Team:Stanford-Brown/SB16 BioSensor FQsensor


Stanford-Brown 2016

BioSensor team member Mike introduces the Fluorophore-Quencher subproject

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.

Biodevice Proof of Concept

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.
Figure 3: Biodevice of ATP Aptamer Fluorophore-Quencher System with Cellulose Cross-linker. Fluorescence was also quantified on the Typhoon scanner for characterization purposes. The depicted timelapse gives qualitative proof that both the FQ sensor and Cellulose Cross-linker are working in tandem, as evidenced by the drastic difference in fluorescence between the experiment and controls, amplified from day 1 to day 7.
Figure 4: A schematic of the biodevice
Figure 5: In order to confirm that our biodevice was working, we measured the fluorescent activity on a cellulose sheet over the course of a week. Each day 5x 1mL 10mM ATP was pipetted onto each 9-well grid to simultaneosly wash off unattached sensors and trigger attached sensors. Fluorescent images were analyzed with Typhoon Scanner (Absorption=495nm, Emission=525nm) and processed quantitatively with Python. We understand that photobleaching and heterogeneous mixing across the wells are uncontrolled for, but this pilot study gives way for proof of concept embedded abiotic nucleic-acid based sensors.
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