Compositions, Devices, Systems, and Methods for Using a Nanopore

• US 20160289758A1
• Filed: 05/23/2016
• Published: 10/06/2016
• Est. Priority Date: 04/04/2007
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Akeson, Mark A., Deamer, David W., Chen, Roger Jinteh Arrigo
• Granted Patent: US 9,797,013 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2523/307 Denaturation or renaturation by electric current/voltage
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C25B 3/29 Coupling reactions
• CPC Class Codes: G01N 27/3278 involving nanosized elements, e.g. nanogaps or nanoparticles nanopores G01N33/48721; magnetic beads G01N27/745
• CPC Class Codes: G01N 27/4166 measuring a particular property of an electrolyte
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

Devices and methods that can detect and control an individual polymer in a mixture is acted upon by another compound, for example, an enzyme, in a nanopore are provided. The devices and methods also determine (˜>50 Hz) the nucleotide base sequence of a polynucleotide under feedback control or using signals generated by the interactions between the polynucleotide and the nanopore. The invention is of particular use in the fields of molecular biology, structural biology, cell biology, molecular switches, molecular circuits, and molecular computational devices, and the manufacture thereof.

Claims

1. A method for controlling insertion of a pore molecule into a thin film of a nanopore device, the thin film separating two pools of a liquid medium, the method comprising:

(a) providing, in one of the two pools, an amount of pore molecules sufficient to form at least a single nanopore in said thin film by insertion of a pore molecule into the thin film;

(b) providing a circuit comprising electrodes in the two pools, thereby providing a potential difference across the thin films, said circuit further connected to voltage control logic; and

(c) detecting insertion of a pore molecule into the thin film by sensing a change in a transport property with the voltage control logic; and

(d) adjusting the potential difference in response to the change in a measured transport property resulting from insertion of the pore molecule, to thereby decrease the possibility of insertion of a second nanopore in said film.

2. A method according to claim 1 wherein insertion of the nanopore is detected by a finite state machine.

3. A method according to claim 2 wherein the adjusting of the potential difference is carried out by the finite state machine.

4. A method according to claim 1 wherein the thin film is a lipid bilayer.

5. A method according to claim 1 wherein the change in transport property is measured as ion current flow.

6. A method according to claim 1 wherein the nanopore device comprises an array of elements comprising thin films and individual cis and trans chambers, wherein a common electrode is provided in the cis chamber and electrodes are provided in each of the trans chambers for providing a potential difference across each thin film, further comprising steps of:

measuring a transport property across each thin film, and detecting the insertion of a nanopore in each film on the basis of a change in the measured transport property; and

individually adjusting the potential difference across the thin film in response to the change in transport property to decrease the possibility of insertion of a second nanopore in a film in a chamber.

7. An apparatus comprising:

(a) a thin film separating a first pool and second pool, the two pools containing an ionic medium in a cis chamber and a trans chamber, respectively, and for containing pore molecules in one pool, providing a nanopore between the two pools;

(b) electrodes arranged to provide a potential difference between electrodes in the first pool and the second pool;

(c) a measurement system configured to measure a transport property across the thin film and to detect the insertion of a nanopore in the film on the basis of a change in the measured transport property, and

(d) control logic configured to adjust the potential difference between the electrodes in response to the change in transport property in order to decrease the possibility of insertion of second nanopore in the film.

8. An apparatus according to claim 7 wherein the transport property is ion current flow through the nanopore.

9. An apparatus according to claim 7 comprising an array of elements comprising thin films, wherein

(a) an electrode is provided in each cis chamber in the array and respective trans chamber in the array, comprising a cis chamber and a plurality of trans chambers wherein each of the thin films separates the cis chamber from a respective trans chamber, wherein a common electrode is provided in the cis chamber and respective electrodes are provided in each of the trans chambers for providing a potential difference across each thin film providing a potential difference across each thin film in the array;

and

(b) the measurement system is configured to individually detect a transport property across each thin film in the array and wherein the control logic is configured to individually adjust the potential difference across each thin film in response to a change in transport property in order to decrease the possibility of insertion of second nanopore in a respective film in the array.