|
|
|
|
|
|
|
|
|
|
|
Results: Detection on Chip
|
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$, 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 with on chip. 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 RNase Alert with our detector. For comparison, we also measured a positive control containing RNase A and a negative control containing only RNase Alert. 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 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 which corresponds to a RNaseAlert concentration lower than 10 nM. To increase the reproducibility we should calibrate our detector again with cleaved RNaseAlert.
|
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.
|
Discussion and conclusion
We classified our detector by creating the cost vs. sensitivity diagram in the figure below. We compared commercial detectors, detectors from other iGEM teams and low-cost detectors from publications to our detector.
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.
|
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.
|
|
|
|
|
