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<OL> <LI> We will convert our current assay to a flow (sequential) injection format. To accomplish this objective, we will convert our current microplate-based colorimetric assay to a flow-based assay using a commercially-available sequential injection instrument. Our current assay format (reagents and colorimetric detection scheme) is amenable to rapid conversion from plate-based to the flow injection platform (using the same reagents and direct colorimetric detection of melamine). Using melamine reference solutions, we will modify the basic sequential injection reaction parameters (temperature, flow cell length, holding time and mixing ratios) to optimize the method to attain highest sensitivity for melamine detection. We expect to detect 50 ppb melamine in reference solutions. <LI> Adapt the enzyme-based sequential injection assay(s) to detect melamine in various food and feed samples.We will use the optimized assay to detect melamine in milk samples using the direct colorimetric and ammonia specific detection methods. Using spiked milk samples, we will systematically eliminate steps our from our (SPE)sample prep procedure to develop a more rapid, streamlined sample prep procedure for sequential injection with sensitive ammonia specific detection. Our goal is to successfully detect 200 ppb of melamine in milk-based samples prepared with reduced sample preparation.
NON-TECHNICAL SUMMARY: Protecting the integrity of the nation's food from contamination by toxins is a very serious challenge for our country. Melamine, in particular, is a toxic substance which has recently emerged as a serious threat to the safety of the U.S. food supply. Recent, widely-reported food and feed contaminations by melamine have fueled an increased demand for improved assays to screen the food supply for melamine contamination. However, the current testing methods all possess significant limitations which stifle their capacity for comprehensive testing of the food supply. We have recently commercialized a simple, rapid enzymatic assay for melamine using a microbial melamine deaminase enzyme. This assay has a very flexible design and can be adapted to a more rugged and sensitive detection platform (than its current microplate format). In this project, we will invent a new sequential injection based enzymatic assay for the detection of melamine in food samples. This new assay will combine the rapid and direct enzymatic test with the highly sensitive, reproducible and rugged nature of sequential injection instruments. The more robust design of the assay will allow better sensitivity of detection with less sample preparation cost and effort than by currently available methods. Our new product will allow food safety analysts to conveniently and cost-effectively detect melamine in a wide variety of food and feed sample types.
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APPROACH: We will systematically convert the existing plate-based melamine assay to a sequential injection format. Our established assay is compatible with easy integration into the sequential injection format. We will purchase a sequential injection machine; this machine will have a computer-controlled syringe pump for accurate flow as well as both defect colorimetric and gas diffusion membrane. We will systematically optimize the basic sequential injection parameters to maximize signal and reduce background signal. We will vary the flow rate, temperature, tubing length, holding time and mixing ratios for the reaction components to enhance the signal to noise ratio. We will also modify our detection method from an indophenol-based method to an ammonia diffusion method. This detection method may be more robust to allow development of a streamlined sample prep procedure to reduce sample prep time.