Microarray Based Multiplex Pathogen Analysis and Uses Thereof

• US 20180251758A1
• Filed: 03/08/2018
• Published: 09/06/2018
• Est. Priority Date: 12/28/2015
• Status: Active Grant

• Current Assignee: PathogenDx, Inc.
• Original Assignee: PathogenDx, Inc.
• Inventors: Hogan, Michael Edward, May, Melissa Rose, Eggers, Frederick Henry
• Granted Patent: US 11,542,498 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Abstract

Provided herein is a 3-dimensional lattice microarray system for DNA sequence detection and analysis. The system has a plurality of bifunctional polymer linkers, on one end of which are attached nucleic acid probes where each have a sequence complementary to signature nucleotide sequences in pathogens, plants or animals. The other end of the bifunctional polymer linker is attached to a solid support by non-covalent or covalent means. Each of the nucleic acid probes have terminal thymidine bases at the 5′ and 3′ ends that permit attachment of the probes to the bifunctional polymer linkers. Also provided is a method for fabricating the microarray system by first attaching the bifunctional polymer linkers to the solid support, followed by photochemical coupling of the nucleic probes to the microarray. A customizable microarray kit is provided that contains the solid support, linkers, probes, solvent mixture and instructions to use the kit.

Claims

1. A covalently bonded, 3-dimensional lattice microarray system comprising:

a solid support with a front surface and a back surface;

a plurality of bifunctional polymer linkers each having a top domain and a bottom end, the bottom end attached to the solid support; and

a plurality of nucleic acid probes attached to the top domain of the bifunctional polymer linker.

2. The microarray system of claim 1, wherein the solid support is borosilicate glass, an inert metal, a thermoplastic acrylic resin, a cycloolefin polymer, a polyethylene terephthalate, a polycarbonate, a nylon, a ceramic, a engineered carbon surfaces or a combination thereof.

3. The microarray system of claim 1, wherein the bifunctional polymer has an adsorptive group at the bottom end to non-covalently attach to the solid support.

4. The microarray system of claim 3, wherein the adsorptive group is a single stranded nucleic acid, an amine-polysaccharide, or an extended planar hydrophobic group.

5. The microarray system of claim 4, wherein the single stranded nucleic acid is OligodT or equivalent thymidine rich nucleic acid.

6. The microarray system of claim 4, wherein the amine-polysaccharide is chitosan.

7. The microarray system of claim 4, wherein the extended planar hydrophobic group is a pyrene, a digoxigenin, or a Cy5 dye.

8. The microarray system of claim 1, the solid support further comprising a plurality of chemically activatable groups attached to the front surface and the plurality of bifunctional polymer linkers are covalently attached by their bottom end to said plurality of chemically activatable groups

9. The microarray system of claim 8, said chemically activatable group is an epoxysilane group, a maleimido group, an amino group or an activated carboxylic acid ester.

10. The microarray system of claim 8, wherein the bifunctional polymer has a first reactive moiety at the bottom end to covalently attach to the chemically activatable groups in the solid support.

11. The microarray system of claim 10, wherein the first reactive moiety is an amino group, a thiol group, an aldehyde group or a carboxylate.

12. The microarray system of claim 8, wherein the molar ratio of the chemically activatable groups to the bifunctional polymer linkers is greater than about 10.

13. The microarray system of claim 1, wherein the bifunctional polymer has a second reactive moiety at the top to covalently attach to the nucleic acid probe.

14. The microarray system of claim 13, wherein the second reactive moiety is a nucleotide base, an amino acid, or an aminosacharride.

15. The microarray system of claim 14, wherein the nucleotide base is thymidine, adenine, guanine, cytidine, uracil or bromodeoxyuridine.

16. The microarray system of claim 14, wherein the amino acid is cysteine, phenylalanine, tyrosine glycine, serine, tryptophan, cystine, methionine, histidine, arginine or lysine.

17. The microarray system of claim 1, wherein the bifunctional polymer linker is an oligonucleotide, an amino polysaccharide, a polyamine or a polyamino acid.

18. The microarray system of claim 17, wherein the oligonucleotide is OligodT or equivalent thymidine rich nucleic acid.

19. The microarray system of claim 1, wherein the bifunctional polymer linker has a fluorescent label covalently attached, wherein the fluorescent label is Cy5 DYLIGHT™

DY647, or ALEXA FLUOR®

647, which are attached to the top domain of the bifunctional linker at its end, or internally.

20. The microarray system of claim 1, each of the plurality of nucleic acid probes having at least three terminal thymidine bases at a 5′

end and at a 3′

end.

21. The microarray system of claim 20, wherein the thymidine base is covalently attached to the second reactive moiety on the bifunctional polymer linker.

22. The microarray system of claim 1, having a molar ratio of the bifunctional polymer linker to the nucleic acid probe between is greater than about 0.1.

