High Quality Ion Channel Analysis on a Chip with the NPC^© Technology

Abstract

In evaluating ion channel function, electrophysiology, e.g., patch clamping, provides the highest information content. For the analysis of ion channel-modulating compounds, one variant of the patch-clamp technique, the whole-cell configuration, is particularly useful. We present here patch-clamp recordings in the whole-cell configuration and single channel recordings performed with planar patch-clamp chips, which are microstructured from borosilicate glass substrate. The chips are used in the Port-a-Patch, an ion channel research/screening instrument that enables automated patch-clamp experiments on a single cell. A software runs the experiment by executing user-determined protocols for cell positioning, as well as for electrical stimulation and current readout. In various electrophysiological experiments, the high quality of recordings and the versatility of the perfusion of the recorded cells are demonstrated. Quantitative pharmacological experiments are performed on sodium channels expressed in HEK cells using solution volumes in the low microliter range. The exceptionally low volume consumption in the experiments make the system attractive for work on rare or expensive compounds. Due to the low volumes necessary, a rapid solution exchange is facilitated, which is shown on RBL cells. The patch-clamp chip enables a rapid and precise perfusion, allowing sophisticated investigations on ion channel function with the Port-a-Patch.

Hourglass SiO2 coating increases the performance of planar patch-clamp

“…Chien, 2003 The optimisation of planar patch-clamp cell sealing involves both biological and technological approaches. Some authors introduced new procedures in order to improve the quality of the cell suspension ( ) or optimize the identification of cells that Bruggemann et al, 2003 production of which offers high accuracy and flexibility of microstructuration and surface functionalisation. This approach has led to a model of PECVD SiO coating defining an hourglass shape of the hole ( ).…”

Glass reflow on 3-dimensional micro-apertures for electrophysiological measurements on-chip

“…Gigaseal formation and pharmacological measurements on cells using planar Si micro-apertures (Asmild et al 2003;Pantoja et al 2004), as well as glass chips (Bruggemann et al 2003;Xu et al 2003;van Stiphout et al 2005) have been successfully demonstrated. Seal resistances with hard plastic substrates did not exceed the range of a few hundred mega-ohms (Schroeder et al 2003).…”

A novel method for patch-clamp automation

“…Similarly, CytoPatch (Cytocentrics GmbH) allows immobilization of selected cells from a flowing suspension on the CytoPatch chip by suction with a success rate of 97% and subsequent formation of gigaseals with a success rate of 68% [20]. NPC-16 utilizes a planar silicone chip that enables solution exchange on a subsecond scale [2]. A common limitation of all of these systems, however, is their limited agonist washout capabilities, which constrains the number of data points that can be obtained from a single cell.…”