title: Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage.
abstract: 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.
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea.
authors.forename: Seonhwa
authors.initials: S
authors.lastname: Park
authors.affiliation: Department of Biological Sciences, KAIST Institute for the BioCentury, Cancer Metastasis Control Center, KAIST, Daejeon 305-701, Republic of Korea.
authors.forename: Yu Mi
authors.initials: YM
authors.lastname: Shin
authors.affiliation: Department of Biological Sciences, KAIST Institute for the BioCentury, Cancer Metastasis Control Center, KAIST, Daejeon 305-701, Republic of Korea.
authors.forename: Ji-Joon
authors.initials: JJ
authors.lastname: Song
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan 609-735, Republic of Korea. Electronic address: hyang@pusan.ac.kr.
authors.forename: Haesik
authors.initials: H
authors.lastname: Yang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods; D001905:Botulinum Toxins / Q000032:analysis* / Q000378:metabolism; D001906:Botulism / Q000382:microbiology; D003014:Clostridium botulinum / Q000201:enzymology* / Q000302:isolation & purification / Q000378:metabolism; D060766:Drinking Water / Q000382:microbiology; D055664:Electrochemical Techniques / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D009498:Neurotoxins / Q000032:analysis* / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010084:Oxidation-Reduction; D059748:Proteolysis
chemical_list: D060766:Drinking Water; D009498:Neurotoxins; D009842:Oligopeptides; D001905:Botulinum Toxins; C096326:botulinum toxin type E
keywords: Botulinum neurotoxin; Electrochemical detection; Leucyl aminopeptidase; Protease sensor; Redox cycling