Simultaneous alternating and direct current readout of protein ion channel blocking events using glass nanopore...

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.