Electrophoresis apparatus having valve system

• US 8,007,725 B2
• Filed: 11/17/2006
• Issued: 08/30/2011
• Est. Priority Date: 11/07/2003
• Status: Active Grant

• Current Assignee: Princeton Biochemicals, Inc.
• Original Assignee: Princeton Biochemicals, Inc.
• Inventors: Guzman, Norberto A.
• Primary Examiner(s): Jung; Unsu
• Application Number:
  US11/601,856
• Publication Number:
  US 20070128714A1
• Time in Patent Office: 1,747 Days
• Field of Search: 204/180.1, 204450-455, 204600-605
• US Class Current: 422/82.01
• CPC Class Codes: G01N 1/405 by adsorption or absorption
• CPC Class Codes: G01N 27/44708 Cooling
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44756 Apparatus specially adapted therefor
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/561 Immunoelectrophoresis

Abstract

An electrophoresis apparatus is generally disclosed for sequentially analyzing a single sample or multiple samples having one or more analytes in high or low concentrations. The apparatus comprises a relatively large-bore transport capillary which intersects with a plurality of small-bore separation capillaries and includes a valve system. Analyte concentrators, having antibody-specific (or related affinity) chemistries, are stationed at the respective intersections of the transport capillary and separation capillaries to bind one or more analytes of interest. The apparatus allows the performance of two or more dimensions for the optimal separation of analytes. The apparatus may also include a plurality of valves surrounding each of the analyte concentrators to localize each of the concentrators to improve the binding of one or more analytes of interest.

Claims

1. A multi-dimensional electrophoresis apparatus, comprising:

a transport passage having an inlet end and an outlet end;

a separation passage having a transport/separation passage overlapping portion which overlaps a portion of the transport passage;

the separation passage being communicable upstream of the transport/separation passage overlapping portion with a separation buffer supply or with an eluting buffer supply;

an analyte concentrator containing one or more immobilized affinity ligands in the transport/separation passage overlapping portion to concentrate at least one analyte of interest from a fluid-containing sample introduced into the transport passage from a sample supply;

the analyte concentrator being positionable upstream of an analyte detector zone wherein the at least one analyte of interest eluted from the one or more immobilized affinity ligands and delivered thereto via the separation passage can be identified and characterized;

one of the anode or cathode sides of the multi-dimensional electrophoresis apparatus being generally at the eluting buffer supply or at the separation buffer supply side and the other side being downstream of the analyte concentrator; and

localizing means associated with the transport passage for localizing the analyte concentrator to thereby increase the concentration of the at least one analyte of interest from the sample at the analyte concentrator wherein the intersection has a staggered configuration such that the transport/separation passage overlapping portion forms an elongated separation passage portion containing the one or more immobilized affinity ligands.

2. The multi-dimensional electrophoresis apparatus of claim 1 further comprising passage bulge members or frit structures that retain a matrix and/or beads that contain the one or more immobilized affinity ligands in the analyte concentrator.

3. The multi-dimensional electrophoresis apparatus of claim 1 wherein the one or more affinity ligands are immobilized to the transport/separation passage overlapping portion corresponding to the capturing area of the analyte concentrator in an orientation that facilitates the binding of the at least one analyte of interest present in the fluid-containing sample.

4. The multi-dimensional electrophoresis apparatus of claim 1 wherein an inlet end of the separation passage is alternatively in fluid communication with the separation buffer supply, the eluting buffer supply and a cleaning solution supply.

5. The multi-dimensional electrophoresis apparatus of claim 1 wherein the at least one analyte of interest includes a small molecular weight substance, a biomolecule, a simple molecule and/or a complex molecule.

6. The multi-dimensional electrophoresis apparatus of claim 1 wherein the analyte concentrator includes free-floating, non-bound beads and antibody fragments covalently attached to surfaces of the beads or intact antibodies covalently attached to surfaces of the beads.

7. The multi-dimensional electrophoresis apparatus of claim 1 wherein the analyte concentrator includes free-floating, non-bound beads and antibody fragments non-covalently attached to surfaces of the beads or intact antibodies non-covalently attached to surfaces of the beads.

8. The multi-dimensional electrophoresis apparatus of claim 1 wherein passage bulging members or frit structures retain free-floating, non-bound beads in the analyte concentrator.

9. The multi-dimensional electrophoresis apparatus of claim 1 wherein:

the analyte concentrator includes a matrix comprising sol-gel, monolithic or interconnected beads linked to each other and to an inner surface of the analyte concentrator; and

the analyte concentrator is free of bulging retainer members and frits.

10. The multi-dimensional electrophoresis apparatus of claim 1 wherein the localizing means comprises a valve system,

the valve system comprising:

a first valve on the first separation passage downstream of the first analyte concentrator,

a second valve on the transport passage upstream of the first analyte concentrator,

a third valve on the transport passage downstream of the first analyte concentrator,

a fourth valve on the first separation passage upstream of the first analyte concentrator,

wherein the valve system allows the first and fourth valves to be closed and the second and third valves to be opened to pass the sample and a washing buffer through the transport passage to permit the at least one analyte of interest present in the fluid-containing sample to be bound to the one or more immobilized affinity ligands of the analyte concentrator.

11. The multi-dimensional electrophoresis apparatus of claim 10 wherein the valve system allows the first and fourth valves to be opened and the second and third valves to be closed so that analytes bound to the one or more immobilized affinity ligands within the analyte concentrator can be released with a plug of an elution buffer from the eluting buffer supply.

