Hairpin sensors using quenchable fluorescing agents

• US 20030013109A1
• Filed: 06/21/2002
• Published: 01/16/2003
• Est. Priority Date: 06/21/2001
• Status: Abandoned Application

• Current Assignee: Evident Technologies Incorporated
• Original Assignee: Evident Technologies Incorporated
• Inventors: LoCascio, Michael, Ballinger, Clinton T., Landry, Daniel P.
• Application Number:
  US10/176,055
• Publication Number:
  US 20030013109A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00576 fluorophore
• 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 2219/00626 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 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

The present invention provides for a device and method for detecting genetic material. The device includes at least one hairpin sensor or, preferably two or more hairpin sensors, spatially and/or spectrally multiplexed on a conductive or semi-conductive substrate or particle. The at least one hairpin sensor includes a quenchable fluorescing agent bound to a hairpin loop assembly and the hairpin loop assembly includes a probe complementary to a nucleotide sequence of interest. The method includes providing at least one hairpin sensor, exposing the at least one hairpin sensor to a sample of interest, and detecting fluorescence produced by the quenchable fluorescing agent. The fluorescence indicates the binding of a target nucleotide sequence to the complementary probe of the hairpin loop assembly.

Claims

1. A hairpin sensor comprising:

a hairpin loop assembly including,

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm; and

a quenchable fluorescing agent joined, directly or indirectly, to the end of the second inverse repeat arm of the hairpin loop assembly opposite the complementary probe.

2. The hairpin sensor of claim 1, further comprising a functional group joined to the end of the first inverse repeat arm opposite the complementary probe, the functional group selected from the group consisting of amino, carboxyl, thiol, and hydroxyl.

3. The hairpin sensor of claim 1, further comprising a first spacer joined to the end of the first inverse repeat arm opposite the complementary probe.

4. The hairpin sensor of claim 3, further comprising a functional group joined to the end of the first spacer opposite the first inverse repeat arm, the functional group selected from the group consisting of amino, carboxyl, thiol, and hydroxyl.

5. The hairpin sensor of claim 1, further comprising a ligand positioned between the second inverse repeat arm and the quenchable fluorescing agent, the ligand selected from the group consisting of mercapto, hydroxyl, amino, nitrile, and carboxyl, carboxylic acid, organic acid, and amino acid.

6. The hairpin sensor of claim 1, further comprising a second spacer positioned between the second inverse repeat arm and the quenchable fluorescing agent.

7. The hairpin sensor of claim 6, further comprising a ligand positioned between the second spacer and the quenchable fluorescing agent, the ligand selected from the group consisting of mercapto, hydroxyl, amino, nitrile, and carboxyl, carboxylic acid, organic acid, and amino acid.

8. The hairpin sensor of claim 1, wherein the quenchable fluorescing agent comprises a semiconductor nanocrystal.

9. The hairpin sensor of claim 1, wherein the quenchable fluorescing agent comprises a rhodamine B-labeled dye.

10. A microarray comprising:

at least one hairpin sensor including,

a hairpin loop assembly characterized by,

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm, the end of the first inverse repeat arm opposite the complementary probe bound, directly or indirectly, to a support; and

a quenchable fluorescing agent joined directly or indirectly to the end of the second inverse repeat arm of the hairpin loop assembly opposite the complementary probe.

11. The microarray of claim 10, wherein the support is capable of accepting a charge.

12. The microarray of claim 10, wherein the at least one hairpin sensor comprises two or more hairpin sensors.

13. The micro array of claim 12, wherein the two or more hairpin sensors include complementary probes that are the same and respective quenchable fluorescing agents that are the same.

14. The microarray of claim 12, wherein the two or more hairpin sensors include complementary probes that are different and respective quenchable fluorescing agents that are the same.

15. The microarray of claim 14, wherein the two or more hairpin sensors are arranged in a spatially-defined pattern.

16. The microarray of claim 10, wherein the two or more hairpin sensors include complementary probes that are different and respective quenchable fluorescing agents that are different.

17. The microarray of claim 16, wherein the two or more hairpin sensors are arranged in a spatially-defined pattern.

19. The method of claim 18, wherein the at least one hairpin sensor comprises two or more hairpin sensors.

20. The method of claim 19, wherein the two or more hairpin sensors are arranged in a spatially-defined pattern on the support.

21. The method of claim 20, further comprising identifying the target nucleotide sequence by the location of the complementary probe to which the target nucleotide sequence binds.

