Molecular recognition measurements using AFM are based on the interaction between two molecules. One molecule is attached to the tip of the AFM whereas the second molecule is attached to the sample surface (see groups A and B of Figure 1). Tip functionalization is the several chemical steps that lead to attaching the molecules A to the tip of the AFM.
Figure 1: The AFM tip is extended toward and then retracted from the surface as the deflection of the cantilever is monitored as a function of distance. The retraction 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 attached to the AFM tip, whereas receptor molecules (B) are present on the sample surface. Use of a linker molecule (e.g. PEG) results in a characteristic curved unbinding peak as the linker stretches, enabling easier identification of specific unbinding interactions between A and B (see representative curve in inset).
In figure 1 right half is a force-distance curve. The tip is first brought near the surface until it makes contact, thereby exerting a positive load on the surface. The tip is retracted back and during this action, a downward peak is likely to happen in the retraction curve. This indicates that adhesion has taken place between the sample and the tip. The tip-sample adhesion can be calculated if the deflection sensitivity and spring constant of the cantilever are known.
Adhesion between the tip and the sample is usually observed while utilizing non-functionalized tips. Differentiating between the desired specific interaction and non-specific interactions is often a challenge when a functionalized tip is used for molecular recognition measurements. In order to overcome this challenge, intermediate molecules called linkers or spaces are used between the molecule A and the AFM tip. The linker’s flexibility provides mobility to the ligand molecule to access the binding receptor.
Factors to be Considered During Tip Functionalization
Although several techniques have been utilized to attach molecules to AFM probes, a number of issues must be taken into consideration:
- The selection of a suitable AFM probe is critical, the key factors being sharpness of the tip and the cantilever’s spring constant.
- The selection of tip functionalization chemistry is important since the ligand molecule must be connected to the tip so that the binding strength between the tip and the molecule is more than the interaction between the surface receptor and the ligand molecule.
- Techniques to decrease the ligand’s surface density are crucial for measuring single binding events.
- Factors such as temperature, buffer composition, and pH must be appropriate during measurement and tip functionalization so that the binding activity of the binding molecules is not altered.
Steps Involved in Tip Functionalization
Tip functionalization always begins with a silicon nitride or silicon tip on an AFM probe. Two common approaches, namely amination through a thiol-based self-assembled monolayer (SAM) and direct tip amination by esterification or silanization are used for selecting a starting point for tip functionalization.
Amination Through Esterification and Silanization
The esterification and silanization processes functionalize the probe directly. The silanization reaction takes place between a trichlorosilane group in the silane reagent and a trichlorosilane group in the silane reagent. This leads to the development of an organosilane layer, thereby forming Si-O-Si covalent bonds between the hydrogen bonds and silane molecules and the tip (see Figure 2A). Amination can be carried out through esterification by the reaction of ethanolamine and surface silanol groups.
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.
Animation Through Self-Assembled Monolayer
An SAM is generated through the adsorption of alkanethiol molecules to a gold coated tip as shown in Figure 2C. Gold-coated probes can be recycled by eradicating all the attached molecules. Thiol groups also have a high affinity with gold and ensure in forming a stronger tip-ligand interaction than the ligand-receptor interaction. SAM’s acyl chains generate a close- packed structure that enhances the robustness of tip functionalization. However, this technique needs tip-side gold coated AFM probes, which have large radii and are not universally available.
Introduction of Linker Molecule
The next stage is the introduction of the linker molecules. This stage also offers systematic control of the ligand molecules’ surface density. This can be attained by utilizing mixed SAM that contains two kinds of molecules with various terminal groups. This technique is used for studying the interaction between cyclodextrin and ferrocene molecules by utilizing a mixed SAM. At this stage, if the gold-thiol SAM approach is selected then appropriate reagents such as PEG (polyethylene glycol) / NTA (N-nitrilotriacetic acid) can be used to incorporate linker molecules and form SAM. In Figure 3, the majority of SAM consists of triethylene-glycol-alkyl-thiol whereas the remainder consists of NTA-triethylene-glycol-alkyl- thiol. The tetradentate NTA is likely to generate a hexagonal complex with metal cations. Four chelation bonds are formed with Ni2+ and the two bonds are used for targeting the histidine groups. Thus, a low percentage of NTA-PEG-thiol bonds with the ligand. The PEG-thiol remains inert and limits the density of proteins on the tip surface.
Figure 3: Mixed SAMs are formed on a gold-coated tip. Only a very low percentage of so-called NTA- terminated alkanethiols will establish a chelation with cations, which will also interact with polyhistidine groups belonging to peptides or proteins.
In the other technique, where the silicon nitride or silicon tip is amino-functionalized with ethanoloamine or silanes, a different strategy is used. In this technique, the PEG linker molecule’s one end reacts with the surface amino groups. This allows the other end to bind with the protein. Companies use a variety of heterobifunctional PEG linkers.
Commonly used PEG linkers are shown as Figure 4 and listed in Table 1.
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).
Table 1. Common binding targets and matching reactive groups
||Reactive group on PEG
(reaction requires activation with EDC)
silane treated tip
ethanolamine treated tip
avidin modified proteins
The final stage of tip functionalization is the reaction of the terminal group with amino acids in the protein of ligand molecule. In this stage, known points should not be targeted within the ligand’s functional binding site to avoid changes in the functionality.
This note has analyzed major applications and strategies of tip functionalization. The above-described methods are used for force-volume molecular recognition mapping and single point force measurements. Initial set of results indicate that tip functionalization will be helpful when used with the PeakForce QNM imaging mode, thus enabling higher resolution, faster and a more quantitative molecular interaction mapping.
This information has been sourced, reviewed and adapted from materials provided by Bruker Nano Surfaces.
For more information on this source, please visit Bruker Nano Surfaces.