A sensitive assay for trypsin using poly(thymine)-templated copper nanoparticles as fluorescent probes.

Area of Science:

• Biochemistry
• Analytical Chemistry
• Nanotechnology

Background:

• Enzyme assays are crucial for diagnostics and research.
• Developing sensitive and simple detection methods for proteases like trypsin remains an active area of research.
• Copper nanoparticles (CuNPs) offer unique optical properties for biosensing applications.

Purpose of the Study:

• To develop a novel, simple, and sensitive fluorescence strategy for trypsin detection.
• To utilize the interaction between copper nanoparticles and trypsin-catalyzed hydrolysis products of cytochrome c for sensing.
• To establish a new biochemical sensing strategy for quantitative trypsin determination.

Main Methods:

• A fluorescence-based assay was designed using polythymine (poly T)-templated copper nanoparticles (CuNPs) as fluorescent probes.
• Cytochrome c (Cyt c) was employed as a substrate for trypsin, and its hydrolysis products were utilized to induce a fluorescence quenching effect.
• The formation of nonfluorescent coordination complexes between released cysteine residues and CuNPs was monitored to quantify trypsin concentration.

Main Results:

• The developed method demonstrated a sensitive fluorescence response to trypsin-catalyzed hydrolysis of Cyt c.
• A linear detection range for trypsin was established from 0.25 μg mL⁻¹ to 1000 μg mL⁻¹.
• A low detection limit of 42 ng mL⁻¹ was achieved, indicating high sensitivity.

Conclusions:

• This study presents a novel fluorescence strategy for trypsin assay based on CuNPs and Cyt c.
• The method leverages the quenching effect induced by cysteine residues released from trypsin-mediated Cyt c hydrolysis.
• This work offers a new approach for biochemical sensing and quantitative determination of trypsin.