Single-Cell Measurements of Purine Release Using a Micromachined Electroanalytical Sensor

Abstract

To study the cellular events surrounding the formation of purines in cardiac ischemia, we have micromachined a micrometer-scale titer chamber containing an integrated electrochemical sensor, capable of measuring analytes produced by a single heart cell. The analytical procedure involves the determination of metabolites via the amperometric detection of enzymically generated hydrogen peroxide, measured at a platinized microelectrode, poised at a suitably oxidizing potential, equivalent to +420 mV vs Ag/AgCl. Signals were recorded as current-time responses and were integrated to give a total charge (Q) attributable to the reaction under investigation. The amount of analyte produced by the cell was subsequently quantified by the addition of a known amount of calibrant. As a consequence, by using a cascade of three enzymes (adenosine deaminase, nucleotide phosphorylase, and xanthine oxidase), we were able to show that, after rigor contracture had been induced in a single myocyte, adenosine (but not inosine) only reached the extracellular space after the cell membrane had been permeabilized by detergent. These data, which could only be obtained unambiguously by using this single-cell methodology, have provided us with information on the origin of ischemic adenosine which challenges the established assumption that purine release is an early retaliatory response from intact anoxic myocytes.

Three-electrode analytical and preparative electrochemistry in micro-volume hanging droplets

“…Within this framework, electrochemistry brings a significant contribution to the analysis in small volumes, notably through the miniaturization of electrodes [1]. On the other hand, the development of microfabrication techniques such as photolithography [2][3][4][5][6][7], screen printing [8,9], plasma etching [10], and laser ablation [11,12] allowed the preparation of volume-limited structures. In this context, two main approaches have been developed aimed at running electrochemistry in small volumes.…”

Monitoring in Real Time with a Microelectrode the Release of Reactive Oxygen and Nitrogen Species by a Single Macrophage Stimulated by its Membrane Mechanical Depolarization

“…[1] Owing to its physical dimensions and physicochemical properties, a microelectrode can be easily positioned in the close environment of an isolated living cell to detect the temporal release of important biological species such as messengers, [2][3][4][5] effectors, [6][7][8][9] or aggressors [10][11][12] towards other cells or microorganisms. The principle of this analytical situation, termed an "artificial synapse", is to minimize the distance and consequently the volume created between the microelectrode detecting surface and the surface of the living cell, in order to obtain a sufficiently high local concentration rise following the release of a minute amount of an electroactive species by the cell.…”

On-chip acidification rate measurements from single cardiac cells confined in sub-nanoliter volumes

“…Furthermore, single cell analysis in small volumes provides the opportunity for more detailed information on cells that are difficult to culture or are rare, and a more sensitive analysis with a higher temporal resolution than measurements on populations of cells. Single cell studies in oil-sealed restricted volumes on amperometric sensors have shown the release of lactate or purine from single cardiac myocytes (Bratten et al, 1998;Cai et al, 2002); however, this approach does not allow small extracellular volumes, the chemical control, and the perfusion of the extracellular space.…”