Methods and devices for detection and acquisition of biomarkers

• US 9,540,684 B2
• Filed: 12/13/2013
• Issued: 01/10/2017
• Est. Priority Date: 12/14/2012
• Status: Active Grant

• Current Assignee: MiNDERA Corporation, The Scripps Research Institute
• Original Assignee: MiNDERA Corporation, The Scripps Research Institute
• Inventors: Mahmood, Tahir A., Dickerson, Tobin J., Mahmood, Nadir A., Capek, Petr
• Primary Examiner(s): Riggs, Larry
• Assistant Examiner(s): DINES, KARLA A
• Application Number:
  US14/106,661
• Publication Number:
  US 20140287942A1
• Time in Patent Office: 1,124 Days
• Field of Search: 506/9
• US Class Current: 1/1
• CPC Class Codes: A61B 10/0045 Devices for taking samples of body liquids devices for taking blood samples A61B5/15
• CPC Class Codes: A61B 10/0233 Pointed or sharp biopsy instruments
• CPC Class Codes: A61B 2010/008 Interstitial fluid
• CPC Class Codes: A61B 2562/046 in a matrix array
• CPC Class Codes: A61B 5/14503 invasive, e.g. introduced into the body by a catheter or needle or using implanted sensors A61B5/1459, A61B5/1464, A61B5/1473, A61B5/1482, A61B5/14865 take precedence
• CPC Class Codes: A61B 5/14514 using means for aiding extraction of interstitial fluid, e.g. microneedles or suction
• CPC Class Codes: A61B 5/1468 using chemical or electrochemical methods, e.g. by polarographic means A61B5/1486 takes precedence
• CPC Class Codes: A61B 5/150015 Source of blood
• CPC Class Codes: A61B 5/150022 for capillary blood or interstitial fluid
• CPC Class Codes: A61B 5/150984 Microneedles or microblades
• CPC Class Codes: A61B 5/685 Microneedles
• CPC Class Codes: A61K 9/0021 Intradermal administration, e.g. through microneedle arrays, needleless injectors mechanical aspects A61M
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2543/101 in situ amplification
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/629 : being a microfluidic device
• CPC Class Codes: C12Q 2600/16 : Primer sets for multiplex assays
• CPC Class Codes: G01N 1/08 : involving an extracting tool, e.g. core bit

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).

Claims

1. A method for detecting one or more biomarkers from a tissue or biological sample of a subject, comprising:

(a) providing a device comprising one or more microneedles to which one or more probes specific for the one or more biomarkers are covalently attached, wherein the one or more probes comprise polynucleotides complementary to the one or more biomarkers;

(b) contacting the microneedle device with the tissue or biological sample of the subject such that the one or more probes complementary to the one or more biomarkers hybridize to the one or more biomarkers in situ, thereby binding the one or more biomarkers to the one or more microneedles; and

(c) detecting the one or more biomarkers that are bound to the one or more probes on the one or more microneedle, wherein at least one biomarker is an mRNA polynucleotide and wherein the one or more biomarkers bound to the one or more probes on the one or more microneedles are present in quantities amenable to detection following elution from the one or more microneedles, and wherein the method is able to detect mRNA biomarkers present in the tissue or biological sample at a concentration of 1.66 pM.

2. The method of claim 1, wherein the device comprises a plurality of probes complementary to a plurality of different biomarkers, and wherein each probe of the plurality of probes is attached to a different microneedle.

3. The method of claim 1, wherein the device comprises a plurality of probes complementary to an identical biomarker, and wherein the plurality of probes complementary to the identical biomarker are attached to two or more microneedles.

4. The method of claim 1, wherein the one or more biomarkers complementary to the one or more probes on the microneedles are detected by conducting a polymerase chain reaction (PCR).

5. The method of claim 4, wherein the PCR is real-time PCR.

6. The method of claim 4, wherein the one or more biomarkers complementary to the one or more probes comprise a DNA biomarker and the method further comprises detecting the DNA biomarker.

7. The method of claim 1, wherein the one or more microneedles comprise polycarbonate, and the one or more probes are attached to the one or more microneedles via a thiol/amino bifunctional linker.

8. The method of claim 1, wherein at least one of the polynucleotides complementary to the one or more biomarkers hybridizes to a polymorphism in the biomarker.

9. The method of claim 1, wherein the one or more probes are DNA polynucleotides.

10. The method of claim 1, wherein the tissue or biological sample is derived from a human.

11. The method of claim 1, wherein the tissue or biological sample is skin.

12. The method of claim 11, wherein at least one of the one or more biomarkers is associated with a skin cancer.

13. The method of claim 12, wherein the skin cancer is melanoma.

14. The method of claim 12, wherein the skin cancer is a non-melanocyte skin cancer.

15. The method of claim 11, wherein at least one of the one or more biomarkers is a biomarker associated with an autoimmune disease.

16. The method of claim 15, wherein the autoimmune disease is psoriasis.

17. The method of claim 11, wherein at least one of the one or more biomarkers is associated with an infectious disease.

18. The method of claim 1, wherein one or more of the microneedles are made of a polymer.

19. The method of claim 1, wherein the detecting the one or more biomarkers comprises conducting a quantitative assay.

20. The method of claim 1, wherein the detecting the one or more biomarkers comprises conducting an amplification assay.

21. The method of claim 1, wherein the detecting the one or more biomarkers comprises conducting a reverse-transcription assay.

22. The method of claim 1, wherein the mRNA polynucleotide is released from cells disrupted by the one or more microneedles.

23. The method of claim 1, wherein the polynucleotides complementary to the one or more biomarkers are between 15 and 100 nucleotides in length.

24. The method of claim 1, wherein the biomarkers bound to the one or more probes are present at a level detectable following dilution of the biomarkers.

25. The method of claim 2, wherein the plurality of probes are identical probes.