Methods and apparatus for conducting multiple measurements on a sample

• US 20030113713A1
• Filed: 09/10/2002
• Published: 06/19/2003
• Est. Priority Date: 09/10/2001
• Status: Active Grant

• Current Assignee: Meso Scale Technologies, LLC
• Original Assignee: Meso Scale Technologies, LLC
• Inventors: DeBad, Jeff D., Wohlstadter, Jacob N., Wilbur, James, Glezer, Eli N., Biebuyck, Hans, Johnson, Kent, Sigal, George, Umek, Robert M., Kenten, John H., Eason, Paula Denney, Tsionsky, Michael
• Granted Patent: US 7,858,321 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/485 involving kinase
• CPC Class Codes: G01N 2333/11 Orthomyxoviridae, e.g. influenza virus
• CPC Class Codes: G01N 2333/135 Respiratory syncytial virus
• CPC Class Codes: G01N 2333/16 HIV-1, HIV-2
• CPC Class Codes: G01N 2333/525 Tumor necrosis factor [TNF]
• CPC Class Codes: G01N 2333/5412 IL-6
• CPC Class Codes: G01N 2333/545 IL-1
• CPC Class Codes: G01N 2333/57 IFN-gamma
• CPC Class Codes: G01N 2333/71 for growth factors; for growth regulators
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/5304 Reaction vessels, e.g. agglutination plates for solid-phase systems G01N33/543
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G01N 33/5438 Electrodes
• CPC Class Codes: G01N 33/54386 Analytical elements
• CPC Class Codes: G01N 33/56944 Streptococcus
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/56988 HIV or HTLV
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/68 involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures

Abstract

Multiplexed test measurements are conducted using an assay module having a plurality of assay domains. In preferred embodiments, these measurements are conducted in assay modules having integrated electrodes with a reader apparatus adapted to receive assay modules, induce luminescence, preferably electrode induced luminescence, in the wells or assay regions of the assay modules and measure the induced luminescence.

Claims

1. An assay module for conducting luminescence assays comprising a plurality of assay domains, said assay domains comprising a first assay domain having a first reagent and a second assay domain having a second reagent, wherein said first assay domain is capable of generating luminescence at least 10 times brighter than said second assay domain and said first assay domain and said second assay domains are not adjacent and/or are separated by at least one other assay domain to reduce luminescence emitted from said first assay domain from interfering with luminescence emitted from said second assay domain.

3. The assay module of claim 1, wherein said first assay domain comprises at least 10 times capture reagent than said second assay domain.

5. The assay module of claim 1, wherein said first assay domain is capable of generating luminescence at least 100 times brighter than said second assay domain.

6. The assay module of claim 1, wherein said first assay domain is capable of generating luminescence at least 1,000 times brighter than said second assay domain.

7. The assay module of claim 1, 3, 5 or 6, wherein said second assay domain has one or more properties that diminish emitted luminescence.

8. The assay module of claim 7, wherein said one or more properties include a capture reagent that diminishes emitted luminescence and/or a target analyte that diminishes emitted luminescence.

9. The assay module of claim 1, 3, 5, 6, 7 or 8, wherein said first assay domain is separated from said second assay domain by at least one other assay domain.

10. The assay module of claim 1, 3, 5, 6, 7, 8 or 9, wherein said first assay domain and said second assay domain are separately addressable.

11. The assay module of claim 1, 3, 5, 6, 7, 8, 9 or 10, wherein said assay module is a multi-well plate comprising a plurality of wells, each of said wells having a copy of said plurality of assay domains.

132. The assay module of claim 1, 3, 5, 6, 7, 8, 9, 10, 11, 19, 20, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 46, 47, 49, 53, 59, 63, 64, 66, 68, 74, 75, 79, 80, 81, 82, 83, 84, 88, 89, 90, 91, 92, 93, 95, 97, 98, 99, 100, 101, 102, 104, 105, 107, 108, 122, 123, 124, 125, 126 or 129, wherein said assay module is a multi-well plate and said assay domains in said assay module are located within a well of said plate.

133. The multi-well plate of claim 132, wherein said well comprises one or more working electrode surfaces and said assay domains are located on said one or more working electrode surfaces.

134. The assay module of claim 1, 3, 5, 6, 7, 8, 9, 10, 11, 19, 20, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 40, 41, 46, 47, 49, 53, 59, 63, 64, 66, 68, 74, 75, 79, 80, 81, 82, 83, 84, 88, 89, 90, 91, 92, 93, 95, 97, 98, 99, 100, 101, 102, 104, 105, 107, 108, 122, 123, 124, 125, 126 or 129, wherein said assay module further comprises one or more working electrode surfaces and said assay domains are located on said one or more working electrode surfaces.

