Nitrogen-doped carbon quantum dots fabricated from cellulolytic enzyme lignin and its application to the determination of cytochrome c and trypsin.

A sensitive and effective strategy for the detection of cytochrome c (Cyt c) and trypsin was developed using biomass nitrogen-doped carbon quantum dots (N-CQDs) as the fluorescence probe. N-CQDs were synthesized through a one-pot hydrothermal method by utilizing cellulolytic enzyme lignin as the carbon source and ammonia as the solvent and nitrogen source. The obtained N-CQDs had good water solubility and stable optical properties. The introduction of nitrogen increased fluorescence quantum yield (QY) to 8.23%, which was almost four times as high as that before nitrogen doping. The N-CQDs were fabricated as a label-free biosensor to detect Cyt c and trypsin. The fluorescence of N-CQDs was quenched with positively charged Cyt c due to electrostatic induction aggregation and static quenching. However, Cyt c tended to be hydrolyzed into small peptides in the presence of trypsin, which caused fluorescence recovery of the N-CQDs/Cyt c complex. A wide linear response range was achieved for Cyt c within 1-50 µM and the developed N-CQDs/Cyt c complex displayed a linear response for trypsin within 0.09-5.4 U/mL. The detection limits were 0.29 µM for Cyt c and 0.013 U/mL for trypsin, respectively. Furthermore, this assay had been applied to Cyt c and trypsin detection in serum samples with the recoveries in the range of 94.6-98.5% and 95.5-102.0%, respectively. The established method was sensitive, selective, easy to operate, and low cost, which proved its potential application in clinical diagnosis. The synthesis and fluorescence mechanism of N-CQDs and the strategy for Cyt c and trypsin detection.