Virtual wells for use in high throughput screening assays

• US 20040018615A1
• Filed: 04/08/2003
• Published: 01/29/2004
• Est. Priority Date: 08/02/2000
• Status: Abandoned Application

• Current Assignee: Tina K. Garyantes
• Original Assignee: Tina K. Garyantes
• Inventors: Garyantes, Tina K.
• Application Number:
  US10/410,504
• Publication Number:
  US 20040018615A1
• Time in Patent Office: Days
• US Class Current: 435/305.2
• CPC Class Codes: B01F 33/3021 the components to be mixed being combined in a single independent droplet, e.g. these droplets being divided by a non-miscible fluid or consisting of independent droplets
• CPC Class Codes: B01F 33/30351 using hydrophilic/hydrophobic surfaces
• CPC Class Codes: B01F 33/30352 using roughness of the surfaces
• CPC Class Codes: B01F 33/3039 with mixing achieved by diffusion between layers
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00536 in the shape of disks
• CPC Class Codes: B01J 2219/00587 High throughput processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00648 : by the use of solid beads
• CPC Class Codes: B01J 2219/00659 : Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 : Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01J 2219/0074 : Biological products
• CPC Class Codes: B01L 2300/042 : Caps; Plugs
• CPC Class Codes: B01L 2300/0829 : Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0858 : Side walls
• CPC Class Codes: B01L 2400/022 : droplet contacts the surface of the receptacle
• CPC Class Codes: B01L 3/0244 : using pins
• CPC Class Codes: B01L 3/0262 : using touch-off at substrate or container
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B01L 3/50853 : with covers or lids
• CPC Class Codes: B01L 3/5088 : confining liquids at a location by surface tension, e.g. virtual wells on plates, wires B01L3/50857 takes precedence
• CPC Class Codes: B01L 3/563 : Joints or fittings in general F16L; Separable fluid transfer means to transfer fluids between at least two containers, e.g. connectors
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries

Abstract

Microtiter-like plates containing virtual wells formed by an arrangement of relatively hydrophilic domains within relatively hydrophobic fields are provided. Assay mixtures are confined to the hydrophilic domains of the virtual wells by the edges of the hydrophobic fields. The use of virtual wells allows one to perform homogeneous and capture and wash high throughput screening assays with assay mixtures having volumes on the order of about 100 nl to 10 μ. Virtual wells also provide a means of easily moving fluids, which is particularly useful for simultaneous additions needed for kinetic studies and flash detection and washing. Methods for controlling evaporation during the dispensing of reagents as well as during incubation of high throughput screening utilizing microtiter-like plates containing virtual wells are also provided.

The present invention also provides an inexpensive, disposable device for transferring small volumes of an entire array of compounds from a first microtiter-like plate to a second microtiter-like plate, preserving the spatial arrangement of the compounds. Methods of manufacturing and using the device are also provided.

Claims

1. A microtiter-like plate comprising:

(a) a bottom having an upper surface comprising a plurality of virtual wells, said virtual wells being relatively hydrophilic domains within a relatively hydrophobic field;

(b) a cover or top configured for enclosing said bottom.

2. The microtiter-like plate of claim 1 where said bottom comprises a sidewall or spacer and said top rests on said sidewall or spacer.

3. The microtiter-like plate of claim 2 where said top has a lower surface comprising a plurality of virtual wells, and said plurality of virtual wells of said bottom and said top are present in an arrangement such that, when said bottom and said top are in close proximity, column-like fluid wells are formed between said bottom and said top, or fluid is transferred, or components are captured.

4. The microtiter-like plate of claim 2 where said top has a sidewall and the difference in the height of said sidewall of said bottom and said sidewall of said top is such that when said top is resting on said bottom sidewall, then said upper surface of said bottom and said lower surface of said top are in close proximity.

5. The microtiter-like plate of claim 3 comprising a spacer that can vary in height so that the distance between said bottom and said top is determined by the height of said spacer.

6. The microtiter-like plate of claim 1 where said hydrophilic domains are selected from the group consisting of:

plain glass;

derivatized glass;

silanized glass;

glass with bio-and non-biopolymers absorbed;

polystyrene or other plastics;

Indium Tin Oxide or other metal oxides;

gold or other metals;

silicon or other crystals; and

ceramics.

7. The microtiter-like plate of claim 1 where said hydrophilic domains are polygons or circles having a diameter of from about 10 μ

m to about 10 mm.

