Optical sensors for rapid, sensitive detection and quantitation of bacterial spores

• US 6,498,041 B1
• Filed: 08/18/2000
• Issued: 12/24/2002
• Est. Priority Date: 08/18/2000
• Status: Active Grant

• Current Assignee: Echo Technologies Incorporated
• Original Assignee: Echo Technologies Incorporated
• Inventors: Tabacco, Mary Beth, Taylor, Laura C.
• Primary Examiner(s): Warden, Jill
• Assistant Examiner(s): Cole, Monique T.
• Application Number:
  US09/641,240
• Time in Patent Office: 858 Days
• Field of Search: 436/79, 436/172, 435/34, 435/39, 435/287.1, 435/287.9, 435/288.7, 250/252.1, 250/361.R, 250/361.C, 250/362, 250/368
• US Class Current: 436/172
• 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 21/7703 using reagent-clad optical fibres or optical waveguides using measurement of total internal reflection or attenuated total reflection G01N21/552; optical fibres or waveguides per se G02B

Abstract

Bacterial spores can be rapidly and sensitively detected and quantified based upon molecular recognition of unique chemicals in the spore coat. Spores can be detected and assayed based upon the calcium concentration in bacterial spore coats using the calcium which is unique to the bacterial spores. Since spores contain a high concentration of calcium relative to other biological materials, fluorescent calcium-sensitive indicators are used to detect Ca⁺² displaced from the spore case or free in solution or from the aerosol phase. Visibly excitable fluorescent dyes provide a sensitive and selective means to monitor changes in spore concentration and avoid difficulties associated with laser or UV-excitation.

Claims

1. A sensor for detecting bacterial spores comprising a fluorescent calcium-sensitive indicator;

a planar coupon in which the calcium-sensitive indicator is fixed, said planar coupon being affixed to the distal end of an optical fiber;

whereby said optical fiber provides excitation light and collects fluorescence emission from the planar coupon.

2. The sensor according to claim 1, wherein the calcium-sensitive indicator is calcein.

3. The sensor according to claim 1, wherein more than one calcium indicator is fixed to the coupon.

4. The sensor according to claim 1, wherein the calcium sensitive indicator is incorporated in a viscous membrane.

5. The sensor according to claim 1 wherein the calcein is incorporated in glycerol.

6. A method for detecting bacterial spores comprising contacting a sensor comprising a planar coupon in which a fluorescent calcium-sensitive indicator is fixed with a sample, monitoring any change in fluorescence intensity of the sensor, and correlating the change in fluorescence intensity to the presence of bacterial spores.

7. The method according to claim 6 wherein the calcium-sensitive indicator is calcein.

8. The method for detecting bacterial spores comprising detecting the calcium concentration in bacterial spore coat using a fluorescent calcium indicator wherein the fluorescent calcium indicator is immobilized in a polymer membrane, and correlating the change in fluorescence intensity to the presence of bacterial spores.

9. The method according to claim 8 wherein the fluorescent calcium indicator is incorporated in glycerol.