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Orientation and characterization of immobilized antibodies for improved

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Orientation and characterization of immobilized antibodies for improved
immunoassays (Review)
Nicholas G. WelchJudith A. Scoble and Benjamin W. MuirPaul J. Pigram

Citation: Biointerphases 12, 02D301 (2017); doi: 10.1116/1.4978435

View online: http://dx.doi.org/10.1116/1.4978435
View Table of Contents: http://avs.scitation.org/toc/bip/12/2
Published by the American Vacuum Society
Orientation and characterization of immobilized antibodies for improved
immunoassays (Review)
Nicholas G. Welch
Centre for Materials and Surface Science and Department of Chemistry and Physics, School of Molecular
Sciences, La Trobe University, Melbourne, VIC 3086, Australia and CSIRO Manufacturing, Clayton,
VIC 3168, Australia
Judith A. Scoble and Benjamin W. Muir
CSIRO Manufacturing, Clayton, VIC 3168, Australia
Paul J. Pigrama)
Centre for Materials and Surface Science and Department of Chemistry and Physics,
School of Molecular Sciences, La Trobe University, Melbourne, VIC 3086, Australia
(Received 19 January 2017; accepted 28 February 2017; published 16 March 2017)
Orientation of surface immobilized capture proteins, such as antibodies, plays a critical role in the
performance of immunoassays. The sensitivity of immunodiagnostic procedures is dependent on
presentation of the antibody, with optimum performance requiring the antigen binding sites be
directed toward the solution phase. This review describes the most recent methods for oriented
antibody immobilization and the characterization techniques employed for investigation of the
antibody state. The introduction describes the importance of oriented antibodies for maximizing
biosensor capabilities. Methods for improving antibody binding are discussed, including surface
modification and design (with sections on surface treatments, three-dimensional substrates, self-
assembled monolayers, and molecular imprinting), covalent attachment (including targeting amine,
carboxyl, thiol and carbohydrates, as well as “click” chemistries), and (bio)affinity techniques
(with sections on material binding peptides, biotin-streptavidin interaction, DNA directed immobi-
lization, Protein A and G, Fc binding peptides, aptamers, and metal affinity). Characterization tech-
niques for investigating antibody orientation are discussed, including x-ray photoelectron
spectroscopy, spectroscopic ellipsometry, dual polarization interferometry, neutron reflectometry,
atomic force microscopy, and time-of-flight secondary-ion mass spectrometry. Future
perspectives and recommendations are offered in conclusion. V C 2017 Author(s). All article content,

except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license
(http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1116/1.4978435]

I. INTRODUCTION facing, sometimes referred to as “end-on”; however, randomly

Immunodiagnostics, protein biochips, and biosensors immobilized antibodies may assume various surface orienta-
employed for antigen detection and quantification from bio- tions, including those loosely referred to as “head-on,” “side-
logical samples often employ recognition proteins such as on,” and “lying-on” (see Fig. 1).7
antibodies.1–4 Assay sensitivity is dependent on immobiliza- Generally, hydrophobic amino acids are internalized in a
tion of the capture antibodies onto a solid support with a suf- correctly folded protein structure, leaving hydrophilic resi-
ficient surface density, a conformation that is representative dues at the antibody surface, with chemically reactive func-
of their native, solution-phase state, and an orientation that tionalities, including amine, carboxyl, and hydroxyl groups.
maximizes their antigen capture potential. Disulfides, such as those that contribute to the hinge region,
Antibodies are biopolymers of approximately 150 kDa can be reduced to make thiol species that can also be conju-
molecular mass and with dimensions of approximately gated. Further, sites for targeted immobilization (including
14  10  4 nm (Refs. 3 and 5) comprised of amino acids natural or non-natural amino acids) can be introduced
whose sequence and composition, like other proteins, define recombinantly to antibodies.8–10
the three-dimensional structure.6 An antibody is comprised of Physical adsorption of antibodies onto traditional immu-
two fragment antigen binding (Fab) regions and a fragment noassay solid supports, such as polystyrene, occurs via
crystallizable region (Fc). The Fab regions, joined by the hinge hydrophobic and electrostatic interactions.11 While this
region, is known as F(ab0 )2 fragment. The Fab regions are dis- method offers the simplest attachment pathway, it is uncon-
similar in their composition, isoelectric point, and physical trollable, and antibodies can be immobilized in a randomly
structure to the Fc region of the antibody (Fc), allowing deter- oriented manner, denatured, or displaced in later steps by
mination of antibody orientation on the surface. Ideally, the washing.12–14 The substrate design has increased the capabil-
immobilized antibody is oriented such that the Fc is substrate ities of immunoassays by improving antibody binding capac-
ity and reducing denaturation at the surface.15 Covalent
attachment of the antibody, via its functional groups, to
a)
Electronic mail: p.pigram@latrobe.edu.au chemically engineered substrates has resulted in further

02D301-1 Biointerphases 12(2), June 2017 1934-8630/2017/12(2)/02D301/13 C Author(s) 2017.

V 02D301-1
02D301-2 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-2

II. IMPROVING ANTIBODY BINDING AT THE

INTERFACE
A. Surface modification and design strategies
Physical adsorption is the simplest method for the immo-
bilization of antibodies to immunoassay solid supports,
such as microtiter plates. However, this method does not
allow control of the antibody orientation and is typically
associated with poor binding and denaturation.18 Microtiter
plate manufacturers utilize polymers such as polystyrene,
polypropylene, polyethylene, and cyclic olefin copolymer
blends, employing surface modification methods to increase
the hydrophilicity of substrates. The increase in hydrophilic-
ity can increase antibody binding (density) and decrease the
amount of denatured protein. Polymer substrates are the tra-
ditional immobilization platform for immunoassay as they
provide a cheap, stable, reproducible substrate that is easy to
manufacture with precision.

1. Plasma treatment and plasma polymers

Plasma treatment is a surface modification method that
uses radio-frequency glow-discharge to generate a plasma of
a gas or monomer vapor. Microtiter plates have been treated
FIG. 1. Antibody orientation, dimensions, and important chemical species with oxygen, nitrogen, and other gas plasmas to create chem-
for targeting. ical functionalities at the surface, thereby reducing the
hydrophobicity of the plastic.19 Similarly, nonreactive gases
improvements in antibody density, though often these meth- such as argon can be used to “activate” the surface by intro-
ods are not site-directed and unfavorable random orientation ducing radicals, which react with atmospheric species upon
can occur.16 In an ideal scenario, antibodies should be exposure to air.20 Plasma treatment offers a method to func-
immobilized in their native form, without the need for intro- tionalize existing substrates’ increasing hydrophilicity21
duced functional groups, in a homogeneous arrangement and reducing denaturation of bound proteins or to provide a
such that their antigen binding sites are free from steric hin- starting point for further chemical treatment and covalent
drance and are oriented so as to maximize complementary grafting of proteins.22 In a recent example, P^aslaru et al.23
binding. A method that truly provides the ideal scenario has plasma treated poly(vinylidene fluoride) (PVDF) with CO2,
yet to be realized; however, recent developments, as dis- N2 and N2/H2 (25/75) gases to attach carboxyl or amine
cussed in this review, offer improved control over antibody functionality for subsequent covalent immobilization of
immobilization and orientation at the interface. proteins. N-ethyl-N-(3-dimethylaminopropyl) carbodiimide
In parallel with the development and evolution of immo- (EDC)/N-hydroxysuccinimide (NHS) chemistry was used to
bilization methods, a significant need has emerged for sur- attach IgG or Protein A (with subsequent IgG binding) to the
face characterization techniques that can accurately identify PVDF treated surfaces. Possible preferential end-on orienta-
antibody orientation at a substrate. Current techniques that tion of IgG was achieved via the PVDF surfaces treated with
rely on indirect analysis, or inference from complex models, N2/H2 and grafted with the Protein A.
make definitive conclusions regarding orientation difficult.17 Plasmas can also be used to produce polymer thin films
Advances in data processing and multivariate analysis have that retain some of the chemistry associated with the mono-
provided an improved level of understanding of complex mer. The radicalized monomer fragments bind to the substrate
surfaces, and direct surface analysis techniques, such as surface, and to one another, creating a ubiquitous surface
time-of-flight secondary ion mass spectrometry (ToF-SIMS), coating referred to as a plasma polymer. This methodology of
provide molecular information that has the potential to deter- creating polymers allows adherent and continuous coatings to
mine antibody orientation with confidence. be formed on a broad range of substrates, including microtiter
This review is structured into two parts. First, a review of plates.24 Plasma polymers have been produced from a diverse
current antibody immobilization strategies, including surface range of monomers, including allylamine,25,26 cyclopropyl-
modification, antibody targeting, and coupling, will be pre- amine,27 bromine,28 polyethylene glycol (PEG), diethylene
sented. Second, advances in characterization techniques for glycol dimethyl ether (diglyme),29–31 and many others,32,33
investigating these systems will be explored, including indi- providing a broad spectrum of chemical functionalities for
rect and direct analyses. The advantages and shortfalls of subsequent protein grafting steps, including the option for pat-
strategies and techniques will be addressed, and the review terning.34–36 Overall, a significant range of polymers have
will conclude with future perspectives and recommendations. been used to improve biomolecule immobilization properties

