Nanoelectrodes for determination of reactive oxygen and nitrogen species inside murine macrophages

Abstract

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 nanometerscale 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.amperometry | atomic force microscopy | oxidative stress | electrochemical nanofabrication | intracellular sensor M acrophage cells are essential for the performance of the immune system. Their activation, either under normal biological conditions or by specific biochemical activators in vitro, results in the production of reactive oxygen and nitrogen species (ROS and RNS) and creation of a large number of vacuoles (phagosomes and phagolysosomes; see Fig. 1A and SI Appendix) (1-3). These vacuoles play an important role in phagocytosisa mechanism used by the immune system to remove pathogens and cell debris. A cell (or debris) is engulfed into a vacuole and subjected to an intense oxidative burst (2), and the indigestible debris and excess ROS and RNS are subsequently evacuated from the macrophage (Fig. 1B).The changes in oxygen and hydrogen peroxide concentrations during the oxidative burst of a stimulated macrophage cell were detected previously using the scanning electrochemical microscope (4). Extensive studies with amperometric microelectrodes positioned in the cell proximity showed that the basal release is due to a cocktail composed of several ROS and RNS evolving from the primary production of O 2•− and NO (5-8). However, the concept that ROS and RNS released inside phagolysosomes may diffuse across the vacuole membrane and leak in the cell cytoplasm remains controversial (9-12). In fact, NO and the transisomer of protonated peroxynitrite ion are capable of crossing biological membranes due to their lipophilicity (13,14). This underscores the importance of probing for the intracellular presence of ROS and RNS in activated macrophages.For electrochemical measurements inside an activated macrophage one needs nanometer-sized electrode...

Electrochemical Nanoprobes for Single-Cell Analysis

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