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Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 2/1/2016 Publication Date: 2/4/2016 Publication URL: http://handle.nal.usda.gov/10113/63152 Citation: Rasooly, R., Do, P.M., Hernlem, B.J. 2016. Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1. Journal of Agricultural and Food Chemistry. 80:405-410. Interpretive Summary: Aflatoxin B1 (AFB1) is a toxic, disease and cancer causing chemical produced by fungi that frequently contaminate food. Testing for AFB1, especially for the active form of the toxin, usually requires costly equipment and live animals. This limits testing in countries with few resources. We built and tested a low cost device using a CCD camera to measure AFB1. We find that this device compares in results to equipment costing 100 times more. The device can measure AFB1 directly and also, with special fluorescence producing cells, measure activity of the toxin. This approach needs no live animals. The impact of this research is to promote food safety by making testing cheaper, more portable and available where resources are limited. Technical Abstract: Aflatoxin B1 (AFB1) producing fungi contaminate food and feed and are a major health concern. To minimize the sources and incidence of AFB1 illness there is a need to develop affordable, sensitive mobile devices for detection of active AFB1. In the present study we used a low cost fluorescence detector and describe two quantitative assays for detection of detoxified and active AFB1 demonstrating that AFB1 concentration can be measured as intensity of fluorescence. When the assay plate containing increasing concentrations of AFB1 is illuminated with a 366 nm ultraviolet lamp, AFB1 molecules absorb the photons and emit blue light with peak wavelength of 432 nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1which poses a threat to health, and the detoxified AFB1 which exhibits no toxicity. To measure the toxin activity, we used a cell based assay that makes quantification more robust and is capable of detecting multiple samples simultaneously. It is an alternative to the qualitative duckling bioassay which is the “gold-standard” assay currently being used for quantitative analysis of active AFB1. AFB1 was incubated with transduced Vero cells expressing the GFP gene. After excitation with blue light at 475 nm, cells emitted green light with emission peak at 509 nm. The result shows that AFB1 inhibits protein synthesis in a concentration dependent manner resulting in proportionately less GFP fluorescence in cells exposed to AFB1. The result also indicates strong positive linear relationship with R2=0.90 between the low cost CCD camera and a fluorometer which costs 100 times more than a CCD camera. This new analytical method for measuring active AFB1 is low in cost and combined with in vitro assay is quantitative. It also does not require the use of animals and may be useful especially for laboratories in regions with limited resources. |
