Surface Chemistry - Designing a Specific Surface

The Challenge

Immobilization of proteins is a major task in the development of diagnostic devices. For our approach we require a chemical surface on a glass slide that allows the immobilization of our antigens at distinct spots. It has to be strong enough to prevent the antigens from being washed away during the iRIf measurement. Therefore, a covalent binding to the surface is preferable. Since we also incubated the coated slide with cell-free expression mix the surface has to selectively bind our target proteins. To fulfill these criteria we worked with different tag systems.

Though there are many different possibilities to bind a protein to a chemical surface, a lot of testing is required in order to find the surface that fits best to ones needs. Therefore, we established different types of chemical surfaces. They vary in their binding specificity as well as in their layer thickness.

Unspecific Surfaces

PDITC surface

Figure 1: Binding meachanism of proteins to the PDITC surface.

To enable the detection of an antibody-antigen interaction in iRIf, purified antigens can be immobilized on the surface of the iRIf slide with PDITC (p-phenyldiisothiocyanate) by pipetting. The two isothiocyanate groups of PDITC serve as a linker for amino groups (see the binding mechanism shown in figure 1). Therefore, it is often used to produce a surface that is able to bind proteins covalently.

In order to coat the iRIf slides with a PDITC surface we first activated them with oxygen plasma. This generates highly reactive hydroxy groups on top of the glass. If a silane is added to the activated glass slide the hydroxy groups bind to the silicium atoms. The silane APTES (3-aminopropyltriethoxysilane) provides an amino group that can link PDITC to the surface. With this system (figure 2) we were not only able to immobilize our purified antigens but also to set up a specific Ni-NTA surface on its basis.

Figure 2: PDITC surface.

Additionally, the chemistry of PDITC can also be used for the immobilization of DNA. We linked the DNA constructs for cell-free expression to an amino group which enabled the binding to PDITC. On the PDMS slide (polydimethylsiloxane) representing one part of our two-slide-system a PDITC surface can be created, as shown in figure 3, thus allowing the immobilization of the modified DNA. This allows the direct expression of proteins in our flow chamber ¹⁾.

Figure 3: Scheme of the APTES/PDITC surface on PDMS.

GOPTS Surface

The GOPTS (3-glycidoxypropyltrimethoxysilane) surface (figure 4) is produced in a very similar manner to the PDITC surface. By plasma activation reactive hydroxy groups are generated on the glass surface. These are able to bind the silane GOPTS. In contrast to the PDITC surface the silane GOPTS carries an epoxy instead of an amino group. The epoxy group is a extremly strained three atom triangle and therefore highly reactive. This allows for the coupling of a variety of different ligands with different functional groups ²⁾. The immobilization of proteins is achieved by the covalent binding of amines, thioles or hydroxy groups of a protein to the epoxy groups on the layer on top of the glass surface. As there is no need for an additional linker (like PDITC) between silane and protein, the GOPTS surface can be produced a lot faster.³⁾.
In our experiments, the GOPTS surface was able to successfully immobilize proteins, however, the PDITC surface showed better binding capacities and worked better as basis for the Ni-NTA/His-tag system (see Results: Binding on Surface), so that we focused on this surface type.

Figure 4: GOPTS surface.

Specific Surfaces With Tag-Systems

For the application of the DiaCHIP, antigens are produced on demand by applying cell-free expression mix to the microfluidic chamber. Therefore, a surface that specifically binds our freshly expressed antigens and not all the other proteins present in the cell-free mix is required. This is why we started developing specific surfaces for different tag-systems.

Ni-NTA - His-Tag System

The Nickel-NTA (Nitrilotriacetic acid) system is commonly used for protein purification. Here, the coordination of the imidazole residue of histidine to nickel ions is utilized to bind proteins with a His-tag to Ni-NTA covered columns reversibly. The proteins can be eluted with imidazole which competes with the Histidine-tag ⁴⁾. The same coordination can be used to fix proteins on a surface. We used this interaction to establish a specific Ni-NTA surface on the glass slide of our DiaCHIP. In order to bind our antigens to the Ni-NTA surface, all of them were cloned with a His-tag.

We established our protocol for the preparation of this Ni-NTA surface based on a PDITC surface. We were able to successfully immobilize cell-free expressed His-GFP with this surface (see Results: Selective Surface). NTA with a lysine residue (AB-NTA) was immobilized on the unspecific PDITC surface and loaded with Ni ions ⁵⁾. The general setup of the surface is shown in figure 5.

Figure 5: Ni-NTA surface.

Halo Surface

The Halo surface is based on the HaloTag system developed by Promega ⁶⁾. The system consists of a 34 kDa protein called the HaloTag that binds covalently to a chloroalkane ligand. The binding of these two parts is fast, highly specific and irreversible due to slight modifications in the reactive center of the HaloTag protein that prevent dissociation. The drawback of this system is the relatively huge tag, which has to be fused to the target protein. The HaloTag can compromise folding, solubility or functionality of the target protein ^{7) 8)} .

To immobilize proteins specifically on a glass surface we coated the surface with the HaloTag ligand (figure 6). The ligand is bound to the surface after oxygen plasma activation - as described for PDITC and GOPTS surfaces. The HaloTag, which is fused to the protein meant to be immobilized, binds to its ligand and therefore attaches the protein of interest to the surface.
We experimented with a variety of different HaloTag ligands, which differed in their length of the alkane chain and the method for surface attachment. The surface consisting of (3-Chloropropyl)triethoxysilane shown in figure 5 exhibited the most promising results. We were able to immobilize Halo-tagged proteins on the surface but the main challenge to overcome for this surface was minimizing unspecific binding (see Results: Halo-Surface). The optimizations necessary for this surface to be specific enough for our purposes could not be realized due to time limitations.

Figure 6: Halo surface.

Spy Surface

The Spy surface is based on the SpyTag/SpyCatcher system. It was created by splitting the CnaB2 domain of the FbaB-protein from Streptococcus pyogenes in two parts, followed by rational modifications of the fragments. The SpyTag binds to the SpyCatcher due to the spontaneous formation of an isopeptide bond within minutes. Due to the covalent nature of this hybridization the binding is irreversible ⁹⁾. In order to immobilize proteins on a surface using the Spy system we fused the target proteins to the SpyTag. Then the purified SpyCatcher was bound to a protein binding surface like GOPTS or PDITC. Because of the binding of SpyTag and SpyCatcher the desired protein is steadily immobilized on the surface.
Due to troubles during cloning and purification of the SpyCatcher we were not able to establish this surface.