Particle based assay system

• US 20020155490A1
• Filed: 04/17/2002
• Published: 10/24/2002
• Est. Priority Date: 04/18/2001
• Status: Active Grant

• Current Assignee: Ambrigen LLC
• Original Assignee: Ambrigen LLC
• Inventors: Fenton, Paul, Adaniya, George A., Skinner, Nigel G.
• Granted Patent: US 7,572,642 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: G01N 33/54346 Nanoparticles
• CPC Class Codes: G01N 33/54373 involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The invention provides a particle comprising a surface, wherein a portion of the surface is capable of emitting electromagnetic radiation and another portion of the surface is capable of emitting a differential electromagnetic radiation (either different intensity, different frequency or no radiation), and wherein the arrangement of said portions of the surface defines a spatially distributed code for identifying the particle. The invention also provides method of manufacturing a particle having an identifying code comprising providing a particle with a functionalized surface which comprises functional binding moieties and selectively removing a plurality of the functional binding moieties from the surface to create a pattern of functionalized and differentially-functionalized zones on the surface. Various liquid-based assay methods employing the particle of the invention and a kit comprising the particle of the invention are also disclosed.

Claims

1. A particle comprising a surface, wherein a portion of the surface or sub-surface is capable of emitting a first electromagnetic radiation and another portion of the surface is capable of emitting a differential electromagnetic radiation, and wherein the arrangement of said portions of the surface defines a spatially distributed code for identifying the particle.

2. The particle of claim 1, wherein the first electromagnetic radiation is of a wavelength from ultraviolet through to infra-red.

3. The particle of claim 1, wherein the first electromagnetic radiation is of a wavelength between 400 nm to 1 μ

m.

4. The particle of claim 1, wherein the differential electromagnetic radiation is chosen from the group consisting of electromagnetic radiation of a different intensity than the first electromagnetic radiation, electromagnetic radiation of a different frequency than the first electromagnetic radiation, the near absence of electromagnetic radiation or the absence of electromagnetic radiation.

5. The particle of claim 1, wherein the electromagnetic radiation is of narrow bandwidth.

6. The particle of claim 5, wherein the bandwidth is between 1 and 100 nm.

7. The particle of claim 1, wherein the electromagnetic radiation is derived from optical excitation of the particle.

8. The particle of claim 1, wherein the electromagnetic radiation is derived from non-optical excitation of the particle.

9. The particle of claim 8, wherein the non-optical excitation is chosen from the group consisting of electrical, magnetic, physical, chemical, biological, electrochemical, and combinations thereof.

10. The particle of claim 1, wherein the electromagnetic radiation is derived from a combination of optical and non-optical excitation of the particle.

11. The particle of claim 1, wherein the particle has a shape chosen from the group consisting of cylindrical, spherical, conical, elliptical, bar-like, slab-like, ribbon-like, ovoid, spiral, amoeba-like, tube-like, and combinations thereof.

12. The particle of claim 11 having a dimension chosen from the group consisting of a width dimension,a length dimension, a radial dimension and combinations thereof.

13. The particle of claim 12 wherein the ratio of width to length is from about 1:

2 to about 1;

10.

14. The particle of claim 12, wherein the ratio of width to length is from about 1:

3 to about 1;

5.

15. The particle of claim 1, wherein the surface of the particle is chosen from the group consisting of flat, curved, rough, smooth, undulating and combinations thereof.

16. The particle of claim 1, wherein the particle is formed from a material chosen from the group consisting of polymers, composites, inorganics, natural products, and combinations thereof.

17. The particle of claim 16, wherein the polymers are chosen from the group consisting of polypropylene, polyethylene, polyacetylene, polypyrrole, conducting polymers, and combinations thereof.

18. The particle of claim 16, wherein the composites are chosen from the group consisting of glass fiber composites, carbon fiber composites, and combinations thereof.

19. The particle of claim 16, wherein the natural products are chosen from the group consisting of silk, wax, rubber, resins, and combinations thereof.

20. The particle of claim 1, wherein one or more portions of the surface capable of emitting said first electromagnetic radiation and one or more portions of the surface capable of emitting said differentialelectromagnetic radiation are arranged in an ordered pattern.

45. A labeled oligonucleotide library comprising a plurality of oligonucleotide compounds attached to a plurality of particles according to claims 1 or 21.

65. A kit for the detection of binding between a probe and a target comprising at least one particle as defined in claims 1 or 21 and a flow cytometric device.

