Systems, tools and methods of assaying biological materials using spatially-addressable arrays

• US 20030003495A1
• Filed: 07/11/2002
• Published: 01/02/2003
• Est. Priority Date: 11/08/1999
• Status: Active Grant

• Current Assignee: Glenda C. Delenstarr
• Original Assignee: Glenda C. Delenstarr
• Inventors: Delenstarr, Glenda C.
• Granted Patent: US 7,052,841 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2523/101 Crosslinking agents, e.g. psoralen
• CPC Class Codes: C12Q 2537/137 a displacement step
• CPC Class Codes: C12Q 2565/133 conformational analysis
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

Systems, tools and methods of assaying biological material are used to perform complex sandwich hybridization assays. The tools used comprise biological solution probes that are customized for each assay. The solution probe comprises a first region for hybridizing to a probe, in a generic set of capture probes on a universal assay apparatus, and a second region for hybridizing to a target in a sample. The solution probe assembles the target to the assay apparatus by hybridizing the second region to the target and the first region to the capture probe. In array assays, one or more biological samples, having one or more targets per sample, can be multiplexed on the same universal array comprising the generic set of capture probes in an array pattern of features on the substrate. The customized solution probe addresses and assembles a predetermined target-sample combination onto the array at a corresponding capture probe address location. The systems, tools and methods have specificity and sensitivity by systematically providing a reduced likelihood of cross-hybridizations and intramolecular structures in the probes. Specificity and sensitivity of the assay are provided by the incorporation of a chemically modified monomer in the capture probe and a similarly modified monomer complement in the first region of the solution probe. The modified monomers preferentially hybridize with each other. When the probe and respective probe region are oligonucleotides, the complementary modified nucleotides have a reversed polarity relative to the polarity of the respective probe and probe region. The complementary reversed polarity nucleotides form a thermodynamically more stable hybridization to each other than a hybridization between the reversed polarity nucleotide and a complementary nucleotide whose polarity is not similarly reversed.

Claims

1. An assay system for multiplexing on a single array one or more biological samples having one or more biological targets per sample, comprising:

an array apparatus that comprises a first plurality of biological probes on a substrate in an array pattern of features, wherein each first probe of the first plurality is different from others in the first plurality, each different first probe is located at a different feature location of the array and is a different address on the array; and

a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region, wherein the first region of each second probe is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes.

2. The assay system of claim 1, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises the one target per sample to be multiplexed, and wherein the second region of each second probe is the same and is complementary to the one target per sample in the plurality of different samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different samples.

3. The assay system of claim 1, wherein the one or more biological samples comprises the one sample and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets.

4. The assay system of claim 1, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample in the plurality of samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets in the different samples.

5. The assay system of claim 1, wherein the first plurality of biological probes comprises a group selected from oligonucleotides, antigens, antibodies, receptors or ligands.

6. The assay system of claim 5, wherein the first regions of the second plurality of biological probes comprise a group complementary to the first plurality of probes selected from oligonucleotides, antigens, antibodies, receptors or ligands.

7. The assay system of claim 1, wherein each first probe and each complementary first region comprises a member of a specific binding pair that cross-links to each other during the assay.

8. The assay system of claim 1, wherein the second regions of the second plurality of probes comprise a group complementary to the one or more biological targets and is selected from oligonucleotides, cDNAs, PCR products, antigens, antibodies, receptors or ligands.

9. The assay system of claim 1, wherein the first plurality of probes comprises multiple copies of each first probe at each respective feature location, and wherein the second plurality of probes comprises multiple copies of each second probe.

10. The assay system of claim 1, wherein each first probe of the first plurality of probes comprises a first sequence of monomers, and wherein the first region of each second probe of the second plurality of probes comprises a second sequence of monomers, and wherein the first sequence of monomers comprises a first modified monomer and the second sequence of monomers comprises a second similarly modified monomer that is complementary to the first modified monomer, wherein the modification causes the complementary first modified monomer and second modified monomer to preferentially hybridize or bind with each other instead of with a complementary monomer that is not similarly modified.

