Electrophoresis extraction device

• US 8,268,247 B2
• Filed: 05/05/2009
• Issued: 09/18/2012
• 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): Yu, Melanie J
• Assistant Examiner(s): Crawford, Erik B
• Application Number:
  US12/436,034
• Publication Number:
  US 20090301885A1
• Time in Patent Office: 1,232 Days
• Field of Search: 422/50, 422/68.1, 422/81, 422/82.01, 422/82.05, 422/82.07, 422/82.08, 435/5, 435/7.1
• US Class Current: 422/82.01
• CPC Class Codes: G01N 27/44743 Introducing samples
• CPC Class Codes: G01N 33/543 with an insoluble carrier for immobilising immunochemicals

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. An electrophoresis extraction device, comprising:

a base having a transport passage, a transport passage inlet port, a transport passage outlet port, the transport passage communicating with the transport passage inlet port and the transport passage outlet port,

the base having a first extraction device comprising a first separation passage first port, a first separation passage second port, and a first separation passage communicating with the first separation passage first port and the first separation passage second port,

the base having a second extraction device comprising a second separation passage first port, a second separation passage second port, a second separation passage communicating with the second separation passage first port and the second separation passage second port and an exit passage into which the second port of the first and second separation passages merge to create a single outlet passage;

a first reservoir for a separation buffer supply, eluting buffer supply or cleaning solution supply communicating with the first separation passage first port, a second reservoir for a separation buffer supply, eluting buffer supply or cleaning solution supply communicating with the second separation passage first port,

the first and second separation passages within the corresponding extraction devices each including an elongated passage portion;

between the transport passage inlet port and the transport passage outlet port, the transport passage intersecting as a staggered configuration intersection with the elongated passage portion of each of the first and second separation passages within each corresponding extraction device;

the elongated passage portion of the first separation passage within the first extraction device defining an area for holding a first means for capturing and concentrating a first analyte of interest in a sample,

the elongated passage portion of the second separation passage within the second extraction device defining an area for holding a second means for capturing and concentrating a second analyte of interest in the sample,

the sample passing from the transport passage inlet port through the staggered configuration intersection with the elongated passage portion of each of the first and second separation passages within each corresponding extraction device to the transport passage outlet port and the concentrated first and second analyte of interest to be released from the elongated passage portion of each of the first and second separation passages by alternating fluid communication of the separation buffer supply and then the eluting buffer supply from the respective first reservoir and second reservoir and the first and second analyte of interest being delivered out the respective second port of the first and second separation passages to the exit passage separately, independently and sequentially by pressure, electrophoresis migration or a combination of electro-osmotic flow and pressure and to an analyte detection system associated with the exit passage;

a first auxiliary buffer passage communicating with the first separation passage and a second auxiliary buffer passage communicating with the second separation passage, the first and second auxiliary buffer passages positioned downstream of the respective means for capturing and concentrating a first analyte of interest and a second analyte of interest and before the second ports of the first and second separation passages merge into the single outlet passage; and

a valve system operatively associated with the transport passage and the first and second elongated passage portions of the corresponding extraction devices for independently controlling the flow of the sample in the transport passage and separately and independently forming a microenvironment in the first and second separation passages to mix reagents to optimize the binding and capture of the first and second analytes of interest by the respective means for capturing and concentrating the first and second analytes of interest,

wherein the elongated passage portion of the first and second separation passages of the base of each of the corresponding extraction devices defining an extraction device or analyte concentrator-microreactor device.

2. The electrophoresis extraction device of claim 1 wherein the first and second analytes of interest are simple or complex analytes,

the first means for capturing and concentrating includes a first affinity ligand component and a different second affinity ligand component, and the first and second affinity ligand components are attracted and bind to different portions of the first analyte of interest which thereby increases the capturing of the first analyte of interest by the first means for capturing and concentrating,

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

3. The electrophoresis extraction device of claim 1 wherein each of the means for capturing and concentrating includes a combination of an antibody, a lectin and/or another affinity ligand bound to a matrix within the first and second extraction devices to capture the respective first and second analyte of interest by a binding process selected from the group consisting of (a) through a selective peptide sequence and/or epitope or through a selective sugar present on the respective analyte of interest or (b) through both a selective peptide sequence and/or epitope and a selective sugar present on the respective analyte of interest and (c) through any other selective chemical group and/or site, region, motif-specific present on the respective analyte of interest.

