Array cytometry

• US 20030022393A1
• Filed: 03/27/2002
• Published: 01/30/2003
• Est. Priority Date: 04/25/1996
• Status: Active Grant

• Current Assignee: BioArray Solutions Limited (Werfen SA)
• Original Assignee: BioArray Solutions Limited (Werfen SA)
• Inventors: Seul, Michael, Li, Alice Xiang
• Granted Patent: US 7,056,746 B2
• Time in Patent Office: Days
• US Class Current: 436/518
• 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. An assembly of cells comprising:

a substrate; and

a dynamically formed planar assembly of cells in a designated area on the substrate.

2. The assembly of cells accordingly to claim 1, wherein said assembly is formed by a method comprising the following steps:

(a) providing a first electrode positioned in a first plane and a second electrode positioned in a second plane different from the first plane;

(b) providing a plurality of cells suspended in an electrolyte solution, the cells and the electrolyte solution being located between the first and the second electrode, wherein the second electrode comprises either

(i) a light-sensitive electrode, such that when an AC electric field is generated at an interface between the second electrode and the electrolyte solution and the second electrode is illuminated with a predetermined light pattern, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the pattern of illumination or

(ii) a electrode having a surface or an interior, the surface or interior having been modified to produce spatial modulation in properties of the second electrode that affects the local distribution of an electric field generated at an interface between the second electrode and the electrolyte solution, such that when an AC electric field is generated at the interface, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the spatial modulation in the properties of the second electrode; and

(c) forming a planar assembly of cells in a designated area of the second electrode by applying an AC electric field at the interface between the second electrode and the electrolyte solution and, if the second electrode comprises a light-sensitive electrode, illuminating the second electrode with a predetermined light pattern.

3. The method of claim 2, wherein the first and the second electrode each comprises a planar electrode that are parallel to each other and separated by a gap, with the electrolyte solution and the cells being located in said gap, wherein the electric field is generated by applying an AC voltage between the electrodes.

4. The method of claim 3, wherein the first electrode comprises an optically transparent electrode that allows optical imaging of the cell assembly.

5. The method of claim 2, wherein the first and the second electrode are contained in an assay cartridge, said cartridge having a fluidic connection that permits introduction of a fluid into the cartridge and exit of the fluid from the cartridge.

6. The method of claim 2, wherein the second electrode comprises a silicon electrode.

7. The method of claim 2, wherein the second electrode comprises a silicon electrode coated with a dielectric layer.

8. The method of claim 2, wherein the second electrode comprises a light-sensitive electrode, said electrode having been patterned by spatially modulated oxide growth, surface chemical patterning or surface profiling, such that the location of the cellular assembly is controlled by said patterning in addition to the illumination.

9. The method of claim 2, wherein the second electrode comprises an electrode patterned by spatially modulated oxide growth, surface chemical patterning or surface profiling, such that the location of the cellular assembly is controlled by said patterning.

10. The method of claim 2, wherein the designated area corresponds to an area of low interfacial impedance.

11. The method of claim 2, wherein the cells in the assembly are immobilized by chemical or physical means.

12. The method of claim 2, wherein the cellular assembly has a density of at least about 10⁴ cells/mm².

13. A method of detecting a binding interaction between a cell and an analyte, the method comprising:

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

contacting the cells with an analyte to allow binding of the analyte to the cells; and

detecting said binding.