2. A method of effecting assays for a plurality of analytes in an assay module comprising a plurality of assay domains and said assay domains comprising a first assay domain having a first reagent for measuring a first analyte and a second assay domain having a second reagent for measuring a second analyte, wherein said first assay domain is capable of generating luminescence at least 10 times brighter than said second assay domain and said first assay domain and said second assay domains are not adjacent and/or are separated by at least one other assay domain to reduce luminescence emitted from said first assay domain from interfering with luminescence emitted from said second assay domain.

4. The method of claim 2, wherein said first assay domain comprises at least 10 times more reagent than said second assay domain.

12. A method of making an assay module for conducting luminescence assays comprising a plurality of assay domains, said method comprising forming said assay domains in a pattern configured to reduce interference between said assay domains.

13. The method of claim 12, wherein said pattern is configured to reduce interference between luminescence emitted from said assay domains.

14. The method of claim 12 or 13, wherein said pattern is configured to reduce interference between reactions occurring at said assay domains.

15. The method of claim 12, 13 or 14, wherein said forming said assay domains comprises placing a first assay domain so as to be not adjacent to a second assay domain.

16. The method of claim 12, 13 or 14, wherein said forming said assay domains comprises placing a first assay domain so as to be not adjacent to a second assay domain, wherein a reaction occurring at or a reagent immobilized at said first assay domain interferes with:

(i) luminescence generated at said second assay domain, (ii) a reaction occurring at said second assay domain and/or (iii) a reagent immobilized at said second assay domain.

17. The method of claim 12, 13, 14, 15 or 16, wherein said pattern is configured to optimize the performance of said luminescence assay.

18. The method of claim 12, 13, 14, 15, 16 or 17, wherein said pattern includes the arrangement, the spacing, the size and concentration of said assay domains.

19. An assay module comprising a plurality of assay domains for measuring a panel of analytes, said panel comprising a plurality of analytes that are members of an analyte category selected from cytokines and/or their receptors;

growth factors and/or their receptors;

second messengers;

drugs of abuse;

therapeutic drugs;

auto-antibodies;

allergen specific antibodies;

tumor markers;

cardiac markers;

markers associated with hemostasis;

markers of acute viral hepatitis infection;

markers of Alzheimers Disease;

markers of osteoporosis;

markers of fertility;

markers of congestive heart failure;

markers of thyroid disorders;

markers of prostrate cancer;

nucleic acid arrays for measuring mRNA levels of mRNA coding for cytokines, growth factors, components of the apoptosis pathway, expression of the P450 enzymes or expression of tumor related genes;

nucleic acid arrays for genotyping individuals, pathogens, or tumor cells.

20. An assay module of claim 19, wherein said assay domains comprise:

(i) analytes or analogs of analytes;

(ii) binding reagents that bind analytes; and

/or (iii) substrates for catalytic activity of analytes.

21. A method of performing an assay for a plurality of analytes using an assay module, said method comprising:

(a) contacting a sample with a plurality of assay domains within said assay module, wherein said plurality of assay domains are capable of measuring a panel of analytes, said panel comprising a plurality of analytes that are members of an analyte category selected from cytokines and/or their receptors;

growth factors and/or their receptors;

second messengers;

drugs of abuse;

therapeutic drugs;

auto-antibodies;

allergen specific antibodies;

tumor markers;

cardiac markers;

markers associated with hemostasis;

markers of acute viral hepatitis infection;

markers of Alzheimers Disease;

markers of osteoporosis;

markers of fertility;

markers of congestive heart failure;

markers of thyroid disorders;

markers of prostrate cancer;

nucleic acid arrays for measuring mRNA levels of mRNA coding for cytokines, growth factors, components of the apoptosis pathway, expression of the P450 enzymes or expression of tumor related genes;

nucleic acid arrays for genotyping individuals, pathogens, or tumor cells; and

(b) measuring said analytes at said assay domains.

22. A method of performing an electrochemiluminescence assay using an electrochemiluminescence assay module, said method comprising:

(a) contacting a sample with a plurality of assay domains in said module, said plurality of assay domains capable of measuring a panel of analytes, said panel comprising a plurality of analytes that are members of an analyte category selected from cytokines and/or their receptors;

growth factors and/or their receptors;

second messengers;

drugs of abuse;

therapeutic drugs;

auto-antibodies;

allergen specific antibodies;

tumor markers;

cardiac markers;

markers associated with hemostasis;

markers of acute viral hepatitis infection;

markers of Alzheimers Disease;

markers of osteoporosis;

markers of fertility;

markers of congestive heart failure;

markers of thyroid disorders;

markers of prostrate cancer;

nucleic acid arrays for measuring mRNA levels of mRNA coding for cytokines, growth factors, components of the apoptosis pathway, expression of the P450 enzymes or expression of tumor related genes;

nucleic acid arrays for genotyping individuals, pathogens, or tumor cells and said assay domains comprising or being adjacent one or more working electrodes;

(b) inducing electrochemiluminescence at said plurality of assay domains; and

(c) detecting emitted electrochemiluminescence.

