Plasma membrane anchored nanosensor for quantifying endogenous production of H(2)O(2) in living cells.

Affiliation

Hosogi S(1), Marunaka Y(2), Ashihara E(3), Yamada T(4), Sumino A(5), Tanaka H(6), Puppulin L(7).
Author information:
(1)Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, 607-8414, Japan; Department of Molecular Cell Physiology, Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi-Hirokoji, Kyoto, 602-8566, Japan.
(2)Department of Molecular Cell Physiology, Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi-Hirokoji, Kyoto, 602-8566, Japan; Research Center for Drug Discovery and Pharmaceutical Development Science, Research Organization of Science and Technology, Ritsumeikan University, Kusatsu, 525-8577, Japan; Research Institute for Clinical Physiology, Kyoto Industrial Health Association, 67 Kitatsuboi-cho, Nishino-kyo, Nakagyo-ku, Kyoto, 604-8472, Japan.
(3)Department of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto, 607-8414, Japan.
(4)Department of Pulmonary Medicine, Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi-Hirokoji, Kyoto, 602-8566, Japan.
(5)WPI Nano Life Science Institute
(WPI-NanoLSI), Kanazawa University, Kakumamachi, Kanazawa, 920-1192, Japan; Institute for Frontier Science Initiative, Kanazawa University, Kakumamachi, Kanazawa, 920-1192, Japan.
(6)Department of Pathology and Cell Regulation, Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi-Hirokoji, Kyoto, 602-8566, Japan.
(7)WPI Nano Life Science Institute
(WPI-NanoLSI), Kanazawa University, Kakumamachi, Kanazawa, 920-1192, Japan; Department of Pathology and Cell Regulation, Graduate School of Medical Sciences, Kyoto Prefectural University of Medicine, Kajii-cho, Kawaramachi-Hirokoji, Kyoto, 602-8566, Japan. Electronic address: [Email]

Abstract

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.