The Fluorophore-Quencher (FQ) system operates on the basic biosensing platform: use an aptamer as a sensing domain and something else as an expression platform to signal that the aptamer had bound its target. In the case of the fluorophore-quencher, we synthesized a fluorescent molecule (fluorophore) directly onto the 5’ end of the aptamer, and a biotinylated the 3’ end. Unlike IRES, the FQ system is based on DNA, which is as versatile for folding for binding, and it is a much more stable biomolecule, allowing it withstand the harsher conditions of UV radiation in space. From there, we ordered an oligonucleotide that was complementary to the 5’ end of the aptamer and had a quencher synthesized onto the end of it. When both strands were allowed to incubate in a Streptavidin-coated plate, the result was the complex on the far left of Figure 1, both oligonucleotidess have bound together to form a quenched sensor complex that is attached to the Streptavidin-coated surface. When target ligand is introduced, the aptamer undergoes conformational change as it folds around the ligand. This conformational change sterically displaces the quencher oligonucletoide and exposes the fluorophore, thus emitting detectable signal.

Once we were able to achieve a working biosensor prototype, our next step was to utilize this in a scenario applicable to embedding in our bioballoon. We decided that cellulose sheets would serve as a satisfactory surface for proof of concept, knowing that later down the road, we could use different binding domains for latex, elastin, collagen, or p-aramid fibers. Conveniently the 2014 Stanford-Brown-Spelman iGEM team had created a Cellulose Cross Linker BioBrick BBa_K1499004 that needed further characterization. We filled this need by purifying the protein (validating the presence of its HisTag), and binding our fluorophore sensor to the linker protein (with quencher). We then distributed this incubated concoction to wax-coated cellulose filter paper to measure the binding activity to the paper over a week. Initially the mixture was applied to the paper and per recommendation 2-3 days is necessary for the cellulose binding domain to take effect. After this initial binding period, 5 x 1mL of milliQ water (with 1mM ATP) were washed over each 9-well sample each day for a week. The positive control had the FQ system, but no linker. The negative control had no FQ either.