Label-free fluorometric detection of chymotrypsin activity using graphene oxide/nucleic-acid-stabilized silver nanoclusters hybrid materials

• Published in: Biosensors & bioelectronics 2017-02, Vol.88, p.210-216
• Main Authors: Li, Shuangqin, Fu, Yuewei, Ma, Xuejuan, Zhang, Yaodong
• Format: Article
• Language: English
• Subjects: Amino acids; Biosensing Techniques - methods; Chymotrypsin; Chymotrypsin - blood; Fluorescence; Fluorescence Resonance Energy Transfer - methods; Fluorimetry; Fragments; Fretting; Graphene; Graphene oxide; Graphite - chemistry; Humans; Label-free; Limit of Detection; Models, Molecular; Nanostructure; Nanostructures - chemistry; Oxides - chemistry; Peptides; Silver - chemistry; Silver nanocluster
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2016.08.029

Pancreatic function tests are used to determine the presence of chronic pancreatitis, particularly in the early stage of the disease. Chymotrypsin is an indicator of pancreatic function and is thus related to pancreatic diseases. A new fluorescent biosensing method for assay of chymotrypsin activity was developed using DNA (dC12)-templated silver nanoclusters and graphene oxide (GO). A peptide probe was also designed using chymotrypsin-cleavable amino acid sequence and a cysteine terminus. The peptide probe formed Ag-S bond to dC12-AgNCs to enhance the fluorescence of dC12-AgNCs. After the addition of GO, the peptide was adsorbed to the negative GO surface and the fluorescence of dC12-AgNCs was quenched by FRET. The peptide was then degraded into amino acid fragments upon addition of chymotrypsin; these fragments were released from the GO surface, and the FRET was terminated. The developed label-free method features lower cost and higher sensitivity to chymotrypsin activity assay compared with conventional fluorescence analysis. The method can be used to analyze chymotrypsin (as low as 3ng/mL, signal/noise =3) across a dynamic range of 0.0–50.0ng/mL. The proposed biosensing strategy can also be extended to other proteases by using different peptide substrates. •A label-free fluorescent method for biosensing chymotrypsin has been developed.•The peptide tagged with a cysteine can largely enhance the fluorescence of silver nanoclusters.•The method has a low detection limit of 3ng/mL chymotrypsin.•The biosensing strategy can be extended to other proteases by using different peptide substrates.