Protease Chain Reaction (Pro-CR)

The primary goal of Pro-CR is to combine single-molecule level detection with secondary signal amplification, resulting in a diagnostic technology that delivers 1.) unparalleled sensitivity and 2.) precise measurement.


Step One: 
Sensitive, Linear Detection
Step Two: 
Initial Amplification of Signal
Step Three: 
Exponential Self-Amplification of Signal

 
 
A small amount of trigger molecule is added to a solution of protein complex A. Protein complex A consists of 1.) a protease and 2.) a complementary inhibitor with a secondary site specific to the target molecule. Each trigger molecule catalyzes the separation of the inhibitor from the protease in complex A. If complex A = IPa (inhibitor bound to protease), and T=trigger molecule:

1.) IPa + T --> TIdeg + Pa

The trigger molecule also degrades the inhibitor. Therefore, the amount of uninhibited protease A is linearly correlated to the number of trigger molecules initially added to solution.
  


 The uninhibited protease A is then added to a solution of protein complex B. Protein complex B also includes a (different) protease, bound to its complementary inhibitor; however, this inhibitor has a secondary site specific to both protease A and protease B. 

2.) Pa + IPb --> Pa + Pb + Ideg

Each protease A releases many protease B, and degrades the B inhibitor, so the result is an exponential increase in uninhibited protease B.
  
 
 
The final step is exponential self-amplification of protease B. Since the inhibitor component of protein complex B is specific to its own protease (in addition to protease A, as discussed above), protein complex B is capable of self-amplification. 

3.) Pb + IPb --> 2Pb + Ideg

Steps two and three in combination cause an explosion of biofluorescent signal. The time at which this occurs is precisely correlated to the original concentration of trigger molecule, at a level of attomolar accuracy.