Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

Area of Science:

• Biophysics
• Nanotechnology
• Biosensor technology

Background:

• Functional analysis of ion channel activity is crucial for understanding cellular processes.
• Existing methods like electrophysiology have limitations in sensitivity and throughput.
• Artificial lipid bilayers and microwell platforms offer potential for novel biosensing approaches.

Purpose of the Study:

• To develop a highly sensitive biosensor for analyzing ion channel activity.
• To demonstrate the utility of a microwell array with confined fluorescent probes sealed by a lipid bilayer.
• To enable high throughput and parallel testing of functional membrane proteins.

Main Methods:

• Formation of an artificial lipid bilayer over microwells on a silicon substrate.
• Rupturing giant unilamellar vesicles to form the bilayer.
• Confinement of calcium (Ca2+) ion indicators (fluo-4) within microwells sealed by the lipid bilayer.
• Analysis of Ca2+ ion transport through α-hemolysin channels by monitoring fluorescence intensity changes.

Main Results:

• A stable artificial lipid bilayer was formed over microwells, preventing membrane collapse.
• The system successfully confined fluorescent probes (fluo-4) for Ca2+ ion detection.
• The biosensor achieved a high detection limit (tens of ions/s/μm2), surpassing standard electrophysiology.
• The microwell system demonstrated potential for mimicking local ion concentration changes within cells.

Conclusions:

• A microwell array with confined fluorescent probes and a lipid bilayer is a viable basic component for a highly sensitive biosensor array.
• This platform enables high throughput and parallel testing of functional membrane proteins.
• Further improvements in signal-to-noise ratio are needed for single-channel analysis.