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Attachment of Molecules to Desired Surfaces

Studies of the adhesive properties in living cells require the application of protocols that are used to modify surfaces of both cantilevers and substrates. The group of molecules specifically recognized by the cell surface receptors includes almost all molecules present in nature. The molecules of interest can be broadly grouped into (1) nucleic acids (DNA, RNA), (2) proteins (antibodies, enzymes, and receptors), (3) small molecules (e.g., peptides, metabolites), and (4) other biomolecules, like carbohydrates or lipids. Their attachment to various surfaces depends strongly on their surface properties, on the chemistry of substrate, and/or on the liquid medium composition.

AFM probe functionalization

Such molecules like proteins exhibit different structural properties, manifesting in highly heterogeneous hydrophobicity, and charge distribution. This can make their deposition on the AFM probe very difficult. In particular, for all of them, it is essential to preserve biological activity during the experiment. Additional complications can arise when a correct orientation of the bound molecule is required. The immobilization protocols used to attach molecules employ both adsorption and covalent binding. Although many various protocols have been developed, several basic issues always have to be considered:

  • (1) Choice of an appropriate AFM probe in terms of a spring constant value (due to detection limit described in Chapter 3); a tip sharpness, surface chemistry (e.g., silicon or silicon nitride or gold coating).
  • (2) Knowing the surface chemistry of the AFM probe and the properties of the molecule to be attached, the corresponding functionalization procedure should be selected. It is important to remember that during force-induced unbinding experiments, it is desirable that the unbinding should occur between a pair of two interacting molecules that are studied (the strength of the molecule attachment to the tip surface should be larger than the interaction between the studied molecules);
  • (3) The density of molecules on the surface of the AFM probe should be considered, since low concentrations can not only reduce multiple interactions but also eliminate the occurrence of single unbinding events.
  • (4) The decision whether to use or not polymeric spacers should be taken after considerations whether spacer may help to recognize specific unbinding events.
  • (5) During the AFM probe functionalization (and also measurements), environmental factors such as buffer compositions, pH or temperature should be maintained to assure binding activity unchanged.
  • (6) When molecules are needed to be attached on a support like mica or glass surface, their roughness should be smaller than the diameter of molecules.

The choice of chemical reagents depends strongly on binding targets (several examples are provided in Table 5.1).

Table 5.1 Common binding targets used in the AFM probe functionalization

Target functional

Reactive

group

Found in

group

Bond type

  • -COOH
  • (carboxyl)

Aspartate (AA) Glutamate (AA)

NH2

OH

Amide

Ester

  • -hn2
  • (amine)

Lysine (AA)

Silane treated surface Ethanolamine treated surface

NHS-ester

Carboxyl

Amide

Ester

Target functional group

Found in

Reactive

group

Bond type

  • -SH
  • (sulfhydryl)

Cysteine (AA) Thiol

Maleimide

carboxyl

Thio-ester

Thio-ether

  • -CHO
  • (carbonyl)

Oxidized

carbohydrates

Hydrazide

Hydrazone

  • -OH
  • (hydroxyl)

Serine (AA) Theronine (AA)

Carboxyl

ester

Avidin

Avidin modified proteins

Biotin

Avidin- Biotin bond

AA: amino acid.

Actually, there are two popular approaches that either directly or indirectly attach molecules to the surface of the AFM tip (Fig. 5.12).

Two ways of the AFM tip functionalization

Figure 5.12 Two ways of the AFM tip functionalization. The desired molecules are either (a) directly attached to the probe surface through cross-linking agents as glutaraldehyde (GL), or (b) indirectly through a polymeric spacer (e.g., PEG).

The first step in the approach enabling the direct immobilization of molecules to the AFM probe, is the surface silanization with 3-amino-propyltriethoxylsilane (APTES[1]), which enriches the surface with amino groups [36]. Then, the silanized surface is activated using a cross-linking agent, such as glutaraldehyde, which binds a protein through amine group (Fig. 5.12a). Such a way of AFM tip functionalization results in a randomly attached protein molecules without knowing and controlling their density and orientation. Thus, only a small percentage of molecules will be oriented in such a way that they can interact with the corresponding molecule (receptor) present on a cell surface. The main advantages of this approach are the simplicity and generality. The AFM probes can be functionalized during a relatively short time (less than 2-3 h), which provides always freshly prepared AFM probes used during the measurements. The main drawback is a lack of protein flexibility, which leads to low efficiency of measurements and difficulties in identification of the specificity interactions, since very often non-specific forces are difficult to be excluded from the analysis.

