Single-Entity Resolution Single-Cell Nanosensor Reveals Reactive Oxygen Species at Stress Granules Are Formed by Interfacial Redox Chemistry

The electrochemical activities of biomolecular condensates represent a new fundamental functioning mechanism in biochemistry and cell biology. However, our understanding of the underlying molecular mechanism and the interfacial field-dependent chemical activities remains limited. This is due to the lack of technology to probe such activities in real time and at a single-condensate level. Stress granules (SGs) are membraneless organelles that form in the cytoplasm to adapt to cell stress, which are found to encapsulate reactive oxygen species (ROS) in our lab. Here, we design and implement a collision-based electrochemical nanosensor that enables probing of the redox activities of SGs at a single-condensate level in live cells. We show that ex-vivo separated SGs drive the redox reactions depending on their own interfacial potentials and the constituents of the solution system. Surprisingly, we found that water molecules, rather than solvated oxygen (the main source of ROS produced by a conventional enzyme reaction in cells), are the main chemical origin of the redox activity of SGs. Finally, we demonstrate the application of this electrochemical nanosensor in real-time probing of the generation of hydrogen peroxide from SGs in mammalian cells and show that the electrochemical environment of the cells can regulate the redox activity of SGs. This work uncovers the likely mechanisms encoding nonenzymatic redox activities of SGs and demonstrates a key fundamental technological capability that can be highly useful in exploring the intracellular electroactive pathways of macroscale assemblies.