Nanomaterials functionalized signal-ON/OFF electrogenerated chemiluminescence biosensor for quantization of trypsin based on target-induced cleavage of peptide

Trypsin (TPN) is an important proteolytic enzyme in the digestive system and its abnormal levels are indicative of some pancreatic diseases. As an endopeptidase, TPN can cleave substrate peptide mainly by catalyzing the hydrolysis of the carboxyl side peptide bond of lysine (K) or arginine (R) residues. Based on this hydrolysis cleavage effect, two kinds of nanomaterials functionalized electrogenerated chemiluminescence (ECL) biosensors for the determination of TPN were designed as follows: A signal-ON ECL biosensor was fabricated by attaching substrate peptide (HWR*GWVC, “*” representing the cleavage site, abbreviated as HGC) labeled with ferrocene carboxylic acid (as quencher) onto the surface of NH₂-MIL-53(Al) film which was incorporated with ECL emitting species (bis (2,2′-bipyridine)-4′-methyl-4-carboxybipyridine-ruthenium (N-succinimidylester)-bis (hexafluorophosphate) (Ru complex)). The presence of tryptic cleavage event can eventually lead the Fc complex to leave the electrode and results in the increase of the ECL intensity. HGC labeled with Ru-Ti₃C₂T_x-AuNP complex was used as capture probe and signal probe, which was attached onto NH₂-MIL-53(Al) film modified glassy carbon electrode. A signal-OFF ECL biosensor was built as described above. The presence of TPN lead the Ru-Ti₃C₂T_x-AuNPs to leave the electrode, which resulted in the decreasing ECL intensity. As expected, the fabricated ECL biosensing methods provide excellent sensitivity and selectivity toward the TPN activity. Thus, this strategy shows great potential application in the clinic for diagnosis of TPN-indicating diseases as well as the screening of TPN inhibitor-based anti-cancer drugs.

Graphical abstract