22. The method of claim 19, wherein the two or more hairpin sensors include complementary probes that are different.

23. The method of claim 19, wherein the two or more hairpin sensors include quenchable fluorescing agents that are different.

25. The kit of claim 24, wherein the support is capable of accepting a charge.

27. The hairpin sensor system of claim 26, wherein the particle is conductive or semi-conductive.

28. The hairpin sensor system of claim 26, further comprising a functional group joined to the end of the first inverse repeat arm opposite the complementary probe, the functional group selected from the group consisting of amino, carboxyl, thiol, and hydroxyl.

29. The hairpin sensor system of claim 26, further comprising a first spacer joined to the end of the first inverse repeat arm opposite the complementary probe.

30. The hairpin sensor system of claim 29, further comprising a functional group joined to the end of the first spacer opposite the first inverse repeat arm, the functional group selected from the group consisting of amino, carboxyl, thiol, and hydroxyl.

31. The hairpin sensor system of claim 26, further comprising a ligand positioned between the second inverse repeat arm and the quenchable fluorescing agent, the ligand selected from the group consisting of mercapto, hydroxyl, amino, nitrile, and carboxyl, carboxylic acid, organic acid, and amino acid.

32. The hairpin sensor system of claim 26, further comprising a second spacer positioned between the second inverse repeat arm and the quenchable fluorescing agent.

33. The hairpin sensor system of claim 32, further comprising a ligand positioned between the second spacer and the quenchable fluorescing agent, the ligand selected from the group consisting of mercapto, hydroxyl, amino, nitrile, and carboxyl, carboxylic acid, organic acid, and amino acid.

34. The hairpin sensor system of claim 26, wherein the quenchable fluorescing agent comprises a semiconductor nanocrystal.

35. The hairpin sensor system of claim 26, wherein the quenchable fluorescing agent comprises a rhodamine B-labeled dye.

36. The hairpin sensor system of claim 26, wherein the at least one hairpin sensor assembly comprises two or more hairpin sensor assemblies.

37. The hairpin sensor system of claim 36, wherein the two or more hairpin sensor assemblies include complementary probes that are the same and respective quenchable fluorescing agents that are the same.

38. The hairpin sensor system of claim 36, wherein the two or more hairpin sensors include complementary probes that are different and respective quenchable fluorescing agents that are different.

40. The method of claim 39, wherein the at least one hairpin sensor assembly comprises two or more hairpin sensor assemblies.

18. A method for detecting a target nucleotide sequence in a sample comprising:

providing at least one hairpin sensor immobilized on a substrate, the at least one hairpin sensor comprising

a hairpin loop assembly including,

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm, the end of the first inverse repeat arm opposite the complementary probe bound, directly or indirectly, to a support; and

a quenchable fluorescing agent joined, directly or indirectly, to the second inverse repeat arm of the hairpin loop assembly;

exposing the at least one sensor to a sample of interest; and

detecting fluorescence produced by the quenchable fluorescing agent,

wherein the fluorescence indicates the binding of the target nucleotide sequence to the complementary probe.

24. A kit for detecting a target nucleotide sequence in a sample comprising:

a hairpin sensor characterized by,

a hairpin loop assembly including,

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm; and

a quenchable fluorescing agent joined, directly or indirectly, to the second inverse repeat arm of the hairpin loop assembly; and

a support.

26. A hairpin sensor system including at least one hairpin sensor assembly, the at least one hairpin sensor assembly comprising:

a hairpin loop assembly including

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm, wherein the end of the first inverse repeat arm opposite the complementary probe is bound, directly or indirectly, to a particle; and

a quenchable fluorescing agent joined, directly or indirectly, to the end of the second inverse repeat arm opposite the complementary probe.

39. A method for detecting a target nucleotide sequence in a sample comprising:

providing a hairpin sensor system, the hairpin sensor system including at least one hairpin sensor assembly, the at least one hairpin sensor assembly comprising;

a hairpin loop assembly including,

a complementary probe positioned between a first inverse repeat arm and a second inverse repeat arm, wherein the first inverse repeat arm is bound, directly or indirectly, to a particle; and

a quenchable fluorescing agent joined, directly or indirectly, to the

second inverse repeat arm of the hairpin loop assembly;

exposing the hairpin sensor system to a sample of interest; and

detecting fluorescence produced by the quenchable fluorescing agent attached to the bound complementary probe.