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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 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...

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans-insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans-insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.

Amperometric currents displaying a pre-spike feature (PSF) may be treated so as to lead to precise information about initial fusion pores, viz., about the crucial event initiating neurotransmitter vesicular release in neurons and medullary glands. However, amperometric data alone are not self-sufficient, so their full exploitation requires external calibration to solve the inverse problem. For this purpose we resorted to patch-clamp measurements published in the literature on chromaffin cells. Reported pore radii were thus used to evaluate the diffusion rate of neurotransmitter cations in the partially altered matrix located near the fusion pore entrance. This allowed an independent determination of each initial fusion pore radius giving rise to a single PSF event. The statistical distribution of the radii thus obtained provided for the first time an experimental access to the potential energy well governing the thermodynamics of such systems. The shape of the corresponding potential energy well strongly suggested that, after their creation, initial fusion pores are essentially controlled by the usual physicochemical laws describing pores formed in bilayer lipidic biological membranes, i.e., they have an essentially lipidic nature.