Step 3: Fluorescent Signaling
The addition of a cleavable signaling peptide creates a highly amplified signal.




The key to detection is the addition of a signaling peptide. Intact, it does not provide any fluorescent signal. However, it can be cleaved by free protease which activates the fluorescence. 




The combination of the rapid self-amplification of the proteases and simultaneous peptide cleavage creates a single discrete signal, represented above.