule (ligand)
and thereby provides a robust sensor surface for detection of analyte in solution. The
concept was demonstrated using His-tagged IgG-Fc as the ligand and protein A as the
analyte. Surprisingly, as concluded from IR spectroscopy and surface plasmon resonance
(SPR) analysis, the binding ability of this bivalent ligand was found to be more than two
times higher with random orientation obtained by amine coupling than with homogeneous
orientation obtained by CAP. It is suggested that a multivalent ligand is less sensitive to
orientation effects than a monovalent ligand and that island formation of the
alkanethiolates used for CAP results in a locally high ligand density and steric hindrance.
Paper II describes the development of nanoscale hydrogel structures. These were
photografted on a SAM pattern obtained by dip-pen nanolithography (DPN) and
subsequent backfilling. The hydrogel grew fast on the hydrophilic patterns and slower on
the hydrophobic background, which contained a buried oligo(ethylene glycol) (OEG)
chain. Using IR spectroscopy, it was found that the OEG part was degraded during UV
light irradiation and acted as a sacrificial layer. In this process other OEG residues were
exposed and acted as new starting points for the self-initiated photografting and
photopolymerization (SIPGP). A biotin derivative was immobilized to the hydrogel
density pattern and interaction with streptavidin was demonstrated by epifluorescence
microscopy.
List of papers
Paper I Ericsson, E.M., Enander, K., Bui, L., Lundström, I., Konradsson, P., Liedberg, B.
Controlled Orientation and Covalent Attachment of Proteins on Biosensor Surfaces by
Chelation Assisted Photoimmobilization. In manuscript, 2013.
Contribution
EE planned and performed the major part of the lab work and data evaluation and did the
major part of the writing. KE was involved in the final experimental design and the
manuscript writing. LB did the molecular synthesis and participated in initial lab work. BL
was involved in the writing. The CAP concept was developed in theory by IL, BL and PK.
Paper II Rakickas, T., Ericsson, E.M., Ruželė, Ž., Liedberg, B., Valiokas, R.
Functional Hydrogel Density Patterns Fabricated by Dip-Pen Nanolithography and
Photografting. Small, 2011, 7(15), 2153-2157. DOI: 10.1002/smll.201002278
Contribution
EE performed the middle steps of sample preparation (hydrogel photografting and
carboxylation), all IRAS measurements and analysis. EE wrote the text on this with the
help of BL. Experimental planning was done together with TR and RV. TR performed all
other lab work except molecular synthesis performed by ŽR. TR and RV wrote the major
part of the manuscript.
Conference contribution
Ericsson, E.M., Bui, L., Andersson, O., Enander, K., Lundström, I., Konradsson, P.,
Liedberg, B. Chelation Assisted Photoimmobilization – CAP. A Surface Chemistry for
Controlled Orientation and Covalent Attachment of Proteins. Poster. Europt(r)ode X,
European conference on optical chemical sensors and biosensors. Prague, Czech Republic,
2010.
Other contributions not related to the thesis
Ericsson, E.M., Faxälv, L., Weissenrieder, A., Askendal, A., Lindahl, T.L., Tengvall, P.
Glycerol Monooleate – Blood Interactions. Colloids and Surfaces B: Biointerfaces, 2009.
68 (1): pp. 20-26. DOI: 10.1016/j.colsurfb.2008.09.016
Peng, X., Jin, J., Ericsson, E.M., Ichinose, I. Ultrathin Free-Standing Films of
Nanofibrous Composite Materials. Journal of the American Chemical Society, 2007. 129
(27): pp. 8625-8633. DOI: 10.1021/ja0718974
Abbreviations
AFM Atomic Force Microscopy
BP Benzophenone
BPNTA The alkane disulfide with BP and NTA terminations used for CAP
CAP Chelation Assisted Photoimmobilization
DPN Dip-Pen Nanolithography
EDC N-Ethyl-N'-3-Dimethylaminopropyl Carbodiimide
EDTA Ethylene Diamine Tetraacetic Acid
EG Ethylene Glycol
Fab Antigen binding fragment of immunoglobulin
Fc Crystallizable fragment of immunoglobulin
IgG ImmunoGlobulin G
His Histidine
His-IgGFc IgG-Fc modified with hexahistidine tags
IMAC Immobilized Metal ion Affinity Chromatography
IRAS Infrared Reflection Absorption Spectroscopy
iSPR Imaging Surface Plasmon Resonance
LFM Lateral Force Microscopy
NHS N-Hydroxy Succinimide
NTA NitriloTriacetic Acid
OEG Oligo(Ethylene Glycol)
PBS Phosphate Buffered Saline
PEG Poly(Ethylene Glycol)
QD Quantum Dot
SAM Self-Assembled Monolayer
SIPGP Self-Initiated Photografting and Photopolymerization
SPR Surface Plasmon Resonance
Contents
1. Introduction _________________________________________________ 1
2. Challenges in biosensor development ___________________________ 3 2.1 Denaturation _____________________________________________ 3 2.2 Nonspecific binding ________________________________________ 3 2.3 Orientation of the recognition molecule _________________________ 4 2.4 Miniaturization ____________________________________________ 6
3. Surface modification __________________________________________ 7 3.1 Self-assembled monolayers _________________________________ 7 3.2 Dip-pen nanolithography ___________________________________ 10 3.3 Hydrogel photografting ____________________________________ 10
4. Protein immobilization _______________________________________ 13 4.1 Amine coupling __________________________________________ 13 4.2 Metal ion affinity _________________________________________ 15 4.3 Photoimmobilization ______________________________________ 16 4.4 Chelation Assisted Photoimmobilization _______________________ 16
5. Biomolecular interaction ______________________________________ 19 5.1 Antibodies ______________________________________________ 19 5.2 Biotin – avidin ___________________________________________ 20
6. Experimental techniques _____________________________________ 23 6.1 Contact angle goniometry __________________________________ 23 6.2 Null ellipsometry _________________________________________ 24 6.3 Infrared spectroscopy _____________________________________ 26 6.4 Surface plasmon resonance ________________________________ 30 6.5 Atomic force microscopy ___________________________________ 33 6.6 Epifluorescence microscopy ________________________________ 35
7. Demonstration and evaluation of CAP __________________________ 37
8. Photografting hydrogels on patterned substrates _________________ 43
9. Future perspectives __________________________________________ 45
10. References _________________________________________________ 47
1
1. Introduction
Biosensors are used today not only in health care but also in the food industry and for
environmental or security monitoring. Hence there are many incentives to further improve
the sensitivity, selectivity and stability of these devices. The work presented in this thesis
is a contribution to this development. It concerns the design and characterization of
different surface chemistries with potential use in the area of optical biosensing.
One of the first biosensors described was an enzyme electrode for measurement of
glucose [1] which later developed into the blood glucose meter used by diabetics today.
Since then the development of biosensors has continued and diversified and the
International Union of Pure and Applied Chemistry (IUPAC) now defines a biosensor
(Figure 1) as a self-contained integrated device that provides quantitative analytical
information using a biological recognition element (biochemical receptor) in contact with
a transduction element [2]. Furthermore, a distinction is made between biosensors,
bioanalytical systems and single-use biosensors. A bioanalytical system requires
additional processing steps (such as addition of reagents) and a single-use biosensor (e.g.
pregnancy test, glucose meter test strip) is disposable and cannot monitor analyte
concentration continuously. Using this definition, the work presented in this thesis should
be classified as work concerning surface modifications for bioanalytical systems rather
than for actual biosensors. However, the scientific community usually adopts the more
general definition of a biosensor as an analytical device combining a biochemical element
with a transducer element for converting the response into an electronic signal.
The mode of biosensor transduction could be e.g. optical, electrochemical or
piezoelectrical, meaning one could measure photons, electrons or resonance frequency,
respectively. In this thesis, mainly optical detection techniques have been used for surface
characterization as well as for detection of biomolecular interaction. The developed
surface chemistries are, however, compatible with other modes of transduction.
Finally it should be noted that in this thesis, as is conventional in the field of
biodetection, the biochemical recognition element is referred to as the ligand and the
biomolecule to be detected in the sample solution is referred to as the analyte.
2
Figure 1. The definition of a biosensor is a biological recognition element in contact with a transducer element which detects a physical, chemical or electrical change when the recognition element binds analyte from solution.
Transducer element
Biological recognition element
Recognized analyte
Mixed solution containing analyte
Data evaluationSignal
3
2. Challenges in biosensor development
A protein is formed by one or several chains of amino acids joined by peptide bonds. The
chains are folded into a tertiary structure that is usually essential for the function of the
protein in for example catalysis, cell signaling or immune response. In order to develop
well-functioning biosensors, the binding capability of the immobilized protein needs to be
retained and nonspecific (unwanted) binding of proteins must be reduced. A related issue
is that of protein orientation. The immobilized ligand can fail to act as a recognition
element if the analyte cannot reach it due to steric hindrance.
2.1 Denaturation A protein in its native form has a hydrophobic core which is protected from interaction
with water in the physiological environment. If the conditions are changed, the protein
may unfold, whereby the binding site (where biomolecular interaction occurs) may be
disrupted and the protein may lose its binding ability. A protein can unfold as a response
to a change in the physical environment (such as temperature, pressure) or chemical
environment (solvent, pH). Sometimes the denaturation is reversible [3].
In Paper I, the protein immobilized to the sensor surface denatured upon drying
causing loss of function. In this case, denaturation was avoided by incubation in a solution
of trehalose dihydrate before drying. Trehalose is naturally produced in mammal cells as a
response to stress and prevents protein denaturation. Therefore it is commonly used for
protein stabilization although the mechanisms for this are not yet fully understood [4].
When spotting proteins in a microarray format, glycerol can be used to protect the protein
from drying as the solvent quickly evaporates from the spotted droplets [5].
2.2 Nonspecific binding Spontaneous protein adsorption to biosensor surfaces is unwanted since it lowers the
sensitivity and might obstruct the monitoring of the biomolecular interaction to be studied.
Nonspecific binding occurs if the conditions are such that the adsorbed state of the protein
is more energetically favorable than the aqueous state [6] and it is generally driven by
electrostatic or hydrophobic interactions between proteins and the surface. Hydrophobic
interactions are a problem especially when using complex biological fluids such as blood
4
plasma which contains a number of sticky proteins, e.g. fibrinogen. To reduce nonspecific
binding, either the buffer solution or the sensor surface can be modified to minimize the
unwanted interactions.
One alternative is to modify the buffer solution by increasing the salt concentration
or adjusting the pH to minimize electrostatic interactions. Another alternative is adding
surfactants to reduce the hydrophobic interactions [7]. Several amino acids can bind metal
ions, and adding a chelating agent that acts as a metal ion scavenger can reduce metal ion
dependent nonspecific binding.
