Signal detection methods and apparatus

• US 6,225,625 B1
• Filed: 06/01/1995
• Issued: 05/01/2001
• Est. Priority Date: 06/07/1989
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Pirrung, Michael C., Fodor, Stephen P. A., Stryer, Lubert, Read, J. Leighton
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/456,598
• Time in Patent Office: 2,161 Days
• Field of Search: 250/302, 250/458.1, 435/6, 435/7.1, 536/22.1, 536/25.32
• US Class Current: 250/302
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00434 Liquid crystal masks
• CPC Class Codes: B01J 2219/00436 Maskless processes
• CPC Class Codes: B01J 2219/00459 Beads
• CPC Class Codes: B01J 2219/00468 by manipulation of individual beads
• CPC Class Codes: B01J 2219/00475 Sheets
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00531 essentially square
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential 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 2219/00608 DNA chips
• 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 2219/00626 : Covalent
• CPC Class Codes: B01J 2219/00637 : by coating it with another layer
• CPC Class Codes: B01J 2219/00639 : the compounds being trapped in or bound to a porous medium
• 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 2219/00689 : using computers
• CPC Class Codes: B01J 2219/00695 : Synthesis control routines, e.g. using computer programs
• CPC Class Codes: B01J 2219/00711 : Light-directed synthesis
• CPC Class Codes: B01J 2219/00722 : Nucleotides
• CPC Class Codes: B01J 2219/00725 : Peptides
• CPC Class Codes: B01L 7/52 : with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07C 229/14 : to carbon atoms of carbon skeletons containing rings
• CPC Class Codes: C07C 229/16 : to carbon atoms of hydrocarbon radicals substituted by amino or carboxyl groups, e.g. ethylenediamine-tetra-acetic acid, iminodiacetic acids
• CPC Class Codes: C07D 263/44 : Two oxygen atoms
• CPC Class Codes: C07D 317/62 : with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to atoms of the carbocyclic ring
• CPC Class Codes: C07H 19/04 : Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 : with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/042 : characterised by the nature of the carrier
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C07K 1/062 : for alpha- or omega-carboxy functions
• CPC Class Codes: C07K 17/06 : attached to the carrier via a bridging agent
• CPC Class Codes: C07K 17/14 : Peptides being immobilised on, or in, an inorganic carrier
• CPC Class Codes: C07K 7/06 : having 5 to 11 amino acids
• CPC Class Codes: C08G 69/00 : Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule products obtained from isocyanates or isothiocyanates C08G18/00; polyamide-imides C08G73/14
• CPC Class Codes: C12Q 1/6809 : Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 : characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6874 : involving nucleic acid arrays, e.g. sequencing by hybridisation
• 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
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers
• CPC Class Codes: G01N 15/1433 : using image recognition
• CPC Class Codes: G01N 21/253 : for batch operation, i.e. multisample apparatus analytical automats G01N35/00
• CPC Class Codes: G01N 21/6428 : Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6452 : Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G03F 7/00 : Photomechanical, e.g. photolithographic, production of textured or patterned surfaces, e.g. printing surfaces; Materials therefor, e.g. comprising photoresists; Apparatus specially adapted therefor using photoresist structures for special production processes, see the relevant places, e.g. B44C, H01L, e.g. H01L21/00, H05K
• CPC Class Codes: G03F 7/26 : Processing photosensitive materials; Apparatus therefor G03F7/12 - G03F7/24 take precedence
• CPC Class Codes: G03F 7/265 : Selective reaction with inorganic or organometallic reagents after image-wise exposure, e.g. silylation
• CPC Class Codes: G03F 7/38 : Treatment before imagewise removal, e.g. prebaking G03F7/265 takes precedence
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00
• CPC Class Codes: Y02P 20/55 : Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 435/961 : including a step of forming, releasing, or exposing the antigen or forming the hapten-immunogenic carrier complex or the antigen per se
• CPC Class Codes: Y10S 435/968 : High energy substrates, e.g. fluorescent, chemiluminescent, radioactive
• CPC Class Codes: Y10S 435/969 : Multiple layering of reactants
• CPC Class Codes: Y10S 435/973 : Simultaneous determination of more than one analyte
• CPC Class Codes: Y10S 436/807 : Apparatus included in process claim, e.g. physical support structures
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 428/31971 : Of carbohydrate
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

A method and apparatus for preparation of a substrate containing a plurality of sequences. Photoremovable groups are attached to a surface of a substrate. Selected regions of the substrate are exposed to light so as to activate the selected areas. A monomer, also containing a photoremovable group, is provided to the substrate to bind at the selected areas. The process is repeated using a variety of monomers such as amino acids until sequences of a desired length are obtained. Detection methods and apparatus are also disclosed.