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02D301-3 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-3

of substrates for use in microarray and protein assay applica- the CH3 surface. However, this antibody was found to have
tions.33,37 Hasan and Pandey38 provide an excellent review of no antigen recognition indicating denaturation or poor ori-
polymers and plasma techniques for producing materials for entation, with the latter supported by ToF-SIMS findings.
protein immobilization. Chen et al.44 demonstrated preferred orientation of mouse
IgG1 (and to a lesser extent IgG2a) by exploiting the anti-
2. Three-dimensional substrates body dipole and the use of charged SAM surfaces. IgG1
Three-dimensional substrates are attractive as they offer adsorbed to a NH2 terminated SAM produced from 11-
increased surface area for antibody binding and can mini- amino-1-undecanethiol had a higher antigen/antibody ratio
mize steric hindrance that may prevent antigen capture. than a COOH terminated SAM produced from 16-
Porous three-dimensional substrates have been prepared mercaptohexadecanoic acid. Vashist et al.46 provide a good
from a variety of materials, including various polymers, sili- review of antibody immobilization using silane SAMs on
con, glass slides, metals, and gels. Wang and Feng15 provide various substrates for improved surface densities.
an excellent review on three-dimensional substrates with a
focus on the orientation of proteins. 4. Molecularly imprinted polymers
Similarly, gels and sol–gels manufactured from agarose Molecular imprinting is a polymerization technique that
or dextran have found improved antibody binding due to uses a molecular template to produce target-specific binding
their high surface area.1,39 In a recent study, Orlov et al.40 regions.47 Once formed, the target-specific binding sites in
employed three-dimensional immunochromatographic nitro- the polymer substrate may selectively immobilize the target,
cellulose membranes impregnated with magnetic nanopar- such as an antibody, from a complex matrix. Bereli et al.48
ticles for use in a strip sensor immunoassay, providing a imprinted a poly(hydroxyethyl methacrylate) cryogel with
solid phase with a large surface area for antibody immobili- the Fc portion of anti-human-IgG to create an antibody ori-
zation. The sensor had a limit of detection of approximately enting substrate. The Fc portion was then flushed from the
740 fM and had a strong correlation with a standard enzyme- cryogel, which was subsequently activated with carbodii-
linked immunosorbent assay (ELISA) for the detection of mide for whole antibody binding. Anti-human-IgG was
prostate specific antigen from serum, though with improved then used as a capture antibody to bind IgG from human
dynamic range (3.5 orders). One example from Feng et al.41 plasma and the imprinted cryogel was demonstrated to be at
utilized repeat units of Protein A genetically fused to the least three times better than using a nonimprinted cryogel.
nickel chelating His-tag. Adjacently immobilized (via a This was despite similar amounts of the capture anti-human-
nickel matrix substrate) the fused proteins order as columns IgG being immobilized, via the nonspecific carbodiimide
protruding from the substrate and could immobilize five anti- method, indicating preferential antibody orientation. In
bodies via their Fc regions. This three-dimensional protein addition to providing orientating substrates, molecularly
construct had a 64-fold increase in antigen detection sensi- imprinted polymers utilize gel structures with aqueous envi-
tivity relative to standard IgG immobilization. ronments that are thought to reduce the probability of protein
denaturation.49
3. Self-assembled monolayers
B. Covalent binding targets
The formation of a self-assembled monolayer (SAM) pro-
vides another means of modifying surface chemistry to pro- While substrate design is important, the ability to reliably
mote antibody adsorption or to produce functional groups attach the antibody to a solid support underpins the success
for subsequent covalent attachment.42 SAMs are typically of an immunoassay. The covalent coupling of capture anti-
formed from molecules that contain active functional head- bodies ensures robust immobilization and can improve den-
groups at either end of a hydrocarbon chain and a linear car- sity and orientation outcomes at the substrate. However, for
bon chain which promote self-assembly when they attach to oriented immobilization, site-directed attachment to the anti-
a surface. The anchoring head-group has an affinity for the body is required. The covalent attachment can proceed via
surface, while the other provides a solution-facing chemistry various chemistries dependent on the substrate functionality,
for protein adsorption or attachment. The central hydrocar- target group on the antibody, and the physical restraints of
bon chains provide stabilization of the SAM by interchain the system, i.e., pH, temperature, and degree of conjuga-
hydrophobic interactions.38 tion.33 This section will discuss functional groups on anti-
The gold–thiol interaction has been exploited most bodies for covalent attachment, including common targets
commonly by utilizing alkanethiols to provide a linker such as amine, carboxyl, thiol, and carbohydrate moieties
that can bind gold substrates via the thiol group, and offer (see Fig. 2 for overview). The covalent attachment methods
customizable chemistry for adsorption or coupling anti- of proteins are covered in good detail in reviews by Rao
bodies to SAMs.43,44 Lebec et al.45 used alkanethiol SAMs et al.50 and Liu and Yu.16
formed from 11-mercaptoundecanoic acid (11-MUA) and
1-undecanethiol on gold substrates to produce COOH and 1. Amine and carboxyl groups
CH3 surface chemistries, respectively. Antibodies were Amine and carboxyl groups are ubiquitous throughout an
adsorbed to both substrate types with an increase shown for antibody’s structure and are common at the antibody’s

Biointerphases, Vol. 12, No. 2, June 2017

02D301-4 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-4

FIG. 2. Oriented antibody immobilization strategies. (a) EDC/NHS coupling of antibody amine and carboxyl groups to surface carboxyl and amine groups. (b)
Reduction of antibody disulfides, with TCEP or 2-MEA, to reactive thiols for binding gold substrates. (c) Periodate oxidation of carbohydrates in the Fc region
of antibodies followed by coupling to hydrazide surface chemistry.

surface due to their polar nature. Amino acids such as lysine steric hindrance may limit antigen binding. Exploitation of
with reactive primary amine side chains, and aspartate and the gold–thiol bond makes this immobilization strategy useful
glutamate with carboxyl side chains, can be targeted for for gold substrates and nanoparticles in techniques such as
covalent attachment. Due to their prevalence throughout the surface plasmon resonance (SPR).55 UV-excitation has also
antibody surface, site-directed covalent antibody immobili- been used to initiate photoreduction of disulfide bridges in
zation targeting these groups is difficult. Amine and carboxyl hinge regions of antibodies according to an approach known
coupling (between the substrate and protein) is commonly as the photonic immobilization technique.56 Employing UV
attained with carbodiimide chemistry that utilizes EDC com- pulses at 258 nm and 10 kHz, free thiols can be produced that
bined with succinimidyl esters (such as NHS).33 This method are then able to bind gold substrates for quartz crystal micro-
is known as EDC/NHS coupling and results in robust amide balance (QCM) measurements.57 Antibody fragments, such as
bond formation. EDC/NHS chemistry has been employed as Fab0 , can also be produced with reactive thiols and used in
a covalent attachment methodology for immobilization of immunosensors;5,42 however, this review aims to focus pri-
proteins and also as a method for the preparation of sub- marily on whole antibody immobilization and will not cover
strates. Carrigan et al.51 used EDC/NHS in two ways, (1) for antibody fragments specifically.
the cross-linking of polyethylenimine and carboxymethyl Alternatively, primary amine groups can be converted
cellulose, and (2) for activation of this substrate for protein to thiol functionality using, for example, 2-iminothiolane
binding targeting amine and carboxyl functionality. Sun (Traut’s reagent) for subsequent immobilization using the
et al.52 took a novel approach to EDC/NHS coupling by same maleimide or iodoacetyl chemistry.58 For example, lipid
introducing NHS reactive groups to random sites on an anti- PEG, functionalized with maleimide, was incorporated into
0
body then using an electric field to preferentially orient the lipid nanocapsules for coupling thiolated antibodies or Fab .59
antibody, via its intrinsic dipole, before reaction to the free
amines on cysteine immobilized to a gold electrode. 3. “Click” chemistry
More recently, “click chemistry” exploiting 1,3-dipolar
2. Thiol groups cycloaddition between an azide and an alkyne has demon-
Disulfide-bridged cysteines, such as those present in the strated utility for the conjugation of single-domain camelid
hinge region of antibodies, can also be targeted by reducing antibodies, known as VHH, to a dextran substrate.9 This
agents such as tris(2-carboxyethyl)phosphine (TCEP) or 2- technique produced an oriented system via the site-specific
mercaptoethylamine (2-MEA) to form reactive thiols,8,53 insertion of azidohomoalanine into the VHHs.
which may subsequently react with maleimide or iodoacetyl
activated surfaces. However, as the cysteines are internal to 4. Carbohydrate groups
the antibody tertiary structure, covalent attachment via this Antibodies are glycosylated at the Fc region and can pro-
method can disrupt the conformation of the antibody54 while vide a target for site-directed immobilization.60,61 Periodate