Another solution is to cover the remaining exposed part of the surface after ligand
immobilization with a biologically inert protein. Bovine serum albumin (BSA) is
commonly used to block exposed surface areas so that the analyte only binds specifically
to the immobilized ligand.
Generally, protein resistance is improved by the choice of chemical modification of
the surface, such as self-assembled monolayers (SAMs, section 3.1) or hydrogels (section
3.3). For example, SAMs containing sugar structures such as galactose and maltose [8, 9]
have been reported to yield low nonspecific binding. For both SAMs and hydrogels
however, oligo- or poly-(ethylene glycol) (OEG or PEG) is most commonly used [9-12]
because of its excellent protein resistance properties. This is also the case in the two
papers presented in this thesis.
The OEG molecular structure easily absorbs and retains water by forming hydrogen
bonds (one water molecule per EG unit). It is believed that the protein resistance results
from this water retaining ability which in turn depends on the molecular conformation [13].
Importantly, although OEG-containing surfaces have shown outstanding protein resistance
properties in single-protein systems it does not automatically mean that they have the
same properties in contact with complex biological media such as blood plasma [14].
2.3 Orientation of the recognition molecule The hypothesized impact of ligand orientation on analyte binding capacity is a hot topic of
discussion in biosensor research. One intuitively understands that out of an assembly of
randomly oriented ligands on a sensor surface, there will be a number of them that cannot
bind analyte for steric reasons (Figure 2). In reality the situation can be more complex, as
discussed in Paper I.
5
Random orientation results from nonsite-specific immobilization methods, e.g.
amine coupling (section 4.1). Controlled ligand orientation on surfaces can be achieved by
allowing a protein with a specific region or tag (e.g. a carbohydrate residue, the Fc region
of IgGs or a biotin-tag) to interact with its surface immobilized affinity partner (lectin,
protein A/G and avidin, respectively) [15, 16]. Using thiol coupling, antibody F(ab’)
fragments can be oriented via the thiol group located on the opposite side of the antigen
binding site.
Uniform orientation has often been preferred to random orientation based on the
difference in absolute analyte responses, without taking into account possible differences
in ligand immobilization level. However, there are studies where normalized ligand
immobilization levels have been considered. These reports show that the orientation of
immobilized antibody F(ab’) fragments can result in an increased antigen binding
efficiency compared to when the fragments are randomly bound [17, 18]. Depending on
the detection method used, a factor 2.7-11 difference was reported in these two studies.
Bonroy et al [19] also used F(ab’) fragments but came to the opposite conclusion. In this
case random orientation resulted in higher analyte binding efficiency with a factor 1.3 and
a possible explanation given was crowding of the oriented ligand, since its immobilization
level was twice that of randomly bound ligand. Using full-length antibodies Caruso et al
[20] showed that depending on the antibody-antigen system, orientation could increase the
analyte binding efficiency by a factor 1-2. Hence, it seems that larger differences are seen
for monovalent ligands such as F(ab’) fragments than for bivalent ligands such a
antibodies. Furthermore, a study on immobilization of full-length antibodies in a three-
dimensional matrix concluded that site-specific immobilization did not increase ligand
activity compared to random immobilization methods [21]. Thus, the problem might be
more relevant for two-dimensional sensor surfaces. In general, the results seem to be
closely associated with both the ligand and the surface chemistry used. Therefore,
normalization of the analyte binding signal based on ligand quantification will continue to
be important when choosing the most proper surface chemistry and immobilization
strategy for a specific ligand.
Paper I in this thesis contributes to this discussion by comparing nonspecific and
site-specific immobilization methods to quantitatively evaluate the difference in analyte
binding capacity between randomly and homogenously oriented ligand.
6
Figure 2. Illustration of the ligand orientation problem. A random orientation of ligand (left) results in some ligand molecules being unavailable for analyte binding. Therefore a controlled, homogenous orientation (right) is often desired. 2.4 Miniaturization Miniaturization of biosensors is necessary to obtain high-throughput and low sample
consumption. An attractive future application of biosensors is in the form of biochips,
containing recognition elements in an array of thousands or possibly millions of micro- or
nanometer sized structures.
There are several chemical strategies [22] and patterning methods [23] available for
biochip fabrication. Microarrayers are used for direct spotting of a large number of
recognition elements onto pretreated surfaces [24]. Photolithography and direct
photopatterning (without photoresist) utilize patterning by light. The size of the obtained
structures is limited by diffraction and in general resolution below that of the wavelength
of the light used cannot be obtained. In future applications other techniques, e.g. parallel
dip-pen nanolithography (DPN) could provide better resolution.
Paper I describes a surface photochemistry that can be used for protein patterning.
DPN has been used in Paper II to create nanometer-sized structures, demonstrating a
possible method for biochip fabrication.
7
3. Surface modification
When the first biosensors were developed, simple approaches to protein immobilization
were used, e.g. spontaneous protein adsorption onto a clean metal surface by hydrophobic
interaction. This typically leads to unfolding of the protein (denaturation) and partial loss
of function. Therefore, more gentle protein immobilization strategies have been developed
by modifying the surface chemically with some sort of linker layer. Another reason for
surface modification is to increase control of the surface properties and to provide a means
of miniaturization in an array format.
3.1 Self-assembled monolayers Self-assembly is the spontaneous formation of complex structures of molecules that are
held together by non-covalent intermolecular interactions. A self assembled monolayer
(SAM) is a single layer of molecules on a solid substrate, stabilized by hydrogen bonds,
Coulombic interactions (electric forces), van der Waals forces (dispersion forces) or
hydrophobic effects. SAMs can be formed from a wide variety of compounds – such as
fatty acids, organosilicon or organosulphur compounds – and on a wide range of surface
materials – such as aluminum oxide, silver, silicon oxide, glass and gold, to name a few
[25]. Alkylsilanes form a polysiloxane bond to silicon oxide surfaces and this technique
has been widely used although it suffers from moisture sensitivity.
SAMs prepared from alkanethiol (sulfhydryl) molecules (Figure 3) chemisorbed on
a gold surface was first described by Nuzzo and Allara [26]. The gold-sulfur binding is
very strong and alkanethiols or alkane disulphides in solution spontaneously form a SAM
on gold. After initial adsorption, which takes only a few seconds, an organization phase of
several hours follows. For thiols with sufficiently long alkyl chains (more than 12
methylene units), perfectly organized monolayers can be obtained. Gauche defects occur
when the backbone chain is not fully extended, which disrupts the SAM packing. Due to
intermolecular forces, defects are rare and occur mostly in the top part of the SAM.
Because of the lattice structure of gold, typically Au(111), and the overlayer structure of
the adsorbed thiolate molecules, there is a certain available area per molecule. Since the
molecules are too small to occupy the whole area if they would be arranged
perpendicularly to the surface, they tilt about 30º from the surface normal. Therefore, the
8
thickness of the SAM is smaller than the length of the adsorbed molecules. The
terminating group of the alkanethiolate affects different surface properties, such as
hydrophilicity [27], and can be used for further functionalization of the SAM, e.g. by
covalent coupling or by grafting polymer chains to it via surface-initiated polymerization
[28].
For biosensors, different functionalities are needed for protein immobilization and
reduction of nonspecific binding and therefore the formation of mixed monolayers
assembled from a solution of different alkanethiols is more relevant than single-
component SAMs. It has been proposed that mixed monolayers are composed of phases or
islands separating the different adsorbates. Early work concluded that no macroscopic
islands were formed [29] and later studies have shown evidence of phase-separation in
mixed SAMs [30-32].
The use of a SAM on a gold surface is one of the common denominators in the two
included papers. All molecules used for self-assembly are presented in Figure 4. In Paper I,
mixed monolayers were prepared by coadsorption of alkanethiols or alkane disulphides. In
Paper II, more sophisticated methods (i.e. microcontact printing, dip-pen nanolithography
and subsequent back-filling) were used for producing separate regions of single-
component SAMs. In both cases, ultraviolet (UV) light were used for further surface
modification. Alkanethiolate SAMs on gold are photooxidized upon UV light irradiation
in air, probably due to the photolysis of oxygen to reactive ozone [28]. Therefore, UV
light experiments were performed under nitrogen gas flow.
Figure 3. Schematic drawing of a SAM of hexadecanethiol (HDT) on a gold substrate.
CH3
S
CH3
S
CH3
S
CH3
S
CH3
S
CH3
S
CH3
S
CH3
S
Au
9
Figure 4. The molecules I-V were used for self-assembly in Paper I and are henceforth referred to as the (I) EG3, (II) NTA and (III) BP alkanethiols and the (IV) EG3 and (V) BPNTA alkane disulphides. The molecules VI-VIII were used in Paper II and are referred to as (VI) MHA, (VII) HDT and (VIII) EG6C16, respectively.
O
OOOOOOOO
HN
OS
NH
O O O NH
OOS N
OH
O
OH
O
HO O
O
OOOOOOOO
HN
OHS
NH
O O O NH
OOHS N
OH
O
OH
O
HO O
NH
O O OHO
S
NH
O O OHO
S
NH
O O OHO
HS(I)
(II)
(IV)
(V)
(III)
HS
HS(VI)
(VII)
(VIII)
CH3
OH
O
HS
HN
OO
NHO
O
CH313313
10
3.2 Dip-pen nanolithography Using the setup for atomic force microscopy (AFM, section 6.5) Piner et al [33] developed
dip-pen nanolithography (DPN) for direct writing of thiol molecules on a gold substrate
(Figure 5). In this method, the AFM tip is used as a pen and it is dipped in a solution (the
ink) containing molecules that has affinity for the substrate (the paper). When the tip is
brought in close contact with the substrate, molecules are transferred to the substrate by
capillary force. In this way, molecular patterns can be written on the surface. The
difference from microcontact printing and other lithographic techniques [34] is that DPN
is slower but simpler and offers the possibility to print different molecules in different
areas. DPN has been used to produce nanoscale arrays of polymers, DNA and even
proteins on different substrates [35].
In Paper II, DPN was used to write a pattern of an alkanethiol (VI, Figure 4)
followed by backfilling of another alkanethiol (VII or VIII, Figure 4). Onto this SAM
pattern a hydrogel was then grafted.
Figure 5. Illustration of the DPN principle, adapted from [33]. The tip is dipped in a solution containing molecules which are then transferred to the substrate.
3.3 Hydrogel photografting Polymer coatings can be produced either by tethering polymer chains to a substrate
(“grafting to”) or by allowing polymerization of monomers to occur on the substrate
(“grafting from”). While the first strategy provides control of the resulting surface
structure, it is difficult to make thick coatings [36]. Therefore the latter approach is often
used, although it too has weaknesses. For example, as monomers polymerize on the
surface, the diffusion is limited and hence the resulting structure becomes dense with a
large distribution of the molecular weights of the polymer chains.
