Device and methods for detecting and quantifying one or more target agents

• US 20090036315A1
• Filed: 02/07/2007
• Published: 02/05/2009
• Est. Priority Date: 02/07/2006
• Status: Abandoned Application

• Current Assignee: Antara Biosciences Incorporated
• Original Assignee: Antara Biosciences Incorporated
• Inventors: Suhy, David A., Lobban, Peter E., Kozlowski, Mark T., Norris, Michael C., Jen, I-Min M., Shen, Naiping, Ammini, Chandramohan V., Labgold, Marc R., Jokhadze, George G.
• Application Number:
  US11/703,103
• Publication Number:
  US 20090036315A1
• Time in Patent Office: Days
• US Class Current: 506/1
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms

Abstract

The present invention provides a device and methods for the detection and quantification of one or more target agents in a sample by rapid and specific electrochemical detection. The present invention includes kits, devices and compositions capable of performing rapid, specific and accurate detection of one or more target agents in a sample.

Claims

1. A method of determining a presence of a target agent in a sample comprising:

(a) mixing said sample with capture-associated oligos conjugated to capture moieties specific for said target agent, thereby producing a first mixture comprising reacted capture-associated oligo complexes that are associated with said target agent and unreacted capture-associated oligo complexes that are not associated with said target agent;

(b) contacting said first mixture with immobilized binding partners, wherein said immobilized binding partners facilitate separation of said unreacted capture-associated oligo complexes from said reacted capture-associated oligo complexes to produce a second mixture comprising said unreacted capture-associated oligo complexes and a third mixture comprising said reacted capture-associated oligo complexes;

(c) providing a detection device comprising oligos complementary to said capture-associated oligos, wherein said detection device produces a signal if there is a hybridization event between said capture-associated oligos and said oligos complementary to said capture-associated oligos;

(d) introducing said third mixture to said detection device; and

(e) detecting said signal, wherein said signal is indicative of said presence of said target agent in said sample.

2. The method of claim 1, wherein said capture-associated oligo is a capture-associated universal oligo.

3. The method of claim 1, wherein said capture-associated oligo is conjugated to said capture moiety through a scaffold.

4. The method of claim 3, wherein said scaffold is attached to a plurality of capture moieties.

5. The method of claim 3, wherein said scaffold is attached to a plurality of capture-associated oligos.

6. The method of claim 3, wherein said reacted capture-associated oligo complexes are reacted loaded scaffolds and said unreacted capture-associated oligo complexes are unreacted loaded scaffolds.

7. The method of claim 3, wherein said scaffolds are composed of material selected from the group consisting of:

gold, aluminum, copper, platinum, silica, titanium dioxide, carbon nanotubes, polystyrene particles, polyvinyl particles, acrylate and methacrylate particles, glass particles, latex particles, Sepharose beads and other like particles, polymer coated magnetic beads, semiconducting materials, and radio frequency identification substrates.

8. The method of claim 1, wherein neither said capture-associated oligos nor said oligos complementary to said capture-associated oligos hybridize to nucleic acid sequences present in said sample.

9. The method of claim 1, wherein said capture moiety is selected from the group consisting of antibodies, antigens, proteins, ligands, receptors, nucleic acids, toxins, immunoglobulins, metabolites, and hormones.

10. The method of claim 1, wherein said detection device is an electrochemical detection device comprising electrodes and a circuit, and further wherein said oligo complementary to said capture-associated oligo is an electrode-associated universal oligo.

11. The method of claim 10, wherein an electrochemical hybridization detector is used to enhance signal production by said electrochemical detection device.

12. The method of claim 11, wherein said electrochemical hybridization detector is an agent that binds more strongly to double-stranded nucleic acid than single-stranded nucleic acid.

13. The method of claim 12, wherein said electrochemical hybridization detector is an agent that binds differently to double-stranded nucleic acid than it does to single-stranded nucleic acid in such a way that an electrochemical signal produced from a double-stranded nucleic acid bound to said agent is enhanced relative to a single-stranded nucleic acid bound to said agent.

14. The method of claim 11, wherein said electrochemical hybridization detector is selected from the group comprising a minor groove binder, a major groove binder, an intercalator, and a transition metal complex.

15. The method of claim 14, wherein said electrochemical hybridization detector is an intercalating agent, and said intercalating agent is selected from the group consisting of:

ethidium, ethidium bromide, acridine, aminoacridine, acridine orange, proflavin, ellipticine, actinomycin D, daunomycin, mitomycin C, Hoechst 33342, Hoechst 33258, aclarubicin, DAPI, Adriamycin, pirarubicin, actinomycin, tris (phenanthroline) zinc salt, tris(phenanthroline) ruthenium salt, tris(phenanthroline) cobalt salt, di(phenanthroline) zinc salt, di(phenanthroline) ruthenium salt, di (phenanthroline) cobalt salt, bipyridine platinum salt, terpyridine platinum salt, phenanthroline platinum salt, tris(bipyridyl) zinc salt, tris(bipyridyl) ruthenium salt, tris (bipyridyl) cobalt salt, di(bipyridyl) zinc salt, di(bipyridyl) ruthenium salt, di(bipyridyl) cobalt salt, and intercalators containing metal ions.

