The procedure performed for PRIA was based on his-tagged repressor proteins immobilized on Ni-NTA agarose. Herefore, we constructed a device for protein expression (LacI-His) which we liked to immobilize on Ni-NTA agarose in a reaction tube. Plasmid DNA containing the operator site for specific binding of the repressor was added to the Ni-NTA agarose, unbound plasmid was washed out due to oversaturation of the DNA and the remained plasmid could be eluted by addition of the analyte to the wash buffer. To prove if there is no significant loss of protein, we elute the protein with an imidazole buffer. This imidazole step was to prove that the loss of protein during the procedure was not significant. We proved this by analysis of the samples via SDS-Page (See Figure 3) We analyzed the released DNA in the supernatant with simple gel electrophoresis. On a future test strip the immobilized DNA-protein complex would be provided. So the user just needs to add the potentially contaminated water plus sodium chloride and buffer solution which we would provide in the kit.

Tap water with the analyte alone could not disrupt the binding between the plasmid and the protein. Thus, the user would be required to add a certain amount of salt to the sample. Common sodium chloride is suitable for that purpose, as we tested (See Figure xx). Nevertheless, the solution should be slightly buffered with TRIS-HCl or sodium/potassium phosphate.

To further reduce the time for the assay we also tested whether the complex could be formed prior to addition of the complex to the Ni-NTA agarose. In this case the bond between repressor and plasmid could not be disrupted at all. In the agarose gel DNA was visible after the elution of the protein with imidazole that was performed to verify the presence of the protein at the end of the assay. Furthermore, it could still detected on the Ni-NTA agarose.

We aimed at the development of a paper-based system and optimized the procedure for immobilized protein. So we focused on the approach which was based on repressors fused to a cellulose binding domain (BBa_1321340). We cloned and expressed fusion proteins for our LacI-lacO model system, the repressor ArsR for the detection of arsenic and for the protein BlcR, the repressor of the blc-operon, which we were working with in order to detect date rape drugs. When we added the proteins onto various types of paper, their presence could be confirmed by staining the paper with Coomassie brilliant blue and destaining with destaining solution used for SDS-PAGEs. The binding was unspecific. In this way we are able to detect any protein. Besides, most CBDs bind to microcrystalline cellulose or cotton (Lethiö et al.). We were not able to find a hint about the binding of CBDs to common paper. That is why we focused on the second approach with immobilized DNA.

Based on a method proposed by Araújo et al., DNA was immobilized on filter paper previously activated with p-phenylene-diisothiocyanate (PDITC). To accomplish this, amino-labeled DNA is required. We made some adaptations to the original protocol in order to immobilize dsDNA instead of ssDNA. We omitted a 30 min washing step with 4x SSC buffer, since this serves for the denaturing denaturation of DNA. Furthermore, we hybridized the amino-labeled operator strand with the complementary strand that was Cy3 labeled to be able to quantify the DNA immobilized on the paper. This was performed via a simple annealing at 55 °C of these two oligonucleotides. So by detecting the Cy3-labeled DNA via a simple annealing of two oligonucleotides. So by detecting the Cy3 label via the Typhoon fluorescence scanner, we could be sure, that the immobilized DNA was the correct dsDNA.

We dissolved PDITC, the substance for activation of paper for DNA immobilization, in pure ethanol and DMSO. We compared which solvent is better for the activation. Herefore, we washed the immobilized DNA on paper, which was activated differently, and compared the DNA intensity remained on paper after washing. The immobilization works even better if ethanol is used as a solvent for the PDITC. The loss of signal can be mainly attributed to the washing out of DNA, since a control showed that the decrease of fluorescence of the Cy3 dye due to repeated scanning is minimal. We applied three different liquids for washing:

• an antibody stripping buffer that is normally used to disrupt all protein-protein interactions in a western blot. This resulted in strong blurring of the DNA spots.
• water
• the binding buffer that was normally used in PRIA

Compared to the remaining fluorescence signal on the activated papers the signal on the not activated paper decreases strongly. This indicated, that the immobilization of DNA on activated paper was successful. Additionally, we tested how stable Cy3-labeled DNA is against multiple scan light and washing. Herefore, we quantified the spot intensity of immobilized DNA. As you see in the figures below, the signal did not decrease so much. So the fluorescence signal is stable enough for the strategy.