12. The multi-dimensional electrophoresis apparatus of claim 1 wherein the transport and separation passages are both capillaries.

13. The multi-dimensional electrophoresis apparatus of claim 1 wherein the transport and separation passages are both at least substantially channels.

14. The multi-dimensional electrophoresis apparatus of claim 1 wherein the inlet end of the transport passage can be alternatively in fluid communication with a supply of a cleaning solution, a supply of a conditioning buffer, a supply of the sample and a supply of a washing solution.

15. The multi-dimensional electrophoresis apparatus of claim 1 wherein the separation passage defines a first separation passage, the analyte concentrator defines a first analyte concentrator, the one or more immobilized affinity ligands define one or more first immobilized affinity ligands, the at least one analyte of interest defines a first analyte of interest, the localizing means defines first localizing means, the valve system defines the first valve system and the transport/separation passage overlapping portion defines a transport/separation first passage overlapping portion;

the first valve system comprising;

a first valve on the first separation passage downstream of the first analyte concentrator,

a second valve on the transport passage upstream of the first analyte concentrator,

a third valve on the transport passage downstream of the first analyte concentrator,

a fourth valve on the first separation passage upstream of the first analyte concentrator, and further comprising;

a second separation passage having a transport/separation second passage overlapping portion which overlaps a different portion of the transport passage;

the second separation passage being communicable upstream of the second passage overlapping portion with a separation buffer supply and an eluting buffer supply;

a second analyte concentrator containing one or more second immobilized affinity ligands in the second passage overlapping portion to concentrate a second analyte of interest from the sample; and

second localizing means associated with the second analyte concentrator for localizing the second analyte concentrator to thereby increase the concentration of the second analyte of interest from the sample at the second analyte concentrator, the second localizing means comprising a second valve system, the second valve system comprising a fifth valve on the second separation passage at an inlet first end of the second analyte concentrator;

a sixth valve on the second separation passage at an outlet second end of the second analyte concentrator; and

a seventh valve on the transport passage downstream of the second analyte concentrator wherein separation of analytes is optimized separately, independently and sequentially in the first and second separation passages.

16. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first and second analyte concentrators have independent time, temperature and movement or interactions of molecules and reagents therein;

the first localizing means allows for individual optimal conditions for concentrating the first analyte of interest by the one or more first immobilized affinity ligands; and

the second localizing means allows for individual optimal conditions for concentrating the second analyte of interest by the one or more second immobilized affinity ligands.

17. The multi-dimensional electrophoresis apparatus of claim 15 wherein the transport passage has a larger diameter bore than the bores of the first and second separation passages.

18. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

an inlet of the first separation passage is in fluid communication alternatively with the separation buffer supply and the eluting buffer supply allowing a plug of eluting buffer to release the bound first analyte of interest from the first affinity ligands into the first separation passage and allowing the separation buffer to separate the released first analyte of interest in the first separation passage, and the first separation passage being alternatively in fluid communication with the separation buffer supply and the eluting buffer supply; and

/or

an inlet of the second separation passage is in fluid communication alternatively with the separation buffer supply and the eluting buffer supply allowing a plug of eluting buffer to release the bound second analyte of interest from the second affinity ligands into the second separation passage and allowing the separation buffer to separate the released second analyte of interest in the second separation passage, and the second separation passage being alternatively in fluid communication with the separation buffer supply and the eluting buffer supply.

19. The multi-dimensional electrophoresis apparatus of claim 18 wherein:

the composition of the separation buffer in the separation buffer supply providing separation buffer to the second separation passage is the same as or is different from the composition of the separation buffer in the separation buffer supply providing separation buffer to the first separation passage; and

/or

the composition of the eluting buffer in the eluting buffer supply providing a plug of the eluting buffer to the second separation passage is the same as or is different from the composition of the eluting buffer in the eluting buffer supply providing a plug of the eluting buffer to the first separation passage.

20. The multi-dimensional electrophoresis apparatus of claim 15 wherein an inlet end of the transport passage can be in fluid communication with a cleaning solution supply followed by a conditioning buffer supply for cleaning and conditioning the first analyte concentrator with the first antibody immobilized therein and the second analyte concentrator with the second antibody immobilized therein, after introduction of the sample containing the first and second analyte of interest, to be captured by the respective first and second immobilized antibodies, followed by a washing buffer to remove excess amount of sample and unwanted materials bound non-specifically to the transport passage and/or to the analyte concentrator.

21. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

passage bulging members or frit structures retain beads that contain the one or more first immobilized affinity ligands, and the one or more first immobilized affinity ligands are immobilized in the first analyte concentrator; and

/or

passage bulging members or frit structures retain beads that contain the one or more second immobilized affinity ligands, and the one or more second immobilized affinity ligands are immobilized in the second analyte concentrator.

22. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the valves on the transport passage control flow of a cleaning fluid, a conditioning buffer, the sample and a washing buffer through the transport passage and the valves on the first and second separation passages control flow of conditioning, separation and washing fluids through the first and second separation passages, respectively, such that the first and second analyte concentrators can be localized by the valves on the transport passage and the valves on the respective first and second separation passages to increase the attracting and binding of the first analyte of interest to the one or more first immobilized affinity ligands and the second analyte of interest to the one or more second immobilized affinity ligands.

23. The multi-dimensional electrophoresis apparatus of claim 22 wherein the valves at least in part controls fluid flow from the first and second analyte concentrators via the first and second separation passages, respectively, to the analyte detector zone.