16. The method of claim 11, wherein said electrochemical hybridization detector is conjugated onto said capture-associated oligos.

17. The method of claim 16, wherein said electrochemical hybridization detector is an electroactive marker, and said electroactive marker is selected from the group consisting of:

ferrocene derivatives, ferritin derivatives, anthraquinone, silver, silver derivatives, gold, gold derivatives, osmium, osmium derivatives, ruthenium, ruthenium derivatives, cobalt, and cobalt derivatives.

18. The method of claim 16, wherein said electrochemical hybridization detector is a detection moiety, and whereby said method further comprises creating a circular structure by molecular interactions between said capture-associated oligos and said electrode-associated oligos.

19. The method of claim 11, wherein a combination of electrochemical hybridization detectors are used to enhance signal production by said electrochemical detection device.

20. The method of claim 10, wherein said electrochemical detection device comprises a surface on which said electrode-associated oligos are immobilized, wherein said surface is a substrate selected from the group consisting of:

fiberglass, Teflon™

, ceramics, glass, silicon, mica, plastic, acrylics, polystyrene and copolymers of styrene, polypropylene, polyethylene, polybutylene, polycarbonate, polyurethanes, GETEK, polypropylene oxide, and mixtures thereof.

21. The method of claim 10, wherein said electrochemical detection device comprises a surface that is an oligo chip comprising a plurality of electrodes, where at least one electrode is independently addressable, and where said electrode comprises materials selected from the group consisting of:

gold, aluminum, platinum, palladium, rhodium, ruthenium, silicon, titanium, platinum oxide, titanium oxide, tin oxide, indium tin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenum oxide (Mo₂, O₆), tungsten oxide (WO₃) and ruthenium oxides;

carbon, graphite, pyrolytic graphite, carbon fiber, carbon paste, Si, Ge, ZnO, CdS, TiO₂ and GaAs.

22. The method of claim 10, wherein said electrochemical detection device comprises a semi-flexible polymer substrate and a conductive metal layer.

23. The method of claim 22, wherein said conductive metal layer comprises gold.

24. The method of claim 22, wherein said conductive metal layer comprises platinum.

25. The method of claim 22, wherein said semi-flexible polymer substrate is polyethylene terephthalate.

26. The method of claim 10, wherein said electrochemical detection device further comprises a functional element.

27. The method of claim 26, wherein said functional element is a microsensor.

28. The method of claim 26, wherein said functional element is a microheater.

29. The method of claim 10, wherein said electrochemical detection device comprises ninety-six electrodes.

30. The method of claim 10, wherein at least one of said electrodes is a flat planar electrode with addressable locations for synthesis and/or detection.

31. The method of claim 10, wherein at least one of said electrodes is independently addressable, whereby a voltage is applied to each electrode, wherein said voltage is the same voltage, and said electrochemical detection device comprises at least one switch circuit, a decoder circuit, or timing circuit to apply the voltage to the individual electrodes and to receive the output signal from the electrodes.

32. The method of claim 10, wherein at least one of said electrodes is coated with a biocompatible substance selected from the group consisting of:

dextran, carboxylmethyldextran, hydrogels, polypeptides, polynucleotides, biocompatible matrices, bio-inert matrices, and mixtures thereof.

33. The method of claim 1, wherein said immobilized binding partners comprise an epitope of said target agent, wherein said epitope specifically reacts with said capture moieties.

34. The method of claim 1, wherein said immobilized binding partners specifically interact with said capture moiety when said capture moiety has not bound said target agent, thereby immobilizing unreacted capture-associated oligo complexes in an immobilized phase and leaving said reacted capture-associated oligo complexes in a solution phase, wherein said solution phase comprises said third mixture.

35. The method of claim 1, further comprising

(a) specific binding of said immobilized binding partners with said capture moieties when said capture moieties have bound said target agent, thereby immobilizing said reacted capture-associated oligo complexes in an immobilized phase and leaving said unreacted capture-associated oligo complexes in a solution phase, wherein said second mixture comprises said solution phase;

and

(b) separating said second mixture from said immobilized phase, wherein said third mixture comprises said reacted capture-associated oligo complexes in said immobilized phase.

36. The method claim 35, further comprising liberating said capture-associated oligos from said immobilized phase prior to introducing said third mixture to said detection device in step (d).

