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The goal of this work is to develop novel commercially-feasible products and platforms to detect low molecular weight toxic/banned substances which cannot currently be detected in a high-throughput manner (such as histamine and pentachlorophenol) in the nation's food and feed supplies. The identification of low molecular weight banned substances in food samples poses a serious challenge to food safety groups since it is difficult to develop the antibodies required to produce high-throughput screening assays such as ELISA. Nature has provided a wide variety of microbial enzymes which bind to and transform low molecular substances in complex mixtures with high affinity and selectivity. In this project, we will adapt enzymatic-based detection methods to identify specific low molecular weight banned substances from food and feed samples with high sensitivity and specificity.
<P>Specifically, our first objective is to develop and optimize a rapid, fluorescence-based enzyme assay to detect histamine in seafood samples with high sensitivity. To accomplish this objective, we will test and optimize different pairs of electron carriers and fluorescent-dyes to maximize signal and minimize background noise (while simultaneously optimizing compatibility with the sample prep procedure). The assay will use a generalized redox-based detection strategy which could be adapted for future use with other redox enzymes which metabolize low molecular weight banned substances. Our expected output for our first research objective (milestone for success) will be to develop a sensitive plate based high-throughput fluorescence assay to detect unmodified histamine in food samples with a sensitivity of at least 0.1 ppm.
<P>Our second technical objective is to develop and optimize a second enzymatic assay to detect another low molecular weight banned substance (pentachlorophenol). There is currently no high-throughput method available to detect pentachloropenol in food and feed samples. We will develop a novel oxidoreductase based high-throughput enzymatic assay to detect pentachlorophenol in food and feed samples. Our expected output for our second research objective (milestone for success) will be to to develop a sensitive plate based high-throughput fluorescence assay to detect unmodified histamine in food samples with a sensitivity of at least 1 ppm. In developing these two tests we will create a novel commercially-valuable high-throughput platform to for the detection of banned substances.
NON-TECHNICAL SUMMARY: It is important to test the food supply for known toxic or banned substances which can impinge public safety. While a number of high-throughput tests have been developed to screen the food supply for higher molecular weight banned substances, it has been difficult to develop methods to comprehensively screen the food supply for low molecular weight banned substances such as histamine or pentachlorophenol. We must rely on low throughput methods to detect these banned substances in the food supply. Consequently, only a small percentage of the relevant food supply is being screened for low toxic substances such as histamine. Enzymes could potentially be a valuable solution to this problem since they can detect low molecular compounds with high precision and they can readily be adapted for high throughput assays using relatively inexpensive equipment such as a microplate reader. In our Phase I research we will develop enzyme based tests for banned substances for which there are currently no sensitive high-throughput detection assays (histamine and pentachloropenol). We will first use oxidoreductase enzymes to develop a fluorescence-based microplate assay to rapidly detect the presence of low (< 1 ppm) levels of histamine in seafood samples. Secondly, we will use another enzyme to develop a sensitive microplate-based test to detect the presence of pentachlorophenol in food samples. By developing tests to detect these two toxic compounds in food samples, we will have demonstrated the feasibility of this approach as a new commercial platform for the development of cost-effective tests for the high throughput detection of low molecular banned substances which are currently difficult to detect (in practice) in our food supply.
<P>APPROACH: Detection of low molecular weight banned substances in the food supply can be very challenging since high-throughput methods (ELISA, proximity ligation assay, etc.) usually require the the development of good antibodies against the analyte. A wide number of time-honored analytical tests utilize coupled oxidoreductase enzymatic assays to detect substances in biological samples. In principle, these enzymes used in these assays are very capable of binding and detecting low molecular weight substances. In our Phase I research, we will develop and optimize novel assays using microbial oxidoreductase enzymes to detect banned substances in samples from food and feed. From the scientific literature, we have selected enzymes (candidates) which could be used to detect low molecular toxic substances such as histamine or pentachlorophenol. These enzymes will be used to transform these substances to produce oxidized or reduced cofactors which can be used in concert with diaphorase or other commercially-available oxidoreductases to create a detectable colorimetric or fluorescent signal. As needed, we will clone, subclone, express (in E. coli) and purify (using column chromatography) these candidate enzymes to obtain milligram quantities of protein for our testing purposes. We will use a plate reader to develop a simple fluorescence-based assay to detect and measure the chemical transformation of purified analytes by each enzyme. We will vary the reaction components including enzyme, analyte, , fluorescent dye, redox cofactor (e.g. NAD, mPMS, phenazine methosulfate, phenazine ethosulfate, methyl viologen and Meldola Blue) to identify the optimal concentrations for each component. Since the rate of electron transfer is often pH and salt dependent, we will also vary the pH (7 -9) and salt composition for the reaction to find the optimal pH and salt composition for the reaction. By varying these parameters we will optimize the assay to create a linear standard curve for the analyte and detect increasing lower amounts of analyte in samples derived from food. We will also focus on developing optimized sample prep procedures to obtain high recoveries using simple methods that are compatible with enzymatic assays (e.g. homogenization, aqueous extraction, mild heating, centrifugation, etc.). Using food samples spiked with analyte, we will measure/verify the quantitative recovery of the analyte using our modified sample prep procedure. In this manner our goal is to use our novel optimized assay to detect 0.1 to 1 ppm of low molecular weight analyte in food samples using our optimized enzymatic assays. We will focus on simple sample preparation methods (e.g., homogenization, aqueous extraction, mild heating, centrifugation, etc.