A protamine-mediated FRET system for sensitive fluorescent monitoring of trypsin activity

The development of methodologies capable of real-time, sensitive monitoring of protease activity is of paramount importance for both clinical diagnostics and fundamental research. The present study innovatively constructed a protamine (PRTM)-mediated Förster resonance energy transfer (FRET) system, which exhibited a high level of sensitivity for trypsin concentration. The FRET system was constructed by the mixture of protamine, a tetraphenylethene (TPE) derivative (TPE-2C₄), and a commercially available dye, 4-(4-diethylaminostyryl)-1-methylpyridinium iodide (4-Di-2-Asp). The negatively charged TPE-2C₄ was electrostatically absorbed by protamine, inducing fluorescence emission with a maximum wavelength of 480 nm. Subsequently, 4-Di-2-Asp is efficiently encapsulated to the TPE-2C₄/PRTM aggregate, thereby establishing highly efficient FRET between aggregated TPE-2C₄ (donor) and 4-Di-2-Asp (acceptor). This resulted in a shift of the system's emission from 480 nm to 600 nm. Following the introduction of trypsin, the enzyme catalyzes the hydrolysis of protamine, thereby leading to the disintegration of the entire aggregates and the disruption of the FRET process. The FRET 'turn-off' response exhibited a linear correlation with trypsin concentration over the range of 0-2 μg/mL, with an LOD of 9.11 ng/mL in the buffer solution. This correlation was further validated in 10% artificial urine. Consequently, by monitoring the reduction in fluorescence intensity, sensitive quantitative monitoring of trypsin activity can be achieved with discernible changes.