Methods and devices for detection and acquisition of biomarkers

Patent metadata

Field	Value
Publication number	US-10995366-B2
Application number	US-201615367015-A
Country	US
Kind code	B2
Filing date	Dec 1, 2016
Priority date	Dec 14, 2012
Publication date	May 4, 2021
Grant date	May 4, 2021

Abstract

The present invention provides devices and methods for detecting and capturing molecular biomarkers from a subject in situ. Specifically, the devices contain an array of microneedles to which are attached probes specific for one or more biomarkers of interest. The devices can be used directly on a subject (e.g., via skin piercing) in detecting the biomarkers in the body of the subject (e.g., tissues, blood stream).

First claim

What is claimed is:

1. A device for extracting one or more mRNA biomarkers from an in situ tissue or a biopsied tissue of a subject, the device comprising two or more polymeric microneedles covalently attached to one or more nucleic acid probes specific for the one or more mRNA biomarkers, wherein a center-to-center distance between least two adjacent microneedles of the two or more polymeric microneedles is greater than 400 μm.

2. The device of claim 1 , wherein the one or more nucleic acid probes specific for the one or more mRNA biomarkers comprise at least 50 nucleic acid probes specific for the one or more mRNA biomarkers.

3. The device of claim 2 , wherein the at least 50 nucleic acid probes specific for the one or more mRNA biomarkers are covalently attached to at least one of the two or more polymeric microneedles.

4. The device of claim 1 , wherein the device comprises one or more nucleic acid probes specific for a plurality of different mRNA biomarkers.

5. The device of claim 1 , wherein the device comprises two or more polymeric microneedles attached to two or more nucleic acid probes, wherein the two or more nucleic acid probes comprise a same sequence.

6. The device of claim 1 , wherein the one or more nucleic acid probes specific for the one or more mRNA biomarkers are polynucleotides complementary to the mRNA biomarkers.

7. The device of claim 6 , wherein the two or more polymeric microneedles are polycarbonate, and the one or more nucleic acid probes are attached to the two or more polymeric microneedles via thiol/amino bifunctional linkers.

8. The device of claim 1 , wherein the one or more nucleic acid probes are attached to the two or more polymeric microneedles via linkers.

9. The device of claim 8 , wherein the linkers comprise a bifunctional linker.

10. The device of claim 8 , wherein the linkers comprise a thiol/amino bifunctional linker.

11. The device of claim 8 , wherein the linkers comprise a thiol linker.

12. The device of claim 8 , wherein the linkers comprise a maleimide linker.

13. The device of claim 1 , wherein the two or more polymeric microneedles are attached to a substrate.

14. The device of claim 1 , wherein the one or more nucleic acid probes are not fluorescently labeled.

15. The device of claim 1 , wherein the one or more nucleic acids probes comprise DNA.

16. The device of claim 1 , wherein the one or more mRNA biomarkers comprise an mRNA associated with a skin condition.

17. The device of claim 16 , wherein the one or more mRNA biomarkers comprise an mRNA associated with a skin condition selected from the group consisting of melanoma, non-melanocyte skin cancer, autoimmune disease, psoriasis, infection, and a combination thereof.

18. The device of claim 16 , wherein the one or more mRNA biomarkers comprise an mRNA associated with melanoma.

19. The device of claim 16 , wherein the one or more mRNA biomarkers comprise an mRNA associated with psoriasis.

20. The device of claim 1 , wherein the two or more polymeric microneedles comprise at least 25 microneedles.

21. The device of claim 1 , wherein the two or more polymeric microneedles comprise at least 50 microneedles.

22. A method for extracting and detecting one or more mRNA biomarkers from an in situ tissue or biological sample of a subject, comprising (a) providing a device for extracting one or more mRNA biomarkers from an in situ tissue or a biopsied tissue of a subject, the device comprising one or more polymeric microneedles covalently attached to one or more nucleic acid probes specific for the one or more mRNA biomarkers; (b) contacting the one or more polymeric microneedles of the device with the in situ tissue or biological sample of the subject; (c) extracting one or more mRNA biomarkers that are bound to the one or more nucleic acid probes; and (d) detecting the one or more mRNA biomarkers by sequencing.

23. The method of claim 22 , wherein the device comprises one or more nucleic acid probes specific for a plurality of different mRNA biomarkers.

24. The method of claim 22 , wherein the device comprises two or more polymeric microneedles and at least one of the two or more polymeric microneedles is attached to two or more nucleic acid probes, wherein the two or more nucleic acid probes comprise a same sequence.

25. The method of claim 22 , wherein the one or more polymeric microneedles are formed on a substrate.

26. The method of claim 22 , wherein the in situ tissue is the subject's bloodstream, and the one or more polymeric microneedles are contacted with the subject's bloodstream by piercing skin of the subject.

27. The method of claim 22 , wherein the one or more mRNA biomarkers comprise an mRNA associated with a skin condition.

28. The method of claim 27 , wherein the skin condition is melanoma, non-melanocyte skin cancer, autoimmune disease, psoriasis, infection, or a combination thereof.

29. The method of claim 28 , wherein the skin condition is melanoma.

30. The method of claim 28 , wherein the skin condition is psoriasis.

31. The method of claim 30 , wherein the detecting the one or more mRNA biomarkers by sequencing comprises detecting one or more mRNA biomarkers released by the disruption of the cell membranes.

32. The method of claim 22 , wherein the contacting the one or more polymeric microneedles of the device with the in situ tissue or biological sample of the subject disrupts cell membranes.

33. The method of claim 22 , wherein the contacting the one or more polymeric microneedles of the device with the in situ tissue of the subject causes disruption of cell membranes.

34. The method of claim 22 , wherein the in situ tissue or biological sample of the subject comprises skin.

35. The method of claim 22 , wherein the contacting the one or more polymeric microneedles of the device with the in situ tissue or the biological sample of the subject comprises contacting the one or more polymeric microneedles of the device with the in situ tissue of the subject.

36. The method of claim 22 , wherein the in situ tissue or biological sample of the subject comprises ocular tissue, retinal tissue, tumor tissue, or tissue biopsy.

37. The method of claim 22 , wherein the contacting the one or more polymeric microneedles of the device with the in situ tissue or biological sample of the subject is conducted by manual pressure.

38. A device for extracting one or more cellular mRNA biomarkers from an in situ tissue or a biopsied tissue of a subject, comprising two or more polymeric microneedles covalently attached to one or more nucleic acid probes specific for the one or more cellular mRNA biomarkers.

Assignees

• Mindera Corp
• Scripps Research Inst

Inventors

• Mahmood Tahir A
• Dickerson Tobin J
• Mahmood Nadir A
• Capek Petr

Classifications

• C12Q2543/10

  the purpose being "in situ" analysis · CPC title

• G01N33/54366 Primary

  Apparatus specially adapted for solid-phase testing · CPC title

• A61B5/685

  Microneedles · CPC title

• C12Q1/6837

  using probe arrays or probe chips (C12Q1/6874 takes precedence) · CPC title

• A61B5/150015

  Source of blood · CPC title