Detection of biological molecules by differential partitioning of enzyme substrates and products

• US 20040106164A1
• Filed: 09/22/2003
• Published: 06/03/2004
• Est. Priority Date: 09/20/2002
• Status: Active Grant

• Current Assignee: Queen's University At Kingston
• Original Assignee: Queen's University At Kingston
• Inventors: Brown, R. Stephen, Tabash, Samir P., Hussain, Moe, Marcotte, Eric J.P., Wynne-Edwards, Robin A., Hall, Kevin R., Ley, Arthur N., Hodson, Peter V., St. Marseille, John G., Bowers, Raymond J., Akhtar, Parveen, Kozin, Igor S.
• Granted Patent: US 7,402,426 B2
• Time in Patent Office: Days
• US Class Current: 435/14
• CPC Class Codes: B82Y 15/00 Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
• CPC Class Codes: C12Q 1/00 Measuring or testing processes involving enzymes, nucleic acids or microorganisms measuring or testing apparatus with condition measuring or sensing means, e.g. colony counters, C12M1/34; Compositions therefor; Processes of preparing such compositions
• CPC Class Codes: C12Q 1/04 Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor C12Q1/6897 takes precedence
• CPC Class Codes: G01N 2021/6484 Optical fibres
• CPC Class Codes: G01N 21/6428 Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" in vivo A61B5/00; immunoassay G01N33/53
• CPC Class Codes: G01N 21/6486 Measuring fluorescence of biological material, e.g. DNA, RNA, cells G01N21/6428 takes precedence
• CPC Class Codes: G01N 33/56905 Protozoa
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/56916 Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
• CPC Class Codes: G01N 33/56983 Viruses
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: Y10S 435/808 Optical sensing apparatus

Abstract

This invention relates to a method and apparatus for detecting a biological molecule associated with enzyme activity in a sample. The invention is applicable to detecting a microorganism associated with an enzyme in a sample such as water, food, soil, or a biological sample. According to a preferred embodiment of the method of the invention, a sample containing an enzyme of interest or a microorganism associated with the enzyme is combined with a suitable substrate, and a fluorescent product of the enzyme-substrate reaction is selectively detected. The fluorescent product is detected with a partitioning element or optical probe/partitioning element of the invention. In one embodiment the partitioning element provides for partitioning of only the fluorescent product molecule into the probe. The invention also provides an automated system for monitoring for biological contamination of water or other samples.

Claims

1. A method for detecting a biological molecule associated with activity of at least one enzyme in a sample, comprising:

combining at least one enzyme with at least one substrate under conditions which allow for the enzyme to react with the substrate;

providing a partitioning element for partitioning of said biological molecule thereinto; and

detecting fluorescence of said biological molecule in said partitioning element;

wherein said detected fluorescence is indicative of activity of said enzyme in the sample.

2. The method of claim 1, wherein said partitioning element comprises a polymer film.

3. The method of claim 1, wherein said conditions which allow for the enzyme to react with the substrate comprise aqueous conditions.

4. The method of claim 1, wherein said biological molecule is said substrate and said detecting fluorescence comprises detecting a change in amount of fluorescence.

5. The method of claim 1, wherein said biological molecule is a product of said enzyme-substrate reaction.

6. The method of claim 1, wherein said enzyme activity is associated with a microorganism.

7. The method of claim 6, wherein said microorganism is a biological contaminant.

8. The method of claim 1, wherein said at least one enzyme is selected from β

-glucuronidase and β

-galactosidase.

9. The method of claim 7, wherein said microorganism is selected from E. coli and total coliform.

10. The method of claim 1, wherein said at least one substrate is selected from pyrene-β

-D-glucuronide, anthracene-β

-D-glucuronide, pyrromethene-β

-D-glucuronide, pyrene-β

-D-galactopyranoside, and anthracene-β

-D-galactopyranoside.

11. The method of claim 1, wherein said sample is selected from water, a biological sample, food, and soil.

12. The method of claim 1, wherein said enzyme and substrate are combined in a cartridge comprising said partitioning element.

13. An optical probe for detecting fluorescence of a molecule, comprising:

an optical waveguide; and

a partitioning element disposed on one end of the optical waveguide;

wherein said molecule is selectively partitioned into the partitioning element, such that fluorescence of the molecule is coupled into the waveguide.

14. The optical probe of claim 13, wherein the fluorescent molecule is selected from an enzyme substrate and a product of an enzyme-substrate reaction.

15. The optical probe of claim 13, wherein the partitioning element is a polymer film.

16. The optical probe of claim 15, wherein the polymer film comprises polydimethylsiloxane (PDMS).

17. The optical probe of claim 13, wherein the optical waveguide is an optical fiber.

18. An apparatus for detecting presence of a molecule, comprising:

the optical probe of claim 13;

an excitation light source; and

a detector for detecting fluorescence of said molecule;

wherein said detected fluorescence is indicative of presence of said molecule.

