Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 374

wafer. Carboxymethylpullulan (CMP) and chitosan multilayers were grown on brushes of
Ni nanowires; subsequent grafting of an enzyme (GOx was chosen as the model system)
was performed by conjugating free amine side groups of chitosan with carboxylic groups
of the enzyme (EDC and NHS as chemical coupling agents). The nanowires are finally
released by a gentle ultrasonic treatment. The process is very advantageous over the direct
encapsulation of released nanowires because all time-consuming steps related to nano-
wire centrifugation, rinsing, and redispersion are eliminated. This easy and efficient route
to the biochemical functionalization of magnetic nanowires can find widespread use in
the preparation of a broad range of nanowires with tailored surface properties [111].
8.5.4.1.6 Magnetic Chitosan Beads by Photochemical Polymerization
Magnetic chitosan beads can be prepared via photochemical polymerization in Fe 3 O 4
magnetite aqueous suspension under UV irradiation. Chitosan chains can be grafted from
Fe 3 O 4 nanoparticles via recombination of the chitosan free radicals and then the surface of
Fe 3 O 4 nanoparticles was coated by a cross-linked chitosan shell via further cross-linking
with N , N ′-methylene-bis-(acrylamide) (MBA). The magnetic chitosan beads were of regu-
lar spherical shape, had a mean diameter of 86 nm, and exhibited superparamagnetic
property. Pullulanase was covalently immobilized on magnetic chitosan beads by cross-
linking with GA. The K m value of immobilized pullulanase was 3.89 mg/mL, which was
three times higher than that of free pullulanase. This result indicated that the immobi-
lized process slightly decreased the affinity of pullulanase on magnetic chitosan beads to
the substrate. On the other hand, the activity of immobilized pullulanase decreased slowly
with time as compared with that of free pullulanase. However, this immobilization remark-
ably improved the temperature and operational stability, which made it more attractive in
application aspects [112].
8.5.4.2 Metal Oxide (Except for Iron Oxide)-Chitosan Nanocomposite
Metal oxide (except for iron oxide) semiconductors such as zinc oxide (ZnO), cerium oxide
(CeO 2 ), tin oxide (SnO 2 ), titanium oxide (TiO 2 ) and zirconium oxide (ZrO 2 ) have been found
to exhibit interesting properties such as large surface-to-volume ratio, high surface reac-
tion activity, high catalytic efficiency, and strong adsorption ability. Furthermore, they
have the unique ability to promote faster electron transfer between the electrode and the
active site of the desired enzyme. Biosensing properties of metal oxide nanoparticles can
be improved by incorporating these into chitosan in order to prepare metal oxide-chitosan
hybrid nanobiocomposites.
8.5.4.2.1 Chitosan-ZnO Nanocomposites
Zinc oxide (ZnO) nanoparticles have been used for the fabrication of the transducer sur-
face because of their unique ability to promote faster electron transfer between the elec-
trode and the active site of the desired enzyme. This has been attributed to their remarkable
properties such as wide band gap (3.37 eV), high surface area, high catalytic efficiency,
nontoxicity, chemical stability, strong adsorption ability (high isoelectric point (IEP), ~9.5),
and the immobilization of low-IEP (~5.0) proteins via electrostatic interactions. Recently,
ZnO-chitosan nanobiocomposite films have been proposed for amperometric immu-
nosensors for human IgG. Moreover, ZnO-chitosan composite films can be used for appli-
cation to H 2 O 2 , phenol, and cholesterol biosensors, respectively. Solanki et al. reported the
results of their studies on immobilization of Ur and GLDH onto ZnO-chitosan nanobio-
composite films deposited onto an ITO glass substrate for the fabrication of urea sensors.