Page 29 - Read Online

Page 8 of 44 Jung et al. Soft Sci 2024;4:15 https://dx.doi.org/10.20517/ss.2024.02

Table 3. Immobilization methods for enzymes
Elements Methods Advantages Disadvantages Ref.
Physical Adsorption Simple process, maintains enzyme structure, Easy diffusion Weak binding and stability, enzyme leaching, [118]
desorption due to temperature, pH, and analyte
properties
Entrapment Simple process, minimal changes in enzyme structure, High Enzyme leaching, mass transfer resistance, [119]
stability, minimal enzyme demand, various matrix choices, limited diffusion, relatively low enzyme loading
ability to optimize the microenvironment capacity
Chemical Covalent High stability, enhanced tolerance of immobilized enzymes, Enzyme structure modified, harsh conditions [120]
minimized catalyst leakage during immobilization, reduction of mass
transfer, irreversible attachment
Crosslinking Good stability and durability, simple method, cost- Denaturing or altering the structure, delays in [121]
effectiveness mass transport

support (or surface of the electrode). One of the prevalent methods is chemical covalent bonding
[137]
[Figure 2E], wherein stable functional groups on enzyme molecules interact with a support matrix . The
functional group on the enzyme should be non-essential for enzymatic activity, typically involving binding
through side chains of the ε-amino, thiol, and carboxylic groups [137,138] . The covalent bonding process
typically involves activating the support using glutaraldehyde or carbodiimide as linker molecules, followed
by enzyme covalent coupling to the activated sites. Linker molecules serve as multifunctional reagents that
act as bridges between the support and enzyme through covalent bonding.

This chemical bonding method has advantages, such as minimal anticipation of conformational changes
when linking non-functional amino acids to the support and enhanced tolerance of immobilized enzymes
to severe physical and chemical conditions (e.g., temperature, denaturants, and organic solvents). On the
other hand, there are notable concerns regarding the harsh conditions during immobilization and the
possibility of similar acids at the active site coinciding with the support linkage site, potentially leading to
[138]
drastic changes in conformation and diminished catalytic properties of the enzyme .
Crosslinking stands out as another chemical method for enzyme immobilization, presenting an irreversible
strategy involving establishing intermolecular crosslinks among enzymes [Figure 2E]. This process entails
the creation of a robust enzyme network by forming numerous covalent bonds. Employing bi-or
multifunctional reagents, such as glutaraldehyde [139,140] , glyoxal [141,142] , and others [143,144] , achieves the desired
crosslinking effect. Using cross-linkers in covalently linking enzymes to electrodes ensures heightened
durability and stability, surpassing the efficacy of van der Waals or hydrophobic interactions and preventing
enzyme leaching. Its widespread adoption in industrial applications is attributed to the simplicity and cost-
effectiveness of the method. However, a lack of meticulous regulation in the procedure may lead to
substantial enzyme loss. Furthermore, using multifunctional reagents in crosslinking introduces the risk of
denaturing or altering the enzyme structure, potentially resulting in a loss of enzymatic activity. Lastly, this
method contends with diffusional delays in mass transport within the system, contributing to slow reaction
rate and extended equilibrium times [145,146] .

Ionic binding, a less commonly employed strategy for enzyme immobilization in wearable electronics,
capitalizes on ionic interactions between the charged surface of the support matrix and amino acids carrying
opposite charges on the enzyme surface [Figure 2E]. The quantity of enzyme bound to the support matrix is
positively correlated with the surface charge density of the materials. The support matrix materials,
including polysaccharide derivatives (e.g., diethylaminoethyl cellulose , carboxymethyl cellulose , and
[147]
[148]
[149]
[150]
[151]
chitosan ), synthetic polymers (e.g., polystyrene derivatives and polyethylene vinyl alcohol ), and
inorganic substances such as silica gel [152,153] , are utilized in this method. In certain instances, physical