Chemical apparatus and process disinfecting – deodorizing – preser – Analyzer – structured indicator – or manipulative laboratory... – Means for analyzing liquid or solid sample
Reexamination Certificate
1999-11-05
2001-07-03
Chin, Christopher L. (Department: 1641)
Chemical apparatus and process disinfecting, deodorizing, preser
Analyzer, structured indicator, or manipulative laboratory...
Means for analyzing liquid or solid sample
C422S051000, C422S068100, C422S082050, C422S050000, C422S052000, C422S082080, C436S073000, C436S074000, C436S086000, C436S063000, C436S164000, C436S085000, C436S172000, C436S166000, C436S526000, C436S530000, C436S532000, C436S533000, C436S534000, C436S501000, C436S546000, C436S800000, C436S805000, C436S806000, C435S007100, C435S007500, C435S007930, C435S007940, C435S007950, C435S287100, C435S287200, C435S288700, C435S289100, C435S290100, C435S808000, C356S244000, C356S300000, C356S301000, C356S303000, C356S
Reexamination Certificate
active
06254830
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
A fiber-optic biosensor for assaying an analyte of interest.
2. Description of the Related Art
Concern over virulent pathogens contaminating the food supply has the potential to put large segments of the population at risk. The scientific estimates project that between 24 million and 81 million people become ill from foodborne diarrheal disease each year in the United States. The impact of this food contamination costs between $5 billion and $17 billion in medical care costs and lost productivity. Most toxicologists and food scientists agree these figures probably represent about 75% of the whole food safety risks, confirming that microbial pathogens are considered a serious hazard.
The impact due to Salmonella has been estimated at >$4 billion from more than 3 million cases/year. The lack of success in effectively controlling the growth of these pathogens in foods suggests that the best approach would be early and rapid detection. Approximately 5 million analytical tests are performed annually in the U.S., which makes detection a high priority for diagnostic technology.
Traditional methods to identify and quantitate contaminants in foods include physicochemical, biological and serological tests. Most of these approaches lack sufficient sensitivity, selectivity, and take days to perform. There have been many attempts to provide faster and convenient detection methods. Most of these detection systems, although referred to as “rapid methods”, still rely on culturing procedures to selectively amplify microbial populations.
The development of food immunoassays to improve this process has provided increased speed, simplicity, and effectiveness. However, these current procedures are expensive, usually require an enrichment/concentration step and still require hours to days for a final result. The adaptation of this immunoassay technology to biosensors has the potential to take immunoassays into the realm of rapid and reusable biosensors. The application of biosensors to the detection of Salmonella has the potential to contribute directly to the production and processing of safer and healthier food.
Biosensors are analytical devices which incorporate biologically active material in intimate contact with a transduction element to selectively and repeatedly detect analytes in products like food and food raw materials. The effective development of biosensors has become multidisciplinary, relying on biochemistry and biotechnology to provide the sensing elements through immobilization techniques and membrane technology, but also requires expertise in microelectronics, optical, acoustical and advanced signal processing. This dramatic combination can be effectively applied in the area of food safety, for the elimination of serious health risks by rapid detection of specific pathogens.
A compact fiber-optic evanescent-wave biosensor system which features an all-fiber optical design and red semiconductor-laser excitation has been developed. C. Zhou, P. Pivarnik, S. Auger, A Rand and S. Letcher, “A compact fiber optic immunosensor for Salmonella based on evanescent wave excitation”,
Sensors and Actuators
B 42, pp. 169-175, 1997. Tapered fiber tips with immobilized antibodies for Salmonella attached were studied in different shapes and treatments and optimized. The system response for Salmonella concentration was established and could determine as low as 10
4
colony forming units (CFU/mL) in 1 hour with a sandwich immunoassay format. Acoustic enhancement of the fiber-optic biosensor with ultrasonic manipulation of suspended particles has been shown to be an effective way to increase the sensitivity. C. Zhou, P Pivarnik, A. Rand, and S. Letcher, “Acoustic standing-wave enhancement of a fiber optic Salmonella biosensor”,
Biosensors and Bioelectronics
13, pp 495-500, 1998. Polystyrene microspheres (6-mm diameter) coated with immobilized antibodies were allowed to capture the antigens (Salmonella), which in turn captured antibodies labeled with fluorescent dye molecules. Then the entire structure was moved to the center of the acoustic cell, where the optical fiber with its cladding removed was located. The fluorescent signal was greatly increased over the signal without acoustic positioning. Multiple use of the system was also rapid because the fiber tip could be reused indefinitely (no antibodies were attached)—the flow cell just needed to be flushed out with a buffer solution and was ready for reuse.
