Microstructures for use in biological assays and reactions

• US 6,585,939 B1
• Filed: 02/25/2000
• Issued: 07/01/2003
• Est. Priority Date: 02/26/1999
• Status: Expired due to Term

• Current Assignee: Beckman Coulter Incorporated (Danaher Corporation)
• Original Assignee: Orchid Biosciences Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Dapprich, Johannes
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Handy, Dwayne K.
• Application Number:
  US09/512,907
• Time in Patent Office: 1,222 Days
• Field of Search: 422/55, 422/58, 422/68.1, 422/81, 422/82.05, 422/82.06, 422/99, 422/100, 422/129, 422/130, 422/131, 436/164, 436/174, 436/180, 435/283.1, 435/287.1, 435/287.8, 264/239, 264/240, 264/241, 264/250, 264/297.1, 264/297.4, 264/259, 264/299, 264/313
• US Class Current: 422/503
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00353 Pumps
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00389 Feeding through valves
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel 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 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• 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 2219/00691 using robots
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00743 Cells
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/0861 : Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2300/0887 : Laminated structure
• CPC Class Codes: B01L 2300/123 : Flexible; Elastomeric
• CPC Class Codes: B01L 2400/0481 : squeezing of channels or chambers
• 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 3/505 : flexible containers not provided for above
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B29C 65/483 : Reactive adhesives, e.g. chemically curing adhesives
• CPC Class Codes: B29C 66/54 : Joining several hollow-preforms, e.g. half-shells, to form hollow articles, e.g. for making balls, containers; Joining several hollow-preforms, e.g. half-cylinders, to form tubular articles
• CPC Class Codes: B29C 66/543 : joining more than two hollow-preforms to form said hollow articles
• CPC Class Codes: B29C 66/71 : characterised by the composition of the plastics material of the parts to be joined welding bar compositions B29C65/125
• CPC Class Codes: B29K 2027/06 : PVC, i.e. polyvinylchloride
• CPC Class Codes: B29K 2075/00 : Use of PU, i.e. polyureas or polyurethanes or derivatives thereof, as moulding material
• CPC Class Codes: B29K 2083/00 : Use of polymers having silicon, with or without sulfur, nitrogen, oxygen, or carbon only, in the main chain, as moulding material
• CPC Class Codes: B29L 2031/756 : Microarticles, nanoarticles
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6834 : Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2021/0321 : One time use cells, e.g. integrally moulded
• CPC Class Codes: G01N 2021/0325 : Cells for testing reactions, e.g. containing reagents
• CPC Class Codes: G01N 2021/0346 : Capillary cells; Microcells
• CPC Class Codes: G01N 21/03 : Cuvette constructions
• CPC Class Codes: Y10T 436/25 : including sample preparation
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

The present invention relates to microstructures which a fabricated from a cast or molded polymer material, such as polydimethylsiloxane (PDMS). The invention describes and claims methods and compositions from which a variety of microstructures may be prepared. The microstructures are particularly useful for use in assays and reactions in the relating to biological assays, including applications in medical diagnostics. Such assays and reactions included, but are by no means limited to, polynucleotide sequencing and polynucleotides analysis, including analysis of single nucleotide polymorphisms, and for polynucleotide amplification such as PCR.

The microstructures of the invention may contain many features that are useful for such applications. For example, the microstructures may comprise wells or channels for biological fluids and/or reagents, as well as pumps and valves for directing such fluids, e.g., in a biological assay. The microstructures may also comprise a variety of optical devices, including lenses, mirrors, optical waveguides, diffractions gratings, and optical absorbers and/or scatters such as “quantum dots.”

Claims

1. A microstructure comprising:

a first rigid flat substrate having a first surface and a second surface;

a first flexible layer coupled to said first surface, said first flexible layer having a first plurality of one or more cells or channels;

a second flexible layer coupled to said second surface of the first rigid flat substrate to form the composite microstructure, said second flexible layer having a second plurality of one or more cells or channels.

2. The microstructure of claim 1, wherein said first rigid flat substrate comprises a layer of glass, silicon, or metal.

3. The microstructure of claim 1, wherein said first flexible layer and said second flexible layer is fabricated from polymethylsiloxane.

4. The microstructure of claim 1, wherein the first and second flexible layers are attached by means of an adhesive layer.

5. The microstructure of claim 1, wherein the first and second flexible layers are self-sealing against the first and second surface.

6. The microstructure of claim 1, said microstructure further comprising a linear cavity or channel, and means for progressively squeezing the linear cavity or channel.

7. The microstructure of claim 1, further comprising fluidic connections between said first plurality of one or more cells or channels and said second plurality of one or more channels.

