Proximity hybridization triggered hybridization chain reaction for label-free electrochemical homogeneous aptasensors

A label-free homogeneous electrochemical aptasensor was developed for detection of thrombin based on proximity hybridization triggered hybridization chain reaction induced G-quadruplex formation. Thrombin promoted the formation of a complex via the proximity hybridization of the aptamer DNA strands, which unfolded the molecular beacon, the stem part of molecular beacon as a primer to initiate the hybridization chain reaction process. Thus, with the electrochemical indicator hemin selectively intercalated into the multiple G-quadruplexes, a significant electrochemical signal drop is observed, which is dependent on the concentration of the target thrombin. Thus, using this"signal-off" mode, label-free homogeneous electrochemical strategy for sensitive thrombin assay with a detection limit of 44 fM is realized. Furthermore, this method also exhibits additional advantages of simplicity and low cost, since both expensive labeling and sophisticated probe immobilization processes are avoided. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis.