Team:Freiburg/Project/Surface Chemistry

""

Surface Chemistry - Designing a Specific Surface

könnt ihr hier eventuell solche bilder wie in der Präsi verwenden, wo die einzelnen Schichten der Chemie eingezeichnet sind? An die Chemiker, kann man eventuell noch den Reaktionsmechanismus darstellen, wie letztlich ein molekül an der Oberfläche bindet? Es wäre auch cool zu sehen, wie ein Protein oder eine DNA an die Oberflächen binden kann. Z.B. Macht doch an die Schlangenlinie des HIS tags noch ein schematisches Protein dran. Ich glaube dass ein nicht Chemiker sonnst schwierigkeiten hat es zu verstehen.(Stefan)

The Challenge

Immobilization of proteins is a major task in the development of diagnostic devices. For our approach we needed a chemical surface on the glass slide that allowed the immobilization of our antigens at a distinct spot, strong enough to prevent the antigens from being washed away during the iRIf measurement. Therefore a covalent binding would be preferable. However because we use cell-free expression a surface that is able to bind the generated antigens selectively is required. 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 your needs. Therefore we built up different types of chemical surfaces. They varied in their specificity and the layer thickness.

Unspecific Surfaces

PDITC surface

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

To enable a detection of an antibody-antigen-interaction in iRIf, the purified antigens can be immobilized on the surface of the iRIf slide with PDITC (p-phenyldiisothiocyanate). 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 for the production of a surface, that is able to bind proteins covalently.

In order to bind PDITC on the surface of the iRIf slides we first activated them with oxygen plasma. This creates very reactive hydroxyl groups on the glass. If a silane is added to the activated glass slide the hydroxyl groups bind to the silicium atoms. By using the silane APTES (3-aminopropyltriethoxysilane), an amino group that can bind the PDITC to the surface is created. With this surface (figure 2) we were not only able to immobilize our purified antigens but also to establish a specific Ni-NTA surface on its basis.

Figure 2: PDITC surface

Additionally the PDITC chemistry can also be used for the immobilization of DNA. We linked the DNA constructs for cell-free expression to an amino group which enabled binding to PDITC. On the PDMS slide (polydimethylsiloxane), which represents the upper part of our two-slide-system, a PDITC surface can be created, as shown in fig. 3, thus allowing to the immobilization of DNA. This way, we can directly express 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 hydroxyl groups are created on the glass surface. These bind silanes that are added later. In contrast to the PDITC surface the silane GOPTS carries an epoxy- instead of an amino group. The epoxy group is a highly strained three atom triangle and therefore very reactive. This allows for the coupling of a variety of different ligands with different functional groups.4) 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 glass surface. Because there is no need for an additional linker (like PDITC) between silane and protein, the GOPTS surface can be produced much faster. Furthermore, GOPTS surfaces normally exhibit less unspecific binding than other silane surfaces due to the formation of a very dense layer.5)
In our experiments the GOPTS surface was able to immobilize proteins, however, the PDITC surface showed better binding capacities and worked better as basis for the Ni-NTA/His-tag system, so that we focused on this surface type.

Figure 4: GOPTS surface

Specific Surfaces with Tag Systems

In our approach, we produce the antigens we want to test against on demand by cell-free expression. Therefore, a surface that only 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 conquers with the Histidine-tag.2) 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 10xHis-tag.

We established our own protocol for the preparation of this Ni-NTA surface based on a PDITC surface. NTA with a Lysine-residue (AB-NTA) was immobilized on the unspecific PDITC surface and loaded with Ni-ions. The general setup of the surface is shown in figure 5.3)

Figure 5: Ni-NTA surface

Halo surface

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

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. The HaloTag which is fused to the protein we want to immobilize binds to its ligand and therefore attaches the protein of interest on the surface.
We experimented with a variety of different HaloTag ligands, which differed in length of the alkane chain and surface attachment method. The surface consisting of (3-Chloropropyl)triethoxysilane shown in figure 5 showed 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 protein binding. The necessary optimizations for this surface to be specific enough to be used in our project, could not be performed due to time limitations.

Figure 6: Halo surface

Spy surface

The Spy surface is based on the SpyTag/Catcher system. It was created by splitting the CnaB2 domain of the FbaB-protein from Streptococcus pyogenes in two followed by rational modifications of the fragments. The SpyTag binds to the SpyCatcher through the spontaneous formation of an isopeptide bond within minutes. Due to the covalent nature of this hybridization the binding is irreversible. 8) In order to immobilize proteins on a surface using the Spy system we tagged the desired protein with the SpyTag. Then the purified SpyCatcher is bound to a protein binding surface like GOPTS or PDITC. Throughout the binding of SpyTag and SpyCatcher the desired protein is kept at the surface and steadily immobilized.
Due to troubles during the cloning and purification of the SpyCatcher we were not able to test the Spy surface.

References

1) J. Hoffmann et al., 2012. Universal protocol for grafting PCR primers onto various lab-on-a-chip substrates for solid-phase PCR. RCS Adv.
2) J. A. Bornhorst et al., 2000. ] Purification of Proteins Using Polyhistidine Affinity Tags. Methods Enzymol.
3) Y. Asano et al., 2006. Application of an enzyme chip to the microquantiWcation of L-phenylalanine. Anal. Biochem.
4) M. Bañuls et al., 2013. Chemical surface modifications for the development of silicon-based label-free integrated optical (IO) biosensors: A review. Analytica Chimica Acta.
5) J.Phieler et al., 2000. A high-density poly(ethylene glycol) polymer brush for immobilization on glass-type surfaces. Biosensors & Bioelectronics.
6) LP Encell et al., 2012. Development of a dehalogenase-based protein fusion tag capable of rapid, selective and covalent attachment to customizable ligands. Curr Chem Genomics.
7) C. England et al., 2015. HaloTag Technology: A Versatile Platform for Biomedical Applications. Bioconjugate Chem.
8) B. Zakeri et al., 2012. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion. Proc Natl Acad Sci U S A.