title: A screen-printed endotoxin sensor based on amperometry using a novel p-aminophenol conjugated substrate for a Limulus amebocyte lysate protease reaction.
abstract: We developed a novel protease detection method based on amperometry using a p-aminophenol (pAP) conjugated substrate. We prepared Boc-Leu-Gly-Arg-pAP (LGR-pAP) as a novel substrate for a clotting enzyme, which is a protease activated by an endotoxin-induced Limulus amebocyte lysate (LAL) cascade reaction. The basic study using cyclic voltammetry revealed that the oxidation peak potentials of LGR-pAP and pAP were sufficiently separated from each other (0.25 V) to conduct amperometric detection of protease activity. We combined simple amperometric detection with a screen-printed electrode chip to produce a practical protease sensor. As an application of the sensor, we demonstrated quantitative endotoxin sensing. The endotoxin activated zymogens contained in the LAL to generate pAP, which was then electrochemically detected by potential step chronoamperometry (PSCA). The observed oxidation current increased with the concentration of endotoxin in the LAL assay solution. This PSCA detection was performed with a disposable chip sensor consisting of a screen-printed electrode and a fluidic channel with a hydrophilic cover. This chip sensor successfully detected 10-1000 EU L(-1) endotoxin within 60 min. This novel amperometric measurement with a screen-printed electrode not only provides compact, low-cost, and easy-to-use sensors for on-site monitoring of endotoxin, but also shows promise for use in other in vitro protease assays for biochemical research, diagnosis, and drug development.
authors: Micro System Integration Center, Tohoku University, 6-6-11 Aoba, Aramaki, Aoba, Sendai 980-8579, Japan. inoue@bioinfo.che.tohoku.ac.jp matsue@bioinfo.che.tohoku.ac.jp.
authors: Kumi Y
authors: KY
authors: Inoue
authors: Shinichiro
authors: S
authors: Takano
authors: Satoko
authors: S
authors: Takahashi
authors: Yosuke
authors: Y
authors: Ishida
authors: Kosuke
authors: K
authors: Ino
authors: Hitoshi
authors: H
authors: Shiku
authors: Tomokazu
authors: T
authors: Matsue
mesh_terms: D000627:Aminophenols / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D055664:Electrochemical Techniques / Q000379:methods*; D004731:Endotoxins / Q000737:chemistry* / Q000378:metabolism; D006737:Horseshoe Crabs; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D000627:Aminophenols; D004731:Endotoxins; D010447:Peptide Hydrolases; C026729:4-aminophenol