Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However， electrochemical detection of proteases， such as botulinum neurotoxin type E (BoNT/E)， that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase， L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to... More
Facile electrochemical methods for measuring protease concentration or protease activity are essential for point-of-care testing of toxic proteases. However， electrochemical detection of proteases， such as botulinum neurotoxin type E (BoNT/E)， that cleave a peptide bond between two specific amino acid residues is challenging. This study reports a facile and sensitive electrochemical method for BoNT/E detection. The method is based on a two-step proteolytic cleavage using a target BoNT/E light chain (BoNT/E-LC) and an externally supplemented exopeptidase， L-leucine-aminopeptidase (LAP). BoNT/E-LC cleaves a peptide bond between arginine and isoleucine in IDTQNRQIDRI-4-amino-1-naphthol (oligopeptide-AN) to generate isoleucine-AN. Subsequently， LAP cleaves a bond between isoleucine and AN to liberate a free electroactive AN species. The liberated AN participates in electrochemical-chemical-chemical (ECC) redox cycling involving Ru(NH3)6(3+)， AN， and a reducing agent， which allows a high signal amplification. Electrochemical detection is carried out without surface modification of indium-tin oxide electrodes. We show that dithiothreitol is beneficial for enhancing the enzymatic activity of BoNT/E-LC and also for achieving a fast ECC redox cycling. An incubation temperature of 37°C and the use of phosphate buffered saline (PBS) buffer resulted in optimal signal-to-background ratios for efficient BoNT/E detection. BoNT/E-LC could be detected at concentrations of approximately 2.0 pg/mL， 0.2， and 3 ng/mL after 4h， 2h， and 15 min incubation in PBS buffer， respectively， and approximately 0.3 ng/mL after 2-h incubation in bottled water. The method developed could be applied in fast， sensitive， and selective detection of any protease that cleaves a peptide bond between two specific amino acid residues.
