Molecular Junction with a Single Biomolecule: Self-Restoring Device and Phosphorylation Assay

Single-molecule junctions, where a single molecule is trapped in a nanogap between metal electrodes, play pivotal roles in nanoscience and nanotechnology. Measuring biomolecules in the single-molecule junctions offers a unique means to develop single-molecule devices with sophisticated functionalities, to realize novel biomedical and pharmaceutical applications, and so on.<br/>Regarding the device development, the structural, physical, and chemical properties of DNA at the nanoscale have attracted attention to the prospect of using DNA as a building block in single-molecule devices, which leads to extensive investigations into electron transport through a single DNA molecule. Although significant advances have been made in understanding the transport phenomena through a single DNA molecule, the previous studies mainly focused on the transport phenomena in static DNA structure at thermodynamic equilibria. Against this, we expect that electron transport through DNA under the deliberate control of its structure paves the way for DNA electronic devices with functional controllability in a dynamic manner. To prove this, we investigated the electron transport through the single-molecule junction of DNA that orthogonally clamps a metal nanogap. This DNA single-molecule junction differs from conventional ones in the DNA configuration; the present and conventional junctions contain a DNA molecule oriented in perpendicular and parallel directions to the axis of the nanogap, respectively. The STM measurement of the unzipping dynamics revealed that the present DNA junction enables spontaneous restoration of the molecular junction after its electrical failure and thereby improves the reproducibility of the junction formation. Our study demonstrated that a DNA dynamic structural change could be applied to a single-molecule junction by using the perpendicular configuration.<br/>Single-molecule detection of biologically relevant substances has attracted rapidly growing interest. One successful example of single-molecule technology is the detection of DNA achieved using nanogaps between metal electrodes or nanopore devices. Given their primary role in diverse biological functions, proteins have been regarded as the next challenging target in single-molecule studies. In particular, the detection of post-translational modifications (PTMs) of proteins at the single-molecule level is in high demand, since PTMs constitute an essential regulatory mechanism involved in almost all cellular events. Single-molecule studies would revolutionize medical diagnoses through PTM detection primarily because of their high sensitivity and concomitant high-throughput assays. However, the detection of PTMs at the single-protein level is not trivial, since the chemical structure of an amino acid residue is only slightly altered upon modification. Stringent selectivity or specificity for a PTM of interest is consequently vital for single-molecule detection. In this study, we focused on phosphorylation, which is a biologically important PTM, and demonstrated specific single-molecule detection of peptide phosphorylation by electrical conductance measurements. First, we found that a single orthophosphate anion exhibits a conductance value significantly higher than the peptides and their amino acid residues. This unique electronic property of the phosphate serves as a specific marker, enabling the detection of phosphorylation on a single peptide molecule. Based on this unique electrical signature of phosphate, an <i>in</i> <i>situ</i> single-molecule assay of enzymatic phosphorylation was achieved with 95% accuracy and 91% specificity for signal discrimination between phosphorylated and non-phosphorylated peptides. The present study demonstrates novel and specific single-molecule detection of PTMs, which will help elucidate their biological functions and facilitate the development of novel medical diagnostics.