11. The assay system of claim 1, wherein the first plurality of probes comprises oligonucleotide first probes and the first regions of the second plurality of probes comprises oligonucleotide regions, wherein the oligonucleotide first probes comprise:

a plurality of first nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, and

a first modified nucleotide that has a reversed polarity with respect to the polarity of the plurality of first nucleotides, and

wherein the oligonucleotide regions of the second probes comprise;

a plurality of second nucleotides having the same polarity as the polarity of the plurality of first nucleotides, the plurality of second nucleotides being complementary to the plurality of first nucleotides, and

a second modified nucleotide that has a reversed polarity with respect to the polarity of the plurality of second nucleotides, the second reversed polarity nucleotide being complementary to the first reversed polarity nucleotide in the first probe, and

wherein the complementary first reversed polarity nucleotide and the second reversed polarity nucleotide preferentially hybridize with each other instead of with a complementary nucleotide whose polarity is not similarly reversed, such that specificity and sensitivity is provided to the assay.

12. The assay system of claim 1, wherein the first plurality of probes is generic to the one or more biological samples and to the one or more biological targets per sample being assayed, and wherein the second plurality of probes is custom to the one or more biological targets being assayed and to the first plurality of probes.

14. The assay method of claim 13, wherein the step of assembling comprises the steps of:

incubating the one or more biological samples with the second plurality of probes to hybridize the biological targets per sample with complementary second regions of the second probes; and

incubating the second plurality of probes with the first plurality of probes on the array apparatus to hybridize the first regions of the second plurality of probes to corresponding complementary first probes.

15. The assay method of claim 13, wherein the step of removing comprises the step of washing any unhybridized second probes and any unhybridized targets off the array apparatus surface.

16. The assay method of claim 13, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises the one target per sample to be multiplexed, wherein the second region of each second probe is the same and complementary to the one target per sample, and wherein the step of assembling comprises the steps of:

separately preincubating a different second probe with each different sample of the plurality of different samples so that each different second probe hybridizes to the combination of target-sample, and

incubating together the different hybridized second probes with the array apparatus to hybridize the different first regions of the different hybridized second probes with complementary first probes on the array corresponding to the target-sample combinations.

17. The assay method of claim 13, wherein the one or more biological samples comprises the one sample and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, wherein the second region of each second probe is different and complementary to a different one of the plurality of different targets per sample, and wherein the step of assembling comprises the steps of:

incubating the second plurality of probes with the sample so that each different second probe hybridizes to complementary one of the different targets, and

incubating together the different second probes with the array apparatus to hybridize the different first regions of the different second probes with complementary first probes on the array corresponding to the target-sample combinations.

18. The assay method of claim 13, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, wherein the second region of each second probe is different and complementary to a different one of the plurality of different targets per sample, and wherein the step of assembling comprises the steps of:

separately preincubating different second probes that are complementary to the different targets of each different sample so that each different second probe hybridizes to the combination of target-sample, and

incubating together the different hybridized second probes with the array apparatus to hybridize the different first regions of the different hybridized second probes with complementary first probes on the array corresponding to the target-sample combinations.

19. The assay method of claim 13, wherein each first probe of the first plurality of probes comprises a first sequence of monomers, and wherein the first region of each second probe of the second plurality of probes comprises a second sequence of monomers, and wherein the first sequence of monomers comprises a first modified monomer and the second sequence of monomers comprises a second similarly modified monomer that is complementary to the first modified monomer, and wherein in the step of assembling, the first modified monomer preferentially hybridizes to the complementary second modified monomer instead of to a complementary monomer that is not similarly modified, such that the method provides specificity and sensitivity to the assay.

20. The assay method of claim 13, wherein the first plurality of probes comprises oligonucleotide first probes and the first regions of the second plurality of probes comprises oligonucleotide regions, wherein the oligonucleotide first probes comprise:

a plurality of first nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, and

a first modified nucleotide that has a reversed polarity with respect to the polarity of the plurality of first nucleotides, and

wherein the oligonucleotide regions of the second probes comprise;

a plurality of second nucleotides having the same polarity as the polarity of the plurality of first nucleotides, the plurality of second nucleotides being complementary to the plurality of first nucleotides, and

a second modified nucleotide that has a reversed polarity with respect to the polarity of the plurality of second nucleotides, the second reversed polarity nucleotide being complementary to the first reversed polarity nucleotide in the first probe, and

wherein in the step of assembling, the first plurality of probes are hybridized to the second plurality of probes and the first reversed polarity nucleotide preferentially hybridizes to the complementary second reversed polarity nucleotide instead of to a complementary nucleotide whose polarity is not similarly reversed, such that the method provides specificity and sensitivity to the assay.

