Integrated fluorescence-enhanced biosensing and targeted fishing platform for screening biomacromolecular inhibitors: Using chymotrypsin as a model

Regulation and screening of biomacromolecule (e.g., enzyme) activity is one of the core tasks in disease treatment and drug discovery. Natural products are an important source of enzyme inhibitors, but their complex composition makes it difficult to develop effective candidate drug molecules. As a typical biomacromolecular target, chymotrypsin (CHT) exhibits aberrant activity closely associated with diseases such as wound healing, chronic pancreatitis, and emphysema. Therefore, efficient screening of enzyme inhibitors from natural products holds significant importance for clinical diagnosis, treatment, and drug development. This study establishes an integrated screening platform for enzyme inhibitors by combining a Cu²⁺-casein-mediated fluorescence-enhanced biosensing system with targeted ligand capture technology, enabling highly sensitive screening and precise identification of chymotrypsin inhibitors from natural products. In this platform, Cu²⁺-casein functions as a biomacromolecular substrate while also acting as a peroxidase-mimetic enzyme, significantly enhancing the fluorescence signal amplification effect of o-phenylenediamine oxidation. When chymotrypsin hydrolyzes casein, the structure of casein is disrupted, reducing its copper ion-binding capacity. Which leads to decreased catalytic activity and subsequent changes in fluorescence intensity, allowing quantitative detection of chymotrypsin activity with a detection limit as low as 13.6 ng mL⁻¹. In the targeted fishing module, immobilized chymotrypsin (CHT@NH₂-SiO₂) is employed as an affinity medium, and combined with HPLC and LC-MS analysis, enables precise identification of active components in complex samples. This integrated strategy mitigates certain limitations of individual techniques, providing a feasible approach for efficient, high-throughput screening of inhibitors targeting biomacromolecules from complex systems.