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Updated: Oct 25, 2025
Use of the Protease Fluorescent Detection Kit to Determine Protease Activity
Published on: August 4, 2009
Fluorometry detection for trypsin via inner filter effect between cytochrome C and in-situ formed fluorescent
Zhixia Yao1, Yaosheng Liu1, Yongxing Diao1
1State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin, 130022, People's Republic of China; University of Science and Technology of China, Hefei, Anhui, 230026, People's Republic of China.
Abstract:
A fluorometry assay for trypsin sensitive determination has been presented. The fluorescence of the system at 370/445 nm is derived from thiochrome obtained by in-situ oxidation of thiamine. Based on the inner filter effect, cytochrome C (Cyt C) can quench the fluorescence at 445 nm effectively. Cyt C is specifically hydrolyzed by trypsin through an enzymatic reaction, giving rise to the enhancement of the fluorescence intensity. The change value of fluorescence intensity is proportional to trypsin concentration, which is successfully used for trypsin quantitative detection. This method exhibits good repeatability and selectivity with a detection limit of 0.15 μg mL-1 and a quantification limit of 0.50 μg mL-1 for trypsin sensing. Moreover, it is applied to detect trypsin in practical serum and urine samples with accurate results. The proposed assay is not only a promising candidate for trypsin determination in practical application but also a potentially valuable tool in urine comprehensive analysis and disease diagnosis.
