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A General and Efficient Cantilever Functionalization Technique for AFM
A Molecular
General andRecognition
Efficient Cantilever
Studies Functionalization Technique for AFM
Molecular Recognition Studies
Carleen M. Bowers,1 David A. Carlson,1 Alexander A. Shestopalov,2 Robert L. Clark,2 Eric J. Toone1
1
Department of Chemistry, Duke University, Durham, NC 27708
2
Hajim School of Engineering and Applied Sciences, University of Rochester, Rochester, NY 14627

Received 28 January 2012; accepted 2 February 2012

Published online 24 March 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.22061

ABSTRACT: biomolecules are bound to the tip by a strong Si C

Atomic force microscopy (AFM) is a versatile technique bond, completely uniform with regard to both epitope

for the investigation of noncovalent molecular density and substrate orientation, and highly suitable for

associations between ligand–substrate pairs. Surface force microscopy studies. We show that this attachment

modification of silicon nitride AFM cantilevers is most technique can be used to measure the unbinding profiles

commonly achieved using organic trialkoxysilanes. of tip-immobilized lactose and surface-immobilized

However, susceptibility of the Si O bond to hydrolysis galectin-3. Overall, the proposed technique is general,

and formation of polymeric aggregates diminishes operationally simple, and can be expanded to anchor a

attractiveness of this method for AFM studies. wide variety of epitopes to a silicon nitride cantilever

Attachment techniques that facilitate immobilization of a using a stable Si C bond. # 2012 Wiley Periodicals, Inc.

wide variety of organic and biological molecules via the Biopolymers 97: 761–765, 2012.

stable Si C bond on silicon nitride cantilevers would be Keywords: molecular recognition force spectroscopy;

of great value to the field of molecular recognition force atomic force microscopy; silicon nitride; cantilevers;

spectroscopy. Here, we report (1) the formation of stable, hydrosilylation; UV-induced organic monolayers

highly oriented monolayers on the tip of silicon nitride

cantilevers and (2) demonstrate their utility in the
investigation of noncovalent protein–ligand interactions
using molecular recognition force spectroscopy. The
monolayers are formed through hydrosilylation of
hydrogen-terminated silicon nitride AFM probes using a
protected a-amino-x-alkene. This approach facilitates
This article was originally published online as an accepted
preprint. The ‘‘Published Online’’ date corresponds to the
preprint version. You can request a copy of the preprint by
emailing the Biopolymers editorial office at biopolymers@wiley.
com
INTRODUCTION

W
ith a unique ability to detect piconewton scale
the subsequent conjugation of biomolecules. The resulting intermolecular forces, atomic force microscopy
(AFM) has emerged as a broadly applicable
tool for the investigation of intermolecular
Additional Supporting Information may be found in the online version of this interactions between ligands and receptors in
article.
Correspondence to: Eric J. Toone; e-mail: eric.toone@duke.edu
both biological and abiological systems.1 AFM has been utilized
Carleen M. Bowers and David A. Carlson contributed equally to this work in the study of binding interactions between DNA strands,2,3
Contract grant sponsor: NSF
Contract grant number: CMMI-1000724
antibodies and antigens,4 and proteins and carbohydrates.5,6
V
C 2012 Wiley Periodicals, Inc. Because interactions are probed between single species, ensemble

Biopolymers Volume 97 / Number 10 761

762 Bowers et al.

FIGURE 1 Top: Hydrosilylation and subsequent modification of silicon nitride AFM probes.
Bottom: XPS spectra of Boc-modified and amino-terminated monolayers on silicon nitride.

