Method and device for biochemical detection and analysis of subcellular compartments from a single cell

• US 20050048581A1
• Filed: 08/25/2004
• Published: 03/03/2005
• Est. Priority Date: 08/25/2003
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Chiu, Daniel T., Kuo, Jason S., Edgar, John Scott, Allen, Peter B., Sun, Bingyun, Kuyper, Christopher L., He, Mingyan, Fiorini, Gina S., Lim, David S. W., Shelby, James Patrick, Jeffries, Gavin, Mutch, Sarah, Lorenz, Robert M.
• Granted Patent: US 7,767,435 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2200/0673 Handling of plugs of fluid surrounded by immiscible fluid
• CPC Class Codes: B01L 2200/16 Reagents, handling or storing thereof
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/089 Virtual walls for guiding liquids
• CPC Class Codes: B01L 2400/0415 electrical forces, e.g. electrokinetic
• CPC Class Codes: B01L 2400/0427 Electrowetting
• CPC Class Codes: B01L 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/502784 specially adapted for droplet or plug flow, e.g. digital microfluidics automatic analysis using a stream of discrete samples in a tube system G01N35/08
• CPC Class Codes: B82Y 10/00 Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C12N 15/1003 Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
• CPC Class Codes: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/147 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1484 microstructural devices
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor

Abstract

A method and system for performing biochemical detection or analysis on micro- and nano-scale subcellular component within a single biological cell is provided. An integrated platform device and method to perform the biochemical analysis is also provided.

Claims

1. A method comprising:

contacting a subcellular component of a single biological cell with a chemical reactant;

separating a molecular component from the subcellular component; and

detecting the molecular component of the biological cell.

2. The method of claim 1 further comprising contacting the molecular component with the chemical reactant.

3. The method of claim 2 further comprising analyzing the molecular component of the biological cell.

4. The method of claim 1 further comprising isolating the subcellular component from the biological cell.

5. The method of claim 4, wherein said contacting step further comprising encapsulating the subcellular component within a microdroplet.

6. The method of claim 1, further comprising permeating a membrane to the chemical reactant.

7. The method of claim 6, wherein said contacting step further comprises contacting the chemical reactant with the molecular component.

8. The method of claim 4, wherein said isolating step further comprises using a laser for optical trapping and cellular surgery.

9. The method of claim 8, wherein said isolating step further comprises using a plurality of lasers.

10. The method of claim 8 wherein the optical trapping is an optical vortex trap or an optical gradient trap.

11. The method of claim 1 wherein the subcellular component is an organelle or subcellular compartment.

12. The method of claim 1, wherein the molecular component is a single molecule.

13. The method of claim 1 wherein the molecular component is a colloidal entity, a small ion, a nanoparticle, a nucleic acid, an amino acid, a polynucleotide, a polypeptide, protein, or metabolic precursor or metabolite thereof.

14. The method of claim 1 wherein the molecular component is a macromolecular assembly, a signaling complex, a lipid- or membrane-containing structure, a carbohydrate-containing structure, a nucleic acid-containing structure, or a polypeptide-containing structure.

15. The method of claim 1, wherein the biological cell is an animal cell, a plant cell or a bacterium.

16. The method of claim 15, wherein the animal cell is a mammalian cell.

17. The method of claim 1, wherein the chemical reactant is a detectable marker.

18. The method of claim 5, wherein said contacting step further comprises encapsulating the chemical reactant in a microdroplet, and fusing the chemical reactant microdroplet with the subcellular component microdroplet.

19. The method of claim 18, further comprising contacting the molecular component with the chemical reactant.

20. The method of claim 5 wherein the microdroplet is an aqueous microdroplet.

21. The method of claim 20 wherein the microdroplet has a volume from about 10⁻

9 liters to about 10<sup>−

18</sup> liters.

22. The method of claim 5 further comprising

contacting the subcellular component in the aqueous microdroplet with an organic phase;

adjusting the starting volume of the organic phase and the period of time of contacting the aqueous microdroplet with the organic phase; and

controlling the concentration of the molecular component in the aqueous microdroplet.

23. The method of claim 1 wherein said detecting step further comprises immuno-labeling, immuno-magnetic labeling, quantum dot attachment, or labeling with a chemically reactive group.

24. The method of claim 17 wherein the detectable marker is a reactive dye tag, a fluorescent tag, a non-fluorescent tag, or a contrast agent.

25. The method of claim 1 wherein said separating step further comprises liquid-liquid partition, precipitation, adsorption, chromatography, high-performance liquid chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, protein affinity chromatography, hydroxyapatite chromatography, thiophilic chromatography, hydrophobic charge induction chromatography, immobilized boronic acid ligand chromatography, dye interaction chromatography, metal chelate affinity chromatography, immunoaffinity chromatography, ion-exchange chromatography, capillary electrophoresis, capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, micellar electrokinetic capillary chromatography, or capillary electrochromatography.

