Optically decodable microcarriers, arrays and methods

• US 20040248163A1
• Filed: 03/02/2004
• Published: 12/09/2004
• Est. Priority Date: 03/07/2003
• Status: Active Grant

• Current Assignee: Rutgers University
• Original Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: Kramer, Fred R., Marras, Salvatore A. E., Tyagi, Sanjay, Trunfio, Hiyam Elhajj
• Granted Patent: US 7,741,031 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides

Abstract

A coding scheme for microcarriers suitable for use in distributed arrays includes labeling the carriers with quenched signaling hairpin molecules with any one of three to eight distinguishable fluorophores wherein the hairpins are of at least two types, most preferably three types, that open and fluoresce differentially as a chemical or physical condition, for example temperature, is changed. Mixtures of microcarriers having immobilized capture probes can be decoded by measuring fluorescence from said fluorophores under conditions under which only one type of hairpin is open, under which two types of hairpin are open, and so on. Mixtures of coded microcarriers with capture probes are used in assays for nucleic acids utilizing microarray methods.

Claims

1. A coded microcarrier having immobilized on its surface a plurality of quenched, labeled signaling hairpin molecules each comprising an interacting affinity pair separated by a linking moiety, one member of the affinity pair having bound thereto at least one fluorophore and the other member of the affinity pair having bound thereto at least one quencher, wherein interaction of the affinity pair of each hairpin molecule is disruptable by a physical or chemical change in a condition of its environment, wherein the disruption of the interaction of at least one affinity pair occurs at a first level of said condition and the disruption of the interaction of at least another affinity pair occurs at a second level of said condition, and where said disruptions are optically differentiable.

2. A microcarrier according to claim 1 wherein said affinity pair comprises complementary nucleotide sequences

3. A microcarrier according to claim 2 wherein said linking moiety is an oligonucleotide sequence

4. A mixture of a plurality of coded microcarriers according to claim 1 suitable for use in a distributed microarray, wherein the individual microcarriers each have immobilized thereon a capture probe and wherein the coding scheme for identifying individual microcarriers in said mixture comprises a combination of from three to eight spectrally deconvariable fluorophores and at least three affinity pairs disruptable at detectably different levels of said condition.

5. The mixture according to claim 4, wherein said affinity pair comprises complementary nucleotide sequences.

6. The mixture according to claim 4 wherein individual microcarriers are immobilized at the ends of fibers in a fiber-optic bundle.

7. The mixture according to claim 4 which contains a plurality of identically coded microcarriers having immobilized thereon the same capture probe.

8. The mixture according to claim 4 wherein the capture probes are molecular beacon probes.

9. A hybridization assay for a multiplicity of nucleic acid sequences in an analyte utilizing a mixture of claim 4, including steps of:

a) contacting said mixture and said analyte,

b) forming a distributed array of said microcarriers,

c) determining which microcarriers have capture probes hybridized to nucleic acid sequences of said analyte, and

d) optically decoding said microcarriers to identify the sequences of their capture probes.

10. The assay according to claim 9 wherein step a) precedes step b).

11. The assay according to claim 9 wherein the step of decoding includes disrupting said affinity pair by increasing temperature.

12. The assay according to claim 9 wherein the step of decoding includes disrupting said affinity pair by addition of a denaturant.

13. The assay according to claim 9 wherein said distributed array is a planar array.

14. The assay according to claim 13 wherein the planar array comprises microcarriers affixed to the ends of fibers of a fiber-optic bundle.

15. The assay according to claim 9 wherein said distributed array is a linear array.

16. The assay according to claim 15 wherein steps c) and d) include flow cytometry.