A Non-Peptidic Fluorescent Probe for In Vitro Detection of Chymotrypsin

The work presents the development of a non-peptide fluorescent probe, NQ , for in vitro detection of chymotrypsin (Chy). Unlike traditional peptide-based probes that suffer from instability and enzymatic degradation, NQ incorporates hydrocinnamoyl chloride as a recognition group, offering enhanced stability under physiological conditions. Upon enzymatic cleavage by Chy, the probe releases a fluorogenic product (NQOH), generating a turn-on fluorescence signal. NQ exhibits a rapid initial response, with a distinguishable fluorescence signal generated within 10 min, and a low detection limit of 7.64 ng/mL. Although the enzymatic reaction reaches saturation over a period of 30-60 min, the rapid initial turn-on allows for rapid qualitative screening. A notable feature is the probe's large Stokes shift (177nm) combined with an efficient non-peptidic recognition group. This synergistic design significantly improves detection accuracy by minimizing background interference and resisting non-specific degradation in complex biological environments. The probe showed effective performance in HepG2 cell models, confirming its suitability for biological applications. This study underscores the value of non-peptide probe designs for fast and precise enzyme detection in complex biological environments.