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02D301-5 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-5

oxidation can be used to oxidize diols in carbohydrates into (c02,78 PM-OMP25,79 PMMA-tag80), polycarbonate [PC-
aldehyde groups that can react with amines and hydra- OMP6 (Ref. 79)], poly-L-lactide [c22 (Ref. 81)], gold [GBP
zides.62 Diols can also be targeted by boronic acids to form (Ref. 82)], and the well known nickel and copper specific
boronate ester intermediates reversibly.63 Song et al.64 dem- His-tag.83,84
onstrated end-on immobilization of antibodies using 3-
aminophenylboronic acid to first couple a NHS-derivatized 2. Biotin–streptavidin interaction
surface and second bind the Fc carbohydrate. Due to the Oriented immobilization can be realized by exploiting the
reversible nature of boronate esters, Adak et al.61 employed biotin–avidin/streptavidin interaction.85 Antibodies can be
a single molecule with two functional groups to first form a easily conjugated to biotin using biotin-NHS chemistry that
boronate ester with the carbohydrate, and second to cova- targets amines; however, this results in randomly biotiny-
lently attach the antibody via photoinitiated cross-linking. lated antibodies. Paek’s group86 compared randomly biotiny-
UV exposure causes the (trifluoromethyl)phenyldiazirine lated IgG using biotin-NHS, with IgG biotinylated at the
functional group to form reactive carbenes that irreversibly hinge disulfides via competitive maleimide chemistry for
bind the antibody. Alternatively, Huang et al.65 oxidized immobilization to streptavidin treated microwells and glass
the carbohydrate of anti-alpha-fetoprotein to an aldehyde slides. The authors found a two-fold improvement in antigen
and covalently linked the protein to a 3-aminopropyltri detection for the hinge disulfide biotinylated IgG, relative to
ethoxysilane (APTES) modified silicon substrate. The the random system. The group also demonstrated a twofold
amount of antibody immobilized increased by 32% and the improvement for the same system by employing a gold sub-
antigen binding amount by 16% relative to physical adsorp- strate with a thiol SAM and biotin–streptavidin linker.87
tion. Yuan et al.66 used periodate oxidation of carbohydrates
on the anti-CD34 antibody to promote oriented immobiliza- 3. DNA directed immobilization
tion. First, stainless steel substrates were coated with ethyl-
DNA-directed immobilization (DDI) of proteins is
ene vinyl acetate, then treated with O2 plasma, and silanated
another affinity method that can produce oriented systems.
with APTES to create amine groups (labeled SCA-SS).
This method requires the binding of short nucleotide sequen-
Amines were then coupled with the oxidized carbohydrates
ces to the substrate and to the protein of interest, allowing
and successful binding was assessed via cell uptake by the
direct binding between the two. However, to truly achieve
anti-CD34 antibody. Prieto-Simon et al.67 used thiolated
an oriented system, care must be taken to ensure that the
hydrazide SAM linkers or electrografting of diazonium salts
covalent attachment of the DNA to the antibody occurs in a
to immobilize periodate-oxidized carbohydrates of anti-
site-direct manner. Wacker et al.88 investigated antibody
bodies via hydrazide chemistry onto functionalized gold
immobilization and fluorescent immunoassay performance
substrates.
of antibodies bound via DDI, physical adsorption, and by
streptavidin–biotin interactions. IgG immobilized with DDI
C. Affinity immobilization techniques
was found to require 100-fold less antibody for the same
While covalent methods provide robust antibody immobi- fluorescence detection of the analyte; however, orientation
lization and can achieve site-directed immobilization,68 of the antibody was not independently determined, and site-
they may be unsuited due to the high prevalence of a particu- directed biotinylation of the IgG was not stated. More
lar functional group in the antibody (amine and carboxyl), recently, Seymour et al.89 compared anti-ebola virus glyco-
or may cause conformation changes upon attachment protein (EBOV GP) antibody immobilized directly to NHS
(thiol), without the use protein engineering. Affinity immobi- containing copolymer or via DDI. The DDI immobilized
lization techniques provide alternative and potentially antibody was found to be an order of magnitude more sensi-
favorable strategies to promote site-directed antibody tive to the EBOV GP antigen. Glavan et al.90 synthesized
immobilization.15 single-stranded DNA onto paper substrates and investigated
antihuman C-reactive protein (hCRP) immobilized via DDI
1. Material binding peptides for binding hCRP from serum in a sandwich ELISA.
Peptide sequences that will preferentially immobilize to However, DNA conjugation to the antibody utilized non-
substrates, metal ions, and other biomolecules have been site-directed NHS chemistry.
developed for enhanced protein orientation.69 These peptides Boozer et al.91 prepared mixed SAMs containing ssDNA
can be incorporated as tags, into proteins of interest, via thiols and oligo(ethylene glycol) thiols on gold SPR chips.
chemical conjugation or genetic fusion. Phage screening The complementary DNA strand was cross-linked using
techniques have been developed to produce peptides with NHS chemistry to the antibodies and immobilized by the
specificity to a broad range of materials and proteins.70 A SAM. This system generated a 50-fold improvement on
large number of material binding peptides exist, including their previous work92 using biotinylated immobilization.
those specific to polystyrene [PS-tag,71 Lig1 (Refs. 72 and However, by binding DNA to the antibody nonspecifically
73)] and hydrophilic polystyrene (Phi-PS) [PS19-1 and with NHS chemistry, it is more likely that the improvement
PS19-6 (Ref. 74)], silicon (Si-tag75,76), glass slides or silica arises due to the antibody being separated from the surface,
resin [R9 (Ref. 77)], poly(methylmethacrylate) (PMMA) than through orientation.

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02D301-6 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-6

4. Protein A and Protein G nanocarriers with solution-phase orientation. The inclusion

Protein A and Protein G are small proteins, derived from of the peptide gave a 1.4-fold improvement in signal during
bacteria, which can specifically bind the Fc portion of anti- the immunoassay. Lee et al.113 developed photoactivatable
bodies allowing oriented systems to be obtained.93–96 The Fc-specific antibody binding proteins (FcBPs) expressed in
IgG binding domain of Protein A, known as the Z-domain or Escherichia coli that undergo photo-crosslinking (via photo-
ZZ-domain, is also used as a smaller synthetic option for Fc methionine) with antibodies upon UV irradiation. FcBPs
binding.97 This technique offers a method for truly obtaining were immobilized on maleimide-coated slides and the epi-
oriented antibodies as binding can only occur via the Fc por- dermal growth factor receptor (EGFR)-hmAb antibody
tion. Due to its effectiveness, Protein A, or its derivatives, cross-linked with UV exposure. Dose-dependent antigen
has been exploited with many surface immobilization strate- (EGFR) binding was observed at above 110 fmol.
gies including biotin-streptavidin,98 SAMs,99,100 EDC/NHS Aptamers are single chain DNA, or RNA, oligonucleoti-
chemistry,100,101 glutaraldehyde,102 tyrosinase chemistry,102 des that fold to form complex three dimensional structures
non-natural amino acid insertion,10 gold binding peptide82 or and can specifically immobilize proteins of interest.114
polystyrene affinity ligand fusion,73 and additional protein Miyakawa et al.115 developed an RNA aptamer that selec-
linkers.103 The ZZ-domain has also been coupled to carbohy- tively binds the Fc portion of human IgG1 through IgG4, but
drate binding modules for selective immobilization to cellu- not other nonhuman IgGs. SPR was used to assess the bind-
lose coated slides104 and to paper105 substrates, and also the ing site of the aptamer on IgG, and it was found that the site
metal affinity His-tag.106,107 It is important to note that sev- was similarly positioned to that of the Protein A binding site,
eral issues may arise with such systems: (1) the Protein A making it suitable for promoting antibody orientation. Ma
capture of the Fc is reversible, (2) Protein A has been et al.116 produced a DNA aptamer capable of binding the Fc
reported to bind Fab regions and albumin (although to a domain of multiple mouse subclasses. This area has potential
much lesser extent), and (3) it is required that the Fc binding to develop aptamers capable of universal Fc binding sub-
site of the Protein A is correctly oriented at the substrate to strates, hence promoting antibody orientation and immuno-
permit antibody binding.15,16 More recently, Yang et al.108 diagnostic sensitivity.
engineered a photoactivatable Z-domain variant that incor-
porated the UV-active amino acid benzoylphenylalanine and 6. Nucleotide binding site
a biotin molecule. The Z-domain preferentially bound the Fc Antibodies, even across different isotypes, contain largely
portion of IgG that was irreversibly attached using UV- conserved sequences between the heavy and light chains of
activation of the benzoylphenylalanine. When immobilized the Fab region known as a nucleotide binding site (NBS).
to a streptavidin coated substrate, this system had a fivefold Targeting the NBS using a small molecule, indole-3-butyric
lower antigen detection limit than randomly NHS- acid, and UV irradiation, Alves et al.117 were able to immo-
biotinylated IgG. bilize antibodies selectively, offering a 7.9-fold increase in
antigen sensitivity, compared with physical adsorption. The
5. Fc-binding peptides and aptamers group also demonstrated the utility of this technique with
Short peptides with specificity to the Fc domain of anti- Fab fragments.118,119
bodies have been used to promote oriented immobilization.
Jung et al.109 used an Fc-binding peptide to immobilize 7. Metal affinity
human, rabbit, goat, and mouse antibodies, with strong Perhaps the simplest option is to take advantage of the
selectivity for human IgG1 and IgG2. Anti-C-reactive pro- endogenous metal binding properties of antibodies. In addi-
tein (anti-CRP) antibody immobilized with the Fc-binding tion to recombinant peptide tags for metal coordination,
peptide was compared with randomly immobilized (EDC/ native tag-free IgG have been purified using metal affinity.
NHS coupling) antibody immobilized to SPR chip substrates The interaction of histidine and cysteine with metals, particu-
and found that a 1.6-fold increase in the CRP/anti-CRP ratio larly copper and zinc, has been exploited for protein fraction-
when employing the Fc-binding peptide. More recently, Tsai ation and IgG purification using immobilized metal-affinity
et al.110 demonstrated that molecular dynamics can be used chromatography.120,121 Hale122 then went on to demonstrate
to design a short peptide (RRGW) with high specificity to that Co(II) loaded resin could be used to irreversibly bind
the mouse IgG2a antihuman prostate specific antigen (PSA) IgG in an oriented manner via the Fc region. Todorova-
antibody. PSA binding was monitored via SPR demonstrat- Balvay et al.123 used computational modeling and immobi-
ing good antibody orientation. lized metal-ion affinity chromatography to investigate the
Yoo and Choi111 used a phage biopanning to screen for transition metals copper (II), nickel (II), zinc (II) and cobalt
peptides specific to the Fc portion of rabbit anti-goat IgG. (II), to determine a native metal-binding target in the Fc por-
The peptide (KHRFNKD) immobilized with biotin to an tion of whole human IgG1. The histidine cluster His
avidin-QCM surface showed improved IgG binding relative 433–X–His 435 was found to be surface accessible to affinity
to physical adsorption. Dostalova et al.112 used the Fc bind- binding using these metals without the need for recombinant
ing peptide HWRGWVC to immobilize antiprostate specific tags. Muir et al.124 prepared metal coordinating polymer sub-
membrane antigen antibodies to gold coated doxorubicin strates and screened a large range of transition metals to