14. A method of detecting a binding interaction between a cell and an analyte, said method involving an assembly of cells according to claim 2 and comprising the following steps:

(a) providing a first electrode positioned in a first plane and a second electrode positioned in a second plane different from the first plane;

(b) providing a plurality of cells suspended in an electrolyte solution, the cells and the electrolyte solution being located between the first and the second electrode, wherein the second electrode comprises either

(i) a light-sensitive electrode, such that when an AC electric field is generated at an interface between the second electrode and the electrolyte solution and the second electrode is illuminated with a predetermined light pattern, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the pattern of illumination or

(ii) a electrode having a surface or an interior, the surface or interior having been modified to produce spatial modulation in properties of the second electrode that affects the local distribution of an electric field generated at an interface between the second electrode and the electrolyte solution, such that when an AC electric field is generated at the interface, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the spatial modulation in the properties of the second electrode;

(c) contacting the cells with an analyte to allow binding of said analyte to the cells;

(d) forming a planar assembly of cells in a designated area of the second electrode by applying an AC electric field at the interface between the second electrode and the electrolyte solution and, if the second electrode comprises a light-sensitive electrode, illuminating the second electrode with a predetermined light pattern, wherein the planar assembly of the cells are formed either before or after the contacting of the cells with the analyte; and

(e) detecting the binding of the analyte to the cells.

15. The method of claim 14, wherein the analyte is directed to a specific cellular marker, and step (e) comprises detecting the presence of the cellular marker on the surface of the cells.

16. The method of claim 15, wherein the bioassay is directed to cell typing, with the presence of the marker on the cell surface indicating the cell type.

17. The method of claim 14, wherein the analyte comprises a ligand directed to a specific cellular receptor and step (e) comprises detecting the presence of the receptor on the surface of the cells.

18. The method of claim 14, wherein the analyte comprises an antibody directed to a specific cellular antigen and step (e) comprises detecting the presence of the antigen on the surface of the cells.

19. The method of claim 14, wherein the analyte is attached to a bead, said bead being associated with a chemically or physically distinguishable characteristic that uniquely identifies the analyte attached thereto.

20. The method of claim 19, wherein said bead is associated with a fluorescent tag that uniquely identifies the analyte attached thereto.

21. The method of claim 14, wherein a plurality of analytes is tested simultaneously for binding with the cells, with each analyte being attached to a bead that is distinguishable by chemical or physical characteristic, and the detecting step comprising optically imaging the planar assembly of cells and decoding the beads bound to the cells to determine the respective identities of analytes bound to the cells.

22. The method of claim 14, wherein the analyte comprises a ligand or an antibody attached to a bead, said bead being associated with a chemically or physically distinguishable characteristic that uniquely identifies the analyte attached thereto.

23. The method of claim 22, wherein step (d) results in formation of a mixed assembly comprising the cells and the beads in a designated area on the second electrode, the proximity of the cells with the ligand or the antibody attached to the bead allowing the binding interaction therebetween, and wherein the method further comprises disassembling said 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 of absence of a cellular receptor or an antigen specific for the ligand or the antibody.

24. The method of claim 22, further comprising providing a planar array of said beads in a designated area on the second electrode, wherein the cells are assembled in the same designated area as the bead array and the binding interaction is detected by selective adhesion of the cells to the beads in the array.

25. A method of detecting a cellular response to an analyte, the method comprising providing a dynamically formed planar assembly of cells according to claim 1;

contacting the cells with an analyte; and

detecting a cellular response to the analyte.

26. A method of detecting a cellular response to an analyte, the method involving an assembly of cells according to claim 2 and comprising the following steps:

(a) providing a first electrode positioned in a first plane and a second electrode positioned in a second plane different from the first plane;

(b) providing a plurality of cells suspended in an electrolyte solution, the cells and the electrolyte solution being located between the first and the second electrode, wherein the second electrode comprises either

(i) a light-sensitive electrode, such that when an AC electric field is generated at an interface between the second electrode and the electrolyte solution and the second electrode is illuminated with a predetermined light pattern, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the pattern of illumination or

(ii) a electrode having a surface or an interior, the surface or interior having been modified to produce spatial modulation in properties of the second electrode that affects the local distribution of an electric field generated at an interface between the second electrode and the electrolyte solution, such that when an AC electric field is generated at the interface, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the spatial modulation in the properties of the second electrode;

(c) contacting the cells with an analyte;

(d) fonning a planar assembly of cells in a designated area of the second electrode by applying an AC electric field at the interface between the second electrode and the electrolyte solution and, if the second electrode comprises a light-sensitive electrode, illuminating the second electrode with a predetermined light pattern, wherein the planar assembly of the cells are formed either before or after the contacting of the cells with the analyte; and

(e) detecting a cellular response to the analyte.

