Carbon dots doped with nitrogen as an ultrasensitive fluorescent probe for thrombin activity monitoring and inhibitor screening

A simple and sensitive fluorometric assay based on nitrogen-doped carbon dots (N-CDs) was developed for the determination of thrombin (TB) activity in human serum samples and living cells. The novel N-CDs were pre-pared by a facile one-pot hydrothermal method using 1,2-ethylenediamine and levodopa as precursors. Such N -CDs exhibited green fluorescence with excitation/emission peaks at 390/520 nm and a high fluorescence quantum yield of approximately 39.2%. H-D-Phenylalanyl-L-pipecolyl-Larginine-p-nitroaniline-dihydrochloride (S-2238) was hydrolyzed by TB to produce p-nitroaniline which was capable of quenching the fluorescence of N -CDs due to an inner filter effect. This assay was used to detect TB activity with a low detection limit of 11.3 fM. The proposed sensing method was then expanded to the TB inhibitor screening and exhibited excellent appli-cability. As a typical TB inhibitor, argatroban was determined in a concentration as low as 1.43 nM. The method has also been successfully employed for the determination of TB activity in living HeLa cells. This work showed significant potential for TB activity assay in clinical and biomedicine applications.

Automated summary

A simple and sensitive fluorometric assay based on nitrogen-doped carbon dots (N-CDs) was developed for the determination of thrombin (TB) activity in human serum samples and living cells. The N-CDs exhibited green fluorescence and a high fluorescence quantum yield. TB hydrolyzed a specific substrate, leading to fluorescence quenching of the N-CDs. The assay showed a low detection limit for TB activity and could be used for TB inhibitor screening and activity determination in living cells.