Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 379

for biomolecules. In order to overcome this problem, polymers, such as PEO, polyhydroxyl,
hydrophobic poly(vinylpyridine), and PVA, were hybridized into a silica sol-gel matrix.
Chitosan as a natural polymer product was also used to prepare a sol-gel/chitosan com-
posite matrix for enzyme immobilization [16]. For the chitosan-silica hybrid system, many
silylating agents such as tetraethoxysilane (TEOS), tetramethoxysilane (TMOS), tetrakis(2-
hydroxy ethyl) orthosilicates (THEOS), γ-glycidoxypropyltrimethoxysilane (GPTMS),
(3-aoryloxypropyl) dimethoxy methyl silane (APDMOS), and 3-amino propyl tri ethoxy
silane (APTES) have been used as cross-linking agents in the formation of chitosan-silica
hybrid materials.
In addition, silica gel itself had been widely used in many chromatographic adsorbents,
especially when it was used as an affinity chromatographic support. Coating a high-den-
sity cross-linked chitosan layer on porous silica gel beads can be used to inhibit the non-
specific interaction. On the other hand, chitosan can be easily cross-linked by reagents
such as GA to form rigid aquagels. However, severe shrinkage and deformation could not
be easily avoided upon drying the chitosan carriers into the corresponding gels. This can
be improved in conjunction with other solid powders to increase its density and strengthen
its physical properties and thus to expand its applications.
8.5.4.4.1 Cross-Linking by Silylating Agents
Yang et al. prepared porous gels of chitosan-SiO 2 by coupling chitosan with TMOS through
the sol-gel approach and subsequently cross-linking with formaldehyde solution. Since
TMOS was water-insoluble, the mixture of chitosan acetic acid solution and TMOS initially
composed of two phases was made uniform by stirring vigorously till the SiO 2 -containing
phase was distributed evenly in the aqueous solution while the hydrolysis reaction took place.
GOx was selected as a model enzyme to assess its potential applicability for the enzyme
immobilization purpose. The immobilized enzyme was more stable than the free one [128].
A hybrid composite material based on the cross-linking of natural polymer chitosan
with APDMOS was developed for the fabrication of an amperometric H 2 O 2 biosensor. The
cross-linking product butyrylchitosan has the property of a hydrogel because of the inter-
actions of hydrophilic butyrylchitosan and hydrophobic APDMOS, and the amino group
has an “intelligent” pH property in the polymer. In the pH 7.4 PBS, almost all the amino
are nonprotonation, and the film can exist stably. The composite film was used to immobi-
lize HRP on a gold disc electrode. The biosensor retained approximately 75% of its original
activity after about 60 days of storage in a phosphate buffer at 4°C [129].
TMOS or APDMOS as the precursor suffers from poor water solubility, which is unfavor-
able for the gel formation. To increase the solubility, methanol or ethanol is usually added.
As a result, the resultant matrix has poor biocompatibility. Wang and Zhang developed a
new amperometric H 2 O 2 biosensor based on the sol-gel process of THEOS, a completely
water-soluble silica precursor, in the presence of chitosan macromolecules as the nucleating
centers for precipitated silica owing to the formation of hydrogen bonds between hydroxyl
groups and silanols. When the silica precursor THEOS was mixed with aqueous chitosan
solution, no phase separation or precipitation was observed, showing good compatibility.
Moreover, it was found that such mixing was sufficient to promote the synthesis of silica
hybrid material. The sol-gel process could proceed at ambient temperature without the
addition of any catalysts. In the absence of chitosan, however, the sol-gel transition did not
occur even at reduced temperature. It seems that chitosan has a catalytic effect on the sol-
gel process of THEOS. Its gelation time and dynamic rheological properties could be modu-
lated by the amount of added THEOS. HRP entrapped in the hybrid matrix could retain its
native biocatalytic activity and provide a fast amperometric response to H 2 O 2 [130].