Epitope Binning Assay Using an Electron Transfer-Modulated Aptamer Sensor.

Surface plasmon resonance and quartz crystal microbalance are workhorses of protein-DNA interaction research for over 20 years, providing ways to quantitatively determine the protein-DNA binding. However, the cost, necessary technical expertise, and severe nonspecific adsorption poses barriers to their use. Convenient and effective techniques for the measurement of protein-DNA binding affinity and the epitope binning between DNA and proteins for developing highly sensitive detection platform remain challenging. Here, we develop a binding-induced alteration in electron transfer kinetics of the redox reporter labeled (methylene blue) on DNA aptamer to measure the binding affinity between prostate-specific antigen (PSA) and aptamer. We demonstrate that the binding of PSA to aptamer decreases the electron transfer rate of methylene blue for ∼45%. Further, we identify the best pairwise selection of aptamers for developing sandwich assay by sorting from 10 pairwise modes with the PSA detection limit of 500 ng/mL. Our study provides promising ways to analyze the binding affinity between ligand and receptor and to sort pairwise between aptamers or antibodies for the development of highly sensitive sandwich immunoassays.