21. The assay method of claim 14, wherein each first probe and each complementary first region further comprises a member of a specific binding pair, and wherein the step of incubating further comprises the step of cross-linking the members of the pair.

86. The assay method of claim 14, wherein the steps of incubating are performed simultaneously.

87. The assay method of claim 14, wherein the step of incubating the second plurality of probes with the first plurality of probes is performed before the step of incubating the one or more biological samples with the second plurality of probes.

88. The assay method of claim 17, wherein the steps of incubating are performed simultaneously.

96. The assay method of multiplexing of claim 13, wherein the assay results are analyzed at a first location, the method further comprising communicating the assay results or a conclusion based on the assay results to a second location remote from the first location.

97. The assay method of multiplexing of claim 96, wherein the one or more samples was obtained from a third location remote from the first location or the second location.

100. The assay system of claim 1, where the first regions of the second plurality of probes are hybridized to the complementary first probes of the first plurality of probes.

13. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, comprising the steps of:

providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the first plurality being different, each different first probe being located on a different feature location of the array, wherein each different first probe is a different address on the array apparatus;

providing a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region that is complementary to a different one of the first probes, the second region of each second probe being complementary to a target from the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;

assembling the different target-sample combinations to the array with the second set of probes;

removing unassembled biological materials from the array; and

analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations.

22. A sandwich hybridization system for assaying biological material having specificity and sensitivity comprising:

an apparatus having a first plurality of biological probes attached to a substrate, each first probe of the first plurality comprising a first sequence of monomers, wherein at least one first monomer of the first sequence is chemically modified; and

a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, the first region comprising a second sequence of monomers, wherein at least one second monomer is modified similarly to the first modified monomer, the second sequence of monomers and the second modified monomer are respectively complementary to the first sequence of monomers and first modified monomer in the first probe, and wherein the second region is complementary to the biological material to be assayed, wherein the first modified monomer preferentially hybridizes to the complementary second modified monomer during the assay instead of to a complementary monomer that has not been similarly modified.

23. The assay system of claim 22, wherein the first biological probes comprises a group selected from oligonucleotides, antigens, antibodies, receptors or ligands.

24. The assay system of claim 23, wherein the first region of the second biological probes comprises a group complementary to the first probes selected from oligonucleotides, antigens, antibodies, receptors or ligands.

25. The assay system of claim 22, wherein the biological material to be assayed comprises a group selected from oligonucleotides, mRNA, antigens, antibodies, receptors or ligands.

26. The assay system of claim 25, wherein the second region of the second probe comprises a group complementary to the biological material to be assayed and is selected from oligonucleotides, cDNAs, PCR products, antigens, antibodies, receptors or ligands.

27. The assay system of claim 22, wherein the each first probe further comprises a first member of a specific binding pair, and first region of each second probe comprises a second member of the specific binding pair, the first member and the second member cross-link during the assay.

29. The assay system of claim 28, wherein the oligonucleotide first probes and the oligonucleotide first regions each have a sufficient number of complementary nucleotides to form hybridizations strong enough to withstand assay conditions after hybridization.

30. The assay system of claim 29, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 5 to 50 nucleotides in length L with from one to ⅔

L reversed polarity nucleotides.

31. The assay system of claim 30, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 30 nucleotides in length.

32. The assay system of claim 31, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 25 nucleotides in length.

33. The assay system of claim 29, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 5 to 50 nucleotides in length L with from ¼

L to ½

L reversed polarity nucleotides.

34. The assay system of claim 33, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 30 nucleotides in length.

35. The assay system of claim 34, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 25 nucleotides in length.

36. The assay system of claim 29, wherein the oligonucleotide first probes and the oligonucleotide first regions each further comprise a member of a specific binding pair that cross-link to each other to strengthen the hybridization.