Although the use of evanescent wave and tapered fibers provided average sensitivity and measurement potential, these systems did not achieve rapid and direct pathogen detection for food use. Currently the best available commercial system has a sensitivity of 10
4
Colony Forming Units/ml (CFU/ml) and measurement potential of 10
7
to 10
8
CFU/ml . The time to complete an assay is usually 3 to 4 hours.
SUMMARY OF THE INVENTION
The present invention comprises a fiber optic biosensor and method for the detection of pathogens, i.e. bacteria, viruses and/or toxins. The fiber-optic biosensor comprises a laser in communication with an excitation fiber. The excitation fiber is in communication with a collection fiber and the excitation and collection fibers are in communication with a magnetic focusing probe. The collection fiber is in communication with a transmission fiber which is in communication with means for detecting, collecting and measuring fluorescent signals. The present invention reduces the background fluorescence from unbound antibodies and eliminates several rinsing steps thereby resulting in a simple rapid assay.
Broadly the invention comprises magnetic focusing of paramagnetic microspheres with a fiber optic biosensor. Microspheres with immobilized antibodies interact throughout the analyte containing the target antigens, which, in turn, capture fluorescent-labeled antibodies in a standard sandwich assay. The bound antigen/antibody/fluorescent antibody complexes are magnetically attracted to the magnetic focusing probe of the fiber optic biosensor which contains the sensing volume of the excitation and collection fibers, while the uncaptured labeled fluorescent antibodies remain in bulk solution thereby reducing background fluorescence.
In contrast to the prior art fiber-optic immunoassays that are acoustically focused and have several steps for detection, the present invention provides an immunoassay that uses magnetic focusing and only requires three steps, specifically; 1) immunocapture and labeling, 2) rinsing and 3) focusing and measurement of fluorescent signal. Furthermore, the fiber-optic immunosensor of the present invention is more efficient than prior art fluorescent immunosensors because prior art fluorescent immunosensors do not concentrate the bound antigen/antibody/fluorescent antibody complexes into the optically active region (field of view) of the fiber.
In a preferred embodiment of the invention a primary antibody specific for the antigen (pathogen) to be detected is coated on a magnetic bead and a secondary antibody is conjugated to a marker. A food sample is prepared and added to the medium. If the expected pathogen, such as
Salmonella Typhimurium
is present, the pathogen binds to both antibodies forming a magnetic complex. The magnetic complex is attracted to the magnetic focusing probe of the fiber-optic immunosensor containing the excitation and collecting fibers. The fibers are attached to the transmission fiber. The transmission fiber transmits the fluorescent signal received from the contacting fiber to means for detecting, collecting, and measuring the fluorescent signal. The means for detecting, collecting and measuring the fluorescent signal can be a fiber optic spectrometer in communication with a computer or a PIN detector in communication with an optical power meter.
In another aspect of the invention, a sample from the blood of mammals, fish or fowl is prepared and added to a medium comprised of primary antibodies specific for the anitgens (pathogens) to be detected whereby the primary antib
Cao He
Letcher Stephen V.
Pivarnik Philip
Rand A. Garth
Chin Christopher L.
Do Pensee T.
Samuels Gauthier & Stevens
The Board of Governors for Higher Education, State of Rhode Isla
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