# Mini-Review: Recent Technologies of Electrode and System in the Enzymatic Biofuel Cell (EBFC)
## 2 The Enzyme, Support, and Substrate in EBFC  
Enzymes are placed or immobilized on the surface of support materials, and the efficient immobilization of the enzyme is challenging. There are various ways to immobilize the enzyme on the electrode surface, such as encapsulation [3], physical adsorption, entrapment in conduction polymer [29], crosslinking [30], layer-by-layer assembly [31], and covalent attachment [32], in which the enzyme can be reusable, cost-effective, and recyclable [33]. For more understanding, the reader can view the technologies of immobilization from this reference [34]. Figure 1 shows the differences in physical adsorption, covalent attachment, and encapsulation of the enzyme immobilization [35]. The activity and stability of immobilized enzymes depend on the conditions and microenvironment in which enzyme molecules are confined [36]. Not only that, the materials to be used to immobilize the enzyme need to consider the hardness and electrical conductivity of the materials [33]. A novel approach by [PERSON] et al. [37] used the defect and dislocation of nickel-doped MoSe\({}_{2}\) nanoplates for enzyme entrapment for both the bioanode and biocathode. In contrast, [PERSON] et al. [21] used the interaction between NiMoSe\({}_{2}\) nanoplates and a polypyrrole matrix-single walled carbon nanotube to immobilize the biomolecules. The use of 2D NiMoSe\({}_{2}\), which has a high defect, can help increase enzyme loading and entrap the enzyme, and CNT and polypyrrole help conductivity on the electrode surface. The excellent performance and reversibility in using this material have produced high-speed kinetics with a value of 15.6 s\({}^{-1}\). A convenient immobilization method recommended for interaction between enzyme and support [30] needs to increase the enzyme's storage, solvent, pH, and thermal stability [33; 38; 33]. However, some factors have been detected behind enzyme-support interactions that lead to low power and energy density. There is the inadequate and inefficient interaction [16] as well as imperfect [20] and low enzyme loading between enzyme and support that reduces electron transfer [39], leaches out enzyme [26], leads to degradation in long-term operation, causes potential enzyme poisoning when using non-biocompatibilitysupport materials, reduced lifetime [21], and limited enzyme stability [24; 39]. Various materials and methods are used to alleviate this problem that give different chemical, mechanical, and electrical properties [39; 40]; for example, [PERSON] et al. [41] reduced the leached out enzyme problem by linking the interaction between hemin and carbon nanotubes through functionalized amine groups to form amide bonds, as shown in Figure 2. Meanwhile, they conjugated the GOx enzyme with two poly (dimethyl-diallyl ammonium chloride), producing a sandwich structure, which maintains GOx loading. The stability of this chemical bonding has kept 82.1% activity for four weeks. Figure 2 also shows that various interaction experiments produce different electrochemical reaction activities. In addition to covalent bonds formed through the amino group, crosslinking agents are also possible through carboxyl (-COOH) groups [42]. [PERSON] et al. [43] produced polyethyleneimine (PEI), in which the redox polymer is used to entrap and bind GOx enzyme with F-N/CNT and PEI, thus making physical interaction through electrostatic attraction. PEI offers advantages in increasing electron transfer because it has a lower glass transition that mobilizes between the polymer structures and has high stability, sensitivity [42], and conductivity [16]. In addition, [PERSON] et al. [18] minimized enzyme leaching by producing laser-induced graphene with CNTs in microfluidic EBFCs. The high porosity and low cost of this material increase the active site for the enzyme and give 1.37 times higher power density than without CNTs. [PERSON] et al. [1] produced carbon composites by combining MWCNT with Kraton (non-perfluorinated sulfonated penta block copolymer) to form an excellent matrix for enzyme immobilization.