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

• US 7,767,435 B2
• Filed: 08/25/2004
• Issued: 08/03/2010
• Est. Priority Date: 08/25/2003
• Status: Expired due to Fees

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: 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., Chiu, Daniel T., Kuo, Jason S.
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Assistant Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US10/926,656
• Publication Number:
  US 20050048581A1
• Time in Patent Office: 2,169 Days
• Field of Search: 422/50, 422/55, 422/58, 422/61, 422/68.1, 422/100, 422/101, 435/4, 435/286.5, 435/287.1, 435/287.9
• US Class Current: 435/286.5
• 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 device comprising:

a substrate to contact a single biological cell;

means to remove a subcellular component of the biological cell;

a chemical reactant selected to facilitate detection of a molecular component present in the subcellular component removed from the biological cell;

a force generator to manipulate the at least one of the biological cell, the molecular component, and the subcellular component;

a separation microchannel on the substrate to separate the molecular component from the subcellular component; and

a controller, the controller comprising a processor and a memory logically coupled to the processor, the memory storing data and machine readable and executable instructions that when executed by the processor, cause a plurality of functions to be carried out, including using the force generator to encapsulate at least one of the subcellular component and the molecular component into an aqueous microdroplet.

2. The device of claim 1, further comprising a detector to identify or analyze the molecular component.

3. The device of claim 1, wherein the controller is further configured to implement the function of reducing a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component.

4. The device of claim 3, wherein said means to remove the subcellular component from the biological cell comprises a laser.

5. The device of claim 4, wherein the controller is further configured to implement the following functions:

using the force generator to generate a second microdroplet within a microfluidic channel, said second microdroplet encapsulating a chemical reactant; and

using the force generator to fuse the aqueous microdroplet encapsulating the at least one of the subcellular component and the molecular component with the second microdroplet, thereby exposing the at least one of the subcellular component and the molecular component to the chemical reactant.

6. The device of claim 1, further comprising:

a reactant droplet chamber configured to generate a microdroplet encapsulating the chemical reactant, the chemical reactant having been selected based on its ability to facilitate detection of the molecular component; and

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

7. The device of claim 4, wherein the laser is used in connection with at least one trap selected from the group consisting of an optical vortex trap, and an optical gradient trap.

8. The device of claim 7, further comprising a plurality of lasers for optical trapping and cell surgery.

9. The device of claim 1 wherein the substrate is a polymeric material.

10. The device of claim 9 wherein the substrate comprises at least one material selected from the group consisting of a polymeric material, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, polyurethane, cyclic olefin copolymer, perfluoropolyether, polystyrene, polyvinylchloride, polyethyleneterephthalate glycol, and a combination thereof.

11. The device of claim 1 wherein the chemical reactant is a detectable marker.

12. The device of claim 11 wherein the detectable marker is based upon at least one labeling technique selected from the group consisting of immuno-labeling, immuno-magnetic labeling, quantum dot attachment, and labeling with a chemically reactive group.

13. The device of claim 11 wherein the detectable marker comprises at least one element selected from the group consisting of a reactive dye tag, a fluorescent tag, a non-fluorescent tag, and a contrast agent.

14. The device of claim 1 wherein the aqueous microdroplet is generated in an organic phase.

15. The device of claim 14 wherein the organic phase comprises at least one component selected from the group consisting of oil, soybean oil, mineral oil, n-hexadecane, decanol, nonanol, a perfluoro-based solution, perfluorononane, perfluorodecane, and acetophenone.

16. The device of claim 3 wherein the force comprises at least one force selected from the group consisting of a mechanical force, an electric field force, a magnetic field force, a hydrodynamic force, a surface tension force, an interfacial tension force, a thermal force, and a combination thereof.

17. The device of claim 1 wherein the chemical reactant is based upon at least one labeling technology selected from the group consisting of immuno-labeling, immuno-magnetic labeling, quantum dot attachment, and labeling with a chemically reactive group.