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02D301-7 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-7

assess their antibody binding capabilities. This work covered (DGpp) as a substrate for binding [Cr(OH)6]3 with subse-
a library of 1600 different metal immobilized surface chemis- quent antibody immobilization. When equivalent amounts of
tries and identified chromium perchlorate with ethylenedi- antibody were immobilized on the DGpp and the chromium
amine as the “lead” combination. Immobilization of functionalized DGpp substrates, a tenfold improvement in
antitumor necrosis factor alpha (anti-TNFa) to Luminex ELISA signal intensity was observed for the chromium func-
beads with chromium perchlorate and ethylenediamine was tionalized system indicating an oriented system. ToF-SIMS
compared with carbodiimide coupling chemistry. The analysis identified that chromium may be binding the anti-
chromium-mediated immobilization has an approximately body through lysine, methionine, arginine, and histidine
ninefold improvement in TNFa antigen sensitivity relative to residues.
the carbodiimide method. Pingarron’s group used a metallic-
complex chelating polymer (Mix&GoTM) to achieve oriented III. IMMOBILIZED ANTIBODY CHARACTERIZATION
immobilization of native anti-adiponectin antibody OVERVIEW
on carboxyphenyl multiwalled carbon nanotubes125 and An immunoassay is the most widely employed method to
graphene oxide-carboxymethyl cellulose hybrid.126 Recently, assess the state of an immobilized antibody, in that it repre-
Welch et al.127 employed the chromium complex, sents the practical application of successful immobilization.
[Cr(OH)6]3, buffered with ethylenediamine as a wet chemi- ELISAs require that immobilized antibodies maintain the
cal modification to ten commercial microtiter plates, postpro- correct orientation, unperturbed conformation, and adequate
duction, to improve antibody immobilization and ELISA density at the substrate surface to maximize signal produc-
performance. For an anti-EGFR, x-ray photoelectron spec- tion and thus antigen quantification. However, as this is an
troscopy (XPS) analysis indicated that the chromium modi- indirect analysis technique, it only allows the antibody state
fied microtiter plate bound twice the amount of antibody and orientation to be determined by inference. As a result,
relative to the unmodified plate, and the ELISA signal more complementary and independent methods for assessing the
than tripled indicating improved antibody orientation. The antibody have been developed. While there are a number of
chromium modification was demonstrated for use with five techniques for quantifying the density of adsorbed pro-
other antigen capture ELISAs. Welch et al.128 also demon- teins,129 the number of techniques that can probe antibody
strated optimization of the chromium complex by varying the orientation is much smaller. Table I presents an overview of
metal salt and buffering base compounds and ratios. The opti- current characterization techniques, and schematic represen-
mized complex (1:1 chromium perchlorate hexahydrate to tations are shown in Fig. 3.
ethylenediamine) was used to improve the antigen detection
limits of a bovine tumor necrosis factor alpha (TNFa) ELISA A. X-Ray photoelectron spectroscopy
by an order of magnitude relative to untreated plates. In a XPS is a spectroscopic technique that uses incident x-ray
recent study, Welch et al.29 employed a traditionally low photons to probe the elemental and chemical composition of
fouling diethylene glycol dimethyl ether plasma polymer the top 10 nm of the sample surface. This technique can be

TABLE I. Overview of surface analysis techniques employed for investigating antibody.

Technique Input Output Information Comments References

A) XPS Monochromatic Photoelectrons Elemental and chemical Quantification of anti- 23, 29, 127, 128,
x-rays body surface density and 131
B) SE Elliptically polar- Change in light Thickness, refractive Model based analysis, 132–137
ized light phase or intensity index, surface roughness inferred state of antibody
C) Dual polarization Laser light Evanescent wave Mass, film thickness, Inferred state of antibody 64, 138, and 139
interferometry change refractive index, density based on mass and film
(DPI) thickness
D) SPR Monochromatic Change in Refractive index, film Inferred state based on 55 and 140–142
multiangle laser reflected and thickness antibody and antigen
light absorbed light adsorption characteristics
E) NR Neutron beam Change in reflec- Refractive index, film Model based analysis, 143–146
tion of neutron thickness, surface inferred state of antibody
beam roughness
F) AFM Feedback driven z-height in 2D and Surface roughness, phase Correctly oriented anti- 56, 142, and
cantilevered tip tip/surface force information, imaging bodies have 14 nm height 147–149
G) QCM Resonance fre- Change in fre- Mass of adsorption, Inferred state of antibody 150 and 151
quency of quency and bioaffinity based on adsorption and
microbalance amplitude mass
H) ToF-SIMS Ionized metal Ionized sample Semiquantitative elemen- Amino acid composition 45, 130, and
clusters, “primary- fragments, tal, chemical, and of F(ab0 )2 and Fc varies 152–157
ions” “secondary-ions” molecular and can be distinguished

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02D301-8 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-8

FIG. 3. Illustrative schematic of antibody orientation characterization techniques: (a) X-ray photoelectron spectroscopy, (b) spectroscopic eMipsometry, (c)
dual polarization interferometry, (d) surface plasmon resonance, (e) neutron reflectometry, (f) atomic force microscopy, (g) quartz crystal microbalance, and
(h) time-of-flight secondary-ion mass spectrometry.

used to quantify the amount of nitrogen present, which in MEA, immobilized to a gold substrate, bind 2.5 times the
turn can be correlated to the amount of immobilized anti- amount of antigen as compared with their intact whole anti-
body.29,127,128,130 Antibody orientation can then be inferred body counterparts immobilized randomly and covalently to
based on the immunoassay signal. Radadia et al.131 com- SAMs. Bae et al.135 compared IgG immobilized to thio-
pared XPS and ELISA results to infer stability of antibody lated Protein G (to represent an oriented antibody) with
immobilized to glass and diamond films. P^aslaru et al.23 pre- chemically bound IgG to an 11-MUA SAM, to represent
pared plasma treated PVDF membranes for adsorption or randomly oriented binding. SE was used to estimate the
grafting of Protein A (followed by IgG) or IgG alone and IgG film thickness and together with atomic force micros-
used XPS to investigate sulfur and nitrogen content in the copy (AFM) and SPR inference could be made regarding
samples. The nitrogen concentration was used to quantify different orientations of the antibodies. Wang and Jin136
overall protein content, and sulfur concentration (present due first utilized Protein A adsorbed to silicon to immobilize
to disulfide bonds in the IgG) was used to monitor IgG anti-IgG and compared with anti-IgG adsorbed to the sili-
binding. con. In a kinetic manner, SE was used to investigate the
film thickness and found an increase in the anti-IgG bound
B. Spectroscopic ellipsometry by Protein A, inferring a preferentially oriented system.
Spectroscopic ellipsometry (SE) is a surface sensitive Wang then went on to investigate three different silane
optical technique that monitors the polarization change in modifications to silicon for IgG binding and identified with
light reflected from the sample surface (typically a metal or SE that APTES/methyltriethoxysilane functionalized sili-
ceramic).132,133 Polarization changes occur due to varia- con covalently bound IgG using glutaraldehyde gave the
tions in the dielectric or refractive index properties of the largest increase in antibody and antigen binding as com-
sample. Balevicius et al.134 used total internal reflection pared to either APTES and glutaraldehyde, or APTES
ellipsometry to demonstrate that antibodies reduced with 2- alone.137

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02D301-9 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-9