8. The microtiter-like plate of claim 1 where said hydrophobic field is selected from the group consisting of:

polyfluorocarbons;

TEFLON®

or TEFLON®

beads;

perfluoropropene;

paraffin or other waxes or oils;

polyethylene or other hydrocarbons;

chlorodimethyl octyl silane or other silanizing agents;

polypropylene or other hydrophobic polymers;

bifunctional materials containing beads or other hydrophobic protrusions such as a polyfluorocarbon or polyfluorocarbon coated beads; and

hydrocarbon or hydrocarbon-coated beads.

9. The microtiter-like plate of claim 8 where said hydrophobic field is layered on a smooth or microscopically rough surface where the surface is rough to about from 50 to 5,000 nm.

10. A combination of a microtiter-like plate containing virtual wells and cells, said cells being present in said virtual wells, where the combination is suitable for use in a screening assay.

11. A method of transferring fluid to a plurality of virtual wells comprising:

(a) providing a first plate or lid comprising a plurality of virtual wells to which said fluid is to be transferred;

(b) providing a second plate or lid on which said fluid is present;

(c) moving said first plate or lid and said second plate or lid into close proximity so that fluid is transferred from said second plate or lid to said plurality of virtual wells of said first plate or lid.

12. A method of adding fluid to a plurality of virtual wells comprising:

(a) providing a plate or lid containing a plurality of virtual wells to which said fluid is to be added;

(b) moving said plate or lid into close proximity to a fluid reservoir so that fluid is added to said plurality of virtual wells of said plate or lid from said reservoir.

13. A method of nearly simultaneously adding fluid to a plurality of virtual wells so as to enable detection of flash reagents or to enable kinetic studies comprising:

(a) providing a plate or lid containing a plurality of virtual wells to which said fluid is to be added;

(b) providing a second plate or lid on which said fluid is present;

(c) moving said first plate or lid and said second plate or lid into close proximity so that all or most of the fluid is transferred from said second plate or lid simultaneously or nearly simultaneously to said plurality of virtual wells of said first plate or mixed with fluid already in said first plate or lid.

14. The method of claim 13 where step (c) is practiced while said first plate is in front of a detector.

15. A method of removing fluid from a plurality of virtual wells comprising:

(a) providing a first plate or lid comprising a plurality of virtual wells in which said fluid is present;

(b) providing a second plate or lid onto which said fluid is to be transferred;

(c) moving said first plate or lid and said second plate or lid into close proximity so that fluid is transferred from said plurality of virtual wells of said first plate or lid to said second plate or lid, thus removing some or all of said fluid from said virtual wells of said first plate or lid.

16. A method of limiting evaporation during pipetting of assay reagents comprising:

(a) providing a microtiter plate where the microtiter plate has been cooled to the dew point;

(b) pipetting or dispensing assay reagents into said wells of said microtiter plate while the temperature of the microtiter plate is kept at or near the dew point.

17. The method of claim 16 where the plate is monitored to determine whether it is at the dew point by the use of a sensor based on resistivity or by measuring temperature and relative humidity so that the temperature of the plate is automatically controlled at the dew point.

18. A method for limiting evaporation in a microtiter-like plate containg virtual wells during incubation with a lid wherein a fluid reservoir is present in the plate so that any gas that moves into the plate is humidified before it reaches the virtual wells.

19. A method of screening comprising:

(a) adding a series of reagents to a plurality of virtual wells in a microtiter-like plate or lid;

(b) adding a spatially defined array of compounds to the plurality of virtual wells before or after (a);

(c) incubating the reagents and compounds;

(d) reading a diagnostic signal from the virtual wells.

20. The method of claim 19 where the total volume in the virtual wells after step (b) is about 100 nl to about 10 μ

l.

21. The method of claim 19 where the microtiter-like plate has a top and a bottom, and the method furthermore comprises, after step (c) and before step (d), the additional steps of:

(i) separating the top and bottom of the microtiter-like plate and adding a new top or bottom that is engineered to bind one or more of the reagents in the virtual wells;

(ii) incubating as desired to allow binding of the reagents to the new top or bottom;

(iii) washing the bound reagents as desired;

(iv) repeating steps (a)-(b) and (i)-(iii) above as desired.

(v) reading a diagnostic signal from the virtual wells or from the new top or bottom that has bound the reagent or reagents.

22. The method of claim 19 comprising repeating step (d) one or more times.

23. A method of screening to identify a compound capable of modulating a preselected biological activity exhibited by cells comprising:

(a) providing cells in the virtual wells of a microtiter-like plate;

(b) exposing the cells to a compound or collection of compounds suspected of being capable of modulating the preselected biological activity to be exhibited by the cells;

(c) determining whether the preselected biological activity has been modulated.