18. The device of claim 1 wherein the separation microchannel isolates the molecular component using at least one technique selected from the group consisting of 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, and capillary electrochromatography.

19. The device of claim 2 wherein the detector comprises at least one device selected from the group consisting of a single-molecule confocal fluorescence microscope, and a mass spectrometer.

20. The device of claim 2 wherein the detector comprises at least one detection technology selected from the group consisting of 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, alternating current impedance spectrometry, voltammetry, electrochemical detection, and conductivity measurement.

38. The device of claim 1, wherein the controller is further configured to implement the function of reducing a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component by introducing the aqueous microdroplet into a volume filled with an organic liquid, and keeping the aqueous microdroplet in the organic liquid for a period of time sufficient to shrink the aqueous microdroplet by a desired amount, thereby increasing a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet.

21. A system comprising:

a substrate to contact a single biological cell;

means to remove a subcellular component of the biological cell;

a reactant chamber configured to provide a chemical reactant selected to facilitate the detection of a molecular component present in the subcellular component removed from the biological cell;

a force generator to manipulate the at least one of the biological cell, the molecular component, and the subcellular component;

a separation microchannel on the substrate to separate the molecular component of the subcellular component;

a controller, the controller comprising a processor and a memory logically coupled to the processor, the memory storing data and machine readable and executable instructions that when executed by the processor, cause a plurality of functions to be carried out, including;

using a force generator to encapsulate at least one of the subcellular component and the molecular component into an aqueous microdroplet; and

reducing a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component.

22. The system of claim 21, further comprising a detector to analyze the molecular component.

23. The system of claim 21 wherein the force generator produces at least one force selected from the group consisting of a mechanical force, an electric field force, a magnetic field force, a hydrodynamic force, a surface tension force, an interfacial tension force, a thermal force, and a combination thereof.

24. The system of claim 21, further comprising a microfluidic channel on the substrate to receive the subcellular component.

25. The system of claim 21, wherein the chemical reactant is further selected to permeate a membrane of the cell.

26. The system of claim 21, wherein the laser is used in connection with at least one trap selected from the group consisting of an optical vortex trap, and an optical gradient trap.

27. The system of claim 21 wherein the substrate is a polymeric material.

28. The system of claim 27 wherein the substrate comprises at least one component selected from the group consisting of a polymeric material, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polyester (PET), polytetrafluoroethylene (PTFE), polycarbonate, polyurethane, cyclic olefin copolymer, perfluoropolyether, polystyrene, polyvinylchloride, polyethyleneterephthalate glycol, and a combination thereof.

29. The system of claim 21, further comprising a plurality of lasers for optical trapping and cell surgery.

30. The system of claim 21, wherein the controller is further configured to control generation of the aqueous microdroplet and a size of the aqueous microdroplet.

31. The system of claim 21 wherein the chemical reactant is a detectable marker.

32. The system of claim 31 wherein the detectable marker is based on at least one labeling technology selected from the group consisting of immuno-labeling, immuno-magnetic labeling, quantum dot attachment, and labeling with a chemically reactive group.

33. The system of claim 31 wherein the detectable marker comprises at least one element selected from the group consisting of a reactive dye tag, a fluorescent tag, a non-fluorescent tag, and a contrast agent.

34. The system of claim 21 wherein the aqueous microdroplet is generated in an organic phase.

35. The system of claim 34 wherein the organic phase comprises at least one element selected from the group consisting of oil, soybean oil, mineral oil, n-hexadecane, decanol, nonanol, perfluoro-based solution, perfluorononane, perfluorodecane, and acetophenone.

