Array Cytometry

• US 6,387,707 B1
• Filed: 05/28/1999
• Issued: 05/14/2002
• Est. Priority Date: 04/25/1996
• Status: Expired due to Term

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Li, Alice Xiang, Seul, Michael
• Primary Examiner(s): Le, Long V.
• Assistant Examiner(s): Do, Pensee T.
• Application Number:
  US09/320,274
• Time in Patent Office: 1,082 Days
• Field of Search: 435/4, 435/6, 435/7.1, 435/7.2, 435/7.21, 435/7.23, 435/7.92, 435/7-3-, 422/50, 422/57, 422/60, 422/63, 422/67, 422/68.1, 427/2.12, 427/2.13, 427/483, 427/59, 427/77, 436/519, 436/538, 436/806, 436/807
• US Class Current: 436/164
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0668 Trapping microscopic beads
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• 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 2400/0454 radiation pressure, optical tweezers
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• 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: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2563/155 : Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C12Q 2565/501 : being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/607 : being a sensor, e.g. electrode
• CPC Class Codes: C40B 40/00 : Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G01N 15/14 : Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 15/149 : specially adapted for sorting particles, e.g. by their size or optical properties
• CPC Class Codes: G01N 30/0005 : Field flow fractionation
• CPC Class Codes: G01N 33/54313 : the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/5438 : Electrodes
• CPC Class Codes: G02B 3/0012 : characterised by the manufacturing method
• CPC Class Codes: G02B 3/0056 : arranged along two different directions in a plane, e.g. honeycomb arrangement of lenses G02B3/0043 takes precedence; miniaturised objectives for electronic devices employing wafer level optics G02B13/0085
• CPC Class Codes: H05H 3/04 : Acceleration by electromagnetic wave pressure
• CPC Class Codes: Y10S 436/806 : Electrical property or magnetic property
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures

Abstract

A method and apparatus for the manipulation of colloidal particulates and biomolecules at the interface between an insulating electrode such as silicon oxide and an electrolyte solution. Light-controlled electrokinetic assembly of particles near surfaces relies on the combination of three functional elements: the AC electric field-induced assembly of planar aggregates; the patterning of the electrolyte/silicon oxide/silicon interface to exert spatial control over the assembly process; and the real-time control of the assembly process via external illumination. The present invention provides a set of fundamental operations enabling interactive control over the creation and placement of planar arrays of several types of particles and biomolecules and the manipulation of array shape and size. The present invention enables sample preparation and handling for diagnostic assays and biochemical analysis in an array format, and the functional integration of these operations. In addition, the present invention provides a procedure for the creation of material surfaces with desired properties and for the fabrication of surface-mounted optical components. This invention is also for a method and apparatus to direct the lateral motion and induce the assembly into planar arrays of cells on semiconductor surfaces in response to temporally and spatially varying electric fields and to projected patterns of illumination.

Claims

1. A method of forming an assembly of cells in a designated area on a light-sensitive electrode, comprising:

providing a plurality of cells suspended at an interface between said light-sensitive electrode electrolyte solution;

generating an electric field at the interface; and

illuminating the interface with a predetermined light pattern to form a planar assembly of substantially one layer of cells in a designated area on the light-sensitive electrode, wherein the designated area is defined by the pattern of illumination.

2. The method of claim 1, further comprising an additional electrode, the additional electrode and the light-sensitive electrode being substantially planar and aligned to one another and separated by a gap, wherein the additional electrode comprises an optically transparent electrode and wherein the gap contains the plurality of cells.

3. The method of claim 1, wherein the electrode has a surface or an interior, the surface or interior having been modified to produce spatial modulations in properties of the electrode, said properties affecting the local distribution of the electric field at said interface, wherein the surface or interior have been modified by spatially modulated oxide growth, surface chemical patterning or surface profiling.

4. The method of claim 1, further comprising the step of spatially or temporally varying the light pattern to cause the alteration of the assembly, said alteration being selected from the group consisting of disassembly, disassembly followed by reassembly, repositioning of the assembly, reconfiguration of the assembly, and segmentation of the assembly.

5. The method of claim 1, further comprising the step of varying the frequency or the voltage of the electric field to cause the alteration of the assembly, said alteration being selected from the group consisting of disassembly, disassembly followed by reassembly, repositioning of the assembly, reconfiguration of the assembly, and segmentation of the assembly.

6. The method of claim 1, in which the plurality of cells comprises more than one type of cells, the method further comprising the step of fractionating one type of cells from another to induce a displacement of the plurality of cells within the assembly, fractionation arising as a result of differences in mobility of said types of cells.

7. The method of claim 1, further comprising the step of maintaining the cells in the assembly, wherein the maintenance step comprises maintaining the electric field and the predetermined light pattern, or immobilizing the cells by chemical or physical means.

8. The method of claim 7, in which the cellular assembly is immobilized on the electrode by chemically linking the cells or confining the cells.

9. An assembly of cells formed in a designated area on a light-sensitive electrode, comprising

the light-sensitive electrode; and

a planar assembly of cells comprising substantially one layer of cells in said designated area on the light-sensitive electrode, wherein the assembly is formed according to the method of claim 1.

