Target analyte sensors utilizing microspheres

• US 6,859,570 B2
• Filed: 08/08/2001
• Issued: 02/22/2005
• Est. Priority Date: 03/14/1997
• Status: Expired due to Term

• Current Assignee: Trustees of Tufts College
• Original Assignee: Tufts University
• Inventors: Michael, Karri L., Walt, David R.
• Primary Examiner(s): Sanghavi, Hemang
• Application Number:
  US09/925,292
• Publication Number:
  US 20030016897A1
• Time in Patent Office: 1,294 Days
• Field of Search: 385/12, 385/31, 385/35, 385/38, 385/147, 385/901, 385/115, 385/13, 385/33, 359/900, 359/308, 359/236, 435/808, 435/4, 435/283.1, 435/287.1, 435/DIG.40, 435/DIG.43, 436/518, 436/524, 436/255, 436/164, 436/172, 436/800, 436/805, 428/209, 428/446, 428/690, 250/556
• US Class Current: 385/12
• 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 2219/00317 Microwell devices, i.e. having large numbers of wells
• 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 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• 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: G01N 15/1433 using image recognition
• CPC Class Codes: G01N 15/1456 without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
• CPC Class Codes: G01N 15/1459 the analysis being performed on a sample stream
• CPC Class Codes: G01N 15/1468 with spatial resolution of the texture or inner structure of the particle
• CPC Class Codes: G01N 2015/1438 Using two lasers in succession
• CPC Class Codes: G01N 2035/0097 monitoring reactions as a function of time
• 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: 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 33/543 with an insoluble carrier for immobilising immunochemicals
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: Y10S 359/90 : Methods
• CPC Class Codes: Y10S 435/808 : Optical sensing apparatus

Abstract

A microsphere-based analytic chemistry system and method for making the same is disclosed in which microspheres or particles carrying bioactive agents may be combined randomly or in ordered fashion and dispersed on a substrate to form an array while maintaining the ability to identify the location of bioactive agents and particles within the array using an optically interrogatable, optical signature encoding scheme. A wide variety of modified substrates may be employed which provide either discrete or non-discrete sites for accommodating the microspheres in either random or patterned distributions. The substrates may be constructed from a variety of materials to form either two-dimensional or three-dimensional configurations. In a preferred embodiment, a modified fiber optic bundle or array is employed as a substrate to produce a high density array. The disclosed system and method have utility for detecting target analytes and screening large libraries of bioactive agents.

Claims

1. A composition comprising:

a) a substrate with a surface comprising discrete sites at a density of at least 100 discrete sites per 1 mm², said discrete sites comprising wells; and

b) a population of microspheres randomly distributed in said wells, said population comprising at least a first and a second subpopulation, said microspheres comprising a bioactive agent, and wherein said sites can have only a single microsphere.

3. A composition according to claim 1 or 2 wherein said substrate is a fiber optic bundle.

4. A composition according to claim 1 or 2 wherein said substrate is selected from the group consisting of glass and plastic.

5. A composition according to claim 1 wherein said population of microspheres comprises at least a first and a second subpopulation, wherein the microspheres of said first subpopulation of microspheres are a different size than the microspheres of said second subpopulation.

6. A composition according to claim 1 or 2 wherein said bioactive agent comprises a protein.

7. A composition according to claim 6 wherein said protein is selected from the group consisting of enzymes and antibodies.

8. A composition according to claim 1 or 2 wherein said bioactive agent is a nucleic acid.

11. A composition according to claim 1, 9, 5, or 10 wherein said first and said second subpopulations comprise a first and a second bioactive agent, respectively.

12. The composition according to claim 11, wherein said first and second subpopulations further comprise a first and a second optical signature, respectively.

13. A composition according to claim 12 wherein said at least one of said optical signatures comprises at least one chromophore.

14. A composition according to claim 12 wherein said at least one of said optical signatures comprises at least one fluorescent dye.

15. A composition according to claim 14 wherein said fluorescent dye is entrapped within said microspheres.

16. A composition according to claim 14 wherein said fluorescent dye is attached to said microspheres.

17. A composition according to claim 12 wherein said optical signature comprises at least two fluorescent dyes.

21. A composition according to claim 1 or 2 wherein said bead is covalently associated with the well.

22. A composition according to claim 1 or 2 wherein said bead is non-covalently associated with the well.

23. A composition according to claim 4 wherein said substrate is glass.

24. A composition according to claim 4 wherein said substrate is plastic.

25. A composition according to claim 7 wherein said protein is an enzyme.

26. A composition according to claim 7 wherein said protein is an antibody.

2. A composition comprising:

a) a substrate with a patterned surface comprising discrete sites, said substrate comprising discrete sites at a density of at least 100 discrete sites per 1 mm²; and

b) a population of microspheres, randomly distributed on said sites, wherein each microsphere comprises a bioactive agent; and

wherein said sites can have only a single microsphere.

9. A composition according to claim 2, wherein said population of microspheres comprises at least a first and a second subpopulation.

10. A composition according to claim 9, wherein the microspheres of said first subpopulation of microspheres are a different size than the microspheres of said second subpopulation.

18. A composition according to claim 9 wherein said bioactive agent comprises a protein.

19. A composition according to claim 18 wherein said protein is selected from the group consisting of enzymes and antibodies.

20. A composition according to claim 9 wherein said bioactive agent is a nucleic acid.

27. A composition according to claim 19 wherein said protein is an enzyme.

28. A composition according to claim 19 wherein said protein is an antibody.

29. A method of determining the presence of at least a first and second target analyte in a sample comprising:

a) contacting said sample with a composition comprising;

i) a substrate with a patterned surface comprising discrete sites; and

ii) a population of microspheres comprising at least a first and a second subpopulation, wherein said first subpopulation comprises a first bioactive agent and said second subpopulation comprises a second bioactive agent, wherein said microspheres are randomly distributed on said surface such that said discrete sites contain only one microsphere; and

b) determining the presence of said first and second target analyte.

30. A method according to claim 29 wherein said substrate is a optical fiber bundle and said microspheres are located within wells at a first terminal end of said bundle.

31. A method according to claim 29 further comprising identifying the location of said first and second bioactive agent on said substrate.

32. The method according to claim 29, wherein said discrete sites are wells.

33. The method according to claim 29, wherein said substrate is selected from the group consisting of glass and plastic.

39. A method according to claim 29 or 34, wherein said bead is covalently attached to the well.

40. A method according to claim 29 or 34, wherein said bead is non-covalently attached to the well.

41. A method according to claim 29 or 34, wherein said bioactive agent is a nucleic acid.

42. A method according to claim 33 or 37 wherein said substrate is glass.

43. A method according to claim 33 or 37 wherein said substrate is plastic.

44. A method according to claim 29 or 34 when said bioactive agent is a protein.

34. A method of making a composition comprising:

a) providing a patterned surface comprising individual sites on a substrate;

b) randomly distributing microspheres on said surface such that said individual sites contain microspheres, wherein said sites can have only a single microsphere, and wherein said microspheres comprise at least a first and a second subpopulation comprising;

i) a first and second bioactive agent, respectively; and

ii) a first and second optical signature, respectively;

c) detecting said first and second optical signatures while said microspheres are distributed on said surface; and

d) correlating the location of at least one individual site on the array with the bioactive agent at that particular site.

35. A method according to claim 34, wherein said distributing comprises serially adding said subpopulations to said sites.

36. A method according to claim 34, wherein said substrate is a fiber optic bundle.

37. A method according to claim 34, wherein said substrate is selected from the group consisting of glass and plastic.

38. A method according to claim 34, wherein said sites are wells.