Binding-induced nicking site reconstruction strategy for quantitative detection of membrane protein on living cell

Here, a binding-induced nicking site reconstruction strategy has been fabricated for quantitative detection of membrane protein on living cell. Taking protein tyrosine kinase-7 (PTK7) as model analyst, first, an aptamer probe was designed with an aptamer sequence, a trigger sequence and a nicking site. In the absence of PTK7, the aptamer sequence could partially hybridize with the trigger sequence, forming a stem-loop structure. And the two complementary sequences of the nicking site were separated, which could not be recognized by nicking enzyme. In the presence of PTK7, the aptamer probe and PTK7 binding caused the reconstruction of the probe, leading to the hybridization of the two separated nicking site sequences. Then, the nicking site could be identified and nicked, yielding the release of the trigger sequence. Next, the trigger sequence could initiate the homogeneous cascade amplification, producing multiple G-quadruplex structures. By inserting the N-Methyl Mesoporphyrin IX (NMM), enhanced fluorescence signal could be acquired. Through the binding-induced nicking site reconstruction, the trigger sequence could be released on the surface of living cell and became more accessible. By combining the cascade rolling circle amplification (RCA) and hybridization chain reaction (HCR), high sensitivity was achieved with a detection limit of 0.3 fM. Moreover, Quantitative assay of PTK7 on living cancer cells and normal cells were performed, suggesting that the proposed method was sensitive enough to detect changes in PTK7 expression. Thus, this strategy provided a novel and reliable method for membrane protein expression assay on living cell.