37. The method of claim 1, further comprising

(a) specific binding of said immobilized binding partners with said target agent or capture moiety/target agent complex, thereby immobilizing said reacted capture-associated oligo complexes in an immobilized phase and leaving said unreacted capture-associated oligo complexes in a solution phase, wherein said second mixture comprises said solution phase;

and

(b) separating said second mixture from said immobilized phase, wherein said third mixture comprises said reacted capture-associated oligo complexes in said immobilized phase.

38. The method claim 37, further comprising liberating said capture-associated oligos from said immobilized phase prior to introducing said third mixture to said detection device in step (d).

39. The method claim 1 wherein said immobilized binding partners are immobilized on a particle.

40. The method of claim 39, wherein said particle is a bead.

41. The method of claim 1, wherein said immobilized binding partners are selected from the group consisting of ligands of said capture moieties, substrates for enzymes, receptors for signaling molecules, antigens specific for said capture moieties, antibodies specific for said capture moieties, and nucleic acids complementary to said capture moieties.

42. The method of claim 1, further comprising adding to the mixture an agent to reduce background signal.

43. The method of claim 42, wherein said agent to reduce background signal is selected from the group consisting of:

a single-stranded nuclease, mung bean nuclease, nuclease P1, exonuclease I, exonuclease VII, S1 nuclease and single-stranded DNA binding proteins.

44. The method of claim 1, further comprising adding to the mixture an enzyme to increase strength of said signal.

45. The method of claim 1, further comprising separating said capture-associated oligos from said reacted capture-associated oligo complexes to produce released capture-associated complexes, wherein said third mixture comprises said released capture-associated complexes.

46. The method of claim 45, wherein said separating involves use of a digestive enzyme.

47. The method of claim 46, wherein said digestive enzyme is an endonuclease.

48. The method of claim 45, wherein said separating involves photocleavage.

49. The method of claim 1 wherein said target agent is an antibody and said immobilized binding partners are selected from the group consisting of protein A, protein G, a thiophilic resin, and an anti-class-specific antibody specific for a class of antibodies comprising said target agent.

50. The method of claim 1, further comprising

(i) adding quantifying oligos to said third mixture, wherein each of said quantifying oligos is present in a known concentration, and further wherein said detection device further comprises oligos complementary to said quantifying oligos;

(ii) detecting quantifying signals for each of said quantifying oligos upon hybridization to said oligos complementary to said quantifying oligos; and

(iii) comparing said quantifying signals to the signal detected in (e) to determine an amount of said target agent in said sample.

51. The method of claim 50, wherein at least two of said quantifying oligos are present in different concentrations.

52. The method of claim 50, wherein at least three of said quantifying oligos are present in graduated concentrations.

53. The method of claim 1, wherein there are multiple capture-associated oligos and multiple capture moieties in said first mixture.

54. The method of claim 53, wherein

(a) said sample comprises multiple target agents;

(b) each of said multiple capture moieties is specific for a different one of said multiple target agents in said sample;

(c) each of said multiple capture moieties is conjugated to a different one of said multiple capture-associated oligos;

(d) said third mixture comprises multiple reacted capture-associated oligo complexes; and

(e) said detection device comprises oligonucleotides complementary to each of said multiple capture-associated oligos, thereby allowing simultaneous detection of said multiple target agents in said detection device.

55. The method of claim 54, wherein said multiple target agents include members selected from at least two of the classes consisting of proteins, ligands, receptors, nucleic acids, toxins, immunoglobulins, metabolites, and hormones.

56. The method of claim 1, wherein said detection device does not produce a signal indicating that said target agent is absent from said sample.

65. The method claim 1, wherein said immobilized binding partners are immobilized on a particle.

66. The method of claim 65, wherein said particle is a bead.

57. A method of determining a presence of a target agent in a sample comprising:

(a) mixing said sample with capture-associated oligos conjugated to capture moieties specific for said target agent, thereby producing a first mixture comprising reacted capture-associated oligo complexes that are associated with said target agent and unreacted capture-associated oligo complexes that are not associated with said target agent;

(b) contacting said first mixture with immobilized binding partners, wherein said immobilized binding partners specifically associate with said target agent or capture moiety/target agent complex, thereby immobilizing said reacted capture-associated oligo complexes in an immobilized phase and leaving said unreacted capture-associated oligo complexes in a solution phase;

(c) separating said solution phase from said immobilized phase;

(d) hybridizing an intermediary oligo to said capture-associated oligo, wherein said intermediary oligo comprises a first region complementary to said capture-associated oligo and a second region, and further wherein hybridization of said intermediary oligo to said capture-associated oligo creates a restriction endonuclease recognition site;

(e) adding a restriction endonuclease that cleaves at said restriction endonuclease recognition site, thereby releasing a portion of said intermediary oligo comprising said second region;

(f) providing a detection device comprising oligos complementary to said second region of said intermediary oligo, wherein said detection device produces a signal if there is a hybridization event between said second region and said oligos complementary to said second region;

(d) introducing said portion of said intermediary oligo released in (e) to said detection device; and

(e) detecting said signal, wherein said signal is indicative of said presence of said target agent in said sample.

