PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity

Abstract Developing rapid and highly sensitive methods for alkaline phosphatase (ALP) activity analysis is significant for the clinical diagnosis and treatment of diseases. Here, a Pyrococcus furiosus Argonaute ( Pf Ago)-based biosensor is presented for ALP activity detection in which the ALP-catalyzed hydrolysis of 3’-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon -mediates the fluorescent reporter cleavage as a guide sequence of Pf Ago. Under the dual amplification mode of Pf Ago-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied in ALP activity analysis with a detection limit (LOD) of 0.0013 U L −1 (3σ) and a detection range of 0.0025 U L −1 to 1 U L −1 within 90 min. The Pf Ago-based method exhibits satisfactory analytic performance in the presence of the potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid, highly sensitive, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.