# Mini-Review: Recent Technologies of Electrode and System in the Enzymatic Biofuel Cell (EBFC)
## 2 The Enzyme, Support, and Substrate in EBFC
[53] investigate various mediators, namely, polymerized phenothiazines (thionine, methylene green, methylene blue, and toluidine blue). Among the mediators, the poly(methylene green) shows a clear redox-mediating ability with a current density of 3 mA/cm\({}^{2}\). The current produced depends on the concentration of enzyme and glucose and the polymer loading on the electrode surface. The use of MET was detected to have problems such as toxicity of the mediator, leakage,  
Figure 2: CV curves of catalysts using amine-CNT/hemin, PDDA layers and GOx. (**a**) (Aminen-CNT/hemin)/GOx, (**b**) (amine-CNT/hemin)/PDDA/GOx, (**c**) (amine-CNT/hemin)/GOx/PDDA, and (**d**) (amine-CNT/hemin)/PDDA/GOx/PDDA, without the injection of glucose (black dashed line) and with the injection of 30 mM glucose (red solid line). For the tests, 0.01 M PBS (pH 7.4) was used as an electrolyte under air condition [41]. Copyright 2020 Elsevier. producing a low open-circuit voltage (OCV), mediator mobilization [1], being expensive, and instability of the metal ion-based redox [43], even though using MET can produce higher EBFC power than DET [15]. The production of mediators for the interaction between the enzyme and the electrode surface needs to consider the same structure between the mediator and the co-factor and add a polymerizable vinyl group to the mediator if using a mediator-containing polymeric network [54]. [PERSON] et al. [55] synthesized a mediator-modified working electrode through poly(methyl methacrylate-co-vinyl ferrocene) by free radical polymerization. The mediator-modified electrode showed good electrochemical reaction by the GOx and BOD for the glucose oxidation and oxygen reduction reactions, respectively, by optimizing the cell parameters such as polymer amount, temperature, and cell voltage EBFC. The DET is when electrons are transferred directly from enzymes to conductive supports on electrodes [46]. The electrode is the direct redox acceptor. However, the barrier leading to low DET is that the redox center found in the enzyme is located deeply in 3D protein matrices [15; 28]. Among the ways to increase electron transfer through MET and DET is to use small molecules of the active mediators and develop highly conductive materials with a high active site for enzyme loading, respectively [56]. [PERSON] et al. [57] created a double layer of carbon electrode in which the mesoporous carbon nanoparticle and carbon-coated magnetic nanoparticle, referring to the first and second layers of carbon electrode, activated both MET and DET, respectively. The carbon-coated magnetic nanoparticle forms the immobilized enzyme through cascade formation, as shown in Figure 4. [PERSON] et al. [58] produced Fe-based nanoparticles (diameter of the nanoparticle was 25 nm) and coated them on the carbon layer to trigger the DET transfer to reduce the leakage issues and prevent the destructive enzyme modification paths. Fe-based nanoparticles  
Figure 3: Schematic illustration of (**A**) the structures of the main building block materials and (**B**) the assembled enzymatic biofuel cell [48]. Copyright 2019 Royal Society of Chemistry. with a magnetic field gradient feature help the DET process from enzyme to electrode surface while increasing the lifetime of the EBFC. However, some studies use an additive or adhesive agent to help electron transfer, such as Nafion [33; 59], n-type semiconductor polymer [25], and ferritin (Frt) [15]. Electrodes need to have specific characteristics so that the enzyme can be fully utilized. To achieve biocompatibility and biodegradability between enzyme and electrode in EBFC, the electrode used must have low toxicity or chemical inertness and high reproducibility. High surface activity with a large surface-to-volume ratio is required so that the electrode in EBFC can achieve high energy conversion efficiency [35]. The selection of materials for electrodes requires abundance, low cost [20], ease to produce, and scalability, so that the manufacturing and commercialization process becomes easy.