Claims

1. An apparatus for detection of fluorescent marked locations on a substrate comprising:

a) a substrate that comprises a plurality of different polymer sequences coupled to a surface of said substrate, wherein said plurality of different polymer sequences comprises a plurality of different oligonucleotide sequences, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10<sup>−

2</sup> cm² or less;

b) a light source for directing light at said surface of said substrate;

c) a detector for detecting light fluoresced from said surface in response to said light source;

d) a translator for translating said substrate relative to said light source whereby a plurality of said known locations of said surface of said substrate are exposed to said light; and

e) a data storage system for storing fluoresced light intensity as a function of location on said surface, said data storage system coupled to said translator and said detector.

2. The apparatus as recited in claim 1, wherein said plurality of different oligonucleotide sequences comprises greater than 10 different oligonucleotide sequences on said surface of said substrate.

3. The apparatus as recited in claim 1, wherein said plurality of different oligonucleotide sequences comprises at least 100 different oligonucleotide sequences on on said surface of said substrate.

4. The apparatus as recited in claim 1, wherein said plurality of different oligonucleotide sequences comprises at least 1000 different oligonucleotide sequences on said surface of said substrate.

5. The apparatus as recited in claim 1, wherein said plurality of different oligonucleotide sequences comprises greater than 10,000 different oligonucleotide sequences on said surface of said substrate.

6. The apparatus as recited in claim 1, wherein said plurality of different oligonucleotide sequences comprises greater than 100,000 different oligonucleotide sequences on said surface of said substrate.

7. The apparatus as recited in claim 1, wherein each of said different oligonucleotide sequences is in a known location on said surface of said substrate has an area of less than 10,000 μ

m².

8. An apparatus for detecting fluorescent marked locations on a surface of a substrate, comprising:

a point light source for generating an excitation light;

a substrate that comprises a plurality of different polymers coupled to a surface of said substrate, wherein said plurality of different polymers comprises a plurality of different oligonucleotide sequences, each of said different polymer sequences being coupled in a different known location of said surface, each of said known locations having an area of 10<sup>−

2</sup> cm² or less;

an objective lens for focusing said point light source at said surface of said substrate, whereby said fluorescently marked locations emit a fluoresced light in response to said excitation light;

an x-y translation stage for moving said substrate relative to said excitation light;

a dichroic mirror for reflecting light having a wavelength of said excitation light and passing light having a wavelength of said fluoresced light;

a photomultiplier and photon counter for detecting said fluoresced light; and

an appropriately programmed computer for recording said fluoresced light as a function of a position on said surface of said substrate from which said fluoresced light was emitted.

9. The apparatus as recited in claim 8, wherein said plurality of different oligonucleotide sequences comprises greater than 10 different oligonucleotide sequences on said surface of said substrate.

10. The apparatus as recited in claim 8, wherein said plurality of different oligonucleotide sequences comprises at least 100 different oligonucleotide sequences on said surface of said substrate.

11. The apparatus as recited in claim 8, wherein said plurality of different oligonucleotide sequences comprises at least 1000 different oligonucleotide sequences on said surface of said substrate.

12. The apparatus as recited in claim 8, wherein said plurality of different oligonucleotide sequences comprises greater than 10,000 different oligonucleotide sequences on said surface of said substrate.

13. The apparatus as recited in claim 8, wherein said plurality of different oligonucleotide sequences comprises greater than 100,000 different oligonucleotide sequences on said surface of said substrate.

14. The apparatus as recited in claim 8, wherein each of said different oligonucleotide sequences is in a known location on said surface of said substrate having an area of less than 10,000 μ

m².

15. A method of detecting the presence of a fluorescent marker on a surface of a substrate, the method comprising:

directing an excitation light at the surface of the substrate; and

detecting light fluoresced from the surface of the substrate at a plurality of locations on said substrate,

wherein said surface of said substrate comprises a plurality of different oligonucleotides covalently bound thereto, each of said different oligonucleotides being bound at a different known location on the substrate, and said fluorescent marker comprises a fluorescently labeled target nucleic acid that is capable of hybridizing with one or more of said plurality of different oligonucleotides.