23. A method of effecting a specific-binding assay of a ligand in an assay module comprising a plurality of binding domains for measuring a panel of analytes, said panel comprising a plurality of analytes that are members of an analyte category selected from cytokines and/or their receptors;

growth factors and/or their receptors;

second messengers;

drugs of abuse;

therapeutic drugs;

auto-antibodies;

allergen specific antibodies;

tumor markers;

cardiac markers;

markers associated with hemostasis;

markers of acute viral hepatitis infection;

markers of Alzheimers Disease;

markers of osteoporosis;

markers of fertility;

markers of congestive heart failure;

markers of thyroid disorders;

markers of prostrate cancer;

nucleic acid arrays for measuring mRNA levels of mRNA coding for cytokines, growth factors, components of the apoptosis pathway, expression of the P450 enzymes or expression of tumor related genes;

nucleic acid arrays for genotyping individuals, pathogens, or tumor cells.

24. The method of claim 23, wherein said panel is a panel of growth factor receptors.

25. An assay module comprising a plurality of assay domains, said assay domains comprising a first assay domain comprising a first reagent capable of binding a first form of an analyte and a second assay domain comprising a second reagent capable of binding a second form of said analyte.

28. The assay module of claim 25, wherein said second form is a cleaved portion of said first form.

29. The assay module of claim 25, wherein said second form is an ubiquitinated form of said first form.

30. The assay module of claim 25, wherein said second form of said analyte is a phosphorylated form of said first form.

31. The assay module of claim 25, wherein said second form is a bound form of said first form.

32. The assay module of claim 25, wherein said second assay domain is also capable of binding said first form.

33. The assay module of claim 25, wherein said first form is un-glycosylated protein and said second form is glycosylated protein.

34. The assay module of claim 25, wherein said first form is free PSA and said second form of said analyte is bound PSA.

35. The assay module of claim 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34, further comprising a detection reagent capable of binding with said first form and said second form.

26. A method for measuring two forms of an analyte comprising:

(a) introducing a sample into an assay module comprising a plurality of assay domains, a first assay domain comprising a first reagent capable of binding a first form of an analyte and a second assay domain comprising a second reagent capable of binding a second form of said analyte; and

(b) measuring the binding of the analyte to said first and second assay domains to measure the amounts of said first and second forms.

27. The method of claim 26, wherein said second form is a cleaved portion of said first form.

36. An assay module for conducting an assay comprising:

(a) a first assay domain having a first reagent capable of binding a first analyte;

(b) a second assay domain having a second reagent capable of binding a modified form of said first analyte;

(c) a third assay domain having a third reagent capable of binding a second analyte; and

(d) a fourth assay domain having a fourth reagent capable of binding a modified form of said second analyte.

37. The assay module of claim 36, wherein said assay module further comprises a labeled reagent capable of binding said first analyte, said modified form of said first analyte, said second analyte and a modified form of said second analyte.

38. A method of measuring two or more forms of an analyte in a single sample in an assay module having a first assay domain comprising a first reagent capable of binding a first form of said analyte and a second assay domain comprising a second reagent capable of binding a second form of said analyte, said method comprising:

(a) contacting said sample with said first assay domain and said second assay domain and a labeled reagent capable of binding said first form and said second form of said analyte; and

(b) detecting signal emitted from said labeled reagent.

39. The method of claim 38, further comprising a wash step to remove unbound labeled reagent.

40. An assay module for conducting an assay comprising:

(a) a first assay domain having a first reagent capable of binding a first analyte;

(b) a second assay domain having a second reagent capable of binding a modified form of said first analyte;

(c) a third assay domain having a third reagent capable of binding a second analyte; and

(d) a fourth assay domain having a fourth reagent capable of binding a modified form of said second analyte.

41. The assay module of claim 40, wherein said assay module further comprises a labeled reagent capable of binding said first analyte, said modified form of said first analyte, said second analyte and a modified form of said second analyte.

42. A method of measuring two or more forms of an analyte in a single sample in an assay module having a first assay domain comprising a first reagent capable of binding a first form of said analyte and a second assay domain comprising a second reagent capable of binding a second form of said analyte, said method comprising:

(a) contacting said sample with said first assay domain and said second assay domain and a labeled reagent capable of binding said first form and said second form of said analyte; and

(b) detecting signal emitted from said labeled reagent.

43. The method of claim 42, further comprising a wash step to remove unbound labeled reagent.

44. A method of measuring two or more epitopes of an analyte in a single sample in a assay module having a first assay domain comprising a first reagent capable of binding a first epitope of said analyte and a second assay domain comprising a second reagent capable of binding a second epitope of said analyte, said method comprising contacting said sample with said first assay domain and said second assay domain.

45. The method of claim 44, further comprising contacting said sample with one or more labels capable of binding said analyte and detecting a signal emitted by said one or more labels.

46. An assay module for conducting an assay for an analyte, said assay module comprising:

(a) a first assay domain comprising a first reagent capable of binding said analyte; and

(b) a second assay domain comprising said first reagent or a second reagent capable of binding said analyte;

wherein said first assay domain and said second binding domain have different properties, said different properties selected from the group consisting of surface area, reagent concentration, heat stability, pH sensitivity, wettability, epitope specificity, binding kinetics, non-specific binding properties, and binding constants.

