Electrochemical sensor using intercalative, redox-active moieties

• US 20040063126A1
• Filed: 08/14/2003
• Published: 04/01/2004
• Est. Priority Date: 04/09/1997
• Status: Active Grant

• Current Assignee: Elizabeth M. Boon, Jacqueline K. Barton, Michael G. Hill, Shana O. Kelley
• Original Assignee: Elizabeth M. Boon, Jacqueline K. Barton, Michael G. Hill, Shana O. Kelley
• Inventors: Barton, Jacqueline K., Boon, Elizabeth M., Kelley, Shana O., Hill, Michael G.
• Granted Patent: US 7,202,037 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/113 the label being electroactive, e.g. redox labels
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

Compositions and methods for electrochemical detection and localization of genetic point mutations, common DNA lesions and other base-stacking perturbations within oligonucleotide duplexes adsorbed onto electrodes and their use in biosensing technologies are described. An intercalative, redox-active moiety (such as an intercalator or nucleic acid-binding protein) is adhered and/or crosslinked to immobilized DNA duplexes at different separations from an electrode and probed electrochemically in the presence or absence of a non-intercalative, redox-active moiety. Interruptions in DNA-mediated electron-transfer caused by base-stacking perturbations, such as mutations or binding of a protein to its recognition site are reflected in a difference in electrical current, charge and/or potential.

Claims

1. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.

2. The method of claim 1, wherein the first single stranded nucleic acid probe sequence contains a thiol terminated 5′

end.

3. The method of claim 2, wherein the thiol-terminated 5′

end is a thiol terminated alkyl chain.

4. The method of claim 1, wherein the second single stranded nucleic acid target sequence is DNA.

5. The method of claim 1, wherein the second single stranded nucleic acid target sequence is RNA.

6. The method of claim 1, wherein the intercalative, redox-active moiety is methylene blue.

7. The method of claim 1, wherein the non-intercalative, redox-active moiety is ferricyanide.

8. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded, derivatized nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded derivatized nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded, derivatized nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded, derivatized nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded, derivatized nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.

9. The method of claim 8, wherein the derivatized nucleic acid probe sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

10. The method of claim 9, wherein the oligonucleotide is a DNA sequence.

11. The method of claim 8, wherein the second single stranded nucleic acid target sequence is DNA.

12. The method of claim 8, wherein the second single stranded nucleic acid target sequence is RNA.

13. The method of claim 8, wherein the electrode is gold.

14. The method of claim 8, wherein the electrode is carbon.

15. The method of claim 8, wherein a salt is added to the duplex nucleic acid, before the duplex nucleic acid is deposited onto the electrode.

16. The method of claim 15, wherein the salt is MgCl₂.

17. The method of claim 15, wherein the salt added has a concentration of greater than 10 mM.

18. The method of claim 17, wherein the salt has a concentration of 100 mM.

19. The method of claim 8, wherein the monolayer of first single stranded derivatized nucleic acid probe sequences is from about 35 pmol/cm² to about 60 pmol/cm².

20. The method of claim 19, wherein the monolayer of first single stranded derivatized nucleic acid probe sequences is about 50 pmol/cm².

21. The method of claim 8, wherein the monolayer of first single stranded derivatized nucleic acid probe sequences is from about 0.5 pmol/cm² to about 35 pmol/cm².

22. The method of claim 21, wherein the monolayer of first single stranded derivatized nucleic acid probe sequences is about 1 pmol/cm².

23. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified filn;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety, wherein the intercalative, redox-active moiety is intercalatively stacked and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.

24. The method of claim 23, wherein the intercalative, redox-active moiety is methylene blue.

25. The method of claim 23, wherein the non-intercalative, redox-active moiety is ferricyanide.

26. The method of claim 23, wherein the second single stranded nucleic acid target sequence is DNA.

27. The method of claim 23, wherein the second single stranded nucleic acid target sequence is RNA.

28. The method of claim 23, wherein the intercalative, redox-active moiety is not covalently bound to the nucleic acid-modified film.

29. A method of detecting one or more single-base mismatches in a target nucleic acid sequence, comprising:

a) contacting a first single stranded, derivatized nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded derivatized nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence mismatches is indicative of the presence or absence of one or more single-base sequence mismatches in the target sequence.

30. The method of claim 29, wherein the single-base mismatch is a guanine-adenine pairing.

31. The method of claim 29, wherein the single-base mismatch is an adenine-adenine pairing.

32. The method of claim 29, wherein the single base mismatch is a guanine-guanine pairing, a thymine-thymine pairing, a cytosine-cytosine pairing, a guanine-thymine pairing, a cytosine-thymine pairing, or a cytosine-adenine pairing.

