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Imaging of compartmentalised intracellular nitric oxide, induced during bacterial phagocytosis, using a metalloprotein–gold nanoparticle conjugate

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Abstract

Nitric oxide (NO) plays an essential role within the immune system since it is involved in the break-down of infectious agents such as viruses and bacteria. The ability to measure the presence of NO in the intracellular environment would provide a greater understanding of the pathophysiological mechanism of this important molecule. Here we report the detection of NO from the intracellular phagolysosome using a fluorescently tagged metalloprotein–gold nanoparticle conjugate. The metalloprotein cytochrome c, fluorescently tagged with an Alexa Fluor dye, was self-assembled onto gold nanoparticles to produce a NO specific nanobiosensor. Upon binding of NO, the cytochrome c protein changes conformation which induces an increase of fluorescence intensity of the tagged protein proportional to the NO concentration. The nanobiosensor was sensitive to NO in a reversible and selective manner, and exhibited a linear response at NO concentrations between 1 and 300 μM. In RAW264.7γ NO macrophage cells, the nanobiosensor was used to detect the presence of NO that had been endogenously generated upon stimulation of the cells with interferon-γ and lipopolysaccharide, or spontaneously released following treatment of the cells with a NO donor. Significantly, the nanobiosensor was shown to be taken up by the macrophages within phagolysosomes, i.e., the precise location where the NO, together with other species, destroys bacterial infection. The nanobiosensor measured, for the first time, increasing concentrations of NO produced during combined stimulation and phagocytosis of Escherichia coli bacteria from within localised intracellular phagolysosomes, a key part of the immune system.

Graphical abstract: Imaging of compartmentalised intracellular nitric oxide, induced during bacterial phagocytosis, using a metalloprotein–gold nanoparticle conjugate

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Publication details

The article was received on 31 May 2017, accepted on 02 Aug 2017 and first published on 29 Sep 2017


Article type: Paper
DOI: 10.1039/C7AN00898H
Citation: Analyst, 2017, Advance Article
  • Open access: Creative Commons BY license
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    Imaging of compartmentalised intracellular nitric oxide, induced during bacterial phagocytosis, using a metalloprotein–gold nanoparticle conjugate

    R. Leggett, P. Thomas, M. J. Marín, J. Gavrilovic and D. A. Russell, Analyst, 2017, Advance Article , DOI: 10.1039/C7AN00898H

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