47. An assay module for optimizing the design of an assay domain for conducting an assay for an analyte, said module comprising:

(a) a first assay domain comprising a first reagent capable of binding said analyte; and

(b) a second assay domain comprising said first reagent;

wherein said first assay domain and said second binding domain having different properties, said different properties selected from the group consisting of surface area, reagent concentration, heat stability, pH stability, hydrophobic or hydrophilic properties, co-reactants, epitope specificity, binding kinetics, non-specific binding properties, binding constants, and/or thermodynamics.

48. A method for detecting or correcting for a source of assay error in an assay for an analyte comprising:

(a) introducing a sample comprising analyte into an assay module comprising

(i) a first assay domain comprising a first reagent capable of binding said analyte; and

(ii) a second assay domain comprising said first reagent;

wherein said first assay domain and said second binding domain having different properties, said different properties selected from the group consisting of surface area, reagent concentration, heat stability, pH stability, hydrophobic or hydrophilic properties, co-reactants, epitope specificity, binding kinetics, non-specific binding properties, binding constants, and/or thermodynamics; and

(b) comparing the binding of the analyte to the first and second assay domains so as to detect or correct for sources of assay error.

49. An assay module for identifying a biological material, said module comprising:

(a) a first assay domain comprising reagents capable of binding with components of a first biological material and components of a second biological material; and

(b) a second assay domain comprising second reagents capable of binding with said components of said first biological material and said components of said second biological material;

wherein the ratio of the binding to the first assay domain relative to the second assay domain is indicative of the identity of the biological material.

50. A method of identifying a biological material comprising:

(a) introducing a sample of said biological material into an assay module comprising;

(i) a first assay domain comprising reagents capable of binding with first components of a first biological material and first components of a second biological material; and

(b) a second assay domain comprising second reagents capable of binding with second components of said first biological material and second components of said second biological material;

wherein the ratio of the binding to the first assay domain relative to the second assay domain is indicative of the identity of the biological material; and

(b) measuring the ratio of the binding of said biological material to said first and second assay domains so as to determine the identity of the biological material.

51. A method for conducting an assay for two or more pathogens having similar properties, said method comprising:

(a) introducing a sample into an assay module having a first assay domain comprising reagents capable of binding with said first pathogen and said second pathogen and a second assay domain comprising second reagents capable of binding with said first pathogen and said second pathogen, wherein the presence of said first pathogen results in said first assay domain emitting a greater signal then said second assay domain; and

(b) detecting first signal emitted from said first assay domain and second signal emitted from said second assay domain.

52. The method of claim 51, further comprising deconvolving the amount of said first pathogen and said second pathogen from said first signal and said second signal.

53. An assay module comprising:

(a) a first assay domain comprising reagents capable of binding with an analyte; and

(b) a second assay domain comprising said reagents capable of binding with said analyte,

wherein said first assay domain and said second assay domain comprise substantially the same concentration of said reagents and are independently addressable.

54. A method of determining the rate of a reaction comprising:

(a) contacting a sample with an assay module having a plurality of assay domains, said assay domains each having a concentration of reagents capable of binding an analyte and being independently addressable;

(b) sequentially addressing and detecting a signal from said assay domains.

55. The method of claim 54, wherein said reaction is an enzymatic reaction.

56. The method of claim 55, wherein said domains comprise binding reagents for the product of the enzyme reaction.

57. The method of claim 54, wherein said reaction is a binding reaction of two binding partners and said domains comprise one of said binding partners.

58. A method of optimizing the production of an assay module for use in detecting one or more analytes comprising:

(a) forming a prototype assay module having a plurality of assay domains, wherein said plurality of assay domains are capable of binding with said one or more analytes and having different parameters, said different parameters selected from the group consisting of;

(i) binding reagent;

(ii) co-reactant;

(iii) pH;

(iv) location within well;

(v) reagent concentration;

(vi) domain location;

(vii) assay reagent affinity;

(viii) assay reagent specificity;

(ix) sensitivity to interferents and (x) sensitivity to optical distortion;

(b) performing a test assay using a test sample containing said one or more analytes using said prototype assay module; and

(c) determining which assay domain achieved the best performance.

59. An assay module for detecting a first analyte and/or a second analyte comprising

(a) a first assay that binds to said first analyte and said second analyte, but binds better to said first analyte then to said second analyte;

and

(b) a second assay domain that binds to said first analyte and said second analyte, but binds to said second analyte better than said first analyte

60. A method of detecting at least a first analyte and a second analyte in a sample, wherein said first analyte and said second analyte have similar binding properties, said method comprising:

(a) contacting said sample with a first assay domain and a second assay domain, where said first assay domain binds to said first analyte and said second analyte, but binds better to said first analyte then to said second analyte and where said second assay domain binds to said first analyte and said second analyte, but binds to said second analyte better than said first analyte;

(b) inducing said first assay domain to emit a signal;

(c) measuring said signal from said first assay domain; and

(d) deconvolving the amount of said first analyte and/or said second analyte.

