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Surchem methods

PDITC surface

To enable a detection of antigens with iRIf, these anntigens have to be immobilized on the surface of the iRIf surface. PDITC (p-phenyldiisothiocyanate) can link amino groups through its two isothiocyanate groups and therefore is often used to produce a surface that can bind proteins. To get the PDITC on the surface of the iRIf slides we first plasma activated them. 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 atom. To get an amino group which could bind to PDITC to the surface we used the silane APTES (3-aminopropyltriethoxysilane). With this surface we weren’t only able to immobilize our purified antigens but also were able to establish a specific Ni-NTA surface on its basis.
Additionally the PDITC chemistry can also be used for the immobilization of DNA. We used this to immobilize the DNA on PDMS (polydimethylsiloxane) slides, which builds the upper part of our two-slides-system.¹⁾

PDITC surface

Ni-NTA - His Tag system

The Nickel-NTA (Nitrolotriacetic acid) system is very 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 reversible to a Ni-NTA covered columns. The proteins can be eluted with Imidazole which conquers with the Histidine-tag.²⁾ The same coordination can be used to fuse proteins to a surface. We used this to produce specific surfaces on the glass slide of our DiaChip. In our approach we are producing the antigens against which we want to test on demand by cell free expression. After expression we want only our antigens bound to the surface and not all the other proteins present in the cell free mix. This is what we need the specific surface for. All our antigens are cloned with a 10xHis-tag, so that they bind to a Ni-NTA surface.
We managed to establish our own protocol for the preparation of this Ni-NTA surface based on a PDITC surface. On the unspecific PDITC surface NTA with a Lysine-residue (AB-NTA) was immobilized and loaded with Ni-ions, the general setup of the surface is shown in the graphic.³⁾

Ni-NTA surface

info

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.