Nanopore and carbon nanotube based DNA sequencer and a serial recognition sequencer

• US 8,628,649 B2
• Filed: 03/18/2009
• Issued: 01/14/2014
• Est. Priority Date: 03/18/2008
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Lindsay, Stuart, He, Jin, Zhang, Peiming, Reinhart, Kevin
• Primary Examiner(s): Noguerola, Alex
• Application Number:
  US12/920,809
• Publication Number:
  US 20110120868A1
• Time in Patent Office: 1,763 Days
• Field of Search: 204/603, 204/452, 204/409, 204/412, 204/450, 204/600, 204/403.01, 205/777.5
• US Class Current: 204/452
• 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: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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 present invention is directed to systems, devices and methods for identifying biopolymers, such as strands of DNA, as they pass through a constriction such as a carbon nanotube nanopore. More particularly, the invention is directed to such systems, devices and methods in which a newly translocated portion of the biopolymer forms a temporary electrical circuit between the nanotube nanopore and a second electrode, which may also be a nanotube. Further, the invention is directed to such systems, devices and methods in which the constriction is provided with a functionalized unit which, together with a newly translocated portion of the biopolymer, forms a temporary electrical circuit that can be used to characterize that portion of the biopolymer.

Claims

1. A molecular recognition chip configured to identify at least one portion of a target molecule, the chip comprising:

a substrate;

a first nanotube passing through at least a portion of the substrate, the first nanotube provided with a nanopore and configured and dimensioned to permit the translocation of a target molecule therethrough;

a second nanotube supported by the substrate and disposed in a fixed relationship with respect to said first nanotube so as to form a first gap with the first nanotube; and

an electrical circuit configured to detect an electrical current between the first and second nanotubes, upon passage of the target molecule past said gap.

2. The chip according to claim 1, wherein:

the first nanotube comprises a first recognition element connected to an end thereof; and

the second nanotube comprises a second recognition element connected to a first end thereof.

3. The chip according to claim 2, wherein the first recognition element comprises a guanidinium moiety;

and the second recognition element comprises a base reader.

4. The chip according to claim 2, wherein the first recognition element comprises a base and the second recognition element comprises a base-pair reader.

5. The chip according to claim 1, wherein:

the first gap is between 1.0 nm-5.0 nm.

6. A method of sequencing DNA comprising;

providing four types of molecular recognition chips, each molecular recognition chip comprising;

a substrate;

a first nanotube passing through at least a portion of the substrate, the first nanotube provided with a nanopore and configured and dimensioned to permit the translocation of a target molecule therethrough;

a second nanotube supported by the substrate and disposed in a fixed relationship with respect to said first nanotube so as to form a first gap with the first nanotube; and

an electrical circuit configured to detect an electrical current between the first and second nanotubes, upon passage of the target molecule past said gap, wherein;

at least one of the first and second nanotubes of each is functionalized to recognize one of four DNA bases;

placing said plurality of types of molecular recognition chips in an apparatus configured to accommodate the DNA, with copies of the DNA to be sequenced present on one side of each such chip;

electrophoresing the DNA through the nanopores of the first nanotubes of each chip;

recording current as a function of time as the DNA emerges from the nanopore of the first nanotube and forms temporary hydrogen bonds with molecules connected to the first and second nanotubes, thereby creating a detectable tunneling current; and

aligning data obtained from at least four different types of molecular recognition chips.

7. The method according to claim 6, further comprising:

recording information reflective of said electrical current as a function of time; and

obtaining at least one parameter reflective of an identification of each nucleotide in the DNA, from said recorded information;

wherein said at least one parameter comprises one from the group consisting of charge, duration of current signal, shape of current signal and decay of current, and

further comprising comparing said at least one parameter with a predetermined threshold to determine whether a particular nucleotide in the DNA has been recognized; and

detecting electrical current from a same portion of a predetermined number of copies of said DNA molecule to thereby improve recognition accuracy of said portion.

8. A method of fabricating a molecular recognition chip configured to identify at least one portion of a target molecule, the method comprising:

providing a silicon substrate;

forming a first insulating layer over the silicon substrate providing one or more vertically oriented first nanotubes on the first insulating layer;

forming a first metal contact layer over the first insulating layer;

forming a second insulating layer over the first metal contact layer;

etching back the resultant structure to remove an extra portion of the first nanotube which protrudes above the surface of the second insulating layer;

etching the underside of the silicon substrate to form a window so as to expose a lower end of the first nanotube;

functionalizing the first nanotube with a carboxylic acid group;

reacting the carboxylic acid functionalized first carbon nanotube with a guanidinium group to thereby form a guanidinium-functionalized first carbon nanotube;

connecting a sacrificial nucleotide to the guanidinium-functionalized first carbon nanotube;

providing at least one second nanotube having a recognition element complementary to said sacrificial nucleotide, in the proximity of the guanidinium-functionalized vertically oriented nanotube so that the sacrificial nucleotide forms at least one bond with said recognition element;

forming a protective passivation layer over the structure, the passivation layer covering at least the region in which the first and second nanotubes are present;

forming a ring-shaped second metal contact layer on the structure, the second metal contact layer contacting an end of the second carbon nanotube; and

removing the passivation layer and the sacrificial nucleotide, to thereby leave a gap between the vertically oriented nanotube and second nanotube.

9. A device for obtaining an electrical signal characteristic of a DNA base pair comprising

a DNA nucleotide base reader capable of binding to a DNA nucleotide base with a complimentary pattern and spacing of Hydrogen bond donors and acceptors attached to a first electrode and a DNA base pair reader attached to a second electrode, wherein the DNA base pair reader is capable of forming three hydrogen bonds with the DNA base pair comprising the DNA nucleotide base reader and the DNA nucleotide base.