4. The electrophoresis extraction device of claim 1 wherein the first and second means for capturing and concentrating permits capture of the respective first and second analyte of interest present in simple or complex mixtures of the sample for purification, desalting and enrichment, wherein the respective as analyte of interest is a simple or complex small molecule or macromolecule, a cell structure, a subcellular structure, a phage, a virus, a prion, or a globular structure;

retained within the respective first and second extraction device.

5. The electrophoresis extraction device of claim 4 wherein the means for capturing and concentrating isolates, purify and concentrates the respective analyte of interest from the simple or complex mixture followed by release of the retained analytes of interest from the first and second extraction devices and separation of the released analytes of interest in the separation passages.

6. The electrophoresis extraction device of claim 1 wherein the sample is introduced by pressure from a sample reservoir into and through the transport passage inlet and outlet ports and to a waste reservoir.

7. The electrophoresis extraction device of claim 1 wherein each of the means for capturing and concentrating includes beads interconnected to each other by at least one chemical bond within a matrix and to inner walls of the elongated passage portions and one or more affinity ligands attached to the beads.

8. The electrophoresis extraction device of claim 1 wherein each of the means for capturing and concentrating includes a matrix formed by beads having antibody fragments covalently or non-covalently attached to surfaces of the beads or intact antibodies covalently or non-covalently attached to surfaces of the beads.

9. The electrophoresis extraction device of claim 8 wherein the means for capturing and concentrating includes an altered condition in the elongated passage portions to improve binding and capture of the analytes of interest to the intact antibodies or the antibody fragments.

10. The electrophoresis extraction device of claim 9 wherein the altered condition is temperature.

11. The electrophoresis extraction device of claim 9 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing.

12. The electrophoresis extraction device of claim 1 wherein the means for capturing and concentrating are adapted to perform microreactions of online enzymatic treatments of proteins to cleave the proteins to generate peptides.

13. The electrophoresis extraction device of claim 9 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing which is adapted to accelerate the work of proteases immobilized to the matrix of each of the means for capturing and concentrating and reduce the time required to cleave a protein into peptide components thereof.

14. The electrophoresis extraction device of claim 1 wherein each of the means for capturing and concentrating includes a matrix formed by microstructures selected from a group consisting of beads, platelets, chips, fibers, polymers, globules and filaments whereby affinity ligands can be immobilized to their surfaces covalently or non-covalently.

15. The electrophoresis extraction device of claim 1 wherein the valve system includes valves which are motor operated and controlled remotely by a processor based on a predetermined set of instructions.

16. The electrophoresis extraction device of claim 1 wherein the staggered configuration intersection includes bulging members configured to retain the means for capturing and concentrating in the elongated passage portion of the first and second separation passages.

17. The electrophoresis extraction device of claim 1 wherein the staggered configuration intersection includes frit structures configured to retain the means for capturing and concentrating in the elongated passage portion of the first and second separation passages.

18. The electrophoresis extraction device of claim 1 wherein the staggered configuration intersection includes means other than bulging members or frit structures for retaining the means for capturing and concentrating in the elongated passage portion of the first and second separation passages.

19. The electrophoresis extraction device of claim 1 wherein the analyte detection system is a component of an electrophoresis apparatus;

and further comprising first coupling means for coupling the transport passage inlet and outlet ports to a transport passage of the electrophoresis apparatus, second coupling means for coupling the first and second ports of the first and second separation passages to a separation passage of the electrophoresis apparatus and the analyte detection system is operatively associated with the separation passage.

20. The electrophoresis extraction device of claim 19 wherein the first coupling means includes threaded connectors and the second coupling means includes threaded connectors.

21. The electrophoresis extraction device of claim 1 wherein the base includes threaded openings, each associated with a respective one of the ports.

22. The electrophoresis extraction device of claim 1 wherein the base includes openings for inserting the means for capturing and concentrating into and replacing the means for capturing and concentrating in the elongated passage portion of each of the first and second passages.