C. Dual polarization interferometry observed by the film thickness.143 Then using anti-PSA
DPI is an optical waveguide technique that utilizes immobilized to silicon-oxide substrate, the combination of
changes in the evanescent wave of the sample beam of laser NR and DPI was used to demonstrate flat-on antibody orien-
light relative to a reference beam. The combined beams cre- tation.145 NR and SE were also used complementarily to
ate an interference pattern that provides information regard- assess the mass of antibody immobilized at different concen-
ing mass, film thickness, refractive index, and density.138 trations.144 Schneck et al.146 used NR to confirm the orienta-
Song et al.139 used DPI to investigate anti-PSA antibody tion of anti-(polyethylene glycol) (anti-PEG) antibodies
(anti-PSA) immobilized covalently via lysines to thiolated immobilized to PEG polymer brushes of varying their graft-
DPI chips, or captured via the Fc using immobilized Protein ing density. They noted that increased grafting density
G. The thickness of the two systems was monitored as the caused the distance between the two Fab regions to decrease
antibody bound the PSA antigen and a detection limit of and overall orients the antibodies such that the Fc region
10 pg/ml was achieved. Song went on to investigate different faced away from the PEG brush substrate into the bulk
methods for anti-PSA immobilization with DPI, comparing solution.
boronate chelation, TCEP reduction with maleimide cova-
lent linkage, Protein G, and random immobilization methods F. Atomic force microscopy
to PEG:Thiol or amine modified DPI chips.64 They found AFM is a topographic analysis technique that employs
that while the mass of anti-PSA loaded onto the DPI chip the scanning of a nanoscale tip across a sample surface. The
was lower for TCEP than boronate chelation, the antigen tip is bound to an oscillating cantilever and by accurately
sensitivity was 20 times higher inferring preferential orienta- monitoring the cantilever’s change in resonance, a nanome-
tion with the TCEP method using amine modified chips. ter scale resolution image of the surface can be achieved.
When surface bound, the asymmetrical dimensions of anti-
D. Surface plasmon resonance bodies (14  10  4 nm3) allow changes to the surface topog-
SPR is an optical technique used to monitor protein raphy, i.e., film thickness and surface roughness, to be
adsorption and surface interactions. Monochromatic laser measured and can be representative of different antibody ori-
light is used to stimulate a surface plasmon wave in the sens- entations. Chen et al.142 used AFM and SPR to confirm end-
ing surface and the reflected light angle is measured. Upon on antibody orientation to the ProLinkerTM SAM by plotting
absorption on the sensing surface, the refractive index of the the height profile of the immobilized antibody and monitor-
material changes causing the reflected light angle and the ing antigen uptake. Coppari et al.147 investigated a monoclo-
surface plasmon resonance angle to change accordingly. A nal antibody adsorbed on mica and, by combining height
recent review by Mauriz et al.55 covers SPR-based assays in traces and images, were able to determine different antibody
great detail. orientations with AFM. By incorporating molecular dynamic
Vashist et al.140 utilized SPR as an assay method to inves- simulations with AFM images, Vilhena et al.148 demon-
tigate antibody immobilization strategies; random, covalent strated flat-on, head-on, side-on, and end-on orientations of
(EDC/NHS), oriented with adsorbed Protein A followed by IgG adsorbed to graphene. Funari et al.56 characterized phys-
antibody binding, covalent-oriented Protein A followed by isorbed and irradiation-coupled antibodies on extremely
antibody binding, and last covalent-CM5-dextran binding. smooth (root-mean-squared roughness 0.15 6 0.01 nm) gold-
SPR determined that the mass of antibody immobilized coated silicon wafers. The irradiation causes photo-reduction
was greatest for the CM5 system. However, the covalent- of disulfide bridges that yield free-thiols for binding gold.
oriented system bound the greatest amount of antigen and Using AFM, the authors found that the irradiated antibodies
indicated preferential orientation. Zhang et al.141 used SPR had a smaller contact area with the surface and a larger
to investigate antigen binding capabilities of gold–graphene height distribution indicating a side-on orientation relative to
oxide (Au/GO) composites compared with gold coated with the physisorbed system being flat-on. Marciello et al.149
Protein A. By tracking the resonant wavelength change as a used AFM to investigate the orientation of antibodies at the
function of antigen concentration, the authors demonstrated surface of lipase-coated magnetic nanoparticles. The authors
the oriented Protein A on Au/GO system had improved anti- assessed the surface of the nanoparticles and after immobili-
gen sensitivity compared with Protein A on Au alone. zation. Two immobilized antibody systems were investi-
gated; the optimized sample, S1, with a recovered immune
E. Neutron reflectometry activity (from immunoassay) of 80%, and S2, with a lower
Neutron reflectometry (NR) is a diffraction technique recovered immune activity (about 3%). The peak-to-valley
used to investigate film thickness. Neutrons are reflected off height determined with AFM of S1 was found to be 9 nm as
the sample surface and assessed as a function of change in compared with only 5 nm for S2, indicating preferential ori-
angle or wavelength. Lu’s team have used NR to investigate entation of the antibody on S1.
several antibody binding and orientation effects.143–145 NR
was used in conjunction with AFM to determine a flat-on G. Quartz crystal microbalance
orientation of anti-b-hCG antibody (specific to the b unit QCM is a mass sensitive technique that monitors the
of human chorionic gonadotrophin) to silicon-oxide, as change in frequency and damping of a resonant quartz

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02D301-10 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-10

piezoelectric crystal. As the material is adsorbed to the crys- the F(ab0 )2 and Fc portions, and these were correlated against
tal, the resonance frequency decreases, allowing the amount the antibody amino acid composition. A ratio of the impor-
of material to be determined very precisely. Thus, if the tant amino acids could then be used as predictors for anti-
mass of the antibody and its complementary antigen are body orientation. More recently, Welch et al. has employed
known, then antibody orientation can be inferred from anti- larger peak lists incorporating over 700 mass fragments for
gen binding. Recently, Deng et al.150 have developed a characterizing adsorbed whole antibody and antibody frag-
QCM chip modification that is used to orient biotin-labeled ments.156 The increased peak list significantly improved the
antibodies for use in an immunoassay. QCM was used to ability to identify and classify the samples using multivariate
quantify antibody binding and antigen coupling. Compared analysis techniques. One potential drawback is that ToF-
with the control surface, the biotinylated graphene oxide- SIMS operates in an ultrahigh vacuum environment (UHV)
avidin surface modification was found to bind slightly lower that is likely to denature proteins.157 One possible strategy
amounts of antibody, although it demonstrated improved for avoiding UHV induced denaturation is by the fixation of
antigen capture, suggestive of antibody orientation. proteins at surfaces with trehalose. Trehalose is a disaccha-
Dissipation monitoring during QCM can provide insight ride that has been used to fix the state of proteins at interfa-
into the density and permeability of immobilized (bio)mole- ces to preserve their conformation and minimize changes to
cules at the interface.51,158 Via this method, protein orienta- their orientation.153,162 However, coating samples prior to
tion may be investigated; for instance, an antibody ToF-SIMS analysis, or other surface analysis techniques
immobilized close to the surface in a flat-on orientation will such as XPS, may cause difficulties in subsequent data
have a low viscoelastic dissipation, while in contrast, an anti- analysis.
body in head-on or end-on orientation will have a higher vis-
coelastic dissipation. I. Multivariate analysis
Another QCM mass-based immunoassay was proposed It is now a common practice to employ multivariate anal-
by Akter et al.151 who employed Protein A as the antibody ysis techniques such as PCA to reduce the dimensionality of
orienting component. Using QCM, the authors were able to complex data sets, such as those derived from ToF-SIMS.
monitor antibody binding, antigen selectivity, and demon- PCA is used to identify the variables that contribute to the
strate the benefits of their precipitation mass amplification largest amount of variance in the dataset. In the case of ToF-
system as applicable in immunoassay. SIMS analysis, the intensity or number of counts obtained
for each mass fragment comprise the input variables for
H. Time-of-flight secondary-ion mass spectrometry PCA. PCA has been used to investigate antibody orientation
ToF-SIMS employs a focused beam of ionized metal on various substrates to good effect.163,164 Liu et al.165
atoms (or clusters of atoms), large molecules such as fuller- immobilized Fab and Fc fragments to both gold and
enes (C60), or gas clusters, to bombard the sample and polymer-coated slide substrates and used PCA to investigate
remove fragmented material from the surface. A small per- each of the four systems. Principal component 1 (PC1) sepa-
centage of the fragments are ionized, known as secondary- rated the samples based on substrate, and principal compo-
ions, which are collected as a representative sample of the nent 2 (PC2) separated samples based on antibody fragment.
elemental and molecular species from the top few mono- The loadings plot for PC2, showing the contributions from
layers of the surface (on the order of 10 Å).159 The 14 nm each of the amino acid variables, correlated strongly with
long axis of antibodies, combined with this limited sampling natural amino acid composition differences in the antibody
depth, provides differentiation between orientations of fragments. Park et al.164 interrogated randomly and site-
immobilized antibody due to different amino acid composi- directed IgG and F(ab0 )2 with ToF-SIMS and PCA. Using
tions in the portions being analyzed. known amino acid related mass fragments, PC1 showed that
ToF-SIMS analysis of known peptides has been used to site-directed IgG and F(ab0 )2 were more commonly in the
determine most common mass fragments arising from partic- end-on orientation than the randomly immobilized proteins.
ular amino acids.152,160,161 These amino acid specific lists, Kosobrodova et al.166 investigate antibodies immobilized to
typically of up to 40 mass fragments, allow differentiation untreated and plasma treated polycarbonate with ToF-SIMS
between protein and substrate.153,154 Foster et al.130 analyzed and PCA and found that the F(ab0 )2 component of the anti-
bovine IgG adsorbed to gold and sodium styrenesulfonate body was preferentially exposed on the plasma treated
coated gold surfaces. As the prevalence of serine is higher in surface.
the F(ab0 )2 region of the IgG, and aspartate and valine preva- Artificial neural networks (ANN) are another class of
lence higher in the Fc region, then a ratio of these amino multivariate analysis techniques and classify and group sam-
acids mass fragment intensities could be used to assess the ples based on their similarities and differences across the
relative orientation of IgG immobilized to the substrates. input variables. Sanni et al.167 employed ANN to differenti-
Wang et al.155 investigated anti-hCG IgG, F(ab0 )2, and Fc ate between 13 different protein films, including two types
immobilized on gold and also orientation promoting SAMs of antibodies, using nominal mass values of all mass frag-
(COOH and NH2). Principal component analysis (PCA) ments available in the spectra. ANN was able to identify the
identified amino acid fragments that were more prevalent in key mass fragments associated with each of the proteins to

