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Jung et al. Soft Sci 2024;4:15 https://dx.doi.org/10.20517/ss.2024.02 Page 7 of 44

Immobilization of enzyme toward the fabrication of wearable biosensors
Enzyme immobilization refers to the process of constraining the mobility of an enzyme either entirely or
significantly within a given space. The utilization of immobilized enzymes presents several advantages,
including facile separation from the target analyte upon completion of the reaction, the ability to catalyze
reactions iteratively, and the potential for multiple reuses . However, biocatalysts immobilized on
[116]
electrodes encounter challenges such as limited substrate accessibility and constrained mass transfer. The
overall properties and efficacy of an enzyme are contingent upon factors such as the enzyme type, the
[117]
immobilization matrix employed, and the methodology applied for enzyme immobilization .
Consequently, ensuring effective immobilization of the enzyme molecule onto the electrode is crucial for
facilitating electron transport on the electrode surface. Table 3 summarizes the various immobilization
methods for enzymes, highlighting their respective advantages and limitations in enhancing electrode
performance. Methods for immobilizing enzymes can be either physical [Figure 2D] or chemical
[Figure 2E].

As illustrated in Figure 2D, the physical adsorption method is widely employed to develop enzyme-based
biosensors due to its simplicity. Various studies have reported using this method, which involves
straightforwardly applying the enzyme solution onto the electrode surface through soaking or drop-casting.
The process typically includes an incubation period of overnight or 24 h to facilitate the occurrence of
physical adsorption [122-125] . In this method, non-covalent linkages such as van der Waals forces, hydrophobic
interactions, and hydrogen bonding allow the enzyme to adsorb onto the electrode surface without
[118]
requiring pre-activation of the surface .
Despite maintaining the natural conformation of enzymes, this method faces notable drawbacks, including
enzyme leakage and desorption triggered by changes in temperature, pH, and the nature of the analyte
solution .
[126]
An additional example of a physical method is entrapment [Figure 2D]. In this method, enzymes are
physically confined within a porous polymer matrix by linking side chains of the enzyme surface (amino
acids) to the polymer surface. Importantly, the enzymes are not directly affixed to the electrode surface,
allowing only the traverse of the targeted analyte and products . The entrapment process involves mixing
[106]
the enzyme into a monomer solution, followed by the polymerization of the monomer solution through a
chemical reaction or by altering experimental conditions. Various procedures are employed in an
entrapment method, depending on the entrapment type, such as electropolymerization,
photopolymerization [127,128] , the sol-gel process [129,130] , and microencapsulation [131,132] .

This method provides several advantages, including simplicity of process, preservation of intrinsic enzyme
characteristics, enhanced stability, reduced enzyme leaching and denaturation, absence or required chemical
modification, minimal enzyme demand, various matrix choices (e.g., chitosan, alginate, poly-acrylamide,
etc.), and the ability to optimize the microenvironment for the enzyme by modifying the encapsulating
materials to achieve the optimal pH and/or polarity [118,131,133] . However, there are some drawbacks, such as
enzyme leakage due to the pore size of the support matrix, the occurrence of mass transfer resistance due to
increased gel matrix thickness, limited diffusion of the targeted analyte due to size restrictions, and a
relatively low enzyme loading capacity [120,134-136] .

In addition to physical immobilization methods, chemical immobilization techniques are also explored to
enhance the characterization of immobilized enzymes. Immobilizing enzymes through chemical methods
involves irreversible processes where covalent or ionic bonds are established between the enzyme and the