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Low cost quantitative digital imaging as an alternative to qualitative in vivo bioassays for analysis of active aflatoxin B1

Rasooly, Reuven, Do, Paula M., Hernlem, Bradley J.
Biosensors & bioelectronics 2016 v.80 pp. 405-410
aflatoxin B1, analytical methods, bioassays, blue light, cameras, digital images, ducklings, feed contamination, fluorescence, food contamination, genes, green fluorescent protein, green light, in vitro studies, in vivo studies, light intensity, photons, protein synthesis, quantitative analysis, toxicity, toxigenic strains, wavelengths
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 366nm ultraviolet lamp, AFB1 molecules absorb photons and emit blue light with peak wavelength of 432nm. The fluorescence intensity increased in dose dependent manner. However, this method cannot distinguish between active AFB1 which 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 green fluorescence protein (GFP) gene. After excitation with blue light at 475nm, cells emitted green light with emission peak at 509nm. The result shows that AFB1 inhibits protein expression 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.