58. The method of claim 57, wherein said capture moiety is selected from the group consisting of antibodies, proteins, ligands, receptors, nucleic acids, toxins, immunoglobulins, metabolites, and hormones.

59. The method of claim 57, wherein said detection device is an electrochemical detection device comprising electrodes and a circuit.

60. The method of claim 59, wherein an electrochemical hybridization detector is used to enhance signal production by said electrochemical detection device.

61. The method of claim 60, wherein said electrochemical hybridization detector is selected from the group comprising a minor groove binder, a major groove binder, an intercalator, and a transition metal complex.

62. The method of claim 60, wherein said electrochemical hybridization detector is conjugated onto said second region of said intermediary oligo.

63. The method of claim 62, wherein said electrochemical hybridization detector is ferrocene or a derivative thereof.

64. The method of claim 60, wherein a combination of electrochemical hybridization detectors are used to enhance signal production by said electrochemical detection device.

67. The method of claim 57, wherein said immobilized binding partners are selected from the group consisting of ligands of said capture moieties, antibodies specific for said capture moieties, and nucleic acids complementary to said capture moieties.

68. The method of claim 57, wherein said target agent is an antibody and said immobilized binding partners are selected from the group consisting of protein A, protein G, a thiophilic resin, and an anti-class-specific antibody specific for a class of antibodies comprising said target agent.

69. The method of claim 57, wherein said detection device does not produce a signal indicating that said target agent is absent from said sample.

70. A method of determining a presence of a target agent in a sample comprising:

(a) mixing said sample with capture-associated oligos conjugated to capture moieties specific for said target agent, thereby producing a first mixture comprising reacted capture-associated oligo complexes that are associated with said target agent and unreacted capture-associated oligo complexes that are not associated with said target agent;

(b) contacting said first mixture with immobilized binding partners, wherein said immobilized binding partners facilitate separation of said unreacted capture-associated oligo complexes from said reacted capture-associated oligo complexes to produce a second mixture comprising said unreacted capture-associated oligo complexes and a third mixture comprising said reacted capture-associated oligo complexes;

(c) adding an oligonucleotide comprising a polymerase recognition sequence to said third mixture, wherein said reacted capture-associated oligo complexes in said third mixture comprise a complement to said polymerase recognition sequence, thereby producing a double-stranded polymerase recognition site;

(d) adding a polymerase and nucleotides to said third mixture under conditions to allow amplification of said capture-associated oligo to produce amplified oligos;

(e) providing a detection device comprising oligos complementary to said amplified oligos, wherein said detection device produces a signal if there is a hybridization event between said amplified oligos and said oligos complementary to said amplified oligos;

(d) introducing said third mixture to said detection device; and

(e) detecting said signal, wherein said signal is indicative of said presence of said target agent in said sample.

71. The method of claim 70, wherein said polymerase is selected from the group consisting of T3 polymerase, 17 polymerase, SP6 polymerase, T7 polymerase Y639F and T7 polymerase S641A.

72. The method of claim 70, wherein said polymerase is an RNA polymerase that has been modified to allow linear amplification using deoxyribonucleotides.

73. The method of claim 70, wherein said capture-associated oligo is a capture-associated universal oligo.

74. The method of claim 70, wherein said capture moiety is selected from the group consisting of antibodies, proteins, ligands, receptors, nucleic acids, toxins, immunoglobulins, metabolites, and hormones.

75. The method of claim 70, wherein said detection device is an electrochemical detection device comprising electrodes and a circuit, and further wherein said oligo complementary to said capture-associated oligo is an electrode-associated universal oligo.

76. The method of claim 75, wherein an electrochemical hybridization detector is used to enhance signal production by said electrochemical detection device.

77. The method of claim 76, wherein said electrochemical hybridization detector is selected from the group comprising a minor groove binder, a major groove binder, an intercalator, and a transition metal complex.

78. The method of claim 76, wherein said electrochemical hybridization detector is conjugated onto said capture-associated oligos.

79. The method of claim 76, wherein a combination of electrochemical hybridization detectors are used to enhance signal production by said electrochemical detection device.

80. The method of claim 70, wherein said immobilized binding partners comprise an epitope of said target agent, wherein said epitope specifically reacts with said capture moieties.

81. The method of claim 70, wherein said immobilized binding partners specifically interact with said capture moiety when said capture moiety has not bound said target agent, thereby immobilizing unreacted capture-associated oligo complexes in an immobilized phase and leaving said reacted capture-associated oligo complexes in a solution phase, wherein said solution phase comprises said third mixture.