averaging is absent and the reproducibility and interpretation of
intermolecular force measurements is critically dependent on
the stable and uniform adsorption of specifically oriented bind-
ing partners, on both the flat surface of a stage and the sharp tip
of a cantilever. Although simple adsorption is frequently utilized,
covalent immobilization is the only approach that meets the
stringent requirements of stability and orientation.
Cantilever tips are most commonly constructed of silicon
or silicon nitride: because of its mechanical properties, silicon
nitride is generally preferred for molecular recognition stud-
ies.7 Surface modification of silicon nitride is most commonly
achieved using the reaction of organic silanes with a native ox-
ide layer.8 Although such monolayers form readily using either
solution or vapor phase deposition, several inherent limita-
tions, including polydisperse surface densities, the susceptibil-
ity of Si O bond to hydrolysis, and the formation of poly-
meric aggregates on surfaces, diminish their attractiveness for
AFM studies.9–11 Immobilization techniques that facilitate
attachment of a wide variety of organic and biological mole-
cules provide uniform surfaces, both with regard to the orien-
tation of the immobilized species and the density of epitopes,
and that withstand prolonged exposure to aqueous environ-
ments over a range of pH values would be of great value to the
field of molecular recognition force spectroscopy.
Monolayers covalently bound to silicon or silicon nitride
via Si C bonds are considerably more stable than silane