38. The method of claim 37, wherein the first sequence of monomers comprises:

a first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity; and

wherein the first modified monomer comprises;

a first nucleotide with a reversed polarity relative to the polarity of the first sequence of nucleotides; and

wherein the second sequence of monomers comprises;

a second sequence of nucleotides complementary to the first sequence that has the same (3′

→

5′

) or (5′

→

3′

) polarity as the polarity of the first sequence of nucleotides; and

wherein the second modified monomer comprises;

a second nucleotide with reversed polarity relative to the polarity of the second sequence, the second reversed polarity nucleotide being complementary to the first reversed polarity nucleotide, and

wherein in the step of hybridizing the second set of probes with the first set of probes, the complementary first reversed polarity nucleotide and second reversed polarity nucleotide form a more thermodynamically stable hybridization with each other than either of them with a complementary nucleotide whose polarity is not similarly reversed.

39. The assay method of claim 37, wherein the first sequence of monomers and the second sequence of monomers each comprise a member of a specific binding pair, and wherein the step of hybridizing the second set of probes with the first set of probes comprises the step of cross linking the members of the binding pair.

89. The sandwich hybridization method of claim 37, wherein the hybridizations in the step of hybridizing the biological material to the second set of probes and hybridizing the second set of probes to the first set of probes on the apparatus are performed simultaneously.

28. A sandwich hybridization system for assaying biological material having specificity and sensitivity comprising:

an apparatus having a first plurality of oligonucleotide probes attached to a substrate, each first probe of the first plurality comprising;

a first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity,

at least one first nucleotide that has a reversed polarity relative to the polarity of the first sequence; and

a second plurality of biological probes, each second probe of the second plurality comprising a first oligonucleotide region and a second region, the first oligonucleotide region comprising;

a second sequence of nucleotides having the same polarity as the polarity of the the first sequence, and are respectively complementary to the first sequence of nucleotides, and

at least one second nucleotide that is complementary to the at least one reversed polarity first nucleotide, the second nucleotide having a reversed polarity relative to the polarity of the second sequence; and

the second region is complementary to the biological material to be assayed, and

wherein the reversed polarity first nucleotide and the complementary reversed polarity second nucleotide preferentially hybridizes to each other during the assay than to a complementary nucleotide whose polarity is not similarly reversed.

37. A sandwich hybridization method of assaying biological materials with specificity and sensitivity comprising the steps of:

providing an apparatus having a first set of biological probes on a surface of a substrate, each first probe of the first set comprising a sequence of monomers, wherein at least one first monomer of the first sequence is chemically modified;

providing a second set of biological probes, each second probe of the second set comprises a first region and a second region, the first region of each second probe comprises a second sequence of monomers, wherein at least one second monomer of the second sequence is chemically modified similarly to the first modified monomer, the second sequence of monomers and second modified monomer being complementary respectively to the first sequence and first modified monomer, and wherein the second region of each second probe is complementary to a biological material to be assayed,

hybridizing the biological material to the second set of probes and hybridizing the second set of probes to the first set of probes on the apparatus, wherein the first modified monomer preferentially hybridizes to the complementary second modified monomer instead of to a complementary monomer that has not been similarly modified;

removing unhybridized biological material from the apparatus; and

analyzing assay results.

40. A assay system with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample on a single array comprising:

an array apparatus that comprises a first plurality of biological probes on a substrate in an array pattern of features, wherein each first probe of the first plurality comprises a different first sequence of monomers, wherein at least one first monomer of the first sequence comprises a chemical modification, and wherein each different first probe is located at a different feature location of the array and is a different address on the array apparatus; and

a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region, wherein each different first region comprises a second sequence of monomers, the second sequence comprising at least one second monomer having a chemical modification similar to, and being complementary to, the first modified monomer, wherein each different first region is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes , and wherein the first modified monomer and the second modified monomer preferentially hybridize together instead of to a monomer that has not been similarly modified.

41. The assay system of claim 40, wherein one target per sample in a plurality of different samples are to be multiplexed, and wherein the second region of each second probe is the same and is complementary to the one target per sample in the plurality of different samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different samples.

42. The assay system of claim 40, wherein a plurality of different targets per sample in one sample are to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets.

43. The assay system of claim 40, wherein a plurality of different targets per sample in a plurality of different samples are to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample in the plurality of samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets in the different samples.

44. The assay system of claim 40, wherein the first plurality of biological probes comprises a group selected from oligonucleotides, antigens, antibodies, receptors or ligands.

45. The assay system of claim 44, wherein the first regions of the second plurality of biological probes comprise a group complementary to the first plurality of probes selected from oligonucleotides, antigens, antibodies, receptors or ligands.

