DC Field | Value | Language |
---|---|---|
dc.contributor.author | Park, Seonhwa | ko |
dc.contributor.author | Shin, Yu Mi | ko |
dc.contributor.author | Song, Ji-Joon | ko |
dc.contributor.author | Yang, Haesik | ko |
dc.date.accessioned | 2015-07-23T01:42:18Z | - |
dc.date.available | 2015-07-23T01:42:18Z | - |
dc.date.created | 2015-07-21 | - |
dc.date.created | 2015-07-21 | - |
dc.date.issued | 2015-10 | - |
dc.identifier.citation | BIOSENSORS & BIOELECTRONICS, v.72, pp.211 - 217 | - |
dc.identifier.issn | 0956-5663 | - |
dc.identifier.uri | http://hdl.handle.net/10203/200149 | - |
dc.description.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 degrees 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 4 h, 2 h, 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. | - |
dc.language | English | - |
dc.publisher | ELSEVIER ADVANCED TECHNOLOGY | - |
dc.subject | LEUCINE AMINOPEPTIDASE | - |
dc.subject | ENZYMATIC-ACTIVITY | - |
dc.subject | RAPID DETECTION | - |
dc.subject | IMMUNOASSAY | - |
dc.subject | TOXIN | - |
dc.subject | PROTEASE | - |
dc.subject | BIOSENSORS | - |
dc.subject | INHIBITOR | - |
dc.subject | MECHANISM | - |
dc.subject | BINDING | - |
dc.title | Facile electrochemical detection of botulinum neurotoxin type E using a two-step proteolytic cleavage | - |
dc.type | Article | - |
dc.identifier.wosid | 000356734000031 | - |
dc.identifier.scopusid | 2-s2.0-84929149059 | - |
dc.type.rims | ART | - |
dc.citation.volume | 72 | - |
dc.citation.beginningpage | 211 | - |
dc.citation.endingpage | 217 | - |
dc.citation.publicationname | BIOSENSORS & BIOELECTRONICS | - |
dc.identifier.doi | 10.1016/j.bios.2015.05.016 | - |
dc.contributor.localauthor | Song, Ji-Joon | - |
dc.contributor.nonIdAuthor | Park, Seonhwa | - |
dc.contributor.nonIdAuthor | Shin, Yu Mi | - |
dc.contributor.nonIdAuthor | Yang, Haesik | - |
dc.description.isOpenAccess | N | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordAuthor | Protease sensor | - |
dc.subject.keywordAuthor | Botulinum neurotoxin | - |
dc.subject.keywordAuthor | Leucyl aminopeptidase | - |
dc.subject.keywordAuthor | Electrochemical detection | - |
dc.subject.keywordAuthor | Redox cycling | - |
dc.subject.keywordPlus | LEUCINE AMINOPEPTIDASE | - |
dc.subject.keywordPlus | ENZYMATIC-ACTIVITY | - |
dc.subject.keywordPlus | RAPID DETECTION | - |
dc.subject.keywordPlus | IMMUNOASSAY | - |
dc.subject.keywordPlus | TOXIN | - |
dc.subject.keywordPlus | PROTEASE | - |
dc.subject.keywordPlus | BIOSENSORS | - |
dc.subject.keywordPlus | INHIBITOR | - |
dc.subject.keywordPlus | MECHANISM | - |
dc.subject.keywordPlus | BINDING | - |
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