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

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.