A Dual-Detection AIE-Based Fluorescent Probe for Sensitive Detection of Protamine and Trypsin in Biological Media

Accurate detection of biomolecules such as protamine and trypsin is critical due to their significant clinical and diagnostic roles. Protamine sulfate, a highly cationic peptide, is routinely used to reverse the anticoagulant effects of heparin, necessitating precise monitoring to avoid adverse outcomes. Trypsin, a serine protease, is essential in digestion and implicated in disorders such as pancreatitis, cystic fibrosis, and various cancers, making it a valuable disease biomarker. In this work, we introduce a fluorescence-based sensing platform utilizing commercially available carboxylated tetraphenylethylene (CTPE), an aggregation-induced emission (AIE) luminogen. This is a unique demonstration of dual protamine/trypsin detection using a readily available AIE luminogen, eliminating the need for custom synthesis and simplifying real-world deployment. CTPE forms highly emissive aggregates in aqueous buffer via electrostatic complexation with protamine, leading to significant fluorescence enhancement due to restricted intramolecular motion. This enables highly sensitive protamine detection with a detection limit of 0.7 nM, demonstrating successful application in diluted human serum. Comprehensive photophysical analyses─including absorption, steady-state and time-resolved fluorescence spectroscopy─confirmed the formation and disassembly dynamics of the CTPE-PrS complex. Upon trypsin-mediated enzymatic cleavage of protamine, the aggregates dissociate, triggering measurable fluorescence quenching. This system achieves trypsin detection down to 11.8 pM, maintaining a linear response from 0 to 20 nM, even in 20% human urine, underscoring its robustness in complex biological matrices. This dual-analyte sensing platform, based on a readily available AIE fluorophore, offers a sensitive, selective, and scalable approach for real-sample analysis and enzyme activity monitoring.