A fluorescence turn on trypsin assay based on aqueous polyfluorene

Area of Science:

• Biochemistry
• Analytical Chemistry
• Polymer Science

Background:

• Trypsin is a key enzyme in biological processes, and its activity is crucial in various physiological and pathological conditions.
• Developing sensitive and real-time assays for enzymatic activity is essential for biological research and drug discovery.
• Existing methods for trypsin detection may require labeled substrates or lack continuous monitoring capabilities.

Purpose of the Study:

• To develop a novel, continuous, and real-time "turn-on" fluorescence assay for trypsin enzymatic activity.
• To utilize the electrostatic interaction between an anionic polymer (P1) and a cationic polypeptide (Arg6) for assay development.
• To demonstrate the utility of the assay for screening trypsin inhibitors.

Main Methods:

• A novel anionic water-soluble conjugated polymer (P1) was synthesized and characterized.
• The electrostatic interaction between P1 and a positively charged polypeptide (Arg6) was exploited to quench P1 fluorescence.
• Trypsin enzymatic activity was measured by the recovery of P1 fluorescence upon Arg6 fragmentation under alkaline conditions.

Main Results:

• The developed assay demonstrated a continuous and real-time "turn-on" fluorescence response to trypsin activity.
• A low limit of detection (LOD) of 0.17 nM for trypsin was achieved.
• The assay successfully screened for inhibitors of trypsin, showing decreased fluorescence recovery in their presence.

Conclusions:

• The developed fluorescence assay provides a sensitive, real-time, and label-free method for quantifying trypsin activity.
• The use of a water-soluble conjugated polymer (P1) and a polypeptide (Arg6) offers a simple and effective platform for enzyme activity monitoring.
• This method holds significant potential for biochemical research, diagnostics, and high-throughput screening of enzyme inhibitors.