APPARATUS AND METHOD FOR ELECTRICAL DETECTION OF OLIGONUCLEOTIDES THROUGH PORE BLOCKADES

• US 20140248711A1
• Filed: 02/25/2014
• Published: 09/04/2014
• Est. Priority Date: 09/01/2011
• 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: Monbouquette, Harold G., Schmidt, Jacob J.
• Granted Patent: US 9,428,806 B2
• Time in Patent Office: Days
• US Class Current: 436/501
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/4473 by electric means
• 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

Systems and methods for specific nucleic acid (NA) sequence detection that do not rely on polymerase chain reaction (PCR) for target sequence amplification and do not require any special reagents other than a complementary sequence capture probe conjugated to spherical beads.

Claims

1. An apparatus for sequence-specific nucleic acid detection in a sample, the apparatus comprising:

a first chamber;

a second chamber;

a membrane separating said first and second chambers, said membrane having a pore extending through the membrane between the first and second chambers;

a first electrode disposed in the first chamber; and

a second electrode disposed in the second chamber;

wherein when a liquid is introduced into said first and second chambers, wherein when a source of direct electric current is connected to said electrodes with a negative polarity being connected to the first electrode, and wherein when a bead which is conjugated with one or more strands of capture probe nucleic acid that is complementary to a desired target sequence to be detected is subsequently introduced into the liquid in the first chamber, the presence of said target nucleic acid is indicated by a decrease in electric current between the electrodes in relation to electric current between the electrodes prior to introduction of the beads into the first chamber, said decrease in electric current resulting from movement of the bead and the bead causing blockage of the pore.

2. An apparatus as recited in claim 1:

wherein the pore has a length between a first end and a second end;

wherein the pore has a diameter that decreases from said first end toward said second end;

wherein the bead has a diameter less than the diameter at the first end of the pore; and

wherein the pore has a diameter less than the diameter of the bead at a point along the length of the pore.

3. An apparatus as recited in claim 1, wherein the pore has a diameter ranging from approximately 10 nm to approximately 5 μ

m.

4. An apparatus as recited in claim 1 or 2, wherein the pore comprises an opening extending through said membrane.

5. An apparatus as recited in claim 1 or 2, wherein the pore comprises a pipette extending through said membrane.

6. An apparatus as recited in claim 1 or 2, wherein the bead comprises unmodified or functionalized polystyrene.

7. An apparatus as recited in claim 6, wherein the bead has a surface functionalized with amine or carboxyl groups to facilitate cross-linking to a terminal carboxyl or amine group, respectively, added to the terminus of nucleic acid capture probes.

8. An apparatus as recited in claim 1, wherein the strands of capture probe nucleic acid comprise a peptide nucleic acid.

9. An apparatus as recited in claim 1, wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water.

10. An apparatus as recited in claim 9, wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other electrolytes.

11. An apparatus as recited in claim 1, wherein a single molecule of a desired target sequence can be detected.

12. An apparatus for sequence-specific nucleic acid detection in a sample, the apparatus comprising:

a first chamber;

a second chamber;

a membrane separating said first and second chambers, said membrane having a pore extending through the membrane between the first and second chambers;

wherein the pore has a length between a first end and a second end;

wherein the pore has a diameter that decreases from said first end toward said second end;

wherein the bead has a diameter less than the diameter at the first end of the pore;

wherein the pore has a diameter less than the diameter of the beads at a point along the length of the pore;

a first electrode disposed in the first chamber; and

a second electrode disposed in the second chamber.

13. An apparatus as recited in claim 12, wherein when a liquid is introduced into said first and second chambers, wherein when a source of direct electric current is connected to said electrodes with a negative polarity being connected to the first electrode, and wherein when a bead which is conjugated with one or more strands of capture probe nucleic acid that is complementary to a desired target sequence to be detected is subsequently introduced into the liquid in the first chamber, the presence of said target nucleic acid is indicated by a decrease in electric current between the electrodes in relation to electric current between the electrodes prior to introduction of the beads into the first chamber, said decrease in electric current resulting from movement of the bead and the bead causing blockage of the pore.