61. The method of claim 60, wherein said second assay domain sequesters said second analyte.

62. The method of claim 60 or 61, further comprising measuring signal from said second assay domain.

63. An assay module for conducting an assay on a sample comprising an analyte and at least one interferent, said module comprising:

(a) an assay domain capable of binding said analyte; and

(b) a sequestering domain capable of binding said interferent.

64. The assay module of claim 63, wherein said module is adapted to measure said interferent bound to said sequestering domain.

65. A method for measuring an analyte in the presence of an interferent comprising

(a) introducing the sample into an assay module comprising

(i) an assay domain capable of binding said analyte;

and

(ii) a sequestering domain capable of binding said interferent; and

(b) detecting the binding of said analyte to said assay domain.

66. An assay module for measuring an enzymatic reaction comprising:

(a) a first assay domain capable of binding to a substrate of said enzyme; and

(b) a second assay domain capable of binding to a product formed by the action of said enzyme on said substrate.

67. A method for measuring an enzymatic reaction comprising:

(a) incubating said a sample of the enzyme with a substrate to form a reaction solution;

(b) contacting said reaction solution with a plurality of assay domains in an assay module, said assay domains including

(i) a first assay domain capable of binding to said substrate; and

(ii) a second assay domain capable of binding to the product formed by the action of the enzyme on said substrate; and

(c) detecting the binding of said substrate and product to said first and second binding domains so as to measure the enzyme reaction.

68. An assay module for measuring an enzymatic reaction comprising:

(a) a first assay domain capable of binding to a substrate or product of said enzyme; and

(b) a second assay domain capable of binding to said enzyme.

69. A method for measuring an enzymatic reaction comprising:

(a) introducing an enzyme into an assay module comprising

(i) a first assay domain capable of binding one of a substrate or product of said enzyme; and

(ii) a second assay domain capable of binding said enzyme;

(b) binding said enzyme to said second assay domain;

(c) washing said assay module to purify said enzyme;

(d) contacting said enzyme with said substrate so as to form said product; and

(e) measuring the binding of said one of said substrate or product to said first assay domain so as to measure said enzymatic reaction.

70. A method for measuring an enzyme comprising:

(a) introducing an enzyme into an assay module comprising

(i) a first assay domain capable of binding one of a substrate or product of said enzyme; and

(ii) a second assay domain capable of binding said enzyme;

(b) binding said enzyme to said second assay domain;

(c) contacting said enzyme with said substrate so as to form said product;

(d) measuring the binding of said one of said substrate or product to said first assay domain so as to measure the activity of said enzyme; and

(e) measuring the binding of said enzyme to said second assay domain to measure the amount of enzyme.

71. A method for measuring an enzymatic reaction comprising:

(a) introducing an enzyme into an assay module comprising

(i) a first assay domain capable of binding one of a substrate or product of said enzyme; and

(ii) a second assay domain capable of binding said enzyme;

(b) binding said enzyme to said second assay domain to form an immobilized enzyme;

(c) contacting said enzyme with said substrate so as to form said product;

(d) measuring the binding of said one of said substrate or product to said first assay domain so as to measure said enzymatic reaction; and

(e) assaying for a modification of said immobilized enzyme.

72. The method of claim 71, further comprising contacting said enzyme with a binding reagent specific for said modification.

73. The method of claim 71, further comprising contacting said enzyme with a modifying activity, wherein said step of assaying for a modification measures said modifying activity.

74. An assay module having for measuring an enzymatic reaction of a substrate to a product by an enzyme, said module comprising:

(a) a first assay domain capable of binding to a first reactant selected from the group consisting of;

(i) enzyme;

(ii) substrate;

(iii) product;

(iv) reaction intermediate;

(v) reactant by-product and/or (vi) reaction interferent; and

(b) a second assay domain capable of binding to a second reactant selected from the group consisting of;

(i) enzyme;

(ii) substrate;

(iii) product;

(iv) reaction intermediate;

(v) reactant by-product and/or (vi) reaction interferent;

wherein said first reactant and said second reactant are different thereby measuring at least two reactants of said enzymatic reaction.

75. An assay module for measuring a cleavage reaction, said module comprising:

(a) a first assay domain comprising a substrate for a cleavage reaction; and

(b) a second assay domain capable of binding to a product of said cleavage reaction.

76. A method of measuring a cleavage reaction in an assay module, the assay module comprising:

(a) a first assay domain comprising a substrate for said cleavage reaction; and

(b) a second assay domain capable of binding to a product of said cleavage reaction.

77. A method of determining the effect of an inhibitor on a plurality of enzymes comprising contacting a sample containing said inhibitor and said plurality of enzymes with a plurality of assay domains having substrates for said enzymes immobilized thereon and detecting the effect of said inhibitor on said plurality of enzymes.

