NIR-Sensitive Squaraine Dye-Peptide Conjugate for Trypsin Fluorogenic Detection

Trypsin enzyme has gained recognition as a potential biomarker in several tumors, such as colorectal, gastric, and pancreatic cancer, highlighting its importance in disease diagnosis. In response to the demand for rapid, cost-effective, and real-time detection methods, we present an innovative strategy utilizing the design and synthesis of NIR-sensitive dye-peptide conjugate ( ) for the sensitive and selective monitoring of trypsin activity by fluorescence ON/OFF sensing. The current research deals with the design and synthesis of three unsymmetrical squaraine dyes , , and along with a dye-peptide conjugate as a trypsin-specific probe followed by their photophysical characterizations. The absorption spectral investigation conducted on both the dye alone and its corresponding dye-peptide conjugates in water, utilizing and respectively, reveals enhanced dye aggregation and pronounced fluorescence quenching compared to observations in DMSO solution. The absorption spectral investigation conducted on dye only and corresponding dye-peptide conjugates in water utilizing and , respectively, reveals not only the enhanced dye aggregation but also pronounced fluorescence quenching compared to that observed in the DMSO solution. The trypsin-specific probe demonstrated a fluorescence quenching efficiency of 61.8% in water attributed to the combined effect of aggregation-induced quenching (AIQ) and fluorescence resonance energy transfer (FRET). FRET was found to be dominant over AIQ. The trypsin-mediated hydrolysis of led to a rapid and efficient recovery of quenched fluorescence (5-fold increase in 30 min). Concentration-dependent changes in the fluorescence at the emission maximum of the dyes reveal that works as a trypsin enzyme-specific fluorescence biosensor with linearity up to 30 nM along with the limit of detection and limit of quantification of 1.07 nM and 3.25 nM, respectively.