14. An apparatus as recited in claim 12, wherein the pore has a diameter ranging from approximately 10 nm to approximately 5 μ

m.

15. An apparatus as recited in claim 12, wherein the pore comprises an opening extending through said membrane.

16. An apparatus as recited in claim 12, wherein the pore comprises a pipette extending through said membrane.

17. An apparatus as recited in claim 12, wherein the bead comprises unmodified or functionalized polystyrene.

18. An apparatus as recited in claim 17, wherein the bead has a surface functionalized with amine or carboxyl groups to facilitate cross-linking to a terminal carboxyl or amine group, respectively, added to the terminus of nucleic acid capture probes.

19. An apparatus as recited in claim 13, wherein said strands of capture probe nucleic acid comprise a peptide nucleic acid.

20. An apparatus as recited in claim 13, wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water.

21. An apparatus as recited in claim 20, wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other electrolytes.

22. An apparatus as recited in claim 12, wherein a single molecule of a desired sequence can be detected.

23. A method for detecting the presence of nucleic acids with a target sequence from a sample, the method comprising:

(a) providing a detection apparatus, said detection apparatus comprising;

(i) a first chamber;

(ii) a second chamber;

(iii) a membrane separating said first and second chambers, said membrane having a pore extending through the membrane between the first and second chambers;

(iv) a first electrode disposed in the first chamber; and

(v) a second electrode disposed in the second chamber;

(b) introducing a liquid into said first and second chambers;

(c) applying a direct electric current to said first and second electrodes with a negative polarity being connected to the first electrode;

(d) measuring electric current between the electrodes as a reference current;

(e) introducing, into said first chamber, a bead which is conjugated with one or more strands of capture probe nucleic acid that are complementary to a desired target sequence to be detected; and

(f) measuring electric current between the electrodes after introduction of the bead, wherein a decrease in measured electric current indicates the presence of one or more strands of target nucleic acid that are complimentary to the probe sequence conjugated to the bead.

24. A method as recited in claim 23, wherein said decrease in measured electric current results from movement of the bead and the bead causing blockage of the pore.

25. A method as recited in claim 23:

wherein the pore has a length between a first end and a second end;

wherein the pore has a diameter that decreases from said first end toward said second end;

wherein the beads have a diameter less than the diameter at the first end of the pore; and

wherein the pore has a diameter less than the diameter of the beads at a point along the length of the pore.

26. A method as recited in claim 23, wherein the pore has a diameter ranging from approximately 10 nm to approximately 5 μ

m.

27. A method as recited in claim 23, wherein the pore comprises an opening extending through said membrane.

28. A method as recited in claim 23, wherein the pore comprises a pipette extending through said membrane.

29. A method as recited in claim 23, wherein said bead comprises unmodified or functionalized polystyrene.

30. A method as recited in claim 29, wherein the bead has a surface functionalized with amine or carboxyl groups to facilitate cross-linking to a terminal carboxyl or amine group, respectively, added to the terminus of nucleic acid capture probes.

31. A method as recited in claim 23, wherein said strands of capture probe nucleic acid comprise peptide nucleic acids.

32. A method as recited in claim 23, wherein the liquid comprises an electrolyte solution comprising approximately 0.01 M to approximately 1.0 M of an electrolyte in buffered water.

33. A method as recited in claim 32, wherein said electrolyte is selected from the group comprising of NaCl, KCl, and other strong electrolytes.

34. A method as recited in claim 23, further comprising hydrostatically, osmotically, or electro-creating a fluid flow through said membrane from the second chamber to the first chamber through a negative surface charge on the membrane and pore.

35. A method as recited in claim 23, further comprising detecting a single molecule of a desired sequence.