Measuring specificbinding interactions

Functionalizing
the AFM tip
Probing cells forbinding interactions

Application Note #130
Common Approaches to Tip
Functionalization for AFM-Based
Molecular Recognition Measurements
Though most often considered as a nanoscale imaging
technique, atomic force microscopy (AFM) is also
uniquely well suited to measuring force interactions at thepiconewton and nanonewton scale. This capability has been
widely used to measure the nanomechanical properties ofmaterials ranging from polymers to living cells and even tocharacterize single molecules. These measurements arenormally made using common, unmodified AFM probes.
However, another type of measurement is made possibleby the chemical functionalization of AFM probes. This
enables the interaction between two specific molecules
to be investigated. Most commonly, these molecules arebiomolecules and the interactions measured are specificbinding interactions, for instance between an antibody
and an antigen. Such interactions are critical to life andit’s known that many diseases have their cause at themolecular level, sometimes resulting in a malfunctioning ofmolecular recognition and directly impacting cell behavior.
Thus, understanding the specific molecular forces between
individual receptors and ligands can be of great relevancein biomedical research.1-10 AFM force measurements canbe used to estimate the specific unbinding forces between
such molecules with piconewton accuracy by attaching
one of the molecules (referred to as the ligand) to the AFM
tip and probing corresponding receptors on surfaces ofinterest. This technical note reviews common approaches tofunctionalizing AFM tips for this type of research.

PeakForce QNM imageof binding sites

Principles of molecular recognition measurements
AFM molecular recognition measurements are based on
the interaction between two molecules: one attached to
the AFM tip and the other bound to the surface of interest(groups A and B respectively in figure 1). What we refer toas tip functionalization are all the chemical steps that lead tothe fixation of molecules A to the AFM tip. The right half offigure 1 shows a typical approach/retract cycle, also calleda force-distance curve. First the tip is brought down to thesurface until it contacts and exerts a positive load on thesurface (blue trace). Then the tip is retracted and moves
back to its original position (red trace). During this retraction,
a downward peak may occur in the retraction curve thatindicates adhesion between the tip and the sample. If thespring constant (in N/m) and the deflection sensitivity (innm/V) of the cantilever are known, one can calculate themaximum tip-sample adhesion (in N).
Tip-sample adhesion is often observed when using “bare”(non-functionalized) tips, though in this case it corresponds
to a non-specific interaction. When the tip is functionalized
for molecular recognition measurements one challenge
becomes the ability to distinguish between these nonspecific interactions and the desired specific interaction.
For this reason it is common to use intermediate molecules
called spacers or linkers between the tip and molecule A,
which have a key role in helping to enable and recognize

specific interactions. The greater flexibility of the linker
provides the ligand molecule some mobility to best accessthe binding receptor. It also introduces a characteristic
curved adhesion peak (see inset force curve in figure 1)
due to entropic stretching of the linker molecule, whichhelps distinguish the desired specific interactions from
non-specific interactions.

Principle of molecular recognition force measurements

Overview of tip functionalization strategies
Though many different approaches have been used toattach molecules to AFM probes, several basic issues must
always be considered:
1) One must choose an appropriate AFM probe. Usually themost important consideration is the spring constant of thecantilever, but sometimes tip sharpness is also important.
Molecular binding interactions are usually quite small, so
typically very soft cantilevers are used (k < 0.1 N/m). If
the interaction is to be measured quantitatively, the spring
constant must be calibrated using an appropriate method.11
2) One must select the tip functionalization chemistry,
keeping in mind that the ligand molecule must be attachedto the tip such that the binding strength between it and thetip is greater than the interaction between the ligand and thesurface receptor. Covalent binding or strong chemisorption(e.g. gold-thiol) approaches are typically used to ensure that
this condition is met.

Figure 1: The AFM tip is extended toward and then retracted fromthe surface as the deflection of the cantilever is monitored as afunction of distance. The retract part of the curve (in red) will show
any adhesion force between the tip and the sample. In molecular
recognition force measurements, ligand molecules (A) are attachedto the AFM tip, whereas receptor molecules (B) are present on thesample surface. Use of a linker molecule (e.g. PEG) results in acharacteristic curved unbinding peak as the linker stretches, enablingeasier identification of specific unbinding interactions between A andB (see representative curve in inset).

