Allosteric kissing complex-based electrochemical biosensor for sensitive, regenerative and versatile detection of proteins

Herein, an allosteric kissing complex-based electrochemical biosensor was ingeniously proposed for the simple, sensitive, regenerative and versatile detection of proteins. Two hairpins (Hp1 and Hp2) were designed and the Hp1 was immobilized on the electrode surface, which could form a kissing complex with Hp2 through the apical loop-loop or kissing interaction of the RNA-RNA base sequences. The Hp2 possesses the appended single-stranded tails on each end, which hybridize with the recognition element-conjugated DNA strands to construct a protein responsive switch of Hp2 scaffold. After kissing complex formation between the Hp2 scaffold and the immobilized Hp1, the streptavidin-labeled alkaline phosphatase (SA-ALP) can be introduced onto the electrode surface for the generation of electrochemical signal. In the presence of target protein, its binding to the recognition elements linked onto the Hp2 scaffold endows the steric strain to open the Hp2 stem, propagated by the disruption of the kissing complex structure, resulting into a decreased electrochemical signal related with the protein quantification. Also, the Hp1 immobilized electrode can be directly regenerated after protein-induced kissing complex dissociation. The current kissing complex-based electrochemical biosensing strategy can be easily extended for the detection toward different protein targets of interest by simply changing the recognition elements conjugated onto the Hp2 scaffold. The sensitive and selective detection toward proteins could be achieved with the detection limits toward Anti-Dig antibody and thrombin of about 1 ng/mL and 10 pM, respectively. The developed kissing complex-based protein biosensing strategy should be a beneficial supplement in current biosensor field, providing a promising means for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.