Abstract
A new fluorescence resonance energy transfer (FRET) biosensor to evaluate the presence of biotin has been developed and optimized. The biosensor is based on the direct evaluation of BirA enzyme from Escherichia coli homodimerization as a result of biotin binding. BirA monomer was genetically fused either to fluorescent protein mTurquoise2 (mTur) or to cp173Venus (Venus). In the presence of biotin, BirA dimerization brings mTur and Venus molecules close enough for an energy transfer. The analytical performances of the biosensor in the presence biotin using the purified fusion proteins which expressed in Escherichia coli have been evaluated and optimized. The biotin assay can be performed in 96-well microplate format with a 10 min incubation. The FRET signal increased with the biotin concentration in the range of 0.4 nmol/L - 4 nmol/L, and the linear equation was Y = 0.235 + 0.333X (R2 = 0.991), which was slightly affected by other water-soluble vitamins and trace metal elements. The FRET biosensor was used to detect biotin in infant formula samples, and the results were in good agreement with those obtained from the microbial method, indicating that the developed FRET biosensor was applicable to the biotin detection in food.
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