1a. Objectives (from AD-416):
The goals of this project are to enhance food safety through the development of tools to more effectively monitor for natural toxins, and to reduce exposure to such toxins. The first goal will be addressed by improving methods for toxin detection, in particular methods to detect multiple toxins and their metabolites simultaneously. To meet this goal requires the development of materials with performance characteristics capable of being used in multiplexed assays and materials that can detect toxins that are currently poorly detected (such as the masked mycotoxins). Development of these materials is integrated with a second goal, the removal of toxins from foods, thereby reducing exposure. To meet these goals we have four objectives. Objective 1. Improve detection of foodborne toxins through development of novel technologies based upon biosensor platforms and new component materials. Sub-objective 1.1. Development and evaluation of multiplexed assay platforms. Sub-objective 1.2. Development and evaluation of materials that can function in multiplexed assays. Objective 2. Improve detection of foodborne toxins through development of direct detection technologies based upon novel mass-spectrometric platforms. Sub-objective 2.1. Develop novel ambient ionization mass spectrometric techniques for detecting single or closely related groups of foodborne toxins. Sub-objective 2.2. Expand techniques such that they can detect multiple toxins simultaneously (multiplexed assays). Objective 3. Improve the ability to detect and measure “masked” mycotoxins and biomarkers of mycotoxin exposure in commodities and foods. Sub-objective 3.1. Application of novel mass spectrometric tools to detect masked toxins. Sub-objective 3.2. Development of novel bio-based materials for masked mycotoxin detection. Objective 4. Improve toxin detection methods and reduce exposure through the development and application of synthetic materials. Sub-objective 4.1. Develop synthetic receptor materials using computational methods and materials science/synthetic strategies. Sub-objective 4.2. Characterize and apply synthetic receptor materials in methods to reduce exposure to toxins.
1b. Approach (from AD-416):
Food crops are commonly infested with fungi, both in the field and in storage. Certain fungi produce toxins (mycotoxins) that can adversely affect human health and the health of domestic animals. By permitting the timely diversion of contaminated ingredients from the food supply, detection of foodborne toxins can directly improve food safety and the safety of animal feed. Monitoring for the presence of such naturally occurring toxins is widespread and occurs at many of the stages between the producer and the consumer. Increasing the efficiency and improving the accuracy of monitoring results in more appropriate and efficient diversion of contaminated products. The need to monitor for greater numbers of mycotoxins is a trend that will continue, in particular because of recent concern over the so called “masked” mycotoxins. This project seeks to address the need for improved toxin detection by developing rapid, multi-toxin detection methods. Two major approaches will be used: development of advanced biosensor techniques and development of novel chemical methods based upon mass spectrometry. In support of the former, novel biological binding materials will be developed. Certain toxin-binding materials may also have the potential to be used to remove toxins from foods, and this will be approached through the development and application of novel synthetic materials to reduce exposures.
3. Progress Report:
Objective 2. Improve detection of foodborne toxins through development of direct detection technologies based upon novel mass-spectrometric platforms. The field of ambient ionization mass spectrometry, wherein compounds for analysis are ionized outside of traditional mass spectrometry, continues to develop rapidly. A technique was developed wherein light from an infrared laser was used in combination with a novel technology known as direct analysis in real time-mass spectrometry (DART-MS) for detection of the carcinogenic mycotoxin aflatoxin B1 (AFB1). The newly developed technique was used by an ARS scientist in Peoria, Illinois, to detect AFB1 from glass plates. The mass spectral behavior of the DART ionization from glass surfaces was compared to the mass spectral behavior in similar experiments from samples deposited on a paper substrate. While these experiments were conducted on manually deposited surfaces, the technique can be readily adapted to automated micro-spotting with a robotic micro-spotting workstation. Analysis from glass surfaces can be a convenient way to introduce large numbers of samples for mass spectral analysis with minimal sample preparation. This research supports milestone 2.1. Further experiments were also conducted with an alternative ambient ionization technique known as desorption electrospray ionization (DESI). An ARS scientist in Peoria, Illinois, recently modified a DESI-MS technique by directly applying ionization voltage to the substrate surface, replacing the separate electrospray emitter. This modification of the DESI technique, commonly called “paperspray,” was applied to the analysis of fumonisin mycotoxins. The paperspray analysis of fumonisins gave good sensitivity. This is in contrast to DART-MS which has shown very low sensitivity for fumonisin analysis. This first application of the paperspray technique to mycotoxins could be complementary to the DART-MS technique, as devices for high throughput paperspray analysis are