Difference between revisions of "Team:Stanford-Brown/SB16 BioSensor FQsensor"

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The Fluorophore-Quencher system is a variation on the same idea that the IRES system was predicated on: 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. From there, we ordered an oligo that was complementary to the 5’ end of the aptamer and had a quencher synthesized onto the end of it. When both oligos were allowed to incubate in a streptavidin plate, the result was the complex in Figure 1 part A, where the sensor is attached to a solid surface, and its fluorescence is quenched. When target ligand is introduced, the aptamer undergoes conformational change as it folds around the ligand. This conformational change sterically displaces the quencher oligo and exposes the fluorophore, thus emitting detectable signal.
 
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<div class="col-sm-12 pagetext">The Fluorophore-Quencher system is a variation on the same idea that the IRES system was predicated on: 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. From there, we ordered an oligo that was complementary to the 5’ end of the aptamer and had a quencher synthesized onto the end of it. When both oligos were allowed to incubate in a streptavidin plate, the result was the complex in Figure 1 part A, where the sensor is attached to a solid surface, and its fluorescence is quenched. When target ligand is introduced, the aptamer undergoes conformational change as it folds around the ligand. This conformational change sterically displaces the quencher oligo and exposes the fluorophore, thus emitting detectable signal.
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Revision as of 18:48, 7 October 2016


Stanford-Brown 2016

Introduction

The Fluorophore-Quencher system is a variation on the same idea that the IRES system was predicated on: 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. From there, we ordered an oligo that was complementary to the 5’ end of the aptamer and had a quencher synthesized onto the end of it. When both oligos were allowed to incubate in a streptavidin plate, the result was the complex in Figure 1 part A, where the sensor is attached to a solid surface, and its fluorescence is quenched. When target ligand is introduced, the aptamer undergoes conformational change as it folds around the ligand. This conformational change sterically displaces the quencher oligo and exposes the fluorophore, thus emitting detectable signal.
We tested this system using the ATP aptamer as our sensing domain, with the following observed results:

Data

Sample
We conclude from this data that this sensor worked well with high ATP concentrations, but with further research could almost certainly be optimized to work better at lower concentrations. Advantages of this system include its customizability (theoretically it should work to detect any molecule for which there exists an aptamer) and its ability to retain sensory functionality while bound to a solid surface. Disadvantages include only detecting high concentrations of target ligand (at least currently, though this could theoretically be at least somewhat mitigated with further efforts at system optimization), and the fact that this system is fundamentally synthetic, limiting its ability to be easily and cheaply replicated in vivo like other bio-bricked parts. Instead, expensive new constructs have to be ordered to build each new sensor.