46. The assay system of claim 40, wherein the one or more biological targets comprises a group selected from oligonucleotides, mRNA, antigens, antibodies, receptors or ligands.

47. The assay system of claim 46, wherein the second regions of the second plurality of probes comprise a group complementary to the one or more biological targets selected from oligonucleotides, cDNAs, PCR products, antigens, antibodies, receptors or ligands.

48. The assay system of claim 40, wherein the first plurality of probes comprises multiple copies of each first probe at each respective feature location, and wherein the second plurality of probes comprises multiple copies of each second probe.

49. The assay system of claim 45, wherein the first plurality of probes are oligonucleotides and first sequence of monomers comprises:

a first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity; and

the first modified monomer comprises;

a first nucleotide with a reversed polarity relative to the polarity of the first sequence; and

wherein the first regions of the second plurality of probes are oligonucleotides

and the second sequence of monomers comprises;

a second sequence of nucleotides having the same polarity as in the polarity of the first sequence of nucleotides, the second sequence of nucleotides being complementary to the first sequence; and

the second modified monomer comprises;

a second nucleotide with a reversed polarity relative to the polarity of the second sequence, the reversed polarity second nucleotide being complementary to the reversed polarity first nucleotide; and

wherein the complementary first reversed polarity nucleotide and second reversed polarity nucleotide form a more thermodynamically stable hybridization with each other than with a complementary nucleotide whose polarity is not similarly reversed.

50. The system of claim 40, wherein the first sequence of monomers of the first probes and the second sequence of monomers of the first region of the second probes each further comprises a member of a specific binding pair.

51. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, the method having specificity and sensitivity and comprising the steps of:

providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the plurality being different from others in the first plurality by comprising a different first sequence of monomers, wherein the first sequence of monomers comprises at least one chemically modified monomer, each different first probe being located on a different feature location of the array apparatus, wherein each different first probe -is a different address on the array apparatus;

providing a second plurality of biological probes, each second probe of the second plurality comprising a first region and a second region, each second probe being different from others in the second plurality by comprising a different first region that is complementary to a different one of the first probes, the first region comprising a second sequence of monomers, wherein the second sequence of monomers comprises at least one monomer similarly modified as and being complementary to the first modified monomer, the second region of each second probe being complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;

assembling the different target-sample combinations to the array with the second set of probes, comprising the steps of;

incubating the one or more biological samples with the second plurality of probes to hybridize the biological targets per sample with complementary second regions of the second probes; and

incubating the second plurality of probes with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the second plurality of probes to corresponding complementary first probes by hybridizing the second modified monomer of each first region to its complementary first modified monomer of each first probe instead of with a complementary monomer that has not been similarly modified;

removing unassembled biological materials from the array; and

analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations.

52. The assay method of multiplexing having sensitivity and specificity of claim 51, wherein the target-sample combinations are selected from the one target per sample in a plurality of different samples, a plurality of different targets per sample in the one sample, or a plurality of different samples having a plurality of different targets per sample.

90. The assay method of claim 51, wherein the steps of incubating are performed simultaneously.

53. A assay system with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample on a single array comprising:

an array apparatus that comprises a first plurality of oligonucleotide probes on a substrate in an array pattern of features, wherein each first probe of the first plurality comprises;

a first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, at least one first nucleotide has a reversed polarity relative to the polarity of the first sequence, and

wherein each first sequence is different from other first sequences, each different first probe is located at a different feature location of the array, and each different first probe is a different address on the array apparatus; and

a second plurality of biological probes, each second probe of the second plurality comprising an oligonucleotide first region and a second region, each second probe being different from others in the second plurality by comprising a different oligonucleotide first region complementary to a different one of the first oligonucleotide probes, wherein each different oligonucleotide first region comprises;

a second sequence of nucleotides having the same polarity as the polarity of the first plurality of probes, at least one second nucleotide having a reversed polarity relative to the polarity of the second sequence, and

wherein the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes,

wherein the first reversed polarity nucleotide and the second reversed polarity nucleotide preferentially hybridize to each other by forming a more thermodynamically stable hybridization than to a complementary nucleotide whose polarity is not similarly reversed.

54. The assay system of claim 53, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises the one target per sample to be multiplexed, and wherein the second region of each second probe is the same and is complementary to the one target per sample in the plurality of different samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different samples.

