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Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

Saha SC, Henderson AJ, Powl AM, Wallace BA, de Planque MR, Morgan H - PLoS ONE (2015)

Bottom Line: The platform allows up to 6 bilayers to be analysed simultaneously.Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips.The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds.

View Article: PubMed Central - PubMed

Affiliation: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, United Kingdom.

ABSTRACT
This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (Na(v)Sp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

No MeSH data available.


Related in: MedlinePlus

Bilayer platform for parallel channel recording.(a) Photograph of the BLM recording platform with three bilayer chips, each with two bilayers. (b) A daughter board showing two dual core ASIC amplifiers interfacing with the microfluidic chips. The integrated platform comprises three identical daughter boards, each of which can record from up to two bilayers, together with signal processing electronics. Data is transmitted to a computer via a USB connection. (c) Close up view of the bilayer aperture with Ag/AgCl electrode and flow channel. (d) Cross section X-X’ of the chip showing the dry film resist that forms the aperture together with the integrated Ag/AgCl electrodes, one common and one for each bilayer.
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pone.0131286.g001: Bilayer platform for parallel channel recording.(a) Photograph of the BLM recording platform with three bilayer chips, each with two bilayers. (b) A daughter board showing two dual core ASIC amplifiers interfacing with the microfluidic chips. The integrated platform comprises three identical daughter boards, each of which can record from up to two bilayers, together with signal processing electronics. Data is transmitted to a computer via a USB connection. (c) Close up view of the bilayer aperture with Ag/AgCl electrode and flow channel. (d) Cross section X-X’ of the chip showing the dry film resist that forms the aperture together with the integrated Ag/AgCl electrodes, one common and one for each bilayer.

Mentions: In this study, we use a miniature microfluidic ion-channel screening platform to characterise the NavSp pore construct. Bilayers were made across cavities fabricated in a microfabricated polymer support made on small glass chips. Each of these chips holds two independent bilayers and a miniature platform has been developed to accommodate three chips, allowing six bilayers to be continuously measured in parallel, as shown in Fig 1(A). The microfluidic chips interface with custom-designed integrated amplifiers (ASICs) and recording hardware/software as shown in Fig 1(B). The glass chips contain integrated Ag/AgCl electrodes together with edge connectors that make direct contact to the ASICs, significantly reducing contact resistance and noise. The bilayers are formed across miniature apertures made in an epoxy photoresist and the design provides fluid access on both sides, via a microfluidic channel (Fig 1C and 1D) that provides a simple means of quickly introducing compounds to the bilayer using passive pumping from droplets, eliminating syringe pumps and associated tubing [11]. In this work, the ion channels are inserted into the bilayer by the addition of proteoliposomes which spontaneously fuse with the lipid bilayer, leading to incorporation of the pores. To increase the probability of fusion the bilayers were formed across relatively large apertures, with a diameter of 75 μm. For the NavSp pore, spontaneous vesicle fusion and incorporation of active protein was infrequent although channel incorporation was facilitated, to some extent, by breaking and reforming the bilayers in the presence of proteoliposomes. However, the simultaneous acquisition of data from all 6 bilayers significantly increased the probability of observing active channels. Single-channel current and channel open probabilities were determined in the presence of the channel blocker nifedipine, enabling the determination of IC50 curves for this drug.


Characterization of the Prokaryotic Sodium Channel NavSp Pore with a Microfluidic Bilayer Platform.

Saha SC, Henderson AJ, Powl AM, Wallace BA, de Planque MR, Morgan H - PLoS ONE (2015)

Bilayer platform for parallel channel recording.(a) Photograph of the BLM recording platform with three bilayer chips, each with two bilayers. (b) A daughter board showing two dual core ASIC amplifiers interfacing with the microfluidic chips. The integrated platform comprises three identical daughter boards, each of which can record from up to two bilayers, together with signal processing electronics. Data is transmitted to a computer via a USB connection. (c) Close up view of the bilayer aperture with Ag/AgCl electrode and flow channel. (d) Cross section X-X’ of the chip showing the dry film resist that forms the aperture together with the integrated Ag/AgCl electrodes, one common and one for each bilayer.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493117&req=5

pone.0131286.g001: Bilayer platform for parallel channel recording.(a) Photograph of the BLM recording platform with three bilayer chips, each with two bilayers. (b) A daughter board showing two dual core ASIC amplifiers interfacing with the microfluidic chips. The integrated platform comprises three identical daughter boards, each of which can record from up to two bilayers, together with signal processing electronics. Data is transmitted to a computer via a USB connection. (c) Close up view of the bilayer aperture with Ag/AgCl electrode and flow channel. (d) Cross section X-X’ of the chip showing the dry film resist that forms the aperture together with the integrated Ag/AgCl electrodes, one common and one for each bilayer.
Mentions: In this study, we use a miniature microfluidic ion-channel screening platform to characterise the NavSp pore construct. Bilayers were made across cavities fabricated in a microfabricated polymer support made on small glass chips. Each of these chips holds two independent bilayers and a miniature platform has been developed to accommodate three chips, allowing six bilayers to be continuously measured in parallel, as shown in Fig 1(A). The microfluidic chips interface with custom-designed integrated amplifiers (ASICs) and recording hardware/software as shown in Fig 1(B). The glass chips contain integrated Ag/AgCl electrodes together with edge connectors that make direct contact to the ASICs, significantly reducing contact resistance and noise. The bilayers are formed across miniature apertures made in an epoxy photoresist and the design provides fluid access on both sides, via a microfluidic channel (Fig 1C and 1D) that provides a simple means of quickly introducing compounds to the bilayer using passive pumping from droplets, eliminating syringe pumps and associated tubing [11]. In this work, the ion channels are inserted into the bilayer by the addition of proteoliposomes which spontaneously fuse with the lipid bilayer, leading to incorporation of the pores. To increase the probability of fusion the bilayers were formed across relatively large apertures, with a diameter of 75 μm. For the NavSp pore, spontaneous vesicle fusion and incorporation of active protein was infrequent although channel incorporation was facilitated, to some extent, by breaking and reforming the bilayers in the presence of proteoliposomes. However, the simultaneous acquisition of data from all 6 bilayers significantly increased the probability of observing active channels. Single-channel current and channel open probabilities were determined in the presence of the channel blocker nifedipine, enabling the determination of IC50 curves for this drug.

Bottom Line: The platform allows up to 6 bilayers to be analysed simultaneously.Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips.The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds.

View Article: PubMed Central - PubMed

Affiliation: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, United Kingdom.

ABSTRACT
This paper describes the use of a newly-developed micro-chip bilayer platform to examine the electrophysiological properties of the prokaryotic voltage-gated sodium channel pore (Na(v)Sp) from Silicibacter pomeroyi. The platform allows up to 6 bilayers to be analysed simultaneously. Proteoliposomes were incorporated into suspended lipid bilayers formed within the microfluidic bilayer chips. The chips provide access to bilayers from either side, enabling the fast and controlled titration of compounds. Dose-dependent modulation of the opening probability by the channel blocking drug nifedipine was measured and its IC50 determined.

No MeSH data available.


Related in: MedlinePlus