21. A particle comprising a surface wherein the surface comprises at least one modified portion comprising a plurality of functional binding moieties and at least one unmodified portion which is substantially free of functional binding moieties, wherein the modified portion(s) further comprise an electromagnetic radiation emitting species, and wherein the arrangement of modified and unmodified portions on the surface forms a pattern amenable to detection.

22. The particle of claim 21, wherein the pattern defines a spatially distributed code for identifying the particle.

23. The particle of claim 21, wherein the electromagnetic radiation emitting species is attached to at least one of the plurality of said functional binding moieties.

24. The particle of claim 21, wherein the electromagnetic radiation emitting species is attached to a compound attached to at least one of the plurality of said functional binding moieties.

25. The particle of claim 21, wherein one or more functional binding moieties comprise a probe to which is bound a target species and wherein the electromagnetic radiation emitting species is attached to the target species.

26. The particle of claim 21, wherein the electromagnetic radiation emitting species is chosen from the group consisting of fluorescent, electrochemiluminescent, chemiluminescent, and biochemiluminescent species.

27. The particle of claim 26, wherein the fluorescent species is chosen from the group consisting of fluoroscein isothiocyanate (FITC), Texas Red, Cyanin 5 and Cyanin 5.5.

28. The particle of claim 26, wherein the electrochemiluminescent species is a ruthenium tris bipyridyl salt.

29. The particle of claim 26, wherein the chemiluminescent species is chosen from the group consisting of CN, HF, HCF, HCHO, acridinium compounds, phthalhydrazides, oxalate esters, stabilized dioxetanes and dioxetanones.

30. The particle of claim 26, wherein the biochemical luminescent species is chosen from the group consisting of green fluorescent protein (GFP), luciferase, luminol, aequorin and obelin.

48. A particle according to claim 21 further comprising a plurality of oligonucleotide compounds attached to the plurality of functionalized binding moieties.

58. A method for determining the amount of a target compound in a test sample comprising the steps of incubating a mixture of a test sample suspected of containing the target compound with at least one particle according to claim 21, wherein the particle(s) comprise a plurality of an antibody which is specific for the target compound, under conditions appropriate to form target/antibody complexes, wherein the electromagnetic radiation emission occurs upon formation of the target/antibody complex, and measuring the amount of electromagnetic radiation present in said mixture thereby determining the amount of target compound in said test sample.

31. A method of manufacturing a particle having an identifying code comprising providing a particle with a functionalized surface which comprises functional binding moieties and selectively removing a plurality of the functional binding moieties from the surface to create a pattern of functionalized and differentially-functionalized zones on the surface.

32. The method of claim 31, further comprising the step of contacting the particle with a plurality of electromagnetic radiation emitting moieties under conditions appropriate to allow for the covalent binding of the electromagnetic radiation emitting moieties to the plurality of the functionalized binding moieties.

33. The method of claim 32, wherein the particle is contacted with the electromagnetic radiation emitting moieties after the selective removal of a plurality of functional binding moieties.

34. The method of claim 32, wherein the particle is contacted with the electromagnetic radiation emitting moieties before the selective removal of a plurality of functional binding moieties.

35. The method according to claim 31, wherein the particle includes a cross-section comprising a geometric shape.

36. The method according to claim 31, wherein the particle includes a substantially cylindrical cross-section, and wherein the functionalized zones and the nonfunctionalized zones are positioned substantially around the circumference of the cross-section.

37. The method according to claim 31, wherein the selective removal of functional binding moieties from the surface comprises contacting the particle with laser light.

38. The method according to claim 37, wherein the laser light is produced by a UV-excimer laser.

39. The method according to claim 37, wherein the laser light is produced by a pulsed laser.

40. A method of manufacturing a particle having an identifying code comprising providing a particle having a differentially-functionalized surface and selectively adding a plurality of functional binding moieties to the surface to create a pattern of functionalized and differentially-functionalized zones on the surface.

41. The method of claim 40, further comprising the step of contacting the particle with a plurality of electromagnetic radiation emitting moieties under conditions appropriate to allow for the covalent binding of the electromagnetic radiation emitting moieties to the plurality of the functionalized binding moieties.

42. The method according to claim 40, wherein the particle includes a cross-section comprising a geometric shape.

43. The method according to claim 40, wherein the particle includes a substantially cylindrical cross-section, and wherein the functionalized zones and the nonfunctionalized zones are positioned substantially around the circumference of the cross-section.

