Method and apparatus for performing biological reactions on a substrate surface

• US 6,589,778 B1
• Filed: 12/15/1999
• Issued: 07/08/2003
• Est. Priority Date: 12/15/1999
• Status: Expired due to Fees

• Current Assignee: Amersham Biosciences (GE Healthcare Technologies, Inc.)
• Original Assignee: Amersham Biosciences (GE Healthcare Technologies, Inc.)
• Inventors: Hawkins, George W.
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US09/464,490
• Time in Patent Office: 1,301 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/287.2, 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 2200/10 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 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/1822 using Peltier elements
• CPC Class Codes: B01L 2300/1827 using resistive heater
• CPC Class Codes: B01L 2300/1866 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 2400/0677 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 3/50853 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

Abstract

The present invention provides an apparatus for performing biological reactions on a substrate layer having a multiplicity of oligonucleotide binding sites disposed thereon. The invention provides a hybridization chamber wherein nucleic acid hybridization is performed by reacting biological material on a biochip comprising a substrate having an array of oligonucleotide binding sites. The binding sites are associated with an array of 3-dimensional polyacrylamide pads for anchoring the reactants. The arrays are covered with a flexible layer that permits mixing of the hybridization solution on the biochip and detection of hybridization in situ. Fluid inlet and outlet ports in the chamber provide for control of fluid flow into and out of the chamber.

Claims

1. An apparatus for performing biological reactions, comprising:

(a) a glass microscope slide having a first surface and a second surface opposite thereto;

(b) an array of biomolecules positioned on the first surface of the slide, wherein each biomolecule within the array is anchored to the first surface by a polyacrylamide gel pad;

(c) a layer of polyvinylidene chloride affixed to the first surface of the slide by a layer of double-sided adhesive, wherein the adhesive layer is deposited on the first surface of the slide and encloses an area thereupon;

(d) a first and second port extending through the slide from the first surface to the second surface thereof each having a first opening and a second opening, wherein the first opening of each port is provided within the area on the first surface of the slide bounded by the adhesive and covered by the flexible layer and the second opening of each port is provided on the second surface of the slide, and wherein each port is in the shape of a truncated cone having a small-diameter end and a large diameter end, and wherein the small diameter end is the first opening and the large diameter end is the second opening;

(e) a layer of foil tape positioned over the second opening of each port;

(f) a layer of a polyethylene glycol positioned between the first surface of the slide and the layer of polyvinylidene chloride;

(g) a reflective layer positioned between the array and the first substrate surface;

(h) a layer of parylene positioned between the reflective layer and the layer of polyvinylidene chloride; and

(i) a resistive heater.

2. An apparatus for performing biological reactions, comprising:

(a) a glass microscope slide having a first surface and a second surface opposite thereto,

(b) an array of biomolecules positioned on the first surface of the slide, wherein each biomolecule within the array is anchored to the first surface by a polyacrylamide gel pad;

(c) a layer of polyvinylidene chloride affixed to the first surface of the slide by a layer of double-sided adhesive, wherein the adhesive layer is deposited on the first surface of the slide and encloses an area thereupon;

(d) a first and second port extending through the slide from the first surface to the second surface thereof each having a first opening and a second opening, wherein the first opening of each port is provided within the area on the first surface of the slide bounded by the adhesive and covered by the flexible layer and the second opening of each port is provided on the second surface of the slide, and wherein each port is in the shape of a truncated cone having a small-diameter end and a large diameter end, and wherein the small diameter end is the first opening and the large diameter end is the second opening;

(e) a layer of foil tape positioned over the second opening of each port;

(f) a layer of a polyethylene glycol positioned between the first surface of the slide and the layer of polyvinylidene chloride;

(g) a reflective layer positioned between the array and the first substrate surface;

(h) a layer of parylene positioned between the reflective layer and the layer of polyvinylidene chloride;

(i) a resistive heater;

a case having a lid and a base, wherein the lid further comprises a display unit;

(k) a cavity disposed in the base; and

(l) a carriage comprising a scanner and a roller, wherein the carriage is provided in the cavity, and wherein the slide is removably positioned above the carriage such that the first slide surface is in operative contact with the carriage.