55. The assay system of claim 53, wherein the one or more biological samples comprises the one sample and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets.

56. The assay system of claim 53, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, and wherein the second region of each second probe is different and is complementary to a different one of the plurality of targets per sample in the plurality of samples, such that each solution probe addresses the different combinations of target-sample to the corresponding first probes on the array according to the different targets in the different samples.

57. The assay system of claim 53, wherein each first probe and the first region of each second probe each further comprises (5′

→

3′

) and (3′

→

5′

) internucleotide linkage and an inverted polarity (3′

→

3′

) or (5′

→

5′

) internucleotide linkage, the inverted polarity linkage of each first probe being between the reversed polarity first nucleotide and a nucleotide of the first sequence, and the inverted polarity linkage of the first region of each second probe being between the reversed polarity second nucleotide and a nucleotide of the second sequence.

58. The assay system of claim 57, wherein the second regions of the second plurality of probes comprise a group complementary to the one or more biological targets and is selected from oligonucleotides, cDNAs, PCR products, antigens, antibodies, receptors or ligands.

59. The assay system of claim 53, wherein the first plurality of probes comprises multiple copies of each first probe at each respective feature location, and wherein the second plurality of probes comprises multiple copies of each second probe.

60. The assay system of claim 53, wherein the oligonucleotide first probes and the oligonucleotide first regions have a sufficient number of complementary nucleotides to form hybridizations between complementary nucleotides that are strong enough to withstand assay conditions after hybridization.

61. The assay system of claim 60, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 5 to 50 nucleotides in length L with from one to ⅔

L reversed polarity nucleotides.

62. The assay system of claim 60, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 5 to 50 nucleotides in length L with from ¼

L to ½

L reversed polarity nucleotides.

63. The assay system of claim 61, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 30 nucleotides in length L.

64. The assay system of claim 62, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 30 nucleotides in length L.

65. The assay system of claim 63, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 25 nucleotides in length L.

66. The assay system of claim 64, wherein the oligonucleotide first probes and the oligonucleotide first regions are from 10 to 25 nucleotides in length L.

67. The assay system of claim 60, wherein the oligonucleotide first probes and the oligonucleotide first regions each further comprise a member of a specific binding pair that cross-link to each other.

68. The assay system of claim 53, wherein the first plurality of probes is generic to the one or more biological samples and to the one or more biological targets per sample being assayed, and wherein the second plurality of probes is custom to the one or more biological targets being assayed and to the first plurality of probes.

70. The assay method of claim 69, wherein the step of assembling comprises the steps of:

incubating the one or more biological samples with the second plurality of probes to hybridize the biological targets per sample with complementary second regions of the second probes; and

incubating the second plurality of probes with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the second plurality of probes to corresponding complementary first probes by forming a more thermodynamically stable hybridization between the complementary first reversed polarity nucleotide and the second reversed polarity nucleotide than between either of the reversed polarity nucleotides and a complementary nucleotide whose polarity is not similarly reversed.

71. The assay method of claim 69, wherein the step of removing comprises the step of washing any unhybridized second probes and any unhybridized targets off the array apparatus surface.

72. The assay method of claim 69, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises the one target per sample to be multiplexed, wherein the second region of each second probe is the same and complementary to the one target per sample, and wherein the step of assembling comprises the step of:

separately preincubating a different second probe with each different sample of the plurality of different samples so that each different solution probe hybridizes to the combination of target-sample, and

incubating the hybridized second plurality of probes with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the hybridized second plurality of probes to corresponding complementary first probes by forming a more thermodynamically stable hybridization between the complementary first reversed polarity nucleotide and the second reversed polarity nucleotide than between either of the reversed polarity nucleotides and a complementary nucleotide whose polarity is not similarly reversed.

73. The assay method of claim 69, wherein the one or more biological samples comprises the one sample and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, wherein the second region of each second probe is different and complementary to a different one of the plurality of different targets per sample, and wherein the step of assembling comprises the step of:

incubating the second plurality of probes with the sample so that each different solution probe hybridizes to a complementary one of the different targets, and

incubating the second plurality of probes with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the second plurality of probes to corresponding complementary first probes by forming a more thermodynamically stable hybridization between the complementary first reversed polarity nucleotide and the second reversed polarity nucleotide than between either of the reversed polarity nucleotides and a complementary nucleotide whose polarity is not similarly reversed.