23. The electrophoresis extraction device of claim 1 wherein the base includes four side-edge threaded openings for receiving therein a respective transport and/or separation passage connector.

24. The electrophoresis extraction device of claim 1 wherein the elongated passage portion of each of said first and second separation passages is embedded and enclosed in the base.

60. The electrophoresis extraction device of claim 8 wherein the attachment of the antibody fragments or intact antibodies are carried out having the correct orientation for an optimal binding and capture of the analyte of interest.

61. The electrophoresis extraction device of claim 1 wherein the means for capturing and concentrating allows the capture and concentration of the first and second analytes of interest using affinity ligands and depending on the affinity ligands in use the elution process can be carried out as a single elution or a sequential elution using one or more eluting buffers in a sequential order.

62. The electrophoresis extraction device of claim 1 wherein the detection system comprises one or more detectors functioning independently or simultaneously, selected from a group consisting of an ultraviolet, fluorescence, laser-induced fluorescence, mass spectrometer, conductimeter, electrochemical, nuclear magnetic resonance, and circular dichroism detector.

25. An electrophoresis extraction device, comprising:

a base having a transport passage, a transport passage inlet port, a transport passage outlet port, the transport passage communicating with the transport passage inlet port and the transport passage outlet port,

the base having a first extraction device comprising a first separation passage first port, a first separation passage second port, and a first separation passage communicating with the first separation passage first port and the first separation passage second port,

the base having a second extraction device comprising a second separation passage first port, a second separation passage second port, a second separation passage communicating with the second separation passage first port and the second separation passage second port and an exit passage into which the second port of the first and second separation passages merge to create a single outlet passage;

a first reservoir for a separation buffer supply, eluting buffer supply or cleaning solution supply communicating with the first separation passage first port, a second reservoir for a separation buffer supply, eluting buffer supply or cleaning solution supply communicating with the second separation passage first port,

the first and second separation passages within the corresponding extraction device each including an elongated passage portion;

between the transport passage inlet port and the transport passage outlet port, the transport passage intersecting as a staggered configuration intersection with the elongated passage portion of each of the first and second separation passages within each corresponding extraction device;

the elongated passage portion of the first separation passage within the first extraction device defining an area for holding a means for capturing and concentrating a first analyte of interest in a sample,

the elongated passage portion of the second separation passage within the second extraction device defining an area for holding a means for capturing and concentrating a second analyte of interest in a sample,

the sample passing from the transport passage inlet port through the staggered configuration intersection with the elongated passage portion of each of the first and second separation passages within each corresponding extraction device to the transport passage outlet port and the concentrated first and second analyte of interest to be released from the elongated passage portion of each of the first and second separation passages by alternating fluid communication of the separation buffer supply and the eluting buffer supply and then back to the separation buffer supply from the respective first reservoir and second reservoir and the first and second analyte of interest being delivered out the respective second port of the first and second separation passages to the exit passage separately, independently and sequentially by pressure, electrophoresis migration or a combination of electro-osmotic flow and pressure and to an analyte detection system associated with the exit passage;

a first auxiliary buffer passage communicating with the first separation passage and a second auxiliary buffer passage communicating with the second separation passage, the first and second auxiliary buffer passages positioned downstream of the respective means for capturing and concentrating a first analyte of interest and a second analyte of interest and before the second ports of the first and second separation passages merge into the single outlet passage; and

a valve system operatively associated with the transport passage and the first and second elongated passage portions of the corresponding extraction devices for independently controlling the flow of sample in the transport passage and separately and independently forming a microenvironment in the first and second separation passages to mix reagents to optimize the binding and capture of the first and second analytes of interest by the respective means for capturing and concentrating the first and second analytes of interest,

wherein the elongated passage portion of the first and second separation passages of the base of each of the corresponding extraction devices defining an extraction device or analyte concentrator-microreactor device,

the extraction device being a component of an electrophoresis apparatus;

a connector which operatively connects the exit passage of each extraction device to a separation passage of the electrophoresis apparatus separately and independently; and

the individual separation passages merging to a single exit passage being operatively associated with the analyte detection system.

26. The electrophoresis extraction device of claim 25 wherein the connector defines a first connector, and further comprising a second connector which operatively connects the transport passage outlet port to a transport passage of the electrophoresis apparatus.