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02D301-11 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-11

distinguish between them. More recently, Welch et al.156 Nevertheless, ToF-SIMS has the potential to characterize
employed ANNs to discriminate between an antibody and its and differentiate antibody properties including orientation
proteolysis fragments adsorbed to silicon substrates, based and denaturation state, yielding molecularly specific infor-
solely on their ToF-SIMS spectra. The ANN analysis mation from the uppermost surface. The information-rich
method holds promise for investigating antibody orientation data greatly stands to benefit from interrogation with multi-
at interfaces due to its ability to incorporate a broad range of variate analysis techniques PCA and ANN. Not only can this
mass fragments and investigate complex relationships combination offer new insight into the state of immobilized
between variables. proteins, but it is also directly applicable to new material dis-
covery and will greatly assist in the development and optimi-
IV. CONCLUSIONS AND PERSPECTIVES zation of immunoassay performance. In parallel with this
In summary, existing and new methods for oriented anti- approach, recent developments in molecular modeling, and,
body immobilization have been developed over the past few in particular, coarse-grain modeling,171,172 may provide a
years with good progress made on improving the established method for assessing antibody orientation characteristics on
methods. Nevertheless, some shortcomings and challenges novel substrates in silico before fabrication, or to aid sub-
still exist. Oriented systems based on novel surface chemis- strate design.
try, protein engineering, or both, require complex and time- In this article, we have discussed the most up-to-date and
consuming production steps, which may also be expensive. state-of-the-art methods used for the immobilization of anti-
Ideally, antibodies truly representative of their native bodies in an oriented manner, particularly for the improve-
solution-phase state, i.e., without protein engineered tags, ment of immunoassay performance. Further, we have
would be immobilized to a simple, cheap, and easy to pro- discussed the current range of surface analysis techniques
being used for investigating the orientation of antibody sys-
duce substrate, homogeneously arranged and site-directed as
tems and have highlighted the importance of multivariate
to maximize their antigen capture. However, it is likely that
analysis tools in the interrogation and analysis of the experi-
smaller antibody fragments and aptamers may be favored
mental data produced.
over whole antibodies in the future as they can be prepared
recombinantly and are easily modified genetically or chemi- 1
Q. L. Yu, Q. H. Wang, B. M. Li, Q. Y. Lin, and Y. X. Duan, Crit. Rev.
cally with the ability to maximize capture events due to Anal. Chem. 45, 62 (2015).
increased packing and binding site density.168 Additionally, 2
Y. S. Liu and C. M. Li, Anal. Lett. 45, 130 (2012).
3
camelid antibodies with one single domain for antigen bind- B. Lu, M. R. Smyth, and R. O’Kennedy, Analyst 121, 29R (1996).
4
E. K. Wujcik, H. Wei, X. Zhang, J. Guo, X. Yan, N. Sutrave, S. Wei, and
ing (known as VHH) have attracted attention due to their Z. Guo, RSC Adv. 4, 43725 (2014).
high solubility and stability, and may provide an opportunity 5
V. Crivianu-Gaita and M. Thompson, Biosens. Bioelectron. 70, 167
for incorporation into sensors.8 Also, peptides and aptamers 6
(2015).
C. Menti, J. A. Henriques, F. P. Missell, and M. Roesch-Ely, Appl.
provide a highly customizable method for producing cova-
Microbiol. Biotechnol. 100, 6149 (2016).
lent attachment of antibodies, or fragments thereof, either by 7
H. Chen, F. Liu, F. Qi, K. Koh, and K. Wang, Int. J. Mol. Sci. 15, 5496
active targeting of the protein, or the substrate, or both. In (2014).
8
the future, it may be seen that metallic thin films are used to A. K. Trilling, J. Beekwilder, and H. Zuilhof, Analyst 138, 1619 (2013).
9
A. K. Trilling, T. Hesselink, A. van Houwelingen, J. H. Cordewener, M.
produce homogeneous substrates that can be targeted simply A. Jongsma, S. Schoffelen, J. C. van Hest, H. Zuilhof, and J. Beekwilder,
by endogenous antibody epitopes or material binding pepti- Biosens. Bioelectron. 60, 130 (2014).
10
des coupled to Fc specific aptamers or peptides. In the case J. Z. Hui, S. Tamsen, Y. Song, and A. Tsourkas, Bioconjugate Chem. 26,
of the latter, such a system could be near universally applica- 1456 (2015).
11
Y. Jung, J. Y. Jeong, and B. H. Chung, Analyst 133, 697 (2008).
ble to native state antibodies. 12
J. E. Butler, Methods 22, 4 (2000).
It is clear that surface analysis techniques and multivari- 13
J. E. Butler, L. Ni, W. R. Brown, K. S. Joshi, J. Chang, B. Rosenberg,
ate analysis tools will play a prevalent role in identifying, and E. W. Voss, Mol. Immunol. 30, 1165 (1993).
14
J. E. Butler, L. Ni, R. Nessler, K. S. Joshi, M. Suter, B. Rosenberg, J.
investigating, and predicting antibody orientation at sub-
Chang, W. R. Brown, and L. A. Cantarero, J. Immunol. Methods 150, 77
strates of interest. Characterization of antibody orientation in (1992).
15
situ or in the native “wet” environment permits a more accu- C. Wang and B. Feng, Mol. Biol. 49, 1 (2015).
16
rate presentation of the antibody state without the potential Y. S. Liu and J. Yu, Microchim. Acta 183, 1 (2016).
17
W. Norde, T. A. Horbett, and J. L. Brash, Proteins at Interfaces III: State
for confirmation changes. XPS and ToF-SIMS require UHV of the Art, edited by T. Horbett, J. L. Brash, and W. Norde (American
conditions and are ex situ. AFM is typically performed in a Chemical Society, Washington, 2012), Vol. 1120, pp. 1–34.
18
dry state (however progress has been made regarding wet W. Kusnezow and J. D. Hoheisel, J. Mol. Recognit. 16, 165 (2003).
19
analysis) and is performed ex situ. QCM, SPR, NR, SE, and M. M. Bilek and D. R. McKenzie, Biophys. Rev. 2, 55 (2010).
20
K. S. Siow, L. Britcher, S. Kumar, and H. J. Griesser, Plasma Processes
DPI can be performed in solution and with the correct appa- Polym. 3, 392 (2006).
ratus in situ also. Additionally, complementary techniques 21
F. Awaja, M. Gilbert, G. Kelly, B. Fox, and P. J. Pigram, Prog. Polym.
such as sum frequency generation promise to provide in situ Sci. 34, 948 (2009).
22
K. Nakanishi, H. Muguruma, and I. Karube, Anal. Chem. 68, 1695
characterization of protein orientation via changes in the
(1996).
infrared absorption patterns; however, complex analysis is 23
E. P^aslaru, M. C. Baican, E. G. Hitruc, M. T. Nistor, F. Poncin-Epaillard,
still required.169,170 and C. Vasile, Colloids Surf., B 115, 139 (2014).

