Controlled electrophoresis method

• US 8,030,092 B2
• Filed: 11/17/2006
• Issued: 10/04/2011
• 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:
  US11/601,855
• Publication Number:
  US 20070134812A1
• Time in Patent Office: 1,782 Days
• Field of Search: None
• US Class Current: 436/518
• 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 controlled electrophoresis method, comprising:

introducing a sample into a transport passage of a multi-dimensional electrophoresis apparatus;

the multi-dimensional electrophoresis apparatus including a first separation passage intersecting the transport passage at a first overlapping portion, a first analyte concentrator at the first overlapping portion to concentrate a first analyte of interest, a second separation passage intersecting the transport passage at a second overlapping portion, and a second analyte concentrator at the second overlapping portion to concentrate a second analyte of interest;

passing the introduced sample in the transport passage into the first overlapping portion so that the first analyte of interest in the sample is concentrated in the first analyte concentrator;

passing the introduced sample in the transport passage into the second overlapping portion so that the second analyte of interest in the sample is concentrated in the second analyte concentrator;

localizing the first analyte concentrator and thereby increasing the concentration of the first analyte of interest by the first analyte concentrator;

localizing the second analyte concentrator and thereby increasing the concentration of the second analyte of interest by the second analyte concentrator;

releasing the first analyte of interest concentrated in the first analyte concentrator into the first separation passage;

releasing the second analyte of interest concentrated in the second analyte concentrator into the second separation passage;

delivering the released first analyte of interest in the first separation passage to an analyte detection zone which identifies and characterizes the first analyte of interest; and

delivering the released second analyte of interest in the second separation passage to the analyte detection zone which also identifies and characterizes the second analyte of interest wherein;

an intersection of the transport passage and the first separation passage has a staggered configuration such that the first overlapping portion is an elongated passage portion of the first separation passage and contains the first analyte concentrator; and

/or

an intersection of the transport passage and the second separation passage has a staggered configuration such that the second overlapping portion is an elongated passage portion of the second separation passage and contains the second analyte concentrator.

2. The method of claim 1 wherein:

the transport passage being communicable at an inlet end with a sample supply,

the first and second separation passages being communicable upstream of the first and second overlapping portions with a separation buffer supply or with a supply of an elution buffer or solution,

a valve system for controlling the flow of the sample in the transport passage from the inlet end of the transport passage to an outlet end of the transport passage,

the valve system controlling the flow of the separation buffer supply or the supply of elution buffer or solution in a sequential order from an inlet end of the separation passage to an outlet end of the separation passage,

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,

a fifth valve on the second separation passage upstream of the second analyte concentrator,

a sixth valve on the second separation passage downstream of the second analyte concentrator, and

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

3. The method of claim 2 wherein the localizing the first analyte concentrator includes closing the first valve, the second valve, the third valve and the fourth valve.

4. The method of claim 2 wherein the localizing the second analyte concentrator includes closing the third valve, the fifth valve, the sixth valve and the seventh valve.

5. The method of claim 2 wherein the passing the introduced sample into the first overlapping portion and into the second overlapping portion include the second, third and seventh valves being open and the first, fourth, fifth and sixth valves being closed.

6. The method of claim 2 wherein the releasing the first analyte of interest includes opening the first and fourth valves.

7. The method of claim 2 wherein:

the releasing the first analyte of interest includes the second and third valves being closed and the first and fourth valves being open; and

the releasing the second analyte of interest includes the third and seventh valves being closed and the fifth and sixth valves being open.

8. The method of claim 2 wherein:

the first analyte concentrator includes one or more first immobilized affinity ligands to capture the first analyte of interest; and

the second analyte concentrator includes one or more second immobilized affinity ligands to capture the second analyte of interest.

9. The method of claim 8 wherein:

the one or more first affinity ligands are immobilized to the first overlapping portion in an orientation that facilitates the binding of the first analyte of interest thereto; and

/or

the one or more second affinity ligands are immobilized to the second overlapping portion in an orientation that facilitates the binding of the second analyte of interest thereto.

