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.