Team:CCU Taiwan/Demonstrate

Confirmation of cis-cleavage

To prove our experiment is feasible, we have to first confirm the sequences that we designed can be detected. So, we designed positive/negative controls of the dsDNA target to ensure that there would be no false positive reactions.

Figure. 1. CRISPR - Cas12a system tested with POS / NEG linear controls

According to Figure 1, we can confirm that in the presence of the dsDNA and the Cas12a, the Cas12a won’t be activated by the negative control. On the other hand, Cas12a can be triggered and cis cleavage of the dsDNA can be carried out in the presence of the positive control plasmid, which is cleaved into two pieces appearing at 1.7 kb and 0.6 kb.

Confirmation of trans-cleavage

To ensure Cas12a protein is able to cleave all the ssDNA and determine sufficient reaction time, we observe the results of the cleavage of ssDNA at different time points.

Figure. 2. CRISPR - Cas12a system treated with POS control and ssDNA, measured at different time points

As you can see in Figure 2, Cas12a protein cleaves almost all the ssDNA by around 40 minutes. Thus, we can confirm that the reaction rate can be detected by the device in real-time.

Measurement of the fluorescent probe, PicoGreen

This experiment has two purposes. First, it will prove the ssDNA can complement successfully, since excitation cannot be triggered if it does not complement successfully. Second, it will show that the fluorescent probe can intercalate into the dsDNA and carry out the excitation.

We designed ssDNA to complement the ssDNA attached to the magnetic beads. After the complementation reaction, we add PicoGreen to intercalate into the dsDNA.

As Figure 3 shows, compared with the fluorescence of the ssDNA attached to the magnetic beads, the conjugated dsDNA has greater fluorescence intensity.

Figure 3. Fluorescence intensity of PicoGreen

On-site test operation

In this project, we provided a device prototype that works with 3 software programs. In this video, we demonstrate the operation of the entire test. First, the operator enters the serial number of the test subject. Then, the device will automatically add reagents, isolate magnetic beads and carry out the fluorescence test. In this case, we also displayed the process of measuring the fluorescence. Finally, the result will be uploaded to a cloud database and displayed on the phone.
*The demonstration is done without a test sample.

Function of the fluorometer

In this video, we demonstrate the operation of our fluorometer using a blank solution and a sodium fluorescein salt sample. Our self-designed fluorometer can easily detect the signals of fluorescence from the sodium fluorescein salt. However, the fluorescence intensity of PicoGreen is lower and cannot yet be detected by our device. Hence, we provide a future improvement plan for the fluorometer, to make it capable of detecting the PicoGreen fluorescence signal.