Highly sensitive electrochemical nuclear factor kappa B aptasensor based on target-induced dual-signal ratiometric and polymerase-assisted protein recycling amplification strategy

In this work, an amplified electrochemical ratiometric aptasensor for nuclear factor kappa B (NF-κB) assay based on target binding-triggered ratiometric signal readout and polymerase-assisted protein recycling amplification strategy is described. To demonstrate the effect of “signal-off” and “signal-on” change for the dual-signal electrochemical ratiometric readout, the thiol-hairpin DNA (SH-HD) hybridizes with methylene blue (MB)-modified protection DNA (MB-PD) to form capture probes, which is rationally introduced for the construction of the assay platform. On the interface, the probes can specifically bind to target NF-κB and expose a toehold region which subsequently hybridizes with the ferrocene (Fc)-modified DNA strand to take the Fc group to the electrode surface, accompanied by displacing MB-PD to release the MB group from the electrode surface, leading to the both “signal-on” of Fc (IFc) and “signal-off” of MB (IMB). In order to improve the sensitivity of the electrochemical aptasensor, phi29-assisted target protein recycling amplification strategy was designed to achieve an amplified ratiometric signal. With the above advantages, the prepared aptasensor exhibits a wide linear range of 0.1 pg mL−1 to 15 ng mL−1 with a low detection limit of 0.03 pg mL−1. This strategy provides a simple and ingenious approach to construct ratiometric electrochemical aptasensor and shows promising potential applications in multiple disease marker detection by changing the recognition probe.