currently being developed. This research supports milestone 2.2. Objective 3. Improve the ability to detect and measure “masked” mycotoxins and biomarkers of mycotoxin exposure in commodities and foods. Fumonisins are a group of toxins produced by fungi (mycotoxins) that are routinely found worldwide in commodities such as maize. They cause a variety of diseases in domestic animals and have been implicated in several diseases in humans, in particular esophageal cancer in adults and neural tube defects in newborns. Monitoring of these toxins is conducted in order to effectively divert infested commodities from the human food and animal feed supplies. However, such monitoring is complicated by the fact that fumonisins can form derivatives with other food components which can prevent their detection with commonly used screening techniques. These so-called "masked" toxins are an indeterminate hazard and, because of this, rapid methods for their detection are desired. ARS scientists in Peoria, Illinois, have developed an antibody-based screening assay that detects several of the masked forms of the fumonisin mycotoxins. The assay will be a useful tool for determining the extent to which the masked fumonisins are present in maize and whether or not such forms represent an additional hazard to human or animal health. Furthermore, to confirm the presence of such fumonisin-carbohydrate complexes in corn, a liquid chromatography high resolution mass spectrometry (LC-HRMS) method was developed for their detection. The LC-HRMS method has facilitated the production of small amounts of purified fumonisin-carbohydrate analogs that will be useful as analytical standards, allowing confident identification of such toxins in naturally contaminated maize. Evaluation of commercial kits for detecting newly discovered toxins. Deoxynivalenol (DON) is a toxin produced by certain fungi that cause Fusarium Head Blight (FHB), a disease of cereal crops that results in substantial economic losses worldwide. DON is both a mycotoxin, capable of causing disease in animals, and a compound that facilitates fungal infection of the host cereal crop (a virulence factor). Recently researchers in Europe discovered a group of toxins with similarities to DON. These toxins, known as “NX” toxins, are sufficiently similar to DON in that they might be detected using the commercial screening assays (test kits) commonly used within the industry to detect DON. To determine whether such kits might “cross-react” with the NX toxins, scientists from the University of Natural Resources and Life Sciences (Vienna, Austria) and an ARS scientist in Peoria, Illinois, investigated six commercially available test kits, and two immunoassays developed by ARS for their ability to detect DON and the NX-toxins. None of the commercial test kits were able to detect the NX-toxins, whereas one of the ARS-developed immunoassays did. This suggests that the NX-toxins are likely avoiding detection when grain is tested with the commercial kits, but that their detection is possible with the appropriate assay. The significance of the inability of commercial test kits to detect the NX-toxins will depend upon how widespread these toxins are eventually found to be. Objective 4. Improve toxin detection methods and reduce exposure through the development and application of synthetic materials. Ochratoxin A (OTA) is a toxic natural product that can contaminate a wide variety of foods and beverages, including fruit juices. A material to selectively isolate OTA from beverages was designed using computational techniques, including density functional theory and semi-empirical methods. The material was synthesized and then characterized by spectroscopic methods. The features of the material were observed on the nanoscale. The material removed components of samples that interfered with detection and improved detection accuracy. The results of this study will be useful to researchers looking for convenient methods to detect OTA in beverages. Reliable detection methods for the pesticide cryomazine may facilitate attempts to reduce exposure to this pesticide from contaminated foods. Computational methods were developed and applied to identify the chemical properties of cyromazine that are related to detection. These findings will be useful to scientists, the food industry, and regulators looking for economical methods to improve cyromazine detection.
1. A biosensor for the simultaneous detection of multiple Fusarium mycotoxins in wheat. Fungi of the genus Fusarium can infest cereal crops and routinely cause significant losses to U.S. agriculture. Certain Fusaria produce toxins that further reduce the value of the crop that remains. Such toxins include deoxynivalenol (also known as DON or vomitoxin), zearalenone, and T-2 toxin. As part of the efforts to divert contaminated material from human food and animal feed supplies, routine monitoring is done for one or more of these toxins. However, most of the screening methods that are currently being used detect only one type of toxin. ARS scientists in Peoria, Illinois, developed a method for detecting all three types of toxins simultaneously in wheat. The method uses a novel biosensor technology known as imaging surface plasmon resonance (iSPR). By allowing for all three types of toxins to be monitored simultaneously, the method reduces the time required to test for all three groups of toxins, an important benefit for screening many samples during and after harvest. This helps to improve the safety of the human food and animal feed supplies by facilitating the timely diversion of contaminated grain.
