Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 109, Issue 29, Pages 11534-11539Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1201552109
Keywords
amperometry; atomic force microscopy; oxidative stress; electrochemical nanofabrication; intracellular sensor
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Funding
- National Science Foundation [CHE-0957313, CHE-1026582]
- Centre National de la Recherche Scientifique, Ecole Normale Superieure, Universite Pierre et Marie Curie Paris 06 [UMR8640]
- French Ministry of Research
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1026582, 0957313] Funding Source: National Science Foundation
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Reactive oxygen and nitrogen species (ROS and RNS) produced by macrophages are essential for protecting a human body against bacteria and viruses. Micrometer-sized electrodes coated with Pt black have previously been used for selective and sensitive detection of ROS and RNS in biological systems. To determine ROS and RNS inside macrophages, one needs smaller (i.e., nanometer-sized) sensors. In this article, the methodologies have been extended to the fabrication and characterization of Pt/Pt black nanoelectrodes. Electrodes with the metal surface flush with glass insulator, most suitable for quantitative voltammetric experiments, were fabricated by electrodeposition of Pt black inside an etched nanocavity under the atomic force microscope control. Despite a nanometer-scale radius, the true surface area of Pt electrodes was sufficiently large to yield stable and reproducible responses to ROS and RNS in vitro. The prepared nanoprobes were used to penetrate cells and detect ROS and RNS inside macrophages. Weak and very short leaks of ROS/RNS from the vacuoles into the cytoplasm were detected, which a macrophage is equipped to clean within a couple of seconds, while higher intensity oxidative bursts due to the emptying of vacuoles outside persist on the time scale of tens of seconds.
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