CRISPR/Cas12a-regulated homogeneous electrochemical aptasensor for amplified detection of protein

Electrochemical aptasensors have been wildly used in the detection and quantification of protein biomarkers, but their practical applications are limited by inefficient signal amplification and laborious and time-consuming probe surface-immobilization procedure. Here, we combine the homogeneous electrochemical aptasensor with the CRISPR/Cas12a strategy to overcome such limitations. The binding-induced DNA strand displacement (BIDSD) strategy is designed to transform thrombin-aptamer interaction into nucleic acid output, which further triggers the rolling circle amplification (RCA) to regulate the deoxyribonuclease activity of CRISPR/Cas12a. Based on the difference in affinity of graphene to single-stranded DNA and double-stranded DNA, this electrochemical aptasensor is free of probe surface-immobilization procedure, which greatly simplifies operation steps and facilitates its flexibility. By integrating BIDSD with two amplification strategies of RCA and CRISPR/Cas12a, this electrochemical aptasensor achieves specific and sensitive detection of thrombin with a calculated limit of detection of 1.26 fM, which provides a paradigm for the sensitive detection of protein biomarker.