Enzyme Immobilization on Chitosan-Based Supports - Chitosan Based Hydrogels: Functions and Applications - page 383

acid response could be reduced using more negative potentials where the response to
glucose increases [142].
Zou and coworkers describe a new type of amperometric cholesterol biosensor based on
the enhancement effect of MWCNTs and sol-gel chitosan-silica hybrid composite films.
The MWCNTs were treated with concentrated nitric acid and sulfuric acid to increase the
solubility and dispersibility in solution. These authors chose chitosan reacting with meth-
yltrimethoxysilane (MTOS) and the introduction of MWCNTs to form organic-inorganic
hybrid composite films for the immobilization of cholesterol oxidase. The results show that
analytical performance of the biosensor can be improved greatly after the introduction of
the MWCNTs. This method has been used to determine the free cholesterol concentration
in real human blood samples [143].
In order to avoid enzyme loss, cholesterol esterase and cholesterol oxidase have been
immobilized using GA as a cross-linker onto sol-gel-derived silica/chitosan/MWCNT-
based nanobiocomposite films deposited onto ITO glass for the estimation of esterified
cholesterol [144]. It has been shown that this nanobiocomposite electrode can be used to
estimate total cholesterol in serum samples.
8.5.4.6 Chitosan-Clay Composite Material
Clay is a stable aluminosilicate with a high cation-exchange capacity and different types of
charges (permanent charges on the faces, pH-dependent charges at the edges), and exfoli-
ated clay particles have a platelet shape with nanoscopic size. Compared with organic
polyelectrolytes, clay has the advantages of high chemical stability, good adsorption prop-
erty due to its appreciable surface area, special structural features, and unusual intercala-
tion property.
The use of clay in making nanocomposites has recently been increased because of its cheap
and easy availability. Chitosan beads or films aggregated with clay could enhance densities
and mechanical strengths, thereby extending their application possibilities. Nanocomposites
based on the intercalation of chitosan in clay are robust and stable three-dimensional (3D)
materials by means of cationic exchange and hydrogen bonding processes.
8.5.4.6.1 Chitosan-Montmorillonite (MMT) Beads
Montmorillonite (MMT) as a natural cationic clay is the most widely used layered silicate
in polymer nanocomposites. Chang and Juang prepared chitosan-clay (MMT) composite
beads for immobilization of α-amylase, β-amylase, and glucoamylase by mixing an equal
weight of activated clay and chitosan and cross-linked with GA. It was shown that the
relative activities of immobilized enzymes are higher than free enzymes over broader pH
and temperature ranges [145]. They also compared the properties of the β-glucosidase
immobilized on wet (without freeze-drying) and dried (with freeze-drying) chitosan-
clay composites. Although the reactivity of dried-composite enzyme was higher than that
of the wet-composite enzyme, the wet-composite enzyme appeared much more reliable
because a more stable behavior was observed from batch to batch. In addition, more logi-
cal results and good storage stability were reported for wet-composite enzyme. Therefore,
the wet-composite immobilized enzyme was more reliable if industrial applications were
considered [146,147].
8.5.4.6.2 Chitosan-Synthetic Clay Composite Film
Laponite is a synthetic cationic clay of the formula of (Mg 5.5 Li 0.5 )Si 4 O 10 (OH) 2 (Na + 0.73 ⋅ n H 2 O).
Positively charged chitosan is expected to aggregate with negatively charged laponite