26. The method of claim 1, wherein said detecting step further comprises single-molecule confocal fluorescence microscopy or mass spectrometry.

27. The method of claim 1 wherein said detecting step further comprises immunoassay, enzyme-linked immunosorbant assay (ELISA), Western blot analysis, immunoligand assay, mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), surface-enhanced laser desorption mass spectrometry (SELDI-MS), matrix-assisted laser desorption mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS), radiation-based spectroscopy, laser-induced fluorescence (LIF), two-photon excited fluorescence, surface-enhanced Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), electric field-based detection, ac impedance spectrometry, voltammetry, electrochemical detection, or conductivity measurement.

28. A method for concentrating a subcellular component in an aqueous microdroplet comprising:

contacting the subcellular component in the aqueous microdroplet with an organic phase; and

concentrating the subcellular component in the aqueous microdroplet.

29. The method of claim 28, further comprising controlling the concentration of the subcellular component in the aqueous microdroplet by adjusting the starting volume of the organic phase, adjusting the period of time of contacting, or adjusting the composition of the aqueous and organic phases.

30. The method of claim 28, further comprising controlling the concentration of the subcellular component in the aqueous microdroplet using a laser to shrink or expand the volume.

31. The method of claim 28 wherein the organic phase is oil, soybean oil, mineral oil, n-hexadecane, decanol, nonanol, perfluoro-based solution, perfluorononane, perfluorodecane, or acetophenone.

32. A device comprising:

a substrate to contact a single biological cell;

a chemical reactant to contact a subcellular component of the biological cell; and

a separation microchannel on the substrate to isolate a molecular component from the subcellular component.

33. The device of claim 32, further comprising a detector to identify or analyze the molecular component.

34. The device of claim 32, further comprising a force generator to manipulate the subcellular component.

35. The device of claim 34, further comprising:

a laser to isolate a subcellular component from the biological cell;

a microfluidic channel on the substrate to receive the subcellular component; and

a reactant chamber to generate a microdroplet within the microfluidic channel.

36. The device of claim 35, further comprising:

a first microdroplet generated within the microfluidic channel, said first microdroplet encapsulating the subcellular component;

a second microdroplet generated within the microfluidic channel, said second microdroplet encapsulating a chemical reactant; and

the force generator to fuse the first microdroplet and the second microdroplet.

37. The device of claim 32, further comprising:

a reactant droplet chamber to generate a microdroplet with the chemical reactant;

the chemical reactant to permeate a membrane of the cell; and

a microfluidic channel on the substrate to receive the subcellular component.

38. The device of claim 35, wherein the laser is an optical vortex trap or an optical gradient trap.

39. The device of claim 38, further comprising a plurality of lasers for optical trapping and cell surgery.

40. The device of claim 32 wherein the substrate is a polymeric material, an inorganic material, or a combination thereof.

41. The device of claim 40 wherein the substrate is a polymeric material, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, polyurethane, cyclic olefin copolymer, perfluoropolyether, polystyrene, polyvinylchloride, polyethyleneterephthalate glycol, or a combination thereof.

42. The device of claim 40 wherein the substrate is an inorganic material, glass, silicon, GaAs, silicon nitride, metals, or a combination thereof.

43. The device of claim 32 wherein the chemical reactant is a detectable marker.

44. The device of claim 43 wherein the detectable marker is generated by immuno-labeling, immuno-magnetic labeling, quantum dot attachment or labeling with a chemically reactive group.

45. The device of claim 43 wherein the detectable marker is a reactive dye tag, a fluorescent tag, a non-fluorescent tag or a contrast agent.

46. The device of claim 32 wherein the microdroplet is an aqueous solution generated in an organic phase.

47. The device of claim 46 wherein the organic phase is oil, soybean oil, mineral oil, n-hexadecane, decanol, nonanol, perfluoro-based solution, perfluorononane, perfluorodecane, or acetophenone.

48. The device of claim 32 wherein the force is mechanical force, electric field force, magnetic field force, hydrodynamic force, surface tension force, interfacial tension force, thermal force, or a combination thereof.

49. The device of claim 32 wherein the chemical reactant is immuno-labeling, immuno-magnetic labeling, quantum dot attachment, or labeling with a chemically reactive group.

50. The device of claim 32 wherein the separation microchannel isolates the molecular component by liquid-liquid partition, precipitation, adsorption, chromatography, high-performance liquid chromatography, hydrophobic interaction chromatography, size-exclusion chromatography, protein affinity chromatography, hydroxyapatite chromatography, thiophilic chromatography, hydrophobic charge induction chromatography, immobilized boronic acid ligand chromatography, dye interaction chromatography, metal chelate affinity chromatography, immunoaffinity chromatography, ion-exchange chromatography, capillary Electrophoresis, capillary zone electrophoresis, capillary isoelectric focusing, capillary gel electrophoresis, micellar electrokinetic capillary chromatography, or capillary electrochromatography.

