Virtual wells for use in high throughput screening assays

• US 6,565,813 B1
• Filed: 08/02/2000
• Issued: 05/20/2003
• Est. Priority Date: 02/04/1998
• Status: Expired due to Fees

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck & Co., Inc.
• Inventors: Garyantes, Tina
• Primary Examiner(s): Markoff, Alexander
• Application Number:
  US09/601,708
• Time in Patent Office: 1,021 Days
• Field of Search: 422/100, 422/102, 422/99, 422/104, 435/288.3, 435/288.4, 359/398
• US Class Current: 422/553
• 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 33/3039 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 2219/00315 Microtiter plates
• 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 2219/00617 by chemical means
• 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 2219/00637 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 2300/0819 : Microarrays; Biochips
• CPC Class Codes: B01L 2300/0829 : Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0858 : Side walls
• CPC Class Codes: B01L 2300/0893 : having a very large number of wells, microfabricated wells
• 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: C12M 23/12 : Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 41/46 : of cellular or enzymatic activity or functionality, e.g. cell viability
• 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 μl. 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 bottom having an upper surface comprising a plurality of virtual wells, said virtual wells being relatively hydrophillic domains within a relatively hydrophobic field; and

a cover or top configured for enclosing said bottom, wherein said cover or 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.

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 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.

4. The microtiter-like plate of claim 1 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.

5. 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.

6. 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.

7. 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.

8. The microtiter-like plate of claim 7 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.

9. 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.

10. 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; and

c) shaking said plate or lid.

11. 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.

12. The method of claim 11 where step (c) is practiced while said first plate is in front of a detector.

13. 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.

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

15. A method of screening using a microtiter-like plate having a top and bottom comprising:

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

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

c) moving said top into close proximity with said bottom so that column-like fluid wells are formed between said bottom and said top, or fluid is transferred, or components are captured;

d) incubating the reagents and compound; and

e) reading a diagnostic signal from the virtual wells.

16. The method of claim 15 where the total volume in the virtual wells after step (b) is about 100 nl to about 10 μ

l.

17. The method of claim 15 where the method furthermore comprises, after step d) and before step e), 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.

18. The method of claim 15 comprising repeating step (d) one or more times.

19. 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, said microtiter-like plate comprising a top and bottom, and a sidewall or spacer that vary in height so that the distance between said bottom and said top is determined by the height of said sidewall or spacer;

(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; and

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

20. The method of claim 19 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.

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

(a) providing on a first plate or lid 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 by moving a second plate or lid in close proximity with said first plate or lid, said second plate or lid having said substance disposed thereon; and

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