24. The multi-dimensional electrophoresis apparatus of claim 22 wherein:

the valves on the first separation passage control at least in substantial part flow of elution buffer and the released first analyte of interest through the first separation passage; and

/or

the valves on the second separation passage control at least in substantial part flow of elution buffer and the released second analyte of interest through the second separation passage.

25. The multi-dimensional electrophoresis apparatus of claim 22 further comprising:

auxiliary means coupled to the first and second separation passages for providing separation buffer to the respective first and second separation passages away and downstream from the first and second analyte concentrators, respectively; and

valves on the auxiliary means that control the flow of the separation buffer from the auxiliary means to the first and second separation passages.

26. The multi-dimensional electrophoresis apparatus of claim 25 wherein the auxiliary means are electrolyte providers providing an electrolyte solution and/or an electrolyte solution containing organic solvents, detergents, or other modifier agents.

27. The multi-dimensional electrophoresis apparatus of claim 25 wherein the first and/or second analyte concentrators operate as microreactors and can perform on-line enzymatic cleavage of proteins to generate peptides.

28. The multi-dimensional electrophoresis apparatus of claim 25 wherein the first and/or second analyte concentrators operate as microreactors and can perform chemical and/or biochemical reactions.

29. The multi-dimensional electrophoresis apparatus of claim 15 wherein all of the valves of the valve system are preferentially motor-operated and remotely controlled by a processor based on a predetermined set of instructions.

30. The multi-dimensional electrophoresis apparatus of claim 15 wherein the first and second valve systems are positionable in a first condition wherein the second, third and seventh valves are opened and the first, fourth, fifth and sixth valves are closed to allow the sample to flow in the transport passage and through the first and second analyte concentrators independently and sequentially.

31. The multi-dimensional electrophoresis apparatus of claim 30 wherein the first and second valve systems are positionable in a second condition after the first condition wherein the first, second, third, fourth, fifth, sixth and seventh valves are closed to allow the first and second analyte concentrators to increase the binding of the first analyte of interest to the one or more first immobilized affinity ligands and the second analyte of interest to the one or more second immobilized affinity ligands.

32. The multi-dimensional electrophoresis apparatus of claim 31 wherein the first and second valve systems are positionable in a third condition wherein the second, third and seventh valves are closed, and the first and fourth valves and the fifth and sixth valves are opened to allow concentrated analytes in the first and second analyte concentrators to be independently and sequentially released therefrom towards the analyte detector zone.

33. The multi-dimensional electrophoresis apparatus of claim 32 further comprising a single detector in the analyte detector zone operative for both the first and second separation passages by way of a single exit separation passage.

34. The multi-dimensional electrophoresis apparatus of claim 32 wherein the valve system when in the third condition allows conditioning, separation, elution and/or washing buffer solutions to pass through the first and/or second separation passages towards the analyte detector zone and in a sequential order when sample previously introduced through the transport passage and analytes of interest bound to the analyte concentrators are released of the first and/or second analyte concentrators.

35. The multi-dimensional electrophoresis apparatus of claim 15 wherein the temperature at the first analyte concentrator can be controlled when localized by the first localizing means at a first temperature associated with the first analyte of interest and the temperature at the second analyte concentrator can be controlled when localized by the second localizing means at a different second temperature associated with the second analyte of interest.

36. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first and second separation passages have independently-controlled temperature-control systems; and

the temperature-control systems can heat or cool the first and second separation passages when they are in a linear configuration or in a coiled configuration.

37. The multi-dimensional electrophoresis apparatus of claim 15 further comprising an analyte detection system positioned at the analyte detector zone.

38. The multi-dimensional electrophoresis apparatus of claim 37 wherein the analyte detection system is an ultraviolet, fluorescence, laser-induced fluorescence, electrochemical, radioactive conductivity, mass spectrometer, circular diochroism or nuclear magnetic resonance detection system.

39. The multi-dimensional electrophoresis apparatus of claim 37 further comprising an exit outlet passage into which the first and second separation passages merge and flow, and the analyte detection system being operatively positioned proximate to and relative to the exit outlet passage.

40. The multi-dimensional electrophoresis apparatus of claim 39 wherein the first and second localizing means at least in part control sequential fluid flow from the first and second separation passages to the exit outlet passage.

41. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

an inlet of the first separation passage is in fluid communication alternatively with a conditioning buffer supply, with a separation buffer supply, with an eluting buffer supply and with a washing buffer supply; and

/or

an inlet of the second separation passage is in fluid communication alternatively with a conditioning buffer supply, with a separation buffer supply, with an eluting buffer supply and with a washing buffer supply.

42. The multi-dimensional electrophoresis apparatus of claim 15 further comprising:

a first auxiliary buffer passage through which a separation buffer can be provided to the first separation passage, and positioned at a location downstream of the first analyte concentrator and upstream of the analyte detector zone; and

a second auxiliary buffer passage through which a separation buffer can be provided to the second separation passage, and positioned at a location downstream of the second analyte concentrator and upstream of the analyte detector zone.

43. The multi-dimensional electrophoresis apparatus of claim 42 further comprising auxiliary valves that control the flow of separation buffer from the first auxiliary buffer passage to the first separation passage and the flow of separation buffer from the second auxiliary buffer passage to the second separation passage.

