Biomedical Engineering Reference
In-Depth Information
8.3.4.2 Chemical Cross-Linking by Multiagents
The method of immobilization involving the amino and hydroxyl groups of chitosan is
named “binary immobilization” [22]. The enzyme immobilization via chemical cross-
linking of multiagents generally emerges MPA.
8.3.4.2.1 Carbodiimide and Glutaraldehyde
Hung et al. reported that lipase was immobilized on chitosan beads by a binary method in
which lipase was first linked to the hydroxyl groups of chitosan activated with EDC fol-
lowed by cross-linking more lipase to the amino group of chitosan using GA. The binary
immobilization method yielded the highest protein loading and activity in comparison
with the immobilized lipase prepared by activation with EDC and by cross-linking with
GA. Broader pH tolerance and higher heat stability could be achieved by this method.
Immobilized lipase retained 74% residual activity after ten hydrolysis cycles and 67% after
7 days of storage [47].
de Oliveira and Vieira describe the procedures and performance of four different meth-
ods (physical adsorption, EDC, GA, and EDC-GA) for gilo (as a source of peroxidase) crude
extract immobilization on a chitosan biopolymer-CPE. The highest biosensor performance
was obtained after the immobilization of peroxidase in chitosan by the activation of
hydroxyl groups with EDC and cross-linking with GA, which was incorporated in a CPE.
The lifetime of this biosensor was 6 months (at least 300 determinations) [48].
8.3.4.2.2 Glutaraldehyde and Epichlorohydrin
de Oliveira et al. also developed a new biosensor for gilo immobilization on a chitosan bio-
polymer that was chemically cross-linked with ECO and GA.
Figure 8.5
shows a suggestion
for the immobilization of peroxidase on (a) chitosan by cross-linking with GA and ECO.
The bifunctional GA reacts with the amine groups of chitosan to form Schiff bases, and
with the addition of the ECO, through the opening of the epoxide ring links are formed with
carbon atoms and the chloride group is discharged. The chemical cross-linking of chitosan
with GA-ECO and peroxidase immobilization shows strong interactions, long-term stability,
thermostability, and high sensitivity compared with other recently constructed biosensors.
The lifetime of this biosensor was 8 months (at least 500 determinations) [49].
8.3.4.2.3 Glycidol and Glutaraldehyde
The influence of activation agents (Gly, GA, and ECO) and immobilization time (5, 24, and
72 h) on hydrolytic activity, thermal and alkaline stabilities of CALB was evaluated by
Rodrigues et al. The best derivative, 58-fold more stable than the soluble enzyme, was
obtained when CALB was immobilized on chitosan activated in two steps, using Gly and
GA, in 72 h immobilization time. The stabilization degree of the derivative increased with
the immobilization time, an indication that a multipoint covalent attachment between the
enzyme and the support had really occurred. However, the longer the immobilization
time, the lower the derivative activity. Therefore, the immobilization time that should be
chosen to produce the derivative is a trade-off between these two parameters [21].
8.3.4.3 Cross-Linking Chitosan as Affinity Support
8.3.4.3.1 Metal Affinity Support
Immobilized metal affinity chromatography (IMAC) has been developed as a popular
chromatographic tool for the purification of enzymes and proteins. Immobilized metal
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