LABELS FOR ELECTRONIC DETECTION OF INDIVIDUAL MOLECULES AND METHODS FOR THEIR DETECTION

• US 20070292877A1
• Filed: 06/12/2007
• Published: 12/20/2007
• Est. Priority Date: 06/15/2006
• Status: Active Grant

• Current Assignee: Krassen Dimitrov
• Original Assignee: Krassen Dimitrov
• Inventors: Dimitrov, Krassen
• Granted Patent: US 7,964,347 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/587 Nanoparticles

Abstract

The invention provides labels for electronic detection of individual molecules. The labels are comprised of elements with different electrical properties that affect the electric current flowing through a nanoelectrode. The labels are of polymeric or filamentous structure where the elements are arranged linearly along their length. The arrangement of the elements is predetermined and combinatorial, so that a high diversity of labels can be generated in a manner that resembles barcoding on a nanoscale level. Methods for the synthesis of said barcode labels and for the binding of the barcode labels to individual molecules, their movement past a nanoelectrode, and their detection are also provided.

Claims

1. A label for the electronic detection of individual target analyte molecules comprising a target specific probe capable of binding the target analyte molecule attached to a polymeric molecule or filament, wherein the polymeric molecule or filament comprises one or more elements with different electrical properties as detected by a nanoelectrode.

2. The label according to claim 1 wherein the target analyte molecule is a nucleic acid, a protein, a polysaccaharide, a polypeptide, a carbohydrate or a small molecule.

3. The label according to claim 2 wherein the target analyte molecule is a nucleic acid.

4. The label according to claim 1 wherein the target specific probe is a nucleic acid molecule complementary to the target analyte molecule, a nucleic acid mimetic, a locked nucleic acid, a nucleic acid aptamer, a peptide, a protein, a receptor, a ligand, an antibody, an antibody fragment or a lecitin molecule.

5. The label according to claim 4 wherein the target specific probe is a nucleic acid molecule complementary to the target analyte molecule.

6. The label according to claim 1 wherein the polymeric molecule is a nucleic acid molecule, a protein, a peptide or a polyacrylic copolymer.

7. The label according to claim 8 wherein the polymeric molecule is a nucleic acid molecule.

8. The label according to claim 1 wherein the polymeric material or filament comprises from two to six elements with different electrical properties as detected by a nanoelectrode.

9. The label according to claim 8 wherein the polymeric material or filament comprises two alternating elements with different electrical properties as detected by a nanoelectrode.

10. The label according to claim 9 wherein the first of the two elements causes a greater decrease in current in the nanoelectrode than the second of the two elements.

11. The label according to claim 10 wherein the polymeric material is a nucleic acid molecule which has been modified to contain one or more biotin residues, the first of the two elements comprises a strepavidin-coated nanoparticle and the second of the two elements comprises an unmodified portion of the nucleic acid molecule.

12. The label according to claim 1 further comprising one or more additional elements capable of providing sufficient stretching of the label to allow each of the elements with different electrical properties to be separately detected by the nanoelcetrode.

13. The label according to claim 12 having one additional element comprising a magnetic nanosphere.

14. The label according to claim 12 having a first additional element comprising a superparamagnetic nanosphere and a second additional element comprising a diamagnetic nanosphere.

15. The label according to claim 12 having a first additional element comprising a magnetic nanosphere and a second additional element comprising a hydrophobic moiety.

16. A composition comprising two or more labels, each of which comprises a target specific probe capable of binding a target analyte molecule attached to a polymeric molecule or filament wherein the polymeric molecule or filament comprises one or more elements with different electrical properties as detected by a nanoelectrode, and wherein the target specific probe of each label is different from each of the other labels.

17. A kit for the electronic detection of individual target analyte molecules comprising:

(a) one or more labels each of which comprises a target specific probe capable of binding the target analyte molecule attached to a polymeric molecule or filament which comprises at least two elements with different electrical properties;

(b) a nanoelectrode capable of detecting the elements with different electrical properties; and

(c) a redox mediator capable of undergoing redox reactions at a surface of the nanoelectrode.

18. The kit according to claim 17 wherein the target specific probe is a nucleic acid molecule complementary to the target analyte molecule and the polymeric molecule is a nucleic acid molecule.