Biopolymers

FIGURE 2 Modification of H-terminated silicon with G3 via alkylation and carbene addition.
monolayers and have been shown to withstand prolonged ex-
posure to aqueous environments and prevent oxidation of
the underlying silicon nitride.12,13 A common approach to
the formation of such monolayers involves hydrosilylation,
during which an unsaturated bond inserts into a silicon-
hydride bond.7,13–18 Hydrosilylation of alkenes on silicon
cantilevers has been reported for applications in high resolu-
tion imaging and sensing.12,19 Molecular recognition studies
on soft or fragile samples, however, require a silicon nitride
cantilever with a low spring constant so that the applied force
is small.20 Here, we report the formation of stable, monodis-
perse, highly oriented monolayers highly suitable for force-
based molecular recognition studies using silicon nitride can-
tilevers. The monolayers are formed through hydrosilylation
of hydrogen-terminated silicon nitride AFM probes using a
protected a-amino-x-alkene; this functionalized hydrosila-
tion substrate facilitates subsequent conjugation of biomole-
cules. The resulting monolayers are robust, bound to the
underlying surface by a strong Si C bond, completely uni-
form with regard to both epitope density and substrate ori-
entation, and highly suitable for force microscopy studies.
RESULTS AND DISCUSSION
To demonstrate the approach, we modified silicon nitride
probes with oriented lactose in four sequential steps (Figure
1). Boc-terminated amines were immobilized on silicon
nitride probes using a photochemical insertion of alkene 1
Biopolymers
AFM Molecular Recognition Studies 763
into H-terminated silicon nitride; deprotection under acidic
conditions yielded an amino-terminated surface. The spacing
of silicon atoms in the underlying substrate produces a disor-
dered monolayer, and methylene surface area is presumably
exposed to solvent. To prevent nonspecific adsorption of
biomolecules to this surface area, the amino-terminated
substrate was reacted with heterobifunctional poly(ethylene
glycol) (PEG) linker 2. Finally, incubation of the surface with
thiol 3 yielded a lactose-modified surface via conjugate
addition to the pendant maleimide. The resulting probes
were imaged using scanning electron microscope (SEM) to
confirm that the tip morphology and radius of curvature
remained unaffected by the protocol; details and images are
included in the Supporting Information.
X-ray photoelectron spectrometer (XPS) analysis of sili-
con nitride surfaces confirmed successful modification of sili-
con nitride with Boc-protected a,x-amino alkenes. XPS
spectra of the Boc-terminated substrate showed an increase
in carbon compared to blank silicon nitride and also showed
a C1s peak at 287 eV resulting from the carbamate carbonyl
(Figure 1). XPS spectra of deprotected substrates lacked this
peak and showed a diminished carbon signal that correlates
with the calculated carbon concentration for completely
deprotected surface. Goniometry analysis in water of the t-
BOC protected amine substrate (Yadv. 5 758, Yrec 5 658) is
reflective of the hydrophobic nature of the t-BOC protecting
group. Water contact angle measurements of the deprotected
amino-terminated substrate (Yadv 5 688, Yrec 5 458) are
764 Bowers et al.
lower due to the decrease in hydrophobicity and protonation
of the amino groups by trifluoroacetic acid (TFA). Both the
value of the contact angle and hysteresis are consistent with a
disordered monolayer resulting from the Si-atomic spacing.
Contact angle measurements are consistent with those previ-
ously reported.17
We have previously reported a method for patterning bio-
logical species on passivated silicon, and we utilized a similar
strategy here to immobilize His6-tagged galectin-3 (G3) on sil-
icon (Figure 2).21 The technique simultaneously protects sili-
con from degradation in biological environments and provides
chelation sites for the uniform, highly oriented immobiliza-
tion of proteins. H-terminated silicon was chlorinated with
PCl5 then alkylated with propenyl Grignard 4. The alkylated
substrate was then reacted with diazirine 5 to yield an NHS
modified monolayer (S4), which was sequentially reacted with
lysine-N,N-diacetic acid (6), then incubated with Ni21 and
histidine tagged G3 providing the final surface (S6).
The final molecular assemblies of Figures 1 and 2 were used
for force spectroscopy experiments on a custom 3-axis AFM
using a previously reported force minimization protocol.22 The
surface-immobilized carbohydrate binding protein G3 binds
immobilized lactose with an affinity of 6400M21. At the C-
terminus, a 137 amino acid carbohydrate recognition domain
(CRD) is oriented away from the surface and a disordered N-
terminal 120 amino acid collagen-like-repeat (CLR) is anch-
ored by an N-terminal His6 tag. When force is exerted on a
bound G3-lactose complex the total estimated length, from tip
to surface, of immobilized lactose bound to a properly folded
CRD with fully extended CLR and linkers is 63 nm.
Our goal was to develop a general functionalization
method for force spectroscopy that provides robust, mono-
disperse, highly oriented molecular systems that are stable to
aqueous environments and do not degrade under mechanical
strain. To evaluate our surfaces against these requirements,
200 force/distance curves were generated at each of 20 differ-
ent locations across the sample surface. All rupture forces
[35 pN were considered resolved above noise. Unbinding
events at extensions below 10 nm were considered nonspe-
cific adhesion. Our previously reported data collection rou-
tine was utilized: this approach minimizes contact forces and
ensures that ruptures originate from specific, blockable inter-
actions. Only force/distance curves with contact forces
between 40 and 300 pN were considered.22
Figure 3 depicts a representative force distance/curve for a
bound G3-lactose interaction. The probability of observing a
rupture event (qbind 5 number of rupture-containing pulls/
total pulls) over 200 pulls was 0.41. To confirm that the
observed binding events represented lactose–G3 interaction,
soluble b-methyl lactose (10 mM in PBS pH 7.4) was intro-
FIGURE 3 Representative force vs. extension plot of a typical
rupture event; approach (green) and retract (black) curves shown.
Binding probabilities for unblocked (PBS, pH 7.4) and blocked
(b-methyl lactose, 10 mM in PBS, pH 7.4) systems (inset).
duced into the AFM liquid cell to competitively inhibit inter-
actions between surface-bound G3 and immobilized lactose.
Upon introduction of this blocking agent, qbind decreased to
0.10. Previously, we investigated the probability of binding
between G3 and lactose immobilized through traditional
silane-based methods. Here, we were able to achieve a higher
binding probability (qbind 5 0.41 vs. 0.33), presumably
reflective of more uniform surfaces, and similar blocking
efficiency.
In summary, we have demonstrated a stable and efficient
covalent immobilization of binding partners to a silicon
nitride AFM cantilevers using hydrosilylation chemistry. Our
work provides a general and operationally simple method by
which to covalently immobilize a wide variety of organic and
biological molecules with complete control over both epitope
density and orientation, and eliminates the need for optimi-
zation of each individual system.1,8 The technique utilizes ro-
bust Si C bonds and, in conjunction with our previously
reported technique for the formation of stable, highly ori-
ented monolayers of histidine tagged proteins on silicon,
simplifies both the construction and interpretation of AFM-
based molecular recognition studies.
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Reviewing Editor: Gary D. Glick