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

• US 7,402,426 B2
• Filed: 09/22/2003
• Issued: 07/22/2008
• Est. Priority Date: 09/20/2002
• Status: Active Grant

• Current Assignee: Queen's University At Kingston
• Original Assignee: Queen's University At Kingston
• Inventors: Marcotte, Eric J. P., Brown, R. Stephen, Tabash, Samir P., Hussain, Moe, 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.
• Primary Examiner(s): Beisner; William H.
• Assistant Examiner(s): BOWERS, NATHAN ANDREW
• Application Number:
  US10/665,718
• Publication Number:
  US 20040106164A1
• Time in Patent Office: 1,765 Days
• Field of Search: 422/82.07, 422/82.06, 435/288.7, 435/808, 435/287.9, 435/288.3, 435/288.1, 600/317, 600/342
• US Class Current: 435/288.7
• 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 system for detecting presence of an organism having at least one enzyme in a sample, comprising:

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

a solid partitioning element that allows partitioning of only one of said biological molecule and said at least one substrate thereinto, the partitioning element not including an indicator agent that interacts with said biological molecule or said at least one substrate;

an excitation light source that irradiates said biological molecule or said at least one substrate partitioned into said partitioning element;

a detector that detects fluorescence of said biological molecule or said at least one substrate partitioned into said partitioning element; and

a control unit;

wherein said detected fluorescence is indicative of presence of said organism in the sample.

2. The system of claim 1, 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.

3. The system of claim 1, wherein the vessel comprises a removable cartridge for containing the sample and the substrate.

4. The system of claim 3, wherein said partitioning element is disposed in said removable cartridge.

5. The system of claim 1, further comprising a communications unit that relays data relating to fluorescence detection to a communications network.

6. The system of claim 1, wherein said organism is a biological contaminant.

7. The system of claim 1, wherein the sample is selected from water, a biological sample, food, and soil.

8. The system of claim 1, wherein said enzyme is selected from β

-glucuronidase and β

-galactosidase.

9. The system of claim 6, wherein said organism is selected from Escherichia coil and total coliform bacteria.

10. The system 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 system of claim 1, further comprising means for calibrating said partitioning element and/or optical components of the system or for monitoring said fluorescence detection, or both.

12. The system of claim 11, wherein said means for calibrating said partitioning element and/or optical components and/or for monitoring 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/or optical components of the system or to monitor fluorescence detection of the system.

13. The system of claim 1, wherein the biological molecule partitions into the partitioning element and the at least one substrate does not partition into the partitioning element.

14. The system of claim 1, wherein the partitioning element comprises a polymer film.

15. The system of claim 14, wherein the polymer film comprises a hydrophobic polymer.

16. The system of claim 14, wherein the polymer film comprises polydimethylsiloxane (PDMS).