A novel electrochemical biosensor for highly selective detection of protease biomarker from Bacillus licheniformis with D-amino acid containing peptide.

A simple, selective and sensitive electrochemical biosensor has been developed to detect protease biomarker from Bacillus licheniformis, a recognized model of the biochemical warfare agent Bacillus anthracis. In this assay, the biosensor is constructed using a d-amine acid containing substrate peptide via self-assembly of cysteine residual at the C-terminal. A biotin modifier is labelled at the N-terminal of the substrate peptide. This enables sensitive electrochemical detection of the intact substrate peptide using a streptavidin-conjugated alkaline phosphatase, which catalyzes the conversion of electrochemically inactive 1-naphthyl phosphate into electrochemically active phenol. In the presence of the protease biomarker, the peptide is cleaved, and the biotin moiety is removed away from the electrode surface, which results in a decreased electrochemical signal corresponding to the concentration of the protease biomarker. This electrochemical biosensor is simple, sensitive and cost effective. The introduction of d-amino acids into the peptide substrate enables high species selectivity and eliminates the steps for enzyme isolation and purification. Under optimized conditions, the protease can be determined in the concentration range from 0.5 to 100 μg mL(-1) with a detection limit to 0.16 μg mL(-1).