Multiplexed diagnostic and therapeutics

• US 7,413,852 B2
• Filed: 12/11/2002
• Issued: 08/19/2008
• Est. Priority Date: 12/31/1996
• Status: Expired due to Term

• Current Assignee: HTG Molecular Diagnostics, Inc.
• Original Assignee: High Throughput Genomics, Inc.
• Inventors: Balch, William J.
• Primary Examiner(s): Chin; Christopher L.
• Application Number:
  US10/316,077
• Publication Number:
  US 20040023249A1
• Time in Patent Office: 2,078 Days
• Field of Search: 422/56, 422/58, 435/6, 435/287.2, 436/512, 436/518, 436/527
• US Class Current: 435/6.11
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• 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 2219/0061 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 2219/00621 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 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0893 having a very large number of wells, microfabricated wells
• CPC Class Codes: B01L 3/0262 using touch-off at substrate or container
• CPC Class Codes: B01L 3/0268 : using pulse dispensing or spraying, eg. inkjet type, piezo actuated ejection of droplets from capillaries
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• 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 2035/00237 : Handling microquantities of analyte, e.g. microvalves, capillary networks
• CPC Class Codes: G01N 2035/1034 : Transferring microquantities of liquid
• CPC Class Codes: G01N 2035/1039 : Micropipettes, e.g. microcapillary tubes
• CPC Class Codes: G01N 2035/1041 : Ink-jet like dispensers
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 35/0099 : comprising robots or similar manipulators robots per se B25J
• CPC Class Codes: G01N 35/028 : having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1065 : Multiple transfer devices
• CPC Class Codes: G01N 35/1074 : arranged in a two-dimensional array
• CPC Class Codes: Y10S 435/808 : Optical sensing apparatus
• CPC Class Codes: Y10S 436/80 : Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10S 436/804 : Radioisotope, e.g. radioimmunoassay
• CPC Class Codes: Y10S 436/805 : Optical property
• CPC Class Codes: Y10S 436/809 : Multifield plates or multicontainer arrays
• CPC Class Codes: Y10T 436/11 : Automated chemical analysis
• CPC Class Codes: Y10T 436/115831 : Condition or time responsive

Abstract

A method and apparatus for analyzing molecular structures within a sample substance using an array having a plurality of test sites upon which the sample substance is applied. The invention is also directed to a method and apparatus for constructing molecular arrays having a plurality of test sites. The invention allows for definitive high throughput analysis of multiple analytes in complex mixtures of sample substances. A combinatorial analysis process is described that results in the creation of an array of integrated chemical devices. These devices operate in parallel, each unit providing specific sets of data that, when taken as a whole, give a complete answer for a defined experiment. This approach is uniquely capable of rapidly providing a high density of information from limited amounts of sample in a cost-effective manner.

Claims

1. A reaction substrate useful for analyzing one or more targets in a sample comprising before the addition of the sample multiple regions, at least two of which are substantially identical, each region comprising an array of at least eight different biosites, each comprising a different type of capture probe bound to said substrate, each capture probe bound to a corresponding bi-functional target probe comprising two domains, a first domain comprising a capture probe specific domain which specifically binds a corresponding capture probe, and a second domain comprising a target specific domain.

2. The reaction substrate of claim 1, wherein said reaction substrate is about 50 μ

m to about 300 μ

m in thickness.

3. The reaction substrate of claim 1, wherein said capture probe is a first oligonucleotide, each capture probe specific domain is a second oligonucleotide, and each target analyte specific domain is a third oligonucleotide.

4. The reaction substrate of claim 1, wherein each capture probe is a hapten, each capture specific domain is a hapten binding polypeptide, and each target analyte specific domain is an oligonucleotide.

5. The reaction substrate of claim 4, wherein each hapten binding polypeptide is selected from the group consisting of an antibody, a Fab, an F(ab′

)₂, an Fv, an SCA and a CDR.

6. The reaction substrate of claim 3, wherein each target analyte specific domain is an oligonucleotide nucleic acid amplification primer.

7. The reaction substrate of claim 1, wherein at least one reaction substrate is contained in at least one reaction chamber.

8. The reaction substrate of claim 7, wherein a plurality of reaction substrates are contained within each reaction chamber.

9. The reaction substrate of claim 8, further including a reaction vessel, wherein a plurality of reaction chambers are contained within said reaction vessel.

10. The reaction substrate of claim 8, wherein said reaction vessel comprises about 2 to about 10,000 reaction chambers.

11. The reaction substrate of claim 1, wherein said reaction substrate is optically clear.

12. The reaction substrate of claim 1, wherein each region comprises at least about 64 different capture probes.

13. The reaction substrate of claim 1, having at least about 96 to about 1536 substantially identical regions.

14. The reaction substrate of claim 1, wherein each of said regions comprises about 30 to about 100 capture probes, each specific for a different target.

15. The reaction substrate of claim 1, having about 96 substantially identical regions, wherein each region comprises about 16, 36, 46 or 100 different oligonucleotide capture probes.

16. The reaction substrate of claim 1, having about 384 substantially identical regions, wherein each region comprises about 9, 16, or 25 different oligonucleotide capture probes.

17. The reaction substrate of claim 1, having about 1536 substantially identical regions, wherein each region comprises about 4 or 9 different oligonucleotide capture probes.

18. The reaction substrate of claim 1, wherein said regions are further subdivided into smaller subregions.

19. The reaction substrate of claim 1, wherein the region further comprises controls for hybridization efficiency or specificity.

20. The reaction substrate of claim 1, wherein the region further comprises controls for the capacity of a locus to bind target.

21. The reaction substrate of claim 1, wherein said probe is a peptide or protein molecule.

22. The reaction substrate of claim 1, wherein said array of biosites comprises from about 8 to about 10,000 biosites.

23. The reaction substrate of claim 1, wherein said array of biosites comprises from about 8 to about 100,000 biosites.

24. A method of determining the presence of a nucleic acid target in a sample comprising contacting a nucleic acid comprising a target sequence or a complement thereof with the reaction substrate of claim 1.

25. A method of determining the presence of a nucleic acid target in a sample comprising contacting a nucleic acid comprising a target sequence or a complement thereof with the reaction substrate of claim 1.

26. A method of claim 25 comprising contacting said substrate with a medium to which was added said nucleic acid sequence or complement and corresponding target probe(s).

27. A method of claim 25 comprising forming on said substrate said capture probe/target probe complex and then contacting the latter with said nucleic acid comprising a target sequence or a complement thereof.