Simultaneous alternating and direct current readout of protein ion channel blocking events using glass nanopore membranes.
Alternating current (ac) phase-sensitive detection is used to measure the conductance of the ion channel alpha-hemolysin (alphaHL), while simultaneously applying a direct current (dc) bias to electrostatically control the binding affinity and kinetics of charged molecules within the protein lumen. Ion channel conductance was recorded while applying a 10-20 mV rms, 1-2 kHz bias across a single alphaHL protein inserted in a 1,2-diphytanoyl-sn-glycero-3-phosphocholine lipid bilayer that is suspended across the orifice (100-500 nm radius) of a glass nanopore membrane. Step changes in the ac ion channel conductance with a temporal response (t(10-90)) of 1.5 ms and noise amplitude of approximately 2 pA were obtained using a low-noise potentiostat and a lock-in amplifier. These conditions were used to monitor the reversible and stochastic binding of heptakis-(6-O-sulfo)-beta-cyclodextrin and a nine base pair DNA hairpin molecule to the ion channel. Alternating current methodology allows the binding kinetics and affinity between the protein ion channel and analyte to be investigated as a function of the dc bias, including ion channel conductance measurements in the absence of a dc bias.
Ervin
Eric N
EN
Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112, USA.
Kawano
Ryuji
R
White
Ryan J
RJ
White
Henry S
HS
D001483:Base Sequence; D004277:DNA, Single-Stranded; D005898:Glass; D007700:Kinetics; D008567:Membranes, Artificial; D036103:Nanotechnology; D011506:Proteins / Q000737:chemistry*
D004277:DNA, Single-Stranded; D008567:Membranes, Artificial; D011506:Proteins