2. A novel way to detect toxins using rare-earth metals. Cyclopiazonic acid (CPA) is a neurotoxin that is made by some of the same fungi that make the more widely known aflatoxins. The toxin acts by altering calcium levels within cells. CPA has been found as a natural contaminant in cheeses, figs, maize, rice, peanuts, millet, feeds, and meat. Data on exposure of people to CPA is limited, in part because the tools for its measurement are also limited. An ARS scientist in Peoria, Illinois, has developed a method that can render CPA fluorescent, which greatly facilitates its detection. The extent to which the enhancement of fluorescence can be used in practical assays for CPA continues to be explored. The method, which is based upon the metal Terbium, has been used to determine the relative binding of 10 metals to CPA. Determining how CPA binds with metals is important in revealing how this toxin interacts with its target enzyme, and therefore the mechanism wherein it exerts its toxic effects. Knowing the mechanism of action will help efforts to mitigate the potential effects of exposure.
3. A method for detecting the “masked” derivative of T-2 toxin in wheat. T-2 toxin is produced by certain fungi that infest oats, wheat, barley, rye, maize, and rice. Plants protect themselves from T-2 toxin by metabolizing it, adding a sugar. The metabolite (T2-glucoside) is not picked up by routine tests for T-2 toxin. Digestion may lead to regeneration of the toxin (T-2). To improve monitoring, methods should be able to detect both the original toxin and its metabolite. A novel method for detecting both compounds was developed by ARS scientists in Peoria, Illinois, using a new technology, imaging surface plasmon resonance (iSPR). The method was able to detect very low levels (4 ppb) of the toxin and was validated so that it could meet the criteria for testing of grain for export, at a level established by the European Union (100 ppb). Development and validation of the method ensures both the toxin and its metabolite are detected at levels that ensure food safety and support the export of U.S. commodities.
4. Improved detection of the mycotoxin citrinin. Citrinin is a potential liver toxin and carcinogen produced by fungi that occasionally contaminate agricultural commodities, including corn and other grains. Detection of citrinin is important in removing contaminated commodities from the food supply and prevent exposure. Experimental and computational methods were employed to improve the reliability of fluorescence detection methods that are frequently applied to monitor citrinin contamination in grains. ARS scientists in Peoria, Illinois, described the changes in chemical structure of citrinin that influence selective and accurate fluorescence detection. Acidity is a major factor influencing fluorescence of citrinin, and this knowledge is useful for analytical scientists seeking to develop more reliable methods to detect citrinin in agricultural commodities. These findings are important to scientists, the food industry, and regulators looking for more accurate and economical methods to detect citrinin contamination.
Hossain, M.Z, McCormick, S.P., Maragos, C.M., 2018. An imaging surface plasmon resonance biosensor assay for the detection of T-2 toxin and masked T-2 toxin-3-glucoside in wheat. Toxins. 10(3)119.
Berthiller, F., Cramer, B., Iha, M.H., Krska, R., Lattanzio, V.M.T., MacDonald, S., Malone, R.J., Maragos, C., Solfrizzo, M., Stranska-Zachariasova, M., Stroka, J., Tittlemier, S.A. 2018. Developments in mycotoxin analysis: an update for 2016-2017. World Mycotoxin Journal. 11(1):5-32.
Appell, M., Hurst, W.J., Finley, J.W., deMan, J.M. 2018. Amino acids and proteins. In: deMan, J.M., Finley, J.W., Hurst, W.J., Lee, C.Y., editors. Principles of Food Chemistry. 4th edition. New York, NY: Springer. p. 117-164.
Appell, M., Evans, K.O., Compton, D.L., Wang, L.C., Bosma, W.B. 2018. Spectroscopic and time-dependent density functional investigation of the role of structure on the acid-base effects of citrinin detection. Structural Chemistry. 29(3):715-723. https://doi.org/10.1007/s11224-017-1065-1.
Ma, L., Zhang, Y., Maragos, C.M. 2017. Interaction of zearalenone with bovine serum albumin as determined by fluorescence quenching. Mycotoxin Research. 34(1):39-48.
Wetterhorn, K.M., Gabardi, K., Michlmayr, H., Malachova, A., Busman, M., McCormick, S.P., Berthiller, F., Adam, G., Rayment, I. 2017. Determinants and expansion of specificity in a trichothecene UDP-glucosyltransferase from Oryza sativa. Journal of Biochemistry. 56(50):6585-6596.
Busman, M. 2018. Quantitation of mycotoxins using direct analysis in real time mass spectrometry (DART-MS). Journal of AOAC International. 101(3):643-646. https://doi.org/10.5740/jaoacint.17-0338.
Maragos, C.M. 2018. Complexation of the mycotoxin cyclopiazonic acid with lanthanides yields luminescent products. Toxins. 10(7):285. https://doi.org/10.3390/toxins10070285.