78. The method of claim 77, wherein said plurality of enzymes comprise a plurality of kinases.

138. The method of claim 77 or 78, wherein said plurality of assay domains are located on one or more working electrodes.

79. An assay module having a plurality of assay domains, said assay domains having enzymes immobilized thereon.

80. The assay module of claim 79, wherein said assay domains include a first assay domain having a first enzyme immobilized thereon and a second assay domain having a second enzyme immobilized thereon.

81. The assay module of claim 79 or 80, further comprising a binding reagent capable of binding products of said enzymes, said binding reagent being immobilized in binding domains located adjacent said assay domains.

82. The assay module of claim 79 or 80, further comprising a binding reagent capable of binding products of said enzymes, said binding reagent being co-immobilized on said assay domains with said enzymes.

83. The assay module of claim 79, 80, 81 or 82, wherein said assay domains are contained within a containment region adapted to reduce the diffusion of products produced by said enzymes immobilized on said assay domains.

84. The assay module of claim 79, 80, 81 or 82, wherein said assay domains are sunken within a dielectric layer to reduce the diffusion of products produced by said enzymes immobilized on said assay domains.

85. A method of measuring the activity of a plurality of enzymes comprising:

(a) introducing a solution comprising substrates of said enzymes into an assay module comprising;

(i) a plurality of assay domains having said enzymes immobilized thereon; and

(ii) a binding reagent capable of binding the products resulting from the action of said enzymes on said substrates;

wherein the enzymes act on said substrates to form said products and said binding reagent is co-immobilized with said enzymes in said assay domains or said binding reagent is immobilized in binding domains located adjacent to said assay domains.

(b) measuring the binding of said products to said binding reagent to measure the activity of said plurality of enzymes, wherein, the extent of binding to binding reagent located within or adjacent to a specific binding domain is indicative of the enzyme activity within that binding domain.

86. A method of conducting an assay comprising contacting a sample containing a plurality of enzymes with an assay module having a plurality of assay domains comprising substrates corresponding to said enzymes immobilized thereon and detecting the enzymatic reaction of said substrates at said assay domains.

87. An assay domain having immobilized thereon an enzyme and a reagent capable of binding a product of an enzymatic reaction initiated by said enzyme.

139. The assay domain of claim 87, wherein said assay domain is located on a working electrode.

88. An assay module comprising a plurality of assay domains, said plurality of assay domains comprising:

(a) a first assay domain capable of binding an analyte A, an analyte E and an analyte C;

(b) a second assay domain capable of binding an analyte B, an analyte C and an analyte F;

(c) a third assay domain capable of binding an analyte B, an analyte D and an analyte E; and

(d) a fourth assay domain capable of binding an analyte A, an analyte D and an analyte F.

89. The assay module of claim 88, wherein said first assay domain, said second assay domain and said third assay domain are capable of binding an analyte G;

said second assay domain, said third assay domain and said fourth assay domain are capable of binding an analyte H;

said first assay domain, third assay domain, and said fourth assay domain are capable of binding an analyte I.

90. The assay module of claim 88, wherein said first assay domain is capable of binding an analyte W, said second assay domain is capable of binding an analyte X, said third assay domain is capable of binding an analyte Y and said fourth assay domain is capable of binding an analyte Z.

91. An assay module comprising a plurality of assay domains, said plurality of assay domains comprising:

(a) a first assay domain capable of binding an analyte A, an analyte E, an analyte F and an analyte H;

(b) a second assay domain capable of binding an analyte A, an analyte B, an analyte G and an analyte I;

(c) a third assay domain capable of binding an analyte B, an analyte C, analyte H and an analyte J;

(d) a fourth assay domain capable of binding an analyte C, an analyte D, an analyte F and an analyte I; and

(e) a fifth assay domain capable of binding an analyte D, an analyte E, an analyte G and an analyte J.

92. The assay module of claim 91, wherein:

(a) said first assay domain is capable of binding an analyte K;

(b) said second assay domain is capable of binding an analyte L;

(c) said third assay domain is capable of binding an analyte M;

(d) said fourth assay domain is capable of binding an analyte N; and

(e) said fifth assay domain is capable of binding an analyte O.

93. An assay module comprising M assay domains and Z different assay reagents, wherein:

(i) M is an integer greater than three and Z is an integer greater than M and less than or equal to (M!)/[2!(M−

2)!]; and

(ii) said assay reagents are distributed among said assay domains so that between I and M−

1 assay reagents are immobilized in each domain and at least one assay reagent is immobilized in a unique pair of domains.

94. A method of screening for a substance that interacts with an assay reagent, said method comprising:

(a) introducing said substance into an assay module comprising M assay domains and Z different assay reagents, wherein

(i) M is an integer greater than three and Z is an integer greater than M and less than or equal to (M!)/[2!(M−

2)!];

(ii) said assay reagents are distributed among said assay domains so that between 1 and M-1 assay reagents are immobilized in each domain and at least one assay reagent is immobilized in a unique pair of domains; and

(b) determining which assay reagents interacted with said substance.

