Bile salt sensitive fluorescent probes for visualization of enzymatic proteolysis of hydrogels: Development and...

Protein digestion is driven by interactions with enzymes and bile salts. To visualize and measure cleavage within hydrogels, fluorescent probes were developed by conjugating labeled whey protein isolate (WPI) to microbeads. These had favorable limits of detection (LOD) and range of operation. LODs were 11.3 nmol/mL Fluorescein isothiocyanate (FITC), or 26.4 nmol/mL WPI, equivalent to 5.3 ng/mL trypsin. Probes displayed bile's effect of promoting proteolysis in solution - a unique property. Protein cleavage was measured as a function of gel penetration depth, demonstrating utility for optimizing design of protein gels for digestibility or release of nutrients/drugs. Effects of gel density and bile salts were examined, and while clear density effects were observed, bile salts did not influence hydrogel digestion - suggesting an important relationship between structure and protease accessibility. Protein hydrogel interactions with bile salts were previously unknown. These tools provide new methods for characterizing food and drug delivery materials.