24. The method of claim 23 where the preselected biological activity is selected from the group consisting of:

changes in membrane potential of the cells;

increases or decreases in metabolites or ions such as ATP, cAMP, cGMP, phospholipids, calcium;

changes in the transcription of certain genes;

changes in fluorescent or chemiluminescent behaviour;

changes in pH;

changes in enzymatic activity;

changes in the activity of receptor proteins;

changes in the activity of ion channels;

changes in the translational control of certain mRNAs;

changes in the translocation of certain proteins into or out of subcellular locations;

cell growth or inhibition of growth;

pigment dispersion or aggregation; and

antibody binding.

25. A method of high throughput screening to identify a substance capable of binding to or modulating the activity of a protein or a nucleic acid comprising:

(a) providing said protein or said nucleic acid, in solution, in membranes, or in cells, in virtual wells;

(b) exposing said protein or said nucleic acid to a substance suspected of being capable of binding to or modulating the activity of said protein or said nucleic acid;

(c) determining whether said substance modulates the activity of said protein or said nucleic acid.

45. A method of high throughput screening comprising:

(a) removing a volume of fluid from a storage plate by use of the device of claim 27;

(b) transferring said volume to an assay plate by use of the device of claim 25;

(c) detecting a diagnostic signal in said assay plate.

26. A device for transferring fluids where the device comprises a plurality of pins in a microtiter-like plate or lid. where the pins have a circular or other face having a diameter of from 50 to 1,000 μ

m;

where the pins have a depth of 0.3 to 10 mm; and

where the device transfers a volume of fluid between 100 pl and 1 μ

l.

27. The device of claim 26 where the pins have been produced by a process selected from the group consisting of:

micromachining into the surface of a material selected from the group consisting of glass, metal, plastic, silicon and other crystalline materials by a process selected from the group consisting of anisotropic, isotropic, plasma, and reactive ion etching;

electron discharge machining into the surface of a material selected from the group consisting of metal and other conductive materials;

laser cutting into the surface of a material selected from the group consisting of glass, silicon or other crystalline material;

metal or other conductive materials; and

plastic; and

molding from a material selected from the group consisting of plastic, glass, and metal.

28. The device of claim 26 where the pins have been modified so that their tips are hydrophilic and their shaft or portion of their shaft is hydrophobic.

29. The device of claim 27 where the material is Monel or other non corrosive metal, the process is wire EDM, the pins have a square or other closed polygon face having a diameter of about 200-400 μ

m, and the device transfers a volume of about 10 nl.

30. The device of claim 29 where the face has a surface irregularity.

31. The device of claim 27 where the material is glass or other etchable material, the process is anisotropic wet etching, the pins have a circular or other shaped face having a diameter of 200-400 μ

m, and the device transfers a volume of about 10 nl.

32. The device of claim 27 where the material is 111 silicon or other crystaline material, the process is an isotropic wet etch, the pins have a square or other shape face having a diameter of about 200-400 μ

m, and the device transfers a volume of about 10 nl.

33. A combination of the device of claim 26 and a microtiter plate where the device acts as a lid for the microtiter plate.

34. The combination of claim 33 where the microtiter plate contains virtual wells.

35. The combination of claim 33 where the device acts as an evaporation control barrier for the microtiter plate.

36. A method of transferring fluid from a first microtiter plate to a second microtiter plate that comprises:

(a) providing a plurality of fluids present in a spatial array in the wells of a first microtiter plate;

(b) providing a device having pins arranged in a spatial array similar to the spatial array of the wells in the first microtiter plate;

(c) moving the device into close proximity to the first microtiter plate so that the spatial array of pins in the device matches the spatial array of wells of the first microtiter plate so that fluid is transferred from the wells of the first microtiter plate to the pins of the device;

(d) moving the device into close proximity to a second microtiter plate having wells arranged in a spatial array similar to the spatial array of the first microtiter plate and the spatial array of the pins of the device so that fluid is transferred from the pins of the device to the wells of the second microtiter plate;

where the spatial array of the fluid in the first microtiter plates is transferred to the second microtiter plate;

where the device has a plurality of pins where the pins have been produced by a process selected from the group consisting of;

micromachining into the surface of a material selected from the group consisting of glass, metal, plastic, silicon and other crystalline materials by a process selected from the group consisting of anisotropic, isotropic, plasma, and reactive ion etching;

electron discharge machining into the surface of a material selected from the group consisting of metal and other conductive materials;

laser cutting into the surface of a material selected from the group consisting of glass, silicon or other crystalline material;

metal or other conductive materials; and

plastic;

molding from a material selected from the group consisting of plastic, glass, and metal;

where the pins have a circular or other shaped face having a diameter of from 50 to 1 mm;

where the pins have a depth of 0.3 to 10 mm; and

where the device transfers a volume of fluid between 100 pl and 1 μ

I.

