A dual-labeled fluorescent probe for visualization of dextranase activity in a simulated food digestion system

Molecular bioimaging of enzyme activity is rapidly emerging as a powerful strategy for accurate disease diagnostics. This work aims to prove that bioimaging of enzyme activity in food digestion with a fluorescent probe is feasible. In this study, a dual-labeled fluorescent probe with dextran-tetramethylrhodamine (TMR)-biotin conjugate (DTB) as the enzyme-cleavable unit, and biotin-(5-fluorescein) conjugate (FB) as the reference unit, was developed. It was immobilized in the agarose gel (the model food matrix) for the fluorescence quantification of dextranase activity. The probe manifested significantly ratiometric fluorescent signals (I_green/I_red) in response to the enzyme-active reaction. Linear relationships of I_green/I_red were obtained against the dextranase concentration ratio (C/C₀). I_green/I_red increased more rapidly with a greater dextranase diffusion rate, also supported by the more significant diffusion coefficient of fluorescently labeled dextranase in 0.5 wt% agarose gel (1.87 × 10^-6 cm² s^-1). Our work provides more mechanistic evidence for enzyme activity imaging in food digestion.