Fluorescent Probe Based On Microbeads For Visualization Of Enzyme Activity In Simulated Food Digestion Process

Molecular bioimaging of enzyme activity is rapidly emerging as a powerful strategy for accurate disease diagnostics.This work is a proof of concept to verify that visualization of enzyme activity with a fluorescent probe(one of the molecular imaging methods)in a distinct scenario,specifically food digestion is practicable.The single-labeled fluorescent probe was assembled by conjugating dextran-TMRbiotin conjugate(DTB).the enzyme cleavable unit with the beads.Results showed that the probe with a stable binding density undergoes significant signal change under hydrolysis,and Confocal Laser Scanning Microscope(CLSM)enables rapid fluorescence quantification.The probe was then immobilized in the agarose gel for imaging of dextranase activity,and results showed the fluorescence signal of the probe at the distal end(20 mm),with no enzymatic reaction,decreased significantly with time.Photobleaching was assumed to be responsible.In addition,various factors like experimental operation may also interfere with the fluorescence signal.Therefore,the single-labeled fluorescent probe gives poor results in quantifying the enzyme activity.The probe was further optimized to achieve fluorescence quantification with higher accuracy.The dual-labeled fluorescent probe was assembled by conjugating the enzyme cleavable unit DTB and the reference unit biotin-(5-fluorescein)(FB)simultaneously to beads.The probe manifested significantly ratiometric fluorescent signals(Igreen/Ired)in response to the enzyme-active reaction.Linear relationships of Igreen/Ired were obtained against the dextranase concentration ratio(C/C0).Igreen/Ired increased more rapidly with a greater dextranase diffusion rate,also supported by the fitted diffusion coefficient of dextranase.Furthermore.Ired was modified with the photobleaching curves,which allows for direct monitoring of dextranase activity.Our work provides more mechanistic evidence for enzyme activity imaging in food digestion,laying the foundation for more in-depth and complex food studies.