44. The multi-dimensional electrophoresis apparatus of claim 43 wherein the auxiliary valves are operable so that the separation buffer solution from a buffer supply of the first auxiliary buffer passage does not interact with the first analyte concentrator, and the separation buffer solution from a buffer supply of the second auxiliary buffer passage does not interact with the second analyte concentrator.

45. The multi-dimensional electrophoresis apparatus of claim 43 wherein the auxiliary valves include a first auxiliary buffer valve at an intersection of the first separation passage and the first auxiliary buffer passage and a second auxiliary buffer valve at an intersection of the second separation passage and the second auxiliary buffer passage.

46. The multi-dimensional electrophoresis apparatus of claim 43 wherein:

the first separation passage and the first auxiliary buffer passage form a first junction;

the auxiliary valves include a first auxiliary buffer valve positioned at the first auxiliary buffer passage between the first junction and a buffer supply of the first auxiliary buffer passage;

the second separation passage and the second auxiliary buffer passage form a second junction; and

the auxiliary valves include a second auxiliary buffer valve positioned at the second auxiliary buffer passage between the second junction and a buffer supply of the second auxiliary buffer passage.

47. The multi-dimensional electrophoresis apparatus of claim 42 further comprising an exit outlet passage into which the first and second separation passages merge and relative to which an analyte detection system in the analyte detector zone is operatively aligned.

48. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the first valve system associated with the first analyte concentrator

includes a first auxiliary valve at an intersection of the first separation passage and the first auxiliary buffer passage.

49. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the second valve system associated with the second analyte concentrator

includes a second auxiliary valve at an intersection of the second separation passage and the second auxiliary buffer passage.

50. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the first separation passage and the first auxiliary buffer passage form a junction;

the first valve is positioned on the first separation passage between the junction and the first analyte concentrator; and

an auxiliary buffer valve is positioned on the first auxiliary buffer passage between the junction and a buffer supply of the first auxiliary buffer passage.

51. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the second separation passage and the second auxiliary buffer passage form a junction;

the sixth valve is positioned on the second separation passage between the junction and the second analyte concentrator; and

an auxiliary buffer valve is positioned on the second auxiliary buffer passage between the junction and a buffer supply of the second auxiliary buffer passage.

52. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the first localizing means includes a first auxiliary valve at a junction of the first separation passage and the first auxiliary buffer passage, the first auxiliary valve controlling at least in substantial part fluid flow in the first separation passage and in the first auxiliary buffer passage; and

/or

the second localizing means includes a second auxiliary valve at a junction of the second separation passage and the second auxiliary buffer passage, the second auxiliary valve controlling at least in substantial part fluid flow in the second separation passage and in the second auxiliary buffer passage.

53. The multi-dimensional electrophoresis apparatus of claim 42 further comprising auxiliary valves associated with the first and second auxiliary buffer passages;

and

wherein the auxiliary valves are operable such that (a) a buffer solution that has been introduced into the first separation passage from the first auxiliary buffer passage does not interact with the first analyte concentrator, (b) a buffer solution that has been introduced into the second separation passage from the second auxiliary buffer passage does not interact with the second analyte concentrator, and (c) the buffer solutions are introduced separately and independently into the first and second separation passages.

54. The multi-dimensional electrophoresis apparatus of claim 42 wherein:

the first localizing means includes a first auxiliary valve at a junction of the first separation passage and the first auxiliary buffer passage, the first auxiliary valve controlling fluid flow in the first separation passage and in the first auxiliary buffer passage;

the second localizing means includes a second auxiliary valve at a junction of the second separation passage and the second auxiliary buffer passage, the second auxiliary valve controlling fluid flow in the second separation passage and in the second auxiliary buffer passage;

the first analyte of interest when released from the first analyte concentrator is separated in the first separation passage by electro-osmotic flow, electrophoresis migration, mechanical pressure or vacuum, or a combination of electro-osmotic flow and mechanical pressure; and

the second analyte of interest when released from the second analyte concentrator is separated in the second separation passage by electro-osmotic flow, electrophoresis migration, mechanical pressure or vacuum, or a combination of electro-osmotic flow and mechanical pressure.

55. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first and second localizing means control at least in substantial part the flow of a cleaning buffer, a conditioning buffer, the sample and a washing fluid from an inlet of the transport passage to an outlet of the transport passage, allowing the first and second analytes of interest in the sample to bind to the first and second analyte concentrators, respectively;

after the binding of the first analyte of interest to the first analyte concentrator, at least one valve of the multi-dimensional electrophoresis apparatus is switchable to allow for separation of the first analyte of interest in the first separation passage; and

after the binding of the second analyte of interest to the second analyte concentrator, at least one valve of the multi-dimensional electrophoresis apparatus is switchable to allow for separation of the second analyte of interest in the second separation passage.

56. The multi-dimensional electrophoresis apparatus of claim 55 wherein after the first and second analytes of interest have been identified and characterized by one or more detectors at the analyte detector zone, the transport passage is communicable with a cleaning buffer to clean at least the transport passage and the first and second separation passages are communicable with cleaning buffers to clean at least the first and second separation passages in order to make the multi-dimensional electrophoresis apparatus ready for further cycle usage.