10. The method of claim 8 wherein:

the localizing the first analyte concentrator includes closing the transport passage using the valve system at inlet and outlet ends of the first analyte concentrator and closing the first separation passage to provide the first analyte of interest in the sample a longer time to be exposed to the one or more first immobilized affinity ligands to thereby increase binding of the first analyte of interest; and

/or

the localizing the second analyte concentrator includes closing the transport passage using the valve system at inlet and outlet ends of the second analyte concentrator and closing the second separation passage to provide the second analyte of interest in the sample a longer time to be exposed to the one or more second immobilized affinity ligands to thereby increase binding of the second analyte of interest.

11. The method of claim 8 wherein:

the localizing the first analyte concentrator includes moving interacting reagents, sample constituents, cells, subcellular components, particles and/or globules in the first analyte concentrator to increase reversible binding of the first analyte of interest to the one or more first immobilized affinity ligands; and

/or

the localizing the second analyte concentrator includes moving interacting reagents, sample constituents, cells, subcellular components, particles and/or globules in the second analyte concentrator to increase reversible binding of the second analyte of interest to the one or more second immobilized affinity ligands.

12. The method of claim 8 wherein:

the first analyte concentrator operates as a microreactor, and further comprising using the first analyte concentrator to perform on-line enzymatic cleavage of proteins to generate peptides; and

/or

the second analyte concentrator operates as a microreactor, and further comprising using the second analyte concentrator to perform on-line enzymatic cleavage of proteins to generate peptides.

13. The method of claim 8 wherein:

the multi-dimensional electrophoresis apparatus includes an exit outlet passage into which the first and second separation passages merge and flow;

the analyte detection zone is one or more detector systems positioned at the exit outlet passage;

the delivering the released first and second analytes of interest include controlling separate and sequential fluid flow from the first and second separation passages into the exit outlet passage; and

the controlling sequential flow includes operating at least one exit valve of the multi-dimensional electrophoresis apparatus.

14. The method of claim 8 wherein:

the valves on the transport passage are adapted to be opened and the valves on the first and second separation passage are adapted to be closed to allow the sample followed by a cleaning buffer to pass through the first and second analyte concentrators towards an outlet end of the transport passage in order to make the multi-dimensional electrophoresis apparatus ready for further cycle usage;

the valves on the transport passage are adapted to be closed and the valves on the first and second separation passage are adapted to be opened to allow separation buffer solutions to pass through the first and second analyte concentrators, independently and sequentially, and in the first and second separation passages towards the analyte detection zone; and

the releasing steps include passing a plug of an elution buffer or solution through the first and second analyte concentrators, respectively, independently and sequentially and using the valve system.

15. The method of claim 8 wherein:

the first analyte concentrator comprises a first concentrator-microreactor; and

/or

the second analyte concentrator comprises a second concentrator-microreactor.

16. The method of claim 15 further comprising:

using the first concentrator-microreactor to bind the first analyte of interest from the sample passed through the transport passage directly to the one or more first affinity ligands being immobilized to an inner wall of the first concentrator-microreactor; and

/or

using the second concentrator-microreactor to bind the second analyte of interest from the sample passed through the transport passage directly to the one or more second immobilized affinity ligands being immobilized to an inner wall of the second concentrator-microreactor.

17. The method of claim 15 further comprising:

performing one or more chemical and/or biochemical reactions in the first concentrator-microreactor and/or the second concentrator-microreactor; and

wherein the chemical and/or biochemical reactions are peptide synthesis, nucleic acid synthesis and/or small molecular weight substances synthesis.

18. The method of claim 15 further comprising:

performing within the first analyte concentrator a chemical reaction or multi-component chemical reactions with one or more components in the sample in the first concentrator-microreactor; and

performing within the second analyte concentrator a chemical reaction or multi-component chemical reactions with one or more components in the sample in the second concentrator-microreactor and independently of the reaction or reactions in the first concentrator-microreactor.

19. The method of claim 15 further comprising:

performing within the first analyte concentrator a biochemical reaction or multi-component biochemical reactions with one or more components in the sample in the first concentrator-microreactor; and

performing within the second analyte concentrator a biochemical reaction or multi-component biochemical reactions with one or more components in the sample in the second concentrator-microreactor and independently of the reaction or reactions in the first concentrator-microreactor.

20. The method of claim 2 further comprising:

before the introducing the sample, introducing a conditioning buffer into the transport passage and passing the conditioning buffer through the first and second analyte concentrators.

21. The method of claim 20 wherein the conditioning buffer improves the binding of the first analyte of interest to one or more affinity ligands immobilized to a matrix in the first analyte concentrator and the binding of the second analyte of interest to one or more affinity ligands immobilized to a matrix in the second analyte concentrator.

22. The method of claim 1 wherein the localizing the first analyte concentrator is after the passing into the first overlapping portion and the localizing the second analyte concentrator is after the passing into the second overlapping portion.

23. The method of claim 1 wherein:

the releasing the first analyte of interest includes introducing a plug of an elution buffer or solution from a first supply of an elution buffer or solution into the first separation passage upstream of the first analyte concentrator;

the releasing the second analyte of interest includes introducing a plug of an elution buffer or solution from a second supply of an elution buffer or solution into the second separation passage upstream of the second analyte concentrator; and

the introducing of the plug of an elution buffer or solution from the first supply of an elution buffer or solution into the first separation passage and the introducing of the plug of an elution buffer or solution from the second supply of an elution buffer or solution into the second separation passage are conducted separated and independently.

24. The method of claim 1 wherein:

introducing a sample into the transport passage;

passing the introduced sample by electro-osmotic flow, pressure or vacuum past a first portion of the transport passage that intersects with the overlapping first separation passage so that the first analyte of interest in the sample is captured by the first analyte concentrator in the first portion;

passing the introduced sample by electro-osmotic flow, pressure or vacuum past a second portion of the transport passage that intersects with the overlapping second separation passage so that the second analyte of interest in the sample is captured by the second analyte concentrator in the second portion;

controlling temperature of the first analyte concentrator to a first temperature associated with the first analyte of interest to thereby increase capturing of the first analyte of interest;

controlling temperature of the second analyte concentrator to a different second temperature associated with the second analyte of interest to thereby increase capturing of the second analyte of interest;

the releasing the first analyte of interest from the first analyte concentrator to the first separation passage is performed by introducing a plug of an elution buffer or solution from a first supply of an elution buffer or solution into the first separation passage upstream of the first analyte concentrator;

separating the released first analyte of interest within the first separation passage by electrophoresis migration, electro-osmotic flow, pressure, or a combination of electro-osmotic flow and pressure to an analyte detection zone which identifies and characterizes the first analyte of interest;

the releasing the second analyte of interest from the second analyte concentrator to the second separation passage is performed by introducing a plug of an elution buffer or solution from a second supply of an elution buffer or solution into the second separation passage upstream of the second analyte concentrator;

separating the released second analyte of interest within the second separation passage by electrophoresis migration, electro-osmotic flow, pressure, or a combination of electro-osmotic flow and pressure to the analyte detection zone which identifies and characterizes the second analyte of interest; and

wherein the controlling temperature of the first analyte concentrator includes closing valves surrounding the first analyte concentrator to thereby isolate the first analyte concentrator; and

/or

the controlling temperature of the second analyte concentrator includes closing valves surrounding the second analyte concentrator to thereby isolate the second analyte concentrator.

25. The method of claim 24 further comprising:

before the releasing the first analyte of interest, subjecting the first analyte concentrator to acoustic mixing; and

/or

before the releasing the second analyte of interest, subjecting the second analyte concentrator to acoustic mixing.

26. The method of claim 1 wherein:

the multi-dimensional electrophoresis apparatus includes preferentially motor-controlled valves; and

the releasing and the delivering include operating at least some of the motor-controlled valves remotely via a processor of the multi-dimensional electrophoresis apparatus.

27. The method of claim 1 wherein:

the multi-dimensional electrophoresis apparatus includes an exit outlet passage into which the first and second separation passages merge and flow;

the exit outlet passage is positioned generally at the analyte detection zone;

the delivering steps are in part via the exit outlet passage; and

the delivering steps include delivering the released first and second analytes of interest separately and sequentially from the first and second separation passages, respectively, and into the exit outlet passage.

28. The method of claim 27 wherein the multi-dimensional electrophoresis apparatus includes a first exit valve on the first separation passage and proximate to the exit outlet passage and a second exit valve on the second separation passage and proximate to the exit outlet passage;

and

further comprising controlling the operation of the first and second exit valves to thereby control the timing of a cyclical release of the first analyte of interest from the first separation passage into the exit outlet passage and a release of the second analyte of interest from the second separation passage into the exit outlet passage.

29. The method of claim 1 wherein one of the anode or cathode sides of the multi-dimensional electrophoresis apparatus is positioned generally at a buffer supply of inlet ends of at least one of the first and second separation passages and the other side is positioned generally downstream of the analyte detection zone.

30. The method of claim 1 further comprising:

introducing a buffer solution via a first auxiliary buffer passage into the first separation passage at a location that is spaced a distance downstream of the first analyte concentrator and a distance upstream of the analyte detection zone, wherein the introducing the buffer solution into the first separation passage includes operating one or more auxiliary valves of the multi-dimensional electrophoresis apparatus so that the buffer solution from the first auxiliary buffer passage does not interact with the first analyte concentrator; and

/or

introducing a buffer solution via a second auxiliary buffer passage into the second separation passage at a location that is spaced a distance downstream of the second analyte concentrator and a distance upstream of the analyte detection zone, wherein the introducing the buffer solution into the second separation passage includes operating one or more auxiliary valves of the multi-dimensional electrophoresis apparatus so that the buffer solution from the second auxiliary buffer passage does not interact with the second analyte concentrator.

31. The method of claim 1 wherein:

the transport passage is a transport capillary, the first separation passage is a first separation capillary, the second separation passage is a second separation capillary, the first analyte concentrator is a first analyte concentrator-microreactor, and the second analyte concentrator is a second analyte concentrator-microreactor; and

the multi-dimensional electrophoresis apparatus includes a first auxiliary capillary through which a separation buffer is provided to the first separation capillary downstream of the first analyte concentrator-microreactor and upstream of the analyte detection zone, a second auxiliary capillary through which a separation buffer is provided to the second separation capillary downstream of the second analyte concentrator-microreactor and upstream of the analyte detection zone, an exit outlet capillary into which the first and second separation capillaries flow and at which the analyte detection zone is generally positioned, and a valve system controls fluid flow in the capillaries.

32. The method of claim 31 wherein one of the anode or cathode sides of the multi-dimensional electrophoresis apparatus is generally at a buffer supply of at least one of the inlet ends of the first and second separation capillaries and the other side is downstream of the exit outlet capillary.

33. The method of claim 31 further comprising an exit valve on the first separation capillary between the first valve and the exit outlet capillary and an exit valve on the second separation capillary between the sixth valve and the exit outlet capillary.

34. The method of claim 31 further comprising:

before the releasing the first analyte of interest and with the first analyte concentrator-microreactor in a localized state, subjecting the first analyte concentrator-microreactor and/or interacting reagents, sample constituents, cells, subcellular components, particles and/or globules in the first analyte concentrator-microreactor to gentle shaking, microwave pulsing and/or acoustic mixing; and

/or

before the releasing the second analyte of interest and with the second analyte concentrator-microreactor in a localized state, subjecting the second analyte concentrator-microreactor and/or interacting reagents, sample constituents, cells, subcellular components, particles and/or globules in the second analyte concentrator-microreactor to gentle shaking, microwave pulsing and/or acoustic mixing.

