*Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

gelation of the alginate, after which the liposome was removed by treatment with detergent to obtain nanoparticles of alginate as shown in **Figure 8** [63, 62].

Alginate nanoparticles have been used in immunotherapy immobilisation (by encapsulation) of antigens [64, 65]. In a particular experiment aimed at the targeted delivery of antigens to dendritic cells, Zhang *et al*. prepared mannose-functionalised alginate (MAN-ALG) NPs using a Ca2+ external gelation approach [65]. MAN-ALG was used for dendritic cell targeting while the model antigen, ovalbumin, was conjugated to the partially oxidised alginate (ALG-OVA) separately by a pH-sensitive Schiff base bond conjugation. MAN-ALG and ALG-OVA were used to prepare the NPs used for an *in vitro* study, where the NPs were found to enhance the dendritic antigen uptake and cytosolic release. More recently a composite of alginate was used to prepare NPs for the immobilisation of glucose oxidase used for glucose-sensitive insulin delivery systems in mice [66] (**Table 1**).



#### **Table 1.**

*Alginate composites with functional properties or enhanced immobilisation systems.*

## **5. General immobilisation approaches**

There are broadly two categories of biomolecules immobilisation – physical and chemical methods. Physical methods include entrapment, adsorption, and microencapsulation, the chemical ones are covalent attachment, crosslinking, ionic bonding, and conjugation by affinity interactions (**Figure 9**).

*Adsorption on insoluble matrices*: this immobilisation involves the biomolecules' attachment to an insoluble support material via noncovalent linkages such as hydrogen bonding, electrostatic or hydrophobic interactions, and van der Waals forces without any need for pre-activation of the support. Alginate and its composites are among the commonly used immobilisation matrices. This simple and mild approach ensures that biomolecules added directly to the surface of active hydrogel adsorbents are adsorbed without considerable conformational/activity perturbation. Parameters influencing this approach include the pH, nature of the solvent, ionic strength, concentration of the biomolecules, and its adsorbents [79–81]. For instance, when the biomolecule(s) to be immobilised are proteinaceous, the pH and ionic strength control becomes critical, as the net charge of proteins changes according to the pH of the solution, thereby altering the kind of electrostatic interactions (**Figure 10**).

#### **5.1 Covalent immobilisation to insoluble matrices**

Covalent immobilisation is a chemical means of immobilising biomolecules onto the insoluble matrix by means of covalent bonds such as peptide and disulphide bonds, and Schiff base. The biomolecules get attached to the reactive groups (e.g., hydroxyl, amide, amino, carboxyl groups) present on the hydrogel matrix or via the spacer arm, which is attached to the matrix [82]. While this immobilisation method benefits from the non-leakage of the immobilised species, it is associated with a higher *Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

#### **Figure 9.**

*Categorisation of immobilisation methods based on the kind of interaction between the biomolecules and the immobilisation matrix.*

#### **Figure 10.**

*Pictorial representation of common immobilisation strategies.*

tendency to modify the activity of the immobilised biomolecules depending on the choice of immobilisation reagents and conditions [83]. Unreacted reagents used in this approach must be removed (by filtration, centrifugation, or dialysis) or quenched by another reagent. For instance, the unreacted 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide (EDC) used in the activation of carboxyl groups of alginates for covalent conjugation with amino-containing biomolecules must be quenched by 2 mercaptoethanol [84, 85] or removed by either dialysis [47], centrifugation [86] or centrifugal ultrafilter [87]. Some of these immobilisation treatments are incompatible with alginate in terms of stability to solvent and chemicals, coupled with the limited

availability of surface chemistries on alginate, that reactive towards the common functional groups found in biomolecules.

Specifically, because the carboxyl functional group on alginate is involved in both the formation of hydrogel (in the presence of divalent cations) and bioconjugation reactions, additional efforts were made to compose alginate with anchor points using other polymers, such as chitosan, gelatine, and polyvinyl alcohol, as well as between surface-functionalized nanoparticles and quantum dots. Also, fully, or partially oxidised alginate can be used for the covalent immobilisation of biomolecules via Schiff base formation. In 2015, Hou and colleagues reported the covalent immobilisation of *Candida rugosa* lipase onto the magnetic bio-composite of polydopamine/alginate [88]. In this work, the oxidised form of alginate – alginate dialdehyde (ADA) was used in conjunction with polydopamine-coated magnetic nanoparticles as the immobilisation support, where the enzyme is covalently bonded to the ADA via Schiff base formation. While the research benefited from the ease of separation because of the magnetic responsiveness, a significant finding was the enhancement of temperature and pH stability of the immobilised lipase [88]. Another important immobilisation strategy was evaluated by Abd El-Ghaffar and Hasmem using a composite of chitosan grafted with polymethyl methacrylate (PMMA-g-CS) and calcium alginate to immobilise chymotrypsin [33]. Firstly, the enzyme chymotrypsin was bonded to the PPMA-g-CS by covalence and then encapsulated within calcium alginate [33]. The advantage of this approach is the freedom to immobilise as many molecules as possible onto the support since alginate is not directly involved in any chemical binding with the biomolecules, thereby retaining the hydrogelation capability of the alginate. Here, the alginate serves to provide insoluble porous aqueous support for the immobilised enzyme.

