Simultaneous Determination of L-Phenylalanine, Phenylethylamine, and Phenylacetic Acid Using Three-Color Whole-Cell Biosensors within a Microchannel Device

The neurotransmitter phenylethylamine (PEA) is highly susceptible to oxidation to produce phenylacetic acid (PA). The fact that PEA and PA are both metabolites of phenylalanine (Phe) in humans makes them important indicators in the diagnosis of phenylketonuria. In this work, three-color whole-cell biosensors were developed to simultaneously detect these analytes (Phe, PEA, and PA). The tyrosine-responsive promoter was used to control the production of green fluorescent protein signals in response to Phe levels. The FeaR regulon was first used to indicate the presence of PEA, whereas the Paa regulon was used for the detection of PA. The combination of three sensor strains together made it possible to semiquantify the three analytes according to unique color outputs without cross-interference. We sought to optimize various modular components (ribosomal binding sites and fluorescent proteins) to ensure the rapid generation of fluorescent signals. Finally, the biosensors were implemented within a microchannel device to reduce sample consumption in point-of-care assays.