A CRISPR-enabled fluorometric biosensor for the sensitive detection of heparin antidote protamine based on programmable nuclease Cas12a

Protamine, an arginine-rich polycationic peptide, is a natural food preservative and the only approved antidote to heparin overdose. The detection of protamine is meaningful; however, the detection of protamine is difficult due to the lack of aromatic amino acids in its composition . Herein, we proposed a fluorometric biosensor for the sensitive detection of protamine based on the protamine-mediated regulation of CRISPR/Cas12a collateral cleavage. In the absence of protamine, the DNA probe specifically hybridizes with the Cas12a-crRNA complex, causing the activation of Cas12a. In the presence of protamine, positively charged protamine tightly binds to negatively charged DNA probes through electrostatic interaction , so that the DNA probe cannot hybridize with the Cas12a-crRNA complex. Using a short ssDNA modified with fluorophore and quencher as a reporter, the information of protamine concentration is transduced into DNA information and sensed in a signal turn-off manner. The linear range of this method in protamine detection is 0.04–3.2 μg/mL, yielding a detection limit of 0.03 μg/mL, and the entire sampling-to-response time is about 1 h. This biosensor provides a rationale for using the programmable nuclease Cas12a to detect protamine, and further advances the application of CRISPR technology in bioanalyses.