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| | + | <div style="margin-top: 40px"><font size=7 color=#51a7f9><b style="color: #51a7f9">Results: Detection on Chip</b></font></div> |
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| − | <div style="margin-top: 40px"><font size=7 color=#51a7f9><b style="color: #51a7f9">Results: Detection on Chip</b></font></div>
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| − | <h3>Portable Fluorescence Detector</h3> | + | <h3>Feasibility of detection on paper</h3> |
| | <p> | | <p> |
| − | Having our RNaseAlert based readout functioning on paper, we created a portable paper-based fluorescence detector to make this readout fit for in field usage. Our detector costs less than 15$, is reusable and can measure time lapses with a sensitivity in the range of a commercial plate reader. To have reproducible measurements we calibrated our detector by measuring dilution series of fluorescein. Our detector therefore measures fluorescence in equivalent fluorescein concentrations. As a first proof of principle we reproduced the plate reader experiments for Cas13a on paper. We were able to measure a time trace of target-activated Cas13a digesting RNaseAlert with our detector. For comparison, we also measured a positive control containing RNase A and a negative control containing only RNaseAlert. The data are displayed in <b>Figure 1</b>.
| + | 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). </p><p> |
| − | </p> | + | 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 <b>(Figure 1)</b>. 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. </p> |
| | <div class="captionPicture"> | | <div class="captionPicture"> |
| − | <img src="https://static.igem.org/mediawiki/2017/e/e2/T--Munich--Hardware_kinetic.png"> | + | <img src="https://static.igem.org/mediawiki/2017/a/a9/T--Munich--DetectionChip_Detection_Circuit.png"> |
| | <p> | | <p> |
| − | <b>Figure 1</b>: Time lapse measurement of Cas13a digesting RNase Alert on paper using our detector. The | + | <b>Figure 1</b>: 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. |
| − | positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are
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| − | connected with lines for the readability. Error bars represent the measurement uncertainties of the detector.
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| | </p> | | </p> |
| | </div> | | </div> |
| − | <p>
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| − | The data show typical curves of enzyme kinetics. It can be seen that RNase A is more active than Cas13a. The negative control shows that our detector was free of RNase contaminations. This proves that our detector is in fact able to quantitatively measure different levels of enzyme activity and can easily distinguish between the negative control and active Cas13a. By assuming that RNase A digested all RNaseAlert, we conclude that 185 nM of RNaseAlert have an equivalent fluorescence to 10 µM fluorescein. Our detection limit for RNaseAlert is therefore around 50 times lower than the limit for fluorescein, which corresponds to a RNaseAlert concentration lower than 10 nM. When characterizing Cas13a, we chose a cut-off of 15% of the total RNaseAlert cleaved to accept a signal as positive, which corresponds to a concentration of roughly 28nM, so our detector limit is good enough for our diagnosis test.
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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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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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