36. A device comprising:

a substrate to contact a single biological cell;

means to selectively remove a portion of the single biological cell, the portion comprising a subcellular component of the biological cell that is transported across a cellular membrane of the single biological cell, while the single biological cell is disposed in a fluid volume defined by the substrate;

means to separate a molecular component from the subcellular component, while the subcellular component is disposed in a fluid volume defined by the substrate, where the molecular component is part of the subcellular component;

means to analyze the molecular component; and

a controller, the controller comprising a processor and a memory logically coupled to the processor, the memory storing data and machine readable and executable instructions that when executed by the processor, cause a plurality of functions to be carried out, including using a force generator to encapsulate at least one of the subcellular component and the molecular component into an aqueous microdroplet.

37. The device of claim 36, wherein the controller is further configured to implement the function of reducing the volume of the aqueous microdroplet by introducing the aqueous microdroplet into a fluid volume defined by the substrate, the fluid volume being filled with an organic liquid, and keeping the aqueous microdroplet in the organic liquid for a period of time sufficient to shrink the aqueous microdroplet by a desired amount, thereby increasing a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet.

39. The device of claim 36, further comprising at least one force generator to manipulate at least one of the subcellular component and a microdroplet, and the controller is further configured to implement the functions of:

introducing a detection marker into the biological cell, the detection marker marking at least one of the molecular component and the subcellular component; and

using said means to selectively remove the portion of the single biological cell to remove the subcellular component from the biological cell after the detection marker has been introduced.

40. A device comprising:

a substrate configured to contact a single biological cell;

a separation microchannel on the substrate to separate the molecular component from the subcellular component; and

a controller, the controller comprising a processor and a memory logically coupled to the processor, the memory storing data and machine readable and executable instructions that when executed by the processor, cause a plurality of functions to be carried out, including using a force generator to encapsulate at least one of the subcellular component and the molecular component into an aqueous microdroplet.

41. The device of claim 40, wherein the controller is further configured to implement the function of reducing a volume of the aqueous microdroplet by introducing the aqueous microdroplet into a volume filled with an organic liquid, and keeping the aqueous microdroplet in the organic liquid for a period of time sufficient to shrink the aqueous microdroplet by a desired amount, thereby increasing a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet.

42. A system comprising:

a substrate configured to contact a single biological cell;

a reactant chamber configured to employ a chemical reactant selected to facilitate the detection of a molecular component present in a subcellular component removed from the biological cell;

a separation microchannel on the substrate to separate the molecular component from the subcellular component; and

a controller configured to implement the functions of;

using a force generator to encapsulate at least one of the subcellular component and the molecular component into an aqueous microdroplet; and

reducing a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component.

43. The system of claim 42, wherein the controller is further configured to implement the function of reducing a volume of the aqueous microdroplet by introducing the aqueous microdroplet into a volume filled with an organic liquid, and keeping the aqueous microdroplet in the organic liquid for a period of time sufficient to shrink the aqueous microdroplet by a desired amount, thereby increasing a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet.

44. A device comprising:

a substrate to contact a single biological cell;

means to remove a subcellular component of the biological cell;

means to separate a molecular component from the subcellular component;

means to analyze the molecular component;

means to encapsulate at least one component selected from a group of components consisting of the subcellular component and the molecular component into an aqueous microdroplet; and

means to reduce a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet.

45. A device comprising:

a substrate to contact a single biological cell;

means to transport a subcellular component of the biological cell across a cellular membrane of the single biological cell, thus removing the subcellular component from the single biological cell, while the single biological cell is disposed in a fluid volume defined by the substrate;

means to separate a molecular component from the subcellular component, while the subcellular component is disposed in a fluid volume defined by the substrate, where the molecular component is part of the subcellular component;

means to encapsulate at least one component into an aqueous microdroplet, the at least one component being selected from a group of components consisting of the subcellular component and the molecular component, while the at least one component is disposed in a fluid volume defined by the substrate;

means to reduce a volume of the aqueous microdroplet to increase a relative concentration of the at least one of the subcellular component and the molecular component in the aqueous microdroplet; and

means to analyze the molecular component.