3) In cases where it is desired to measure single binding
events, techniques should be considered that limit thesurface density of the ligand on the tip. These approaches
can reduce the occurrence of multiple molecularinteractions during a single tip-sample interaction.
4) One should consider the use of a linker or spacer
molecule for the benefits it can provide in promoting andrecognizing specific binding events.
5) One must ensure that environmental factors (buffer
composition, pH, temperature) during tip functionalizationand the measurement itself are appropriate, such that thebinding molecules maintain their binding activity.
This technical note will focus on the options available for thetip functionalization itself (i.e., issues 2–4).

Amination of AFM tips

Figure 2: The first step of tip functionalization is generally to introduce amine groups (shown here as “X”) to the tip surface. Three methods
are widely used: A) treatment with silanes; B) esterification with ethanolamine; and C) formation of a SAM using thiol-gold chemistry.
2

Choosing a starting point for tip functionalization
Tip functionalization is almost always a multi-step process,but fundamentally it always starts with a silicon or silicon
nitride tip on an AFM probe. From this point two generalapproaches appear most commonly in the literature: 1)
direct amination of the tip by silanization or esterification,or 2) amination via a thiol-based self-assembled monolayer(SAM) on a gold-coated tip. These approaches are depicted
schematically in figure 2.
The silanization and esterification approaches offer theadvantage that the probe can be functionalized directly, or atleast after simple cleaning of the tip surface, without otherprior surface preparation. The silanization reaction occurs
between silanol groups on the AFM tip and a trichlorosilane
group in the silane reagent, which lead to the formationof an organosilane layer.12 This results in Si-O-Si covalentbonds between the tip and silane molecules and hydrogenbonding interactions between the aliphatic chains of thesilane molecules (figure 2A). In this figure “X” represents
a reactive group (e.g., most commonly amino, NH2) thatwill be used for the next step of the functionalization. Notethat it is also possible to modify this terminal group after
the formation of the SAM, for example, converting a vinylgroups to hydroxyl groups.13 Alternatively, amination may
be performed via esterification by reaction of surface silanolgroups with ethanolamine [HO-(CH2)2 -NH2] (figure 2B).14
Though one of the most commonly used reagentsfor amination of the tip is APTES (3-aminopropyl
triethoxysilane)15, some work suggests that esterification
with ethanolamine or an alternative silane, aminophenyltrimethoxysilane (APhS), may lead a slightly higher densityof amino groups than APTES treatment.16 Though usedeffectively in many examples, the silanization reactions
can be difficult to control reproducibly, in part because thesilane reagents themselves are sensitive to contamination,
easily crosslink, and can have a short shelf life. This canlead to wide variations in the surface density of the reactivefunctionalization groups, which is particularly undesirable
when the experimental strategy calls for close control ofthis parameter in order to favor single-molecule interactions.
In this regard the ethanolamine esterification approachis preferred.
The alternative approach is formation of a SAM via
adsorption of alkanethiol molecules to a gold coated tip(figure 2C). This strategy presents three main advantages.
First, gold is a rather inert metal and gold-coated probes
can be easily recycled by removing all attached molecules(e.g., with detergents and UV-ozone treatments). Second,
thiol groups have a high affinity with gold, forming strong
bonds that help ensure that the tip-ligand interaction isstronger than the ligand-receptor interaction (i.e. issue#2 above). Third, the acyl chains of the SAM form a very

dense, close-packed structure that rigidifies it and increases
the robustness of the tip functionalization. A notabledisadvantage of this technique is that it requires tip-side
gold coated AFM probes, which are not universally available
commercially and have larger tip radii due to the gold layer.
Certain approaches have been found to assist in theformation of reproducible, robust alkanethiol SAMs.
Though both sputtering and thermal evaporation can
produce thin layers of gold (generally less than 100nm), it
is known that the roughness of the gold layer will directly
impact formation and quality of the thiol SAM.17 Thermal
evaporation followed by carefully controlled annealing is
known to produce atomically flat gold crystals (terraces)
whereas sputtering or similar techniques will produce larger,
rougher aggregates. Note that another thin metal layer
between the gold layer and the silicon or silicon nitride tip
is also required for the gold to adhere, for which chromium
is most often used.18 The choice of alkanethiol moleculeis also important, where it is recommended to use chain
lengths between 10 and 18 carbon atoms to enhancehydrogen bonding and thus stability.19 The speed at which
SAMs form is highly dependent on the solvent used, thetemperature, and the length of the acyl chains (where longer
chains assemble faster than short ones).
It should be noted that both of these approaches have beenused successfully in many molecular recognition studies.
In addition to the considerations already presented, one
path or the other may be preferred due to your expertise or
the availability of the necessary reagents and equipment.
Whichever approach is chosen, these initial steps are
crucial for preparing the tip for the following steps thatcomplete the functionalization a linker molecule and thedesired ligand.

