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:

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:

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:

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.

To achieve higher diagnostic accuracy, we will ultimately use Total Internal Re- flection Fluorescence Microscopy (TIRFM) to perform smFRET measurements on processed blood samples and utilize our self-developed automated analysis program for fluorescence molecular imaging to analyze multiple data points.

Therefore, we also need to verify the feasibility of our system in this fluores- cence detection method and the applicability of our platform to real clinical samples by constructing the following concentration gradient for verification:

Results of numerical analysis for miRNA extracted from cells and tissues: