Team:Stanford-Brown/SB16 BioSensor FQsensor

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 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.

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

Data

Figure 2: ATP FQ Sensor. The x-axis is ATP concentration, and the y-axis is fluorescence. Fluorescence was measured using the SpectraMax GeminiXS Fluorescence Plate Reader.

The graph to the left is 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