Microsensor technology for measuring H⁺ flux in buffered media

Nearly all experiments designed to quantify phenomena in biological systems are conducted in the presence of buffers.Most analytical techniques measure free proton activity, and do not account for the activity of protons associated with buffer molecules, potentially leading to an underestimation of biophysical proton transport. In applications where total proton flux activity at the level of biological membranes is of interest, a dynamic biophysical approach is required. This paper presents a simplifiedmethod and data filtering algorithm for measuring electrophysiological, transmembrane H⁺ flux activities in bufferedmedia based on the self-referencing microsensor technique. A simplified mathematical model was developed that accounts for chemical H ⁺ buffering in a background matrix of buffer molecules. The model and buffer correction algorithmwere validated for various biochemically relevant buffers over the 6.1-10.7 pH range. The buffer correction algorithm was used to filter the output of a self-referencing ion-selective H⁺ sensor for the measurement of proton flux. Special emphasis has been placed on the use of pH sensitive microelectrodes when operating as self-referencing flux sensors, but the analytical method is also applicable to the use of static electrodes and pH sensitive dyes.