More recently, the amino silane-alginate hybrid hydrogel was prepared by Kurayama *et al.* for enzyme immobilisation [89] as an improvement over the previous attempts of preparing alginate microcapsule and then reacting with 3-aminopropyltriethoxysilane (APTES) via electrostatic interaction between the negatively charged carboxyl group of alginate and the positively charged amino group of the APTES [90, 91]. Kurayama *et al.* reported a facile one-step method of immobilising an enzyme on APTES-alginate hybrid beads by simply dripping a solution of sodium alginate containing the enzyme into a crosslinker solution containing CaCl2 and APTES [89]. The hybrid bead was used to immobilise formate dehydrogenase as a model enzyme resulting in an immobilisation yield of 100% and nine cycles of reuse without loss of enzyme activity. This approach is desirable for enzyme immobilisation for its simplicity and efficiency. The presence of APTES in the hybrid beads facilitates electrostatic interactions between the hydrogel and the enzyme, thereby enhancing the retention of the entrapped enzyme within the gel matrix, as evidenced by the many cycles of enzyme reuse. APTES has been used to functionalised magnetic nanoparticles to facilitate the surface reactivity of the nanoparticles towards carboxyl or amine-containing biomolecules using carbodiimide coupling or glutaraldehyde crosslinking, respectively. The new hybrid APTES-alginate can be a platform for immobilising two or more biomolecules having either carboxyl or amino functional groups by selective bonding properties. In another experiment, alginatemontmorillonite composite beads were prepared as an efficient carrier for pectinase immobilisation by Mohammadi *et al*. [92]. Being reputed for their high surface area, high ion exchange, and high adsorption ability, montmorillonite (MMT) fillers have been applied in various nanocomposite systems [93]. Therefore, the authors expected that incorporating MMT into alginate could offer a better immobilisation platform for an industrial enzyme – pectinase. The alginate-MMT beads were crosslinked with glutaraldehyde, after which pectinase was covalently immobilised via glutaraldehydemediated coupling on the beads, displaying a characteristically higher activity than the free enzyme [92].

## **5.2 Immobilisation by microencapsulation and entrapment**

Encapsulation and entrapment are terms that are in most cases broadly used interchangeably to refer to the act of enclosing substances with semi-permeable structures. However, they are technically not the same. Entrapment involves crosslinking the biomolecules to a polymer, such as an alginate, to cover the biomolecule within the porous polymer lattice. The distinguishing principle behind this technique is the formation of a cross-linked polymeric network around the material to be trapped, which is usually performed by mixing the monomers, a cross-linking agent, and the material to be entrapped in a buffered solution and then adding a catalyst system, to initiate the polymerisation [94]. The entrapment allows for the permeation of appropriately sized substrate and release of products in an enzyme study while the porosity can also be adjusted to selectively retain other biomolecules of interest. Encapsulation involves enveloping the cell suspension (or other biological species) within a membrane system in such a way that the membrane creates an intracellular environment for the encapsulated entities, preventing them from leaking out or coming into direct contact with the external environment [95]. Thus, encapsulation offers a flexibility of enclosing any concentration, or volume of cells or biomolecules within membrane envelopes of different configurations. For this reason, encapsulation has been fondly applied in targeted and controlled substance release (**Figure 11**) and the immobilisation of biocatalysts in industrial processes and bioremediation.

This immobilisation approach benefits from the simplicity of the process. Major setbacks that continue to motivate additional research interest are the diffusional constraints where there could be undesirable leakage of the entrapped entity in the

#### **Figure 11.**

*Application of alginate-based encapsulation system in the immobilisation of biomolecules for targeted substance delivery in biomedical application.*

event of changing mechanical properties of the matrix; also, only small-sized substrates/products can be used [82, 96]. Alginate composites have been shown thus far to address the significant setbacks associated with immobilisation by encapsulation.

Alginate-based supports are usually prepared in a gel form by crosslinking between the carboxyl group of the α-l-guluronic acid with a solution of divalent cation crosslinkers such as calcium chloride, barium chloride, or poly(l-lysine). Because of the instability of calcium alginate gel in the presence of high concentrations of phosphate and citrate ions as well as ethylenediaminetetraacetic acid (EDTA), typically found in standard buffer solutions and enzyme reaction medium, composites of alginate became attractive alternatives to overcome such limitations. Taqieddin *et al*. prepared a composite of alginate/chitosan for immobilising β-galactosidase by coreshell microcapsule technology, where alginate was used to encapsulate the enzyme, serving as the core, and chitosan as the semipermeable shell [17]. In this study, using different divalent cations, Ca2+ and Ba2+ liquid and solid alginate cores were obtained, with 60 and 100% loading efficiencies, respectively. One advantage of this approach is the freedom to control the transport of substrates, products, and cofactors by controlling the outer chitosan shell while the biomolecules are stably immobilised in the inner core. This alginate/chitosan core-shell technology was revisited in 2021 by Mirdamadian and colleagues in a slightly different configuration where chitosan served as the core and alginate, the permeable reactive barrier [97]. In their study, the microcapsule core of chitosan was prepared by crosslinking with sodium tripolyphosphate, encapsulating the calcium peroxide (CaO2) nanoparticles, and coating the horseradish peroxidase (HRP)-containing alginate layer crosslinked with calcium [97]. The novelty in this approach has to do with microcapsule immobilisation of the enzyme and oxygen-releasing nanoparticles together but at different layers to produce permeable barriers for the bioremediation of phenol in contaminated waters.

