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