# DNA‐Guided Robust Single‐Protein Electronic Readout

## TL;DR

A DNA-based method improves the accuracy of measuring electrical properties of single proteins by controlling their orientation.

## Contribution

A DNA origami platform enables reproducible single-protein electronic measurements with controlled orientation.

## Key findings

- Bivalently anchored thrombin shows reduced conductance variability compared to randomly adsorbed proteins.
- The platform detects subtle conductance changes in thrombin upon binding with Na+ or an inhibitor.
- The method is generalizable and works with proteins like streptavidin in complex mixtures.

## Abstract

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.

Electronic measurements of single proteins can reveal fundamental insights into their structure, dynamics, and interactions, but reproducibility is severely limited by the intrinsic structural heterogeneity of proteins combined with uncontrolled molecular orientation and electrode contact geometries. This work addresses the long‐standing challenge of achieving precise, label‐free, and reproducible single‐protein conductance measurements by introducing a DNA‐origami‐based platform that promotes preferred orientations of individual proteins for high‐confidence electrical characterization.