95. An assay module comprising M assay domains and Z different assay reagents, wherein

(i) M is an integer greater than three and Z is an integer greater than M and less than or equal to (M!)/[Z!(M−

Z)!]; and

(ii) said assay reagents are distributed among said assay domains so that greater than one assay reagents are immobilized in each domain and at each assay reagent is immobilized in a unique set of between one and Z domains.

96. A method of screening for a substance that interacts with an assay reagent, comprising:

(a) introducing said substance into an assay module comprising M assay domains and Z different assay reagents, wherein

(i) M is an integer greater than three and Z is an integer greater than M and less than or equal to (M!)/[Z!(M−

Z)!];

(ii) said assay reagents are distributed among said assay domains so that at least one assay reagents are immobilized in each domain and each assay reagent is immobilized in a unique set of between 1 and Z domains; and

(b) determining which assay reagents interacted with said substance.

97. An assay module comprising a plurality of assay domains, wherein said plurality of assay domains comprises one or more assay domains comprising one or more binding reagents and further comprising a first control domain comprising pre-bound label, a second control domain providing a defined reaction and a third control domain consisting essentially of blocking agent.

98. The assay module of claim 97, wherein said first control domain controls for one or more of the following:

(i) chemical interference with ECL generation;

(ii) optical interference with light transmission; and

/or (iii) module variation.

99. The assay module of claim 97 or 98, wherein said second control domain controls for one or more of the following:

(i) non-specific binding or biochemical activity;

(ii) chemical interference with ECL generation;

(iii) optical interference with light transmission;

(iv) pipetting errors;

(v) timing errors;

(vi) variations in mixing;

(vii) variations in temperature; and

(viii) assay module variation.

100. The assay module of claim 97, 98 or 99, wherein said third control domain controls for non-specific binding.

101. An assay module comprising a first assay domain and a second assay domain, wherein said first assay domain comprises a binding reagent capable of binding with an analyte and said second assay domain comprises said analyte or an analogue of said analyte.

102. An assay module comprising a first assay domain and a second assay domain, wherein said first assay domain binds with an analyte and said second assay domain competes with said analyte.

103. A method for assaying for an analyte of interest comprising

(a) introducing a sample comprising said analyte into an assay module comprising two assay domains, a first domain that binds said analyte and a second domain that competes with said analyte;

and

(b) conducting in said assay module a sandwich assay and a competitive assay for said analyte.

135. The method of claim 2, 4, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 26, 38, 39, 42, 43, 44, 45, 48, 50, 51, 52, 54, 55, 56, 57, 58, 60, 61, 62, 65, 67, 69, 70, 71, 72, 73, 76, 85, 86, 94, 96, 103, 106, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 127, 128, 130 or 131, wherein said assay module is a multi-well plate and said assay domains in said assay module are located within a well of said plate.

136. The method of claim 135, wherein said well comprises one or more working electrode surfaces and said assay domains are located on said one or more working electrode surfaces.

137. The method of claim 2, 4, 12, 13, 14, 15, 16, 17, 18, 21, 22, 23, 24, 26, 38, 39, 42, 43, 44, 45, 48, 50, 51, 52, 54, 55, 56, 57, 58, 60, 61, 62, 65, 67, 69, 70, 71, 72, 73, 76, 85, 86, 94, 96, 103, 106, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 127, 128, 130 or 131, wherein said assay module further comprises one or more working electrode surfaces and said assay domains are located on said one or more working electrode surfaces.

104. An assay module comprising a first assay domain and a second assay domain, wherein said first assay domain comprises a binding reagent capable of forming a sandwich complex with an analyte and said second assay domain comprises binding reagents which compete with said analyte.

105. An assay module comprising a first assay domain and a second assay domain having binding affinity for the analyte, wherein said first assay domain has a binding affinity for an analyte at least 10 times greater than the binding affinity of said second assay domain.

106. A method for assaying for an analyte of interest comprising

(a) introducing a sample comprising said analyte into an assay module comprising two assay domains, a first assay domain and a second assay domain having binding affinity for the analyte, wherein said first assay domain has a binding affinity for the analyte at least 10 times greater than the binding affinity of said second assay domain;

and

(b) conducting at said first and second domains, sandwich assays for said analyte.

107. An assay module comprising a first assay domain and a second assay domain, wherein said first assay domain comprises a first substrate to an enzyme and said second assay domain comprises a second substrate to said enzyme, wherein said first substrate has, a slower turnover rate and/or higher Vmax than said second substrate.

108. An assay module comprising one or more assay domains comprising assay reagents capable of binding a product of an enzymatic reaction and one or more sequestration domains comprising sequestrants capable of capturing one or more enzymes of said enzymatic reaction.

