Electrochemical biosensor based on base-stacking-dependent DNA hybridization assay for protein detection

An antibody-based electrochemical biosensing platform has been developed and used for the detection of protein. In the presence of the target, an antibody pair binds to the protein simultaneously, which causes two oligo-DNAs conjugated with the antibody pair to hybridize to each other and become a big “stem–loop” structure. Subsequently, the longer oligo-DNA of the stem, with a methylene blue (MB) label at the terminal, hybridizes stably with capture DNA owing to the enhancement of base stacking. The strong redox current signal of MB is used for protein quantification. Using α-fetoprotein (AFP) as a model, the proposed method could detect AFP at a concentration as low as 2 pg ml⁻¹ with a dynamic range of 4 orders of magnitude, which approaches traditional assays such as enzyme-linked immunosorbent assay.