57. The multi-dimensional electrophoresis apparatus of claim 56 further comprising:

a first auxiliary buffer passage communicating with the first separation passage between the first analyte concentrator and the analyte detector zone, and wherein after the first analyte of interest has been identified and characterized by the one or more detectors, the first auxiliary buffer passage is communicable with a cleaning buffer to clean the first auxiliary buffer passage in order to make the multi-dimensional electrophoresis apparatus ready for further cycle usage; and

/or

a second auxiliary buffer passage communicating with the second separation passage between the second analyte concentrator and the analyte detector zone, and wherein after the second analyte of interest has been identified and characterized by the one or more detectors, the second auxiliary buffer passage is communicable with a cleaning buffer to clean the second auxiliary buffer passage in order to make the multi-dimensional electrophoresis apparatus ready for further cycle usage.

58. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first analyte of interest released from the first analyte concentrator is separated in the first separation passage by at least one mode of capillary electrophoresis; and

the second analyte of interest released from the second analyte concentrator is separated in the second separation passage by at least one mode of capillary electrophoresis.

59. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first analyte concentrator includes a first matrix assembly having a surface to which the one or more first affinity ligands are immobilized; and

the second analyte concentrator includes a second matrix assembly having a surface to which the one or more second affinity ligands are immobilized.

60. The multi-dimensional electrophoresis apparatus of claim 59 wherein:

the first matrix assembly includes a plurality of microstructures; and

/or

the second matrix assembly includes a plurality of microstructures.

61. The multi-dimensional electrophoresis apparatus of claim 59 wherein:

the first analyte concentrator retains the first matrix assembly by pressure-resistant porous end walls or frits disposed in the transport passage and the first separation passage; and

/or

the second analyte concentrator retains the second matrix assembly by pressure-resistant porous end walls or frits disposed in the transport passage and the second separation passage.

62. The multi-dimensional electrophoresis apparatus of claim 59 wherein:

the first matrix assembly includes a fixed architecture defined by beaded or other microstructures interconnected to each other and to an inner surface of the first analyte concentrator in which no frit structures are required; and

/or

the second matrix assembly includes a fixed architecture defined by beaded or other microstructures interconnected to each other and to an inner surface of the second analyte concentrator in which no frit structures are required.

63. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first separation passage is capable of separating the first analytes of interest by at least one mode of capillary electrophoresis after the first analyte of interest that was retained by the one or more first immobilized affinity ligands is released from the one or more first immobilized affinity ligands; and

the second separation passage is capable of separating the second analyte of interest by at least one mode of capillary electrophoresis after the second analyte of interest that was retained by the one or more second immobilized affinity ligands is released from the one or more second immobilized affinity ligands.

64. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first separation passage is capable of separating the first analyte of interest by electrophoresis migration, electro-osmotic flow, mechanical pressure or vacuum, or a combination of electro-osmotic flow and mechanical pressure, after the first analyte of interest that was retained by the one or more first immobilized affinity ligands is released from the one or more first immobilized affinity ligands; and

the second separation passage is capable of separating the second analyte of interest by electrophoresis migration, electro-osmotic flow, mechanical pressure or vacuum, or a combination of electro-osmotic flow and mechanical pressure, after the second analyte of interest that was retained by the one or more second immobilized affinity ligands is released from the one or more second immobilized affinity ligands.

65. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the one or more first immobilized affinity ligands include a first affinity ligand component and a different second affinity ligand component;

the first and second affinity ligand components being attracted to different portions of the first analyte of interest; and

/or

the one or more second immobilized affinity ligands include a third affinity ligand component and a different fourth affinity ligand component;

the third and fourth affinity ligand components being attracted to different portions of the second analyte of interest.

66. The multi-dimensional electrophoresis apparatus of claim 15 wherein the first and second valve systems allow first and second of the elution buffer plugs to be passed through the first and second analyte concentrators, respectively, independently and sequentially to elute the first and second analytes of interest released to the first and second separation passages, respectively, and to the analyte detector zone and after having been separated by at least one mode of capillary electrophoresis.

67. The multi-dimensional electrophoresis apparatus of claim 66 further comprising a single power supply for the first and second separation passages that can be used in alternative or sequential operations.

68. The multi-dimensional electrophoresis apparatus of claim 66 further comprising a single power supply having high-voltage relays for the first and second separation passages.

69. The multi-dimensional electrophoresis apparatus of claim 15 wherein the first analyte concentrator comprises a first concentrator-microreactor.

70. The multi-dimensional electrophoresis apparatus of claim 69 wherein the first concentrator-microreactor includes a matrix containing a first immobilized affinity ligand which binds the first analyte of interest from the sample to the first affinity ligand immobilized to the matrix.

71. The multi-dimensional electrophoresis apparatus of claim 69 wherein:

the first concentrator-microreactor binds the first analyte of interest from the sample directly to the one or more first immobilized affinity ligands; and

the one or more first immobilized affinity ligands are immobilized to an inner wall of the first concentrator-microreactor.

72. The multi-dimensional electrophoresis apparatus of claim 69 wherein:

the first concentrator-microreactor includes a matrix containing the one or more first immobilized affinity ligands; and

the one or more first affinity ligands immobilized to the first concentrator-microreactor binds the first analyte of interest from the sample for concentration and/or microreaction purposes, and

the one or more first affinity ligands are immobilized to an inner wall of the first concentrator-microreactor.

73. The multi-dimensional electrophoresis apparatus of claim 69 wherein the second analyte concentrator comprises a second concentrator-microreactor.

74. The multi-dimensional electrophoresis apparatus of claim 73 wherein:

the one or more second affinity ligands immobilized to the second concentrator-microreactor binds the second analyte of interest from the sample for concentration and/or microreaction purposes; and

the one or more second affinity ligands are immobilized to an inner wall of the second concentrator-microreactor.