Biointerphases, Vol. 12, No. 2, June 2017

02D301-12 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-12

24 63
S. H. North, E. H. Lock, C. J. Cooper, J. B. Franek, C. R. Taitt, and S. G. F. Duval, T. A. van Beek, and H. Zuilhof, Analyst 140, 6467 (2015).
64
Walton, ACS Appl. Mater. Interfaces 2, 2884 (2010). H. Y. Song, J. Hobley, X. D. Su, and X. D. Zhou, Plasmonics 9, 851
25
E. J. Anglin, C. Salisbury, S. Bailey, M. Hor, P. Macardle, M. Fenech, H. (2014).
65
Thissen, and N. H. Voelcker, Lab Chip 10, 3413 (2010). C. H. Huang, Q. L. Yang, F. Y. Song, N. Chen, X. L. Liao, B. Yao, S. T.
26
F. Basarir, N. Cuong, W. K. Song, and T. H. Yoon, Macromol. Symp. Zhang, Y. Y. Chen, and G. Jin, Integr. Ferroelectr. 171, 70 (2016).
66
249, 61 (2007). Y. Yuan, M. Yin, J. C. Qian, and C. S. Liu, Soft Matter 7, 7207 (2011).
27 67
A. Manakhov, E. Makhneva, P. Skladal, D. Necas, J. Cechal, L. Kalina, B. Prieto-Sim on, C. Saint, and N. H. Voelcker, Anal. Chem. 86, 1422
M. Elias, and L. Zajickova, Appl. Surf. Sci. 360, 28 (2016). (2014).
28 68
R. T. Chen, B. W. Muir, L. Thomsen, A. Tadich, B. C. Cowie, G. K. A. Makaraviciute and A. Ramanaviciene, Biosens. Bioelectron. 50, 460
Such, A. Postma, K. M. McLean, and F. Caruso, J. Phys. Chem. B 115, (2013).
69
6495 (2011). K. Hernandez and R. Fernandez-Lafuente, Enzyme Microb. Technol. 48,
29
N. G. Welch, R. M. Madiona, C. D. Easton, J. A. Scoble, R. T. Jones, B. 107 (2011).
70
W. Muir, and P. J. Pigram, Biointerphases 11, 041004 (2016). J. Pande, M. M. Szewczyk, and A. K. Grover, Biotechnol. Adv. 28, 849
30
Y. Li, N. P. Reynolds, K. E. Styan, B. W. Muir, J. S. Forsythe, and C. D. (2010).
71
Easton, Appl. Surf. Sci. 361, 162 (2016). J. B. Tang, X. F. Sun, H. M. Yang, B. G. Zhang, Z. J. Li, Z. J. Lin, and Z.
31
Y. Li, B. W. Muir, C. D. Easton, L. Thomsen, D. R. Nisbet, and J. S. Q. Gao, Anal. Chim. Acta 776, 74 (2013).
72
Forsythe, Appl. Surf. Sci. 288, 288 (2014). B. Feng, Y. Dai, L. Wang, N. Tao, S. Huang, and H. Zeng, Biologicals
32
C. M. Chan, T. M. Ko, and H. Hiraoka, Surf. Sci. Rep. 24, 1 (1996). 37, 48 (2009).
33 73
J. M. Goddard and J. H. Hotchkiss, Prog. Polym. Sci. 32, 698 (2007). B. Feng, C. Wang, X. Xie, X. Feng, Y. Li, and Z. Cao, Biochem.
34
G. J. S. Fowler, G. Mishra, C. D. Easton, and S. L. McArthur, Polymer Biophys. Res. Commun. 450, 429 (2014).
74
50, 5076 (2009). Y. Kumada, D. Kuroki, H. Yasui, T. Ohse, and M. Kishimoto, J. Biosci.
35
G. Mishra, C. D. Easton, G. J. S. Fowler, and S. L. McArthur, Polymer Bioeng. 109, 583 (2010).
75
52, 1882 (2011). K. Taniguchi, K. Nomura, Y. Hata, T. Nishimura, Y. Asami, and A.
36
G. Mishra, C. D. Easton, and S. L. McArthur, Langmuir 26, 3720 (2010). Kuroda, Biotechnol. Bioeng. 96, 1023 (2007).
37 76
P. K. Chu, J. Y. Chen, L. P. Wang, and N. Huang, Mater. Sci. Eng., R 36, T. Ikeda, Y. Hata, K. Ninomiya, Y. Ikura, K. Takeguchi, S. Aoyagi, R.
143 (2002). Hirota, and A. Kuroda, Anal. Biochem. 385, 132 (2009).
38 77
A. Hasan and L. M. Pandey, Polym.-Plast. Technol. Eng. 54, 1358 S. M. Fuchs and R. T. Raines, Protein Sci. 14, 1538 (2005).
78
(2015). T. Serizawa, T. Sawada, and H. Matsuno, Langmuir 23, 11127 (2007).
39 79
R. Gupta and N. K. Chaudhury, Biosens. Bioelectron. 22, 2387 (2007). Y. Kumada, S. Murata, Y. Ishikawa, K. Nakatsuka, and M. Kishimoto,
40
A. V. Orlov, V. A. Bragina, M. P. Nikitin, and P. I. Nikitin, Biosens. J. Biotechnol. 160, 222 (2012).
80
Bioelectron. 79, 423 (2016). Y. Kumada, B. Kang, K. Yamakawa, M. Kishimoto, and J. Horiuchi,
41
B. Feng, S. Huang, F. Ge, Y. Luo, D. Jia, and Y. Dai, Biosens. Biotechnol. Prog. 31, 1563 (2015).
81
Bioelectron. 28, 91 (2011). H. Matsuno, J. Sekine, H. Yajima, and T. Serizawa, Langmuir 24, 6399
42
J. L. Acero Sanchez, A. Fragoso, H. Joda, G. Suarez, C. J. McNeil, and (2008).
82
C. K. O’Sullivan, Anal. Bioanal. Chem. 408, 5337 (2016). E. de Juan-Franco, A. Caruz, J. R. Pedrajas, and L. M. Lechuga, Analyst
43
J. C. Love, L. A. Estroff, J. K. Kriebel, R. G. Nuzzo, and G. M. 138, 2023 (2013).
83
Whitesides, Chem. Rev. 105, 1103 (2005). E. Hochuli, W. Bannwarth, H. Dobeli, R. Gentzand, and D. Stuber, Bio-
44
S. F. Chen, L. Y. Liu, J. Zhou, and S. Y. Jiang, Langmuir 19, 2859 Technology 6, 1321 (1988).
84
(2003). E. Hochuli, H. Dobeli, and A. Schacher, J. Chromatogr. 411, 177 (1987).
45 85
V. Lebec, S. Boujday, C. Poleunis, C. M. Pradier, and A. Delcorte, J. Turkova, J. Chromatogr. B 722, 11 (1999).
86
J. Phys. Chem. C 118, 2085 (2014). I. H. Cho, E. H. Paek, H. Lee, J. Y. Kang, T. S. Kim, and S. H. Paek,
46
S. K. Vashist, E. Lam, S. Hrapovic, K. B. Male, and J. H. Luong, Chem. Anal. Biochem. 365, 14 (2007).
87
Rev. 114, 11083 (2014). I. H. Cho, J. W. Park, T. G. Lee, H. Lee, and S. H. Paek, Analyst 136,
47
A. Bossi, S. A. Piletsky, E. V. Piletska, P. G. Righetti, and A. P. Turner, 1412 (2011).
88
Anal. Chem. 73, 5281 (2001). R. Wacker, H. Schroder, and C. M. Niemeyer, Anal. Biochem. 330, 281
48
N. Bereli, G. Erturk, M. A. Tumer, R. Say, and A. Denizli, Biomed. (2004).
89
Chromatogr. 27, 599 (2013). E. Seymour, G. G. Daaboul, X. Zhang, S. M. Scherr, N. L. Unlu, J. H.
49
M. Cretich, F. Damin, G. Pirri, and M. Chiari, Biomol. Eng. 23, 77 Connor, and M. S. Unlu, Anal. Chem. 87, 10505 (2015).
90
(2006). A. C. Glavan, J. Niu, Z. Chen, F. Guder, C. M. Cheng, D. Liu, and G. M.
50
S. V. Rao, K. W. Anderson, and L. G. Bachas, Mikrochim. Acta 128, 127 Whitesides, Anal. Chem. 88, 725 (2016).
91
(1998). C. Boozer, J. Ladd, S. Chen, Q. Yu, J. Homola, and S. Jiang, Anal.
51
S. D. Carrigan, G. Scott, and M. Tabrizian, Langmuir 21, 5966 (2005). Chem. 76, 6967 (2004).
52 92
Y. Sun, H. Y. Du, C. L. Feng, and Y. T. Lan, J. Solid State Electrochem. C. Boozer, Q. M. Yu, S. F. Chen, C. Y. Lee, J. Homola, S. S. Yee, and S.
19, 3035 (2015). Y. Jiang, Sens. Actuator, B 90, 22 (2003).
53 93
H. Sharma and R. Mutharasan, Anal. Chem. 85, 2472 (2013). L. Bjorck and G. Kronvall, J. Immunol. 133, 969 (1984).
54 94
A. A. Karyakin, G. V. Presnova, M. Y. Rubtsova, and A. M. Egorov, P. L. Ey, S. J. Prowse, and C. R. Jenkin, Immunochemistry 15, 429 (1978).
95
Anal. Chem. 72, 3805 (2000). M. Iijima, N. Yoshimoto, T. Niimi, A. D. Maturana, and S. Kuroda,
55
E. Mauriz, M. C. Garcia-Fernandez, and L. M. Lechuga, Trac-Trends Analyst 138, 3470 (2013).
96
Anal. Chem. 79, 191 (2016). S. L. Guo, P. C. Chen, M. S. Chen, Y. C. Cheng, J. M. Lin, H. C. Lee,
56
R. Funari, B. Della Ventura, C. Altucci, A. Offenhausser, D. Mayer, and and C. S. Chen, PLoS One 7, e51370 (2012).
97
R. Velotta, Langmuir 32, 8084 (2016). B. Nilsson, T. Moks, B. Jansson, L. Abrahmsen, A. Elmblad, E.
57
B. Della Ventura, L. Schiavo, C. Altucci, R. Esposito, and R. Velotta, Holmgren, C. Henrichson, T. A. Jones, and M. Uhlen, Protein Eng. 1,
Biomed. Opt. Express 2, 3223 (2011). 107 (1987).
58 98
V. Gauvreau, P. Chevallier, K. Vallieres, E. Petitclerc, R. C. Gaudreault, J. W. Chung, J. M. Park, R. Bernhardt, and J. C. Pyun, J. Biotechnol. 126,
and G. Laroche, Bioconjugate Chem. 15, 1146 (2004). 325 (2006).
59 99
A. Beduneau, P. Saulnier, F. Hindre, A. Clavreul, J.-C. Leroux, and J.-P. V. Crivianu-Gaita, M. Aamer, R. T. Posaratnanathan, A. Romaschin, and
Benoit, Biomaterials 28, 4978 (2007). M. Thompson, Biosens. Bioelectron. 78, 92 (2016).
60 100
S. Kumar, J. Aaron, and K. Sokolov, Nat. Protoc. 3, 314 (2008). A. Makaraviciute, A. Ramanavicius, and A. Ramanaviciene, Anal.
61
A. K. Adak, B. Y. Li, L. D. Huang, T. W. Lin, T. C. Chang, K. C. Methods 7, 9875 (2015).
101
Hwang, and C. C. Lin, ACS Appl. Mater. Interfaces 6, 10452 (2014). S. K. Verma, A. Amoah, U. Schellhaas, M. Winterhalter, S. Springer, and
62
P. Peluso et al., Anal. Biochem. 312, 113 (2003). T. A. Kolesnikova, Adv. Funct. Mater. 26, 6015 (2016).

Biointerphases, Vol. 12, No. 2, June 2017

02D301-13 Welch et al.: Orientation and characterization of immobilized antibodies 02D301-13