109. A method of conducting an assay comprising contacting a sample comprising one or more enzymes with a plurality of assay domains in an assay module, said plurality of assay domains including an assay domain capable of binding one or more products of an enzymatic reaction and one or more enzyme capture domains for capturing one or more enzymes of said enzymatic reaction.

110. The method of claim 109, further comprising washing unbound components of said sample from said module.

111. The method of claim 109 or 110, further comprising adding a second sample comprising one or more substrates of said enzymatic reactions.

112. A method of conducting an assay using an assay module comprising a plurality of assay domains, said assay domains including:

(i) an array of enzyme domains capable of binding enzymes including;

a first enzyme domain capable of binding a first enzyme;

a second enzyme domain capable of binding a second enzyme; and

a third enzyme domain capable of binding a third enzyme;

(ii) an array of product domains capable of binding products of said enzymes including;

a first product domain capable of binding a first product corresponding to said first enzyme;

a second product domain capable of binding a second product corresponding to said second enzyme; and

a third product domain capable of binding a third product corresponding to said third enzyme;

wherein said method comprises;

(a) contacting a sample containing a plurality of components including one or more enzymes and one or more substrates corresponding to said one or more enzymes with said array of enzyme domains and said array of product domains; and

(b) detecting said first product at said first product domain, said second product at said second product domain and said third product at said third product domain.

113. The method of claim 112, wherein said enzyme domains are adjacent said corresponding product domains.

114. The method of claim 113, wherein said enzyme domains overlap said corresponding product domains.

115. The method of claim 112, 113 or 114, further comprising detecting said first enzyme at said first enzyme domain, said second enzyme at said second enzyme domain and said third enzyme at said third enzyme domain.

116. The method of claim 112, 113, 114 or 115, wherein said enzymes are kinases and said products are corresponding phosphorylated products of said enzymes.

117. A method of determining a plurality of enzymatic activities of one or more enzymes using an assay module comprising two or more assay domains, said method comprising contacting a sample comprising one or more enzymes with a first domain capable of binding a first product of a first enzymatic reaction and with a second domain capable of binding a second product of a second enzymatic reaction and detecting said first product and said second product at said first and second binding domains.

118. The method of claim 117, wherein said module further comprises one or more enzyme assay domains capable of binding said one or more enzymes.

119. The method of claim 118, further comprising detecting said one or more enzymes at said one or more enzyme assay domains.

120. The method of claim 117, wherein said module further comprises one or more substrate assay domains capable of binding one or more substrates corresponding to said one or more enzymes.

121. The method of claim 120, further comprising detecting said one or more substrates at said one or more substrate assay domains.

122. An assay module comprising a plurality of assay domains, said plurality of assay domains comprising a first assay domain capable of measuring an activity involving protein cleavage and a second assay domain capable of measuring a cleaved product of said activity.

123. The assay module of claim 122, wherein said first assay domain comprises a labeled protein immobilized thereon and said second assay domain comprises a binding reagent capable of binding with a cleaved derivative of said label protein immobilized thereon.

124. An assay module comprising one or more assay domains comprising assay reagents and one or more sequestrating domains comprising sequestration agents for sequestering interfering species.

125. The assay module of claim 124, wherein said one or more sequestrating domains sequest one or more of the following interfering species:

biotin;

anti-Streptavidin;

hemoglobin;

bilirubin;

lipid;

high and low albumin;

HAMA;

anti-ruthenium;

high Rheumatoid Factor;

DLIF'"'"'s for digoxin or digitoxin type molecules;

drugs/co meds;

cross reactive analytes; and

combinations thereof.

126. An assay module comprising one or more assay domains comprising assay reagents and one or more sequestrating domains comprising sequestration agents capable of sequestering a labeled reagent.

127. A method of performing an assay using an assay module having a plurality of assay domains comprising contacting a composition containing a sample and one or more reagents with said plurality of assay domains, said one or more reagents including a labeled reagent capable of binding with an analyte, an analogue of the analyte or a binding partner of said analyte and said plurality of assay domains including a first assay domain capable of binding with said analyte and a second assay domain capable of binding with said labeled reagent.

128. The method of claim 127, further comprising detecting said analyte on said first assay domain.

129. An assay module comprising a plurality of assay domains, said assay domains including a first assay domain capable of binding to an analyte and a second assay domain capable of binding to one or more lyophilized reagents.

130. A method of conducting an assay using an assay module comprising a plurality of assay domains, said assay domains including a first assay domain capable of binding one or more enzymes and a second assay domain capable of binding one or more enzymatic products of said one or more enzymes, said method comprising:

(a) contacting a first sample containing a plurality of components including one or more enzymes with said first assay domain in said module thereby immobilizing said one or more enzymes;

(b) separating unbound components of said first sample from said module;

(c) contacting a second sample containing one or more substrates of said one or more enzymes with said second assay domain; and

(d) detecting said enzymatic product bound at said second assay domain.

131. The method of claim 130, further comprising detecting said one or more enzymes on said first assay domain.