Ultrasensitive thrombin sensing platform based on three-way junction initiated dual signal amplification

The sensitive and precise quantitative determination of thrombin is important for both fundamental research and clinical diagnostics of hypoxic ischemic encephalopathy because it is a key biological molecule in hemostasis and hemolysis. Herein, we depict a sensitive and label-free thrombin detection approach by taking the advantages of aptamer's superior capability to bind with thrombin and the high efficiency of three-way junction initiated dual signal recycle. In this method, a capture probe which is inserted with an aptamer sequence is designed to specifically identify thrombin molecule and facilitate the signal amplification. Based on the DNA polymerase and endonuclease Nb.BbvCI-assisted chain extension, a large amount of single-strand DNA sequences that can fold into G-quadruplex are produced to specifically recognize commercial fluorescent dye thioflavin T for signal generation. Consequently, the approach exhibits a high detection sensitivity with the limit of detection as low as 768 fM, holding a great promise for detection of thrombin and disease diagnosis in the clinic.