10. A method of conducting a bioassay involving an assembly of cells, comprising:

providing a planar assembly of cells comprising substantially one layer of cells in a designated area on the light-sensitive electrode and wherein the assembly of the cells is formed according to the method of claim 1;

contacting the cells with an analyte; and

detecting the binding of the analyte to the cells.

11. The method of claim 10, in which the cells in the assembly of the cells are immobilized prior to contacting the cells with the analyte.

12. The method of claim 11, in which the cells are immobilized by chemically linking the cells or confining the cells.

13. The method of claim 10, in which the analyte is directed to a specific cellular marker, and the bioassay is for determining the presence of the cellular marker on the surface of the cells.

14. The method of claim 13, in which the bioassay is directed to cell typing, with the presence of the marker on the cell surface indicating the cell type.

15. The method of claim 13, wherein the analyte is attached to a label and the detection of the binding of the analyte to the cells is carried out by detecting the presence of the label.

16. The method of claim 13, further comprising the step of removing the analyte that is not bound to the cell before the detection step is carried out.

17. The method of claim 13, wherein the analyte is attached to a label, said label comprising a flourescent tag.

18. The method of claim 13, wherein the analyte is attached to a label, said label comprising a bead which is distinguishable by chemical or physical characteristics.

19. The method of claim 13, in which more than one analyte is tested simultaneously for binding with the cells, with each analyte being attached to an encoded bead that is distinguishable by chemical or physical characteristics, and the detecting step comprising decoding the beads bound to the cells to determine the respective identities of analytes bound to the cells.

20. The method of claim 13, wherein the analyte is a ligand directed to a specific cellular receptor and the bioassay is for determining the presence of the receptor on the surface of the cells.

21. The method of claim 13, wherein the analyte is an antibody directed to a specific cellular antigen, and the bioassay is for determining the presence of the antigen on the surface of the cells.

22. The method of claim 13, in which the presence of more than one antigen is determined using more than one antibody, wherein each antibody is attached to a fluorophore tag that is chemically distinguishable, wherein the detection step comprises multicolor imaging of the cells.

23. A method of assaying the binding of cells with a ligand or an antibody, said method involving a planar assembly of cells and comprising:

providing an assembly of cells prepared according to claim 1, wherein the assembly further comprises a plurality of beads randomly mixed with the cells in the designated area on the light-sensitive electrode, said beads having a ligand or antibody attached to their surfaces, wherein said beads comprise different types of beads distinguishable by the ligand or antibody attached thereto and further distinguishable by a unique chemical or physical characteristic that identifies said bead type;

allowing the binding interaction to occur between the cells and the ligand or antibody;

disassembling the mixed assembly of the cells and the beads; and

detecting the binding interaction by analyzing the formation of clusters composed of the cells and the beads, the binding indicating the presence or absence of a cellular receptor or an antigen specific for the ligand or the antibody.

24. A method of determining a cellular response to an analyte, the method involving an assembly of cells and comprising:

providing a planar assembly of cells formed according to claim 1;

contacting the cells with an analyte;

an d detecting a cellular response to the analyte.

25. The method of claim 24, wherein th e analyte comprises a drug molecule or a ligand.

26. The method of claim 24, in which the cellular response being detected is an expression of a particular gene, said expression being determined by detecting for the presence of an intracellular reporter gene product.

27. The method of claim 26, in which the expression of the intracellular reporter gene yields intracellular fluorescence.

28. The method of claim 24, in which the cellular response being detected is selected from the group consisting of:

morphological change of the cells, change in the cell membrane permeability, and a change in th e chemiotaxis response by monitoring the movement of cells.

29. A method of detecting a cellular response to an analyte, the method involving an assembly of cells and comprising:

providing a planar assembly of cells prepared according to the method of claim 1;

contacting the cells with an analyte; and

detecting a cellular response to the analyte.

30. The method of claim 29, wherein one or more analytes are being tested for its ability to induce the cells to secrete one or more biologically active substances, and the detection step comprises detecting the presence of the biologically active substances.

31. The method of claim 30, wherein the biologically active substance comprises cytokine.

32. A method of determining a cellular response to an analyte, the method comprising:

providing a first electrode positioned in a first plane and a second electrode positioned in a second plane different from the first plane, the second electrode comprising a light-sensitive electrode and the first and the second electrode each comprising a planar electrode and said electrodes being in a substantially parallel alignment and separated by a gap, said gap containing an electrolyte solution in which a plurality of cells is suspended;

providing a planar array of beads on the first electrode, said beads having a ligand attached to their surfaces in a releasable manner, wherein said beads comprise different types of beads distinguishable by the ligand attached thereto and further distinguishable by a unique chemical or physical characteristic that identifies said bead type

illuminating an interface between the light-sensitive electrode and an electrolyte solution with a predetermined light pattern and generating an electric field at said interface to form a planar assembly of the cells on the second electrode, with the gap area separating the beads from the assembled cells; and

releasing the ligand and monitoring a cellular response, the proximity of the cells to the bead array permitting determination of the identity of the ligand inducing the cellular response.