Miniature support for thin films containing single channels or nanopores and methods for using the same

#1. Field of the Invention

#The field of this invention is ion channels or nanopores, particularly methods of measuring the ionic current flowing through ion channels or nanopores.

#2. Background of the Invention

#FIG. 1. Horizontal thin film apparatus according to the subject invention. A 0.8 cm inside diameter U-shaped tube connects two 65 μl baths (A & B) milled into a Teflon support. The baths and the Teflon tube are filled with 1 M KCl buffer. The chamber is connected to an Axopatch 200B amplifier via AgCl electrodes that are pressure fitted into the sides of the two baths. One end of the Teflon U tube has a conical tip that narrows abruptly to a 25 μm conical aperture. Diphytanoyl PC/hexadecane bilayers are formed across this aperture. α-toxin is inserted into the bilayer following addition of 0.04 μg to bath A. Nucleic acids are driven through the toxin channel by an applied voltage of 120 mV (+ at the trans side (bath B)).

#FIG. 2. Typical blockades of monovalent ion current in the α-hemolysin pore caused by A(30)C(70)Gp RNA observed with the device shown in FIG. 1. A single α-hemolysin channel was inserted into the bilayer with an open current of 126 pA at 120 mV in 1 M KCl buffer. Following control measurements in the absence of RNA, A(30)C(70)Gp RNA was added to the cis bath at 100 μg mL−1. Each event represents translocation of a single RNA molecule across the pore. In this experiment, most biphasic events had the orientation 5 pA residual current (95 percent current blockade) first followed by a 19 pA residual current (84 percent blockade). This corresponds to the polyC segment at the 3′ end of the molecule entering the pore first. The opposite orientation (insert) constituted less than 10% of the blockade events. The solid arrows highlight monophasic current blockades of approximately 95% and 84%; the dashed arrow highlights a permablock that required a voltage reversal to be cleared. This experiment is representative of four experiments with A(30)C(70)Gp RNA in which successful preparative cutting of the T7 RNA polymerase product by T1 RNase was confirmed by PAGE.

#FIG. 3. Effect of ribonuclease A addition upon the frequency of biphasic blockades caused by A(30)C(70)Gp RNA observed in the device of FIG. 1. A single α-hemolysin channel was inserted into the bilayer with an open current of 125 pA at 120 mV in 1 M KCl buffer at room temperature. Following control measurements in the absence of RNA, A(30)C(70)Gp RNA was added to the cis bath for a final concentration of 100 μg mL−1. Channel blockades were recorded for ten minutes, and then ribonuclease A (20 μg mL−1) was added to the cis bath. Blockades were then acquired in two minute increments at intervals up to 20 minutes. a) Frequency of 95%-to-84% biphasic blocks similar to those shown in FIG. 2. b) Frequency of monophasic blockades that reduce the channel current by 95% as observed in experiments with polyC alone. c) Frequency of monophasic blockades that reduce the channel current by 84 percent as observed with polyA alone. In all cases, events were counted if the specified blockade amplitude was achieved for a minimum of 50 μs.

#Single-channel thin film devices and methods for their use are provided. The subject devices have a cis chamber connected to a trans chamber via an electrical communication means, e.g. a U-shaped conductor. At the cis end of the electrical communication means is a conical aperture sealed with a thin film having a single nanopore or channel. The subject devices further include a means for applying an electric field between the cis and trans chambers, e.g. cis and trans electrodes. The subject devices find use in a variety of different applications in which the ionic current through a nanopore or channel is monitored. In further describing the subject invention, the subject devices will be described first followed by a review of a number of different representative methods in which the subject devices find use.

#Before the subject invention is described further, it is to be understood that the invention is not limited to the particular embodiments of the invention described below, as variations of the particular embodiments may be made and still fall within the scope of the appended claims. It is also to be understood that the terminology employed is for the purpose of describing particular embodiments, and is not intended to be limiting. Instead, the scope of the present invention will be established by the appended claims.

#In this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs.