Multi-dimensional electrophoresis method

• US 7,736,480 B2
• Filed: 01/24/2006
• Issued: 06/15/2010
• Est. Priority Date: 11/08/1999
• Status: Expired due to Term

• Current Assignee: Princeton Biochemicals, Inc.
• Original Assignee: Princeton Biochemicals, Inc.
• Inventors: Guzman, Norberto A.
• Primary Examiner(s): Noguerola; Alex
• Application Number:
  US11/339,245
• Publication Number:
  US 20060124460A1
• Time in Patent Office: 1,603 Days
• Field of Search: 204600-605, 204451-455
• US Class Current: 204/450
• CPC Class Codes: G01N 27/44704 Details; Accessories
• CPC Class Codes: G01N 27/44726 using specific dyes, markers or binding molecules
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 27/44773 Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

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. 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.

Claims

1. An electrophoresis method, comprising:

passing a sample introduced into a transport passage through a first analyte concentrator in the transport passage which captures a first analyte of interest and through a second analyte concentrator in the transport passage which captures a second analyte of interest;

the first analyte concentrator being in an intersection of the transport passage with a first separation passage;

the second analyte concentrator being in an intersection of the transport passage with a second separation passage;

eluting the captured first analyte of interest from the first analyte concentrator;

eluting the captured second analyte of interest from the second analyte concentrator;

passing the eluted first analyte of interest by at least electrophoresis migration via the first separation passage to a detector system which identifies and characterizes the first analyte of interest; and

passing the eluted second analyte of interest by at least electrophoresis migration via the second separation passage to the detector system which identifies and characterizes the second analyte of interest.

2. The method of claim 1 wherein the passing the eluted first analyte step includes passing the eluted first analyte of interest through an additional analyte concentrator containing a chromophoric reagent and positioned in the first separation passage downstream of the first analyte concentrator.

3. The method of claim 1 wherein the eluting the captured first analyte step uses an eluting solution which contains a chromophoric reagent allowing decoupling and derivatization to occur simultaneously.

4. The method of claim 1 wherein the passing the eluted second analyte step is after the passing the eluted first analyte step.

5. The method of claim 1 wherein the passing the eluted first analyte step includes passing the eluted first analyte through an extra analyte concentrator in the first passage which causes a chemical or biochemical reaction, the extra analyte concentrator being positioned downstream of the first analyte concentrator and upstream of the detector system.

6. The method of claim 1 further comprising before the passing step, preparing the sample by passing a quantity of sample solution through an analyte concentrator containing non-specific affinity ligands and thereby trapping different analytes including the first and second analytes of interest, and after the trapping step, injecting an elution-containing buffer solution through the analyte concentrator and into the transport passage and thereby releasing the trapped analytes into the buffer solution to form a sample.

7. The method of claim 6 wherein the analyte concentrator containing non-specific affinity ligands includes a matrix including free-floating beads retained in the transport passage by end fits.

8. The method of claim 1 further comprising an exit output passage into which the first and second analytes separated independently in respective ones of the first and second separation passages flow, and the detector system identifying and characterizing the analytes in the exit output passage.

9. The method of claim 8 wherein the detector system senses ultraviolet or fluorescent energy to identify and characterize the analytes.

10. The method of claim 9 further comprising the detector system defining a first detector system, and passing the eluted first and second analytes of interest past a second detector system operatively positioned with respect to the exit output passage for a detection process.

11. The method of claim 10 wherein the second detector system comprises an electrochemical, mass spectrometry, circular dichroism or nuclear magnetic resonance detector system.

12. The method of claim 1 wherein the sample defines a first sample, and further comprising sequentially delivering the first sample and a second sample to the separation passages via the transport passage.

13. The method of claim 1 further comprising a sample introduction passage having an analyte concentrator containing non-specific affinity ligands therein, and an outlet end of the introduction passage is in fluid communication with an inlet end of the transport passage.

14. The method of claim 13 further comprising operating a valve between the introduction passage and the transport passage to control flow of the sample into the transport passage.

15. The method of claim 1 wherein the first analyte concentrator includes a plurality of microstructures carrying at least one affinity element adapted to attract the first analyte of interest.

16. The method of claim 1 wherein the passing the eluted first analyte step includes the eluted first analyte passing operatively past the detector system in the first passage and directly to a waste and grounding container, and the passing the eluted second analyte step includes the eluted second analyte passing operatively past the detector system in the second passage and directly to a waste and grounding container.

17. The method of claim 1 wherein the eluting the captured first analyte step includes hydrodynamically introducing a plug of eluting buffer into the first separation passage upstream of the first analyte concentrator.

18. The method of claim 1 wherein the passing the eluted first analyte of interest is by electrophoresis migration or a combination of electrophoresis migration and pressure;

and wherein the passing the eluted second analyte of interest is by electrophoresis migration or a combination of electrophoresis migration and pressure.

19. An electrophoresis method, comprising:

introducing a sample into a transport passage;

passing the introduced sample past a first portion of the transport passage which overlaps with an intersecting first separation passage so that a first analyte of interest in the sample is captured by a first analyte concentrator in the first portion;

passing the introduced sample past a second portion of the transport passage which overlaps with an intersecting second separation passage so that a second analyte of interest in the sample is captured by a second analyte concentrator in the second portion;

releasing the captured first analyte from the first analyte concentrator into the first separation passage;

causing the released first analyte to pass at least by electrophoresis migration to a detector system which identifies and characterizes the first analyte;

releasing the captured second analyte from the second analyte concentrator into the second separation passage; and

causing the released second analyte to pass at least by electrophoresis migration to the detector system which identifies and characterizes the second analyte of interest.

20. The method of claim 19 wherein the releasing the captured first analyte step includes introducing a plug of eluting buffer from a buffer supply into the first separation passage upstream of the first analyte concentrator.

21. The method of claim 20 wherein the introducing the plug step is by hydrodynamic means.

22. The method of claim 20 wherein the causing the released first analyte to pass step includes activating a power supply connected to the buffer supply.

23. The method of claim 19 wherein the introducing a sample step includes the sample having been passed through an analyte concentrator containing non-specific affinity ligands at an inlet end of the transport passage.

24. The method of claim 19 wherein the sample includes a buffer solution passed through an analyte concentrator containing non-specific affinity ligands and eluting concentrated analytes therefrom.

25. The method of claim 19 wherein the causing steps include the released first analyte passing from the first separation passage to an exit output passage and then to the detector system and the released second analyte passing from the second separation passage to the exit output passage and then to the detector system.

26. The method of claim 25 wherein the causing steps include the released first analyte passing to the detector system before the released second analyte passing to the detector system.

27. The method of claim 19 further comprising before the introducing the sample into the transport passage step, preparing the sample.

28. The method of claim 27 wherein an introduction passage communicates with an inlet end of the transport passage and an analyte concentrator containing non-specific affinity ligands is in the introduction passage;

and the preparing the sample step includes communicating an inlet end of the introduction passage with a buffer supply and then communicating the inlet end with a cleaning supply, and the introducing the sample step includes communicating the inlet end with a buffer supply.

29. The method of claim 28 wherein a valve is positioned between the introduction passage and the transport passage, the valve is in a closed position during the communicating with a buffer supply step and during the communicating with a cleaning supply step, and then with a sample supply, and the valve is in an open position during the communicating with a buffer supply step of the introducing the sample step.

30. The method of claim 19 wherein the causing the released first analyte to pass is by electrophoresis migration or a combination of electrophoresis migration and pressure;

and wherein the causing the released second analyte to pass is by electrophoresis migration or a combination of electrophoresis migration and pressure.

31. An electrophoresis method, comprising:

introducing a sample into a transport passage of an electrophoresis apparatus;

the 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 through the first overlapping portion whereby the first analyte of interest in the sample is concentrated in the first analyte concentrator;

passing the introduced sample in the transport passage through the second overlapping portion whereby the second analyte of interest in the sample is concentrated in the second analyte concentrator;

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

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

passing the eluted first analyte of interest in the first separation passage to a detector system which identifies and characterizes the first analyte of interest; and

passing the eluted second analyte of interest in the second separation passage to the detector system which identifies and characterizes the second analyte of interest.

32. The method of claim 31 wherein the passing the eluted first and second analyte steps are both by high-resolution electrophoresis migration or a combination of electrophoresis migration and pressure.

33. The method of claim 31 wherein the eluting the first analyte step includes hydrodynamically introducing a first plug of eluting buffer into the first separation passage upstream of the first analyte concentrator, and the eluting the second analyte step includes hydrodynamically introducing a second plug of eluting buffer into the second separation passage upstream of the second analyte concentrator.

34. The method of claim 31 wherein the elating the second analyte step is after the eluting the first analyte step.

35. The method of claim 31 wherein all of the passages are capillaries.

36. The method of claim 31 wherein the detector system includes mass spectrometry.

37. The method of claim 31 wherein the introducing the sample step includes passing a sample solution through an analyte concentrator containing non-specific affinity ligands and positioned in an introduction passage and thereby capturing analytes in the sample solution, and after the passing the sample solution step, passing a buffer through the analyte concentrator containing non-specific affinity ligands to release the captured analytes into the transport passage.

38. The method of claim 37 wherein the introducing the sample step includes after the passing the sample solution step and before the passing the buffer step, opening a valve which is positioned between the analyte concentrator containing non-specific affinity ligands and the first analyte concentrator.

39. The method of claim 38 wherein the introducing the sample step includes the valve being in a closed position such that the sample solution after passing through the analyte concentrator containing non-specific affinity ligands is directed away from the transport passage.

40. An electrophoresis method, comprising:

providing an electrophoresis apparatus which includes a transport passage, a separation passage, an analyte concentrator at an intersection of the transport and separation passages and adapted to attract an analyte of interest from a sample, and a detector system downstream of the analyte concentrator, the anode side of the electrophoresis apparatus being upstream of the analyte concentrator and the cathode side being downstream of the detector system;

passing a sample in the transport passage through the analyte concentrator which concentrates the analyte of interest from the passing sample;

after the passing the sample step, introducing eluting solution into an upstream end of the separation passage and thereby removing the concentrated analyte from the analyte concentrator;

delivering the eluted analyte via the separation passage to the detector system; and

identifying and characterizing the analyte using the detector system.

41. The method of claim 40 wherein the delivering is by at least electrophoresis migration.

42. The method of claim 40 wherein the electrophoresis apparatus further includes an analyte concentrator containing non-specific affinity ligands at an inlet portion of the transport passage, and the passing the sample step includes injecting an eluting solution through the analyte concentrator containing non-specific affinity ligands and thereby eluting captured sample analytes from the analyte concentrator containing non-specific affinity ligands into the transport passage.

43. The method of claim 40 further comprising:

the separation passage defining a first separation passage, the analyte of interest defining a first analyte of interest, and the analyte concentrator defining a first analyte concentrator;

the electrophoresis apparatus further including a second separation passage and a second analyte concentrator at an intersection of the transport passage and the second separation passage and adapted to attract a second analyte of interest from the sample;

the passing the sample step including passing the sample through the second analyte concentrator which concentrates the second analyte of interest from the passing sample;

after the passing the sample step, introducing eluting solution into an upstream end of the second separation passage to thereby remove the concentrated second analyte from the second analyte concentrator;

delivering the eluted second analyte via the second separation passage to the detector system; and

identifying and characterizing the second analyte using the detector system.

44. The method of claim 43 further comprising the electrophoresis apparatus including an exit output passage into which the first and second separation passages feed;

and the identifying and characterizing steps include identifying and characterizing the first and second analytes of interest when in the exit output passage using the detector system.

45. The method of claim 44 wherein the identifying and characterizing steps include sensing ultraviolet or fluorescent energy.

46. The method of claim 45 wherein the electrophoresis apparatus includes an off-column detector on the exit output passage and for the detection and characterization of the first and second analytes of interest, and the off-column detector comprises an electrochemical, mass spectrometry, circular dichroism or nuclear magnetic resonance detector.

47. The method of claim 43 further comprising:

the electrophoresis apparatus further including a third separation passage and a third analyte concentrator at an intersection of the transport passage and the third separation passage and adapted to attract a third analyte of interest from the sample;

the passing the sample step including passing the sample through the third analyte concentrator which concentrates the third analyte of interest;

after the passing the sample step, introducing eluting solution into an upstream end of the third separation passage to thereby remove the concentrated third analyte from the third analyte concentrator;

delivering the eluted third analyte via the third separation passage to the detector system; and

identifying and characterizing the third analyte of interest using the detection system.

48. The method of claim 47 further comprising the electrophoresis apparatus including an exit output passage into which the first, second and third separation passages feed;

and the identifying and characterizing steps include identifying and characterizing the first, second and third eluted analytes of interest when in the exit output passage with the detector system.

49. The method of claim 47 wherein all of the passages are capillaries.

50. The method of claim 49 wherein all of the capillaries are formed in a microchip.

51. The method of claim 40 wherein the analyte concentrator includes a plurality of microstructures carrying at least one affinity element adapted to attract the analyte of interest from the sample.

52. The method of claim 40 wherein the delivering the eluted analyte step includes passing the eluted analyte through an additional analyte concentrator which contains a chromophoric reagent and is positioned in the separation passage.

53. The method of claim 40 wherein the introducing eluting solution step uses an eluting solution which contains a chromophoric reagent allowing decoupling and derivatization to occur simultaneously.

54. The method of claim 40 wherein the electrophoresis apparatus includes an introduction passage at an inlet end of the transport passage and a valve therebetween, and before the passing the sample step, communicating an inlet end of the introduction passage with a sample supply with the valve in a closed position, and the passing the sample step is with the valve in an open position.

55. The method of claim 54 further comprising after the communicating with a sample supply step, communicating the inlet end with a cleaning supply with the valve in the closed position and then communicating the inlet end with a buffer supply also with the valve in the closed position.

56. The method of claim 40 wherein the delivering is by electrophoresis migration or a combination of electrophoresis migration and pressure.