3. An apparatus for performing biological reactions comprising:

a) a substrate comprising a first and a second surface;

b) an array of biomolecular probes positioned on said first surface;

c) a flexible layer comprising a gas permeable membrane affixed to said first surface by an adhesive layer, forming a reaction volume; and

d) a port extending through said substrate from said second surface to said first surface.

8. An apparatus according to claim 3, 4, or 5 wherein said biomolecular probes are oligonucleotides.

9. An apparatus according to claim 3, 4, or 5 wherein said biomolecular probes are DNA.

10. An apparatus according to claim 3, 4, or 5 wherein said substrate comprises glass.

11. An apparatus according to claim 3, 4, or 5 wherein said substrate comprises a polymer.

12. An apparatus according to claim 3, 4, or 5 wherein said substrate comprises ceramic.

13. An apparatus according to claim 3, 4, or 5 wherein said substrate comprises silicon.

14. An apparatus according to claim 3, 4, or 5 wherein said biomolecular probes are anchored to said first surface using polyacrylamide.

15. An apparatus according to claim 3, 4, or 5 further comprising a heating element positioned under said reaction volume.

16. An apparatus according to claim 15 wherein said heating element is a resistive heater.

17. An apparatus according to claim 3, 4, or 5 comprising a plurality of arrays of said biomolecular probes;

and said flexible layer, said adhesive layer and said first surface comprise a plurality of reaction volumes each containing one of said arrays.

18. An apparatus according to claim 3, 4, or 5 wherein said reaction volume further comprises a water-soluble compound that is a solid at room temperature and a liquid at a second, higher temperature.

19. An apparatus according to claim 3, 4, or 5 further comprising a scanner.

20. An apparatus according to claim 3 or 5 further comprising a sample preparation chip.

4. An apparatus for performing biological reactions comprising:

a) a substrate comprising a first and a second surface;

b) an array of biomolecular probes positioned on said first surface;

c) a flexible layer affixed to said first surface by an adhesive layer, forming a reaction volume;

d) a port extending through said substrate from said second surface to said first surface;

e) a sample preparation chip; and

wherein said port is a first port that extends from said second surface to said reaction volume and said sample preparation chip is in contact with said second surface and wherein said sample preparation chip has a port that is aligned with said first port.

5. An apparatus for performing biological reactions comprising:

a) a substrate comprising a first and a second surface;

b) an array of biomolecular probes positioned on said first surface;

c) a flexible layer affixed to said first surface by an adhesive layer, forming a reaction volume; and

d) a port extending through said substrate from said second surface to said first surface; and

e) a roller, wherein said roller is in contact with said flexible layer.

6. A method of making an apparatus comprising:

a) providing a substrate with a first and a second surface, wherein said first surface comprises a continuous layer of derivatized polyacrylamide;

b) contacting said derivatized polyacrylamide with biomolecular probes;

c) forming an amide bond between said derivatized polyacrylamide and said biomolecular probe;

d) adding a flexible layer comprising a gas permeable membrane to said first surface to form a reaction volume.

7. A method of detecting the presence of a target molecule in a sample comprising:

a) providing an apparatus comprising;

i) a substrate comprising a first and a second surface;

ii) an array of biomolecular probes positioned on said first surface; and

iii) a flexible layer comprising a gas permeable membrane affixed to said first surface by an adhesive layer, forming a reaction volume;

wherein said apparatus comprises at least a first port into said reaction volume;

b) introducing said sample through said first port into said reaction volume under conditions that allow the binding of said target molecule to at least one of said biomolecular probes; and

c) detecting said binding as an indication of the presence of said target molecule.

21. A method according to claim 7 wherein said target molecule is labeled with a fluorescent label.

22. A method according to claim 7 wherein said biomolecular probes are nucleic acids.

23. A method according to claim 7 wherein said target molecule is a nucleic acid.