82. The method of claim 70, further comprising

(a) specific binding of said immobilized binding partners with said capture moieties when said capture moieties have bound said target agent, thereby immobilizing said reacted capture-associated oligo complexes in an immobilized phase and leaving said unreacted capture-associated oligo complexes in a solution phase, wherein said second mixture comprises said solution phase;

and

(b) separating said second mixture from said immobilized phase, wherein said third mixture comprises said reacted capture-associated oligo complexes in said immobilized phase.

83. The method claim 82, further comprising liberating said capture-associated oligos from said immobilized phase prior to introducing said third mixture to said detection device in step (d).

84. The method of claim 70, further comprising

(a) specific binding of said immobilized binding partners with said target agent or capture moiety/target agent complex, thereby immobilizing said reacted capture-associated oligo complexes in an immobilized phase and leaving said unreacted capture-associated oligo complexes in a solution phase, wherein said second mixture comprises said solution phase;

and

(b) separating said second mixture from said immobilized phase, wherein said third mixture comprises said reacted capture-associated oligo complexes in said immobilized phase.

85. The method claim 84, further comprising liberating said capture-associated oligos from said immobilized phase prior to introducing said third mixture to said detection device in step (d).

86. The method claim 70, wherein said immobilized binding partners are immobilized on a particle.

87. The method of claim 86, wherein said particle is a bead.

88. The method of claim 70, wherein said immobilized binding partners are selected from the group consisting of ligands of said capture moieties, antibodies specific for said capture moieties, and nucleic acids complementary to said capture moieties.

89. The method of claim 70, further comprising separating said capture-associated oligos from said reacted capture-associated oligo complexes to produce released capture-associated complexes, wherein said third mixture comprises said released capture-associated complexes.

90. The method of claim 89, wherein said separating involves use of a digestive enzyme.

91. The method of claim 90, wherein said digestive enzyme is an endonuclease.

92. The method of claim 89, wherein said separating involves photocleavage.

93. The method of claim 70, wherein said target agent is an antibody and said immobilized binding partners are selected from the group consisting of protein A, protein G, a thiophilic resin, and an anti-class-specific antibody specific for a class of antibodies comprising said target agent.

94. The method of claim 70, wherein said detection device does not produce a signal indicating that said target agent is absent from said sample.

95. A method for selecting universal oligo pairs comprising:

(a) generating a candidate oligo of length X;

(b) screening said candidate oligo against one or more reference sequences to determine sequence similarity;

(c) discarding said candidate oligo if said sequence similarity is at or above a first threshold;

(d) extending the length of said candidate oligo if said sequence similarity is below said first threshold to produce an extended candidate oligo;

(e) screening said extended candidate oligo against one or more reference sequences to determine sequence similarity;

(e) discarding said extended candidate oligo if said sequence similarity is at or above a second threshold;

(g) extending the length of said extended candidate oligo if said sequence similarity is below said second threshold;

(h) repeating steps (e) through (g) until said extended candidate oligo has a length Y, where Y>

25, thereby producing a candidate oligo of length Y;

(i) placing said candidate oligo of length Y in a first group;

(j) repeating steps (a) through (i) until a desired number of candidate oligos of length Y populate said first group, wherein any oligos in or added to said first group are first group oligos;

(k) generating complementary oligos to said first group oligos;

(l) adding said complementary oligos to said first group, thereby creating first group oligo pairs, each of which comprises one of said candidate oligos of length Y and a complementary oligo thereto generated in step (k);

(m) screening each of said first group oligo pairs for sequence similarity against all other of said first group oligo pairs;

(n) discarding each of said first group oligo pairs that has sequence similarity to another of said first group oligo pairs at or above a third threshold; and

(o) adding each of said first group oligo pairs that is not discarded in step (n) to a second group, wherein each of said first group oligo pairs added to said second group is a universal oligo pair, and wherein said second group is a universal oligo set.

96. The method of claim 95, further comprising screening said universal oligo set for additional parameters selected from melting temperature (T/m), existence of duplexes, existence of a GC clamp, existence of hairpins, existence of sequence repeats, dissociation minimum for a 3′

dimer, dissociation minimum for a 3′

terminal stability range, dissociation minimum for a minimum acceptable loop, maximum number of acceptable sequence repeats, and frequency threshold.

97. The method of claim 95, wherein length X is between 8 and 25.

98. The method of claim 97, wherein length X is between 10 and 18.

99. The method of claim 95, wherein one nucleotide is added in at least one of steps (d) and (g).

100. The method of claim 99, wherein each of A, T, G, and C are added in parallel in said extending step.

101. The method of claim 95, wherein said reference sequences include those at ncbi.nlm.nih.gov/BLAST and said first threshold is any value greater than zero.

