Chemistry: analytical and immunological testing – Optical result – With fluorescence or luminescence
Reexamination Certificate
2000-08-18
2002-12-24
Warden, Jill (Department: 1743)
Chemistry: analytical and immunological testing
Optical result
With fluorescence or luminescence
C436S079000, C435S034000, C435S039000, C435S287100, C435S287900, C435S288700, C250S252100, C250S36100C, C250S36100C, C250S362000, C250S368000
Reexamination Certificate
active
06498041
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to optical sensors for detecting and quantitating bacterial spores.
BACKGROUND OF THE INVENTION
The threat from biological weapons as tools of modern warfare and urban terrorism is increasing. Development of early detection, counter measures, and remediation technology is a high priority in many military, government and private laboratories around the world. Biological warfare (BW) agents of critical concern are bacterial spores, such as
Bacillus anthracis
(anthrax),
Clostridium tetani
(tetanus), and
Clostridium botulinum
(botulism). Spores, produced by certain types of gram positive bacteria in response to starvation, are non-growing, heat-resistant, dehydrated, and resistant to extremes of temperature, pH, desiccation, radiation, and chemical agents.
1, 2
Due to their high stability, spores are difficult to stain using typical cell biology methods and, consequently, are challenging to detect and enumerate. This stability and difficulty with conventional detection methods, in turn, make them an attractive tool for use in BW weapons.
An effective bacterial spore detection method must be rapid, sensitive, selective, and cost-effective. In addition to these criteria, the technology must be easily incorporated into a handheld or field-portable device that has low power requirements, requires little maintenance, and may be operated by untrained personnel. In recent years several detection techniques have been explored including transmittance and reflectance,
3
Pyrolysis/Mass Spectrometry,
4
Pyrolysis-Gas Chromatography-Ion Mobility Spectrometry (Py-GC/IMS),
5
PCR-amplified DNA sequences,
6
f 7 Enzyme-Linked Immunosorbent Assays (ELISAs),
8, 9
Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-OF-MS),
10
and detection of terbium dipicolinate complexes.
11, 12
Although many of these technologies have been demonstrated as viable for spore detection, most require substantial operator expertise and incorporation of these methods into a low-cost field-portable device may not be possible. Moreover, spores are resistant to detection by a variety of other methods, including conventional microscopic staining, DNA probes, and antibody detection.
SUMMARY OF THE INVENTION
It is an object of the present invention to overcome the aforesaid deficiencies in the prior art. It is another object of the present invention to provide a method for detecting and quantifying bacterial endospores.
It is another object of the present invention to provide a system for detecting and quantifying bacterial endospores.
It is yet another object of the present invention to detect and quantify bacteria using visibly excitable fluorescent dyes.
According to the present invention, 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.
The fluorescent calcium indicator is immobilized in a polymer membrane for solid-state sensing. This technique makes it possible to measure sample fluorescence without further washing or filtering steps, thus increasing the speed of data acquisition and reducing possible sources of error. The cost of assaying is much lower than with conventional methods, and no laboratory manipulation or skilled user is required. The technology is applicable to real-time detection of bacterial spores, and the chemistry is immune to the presence of bacteria, viruses, pollen, and fungal spores. Because the process of this invention detects bacterial spores, this process can be used to discriminate potentially pathogenic spore-forming bacteria from non spore-forming genera.
According to the present invention, a visibly excitable fluorescent calcium-sensitive indicator is used with optical fibers and disposable planar glass coupons to provide a simple biosensor. The use of visibly excitable fluorescent dyes provides a sensitive and selective means to monitor changes in spore concentration, and avoids the high cost, power consumption, and complexity associated with laser or UV-excitation. The calcium in the spores is mostly complexed with dipicolinic acid also unique to the spore and not found in the corresponding vegetative cells.
REFERENCES:
patent: 5876960 (1999-03-01), Rosen
Belgrader, P., et al., “A Minisonicator to Rapidly Disrupt Bacterial Spores for DNA Analysis,”Anal. Chem71:4232-4236 (1998).
Beverly, M.B., et al., “A Rapid Approach for the Detection of Dipiconnic Acid in Bacterial Spores Using Pyrolysis/Mass Spectrometry,”Rapid Comm. in Mass Spec.10(4):455-458 (1996).
Tuminello, P.S., et al.,Applied Optics36(13):2818-2824 (1997).
Nudelman, R., et al., “Fluorescence of Dipicolinic Acid as a Possible Component of the Observed UV Emission Spectra of Bacterial Spores,” in Leonelli, J., et al., Air Monitoring and Detection of Chemical and Biological Agents, SPIE Proceedings Seris vol. 3533: 190-195 (1998).
Rosen, D.L., et al., “Bacterial Spore Detection and Determination by Use of Terbium Dipicolinate Photoluminescence,”Analytical Chemistry69(6):1082-1085 (1997).
Pellegrino, P.M., et al., “Bacterial Endospore Detection Using Terbium Dipicolinate Photoluminescence in the Presence of Chemical and Biological Materials,”Anal. Chem.70:1755-1760 (1996).
School, P.F., et al., “The Development of Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry for the Detection of Biological Warfare Agent Aerosols,”Johns Hopkins APL Technical Digest20(3):343-351 (1999).
Quinlan, J.J., et al., “Monoclonal Antibody-Based ELISAS for the Detection of Bacterial Spores,”Journal of Rapid Methods and Automation in Microbiology6:1-16 (1998).
Quinlan, J.J., et al., “Monoclonal Antibodies for Use in Detection of Bacillus and Clostridium Sports,”Applied and Environmental Microbiology63(2):482-487 (1997).
Blake, M., et al., “Immunomagnetic Dectection ofBacillus stearothermphilusSpores in Food and Environmental Samples,”Applied and Environmental Microbiology63(5): 1643-1646 (1997).
Rodriguez-Romo, L.A., et al., “Detection of EnterotoxigenicClostridium perfringensin Spices Used in Mexico by Dot Blotting Using a DNA probe,”Journal of Food Protection61(2):201-204 (1998).
Belgrader, P., et al., “Autonomous System for Pathogen Detection and Identification,” in SPIE vol. 3533: 198-206 (1998).
Sperveslage, J., et al., “Detection of Bacterial Contamination, includingBacillusspores, in Dry Growth Media and in Milk by Identification of Their 16S rDNA by Polymerase Chain Reaction,”Journal of Microbiological Methods26:219-224 (1996).
Herman, L.M.F., et al., “Identification and Detection ofBacillus sporothermoduransSpores in 1, 10, and 100 Milliliters of Raw Milk by PCR,”Applied and Environmental Microbiology63(6):3139-3143 (1997).
Bruno, J.G., et al., “In vitro Selection of DNA Aptamers to Anthrax Spores with Electrochemiluminescence Detection,”Biosensors and Bioelectronics14:457-464 (1999).
Snyder, A.P., et al., “Detection of the Picolinic Acid Biomarker in Bacillus Spores Using a Potentially Field-POrtable Pyrolysis-Gas Chromatography-Ion Mobility Spectrometry System,”Field Analytical Chemistry and Technology, 1(1):49-58 (1996).
Tabacco Mary Beth
Taylor Laura C.
Browdy and Neimark , P.L.L.C.
Cole Monique T.
Echo Technologies, Inc.
Warden Jill
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