Cantilever with tip (”pen”)
Substrate (”paper”)
Writing direction
Molecules in solution ( “ink”)
11
OOH
O
x
In the class of “grafting from” approaches, an emerging technique is self-initiated
photografting and photopolymerization (SIPGP), first described by Wang et al [37, 38].
Since the polymerization of monomers is induced by UV light, no addition of
photoinitiator is required, which is of benefit for biomaterials and other applications where
initiator residues could be harmful. Photopolymerization includes the possibility of
photolithograpy for patterning and miniaturization of polymer architectures.
A hydrogel is a polymer that can absorb water so that the structure swells to several
times its thickness in dry state. In contrast to the two-dimensional SAM-ambient interface,
a hydrogel provides a three-dimensional structure. Hydrogels can be prepared from
monomers with functional groups that can be used for biomolecule immobilization or the
hydrogel can be functionalized after grafting. The larger surface area results in a larger
immobilization capacity compared to SAMs. One example is the commercially available
carboxymethylated dextran which is prepared in a “grafting to” manner by attaching
dextran chains to a SAM on gold [39]. Similarly, PEG chains can be grafted to SAM
covered substrates [40, 41].
In Paper II, a hydrogel (Figure 6a) was prepared by the SIPGP method using a
mixture of two monomers (Figure 6b): poly(ethylene glycol) methacrylate (PEGMA) and
2-hydroxyethyl methacrylate (HEMA). Here, a hydrogel density pattern was obtained by
an underlying SAM pattern created by DPN.
a) b)
Figure 6. a) Illustration of a hydrogel, prepared by SIPGP, on a hydrophilic SAM on a gold substrate. b) The chemical structure PEGxMA with x EG units. If x = 1, the structure is HEMA.
12
13
4. Protein immobilization
There are plenty of strategies for immobilizing proteins on sensor surfaces. The methods
can be classified into three groups [16]: physical immobilization, covalent immobilization
and bioaffinity immobilization.
Physical immobilization is driven by intermolecular forces (electrostatic, polar or
hydrophobic) between protein and surface. It results in a weak attachment of ligands with
heterogenous distribution and random orientation.
Covalent immobilization utilizes surface bound functional groups that can react with
exposed amino acid side chains on the protein surface. Amine coupling (section 4.1) and
thiol coupling are commonly used. Photochemical methods (section 4.3) involve
photolabile agents forming covalent bonds upon UV light activation. Another example is
click chemistry, involving cycloaddition of an azide and an alkyne.
Bioaffinity immobilization methods rely on recombinant affinity tags (section 4.2),
the naturally occurring biotin-avidin system (section 5.2). Antibody immobilization can be
mediated by protein A/G and glycoproteins can be immobilized via their carbohydrate
moieties to surface bound lectins (sugar-binding proteins). DNA-directed immobilization
uses oligonucleotide-tagged proteins to convert a DNA array into a protein array by DNA
hybridization.
4.1 Amine coupling Amine coupling involves activation of a carboxylate group that can react with a primary
amine so that an amide bond is formed. This is a frequently used method for covalent
attachment of proteins to SAMs [42] and hydrogels [39]. The activation agents used in
water solution are 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS). EDC reacts with a carboxylic acid to form an active
ester intermediate which is then displaced by an NHS ester. This more reactive ester then
rapidly couples with primary amines [43]. EDC could be used alone but with less
efficiency due to the lower reactivity and the susceptibility to hydrolysis of the EDC-ester.
In the case of protein immobilization to surfaces (Figure 7), the carboxylates are
surface bound and react with the amine side chains of lysines in the protein. Following
activation with EDC/NHS the protein solution is added. The solution should have a pH
14
below pI of the protein to facilitate electrostatic interaction (preconcentration) between the
protein and the negatively charged carboxylated surface prior to amine coupling. After
amine coupling, deactivation with ethanolamine is performed to remove residual NHS-
esters on the surface.
For amine coupling at low pH (for acidic proteins), sulfo-NHS is preferred over
NHS [44] because the sulfate group keeps a negative charge on the surface whereas the
common NHS-ester is uncharged. The drawback is that sulfo-NHS-activated esters also
react with sulfhydryl and hydroxyl groups to form thioesters and esters, respectively [43].
In this thesis, amine coupling has been used for immobilization of a protein in Paper
I and a smaller amine-functionalized biomolecule, biotin, in Paper II.
Figure 7. The amine coupling reaction starting with the formation of an EDC-ester, which is displaced by an NHS-ester. The NHS-ester reacts with a primary amine in the protein and an amide bond is formed. Following amine coupling, residual NHS-esters are deactivated using ethanolamine. Adapted from [43].
Surface bound carboxylic acid
NHS
Deactivation with ethanolamine
Amine coupling via primary amine in ligand
R-C-O-
=
O
N+
N
C
N
=
H
N=C=N N-H+
EDC
R-C
R-C-N-O
H
=_ Ligand
O=
H
_
R-C-O-N
O=
=O
=
O
HO-N
=O
=
O-H
O
OH
=-
15
4.2 Metal ion affinity A popular functional group used for achieving controlled orientation upon protein
immobilization is nitrilotriacetic acid (NTA), otherwise commonly used in immobilized
metal ion affinity chromatography (IMAC). The chelating agent NTA has a strong affinity
for bivalent metal ions such as Ni2+ which have six coordination sites. Four of these are
chelated to NTA, leaving two sites free to coordinate other groups. Several amino acids,
including histidine (His), have a moderate affinity (μM-range) to Ni2+-NTA complexes
(Figure 8).
Figure 8. The chemical structure of NTA, its chelation of a metal ion (here Ni2+) and the coordination of two imidazole molecules to the complex. Imidazole is the side chain group of the histidine amino acid and the Ni2+-NTA complex can be used for binding proteins modified with a histidine tag.
Due to the reversibility, the Ni2+-NTA complex is commonly used in affinity purification
of recombinant proteins containing His-tags [45] and the same principle is used for non-
covalent immobilization of His-tagged proteins to biosensor surfaces with surface bound
NTA. Stable binding can be achieved, despite the moderate affinity, if the surface
concentration of NTA is sufficiently high (surface multivalency), which has been shown
to enable continuous rebinding in a dextran matrix [46] and on surfaces presenting bis- or
tris-NTA instead of mono-NTA [47, 48]. The binding stability also depends on the His-tag
length (molecular multivalency) [49] and a double-His6-tag has been shown to provide
stronger binding than a single-His6-tag [50]. Although the interaction of His-tags with
Ni2+-NTA can be made stable it is never irreversible. Upon addition of a competing
molecule such as imidazole or EDTA, or by changing the pH to below the pKa of His (~6),
the His-tagged protein is eluted.
NO
O
Ni2+O
O
O
O
N
HN
N NH
-
-
-
16
4.3 Photoimmobilization Photolabile agents, e.g. arylazides, benzophenones, diazirines and nitrobenzyl groups [22],
can be surface bound in order to allow for protein immobilization upon UV light
activation. Benzophenone (BP) is activated by UV light in the region 350-360 nm and its
reaction mechanism is well-known: The carbonyl oxygen forms a ketyl radical when
exposed to UV light and then abstracts the hydrogen from a C-H bond in a nearby
molecule, followed by C-C bond formation between BP and the attacked molecule (Figure
9).
Advantages of BP include chemical stability, activation in a UV range that does not
damage proteins or cells, and stability in ambient light [51]. The disadvantages are the
bulkiness and hydrophobicity of the molecule and the fact that the C-H bond has to be
within 3.1 Å of the carbonyl oxygen for crosslinking to take place [52]. Benzophenone has
been used in applications for crosslinking of polymers [53] in combination with SAMs of
alkylsilanes [54]. Most interestingly, BP has been used for photoimmobilization of IgGs
or F(ab’) fragments to SAMs of alkanethiolates [55] or alkylsilanes [56] and to
polysterene [57].
Figure 9. The chemical structure of BP and the reaction that results in covalent bond formation when BP attacks a covalent bond upon UV light activation.
4.4 Chelation Assisted Photoimmobilization In Paper I, we introduce a novel immobilization method for controlled orientation and
covalent attachment of proteins on biosensor surfaces. We refer to this method as
Chelation Assisted Photoimmobilization (CAP). It is based on a SAM of alkanethiolates
O
C R3R1
R2
C
O
C R3R1
R2
H
C
C R3R1
R2
OH
H
17
(molecules I-V, Figure 4) on a gold substrate exposing the two agents discussed above:
NTA for orientation and BP for photoimmobilization of proteins. The alkanethiolates also
contain an OEG portion for reduction of nonspecific binding. An OEG terminated
alkanethiol is used for dilution of NTA and BP on the surface.
The principle of CAP is as follows: A His-tagged ligand is attached to the surface in
a proper orientation by NTA and then covalently immobilized upon UV activation of BP
(Figure 10). After UV irradiation, any residual ligand bound to Ni2+-NTA but not
photoimmobilized, is eluted by imidazole which competes with the His-tag in binding to
the Ni2+-NTA complex. The binding site of the oriented and covalently bound ligand is
then available for subsequent interaction with the analyte.
Figure 10. A schematic drawing of the CAP principle. The ligand is coordinated via its His-tag to Ni2+-NTA and then locked in this orientation upon photoactivation of BP. The oriented and covalently bound ligand is then available for analyte interaction.
Ni2+
OEG
UV light
Analyte
Ni2+
OO
OO
O
O
O
OO
O
OHH
HH
HH
H
SAM on Au substrate
NTA
LigandwithHis-tag BP
OO
OO
O
O
O
O
OO
O
H
H
H
HH
H
18
19
5. Biomolecular interaction
In biosensor development, thoroughly characterized and well-understood interaction
model systems are often used for studying ligand-analyte binding. The following sections
describe the biomolecular model systems used for proof of concept experiments in the two
papers presented in this thesis.
5.1 Antibodies Antibodies (immunoglobulins) [58] are proteins that recognize molecules that are foreign
to the body (antigens) in order to activate the immune system. Antibodies are divided in
five classes: A, D, E, G and M. Here, only immunoglobulin G (IgG), the most abundant in
serum, is described.
An IgG consists of two heavy and two light polypeptide chains, connected via
covalent disulphides bonds in a Y-shaped form (Figure 11). The two arms (Fab, so called
because the fragments are antigen binding) each contain a highly variable part, which
provides the specificity for different antigens. The stem (Fc, so called because the
fragment is crystallizable) is a constant sequence similar in all IgGs. In humans, there are
four types of the heavy chain, dividing the IgGs in four subclasses: IgG1- IgG4. The Fc
and Fab regions are connected via disulphide bridges in the hinge region, making the
molecule very flexible.