35. The method of claim 31 further comprising after the passing steps and before the releasing steps, passing a washing buffer or solution through the transport passage and the first and second analyte concentrator-microreactors to wash and remove unwanted material from the transport passage and from the first analyte concentrator-microreactor with the first antibody immobilized to the first analyte microreactor and from the second analyte concentrator-microreactor with the second antibody immobilized to the second microreactor.

36. The method of claim 1 wherein:

the delivering the released first analyte of interest includes applying electro-osmotic flow and/or mechanical pressure/vacuum; and

/or

the delivering the released second analyte of interest includes applying electro-osmotic flow and/or mechanical pressure/vacuum.

37. The method of claim 1 wherein:

the multi-dimensional electrophoresis apparatus includes a microextraction device first base in which the first analyte concentrator-microreactor is enclosed, inlet and outlet transport capillary connectors for the first base, and inlet and outlet first separation capillary connectors for the first base; and

/or

the multi-dimensional electrophoresis apparatus includes a microextraction device second base in which the second analyte concentrator-microreactor is enclosed, inlet and outlet transport capillary connectors for the second base, and inlet and outlet second separation capillary connectors for the second base.

38. The method of claim 1 further comprising after passing the introduced sample through the transport passage into the first overlapping portion and before releasing the first analyte of interest from the first concentrator to the first separation passage, passing washing solution or buffer through the first overlapping portion with a first antibody immobilized to the first analyte concentrator and thereby washing away at least some of salts and other unwanted materials in the first overlapping portion with the first analyte of interest concentrated by the first analyte concentrator.

39. The method of claim 1 further comprising after passing the introduced sample through the transport passage into the second overlapping portion and before releasing the second analyte of interest from the second concentrator to the second separation passage, passing washing solution or buffer through the second overlapping portion with a second antibody immobilized to the first analyte concentrator and thereby washing away at least some of salts and other unwanted materials in the second overlapping portion with the second analyte of interest concentrated by the second analyte concentrator.

40. The method of claim 1 further comprising after the passing steps of fluid-containing sample, passing a washing buffer through the transport passage and the first and second analyte concentrators to wash out constituents of the sample retained at the transport passage, the first analyte concentrator and/or the second analyte concentrator;

and

wherein the passing a fluid-containing sample and a washing buffer includes introducing the fluid-containing sample and the washing buffer using controlled electro-osmotic flow and/or pressure or vacuum.

41. The method of claim 40 further comprising after the passing a cleaning buffer, passing a conditioning buffer followed by a fluid-containing sample through the transport passage and the first and second analyte concentrators to increase binding properties of the one or more first and second affinity ligands immobilized to the first and second analyte concentrators with the analytes of interest present in the fluid-containing sample;

and

wherein the passing a cleaning buffer, a conditioning buffer and a fluid-containing sample uses controlled electro-osmotic flow and/or pressure or vacuum.

42. The method of claim 1 further comprising:

after the passing the introduced sample through the transport passage into the first overlapping portion and before the releasing the first analyte of interest, passing washing buffer through the first analyte concentrator and via the first separation passage to wash out constituents of the sample retained at the first analyte concentrator; and

after the passing the introduced sample through the transport passage into the second overlapping portion and before the releasing the second analyte of interest, passing washing buffer through the second analyte concentrator and via the second separation passage to wash out constituents of the sample retained at the second analyte concentrator.

43. The method of claim 42 wherein:

the releasing the first analyte of interest includes applying a plug of elution buffer or solution to an inlet end of the first separation passage using (a) electrical migration, pressure, vacuum or electro-osmotic flow or (b) a combination of pressure and electro-osmotic flow; and

/or

the releasing the second analyte of interest includes applying a plug of elution buffer or solution to an inlet end of the second separation passage using (c) electrical migration, pressure, vacuum or electro-osmotic flow or (d) a combination of pressure and electro-osmotic flow.

