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

• Published in: Sensors and actuators. B, Chemical Chemical, 2023-01, Vol.374, p.132709, Article 132709
• Main Authors: Ji, Zhirun, Shang, Zhaoyang, Sohail, Muhammad, Wang, Pei, Li, Bingzhi, Zhang, Xing, Chen, Guoxiang
• Format: Article
• Language: English
• Subjects: Amino acids; Antidotes; Biosensors; CRISPR; CRISPR/Cas; Functional nucleic acids; Heparin; Natural & organic foods; Nuclease; Pharmaceutical analysis; Preservatives; Programmable nuclease; Protamine; Response time
• ISSN: 0925-4005; 1873-3077
• DOI: 10.1016/j.snb.2022.132709

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. •The inhibitory effect of protamine on CRISPR/Cas12a activity was reported.•The affinity of protamine-ssDNA binding is higher than that between ssDNA and Cas12a-crRNA.•Highly sensitive and selective protamine detection was realized.