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Results: Detection on Chip
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Portable Fluorescence Detector
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$ and can measure time lapses with a sensitivity competitive to a commercial plate reader. To have reproducible measurements we calibrated our detector by measuring dilution series of fluorescein with our detector. 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 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 the figure below.
Time lapse measurement of Cas13a digesting RNaseAlert on paper using our detector. The
positive control contains RNaseA and RNaseAlert. The negative control contains only RNaseAlert. Data points are
connected with lines for the convenience of the eye. Error bars represent the measurement uncertainties of the detector.
The data show typical curves of enzyme kinetics. It can be seen that RNaseA is more active than Cas13a. The negative control shows that our detector was free of RNase contaminations. This shows 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 which corresponds to a RNaseAlert concentration lower than 10 nM. To increase the reproducibility we should calibrate our detector again with cleaved RNaseAlert.
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Reproducibility
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Discussion and conclusion
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References
- Gootenberg, J. S., Abudayyeh, O. O., Lee, J. W., Essletzbichler, P., Dy, A. J., Joung, J., ... & Myhrvold, C. (2017). Nucleic acid detection with CRISPR-Cas13a/C2c2. Science, eaam9321.
- Esfandiari, L., Wang, S., Wang, S., Banda, A., Lorenzini, M., Kocharyan, G., ... & Schmidt, J. J. (2016). PCR-Independent Detection of Bacterial Species-Specific 16S rRNA at 10 fM by a Pore-Blockage Sensor. Biosensors, 6(3), 37.
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