**5. Immobilization techniques in biosensors**

To create a biosensor, biological elements must be immobilized on the transducer's surface. Depending on the component of biorecognition, the physical-chemical environment, the transducer, and the features of the analyte, two well-known ways are physical and chemical immobilization strategies. One of the most crucial steps in sensor preparation is enzyme immobilization, which guarantees that the biomolecules can retain their biological activity, structure, and usefulness [2].

#### **5.1 Chemical immobilization**

This technique involves creating powerful chemical interactions between the functional groups of the biorecognition element and the transducer surface, such as covalent binding or covalent connection. Chemical immobilization techniques, on the basis of chemical bonding type, are divided into two groups: (a) direct covalent binding and (b) covalent cross-linking [2].

#### *5.1.1 Direct covalent bonding*

The biorecognition component is directly covalently bonded, a technique frequently used to immobilize enzymes, to either the electrode or transducer surface, the inert matrix of the membrane, or both. The production of the functional polymer and covalent immobilization are the two approachable steps. Excellent environmental resilience, less leakage of the biorecognition element (enzyme), and strong bond formation between the biorecognition element (enzyme) and matrix are benefits of direct covalent binding. The inability to replenish the

formed matrix after use and the use of harsh chemicals are two downsides of this method [2].

#### *5.1.2 Covalent cross-linking*

Cross-linking is a technique where biorecognition elements and inactive proteins create intermolecular covalent cross-linkages, such as enzymes. It uses multifunctional chemicals as linkers to connect enzyme molecules in threedimensional (3D) aggregates to the transducer surface. The optimal conditions for cross-linking include pH, temperature, and ionic strength. It offers advantages like faster reaction times, stronger adhesion, and higher enzyme catalytic activity. However, partial protein denaturation limits cross-linking immobilization and results in covalent cross-links between protein molecules rather than between the protein and matrix [2].

### **5.2 Physical immobilization**

This method relies on affixing enzymes to the transducer's surface without using chemical bonds. These immobilizations consist of (a) entrapment and (b) adsorption.

### *5.2.1 Entrapment*

This method involves using 3D matrices to physically entrap biorecognition components through covalent or noncovalent bonds. Enzymes are added to a monomer solution, polymerized by a chemical reaction, or modified experimental conditions. Biorecognition components are connected to organic or inorganic materials in a 3D network. Organic materials include polydimethylsiloxane, photopolymer, gelatin, alginate, cellulose, and acetate phthalate, while inorganic materials include activated carbon and porous ceramic materials. Sol-gel methods, microencapsulation, and electropolymerization are used for this process [2].

#### *5.2.1.1 Sol-gel method*

At low temperatures, the sol-gel method is a method used to capture enzymes. Metal alkoxides are hydrolyzed and condensed to produce a nanoporous material that contains bioelements in a three-dimensional matrix. It is more easily synthesized, thermally and chemically stable, and capable of encapsulating large concentrations of biomolecules under mild immobilizing conditions [2].

## *5.2.1.2 Microencapsulation*

A semipermeable membrane is enclosed by biorecognition components (such as enzymes) by using the microencapsulation technique. The two processes that are preferred are the polymerization of a monomer at the interface of two immiscible substances and the phase separation of enzyme microdroplets in water-immiscible liquid phases. As a result, the polymeric membrane's internal enzyme is obscured [2].
