A ratiometric fluorescent probe with distinct response to Aflatoxin B1 and B2a: probe design, bioimaging, and quantification.

Aflatoxin B1 (AFB1) and its photo-degraded metabolite Aflatoxin B2a (AFB2a) are both highly toxic, yet most analytical methods focus only on AFB1, neglecting AFB2a. We developed a human serum albumin (HSA)-based fluorescent probe that exhibits distinct ratiometric responses to AFB1 and AFB2a, which can be owing to the different binding preference of two analytes that facilitates the distinguished sensing mechanisms: indicator-displacement assay for AFB2a and indicator-analyte co-existence for AFB1. This probe enables quantitative, on-site detection of both toxins in corn-based food extracts via spectral ratio signals accompanied by visible color changes. By incorporating photo-irradiation pretreatment, the method can accurately measure the AFB2a molar concentrations composed by the initially existed and those transformed from AFB1, thereby enabling estimation of the total AFB1 burden in food. Furthermore, the dual-emissive probe allows ratiometric imaging of aflatoxin distribution in zebrafish intestines, mouse intestinal tissues, and cells under dual-channel fluorescence.