Expanding the scope of self-assembled supramolecular biosensors: a highly selective and sensitive enzyme-responsive AIE-based fluorescent biosensor for trypsin detection and inhibitor screening.

Trypsin, a pancreatic enzyme associated with diseases like pancreatic cancer and cystic fibrosis, requires effective diagnostic tools. Current detection systems seldom utilize macrocyclic molecules and tetraphenyl ethylene (TPE) derivative-based supramolecular assemblies, known for their biocompatibility and aggregation-induced emission (AIE) properties, for trypsin detection. This study presents an enzyme-responsive, AIE-based fluorescence 'Turn-On' sensing platform for trypsin detection, employing sulfated-ß-cyclodextrin (S-ßCD), an imidazolium derivative of TPE (TPE-IM), and protamine sulfate (PrS). The anionic S-ßCD and cationic TPE-IM formed a strongly fluorescent supramolecular aggregation complex in an aqueous buffer. However, PrS suppresses fluorescence because of its strong binding affinity with S-ßCD. The non-fluorescent TPE-IM/S-ßCD/PrS supramolecular assembly system exhibits trypsin-responsive properties, as PrS is a known trypsin substrate. Trypsin restores fluorescence in the TPE-IM/S-ßCD system through the enzymatic cleavage of PrS, correlating linearly with trypsin catalytic activity in the 0-10 nM concentration range. The limit of detection is 10 pM. This work contributes to the development of self-assembled supramolecular biosensors using charged TPE derivatives and ß-cyclodextrin-based host-guest chemistry, offering an innovative fluorescence 'Turn-On' trypsin sensing platform. The sensing system is highly stable under various conditions, selective for trypsin, and demonstrates potential for biological analysis and disease diagnosis in human serum. Additionally, it shows promise for the screening of trypsin inhibitors.