A metal-supported biomimetic micromembrane allowing the recording of single-channel activity and of impedance spectra of membrane proteins.
A novel tethered bilayer lipid micromembrane (tBLmicroM) was prepared and characterized. It consists of a mercury cap electrodeposited on a platinum microelectrode, about 20 microm in diameter. The micromembrane was prepared by tethering to the mercury cap a thiolipid monolayer and by then self-assembling a lipid monolayer on top of it. The thiolipid consisted of a disulfidated tetraoxyethylene hydrophilic spacer covalently linked to two phytanyl chains. Upon incorporating OmpF porin in the tBLmicroM, its single-channel activity was recorded by the patch-clamp technique, and its particular features described. An electrochemical impedance spectrum of the tBLmicroM incorporating OmpF porin is also reported. To the best of our knowledge, this tBLmicroM is the first metal-supported biomimetic micromembrane capable of incorporating non-engineered channel proteins in a functionally active state from their detergent solutions, and of allowing the recording of single-channel activity and of impedance spectra of these proteins via ion translocation into the hydrophilic spacer. The limited spaciousness of the spacer prevents a statistical analysis based on current-amplitude or blockage-time histograms. Nonetheless, the robustness, stability, ease of preparation and disposability of the present tBLmicroM may open the way to the realization of a channel-protein microarray platform allowing a high throughput drug screening.
Becucci
Lucia
L
Department of Chemistry, Florence University, Via della Lastruccia 3, 50019 Sesto Fiorentino (Firenze), Italy. lucia.becucci@unifi.it
D'Amico
Massimo
M
Daniele
Salvatore
S
Olivotto
Massimo
M
Pozzi
Andrea
A
Guidelli
Rolando
R
D032701:Biomimetics / Q000379:methods*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D004566:Electrodes; D051638:Ether-A-Go-Go Potassium Channels / Q000378:metabolism; D006096:Gramicidin / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D008555:Melitten / Q000378:metabolism; D021962:Membrane Microdomains / Q000737:chemistry* / Q000378:metabolism*; D008628:Mercury / Q000737:chemistry*; D009124:Muscle Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D018272:Porins / Q000378:metabolism*; D021381:Protein Transport; D011510:Proteolipids / Q000378:metabolism; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D051638:Ether-A-Go-Go Potassium Channels; D006096:Gramicidin; D008051:Lipid Bilayers; D008555:Melitten; D008628:Mercury; D009124:Muscle Proteins; D018272:Porins; D011510:Proteolipids; D000099211:Phospholamban; C109237:OmpF protein; C077258:sarcolipin