title: Immobilization of enzymes at high load/activity by aqueous electrodeposition of enzyme-tethered chitosan for highly sensitive amperometric biosensing.
abstract: 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 H(2)O(2) 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 microA 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-microm-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.
authors.affiliation: Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education of China), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha, PR China.
authors.forename: Yueming
authors.initials: Y
authors.lastname: Tan
authors.forename: Wenfang
authors.initials: W
authors.lastname: Deng
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.forename: Qingji
authors.initials: Q
authors.lastname: Xie
authors.forename: Lihong
authors.initials: L
authors.lastname: Lei
authors.forename: Yunyong
authors.initials: Y
authors.lastname: Li
authors.forename: Zhengfa
authors.initials: Z
authors.lastname: Fang
authors.forename: Ming
authors.initials: M
authors.lastname: Ma
authors.forename: Jinhua
authors.initials: J
authors.lastname: Chen
authors.forename: Shouzhuo
authors.initials: S
authors.lastname: Yao
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D048271:Chitosan; D003432:Cross-Linking Reagents; D055664:Electrochemical Techniques; D004800:Enzymes, Immobilized; D005949:Glucose Oxidase; D005976:Glutaral; D006046:Gold; D006863:Hydrogen-Ion Concentration; D057230:Limit of Detection; D053768:Metal Nanoparticles / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D010984:Platinum; D011791:Quartz; D014867:Water
chemical_list: D003432:Cross-Linking Reagents; D004800:Enzymes, Immobilized; D014867:Water; D011791:Quartz; D010984:Platinum; D006046:Gold; D048271:Chitosan; D005949:Glucose Oxidase; D005976:Glutaral