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Results: Detection on Chip
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We used a step-wise experimental method to determine if our detection circuit could be detected on chip. We first put our bulk solution on paper and followed the fluorescence in a plate reader, we then lyophilized the Cas13a on paper, and finally we checked the bulk reaction on paper in our self-built detector.
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Feasibility of detection on paper
First, we needed to optimize the paper on which the detection was done. We found that our detection circuit was hindered on nitrocellulose paper, and that the best support was glass fiber filter paper blocked with 5% BSA overnight, rinsed with RNase-free water and dried in the oven (70°C, 20 minutes).
As a first simple test, we pipetted our reaction mixture on the paper, sandwiched the strip in coverslips and pinched the paper in a 3D-printed 96-well plate, to avoid any artefacts due to evaporation, curling of the paper or similar effects. We could then measure the fluorescence in a plate reader. We found the kinetics to be similar to bulk, and the detection limit to be between 10nM and 50nM (Figure 1). This is similar to the detection limit in bulk experiments, but as in this particular set of experiment, we did not have a positive control, we cannot normalise the amount of cleaved RNaseAlert and directly compare the titration curves.
Figure 1: The detection circuit is pipetted on paper, with different concentrations of target RNA. The type of paper and its treatment are crucial to the correct processing of the circuit.
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Lyophilization
We then looked into lyophilizing the protein. As we intend our paper strips to be distributable, we need the detection circuit to resist a range of temperatures and humidity, and lyophilization was found to be the optimal method for this purpose (cite Pardee and Gootenberg). For this purpose, we mixed all the detection circuit components except for the target and applied them on paper, froze the paper at -80°C or in liquid nitrogen, and lyophilized the sample. The target was then pipetted on the paper with water to reach the intended final concentration, the paper was treated as described above, and the fluorescence was tracked in the plate reader. We found that the detection efficiency was lower than for pure bulk experiments, and we could only detect 100nM of target. All other concentrations of target gave a signal below that of the negative control (Figure 2), which can happen as shown before due to the design of our negative control. We think that the decrease in detection limit is caused by inactivation of the Cas13a protein during the lyophilization process. An optimisation of the concentrations of Cas13a and crRNA could help improve our detection limit on lyophilized samples.
Figure 2: Fluorescence intensity at 30 minutes, with lyophilized detection circuit, for different target concentrations.
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Reproducibility
We created a detailed documentation of our detector including derivations of equations, a detailed consideration of measurement uncertainties and a complete description of the assembly of our detector. This should enable other iGEM teams to rebuild and use our detector. As we intend our detector to be easy to assemble and use (see our Measurement page), we are confident that it could be used to characterize fluorescence circuits in a reproducible manner. However, seeing the difference in detection limit between fluorescein and RNase Alert, we think our detector should be calibrated with cleaved RNaseAlert.
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Discussion and Conclusion
We classified our detector by creating a cost vs. sensitivity diagram in Figure 2. We compared commercial detectors, detectors from other iGEM teams and low-cost detectors from publications to our detector. Although the other detectors roughly fall along a line of cost vs. sensitivity. Our detector, however, shows a better sensitivity than all iGEM-built detectors we found, and a lower cost than all other detectors from publications or companies.
Figure 2: Cost vs. sensitivity diagram of several fluorescence detectors. We compared commercially available detectors (orange dots), low-cost detectors from publications1-5 (green dots) and detectors from other iGEM teams (blue dots) to our fluorescence detector (red dot).
The detector is an excellent alternative to commercial fluorescence detectors. It is not limited to our specific application but can be used for the detection of any fluorescence signal in biological or chemical systems. We therefore think that our detector can benefit other iGEM teams and research groups that want to make fluorescence based detection fit for in-field applications.
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References
- Cohen, Limor, and David R. Walt. "Single-Molecule Arrays for Protein and Nucleic Acid Analysis." Annual Review of Analytical Chemistry 0 (2017).
- Nakano, Michihiko, et al. "Single-molecule PCR using water-in-oil emulsion." Journal of biotechnology 102.2 (2003): 117-124.
- Taniguchi, Yuichi, et al. "Quantifying E. coli proteome and transcriptome with single-molecule sensitivity in single cells." science 329.5991 (2010): 533-538.
- Rissin, David M., et al. "Single-molecule enzyme-linked immunosorbent assay detects serum proteins at subfemtomolar concentrations." Nature biotechnology 28.6 (2010): 595-599.
- Pardee, Keith, et al. "Rapid, low-cost detection of Zika virus using programmable biomolecular components." Cell 165.5 (2016): 1255-1266.
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