Study on thrombin detection technology based on trans-cleavage activity of CRISPR-Cas12a system

Abstract:

Objective To provide new strategies for the early diagnosis and treatment of a variety of vascular embolic diseases，like cerebral venous sinus thrombosis（CVST），by constructing a rapid and sensitive thrombin detection technology.Methods The screened aptamer，which could recognize thrombin with high specificity，was used to construct the allosteric probe and to convert the thrombin signal into a nucleic acid signal. After the allosteric probe recognized thrombin，aptamer section in allosteric probe bound to thrombin，exposing the Cas12a active moiety. CRISPR-Cas12a system subsequently recognized the active moiety，and then activated its trans-cleavage property to cleave the surrounding single-stranded DNA fluorescent probes disorderly，resulting in separation of a fluorescent group（Cy3） and a quenching group（BHQ） which were originally labeled at both ends of the fluorescent probe. As a result，fluorescence signal of Cy3 reappeared. The intensity of the generated fluorescent signal was positively correlated with the concentration of thrombin present in the system.Results The fluorescence experiment confirmed that the designed allosteric probe showed high recognitive specificity and stability for thrombin. This method exhibited good detection performance in detection，with a limit of 0.23 pmol/L.Conclusion This method combines the advantages of high sensitivity，low cost and good portability，and can provide strong support for the detection and accurate diagnosis of thrombin-related diseases.