102 139
S. R. Ahmed, A. T. Lutes, and T. A. Barbari, J. Membr. Sci. 282, 311 H. Y. Song, X. Zhou, J. Hobley, and X. Su, Langmuir 28, 997 (2012).
140
(2006). S. K. Vashist, C. K. Dixit, B. D. MacCraith, and R. O’Kennedy, Analyst
103
H. Miyao, Y. Ikeda, A. Shiraishi, Y. Kawakami, and S. Sueda, Anal. 136, 4431 (2011).
141
Biochem. 484, 113 (2015). J. Zhang, Y. Sun, Q. Wu, H. Zhang, Y. Bai, and D. Song, Analyst 138,
104
K. Ofir, Y. Berdichevsky, I. Benhar, R. Azriel-Rosenfeld, R. Lamed, Y. 7175 (2013).
142
Barak, E. A. Bayer, and E. Morag, Proteomics 5, 1806 (2005). H. X. Chen, J. Y. Huang, J. Lee, S. Hwang, and K. Koh, Sens. Actuator,
105
A. M. Rosa, A. F. Louro, S. A. Martins, J. Inacio, A. M. Azevedo, and D. B 147, 548 (2010).
M. Prazeres, Anal. Chem. 86, 4340 (2014). 143
H. Xu, X. Zhao, C. Grant, J. R. Lu, D. E. Williams, and J. Penfold,
106
H. M. Yang, S. J. Liang, J. B. Tang, Y. Chen, and Y. Z. Cheng, Anal. Langmuir 22, 6313 (2006).
Biochem. 432, 134 (2013). 144
X. Zhao, F. Pan, L. Garcia-Gancedo, A. J. Flewitt, G. M. Ashley, J. Luo,
107
S. Chebil, A. Miodek, V. Ambike, H. Sauriat-Dorizon, C. Policar, and H. and J. R. Lu, J. R. Soc. Interface 9, 2457 (2012).
Korri-Youssoufi, Sens. Actuator, B 185, 762 (2013). 145
X. Zhao, F. Pan, B. Cowsill, J. R. Lu, L. Garcia-Gancedo, A. J. Flewitt,
108
H. M. Yang, R. M. Bao, C. M. Yu, Y. N. Lv, W. F. Zhang, and J. B. G. M. Ashley, and J. Luo, Langmuir 27, 7654 (2011).
Tang, Anal. Chim. Acta 949, 76 (2017). 146
E. Schneck, I. Berts, A. Halperin, J. Daillant, and G. Fragneto,
109
Y. Jung, H. J. Kang, J. M. Lee, S. O. Jung, W. S. Yun, S. J. Chung, and Biomaterials 46, 95 (2015).
B. H. Chung, Anal. Biochem. 374, 99 (2008). 147
E. Coppari, S. Santini, A. R. Bizzarri, and S. Cannistraro, Biophys.
110
C. W. Tsai, S. L. Jheng, W. Y. Chen, and R. C. Ruaan, Anal. Chem. 86, Chem. 211, 19 (2016).
2931 (2014). 148
J. G. Vilhena, A. C. Dumitru, E. T. Herruzo, J. I. Mendieta-Moreno, R.
111
R. J. Yoo and S. J. Choi, Biochip J. 10, 88 (2016). Garcia, P. A. Serena, and R. Perez, Nanoscale 8, 13463 (2016).
112
S. Dostalova et al., ACS Appl. Mater. Interfaces 8, 14430 (2016). 149
M. Marciello, M. Filice, D. Olea, M. Velez, J. M. Guisan, and C. Mateo,
113
Y. Lee, J. Jeong, G. Lee, J. H. Moon, and M. K. Lee, Anal. Chem. 88, Langmuir 30, 15022 (2014).
9503 (2016). 150
X. Deng, M. Chen, Q. Fu, N. M. Smeets, F. Xu, Z. Zhang, C. D. Filipe,
114
R. Nezlin, Mol. Immunol. 70, 149 (2016). and T. Hoare, ACS Appl. Mater. Interfaces 8, 1893 (2016).
115
S. Miyakawa, A. Oguro, T. Ohtsu, H. Imataka, N. Sonenberg, and Y. 151
R. Akter, C. K. Rhee, and M. A. Rahman, Biosens. Bioelectron. 66, 539
Nakamura, RNA 12, 1825 (2006). (2015).
116
J. Ma et al., Genet. Mol. Res. 12, 1399 (2013). 152
N. T. Samuel, M. S. Wagner, K. D. Dornfeld, and D. G. Castner, Surf.
117
N. J. Alves, T. Kiziltepe, and B. Bilgicer, Langmuir 28, 9640 (2012). Sci. Spectra 8, 163 (2001).
118
N. Mustafaoglu, N. J. Alves, and B. Bilgicer, Biotechnol. Bioeng. 112, 153
Y. P. Kim, M. Y. Hong, J. Kim, E. Oh, H. K. Shon, D. W. Moon, H. S.
1327 (2015).
119 Kim, and T. G. Lee, Anal. Chem. 79, 1377 (2007).
N. Mustafaoglu, N. J. Alves, and B. Bilgicer, Langmuir 31, 9728 (2015). 154
120 L. Baugh, T. Weidner, J. E. Baio, P. C. Nguyen, L. J. Gamble, P. S.
J. Porath, J. Carlsson, I. Olsson, and G. Belfrage, Nature 258, 598 (1975).
121 Stayton, and D. G. Castner, Langmuir 26, 16434 (2010).
J. Porath and B. Olin, Biochemistry 22, 1621 (1983). 155
122 H. Wang, D. G. Castner, B. D. Ratner, and S. Jiang, Langmuir 20, 1877
J. E. Hale, Anal. Biochem. 231, 46 (1995).
123 (2004).
D. Todorova-Balvay, O. Pitiot, M. Bourhim, T. Srikrishnan, and M. 156
N. G. Welch, R. M. Madiona, T. B. Payten, R. T. Jones, N. Brack, B. W.
Vijayalakshmi, J. Chromatogr., B 808, 57 (2004).
124 Muir, and P. J. Pigram, Langmuir 32, 8717 (2016).
B. W. Muir, M. C. Barden, S. P. Collett, A. D. Gorse, R. Monteiro, L. 157
N. G. Welch, R. M. Madiona, J. A. Scoble, B. W. Muir, and P. J. Pigram,
Yang, N. A. McDougall, S. Gould, and N. J. Maeji, Anal. Biochem. 363,
Langmuir 32, 10824 (2016).
97 (2007). 158
125 A. R. Patel, B. A. Kerwin, and S. R. Kanapuram, J. Pharm. Sci. 98, 3108
I. Ojeda, M. Barrejon, L. M. Arellano, A. Gonzalez-Cortes, P. Yanez-
Sedeno, F. Langa, and J. M. Pingarron, Biosens. Bioelectron. 74, 24 (2009).
159
(2015). C. J. Vickerman and D. Briggs, ToF-SIMS Surface Analysis by Mass
126
C. B. Arenas, E. Sanchez-Tirado, I. Ojeda, C. A. Gomez-Suarez, A. Spectrometry (IM Publications, Chichester, 2001).
160
Gonzalez-Cortes, R. Villalonga, P. Yanez-Sedeno, and J. M. Pingarron, M. S. Wagner and D. G. Castner, Langmuir 17, 4649 (2001).
161
Sens. Actuator, B 223, 89 (2016). D. S. Mantus, B. D. Ratner, B. A. Carlson, and J. F. Moulder, Anal.
127
N. G. Welch, C. D. Easton, J. A. Scoble, C. C. Williams, P. J. Pigram, Chem. 65, 1431 (1993).
162
and B. W. Muir, J. Immunol. Methods 438, 59 (2016). N. Xia, C. J. May, S. L. McArthur, and D. G. Castner, Langmuir 18, 4090
128
N. G. Welch, J. A. Scoble, C. D. Easton, C. C. Williams, B. J. Bradford, (2002).
163
L. K. Mamedova, P. J. Pigram, and B. W. Muir, Anal. Chem. 88, 10102 X. Cheng, H. E. Canavan, D. J. Graham, D. G. Castner, and B. D. Ratner,
(2016). Biointerphases 1, 61 (2006).
164
129
Q. Wei, T. Becherer, S. Angioletti-Uberti, J. Dzubiella, C. Wischke, A. J. W. Park, I. H. Cho, D. W. Moon, S. H. Paek, and T. G. Lee, Surf.
T. Neffe, A. Lendlein, M. Ballauff, and R. Haag, Angew. Chem. Int. Ed. Interface Anal. 43, 285 (2011).
165
Engl. 53, 8004 (2014). F. Liu, M. Dubey, H. Takahashi, D. G. Castner, and D. W. Grainger,
130
R. N. Foster, E. T. Harrison, and D. G. Castner, Langmuir 32, 3207 Anal. Chem. 82, 2947 (2010).
166
(2016). E. Kosobrodova, R. T. Jones, A. Kondyurin, W. Chrzanowski, P. J.
131
A. D. Radadia, C. J. Stavis, R. Carr, H. Zeng, W. P. King, J. A. Carlisle, Pigram, D. R. McKenzie, and M. M. Bilek, Acta Biomater. 19, 128
A. Aksimentiev, R. J. Hamers, and R. Bashir, Adv. Funct. Mater. 21, (2015).
167
1040 (2011). O. D. Sanni, M. S. Wagner, D. Briggs, D. G. Castner, and J. C.
132 Vickerman, Surf. Interface Anal. 33, 715 (2002).
S. Ray, G. Mehta, and S. Srivastava, Proteomics 10, 731 (2010).
133 168
H. Elwing, Biomaterials 19, 397 (1998). M. A. Wronska, I. B. O’Connor, M. A. Tilbury, A. Srivastava, and J. G.
134 Wall, Adv. Mater. 28, 5485 (2016).
Z. Balevicius, A. Ramanaviciene, I. Baleviciute, A. Makaraviciute, L.
169
Mikoliunaite, and A. Ramanavicius, Sens. Actuator, B 160, 555 (2011). S. J. Roeters, C. N. van Dijk, A. Torres-Knoop, E. H. G. Backus, R. K.
135
Y. M. Bae, B. K. Oh, W. Lee, W. H. Lee, and J. W. Choi, Biosens. Campen, M. Bonn, and S. Woutersen, J. Phys. Chem. A 117, 6311
Bioelectron. 21, 103 (2005). (2013).
136 170
Z. Wang and G. Jin, J. Biochem. Biophys. Methods 57, 203 (2003). T. Weidner and D. G. Castner, Phys. Chem. Chem. Phys. 15, 12516
137
Z. H. Wang and G. Jin, Colloids Surf., B 34, 173 (2004). (2013).
138 171
J. Escorihuela, M. A. Gonzalez-Martinez, J. L. Lopez-Paz, R. Puchades, D. B. Bush and T. A. Knotts, J. Chem. Phys. 143, 061101 (2015).
172
A. Maquieira, and D. Gimenez-Romero, Chem. Rev. 115, 265 (2015). S. Wei and T. A. Knotts IV, J. Chem. Phys. 139, 095102 (2013).

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