DNA‐Guided Robust Single‐Protein Electronic Readout

Electronic transport properties of single proteins offer powerful insights into their structure and dynamics. However, the orientation variability of electrode-attached protein molecules poses a big difficulty in achieving precise electric measurements and reliable data interpretation. Here, a DNA origami-based method that promotes preferred orientations of target proteins is reported, enabling more reproducible single-molecule electrical characterization. With two orthogonal DNA aptamers installed inside a DNA origami nanocavity, a thrombin protein can be captured with a prescribed orientation between a gold substrate and a conductive AFM tip. Matrix-patterned I-V measurements reveal that bivalently anchored thrombin molecules exhibit significantly reduced conductance variability compared to randomly adsorbed ones. This critical progress then enables the detection of a subtle conductance change of thrombin upon binding with Na⁺ or an inhibitor molecule. The generalizability of this approach is showcased by further applying it to a streptavidin protein. Moreover, the platform allows for the selective recruitment and electrical readout of proteins from a mixed sample, demonstrating the feasibility of single-entity measurements within a complex molecular environment. This work provides a versatile platform for protein-electrode interfacing with deterministic molecular orientation control, highlighting the potential of DNA nanotechnology in single-protein electronic measurements.