44. The method of claim 42 wherein:

the delivering the released first analyte of interest includes separating the released first analyte of interest in the first separation passage by one or more modes of capillary electrophoresis; and

/or

the delivering the released second analyte of interest includes separating the released second analyte of interest in the second separation passage by one or more modes of capillary electrophoresis.

45. The method of claim 42 further comprising:

before the releasing the first analyte of interest, introducing an auxiliary buffer that includes preferentially an organic solvent, or a mixture of organic solvents and/or other chemical additions into the first separation passage downstream of the first analyte concentrator and upstream of the analyte detection zone; and

/or

before the releasing the second analyte of interest, introducing an auxiliary buffer that includes preferentially an organic solvent, or a mixture of organic solvents and/or other chemical additions into the second separation passage downstream of the second analyte concentrator and upstream of the analyte detection zone.

46. The method of claim 1 wherein:

the first analyte concentrator includes a first affinity ligand component and a different second affinity ligand component, and wherein the first and second affinity ligand components are attracted to different portions of the first analyte of interest which thereby increases the capturing of the first analyte of interest by the first analyte concentrator; and

/or

the second analyte concentrator includes a third affinity ligand component and a different fourth affinity ligand component, and wherein the third and fourth affinity ligand components are attracted to different portions of the second analyte of interest which thereby increases the capturing of the second analyte of interest by the second analyte concentrator.

47. The method of claim 1 wherein:

the delivering the released first analyte of interest into the first separation passage includes separating out the analytes at least by one mode of capillary electrophoresis or other technology using electrical migration, electro-osmotic flow, pressure or a combination of pressure and electro-osmotic flow; and

the delivering the released second analyte of interest into the second separation passage includes separating out the analytes by at least one mode of capillary electrophoresis or other technology using electrical migration, electro-osmotic flow, pressure or a combination of pressure and electro-osmotic flow.

48. The method of claim 1 further comprising:

before the introducing the sample, cleaning the transport passage and the first and second separation passages;

after the cleaning and before the introducing the sample, conditioning the transport passage and the first and second separation passages;

the passing steps being after the cleaning and the conditioning;

after the passing steps, washing the transport passage and the first and second analyte concentrators;

before the releasing the first analyte of interest, introducing a separation buffer in the first separation passage;

before the releasing the second analyte of interest, introducing a separation buffer in the second separation passage;

the releasing the first analyte of interest including applying a first plug of the elution buffer or solution from a first supply of elution buffer or solution in the first separation passage;

the releasing the second analyte of interest including applying a second plug of the elution buffer or solution from a first supply of elution buffer or solution in the second separation passage;

the delivering the released first analyte of interest into the first separation passage includes separation by at least one mode of capillary electrophoresis or another procedure that requires electrical migration, electro-osmotic flow, pressure or vacuum, or a combination of electro-osmotic flow and pressure;

the delivering the released second analyte of interest into the second separation passage includes separation by at least one mode of capillary electrophoresis or another procedure that requires electrical migration, electro-osmotic flow, pressure or vacuum, or a combination of electro-osmotic flow and pressure;

acquiring analyte identification-and-characterization data via the analyte detection zone;

the sample defining a first sample; and

after the delivering steps, conditioning the transport passage and the first and second separation passages and then introducing a second sample into the transport passage.

49. The method of claim 48 wherein the applying the first plug of the elution buffer or solution from a supply of an elution buffer or solution and the applying the second plug of the elution buffer or solution from a supply of an elution buffer or solution are conducted separately and sequentially.

50. The method of claim 1 wherein:

the releasing the first analyte of interest includes introducing a plug of the elution buffer or solution from the first supply of elution buffer or solution into the first separation passage upstream of the first analyte concentrator;

or

the releasing the second analyte of interest includes introducing a plug of the elution buffer or solution from the second supply of elution buffer or solution into the second separation passage upstream of the second analyte concentrator.

