Nanopore Sensors for Biomolecular Characterization

• US 20140174927A1
• Filed: 07/26/2012
• Published: 06/26/2014
• Est. Priority Date: 07/27/2011
• Status: Active Grant

• Current Assignee: Board of Trustees of The University of Illinois
• Original Assignee: Board of Trustees of The University of Illinois
• Inventors: Bashir, Rashid, Venkatesan, Bala Murali
• Granted Patent: US 10,175,195 B2
• Time in Patent Office: Days
• US Class Current: 204/452
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2537/164 Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
• 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

Provided herein are methods and devices for characterizing a biomolecule parameter by a nanopore-containing membrane, and also methods for making devices that can be used in the methods and devices provided herein. The nanopore membrane is a multilayer stack of conducting layers and dielectric layers, wherein an embedded conducting layer or conducting layer gates provides well-controlled and measurable electric fields in and around the nanopore through which the biomolecule translocates. In an aspect, the conducting layer is graphene.

Claims

1. A method for characterizing a biomolecule parameter, said method comprising the steps of:

providing a nanopore in a membrane comprising a conductor-dielectric stack, wherein said membrane separates a first fluid compartment from a second fluid compartment and said nanopore fluidly connects said first and said second fluid compartments and said conductor comprises graphene or an atomically thin electrically conducting layer of Molybdenum disulfide (MoS₂), doped silicon, silicene, or ultra-thin metal;

providing the biomolecule to said first fluid compartment;

applying an electric field across said membrane;

driving said biomolecule through said nanopore to said second fluid compartment under said applied electric field; and

monitoring an electrical parameter across the membrane or along a plane formed by the membrane as the biomolecule transits the nanopore, thereby characterizing said biomolecule parameter.

2. The method of claim 1, wherein said biomolecule parameter is selected from the group consisting of:

polynucleotide sequence;

presence of modified nucleotides;

polynucleotide methylation state;

polynucleotide hydroxymethylation state;

methyl-dependent or hyrdoxymethyl-dependent binding protein bound to a one or more methylated or hydroxymethylated sites on a polynucleotide sequence;

presence of a protein-polynucleotide binding event;

polypeptide sequence;

biomolecule secondary structure; and

amino acid sequence.

3. The method of claim 2, wherein said polynucleotide comprises an organic or synthetic nucleic acid.

4. The method of claim 1, wherein said conductor-dielectric stack comprises:

a plurality of graphene layers, wherein adjacent graphene layers are separated by a dielectric layer.

5. The method of claim 4, wherein one or more of said graphene layers comprises a graphene microribbion, graphene nanoribbon, or graphene nanogap through which said nanopore traverses in a direction that is transverse to a longitudinal direction of said graphene microribbion, graphene nanoribbon, or graphene nanogap, said method further comprising:

measuring a time-course of electric potential or transverse current along said graphene microribbon or graphene nanoribbon or across said graphene nanogap during said biomolecule transit through said nanopore, thereby characterizing a sequence, composition, flux, duration or length of said biomolecule; and

wherein said measuring is multiple simultaneous measures at different locations of a translocating biomolecule.

6. The method of any of claims 1-5, further comprising independently electrically biasing one or more of said conducting layers to provide electrical gating of said nanopore.

7. The method of claim 6, wherein said biasing is by electrically connecting an electrode to an individual graphene layer embedded in the graphene-dielectric stack, and said biasing modifies an electric field in the nanopore generated by the applied electric field across the membrane.

8. The method of any of claim 1-5 or 7, wherein said dielectric layer comprises Aluminum Oxide, Tantalum Oxide, Titanium Oxide, Silicon Dioxide, Hafnium Oxide, Zirconium Oxide, Boron Nitride, Silicon Nitride, nanolaminates thereof, or any combination thereof.

9. The method of claim 1, wherein said electrical parameter is selected from one or more of the group consisting of:

current or current blockade through the nanopore;

tunneling current across the nanopore;

electrochemical current through a transverse electrode;

conductance;

resistance;

impedance;

electric potential;

translocation time of said biomolecule through said nanopore; and

biomolecule flux or translocation frequency.

