Nanopore Unitary Permeability Measured by Electrochemical and Optical Single Transporter Recording

Abstract

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.

Nano for bio: Nanopore arrays for stable and functional lipid bilayer membranes (Mini Review)

“…After a long lag time, which depends on the pore size and the ␣-HL concentration, the conductance of the lipid bilayer increased by steps of 9 pA under the indicated conditions. This value is in agreement with the reported single ␣-HL pore current of 7 pA determined at 30 mV and 250 mM KCl, 49 a recent study using variable conditions. The benefit of using nanopore arrays for monitoring single molecule signals may rely on at least two factors: the stabilization of lipid bilayers and the high S/N ratio as reported for small apertures 38 as confirmed by the low observed noise ͑see Fig.…”

Formation of lipid bilayer membrane in a poly(dimethylsiloxane) microchip integrated with a stacked polycarbonate membrane support and an on-site nanoinjector

“…29,30 Among commercially available filter membranes, PC membrane has become one of the most suitable porous supports for the pore-spanning BLM formation, [31][32][33][34][35] owning to its favorable characteristics, such as circular apertures of the greater regularity, smooth surfaces, and high resistivity. But, because of its high pore population, PC-supported BLMs are very susceptible to hydrodynamic pressure.…”

Micro- and nano-characterization of membrane materials

“…More recently, other approaches such as molecular resolution porosimetry for micropores, high-resolution N 2 adsorption, He adsorption at 4.2 K, and molecular simulation for the characterization of porous solids were also developed (Kaneko, 1994). Some other advanced methods have been developed to characterize membrane materials of nano-pore size with electrochemical and optical single transporter recording (Hemmler et al, 2005), fluorescent dextran test (Mulherkar and van Reis, 2004) and atomic force microscopy (Hilal et al, 2005).…”