Some proteins bind to antibodies in regions other than the Fab. The bacterial protein
A and protein G bind to the Fc domain mainly by hydrophobic interaction and while the
affinity is strong at neutral and basic pH, it is weak at acidic pH [59]. These proteins are
therefore commonly used in antibody affinity purification but have also been used in the
preparation of immunosensors. For example, surface immobilized protein A binds the Fc
of IgG resulting in a partial orientation of the IgG (the ligand) with at least one Fab region
exposed for binding the antigen (the analyte) [20, 60]. Although it has more than two
functional binding sites [61], protein A can bind only two IgG molecules at once and one
IgG can bind two protein A molecules.
In Paper I, protein A was used as the model analyte and the Fc region of human IgG
of subclass 4 was used as the model ligand. The ligand was modified with one
hexahistidine tag at each C-terminus [62] and is hereafter referred to as His-IgGFc.
20
Figure 11. The general structure of IgG which has a Y-shape with two arms (Fab) and a stem (Fc). The arrows mark the binding sites of antigen and protein A. His-IgGFc and protein A were used in Paper I as ligand and analyte, respectively.
5.2 Biotin – avidin The interaction between biotin and avidin is one of the strongest known in nature with a
dissociation constant of 10-15 M [63]. Biotin (Figure 12), also known as vitamin H, is a
small molecule which acts as a coenzyme in cell metabolism. While avidin is found in egg
white, streptavidin is a bacterial protein. They are different in primary structure, but both
have four subunits and four binding sites and bind biotin with similar strength.
Streptavidin has a more neutral pI (5-6) compared to avidin (pI = 10) and therefore
exhibits less nonspecific binding caused by electrostatic interactions [43]. Both biotin and
(strept)avidin can be conjugated to other proteins or fluorescent labels without loss of
affinity. The affinity remains high even in a large range of pH and concentrations of buffer
additives or salt. For all these reasons, biotin-streptavidin is a commonly used affinity pair
in different biotechnological applications, such as immunoassays and, as in Paper II, as a
model system in biosensor research.
Protein Abinding sites
Fab
Fc
Antigen binding sites
21
Figure 12. The chemical structure of biotin and a schematic drawing of streptavidin binding four biotin molecules simultaneously.
S
NH
O
HN=
COOH
Biotin Streptavidin
22
23
6. Experimental techniques
Here, the techniques for surface characterization and study of biomolecular interactions
used in the papers included in this thesis are described. Some optical detection techniques
can be used with a photodetector that averages the information over the studied surface
area, or with a camera for imaging of surfaces with lateral resolution. This is the case for
null ellipsometry, infrared reflection-absorption spectroscopy and surface plasmon
resonance (sections 6.2-6.4). In this thesis, only the latter has been used in imaging mode.
6.1 Contact angle goniometry A goniometer is used for measuring angles and in this case to perform wettability
measurements. A liquid drop is added on the substrate to be studied and a camera captures
images of this arrangement which the computer software then uses to calculate the contact
angle. This is the angle between the surface and the tangent line to the curve described by
the drop, starting at the surface-liquid-ambient interface (Figure 13a). Measurements can
be performed in ambient atmosphere and with pure water as liquid. On a hydrophilic
surface the drop will spread out, resulting in a low contact angle value. On a hydrophobic
surface the solid-liquid interface will be minimized yielding a higher contact angle.
a) b) c)
Figure 13. Schematic drawings of the sessile drop method (a) and dynamic sessile drop method (b,c) in contact angle goniometry. The static angle (θstat) gives a measure of the surface hydrophilicity. Using a syringe to add or remove liquid from the drop, the advancing (θadv) and receding (θrec) contact angles can be measured. The hysteresis between these is a measure of surface roughness or heterogeneity.
Using the sessile drop method, a drop of liquid is added to the surface and the static
contact angle is directly obtained. The dynamic sessile drop method involves a syringe
θadv
Liquidadded
θrec
Liquid removed
θstat
24
used to control the volume of the liquid drop (Figure 13b-c). The advancing angle is
measured as the volume of the drop is increased and the receding angle is measured as the
volume is decreased. As the advancing drop is moving over a dry surface whereas the
receding drop is moving over a wet surface, the advancing angle is larger than the
receding angle. The difference between these is called hysteresis [64] and it is interpreted
as a measure of the roughness or heterogeneity of the surface. It can also be due to the fact
that the wetting itself can induce reconstruction of polar/nonpolar groups on the surface
[27, 64]. On the other hand, it has been shown that mixed SAMs on ultrasmooth gold
surfaces do not exhibit hysteresis [65]. This could mean that the hysteresis in many
systems previously studied could be a result of surface roughness rather than due to islets
formed in a heterogeneous, mixed SAM. Hence, although a contact angle measurement is
simple to perform, the data interpretation is not [66]. Importantly, comparisons should be
made between measurements in similar systems rather than drawing conclusions from
single absolute values [65].
In Paper I, contact angle measurements were used to obtain information related to
the composition of both pure and mixed SAMs.
6.2 Null ellipsometry Light can be described by two cosine waves, transverse to each other and to the direction
of propagation. The end points of the vectors representing the waves (the polarization
components) describe an ellipse. In special cases, depending on the phase shift between
the waves, the light becomes linearly polarized or circularly polarized.
Null ellipsometry is a nondestructive technique for determining surface film
thickness. It is a fast and easy to use method that can be performed in air or in a liquid cell.
The original meaning of the term ellipsometry is the measurement of elliptically polarized
light, but it now refers to the analysis of polarized light in order to determine the optical
properties of thin films on reflecting substrates.
As light is reflected off a thin film on a metal substrate it is phase shifted and
attenuated. The phase shift is described by the phase angle Δ (delta). The amplitude
change is described by the tangent of Ψ (psi), which is the ratio of the complex reflection
coefficients of s- and p-polarized light, respectively. The fundamental equation of
ellipsometry gives the relation between the normalized reflection coefficient ρ and the
25
ellipsometric parameters Δ and Ψ, which in turn are related to the complex refractive
index (N), extinction coefficient (κ) and thickness (d) of the film [67]:
ρ = tan Ψ eiΔ = f (N, κ, d)
The PCSA set-up [6, 67] of a null-ellipsometry instrument is illustrated in Figure 14. The
monochromatic light comes from a He-Ne laser and the angle of incidence (θ) can be
varied. The light passes a rotating polarizer (P) and a quarter wave plate compensator (C)
before reaching the sample surface (S). The reflected light passes another rotating
polarizer, the analyzer (A), before reaching the photodetector. The incident linear light is
elliptically polarized by the compensator before reaching the surface. At a certain angle of
the polarizer, the light becomes linearly polarized when it is reflected from the surface.
The analyzer is then rotated to the angle where all reflected light is extinguished and no
light reaches the photodetector. When this co-called “null-condition” is reached, the
ellipsometric parameters Δ and Ψ can be obtained from the rotation angles of the polarizer,
compensator and analyzer. Subsequently, the refractive index or the film thickness can be
calculated.
There are several methods available for these calculations, one of which is the
McCrackin algorithm [68]. The complex refractive index (N = n – i k) applies to all thin
films but if the film can be assumed to be transparent, the imaginary part is zero and the
refractive index is a real number, n. It is often set to 1.465 or 1.5 for transparent organic
thin films. Since the calculation averages the information from a beam area of about 1
mm2 it is difficult to draw conclusions on the distribution or the presence of voids in the
film. A protein film thickness of 1 nm corresponds to a surface density of approximately
1.2 ng/mm2 [6].
In Paper I, ellipsometry was used for basic characterization of untreated SAMs and
for measuring change in surface film thickness after protein immobilization. Small
amounts of protein were detected. As explained above, the area of detection is so large
that the ligand surface density could be locally high and yet result in a low film thickness
on average. In this case, null ellipsometry data were complemented by information from
infrared spectroscopy which will be described next.
26
Figure 14. The PCSA-setup of a null ellipsometer. The ellipsometric parameters used for calculation of film thickness are obtained when the polarizer and analyzer angles are such that no reflected light reaches the detector (the null condition).
6.3 Infrared spectroscopy Infrared (IR) spectroscopy is based on the fact that molecular bonds are excited by IR
radiation of specific wavelengths. This is a nondestructive optical technique used to obtain
information about what type of bonds are present in a molecule (transmission mode) or
how a molecule is oriented with respect to the surface it is attached to (reflection-
absorption mode).
IR spectrometers are based on interferometric measurements and data conversion
using Fourier Transform (FT). The instrument contains a radiation source, an
interferometer, a sample holder and a photodetector.
The Michelson interferometer (Figure 15) contains a beam-splitter, which divides a
light beam from the source into two paths. The beam in one path is reflected against a
fixed mirror, while the other beam is reflected against a moving mirror. After reflection
the beams recombine at the beam-splitter and continue toward the sample, where some
wavelengths are absorbed. The moveable mirror changes the difference in path length so
that the beams interfere constructively or destructively. The detector records an
interferogram which is converted using FT calculations into a spectrum of intensity versus
wavelength (or rather wavenumber, the inverted wavelength) [69]. Most measurements are
performed in the mid-IR region, 4000-400 cm-1 (λ = 5-25 μm).
Surface
Laser
Polarizer
Compensator Analyzer
Photodetector
θ
27
Figure 15. Schematic drawing of the Michelson interferometer used in Fourier transform spectrometers, redrawn from [69]. Lenses have been omitted and the reflected beams have been displaced for clarity. The moving mirror causes a phase shift between the beams reaching the detector. The resulting interferogram, affected by the molecular composition of the sample, is converted by Fourier transform into a spectrum. A transparent sample is shown here but the same principle is used for reflecting samples.
In most IR spectroscopy instruments, the sample is contained either in a vacuum chamber
or in a chamber that is purged by nitrogen gas to minimize interference from atmospheric
H2O and CO2. The intensity is measured for all wavelengths simultaneously during one
scan. Many scans are made during a measurement, which results in a good signal-to-noise
ratio [70]. IR spectroscopy can be used to study liquids, solids and gases. Here the focus is
on solid samples.
In transmission mode, the compound to be studied can be incorporated in a pellet of
an alkali halogenide material (e.g. KBr) which does not absorb light of wavelengths in the
relevant mid-IR region when sintered under high pressure [69]. The sample powder is
finely ground, mixed with KBr and then pressed into a pellet which is mounted in a holder
in the instrument. In a bulk sample all orientations of a molecule are possible and hence
vibrations from all bonds with a non-zero transition dipole moment can be detected. This
however is not the case for IR spectroscopy in reflection-absorption mode.