44. An article of manufacture having a substantially cylindrical shape with a diameter between about 5 μ

m and 200 μ

m, and a length between about 10 μ

m and 2000 μ

m, wherein the surface of the particle comprises at least one substantially circumferential functionalized zone, at least one substantially circumferential differentially-functionalized zone between the end of the particle and the functionalized portion or between functionalized portions, and a combination of the width(s) of the functionalized portion(s) and the width(s) of the differentially-functionalized zone(s) establishes a code for identifying the particle.

46. The labeled oligonucleotide library of claim 45, wherein each particle contains a single species of oligonucleotide compound.

47. The labeled oligonucleotide library of claim 45, wherein the oligonucleotide compounds are synthetic oligonucleotides.

49. The particle of claim 48 wherein the oligonucleotide compounds are of a single known species of oligonucleotide compound.

50. A method of determining the sequence of an unknown oligonucleotide species in a solution comprising providing a labeled oligonucleotide library of claim 45, wherein the oligonucleotide sequence of each oligonucleotide attached to each particle in the library and the characteristic electromagnetic emission intensity profile of each particle in the library is stored and correlated in a database, mixing the solution with the labeled oligonucleotide library under conditions sufficient to permit the unknown oligonucleotide species to hybridize with corresponding particles of the lableled oligonucleotide library, exciting the particles to produce the characteristic electromagnetic emission intensity profile corresponding to the particle(s) in the library, detecting the characteristic electromagnetic emission intensity profile and correlating it to the characteristic emission intensity profile in the database, thereby determining the sequence of the unknown oligonucleotide species.

51. The method according to claim 50, wherein the characteristic emission profile is detected by a charged coupling device (CCD) array, photodiode array, or photomultiplier tube.

52. The method according to claim 50, wherein a laser is used to excite the particles.

53. The method according to claim 50, wherein a flow cytometer is used to mix the particle library with the solution containing the unknown oligonucleotide species.

54. The method according to claim 50, wherein the unknown oligonucleotide species is a single nucleotide polymorphism (SNP).

55. The method according to claim 50, wherein the unknown oligonucleotide species is cDNA cloned from RNA expressed by a normal cell.

56. The method according to claim 50, wherein the unknown oligonucleotide species is cDNA cloned from RNA expressed by a cell that has been subjected to a drug, toxic agent, or other chemical substance.

57. A method for detecting a genetic mutation in a PCR product amplified from a nucleic acid sample containing a target gene of interest, comprising the steps:

(a) selecting an oligonucleotide probe, said oligonucleotide probe including a polymorphic site, said polymorphic site including said genetic mutation or the wild type sequence found at the analogous position of said genetic mutation in a wild type target gene;

(b) coupling said oligonucleotide probe to one of a plurality of particles of a library of particles according to claim 45 to form a labeled probe library;

(c) providing PCR products comprising the region of said target gene amplified by PCR;

(d) mixing the labeled probe library and the PCR products;

(e) incubating said mixture under hybridization conditions, wherein if said PCR products include said polymorphic site, said PCR products bind to a particle(s) of said oligonucleotide probe library;

(f) exciting the particles to produce the characteristic electromagnetic emission intensity profile corresponding to the particle(s) in the library,

(g) detecting the characteristic electromagnetic emission intensity profile of the particle(s); and

(h) detecting said genetic mutation, or absence thereof, as a function of the measured characteristic electromagnetic emission intensity profile of the particle(s).

60. The assay of claim 59, wherein the particle comprises a single electromagnetic radiation emitting species that emits both said electromagnetic radiation upon binding between probe and target and said electromagnetic radiation as a means of identifying the particle in solution.

61. The assay of claim 59, wherein the particle comprises different electromagnetic radiation emitting species to emit said electromagnetic radiation upon binding between probe and target and to emit said electromagnetic radiation as a means of identifying the particle in solution.

62. The assay of claim 59, wherein the electromagnetic radiation emitted as a means of identifying the particle is emitted as a pattern of electromagnetic radiation, wherein the pattern is formed by attaching the electromagnetic radiation emitting species to functionalized zones on the surface of the particle spatially distributed among differentially functionalized zones on the surface of the particle that do not contain the electromagnetic radiation emitting species.

63. The assay of claim 59, wherein the first electromagnetic radiation is of a wavelength from ultraviolet through to infra-red.

64. The assay of claim 59, wherein the first electromagnetic radiation is of a wavelength between 400 nm to 1 μ

m.

59. An assay for the detection of binding between a probe and a target comprising contacting the probe with a solution suspected of containing the target, wherein the probe is attached to a particle which emits electromagnetic radiation upon binding between probe and target and emits electromagnetic radiation as a means of identifying the particle in solution.