74. The assay method of claim 69, wherein the one or more biological samples comprises a plurality of different samples and the one or more biological targets per sample comprises a plurality of different targets per sample to be multiplexed, wherein the second region of each second probe is different and complementary to a different one of the plurality of different targets per sample, and wherein the step of assembling comprises the step of:

separately preincubating the different second probes that are complementary to the different targets of each different sample so that each different solution probe hybridizes to the combination of target-sample, and

incubating the hybridized second plurality of probes together with the first plurality of probes on the array apparatus to preferentially hybridize the first regions of the hybridized second plurality of probes to corresponding complementary first probes by forming a more thermodynamically stable hybridization between the complementary first reversed polarity nucleotide and the second reversed polarity nucleotide than between either of the reversed polarity nucleotides and a complementary nucleotide whose polarity is not similarly reversed.

75. The assay method of claim 69, wherein each first probe and each complementary first regions further comprise a member of a specific binding pair, and wherein the step of incubating further comprises the step of cross-linking the members of the binding pair to strengthen the hybridization between complementary first probes and first regions of the second probes during the assay.

76. The assay system of claim 70, wherein the complementary reversed polarity nucleotides preferentially hybridize, such that the assay is specific by providing a reduced likelihood of cross-hybridizations between oligonucleotide probes and noncomplementary first regions of the set of biological probes, the second regions of the set of biological probes, or the biological material, and such that the assay is sensitive by providing a reduced likelihood of intramolecular structures within any of the oligonucleotide probes and first regions of the set of biological probes.

91. The assay method of claim 70, wherein the steps of incubating are performed simultaneously.

92. The assay method of claim 70, wherein the steps of incubating are performed sequentially.

93. The assay method of claim 70, wherein the step incubating the second plurality of probes with the first plurality of probes is performed before the step of incubating the one or more biological samples with the second plurality of probes.

94. The assay method of claim 73, wherein the steps of incubating are performed simultaneously.

98. The assay method of multiplexing of claim 69, wherein the assay results are analyzed at a first location, the method further comprising communicating the assay results or a conclusion based on the assay results to a second location remote from the first location.

99. The assay method of multiplexing of claim 98, wherein the one or more samples was obtained from a third location remote from the first location or the second location.

69. An assay method of multiplexing on a single array one or more biological samples having one or more biological targets per sample, the method having specificity and sensitivity and comprising the steps of:

providing an array apparatus having a first plurality of biological probes on a substrate in an array pattern of features, each first probe of the plurality being a different oligonucleotide by comprising a different first sequence of nucleotides, wherein the first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, at least one first nucleotide has a reversed polarity relative to the polarity of the first sequence, and wherein each different first probe is located at a different feature location of the array, and each different first probe is a different address on the array apparatus;

providing a second set of biological probes, each second probe of the second set comprising an oligonucleotide first region and a second region, each second probe being different from others in the second set by comprising a different oligonucleotide first region complementary to a different one of the first oligonucleotide probes, each different oligonucleotide first region comprises a different second sequence of nucleotides having the same polarity as the first sequence of nucleotides, at least one second nucleotide having a reversed polarity relative to the polarity of the second sequence, and wherein the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes;

assembling the different target-sample combinations to the array with the second set of probes;

removing unassembled biological materials from the array; and

analyzing assay results comprising the step of determining the presence of the one or more targets in the one or more samples from whether the different target-sample combinations are assembled on the array at the corresponding first probe locations.

77. A kit for multiplexing one or more biological samples having one or more targets per sample in an addressable, self-assembling assay on a single array comprising:

an array apparatus that comprises a first set of biological probes on a substrate in an array pattern of features, wherein each first probe of the first set is different from others in the first set, each different first probe is located at a different feature location of the array corresponding to a different address on the array;

a second set of biological probes, each second probe of the second set comprising a first region and a second region, each second probe being different from others in the second set by comprising a different first region, wherein the first region of each second probe is complementary to a different first probe of the first plurality, and the second region of each second probe is complementary to a target in the one or more samples, such that each second probe addresses a different predetermined combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed to each different address on the array corresponding to the different first probes ; and

instructions for using the apparatus and second set of probes in a multiplexing assay of target-sample combinations selected from one target per sample in a plurality of different samples, a plurality of different targets per sample in one sample, or a plurality of different targets per sample in a plurality of different samples.

