Electrochemical method allows imaging of single reactions

A new method detects single molecules using a chemical reaction and creates images with high sensitivity and high spatial resolution such as those made with superresolution microscopy. Superresolution microscopy creates images of surfaces by detecting individual molecules, but it is done with fluorescence and requires the use of lasers, which can damage some samples and bleach the fluorescent labels.

Jiandong Feng and coworkers at Zhejiang University have imaged individual electrochemical reactions using electrochemiluminescence (ECL), in which an applied voltage drives light-emitting reactions (Nature 2021, DOI: 10.1038/s41586-021-03715-9). ECL “does not require the use of excitation light, which is a benefit to achieve very low background and ultrahigh sensitivity for resolving weak signals,” Feng says.

The researchers used a well-studied ECL reaction between tris(2,2′-bipyridyl)ruthenium(II), also called Ru(bpy)₃²⁺, and tri-n-propylamine (TPrA). In this system, an applied voltage oxidizes Ru(bpy)₃²⁺ to Ru(bpy)₃³⁺ at the surface of an indium tin oxide electrode. Subsequent reduction of the Ru(bpy)₃³⁺ by a free radical of TPrA results in the formation of an excited Ru complex that regenerates Ru(bpy)₃²⁺ and releases a photon, which is collected by a microscope objective and detected with a camera. The reaction is triggered at the electrode surface, but the molecules can diffuse away before emitting a photon, so the reaction and detection sites can be about 20 nm apart.

To detect single reactions requires some special strategies, Feng says. Using dilute solutions ensures a given field is likely to only contain a single molecule to be detected, and fast cameras help make sure a given reaction isn’t missed. Because the reaction only happens at or very close to the electrode, the photons can be used to image the electrode itself or things on its surface. Feng and coworkers monitored the ECL reaction across the surface of the electrode and cells on the electrode. Places where a cell adheres block reagents from reaching the electrode, so ECL only happens where the cell doesn’t adhere. The resulting image is a negative of the cell.

Although the method is limited to electrochemical reactions that release photons, it could still be useful for biological analysis and imaging, because many types of biomolecules could be linked to the Ru complex says Dechen Jiang, an ECL expert at Nanjing University.

“The authors’ findings open the way to a new concept in imaging: a chemistry-based approach to super-resolution microscopy,” Frédéric Kanoufi of the University of Paris and Neso Sojic of the University of Bordeaux write in an accompanying commentary.