Moving mirror
Fixed mirror
Light source
Detector
Beam splitter
Transparent sample
28
Infrared reflection-absorption spectroscopy (IRAS, also known as RAIRS or
IRRAS ) was first described by Francis and Ellison [71] and Greenler [72]. It is now used
to study thin films on metallic substrates. It gives information about both composition and
orientation of the molecules constituting the film. Infrared radiation is polarized and then
focused onto the sample. The sample reflects the light and is then recollimated by a set of
focusing mirrors before reaching the detector. The surface sensitivity is maximized by
adjusting the incident light beam to a grazing angle (i.e. close to parallel to the surface).
There are two reasons for this:
Firstly, the beam passes the thin film twice, once before and once after reflection
from the metallic surface. A grazing angle results in a longer path length in the film and
hence higher surface sensitivity. Secondly, a grazing angle enhances the strength of the
perpendicular component of the electric field, which is most important in IRAS
measurements. The surface selection rule states that only molecular vibrations that give
rise to a transition dipole moment with a component perpendicular to the metal surface
can be detected [70, 71].
When light is reflected from a metal surface the phase shift depends on both the
angle of incidence and the polarization. For incident and reflected light, the components
parallel to the surface will cancel due to a 180º phase shift upon reflection in the grazing
angle geometry. The components perpendicular to the surface, on the other hand, will
interfere constructively [72]. This is the origin of an electric field perpendicular to the
metal surface (Figure 16). The intensity (I) of a vibrational mode is proportional to the
square of the electric field (E) and the transition dipole moment (M) of the molecular
bonds and depends on the angle φ between the surface normal and the dipole moment [6]:
I ~ |E•M|2 = |E|2 • |M|2 • cos2 φ
This means that the intensity for a vibrational mode is at its maximum when the vibration
occurs perpendicular to the surface, whereas a vibration parallel to the surface cannot be
detected at all. Therefore it is important to remember that a molecular bond may be
present on a sample surface although it is not detected by IRAS. Comparing spectra from
IRAS and transmission (bulk) spectroscopy can yield information about the orientation of
molecules on the surface (Figure 17).
29
Figure 16. Illustration of the electromagnetic components (E) of incident (i) and reflected (r) light at the surface in IRAS. Redrawn from [6]. The components parallel (p) to the plane of incidence (Eip and Erp) interferes constructively while the perpendicular (s) components (Eis and Ers) cancel. The resulting electromagnetic field (ER) is therefore perpendicular to the surface and hence only molecular vibrations with dipole moments perpendicular to the surface can be detected in IRAS.
In Paper I, the composition of synthesized molecules was confirmed using IR
spectroscopy in transmission mode, while their packing and orientation in SAMs on gold
were studied by IRAS. Furthermore, IRAS was used to quantify the amount of ligand
immobilized and the technique proved to be more sensitive than ellipsometry. In Paper II,
IRAS was used to study the different steps of preparation of a hydrogel modified substrate.
Ers
Eis
EipErp
EREis
Eip Erp
Ers
30
Figure 17. For the EG3 disulfide (molecule IV, Figure 4) used in Paper I, the amide I peak at 1643 cm-1 is the strongest in the transmission spectrum but not present at all in the RA spectrum, confirming that the molecules in the SAM are well packed with the carbonyl bond of the amide group parallel to the surface. 6.4 Surface plasmon resonance The technique to use Surface Plasmon Resonance (SPR) as a tool for detecting and
characterizing biomolecular interactions was first developed by Liedberg et al [73]. With
this method, biomolecular interactions can be studied in real time and information about
kinetics, affinity and specificity can be obtained.
A surface plasmon is a charge density wave propagating at the interface between a
metal, such as gold, and a dielectric (polarizable) medium, such as water. When light is
totally internally reflected towards a thin gold film, part of the photon energy may set up
an evanescent field at the opposite interface which excites the surface plasmon. This
happens at a certain wavelength and a certain angle of incidence (the SPR angle, θSPR),
determined by the refractive index of the medium outside the film. At θSPR reflected light
is attenuated due to energy coupling into the evanescent field. When allowing
31
biomolecules to bind by interaction on or very close to the surface, the refractive index is
changed, causing a change in θSPR which is observed as a shift of the dip in the intensity of
the reflected light. The penetration depth of the evanescent field is of the order of half a
wavelength of the exciting light. Sensitivity decreases exponentially away from the
surface and is highest in the first hundred nanometers [74].
Figure 18. The Kretschmann set-up of an SPR instrument. Incident light excites a surface plasmon upon reflection off the back side of the gold film in a sensor chip. This occurs at θSPR, at which the minimum in the intensity of the reflected light is detected. As analyte binds to ligand, the refractive index at the surface changes, causing a θSPR shift. The real time detection of the shift is presented in a curve with response versus time.
Many SPR instruments use the so-called Kretschmann configuration (Figure 18), with a
glass prism, the sensor chip (usually a gold film on a glass substrate), a liquid handling
system, a light source and a detector. In the commercially available instrument used in this
work, an integrated microfluidic cartridge (IFC) contains the sample and buffer loops and
several flow channels. As the IFC and the gold side of the sensor chip are pressed together,
individually addressable flow cells are formed where the biomolecular interactions can
take place. The convergent beam from a light emitting diode (LED) is refracted by a glass
prism and then reflected at the glass/gold interface on the backside of the sensor chip. The
light beam is convergent and focused on a small surface area and the divergent reflected
light is detected by a photodiode array (PDA). This set up with a fan shaped beam
requires no moving optical parts to detect the changes in θSPR [74]. A real-time recording
Flow cell
Light source
Gold surface with ligand
GlassSensor chip
Glass prism
Analyte insolution
θSPR
Photodetector
{
32
of θSPR is presented in a sensorgram (Figure 19) with relative response units (RU) as a
function of time. A 0.0001º shift in θSPR corresponds to 1 RU and approximately 1 pg/mm2
of protein [75].
Figure 19. A typical sensorgram showing amine coupling of the ligand His-IgGFc (Paper I) to a carboxylated matrix and subsequent interaction with the analyte protein A. The start of each sample injection is marked with an arrow.
Some advantages with the SPR technique compared to the Enzyme-Linked
ImmunoSorbent Assay (ELISA), which is another commonly used strategy to monitor
biomolecular interactions, are that it is more reproducible, gives an earlier detection of
interaction and the real-time detection allows for kinetic analysis. Molecular labeling is
not needed and the surface can be regenerated and reused. The disadvantage compared to
ELISA is that fewer samples can be analyzed during the same time frame.
By adding a camera to the SPR instrument, an image of the surface can be obtained
[76]. SPR microscopy, or imaging SPR (iSPR), can be used to study surface patterns and
molecular arrays. In general surfaces are prepatterned or the ligand is immobilized offline
33
by spotting. To avoid image distortion in iSPR it is common to fix the angle of incidence
while scanning the wavelength [77]. Instead of detecting θSPR, the wavelength (λSPR)
resulting in a minimum in the intensity of the reflected light is recorded.
In Paper I, a commercial SPR instrument was used to study the specific interaction
between protein A in solution and surface immobilized His-IgGFc. In further work,
related to but not reported in Paper I, an in-house built iSPR instrument [77] was also used
to study photoimmobilization of His-IgGFc and subsequent interaction with protein A
(Chapter 7).
6.5 Atomic force microscopy Scanning Probe Microscopy (SPM) [78] is an umbrella term for a number of techniques
using a tip (probe) for surface characterization, topographic imaging and manipulation.
Atomic Force Microscopy (AFM) is an SPM technique where high-resolution
imaging based on atomic forces can be obtained. In general, the instrument contains a
laser source, a sample holder, a tip on a cantilever and a photodetector. The laser beam is
reflected on top of the cantilever before it reaches the photodetector. As the surface and
the tip are brought close together, the intermolecular interactions cause the cantilever to
deflect, either by attraction or repulsion. The change in cantilever bending angle causes a
change in laser beam position on the detector. This can be presented as a force curve
(force versus tip-sample distance) which provides information about the tip-sample
interaction and thus of the surface properties.
For scanning the tip across the surface, either the sample or the cantilever is placed
on a piezoelectric tube scanner. Most set-ups (Figure 20) use a feed-back loop to keep the
surface-tip distance constant. The voltage change needed for the correct positioning of the
piezoelectric stage is used as the signal for creating a topographic image of the surface.
Measurements can be performed in air or liquid and different modes are used depending
on the properties of the sample and the information asked for. A general problem for all
modes is that image artifacts are produced when the tip fails to "sense" the topography
properly due to its shape and size.
34
Figure 20. The general set-up of AFM. Either the sample or the cantilever can be placed in contact with a piezoelectric tube scanner. When tip and sample interact, the deflection of the cantilever is detected as a change in position of the reflected light. Electronic feedback is used to keep a constant tip-sample distance and to render a topographic image of the sample. In contact mode, high resolution is obtained because the tip interaction with the closest
substrate molecules is so strong that interactions with neighboring molecules make a small
difference. However, soft samples are damaged due to scraping. A special case of contact
mode is friction force mode or lateral force microscopy (LFM) where the tip is dragged
across the surface and the twisting of the tip due to lateral forces is recorded. This can give
information about local differences in density, so called material-specific contrast.
In non-contact mode the tip is positioned at a greater distance from the surface than
in contact mode and thus the resolution is lower. In this mode, imaging is based on long
range forces (magnetic or electrostatic) because in ambient atmosphere a liquid layer is
formed on the sample surface which makes it hard to use short range forces.
Therefore, tapping (intermittent) mode is frequently used for soft samples. It
provides good resolution without damaging the surface because the lateral forces are
weaker compared to the forces in contact mode. The cantilever is oscillated and taps the
Laser
Cantilever with tip
Sample
Position sensitivephotodetector
Feedback controller
Scanner
Sample stage with vibration isolation
35
surface. Any interaction results in a change of the oscillation amplitude, counteracted by a
feedback system, which adjusts the tip-sample distance to keep the oscillation amplitude
constant. This creates an image of the forces during intermittent contact.
In Paper II, AFM was used in lateral force mode and tapping mode (Figure 21) to
study DPN-made arrays of SAMs of different alkanethiols and the hydrogel structures
formed on these.
Figure 21. Figure 3 in Paper II, reproduced with permission. a) LFM image showing material specific contrast of a DPN-made MHA pattern after backfilling with EG6C16. b) Topography image (tapping mode) of the same sample after hydrogel photografting. The inset below shows the height of the structures marked by the horizontal lines.
