NANOPORE SEQUENCING DEVICES AND METHODS

• US 20100331194A1
• Filed: 04/09/2010
• Published: 12/30/2010
• Est. Priority Date: 04/10/2009
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Callebaut, Hans, Sebra, Robert, Turner, Stephen, Foquet, Mathieu, Korlach, Jonas, Bjornson, Keith, Emig, Robin, Sorenson, Jon, Chaudhuri, Bidhan, Flusberg, Benjamin, Fehr, Adrian
• Granted Patent: US 8,986,928 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• 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 2527/113 Time
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/447 using electrophoresis
• CPC Class Codes: G01N 27/44791 Microapparatus sample containers with integrated microfluidic structures B01L3/5027
• 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

The invention relates to devices and methods for nanopore sequencing. The invention includes arrays of nanopores having incorporated electronic circuits, for example, in CMOS. In some cases, the arrays of nanopores comprise resistive openings for isolating the electronic signals for improved sequencing. Methods for controlling translocation of through the nanopore are disclosed.

Claims

1. A device for determining polymer sequence information comprising:

a substrate comprising an array of nanopores;

each nanopore fluidically connected to an upper fluidic region and a lower fluidic region;

wherein each upper fluidic region is fluidically connected through an upper resistive opening to an upper liquid volume.

2. The device of claim 1 wherein the upper liquid volume is fluidically connected to two or more upper fluidic regions.

3. The device of claim 1 wherein each lower fluidic region is fluidically connected through a lower resistive opening to a lower liquid volume, and wherein the lower liquid volume is fluidically connected to two or more lower fluidic regions.

4. The device of claim 1 wherein the substrate is a semiconductor comprising circuit elements.

5. The device of claim 4 wherein either the upper fluidic region or the lower fluidic region for each nanopore or both the lower fluidic region and the upper fluidic region for each nanopore is electrically connected to a circuit element.

6-14. -14. (canceled)

15. A polymer sequencing device comprising:

a) a nanopore layer comprising an array of nanopores, each nanopore having a cross sectional dimension of 1 to 10 nanometers, and having a top and a bottom opening, wherein the bottom opening of each nanopore opens into a discrete reservoir, resulting in an array of reservoirs, wherein each reservoir comprises one or more electrodes, the nanopore layer physically and electrically connected to a semiconductor chip, and

b) the semiconductor chip, comprising an array of circuit elements, wherein each of the electrodes in the array of reservoirs is connected to at least one circuit element on the semiconductor chip.

16-19. -19. (canceled)

20. A method of fabricating a polymer sequencing device comprising:

a) obtaining a semiconductor substrate;

b) processing the semiconductor substrate to create an array of microfluidic features, wherein the microfluidic features are capable of supporting an array of nanopores;

c) subsequently producing circuit elements on the substrate that are electronically coupled to the microfluidic features; and

d) introducing nanopores into the microfluidic features.

21. The method of claim 20 wherein the circuit elements are CMOS circuit elements.

22. The method of claim 20 wherein the CMOS circuit elements comprise amplifiers, analog to digital converters.

23-31. -31. (canceled)

32. A method for determining sequence information about a polymer molecule comprising:

a) providing a device comprising a substrate having an array of nanopores;

each nanopore fluidically connected to an upper fluidic region and a lower fluidic region;

wherein each upper fluidic region is fluidically connected through a an upper resistive opening to an upper liquid volume; and

each lower fluidic region is connected to a lower liquid volume, and wherein the upper liquid volume and the lower liquid volume are each fluidically connected to two or more fluidic regions, wherein the device comprises an upper drive electrode in the upper liquid volume, a lower drive electrode in the lower liquid volume, and a measurement electrode in either the upper liquid volume or the lower liquid volume;

b) placing a polymer molecule to be sequenced into one or more upper fluidic regions;

c) applying a voltage across the upper and lower drive electrodes so as to pass a current through the nanopore such that the polymer molecule is translated through the nanopore;

d) measuring the current through the nanopore over time; and

e) using the measured current over time in step (d) to determine sequence information about the polymer molecule.

33. The method of claim 32 wherein the substrate comprises electronic circuits electrically coupled to the measurement electrodes which at least partially process signals from the measurement electrodes.

34. The method of claim 33 wherein the upper drive electrode and lower drive electrode are each biased to a voltage above or below ground, and at least a portion of the substrate electrically connected to the electronic circuits is held at ground potential.

35. A method for determining sequence information about a polymer molecule comprising:

a) providing a device having an array of nanopores, each connected to upper and lower fluid regions;

wherein the device comprises electronic circuits electrically connected to electrodes in either the upper fluid regions or lower fluid regions or both the upper and lower fluid regions;

b) placing a polymer molecule in an upper fluid region;

c) applying a voltage across the nanopore whereby the polymer molecule is translocated through the nanopore;

d) using the electronic circuits to monitor the current through the nanopore over time, wherein the electronic circuits process the incoming current over time to record events, thereby generating event data; and

e) using the event data from step (d) to obtain sequence information about the polymer molecule.

36-41. -41. (canceled)

42. A method for determining the sequence of a polymer having two or more types of monomeric units in a solution comprising:

a) actively translocating the polymer through a pore;

b) measuring a property which has a value that varies depending on whether and which of the two or more a types of monomeric unit is in the pore, wherein the measuring is performed as a function of time while the polymer is actively translocating; and

c) determining the sequence of the two or more types of monomeric units in the polymer using the measured property from step (b) by performing a process including the steps of;

(i) deconvolution, (ii) peak finding, and (iii) peak classification.

43. The method of claim 42 wherein the polymer is a nucleic acid, the monomeric units are nucleotide bases or nucleotide analogs, and the measured property is current.

44-49. -49. (canceled)