Biomedical Engineering Reference
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respectively. The ionotropic gelation method with two different gelation agents including
sulfate and TPP was used for nanoparticle preparation. The activities of β-galactosidase
covalently attached to GA-activated different sized particles were evaluated and com-
pared. The highest activity was shown by the biocatalyst immobilized on nanoparticles
obtained by means of the ionotropic gelation method with sodium sulfate as a gelation
agent. β-Galactosidase fixed on macro- and microspheres exhibited excellent storage sta-
bility in aqueous solution, with no more than 5% loss of activity after 3 weeks of storage at
4°C and pH 7.0 [37].
8.3.4 Chemical Cross-linking Method
As a cross-linking and surface-activating agent, mostly GA is used. This is due to its reli-
ability and ease of use, but more importantly, due to the availability of amino groups for
the reaction with GA, not only on enzymes but also on chitosan. Other difunctional agents
include carbodiimide, epoxide reactants (Gly and ECO), tris(hydroxymethyl)phosphine
P(CH 2 OH) 3 , and so on.
8.3.4.1 Chemical Cross-Linking by One Agent
8.3.4.1.1 GA as a Cross-Linking Agent
Probably the mildest and most straightforward route for functionalizing primary amine
containing materials is reaction with an aldehyde to form the corresponding imine. The
process is very simple and superior to that of other immobilization processes on synthetic
supports. Cross-linking with dialdehyde such as glyoxal and GA is a key technique in
chitosan chemistry, and leads to more stable chitosan derivatives as the amine groups in
different chains link together via the diimine.
Cetinus et al. reported that chemically cross-linked chitosan beads were prepared by the
addition of an acidic chitosan solution to a mixture of di-ion (diphosphate) and nontoxic
dialdehyde (glyoxal) and then treating GA for stability in both alkaline and acidic media.
As chitosan is soluble in acidic solutions, the continuous prolonged exposure of chitosan
beads, made via counterion precipitation, may result in gel softening and bead disintegra-
tion. Moreover, Schiff base formed between chitosan and glyoxal is essentially reversible;
long time operation under acidic conditions may lead to gradual leakage of glyoxal. GA
irreversible cross-linking via Schiff base may lead to chitosan beads exhibiting high opera-
tional stability. The catalase (CAT) and pepsin immobilized in these chitosan beads exhibit
improved resistance against thermal and pH denaturation [38,39]. As a result, GA was
often used.
The various enzyme immobilization trials by chemical cross-linking have been intro-
duced in Section 8.2.4 [20]. In particular, immobilization of enzyme on support materials
activated with GA has received a great deal of attention. Juang et al. reported that the equi-
librium amount of cross-linking can be described by a pseudo-second-order equation. The
amounts of sorption of reactive dye RR222 and Cu (II), and the activities of immobilized
enzymes (phosphatase and β-glucosidase), onto cross-linked beads were greatly affected
by the degree of cross-linking [29]. Thus the control of support activation with GA is very
important.
The precise control of the conditions during support activation with GA has enabled the
modification of the amino groups of the matrix with one or two GA molecules. It has been
reported that monomers and dimers of GA have different reactivities: while the dimer is
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