Fluorescence Turn-Off Sensing of Protease Activity with Carbon Dots: Papain as a Model System for Surface Accessibility-Driven Interfacial Kinetics.

A fluorescence-based sensing platform for protease activity determination was developed using l-arginine-doped carbon dots (CDs), with papain as a model protease. CDs were synthesized via a rapid microwave-assisted method and purified by either dialysis (D-CDs) or syringe filtration (SF-CDs) to investigate the impact of purification-dependent surface chemistry on enzyme-nanoparticle interactions and sensing kinetics. The detection mechanism is based on fluorescence quenching resulting from surface interactions between Nα-benzoyl-l-arginine p-nitroanilide (BAPNA) and the CDs, with fluorescence recovery occurring upon protease-catalyzed substrate hydrolysis. Both CD systems exhibited rapid fluorescence responses upon enzyme addition; however, distinct time-dependent behaviors were observed. SF-CDs achieved a stable fluorescence signal rapidly, whereas D-CDs displayed a nonmonotonic fluorescence response before stabilization, underscoring the importance of surface accessibility and interfacial interactions in modulating enzyme-induced fluorescence. These kinetic differences produced dual linear response regions, allowing reliable quantification of protease activity across a broad concentration range. The method was successfully applied to quantify protease activity in commercial fruit juice samples, reporting results as papain-equivalent protease activity. Recovery values were matrix-dependent, with clarified matrices showing satisfactory accuracy, while polyphenol-rich samples exhibited signal enhancement due to protein-polyphenol interactions. The assay demonstrated good precision, with intra- and interday relative standard deviations (%RSD) below 3.5%. In summary, this study presents a rapid and sensitive CD-based fluorescence platform for protease activity determination while highlighting purification-dependent surface accessibility as a key factor governing interfacial sensing behavior.