A sensitive and label-free trypsin colorimetric sensor with cytochrome c as a substrate

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Protease sensing is crucial for clinical diagnostics and drug development.
• Simple, sensitive, and rapid detection methods are needed for proteases like trypsin.

Purpose of the Study:

• To develop a simple, rapid, label-free, and sensitive colorimetric sensor for trypsin detection.
• To utilize cytochrome c (cyt c) as an enzyme substrate and 3,3´,5,5´-tetramethylbenzidine (TMB) as a chromogenic reagent for trypsin sensing.

Main Methods:

• Cytochrome c (cyt c) was used as a substrate, and 3,3´,5,5´-tetramethylbenzidine (TMB) as a chromogenic reagent.
• Trypsin hydrolyzes cyt c, enhancing the catalytic effect on H2O2-mediated TMB oxidation, leading to a color change.
• Visual detection limit of 50 ng/mL trypsin; spectrophotometric detection limit of 4.5 ng/mL in the range of 5.0 ng/mL to 2.0 μg/mL.

Main Results:

• The sensor enabled visual detection of trypsin down to 50 ng/mL.
• Spectrophotometric analysis showed a linear response from 5.0 ng/mL to 2.0 μg/mL with a detection limit of 4.5 ng/mL.
• High precision was observed with relative standard deviations of 2.5% and 1.7% for trypsin concentrations of 50.0 ng/mL and 1.0 μg/mL, respectively.

Conclusions:

• A novel, sensitive, and rapid colorimetric sensor for trypsin was successfully developed.
• The sensor demonstrated effective application in human urine samples and inhibitor screening.
• This method holds significant potential for clinical diagnostics and drug development applications.