Constructing a proton titration curve from ion-step measurements, applied to a membrane with adsorbed protein

A new measuring method is described for obtaining a proton titration curve. The curve is obtained from a microporous composite membrane, consisting of polystyrene beads in an agarose matrix, with lysozyme molecules adsorbed to the bead surface. The membrane is incorporated into a sensor system by deposition on a silicon chip with a pH-sensitive ion-sensitive field effect transistor (ISFET) located in the middle of a Ag/AgCl electrode. The actual measurement is performed by creating a stepwise change in the salt concentration of the bathing electrolyte (the ion step) and measuring the ISFET potential versus the Ag/AgCl electrode. This potential shows a transient change in the ion step, which indicates a transient pH change in the membrane. This procedure is repeated at a series of pH values. Equations are presented to calculate the proton titration curve of the membrane from the amplitude and duration of the measured transients. Measurements show qualitative agreement between the curves obtained and equilibrium titration experiments on the same system. (C) 1997 Academic Press.