Attaching flexible linker molecules to the tip
Though it is possible to attach proteins directly to the tip
at this point using the reactions discussed in the next
section, it is generally recommended to first introduce linker
molecules. In addition to the advantages already discussed
regarding promotion and recognition of specific binding
events, this stage of the functionalization process alsopresents the opportunity to more systematically control
the eventual surface density of the ligand molecules. Thiscan be accomplished by using a mixed SAM containing
two different molecules with different terminal groups. For
instance, a SAM containing 99% of molecule HS-R-X’ and
1% of molecule HS-R-X, where X represents a reactive
chemical group able to bind to the ligand and X’ represents
an inert terminal group. This approach is commonly used,
for instance by Vansco et al. to study the interaction
between ferrocene and cyclodextrin molecules by using amixed SAM made primarily of 2-mercaptoethanol with avery low percentage of thiol-modified ferrocene.20

3

Options at this stage depend somewhat on the initialapproach taken to treating the tip. If the gold-thiol SAM
approach is chosen, then it is possible with the appropriate
reagents to directly incorporate linker molecules at thesame time as formation of the SAM. One possibility is touse a PEG (Polyethylene glycol) / NTA (N-nitrilotriacetic acid)strategy.21 This strategy can be combined with use of mixed
SAMs to control ligand surface density, for instance in an
example by Dufrene et al.22 In this example, the majority(typically 90 to 95%) of the SAM was comprised of atriethylene-glycol-alkyl-thiol [PEG-thiol] and the remainder
was a NTA-triethylene-glycol-alkyl-thiol [NTA-PEG-thiol](see figure 3). The tetradentate NTA can form a hexagonal
complex with metal cations like nickel. Four chelation
bonds are established with Ni2+ whereas the two remaining
ones can be used to target histidine groups (see figure 3)
belonging to poly-His-modified proteins (i.e., the ligand).
Thus only this low percentage of NTA-PEG-thiol binds theligand whereas the PEG-thiol remains inert, thereby limiting
the density of proteins on the tip surface. In the Dufrene etal. example this approach was used successfully to study
the specificity of interaction between an Fv fragment ofanti-lysozyme antibodies and lysozyme molecules attached
to a plane support.22
Following the other approach, where the native silicon orsilicon nitride tip is amino-functionalized with silanes or
ethanoloamine, the strategy is slightly different. In this caseone end of the PEG linker molecule must react with thesurface amino groups, leaving the other end free to bind to
the protein of interest. These PEG molecules are commonlyreferred to as heterobifunctional PEG linkers.23,24
Many companies offer a wide range of heterobifunctional
PEG linkers.25 Detailing all the possible reactive groupfunctionalities and their binding targets is not possible here,
but some of the most commonly encountered ones are
summarized in Table 1 and shown schematically in figure 4.
Because the tip is amino-functionalized, one end of theheterobifunctional PEG linkers should generally have an
NHS-ester reactive group. The reactive group on the otherend of the PEG linker should be chosen based on thedesired ligand binding chemistry options, described in thenext section.
The optimal length of PEG linkers is still debated. Though
it’s generally accepted that the PEG linker should not
be less than 12 carbons, it has been demonstrated very
long chains (>35nm) can cause the binding probability
to significantly decrease. 26 The chosen length may be acompromise forced by the limited selection of commerciallyavailable PEG linkers

4

Principle of hexahistidine peptide tags

Figure 3: Mixed SAMs are formed on a gold-coated tip. Only avery low percentage of so-called NTA-terminated alkanethiols will
establish a chelation with cations, which will also interact withpolyhistidine groups belonging to peptides or proteins.