51. The device of claim 32 wherein the detector is a single-molecule confocal fluorescence microscope or a mass spectrometer.

52. The device of claim 32 wherein the detector is immunoassay, enzyme-linked immunosorbant assay (ELISA), Western blot analysis, immunoligand assay, mass spectrometry, electrospray ionization mass spectrometry (ESI-MS), surface-enhanced laser desorption mass spectrometry (SELDI-MS), matrix-assisted laser desorption mass spectrometry (MALDI-MS), secondary ion mass spectrometry (SIMS), radiation-based spectroscopy, laser-induced fluorescence (LIF), two-photon excited fluorescence, surface-enhanced Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), electric field-based detection, ac impedance spectrometry, voltammetry, electrochemical detection, or conductivity measurement.

53. A system comprising:

a substrate to contact a single biological cell;

a reactant generation chamber to provide a chemical reactant to contact a subcellular component of the biological cell; and

a separation microchannel on the substrate to isolate a molecular component of the subcellular component.

54. The system of claim 53, further comprising a detector to analyze the molecular component.

55. The system of claim 53, further comprising a force generator to manipulate the subcellular component.

56. The system of claim 55 wherein the force generator is mechanical force, electric field force, magnetic field force, hydrodynamic force, surface tension force, interfacial tension force, thermal force, or a combination thereof.

57. The system of claim 55, further comprising:

a laser to isolate a subcellular component from the biological cell;

a microfluidic channel on the substrate to receive the subcellular component;

a first microdroplet generated within the microfluidic channel to encapsulate the subcellular component;

a second microdroplet generated within the microfluidic channel to encapsulate a chemical reactant; and

the force generator to fuse the first microdroplet and the second microdroplet.

58. The system of claim 53, further comprising a microfluidic channel on the substrate to receive the subcellular component.

59. The system of claim 53, wherein the chemical reactant permeates a membrane of the cell.

60. The system of claim 57, wherein the laser is an optical vortex trap or an optical gradient trap.

61. The system of claim 53 wherein the substrate is a polymeric material, an inorganic material, or a combination thereof.

62. The system of claim 61 wherein the substrate is a polymeric material, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, polyurethane, cyclic olefin copolymer, perfluoropolyether, polystyrene, polyvinylchloride, polyethyleneterephthalate glycol, or a combination thereof.

63. The system of claim 61 wherein the substrate is an inorganic material, glass, silicon, GaAs, silicon nitride, metals, or a combination thereof.

64. The system of claim 53, further comprising a plurality of lasers for optical trapping and cell surgery.

65. The system of claim 53, further comprising a computing device in electrical communication with the reactant generation chamber to control generation of droplets and size of droplets.

66. The system of claim 65 further comprising computer readable instructions to read an input signal from the chamber and generate an output signal to the chamber.

67. The system of claim 57, further comprising a computing device in electrical communication with the laser to control cell manipulation.

68. The system of claim 67 further comprising computer readable instructions to read an input signal from the laser and generate an output signal to the laser.

69. The system of claim 57, further comprising a computing device in electrical communication with the force generator to control movement of subcellular components and molecular components.

70. The system of claim 69 further comprising computer readable instructions to read an input signal from the force generator and an output signal to the force generator.

71. The system of claim 53, further comprising a computing device in electrical communication with the separation microchannel to isolate the molecular component from the cellular component.

72. The system of claim 71 further comprising computer readable instructions to read an input signal from the separation microchannel and an output signal to the separation microchannel.

73. The system of claim 53, further comprising a computing device in electrical communication with the detector to identify the molecular component.

74. The system of claim 73 further comprising computer readable instructions to read an input signal from the detector and an output signal to the detector.

75. The system of claim 53 wherein the chemical reactant is a detectable marker.

76. The system of claim 75 wherein the detectable marker is generated by immuno-labeling, immuno-magnetic labeling, quantum dot attachment or labeling with a chemically reactive group.

77. The system of claim 75 wherein the detectable marker is a reactive dye tag, a fluorescent tag, a non-fluorescent tag or a contrast agent.

78. The system of claim 53 wherein the microdroplet is an aqueous solution generated in an organic phase.

79. The system of claim 78 wherein the organic phase is oil, soybean oil, mineral oil, n-hexadecane, decanol, nonanol, perfluoro-based solution, perfluorononane, perfluorodecane, or acetophenone.

80. A device for biochemical detection or analysis of a single biological cell comprising

a substrate for contacting the single biological cell;

manipulation means for isolating a subcellular component from the single biological cell;

encapsulation means for isolating a chemical reactant from the subcellular component;

force means for contacting the subcellular component with the chemical reactant;

separation means for isolating a molecular component from the subcellular component; and

detecting means for analyzing the molecular component.