10. The method of claim 1, further comprising the step of functionalizing exposed edges of graphene in the nanopore by attaching a chemical moiety to an exposed nanopore graphene edge;

wherein the chemical moiety has an affinity to a portion of the biomolecule, and the chemical moiety interacting with the portion of the biomolecule changes the monitored electrical parameter as the biomolecule transits the nanopore.

11. The method of claim 10, wherein the chemical moiety is selected from the group consisting of:

proteins, polypeptides and polynucleotides having a sequence that binds to a sequence within the biomolecule; and

chemical constructs having a binding affinity to a specific nucleotide within the biomolecule that is a polynucleotide.

12. The method of claim 11, wherein the specific nucleotide in the biomolecule is labeled with heavy atoms, chemical functional groups, or tags to enhance affinity between the chemical moiety and the specific nucleotide.

13. The method of claim 1, further comprising the step of digesting a biomolecule having a polynucleotide sequence into a plurality of smaller sequences by contacting the biomolecule with an exonuclease that is anchored to the graphene-dielectric stack thereby providing sequencing by digestion.

14. The method of claim 13, wherein at least a portion of the plurality of smaller sequences correspond to individual bases.

15. The method of claim 1, further comprising the step of synthesizing a polynucleotide sequence by adding nucleotides to the biomolecule that is transiting the nanopore, thereby providing sequencing by synthesis.

16. The method of claim 15, wherein the sequencing by synthesis is by a polymerase anchored to the graphene-dielectric stack and the added nucleotides and reagents are from a source of nucleotides in the first or second fluid compartment.

17. The method of claim 16, wherein the sequencing by synthesis further comprises the step of detecting released H⁺ or pyrophosphates during addition of a nucleotide to the biomolecule transiting the pore.

18. The method of claim 17, wherein the detecting released H⁺ or pyrophosphates is by measuring a change in nanopore current.

19. The method of claim 1, wherein the nanopore is a biological nanopore comprising a protein complex with an inner channel that is the nanopore, said protein selected from the group consisting of:

DNA binding proteins including transcription factors, polymerases, nucleases and histones;

alpha hemolysin;

Mycobacterium smegmatis porin A and GP10.

20. The method of any of claims 1-5, wherein a top-most graphene layer is in fluid and electrical contact with a fluid in the first and/or second fluid compartments and the electrical parameter is from an electrochemical measurement by the graphene layer in fluid and electrical contact with the fluid in the first and/or second fluid compartment as the biomolecule transits the pore.

21. The method of any of claims 1-5, wherein the electrical parameter is measured by field effect gating or field effect sensing by a graphene layer electrically insulated from the fluid in the fluid compartment and in the nanopore.

22. The method of claim 1, wherein the biomolecule is a double stranded polynucleotide sequence, the method further comprising the step of unzipping the double stranded polynucleotide sequence and driving a single strand of the double stranded polynucleotide sequence through the nanopore, thereby providing sequencing of said biomolecule.

23. The method of claim 22, wherein the unzipping is by a helicase anchored to the graphene-dielectric stack.

24. The method of claim 4, wherein a buried graphene layer measures an electrochemical current through another graphene layer.

25. A device for characterizing a biomolecule parameter, said device comprising:

a membrane comprising;

a first surface and a second surface opposite said first surface, wherein said membrane separates a first fluid compartment comprising said first surface from a second fluid compartment comprising said second surface;

a graphene/dielectric/graphene/dielectric stack positioned between said first surface and said second surface; and

a nanopore through said membrane that fluidically connects said first compartment and said second compartment;

a power supply in electrical contact with said membrane to provide an electric potential difference between said first fluid compartment and said second fluid compartment; and

a detector to detect an electrical current through said nanopore as a biomolecule transits said nanopore under an applied electric potential difference between said first and second fluid compartments.

26. The device of claim 25, further comprising one or more gate electrodes, wherein each of said one or more gate electrodes is a graphene layer in said stack.

27. The device of claim 26, wherein said graphene layer has a thickness that is less than or equal to 3 nm at the nanopore, and said electrical contact comprises a metal pad in electrical contact with said graphene layer, wherein said metal pad is electrically isolated from any of said first and second fluid compartments.

