To verify that our biochemical circuit operates as intended, we will split the secondary amplification into two parts for validation:
We mixed the components of the primary amplification system separately and performed electrophoresis validation:
Figure 1: Electrophoresis analysis of the primary amplification circuit.
This figure indicates that the reaction process can only be completed and the reporter probe RP generated when Rep, Target, Bst, DDSD, F, and RP are all present, thereby achieving primary signal amplification.
We mixed the components of the primary amplification system separately and performed electrophoresis validation:
Figure 2: Electrophoresis analysis of the secondary amplification circuit.
This figure shows that non-specific cleavage of the reporter probe can only be completed when wt, cas12A, crRNA, and RP are all present, thus achieving secondary signal amplification.
To validate that our system can produce a quantitative response to targets, we set up a concentration gradient of the target in the solution and verified it by examining the fluorescence curves generated by the reaction:
Figure 3: Fluorescence curve of target synthesized in the primary amplification system.
Figure 4: Fluorescence curve of target synthesized in the secondary amplification system.
As shown in the figures, our fluorescence curves can effectively distinguish between different target concentrations, indicating the ability to achieve a quan- titative response to the target.
We validated our system using 20 clinical tissue samples. The results show that the system is sensitive enough to detect targets at very low concentrations (9.3 pM) and fast enough that the whole process takes only 10 minutes. The mean values of the cancer samples and normal samples are shown in the figure, showing significant differences with p-values of less than one in a thousand. The ROC curve in the figure has an AUC value of 0.96, showing very high diagnostic accuracy. This is a fluorescence plot taken continuously with our single molecule platform.
- 0 μM:
Figure 5
- 6.25 μM:
Figure 6
- 25 μM:
Figure 7
- 50 μM:
Figure 8
- 100 μM:
Figure 9
Results of numerical analysis for miRNA extracted from cells and tissues:
Figure 10. hsa-mir-141 target test cell sample 1
Figure 11. hsa-mir-141 target test cell sample 2
Figure 12. hsa-mir-141 target test cell sample 3
Figure 13. hsa-mir-141 target test cell sample 4
Figure 14. hsa-mir-141 target test cell sample 5
Figure 15. hsa-mir-21 target test cell sample 1
Figure 16. hsa-mir-21 target test cell sample 2
Figure 17. hsa-mir-21 target test cell sample 3
Figure 18. hsa-mir-21 target test cell sample 4
Figure 19. hsa-mir-21 target test cell sample 5