Devices, compositions and methods pertaining to microscopic analysis of microorganisms and other analytes of interest

• US 9,976,173 B2
• Filed: 10/09/2013
• Issued: 05/22/2018
• Est. Priority Date: 10/09/2012
• Status: Active Grant

• Current Assignee: Advanex Incorporated
• Original Assignee: Advanex Incorporated
• Inventors: Fuchs, Martin, Meltzer, Michelle C., Deck, Melissa K
• Primary Examiner(s): Myers, Carla J
• Application Number:
  US14/434,759
• Publication Number:
  US 20150275280A1
• Time in Patent Office: 1,686 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• 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 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/6895 for plants, fungi or algae
• CPC Class Codes: C12Q 2525/107 incorporating a peptide nucleic acid
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2565/101 Interaction between at least two labels
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/601 being a microscope, e.g. atomic force microscopy [AFM]

Abstract

This invention pertains to devices, compositions and methods that can be used for the rapid determination of microorganisms, cells and other analytes of interest (e.g. a nucleic acid target) as well as associated properties of said microorganisms, cells and analytes. For example, said devices, compositions and/or methods can be applied to the determination of a trait of a microorganism present in a sample. Said devices, compositions and methods utilize matrix-forming prolonged-dissolution hydrophilic polymer to encapsulate hybridization probes and optionally other assay reagents within two or more reagent zones and/or matrix zones disposed on the surface of a substrate. Each reagent zone and/or matrix zone can be designed as a separate assay. Thus, a plurality of assays can be performed on a single substrate. In some embodiments, devices can be supplied in a form ready for a customer to rapidly perform one or a plurality of assays.

Claims

1. A composition comprising a matrix zone and a polymer cap disposed on the matrix zone;

wherein the matrix zone comprises;

i) at least one hybridization probe comprising a linked fluorescent label;

ii) at least one hybridization probe comprising a linked quencher moiety; and

iii) at least one matrix-forming prolonged-dissolution hydrophilic polymer;

wherein said matrix zone is a gel, semi-solid or solid;

wherein said matrix-forming prolonged-dissolution hydrophilic polymer is selected from the group consisting of;

multi-subunit sugar copolymers, pullulan, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylalcohol(s), polyacrylamide(s), polyethyleneimines(s), pectin(s) or mixtures of any two or more of the forgoing;

wherein the matrix-forming prolonged-dissolution hydrophilic polymer localize the hybridization probe;

wherein the polymer cap comprises polyethylene oxide (PEO) with molecular weight of 300-600 k g/mol.

2. The composition of claim 1, wherein one or both of:

i) said at least one hybridization probe comprising a linked fluorescent label and ii) said at least one hybridization probe comprising a linked quencher moiety, is a peptide nucleic acid probe.

3. The composition of claim 1, wherein said composition is disposed on a substrate.

4. The composition of claim 1, wherein the polymer cap is soluble in ethanol, acetonitrile, methylene chloride, chloroform, benzene or dichloroethane.

5. The composition of claim 1, further comprising a buffer, residual water, and/or a detergent.

27. The composition of claim 1, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is selected from the group consisting of pullulan, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylalcohol(s), polyacrylamide(s), pectin(s), dextran sulphate, or mixtures of any two or more of the forgoing.

28. The composition of claim 1, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is selected from the group consisting of pullulan, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylalcohol(s), or mixtures of any two or more of the forgoing.

29. The composition of claim 1, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is polyvinylalcohol.

30. The composition of claim 1, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is polyvinylalcohol with molecular weight of 31-50 k g/mol.

31. The composition of claim 1, wherein the polymer cap comprises polyethylene oxide (PEO) with molecular weight of 600 k g/mol.

6. An assay device comprising:

i) a first substrate comprising a first surface;

ii) at least two matrix zones disposed on said first surface of said first substrate wherein at least one of said at least two matrix zones comprises;

a) at least one hybridization probe comprising a linked fluorescent label;

b) at least one hybridization probe comprising a linked quencher moiety; and

c) at least one matrix-forming prolonged-dissolution hydrophilic polymer, wherein each matrix zone is a gel, semi-solid or solid; and

iii) a polymer cap disposed on the substrate and over each matrix zone;

wherein each matrix zone is located between the polymer cap and the substrate;

wherein said matrix-forming prolonged-dissolution hydrophilic polymer is selected from the group consisting of;

multi-subunit sugar copolymers, pullulan, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylalcohol(s), polyacrylamide(s), polyethyleneimines(s), pectin(s) or mixtures of any two or more of the forgoing;

wherein the matrix-forming prolonged-dissolution hydrophilic polymer localize the hybridization probe;

wherein the polymer cap comprises polyethylene oxide (PEO) with molecular weight of 300-600 k g/mol.