6.6 Epifluorescence microscopy Fluorescence is frequently used for detection of biomolecular interactions. An
epifluorescence microscope [79] generally consists of a light source, filters, a dichroic
mirror, an objective and a detector. Light with the wavelength desired for excitation is
selected by an excitation filter and directed towards the sample by a dichroic mirror. The
objective focuses the light on the sample, resulting in fluorophore excitation. Light enters
36
back into the same objective and passes the dichroic mirror. The fluorescence wavelengths
desired for detection are selected by an emission filter before reaching the detector,
producing and image of the fluorescence in the sample.
The aromatic amino acids are intrinsic fluorophores and can be used for protein
detection in general. Fluorescent labeling is needed for stronger fluorescence signals, for
specific protein detection and for detection of non-fluorescent biomolecules. Several
fluorophores with higher emission wavelength than the aromatic amino acids are available
for labeling. Furthermore, quantum dots (QDs) can be used.
QDs [80] are nanoparticles consisting of a core of a semiconducting material such as
CdSe or CdTe covered by a shell of ZnS. The luminescent property is due to the small
dimensions and the core material and it is enhanced by the shell material. The
nanoparticles are coated with a polymeric material that provides stabilization and acts as a
scaffold for further functionalization. In Paper II, fluorescence of streptavidin-conjugated
QDs, bound to a hydrogel pattern via biotin-streptavidin interaction, was detected with
epifluorescence microscopy.
37
7. Demonstration and evaluation of CAP
Paper I describes the design and implementation of a novel surface chemistry for
controlled orientation and covalent attachment of proteins, called Chelation Assisted
Photoimmobilization (CAP). The molecules used for SAM preparation and the
immobilization method have been described earlier (see Figure 4 and section 4.4).
Early work in the development of CAP involved finding suitable experimental
parameters. Different ratios of the alkanethiols or alkanedisulphides in solution used for
SAM preparation were evaluated before choosing the 80:20 ratio for the EG3 and BPNTA
disulfides (referred to as 20% BPNTA). Evaluation of the BP photochemistry involved
finding suitable UV irradiation intensity and exposure time. Also, the choice of model
system required some thought.
A few different protein model systems were tested for the evaluation of CAP. Large
nonspecific analyte binding to the CAP surface was sometimes observed, probably by
hydrophobic interaction with BP, by undesired metal binding to Ni2+-NTA or a
combination thereof. For example, anti-hexahistidine was evaluated as an indirect control,
which would ideally bind the His-tag if the ligand was in random orientation, but not if it
was homogenously oriented by NTA. Both rabbit and goat anti-hexahistidine antibodies
were tested but not used further due to a large nonspecific binding which could only
marginally be reduced with buffer additives such as NaCl or detergents. Therefore it
would be interesting to look at further reduction of nonspecific binding by controlling the
surface chemistry. To reduce the hydrophobic contribution from the BP group, a 4-
hydroxy-benzophenone alkanethiol was synthesized, which could be used to render a
more hydrophilic and thus more protein resistant surface. This has not yet been evaluated.
During the work with this thesis, a commercial SPR instrument and an in-house built
iSPR equipment were used in parallel to study different approaches with CAP. With the
commercial instrument, protein immobilization was performed on the lab bench and
docked into the instrument to study only analyte interaction. In the second case, the UV
source was mounted on the iSPR instrument so that all steps of the CAP method could be
monitored. For reasons described and discussed below, the first approach was used in the
final work leading up to Paper I.
38
The steps of the CAP process were studied by iSPR by mounting the sample surface
onto the SPR prism in an open cuvette so that it could be irradiated by UV light from
above. Reagent solutions were added and removed by pipetting. A chromium patterned
quartz mask was used to obtain spots, 300 μm in diameter, upon UV light irradiation. The
mask was placed on top of the drop of ligand solution on the surface. All experiments
started with the background measurements of the untreated SAM. This image was then
subtracted from all subsequent measurements, resulting in images of the SPR wavelength
shift (ΔλSPR). In this case, a shift ΔλSPR = 1 nm corresponds to an organic film of
approximately 5 Å [81].
Three typical iSPR experiments are presented in Figure 22, which will be discussed
in detail here. These experiments were performed on the same type of surface (20%
BPNTA), but the two control experiments utilized either BP or NTA separately.
The top row shows a typical CAP experiment: the NTA groups were first loaded
with Ni2+ (step 1). The surface was incubated with ligand and irradiated by UV light to
activate BP followed by rinsing in PBS (step 2). The surface was irradiated through a
chrome mask, but no pattern was visible at this stage due to the specific but non-covalent
binding of ligand to NTA in the background. After rinsing with imidazole (step 3), non-
covalently bound ligand was eluted and the pattern clearly seen. After analyte incubation a
slight increase in λSPR was detected (step 4) showing qualitatively that the oriented and
covalently bound ligand was active.
The bottom row shows non-covalent ligand immobilization to Ni2+-NTA. The
surface was loaded with Ni2+ and incubated with ligand followed by PBS rinsing (step 2).
Photopatterning was not used since non-covalent immobilization was desired in this
control experiment. The ligand was homogenously distributed on this large scale (1 mm2).
Step 3 was omitted and after analyte incubation and PBS rinsing (step 4), analyte binding
was clearly detected. Imidazole rinsing (step 5) eluted the ligand and hence analyte was
also removed. The remaining protein film was explained partly by nonspecific binding but
also by the repeated manual pipetting which could lead to incomplete rinsing. Future use
of this instrumental set-up would benefit from a flow cell with a UV transparent top and a
proper flow system.
For the BP experiment in the middle row step 1 was omitted since a random ligand
orientation was desired in this control experiment. Spots of photoimmobilized ligand were
39
seen in a background of ligand (step 2) after PBS rinsing. This ligand was eluted by
imidazole (step 3), indicating a Ni2+- NTA dependent binding of the His-tagged ligand
although the surface was not loaded with Ni2+. Additional experiments showed similar
signs of metal ion contamination in PBS buffer. Hence, the photoimmobilized ligand in
the BP experiment was unintentionally oriented, resulting in conditions similar to CAP
and disqualifying this experiment as a control with random ligand orientation.
In continued experiments, resulting in Paper I, random immobilization was therefore
studied on 10% BP surfaces, with no NTA present, thereby eliminating the metal ion
contamination problem. His-tag binding to Ni2+-NTA was studied on 10% NTA surfaces,
with no BP present. Additionally, amine coupling to the carboxyl groups of 10% NTA
was performed in the presence of the chelating agent EDTA, to remove contaminating
metal ions and prevent unwanted orientation of the His-tagged ligand in this case. EDTA
was not used in photoimmobilization experiments due to the risk of BP forming covalent
bonds with EDTA instead of the protein.
Figure 22. Imaging SPR was used to monitor the immobilization of a His-tagged ligand to a 20% BPNTA surface in three different ways: by CAP (top), by photoimmobilization to BP (middle) and by reversible coordination to Ni2+-NTA (bottom). Each image shows the change ΔλSPR (with 2 nm units on the perpendicular axis) after each treatment step compared to the untreated SAM. The imaged surface area is 1 mm2 and each spot is 300 μm in diameter. See the text for details and discussion.
40
For reasons discussed above, work was continued by offline ligand immobilization
followed by monitoring analyte interaction in the commercial SPR instrument. This
however, required other means of quantifying the immobilized ligand amount and for this
purpose, null ellipsometry and IRAS were used. It was found that the low protein amounts
could be readily detected and quantified by IRAS (Figure 23) but not with ellipsometry.
Figure 23. Results of ligand quantification data obtained by IRAS, as presented in Paper I. The two groups show the immobilized ligand amount before and after imidazole treatment, which elutes Ni2+-NTA bound ligand (10% NTA) but leaves covalently bound ligand unaffected. More ligand is immobilized by CAP (20% BPNTA) than by amine coupling to the carboxyl groups of NTA (10% NTA) or photoimmobilization to BP (10% BP).
In order to monitor the ligand orientation effect on analyte responses, the analyte binding
capacity of ligand immobilized by CAP and ligand immobilized in a random fashion by
amine coupling was compared. The analyte responses measured by SPR were normalized
using the ligand quantification data obtained by IRAS. Surprisingly, it was found that the
amine coupled ligand had a more than two times higher analyte binding capacity
compared to the ligand immobilized by CAP. There are two explanations for this
interesting result. Firstly, island formation in the mixed SAM could cause locally high
surface concentrations of immobilized and oriented protein, resulting in reduced
41
performance when using CAP. Also, the ligand immobilization level was three times
higher with CAP, adding to the crowding problem. Secondly, the bivalent ligand used is
probably not as sensitive to orientation effects as a monovalent ligand would be. None the
less, the CAP method works in principle and it would be interesting to evaluate its
performance using a monovalent ligand and in photopatterning applications.
42
43
8. Photografting hydrogels on patterned substrates
In Paper II, a hydrogel pattern was fabricated by controlling the underlying surface
chemistry (molecules VI-VIII in Figure 4). The SIPGP method for hydrogel fabrication
was used in combination with DPN to further miniaturize the hydrogel structures. A
pattern of MHA was “written” with DPN on clean gold followed by backfilling with a
longer alkanethiol containing an OEG portion and a hexadecane termination chain
(EG6C16). A hydrogel was then grafted on top of this patterned surface, resulting in a
hydrogel pattern. Although hydrogel polymerization is initiated preferentially on
hydrophilic surfaces, the hydrogel here formed a thin background layer also on the
hydrophobic EG6C16 regions. As shown by IRAS studies (Paper II, Supporting
Information), the OEG core of the EG6C16 is degraded by the UV wavelength used for
photografting (254 nm). This eventually results in OEG portions being exposed, thereby
initiating hydrogel grafting also in the background. However, this takes longer time than
the grafting initiated at hydrophilic sites, and the background acting as a sacrificial layer
explains the difference in hydrogel thickness.
To demonstrate the ligand immobilization capacity, biotin modified with an amine-
terminated EG2-linker (biotin-amine) was amine coupled to the carboxylated hydrogel
structures (Figure 24). Initial experiments involved delivering amine-biotin directly to the
hydrogel spots via DPN. Due to difficulties in realigning the DPN tip to the preformed
structures a less complicated approach was chosen. The entire sample surface was
incubated in biotin-amine solution and hence the ligand was bound also to the thin
hydrogel background layer. Thereafter the surface was incubated with streptavidin-
conjugated QDs and imaged with an epifluorescence microscope (Figure 25). The specific
binding of streptavidin to biotin could be confirmed by repeated regeneration and
incubation. A stronger fluorescence signal was obtained from the thicker hydrogel spots
than from the thin hydrogel background layer. The thin hydrogel in the background might
be an advantage. It could be useful in studying the effect of surface density of ligands, e.g.
cell surface receptors. Furthermore, by controlling the UV exposure time with a moving
shutter during photografting, a thickness gradient of the hydrogel structures can be easily
produced.