75. The multi-dimensional electrophoresis apparatus of claim 73 wherein:

the second concentrator-microreactor includes a matrix and the one or more second affinity ligands are immobilized to the matrix; and

the one or more second affinity ligands immobilized to the second concentrator-microreactor binds the second analyte of interest from the for concentration and/or microreaction purposes.

76. The multi-dimensional electrophoresis apparatus of claim 73 wherein the first analyte of interest is a first simple molecule of interest and the second analyte of interest is a second simple molecule of interest.

77. The multi-dimensional electrophoresis apparatus of claim 73 wherein the first analyte of interest is a first complex molecule of interest and the second analyte of interest is a second complex molecule of interest.

78. The multi-dimensional electrophoresis apparatus of claim 73 wherein one of the first and second analytes of interest is a simple molecule of interest and the other of the first and second analytes of interest is a complex molecule of interest.

79. The multi-dimensional electrophoresis apparatus of claim 73 wherein:

the first analyte of interest is one or more simple molecules of interest and one or more complex molecules of interest; and

the second analyte of interest is one or more simple molecules of interest and one or more complex molecules of interest.

80. The multi-dimensional electrophoresis apparatus of claim 73 wherein:

the one or more first immobilized affinity ligands includes a first affinity ligand component and a different second affinity ligand component;

the first and second affinity ligand components being attracted to different portions of the first analyte of interest; and

/or

the one or more second immobilized affinity ligands includes a third affinity ligand component and a different fourth affinity ligand component;

the third and fourth affinity ligand components being attracted to different portions of the second analyte of interest.

81. The multi-dimensional electrophoresis apparatus of claim 73 wherein a detector at the analyte detector zone identifies and characterizes simple and/or complex molecules delivered thereto via at least one of the first and second separation passages.

82. The multi-dimensional electrophoresis apparatus of claim 73 wherein the one or more first and second immobilized affinity ligands can perform a chemical reaction or multi-component chemical reactions with one or more components in the sample independently in the first and second analyte concentrator-microreactors, respectively.

83. The multi-dimensional electrophoresis apparatus of claim 82 wherein the chemical reaction is a biochemical reaction.

84. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first separation passage has electrically conductive fluid therein that has buffer conditions that facilitate separation of the first analyte of interest that has been released from the one or more first immobilized affinity ligands in the first analyte concentrator into the first separation passage towards the analyte detector zone; and

/or

the second separation passage has electrically conductive fluid therein that has buffer conditions that facilitate separation of the second analyte of interest that has been released from the one or more second immobilized affinity ligands in the second analyte concentrator into the second separation passage towards the analyte detector zone.

85. The multi-dimensional electrophoresis apparatus of claim 15 wherein the first separation passage has electrically conductive fluid and a gel matrix therein.

86. The multi-dimensional electrophoresis apparatus of claim 85 wherein:

the one or more first affinity ligands are immobilized to the first analyte concentrator;

the electrically conductive fluid has buffer conditions that facilitate binding of the one or more first immobilized affinity ligands with the first analyte of interest; and

/or

the gel matrix and the electrically conductive fluid have conditions that facilitate separation in the first separation passage of the released first analyte of interest from the one or more first immobilized affinity ligands.

87. The multi-dimensional electrophoresis apparatus of claim 85 wherein:

the electrically conductive fluid has buffer conditions, from the inlet of the first separation passage to a junction of the first separation passage with a first auxiliary buffer passage, that facilitate binding of the one or more first immobilized affinity ligands with the first analyte of interest; and

/or

the gel matrix and the electrical conductive fluid introduced through the first auxiliary buffer passage into the first separation passage, downstream of the first analyte concentrator and towards the analyte detector zone, have conditions that facilitate separation of the released first analyte of interest from the one or more first immobilized affinity ligands.

88. The multi-dimensional electrophoresis apparatus of claim 15 wherein the one or more second affinity ligands are immobilized to the second analyte concentrator;

wherein the second separation passage has electrically conductive fluid and a gel matrix therein; and

wherein;

(1) the electrically conductive fluid in the second separation passage has buffer conditions, from the inlet of the second separation passage to a junction of the second separation passage with a second auxiliary buffer passage, that facilitate binding of the one or more second immobilized affinity ligands to the second analyte concentrator with the second analyte of interest; and

/or

(2) the gel matrix and the electrical conductive fluid introduced through a second auxiliary buffer passage into the second separation passage, downstream of the second analyte concentrator towards the analyte detector zone have conditions that facilitate separation in the second separation passage of the released second analyte of interest from the one or more second immobilized affinity ligands.

89. The multi-dimensional electrophoresis apparatus of claim 15 wherein:

the first and second valve systems allow analyte separation processes occurring in the first and second separation passages to be conducted separately and sequentially.

90. The multi-dimensional electrophoresis apparatus of claim 15 wherein the inlet end of the transport passage can be in fluid communication with a washing buffer supply for washing and removing unwanted material bound by non-specific attractions to the transport passage, to the first analyte concentrator with the first analyte of interest captured therein and to the second analyte concentrator with the second analyte of interest captured therein.

107. The electrophoresis apparatus of claim 1 being configured to analyze one or multiple molecular or cellular-subcellular constituents of a single fluid-containing sample, or one or multiple molecular or cellular-subcellular constituents of a plurality of fluid-containing samples.