102. A method for selecting a universal oligo pairs comprising:

(a) generating a candidate oligo of length X;

(b) calculating a GC content of said candidate oligo, wherein if said GC content is outside of a first threshold said candidate oligo is discarded and a new candidate oligo is generated in (a), and wherein if said GC content is inside of said first threshold said candidate oligo is a GC-approved oligo;

(c) screening said GC-approved oligo for a mononucleotide repeat whose length exceeds a second threshold, wherein if said mononucleotide repeat whose length exceeds said second threshold occurs said GC-approved oligo is discarded and a new candidate oligo is generated in (a), and wherein if said mononucleotide repeat whose length exceeds said second threshold does not occur said GC-approved oligo is a repeat-approved oligo;

(d) performing a further screening of said repeat-approved oligo, wherein if said repeat-approved oligo does not pass said further screening such repeat-approved oligo is discarded and a new candidate oligo is generated in (a), and wherein if said repeat-approved oligo passes said further screening said repeat-approved oligo is a further screening-approved oligo;

(e) screening said further screening-approved oligo against one or more reference sequences to determine sequence similarity,

(f) discarding said screening-approved oligo if said sequence similarity is at or above a third threshold;

(g) placing said screening-approved oligo in a first group if said sequence similarity is below said third threshold;

(h) repeating steps (a) through (g) until a desired number of screening-approved oligos populate said first group, wherein any oligos in or added to said first group are first group oligos;

(i) generating complementary oligos to said first group oligos;

(j) adding said complementary oligos to said first group, thereby creating first group oligo pairs, each of which comprises one of said screening-approved oligos and a complementary oligo thereto generated in step (i);

(k) screening each of said first group oligo pairs for sequence similarity against all other of said first group oligo pairs;

(l) discarding each of said first group oligo pairs that has sequence similarity to another of said first group oligo pairs at or above a fourth threshold; and

(m) adding each of said first group oligo pairs that is not discarded in step (l) to a second group, wherein each of said first group oligo pairs added to said second group is a universal oligo pair, and wherein said second group is a universal oligo set.

103. The method of claim 102, wherein said further screening is selected from the group consisting of melting temperature (T_m), existence of duplexes, existence of a GC clamp, existence of hairpins, existence of sequence repeats, dissociation minimum for a 3′

dimer, dissociation minimum for a 3′

terminal stability range, dissociation minimum for a minimum acceptable loop, maximum number of acceptable sequence repeats, and frequency threshold.

104. The method of claim 102, wherein length X is between 40 and 100.

105. The method of claim 103, wherein length X is between 50 and 80.

106. The method of claim 103, wherein said reference sequences include those at ncbi.nlmih.gov/BLAST and said third threshold is any value greater than zero

107. A universal oligo comprising a sequence selected from SEQ ID NO 1 through SEQ ID NO 200.

108. A universal oligo set comprising two or more sequences selected from SEQ ID NO 1 through SEQ ID NO 200.

109. A method for using a universal oligo chip to determine a presence of a target agent in a sample by electrochemical detection, said method comprising:

(a) mixing said sample with capture-associated universal oligos conjugated to capture moieties specific for said target agent, thereby producing a first mixture comprising reacted capture-associated universal oligo complexes that are associated with said target agent and unreacted capture-associated universal oligo complexes that are not associated with said target agent;

(b) contacting said first mixture with immobilized binding partners, wherein said immobilized binding partners specifically interact with said unreacted capture-associated universal oligo complexes, thereby immobilizing said unreacted capture-associated universal oligo complexes in an immobilized phase and leaving said reacted capture-associated oligo complexes in a solution phase;

(c) providing a detection device comprising universal oligos complementary to said capture-associated universal oligos, wherein said detection device produces a signal if there is a hybridization event between said capture-associated universal oligos and said universal oligos complementary to said capture-associated universal oligos;

(d) introducing said solution phase to said detection device; and

(e) detecting said signal, wherein said signal is indicative of said presence of said target agent in said sample.

110. A composition comprising

(a) an electrode;

(b) an electrode-associated oligo hybridized to a capture-associated oligo;

(c) a capture moiety conjugated to said capture-associated oligo; and

(d) a target agent bound to said capture moiety.

111. The composition of claim 110, further comprising a binding partner bound to said capture moiety, said target agent, or a complex thereof.

112. The composition of claim 110, wherein said capture moiety is an antibody.

113. The composition of claim 110, further comprising an electrochemical hybridization detector.

114. A biosensor comprising at least one electrode and current or impedance measuring elements, where said measuring elements are enabled to detect changes in current or impedance in response to the presence of a reaction produced when a detection moiety is brought within proximity to said electrode.

115. The biosensor of claim 114, whereby said at least one electrode is in a disposable format, whereby said electrode can be used for a single electrochemical detection experiment of one or more samples and discarded.

116. The biosensor of claim 114, whereby said at least one electrode has a conductive detection surface, and further whereby said at least one electrode comprises a mixed monolayer comprising anchoring groups conjugated to electrode-associated oligos and diluent groups, which serve as insulators on a surface of said electrode.

117. The biosensor of claim 116, whereby said anchoring groups and said diluent groups are chosen to provide approximately uniform distance between enforcing groups to maximize interaction capabilities.

