The presence of toxins in foods, whether naturally occurring or intentionally added, is a cause of great concern. A rapid, sensitive scheme for the detection of target toxins; botulinum toxin, ricin, abrin and infective prions; will be developed. Nanotechnology allows us to synthesize and functionalize particles on which bioassays will be conducted. Our goal is to demonstrate enhanced sensitivity and fast turn-around in assays for food-borne toxins.
NON-TECHNICAL SUMMARY: The addition of toxins such as botulinum toxin, ricin and abrin to foods is a potential tool in the arsenal of terrorists. A pressing need exists for faster and more sensitive methods of detecting these compounds in our food supply, without raising false alarms that may prove disruptive and costly to the food industry. Nanotechnology can help to satisfay this need. Special nanoparticles with magnetic and optical properties will be used as substrates to carry-out bioassays using antibodies for these toxins.
APPROACH: The method makes use of the huge surface area presented by nanoparticles as a substrate for the attachment of several antibodies against the target toxins for use in an immunoassay. The nanoparticle substrate is luminescent and magnetic, properties that are obtained through a unique synthesis process that combines a lanthanide oxide with a magnetic core material. The antibodies that are immobilized on the luminescent/magnetic carrier particle will scavenge toxins in samples of food. The carrier or substrate particles will be recalled with a magnetic field for interrogation. A sandwich assay will be carried out by adding labeled secondary antibodies to the concentrated collection of magnetic carrier particles. The secondary labels will initially include several spectrally well-spaced conventional fluorophores, but we will also evaluate quantum dots and additional lanthanide oxide nanoparticles. The fluorescence signal from the substrate particle will indicate the amount of antibodies of each kind that were present in the sample, and the fluorescence signal from the secondary label will provide a measure of the amount of toxin that was captured. By using a ratio of signals from secondary labels to primary substrate, the system will have an internal standard and improved signal/noise compared to direct intensity measurements. The concept will be evaluated with a conventional laboratory plate reader, but will also be deployed and evaluated in a microfabricated system for use in the field.
PROGRESS: 2005/09 TO 2008/08<BR>
OUTPUTS: Our nanoparticle synthesis method offers a high yield, low cost and is environmentally friendly. The materials that we have developed are suitable for wide scale application to screening foods for safety. In addition, the nanoparticles that are produced contain only relatively inert non-toxic materials. Our results obtained with a model protein system clearly demonstrated the beneficial role of an internal standard in improving the precision and reliability of our immunoassays. The results of this work have been presented at national annual meeting of SPIE and the American Chemical Society. In addition, patents have been filed to cover aspects of the synthesis and application of magnetic/luminescent nanoparticles to biosensors for food. We have formed a startup company that is working on commercialization of immunoassays with these nanomaterial formats for pesticide detection in orange oil for a major supplier of food flavorings. <BR> <BR>
IMPACT: 2005/09 TO 2008/08<BR>
We have developed a novel spray pyrolysis method for the synthesis of luminescent core/shell particles consisting of magnetic cores of iron oxide doped with cobalt and neodymium (Co:Nd:Fe2O3) that are encapsulated in luminescent shells of europium-doped gadolinium oxide (Eu:Gd2O3). The magnetism of the nanoparticles has been optimized so they are suitable for magnetic separation applications in biochemistry. Using the magnetic/luminescent core/shell particles, we have demonstrated a novel format for an immunoassay with internal calibration. In this format, the nanoparticles are used as a carrier for the immunoassay and their luminescence is used as a reference for the detection of the specific fluorescent signal. The signal from the labels on the secondary antibody is normalized by the emission of the carrier particle. The ratio is a measure of the analyte concentration. Using this approach we eliminate all the experimental errors related to particle handling such as weighing, dispersing in solution and losses during magnetic separation and washing, sticking to the walls of tubes, wells and pipette tips. We successfully demonstrated the simultaneous detection of three model proteins - rabbit, human and mouse IgG using the nanoparticle-based format. The use of the internal standard allowed us to easily quantify each of the analytes independently of the others. A robust, high throughput method with internal fluorescent calibration was developed for the detection of ricin in food samples. The immunoassay was performed with a commercial 96-well magnetic extractor and simple instrumentation (microplate reader) for fluorescence detection and it takes about 2 hours. Similarly, we developed a protocol for the detection of botulinum toxin (current detection limit of 100 ng/ml. A variety of food matrices such as orange and apple juices, milk, eggs, lettuce, pastry, ground beef and turkey were analyzed for ricin with minimal sample treatment which makes the procedures easy and straightforward. We achieved detection limits (e.g. 10 ng/ml in apple juice; 25 ng/ml in milk and 200 ng/g in ground turkey) that are orders of magnitude lower than the permissible oral dose for ricin (10 micrograms/g food for adults), enabling the use of this method for monitoring food. All the assay steps are performed in a micro-capillary tube introducing the different reagents in a consecutive manner using syringe pumps. External electromagnets are used for magnetic separation within the tube between washing and incubation steps. The detection is also performed within the capillary tube, localizing the nanoparticles at the focal point of two optical fibers - one for laser excitation and another for detection. The emission from the nanoparticles complex is detected and analyzed by a spectrometer. The nanoparticle-based immunoassay for ricin (current detection limit is approximately 15 ng/ml) has been incorporated into the microcapillary system. The early prototype shows great promise as a rapid, simple assay for food-borne toxins.