8. The microstructure of claim 7, wherein the connections comprise holes through the rigid flat layer for fluidically coupling said first flexible layer and said second flexible layer.

9. The microstructure of claim 8, wherein one of said holes and one of said first plurality of one or more cells or channels form a capillary break.

10. The microstructure of claim 7, said microstructure further comprising a cavity and a channel, further comprising means for displacing fluid through the channel by increasing or decreasing a pressure in the cavity.

11. The microstructure of claim 10, wherein the means for displacing comprise a means for increasing or decreasing the pressure in the chamber by mechanically contracting or expanding the cavity.

12. The microstructure of claim 10, wherein the means for displacing comprises means for increasing or decreasing tie pressure in the chamber by optical means.

13. The microstructure of claim 1, wherein the microstructure further comprises a lens or mirror molded from a flexible polymer layer.

14. The microstructure of claim 13, wherein the lens or mirror is molded directly to the microstructure.

15. The microstructure of claim 13, wherein the lens or mirror is molded separately and attached to the microstructure.

16. The microstructure of claim 13, wherein the lens or mirror is used in an optical signal detection means.

17. The microstructure of claim 13, wherein the lens or mirror redirects excitation or emission light.

18. The microstructure of claim 1, further comprising a diffractive optical component.

19. The microstructure of claim 18, wherein the diffractive optical component is molded from a flexible polymer layer.

20. The microstructure of claim 19, wherein the diffractive optical component is molded directly to the microstructure.

21. The microstructure of claim 18, wherein the diffractive optical component is molded separately and attached to the microstructure.

22. The microstructure of claim 18, wherein the diffractive optical component is one selected from the group consisting of an input coupler grating, a fresnel lens and holographic component.

23. The microstructure of claim 1, wherein the microstructure further comprises an optical filter.

24. The microstructure of claim 23, wherein the optical filter is one selected from the group of a dye, an optical absorber and an optical scatterer.

25. The micro structure of claim 23, wherein the optical filter is embedded into the flexible polymer layer.

26. The microstructure of claim 23, further comprising one or more reflective layers or structures, wherein said reflective layers or structures and said optical filter form a waveguide.

27. A microfluidic microstructure comprising a flexible polymer layer, wherein the flexible polymer layer is formed by casting a flexible polymer onto a complementary relief pattern by sandwiching the flexible polymer layer between two juxtaposed complementary relief patterns of a mold to form one or more cells or channels in a first surface and a second surface of the flexible polymer layer and attaching the first surface and the second surface of the flexible polymer layer to a respective first rigid flat surface and a second rigid flat surface so that said flexible polymer layer is positioned therebetween.

28. The microstructure of claim 27, wherein the flexible polymer layer is fabricated from polymethylsiloxane, silicone, urethane, latex, or vinyl.

29. The microstructure of claim 27, wherein one or more biological samples are attached to said first surface, such that the biological samples are positioned in said one or more cells or channels.

30. The microstructure of claim 27, said microstructure being attached to the surface by means of a reversible seal.

31. The microstructure of claim 27, wherein one or more biological samples are positioned on one or more cells or channels on a first surface of the flexible polymer layer, and a second surface of the flexible polymer layer is positioned on a detector, such that the one or more biological samples are detected through the flexible polymer layer.

32. The microstructure of claim 27, further comprising a plurality of wells, wherein the flexible polymer layer forms spacers between said wells.

33. The microstructure of claim 27, further comprising an array of wells, said wells being connected to a network of channels.

34. The microstructure of claim 27, wherein said flexible polymer layer has a fluidic connection therethrough.

35. A method of forming a microfluidic microstructure assembly comprising the step of:

forming a first flexible polymer layer between two juxtaposed complementary relief patterns of a mold to form one or more cells or channels in a first surface and a second surface of the first flexible polymer layer;

forming a second flexible polymer layer between two juxtaposed complementary relief patterns of a mold to form one or more cells or channels in a first surface and a second surface of the second flexible polymer layer; and

attaching the first surface of the first flexible polymer layer and the first surface of the second flexible polymer layer to a respective first rigid flat surface and a second rigid flat surface of a first substrate.

36. The method of claim 35 wherein said step of attaching comprises removably attaching the first surface of the first flexible polymer layer and the first surface of the second flexible polymer layer to a respective first rigid flat surface and a second rigid flat surface of the substrate.

37. The method of claim 35 further comprising coupling a second substrate to the second surface of the first substrate.

38. The method of claim 35 further comprising coupling a second substrate to the second surface of a third substrate.