To provide better flexibility of the interacting molecules, the polymeric spacers (linkers) can be applied (Fig. 5.12b). The attached spacers provide the molecules the freedom to move around and also prevents their denaturation [39]. Usually, such spacers carry two different functional groups, namely: a NHS group reacting with amine groups present on AFM probe surface and PDP (2-pyridyldithiopropionyl) or vinyl sulfone groups that covalently bind to thiols present in the desired molecule (e.g., in proteins). Polymeric spacers can be introduced, for example, either by chemisorption of alkanethiols on gold or by covalent coupling of polyethylene glycol (PEG) to silanes. The degree of molecules freedom can be enhanced by attaching, for example, recombinant histidine-tagged proteins onto an AFM tip coated with nitrilotriacetate (NTA)-terminated alkanethiols [40]. The optimal length of PEG spacers is still under debate [41-43]. Thus, it should be adjusted to molecular complex studied. It has been demonstrated that very long chains (above 35 nm [42]) can decrease significantly the binding probability and, simultaneously, the efficiency of the measurements.

The molecules immobilization onto specific surfaces like mica or glass involves described two major categories of mechanisms: adsorption and covalent binding.

Adsorption relies on non-covalent interactions—mainly electrostatic, van der Waals, and dehydration of hydrophobic interfaces [43]. It has a purely physical nature and therefore displays varying levels of reversibility. The adsorption of proteins depends on two main features: their surface charge and their hydrophobic domains. Both properties enable a certain control of protein deposition; however, they can result in randomly oriented molecules. The electrostatic adsorption seems to be sufficient to assure a relatively strong attachment, but it does not have permanent nature and it can be strongly affected by changes of solution pH and ionic strength. Therefore, only a limited number of proteins can be immobilized in this manner. When the hydrophobic attraction is chosen as a main source of adsorption, stronger and less reversible interaction is expected. On the other hand, it may result in loss of functional activity due to partial denaturation, as the protein unfolds to expose hydrophobic interior portion to the hydrophobic surface. Such way of molecule immobilization results in their random orientation and relatively weak attachment, which may significantly elongate the time of measurement.

The covalent binding of specific molecule functional groups to functionalized surfaces, by definition involves formation of essentially irreversible chemical bonds between the molecule and the substrate surface. However, in many cases the covalent binding is enabled only after additional functionalization of the surface and/or biomolecule [44, 45]. A variety of side groups can be easily used for covalent binding—most common ones are amino, carboxylic, hydroxyl, and thiol groups. Thus, this resulted in many strategies for cross-linking of available functional groups. Most of them use specific cross-linkers, for both attachment and physical separation of protein from the surface, thereby allowing larger fraction of the protein functional domains to be exposed to the buffer. Covalent binding generally produces a higher concentration of proteins than the adsorption. Proteins can be also better oriented by additional techniques, such as

  • (1) use of antibodies that bind proteins leaving the antibody binding sites free;
  • (2) use of biotinylation which enriches proteins with the binding site specific to streptavidin-coated surface;
  • (3) cysteine thiol production in the protein fragment far from the binding site, allowing its deposition on gold coated surface;
  • (4) use the sugar molecules that bind to the oligosaccharide’s moieties of proteins.

In biological applications, the most common AFM probes are made of silicon or silicon nitride. Biomolecules are usually immobilized on glass, mica, and gold. Therefore, immobilization requires a development of an appropriate protocol of the attachment. Such way of molecules immobilization results in a very strong attachment and, in certain instances, enables the oriented molecule deposition.

  • [1] When buying APTES, it is very important to ask the AFTES provider for thefresh compound since the water arriving from the air can lead to deactivate theformation of amino groups on the cantilever surface. The solution should be storedin the fridge with a cap wrapped with parafilm.
 
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