8. The assay device of claim 6, further comprises a second substrate comprising a second surface.

9. The assay device of claim 8, further comprises a sample disposed on said second surface.

10. The assay device of claim 6, wherein said hybridization probe comprising a linked fluorescent label at said at least one of said at least two matrix zones is different as compared with the hybridization probes at a second matrix zone of said at least two matrix zones.

11. The assay device of claim 6, wherein, for one or more of said matrix zones, at least one hybridization probe is a peptide nucleic acid probe.

12. The assay device of claim 6, wherein said surface of said substrate comprises from 2 to 20 matrix zones.

13. The assay device of claim 6, wherein at least one matrix zone further comprises a buffer, residual water, and/or a detergent.

14. The assay device of claim 6, wherein the polymer cap is soluble in ethanol, acetonitrile, methylene chloride, chloroform, benzene or dichloroethane.

15. The assay device of claim 6, wherein said first surface of said first substrate is flat or substantially flat.

16. The assay device of claim 6, wherein said first substrate is transparent.

17. The assay device of claim 8, wherein said second surface of said second substrate is flat or substantially flat.

18. The assay device of claim 8, wherein said second substrate is transparent.

19. The assay device of claim 8, wherein the first surface of the first substrate and the second surface of the second substrate are separated by a narrow gap.

20. The assay device of claim 8, further comprising a liquid disposed between said first surface of said first substrate and said second surface of said second substrate.

21. The assay device of claim 20, wherein said liquid further comprises a sample.

22. The assay device claim 20, wherein said liquid is water or an aqueous solution that may optionally comprise detergent and/or buffer.

23. A method comprising:

A) providing the assay device of claim 8;

B) disposing an aqueous liquid between said first surface of said first substrate and said second surface of said second substrate such that said first surface and said second surface are in liquid communication;

wherein the liquid comprises a sample.

24. The method of claim 23 further comprising:

C) waiting for a period of time sufficient for said matrix-forming prolonged-dissolution hydrophilic polymer to re-hydrate and sufficient for a) said at least one hybridization probe comprising a linked fluorescent label and b) said at least one hybridization probe comprising a linked quencher moiety present at said matrix zone to interact with each other and with said sample.

25. The method of claim 24 further comprising:

D) determining one or more conditions of interest of said sample based, at least in part, on how;

a) said at least one hybridization probe comprising a linked fluorescent label; and

b) said at least one hybridization probe comprising a linked quencher moiety interact with each other and with said sample at one or more of said at least two matrix zones.

26. The method of claim 23, wherein the sample is blood culture, bronchoalveolar lavage, urine or other bodily fluids.

32. The assay device of claim 6, wherein the polymer cap comprises polyethylene oxide (PEO) with molecular weight of 600 k g/mol.

33. The assay device of claim 6, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is polyvinylalcohol.

34. The assay device of claim 6, wherein the matrix-forming prolonged-dissolution hydrophilic polymer is polyvinylalcohol with molecular weight of 31-50 k g/mol.

7. An assay device comprising:

i) a substrate comprising a surface;

ii) a matrix film disposed on said surface of said substrate comprising a matrix-forming prolonged-dissolution hydrophilic polymer;

wherein said matrix-forming prolonged-dissolution hydrophilic polymer is selected from the group consisting of;

multi-subunit sugar copolymers, pullulan, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, carboxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylalcohol(s), polyacrylamide(s), polyethyleneimines(s), pectin(s) or mixtures of any two or more of the forgoing;

wherein the matrix-forming prolonged-dissolution hydrophilic polymer localize the hybridization probe;

iii) at least two reagent zones disposed within the matrix film, wherein the reagent zones comprise at least one hybridization probe comprising a linked fluorescent label and at least one hybridization probe comprising a linked quencher moiety; and

iv) a polymer cap disposed over the matrix film;

wherein the polymer cap comprises polyethylene oxide (PEO) with molecular weight of 300-600 k g/mol.