High density array fabrication and readout method for a fiber optic biosensor

• US 6,146,593 A
• Filed: 07/24/1997
• Issued: 11/14/2000
• Est. Priority Date: 05/23/1995
• Status: Expired due to Term

• Current Assignee: Medical Research Council (Government of United Kingdom), Regents of the University of California (University of California)
• Original Assignee: Medical Research Council (Government of United Kingdom), Regents of the University of California (University of California)
• Inventors: Pinkel, Daniel, Albertson, Donna G., Gray, Joe
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/899,000
• Time in Patent Office: 1,209 Days
• Field of Search: 422/68.1, 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 422/68.1
• CPC Class Codes: B01J 2219/00513 Essentially linear supports
• CPC Class Codes: B01J 2219/00524 in the shape of fiber bundles
• 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 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 21/648 using evanescent coupling or surface plasmon coupling for the excitation of fluorescence
• CPC Class Codes: G01N 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B
• CPC Class Codes: G01N 2201/0833 Fibre array at detector, resolving
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: G02B 6/04 formed by bundles of fibres G02B6/24 takes precedence

Abstract

The invention relates to the fabrication and use of biosensors comprising a plurality of optical fibers each fiber having attached to its "sensor end" biological "binding partners" (molecules that specifically bind other molecules to form a binding complex such as antibody-antigen, lectin-carbohydrate, nucleic acid-nucleic acid, biotin-avidin, etc.). The biosensor preferably bears two or more different species of biological binding partner. The sensor is fabricated by providing a plurality of groups of optical fibers. Each group is treated as a batch to attach a different species of biological binding partner to the sensor ends of the fibers comprising that bundle. Each fiber, or group of fibers within a bundle, may be uniquely identified so that the fibers, or group of fibers, when later combined in an array of different fibers, can be discretely addressed. Fibers or groups of fibers are then selected and discretely separated from different bundles. The discretely separated fibers are then combined at their sensor ends to produce a high density sensor array of fibers capable of assaying simultaneously the binding of components of a test sample to the various binding partners on the different fibers of the sensor array. The transmission ends of the optical fibers are then discretely addressed to detectors--such as a multiplicity of optical sensors. An optical signal, produced by binding of the binding partner to its substrate to form a binding complex, is conducted through the optical fiber or group of fibers to a detector for each discrete test. By examining the addressed transmission ends of fibers, or groups of fibers, the addressed transmission ends can transmit unique patterns assisting in rapid sample identification by the sensor.

Claims

1. A method for comparing relative copy number of nucleic acid sequences in two or more collections of nucleic acid molecules, said method comprising:

(a) providing a biosensor wherein the biosensor comprises a plurality of optical fibers, each fiber including a sensor end and a transmission end where the sensor ends of the optical fibers bear target nucleic acids such hat the sensor end of at least one first fiber has attached a first target nucleic acid comprising a first target nucleotide sequence, and the sensor end of at least one second fiber has attached a second target nucleic acid, wherein said first and second target nucleic acids are different and are uniquely addressed;

(b) contacting said biosensor with

(i) a first collection of labelled nucleic acid molecules, and

(ii) at least a second collection of labelled nucleic acid molecules;

wherein the first and second labels are distinguishable from each other; and

(c) comparing the amount of binding, if any, of the first and second collections of labelled nucleic acid molecules to said first target nucleic acid, thereby determining the relative copy number of any sequences in the first and second collections that are substantially complementary to said first target nucleotide sequence.

2. The method of claim 1, wherein the target nucleic acids are DNA.

3. The method of claim 1, wherein the target nucleic acids are cDNA.

4. The method of claim 1, wherein the target nucleic acids are RNA.

5. The method of claim 1, wherein the target nucleic acids are mapped to specific regions in human chromosomes.

6. The method of claim 1, wherein the target nucleic acids are about 400 to about 1,000,000 nucleotides in complexity.

7. The method of claim 1, wherein the complexity of any sequence substantially complementary to the first or second target nucleotide sequences is less than 1% of the total complexity of the sequences in the sample.

8. The method of claim 1, wherein the first and second labels are fluorescent labels.

9. The method of claim 1, wherein the first collection of labeled nucleic acid molecules comprises mRNA or cDNA from a test cell and the second collection of labeled nucleic acid molecules comprises mRNA or cDNA from a reference cell.

10. The method of claim 1, wherein the first collection of labeled nucleic acid molecules is from a test genome and the second collection of labeled nucleic acid molecules is from a normal reference genome.

11. The method of claim 10, wherein the test genome comprises nucleic acid molecules from fetal tissue.

12. The method of claim 10, wherein the test genome comprises nucleic acid molecules from a tumor.

13. The method of claim 1, wherein the first and second collections of labeled nucleic acid molecules are treated to inhibit the binding of repetitive sequences.

14. The method of claim 13, wherein the first and second collections of labeled nucleic acid molecules are mixed with unlabeled blocking nucleic acids comprising repetitive sequences.

15. The method of claim 1, wherein said second target nucleic acid comprises a second target nucleotide sequence, said method additionally comprising comparing the amount of binding, if any, of the first and second collections of labeled nucleic acid molecules to said second target nucleic acid, thereby determining the relative copy number of any sequences in the first and second collections that are substantially complementary to said second target nucleotide sequence.

16. The method of claim 15, wherein said comparing step comprises determining the ratio of binding of the first and second collections of labeled nucleic acid molecules to each target nucleic acid.

17. The method of claim 16, wherein said comparing step additionally comprises comparing the ratio for the first target nucleic acid to the ratio for the second target nucleic acid.

18. The method of claim 15, wherein said first or second collection of labelled nucleic acid molecules is selected from the group consisting of mRNA, cDNA, and genomic DNA.

19. The method of claim 15 wherein the sensor ends of the optical fibers are concave or convex.

20. The method of claim 15 wherein said biosensor comprises a plurality of target nucleic acids attached to said plurality of optical fibers to provide a sensor face with a target nucleic acid concentration of about 30,000 different target nucleic acids per square centimeter.

21. The method of claim 15 wherein said biosensor comprises a plurality of target nucleic acids attached to said plurality of optical fibers to provide a sensor face, wherein said sensor face is non-planar.

22. The method of claim 15 wherein the amount of binding, if any, of the first and second collections of labelled nucleic acid molecules is detected using a transilluminating light source.

23. The method of claim 15, wherein said first or second target nucleic acid is indirectly attached to the sensor end of said first or second fiber, respectively.