Method and apparatus for performing biological reactions on a substrate surface

• US 6,569,674 B1
• Filed: 01/26/2000
• Issued: 05/27/2003
• Est. Priority Date: 12/15/1999
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences (General Electric Company)
• Original Assignee: Amersham Biosciences (General Electric Company)
• Inventors: Hawkins, George W., Johnson, W. Travis, McGarry, Mark W.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/492,013
• Time in Patent Office: 1,217 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/285.1, 435/287.2, 536/22.1, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/287.2
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0684 Venting, avoiding backpressure, avoid gas bubbles
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/0822 Slides
• CPC Class Codes: B01L 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2300/1866 Microwaves
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/049 : vacuum
• CPC Class Codes: B01L 2400/0677 : phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• 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 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502723 : characterised by venting arrangements
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/50853 : with covers or lids
• CPC Class Codes: B01L 9/527 : for microfluidic devices, e.g. used for lab-on-a-chip
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G01N 2021/054 : Bubble trap; Debubbling
• CPC Class Codes: G01N 2035/00158 : Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2035/00267 : Meltable plugs

Abstract

The present invention provides a method and an improved apparatus for removing gas bubbles from a reaction chamber comprising a flexible layer removably affixed to a substrate layer having a multiplicity of oligonucleotide binding sites disposed thereon, in which biological reactions are performed. The invention specifically relates to methods and apparatus for removing gas bubbles from a reaction chamber wherein target molecules contained in a sample fluid are reacted with probe molecules immobilized on a substrate having an array of oligonucleotide binding sites. The arrays are covered with a flexible, gas permeable layer that permits mixing of the sample fluid on the biochip and removal of gas bubbles from the fluid by use of a means for facilitating diffusion of gas bubbles across the flexible, gas permeable layer.

Claims

1. An apparatus for performing biological reactions, comprising:

(a) a substrate having a first surface and a second surface opposite thereto,

(b) an array of biomolecules positioned on the first surface of the substrate,

(c) a flexible, gas permeable layer affixed to the first surface of the substrate by an adhesive, wherein the adhesive is deposited on the first surface of the substrate and encloses an area thereupon, and wherein the flexible, gas permeable layer, the adhesive, and the first substrate surface enclose a reaction chamber;

(d) a port extending through the substrate from the first surface to the second surface thereof and having a first opening and a second opening, wherein the first opening of the port is provided within the area bounded by the adhesive and covered by the flexible, gas permeable layer and the second opening of the port is provided on the second surface of the substrate;

(e) a removable cover positioned over the second opening of the port;

(f) a layer of a water-soluble compound that is a solid at a first temperature and a liquid at a second, higher temperature, the layer being positioned in the reaction chamber between the first surface of the substrate and the flexible, gas permeable layer; and

(g) a means for facilitating diffusion of gas bubbles across the flexible, gas permeable layer, wherein the diffusion-facilitating means creates a pressure gradient or concentration gradient across the flexible, gas permeable layer thereby increasing the rate of diffusion of gas bubbles from the reaction chamber across the flexible, gas permeable layer.

2. The apparatus of claim 1, wherein the flexible, gas permeable layer is a porous membrane.

3. The apparatus of claim 2, wherein the porous membrane is porous polytetrafluoroethylene.

4. The apparatus of claim 2, wherein, when the reaction chamber contains a fluid sample, the porous membrane prevents the sample fluid from passing through the membrane.

5. The apparatus of claim 1, wherein the means for facilitating diffusion of gas bubbles across the flexible, gas permeable layer comprises a vacuum source removably affixed to the flexible, gas permeable layer, and wherein the vacuum source is used to create a pressure gradient across the flexible, gas permeable layer to facilitate diffusion of gas bubbles from the reaction chamber across the flexible, gas permeable layer.

6. The apparatus of claim 5, wherein the vacuum source comprises:

(a) a vacuum pump, and

(b) a chamber seal removably affixed to the flexible, gas permeable layer and removably connected to the vaccum pump, wherein the seal completely surrounds the area on the first substrate surface containing the array of biomolecules.

7. In an apparatus for performing biological reactions, comprising a substrate having a first surface and a second surface opposite thereto, an array of biomolecules positioned on the first surface of the substrate, a flexible layer affixed to the first surface of the substrate by an adhesive, wherein the adhesive is deposited on the first surface of the substrate and encloses an area thereupon;

a reaction chamber enclosed by the flexible layer, the adhesive and the first substrate surface;

a port extending through the substrate from the first surface to the second surface thereof and having a first opening and a second opening, wherein the first opening of the port is provided within the area bounded by the adhesive and covered by the flexible layer and the second opening of the port is provided on the second surface of the substrate;

a removable cover positioned over the second opening of the port; and

a layer of a water-soluble compound that is a solid at a first temperature and a liquid at a second, higher temperature, the layer being positioned between the first surface of the substrate and the flexible layer, the improvement comprising

(a) a flexible layer that is a gas permeable layer and

(b) a means for facilitating diffusion of gas bubbles across the flexible, gas permeable layer, wherein the diffusion-facilitating means creates a pressure gradient or concentration gradient across the flexible, gas permeable layer such that gas bubbles diffuse from the reaction chamber to the environment outside the flexible, gas permeable layer.

8. The improvement of claim 7, wherein the diffusion-facilitating means comprises a vacuum source removably affixed to the flexible, gas permeable layer and wherein the vacuum source is used to apply a vacuum to the flexible, gas permeable layer to remove gas bubbles from the reaction chamber.