Attaching proteins and other ligands
The final step of the tip functionalization is completed byreaction of the reactive terminal group (X) with amino acids
within the desired protein or other ligand. Care should betaken, where possible, to avoid targeting known points
within the functional binding site of the ligand in order toprevent changes in its activity or function. The availabilityof reactive amino acids within the protein can be modified
by site directed mutagenesis or direct modification of theprotein with various reagents, but again, none of themshould be involved in the binding site.

A simple strategy for quick tip functionalization
It is possible to avoid these more complicatedfunctionalization steps by using the so-called“glutaraldehyde strategy.” First, the AFM probes must be
NH2 activated as previously explained. Then they are treatedwith glutaraldehyde, which reacts with the amine groups.
The tips are then exposed to the desired ligand, which binds
to the remaining free end of the glutaraldehyde molecule(figure 5). The main drawback of this strategy is that there is
no way to control the density of proteins on the tip surface
and that the protein attachment is done randomly. Thus
only a random percentage of ligands will be oriented insuch a way that they can interact with their corresponding
receptors. The main benefits are the simplicity andgenerality of the method. AFM probes can be functionalized
in half an hour and have been used to generate excellent
results.27-29

Table 1. Common binding targets and matching reactive groups
Binding target

Reactive group on PEG

Bond formed–COOH (carbonxyl)
found in:aspartate
glutamateamine(reaction requires activation with EDC)
orhydroxyl

amide
or
ester

–NH2 (amine)found in:lysinesilane treated tip
ethanolamine treated tip

NHS –esterorcarboxyl

amide
or
ester

–SH (sulfhydryl)found in:cysteinemaleimideorcarboxyl

thio –etherorthio –ester–CHO (carbonyl)
found in:oxidized carbohydrates

hydrazide

hydrazone–OH (hydroxyl)
found in:serinethreoninecarboxyl

ester

Avidinbiotinavitin-biotin bondfound in:avidin modified proteins

Common binding chemistries

Glutaraldehyde pathway

Figure 4: Typical reactions between chemically modified AFM tips
and some amino acids (Asp = Aspartate, Glu = Glutamate, Ser =Serine, Thr = Threonine, Cys = Cysteine and Lys = Lysine).

Conclusions
This technical note reviewed the major strategies andapplications of tip functionalization. Though the discussionof existing techniques here is not exhaustive, it should
provide a good start for those getting started withchemically modified AFM probes and molecular recognition
experiments. The methods described have been applied
widely for single point force measurements and forcevolume molecular recognition mapping. Preliminary
results suggest that tip functionalization will also be usefulwhen used with the PeakForce QNM imaging mode,
which should enable faster, higher resolution, and morequantitative mapping of molecular interactions.

Figure 5: After silanization of the AFM tips, glutaraldehyde is used
as a binding agent to attach proteins of interest to the tip. This
strategy doesn’t guarantee any specificity of interaction since any
amino moiety of the protein can randomly be used for the covalentbond, but it does offer the advantage of being extremely easy toapply, with the entire functionalization occurring in no more than
30 minutes.

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Authors
Alexandre Berquand, Bruker Nano Surfaces Business, Atomic ForceMicroscopy Unit
Ben Ohler, Bruker Nano Surfaces Business, Atomic Force Microscopy UnitFirst page image caption : The surfaces of malaria infected red blood cells
exhibit knob like structures responsible for cytoadherence. This overlayed image ofsimultaneous topography (red color coded) and CD36 binding site mapping (gold color
coded) on the surface of human malaria infected erythrocytes using PeakForce QNM
in fluid clearly shows that the specific CD36 binding sites locate precisely on theknobs where they have higher chance to contact and form intermolecular bonds withendothelial receptors. The image is captured at 2um x 2um scan size, taken by Ang Li,from Singapore-MIT Alliance for Research and Technology in Singapore.

Bruker Nano Surfaces Business
Santa Barbara, CA · USA+1.805.967.1400/800.873.9750
productinfo@bruker-nano.com
6

www.bruker-axs.com/atomicforcemicroscopy.html

without notice. ©2010 Bruker Corporation. All rights reserved. AN130, Rev. A0

1. P. Hinterdorfer, W. Baumgartner, H.J. Gruber, K. Schilcher, and H.
Schindler, “Detection and Localization of Individual Antibody-Antigen
Recognition Events by Atomic Force Microscopy,” Proc. Natl. Acad. Sci.
U.S.A. 93: 3477–81 (1996).

Bruker Nano Surfaces Business is continually improving its products and reserves the right to change specifications

References