Surface modifications

The boundary where the evanescence wave is created in TIRF microscopy is usually the interface between the quartz surface of a microfluidic device and the solution

inside the sample chamber.150 When proteins and other biological molecules are loaded into the sample chamber, there is a tendency for them and contaminants in solution to non-specifically stick to the quartz surface itself. In order to decrease the likelihood that unwanted fluorophores bind to the surface, the quartz is modified with a self-assembled monolayer (SAM) to produce a dense molecular brush that passivates the surface and prevents access to the quartz and therefore any non- specific binding.

A suitable molecule to use in a SAM is made up of an anchoring group, a backbone and a terminal group. The anchoring group should form a strong covalent bond between the tether and surface. The backbone is usually a hydrocarbon chain and will ideally produce a dense closely packed surface as a result of either cross linking or van der Waal’s interactions between chains. The terminal group is chosen to either specifically bind a target molecule to surfaces or to provide an electrostatic repulsion to any charged protein surfaces which also helps minimise non-specific interactions. Usually the composition of the SAM is designed to produce a dispersion of anchors for the specific immobilisation of the target molecule amongst a crowd of repulsive head groups, as depicted in Figure 1.21. Examples of SAM’s include alkylthiols on gold and silver and alkylsilanes on glass and quartz. Chemical modifications of SAM’s can produce monolayers such as alkyls, alcohols, carboxylic acids, amines, azides, or alkynes that can securely attach a plethora of substrates to a surface.151, 152

Figure 1.21: A schematic of a surface being passivated with a SAM.

SAMs can provide a specific binding point for molecules of interest as well as presenting a dense brush of repulsive head groups to minimise non-specific binding of molecules to the surface.

There are many examples of single molecule fluorescence experiments that employ a quartz surface that has been functionalised with silanes, which in turn has been modified to produce a uniform surface of poly(ethylene glycol), PEG.146, 153-155 A common approach has been to use the interaction between biotin and avidin to exclusively bind biomolecules of interest to the PEG surface.146, 156 Although this interaction is non-covalent it still has a dissociation constant approximately 1 fM and therefore provides a secure and specific anchor for adsorbates.157 Figure 1.22 shows how ssDNA can be modified with a biotin at the 5’ terminus and how this can be used to bind to a streptavidin molecule. By cross-linking branched PEG molecules, a denser layer of hydrocarbons is achieved and fewer non-specific binding events occur. This allows decreased levels of contamination on a surface used in single molecule experiments.151, 158

Figure 1.22: ssDNA can be specifically immobilised on a quartz slide through a biotin with an eight carbon linker to the 5’ end.

(a) The crystal structure of biotin which interacts with hydrophobic and hydrophilic residues to produce an extremely strong interaction. (b) The crystal structure of streptavidin bound to four biotin molecules, one per monomer. (c) A cartoon of C8 ssDNA bound to streptavidin via a 5’ biotin modification with an eight carbon linker. (Pdb 1mep and 1jmc)

Other common materials used to modify a surface are lipid molecules.159, 160 Lipid molecules spontaneously form a layer and adhere to a surface as a result of arrangement of the molecules to optimise favourable interactions between hydrophobic chains, hydrophilic terminal groups and solution molecules. The lipid layer is a 2D fluid bound to a surface that can be made less viscous by using different lipids or by the addition of cholesterol. This provides a static anchor for the immobilisation of an adsorbate. One different and elegant solution to reducing the movement of lipid molecules on a surface has been pioneered by Greene.161 A score is made into the quartz slide and a flow is supplied over the surface. As the lipids are

pushed along the surface, the score acts as a barrier and the lipid molecules are trapped in position against the score. Attached to the lipid molecules are strands of DNA which are pushed flat against the surface, producing a curtain of DNA that is within range of an evanescent wave in a TIRF experiment. It is therefore possible to directly view the action of DNA motors on a micrometre scale or the binding of SSB like proteins to view nucleofilaments such as RPA, as shown in Figure 1.23.162

Figure 1.23: DNA curtains imaged using RPA tagged with m-Cherry.

(a) A schematic showing the experimental setup to produce (b) images of individual ssDNA molecules lying flat against the surface of a slide. Figure modified from Gibb et al.163

Single molecule experiments that investigate proteins that have low affinity to their substrate cannot usually be immobilised by a standard tether, since the concentrations of labelled substrate or protein would saturate the camera at the concentrations required to observe binding events. While an ensemble experiment would just increase the concentration of the proteins or substrate to compensate for large dissociation constants, this is unsuitable for single molecule experiments as it

would lead to large numbers of fluorophores that are not participating in binding events, essentially increasing the background fluorescence. Trapping the protein and its substrate inside a vesicle with a volume of a few attolitres effectively increases the concentrations required for binding to occur, but also localises the areas of high concentrations so that individual binding events can still be resolved.154, 159, 160, 164 It provides a more natural environment in which to study single molecules since the protein and substrate are still in solution and do not interact with the surface. This bypasses any issues that may occur with the orientation or position that the adsorbate adopts as it binds to the surface. The vesicles are unilamellar and are typically 50 nm in diameter. The walls of these nanocontainers can be made porous either through hydrophobic/hydrophilic interactions of the membrane with the solution,164 or through the introduction of membrane proteins that act as a molecular transport channel as shown in Figure 1.24.159

Figure 1.24: Schematics of RecA binding to ssDNA inside a porous lipid nanocontainer.

(a) A DMPC lipid nanopore is immobilised on a PEG surface through biotin/neutravidin interactions. Inside the nanopore are RecA proteins and a dual labelled ssDNA. (b) α-hemolysin in the DMPC membrane allow the passage of ATP and ATPγS across the membrane which influences the binding of RecA to ssDNA. Figure adapted from Cisse et al.159