78. The kit of claim 77 further comprising specificity and sensitivity, wherein each first probe of the first set comprises a first sequence of monomers, wherein at least one first monomer in the first sequence comprises a chemical modification, and wherein each first region of the second set comprises a second sequence of monomers, wherein at least one second monomer in the second sequence comprises a modification similar to the modification of the first modified monomer, the first modified monomer being complementary to the second modified monomer, and wherein the chemical modification causes the first modified monomer and the second modified monomer to preferentially hybridize together during the assay instead of to a complementary monomer that does not comprise the similar modification.

79. The kit of claim 77 further comprising specificity and sensitivity, wherein each first probe of the first set comprises a first sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, wherein at least one first nucleotide in the first sequence has a reversed polarity relative to the polarity of the first sequence, and wherein each first region of the second set comprises a second sequence of nucleotides having the same polarity internucleotide linkages as the first sequence, wherein at least one second nucleotide in the second sequence has the same reversed polarity as the first reversed polarity nucleotide, the first reversed polarity nucleotide being complementary to the second reversed polarity nucleotide, and wherein the reversed polarity nucleotides preferentially hybridize by forming a thermodynamically more stable hybridization to each other during the assay instead of to a complementary nucleotide whose polarity is not similarly reversed.

80. A set of biological probes in solution for sandwich hybridization assays of one or more biological targets from one or more biological samples multiplexed on an array of biological features, the set of biological probes comprising a plurality of different solution probes, each different solution probe comprises:

a different first probe region complementary to a different one of the biological features on the array; and

a second probe region complementary to a biological target in the one or more samples, such that during an assay, the set of solution probes assembles the one or more biological targets from the one or more biological samples to different biological features on the array.

81. The set of biological probes of claim 80, wherein one biological target per sample in a plurality of different biological samples are to be multiplexed, and wherein the second probe region of each solution probe is the same on each different solution probe and the second probe region is complementary to the one target in the plurality of different samples, such that the one biological target is assembled on the array of biological features according to the plurality of different samples.

82. The set of biological probes of claim 80, wherein a plurality of different biological targets per sample in the one biological sample are to be multiplexed, and wherein the second probe region of each solution probe is different on each different solution probe and each different second probe region is complementary to a different one of the plurality of targets per sample, such that each biological target is assembled on the array according to the plurality of different targets.

83. The set of biological probes of claim 80, wherein a plurality of different biological samples having a plurality of different biological targets per sample are to be multiplexed, and wherein the second probe region of each solution probe is different on each different solution probe and each different second probe region is complementary to a different one of the plurality of targets per sample, such that each target is assembled on the array according to the plurality of different targets and the plurality of different samples.

84. The set of biological probes of claim 80, wherein the first region of each different solution probe comprises a sequence of monomers, wherein at least one monomer is chemically modified, such that the first region will preferentially hybridize to a corresponding biological feature on the array that comprises a complementary monomer that has been similarly modified.

85. The set of biological probes of claim 84, wherein the first region comprises a sequence of nucleotides linked to each other with either (3′

→

5′

) or (5′

→

3′

) polarity phosphodiester linkages, and the modified monomer in the sequence comprises a nucleotide with a reversed polarity relative to the polarity of the sequence of nucleotides, the reversed polarity nucleotide in the first region forming a thermodynamically more stable hybridization with a complementary reversed polarity nucleotide on the corresponding biological feature than with a complementary nucleotide whose polarity is not similarly reversed.

95. An array with specificity and sensitivity for multiplexing one or more biological samples, having one or more biological targets per sample comprising:

a substrate with a surface; and

a plurality of oligonucleotide probes on the surface of the substrate in an array pattern of features, wherein the oligonucleotide probes of the plurality are all different from each other, each different probe of the plurality comprises;

a sequence of nucleotides having either a (3′

→

5′

) or (5′

→

3′

) polarity, and

at least one nucleotide having a reversed polarity relative to the polarity of the sequence,

wherein the sequence for each different probe is different, each different first probe is located at a different feature location of the array, and each different first probe is a different address for a different combination of a target-and-a sample from the one or more samples and the one or more targets per sample being assayed.