Constructing a Proton Titration Curve from Ion-Step Measurements, Applied to a Membrane with Adsorbed Protein

Abstract

Previously it was shown that the potential transient is A new measuring method is described for obtaining a proton determined by a proton release or uptake in the membrane titration curve. The curve is obtained from a microporous compos-(7). In this paper the theoretical relationships are derived ite membrane, consisting of polystyrene beads in an agarose ma-between the amplitude and the duration of the transients and trix, with lysozyme molecules adsorbed to the bead surface. The the proton titration curve of the membrane at a certain pH.membrane is incorporated into a sensor system by deposition on a Using these relationships, and measuring the potential transilicon chip with a pH-sensitive ion-sensitive field effect transistor sients at a series of pH values, it will be possible to construct (ISFET) located in the middle of a Ag/AgCl electrode. The actual a titration curve and thus to obtain information on adsorbed measurement is performed by creating a stepwise change in the macromolecules. For biosensor purposes, the relationships salt concentration of the bathing electrolyte (the ion step) and measuring the ISFET potential versus the Ag/AgCl electrode. This can also be applied in the inverse direction; i.e., the device potential shows a transient change in the ion step, which indicates response can be predicted from known titrational behavior a transient pH change in the membrane. This procedure is re-of the different components. In this paper the adsorption peated at a series of pH values. Equations are presented to calcu-of the positively charged protein lysozyme is studied as a late the proton titration curve of the membrane from the amplitude model. and duration of the measured transients. Measurements showThe measurements presented can be regarded as an extenqualitative agreement between the curves obtained and equilib-sion of common practice in protein titration, namely, the rium titration experiments on the same system. ᭧ 1997 Academic Press relative positioning of the titration curves at different ionic Key Words: proton titration; ion-sensitive field effect transistor; strengths by using the pH change that results from a change polystyrene bead; lysozyme; concentration step; Ag/AgCl elecin ionic strength (8,9). In this type of experiment, the pH trode.change is measured at only one initial pH value. In the investigations presented the method is extended by measuring at a series of pH values. The extension is made possible

Measuring Donnan-related phenomena using a solid-state ion sensor and a concentration-step method

“…Thus it is worthwhile to look at the implications of the use of a double-layer model instead of the Donnan model [25]. A paper on this subject has been accepted for publication [30]. Here it suffices to say, that a double-layer model predicts a larger potential change at the pore walls than the Donnan model for the bulk membrane.…”

“…The use of a double-layer model also gives a better fit with the Meyer-Sievers curves, since the theoretical curves flatten. In addition, much lower fixed-charge concentrations are calculated from these measurements [25,30].…”

“…A protontitration curve of the membrane was obtained, passing through a point of net zero charge around pH 9. This relation between the measured transients and the membrane titration curve has been investigated in more detail [25] and a paper on this subject will be published [30]. A general conclusion is that the measurement method is suitable to detect lysozyme adsorption.…”

“…This behaviour can be understood by realizing that at constant tim, K decreases and the apparent proton diffusion coefficient increases with increasing distance from pile=7, where the concentrations of mobile protons or hydroxyl ions are minimal (see Eqs. (30) and (31)). Since the extreme, A~_xt r, is reached from pile=6 to 8, the ISFET-measured amplitudes at these pH values provide reliable information on the fixed-charge concentration.…”