118. The biosensor of claim 116, whereby a plurality of electrode-associated oligos can be located on said biosensor to enable detection of multiple target agents.

119. The biosensor of claim 116, whereby a specific ratio of said anchoring groups and said diluent groups is used for said monolayer on said electrode to enable a uniform monolayer with evenly distributed anchoring group complexes and diluent groups, thereby optimizing the access of the electrode-associated oligo to any capture-associated oligo present in an assay.

120. The biosensor of claim 116, whereby the concentration and type of said anchoring groups and said diluent groups is selected to maximize the ratio of specific current to non-specific current.

121. The biosensor of claim 116, whereby said conductive detection surface is gold.

122. The biosensor of claim 116, whereby said monolayers comprise hexadecanethiolate and have a contact angle with water from 110°

to 115°

.

123. The biosensor of claim 116, whereby said monolayers are hydrophilic and have a contact angel with water of <

10°

.

124. A system for determining a presence of a target agent in a sample comprising:

(a) a sample containing said target agent;

(b) capture-associated oligos conjugated to capture moieties specific for said target agent;

(c) electrode-associated oligos that are complementary to said capture-associated oligos or complements thereto;

(d) immobilized binding partners; and

(e) a surface comprising at least one electrode, whereon said electrode associated oligos are attached.

125. The system of claim 124, further comprising one or more electrochemical hybridization detectors.

126. The system of claim 124, further comprising an electrochemical detection device.

127. A method of doing business wherein the system of claim 124 is queried remotely to collect information on results.

128. A diagnostic tool for detecting a target agent in a sample, comprising:

(a) a capture moiety which binds preferentially to a target agent;

(b) a first nucleic acid associated with said capture moiety, and

(c) a recognition sequence in said first nucleic acid for linear amplification of said first nucleic acid, wherein said first nucleic acid comprises a sequence substantially the same as a sequence of a second nucleic acid associated with an electrode.

129. The diagnostic tool of claim 128, wherein said capture moiety is an antibody.

130. The diagnostic tool of claim 128, wherein said capture moiety is a ligand.

131. The diagnostic tool of claim 128, wherein said recognition sequence is for an RNA phage polymerase.

132. The diagnostic tool of claim 128, wherein said recognition sequence allows amplification using asymmetric polymerase chain reaction.

133. The diagnostic tool of claim 128, wherein said first nucleic acid further comprises a restriction endonuclease recognition sequence.

134. The diagnostic tool of claim 128, wherein said first nucleic acid further comprises a polymerase recognition sequence.

135. A kit for use in detecting the presence of a target agent in a sample, said kit comprising:

a biosensor comprising at least one disposable electrode and current or impedance measuring elements, wherein said at least one disposable electrode comprises a conductive detection surface, and a mixed monolayer comprising anchoring groups conjugated to electrode-associated oligos and diluent groups;

a first single-stranded nucleic acid molecule that is complementary to a second single-stranded nucleic acid molecule, where said first single-stranded nucleic acid molecule is immobilized on said biosensor, and further where said second single-stranded nucleic acid molecule is conjugated to a capture moiety specific for said target agent;

at least one immobilized binding partner; and

at least one container.

136. A kit for use in detecting the presence of a target agent in a sample, said kit comprising:

a biosensor comprising at least one disposable electrode and current or impedance measuring elements, wherein said at least one disposable electrode comprises a conductive detection surface, and a mixed monolayer comprising anchoring groups conjugated to electrode-associated oligos and diluent groups;

a first single-stranded nucleic acid molecule that is complementary to a second single-stranded nucleic acid molecule, where said first single-stranded nucleic acid molecule is immobilized on said biosensor, and further where said second single-stranded nucleic acid molecule is conjugated to a capture moiety specific for said target agent;

at least one immobilized binding partner; and

at least one container.

137. A method of electrochemically detecting and quantifying a presence of a target agent of interest in a sample comprising:

(a) mixing;

(i) said sample with at least one loaded scaffold comprising a capture-associated universal oligo, a capture moiety specific for said target agent of interest and a scaffold; and

(ii) a sample suspected of containing said target agent of interest, thereby producing a mixture comprising reacted loaded scaffolds and unreacted loaded scaffolds;

(b) contacting the mixture of step (a)(ii) with immobilized binding partners to said capture moieties of said loaded scaffolds so as to allow any of said unreacted loaded scaffolds to bind with said immobilized binding partners resulting in an immobilized phase and a solution phase;

(c) separating the immobilized phase and solution phase;

(d) providing an electrochemical detection device comprising electrodes, electrode-associated universal oligos, and a circuit, wherein said detection device produces a signal if there is a hybridization event between said electrode-associated universal oligos and other nucleic acid molecules;

(e) introducing said solution phase from step (c) to the electrochemical detection device from step (d); and

(h) detecting an electrochemical signal generated by capture-associated universal oligos from said reacted loaded scaffolds and electrode-associated universal oligos.

