4.8 Article

Immobilization of enzymes at high load/activity by aqueous electrodeposition of enzyme-tethered chitosan for highly sensitive amperometric biosensing

期刊

BIOSENSORS & BIOELECTRONICS
卷 25, 期 12, 页码 2644-2650

出版社

ELSEVIER ADVANCED TECHNOLOGY
DOI: 10.1016/j.bios.2010.04.040

关键词

Glucose oxidase; Alkaline phosphatase; Chitosan-enzyme precrosslinking and electrodeposition; Amperometric biosensing

资金

  1. National Natural Science Foundation of China [20675029, 90713018, 20335020]
  2. State Special Scientific Project on Water Treatment [2009ZX07212-001-06]
  3. Foundations of Hunan Provincial Education Department [05K009]
  4. State Key Laboratory of Chemo/Biosensing and Chemometrics [200902]

向作者/读者索取更多资源

Electrodeposition has been so widely used to immobilize biomacromolecules, and it is always an important topic to increase the load and activity of the immobilized biomacromolecules. We report here on a new, simple and rather universal method for the highly efficient immobilization of enzymes by aqueous electrodeposition of enzyme-tethered chitosan (CS) for sensitive amperometric biosensing. Glucose oxidase (GOx) is chosen here to examine the proposed protocol in detail. GOx was crosslinked to CS with low-concentration glutaraldehyde (GA, 0.080 wt%), and the electroreduction of added H2O2 increased the electrode-surface pH and triggered the electrodeposition of a GOx-GA-CS composite film. The GOx-GA-CS electrodeposition was monitored by an electrochemical quartz crystal microbalance and is theoretically discussed based on an electrogenerated base-to-acid titration model. The prepared first-generation enzyme electrode (CS-GA-GOx/Pt-nano/Au) exhibits a current sensitivity as high as 102 mu A mM(-1) cm(-2) at 0.70 V vs SCE, being 13 times that of the CS-GOx/Pt-nano/Au prepared similarly but without the GOx-CS precrosslinking. UV-vis spectrophotometric determination of the GOx remains in the supernatant liquids after pH-induced CS precipitation suggested a high enzyme load in the GOx-GA-CS film, and amperometric measurements suggested a negligible decrease in the enzymatic activity of GOx after its reaction with the low-concentration GA. Also, the proposed protocol works well for the precrosslinking manner of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysulfosuccinimide activation, the water-electroreduction-triggered CS electrodeposition, the second-generation biosensing mode, a 5.0-mu m-radius Pt ultramicroelectrode, and immobilization of alkaline phosphatase for phenyl phosphate biosensing. The proposed protocol of pretethering the target biomacromolecules to the electrodeposition precusor for immobilization of the biomacromolecule at high load/activity is recommended for wide applications. (C) 2010 Elsevier B.V. All rights reserved.

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