91. A multi-dimensional electrophoresis apparatus, comprising:

a transport passage having an inlet end and an outlet end;

a first separation passage having a first transport/separation passage overlapping portion which overlaps a portion of the transport passage;

the first separation passage being communicable upstream of the first transport/separation passage overlapping portion with a separation buffer supply or with an eluting buffer supply;

a first analyte concentrator containing one or more first immobilized affinity ligands in the first transport/separation passage overlapping portion to concentrate at least one first analyte of interest from a fluid-containing sample introduced into the first transport passage from a sample supply;

the first analyte concentrator being positionable upstream of an analyte detector zone wherein the at least one analyte of interest eluted from the one or more first immobilized affinity ligands and delivered thereto via the separation passage can be identified and characterized;

a first valve system associated with an intersection of the transport passage and the first separation passage;

a second separation passage having a second transport/separation passage overlapping portion which overlaps a different portion of the transport passage;

the second separation passage being communicable upstream of the second transport/separation passage overlapping portion with a separation buffer supply or with an eluting buffer supply;

a second analyte concentrator containing one or more second immobilized affinity ligands in the second transport/separation passage overlapping portion to concentrate at least one second analyte of interest from the fluid-containing sample;

a second valve system associated with an intersection of the transport passage and the second separation passage;

one of the anode or cathode sides of the multi-dimensional electrophoresis apparatus being generally at the side of the eluting buffer supply or the separation buffer supply and the other side being downstream of the first analyte concentrator and the second analyte concentrator; and

further comprising;

a first auxiliary analyte concentrator on the first separation passage and downstream of the first analyte concentrator, the first auxiliary analyte concentrator having affinity ligands capable of retaining chromophores to bind the first analyte of interest released from the first analyte concentrator to improve the sensitivity and selectivity of the first analyte of interest once the first analyte of interest is released from the auxiliary analyte concentrator into the first separation passage and towards the analyte detector zone; and

/or

a second auxiliary analyte concentrator on the second separation passage and downstream of the second analyte concentrator, the second auxiliary analyte concentrator having affinity ligands capable of retaining chromophores to bind the second analyte of interest released from the second analyte concentrator to improve the sensitivity and selectivity of the second analyte of interest once the second analyte of interest is released from the second auxiliary analyte concentrator into the second separation passage and towards the analyte detector zone.

92. The electrophoresis apparatus of claim 91 being configured to analyze one or multiple molecular or cellular-subcellular constituents of a single fluid-containing sample, or one or multiple molecular or cellular-subcellular constituents of a plurality of fluid-containing samples.

93. The multi-dimensional electrophoresis apparatus of claim 92 wherein:

the one or more first affinity ligands immobilized to the first analyte concentrator-microreactor are capable of performing at least one chemical or biochemical reaction; and

/or

the one or more second affinity ligands immobilized to the second analyte concentrator-microreactor are capable of performing at least one chemical or biochemical reaction.

94. The multi-dimensional electrophoresis apparatus of claim 92 wherein:

the one or more first affinity ligands are immobilized covalently to a matrix assembly of the first analyte concentrator; and

/or

the one or more second affinity ligands are immobilized covalently to a matrix assembly of the second analyte concentrator.

95. The multi-dimensional electrophoresis apparatus of claim 92 further comprising one or more detection systems positioned at the analyte detector zone.

96. The multi-dimensional electrophoresis apparatus of claim 95 wherein the detection system is an ultraviolet fluorescence, a laser-induced fluorescence, mass spectrometry, circular dichroism, nuclear magnetic resonance, radioactive conductivity, electrochemical detection system.

97. The multi-dimensional electrophoresis apparatus of claim 92 further comprising:

an exit outlet passage into which the first and second separation passages merge and flow;

the exit outlet passage being positioned generally at the analyte detector zone; and

at least one exit valve that controls the flow from the first separation passage into the exit outlet passage and from the second separation passage into the exit outlet passage separately and sequentially.

98. The multi-dimensional electrophoresis apparatus of claim 97 wherein:

the at least one exit valve includes a valve on the first separation passage proximate to the exit outlet passage and a valve on the second separation passage proximate to the exit outlet passage; and

the at least one exit valve when in a first condition blocks flow from the first separation passage into the exit outlet passage and allows flow from the second separation passage into the exit outlet passage and when in a second condition blocks flow from the second separation passage into the exit outlet passage and allows flow from the first separation passage into the exit outlet passage.

99. The multi-dimensional electrophoresis apparatus of claim 97 wherein:

the at least one exit valve includes a valve on the first separation passage proximate to the exit outlet passage and a valve on the second separation passage proximate to the exit outlet passage; and

the at least one exit valve when in a first condition allows flow from the first separation passage into the exit outlet passage and blocks flow from the second separation passage into the exit outlet passage and when in a second condition allows flow from the second separation passage into the exit outlet passage and blocks flow from the first separation passage into the exit outlet passage.

100. The multi-dimensional electrophoresis apparatus of claim 92 wherein the other position of the anode or cathode sides is located downstream of the analyte detector zone.