19. The apparatus of claim 18, wherein the fluorescent molecule is selected from an enzyme substrate and a product of an enzyme-substrate reaction.

20. The apparatus of claim 18, wherein said enzyme is associated with a microorganism.

21. The apparatus of claim 18, wherein said enzyme is selected from β

-glucuronidase and β

-galactosidase.

22. The apparatus of claim 20, wherein said microorganism is selected from E. coli and total coliform.

23. The apparatus of claim 19, wherein said substrate is selected from pyrene-β

-D-glucuronide, anthracene-β

-D-glucuronide, pyrromethene-β

-D-glucuronide, pyrene-β

-D-galactopyranoside, and anthracene-β

-D-galactopyranoside.

36. A kit for detecting a biological contaminant in a sample, comprising:

the apparatus of claim 18;

and

a substrate for an enzyme associated with said biological contaminant;

wherein the kit provides an indication of the presence or amount of said biological contaminant in the sample.

37. The kit of claim 36, further comprising a vessel for incubating the sample and the substrate.

38. The kit of claim 36, wherein the sample is selected from water, food, a biological sample, and soil.

39. The kit of claim 36, wherein said biological contaminant is at least one microorganism selected from E. coli and total coliform.

40. The kit of claim 36, wherein the enzyme is at least one enzyme selected from β

-glucuronidase and β

-galactosidase.

41. The kit of claim 36, wherein the substrate is at least one substrate selected from pyrene-β

-D-glucuronide, anthracene-β

-D-glucuronide, pyrromethene-β

-D-glucuronide, pyrene-β

-D-galactopyranoside, and anthracene-β

-D-galactopyranoside.

24. A system for detecting a biological molecule associated with activity of at least one enzyme in a sample, comprising:

a vessel for incubating the sample and at least one substrate such that the enzyme to reacts with the substrate to produce said biological molecule;

a partitioning element that allows partitioning of said biological molecule thereinto;

an excitation light source that irradiates said biological molecule partitioned into said partitioning element;

a detector that detects fluorescence of said biological molecule partitioned into said partitioning element; and

a control unit;

wherein said detected fluorescence is indicative of activity of said enzyme in the sample.

25. The system of claim 24, wherein the control unit performs at least one function selected from controlling operation of said system, storing data relating to fluorescence detection, and outputting data relating to fluorescence detection.

26. The system of claim 24, wherein the vessel comprises a removable cartridge for containing the sample and the substrate.

27. The system of claim 26, wherein said partitioning element is disposed in said removable cartridge.

28. The system of claim 24, further comprising a communications unit that relays data relating to fluorescence detection to a communications network.

29. The system of claim 24, wherein said enzyme is associated with a biological contaminant.

30. The system of claim 24, wherein the sample is selected from water, a biological sample, food, and soil.

31. The system of claim 24 wherein said enzyme is selected from β

-glucuronidase and β

-galactosidase.

32. The system of claim 29, wherein said biological contaminant is selected from E. coli and total coliform.

33. The system of claim 24, wherein said at least one substrate is selected from pyrene-β

-D-glucuronide, anthracene-β

-D-glucuronide, pyrromethene-β

-D-glucuronide, pyrene-β

-D-galactopyranoside, and anthracene-β

-D-galactopyranoside.

34. The system of claim 24, further comprising means to calibrate said partitioning element and optical components of the system or to monitor said fluorescence detection, or both.

35. The system of claim 34, wherein said means to calibrate said partitioning element and to monitor said fluorescence detection comprises:

a fluorophore that partitions into said partitioning element and fluoresces at a different wavelength than said biological molecule;

wherein said fluorescence of said fluorophore is detected by the detector; and

wherein said control unit uses the detected fluorescence to calibrate the partitioning element and optical components of the system or to monitor fluorescence detection of the system.

42. A method for detecting a target species, comprising:

combining an antibody that is specific for said target species with a sample under conditions which allow the antibody to bind the target species;

providing an enzyme which allows quantification of said bound antibody by producing a fluorescent molecule;

providing a partitioning element for partitioning of said fluorescent molecule thereinto; and

detecting fluorescence of said fluorescent molecule in said partitioning element;

wherein said detected fluorescence is indicative of activity of said enzyme in the sample.

43. The method of claim 42, wherein said antibody is conjugated to said enzyme.

44. The method of claim 42, wherein said enzyme is conjugated to a second antibody that is specific to the antibody that is specific for the target species and the conjugate is mixed with the combination of the antibody specific for the target species and the sample.

45. The method of claim 42, wherein fluorescence is detected continuously throughout the enzyme-substrate reaction.

46. The method of claim 42, wherein fluorescence is detected after a set time during the enzyme-substrate reaction.

47. The method of claim 42, wherein the target species is a biological or chemical contaminant.

48. The method of claim 42, wherein the target species is selected from the group consisting of bacteria, protozoa and viruses.