23. The microarray system of claim 1, the nucleic acid probes having a sequence complementary to signature nucleotide sequences in pathogens, plants or animals.

24. A method of manufacturing a 3-dimensional lattice microarray system using the system of claim 1, comprising the steps of:

contacting the solid support with a formulation comprising;

(i) a plurality of nucleic acid probes;

(ii) a plurality of bifunctional polymer linkers;

(iii) a solvent mixture comprising water and a water-miscible liquid having a boiling point above 100°

C.;

allowing a first attachment whereby the bifunctional polymer linkers become attached to the solid support;

evaporating the water in the solvent mixture thereby concentrating the plurality of nucleic acid probes and plurality of bifunctional polymer linkers attached to the solid support;

allowing a second attachment whereby each of the nucleic acid probes become covalently attached to the second reactive moiety on the bifunctional polymer linker; and

washing the solid support with at least once to remove unattached bifunctional polymer linkers and nucleic acid probes.

25. The method of claim 24, said contacting is by piezo-electric printing, contact printing, ink jet printing or pipetting.

26. The method of claim 24, wherein the first attachment is by adsorption.

27. The method of claim 24, wherein the first attachment is by covalent coupling between the chemically activatable groups on the solid support and the first reactive moiety on the bottom end of the f bifunctional polymer linker.

28. The method of claim 27, wherein the molar ratio of the chemically activatable group to the bifunctional polymer linker is greater than about 10.

29. The method of claim 24, wherein the molar ratio of the bifunctional polymer linker to nucleic acid probe is greater than about 0.1.

30. The method of claim 24, wherein the solvent mixture comprises a water to water-miscible liquid volume ratio from about 10:

1 to about 100;

1.

31. The method of claim 30, wherein the water-miscible liquid is glycerol, dimethyl sulfoxide (DMSO), propanediol or a combination thereof.

32. The method of claim 24, wherein the first coupling reaction is enabled at a temperature between 0°

C. and 40°

C.

33. The method of claim 24, wherein the first attachment is enabled for about 5 minutes to about 100 minutes.

34. The method of claim 24, wherein evaporating the water is at a temperature between 20°

C. and 40°

C.

35. The method of claim 24, wherein evaporating the water is for about 1 hour to about 24 hours.

36. The method of claim 24, wherein the second attachment is enabled at a temperature between 0°

C. and 40°

C.

37. The method of claim 24, wherein the second attachment is enabled for about 1 minute to about 10 minutes.

38. The method of claim 24 wherein the second attachment is by photochemical crosslinking.

39. The method of claim 38, the photochemical crosslinking is enabled by exposure to an ultraviolet light or a laser source.

40. The method of claim 39, the ultraviolet light having a wavelength of 254 nm.

41. The method of claim 39, the ultraviolet light having an energy of 300 mjoules.

42. A customizable microarray kit comprising:

a solid support;

a plurality of bifunctional polymer linkers;

a solvent mixture; and

instructions to use the kit.

43. The customizable microarray kit of claim 42, wherein the solid support is borosilicate glass, an inert metal, a thermoplastic acrylic resin, a cycloolefin polymer, a polyethylene terephthalate, a polycarbonate, a nylon, a ceramic, a engineered carbon surfaces or a combination thereof.

44. The customizable microarray kit of claim 42, wherein the bifunctional polymer linker is an oligonucleotide, an amino polysaccharide, a polyamine, a polyamino acid or chemically modified analogs thereof, having an adsorptive group for non-covalent attachment to the solid support.

45. The customizable microarray kit of claim 42, the solid support further comprising a plurality of chemically activatable groups for covalent attachment with the bifunctional polymer linker.

46. The customizable microarray kit of claim 45, wherein the bifunctional polymer linker is an oligonucleotide, an amino polysaccharide, a polyamine, a polyamino acid or chemically modified analogs thereof.

47. The customizable microarray kit of claim 46, wherein the bifunctional polymer linker is OligodT, chitosan, 5′

-OligodT, 5′

-digoxigenin OligodT, 5′

-pyrene OligodT or 5′

-Cy5 OligodT.

48. The customizable microarray kit of claim 42, wherein the bifunctional polymer linker has a fluorescent label covalently attached, wherein the fluorescent label is Cy5 DYLIGHT™

DY647, or ALEXA FLUOR®

647.

49. The customizable microarray kit of claim 42, wherein the solvent mixture comprises a mixture of water and one of glycerol, dimethyl sulfoxide (DMSO) or propanediol at a volume ratio from about 10:

1 to about 100;

1.

50. The customizable microarray kit of claim 42 further comprising a plurality of nucleic acid probes, each having a sequence complementary to signature nucleotide sequences in pathogens, plants or animals.

51. The customizable microarray kit of claim 50, wherein the nucleic acid probe has at least three terminal thymidine bases at a 5′

end and at a 3′

end.