*1.2.2 Hydrophilicity*

One of the most attractive physical properties of alginate employed in substance immobilisation in industrial and biotechnological applications is its hydrophilicity which controls the swelling behaviour of the alginate gel. Thanks to the early works of Haug *et al.* [10], who reported the pKa values of M-blocks and G-blocks to be 3.38 and 3.65, respectively, above which values the carboxyl groups become deprotonated leading to electrostatic repulsion, thus conferring high hydrophilicity to the alginate polymer [7]. By the same token, in an acidic solution, the alginate polymer chain tends to aggregate because of the decrease in hydrophilicity brought about by the protonation of the carboxyl groups [11]. This phenomenon of aggregation/precipitation of alginate at lower pH values has been investigated concerning the structural composition. For instance, alginates with higher amounts of alternating heterogeneous blocks (MG and GM) tend to precipitate at lower pH values than those that are composed of homogenous blocks (MM and GG) [12]. This pH-dependent change in the hydrophilicity of alginate polymers and the consequent reversible swelling has been exploited in encapsulation studies in targeted substance delivery. To exert robust control over the swelling property of alginate, an innovative double-layer hydrogel based on alginate-carboxymethyl cellulose and synthetic polymer (polyacrylamide) was fabricated for a sustained drug delivery system by a simple and mild method [13], which was prepared by ionic crosslinking (pH-sensitive in a weak alkaline environment), while the outer layer was fabricated by chemical crosslinking having physicochemical stability, to prevent inner hydrogel expansion [13]. Thus, modulating the physical properties of alginate polymers enables differential manipulation thereof.

#### **1.3 Physical crosslinking of alginate (ionic crosslinking)**

In the presence of polyvalent positive ions (such as Mg2+, Ca2+, and Ba2+), alginate can undergo intra and inter-chain crosslinking, forming a stable 3D, thermally irreversible, and water-insoluble network called hydrogel [2], in a process called gelation. Gelation entails a simple substitution of sodium ions with divalent cations of alginate. The type of gel formed, and the rate of gel formation depend on the type and concentration of cation as well as their binding kinetics (Mg2+ < < Ca2+ < Zn2+ < Sr2 <sup>+</sup> < Ba2+). Proper control of cation addition can lead to gel formation with controllable homogeneity. Calcium cation-mediated gelation has been widely reported and preferred method. Alginate can form a stable gel with as low a concentration as 1% (w/v) of the polymer in a relatively simple aqueous process at room temperature providing a reticulated matrix that is biocompatible with slow. Degradation rates [14, 15], which have thus led to its extensive use in the immobilisation of cells and biomolecules, retaining their biological activity. The apparent limitation of calcium alginate gel is the burst effect and fast release due to their high porosity [16], explaining the need to develop alginate-composites, and introduce reactive functionalities to minimise the undesirable loss of the immobilised biomolecules.

#### **1.4 De-crosslinking of alginate**

The reversibility of cation-mediated crosslinking of alginate is a limitation when the application requires gels integrity, while on the other hand, reversibility brings flexibility in some applications. The commonly used alginate matrices crosslinked with Ca2+ ions are unstable in the physiological environment or in standard buffer solutions with a high concentration of counterions (such as phosphate and citrate ions) and chelators that can extract Ca2+ from the alginate and liquefy the system [17]. In addition, alginate monomer linkages can be cleaved through free radical oxidation (by oxidative-reductive depolymerisation reactions) [18] and a pH degradation approach. Generally, alginate tends to be more stable around neutral pH values and undergo proton-induced hydrolysis in pH values below 5, leading to the shrinkage of the gel. In contrast, higher pH than 10 initiates degradation via β-alkoxy elimination, leading to alginate gel dissolution [19]. Alginate gel degradation can be achieved by a combination of monovalent cation and adjustment of ionic strength and acidity of the media [20]. The rate of dissolution of alginate can thus be controlled by oxidation [21], reduction of molecular weight of alginate [22], and the use of more vital divalent metals such as barium [17]. Leaching of polymers out of the gel will occur when exposed to a continuous flow of divalent-poor cation medium.

#### **1.5 Chapter overview**

Apart from crosslinking, the carboxyl functional group on this polymer can be modified or activated to become reactive towards other functional groups (such as - NH2) linked to biomolecules, thus, serving as a conjugational point. However, because excessive modification of the carboxyl groups with other (bio)molecules makes them unavailable for the polyvalent cations required for the gelation process, alternative

efforts are being made to incorporate other molecules (such as natural and synthetic polymers, nanoparticles, etc.) into alginate to form alginate composites.

In this review paper, we describe the preparations of alginate composites and discuss their biotechnological applications in the immobilisation of biomolecules such as enzymes, cells, and microbes. The application of alginate as a matrix for the functionalisation of biosensors was also highlighted. Finally, future research courses were provided in using alginate composites for immobilisation technology.
