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Abstract
An integrated microdevice for measuring proton-dependent membrane activity at the surface of Xenopus laevis oocytes is presented. By establishing a stable contact between the oocyte vitelline membrane and an ion-sensitive field-effect (ISFET) sensor inside a microperfusion channel, changes in surface pH that are hypothesized to result from facilitated proton lateral diffusion along the membrane were detected. The solute diffusion barrier created between the sensor and the active membrane area allowed detection of surface proton concentration free from interference of solutes in bulk solution. The proposed sensor mechanism was verified by heterologously expressing membrane transport proteins and recording changes in surface pH during application of the specific substrates. Experiments conducted on two families of phosphate-sodium cotransporters (SLC20 & SLC34) demonstrated that it is possible to detect phosphate transport for both electrogenic and electroneutral isoforms and distinguish between transport of different phosphate species. Furthermore, the transport activity of the proton/amino acid cotransporter PAT1 assayed using conventional whole cell electrophysiology correlated well with changes in surface pH, confirming the ability of the system to detect activity proportional to expression level.
Highlights
For many decades, electrophysiological methods have been at the forefront of investigations of membrane conduction and excitation in living cells. [1] To measure membrane conductance, control of either the transmembrane voltage or current is required
Based on the lateral diffusion model, we have developed a method for sensing proton-dependent membrane transport in Xenopus laevis oocytes by utilizing a novel arrangement of ion-sensitive field-effect (ISFET) technology
We present an integrated microdevice based on ISFET
Summary
Electrophysiological methods have been at the forefront of investigations of membrane conduction and excitation in living cells. [1] To measure membrane conductance, control of either the transmembrane voltage or current is required. In the commonly used two electrode voltage clamp (TEVC) applied to large cells such as Xenopus laevis oocytes, two microelectrodes impale the oocyte to sense and control the membrane potential. This procedure requires delicate glass microelectrodes and a high degree of micromanipulation, either by a human operator or precision robotics. [4] Recently, several non-invasive voltage clamp techniques for Xenopus laevis oocytes have been developed that leave the membrane intact These methods rely on the physical compartmentalization of the membrane into two areas and measurement of trans-cellular currents across the entire oocyte. In the transoocyte voltage clamp (TOVC) an AC voltage is applied across symmetrically distributed membrane impedances. [5] Asymmetric variants of the TOVC have been realized to better define the transmembrane potential across a smaller region of membrane [6,7] and are akin to the loose macropatch. [8]
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