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 on the far left of Figure 1, both oligos have bound together to form a quenched sensor complex that is attached to the stretavidin-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 oligo and exposes the fluorophore, thus emitting detectable signal.
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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.
 
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                         <div class="figure-legend">Figure 2: ATP FQ Sensor (attached to Streptavidin-coated plate and unattached in solution). The x-axis is ATP concentration, and the y-axis is fluorescence fold-ratio. Fluorescence was measured using the SpectraMax GeminiXS Fluorescence Plate Reader and normalized against the negative control well for a fold-ratio. Variance was computed across two trials for the sensor inputs ATP, GTP, dGTP, and dGTP (all molecules within two functional groups of ATP). Because of limited NTP stocks, higher concentrations were reserved for the assay with the FQ system attached to a Streptavidin plate.</div>
 
                         <div class="figure-legend">Figure 2: ATP FQ Sensor (attached to Streptavidin-coated plate and unattached in solution). The x-axis is ATP concentration, and the y-axis is fluorescence fold-ratio. Fluorescence was measured using the SpectraMax GeminiXS Fluorescence Plate Reader and normalized against the negative control well for a fold-ratio. Variance was computed across two trials for the sensor inputs ATP, GTP, dGTP, and dGTP (all molecules within two functional groups of ATP). Because of limited NTP stocks, higher concentrations were reserved for the assay with the FQ system attached to a Streptavidin plate.</div>
 
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Revision as of 02:12, 16 October 2016


Stanford-Brown 2016

Introduction

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.
Figure 1: Aptamer-Based Fluorophore-Quencher Small Molecule Biosensing System

Data

Figure 2: ATP FQ Sensor (attached to Streptavidin-coated plate and unattached in solution). The x-axis is ATP concentration, and the y-axis is fluorescence fold-ratio. Fluorescence was measured using the SpectraMax GeminiXS Fluorescence Plate Reader and normalized against the negative control well for a fold-ratio. Variance was computed across two trials for the sensor inputs ATP, GTP, dGTP, and dGTP (all molecules within two functional groups of ATP). Because of limited NTP stocks, higher concentrations were reserved for the assay with the FQ system attached to a Streptavidin plate.
The graphs to the left are the data we collected from our test ATP sensor. 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 nanomolar 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.
References:
1) Tang, Z., Mallikaratchy, P., Yang, R., Kim, Y., Zhu, Z., Wang, H., & Tan, W. (2008). Aptamer Switch Probe Based on Intramolecular Displacement. J. Am. Chem. Soc., 130(34), 11268-11269. http://dx.doi.org/10.1021/ja804119s
2) Nutiu, R. & Li, Y. (2005). A DNA-Protein Nanoengine for “On-Demand” Release and Precise Delivery of Molecules. Angewandte Chemie, 117(34), 5600-5603. http://dx.doi.org/10.1002/ange.200501214