27. The method of claim 26, wherein the cellular response being detected comprises cellular secretion of one or more biologically active substances, said secretion being induced by said analyte.

28. The method of claim 27, wherein the biologically active substance comprises a cytokine.

29. A method of determining a cellular response to an analyte, the method involving an assembly of cells according to claim 2 and comprising the following steps:

providing a planar array of beads on the first electrode, the beads displaying ligands on their surfaces and each of said beads being associated with a chemical or physical characteristic that uniquely identifies the ligand attached thereto, said ligands being attached to the beads in a releasable manner;

forming a planar assembly of cells, with the gap area separating the beads from the assembled cells; and

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

30. The method of claim 26, wherein the analyte comprises a drug molecule or a ligand.

31. The method of claim 26, 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.

32. The method of claim 31, in which the expression of the intracellular reporter gene yields intracellular fluorescence.

33. The method of claim 26, 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 the chemotaxis response by monitoring the movement of cells.

34. A method of determining an interaction between different cell types, the method comprising the following steps:

providing a dynamically formed planar assembly of cells according to claim 1, wherein the cells of the assembly comprises at least two different cell types, with the proximity of one cell type to another allowing an interaction between the different cell types to occur; and

detecting the cell-cell interaction.

35. A method of determining an interaction between different cell types, the method involving the assembly of cells according to claim 2 and comprising the following steps:

(a) providing a first electrode positioned in a first plane and a second electrode positioned in a second plane different from the first plane;

(b) providing a plurality of cells comprising at least two cell types suspended in an electrolyte solution, the cells and the electrolyte solution being located between the first and the second electrode, wherein the second electrode comprises either

(i) a light-sensitive electrode, such that when an AC electric field is generated at an interface between the second electrode and the electrolyte solution and the second electrode is illuminated with a predetermined light pattern, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the pattern of illumination or

(ii) a electrode having a surface or an interior, the surface or interior having been modified to produce spatial modulation in properties of the second electrode that affects the local distribution of an electric field generated at an interface between the second electrode and the electrolyte solution, such that when an AC electric field is generated at the interface, a planar assembly of cells forms in a designated area of the second electrode, said designated area being defined by the spatial modulation in the properties of the second electrode;

(c) forming a planar assembly of cells in a designated area of the second electrode by applying an AC electric field at the interface between the second electrode and the electrolyte solution and, if the second electrode comprise a light-sensitive electrode, illuminating the second electrode with a predetermined light pattern, wherein the cells in the assembly comprise different types of cells, with the proximity of one cell type to another allowing an interaction between the different cell types to occur; and

(d) detecting the cell-cell interaction.

36. The method of claim 35, wherein the interaction between the different cell types results in:

a morphological change of at least one cell type;

change in a cell surface marker of at least one cell type;

change in a gene expression of at least one cell type;

a change in the membrane permeability of at least one cell type;

or adhesion of the cells to form a cluster.

37. The method of claim 35, wherein step (d) comprises detecting the interaction between the cell types by ascertaining the formation of cell clusters.

38. The method of claim 35, wherein the assembly of the mixed cell types is prepared by the following steps:

forming an assembly of a first cell type in a designated area on the second electrode;

introducing cells of a second type to the electrolyte solution and moving said cells to the designated area on the second electrode in which the first cell type is located, thereby allowing the interaction between the two cell types to occur.

39. The method of claim 35, wherein the assembly of the mixed cell types is prepared by the following steps:

forming an assembly of a first cell type in a first designated area on the second electrode, forming an assembly of a second cell type in a second designated area on the second electrode; and

combining the assembly of the first and the second cell type to allow the interaction between the different cell types to occur.