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In recent years, the importance of botulism has resurfaced not only related to food and livestock safety, but also related to agro- and bioterrorism. Thus, the main objective of this proposal is the development of a microfluidic-based electrochemical biosensor for the rapid, sensitive and specific detection of botulinum toxin. Using the principles of the previously developed optical biosensor for botulinum toxin and an electrochemical biosensor for Dengue virus, the new microbiosensor will allow the detection of less than 15 pg/mL of the toxin. In addition, the optical and the electrochemical biosensors will be used to investigate a wide variety of food samples and samples taken from the farm environment. The biosensor will be further optimized for performance in the various sample matrices.<P> The specific objectives with respect to the time line are: <OL> <LI> Synthesis of ganglioside-tagged liposomes encapsulating electrochemically active markers, superparamagnetic beads coated with anti-botulinum toxin antibodies, and fabrication of a polydimethyl siloxane electrochemical microfluidic device <LI>Optimization of the electrochemical microfluidic biosensor for the detection of botulinum toxin <LI>Application of the optical lateral-flow assay for the detection of botulinum toxin in food and farm samples in order to determine optimal sample preparation procedures, and determine potential interfering substances <LI>Application of the electrochemical microfluidic biosensor to food and farm samples spiked with botulinum toxin.
NON-TECHNICAL SUMMARY: Foodborne diseases continue to be a major concern in food safety and public health world-wide. Botulinum toxin is the most toxic natural compound known and has been listed as a dangerous bioterrorism agent by the Center of Disease Control and Prevention. The research proposed will develop a rapid, sensitive and reliable detection method for botulinum toxin in food and farm samples. It will thus improve the safety of our food, from farm to table and will enhance the safety and health of humans and livestock.
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APPROACH: A very sensitive ganglioside-immunoassay has been developed previously for botulinum toxin (BT) detection using ganglioside-incorporated liposomes for signal amplification. In a sandwich assay, BT was detected as a colored band on a nitrocellulose membrane strip, where the BT bound to ganglioside-liposomes can be captured by immobilized anti-BT antibodies. The intensity of the band can be visually estimated or measured by densitometry, using a computer scanner. The limit of detection of BT in the assay system was found to be 15 pg/mL which is as sensitive as the most sensitive (yet complex) assays currently available, however, the detection limit increases as soon as food matrices were explored. Converting this optical assay to an electrochemical microbiosensor assay will improve the detection limit especially in food and environmental samples since additional washing steps can be integrated into the microbiosensor. An electrochemical RNA microbiosensor developed previously for Dengue virus will thus be adapted for the detection of BT. The microbiosensor is made using standard photo- and soft-lithography procedures and will be made of polydimethyl siloxane. The microchannels will be packaged in a plexiglass housing that will provide connections for the electrochemical transducer. The biosensor will then be adapted for use with food and farm samples. Wash and simple extraction steps that can be integrated directly into the microchannels will be investigated in order to avoid matrix effects and to enhance the sensitivity for low BT concentrations in actual samples.
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PROGRESS: 2005/10 TO 2008/09<BR>
OUTPUTS: Experiments were conducted to further investigate the use of the ganglioside-liposome assay and transfer it into a microfluidic biosensor system. The botulinum detection remained challenging and needs to be investigated under a different project in the future. However, the cholera toxin development made great progress. Experiments were carried out on three different formats. A lateral flow assay was developed for cholera toxin and directly compared to colloidal gold. Colloidal gold is commercially available and can be found in many point of care tests available on the market. Liposomes were found to be at least 10 times more sensitive than the highest quality colloidal gold purchased. Thus, concentrations 10 times lower were easily detectable using the naked eye or a reflectometer. Secondly, experiments were conducted to transfer the cholera toxin assay into a microfluidic biosensor. Initially, fluorescence detection was used in order to better visualize the assay. Here, anti-cholera toxin antibodies were immobilized on superparamagnetic beads, incubated with the toxin and ganglioside-liposomes. The mixture was added into the microfluidic biosensor, capture on the magnet. Unbound liposomes were washed away. Subsequently, liposomes were lysed and visualized using a fluorescence microscope. Concentrations of liposomes, superparamagnetic beads and buffer conditions were optimized using a microtiter plate format and also the microfluidic assay. Subsequently, flow rates and volumes were optimized in the microfluidic device and a dose response curve was generated. Thirdly, the microfluidic assay was adapted to an electrochemical detection format. Experiments are currently underway for detection in fecal samples <BR><BR>
IMPACT: 2005/10 TO 2008/09<BR>
Foodborne diseases including those caused by botulinum and cholera toxin continue to pose a threat to our society. This is independent from naturally occurring or intentional food poisoning. No sensors are currently available that allow a rapid and inexpensive screening of our food prior to consumption, during preparation and during production. Sensors developed in this project however, would provide food producers and consumers with the ability to determine the safety of their products. This would hence significantly increase the safety of our food supply. Formats available include a lateral flow assay for the most simple analysis and an electrochemical microfluidic sensor which is still portable and simple to use for more sensitive and refined analysis in food production plants and doctors offices.