Additionally, this technology addressed the low level of dissolved oxygen limitation associated with the aerobic treatment of phenol-polluted groundwater by encapsulating oxygen-releasing nanoparticles within the core to ensure a continuous *in situ* supply of hydrogen peroxide needed for the HRP reaction. Farias *et al*. also immobilised HRP on calcium alginate beads to remove reactive dyes [98]. A one-step chitosan/alginate core-shell matrix has also been reported, taking advantage of chitosan's pH-responsive sol-gel transition property and the calcium-responsive solgel transition property of alginate [99]. Apart from the simplicity of methodology, environmental friendliness, and mild condition of this approach, this study demonstrated the pH-responsive reversible sol-gel transition of the crosslinked chitosan core, suggesting the possibility to change the core state (liquid or solid) via pH adjustment. It also showed that the alginate thickness could be modulated easily, making the entire technology suitable for controlled substance release through pH and shell thickness controls. Also, monodisperse core-shell alginate (micro)-capsules with oil core generated from droplets millifluidic was published by Martins and colleagues in 2017 using the original alginate inverse gelation method [31]. In this inverse gelation, oil and CaCl2 solution are emulsified and added into the alginate solution so that the Ca2+ ions diffuse from the emulsion drop to the alginate bath, crosslinking the surrounding alginate molecules resulting in core-shell microcapsules. Direct gelation method involves the preparation of alginate and the (bio)molecules to be encapsulated and then dropping the mixture into the bath containing the crosslinker thereby forming alginate beads (**Figure 3**) This approach can be suitable for the immobilisation of enzymes such as lipase that catalyses reaction at the oil-water interfaces.

#### *Alginate-Based Applications in Biotechnology with a Special Mention to Biosensors DOI: http://dx.doi.org/10.5772/intechopen.110737*

A composite of alginate-grafted-β-cyclodextrin has been used to immobilise β-mannanase, an enzyme popularly used to treat coffee and tea waste in the food sector [100]. The choice of β-cyclodextrin, a seven-sugar unit cyclic oligosaccharide was due to its ability to form additional complexes with a wide variety of macromolecules, leading to an enhanced overall stability and adsorption capacity of the resulting matrix. The grafting of cyclodextrin to the alginate resulted in increased pH and temperature optima (typically from 6.0 to 7.0 and 50–55°C, respectively), thermostability, and extended reusability. More studies are needed on the possible interaction between alginate and cyclodextrin and the immobilised species having different net charges. The effect of cyclodextrin on the porosity of the composite gel and the crosslinking is an interesting research aspect to peer into in the future.

#### **5.3 Immobilisation by bio-affinity interactions**

Protein-protein and protein-small molecule binding interactions are among the widely employed immobilisation strategies that have continued to gain popularity in biomedical and biotechnological applications leveraging the selectivity of such interactions. The immobilisation by bio-affinity interaction demonstrates a characteristically high specificity with respect to the identity of the binding partners and the precise location on the matrix/molecules on which the binding takes place. In this context, the binding of the biomolecules to the matrix is by specific ligands such as his-tag on biomolecule to a metal ion-containing matrix, lectin-containing domain to carbohydrate moieties present on the matrix or biotin on the biomolecules to avidin on the matrix (or vice versa) [82]. The ligands can be naturally present on the biomolecules [101] or attached artificially by fusing the nucleotide sequence corresponding to the tag with the gene coding for the protein of interest. Polyhistidine tag is the most well-known genetically encoded affinity tag. His-tag is a sequential hexahistidine residue that can chelate metal ions such as Ni (II), Co (II), Zn (II), and Cu (II). These metal ions can be prepared for immobilisation by treatment with a chelating moiety such as nitrilotriacetic acid [102, 103] or iminodiacetic acid and can be used alone. For example, because alginate is polyanionic, several alginate nickel composites have been prepared from alginate and NiCl2 [104, 105]. Other affinity tags such as biotin and avidin can be attached to the biomolecules by selected chemistries [106]. This selective approach induces minimal conformational changes to the immobilised entities such as cytokines, growth factors, enzymes, mammalian cell lines, and antibodies [107].

#### **5.4 Immobilisation and multipoint stabilisation**

Polyvinyl alcohol/alginate and polyethylene oxide/alginate nanofibers were prepared by electrospinning for the immobilisation of lipase by Doğaç *et al*. [108]. Lipase immobilised on both composite alginate nanofibers showed high enzyme loading, and remarkable thermal, operational, and pH stability properties being stabilised at two levels, first, immobilisation by adsorption followed by glutaraldehyde crosslinking methods.