10. A DNA sequencing device configured to identify at east one portion of a target DNA molecule, the device comprising:

a) a partition having a first side and a second side, and at least one constriction passing between the first and second sides, the constriction being shaped and sized to permit translocation of only a single copy of the DNA target molecule therethrough;

b) first and second sensing electrodes associated with the first side of the partition and being separated from one another by a first gap;

c) a first affinity element comprising a base reader configured to bind to a DNA nucleotide base of a DNA target module with a complimentary pattern and spacing of Hydrogen bond donors and acceptors connected to the first electrode; and

d) a second affinity element comprising a base pair reader configured to bind to both members of a pair comprising the base reader and the nucleotide base of the DNA target molecule connected to the second electrode;

wherein when a particular portion of the DNA target molecule translocates through the constriction, an electrical current passes through the first electrode, the base reader, said particular portion of the DNA molecule, the base pair reader, and the second electrode.

11. The device according to claim 10, wherein said electrical current comprises a tunneling current that passes through:

a first set of hydrogen bonds established between the base reader and the nucleotide base of the DNA target molecule, and a second set of hydrogen bonds established between the base pair reader and the nucleotide base of the DNA target molecule and the base reader.

12. A method of sequencing DNA as the DNA molecule translocates through a constriction, the method comprising:

a) providing an apparatus comprising a DNA sequencing device in accordance with claim 10, the device being located in the apparatus such that a first chamber is located on the first side of the device and a second chamber is located on the second side of the device;

b) introducing the DNA target molecule into the second chamber;

c) electrophorescing the target DNA molecule so that it translocates through the constriction; and

d) detecting an electrical current passing through the first electrode, the base reader, a nucleotide base of the DNA molecule, the base pair reader, and the second electrode.

13. The method according to claim 12, comprising:

threading a leading end of the target DNA molecule through the constriction and into the first chamber, and detecting said electrical current.

14. A method of sequencing ssDNA comprising:

a) providing an apparatus having the DNA sequencing device in accordance with claim 10 arranged such that a first chamber is located on the first side of the DNA sequencing device and a second chamber is located on the second side of the DNA sequencing device;

b) introducing at least four copies of the target DNA molecule that comprises ssDNA to be sequenced into the second chamber;

c) simultaneously electrophorescing the four ssDNA copies so that each copy translocates through one of the four constrictions of the DNA sequencing device; and

d) during electrophoreses, at each constriction, determining whether a nucleotide base has been recognized based on an electrical current passing through the first electrode, the base reader, the nucleotide, the base pair reader and the second electrode, to thereby create four component read sequences, one component read sequence for each constriction.

15. The method according to claim 14, comprising:

aligning the four component read sequences based on one or more preselected blocks of a repeated nucleotide found in at least one of the component read sequences; and

creating a final sequence of reads based on the four component read sequences.

16. A device for characterizing a biopolymer, the device comprising a carbon nanotube having an interior channel through which the biopolymer is driven by electrophoresis;

wherein the carbon nanotube comprises a plurality of gaps cut into the carbon nanotube; and

wherein the plurality of gaps is functionalized to provide a characteristic signal at each of the plurality of gaps.

17. The device of claim 16 wherein the biopolymer is a nucleic acid and the carbon nanotube comprises four gaps cut into the carbon nanotube, wherein each gap is functionalized with a different nucleoside reader.

18. A device for determining the sequence of a nucleic acid comprising:

a) a solid surface to support a carbon nanotube having an interior channel through which the nucleic acid can travel; and

b) an insulating film layer disposed on top of the carbon nanotube;

wherein the insulating film layer comprises an origination, a first, a second, a third, a fourth and a termination well capable of containing an electrolyte;

wherein the carbon nanotube comprises a first, second, third and fourth gap cut into the carbon nanotube;

wherein the first gap contacts the first well, the second gap contacts the second well, the third gap contacts the third well and the fourth gap contacts the fourth well;

wherein each of the first, second, third and fourth gaps of the carbon nanotube has a first gap end and a second gap end;

wherein each of the first gap ends is functionalized with a first recognition element;

wherein each of the second gap ends is functionalized with a second recognition element;

wherein the carbon nanotube has an origination end contacting the origination well and a termination end contacting the termination well;

wherein an origination electrode contacts the origination well and a termination electrode contacts the termination well;

wherein the origination, and termination electrodes do not contact the carbon nanotube; and

wherein the device further comprises a first electrode contacting the carbon nanotube between the origination well and the first well;

a second electrode contacting the carbon nanotube between the first and second well;

a third electrode contacting the carbon nanotube between the second and third well;

a fourth electrode contacting the carbon nanotube between the third and fourth well; and

a fifth electrode contacting the carbon nanotube located between the fourth and termination well.

19. A method of making a device for determining the sequence of a nucleic acid comprising, a solid surface to support a carbon nanotube having a channel through which the nucleic acid can travel;

and a PMMA film layer on top of the carbon nanotube, the method comprising;

a) growing a carbon nanotube on a silicon wafer;

b) depositing a plurality of electrodes on the silicon wafer to contact the carbon nanotube;

c) depositing a plurality of electrodes on the silicon wafer that do not contact the carbon nanotube;

d) creating a plurality of gaps having a first and second gap end in the carbon nanotube, wherein each gap is located between two of the plurality of electrodes from step (b);

e) conjugating a first recognition element o each of the plurality of first gap ends; and

f) conjugating a second recognition element to each of the plurality of second gap ends.