19. The kit according to claim 17 comprising two or more labels wherein the target specific probe of each label is different from each of the other labels.

20. The kit according to claim 17 wherein the polymeric material or filament comprises two elements with different electrical properties as detected by a nanoelectrode.

21. The kit according to claim 17 wherein the label further comprises one or more additional elements capable of providing sufficient stretching of the label to allow each of the elements with different electrical properties to be separately detected.

22. The kit according to claim 21 having one additional element comprising a magnetic nanosphere.

23. The kit according to claim 21 having a first additional element comprising a magnetic nanosphere and a second additional element comprising a hydrophobic moiety.

24. The kit according to claim 17 wherein the nanoelectrode has a critical dimension of about 20 nm to about 100 nm.

25. The kit according to claim 17 wherein the nanoelectrode has a critical dimension of less than about 100 nm.

26. The kit according to claim 17 wherein the nanoelectrode is fabricated from gold, platinum, copper or carbon nanotubes.

27. The kit according to claim 17 further comprising one or more additional nanoelectrodes fabricated to form an array.

28. The kit according to claim 17 wherein the redox mediator is 1,4-benzoquinone, ferrocene carboxylic acid, phenazine methosulphate, tetrathiofulvalene, tetracyanoquinodimethane, ferrocyanides, dimethylviologen, 1,1′

-dimethylferrocene, phenazine ethosulfate or ferrocenylmethanol.

29. The kit according to claim 17 wherein the nanoelectrode is located at the bottom of a channel in a silicon, glass or plastic chip.

30. A method for the detection of individual target analyte molecules in a sample comprising:

(a) contacting one or more labels, each of which comprises a target specific probe capable of binding the target analyte molecule attached to a polymeric molecule or filament which comprises one or more elements with different electrical properties as detected by a nanoelectrode, with a target analyte molecule;

(b) moving label that is bound to the target analyte molecule sufficiently close to a nanoelectrode such that the electric current of the nanoelectrode is changed; and

(c) detecting the changes in electric current produced in the nanoelectrode by the elements with different electrical properties in label that is bound to the target analyte molecule.

31. The method according to claim 30 wherein the label that is bound to the target analyte molecule is moved using optical tweezers, AC electric fields, hydrodynamic forces or fluidic forces.

32. The method according to claim 30 wherein the label that is bound to the target analyte molecule is moved using magnetic forces.

33. The method according to claim 30 wherein the changes in electric current produced in the nanoelectrode by the elements with different electrical properties in label that is bound to the target analyte molecule are detected using a galvanometer with an electronic amplifier.

34. The method according to claim 30 wherein the target specific probe is a nucleic acid molecule complementary to the target analyte molecule and the polymeric molecule is a nucleic acid molecule.

35. The method according to claim 30 comprising two or more labels wherein the target specific probe of each label is different from each of the other labels.

36. The method according to claim 30 wherein the polymeric molecule or filament comprises two elements with different electrical properties as detected by a nanoelectrode.

37. The method according to claim 30 wherein the label further comprises one or more additional elements capable of providing sufficient stretching of the label to allow each of the elements with different electrical properties to be separately detected.

38. The method according to claim 37 having one additional element comprising a magnetic nanosphere.

39. The method according to claim 37 having a first additional element comprising a magnetic nanosphere and a second additional element comprising a hydrophobic moiety.

40. The method according to claim 30 wherein the nanoelectrode has a critical dimension of about 20 nm to about 100 nm.

41. The method according to claim 30 wherein the nanoelectrode has a critical dimension of less than about 100 nm.

42. The method according to claim 30 wherein the nanoelectrode is fabricated from gold, platinum or carbon nanotubes.

43. The method according to claim 30 further comprising one or more additional nanoelectrodes fabricated to form an array.

44. The method according to claim 30 wherein the nanoelectrode is located at the bottom of a channel in a silicon, glass or plastic chip.

45. The method according to claim 44 wherein the chip contains one or more electronic component capable of amplifying the changes in electric current produced in the nanoelectrode.

46. The method according to claim 45 wherein the one or more electronic components comprise a field-effect transistor.

47. The method according to claim 46 wherein the field-effect transistor is a metal-oxide-semiconductor field-effect transistor.