51. The method of claim 1 wherein the multi-dimensional electrophoresis apparatus includes a single power supply for the first and second separation passages that can be used in alternative or sequential operations.

52. The method of claim 1 wherein the multi-dimensional electrophoresis apparatus includes a single power supply having voltage relays for the first and second separation passages.

53. The method of claim 1 wherein the delivering the released first analyte of interest and the delivering the released second analyte of interest includes the transfer of the one or more released first analyte of interest bound to the first analyte concentrator to the first separation passage, the transfer of the one or more released second analyte of interest bound to the second analyte concentrator to the second separation passage and the separation of the one or more released first analyte of interest and one or more released second analyte of interest respectively within the first and second separation passages independently, separate and sequentially, by any of the modes of capillary electrophoresis to the analyte detection zone which detect, quantify, identify and characterizes the first or second analytes of interest by one or more detectors, independent, separate and in a sequential protocol.

54. The method of claim 1 wherein the multi-dimensional electrophoresis apparatus 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.

55. A controlled electrophoresis method, comprising:

introducing a sample into a transport passage of a multi-dimensional electrophoresis apparatus;

the multi-dimensional electrophoresis apparatus including a first separation passage intersecting the transport passage at a first overlapping portion, a first analyte concentrator at the first overlapping portion to concentrate a first analyte of interest, a second separation passage intersecting the transport passage at a second overlapping portion, a second analyte concentrator at the second overlapping portion to concentrate a second analyte of interest, the first separation passage is a first separation capillary, the second separation passage is a second separation capillary, the first analyte concentrator is a first analyte concentrator-microreactor, and the second analyte concentrator is a second analyte concentrator-microreactor; and

the multi-dimensional electrophoresis apparatus includes a microextraction device first base in which the first analyte concentrator-microreactor is enclosed, inlet and outlet transport capillary connectors for the first base, and inlet and outlet first separation capillary connectors for the first base; and

/or

the multi-dimensional electrophoresis apparatus includes a microextraction device second base in which the second analyte concentrator-microreactor is enclosed, inlet and outlet transport capillary connectors for the second base, and inlet and outlet second separation capillary connectors for the second base;

passing the introduced sample in the transport passage into the first overlapping portion so that the first analyte of interest in the sample is concentrated in the first analyte concentrator;

passing the introduced sample in the transport passage into the second overlapping portion so that the second analyte of interest in the sample is concentrated in the second analyte concentrator;

localizing the first analyte concentrator and thereby increasing the concentration of the first analyte of interest by the first analyte concentrator;

localizing the second analyte concentrator and thereby increasing the concentration of the second analyte of interest by the second analyte concentrator;

releasing the first analyte of interest concentrated in the first analyte concentrator into the first separation passage;

releasing the second analyte of interest concentrated in the second analyte concentrator into the second separation passage;

delivering the released first analyte of interest in the first separation passage to an analyte detection zone which identifies and characterizes the first analyte of interest; and

delivering the released second analyte of interest in the second separation passage to the analyte detection zone which also identifies and characterizes the second analyte of interest the transport passage is a transport capillary wherein;

the first overlapping portion is an elongated staggered portion of the first separation capillary in the first base; and

the second overlapping portion is an elongated staggered portion of the second separation capillary in the second base.

56. The method of claim 55 wherein the delivering the released first analyte of interest and the delivering the released second analyte of interest includes the transfer of the one or more released first analyte of interest bound to the first analyte concentrator to the first separation passage, the transfer of the one or more released second analyte of interest bound to the second analyte concentrator to the second separation passage and the separation of the one or more released first analyte of interest and one or more released second analyte of interest respectively within the first and second separation passages independently, separate and sequentially, by any of the modes of capillary electrophoresis to the analyte detection zone which detect, quantify, identify and characterizes the first or second analytes of interest by one or more detectors, independent, separate and in a sequential protocol.