DNA synergistic enzyme-mediated cascade reaction for homogeneous electrochemical bioassay.

Enzyme-mediated cascade reaction is applied to amplify signal and decrease the background because enzyme can catalyze inactive substrates into active substrates to generate the signal. In this work, Au nanoparticles, as signal probe, are used to load DNA probe and ALP for dual signal amplification. Based on enzyme-mediated cascade reaction, a homogeneous biosensor is constructed for bioassay by employing thrombin as target molecule. When the target is present in the solution, ALP catalyzes the PPi into Pi and then reacts with molybdate in conjunction with Pi in the DNA backbone to produce redox precipitates on the surface of the reduced graphene oxide modified electrode with the help of magnetic separation. Compared with the conventional heterogeneous biosensor, the immobilization-free strategy, proposed in this homogeneous biosensor, improves the sensitivity because of its lower steric hindrance. As a result, this biosensor displayed a great sensitivity with a wide linear range from 1 fM to 10 nM and a detection limit of 0.26 fM, providing a promise and easy operating method for various proteins detection.