Plasma membrane anchored nanosensor for quantifying endogenous production of H₂O₂ in living cells

Hydrogen peroxide (H₂O₂) is one of the main second messengers involved in signaling pathways controlling cell metabolism. During tumorigenesis H₂O₂ is generated on the extracellular space by membrane-associated NADPH oxidases and superoxide dismutase to stimulate cell proliferation and preservation of the transformed state. Accordingly, a characteristic feature of malignant cells is overproduction of H₂O₂ in the extracellular milieu and the subsequent absorption in the cytosol. Since the most significant gradients of endogenous extracellular H₂O₂ can be observed only in a very shallow region of the fluid in contact with the plasma membrane, we show here the use of a newly designed nanosensor anchored to the outer cell surface and capable of quantifying H₂O₂ at nanometer distance from the membrane proteins responsible for its production. This biosensor is built upon gold nanoparticles functionalized with a H₂O₂-sensitive boronate compound that is probed using surface enhanced Raman spectroscopy (SERS). The highly localized information obtained on the cell surface by SERS analysis is combined with analytical methods of redox biology to estimate the associated levels of intracellular H₂O₂ responsible for cell signaling. The results obtained from A549 lung cancer cell line show localized spots on the cell surface at concentration up to 12 μM, associated to intracellular concentration up to 5.1 nM.