27. The electrophoresis extraction device of claim 26 wherein the transport passage is operatively communicable with a waste container.

28. The electrophoresis extraction device of claim 26 wherein the transport passage has a larger diameter than the first and second separation passages.

29. The electrophoresis extraction device of claim 26 wherein the transport passage and the first and second separation passages are both capillaries.

30. The electrophoresis extraction device of claim 26 wherein each of the first and second separation passages are channels along at least a substantial portion their lengths, and the transport passage is a channel along at least a substantial portion of its length.

31. The electrophoresis extraction device of claim 26 further comprising a third connector which operatively connects the first separation passage first port to a portion of the first separation passage, a fourth connector which operatively connects the transport passage inlet port to a portion of the transport passage, and a fifth connector which operatively connects the second separation passage second port of the second extraction device to a portion of the second separation passage.

32. The electrophoresis extraction device of claim 31 wherein the portion of transport passage and the first and second separation passages within each of the extraction devices are operatively communicable with a source of conditioning buffer solution to provide within the elongated portion of each of the extraction devices optimal binding and capturing conditions for the analytes of interest present in a sample fluid.

33. The electrophoresis extraction device of claim 31 wherein the portion of the first and second separation passages is operatively communicable within each of the extraction devices with a source of a cleaning solution with the analyte of interest concentrated in the elongated passage portion, the cleaning solution being adapted to wash away at least some salts and unwanted materials in the elongated passage portion of each of the extraction devices.

34. The electrophoresis extraction device of claim 31 wherein the portion of the transport passage is operatively communicable with a source of the sample.

35. The electrophoresis extraction device of claim 26 wherein the sample is introduced by pressure from a sample reservoir into and through the transport passage and to a waste reservoir.

36. The electrophoresis extraction device of claim 26 wherein the elongated passage portion of the first and second separation passages is embedded and enclosed in the base.

37. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating captures the first and second analytes of interest present in simple or complex mixtures of the sample for purification, desalting and enrichment.

38. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating isolates the first and second analytes of interest from simple or complex mixtures of the sample.

39. The electrophoresis extraction device of claim 26 wherein each of the means for capturing and concentrating includes a matrix formed by beads having antibody fragments covalently or non-covalently attached to surfaces of the beads or intact antibodies covalently or non-covalently attached to surfaces of the beads.

40. The electrophoresis extraction device of claim 39 wherein the means for capturing and concentrating includes an altered condition in the elongated passage portion of each of the extraction devices to improve binding of the analyte of interest to the antibodies or the antibody fragments.

41. The electrophoresis extraction device of claim 40 wherein the altered condition is temperature.

42. The electrophoresis extraction device of claim 40 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing.

43. The electrophoresis extraction device of claim 40 wherein the altered condition is a gentle shaking, a microwave pulse or an acoustic micromixing that is adapted to accelerate the work of proteases immobilized to a matrix of the capturing and concentrating meands and reduce the time required to cleave a protein into peptide components thereof.

44. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating performs on-line enzymatic cleavage of proteins to generate peptides.

45. The electrophoresis extraction device of claim 26 wherein the base includes openings for inserting the means for capturing and concentrating into and replacing the means for capturing and concentrating in the elongated passage portion of the first and second separation passages within each of the extraction devices.

46. The electrophoresis extraction device of claim 26 wherein the staggered configuration intersection includes bulging members positioned and sized to retain the means for capturing and concentrating in the elongated passage portion of the first and second separation passages within each of the extraction devices.

47. The electrophoresis extraction device of claim 26 wherein the staggered configuration intersection includes frit structures configured to retain the means for capturing and concentrating in the elongated passage portion of the first and second separation passages within each of the extraction devices.

48. The electrophoresis extraction device of claim 26 wherein the staggered configuration includes means other than bulging members or frit structures for retaining the means for capturing and concentrating in the elongated passage portion of the first and second separation passages within each extraction device.

49. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes microstructures selected from a group consisting of beads, platelets, chips, fibers, polymers, globules and filaments.

50. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes affinity ligands that are immobilized onto surfaces of magnetic beads.

51. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes a matrix assembly having a fixed architecture defined by interconnected polymeric microstructures that are formed from a monolithic lattice or a sol-gel lattice.

52. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes affinity ligands that are selected from a group consisting of whole antibodies, antibody fragments, lectins, aptamers, chemical dyes, protein A, protein G, substrates, enzymes, proteins, peptides, DNA, RNA, oligonucleotides, carbohydrates, cation exchange resins, anion exchange resins, immobilized metal affinity capture resins, mixed-mode resins, ions, amino acids, monosaccharides, fatty acids, vitamins, metabolites, viruses, bacteria, cells and subcellular organelles.

53. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes controlled porous glass beads having antibody fragments or intact antibodies covalently attached to surfaces thereof.

54. The electrophoresis extraction device of claim 26 wherein the extraction devices forms an-analyte concentrator-microreactors.

55. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes at least one of beads, platelets, chips, fibers, polymers, monolithics, sol-gels, filaments, cells, subcellular organelles, viruses, phages, micro-particles, nano-particles and cellular receptors.

56. The electrophoresis extraction device of claim 26 wherein the means for capturing and concentrating includes entrapped cells, subcellular organelles or cellular receptors to study metabolic pathways and degradation products of small molecular weight substances and biomolecules.

57. The electrophoresis extraction device of claim 26 wherein the valve system includes valves which are motor operated and remotely controlled by a processor based on a predetermined set of instructions.

58. The electrophoresis extraction device of claim 26 further comprising means for cleaning-rinsing sample solution from a sample cleaning-rinsing solution supply before and after passing the sample to the transport passage first port.

59. The electrophoresis extraction device of claim 58 wherein the means for cleaning-rinsing removes unwanted analytes of the sample solution that can be bound non-specifically to the means for capturing and concentrating and to the transport passage by a cleaning solution passed through the transport passage.

63. The electrophoresis extraction device of claim 30 wherein a portion of an inlet side and a portion of an outlet side of the transport passage is a capillary tube and the rest of the transport passage is a channel.

64. The electrophoresis extraction device of claim 30 wherein a portion of an inlet side of the first and second separation passages is a capillary tube and the rest of the first and second separation passages is a channel.

65. The electrophoresis extraction device of claim 30 wherein a portion of an outlet side of the first and second separation passages is a capillary tube and the rest of the first and second separation passages is a channel.

66. The electrophoresis extraction device of claim 26 wherein the transport passage is a capillary tube.

67. The electrophoresis extraction device of claim 26 wherein each of the first and second separation passages is a capillary tube.

68. The electrophoresis extraction device of claim 26 wherein a portion of the transport passage and the separation passage are operatively communicable with a conditioning buffer solution to provide optimal binding and capturing conditions for the analytes of interest, and the portion of the separation passage is then communicable with the separation buffer solution and then the elution buffer solution where a plug of the solution is used for releasing the bound analytes of interest.

69. The electrophoresis extraction device of claim 26 further comprising at least one exit valve that controls the flow from the first separation passage into the exit passage and from the second separation passage into the exit passage.

70. The electrophoresis extraction device of claim 69 wherein:

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

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

71. The electrophoresis extraction device of claim 25 wherein a first buffer and a second buffer can be provided to the first and second separation passages through the respective first and second auxiliary buffer passages, the first buffer is used to provide within the elongated portion of each of the extraction devices optimal binding and capturing conditions for the analytes of interest present in a sample fluid and the second buffer is used to obtain optimal separation of the first and second analytes of interest released from the corresponding extraction device.

72. The electrophoresis extraction device of claim 71 wherein the valve system further comprises a first auxiliary valve positioned at the intersection of the first separation passage and the first auxiliary passage and a second auxiliary valve positioned at the intersection of the second separation passage and the second auxiliary passage for controlling flow of the first and second buffers and permit the separation buffer to go in a direction toward the analyte detection system and away from the corresponding extraction devices.

73. An electrophoresis apparatus comprising two or more of the electrophoresis extraction devices of claim 26, wherein the transport passage of a first electrophoresis extraction device of the two or more electrophoresis extraction devices connects to a connector of a second electrophoresis extraction device of the two or more electrophoresis extraction devices.