33. The method of claim 29, wherein the first single stranded derivatized nucleic acid probe sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

34. The method of claim 29, wherein the second single stranded nucleic acid target sequence is DNA.

35. The method of claim 29, wherein the second single stranded nucleic acid target sequence is RNA.

36. A method of detecting one or more single-base sequence lesions in a portion of a gene sequence, wherein the one or more single-base sequence lesions is associated with a disease, comprising:

a) contacting a first single stranded nucleic acid probe sequence, comprising a derivatized portion of the sequence of a gene, with a second single stranded nucleic acid target sequence comprising a wild type portion of the gene, to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the gene sequence wherein the base stacking perturbation or single-base sequence lesion is associated with a disease.

37. The method of claim 36, wherein the portion of the gene sequence is up to 200 nucleotides in length.

38. The method of claim 36, wherein the portion of the gene sequence is from 10 to 50 nucleotides in length.

39. The method of claim 38, wherein the portion of the gene sequence is from 15 to 35 nucleotides in length.

40. The method of claim 36, wherein the gene is a human p53 gene.

41. The method of claim 36, wherein the second single stranded nucleic acid target sequence is DNA.

42. The method of claim 36, wherein the second single stranded nucleic acid target sequence is RNA.

43. A method of detecting one or more naturally occurring DNA lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded nucleic acid probe sequence containing a DNA lesion with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more DNA lesions is indicative of the presence or absence of one or more DNA lesions in the target sequence.

44. The method of claim 43, wherein the DNA lesion is an oxidized nucleotide.

45. The method of claim 44, wherein the oxidized nucleotide is 8-oxo-adenine.

46. The method of claim 43, wherein the DNA lesion is a hydroxy pyrimidine.

47. The method of claim 46, wherein the hydroxy pyrimidine is 5,6-dihydro thymine.

48. The method of claim 43, wherein the DNA lesion is an abasic site.

49. The method of claim 43, wherein the DNA lesion is deamination of a nucleotide.

50. The method of claim 49, wherein the deaminated nucleotide is cytosine.

51. The method of claim 43, wherein the DNA lesion is deoxyuracil paired with guanine.

52. The method of claim 43, wherein the second single stranded nucleic acid target sequence is DNA.

53. The method of claim 43, wherein the second single stranded nucleic acid target sequence is RNA.

54. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an addressable multielectrode array, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the addressable multielectrode array, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the addressable multielectrode array, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified flm in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.

55. The method of claim 54, wherein the multielectrode array comprises gold electrodes.

56. The method of claim 54, wherein the multielectrode array comprises carbon electrodes.

57. The method of claim 54, wherein the electrodes of the multielectrode array are 500 μ

m or less in diameter.

58. The method of claim 54, wherein the second single stranded nucleic acid target sequence is DNA.

59. The method of claim 54, wherein the second single stranded nucleic acid target sequence is RNA.

60. A method of detecting one or more single-base sequence lesions in a target nucleic acid sequence, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an ultramicroelectrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the ultramicroelectrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the ultramicroelectrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge from the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in the target sequence.

61. The method of claim 60, wherein the second single stranded nucleic acid target sequence is DNA.

62. The method of claim 60, wherein the second single stranded nucleic acid target sequence is RNA.

63. A method of detecting one or more single-base sequence lesions in a hybrid of DNA and RNA, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of the one or more single-base sequence lesions in a hybrid of DNA and RNA.

64. The method of claim 63, wherein the first single stranded nucleic acid probe sequence is DNA and the second single stranded nucleic acid target sequence is RNA.

65. The method of claim 64, wherein the RNA is mRNA.

66. The method of claim 64, wherein the DNA is cDNA.

67. A method of detecting one or more single-base sequence lesions in a portion of a derivatized wild type target nucleic acid sample, comprising:

a) contacting a first single stranded nucleic acid probe sequence with a second single stranded nucleic acid target sequence, comprising a portion of a derivatized wild type target nucleic acid sample, to form a duplex, wherein the second single stranded nucleic acid target sequence is hybridized to a monolayer of first single stranded nucleic acid probe sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid probe sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid probe sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid probe sequence and the second single stranded target sequence form a double stranded nucleic acid-modified film;

b) immersing the nucleic acid-modified film in a solution comprising an intercalative, redox-active moiety and a non-intercalative redox-active moiety; and

c) measuring the electrical current or charges of the catalytic reduction of the non-intercalative, redox-active species by the intercalative, redox-active moiety wherein a difference of the electric current or charge to the electrical current or charge of a nucleic acid duplex not having one or more single-base sequence lesions is indicative of the presence or absence of one or more single-base sequence lesions in a portion of the derivatized wild type target nucleic acid sample.