44
Figure 24. Illustration of the surface in Paper II, where a SAM pattern on gold was the platform for hydrogel photografting. After carboxylation of the hydrogel, amine-biotin was immobilized by amine coupling. Thereafter the binding of streptavidin-conjugated QDs was detected by epifluorescence microscopy.
Figure 25. Figure 4 in Paper II, reproduced with permission. a) AFM tapping mode image of the topography of a hydrogel dot array. b-f) Amine-biotin was coupled to the carboxylated hydrogel and binding of streptavidin-conjugated QDs (b, d, f) was detected by epifluorescence microscopy. After regeneration (c, e) subsequent loadings (d, f) showed stronger fluorescence, indicating that some form of preconditioning of the hydrogel is advantageous.
Streptavidin-conjugated QD
Hydrogel structure
SAM pattern on gold
S
NH
O
HN
=
=
O
NH
OO NH2
Amine coupling of amine-modified biotin
45
9. Future perspectives
This thesis describes different methods of preparation of biosensor surfaces and
contributes to the research on the effect of ligand orientation on the analyte binding
capacity and fabrication of hydrogel-based biochips. The common ingredients of the two
included papers are the use of SAMs on gold substrates as a general platform and the
strategy of modifying the surfaces with organic molecules containing an OEG moiety for
reduction of nonspecific binding of proteins. In both contributions photochemistry has
been used, but for different applications: photoimmobilization of proteins by the novel
method CAP on the one hand and hydrogel fabrication by SIPGP on the other.
The CAP combination of the NTA and BP functionalities could be of use in biochip
fabrication. A clear advantage of photochemistry compared to e.g. amine coupling is the
possibility of different methods of patterning. One approach would be repeating a
sequence of protein loading, directed photoimmobilization via a moveable mask and
elution of non-covalent ligand. A more elegant solution would be using parallel flow
channels and parallel light beams. Another approach would involve conventional spotting
of all ligands on a Ni2+-loaded CAP surface, followed by one photoimmobilization step.
CAP could also be used in combination with parallel-DPN, using thousands of tips for
delivering ligands in an array pattern.
Notably, recombinant proteins are often produced with a His-tag to facilitate protein
purification by IMAC and hence there are large numbers of ligands available, eliminating
the need of further protein modification prior to immobilization. A bivalent ligand was
used in Paper I, but monovalent His-tagged ligands would be of more interest, for example
antibody mimetics, single-chain variable fragments (scFv) or aptamers.
The hydrogel pattern described in Paper II also has potential for biochip fabrication.
The thin hydrogel layer background provides a hospitable environment for cell studies and
a possibility of studying ligand density effects.
46
47
10. References
1. Updike, S.J., Hicks, G.P. The Enzyme Electrode. Nature, 1967, 214, 986-988. 2. Thévenot, D.R., Toth, K., Durst, R. A., Wilson, G. S. Electrochemical Biosensors:
Recommended Definitions and Classification. Biosens. Bioelectron., 2001, 16(1-2), 121-131. 3. Fehrst, A., Structure and Mechanism in Protein Sciences: A Guide to Enzyme Catalysis and
Protein Folding. 1999, New York: W. H. Freeman and Company. 4. Jain, N.K., Roy, I. Effect of Trehalose on Protein Structure. Protein Sci., 2009, 18, 24-36. 5. MacBeath, G.,Schreiber, S.L. Printing Proteins as Microarrays for High-Throughput Function
Determination. Science, 2000, 289(5485), 1760-1763. 6. Tengvall, P., Lundstrom, I.,Liedberg, B. Protein Adsorption Studies on Model Organic Surfaces:
An Ellipsometric and Infrared Spectroscopic Approach. Biomaterials, 1998, 19(4-5), 407-22. 7. Wang, Z., Viana, A.s., Jin, G., Abrantes, L.M. Immunosensor Interface Based on Physical and
Chemical Immunoglobulin G Adsorption onto Mixed Self-Assembled Monolayers. Bioelectrochemistry, 2006, 69, 180-186.
8. Hederos, M., Konradsson, P.,Liedberg, B. Synthesis and Self-Assembly of Galactose-Terminated Alkanethiols and Their Ability to Resist Proteins. Langmuir, 2005, 21(7), 2971-80.
9. Prime, K.L.,Whitesides, G.M. Self-Assembled Organic Monolayers - Model Systems for Studying Adsorption of Proteins at Surfaces. Science, 1991, 252(5009), 1164-1167.
10. Prime, K.L., Whitesides, G.M. Adsorption of Proteins onto Surfaces Containing End-Attached Oligo(Ethylene Oxide): A Model System Using Self-Assembled Monolayers. J. Am. Chem. Soc., 1993, 115, 10714-10721.
11. Pale-Grosdemange, C., Simon, E.S., Prime, K.L., Whitesides, G.M. Formation of Self-Assembled Monolayers by Chemisorption of Derivatives of Oligo( Ethylene Glycol) of Structure Hs(Ch2)11(Och2ch2)Moh on Gold. J. Am. Chem. Soc., 1991, 113, 12-20.
12. Hucknall., A., Rangarajan, S., Chilkoti, A. In Pursuit of Zero: Polymer Brushes That Resist the Adsorption of Proteins. Adv. Mater., 2009, 21, 2441-2446.
13. Harder, P., Grunze, M., Dahint, R., Whitesides, G.M.,Laibinis, P.E. Molecular Conformation in Oligo(Ethylene Glycol)-Terminated Self-Assembled Monolayers on Gold and Silver Surfaces Determines Their Ability to Resist Protein Adsorption. J. Phys. Chem. B, 1998, 102(2), 426-436.
14. Benesch, J., Svedhem, S., Svensson, S.C., Valiokas, R., Liedberg, B.,Tengvall, P. Protein Adsorption to Oligo(Ethylene Glycol) Self-Assembled Monolayers: Experiments with Fibrinogen, Heparinized Plasma, and Serum. J. Biomater. Sci. Polym. Ed., 2001, 12(6), 581-97.
15. Sassolas, A., Blum, L., Leca-Bouvier, B.D. Immobilization Strategies to Develop Enzymatic Biosensors. Biotechnol. Adv., 2012, 30, 489-511.
16. Rusmini, F., Zhong, Z., Feijen, J. Protein Immobilization Strategies for Protein Biochips. Biomacromolecules, 2007, 8, 1775-1789.
17. Brogan, K.L., Wolfe, K., Jones, P.A., Schoenfisch, M.H. Direct Oriented Immobilization of F(ab') Antibody Fragments on Gold. Anal. Chim. Acta, 2003, 496, 73-80.
18. Lu, B., Xie, J., Lu, C., Wu, C., Wei, Y. Oriented Immobilization of Fab’ Fragments on Silica Surfaces. Anal. Chem., 1995, 67, 83-87.
19. Bonroy, K., Frederix, F., Reekmans, G., Dewolf, E., De Palma, R., Borghs, G., Declerck, P., Goddeeris, B. Comparison of Random and Oriented Immobilisation of Antibody Fragments on Mixed Self-Assembled Monolayers. J. Immunol. Methods, 2006, 312(1-2), 167–181.
20. Caruso, F., Rodda, E., Furlong, D.N. Orientational Aspects of Antibody Immobilization and Immunological Activity on Quartz Crystal Microbalance Electrodes. J. Colloid Interface Sci., 1996, 178(1), 104-115.
48
21. Johnsson, B., Löfås, S., Lindquist, G., Edström, Å., Müller Hillgren, R-M., Hansson, A. Comparison of Methods for Immobilization to Carboxymethyl Dextran Sensor Surfaces by Analysis of the Specific Activity of Monoclonal Antibodies. J. Mol. Recognit., 1995, 8, 125-131.
22. Jonkheijm, P., Weinrich, D., Schröder, H., Niemeyer, C.M., Waldmann, H. Chemical Strategies for Generating Protein Biochips. Angew. Chem., Int. Ed., 2008, 47, 9618-9647.
23. Valiokas, R. Nanobiochips. Cell. Mol. Life Sci., 2012, 69, 347-356. 24. Zhu, H., Bilgin, M., Bangham, R., Hall, D., Casamayor, A., Bertone, P., Lan, N., Jansen, R.,
Bidlingmaier, S., Houfek, T., Mitchell, T., Miller, P., Dean, R.A., Gerstein, M., Snyder, M. Global Analysis of Protein Activities Using Proteome Chips. Science, 2001, 293, 2101-2105.
25. Ulman, A. Formation and Structure of Self-Assembled Monolayers. Chem. Rev., 1996, 96(4), 1533-1554.
26. Nuzzo, R.G., Allara, D. L. Adsorption of Bifunctional Organic Disulfides on Gold Surfaces. J. Am. Chem. Soc., 1983, 105(13), 4481-4483.
27. Bain, C.D., Troughton, E.B., Tao, Y.T., Evall, J., Whitesides, G.M.,Nuzzo, R.G. Formation of Monolayer Films by the Spontaneous Assembly of Organic Thiols from Solution onto Gold. J. Am. Chem. Soc., 1989, 111(1), 321-335.
28. Love, J.C., Estroff, L.A., Kriebel, J.K., Nuzzo, R.G.,Whitesides, G.M. Self-Assembled Monolayers of Thiolates on Metals as a Form of Nanotechnology. Chem. Rev., 2005, 105(4), 1103-1169.
29. Laibnis, P.E., Nuzzo, R.G., Ehitesides, G.M. Structure of Monolayers Formed by Coadsorption of Two N-Alkanethiols of Different Chain Length on Gold and Its Relation to Wetting. J. Phys. Chem., 1992, 96, 5097-5105.
30. Stranick, S.J., Parikh, A.N., Tao, Y.T., Allara, D.L., Weiss, P.S. Phase Separation of Mixed-Composition Self-Assembled Monolayers into Nanometer Scale Molecular Domains. J. Phys. Chem., 1994, 98, 7636-7646.
31. Brewer, N.J., Leggett, G.J. Chemical Force Microscopy of Mixed Self-Assembled Monolayers of Alkanethiols on Gold: Evidence for Phase Separation. Langmuir, 2004, 20, 4109-4115.
32. Tamada, K., Hara, M., Sasabe, H., Knoll, W. Surface Phase Behavior of n-Alkanethiol Self-Assembled Monolayers Adsorbed on Au(111): An Atomic Force Microscope Study. Langmuir, 1997, 13(6), 1558-1566.
