Devices and Methods for Target Molecule Characterization

• US 20100084276A1
• Filed: 04/07/2008
• Published: 04/08/2010
• Est. Priority Date: 04/06/2007
• Status: Active Grant

• Current Assignee: Arizona Board of Regents (University of Arizona)
• Original Assignee: Arizona Board of Regents (University of Arizona)
• Inventors: Lindsay, Stuart
• Granted Patent: US 9,395,352 B2
• Time in Patent Office: Days
• US Class Current: 205/93
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: G01N 27/028 Circuits therefor measuring impedance per se G01R27/02
• CPC Class Codes: G01N 27/3276 being a hybridisation with immobilised receptors using a FET type sensor G01N27/4145; concerning the hybridisation C12Q1/68
• 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 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 provides a device having at least one constriction that is sized to permit translocation of only a single copy of the molecule. The device has a pair of spaced apart sensing electrodes that border the constriction, which may be a nanopore. The first electrode is connected to a first affinity element and the second electrode is connected to a second affinity element. The first and second affinity elements are configured to temporarily form hydrogen bonds with first and second portions of the target molecule as the latter passes through the constriction.

Claims

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

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 a target molecule therethrough;

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

a first affinity element connected to the first electrode; and

a second affinity element connected to the second electrode;

wherein;

when a particular portion of a target molecule translocates through the constriction, an electrical current passes through the first electrode, the first affinity element, said particular portion, the second affinity element, and the second electrode.

4. The device according to claim 1, wherein:

the first sensing electrode is connected to the first affinity element via a first linker molecule.

5. The device according to claim 4, wherein:

the first sensing electrode is connected to the first linker molecule via an electrode attachment molecule.

7. The device according to claim 1, wherein:

the first affinity element is a phosphate grabber; and

the second affinity element is a base reader.

8. The device according to claim 7, wherein:

the phosphate grabber comprises a guanidinium moiety.

14. The device according to claim 1, wherein said electrical current comprises a tunneling current that passes through:

a first set of hydrogen bonds established between the first affinity element and a first site of said particular portion, and

a second set of hydrogen bonds established between the second affinity element and a second site of said particular portion.

15. A method of making the molecular recognition device of claim 1, the method comprising:

providing a partition having a first side and a second side;

forming spaced apart first and second sensing electrodes on the first side of the partition;

forming a reference electrode and a counter electrode on the second side of the partition;

forming a pore through the partition;

positioning the partition such that a salt solution is on the first side of the partition and a metal plating solution is on the second side of the partition;

applying a current such that the metal plating solution passes through the pore and plates onto the first and second sensing electrodes; and

connecting at least one affinity element to each of the first and second sensing electrodes.

16. The method according to claim 15, comprising:

connecting a first affinity element to the first sensing electrode; and

connecting a second affinity element to the second sensing electrode.

17. The method according to claim 15, comprising:

exposing the first and second sensing electrodes to an equimolar mix of a phosphate grabber and base reader to thereby connect at least one affinity element to each of the first and second sensing electrodes.

18. The method according to claim 17, comprising:

simultaneously exposing the first and second sensing electrodes to a reagent comprising linker molecules.

19. The method according to claim 15, comprising:

exposing the first and second sensing electrodes to a reagent comprising at least one of linker molecules and a affinity element.

20. The method according to claim 19, comprising:

holding at least one of said sensing electrodes at a predetermined potential so as to prevent said at least one of linker molecules and a affinity element from connecting to said at least one of said sensing electrodes.

21. The method according to claim 15, wherein:

forming the spaced apart first and second sensing electrodes comprising depositing a sensing wire on the first side and subsequently removing a portion of said sensing wire to thereby form a gap between opposing portions of said sensing wire.

22. The method according to claim 21, wherein:

forming the pore comprises milling a hole in the partition between said gap formed between opposing portions of said sensing wire.

23. The method according to claim 15, comprising:

plating metal onto the first and second sensing electrodes until a predetermined tunnel current is obtained between the two sensing electrodes.

24. The method according to claim 23, comprising:

stripping the plated metal to thereby enlarge a gap between the first and second sensing electrodes, after said predetermined tunnel has been obtained.

25. The method according to claim 15, wherein:

applying said current reduces both;

(a) a dimension of said pore, and (b) a gap between the first and second sensing electrodes.

2-3. -3. (canceled)

6. (canceled)

9-13. -13. (canceled)

26-35. -35. (canceled)

36. A molecular recognition device configured to sequence ssDNA, the device comprising:

a partition having a first side and a second side, and four constrictions passing between the first and second sides, each constriction being shaped and sized to permit translocation of only a single copy of the ssDNA to be sequenced;

first and second sensing electrodes associated with each constriction on the first side of the partition, the first and second electrodes of each constriction being separated from one another by a first gap;

a first affinity element connected to the first electrode of each constriction, said first affinity element comprising a phosphate grabber; and

a second affinity element connected to the second electrode of each constriction, the second affinity element comprising a base reader with each of four constrictions being provided with a different base reader;

wherein;

when a particular base of the ssDNA translocates through a constriction connected to a base reader corresponding to said particular base, an electrical current passes through the first electrode, the first affinity element, said particular base, the second affinity element, and the second electrode.

37-39. -39. (canceled)

40. A device for the detection of a target molecule comprising:

a) a partition comprising one or more constrictions sized to permit translocation of a target molecule through the one or more constrictions;

b) an electrode pair on the partition in communication with the one or more constrictions;

c) a first affinity element and a second affinity element bound to one or both members of the electrode pair, wherein the first affinity element is capable of binding to a first site on the target molecule as it enters the one or more constrictions; and

wherein the second affinity element is capable of binding to a second site on the target molecule as it enters the one or more constrictions in the presence of the first affinity element bound to the first site on the target molecule;

wherein the electrode pair is arranged so that binding of the target molecule by the first affinity element bound to one member of the electrode pair coupled with binding of the target molecule by the second affinity element to the other member of the electrode pair generates an electrical signal.

41. The device of claim 40, wherein the first and second affinity elements are bound to one or both members of the electrode pair via a linker.

42. The device of claim 41, wherein the electrodes in the electrode pair are spaced from 0.5 to 10 nm apart.

43. The device of claim 41, wherein the target molecule is a nucleic acid.

44. The device of claim 43, wherein the first affinity element is capable of binding to phosphate groups in the nucleic acid backbone.

45. The device of claim 43, wherein the second affinity element is capable of binding to specific bases in the nucleic acid.

46. The device of claim 41, wherein the affinity element comprises a guanidinium moiety.

47. The device of claim 46, wherein the guanidinium moiety comprises guanidinoethyldisulfide.

48-63. -63. (canceled)