37. The method of claim 36 where the device forms a top or lid for the first microtiter plate and the second microtiter plate, and the spatial array of pins in the device matches and automatically aligns to the spatial array of wells of the first microtiter plate and the second microtiter plate.

38. A method of removing fluid from a plurality of wells in a spatial array comprising:

(a) providing a microtiter plate containing a plurality of wells in a spatial array in which fluid is present;

(b) moving a device having pins in a spatial array similar to the spatial array of the wells of the microtiter plate into close proximity to the microtiter plate so that a portion of the fluid is transferred from the wells of the microtiter plate to the pins of the device, the fluid from each well of the microtiter plate being transferred to a single pin, and where the spatial array of fluids in the wells of the microtiter plate is preserved on the pins;

where the device has a plurality of pins where the pins have been produced by a process selected from the group consisting of;

micromachining into the surface of a material selected from the group consisting of glass, plastic, metal, silicon and other crystalline materials by a process selected from the group consisting of anisotropic, isotropic, plasma, and reactive ion etching;

electron discharge machining into the surface of a material selected from the group consisting of metal and other conductive materials;

laser cutting into the surface of a material selected from the group consisting of glass, silicon or other crystalline material;

metal or other conductive materials; and

plastic;

molding from a material selected from the group consisting of plastic, glass, and metal;

where the pins have a circular or other shaped face having a diameter of from 50 to 1 mm;

where the pins have a depth of 0.3 to 10 mm; and

where the device transfers a volume of fluid between 100 pl and 1 μ

l.

39. The method of claim 38 where the wells are virtual wells.

40. The method of claim 38 where the device and the microtiter plate have been configured so that the device fits as a lid on the microtiterplate with tight alignment

41. The method of claim 38 where the method is practiced repeatedly and the pins are not washed after each practice of the method.

42. A method of adding fluid to a plurality of wells in a microtiter plate in a spatial array comprising:

(a) providing a microtiter plate containing a plurality of wells in a spatial array into which the fluid is to be added;

(b) moving a device having pins coated with fluid where the pins are arranged in a spatial array similar to the spatial array of the wells of the microtiter plate into close proximity to the rnicrotiter plate so that fluid is transferred from the pins to the wells of the microtiter plate, the fluid from each pins being transferred to a single well and where the spatial array of fluids on the pins is preserved in the wells;

where the device has a plurality of pins where the pins have been produced by a process selected from the group consisting of;

micromachining into the surface of a material selected from the group consisting of glass, plastic, metal, silicon and other crystalline materials by a process selected from the group consisting of anisotropic, isotropic, plasma, and reactive ion etching;

electron discharge machining into the surface of a material selected from the group consisting of metal and other conductive materials;

laser cutting into the surface of a material selected from the group consisting of glass, silicon or other crystalline material;

metal or other conductive materials; and

plastic;

molding from a material selected from the group consisting of plastic, glass, and metal;

where the pins have a circular or other shaped face having a diameter of from 50 to 1 mm;

where the pins have a depth of 0.3 to 10 mm; and

where the device transfers a volume of fluid between 100 pl and 1 μ

.

43. The method of claim 42 where the wells are virtual wells.

44. The method of claim 42 where the device and the microtiter plate have been configured so that the device fits as a lid on the microtiterplate with tight alignment.

46. A method of manufacturing a device for transferring a spatial array of fluids comprising:

(a) selecting a base material;

(b) subjecting the base material to a process that produces pins on the surface of the base material;

where the base material is selected from the group consisting of;

glass, silicon or other crystalline materials, metal or other conductive materials, and plastic;

where the process is selected from the group consisting of;

micromachining by anisotropic, isotropic, plasma, or reactive ion etching into the surface of the base material;

electron discharge machining into the surface of the base material;

laser cutting into the surface of the base material; and

molding the base material;

with or without further treatment to make the tip hydrophilic and the shaft hydrophobic

where the pins have a circular or other shaped faces having a diameter of from 50 to 1 mm;

where the pins have a depth of 0.3 to 10 mm; and

where the device transfers a volume of fluid between 100 pl and 1 μ

l .

47. A method of running an assay using an assay plate having a top and a bottom where the top and the bottom are active components of the assay system.

48. The method of claim 47 where the top and bottom are used for fluid transfer.

49. The method of claim 47 where the assay is run between the top and the bottom of the plate and fluid touches both the top and the bottom of the plate.