138. A method of electrochemically detecting and quantify a presence of a target agent of interest in a sample comprising:

(a) mixing;

(i) said sample with a loaded scaffold comprising a capture-associated universal oligo, a capture moiety specific for said target agent of interest and a scaffold; and

(ii) a sample suspected of containing said target agent of interest, thereby producing a mixture comprising reacted loaded scaffolds and unreacted loaded scaffolds;

(b) contacting the mixture of step (a) with immobilized binding partners to said target agents or to capture moiety/target agent complexes so as to allow any of said reacted loaded scaffolds to bind with said immobilized binding partners resulting in an immobilized phase and a solution phase;

(c) separating the immobilized phase and solution phase;

(d) providing an electrochemical detection device comprising electrodes, electrode-associated universal oligos, and a circuit, wherein said detection device produces a signal if there is a hybridization event between said electrode-associated universal oligos and other nucleic acid molecules;

(e) liberating said capture-associated universal oligos into a second solution phase from said immobilized phase;

(f) introducing said solution phase from step (e) to the electrochemical detection device from step (d); and

(h) detecting an electrochemical signal generated by capture-associated universal oligos and electrode-associated universal oligos.

139. A method of doing business, said method comprising use of a electrical signal to determine appropriate medical intervention for a patient, said method comprising:

(a) obtaining a sample from a patient whereby a target agent may be present in said sample;

(b) mixing said sample with capture-associated oligos conjugated moieties specific for said target agent, thereby producing a first mixture comprising reacted capture-associated oligo complexes that are associated with said target agent and unreacted capture-associated oligo complexes that are not associated with said target agent;

(c) contacting said first mixture with immobilized binding partners, wherein said immobilized binding partners facilitate separation of said unreacted capture-associated oligo complexes from said reacted capture-associated oligo complexes to produce a second mixture comprising said reacted capture-associated oligo complexes;

(d) providing a detection device comprising oligos complementary to said capture-associated oligos, wherein said detection device produces a signal if there is a hybridization event between said capture-associated universal oligos and said oligos;

(e) introducing said third mixture to said detection device;

(f) detecting said signal, wherein said signal is indicative of said presence of said target agent in said sample; and

(g) determining the appropriate medical intervention for the patient based upon the presence or absence of said target agent in said sample.

140. An electrical signal used to determine appropriate medical intervention for a patient, whereby said electrical signal is indicative of the concentration of a target agent in a sample taken from said patient;

where the concentration of said target agent is calculated by a software algorithm that correlates the magnitude of said electrical signal of the magnitude of a second electrical signal from a pre-determined set of quantifying target agent; and

where said electrical signal is dependent upon the presence of an electrode, an electrode-associated oligo hybridized to a capture-associated oligo, a capture moiety conjugated to said capture-associated oligo and a target agent bound to said capture moiety.

141. A detection device comprising:

one or more electrochemical chips,

electrode-associated oligos complementary to capture-associated oligos,

a signaling generator for producing a signal upon occurrence of a hybridization event between said capture-associated oligos and said electrode-associated oligos, a receiver for receiving said signal a processor for processing said signal, and a display for displaying the results of said processing.

142. A method of determining a presence of a target nucleic acid in a sample wherein said target nucleic acid is not contacted with a detection device, comprising:

(a) providing hybrid oligos, each of which comprises

     1) a region complementary to a target nucleic acid and ii) a capture-associated oligo;

(b) mixing said sample with said hybrid oligos, thereby producing a first mixture comprising reacted hybrid oligo complexes that are associated with said target nucleic acid and unreacted hybrid oligo complexes that are not associated with said target nucleic acid;

(c) contacting said first mixture with a polymerase and nucleotides under conditions to facilitate creation of double-stranded target nucleic acid on said reacted hybrid oligo complexes to create a second mixture;

(d) exposing said second mixture to a hydroxyapatite matrix, wherein said hydroxyapatite matrix facilitates separation of said double-stranded target nucleic acid on said reacted hybrid oligo complexes from single-stranded nucleic acid species in said second mixture;

(e) removing said single-stranded nucleic acid species from said hydroxyapatite matrix and discarding;

(f) removing said double-stranded target nucleic acid on said reacted hybrid oligo complexes from said hydroxyapatite matrix;

(g) separating said capture-associated oligos from said reacted hybrid oligo complexes removed from said hydroxyapatite matrix in step (f);

(h) providing said detection device comprising oligos complementary to said capture-associated oligos, wherein said detection device produces a signal if there is a hybridization event between said capture-associated oligos and said oligos complementary to said capture-associated oligos;

(i) introducing said capture-associated oligos to said detection device; and

(j) detecting said signal, wherein said signal is indicative of said presence of said target nucleic acid in said sample.