Immunoreaction-triggered DNA assembly for one-step sensitive ratiometric electrochemical biosensing of protein biomarker.

A sensitive ratiometric electrochemical readout was designed with an immunoreaction-triggered DNA assembly for one-step, fast and flexible assay of protein biomarker. The sensing interface was prepared by immobilizing a ferrocene (Fc)-labeled hairpin DNA on a gold electrode. In the presence of DNA2-antibody2 (Ab2) and methylene blue (MB)-labeled DNA1-Ab1 probes, the addition of target protein could induce the sandwich immunoreaction among two probes and the protein to trigger the hybridization of DNA1 and DNA2, which subsequently unfolded the hairpin DNA to form a three-arm DNA structure on the sensing interface. The DNA assembly caused the departure of Fc from the electrode and the approach of MB to the electrode, which led to the signal decrease and increase of Fc and MB respectively for ratiometric readout. Using prostate specific antigen (PSA) as a model target, the ratiometric electrochemical assay showed a linear detection range from 0.01 to 200ng/mL with a detection limit of 4.3pg/mL (the mean signal of blank measures+3σ). By changing the affinity probe pairs this method could be easily expanded for other protein analytes, showing promising potential for point-of-care testing and extensive applications in bioanalysis.