Wash-Free, Sandwich-Type Protein Detection Using Direct Electron Transfer and Catalytic Signal Amplification of Multiple Redox Labels.

Direct electron transfer (DET) between a redox label and an electrode has been used for sensitive and selective sandwich-type detection without a wash step. However, applying DET is still highly challenging in protein detection, and a single redox label per probe is insufficient to obtain a high electrochemical signal. Here, we report a wash-free, sandwich-type detection of thrombin using DET and catalytic signal amplification of multiple redox labels. The detection scheme is based on (i) the redox label-catalyzed oxidation of a reductant, (ii) the conjugation of multiple redox labels per probe using a poly-linker, (iii) the low nonspecific adsorption of the conjugated poly-linker due to uncharged, reduced redox labels, and (iv) a facile DET using long, flexible poly-linker and spacer DNA. Amine-reactive phenazine ethosulfate and NADH were used as the redox label and reductant, respectively. N-terminated polylysine was used as the poly-linker for the conjugation between an aptamer probe and multiple redox labels. Approximately 11 redox labels per probe and rapid catalytic NADH oxidation enable high signal amplification. Thrombin in urine could be detected without a wash step with a detection limit of ∼50 pM, which is practically promising for point-of-care testing of proteins.