Team:Freiburg/Results/Immobilization

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Cell-Free Expression of Immobilized DNA

An important advantage of the DiaCHIP is the possibility to ship and store information encoded by DNA. From a DNA template array protein arrays can be produced on demand. To obtain this template, DNA is fixed on a silicone slide forming one side of our microfluidic chamber. Making use of a cell-free expression system, the DNA can then be transcribed and translated into the respective proteins resulting in the final protein array. The coding sequence of the proteins is genetically fused to a tag allowing their binding to a specific surface on the opposite side of the chamber.

Immobilizing DNA on a PDMS Surface

Figure 1: Scheme of the APTES/PDITC surface. The PDMS surface is layered with PDITC. This enables the binding of the amino-labeled DNA.

The core component of the DiaCHIP is the microfluidic chamber composed of a glass slide for protein immobilization and a PDMS (polydimethylsiloxane) flow cell. The silicone PDMS needs to be activated to enable the generation of the DNA template array by binding of the respective sequences. Oxygen plasma is used to first activate the surface of the PDMS slide. This allows to cover it with the silane APTES and finally apply the crosslinker PDITC. The DNA sequence to be immobilized requires an amino group to be covalently immobilized on the PDITC surface. The structure of this surface is schematically visualized in figure 1. The same surface chemistry can be used to immobilize proteins unspecifically.
To obtain an expression cassette for GFP with such an amino group, the target sequence was amplified by PCR using an amino-labeled reverse primer. Additionally, the forward primer was labeled by the fluorescent dye Cy3 to enable visualization by fluorescence microscopy. Successful amplification of the target sequence was verified by agarose gel electrophoresis.

Figure 2: Immobilization of DNA on a PDMS slide. (A) Top view on the slide indicating the spotting pattern. (B) Microarray scanner measurement of Cy3 fluorescence.

Coupling of DNA to the PDMS slide was achieved using a DNA concentration of 25 ng/µl spotted directly onto the slide (figure 2A). The slide was subsequently incubated overnight and the DNA solution was dried afterwards at 60°C. After washing the slide, binding was confirmed by measuring the Cy3 fluorescence in a microarray scanner (figure 2B). The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide, thereby confirming that the spotting of DNA was successful.

Cell-Free Expression of GFP From Spotted DNA

Figure 3: Cell-free expressed GFP confirmed by fluorescence microscopy.

To confirm that DNA was not only bound to the PDMS slide but is also suited for cell-free expression, we flushed the microfluidic chamber described above with our cell-free expression mix. After incubation for two hours at room temperature the expressed GFP was detected using a standard fluorescence microscope (figure 3). More details on the vector design and cloning strategies to generate the needed DNA can be found here.