33. Piner, R.D., Zhu, J., Xu, F., Hong, S., Mirkin, C.A. "Dip-Pen" Nanolithography. Science, 1999, 283, 661-663.
34. Xia, Y.,Whitesides, G.M. Soft Lithography. Angew. Chem., Int. Ed., 1998, 37(5), 551-575. 35. Salaita, K., Wang, Y., Mirkin, C.A. Applications of Dip-Pen Nanolithography. Nature Nanotech.,
2007, 2, 145-155. 36. Bhat, R.R., Tomlinson, M.R., Wu, T., Genzer, J. Surface-Grafted Polymer Gradients: Formation,
Characterization, and Applications. Adv. Polym. Sci., 2006, 198, 51-124. 37. Wang, H., Brown, H.R. Self-Initiated Photopolymerization and Photografting of Acrylic
Monomers. Macromol. Rapid Commun., 2004, 25, 1095-1099. 38. Steenackers, M., Küller, A., Stoycheva, S., Grunze, M., Jordan, R. Structured and Gradient
Polymer Brushes from Biphenylthiol Self-Assembled Monolayers by Self-Initiated Photografting and Photopolymerization (SIPGP). Langmuir, 2009, 25, 2225-2231.
39. Lofas, S., Johnsson, B. A Novel Hydrogel Matrix on Gold Surfaces in Surface-Plasmon Resonance Sensors for Fast and Efficient Covalent Immobilization of Ligands. J. Am. Chem. Soc., Chem. Comm., 1990, (21), 1526-1528.
40. Piehler, J., Brecht, A., Valiokas, R., Liedberg, B., Gauglitz, G. A High-Density Poly(Ethylene Glycol) Polymer Brush for Immobilization on Glass-Type Surfaces. Biosens. Bioelectron., 2000, 15, 473-481.
49
41. Tokumitsu, S., Liebich, A., Herrwerth, S., Eck, W., Himmelhaus, M., Grunze, M. Grafting of Alkanethiol-Terminated Poly(Ethylene Glycol) on Gold. Langmuir, 2002, 18, 8862-8870.
42. Lahiri, J., Isaacs, L., Tien, J.,Whitesides, G.M. A Strategy for the Generation of Surfaces Presenting Ligands for Studies of Binding Based on an Active Ester as a Common Reactive Intermediate: A Surface Plasmon Resonance Study. Anal. Chem., 1999, 71(4), 777-90.
43. Hermanson, G.T., Bioconjugate Techniques. 2nd ed. 2008: Academic Press, Elsevier Inc. 44. Pei, Z., Anderson, H., Myrskog, A., Dunér, G., Ingemarsson, B., Aastrup, T. Optimizing
Immobilization on Two-Dimensional Carboxyl Surface: Ph Dependence of Antibody Orientation and Antigen Binding Capacity. Anal. Biochem., 2010, 398(2), 161-168.
45. Arnold, F.H. Metal-Affinity Separations: A New Dimension in Protein Processing. Nature Biotechnology, 1991, 9(2).
46. Nieba, L., Axmann, S.E., Persson, A., Hamalainen, M., Edebratt, F., Hansson, A., Lidholm, J., Magnusson, K., Karlsson, A.F.,Pluckthun, A. Biacore Analysis of Histidine-Tagged Proteins Using a Chelating NTA Sensor Chip. Anal. Biochem., 1997, 252(2), 217-228.
47. Lata, S.,Piehler, J. Stable and Functional Immobilization of Histidine-Tagged Proteins Via Multivalent Chelator Headgroups on a Molecular Poly(Ethylene Glycol) Brush. Anal Chem, 2005, 77(4), 1096-1105.
48. Valiokas, R., Kienkar, G., Tinazli, A., Reichel, A., Tampé, R., Piehler, J.,Liedberg, B. Self-Assembled Monolayers Containing Terminal Mono-, Bis-, and Tris-Nitrilotriacetic Acid Groups: Characterization and Application. Langmuir, 2008, 24(9), 4959-4967.
49. Knecht, S., Ricklin, D., Eberle, A.N.,Ernst, B. Oligohis-Tags: Mechanisms of Binding to Ni2+-NTA Surfaces. J. Mol. Recognit., 2009, 22(4), 270-9.
50. Steinhauer, C., Wingren, C., Khan, F., He, M., Taussig, M.J.,Borrebaeck, C.A.K. Improved Affinity Coupling for Antibody Microarrays: Engineering of Double-(His)6-Tagged Single Framework Recombinant Antibody Fragments. Proteomics, 2006, 6(15), 4227-4234.
51. Dormán, G., Prestwich, G.D. Benzophenone Photophores in Biochemistry. Biochemistry, 1994, 33(19), 5661-5673.
52. Prestwich, G.D., Dormán, G., Elliott, J.T., Marecak, D.M., Chaudhary, A. Benzophenone Photoprobes for Phosphoinositides, Peptides and Drugs. Photochem. Photobiol., 1997, 65(2), 222-234.
53. Pu, Q.S., Oyesanya, O., Thompson, B., Liu, S.T.,Alvarez, J.C. On-Chip Micropatterning of Plastic (Cylic Olefin Copolymer, COC) Microfluidic Channels for the Fabrication of Biomolecule Microarrays Using Photografting Methods. Langmuir, 2007, 23(3), 1577-1583.
54. Prucker, O., Naumann, C.A., Ruhe, J., Knoll, W.,Frank, C.W. Photochemical Attachment of Polymer Films to Solid Surfaces Via Monolayers of Benzophenone Derivatives. J. Am. Chem. Soc., 1999, 121(38), 8766-8770.
55. Delamarche, E., Sundarababu, G., Biebuyck, H., Michel, B., Gerber, C., Sigrist, H., Wolf, H., Ringsdorf, H., Xanthopoulos, N.,Mathieu, H.J. Immobilization of Antibodies on a Photoactive Self-Assembled Monolayer on Gold. Langmuir, 1996, 12(8), 1997-2006.
56. Rozsnyai, L.F., Benson, D.R., Fodor, S.P.A.,Schultz, P.G. Photolithographic Immobilization of Biopolymers on Solid Supports. Angew. Chem. Int. Ed. Engl., 1992, 31(6), 759-761.
57. Liu, X.H., Wang, H.K., Herron, J.N.,Prestwich, G.D. Photopatterning of Antibodies on Biosensors. Bioconjug. Chem., 2000, 11(6), 755-761.
58. Kuby, J., Immunology. 3rd ed. ed. 1997: W.H. Freeman and Co. 59. Harlow, E., Lane, D., Using Antibodies: A Laboratory Manual. 1999, Cold Spring Harbor, NY:
CSHL Press. 60. Briand, E., Salmain, M., Compère, C., Pradier, C-M. Immobilization of Protein A on Sams for
the Elaboration of Immunosensors. Colloids Surf., B, 2006, 53, 215-224.
50
61. Moks, T., Abrahmsen, L., Nilsson, B., Hellman, U., Sjoquist, J.,Uhlen, M. Staphylococcal Protein A Consists of 5 IgG-Binding Domains. European Journal of Biochemistry, 1986, 156(3), 637-643.
62. Kanmert, D., Brorsson, A.-C., Jonsson, B.-H.,Enander, K. Thermal Induction of an Alternatively Folded State of Human IgG-Fc. Biochemistry, 2011, 50(6), 981-988.
63. Wilchek, M., Bayer, E.A., Livnah, O. Essentials of Biorecognition: The (Strept)Avidin–Biotin System as a Model for Protein–Protein and Protein–Ligand Interaction. Immunol. Lett., 2006, 103, 27-32.
64. Good, R.J., Contact Angle, Wetting and Adhesion: A Critical Review, in Contact Angle, Wettability and Adhesion, K.L. Mittal, Editor. 1993, VSP: Utrecht.
65. Gupta, P., Ulman, A., Fanfan, S., Korniakov, A., Loos, K. Mixed Self-Assembled Monolayers of Alkanethiols on Ultrasmooth Gold Do Not Exhibit Contact-Angle Hysteresis. J. Am. Chem. Soc., 2005, 127, 4-5.
66. Kwok, D.Y.,Neumann, A.W. Contact Angle Measurement and Contact Angle Interpretation. Adv. Colloid Interface Sci., 1999, 81(3), 167-249.
67. Collet, E., Polarized Light: Fundamentals and Applications. 1993, New York: Marcel Dekker, Inc.
68. McCrackin, F.L. A Fortran Program for Analysis of Ellipsometer Measurements. NBS Technical Note 479, 1969.
69. Günzler, H., Gremlich, H-U., Ir Spectroscopy - an Introduction. 2002, Weinheim, Germany: Wiley-VCH.
70. Attard, G., Barnes, C., Surfaces. Oxford Chemistry Primers. Vol. 59. 1998, Oxford: Oxford University Press.
71. Francis, S.A., Ellison, A.H. Infrared Spectra of Monolayers on Metal Mirrors. J. Opt. Soc. Am., 1958, 49(2), 131-138.
72. Greenler, R.G. Infrared Stydy of Adsorbed Molecules on Metal Surfaces by Reflection Techniques. J. Chem. Phys., 1965, 44(1), 310-315.
73. Liedberg, B., Nylander, C.,Lundstrom, I. Surface-Plasmon Resonance for Gas-Detection and Biosensing. Sens. Actuators, 1983, 4(2), 299-304.
74. Schasfoort, R.B.M., Tudos, A.J., Handbook of Surface Plasmon Resonance. 2008: RSC Publishing.
75. Stenberg, E., Persson, B., Roos, H., Urbaniczky, C. Quantitative Determination of Surface Concentration of Protein with Surface Plasmon Resonance Using Radiolabeled Proteins. J. Colloid Interface Sci., 1990, 143(2), 513-526.
76. Rothenhausler, B.,Knoll, W. Surface-Plasmon Microscopy. Nature, 1988, 332(6165), 615-617. 77. Andersson, O., Imaging Surface Plasmon Resonance. 2008, Linköping University, Sweden:
Dissertation No. 1205. 78. Meyer, E., Hug, H.J., Bennewitz, R., Scanning Probe Microscopy - the Lab on a Tip. 2004:
Springer-Verlag. 79. Lakowicz, J.R., Principles of Fluorescence Spectroscopy. 3rd ed. 2006: Springer. 80. Russ Algar, W., Tavares, A. J., Krull, U. J. Beyond Labels: A Review of the Application of
Quantum Dots as Integrated Components of Assays, Bioprobes, and Biosensors Utilizing Optical Transduction. Anal. Chim. Acta, 2010, 673(1), 1-25.
81. Andersson, O., Larsson, A., Ekblad, T., Liedberg, B. Gradient Hydrogel Matrix for Microarray and Biosensor Applications: An Imaging SPR Study. Biomacromolecules, 2009, 10(1), 142-148.