16. A method of determining whether a fluorescently labeled ligand binds to one or more of a plurality of different polymer sequences, wherein said fluorescently labeled ligand comprises a fluorescently labeled target nucleic acid, and said plurality of different polymer sequences on surface of a substrate comprises a plurality of different oligonucleotides, comprising:

providing a plurality of different polymer sequences covalently bound to a surface of a substrate, each of said different polymer sequences being bound at a known location on the surface of the substrate;

contacting the surface of the substrate with the fluorescently labeled ligand;

washing the surface to remove unbound fluorescently labeled ligand from the surface of the substrate; and

detecting binding between the fluorescently labeled ligand and different polymer sequences, said detecting step comprising directing an excitation light at the surface of the substrate, and detecting light fluoresced from the surface of the substrate at a plurality of locations on said surface of said substrate.

17. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 10 different oligonucleotides on said surface of said substrate.

18. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises at least 100 different oligonucleotides on said surface of said substrate.

19. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises at least 1000 different oligonucleotides on said surface of said substrate.

20. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 10,000 different oligonucleotides on said surface of said substrate.

21. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 100,000 different oligonucleotides on said surface of said substrate.

22. The method as recited in claim 16, wherein each of the different polymer sequences is in a known location on said surface of said substrate having an area of less than 10,000 μ

m².

25. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 10 different polynucleotides on said surface of said substrate.

26. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises at least 100 different polynucleotides on said surface of said substrate.

27. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises at least 1000 different polynucleotides on said surface of said substrate.

28. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 10,000 different polynucleotides on said surface of said substrate.

29. The method as recited in claim 16, wherein the plurality of different polymer sequences comprises greater than 100,000 different polynucleotides on said surface of said substrate.

30. The method as recited in claim 16, wherein each of the different polymer sequences is in a known location on a surface of said substrate having an area of less than 10,000 μ

m².

23. A polynucleotide analysis apparatus comprising:

a substrate that comprises a plurality of different polynucleotides, each of said different polynucleotides being coupled in a different known location of a surface of said substrate, each of said known locations having an area of 10<sup>−

2</sup> cm² or less, said substrate comprising more than 10 of such polynucleotides, at least some of said polynucleotides coupled to labeled target molecules;

a light source for directing light at said surface of said substrate;

a detector for detecting light fluoresced from said surface in response to said light source;

a translator for translating said substrate relative to said light source whereby a plurality of said known locations of said substrate are exposed to light; and

a data storage system for storing fluoresced light intensity as a function of location on said substrate, said data storage system coupled to said translator and said detector, said data storage system storing light intensity as a function of location on said surface of said substrate.

24. A method of determining whether a fluorescently labeled ligand binds to one or more of a plurality of different polymer sequences, wherein said fluorescently labeled ligand comprises a fluorescently labeled target nucleic acid, said plurality of different polymer sequences a surface of a substrate and comprise a plurality of different polynucleotides, comprising:

providing at least 1000 different polymer sequences bound to said surface of said substrate, each of said different polymer sequences being bound at a known location on the surface of the substrate, said polymer sequences comprising polynucleotides;

contacting the surface of the substrate with the fluorescently labeled ligand;

washing the surface to remove unbound fluorescently labeled ligand from the surface of the substrate; and

detecting binding between the fluorescently labeled ligand and at least some of the more than 1000 different polymer sequences, said detecting step comprising directing an excitation light at the surface of the substrate, and detecting light fluoresced from the surface of the substrate at a plurality of locations on said surface of said substrate.

31. An apparatus for detecting labeled comprising:

(a) a substrate having a surface comprising at least 10 different nucleic acids at known locations on the surface of the substrate, each of the known locations having an area of 10<sup>−

2</sup> cm² or less, some of the nucleic acids being bound to labeled receptors;

(b) an excitation light source;

(c) a detector adapted to receive a signal from said bound labeled receptors on said surface;

(d) a translator adapted to move said substrate relative to said detector; and

(e) a data collection system adapted to receive input from said detector.

32. The apparatus of claim 31 wherein the surface of the substrate has at least 100 different nucleic acids.

33. The apparatus of claim 31 wherein the surface of the substrate has at least 1,000 different nucleic acids.

34. The apparatus of claim 31 wherein the surface of the substrate has at least 10,000 different nucleic acids.

35. The apparatus of claim 31 wherein the surface of the substrate has at least 400 different nucleic acids/cm².

36. The apparatus of claim 31 wherein the surface of the substrate has at least 1,000 different nucleic acids/cm².