Patch clamping on plane glass—fabrication of hourglass aperture and high-yield ion channel recording

Abstract

Planar patch-clamp has revolutionized ion-channel measurement by eliminating laborious manipulation from the traditional micropipette approach and enabling high throughput. However, low yield in gigaseal formation and/or relatively high cost due to microfabricated processes are two main drawbacks. This paper presents patch clamping on glass substrate-an economical solution without sacrificing gigaseal yield rate. Two-stage CO(2) laser drilling methodology was used to generate an hourglass, funnel-like aperture of a specified diameter with smooth and debris-free surfaces on 150 microm borosilicate cover glass. For 1-3 microm apertures as patch-clamp chips, seal resistance was tested on human embryonic kidney, Chinese hamster ovary, and Jurkat T lymphoma cells with a gigaseal success rate of 62.5%, 43.6% and 66.7% respectively. Results also demonstrated both whole-cell and single channel recording on endogenously expressed ion channels to confirm the capability of different patch configurations.

High-fidelity patch-clamp recordings from neurons cultured on a polymer microchip

“…3(b), the aperture's edges are rounded and have a lip. Although not as pronounced as in the micromachined hourglass shape (Chen et al 2009), this lip may enhance contact area between the cell membrane and polyimide surface, thus improving seal formation and allowing the recording of high-quality signals.…”

“…Gigaseal formation is poorly understood, but published studies on planar patch-clamp systems have suggested the importance of several parameters. The surface making contact with the cell membrane should be smooth, clean and hydrophilic; it should have a high area of contact with the cell membrane, thus favoring an hourglass shape for the aperture (Chen et al 2009, Lehnert et al 2007, Sordel et al 2006. The type of cells, their passage number and minimal debris in the cell suspension also play a role.…”

“…High resistance seal (gigaseal) formation significantly reduces background current noise and permits the recording of quality data (Hamill et al 1981); gigaseal formation is therefore an important parameter in patch-clamp studies. Although a growing body of evidence points to several parameters playing a role in gigaseal formation, its mechanism is still poorly understood (Chen et al 2009, Sordel et al 2006. Also contributing to poor signal quality is the high R-C (access resistance-shunt capacitance) values of some planar patch-clamp devices which reduce the chip's response time (Sordel et al 2006).…”

“…al. 2004, Sordel et al 2006, Stett et al 2003a, silicon nitride (Fertig et al 2000, Mealing et al 2005, Py et al 2008, Schmidt et al 2000, quartz/glass (Chen et al 2009, Fertig et al 2002, Ong et al 2007, poly(dimethylsiloxane) (Chen and Folch 2006, Dahan et al 2008, Lau et al 2006 and polyimide (Kiss et al 2003, Stett et al 2003b. High throughput analysis systems have been commercialized (Bruggemann et al 2003, Dubin et al 2005, Kutchinsky et al 2003.…”

Development of Patch-Clamp Chips for Mammalian Cell Applications

“…(1b)): the glass pipette was replaced by a membrane having microfeatures (called apertures) mimicking the tip of the pipette [2]. Apertures have been fabricated in silicon dioxide (SiO 2 ) [3][4][5], silicon nitride (Si 3 N 4 ) [6], quartz/glass [7,8], poly(dimethylsiloxane) (PDMS) [9,10] and polyimide (PI) [11]. However, the available planar patch-clamp systems have only been applied to isolated cells in suspension.…”