68. The method of claim 67, wherein the second single stranded nucleic acid target sequence is DNA.

69. The method of claim 67, wherein the second single stranded nucleic acid target sequence is RNA.

70. A method of detecting base flipping in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:

a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;

b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;

c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and

d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of base flipping due to protein binding in the duplex.

71. The method of claim 70, wherein the first single stranded derivatized nucleic acid sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

72. The method of claim 71, wherein the oligonucleotide is a DNA sequence.

73. The method of claim 70, wherein the electrode is gold.

74. The method of claim 70, wherein Mg²⁺ is added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode.

75. The method of claim 70, wherein Mg²⁺ is not added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode

76. The method of claim 75, wherein the electrode surface is contacted with mercaptohexanol after the duplex nucleic acid is deposited onto the electrode.

77. The method of claim 70, wherein the electrochemical probe is daunomycin (DM).

78. The method of claim 77, wherein the daunomycin is covalently crosslinked to a guanine residue near the duplex terminus of the first single stranded nucleic acid sequence.

79. The method of claim 70, wherein the protein is methyltransferase HhaI (M.HhaI), Q237W, or uracil-DNA glycosylase.

80. A method of detecting base stacking perturbations in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:

a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;

b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;

c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and

d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of one or more base stacking perturbations in the duplex.

81. The method of claim 80, wherein the first single stranded derivatized nucleic acid sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

82. The method of claim 81, wherein the oligonucleotide is a DNA sequence.

83. The method of claim 80, wherein the electrode is gold.

84. The method of claim 80, wherein Mg²⁺ is added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode

85. The method of claim 80, wherein Mg²⁺ is not added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode

86. The method of claim 85, wherein the electrode surface is backfilled with mercaptohexanol after the duplex nucleic acid is deposited onto the electrode.

87. The method of claim 80, wherein the electrochemical probe is daunomycin (DM).

88. The method of claim 87, wherein the daunomycin is covalently crosslinked to a guanine residue near the duplex terminus of the first single stranded nucleic acid sequence.

89. The method of claim 80, wherein the protein is TATA-box binding protein (TBP) or restriction endonuclease PvuII (R.PvuII).

90. A method of electrical monitoring of DNA enzymatic reactions in a duplex nucleic acid sequence associated with protein binding to the duplex nucleic acid sequence, comprising:

a) contacting a first single stranded derivatized nucleic acid sequence, wherein the first single stranded nucleic acid sequence contains a protein binding sequence and an electrochemical probe binding sequence, with a second single stranded nucleic acid sequence to form a duplex, wherein the second single stranded nucleic acid sequence is hybridized to a monolayer of first single stranded nucleic acid sequences on an electrode, wherein the monolayer is prepared by first attaching a duplex comprising the first single stranded nucleic acid sequence to the electrode, and dehybridizing the duplex, such that the first single stranded nucleic acid sequence remains attached to the electrode, and wherein the hybrid of the first single stranded nucleic acid sequence and the second single stranded sequence form a double stranded nucleic acid-modified film;

b) adding an electrochemical probe to the double stranded nucleic acid-modified film, wherein the electrochemical probe binds to the electrochemical probe binding sequence of the first single stranded nucleic acid sequence;

c) adding a protein to the double stranded nucleic acid modified film, wherein the protein binds to the protein binding sequence of the first single stranded nucleic acid sequence; and

d) measuring the electrical current or charges of the reduction of the electrochemical probe, wherein a difference of the electrical current of charge from the electrical current or charge of a nucleic acid duplex not having a protein bound to the duplex DNA is indicative of the presence or absence of an enzymatic reaction of the duplex.

91. The method of claim 90, wherein the first single stranded derivatized nucleic acid sequence is an oligonucleotide with a thiol-terminated alkyl chain on the 5′

end.

92. The method of claim 91, wherein the oligonucleotide is a DNA sequence.

93. The method of claim 90, wherein the electrode is gold.

94. The method of claim 90, wherein Mg²⁺ is added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode.

95. The method of claim 90, wherein Mg²⁺ is not added to the duplex nucleic acid before the duplex nucleic acid is deposited onto the electrode.

96. The method of claim 95, wherein the electrode surface is backfilled with mercaptohexanol after the duplex nucleic acid is deposited onto the electrode.

97. The method of claim 90, wherein the electrochemical probe is daunomycin (DM).

98. The method of claim 97, wherein the daunomycin is covalently crosslinked to a guanine residue near the duplex terminus of the first single stranded nucleic acid sequence.

99. The method of claim 90, wherein the protein is a restriction endonuclease.

100. The method of claim 99, wherein the restriction endonuclease is PvuII.