101. The multi-dimensional electrophoresis apparatus of claim 92 wherein:

the first valve system includes a first valve on the first separation passage downstream of the first analyte concentrator, a second valve on the transport passage upstream of the first analyte concentrator, a third valve on the transport passage downstream of the first analyte concentrator, and a fourth valve on the first separation passage upstream of the first analyte concentrator;

the first valve system allows the first and fourth valves to be closed and the second and third valves to be opened to pass cleaning buffers, conditioning buffers, and the sample through the transport passage to permit the first analyte of interest in the sample to be bound to the one or more first immobilized affinity ligands to the first analyte concentrator and then passing washing buffers to remove unbound and unwanted materials that are bound non-specifically to the transport passage and the first analyte concentrator; and

the first valve system allows the first and fourth valves to be opened and the second and third valves to be closed so that the first analyte of interest which is bound to the one or more first immobilized affinity ligands can be released from the first analyte concentrator with an elution buffer plug into the first separation passage and towards the analyte detector zone and separated in the first separation passage.

102. The multi-dimensional electrophoresis apparatus of claim 101 wherein:

the second valve system includes a fifth valve on the second separation passage at an inlet first end of the second analyte concentrator;

a sixth valve on the second separation passage at an outlet second end of the second analyte concentrator; and

a seventh valve on the transport passage downstream of the second analyte concentrator;

the first and second valve systems allow the fifth and sixth valves to be closed and the second, third and seventh valves to be opened to pass conditioning buffers, and the sample through the transport passage to permit the second analyte of interest in the sample to be bound to the one or more second immobilized affinity ligands to the second analyte concentrator and then passing washing buffers to remove unbound and unwanted materials that are bound non-specifically to the transport passage and the second analyte concentrator; and

the first and second valve systems allow the fifth and sixth valves to be opened and the second, third and seventh valves to be closed so that the second analyte of interest which is bound to the one or more second immobilized affinity ligands can be released from the second analyte concentrator with an elution buffer plug into the second separation passage and towards the analyte detector zone and separated in the second separation passage.

103. The multi-dimensional electrophoresis apparatus of claim 92 wherein an inlet end of the transport passage can be in fluid communication with a cleaning buffer supply (a) for cleaning and removing excess amount of sample and unwanted materials bound by non-specific attractions to the transport passage and to the first analyte concentrator with the first analyte of interest captured therein and (b) for cleaning and removing excess amount of sample and unwanted materials bound by non-specific attractions to the transport passage and to the second analyte concentrator with the second analyte of interest captured therein.

104. The multi-dimensional electrophoresis apparatus of claim 92 wherein:

an inlet end of the first separation passage can be in fluid communication with a washing buffer supply for washing the first analyte concentrator with the first analyte of interest captured therein; and

/or

an inlet end of the second separation passage can be in fluid communication with a washing buffer supply for washing the second analyte concentrator with the second analyte of interest captured therein.

105. The multi-dimensional electrophoresis apparatus of claim 92 further comprising:

a first frit structure in an inlet of the transport passage into the first analyte concentrator;

a second frit structure in an outlet of the transport passage from the first analyte concentrator;

a third frit structure in an inlet of the first separation passage into the first analyte concentrator;

a fourth frit structure in an outlet of the first separation passage from the first analyte concentrator;

a fifth frit structure in an inlet of the transport passage into the second analyte concentrator;

a sixth frit structure in an outlet of the transport passage from the second analyte concentrator;

a seventh frit structure in an inlet of the second separation passage into the second analyte concentrator; and

an eighth frit structure in an outlet of the second separation passage from the second analyte concentrator.

106. The multi-dimensional electrophoresis apparatus of claim 92 further comprising:

pressure-resistant porous end walls on both sides of connecting terminals of the transport passage with the first analyte concentrator;

pressure-resistant porous end walls on both sides of connecting terminals of the first separation passage with the first analyte concentrator;

pressure-resistant porous end walls on both sides of connecting terminals of the transport passage with the second analyte concentrator; and

pressure-resistant porous end walls on both sides of connecting terminals of the second separation passage with the second analyte concentrator.

108. A multi-dimensional electrophoresis apparatus, comprising:

a transport passage having an inlet end and an outlet end;

a first separation passage having a first transport/separation passage overlapping portion which overlaps a portion of the transport passage;

the first separation passage being communicable upstream of the first transport/separation passage overlapping portion with a separation buffer supply or with an eluting buffer supply;

a first analyte concentrator containing one or more first immobilized affinity ligands in the first transport/separation passage overlapping portion to concentrate at least one first analyte of interest from a fluid-containing sample introduced into the first transport passage from a sample supply;

the first analyte concentrator being positionable upstream of an analyte detector zone wherein the at least one analyte of interest eluted from the one or more first immobilized affinity ligands and delivered thereto via the separation passage can be identified and characterized;

a first valve system associated with an intersection of the transport passage and the first separation passage;

a second separation passage having a second transport/separation passage overlapping portion which overlaps a different portion of the transport passage;

the second separation passage being communicable upstream of the second transport/separation passage overlapping portion with a separation buffer supply or with an eluting buffer supply;

a second analyte concentrator containing one or more second immobilized affinity ligands in the second transport/separation passage overlapping portion to concentrate at least one second analyte of interest from the fluid-containing sample;

a second valve system associated with an intersection of the transport passage and second the separation passage;

one of the anode or cathode sides of the multi-dimensional electrophoresis apparatus being generally at the side of the eluting buffer supply or the separation buffer supply and the other side being downstream of the first analyte concentrator and the second analyte concentrator;

an intersection of the transport passage and the first separation passage has a staggered configuration forming an elongated first separation passage portion in which a concentration area of the first analyte concentrator is positioned; and

/or

an intersection of the transport passage and the second separation passage has a staggered configuration forming an elongated second separation passage portion in which a concentration area of the second analyte concentrator is positioned.