28. The device of claim 27, wherein the gate electrode is electrically connected to a source electrode powered by said power supply.

29. The device of any of claims 25-28, wherein one or more of said graphene layers comprises a nanoribbon through which said nanopore transits in a transverse direction to said nanoribbon longitudinal axis.

30. The device of claim 29, wherein said nanoribbon further comprises electrical contacts for measuring a transverse current along said nanoribbon during transit of a biomolecule through said nanopore and another of said graphene layers is connected to a gate electrode to electrically bias said graphene layer.

31. The device of claim 29, wherein said nanopore has a diameter that is greater than 5% of the nanoribbon width or is selected from a range between 5% and 95% of the nanoribbon width.

32. The device of claim 25, wherein said detector is a tunneling detector comprising a pair of graphene electrodes facing each other and centered within the nanopore in a direction transverse to the biomolecule transit direction in the nanopore, wherein the biomolecule transits between the pair of electrodes.

33. A method of making a membrane comprising nanopores for characterizing a biomolecule parameter, said method comprising the steps of:

forming a passage in a free-standing dielectric membrane;

growing a graphene layer via chemical vapor deposition;

transferring to the free-standing dielectric membrane a portion of said graphene layer;

forming a dielectric layer on the transferred the graphene layer;

repeating the graphene layer step to generate a second graphene layer;

repeating the dielectric layer forming step to generate a second dielectric layer;

forming a nanopore extending from a first surface of said membrane to a second surface of said membrane, wherein said nanopore traverses each of the graphene and dielectric layers, thereby making a membrane comprising a nanopore in a graphene/dielectric/graphene/dielectric stack.

34. The method of claim 33, further comprising independently electrically contacting said first graphene layer, said second graphene layer or both said first graphene layer and said second graphene layer with an electrical contact to provide an electrically gated nanopore.

35. The method of claim 33, wherein the repeating step is repeated to generate three or more graphene layers, with adjacent graphene layers separated by dielectric layers.

36. The method of claim 35, further comprising electrically contacting one or more of said graphene layers with an electrical contact to provide an electrically gated nanopore.

37. The method of claim 33, further comprising embedding a gate electrode in the nanopore membrane to modify a localized electric field in and adjacent to the nanopore.

38. The method of claim 37, wherein the embedded gate electrode comprises any of the graphene layers of the membrane.

39. The method of claim 33, wherein said dielectric layer comprises a dielectric deposited by atomic layer deposition.

40. The method of claim 33 or 39, wherein said dielectric layer comprises aluminum oxide, tantalum oxide, hafnium oxide, zirconium oxide, silicon dioxide, or silicon nitride or a combination of thereof.

41. The method of claim 33, further comprising electrically connecting said graphene layers in Wheatstone Bridge configuration for measuring an electrical parameter in said nanopore, wherein said electrical parameter is one or more of:

differential impedance, tunneling current, resistance, capacitance, current or voltage.

42. The method of claim 33, further comprising electrically biasing one or more graphene layers with an AC voltage signal.

43. The method of claim 42, further comprising monitoring impedances between a central graphene layer and outer graphene layers, or measuring impedance current or voltage between graphene layers.

44. A method for identifying, characterizing or quantifying the methylation or hydromethylation status of a biomolecule, said method comprising the steps of:

providing a nanopore in a suspended membrane that separates a first fluid compartment from a second fluid compartment, said membrane comprising Aluminum Oxide, Tantalum Oxide, Titanium Oxide, Silicon Dioxide, Hafnium Oxide, Zirconium Oxide, Boron Nitride, Silicon Nitride, graphene or nanolaminates thereof, or any combination thereof;

binding specific proteins, oligonucleotides or chemical tags to methylated or hydroxymethylated sites on target biomolecule; and

applying an electric field across the membrane, from the first fluid compartment to the second fluid compartment, to drive the biomolecule through said nanopore.

45. The method of claim 44, wherein said bound protein or tags on said biomolecule are detected by monitoring changes in ionic current, tunneling current, voltage, conductance or impedance.

46. The method of claim 44, further comprising the step of sequentially shearing bound protein or tags from the biomolecule as the biomolecule transits through said nanopore.